Expression Analysis of Carotenoid Biosynthesis Genes in Carrot (Daucus Carota) Using Real Time Quantitative PCR

www.sol2009.org

SOL 2009thThe 6 Solanaceae Genome Workshop

th th8 -13 November, 2009Le Meridien, New Delhi, India

thThe 6 Solanaceae Genome Workshop 2009

SOL 2009

th th8 -13 November, 2009

Le Meridien, New Delhi, India


Council of Scientific & Industrial ResearchIndia

University of Delhi

Indian National Science Academy

Dept. of Biotechnology&

Dept. of Science & TechnologyGovt. of India

University of Hyderabad

National Institute of Plant Genome Research


SOL 2009 1

CONFERENCE CO - CHAIRS

R. P. Sharma, University of Hyderabad J. P. Khurana, Delhi University South Campus Renu Swarup, Advisor, DBT

THE INTERNATIONAL SOL CHAIRS AND CO-CHAIRS

Dani Zamir (Israel) Akhilesh Tyagi (India) Jim Giovannoni (USA)

Satoshi Tabata (Japan) Giovanni Giuliano (Italy) René Klein Lankhorst (The Netherlands)

Sandra Knapp (UK)

PATRONSM. K. Bhan, Secretary, S. E. Hasnain, Vice-Chancellor

Dept of Biotechnology, New Delhi University of Hyderabad, Hyderabad

NATIONAL ORGANIZING COMMITTEE

E. A. Siddiq, Hyderabad Asis Datta, New Delhi S. K. Sopory, New Delhi

Akhilesh Tyagi, New Delhi M. Udaykumar, Bangalore R. P. Sharma, New Delhi

R. P. Sharma, Hyderabad J. P. Khurana, New Delhi Usha Vijayaraghavan, Bangalore

J. Nagaraju, Hyderabad H. P. Singh, New Delhi P. L. Gautam, Chennai

Renu Swarup, New Delhi Meenakshi Munshi, New Delhi A. S. Raghavendra, Hyderabad

Appa Rao Podile, Hyderabad V. Krishamurthy, Rajahmundry S. K. Sharma, New Delhi

Mathura Rai, Varanasi Meenakshi Srinivas, Bangalore S. K. Pande, Shimla

P. S. Ahuja, Palampur Rakesh Tuli, Lucknow P. Anandkumar, New Delhi

Jayarama, Chikmagalur N. K. Singh, New Delhi Shashi Bala Singh, Leh

Debasis Chattopadhyay, New Delhi Sunil Mukherjee, New Delhi Usha Barwale, Jalana

Suren Tikoo, Pune

THE INTERNATIONAL SOL STEERING COMMITTEE MEMBERS

A. K. Tyagi (India) Antonio Granell Richart (Spain) Barbara Baker (USA)

Barry Flinn (Canada) Carl Braun (USA) Byung - Dong Kim (Korea)

Chris Bowler (France) Christiane Gebhardt (Germany) Chuanyou Li (China)

Daisuke Shibata (Japan) Dani Zamir (Israel) Doil Choi (Korea)

Dominique Crouzillat (France) Erik Postma (The Netherlands) Gerard Bishop (UK)

Giovanni Giuliano (Italy) Graham Seymour (UK) Guozhen Liu (China)

Jim Giovanonni (USA) Jonathan Jones (UK) Lukas Mueller (USA)

Lynn Bohs (USA) Marc Zabeau (Belgium) Miguel Botella (Spain)

Mark Stitt (Germany) Yongbiao Xue (China) Mondher Bouzayen (France)

Pierre de Wit (The Netherlands) R. P. Sharma (India) Sandra Knapp (UK)

Steve Tanksley (USA) Sue Rhee (USA) Tom Gerats (The Netherland)

Willem Stiekema (The Netherlands) René Klein Lankhorst (The Netherland)

SPONSORS

Silver Sponsor

Diamond Sponsor

Other Sponsors

Coffee Board, India

Rijk Zwaan B. V., The Netherlands

Monsanto, USA

Bejo Sheetal Seeds Pvt. Ltd.

Lab India


SOL 20092


SOL 2009 3

8th November, 2009

01:00 pm-06:00 pm Registration

07:00 pm-10:00 pm Opening Ceremony / Reception


SOL 20094

Session I. Structural Genomics 9th November, 2009

Session Chairs:NK Singh (IARI, New Delhi, India) Lukas Muller (Boyce Thompson Institute, Ithaca, USA)

Rapporteurs:Major Singh (IIVR, Varanasi, India)PK Jain (IARI, New Delhi, India)

09:00 am-09:35 am Keynote Speaker: SP-1Lalji Singh (Monsanto Plenary Speaker) (Centre for Cellular and Molecular Biology, Hyderabad, India)Genetic diversity in indian populations and its implications in health and disease

09:35 am-10:05 am Roeland van Ham SP-2 (Keygene N.V.; Plant Research International, Wageningen, The Netherlands)

Status of the international tomato genome sequencing project

10:05 am-10:25 am Steve Stack SP-3(Colorado State University, Fort Collins, USA)

The cytology of pachytene fish in tomato

10:25 am-10:45 am Marco van Schriek SP-4(Keygene N.V., The Netherlands)

Whole genome profiling of tomato and potato

10:45 am-11:15 am Coffee/Tea Break

11:15 am-11:35 am Renu Swarup SP-5(DBT, India)

Plant biotechnology scenario in India

11:35 am-11:55 am Byoung-Cheorl Kang SP-6(Seoul National University, Seoul, Korea)

The complete chloroplast genome sequence of pepper (C. annuum L.): comparative analysis of solanaceous plastomes

11:55 am-12:15 pm José M Jiménez-Gómez SP-7(University of California, Davis, USA)Deep transcriptome sequencing and SNP detection in S. lycopersicum and S. pennellii

12:15 pm-12:45 pm Marcus Droege SP-8(Genome Sequencing, Roche Applied Science)With its long read length the 454 genome sequencer FLX systemenables plant genome sequencing

12:45 pm-12:55 pm Dani Zamir (Bejo Sheetal Plenary Speaker) SP-9 (Hebrew University, Rehovot, Israel)SOL-100

12:55 pm-01:00 pm General Discussion

01:00 pm-02:00 pm Lunch

02:00 pm-03:00 pm Poster Session

Session II. Biotic Stresses 9th November, 2009

Session Chairs:Sunil Mukherjee (ICGEB, New Delhi, India)Doil Choi (Seoul National University, Seoul, Korea)

Rapporteurs:Samir SreevastavaPranjal Yadav

03:00 pm-03:20 pm Teresa Mosquera-Vasquez SP-1(Universidad Nacional de Colombia, Colombia)Identification of diagnostic markers to late blight resistance in Solanum phureja for breeding programs

03:20 pm-03:40 pm Subhra Chakraborty SP-2(NIPGR, New Delhi, India) Comparative proteome analysis of differentially expressed proteins in wild type and genetically engineered crop plants for patho-stress and improvednutritional quality

03:40 pm-04:00 pm Swarup Chakrabarty SP-3 (CPRI, Shimla, India)A major gene with typical R protein structure confers genotype-dependent quantitative resistance to late blight

04:00 pm-04:30 pm Coffee/Tea Break

04:30 pm-05:00 pm Keynote Speaker: SP-4Doil Choi (Seoul National University, Seoul, Korea)Nbhb1, Nicotiana benthamiana homeobox 1, is a JA-responsive positive regulator of pathogen-induced plant cell death

05:00 pm-05:20 pm V.G. Malathi SP-5(IARI, New Delhi, India)Diversity of tomato begmoviruses in India and problems in resistance breeding

05:20 pm-05:40 pm Ben Vosman SP-6 (Wageningen University, Wageningen, The Netherlands)

Late blight resistance genes in populations of Solanum bulbocastanum and related species

05:40 pm-06:00 pm Amar Kumar SP-7(University of Delhi, Delhi, India)

Characterisation of transcriptome from various life cycle stages of the potato cyst nematode Globodera pallida to explore parasitism genes

08:00 pm-10:00 pm SGN Database WorkshopLukas Muller (Boyce Thompson Institute, Ithaca, USA)


SOL 2009 5


SOL 20096

Session III. Abiotic Stresses 9th November, 2009

Session Chairs:KC Bansal (IARI, New Delhi, India)Toni Granell (CSIC UPV, Spain)

Rapporteurs:Manas Chaterjee (Jai Research Foundation,Valsad, India)Snehlata Singla (ICGEB, New Delhi, India)

03:00 pm-03:20 pm Byoung-Cheorl Kang SP-1 (Seoul National University, Seoul, Korea)Characterization and genetic analysis of sy-2, a low temperature sensitive mutant, in Capsicum chinense

03:20 pm-03:40 pm Titti Mariani SP-2(Radboud Univ. Nijmegen, Nijmegen, The Netherlands)Developmental and heat stress-regulated expression of genes in tomato anther

03:40 pm-04:00 pm Anitha Kumari SP-3(Wageningen UR Plant Breeding,Wageningen, The Netherlands)QTL mapping for drought tolerance in potato

04:00 pm-04:30 pm Coffee / Tea break

04:30 pm-05:00 pm Keynote Speaker: Sudhir K. Sopory SP-4 (ICGEB, New Delhi, India) Glyoxalase pathway: role in abiotic stress tolerance

05:00 pm-05:20 pm Miguel Botella SP-5(Universidad de Málaga, Málaga, Spain)Transcriptomic characterization of the tos1 mutant and fine mapping of tos1

05:20 pm-05:40 pm Nurit Firon SP-6 (The Volcani Center, A.R.O., Bet Dagan, Israel)Molecular mechanisms involved in the heat-stress response of developing tomato (Solanum lycopersicum L.) pollen grains

05:40 pm-06:00 pm Marcin Pieczynski SP-7(Adam Mickiewicz University, Poznan, Poland)Artificial micro RNAs as a tool for CBP80 gene silencing in potato to obtain plants with enhanced drought tolerance

08:00 pm-10:00 pm SGN Database WorkshopLukas Muller (Boyce Thompson Institute, Ithaca, USA)

Session IV. Functional Genomics 10th November, 2009

Session Chairs:Akhilesh K Tyagi (NIPGR, New Delhi, India) Rene Klein Lankhorst (Wageningen, The Netherlands)

Rapporteurs:Aditya Gupta (Madurai Kamraj University, Madurai, India)Alok Sinha (NIPGR, New Delhi, India)

09:00 am-09:45 am Keynote Speaker: SP-1Christian Bachem (WUR, Wageningen, The Netherland)

Functional genomics of potato tuber life cycle

09:45 am-10:05 am Giovanni Guiliano SP-2 (ENEA, Roma, Italy)

Evolution of flowering time and fruit quality traits in tomato

10:05 am-10:25 am Asma Aouini SP-3(Univ. of Tsukuba, Tsukuba, Japan)

Identification and characterization of tomato cDNA clones sharing homology with Arabidopsis glutamate receptors and animal GABA receptors

10:25 am-10:45 am Niranjan Chakraborty SP-4(NIPGR, New Delhi, India)

Comparative analysis of alterations in potato (Solanum tuberosum L.) during tuber induction, development and maturation


11:15 am-11:35 am Antonio Di Matteo, SP-5(University of Naples, Italy)

Higher fruit ascorbic acid content in a tomato introgression line is associated with increased expression of genes involved in the pectin breakdown

11:35 am-11:55 am Ana-Rosa Ballester SP-6 (Plant Research International, Wageningen, The Netherlands)

Biochemical and molecular analysis of flavonoid mutants in tomato

11:55 am-12:15 pm Christian Chevalier SP-7(Université de Bordeaux, Bordeaux-Aquitaine, France)

Altering the cell cycle towards endoreduplication contributes to the regulation of cell size and final fruit size in tomato

12:15 pm-12:35 pm Wilco Ligterink SP-8 (WUR, Wageningen, The Netherlands)

Unraveling the complex trait of seed quality in tomato by genetical genomics


12:45 pm-02:00 pm Lunch



SOL 2009 7


SOL 20098

Session V. Metabolomics and Proteomics 10th November, 2009

Session Chairs:MV Rajam (University of Delhi South Campus, New Delhi, India)Arnaud Bovy (Centre for BioSystems Genomics,Wageningen, The Netherlands)

Rapporteurs:PK Burma (University of Delhi South Campus, New Delhi, India)Manoj Goel

03:00 pm-03:20 pm Autar Mattoo SP-1(Beltsville Agricultural Research Center, Beltsville, USA)Metabolic profiling of fruits of polyamine tomato transgenics

03:20 pm-03:40 pm Karen G. Welinder SP-2 (Aalborg University, Denmark)The potato tuber vacuole. Proteomics reveals cultivar-specific variants of the abundant multigene families of stress-storage proteins

03:40 pm-04:00 pm Avital Adato SP-3(Weizman Instiute of Science, Rehovot, Israel)Fruit surface flavonoid accumulation in tomato is controlled by an SlMYB12 regulated transcriptional network


04:30 pm-05:00 pm Keynote SpeakerArnaud Bovy SP-4(Centre for BioSystems Genomics,Wageningen, The Netherlands)Metabolomics data fusion to unravel the regulation of flavour-related metabolic pathways in tomato fruit

05:00 pm-05:20 pm Vipen K. Sawhney SP-5(Univ. of Saskatchewan, Saskatoon, Canada)Proteomic analysis of tomato pollen

05:20 pm-05:40 pm Suping Zhou SP-6(Tennessee State University, Nashville, USA)Proteome changes induced by aluminium stress in tomato roots

05:40 pm-06:00 pm Anuradha Pujar SP-7(Cornell University, Ithaca, NY)Solcyc and metacyc: connecting the genome to the metabolic networks

Session VI. Bioinformatics and Computational Biology 10th November, 2009

Session Chairs:TR Sharma ( IARI, New Delhi, India)Heiko Schoof (MPI Cologne, Germany)

Rapporteurs:Saurabh Raghuvanshi (Delhi University South Campus, New Delhi, India) PK Jain (IARI, New Delhi, India)

03:00 pm-03:20 pm Zhang Zhonghua SP-1(Chinese Academy of Agricultural Sciences, Beijing, China)Comparative sequencing and analysis of syntenic BACs on chromosome 11 between tomato and potato: utility of the conserved regions and implications for organization and evolution of their genomes

03:20 pm-03:40 pm David Francis SP-2 (Ohio State University, Ohio, USA)

Comparative analyses of the potato and tomato transcriptomes

03:40 pm-04:00 pm P.C. Sharma SP-3(GGSIPU, New Delhi, India)Tandem repeats based comparative genomics in Solanaceae


04:30 pm-05:00 pm Keynote Speaker: Naama Menda SP-4(Boyce Thompson Institute for Plant Research, Ithaca, USA)The SOL genomics network: new approaches for clade-oriented phenomics

05:00 pm-05:20 pm Saloni Mathur SP-5(Delhi University, New Delhi, India)Comparative analysis of transcriptional regulators in Solanaceae

05:20 pm-05:40 pm T.R. Sharma SP-6(IARI, New Delhi, India) Understanding molecular basis of host–pathogen interaction: a bioinformatics approach

05:40 pm-6:00 pm Gitanjali Yadav SP-7(NIPGR, New Delhi)Plecdom: a program for identification and analysis of plant lectin domains


SOL 2009 9


SOL 200910

Session VII. Development and Signaling 11th November, 2009 Session Chairs:Sudip Chattopadhyay (NIT, Durgapur, India) and Debasis Chattopadhyay (NIPGR, New Delhi, India )Giovanni Giuliano (ENEA, Rome, Italy)

Rapporteurs:Manoj Prasad (NIPGR, New Delhi, India )Y Sreelakshmi (University of Hyderabad, Hyderabad, India)

09:00 am-09:45 am Keynote Speaker:Jim Giovannoni SP-1(Boyce Thompson Institute for Plant Research, Ithaca, USA)

Genetic regulation of ripening and associated ethylene response in tomato fruit

09:45 am-10:05 am Julin Maloof SP-2(University of California, Davis, USA)

Using genomics to study natural variation in tomato leaf shape and light signaling

10:05 am-10:25 am Alain Tissier SP-3(University of Montpellier, MONTPELLIER CEDEX 5, France)

Terpene biosynthesis in the glandular trichomes of the Solanaceae

10:25 am-10:45 am Antonio Granell Richart SP-4 (IBMCP (CSIC UPV), Spain)

Modulation of the GA perception and response pathway provides plasticity to tomato development


11:15 ma-11:35 am Mohamed Hichem Neily SP-5 (Univ Tsukuba, Tsukuba, Japan)

Alterations in carotenoids contents during tomato fruit development as a result of transgenic spermidine synthase expression

11:35 am-11:55 am Anna Czerednik SP-6(Radboud University Nijmegen, The Netherlands)

Changing tomato fruit texture by manipulation of cell cycle regulators

11:55 am-12:15 pm Aniruddh P. Sane SP-7(National Botanical Research Institute, Lucknow, India)

Role of ERFs in tomato plant development and fruit ripening

12:15 pm-12:35 pm Rameshwar Sharma SP-8(University of Hyderabad, Hyderabad, India)

A mammalian IKK/NF-kB-like signaling pathway regulates nitric oxide level in plants


12:45 pm-02:00 pm Lunch

02:00 pm-06:00 pm Excursion I-IV

07:00 pm-10:00 pm Cultural Programme & Conference Banquet

Session VIII. Molecular Breeding and 12th November, 2009 Session Chairs:T Mohapatra (IARI, New Delhi, India)Dani Zamir (Hebrew University, Jerusalem, Israel)

Rapporteurs:KV Ravishankar (IIHR, Bangalore, India)TK Behera (IARI, New Delhi, India)

09:00 am-09:45 am Keynote Speaker: Dani Zamir (Bejo Sheetal Plenary Speaker) SP-1 (The Hebrew University of Jerusalem, Israel)

“Phenom networks" – knowledge representation of complex phenotypes

09:45 am-10:05 am Avatar Handa SP-2(Purdue University, West Lafayette, USA)

Molecular breeding of fruit crops via novel genetic interventions

10:05 am-10:25 am H. Lindqviat-Kreuze SP-3 (CIP, Lima, Peru)

Molecular approaches to identify sources of resistance to late blight in potato

10:25 am-10:45 am Mathura Rai SP-4 (IIVR, Varanasi, India)

Molecular breeding in solanaceous crops in india


11:15 am-11:35 am Erika Asamizu SP-5(University of Tsukuba, Tsukuba, Japan)

Development of micro-tom mutant database “tomatoma” and a tilling platform

11:35 am-11:55 am Uri Krieger SP-6(The Hebrew University of Jerusalem, Israel)

Genes that drive heterosis

11:55 am-12:15 pm Martin Ganal SP-7(Trait Genetics GmbH, Gatersleben, Germany)

Illumina arrays for tomato and pepper genetic analyses and breeding

12:15 pm-12:35 pm Wencai Yang SP-8(China Agricultural University, Beijing, China)

Development of intron-flanking markers for tomato using both tomato and Arabidopsis genomic information

12:35 pm-12:40 pm Glenn Bryan (SCRI, Dundee, United Kingdom)

SOL 2010


12:45 pm-02:00 pm Lunch


Genetic Enhancement


SOL 2009 11


SOL 200912

Session IX. Biodiversity 12th November, 2009

Session Chairs:HP Singh (ICAR, New Delhi, India) SK Sharma (NBPGR, New Delhi, India)

Rapporteurs:Sunil Archak (NBPGR, New Delhi, India)KK Gangopadhyay (NBPGR, New Delhi, India)

03:00 pm-03:20 pm KS Varaprasad SP-1(NBPGR Regional Station, Hyderabad, India)Pepper genetic diversity: an analysis in the Indian context

03:20 pm-03:40 pm J.L. Karihaloo SP-2 (APCoAB, New Delhi, India)Diversity of eggplant and its wild relatives in India

03:40 pm-04:00 pm R Umashanker SP-3(GVKV, Bangalore, India)TBD


04:30 pm-05:00 pm Keynote Speaker: Thomas Stadler SP-4(ETH Zurich, Zurich, Switzerland)Using multilocus sequence data to infer population structure and demographic history in several wild tomato species (Solanum section lycopersicon)

05:00 pm-05:20 pm Jai Gopal SP-5 (CPRI, Simla, India) Potato genetic resources in India

05:20 pm-05:40 pm Rachel Meyer SP-6(New York Botanical Garden, New York, USA)The history of eggplant domestication: phylogeographic relationships amongcandidate progenitors and Asian heirloom varieties

05:40 pm-06:00 pm H. P. Singh SP-7 (ICAR, New Delhi, India)TBD

09:00 pm-10:00 pm SOL Steering Committee Meeting (Members only)

Session X. RNAi and Emerging Areas 12th November, 2009

Session Chairs:Arun Sharma (Delhi University South Campus, New Delhi, India)Jim Giovannoni (Boyce Thompson Institute for Plant Research, Ithaca, USA)

Rapporteurs:Shailendra Vyas (Delhi University South Campus, New Delhi, India)Saloni Mathur (Delhi University South Campus, New Delhi, India)

03:00 pm-03:20 pm Rob Alba SP-1 (Monsanto Company, St. Louis, USA)Transcriptome network analysis of ripening tomato fruit suggests cross-talk between developmental and environmental regulatory elements

03:20 pm-03:40 pm Alexander Vainstein SP-2(Hebrew University, Rehovot, Israel)Scent, color or both: petunia's perspective

03:40 pm-04:00 pm Sunil Mukherjee SP-3 (ICGEB, New Delhi, India)The mechanism of RNAi suppression by MyMIV-AC2 protein


04:30 pm-05:00 pm Keynote Speaker: Asaph Aharoni SP-4 (Weizman Instiute of Science, Rehovot, Israel) Function and evolution of the TPP riboswitch in the plant kingdom

05:00 pm-5:20 pm Kato K SP-5 (University of Tsukuba,Tsukuba, Japan)Development for massive miraculin production using transgenic tomato fruits in plant factory

05:20 pm-05:40 pm Diretto G SP-6 (ENEA, Rome, Italy)Systems biology of transgenic Solanaceae with altered carotenoid contents reveal novel and unintended effects

05:40 pm-06:00 pm M.V. Rajam SP-7 (University of Delhi South Campus, India)RNA interference for the control of a fungal pathogen (Fusarium oxysporum) in tomato

09:00 pm-10:00 pm SOL Steering Committee Meeting(Members only)


SOL 2009 13


SOL 200914

Satellite Session I. Potato 13th November, 2009

Session Chairs:SK Pandey (CPRI, Shimla, India)Christian Bachem (Wagningen University, Wagningen, The Netherlands)

Rapporteurs:SK Chakrabarti (CPRI, Shimla, India)VU Patil (CPRI, Shimla, India)

09:00 am-09:30 am Keynote Speaker: Christian Bachem SP-1 (Wageningen UR, The Netherlands)

Assembly issues in genome sequencing of heterozygous plants

09:30am-09:50 am Sanwen Huang SP-2(Chinese Academy of Agricultural Sciences, China)

Whole genome shotgun sequencing of doubled monoploid potato

09:50 am-10:10 am Glenn Bryan SP-3(SCRI, Dundee, United Kingdom)

Anchoring the draft potato genome to a diploid genetic map

10:10 am-10:50 am Lefebvre Véronique SP-4(INRA, France)

QTL meta-analysis for late blight resistance in potato including the two novel resistance sources Solanum sparsipilum and S. spegazzinii


11:20 am-11:40 am Jadwiga Sliwka SP-5(Plant Breeding and Acclimatization Institute, Mlochów, Poland)

Identification of new sources of resistance to potato late blight within Solanum michoacanum and S. ruiz-ceballosii

11:40 am-12:00 pm Kare Lehman Nielson SP-6 (Aalborg University, Denmark)

Experimental gene annotation: painting active exons in the potato genome

12:00 pm-12:20 pm Helen Tai SP-7(Agriculture and Agri-Food, Canada)

Comparative transcriptome profiling of diploid potato clones with variation in maturation timing

12:20 pm-12:40 pm Agim Ballvora SP-8 (Max-Planck Institute for Plant Breeding Research, Cologne, Germany)

Potato wart: a biotechnological approach for efficient exploitation of natural resistance


12:45 pm-02:00 pm Lunch

02:30 pm-04:00 pm Closing Ceremony

04:00 pm-04:30 pm High Tea

Satellite Session II. Coffee 13th November, 2009 Session Chairs:Ramesh K Aggarwal (CCMB, Hyderabad, India)Jayarama (Coffee Board, Chikamagalur, India) Alan C. Andrade (Embrapa Recursos Genéticos e Biotecnologia, Brasília-DF, Brazil) Philippe Lasherme (UMR RPB Montpellier, France ) Rapporteurs:N Surya Prakash (Coffee Board, Chikamagalur, India)HL Sreenath (Coffee Board, Chikamagalur, India) 09:00 am-09:30 am G. V. Krishna Rau SP-1

(Coffee Board, Bangalore, India) Overview of Indian industry vis-à-vis world coffee: challenges and opportunities

09:30 am-09:50 am Ramesh K Aggarwal SP-2(CCMB, Hyderabad, India)SuryaPrakash/ Jayarama (Coffee Board, Chikamagalur, India)

Indian initiative on coffee genomics: a brief overview

09:50 am-10:10 am Phillipe Lashermes SP-3(UMR RPB Montpellier, France)

International efforts towards sequencing the Coffea genomes (Coffea L.)

10:10 am-10:30 am Alan Andrade SP-4 (Embrapa Recursos Genéticos e Biotecnologia, Brasília-DF, Brazil)

Molecular analysis of drought tolerance mechanisms in Coffea plants

10:30 am-10:50 am Alvaro Gaitan SP-5(CENICAFÉ, Chinchiná (Caldas) - Colombia)

Functional genomics for the major coffee-pest interactions in Colombia

10:50 am-11:20 am Coffee/Tea Break 11:20 am-11:40 am Dr. Allexandre de Kochko SP-6

(Montpellier, France) Towards the first draft genome sequence of Coffea canephora 11:40 am-12:00 pm Maria Del Pilar Moncada SP-7

(CENICAFE, Manizales, Colombia) Advances in Coffea arabica physical mapping

12:00 pm-12:20 pm Giorgio Graziosi SP-8(Università di Trieste, Trieste, Italy)

Development and analysis of an EST databank of Coffea arabica

12:20 pm-12:40 pm Jorge Mauricio Costa Mondego SP-9(Instituto Agronômico de Campinas, Campinas, Brazil)

Bioinformatics analysis of Coffea arabica ESTs


12:45 pm-02:00 pm Lunch




SOL 2009 15


SOL 200916

Satellite Session III. Tobacco 13th November, 2009

Session Chairs:V. Krishnamurthy (Central Tobacco Research Institute, Rajahmundry, And India)Sanjay Kapoor (University of Delhi South Campus, New Delhi, India)Nikolai Ivanov (Philip Morris Intl., Switzerland)

Rapporteurs:Navin Sharma (ITC R&D Centre, Bangalore, India)Pinky Agarwal (University of Delhi South Campus, New Delhi, India)

09:00 am- 9:30 am Keynote Speaker:V. Krishnamurthy SP-1 (Central Tobacco Research Institute, Rajahmundry, Andhra Pradesh, India)

Tobacco: an ideal plant for biotechnological research

09:30 am-9:50 am Ganesh CT SP-2 (ITC R&D Centre, Hyderabad, INDIA)

Genetic structure analysis of fcv tobacco population for breeding benefits

09:50 am-10:10 am Salej Sood SP-3(ITC R&D Centre, Hyderabad, INDIA)

In vitro induction of haploids and regeneration of fertile doubled haploids in fcv tobacco through androgenesis

10:10 am-10:30 am Akhilesh Kumar SP-4 (NRCPB, New Delhi, India)

Functional analysis of CAMV35S, duplicated CAMV35S and ROLC promoter by using gus (β-glucuronidase) assay in tobacco

10:30 am-10:50 am Carlo Pozzi SP-5(Philip Morris Intl, Switzerland)

Tobacco monosomic lines as a molecular genetics tool


11:20 am-11:40 am Deva Kumari SP-6(ITC R&D Centre, Hyderabad, INDIA)

Involvement of transporters and channels in translocation and redistribution ofpotassium in Nicotiana tabacum

11:40 am-12:00 pm Sharmila Chattopadhyay SP-7

(IICB, Kolkata, India) Role of glutathione as a signaling molecule in plant defense 12:00 pm-12:20 pm Sanjay Kapoor SP-8

(University of Delhi South Campus, New Delhi, India) Tobacco BY-2 cells as a tool for functional validation of genes involved in

hormone biosynthesis/signaling from heterologous systems

12:20 pm-12:40 pm Vikas Koundal SP-9(IARI, New Delhi, India)

role of CMV-2b protein in regulating MiR164 dependent developmental processes in tobacco


12:45 pm-02:00 pm Lunch



Satellite Session IV. Brinjal 13th November, 2009

Session Chairs:Usha Barwale (Mahyco, Jalna, India)Desiree Hautea (University of Philippines, Laguna, Philippines)

Rapporteurs:Radha Anandlakshmi (Mahyco, Jalna, India) 09:00 am-9:30 am Keynote Speaker:

Giuseppe Leonardo Rotino SP-1{(CRA-ORL, Montanaso Lombardo (LO), Italy}

Varietal development in eggplant by combining classical and biotechnological approaches

09:30 am-9:50 am Simon Jan de Hoop SP-2 (East West Seeds Company, Philippines)

Eggplant breeding developments in southeast asia

09:50 am-10:10 am K. K. Gangopadhyay SP-3(National Bureau of Plant Genetic Resources, New Delhi-110 012)

The indian brinjal core collection using geographical and morphological descriptors

10:10 am-10:30 am G.L. Rotino SP-4

(Unità di Ricerca per l’Orticoltura, Montanaso Lombardo, Italy) Genome functional studies in Fusarium resistant eggplant inoculated with

Fusarium oxysporum f. sp. melongenae and/or Verticillium dahliae

10:30 am-10:50 am Discussion


11:20 am-11:40 am K.C. Bansal SP-5(IARI, New Delhi, India)

Expression of E. coli OTSb-A operon in chloroplasts for enhanced tolerance to drought and salinity stresses in eggplant (Solanum melongena)

11:40 am-12:00 pm Srinivas Parimi SP-6(Mahyco, Jalna, India)

Insect resistant crops are an integral part of IPM programs: Bt brinjal

12:00 pm-12:20 pm Usha Barwale Zehr SP-7(Mahyco, Jalna, India)

Bt brinjal development and regulatory processes

12:20 pm-12:40 pm Desiree M. Hautea SP-8(University of the Philippines Los Baños, 4031 College, Laguna, The Philippines)

Bt eggplant (or brinjal) for the philippines: a successful case of south-south partnership


12:45 pm- 02:00 pm Lunch




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SOL 200918

Satellite Session V. Pepper 13th November, 2009

Session ChairsBD Kim (Seoul National University, Seoul, Korea)Mathura Rai (IIVR, Varanasi, India)

Rapporteurs:Major Singh (IIVR, Varanasi, India)Sanjeev Kumar (IIVR, Varanasi, India)

09:00 am- 9:30 am Keynote Speaker:BD Kim SP-1(Seoul National University, Seoul, Korea)Characterization of putative capsaicin synthase promoter activity

09:30 am-10.00 am Ilan Paran SP-2(Volcani Center, Israel)The genetic control of flowering time and shoot architecture in pepper

10.00 am-10.20 am Jundae Lee SP-3(Pepper & Breeding Institute, Korea)Valuable molecular markers associated with several important traits for breedingcommercial F1 variety of chilli pepper in Korea

10:20 am-10:40 am Doil Choi SP-4(Seoul National University, Korea)Comparative genomic analysis between pepper and tomato confirmed genetic synteny but revealed the genome differentiation by accumulation of LTRretrotransposons and unique repeat sequences in pepper genome


11:20 am-11:40 am Giri A.P. SP-5 (National Chemical Laboratory, Pune)Diverse PIN-II proteinase inhibitor genes from Capsicum annuum Linn. associate with endogenous and defense related roles

11:40 am-12:00 pm Véronique Lefebvre SP-6(INRA, France)Phy-p5, a major broad-spectrum resistance QTL conferring resistance to Phytophthora in pepper

12.00 pm-12.30 pm General Discussion

01.00 pm- 02:00 pm Lunch



Satellite Session VI. Tomato 13th November, 2009

Session Chairs:JP Khurana (University of Delhi South Campus, New Delhi, India)Lukas Muller (The Boyce Thompson Institute, Ithaca, USA)Joyce Van Eck (The Boyce Thompson Institute, Ithaca, USA)

Rapporteurs:Shailendra Vyas (University of Delhi South Campus, New Delhi, India)Saloni Mathur (University of Delhi South Campus, New Delhi, India)

Tomato Satellite Session (Part-I) ChromosomesChromosome Updates

09:00 am-09:15 am Lukas Mueller, USA 1, 10

09:15 am-09:30 am Sunghwan Jo, Korea 2

09:30 am-09:45 am Chuanyou Li, China 3

09:45 am-10:00 am Gerard Bishop, UK 4

10:00 am-10:15 am Akhilesh Tyagi, India 5

10:15 am-10:30 am Antonio Granell, Spain 9

10:30 am-10:45 am Mara Ercolano, Italy 12

10:45 am-10:50 am Discussion


Tomato Satellite Session (Part-II) - WGS/NGS

11:20 am-11:50 am Roeland van Ham, The Netherlands

11:50 am-12:20 pm Giovanni Giuliano, Italy

12:20 pm-12:50 pm Sanwen Huang, China

12:50 pm-01:00 pm Discussion

01:00 pm-02:00 pm Lunch




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Satellite Session VII. Secondary Metabolism 13th November, 2009 Session Chairs:RS Sangwan (Agriculture Research Organization, Israel)Efraim Lewinsohn (CIMAP, Lucknow, India)

Rapporteurs:Siddhartha Mishra (CIMAP, Lucknow, India)Asha (CIMAP, Lucknow, India)

09:00 am-09:35 am Keynote Speaker:Efraim Lewinsohn SP-1 (Agriculture Research Organization, Israel)

Metabolic engineering of tomato aroma: the sounds of silent metabolism

09:35 am-10.00 am Mark Taylor SP-2(Scottish Crop Research Institute, UK)

Potato tuber quality traits and secondary metabolism

10:00 am-10:20 am Rajender S Sangwan SP-3(CIMAP, Lucknow, India)

Ashwagandha withanomics: leads in withanolide biosynthesis, novel chemotypes and their metabolic connotations in Withania somnifera

10:20 am-10:40 am Janardan Singh SP-4(CIMAP, Lucknow, India)

Medicinal plants of Solanaceae: growing significance in healthcare since antiquity

10:40 am-11:00 am Michal Oren-Shamir SP-5(Department of Ornamental Horticulture, Bet Dagan, Israel)

Secondary metabolism during active anthocyanin degradation in Brunfelsia calycina flowers


11:30 am-11:50 am Idit Ginzberg SP-6(The Volcani Centre, Israel)

Regulation of glycoalkaloids synthesis in potato

11:50 am-12:10 pm Gianfranco Diretto SP-7(Italian Agency for New Technologies, Energy and Environment, Rome, Italy)

Systematic investigation of carotenoid accumulation in transgenic potato tubers: the long and winding road to the “golden” phenotype

12:10 pm-12:30 pm Ercolano M R SP-8 (University of Naples “Federico II”, Via Università, Portici , Italy)

Assessment of genetic and environmental factors involved in tomato flavor

12:30 pm-12:50 pm Koh Aoki SP-9(Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu, Japan)Gene coexpression analysis of tomato and deduced function of zinc-finger protein gene associated with flavonoid biosynthesesis

12:50 pm-02:00 pm Lunch




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Satellite Session VIII. Viruses 13th November, 2009 Session Chairs:Shelly Parveen (IARI, New Delhi, India)Hanu R. Pappu (WA, USA)

Rapporteurs:Supriya Chakarborty (JNU, New Delhi, India)Vikas Koundal (IARI, New Delhi, India) 09:00 am-09:30 am Keynote Speaker:

Jonathan Whitworth SP-1(Aberdeen, USDA, USA)

Potato virus Y symptoms in potato tubers grown in different environments

09:30 am-09:50 am H.R. Pappu SP-2(WA, USA)

Biological and molecular properties of potato virus S (PVS) and the response ofselected potato genotypes to PVS infection

09:50 am-10:10 am R.K. Jain SP-3

(IARI, New Delhi, India) Present status of chronic and emerging tospoviruses affecting solanaceous

crops in india

10:10 am-10:30 am Shelly Praveen SP-4 (IARI, New Delhi, India)

Viral derived RNAi suppressors: role in pathogenesis and synergism

10:30 am-10:50 am Manash Chatterjee SP-5 (Jai Research Foundation,Valsad, India)

High-throughput mutation breeding in Solanaceae for virus resistance


11:20 am-11:40 am M. Krishna Reddy SP-6(IIHR, Bangalore, India)

Virus diagnosis and development of makers for resistance to viruses in chilli (Capsicum annuum)

11:40 am-12:00 pm Supriya Chakarborty SP-7(JNU, New Delhi, India)

Chilli leaf curl disease is caused by interaction among diverse begomoviruses in india

12:00 pm-12:20 pm D. Datta SP-8(IIVR, Varanasi, India)Validation of ToLCV resistance CAPS marker for TY2 in the parental lines of tomato

12:20 pm-12:40 pm M. Prasad SP-9(NIPGR, New Delhi, India)

Identification and molecular mapping of tomato leaf curl New Delhi virus (ToLCNDV) resistant gene(s) in tomato (Solanum lycopersicum L.)

12:40 pm-12:55 pm General discussion

12:45 pm-02:00 pm Lunch



22

INDEX

LECTURES 23

SESSION I STRUCTURAL GENOMICS 24SESSION II BIOTIC STRESSES 34SESSION III ABIOTIC STRESSES 42SESSION IV FUCTIONAL GENOMICS 50SESSION V METABOLOMICS AND PROTEOMICS 59SESSION VI BIOINFORMATICS AND COMPUTATIONAL BIOLOGY 67SESSION VII DEVELOPMENT AND SIGNALING 75SESSION VIII MOLECULAR BREEDING AND GENETIC ENHANCEMENT 84SESSION IX BIODIVERSITY 93SESSION X RNAI AND EMERGING AREAS 101

SATELLITE SESSION I POTATO 109SATELLITE SESSION II COFFEE 118SATELLITE SESSION III TOBACCO 129SATELLITE SESSION IV BRINJAL 139SATELLITE SESSION V PEPPER 148SATELLITE SESSION VII SECONDARY METABOLISM 155SATELLITE SESSION VIII VIRUSES 164ADDENDUM to the ABSTRACT BOOK 174

POSTERS 188

SESSION I STRUCTURAL GENOMICS 189SESSION II BIOTIC STRESSES 196SESSION III ABIOTIC STRESSES 208SESSION IV FUCTIONAL GENOMICS 216SESSION V METABOLOMICS AND PROTEOMICS 224SESSION VI BIOINFORMATICS AND COMPUTATIONAL BIOLOGY 229SESSION VII DEVELOPMENT AND SIGNALING 233SESSION VIII MOLECULAR BREEDING AND GENETIC ENHANCEMENT 246SESSION IX BIODIVERSITY 261SESSION X RNAI AND EMERGING AREAS 265

SATELLITE SESSION I POTATO 269SATELLITE SESSION II COFFEE 273SATELLITE SESSION IV BRINJAL 288SATELLITE SESSION V PEPPER 290SATELLITE SESSION VI TOMATO 296SATELLITE SESSION VII SECONDARY METABOLISM 298SATELLITE SESSION VIII VIRUSES 304

LIST OF AUTHORS AND PARTICIPANTS 310

AUTHOR INDEX 317

23

LECTURES

SESSION I STRUCTURAL GENOMICS

24

GENETIC DIVERSITY IN INDIAN POPULATIONS AND ITS IMPLICATIONS IN HEALTH AND DISEASE

Lalji Singh Centre for Cellular and Molecular Biology, Hyderabad 500 007, India

India represents one of the largest human biodiversity, consisting of 4635 culturally and anthropologically well-defined populations with little or no gene flow between them. Hence, study on Indian populations known for their cultural and genetic diversity, not only provides insight into their complex origin, history and relatedness, but also helps in understanding molecular pathology of genetic diseases. Therefore, our interest has been to study both population history and molecular mechanism of diseases in Indian populations.

Our study on nearly <10,000 individuals belonging to more than 200 ethnic populations of India, using Y chromosome and mtDNA markers, revealed that the indigenous Andaman Islanders probably are the descendants of the first modern humans out of Africa about 65,000 – 70,000 years ago. In our recent study, we analyzed more than 500,000 genetic markers across the genome of 132 individuals from 25 diverse groups, representing 13 states, all 6 language families, traditionally in “upper” and “lower” castes and tribal groups. We discovered two ancestral groups 'Ancestral North Indians' (ANI) which is genetically close to Middle Easterners, Central Asians, and Europeans, whereas the other, the 'Ancestral South Indians' (ASI), is as distinct from ANI and East Asians as they are from each other. We show that ANI ancestry ranges from 39-71% in most Indian groups, and is higher in traditionally upper caste Indo-European speakers. Groups with only ASI Ancestry may no longer exist in mainland India. However, indigenous Andamans Islanders are unique in being ASI-related groups without ANI ancestry.

The finding that nearly all Indian groups descend from mixtures of two ancestral populations applies to traditional “tribes” as to “castes”. The genetics proves that castes grew directly out of tribal-like organizations during the formation of Indian society.

A medically very important finding is that many groups in modern India descend from a small number of founding individuals and have been isolated from gene flow from other groups for thousands of years. It has medical implications for Indian populations. Recessive hereditary diseases-single gene disorders that occur only when an individual carries two malfunctioning copies of the relevant gene - are likely to be common in populations descended from so few 'founder' individuals. Mapping the causal genes will help to address this problem. The widespread history of founder events in Indian populations helps to explain why the incidence of genetic diseases among Indians is different from the rest of the world. For example, an ancient deletion of 25 bp in the cardiac myosin-binding proteins-C gene (MYBPC3) is associated with heritable cardiomyopathies as well as with an increased risk of heart failure. Its prevalence is high (~4%) in the general populations from the Indian subcontinent. However, this mutation is completely absent among the people from the rest of the world.


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STATUS OF THE INTERNATIONAL TOMATO GENOME SEQUENCING PROJECT

Roeland van Ham, and SOL International Tomato Sequencing Project members

Keygene N.V.; Plant Research International, Wageningen, The Netherlands

The international SOL consortium is currently sequencing, assembling and annotating the tomato genome (Solanum lycopersicum, cv. Heinz 1706). We recently set a novel goal of producing a draft sequence using a whole genome shotgun sequencing (WGS) approach in combination with next generation sequencing (NGS) technologies. Application of the latter is a collaborative effort of partners from SOL, EUSOL and industry (Keygene, Roche, and Applied Biosystems), who have all taken part in the production of data and data analysis. We have now produced ~20X coverage of the genome using the 454 technology, ~30X coverage using the SOLiD technology, and a new physical map using a recently developed proprietary technology by Keygene. We will integrate these datasets with existing datasets (~66 Mb of euchromatic sequence, ~3E6 Sanger shotgun reads, BAC-end and fosmid-end sequences) into a whole genome assembly and use sequence-based anchoring of contigs to the physical map in order to produce a draft sequence of the genome by December 2009. This draft genome sequence will be annotated by ITAG. Our initiative complements the ongoing effort of the international Solanaceae Genome Project (SOL) to sequence the euchromatic part of the tomato genome. In doing so, we will provide a new and indispensable resource that, when integrated with the existing resources, will bring us closer to the ultimate goal of the SOL project: to produce a high quality reference genome sequence of tomato.

THE CYTOLOGY OF PACHYTENE FISH IN TOMATO

Stephen M. Stack, Lindsay A. Shearer, Suzanne M. Royer, Lorinda K. Anderson

Department of Biology, Colorado State University, Fort Collins, CO 80523-1878, USA

Because tomato pachytene chromosomes are 10-15X longer than corresponding mitotic metaphase chromosomes, determining the location of FISH foci is much more accurate on pachytene chromosomes. In addition, localization of DNA sequences can be related to at least four distinct features of pachytene chromosomes. First, in spreads of pachytene chromosomes fixed in 1:3 acetic ethanol, heterochromatin and euchromatin are visible, so it is possible to determine if FISH foci are in or near heterochromatin where genetic activity may be suppressed and repeated sequences tend to be more common. Second, in synaptonemal complex (SC) spreads, FISH foci can be related to the length of chromatin loops extending from SCs. Generally it appears that loops are longer in heterochromatin and in chromosome segments rich in repeated sequences. Third, in SC spreads the location of a FISH focus can be related to the frequency of recombination nodules (RNs) to show the amount of recombination in the vicinity of the FISH focus. Fourth, and most importantly for sequencing the tomato genome, FISH for Cot 100 DNA on SC spreads shows the distribution of repeated sequences along tomato SCs. When the location of a FISH focus is superimposed on the pattern of Cot 100 DNA, this shows the frequency of repeated sequences in the vicinity of the FISH focus. This is of particular importance in determining whether BAC inserts and their contigs should be sequenced.


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WHOLE GENOME PROFILING OF TOMATO AND POTATO

Edwin van der Vossen, Marco van Schriek Keygene N.V., The Netherlands The Whole Genome Profiling (WGP) method enables the de novo construction of a complete high quality physical map. This sequence-based physical map forms the framework to construct a complete genome sequence assembly of plant and animal genomes in a fast and cost-effective manner. We have successfully applied this WGP technology for the crops tomato and potato. A 10 X WGP physical tomato map now exists and is being applied for the assembly of the tomato genome. For this the enzyme libraries MboI, HindIII, EcoRI and the random sheared library were applied. The resulting tomato physical map is roughly 2,500 contigs in size and contains over 52,000 BACs. The average contig size is 0.378 Mbp. On average the BAC specific sequence tags are spaced every 3 Kb allowing for sequence scaffold linkage to the BACs and the physical map as a whole. Similar results have been obtained in the potato WGP physical map. We aim to present the whole genome profiling technology and its application for the tomato and potato physical maps in this overview.

PLANT BIOTECHNOLOGY SCENARIO IN INDIA

Renu Swarup

Adviser, Department of Biotechnology, Ministry of Science & technology, Government of India, New Delhi

Biotechnology with its far reaching implications in the areas of sustainable food production, nutrition security, health care and environmental sustainability, is today emerging as a powerful tool which can convert the country's biological wealth into economic wealth. The Indian biotech sector is gaining visibility globally, and, the greatest challenge today is to ensure the affordability and accessibility of the product of biotechnology to the largest sections of our society. Over the last two decades there has been a conscious effort to address the growing challenges in the area of agriculture and plant biotechnology. Both “public good” and “for profit” research need to become mutually reinforcing to achieve this. The main priorities identified for innovation research are- increased productivity and production; tackling biotic and abiotic stress specially drought and salinity, in view of the declining availability of water as an agricultural input; and improvement in terms of nutritional quality and post harvest. Investment in agriculture and plant related biotechnology has resulted in significantly enhanced R&D capability and institutional building over the years. The emphasis now is on converting the research leads into useable products by developing effective management and commercialization strategies. Some of the priority crops for research have been rice, wheat, cotton, maize, mungbean and tomato. In addition other cereal and non cereal crops and horticultural and plantation crops including tea, coffee and spices have also been studied intensively. Both basic and applied research has received equal emphasis and today India figures prominently in the world map especially in the areas of Plant Genomics (rice, tomato coffee, potato etc.) In the area of transgenics, also we are in a fairly advanced stage with Bt cotton already having received clearance for commercial scale cultivation and a number of other transgenics developed indigenously awaiting clearance for commercialization. Some of the products are transgenic mustard hybrid developed with banase/barstar gene, potato with high protein level content (Ama 1 gene), transgenic tomato with reduced oxalate content, increased shelf life and resistance to tomato leaf curl virus, transgenic rice with salinity tolerance gene, Bt brinjal etc. Some of the other priorities for research, in addition to transgenics and genomics, are marker assisted selection and tissue culture.

The country has now prepared a road map for the next 5 years and the thrust is on basic research, applied research and product development. Public private partnership is being encouraged for promotion of innovation and commercialization of technologies and products. To give a further boost to this, a transparent, simplified regulatory system is also being proposed.

To achieve the ultimate goal as defined above, a strong technology and product based bias is essential and for this there will be a special thrust on translational research involving agricultural and basic science institutes. It is the synergy between technology development, validation and diffusion which will drive the programme forward.


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THE COMPLETE CHLOROPLAST GENOME SEQUENCE OF PEPPER (C. ANNUUM L.): COMPARATIVE ANALYSIS OF SOLANACEOUS PLASTOMES

1 2 3 4 3 1Yeong deuk Jo , Jongsun Park , Jungeun Kim , Wonho Song , Cheol-Goo Hur & Byoung-Cheorl Kang 1 2Department of Plant Science, Seoul National University, Seoul 151-921, Korea; Fungal Bioinformatics

3Laboratory, Seoul National University, Seoul 151-921, Korea; Omics And Integration Research Center, Korea 4Research Institute of Bioscience & Biotechnology, 111 Gwahangno, Yuseong-gu, Daejeon 305-806, Korea; Fungal

Bioinformatics Laboratory, Seoul National University, Seoul 151-921, Korea The complete sequence of chloroplast genomes have been analyzed in Solanaceae crops such as tobacco, potato and tomato. However, no reports have been made on chloroplast genome of hot pepper. In this study, we analyzed the complete chloroplast genome sequence of hot pepper. Chloroplast genome sequences of a male-sterile pepper line, FS4401, were obtained using GS-FLX machinery and assembled as seven contigs. The gaps between these contigs were filled by PCR to generate a contig equivalent to complete sequence of pepper chloroplast genome. The genome is 156,781bp in length and includes a pair of inverted repeats (IR) of 25,783 bp. The content and the order of a total of 133 genes in pepper chloroplast genome are identical to those of other Solanaceae species. However, significant number of SNPs and indels were detected between Solanaceae species on both coding and intergenic regions. Evolutionary relationship between chloroplast genomes of crops including Solanaceae species were analyzed by the genome-wide comparison of SNPs and Indels. In addition, phylogenetic analysis using sequences of the complete set of genes in chloroplast genomes of Solanaceae species was performed.

DEEP TRANSCRIPTOME SEQUENCING AND SNP DETECTION IN S. LYCOPERSICUM AND S. PENNELLII

José M Jiménez-Gómez, Seisuke Kimura, Neelima Sinha and Julin N Maloof

Section of Plant Biology, University of California Davis, Davis, CA 95616, USA

Wild tomato species are native to diverse habitats in South America and show great morphological and ecological diversity that has proven useful in breeding programs. However, relatively little is known about nucleotide diversity between tomato species. High throughput sequencing (HTS) technologies allow for rapid acquisition of significant amounts of sequence data. Application of HTS to wild species could help revolutionize the identification and characterization of natural alleles of ecological and breeding interest.

With this objective in mind, we are deeply sequencing the transcriptomes of the S. lycopersicum and S. pennellii strains used as parents of a publicly available well characterized NIL population. Samples were obtained by pooling RNA from different tissues and developmental stages, which were converted to cDNA and normalized to aid detection of rare transcripts.

We are in the process of producing enough sequence data to assemble the transcriptomes by combining de novo and reference based assembly. In addition to other resources, we are developing a dense and accurate set of polymorphisms for genotyping the above-mentioned NIL population. As a proof-of-principle we mined ~5K SNPs and indels from ~620 kb of ESTs found in the SGN databases. The SNP rates per gene region and estimations of the likelihood of the genotypes at each SNP defined in our preliminary study will be used in a Bayesian model to estimate SNP probabilities in the upcoming HTS data.


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WITH ITS LONG READ LENGTH THE 454 GENOME SEQUENCER FLX SYSTEM ENABLES PLANT GENOME SEQUENCING.

Marcus Droege

Genome Sequencing, Roche Applied Science

The Genome Sequencer FLX System (GS FLX), powered by 454 Sequencing, is a next-generation DNA sequencing technology featuring a unique mix of long reads, exceptional accuracy, and high throughput. It has been proven to be the most versatile of all currently available next-generation sequencing technologies, supporting many high profile studies in over 7 applications categories. GS FLX users have pursued innovative research in de novo sequencing, re-sequencing of whole genomes and target DNA regions, metagenomics, and RNA analysis. 454 Sequencing is exceptionally powerful for plant research on the genomics and transcriptomics level, currently sequencing more than 20 complex plant genomes

This presentation will provide a short overview about the 454 Sequencing technology, and will focus on applications possible to address with the Genome Sequencer FLX system, with special emphasis on plant sequencing. It will also provide information on the newest product developments, including the 2010 launch of kits allowing to generate read lengths of >800 - 1000 bases on the today's FLX instrument.

SOL 100

Dani Zamir

Hebrew University, Rehovot, Israel


SOL 2009 33

SESSION II BIOTIC STRESSES

34

IDENTIFICATION OF DIAGNOSTIC MARKERS TO LATE BLIGHT RESISTANCE IN SOLANUM PHUREJA FOR BREEDING PROGRAMS Teresa Mosquera-Vasquez, Carolina Ballesteros, Gustavo Gómez Universidad Nacional de Colombia, Agronomy School, Biotechnology Laboratory, Colombia Solanum phureja is a diploid species belonging to Andean cultivated species group. Its main biodiversity center is located in Southern Colombia. S. phureja is an important food source in Andean countries. Late blight caused by the oomycete Phytopthora infestans is the most important S. phureja disease. However, some accessions have shown resistance to this pathogen. Control of the disease by genetic resistance is a desirable strategy for potato plant breeding. The objective of this research is to identify diagnostic molecular markers associated with resistance to P. infestans. Association mapping is the strategy employed in this research, it includes: population structure, phenotype and genotype characterization, and association analysis. 113 accessions from the Colombian S. phureja collection are being evaluated in field conditions, in two localities under natural inoculum pressure, and laboratory conditions, using complex races from both localities. For population structure we are employing SSR markers, using initially a set named Potato genetic identity kit, (Ghislain, 2009). This population is being genotyped using SSR, SCAR CAPS and SNPs markers, to find polymorphisms associated with resistance. Several molecular markers reported associated to QTL and major genes controlling resistance to late blight have been evaluated. Most of them do not show polymorphism. We found in 17 accessions the presence of fragment size 1400bp, characteristic for R1 gene derived from S. demisum. We are searching the functionality of this gene in S. phureja accessions in order to establish if it is a homologous to gen R1 from S. demisum.


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COMPARATIVE PROTEOME ANALYSIS OF DIFFERENTIALLY EXPRESSED PROTEINS IN WILD TYPE AND GENETICALLY ENGINEERED CROP PLANTS FOR PATHO-STRESS AND IMPROVED NUTRITIONAL QUALITY Rajgourab Ghosh, Sudip Ghosh, Lalit Agrawal, Niranjan Chakraborty, Asis Datta, and Subhra Chakraborty* National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi-110067, India The genetic improvements in crop plants beyond current capabilities are needed not only for more food, but also for higher-quality and safety. Tomato and potato are the most important non-cereal food crops, besides being used as raw material for manufacture of processed foods and other food products. Fungal susceptibility and nutritional quality are the two major areas of concern in these solanaceous crops. Towards this, earlier we have developed fungal tolerant and nutritionally enriched tomato and potato for increased agricultural productivity and improved quality. Proteome study provides additional information about localization of gene function and pathway compartmentalization. To understand the regulatory networks and metabolic pathways involved in stress tolerance and nutritional improvement in these transgenic crops, comparative proteomes have been developed using two-dimensional gel electrophoresis. The differentially displayed proteomes revealed few hundred protein spots that change their intensities by more than 2.5-fold. The LC-ESI-MS/MS analyses led to the identification of proteins of various functional classes that include predicted and novel candidate proteins. Further a model network has been constructed using the protein data-sets, which aim to show how target proteins might work in coordinated fashion. The patho-stress and nutrient responsive proteome revealed a coordinated response involving both regulatory and functional proteins, impinging upon the molecular mechanism of stress adaptation and reserve accumulation in transgenic crops.

A MAJOR GENE WITH TYPICAL R PROTEIN STRUCTURE CONFERS GENOTYPE-DEPENDENT QUANTITATIVE RESISTANCE TO LATE BLIGHT

1 2 3 4 5 1S.K. Chakrabarti , J.M. Bradeen , B.P. Singh , S. Austin-Phillips , K.V. Raman and S.K. Pandey

1 2Central Potato Research Institute, Shimla 171001, Himachal Pradeh, India; University of Minnesota, Department 3of Plant Pathology, 495 Borlaug Hall, 1991 Upper Buford Circle, St. Paul, MN 55108, U.S.A.; Central Potato

4Research Institute Campus, Modipuram, Meerut 250110, Uttar Pradesh, India; University of Wisconsin

5Biotechnology Centre, Madison, USA; Cornell University, Ithaca, USA

Late blight, caused by the oomycete pathogen Phytophthora infestans, is the most devastating potato disease in the world. Control of late blight relies heavily on fungicide application that has serious environmental implications. Race-specific resistance sources from Solanum demissum have been used to breed late blight resistant cultivars through classical breeding. Eleven such race-specific R genes have been identified so far, several of which have been mapped and cloned. Unfortunately, the hypersensitive resistance induced by those R genes was not found to be long lasting due to quick evolution of matching avirulence genes in the pathogen. Recently, a resistance gene (RB) encoding CC–NBS–LRR (coiled coil –nucleotide binding site–leucine-rich repeat) motifs was mapped and cloned from the diploid wild species S. bulbocastanum. RB confers race non-specific horizontal resistance. The late blight susceptible variety Kufri Bahar and a defeated resistant variety Kufri Jyoti were transformed with the RB gene. Higher levels of field resistance were observed in the transgenic lines of the defeated variety Kufri Jyoti than those of Kufri Bahar. It demonstrated that the RB gene conferred higher level of resistance in the genotypic background of Kufri Jyoti than that of Kufri Bahar. Variation in the level of late blight resistance among different transgenic lines was observed. However, this could be due to position effect or variation in transgene copy number. Previously, it was demonstrated in other potato varieties that the level of late blight resistance increased with increasing copy numbers of the RB gene. Though copy numbers of all the transgenic lines of Kufri Jyoti and Kufri Bahar were not estimated, it is unlikely that all the transgenic lines derived from Kufri Jyoti would have higher copy numbers than those of Kufri Bahar. To eliminate the possible effect of insertion position, a well characterized transgenic line of Katahdin (SP951) with a single copy gene insertion was crossed with both Kufri Bahar and Kufri Jyoti and late blight resistance in the F1 progenies was evaluated. Since the locus of the RB gene in F1 progeny will remain the same, it was expected that there will be no variation due to position effect of the RB gene in the progeny and they would all show levels of resistance similar to the paternal clone SP951. On the contrary, most of the clones derived from Kufri Jyoti showed higher levels of field resistance while those derived from Kufri Bahar showed comparatively lower levels of resistance. However, a range of resistance reactions varying from near immunity to complete susceptibility was observed in the progeny of both the varieties in spite of the presence of RB gene. These results clearly demonstrate that, although RB effectively confers late blight resistance in a range of genetic backgrounds, resistance response of the RB gene is dependent on the genotypic background of the recipient clone. Reasons for this phenomenon will be discussed.


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NBHB1, NICOTIANA BENTHAMIANA HOMEOBOX 1, IS A JA-RESPONSIVE POSITIVE REGULATOR OF PATHOGEN-INDUCED PLANT CELL DEATH

Joonseon Yoon, Doil Choi

Seoul National University, Seoul, Korea

1) Induction of cell death is an important component of plant defense against pathogens. There have been many reports on the role of phytohormones in pathogen-induced cell death, but jasmonic acid (JA) has not been implicated as a regulator of the response. Here we report the function of NbHB1, Nicotiana benthamiana homeobox1, in pathogen-induced cell death in connection with JA signaling. (2) Involvement of NbHB1 in cell death was analyzed by gain- and loss-of-function studies using Agrobacterium-mediated transient overexpression and virus-induced gene silencing, respectively. Expression of NbHB1 following pathogen inoculations and various treatments was monitored by reverse transcription-polymerase chain reaction. (3) Transcript levels of NbHB1 were up-regulated by infection with virulent and avirulent bacterial pathogens. Ectopic expression of NbHB1 accelerated cell death following treatment with darkness, methyl jasmonate, or pathogen inoculation. Conversely, when NbHB1 was silenced, pathogen-induced cell death was delayed. NbHB1-induced cell death was also delayed by silencing of NbCOI1, indicating a requirement for JA-mediated signaling. Overexpression of the domain-deleted proteins of NbHB1 revealed that the homeodomain, leucine zipper, and part of the variable N-terminal region were necessary for NbHB1 functionality. (4) Taken together, our data strongly suggest that NbHB1 plays a crucial role in the regulation of cell death during plant-pathogen interactions via the JA-mediated signaling pathway.

DIVERSITY OF TOMATO BEGOMOVIRUSES IN INDIA AND PROBLEMS IN RESISTANCE BREEDING

V.G. Malathi

Plant Virology Unit, Div. of Plant Pathology, IARI, New Delhi -110012 ,INDIA

The major biotic stress challenging the productivity of tomato crop in India is leaf curl disease caused by virus species belonging to the genus Begomovirus of the family Geminiviridae. The complexity of the problem is due to diverse begomoviruses, active vector biotypes, and cultivation of tomato throughout the year. There are nine tomato begomoviruses so far characterized, which fall into three genome categories. Genomic components DNA A, DNA B, and DNA β of begomoviruses associated with leaf curl disease of tomato in Maharashtra, West Bangal, Haryana and Karnataka were cloned through rolling circle amplification of genome and infectivity was established either through agroinoculation or by biolistic delivery of RCA. Leaf curl isolates were identified as belonging to ToLCNDV, ToLCGV, ToLCBV, ToLCJV based on sequence identity of DNA A component. One of the isolates that showed less than 89% sequences identity was designated as new species Tomato leaf curl Aurangabad virus (ToLCAuV). Infectivity study revealed that ToLCBV, ToLCAuV, ToLCGV are monopartite with satellite DNA β. In all the above cases DNA A alone is infectious but severity is increased by DNA β. Interaction between selected tomato genotypes and the six begomoviruses cloned were investigated by inoculation of individual virus through whitefly inoculation. Results on host genotypes – viruses interaction and the effect of different DNA β on viral pathogenicity will be discussed.


SOL 2009 39


SOL 200940

LATE BLIGHT RESISTANCE GENES IN POPULATIONS OF SOLANUM BULBOCASTANUM AND RELATED SPECIES

Ben Vosman, Anoma Lokossou, Hendrik Rietman, Miqia Wang

Wageningen University and Research Centre- Plant breeding, P.O.Box 16, 6700 AA, Wageningen, Netherlands

Allele mining facilitates the discovery of novel resistance (R) genes that can be used in breeding programs and sheds light on the evolution of these genes. Solanum bulbocastanum is well known for its late blight resistance genes and so far 3 genes have been identified and cloned from it. The genes Rpi-blb1, Rpi-blb2 and Rpi-blb3 are located on linkage groups VII, VI and IV respectively. We analysed the allele frequency and allelic diversity of the 3 cloned S. bulbocastanum resistance genes in a set of tuber-bearing Solanum species. Conserved Rpi-blb1 haplotypes are present in the Mexican diploid species S. bulbocastanum and S. cardiophyllum as well as in the polyploid S. Stoloniferum. Rpi-blb2 was present in S. bulbocastanum accessions only and all sequences were identical, suggesting that it has emerged recently. Rpi-blb3 haplotypes were found in the Mexican diploids S. bulbocastanum, S. cardiophyllum and S. pinnatissectum, as well as in the polyploids S. stoloniferum and S.hjertingii. The results for the 3 genes show that their spread is very limited and confined to a small number of species in Central America.

CHARACTERISATION OF TRANSCRIPTOME FROM VARIOUS LIFE CYCLE STAGES OF THE POTATO CYST NEMATODE GLOBODERA PALLIDA TO EXPLORE PARASITISM GENES

1 2 1 1 1 1Amar Kumar , , Sean Chapman , Peter Cock , Liliya Pylypenko , Mark S. Phillips ,

1 1Vivian C. Blok & John T. Jones

1 2Plant Pathology Programme, SCRI, Invergowrie, Dundee, DD2 5DA, Scotland, UK; Botany Department, University of Delhi, Delhi 110007, India.

Potato cyst nematode (PCN) Globodera pallida is a major pest of potato throughout the world. This endoparasitic nematode infects host roots and triggers the formation of specialized feeding sites by reprogramming of the developmental process of root cells. To identify parasitism genes, we have investigated the transcriptome of various life cycle stages of PCN. We have analysed over 9000 ESTs from cDNA libraries and have identified genes encoding secreted proteins that are upregulated after the onset of parasitism. In situ hybridisation has been used to confirm expression in pharyngeal gland cells of PCN. A substantial gene family encoding G. pallida SPRYSEC proteins (secreted SPRY domain-containing proteins) has been identified. Expression of these genes is restricted to the dorsal oesophageal gland cells. Different members of the SPRYSEC family of proteins from G. pallida showed differential subcellular localisation patterns in plants, with some localised to the cytoplasm and others to the nucleus and nucleolus. Differences in subcellular localisation may reflect different functional roles for each individual protein or, more likely, differential localisation of plant proteins targeted by the nematode. It has been reported that at least two members of the SPRYSEC family may be involved in interactions with plant resistance genes in a susceptible tomato line. Our data are therefore consistent with the suggestion that the SPRYSEC proteins suppress host defences and that they achieve this through interaction with a range of host targets.


SOL 2009 41

SESSION III ABIOTIC STRESSES

42

CHARACTERIZATION AND GENETIC ANALYSIS OF SY-2, A LOW TEMPERATURE SENSITIVE MUTANT, IN CAPSICUM CHINENSE

1 1 1 2 2 1Song-Ji An , Jinjie Li , Jin-Kyung Kwon , Sota Koeda , Hosokawa Munetaka , Byoung-Cheorl Kang * 1Department of Plant Science/Horticultural Science, Seoul National University, Seoul 151-921, Republic of Korea;

2Department of Agronomy and Horticultural Science, Graduated School of Agriculture, Kyoto University, Sakyo-

ku, Kyoto 606-8502, Japan A temperature sensitive mutant of Capsicum chinense, sy-2, shows abnormal leaf development when grown below 24°; chlorophyll deficiency and shrunken leaves. To understand the underlying mechanism of this response, we conducted phenotypic characterization and genetic analysis. From microscopic observations, abnormal chloroplast structures and cell collapse were observed in leaves of the sy-2 mutant grown at 20°. Through the histochemical staining, accumulation of reactive oxygen species (ROS) was detected in the chlorophyll deficient sectors, especially in the chloroplasts and cell death was also observed in the sectors. These results suggest that reactive oxygen species (ROS) is involved in abnormal leaf development and the cell death. In addition, the results of gene expression analysis of ROS scavenging genes indicated that the expression level of ROS scavenging genes, APX and GST, were significantly decreased in sy-2 plants grown at 20°. The results of fatty acid analysis showed that the pathway of linoleic acid (18:2) to linolenic acid (18:3) may be blocked. These changes may lead to dramatic physiological disorder and alteration of leaf morphology by losing low temperature tolerance in sy-2 plants. Genetic analysis of F2 populations obtained from a cross between C. chinense sy-2 and C. chinense No. 3341 showed that sy-2 phenotype is controlled by a single recessive gene. The sy-2 is located in pepper chromosome 1.


SOL 2009 43


SOL 200944

DEVELOPMENTAL AND HEAT STRESS-REGULATED EXPRESSION OF GENES IN TOMATO ANTHER M.Giorno, C.Bita, W.Vriezen, C. Mariani Plant Cell Biology IWWR-FNWI, Huygens Building, Radboud Univ. Nijmegen, Heyendaalseweg 135, 6525 AJ Nijmegen, NL Exposure to environmental stresses, such as high temperature, can severely reduce the productivity/yield of tomato plants grown under field conditions. Decreased fruit set due to heat stress (hs) has been associated with several alterations in the morphology of tomato flower structures and with physiological imbalances in stress-protective metabolites, such as carbohydrates, polyamines and proline. The major factor responsible for the failure of tomato fruit to set under suboptimal temperature conditions is considered to be the higher sensitivity of flower developmental processes to temperature changes, with the anthers being reported to be more vulnerable than the female organs. Alterations in tomato anthers, such as a failure of adequate dehiscence and tapetum development, have been found when hs occurs during the early phases of pollen development, for example 7-15 days before anthesis. The high sensitivity of developing pollen could be due to an inadequate heat stress response. To investigate this, we undertook transcriptome analyses by performing a microarray and cDNA-AFLP of genes expressed in young tomato anthers exposed to heat treatment. Our results confirm that genes and proteins of the heat shock response network are expressed in young anthers and in microspores and are finely regulated by heat shock but also by developmental cues.

QTL MAPPING FOR DROUGHT TOLERANCE IN POTATO

Anitha Kumari, Gerard van der Linden, Richard visser

2, 3 Wageningen UR Plant breeding, WUR, PO Box 386, 6700 AJ Wageningen, The Netherlands

Potato is an important food crop, yet it is relatively susceptible to drought. As a first step towards identifying the genetic basis for drought tolerance in potato, we make use of diploid potato populations that have been genetically well characterized (CxE, SHxRH). The CxE population has been extensively evaluated for drought tolerance in two successive years (2008, 2009) under greenhouse conditions by measuring traits like Relative water content of leaf, ? 13C as a measure of Water Use Efficiency, chlorophyll fluorescence, chlorophyll content, plant biomass and tuber yield. The progeny displayed a wide contrast for drought tolerance, with individuals surviving and recovering completely after 3 weeks of drought, and others completely wilted beyond recovery. Most of the traits had high heritabilities. For optimal QTL mapping, we expanded the CxE and SHxRH genetic maps with 480 SNP markers (384 and 768 SNP array, enriched for drought tolerance candidate genes). The SNPs were discovered in public EST databases using the QualitySNP software and detected with the Illumina GoldenGate assay. About 300 SNPs were mapped in both populations, allowing alignment of the CxE and SHxRH maps. This will enable us to make use of the extensive genetic and sequence information of the SHxRH population and the RH genome sequence. QTLs were identified for tuber number, tuber weight, plant height, shoot fresh and dry weight, Leaf ? 13C and relative water content under drought conditions. Several QTLs, but not all were linked to maturity type indicating that tuber formation interacts with drought tolerance and yield, but other factors play an important role as well. In the future, we will zoom in on the drought tolerance QTLs that we identified in CxE and by expression studies and candidate gene identification and analysis.


SOL 2009 45


SOL 200946

GLYOXALASE PATHWAY: ROLE IN ABIOTIC STRESS TOLERANCE

Sneh Lata Singla-Pareek, Ananda Mustafiz and Sudhir K.Sopory

International Center for Genetic Engineering and Biotechnology, Aruna Asaf Ali Road, New Delhi 110067

The glyoxalase system consists of two enzymes, glyoxalase I (EC 4.4.1.5, lactoylglutathione lyase) and glyoxalase II (EC 3.1.2.6, hydroxyacyl glutathione hydrolase). These enzymes act coordinately to convert cytotoxic methylglyoxal (MG) using glutathione (GSH) as a cofactor in a two step reaction. Glyoxalase I first catalyses the formation of S-D-lactoyl glutathione from hemithioacetal, which is formed by a non enzymatic reaction between GSH and MG. Glyoxalase II catalyzes the hydrolysis of this S-D-lactoylglutathione to D-lactate with regeneration of glutathione in the second step. MG is a primary physiological substrate of glyoxalase I and produced mainly from triose phosphate intermediates of glycolysis, glyceraldehyde- 3-phosphate and dihydroxyacetone phosphate.

We have shown that overexpression of glyoxalase I and II in transgenic plants confer salinity stress tolerance in solanaceous model plant, tobacco. The transgenic plants can grow and set viable seeds under 200 mM NaCl stress. The mechanism behind enhanced salt tolerance conferred by the overexpression of glyoxalase pathway enzymes was studied in transgenic vis-a`-vis wild-type (WT) plants. We have found that abiotic stress induces higher level accumulation of MG, however, the transgenic tobacco plants overexpressing glyoxalase pathway enzymes, resist an increase in the level of MG and also maintained a higher reduced to oxidized glutathione (GSH:GSSG) ratio under salinity. By manipulating the expression of glyoxalase I and II we have also been able to develop transgenic rice plants that can withstand higher concentration of NaCl and also drought. A microarray and proteomics analysis revealed changes in the regulation of a number of genes in the transgenic plants.

An analysis of rice genome has revealed the presence of at least 11 genes encoding glyoxalase I and 3 genes encoding glyoxalase II. A quantitative RT-PCR analysis showed their differential expression in response to stress and also in response to increased concentration of MG which our studies have indicated could be acting as a signal molecule under stress conditions. One of the glyoxalase I family member that was upregulated by stress and MG was characterized in detail. Interestingly it was found to be a Ni dependent enzyme rather than Zn dependent enzyme that has so far not been reported in higher plants and animal kingdom. Overexpression of this glyoxalase I also leads to stress tolerance in transgenic tobacco.

Our studies have indicated an important role of glyoxalase genes in abiotic stress tolerance which can be deployed to generate stress tolerant crop plants. Their efficacy has been tested by other groups in Brassica, legumes and also in tomato.

TRANSCRIPTOMIC CHARACTERIZATION OF THE TOS1 MUTANT AND FINE MAPPING OF TOS1

1 1 2 1 1Itziar Castanedo , David Posé , Abdelhafid Bendahmane , Victoriano Valpuesta and Miguel A. Botella

1Departamento de Biología Molecular y Bioquímica, Universidad de Málaga, 29071 Málaga, Spain; 2INRA-CNRS, UMR1165, Unité de Recherche en Génomique Végétale, 2 rue Gaston Crémieux, F-91057 Evry, France.

Osmotic stress severely limits plant growth and agricultural productivity. We have used mutagenesis to identify plant genes that are required for osmotic stress tolerance in tomato. As a result, we have isolated a novel mutant in tomato (tos1) caused by a single recessive nuclear mutation that is hypersensitive to general osmotic stress. Growth measurements demonstrated that the tos1 mutant is less sensitive to intracellular abscisic acid (ABA) and this decreased ABA sensitivity of tos1 is a basic cellular trait expressed by the mutant at all developmental stages analysed. It is not caused by a deficiency in the synthesis of ABA because the tos1 seedlings accumulated more ABA than the wild type (WT) after osmotic stress. In order to get insight about the pathways altered in tos1 we have performed microarray analysis in conditions of high and low water demand. We are also in the process of identifying TOS1 using a map-based cloning strategy through AFLP and chromosome walking.

Towards the identification of genes involved in vitamin C accumulation through a combination of metabolomic, transcriptomic, and reverse genetics approaches


SOL 2009 47


SOL 200948

MOLECULAR MECHANISMS INVOLVED IN THE HEAT-STRESS RESPONSE OF DEVELOPING TOMATO (SOLANUM LYCOPERSICUM L.) POLLEN GRAINS

Nurit Firon, Gil Frank, Etan Pressman, Ron Ophir, Levia Althan, Rachel Shaked

Institute of Plant Sciences, The Volcani Center, A.R.O., Bet Dagan, Israel

Above-optimal temperatures reduce yield in tomato largely because of the high heat-stress (HS) sensitivity of the developing pollen grains. The high-temperature-response, especially in this most HS-sensitive part of the plant, is considered, however, a poorly understood subject. Detailed transcriptomic analysis of heat-stressed maturing tomato microspores was carried out, using a combination of Affymetrix Tomato Genome array and cDNA-AFLP techniques, in order to obtain an overview of molecular mechanisms that participate in microspores' HS-response (HSR). The data obtained reveal the involvement of specific members of the small heat shock protein (HSP) gene family, HSP70 and HSP90, in addition to heat stress transcription factor A2 (HSFA2) and HSFA3, as well as factors other than the classical HS-responsive genes, in microspores HSR. The results indicate HS-regulation of reactive oxygen species scavengers, osmolytes, plant hormones and regulatory genes that were previously implicated in other types of stress. Specific role for ethylene in microspores' HSR is suggested in view of the high HS-induced elevation in a number of ethylene-related genes. The use of cDNA-AFLP enabled the detection of genes representing pollen-specific functions that may be recruited for coping with HS and are missing from the Tomato Affymetrix chip, such as those involved in vesicle-mediated transport and a pollen-specific, calcium-dependent protein kinase (CDPK2). Functional characterization of selected genes, looking into their potential involvement in thermotolerance, will be discussed.

ARTIFICIAL MICRORNAS AS A TOOL FOR CBP80 GENE SILENCING IN POTATO TO OBTAIN PLANTS WITH ENHANCED DROUGHT TOLERANCE.

1 1 1 2 2 3 3M.Pieczyński , D.Bielewicz , J.Dolata , K.Floras , J.Hennig , D.Czyżewska , W.Marczewski , 1 1A.Jarmołowski , Z.Szweykowska-Kulińska

1 2Department of Gene Expression, Adam Mickiewicz University, Poznań; Laboratory of Plant Pathogenesis, 3

Institute of Biochemistry and Biophysics, Warszawa; Plant Breeding and Acclimatization Institute, Młochów,

In our and others previous investigations we have shown that A. thaliana plants with silenced CBP20 or CBP80 genes are drought tolerant in comparison to wild type plants. In this project we are carrying experiments with the aim to obtain transgenic lines of potato plants with higher drought tolerance. We obtained and sequenced CBP20 and CBP80 cDNAs for all four alleles of two potato cultivars Sante and Désirée. Conservative fragments in each cDNA sequences were used as targets for gene silencing. We prepared two types of silencing constructs. The first one is based on RNAi pathway. The second one contains artificial microRNA gene targeting CBP80 potato gene. Transformants were tested for the presence of the transgene in the genome. Using real-time PCR methods we analyzed the changes in the expression level of CBP80 gene. Our results showed that CBP80 gene is silenced up to 99%. Western analyses showed the lack of CBP80 protein in two transformed lines which were transformed with the constructs containing artificial microRNA. Experiments for the establishment of drought tolerance in transformed plants are now in progress.


SOL 2009 49

SESSION IV FUCTIONAL GENOMICS

50

FUNCTIONAL GENOMICS OF POTATO TUBER LIFE CYCLE

Christian Bachem, Bjorn Kloosterman, Richard Visser

Laboratory of Plant Breeding, WUR, Wageningen, The Netherlands

The life cycle of the potato tuber involves a number of distinct processes comprising the initiation of tuberisation, resource accumulation and storage, dormancy and finally sprouting and resource mobilisation. This transition from a sink to a source organ requires the coordinated expression of a large number of genes controlling several global switches in metabolic pathways. We have used a combination of classical genetic and transcriptomic analyses to investigate the processes involved in tuber life cycle. An overview of the control of tuberisation will be given and details of development of tuber quality traits will be discussed.


SOL 2009 51


SOL 200952

EVOLUTION OF FLOWERING TIME AND FRUIT QUALITY TRAITS IN TOMATO

Falcone G., Fantini E., and Giuliano G.

ENEA, Casaccia Research Center, Via Anguillarese 301, 00123 Roma, Italy

The genome of cultivated tomato (S. lycopersicum) has a limited sequence variation due to bottlenecks during domestication and breeding; however, the study of genetic diversity between tomato and related wild species could provide useful tools for the breeding of agronomically useful characters. We took a candidate gene approach to identify genetic differences responsible for the variability of two traits: the photoperiodic flowering response and the different colour of ripe berries. S. lycopersicum ecotypes and closely related wild tomato species were selected. The CRYPTOCHROME and CONSTANS gene families, controlling flowering time in Arabidopsis, have been completely sequenced, but up to now no mutations discriminating day-neutral from short-day species have been identified. Concerning berry colour, we studied the carotenoid biosynthetic pathway. The sequencing of genes from PSY down to LCY-e (alpha-branch) and CHY1/CHY2 (beta-branch) is complete. The sequence analysis has highlighted the presence of numerous mutations that differentiate the colour-fruited species from the green-fruited ones. Some non-synonymous substitutions are candidate to be hypomorphic alleles.

IDENTIFICATION AND CHARACTERIZATION OF TOMATO CDNA CLONES SHARING HOMOLOGY WITH ARABIDOPSIS GLUTAMATE RECEPTORS AND ANIMAL GABA RECEPTORS Asma Aouini, Erika Asamizu, Chiaki Matsukura, Hiroshi Ezura Grad Sch Life Environ Sci Univ Tsukuba, Tsukuba 305-8572, Ibaraki, Japan γ-Aminobutyric acid (GABA) is a four carbon non protein amino acid present in eukaryotes and prokaryotes. To date, GABA has been well studied as a metabolite to be involved in the osmoregulation, the C:N balance and the defense against insects and nematodes. Recent studies have reported evidence for the role of GABA that plays as a signaling molecule and obviously, the presence of GABA receptors in plants. Although, genes that are highly homologous to the animal GABA receptors are not present in the Arabidopsis genome, similarity searches reveal a new family of 20 genes (GLRs: glutamate receptor-like). Hence, It has been proposed that GABA antagonists are interacting with the GABA-like domain of the putative GLRs in plants. In the present study, we investigated 7 cDNA tomato clones which have a GABA binding domain and homologous to AtGLRs. Parsimony and bootstrap analyses provided evidence that these genes are related to two superfamilies of animal neurotransmitter receptors iGLRs (ionotropic GLRs) and subC-GPCRs (sub family C of the G-protein-coupled receptors). We suggest that these genes and members of these families evolved an ancestor via distinct mechanisms. Using real-time PCR, relative quantification revealed a differential expression of these genes with a preference to most of them to be highly expressed in both stem and leaf. Furthermore, functional characterization of 3 cDNA clones in heterologous expression system Saccharomyces cerevisiae is in progress.


SOL 2009 53


SOL 200954

COMPARATIVE ANALYSIS OF ALTERATIONS IN POTATO (SOLANUM TUBEROSUM L.) DURING TUBER INDUCTION, DEVELOPMENT AND MATURATION Lalit Agrawal, Shubhendu Shekhar, Subhra Chakraborty, Asis Datta, and Niranjan Chakraborty* National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi-110067, India. Potato (Solanum tuberosum L.) is the most important non-cereal food crop and ranks fourth in terms of total global food production. Tuberization in potato is a developmental process that serves a double function, as a storage organ and as a vegetative propagation system. It is a multi-step, complex process and the underlying mechanisms governing these overlapping steps are not fully understood. With an aim to understand the molecular basis of tuberization in potato, a comparative proteome has been developed using two-dimensional gel electrophoresis. Proteomic analysis of the differential proteome revealed 250 protein spots that change their intensities for more than 2.5-fold. The LC-ES-MS/MS analyses led to the identification of about a hundred differentially regulated proteins that include predicted and novel tuber-specific proteins. Of the differentially expressed proteins, 7 were exclusively expressed in the stolon, 9 were found to be expressed throughout the tuber life cycle and remaining 49 were expressed during tuber maturation, highlighting their regulatory and storage functions. These data are particularly important, at least in part, due to the fact that despite the existence of more than 225,000 ESTs, the currently available potato sequence data show a severe under-representation of proteins. Furthermore, elevated expression of the ROS-catabolizing enzymes viz., superoxide dismutase, ascorbate peroxidase and catalase indicates their integrated role in the onset of stolon formation and the developmental transition of stolon into tubers. The differential display of the tuber proteins may provide new insight in further understanding of the underlying mechanisms involved in tuber development in potato. It may also facilitate the targeted alteration of metabolic routes in potato tubers for industrial exploitations besides generation of markers for precision selection in breeding program.

HIGHER FRUIT ASCORBIC ACID CONTENT IN A TOMATO INTROGRESSION LINE IS ASSOCIATED WITH INCREASED EXPRESSION OF GENES INVOLVED IN THE PECTIN BREAKDOWN

Antonio Di Matteo, Adriana Sacco, Milena Anacleria, Mario Pezzotti, Massim Delledonne, Luigi Frusciante, Amalia Barone

DiSSPAPA - University of Naples Federico II, Italy

AsA increase in tomato fruit is a desirable target and a better understanding of mechanisms involved in its synthesis and accumulation is required. Quantitative trait loci (QTLs) for AsA content in tomato fruit have been already reported using Solanum pennellii X S. lycopersicum cv. M82 introgression line (IL) population. In this study, red ripe fruit of the IL12-4 confirmed higher AsA concentration than M82 parental line over three consecutive trials. To identify differentially expressed transcripts in tomato pericarp, total RNA from the IL12-4 and the M82 was hybridized on a Combimatrix TomatArray 1.0. The comparative transcriptomic analysis identified 253 sequences as differentially transcribed. Within the differentially expressed transcripts, 61 showed up-regulation and 192 showed down-regulation. Mapping differentially expressed transcripts on metabolic networks allowed evidencing change in the expression of genes involved in the pectin breakdown process suggesting a feeding effect on the AsA pathway through D-galacturonic acid. In addition, cell wall-associated genes showed co-regulation with key genes of the ethylene biosynthesis. This finding demonstrates that transcriptomic changes occurring in the IL12-4 fruit operates through genes involved in the fruit softening and suggest that the whole process of ripening might be affected. Currently, efforts are focusing on functional characterization of candidate genes affecting expression of the fruit AsA content.


SOL 2009 55


SOL 200956

BIOCHEMICAL AND MOLECULAR ANALYSIS OF FLAVONOID MUTANTS IN TOMATO

1,2 1,2 1 1 1,2 3 4 5Ballester AR , Tikunov YM , Molthoff J , de Vos CH , Hall RD , Grandillo S , Martin C , Granell A and 1,2Bovy AG

1. 2. Plant Research International. POB 16, 6700 AA, Wageningen, The Netherlands; Centre for Biosystems 3.

Genomics, POB 98, 6700 PB Wageningen, The Netherlands; CNR-Institute of Plant Genetics, Via Università 133, 4. 5.

Portici 80055, Italy; John Innes Centre, Norwich Research Park, Colney, Norwich, NR4 7UH, UK; Instituto de Biología Molecular y Celular de Plantas, Consejo Superior de Investigaciones Científicas, Universidad Politécnica de Valencia, Camino de Vera s/n, 46022 Valencia, Spain;

Biochemical analysis of an introgression line (IL) population derived from a cross between Solanum lycopersicum cv Moneyberg and the wild species Solanum chmielewskii revealed interesting ILs with variations for the major tomato flavonoids: (i) strongly decreased levels of naringenin chalcone on IL1b results in pink-coloured fruit and (ii) increased flavonol content on IL5b. Our studies are focussing on a candidate gene approach to unravel the basis for variation in flavonoid content in tomato. High levels of rutin and kaempferol-rutinoside detected in the IL5b fruit are related with an increase of CHI gene expression. The genomic organization of both parentals showed a copia-like retrotransposon in the promoter region of the S. chmielewskii gene that could affect the function of this gene. On the other hand, to further characterise the pink phenotype, metabolic and molecular analysis were carried out in Moneyberg and IL1b tomato fruit. Metabolic analysis showed that pink fruit lack the ripening-dependent accumulation of the yellow-coloured flavonoid naringenin chalcone in the fruit peel. The expression of all genes encoding biosynthetic enzymes involved in the production of the flavonol rutin from naringenin chalcone were down-regulated in pink fruit, suggesting that the candidate pink gene encodes a regulatory protein such as a transcription factor rather than a biosynthetic enzyme. Of 26 MYB and bHLH transcription factors putatively involved in regulating transcription of genes in the phenylpropanoid and/or flavonoid pathway, only the expression level of the one MYB gene correlated well with the decrease in the expression of structural flavonoid genes in peel samples of pink- and red-fruited genotypes during ripening. Biochemical and molecular data, gene mapping, segregation analysis and Virus-induced gene silencing (VIGS) experiments demonstrated that this MYB transcription factor plays an important role in regulating the flavonoid pathway in tomato fruit and suggest strongly that the wild allele of this gene leads to the pink fruit colour.

ALTERING THE CELL CYCLE TOWARDS ENDOREDUPLICATION CONTRIBUTES TO THE REGULATION OF CELL SIZE AND FINAL FRUIT SIZE IN TOMATO

Christian Chevalier

Unité Mixte de Recherche 0619 Biologie du Fruit ; Institut National de la RechercheAgronomique, Université de Bordeaux ; Centre de Recherche INRA Bordeaux-Aquitaine, France.

Early fruit development in tomato essentially proceeds from two growth phases, made of cell division and cell expansion respectively. The cell expansion process is characterised by nuclear endoreduplication, thus accounting for the increase in DNA ploidy level. A clear correlation exists between the mean cell size in the pericarp of various tomato genotypes and the mean ploidy level. Therefore endoreduplication is a critical determinant in the control of cell size and consequently in final fruit size and weight of tomato fruit.

The molecular mechanisms that trigger the switch between the cell cycle and the endoreduplication cycle during fruit development are still poorly understood. Nevertheless the arrest of cell divisions during fruit development is associated with the inhibition of M-phase CDK/cyclin complexes. Three distinct mechanisms may account for the impairment of mitotic CDK/cyclin complex activity, and subsequent cessation of cell divisions and the onset of endoreduplication-driven cell expansion: (i) the phosphorylation status of the CDK subunit within the complex; (ii) the fixation of specific CDK inhibitors on the CDK/cyclin complex; (iii) the availability of the cyclin subunit. Using reverse-genetics we investigated the involvement of key factors thought to be associated to these three mechanisms during tomato early fruit development, namely the WEE1 kinase, the CDK inhibitors KRPs, and the CCS52 proteins.

Our most recent data relative to the contribution of the WEE1, KRPs and CCS52 proteins in the control of cell size and final fruit size, through their involvement in the onset of the endoreduplication process, will be presented.


SOL 2009 57


SOL 200958

UNRAVELING THE COMPLEX TRAIT OF SEED QUALITY IN TOMATO BY GENETICAL GENOMICS

Wilco Ligterink, Rashid Kazmi, Noorullah Kahn, Leo Willems, Henk Hilhorst

Laboratory of Plant Physiology, WUR, Wageningen, Netherlands.

The yield and economic success of horticultural crops depends to a large degree on the quality of the seed used to grow these crops. Seed quality attributes include dormancy, germination, seed and seedling vigour, seedling dry weight, normal embryo- and seedling morphology, as well as the ability to develop into a normal plant. The molecular-genetic dissection of the processes that underlie these quality parameters and their relationship with seed and seedling phenotypes will identify the regulatory genes and signaling pathways involved and, thus, provide the means to predict and enhance seed quality. Our aim is to elucidate the mechanisms involved in the acquisition of seed quality and to develop molecular markers to aid in marker assisted breeding. To reach this aim we make use of the natural variation found in tomato RIL and IL populations. We have phenotyped a broad range of seed quality attributes in these populations and have identified QTLs for various traits. Besides extensive phenotyping, the RILs will also be profiled by metabolomics and transcriptomics (mQTL and eQTL study). This combined use of physiology, genetics and genomics, followed by advanced data analysis will allow the construction of regulatory networks involved in the different aspects of

SESSION V METABOLOMICS AND PROTEOMICS

59


SOL 200960

METABOLIC PROFILES OF TRANSGENIC TOMATOES ENGINEERED TO DIFFERENTIALLY ACCUMULATE POLYAMINES PINPOINT DIFFERENT ROLES OF PUTRESCINE AND SPERMIDINE-SPERMINE IN REGULATING METABOLISM

1 2 1 1 3Autar K. Mattoo , Anatoli Sobolev , Tahira Fatima , Vijaya Shukla and Avtar K. Handa

1United States Department of Agriculture, Agricultural Research Service, The Henry A. Wallace Beltsville Agricultural Research Center, Sustainable Agricultural Systems Laboratory, Beltsville, Maryland 20705, USA; 2 3Institute of Chemical Methodologies, CNR, Monterotondo Stazione, Rome, Italy; Department of Horticulture and

Landscape Architecture, Purdue University, W. Lafayette, IN 47907, USA

Polyamines are a class of biogenic amines shown to have multiple in vitro effects on cellular processes of most organisms. Much attention has focused on the diamine putrescine (Put) and higher polyamines, spermidine (Spd) and spermine (Spm) – the three ubiquitous amines. Generally, it has been assumed that these three amines have similar biological effects but with differing amplitudes. We are using transgenic approach to discern the independent roles of Put, Spd and Spm, and have developed transgenic tomato plants homozygous with yeast polyamine biosynthesis genes. NMR-based metabolite profiling suggests that Spd and Spm are perceived as 'signaling' organic-N metabolites by the fruit cells and influence multiple cellular pathways in diverse subcellular compartments: mitochondria, chloroplasts, chromoplasts and cytosol. A clear interaction is seen between Spd-Spm and amino acids such as glutamate, glutamine and asparagine. Spd-Spm bring about a nexus between N signaling and carbon sequestration involving a network of anabolism-related genes. Correlation coefficient analysis was used to show that, in most instances, Spd-Spm effects on similar targets were exactly opposite of Put, suggesting that these biogenic amines have independent roles in plant metabolism. Analysis of transcript and metabolite profiles also revealed that Spd-Spm action includes posttranscriptional regulation. To determine the early events in Spd-Spm signaling, we have identified and sequenced gene clusters in tomato genome which are targets of polyamine action and regulate initial events in RNA processing and protein synthesis.

THE POTATO TUBER VACUOLE. PROTEOMICS REVEALS CULTIVAR-SPECIFIC VARIANTS OF THE ABUNDANT MULTIGENE FAMILIES OF STRESS-STORAGE PROTEINS.

Jørgensen M, Allan Stensballe, Welinder KG.

Aalborg University, Denmark

Potato starch production leaves behind a 'waste' of protein with useful functions and a nutritional value similar to egg protein. The major site of protein storage is the vacuole, which accounts for ca. 75% of the tuber cell volume. We have characterized the potato tuber transcriptome and proteome of northern Europe's most important starch potato, cv. Kuras in a number of studies and developed a simple method for isolating intact vacuoles from mature tubers. The soluble vacuolar proteins were fractionated and analyzed by Superdex 200 gelfiltration. Our extensive nanoLC-MS/MS analyses of tryptic and chymotryptic digests of each fraction have provided a wealth of protein chemical information. The proteomics data allows identification of three distinct vacuolar import pathways based on differential posttranslational modifications. Patatins exemplify one import pathway via the Golgi and requires a six-residue C-terminal propeptide, -ANKASY-COO¯ (J Biol Chem 284, 9764–9769). Kunitz protease inhibitors come from more than 25 different genes and have a well-known NPIR-like vacuolar targeting signal. However, during vacuolar import the inhibitor proteins have been truncated by unspecific proteolysis yielding a variety of different N-terminal residues for each genetic variant. Lipoxygenases constitute a third vacuolar import pathway. They have no known signal peptides and loose no propeptide, and appears to enter directly from the cytosol. Our data also shows that the many genetic and allelic variant forms of the storage protein families are cultivar specific, i.e. Kuras proteins show small differences to proteins translated from other cultivars.


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SOL 200962

FRUIT SURFACE FLAVONOID ACCUMULATION IN TOMATO IS CONTROLLED BY AN SLMYB12 REGULATED TRANSCRIPTIONAL NETWORK

Avital Adato, Asaph Aharoni

Weizman Instiute of Science, Rehovot, Israel

The cuticle covering plant aerial surfaces is a unique structure that has a role in organ development and protection against stress conditions. In the framework of studying reproductive organ surfaces we carried out a detailed analysis of the tomato colorless peel y mutant. The peel of y lacks the yellow flavonoid pigment naringenin chalcone, which accumulates in fruit peel tissue up to ~1% on a dry weight basis. We found that the y mutant fruit cuticle is thinner and exhibits modified wax composition and reduced cutin content and elasticity. Large scale metabolic and transcript profiling revealed broad effects on both primary and secondary metabolism, mostly related to the biosynthesis of flavonoids. These were not restricted to a specific stage of fruit development, or to the fruit in general. Three R2-R3 MYB transcription factors were significantly down-regulated in the y mutant fruit peel. One of these, SlMYB12, was mapped to the genomic region on tomato chromosome 1, which had previously been shown to harbor the y mutation. Specific down-regulation of SlMYB12 by an artificial microRNA and the identification of an additional mutant allele that co-segregates with the colorless-peel phenotype confirmed that a lesion in this regulator underlies the y mutant phenotype. Thus, we provide a new insight to the study of soft fruit cuticular structure and instigate the elucidation of the regulatory network that controls flavonoid accumulation in tomato fruit cuticle.

METABOLOMICS DATA FUSION TO UNRAVEL THE REGULATION OF FLAVOUR-RELATED METABOLIC PATHWAYS IN TOMATO FRUIT

1,2,3 1,2 1, 4 1,2, Yury M. Tikunov , Ric C.H. de Vos , Ana Mª González Paramás , Robert D. Hall1,2

Arnaud G. Bovy

1 2Centre for BioSystems Genomics, POB 98, 6700 AB Wageningen, the Netherlands,; Plant Research International,

3POB 16, 6700 AA Wageningen, the Netherlands, ; Laboratory for Plant Physiology, Wageningen University,

4Arboretumlaan 4, 6703 BD Wageningen, the Netherlands, ; Universidad de Salamanca, Area de Nutrición y Bromatología, Facultad de Farmacia, Campus Miguel de Unamuno, E-37007 Salamanca, Spain

In addition to sugars and organic acids, volatiles play an important role in determining the flavour of tomato fruit. Many volatiles are present in tomato fruit as glycoconjugates and there is accumulating evidence that glycosylation plays an important role in the storage and emission of volatiles. In this study we describe a novel principle of differential regulation of phenylpropanoid volatile emission from ripening tomato fruit (Solanum lycopersicum L.) upon fruit tissue disruption. Application of a multi-instrumental analytical platform for metabolic profiling of fruits from a diverse collection of tomato cultivars revealed that emission of three discriminatory phenylpropanoid volatiles, namely methyl salicylate, guaiacol and eugenol, took place upon disruption of fruit tissue through cleavage of the corresponding disacharide glycoconjugates. However, in certain genotypes, phenylpropanoid volatile emission was arrested due to conversion of the corresponding disacharide precursors into glycoconjugate species of a higher complexity: trisacharides and malonyl-trisacharides. This glycoside conversion was established to occur in tomato fruit during the later phases of fruit ripening and has consequently led to the inability of red fruits of these genotypes to emit key phenylpropanoid volatiles upon fruit tissue disruption. This novel mechanism of volatile emission regulation can pave the way to new strategies for controlling tomato fruit flavor and taste.


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SOL 200964

PROTEOMICS OF MALE STERILITY IN A TOMATO (SOLANUM LYCOPERSICUM) MUTANT

1 2 2 1Inder S. Sheoran , Andrew R.S. Ross , Douglas J.H. Olson and Vipen K. Sawhney

1 2Department of Biology, 112 Science Place, University of Saskatchewan, Saskatoon, SK, S7N 5E2; Plant Biotechnology Institute, National Research Council of Canada, 110 Gymnasium Place, Saskatoon, SK, S7N 0W9, Canada

The proteomic approach was used to identify proteins associated with male sterility in the 7B-1 male-sterile mutant of tomato. Pollen development in the mutant breaks down prior to meiosis in microspore mother cells (MMCs). The wild type (WT) and mutant anthers of comparable size, with tetrads in WT and the lack of meiosis in mutant, were harvested, proteins extracted, and analyzed by 2-DE and differential in-gel electrophoresis (DIGE) using fluorescent cyanine dyes. Over 1800 spots were detected in gels and of these, 215 spots were differentially expressed. 70 spots with higher expression in either WT or mutant anthers were subjected to matrix-assisted laser desorption-time of flight spectrometry (MALDI-TOF MS), and 59 spots representing 48 proteins were identified by using NCBInr and tomato EST databases. The major proteins up-regulated in WT anthers included a number of proteases e.g., subtilase, proteasome subunits and 5B-protein, leucine rich repeat proteins, transcriptional and translational factors, and FtsZ protein. In the 7B-1 mutant, aspartic protease, ACP-reductase, ribonucleoprotein and nucleotide diphosphate kinase were up-regulated. Cystatin, an inhibitor of cysteine protease, showed the major difference and its activity was higher in the mutant compared to WT. Pollen development in tomato anthers involves programmed cell death (PCD) of the tapetum which is delayed in7B-1 mutant anthers. The higher expression of proteases in WT, and higher activity of cystatin in mutant, anthers could be related to delay of tapetum PCD, and hence male sterility. Other proteins up-regulated in WT anthers have designated roles in cell division and pollen development.

INTEGRATED PROTEOMIC AND GENOMIC ANALYSIS OF TOMATO ROOTS IN RESPONSE TO ALUMINUM TOXICITY

Suping Zhou, Roger Sauve

Department of Agricultural Sciences, School of Agriculture and Consumer Sciences, Tennessee State University, 3500 John A Merritt Blvd, Nashville, TN, USA

Like all tomato cultivars in this species, (Solanum lycopersicon cv. Money Maker) is susceptible to aluminum toxicity. The root system (root tips) presents the first barrier for controlling absorption of the toxic ions into the plant system. Our research is focusing on the identification of genes that are involved in Al toxicity. Identification of such genes is based on alterations in gene expression at both the transcript and protein levels. We have found that genes associated with cellular oxidative stress, detoxification, signal transduction, various transporters, cell cycle progression were all affected by Al toxicity. These included genes such as oxalate oxidase and wali7 homologs, multidrug efflux transporters, adenosyl-L-homocysteine hydrolase lactoylglutathione lyase, multidrug and toxic compound extrusion (MATE) proteins, p34cdc2 protein kinase, cyclin, and histone, putrescine synthesis, ABC transporter, etc. Some of these findings have been published in the following articles: "Proteome changes under Al toxicity in tomato roots", JXB, 2009 60(6):1849-1857, and "Identification of genes associated with aluminum toxicity in tomato roots using cDNA microarrays", Plant Stress, 2008 2:113-120. Our on-going research is presently directed to validate their function by over-expression and RNAi knock-down of these genes.


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SOL 200966

SOLCYC AND METACYC: CONNECTING THE GENOME TO THE METABOLIC NETWORKS

Anuradha Pujar, Ron Caspi, Naama Menda, Isaak Tecle, Aureliano Bombarely-Gomez, Peter Karp and Lukas Mueller

Boyce Thompson Institute, Cornell University, Ithaca, NY and Bioinformatics Research Group, SRI International, Menlo Park, CA

MetaCyc is a metabolic encyclopedia of experimentally validated biochemical pathways curated from scientific literature, which spans all organisms, with an emphasis on plants and microbes. The Pathway tools is a complex curation software suite enables curation of reactions, construction of pathways and annotation with one or more representative enzymes, that include information such as substrate specificity, kinetic properties, activators, inhibitors, cofactor requirements, genes if cloned and links to external databases. In addition curators are able to provide concise, review-level summaries and extensive literature citations. The present database release includes more than 1200 pathways from more than 1549 organisms, 7312 reactions, 5127 enzymes, 4748 genes, 7234 chemical compounds, curated from 17916 citations. The MetaCyc database is the reference database on which the pathways are predicted from annotated genomes by PathoLogic called Pathway/genome Databases (PGDB's). The Biocyc Database (biocyc.org) has a collection over 300 PGDB's. Each BioCyc Database describes the genome and predicted metabolic pathways of a single organism, which are then taken up by interested groups for curation. SolCyc is a collection of such PGDBs, developed for the clade oriented Solanceae Genomics Network (SGN) database. It has predicted metabolic pathway databases of significant species belonging to Solanaceae and includes Lycocyc(tomato), Solacyc (eggplant), Nicotianacyc (tobacco),Petuniacyc (Petunia), Capcyc (Capsicum) , Potatocyc (potato). An interactive web interface has been developed for the seamless flow of information from the SGN phenotype and locus database with SolCyc. This facilitates researchers with the capacity to search for underlying metabolic pathway information of genes and phenotypes that has been curated into the SolCyc database.

SESSION VI BIOINFORMATICS AND COMPUTATIONAL BIOLOGY

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COMPARATIVE SEQUENCING AND ANALYSIS OF SYNTENIC BACS ON CHROMOSOME 11 BETWEEN TOMATO AND POTATO: UTILITY OF THE CONSERVED REGIONS AND IMPLICATIONS FOR ORGANIZATION AND EVOLUTION OF THEIR GENOMES

Zhonghua, Zhang, Jun, He

Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China

Tomato and potato share a common progenitor, and their genomes share a high-level of colinearity, differing mostly by a number of chromosome rearrangements. However, the limited syntenic sequences have hampered the comparative analysis between them. In this study, nine pairs of syntenic BAC sequences on chromosome 11, four on the short and five on the long arm, were identified, sequenced and analyzed. The total length of tomato BACs was 0.9 Mb, representing 6.7% of the 13.5 Mb of the euchromatic regions on chromosome 11. Through identifying conserved regions, BAC finishing could be facilitated and gene annotation could be refined. Repetitive elements were the major constituents of the unmatched sequences. Within analyzed gene-rich regions, syntenic regions are often larger in potato than in tomato. Ty1-copia retrotransposons were preferentially located in gene-rich (presumably euchromatic) regions, whereas Ty3-gypsy retrotransposons predominated in gene-poor regions. In addition, a lower level of colinearity and a higher protein evolution rate were observed on the inverted short arm of chromosome 11, suggesting a possible role for chromosome inversions in speciation of tomato and potato. These observations may help to better understand the genome organization and evolution of tomato and potato following their divergence from a common progenitor.

COMPARATIVE ANALYSES OF THE POTATO AND TOMATO TRANSCRIPTOMES

1 2 3 4 5David M. Francis , Allen Van Deynze , John Hamilton , Walter De Jong , Dave Douches , 6 3

Sawen Huang , C. Robin Buell

1 2Dept. of Horticulture and Crop Sciences, The Ohio State University, Wooster, OH 44691; Seed Biotechnology

3Center, University of California, Davis, CA 95616; Department of Plant Biology, Michigan State University, East

4 5Lansing, MI 48824; Department of Plant Breeding, Cornell University, Ithaca, NY 14853; Department of Crop 6and Soil Science, Michigan State University, East Lansing, MI 48824; Institute of Vegetables and Flowers, Chinese

Academy of Agricultural Sciences, Beijing, China

Bioniformatic and computational comparison of sequences across species has empowered basic and applied research. The identification of Conserved Ortholog Set (COS) genes, the ability to predict intron sequences based on reference genomes, and databases organized around metabolic pathways have further facilitated comparative analysis. Next generation sequencing is now providing much more extensive sequence data and genomic resources that extend horizontally to encompass variation within and between species. To assess the genetic diversity within cultivated U. S. potato and tomato germplasm, we constructed normalized cDNA libraries from three tetraploid potato and six inbred tomato varieties using root or tuber, leaf, flowers, fruit and callus tissue. Using Illumina GAII “next generation” sequencing technology, we generated 2.82 Gb and >2.5 Gb of total sequence for each potato and tomato cultivar, respectively. The 60 bp reads, single and paired end, were assembled using Velvet to generate 38 Mb of transcriptome sequence from potato and 32.5 Mb from tomato. Due to the high quality and depth of sequence coverage, we were able to identify >150,000 high quality SNPs within each potato variety and analyses of tomato SNPs are underway. To identify the genomic context of the assemblies and permit higher-level genomic analyses, we aligned the potato transcriptome to the doubled monoploid Solanum phureja DM1-3 516R44 (CIP801092) draft genome sequence released by the Potato Genome Sequencing Consortium (http://potatogenome.net). Efforts are underway to utilize the S. phureja genome sequence as a proxy for the tomato genome sequence and thus perform parallel analyses in tomato. These new sequences give us tools for the further analysis of the Solanaceae genome landscape, including identification of gene duplications between species, a compendium of SNPs within species, a description of haplotype blocks across domesticated lineages, and the proportion of the genome under purifying/diversifying selection.


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SOL 200970

TANDEM REPEATS BASED COMPARATIVE GENOMICS IN SOLANACEAE

Atul Grover, PC Sharma

School of Biotechnology, Guru Gobind Singh Indraprastha University, Kashmere Gate, Delhi.

Tandem repeats constitute a significant portion of eukaryotic genomes. We studied tandem repeats with large motif size (>7bp) in a clustered set of 116811 unigenes representing 13 species of Capsicum, Nicotiana, Petunia and Solanum and two species of the genus Coffea. Distribution of such repeats was also compared in available genomic sequences of potato (5Mb) and tomato (90Mb). Repeats with motif size in multiple of 3bp were twice more abundant with 15, 18 or 21 bp motifs being most abundant. The relative abundance of these three length motifs was random as no particular motif length was universally favoured. In genomic sequences, tandem repeats with repeat unit of length 22 are also abundant. Interestingly, 114 bp motifs or its multiples (114X) constituted 10-25% of all the repeats in seven of the species studied. Preliminary investigations revealed these repeats to be associated with abiotic stress response genes. In general, the average repeat number declined with an increase in the motif size in all the sequences. The mean motif size within a given contig of tomato significantly deviated from the mode within the same contig. Repeat conservation was maintained in expressed sequences within a genus, but found not so obvious across genera. Nevertheless, this helped in building a number of high quality syntenic pairs. Our dataset provides new insights into occurrence of tandem repeats in the expressed regions of eukaryotic genomes.

THE SOL GENOMICS NETWORK: NEW APPROACHES FOR CLADE-ORIENTED PHENOMICS

Naama Menda, Isaak Tecle, Anuradha Pujar, Aureliano Bambarely, Tom York, Adri Mills, Joseph Gosselin, Robert Buels, Lukas Mueller

Boyce Thompson Institute for Plant Research, Tower Road, Ithaca NY 14853, USA

Biological databases provide a centralized infrastructure for data mining and analysis. Open access to the growing amounts genome information, derived annotations, and supporting data, is an evolving scientific platform which has enabled science to ask large scale biological questions using in-silico tools. Although genome sequences have initiated a new era in biology, its value is limited without manual annotations and supporting phenotype data.The SOL Genomics Network (SGN, http://www.sgn.cornell.edu/) is a clade-oriented database (COD), which provides a scalable, comparative framework for genetic, genomic, phenotypic, and taxonomic information of the Solanaceae and closely related Asterids. A main focus of SGN is developing a comprehensive database for Solanaceae phenotypic variation, linked with the extensive Solanaceae genotype data. Efforts for creating such a resource include literature curation, developing a Solanaceae-specific phenotype ontology in collaboration with breeders and other scientists, and building a community database, in which assigned members of the community have privileges for annotating genes and phenotypes online. In the past two years this model has yielded a comprehensive database with many cross links between genes and morphological and biochemical phenotypes, yet the availability of phenotyped collections has remained a limiting factor. We envision the future of phenotype analysis beginning with an innovative way of maintaining information, by moving towards data sharing similar to today's common practice of sharing sequences. In this talk, ways for annotating and analyzing phenotypes will be discussed, using examples from SGN's developed tools and methodologies.


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SOL 200972

COMPARATIVE ANALYSIS OF TRANSCRIPTIONAL REGULATORS IN SOLANACEAE

1 1 1 1 1 2S. Mathur , S. Vyas , A.K. Sharma , P. Khurana , J.P. Khurana and A.K. Tyagi

1Inter-disciplinary Centre for Plant Genomics, Department of Plant Molecular Biology, University of Delhi South 2Campus, Benito Juarez Road, New Delhi-110021, INDIA; Present address- National Institute of Plant Genome

Research, Aruna Asaf Ali Marg, New Delhi-110067, INDIA

The Solanaceae family members exhibit vast diversity in terms of habitat, from hot deserts to the wettest tropical rain forests, as well as habit, from small herbs to large trees. Transcriptional control of gene regulation governs most of the biological processes including development and differentiation of organs, as well as the ability to respond to various environmental cues. Therefore, a comparative assessment of the transcription factors and their associated proteins (collectively called as transcriptional regulators, TRs) among Solanaceae can provide a framework to investigate biodiversity amongst its members. Efforts so far have focused to sequence only few members of Solanaceae. Therefore, in this study, representation of TR families were assessed using EST-clusters for seven Solanaceae members (Solanum lycopersicum, S. tuberosum, Capsicum annum, Ipomea nil, Petunia hybrida, Nicotiana benthamiana, N. tabacum) and compared with the TRs identified from proteins predicted in six other plant species, whose genomes have been fully sequenced. HMM profiles specific for various domains defining a TR family were either downloaded from PFAM or were created from alignments in-house. From more than 80 TR gene families selected for the analyses, AP2-EREBP, MYB/MYB-related and bHLH emerged as the top three TR families in all the thirteen species analysed. The Solanaceae members showed higher proportion of WRKY TR gene family as compared to the other plants. The predicted TRs from tomato are also being localized on tomato BACs available at SGN. Our data would serve as a major resource for TR families in not only Solanaceae but also across different plant species.

UNDERSTANDING MOLECULAR BASIS OF HOST-PATHOGEN INTERACTION: A BIOINFORMATICS APPROACH

T.R. Sharma, Soham Ray and D.K. Gupta

National Research Centre on Plant Biotechnology IARI, Pusa Campus, New Delhi 110012, India

The use of resistance cultivars is very important in any plant disease management programmes. In specialized pathogens a typical gene-for-gene system exists during host-pathogen interactions. This type of interaction involves recognition of host resistance (R) gene protein by a pathogen Avirulence (Avr) gene protein which results in resistance phenotype. In some host-pathogen systems it has been experimentally proved and validated. Since it is a typical protein-protein interaction, one can use bioinformatics approach to understand the basic mechanism of such interactions. For use of bioinformatics tools in such experiments, both R- and Avr- gene sequences should be known. Though it is relatively easy to clone and characterize plant disease resistance genes using map based cloning approach, the cloning and characterization of pathogen Avr genes is very difficult. Now a days many plant and plant pathogen genomes have been fully sequenced and the genome sequence data are available in public domain. Therefore, if we know the sequence of a specific R-gene, its counterpart Avr gene can be identified by in silico protein modelling and protein interactions. Alternatively, if we know the nucleotide sequence of pathogen Avr gene its complementary R-gene can be identified in the plant genome sequence in silico. The protein-protein interaction studies will be presented in the conference by taking a specific example of a cloned R- gene and complete sequence of the plant pathogen genome. The implication of study in understanding interaction between tomato and its pathogens will be discussed during presentation.


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SOL 200974

PLECDOM: A PROGRAM FOR IDENTIFICATION AND ANALYSIS OF PLANT LECTIN DOMAINS

Smriti, Shridhar, Gitanjali Yadav, Debasis Chattopadhyay

Computational Biology Laboratory, National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi 110067, India

PLecDom is a program for detection of Plant Lectin Domains in a polypeptide or EST sequence, followed by a classification of the identified domains into known families. The web server is a collection of plant lectin domain families represented by alignments and profile Hidden Markov Models. PlecDom was developed after a rigorous analysis of evolutionary relationships between available sequences of lectin domains with known specificities. Users can test their sequences for potential lectin domains, catalog the identified domains into broad substrate classes, estimate the extent of divergence of new domains with existing homologs, extract domain boundaries and examine flanking sequences for further analysis. The high prediction accuracy of PLecDom combined with the ease, with which it handles large scale input, enabled us to apply the program to protein and EST data from 48 plant genome-sequencing projects in various stages of completion. Our results represent a significant enrichment of the currently annotated plant lectins, and highlight potential targets for biochemical characterization. The search algorithm requires input in fasta format and is designed to process simultaneous connection requests from multiple users, such that huge sets of input sequences can be scanned in a matter of seconds. PLecDom is available at http://www.nipgr.res.in/plecdom.html.

SESSION VII DEVELOPMENT AND SIGNALING

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GENETIC REGULATION OF FRUIT RIPENING AND ETHYLENE RESPONSE IN TOMATO

1,2 1,2 1,2 1, 2 1,2James Giovannoni , Cornelius Barry , MiYoung Chung , Julia Vrebalov and JeMin Lee

1 2Boyce Thompson Institute for Plant Research, Ithaca, NY 14853 USA; U.S. Department of Agriculture -Agriculture 2Research Service, Robert W. Holley Center, Ithaca, NY 14853, USA.

The ripening and development of fleshy fruits is regulated by environmental, hormonal and developmental cues. Ethylene is the key ripening hormone of climacteric fruits and can influence ripening in many non-climacteric fruits. Our laboratory uses tomato as a model system to understand ripening regulation and has identified a number of necessary ripening genes via positional cloning of loci underlying ripening mutations and transcriptional profiling studies of ripening associated gene expression. To date we have identified six transcription factors that we have shown to be necessary for tomato fruit ripening via transgenic studies including two MADS-box, two NAC domain, an Ethylene Response Factor (ERF) and an APETALA2 gene homolog. One of the MADS-box genes, TAGL1, is especially intriguing in that it suggests a molecular link between fleshy fruit development and eventual ripening via a single gene product. A summary of these gene activities in the context of other reported regulatory and ethylene response genes will be presented.

USING GENOMICS TO STUDY NATURAL VARIATION IN TOMATO LEAF SHAPE AND LIGHT SIGNALING

Julin N. Maloof, José M. Jiménez-Gómez, Seisuke Kimura, Daniel Koenig, Daniele Filiault, Lauren Headland, Daniel Chitwood, Neelima Sinha

Department of Plant Biology, University of California, Davis, 1 Shields Ave, Davis, CA 95616 USA.

Plants acquire the bulk of their energy from light capture by leaves, and leaf shape has direct consequences on the efficiency of light capture and photosynthetic carbon fixation. As a result, leaf shape and plant architecture must be optimized in response to variation in light quality. To understand the genetic programs controlling fundamental developmental processes (and variation within them), genetic networks regulating both environmental response and morphological form must be integrated. I will present our initial characterization of Solanum sp., which revealed wide variation in both leaf morphology and developmental responses to light quality, and I will discuss our future plans to define the genes and networks underlying these responses. These later goals are being accomplished by characterizing the S. lycopersicum X S. pennellii near isogenic lines through phenotypic measurements, dense genotyping, and transcript profiling. Transcriptional and genotypic variation in these lines will be characterized using high-throughput sequencing to acquire genome-wide mRNA sequence and SNP information. Construction of genetic networks regulating leaf morphology and light development will be coupled with genetic and transgenic approaches to identify central regulators of development and developmental variation. The resulting network will then be used as a guide to survey natural variation found in additional wild tomato accessions.


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SOL 200978

TERPENE BIOSYNTHESIS IN TRICHOMES OF TOMATO AND TOBACCO: FUNCTIONAL AND EVOLUTIONARY ASPECTS

Alain Tissier

UMR DIAPC, Université Montpellier 2, Centre IRD de Montpellier, 911 Avenue Agropolis, B.P. 64501, 34394 Montpellier, Cedex 5, FRANCE.

Many species of the Solanaceae possess glandular trichomes at the surface of their aerial parts. They are the site of production of different classes of compounds including sugar esters, terpenoids, methylketones and flavonoids. The composition of the trichome exudate varies in a species- or even subspecies-specific fashion and usually a single type of compound is produced. For example different land races of the wild tomato species Solanum habrochaites produce either sesquiterpenes and sesquiterpene carboxylic acids, or methylketones. The isolate LA1777 of S. habrochaites produces ��-santalenoic, ��-bergamotenoic and ��-bergamotenoic acids as well as their olefinic precursors, and germacrene B and D. The sesquiterpene carboxylic acids (SCAs) have been suspected to play roles in the interaction with insect pests. Although the biosynthesis of germacrene B and D was previously found to proceed via the classical trans,trans-farnesyl diphosphate intermediate, a novel two-step pathway was discovered for the biosynthesis of the olefinic precursors of the SCAs. The first activity, encoded by the zFPS gene, synthesizes cis,cis-farnesyl diphosphate (Z,Z-FPP). The second gene encodes a kaurene synthase-like enzyme, SBS, which uses Z, Z-FPP to make the mix of olefinic precursors of the SCAs. Both proteins are targeted to the plastids suggesting that this sesquiterpene pathway is directly connected to the plastidic non-mevalonate pathway. In parallel, terpene biosynthesis in glandular trichomes was investigated in the related tobacco, Nicotiana tabacum. In particular, biosynthesis of the labdane diterpene cis-abienol was elucidated. As for the biosynthesis of kaurane diterpenoids in other Angiosperms, two steps are required to proceed from geranyl gernayl diphosphate to cis-abienol. The first is catalyzed by an enzyme of the copalyl-diphosphate synthase family, NtCPS2, which produces copal-8-ol diphosphate. This product is then converted to cis-abienol by a kaurene synthase like enzyme, NtABS. Interestingly, NtABS is most closely related to the tomato enzyme SBS. Similarly, the tobacco trichome specific diterpene synthases CBTS, which produce cembratrien-ols are most closely related to the tomato SSTLH1 and SSTLH2 sesquiterpene synthases which make germacrene B and D respectively. These observations and phylogenetic analyses indicate the existence of a distinct lineage for trichome specific terpene synthases which appeared early in Solanaceae evolutionary history.

MODULATION OF THE GA PERCEPTION AND RESPONSE PATHWAY PROVIDES PLASTICITY TO TOMATO DEVELOPMENT

1Cristina Martí, Jose Luis Rambla, Leandro Hueso, Florence Piron , Esperanza Lazaro, David Alabadí, 2 1 2Maria Ines Zanor , Abdel Bendhamande , Alisdair Fernie , and Antonio Granell

1Fruit Quality Genomics and Biotechnology lab, IBMCP (CSIC UPV), Spain; URGV Evry, Cedex, France; 2MPIMPP Golm, Germany.

The mechanism of action underlying the growth-inducing effect of gibberellins (GA) requires first binding to the GA receptors or GIDs and then involved the participation of the signaling molecule and repressor of growth DELLA. The interaction of GID with DELLA in the presence of GA leads to an increase in the affinity of DELLA for specific SCF E3 ubiquitin ligase complex thus favoring the destruction of DELLA by the proteosome and the alleviation of the restrain in growth imposed by this repressor.

In tomato there appears to be a single LsDELLA gene, but at least three GIDs. We will describe the GID DELLA pathway in tomato at the molecular level and the modulation of the levels of expression of these genes along different tomato developmental processes. Genetic engineering of the single LsDELLA in tomato leads to alterations in reproductive structures (Marti et al 2006). We will present our most recent results using transgenic tomato plants and single point EMS mutants in SlDELLA identified by TILLING that suggests that this protein not only participates in the duration and extent of the cell elongation processes accompanying shoot and fruit growth but it is also a key regulator of other aspects of tomato growth and development.


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SOL 200980

ALTERATIONS IN CAROTENOIDS CONTENTS DURING TOMATO FRUIT DEVELOPMENT AS A RESULT OF TRANSGENIC SPERMIDINE SYNTHASE EXPRESSION

1 1 2 2 2Mohamed Hichem Neily , Chiaki Matsukura , Mickaël Maucourt , Annick Moing , Dominique Rolin , 3 1Takaya Moriguchi , Hiroshi Ezura

1 2Grad Sch Life Environ Sci, Univ Tsukuba; UMR619 Biologie du Fruit, Centre INRA de Bordeaux, France,3NAFRO-NIFTS

Carotenoids are lipid-soluble pigments produced strictly by photosynthetic organisms, well known by their valuable benefits for human health in the prevention of numerous diseases. In our study, the content of carotenoids in mature tomato fruits of homozygous transgenic lines harboring apple spermidine synthase MdSPDS1 (Gene bank accession number AB072915) under the control of 35 promoter was analyzed by HPLC. The analysis of the total carotenoids (phytoene, lutein, lycopene, β carotene) revealed that transgenic fruits exhibit significant increasing content especially for lycopene (1.5-2 folds) high with respect to wild type particularly in the pericarp-columella tissue.

To investigate the relationship between carotenoid content and gene expression, key genes of carotenoids metabolic pathway, encoding phytoene synthase 1 (Psy-1), phytoene desaturase (Pds) , 1-deoxy-D–xylulose-5 phosphate synthase (Dxs) , lycopene –epsilon cyclase (Lcy-e) and lycopene beta cyclase cyclase (Lcy-b), were checked by real time PCR. Since ethylene is known to play critical role during ripening and carotenoid accumulation, ethylene production of fruits ripened on the vine at different stages of fruit development was measured and the results have shown clear differences between transgenic fruits and wild type. Such results lend credence to the idea that elevated polyamines might modulate the carotenoid synthesis.

CHANGING TOMATO FRUIT TEXTURE BY MANIPULATION OF CELL CYCLE REGULATORS

1 2 2 1,2Anna Czerednik , Marco Busscher , Ruud de Maagd and Gerco C.Angenent

1Dept. of Plant Cell Biology, Radboud University Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, the Netherlands, 2Plant Research International, Bioscience, Droevendaalsesteeg 1, 6708 PB Wageningen, the Netherlands.

Fruit development starts from the pollination event that triggers the formation of a fruit from the ovary. Fruit growth can be divided into phases of cell division and cell expansion, the latter being associated with endoreduplication, and ends in a mature green fruit. It has been proposed that these growth stages are critical for the quality of the ripe tomato fruit, because they determine the cellular structure of the fruit. Control of fruit development is intimately associated with the cell cycle and therefore, we focused our research on the main players of the cell cycle - cyclin-dependent kinases (CDKs), cyclins (CYC) and several regulators - EBP and the transcription factors E2F and DP. The aim of our research is to elucidate which molecular and cell biological parameters during tomato fruit development co-determine fruit quality aspects, such as firmness, storage ability, fruit texture, resistance to cracking and taste. We have determined the expression pattern of a number of cell cycle related genes from pollination until the mature green stage and based on their expression pattern we selected candidate genes for further investigation. We generated transgenic lines overexpressing and down-regulating these cell cycle related genes under the control of the fruit specific promoter pTPRP. Altering the expression patterns of these genes, we obtained mutant fruits that are affected in pericarp thickness, size and cellular texture. The relation of the observed phenotypes and the regulation of the cell cycle will be discussed.


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SOL 200982

ROLE OF ERFS IN TOMATO PLANT DEVELOPMENT AND FRUIT RIPENING

Rakesh Kumar Upadhyay, Aniruddha P. Sane, Pravendra Nath

Plant Gene Expression Lab, National Botanical Research Institute, Lucknow, India

The AP2/ERF domain family is a large family of transcription factors (characterized by the presence of degenerate 58-59 amino acid DNA binding domain) with varied roles in plant development, ethylene response and plant adaptation to biotic and abiotic stresses. In an effort to identify ERFs that may play a role in fruit ripening, fruit cDNA was used for isolation of ERF members using degenerate primers. Two ERFs designated as SlERF6 (264aa) and SlERF8 (314aa) were studied for expression during fruit development and in response to abiotic stresses. Despite differences in their structures, a lot of similarities in their expression patterns were observed. The expression of both the ERFs increased during fruit development reaching a peak at the mature green stage. Both showed strong ethylene responsiveness in their expression as evident from increased transcription within 20-60 minutes of ethylene treatment to leaves. The expression of both increased in response to drought after 3 days and transiently within 2h after exposure of one month old plants to cold. Expression of these two ERFs in transgenic Arabidopsis and tobacco plants resulted in pleiotropic changes in plant development. Transgenic SlERF6 expressing plants were slow growing, shorter and showed delayed flowering and senescence as compared to controls. In contrast, SlERF8 expressing tobacco plants showed early senescence. Transgenic SlERF8 Arabidopsis and tobacco plants also showed multiple inflorescence shoots indicating a possible effect on apical dominance. The results indicate that both the genes may have multiple roles in plant growth and fruit development and ripening.

A MAMMALIAN IKK/NF-B-LIKE SIGNALING PATHWAY REGULATES NITRIC OXIDE LEVEL IN PLANTS

Sangeeta Negi, Parankusam Santisree, Eros Vasil Kharshiing, Rameshwar Sharma

School of Life Sciences, University of Hyderabad, Hyderabad-500 046, India

Nitric oxide (NO) is involved in diverse plant growth processes, however little is known about signaling pathways regulating NO levels in plants. We isolated a NO-overproducing mutant of tomato (Solanum lycopersicum) where hyper-accumulation of NO, associated with increase in nitric oxide synthase (NOS) like activity, caused shortening of primary root in the seedlings. The scavenging of NO restored root elongation in shr mutant providing a convenient bioassay to analyze the signaling pathway upstream of NO production. Inhibition of NOS-like activity reduced NO levels and stimulated root elongation in the shr mutant seedlings, while inhibition of nitrate reductase (NR) activity could not rescue shr phenotype. Application of pharmacological inhibitors of mammalian IKK/NF-κB signaling pathway reduced NO levels and NOS-like activity and stimulated shr root elongation. The stimulation of NO levels in shr mutant also conferred increased resistance to pathogen Pseudomonas syringae. Our data indicate that plants regulate NO synthesis using a signaling pathway, which is operationally similar to IKK/NF-κB pathway present in mammals.


SOL 2009 83

SESSION VIII MOLECULAR BREEDING AND GENETIC ENHANCEMENT

84

"PHENOM NETWORKS" - KNOWLEDGE REPRESENTATION OF COMPLEX PHENOTYPES

1 2Yaniv Semel , Dani Zamir

1 2Phenom-Networks, 4 Ben Gurion Street, 74032, Nes-Ziona, Israel; Faculty of Agriculture, the Hebrew University of Jerusalem, Rehovot, Israel

Resolving natural phenotypic variation into genetic and molecular components is a major objective in biology. Over the past decade, tomato interspecific introgression lines (ILs), each carrying a single 'exotic' chromosome segment from a wild species, have exposed thousands of quantitative trait loci (QTL) affecting plant adaptation, morphology, yield, metabolism, and gene expression. QTL for fruit size and sugar composition were isolated by map-based cloning, while others were successfully implemented in marker-assisted breeding programs. Our challenge is to integrate the multitude of QTL in plants and animals, into a single Phenom Networks database, which is imperative as the great majority of the raw QTL data is not uploaded into existing databases and thus is lost. Phenom Networks1 is a web-based system that implements statistical methods and algorithms to analyze phenotypic data of tomato, roses and lisianthus and provides interactive graphical and statistical outputs. Such an integrated phenomic database unravels some unifying principles about the architecture of complex traits and paves the road for genomics assisted breeding.

1Web address http://phnserver.phenome-networks.com/icis/


SOL 2009 85


SOL 200986

MOLECULAR BREEDING OF FRUIT CROPS VIA NOVEL GENETIC INTERVENTIONS

Avtar K. Handa

Center for Plant Environmental Stress Physiology, Horticulture and Landscape Architecture Department, Purdue University, West Lafayette, IN 47907 USA

The unintended senescence induced by organ separation and the genetically programmed leaf senescence and ripening associated fruit textural changes are the major causes of postharvest losses of most fresh produce. Although several plant growth regulators seem to play roles in these processes, their molecular understanding remains at its infancy and modulation of their deleterious effects challenging. We have been coupled the candidate genes approach and molecular breeding to understand these processes and improve fruit quality attributes. The results I will present will include: (1) Modulation of polyamines levels greatly enhances tomato fruit shelf life and delays fruit shriveling by a mechanism independent of rate of water loss from fruit. (2) A novel small heat shock protein enhances biomass accumulation, alters flower architecture and enhances fruit juice quality. (3) Identification of a novel tomato cell wall architecture mutant altered in rhamnogalacturonan I structure by cell wall chemistry and characterization of effects of this mutation on fruit ripening and response to environmental cues. Collectively these results will demonstrate that the candidate gene approach provides a robust model for genetic enhancement of plants species including Solanaceae.

MOLECULAR APPROACHES TO IDENTIFY SOURCES OF RESISTANCE TO LATE BLIGHT IN POTATO

Hannele Lindqvist-Kreuze

International Potato Center (CIP)

Late blight continues to be a major threat for potato production in developing countries and therefore breeding for durable late blight resistance is one of the highest priorities of the International Potato Center (CIP). CIP holds in trust germplasm and has developed advanced breeding materials with promising levels of late blight resistance. Two examples of wild species are Solanum cajamarquence and S. paucissectum both of which contain accessions that display R-gene dependent and/or quantitative resistance. The advanced tetraploid breeding population B3 displays high levels of quantitative resistance under high epidemic pressure in a wide range of tropical and subtropical agro ecologies. To understand the genetic components of resistance in these materials we take the advantage of our diploid experimental populations PCC1 (resistant S. paucissectum / susceptible S. chomatophilum), and B3C1HP (resistant haploidized B3 clone/ susceptible S. phureja). We have identified molecular markers associated with late blight resistance by 10K potato cDNA microarray and tested several different types of markers for association with the resistance phenotype in our mapping populations. Currently, we are attempting to identify Resistance Gene Analogs (RGA) within the late blight QTL by high throughput sequencing. Finally we wish to use these molecular markers to understand the genetic base of the resistance as a means to orient its transfer across populations.


SOL 2009 87


SOL 200988

MOLECULAR BREEDING IN SOLANACEOUS CROPS IN INDIA

Mathura Rai, Major Singh, Rajesh Kumar, Sanjeev Kumar and D. Dutta

Indian Institute of Vegetable Research, P.B. No. 01, P.O. Jakhini (Shahanshahpur), Varanasi 221 305 India

Solanaceous vegetables constitute a group of vegetables like tomato, eggplant, and chilli, which are well known for their nutritional and economic values. In case of Solanaceous vegetables, DNA-based molecular markers have been extensively used for a variety of purposes, especially for the study of marker-trait associations, with a goal to transfer desirable alleles of genes/QTLs controlling important traits to elite cultivars. Another important area, where DNA markers technology is proving its effectiveness is the pyramiding of genes for biotic and abiotic stress resistance. In India, molecular breeding efforts got a quantum jump with the launch of Solanaceous Genome Project, with the prime objective of sequencing of the tomato genome and understanding the functions of interconnected biological processes. Efforts are on to effectively utilize marker aided tools to breed novel vegetable varieties. At IIVR, Varanasi the development of recombinant inbred lines (RILs) is in the advanced stage for traits like leaf curl resistance, lycopene content, early blight resistance in tomato, pungency and fruit morphology in chilli, and fruit size and shape in eggplant. Two sources of ToLCV resistance, viz., L. hirsutum-derived Ty2 and L. chilense-derived Ty1 have been used for pyramiding in a popular variety DVRT-1, which is in the advance stage of selection. Markers for hybrid seed purity testing in tomato, eggplant and chilli have been identified. Significant achievements have been made in the identification of CMS lines and virus resistance lines, and development of a reliable detection system of pepper leaf curl virus in chilli exploiting DNA markers. In the light of fast acceptance and integration of diverse uses of DNA-based molecular markers by the plant breeders, the new generation markers are more likely to become an integral component of conventional plant breeding programmes.

DEVELOPMENT OF MICRO-TOM MUTANT DATABASE “TOMATOMA” AND A TILLING PLATFORM

1 1 1 1 1Erika Asamizu , Yoshihiro Okabe , Takeshi Saito , Tohru Ariizumi , Chiaki Matsukura , 1 1 2 3 1

Tsuyoshi Mizoguchi , Naoya Fukuda , Yukiko Yamazaki , Koh Aoki , Hiroshi Ezura

1 2Graduate School of Life and Environmental Sciences, University of Tsukuba; National Institute of Genetics,

3Kazusa DNA Research Institute

With an aim of establishing functional genomics tools using the model miniature tomato cv. Micro-Tom, we are developing a large-scale mutagenized population. Well-defined phenotyping data is prerequisite to initiate forward genetic screen. We are developing a mutant database named “TOMATOMA” and accumulating the phenotype information. As of July 2009, 787 EMS and 67 gamma ray mutagenized lines with visible mutant phenotypes have been scored. We have obtained many mutants with altered internodes length, leaf shape and color, flower morphology and color. We have also obtained a series of fruit mutants, both shape (long, round, heart-shaped and eccentric) and color (deep green, orange and pink). In order to enable cross searches among other databases, Solanaceae phenotype ontology (SP) information is added to each mutant. We are planning to publicize “TOMATOMA” in the coming year.The genome sequence of tomato is going to be deciphered within next few years, and comprehensive cDNA information is already available. In order to establish a reverse genetic research system, we are developing a TILLING platform using the Micro-Tom EMS mutagenized population. We made a DNA pool from 1,500 mutant lines and the mutation frequency was determined by screening 5 genes. It was revealed that the current pool contains one mutation per 1,163 kb, which is a relatively mild mutation frequency. We could find 0 to 5 mutated allele(s) (average 2.4 alleles) for each gene screened. We are now developing additional TILLING pool from new mutant populations with a range of EMS treatment, 0.7-1.5%.


SOL 2009 89


SOL 200990

GENES THAT DRIVE HETEROSIS

1 2 1Uri Krieger , Zach B. Lippman and Dani Zamir

1 2The Hebrew University of Jerusalem, Israel; Zachary B. Lippman, Cold Spring Harbor Laboratory, NY11724,

USA

Hybrid vigor, or heterosis, describes the phenotypic superiority of a hybrid over its parents with respect to traits such as growth rate, reproductive success and yield. Despite intense research on heterosis over the years, the greatest challenge still centers on identifying which genes are involved, and unequivocally linking specific molecular changes with heterotic phenotypes. Our research employs tomato (Solanum lycopersicum) as a yield-heterosis model to identify heterotic effects due to overdominance. Due to its 'selfing' mode of reproduction, accompanied by only modest effects of 'inbreeding depression', tomato enables greater genetic and phenotypic power in the search for true overdominant genes. In addition to dissecting overdominant QTL from wild species, we have explored a new approach to search for true overdominant genes, which is based on classical studies showing that classically defined “recessive” mutations can be overdominant. We selected isogenic mutants in the background of the tomato cultivar M82, and searched for overdominant effects on yield from more than 70 mutant hybrids over two years. Here we report that SINGLE FLOWER TRUSS (SFT), encoding a major component of the mobile flowering signal “florigen”, increases yield by as much as 80% when sft mutants are heterozygous. We show that sft single gene heterosis is robust, showing strong effects in multiple independent mutations, different genetic backgrounds, and diverse environments. Our phenomics analysis revealed that several component traits driving yield are improved in sft overdominance, and these effects depend on the SFT repressor gene, SELF PRUNING. Despite the remarkable shifts in growth observed for sft/+ heterozygotes, there is little change in expression of SFT or its downstream target APETALA1. We propose that screening for developmental mutations with heterozygous dosage effects is a powerful approach to identify heterosis genes with the potential to increase crop yields.

ILLUMINA ARRAYS FOR TOMATO AND PEPPER GENETIC ANALYSES AND BREEDING

Martin Ganal, Gregor Durstewitz, Andreas Polley, Hartmut Luerssen

TraitGenetics GmbH, Gatersleben, Germany

Using comparative sequencing of 5000 amplicons derived from tomato unigenes and 3500 amplicons derived from pepper unigenes in a set of 16 lines and pools, we have identified several thousand SNP markers in elite tomato and pepper varieties. The identified SNPs were subsequently analysed for their allele frequency in tomato and pepper breeding material and their suitability for the Illumina Golden Gate assay system. For each species, we have subsequently assembled an optimised 1536-plex and tested the multiplex on a set of 90 current varieties and breeding lines. In both cases, more than 90% of the markers were functional and could be scored in the respective material. Based on these data, we will present data on the level of polymorphism of these markers in the different usage types. Furthermore, we have used these markers to generate high density genetic maps in segregating populations with nearly 1000 mapped markers in both cases. These assays together with the associated allele frequencies, map positions and other information will provide a valuable resource for tomato and pepper breeding.


SOL 2009 91


SOL 200992

DEVELOPMENT OF INTRON-FLANKING MARKERS FOR TOMATO USING BOTH TOMATO AND ARABIDOPSIS GENOMIC INFORMATION

1 1 2 1 1 1Yuanyuan Wang , Jia Chen , David M. Francis , Huolin Shen , Tingting Wu , and Wencai Yang ,*

1Department of Vegetable Science, College of Agronomy and Biotechnology, China Agricultural University, Beijing 2100193, China; Department of Horticulture and Crop Science, The Ohio State University/OARDC, 1680 Madison

Ave, Wooster, OH 44691, USA

High density molecular maps have been constructed for tomato. However, almost all these maps are built using populations derived from interspecific crosses. Previous studies suggest that only 5% of the markers developed by this approach are polymorphic within cultivated tomatoes. Thus, there is a lack of markers that can be used to characterize important traits in cultivated tomatoes. In this study, we used both tomato and Arabidopsis genomic information to discover polymorphisms in introns of conserved genes. Primers flanking predicted intron regions were designed for 201 unigenes (SGN 200607#2). PCR was conducted using genomic DNA from 12 tomato accessions including two wild species PI128216 (Solanum pimpinellifolium) and PI114490 (S. lycopersicum .var. cerasiforme), and 10 cultivated tomatoes (S. lycopersicum) OH9242, OH88119, Baiguoqiangfeng, Shijifeng, Meifen 1, Zhongshu 5, Micro-Tom, Moneymaker, Liger 87-5, and Rio Grande. Sequence analysis of 112 PCR products from 84 unigenes revealed that 42 unigenes had 144 single nucleotide polymorphisms (SNP) and 36 intron length polymorphisms (ILP) between S. pimpinellifolium and S. lycopersicum, and 48 SNPs and 17 ILPs were polymorphic within 10 cultivated tomatoes. The SNP frequency in introns for cultivated tomatoes was 1 SNP every 1475 bp, which was 2.7-5.7 times higher than in EST. This suggested that developing intron-flanking markers was a promising approach to discover sufficient markers for cultivated tomatoes.

SESSION IX BIODIVERSITY

93


SOL 200994

PEPPER GENETIC DIVERSITY: AN ANALYSIS IN THE INDIAN CONTEXT

1K.S.Varaprasad*, S.R.Pandravada, B.Sarath Babu and S.K.Sharma

National Bureau of Plant Genetic Resources, Regional Station, Hyderabad-500 030, Andhra Pradesh, India 1NBPGR, New Delhi- 110 012, India

India is a secondary centre of diversity for chilli pepper. Indigenous and exotic pepper diversity is analyzed using tools including GIS in the Indian context to review the status and focus on strengths and gaps. Over 1,000 accessions including 200 landraces and 90 improved varieties mainly belonging to Capsicum annum are available in the country. Seventy percent of global holdings (7,000 accessions) belong to C. annum and the rest to 9 other species. Pepper mainly used as a spice. However, several landraces are cultivated for various purposes such as paprikas and shimla mirch as vegetable, Boora mirapa and Bongu mirchi of south India for pickles, Naga Jolokia with a record pungency of 1,001,304 SHU of North East India with potential for medicinal/ aromatic/ pharmaceutical/ defense utility, Byadige, Warangal and Pachcha mirapa (yellow chilli) of South India, Nagauri of Rajasthan and Degchi of Kashmir as dye. Germplasm has wide range for capsaicin (0.05 - 1.8 %), capsanthin (22.3 - 268.3 ASTA units), xanthophylls and seed oil (12- 27%). Resistant/ tolerant sources are known for diseases like powdery mildew, bacterial wilt, fruit rot, several viruses (TMV, CMV, PVMV, CVMV and PBNV), leaf curl complex thrips, mites and nematodes. Scope for trait specific germplasm collection, introduction of wild species diversity and coloured bell peppers, varietal protection, conservation strategies, core set development and potential of gene pools in the context of improvement for aflatoxin resistance, nutraceutical value and industrial perspective are discussed.

DIVERSITY OF EGGPLANT AND ITS WILD RELATIVES IN INDIA

1J. L. Karihaloo , Shilesh Tiwari, S. Kalia, Sonika Singh, Manjeet Kaur and Ambika Baldev Gaikwad

National Bureau of Plant Genetic Resources, New Delhi- 110 012, India1Present address: Asia-Pacific Consortium on Agricultural Biotechnology, New Delhi-110012, India

Solanum melongena L. or eggplant (Genus Solanum, subgenus Leptostemonum) is one of the most important vegetable crops of India being grown and consumed almost throughout the country. Studies made on morphological diversity of eggplant land races reveal the existence of large diversity in vegetative and fruit characters ranging from forms similar to wild related species to advanced horticultural types. However, comprehensive information about the regional distribution of diversity is still lacking. Besides eggplant, several species of subgenus Leptostemonum exist here in wild, cultivated or naturalized form, some of which have medicinal or edible value while others are sources of valuable genes for eggplant breeding. These also include S. incanum, S. insanum and some weedy forms that together with S. melongena comprise a complex of progenitors and derivatives, the eggplant complex. Cytogenetic analysis revealed the complex to represent a single biological species. Molecular genetic analysis using a variety of marker systems revealed interesting information about the phylogenetic relationships of section Solanum and among the taxa of eggplant complex from South-west Asia and India. Besides, molecular markers were useful in establishing the identity of some mislabelled species.


SOL 2009 95


SOL 200996

R Umashanker

GVKV, Bangalore, India

TBD

USING MULTILOCUS SEQUENCE DATA TO INFER POPULATION STRUCTURE AND DEMOGRAPHIC HISTORY IN SEVERAL WILD TOMATO SPECIES (SOLANUM SECTION LYCOPERSICON)

1 2 2 2Thomas Städler , Aurelien Tellier , Carlos Merino and Wolfgang Stephan

1Plant Ecological Genetics, Institute of Integrative Biology, ETH Zurich, Universität-strasse 16, CH-8092 Zurich,

2Switzerland ; Evolutionary Biology, Department Biologie II, University of Munich (LMU), Gross-haderner Strasse 2, D-82152 Planegg-Martinsried, Germany

Wild tomato species are particularly attractive model systems for studying the evolutionary divergence among closely related taxa, including the genetic basis of reproductive isolation and adaptations to a variety of biotic and abiotic conditions. Before population-genetic data on candidate genes can be properly evaluated, however, it is imperative to establish baseline data on neutrally evolving reference loci. Under the guiding framework of coalescent theory, sequence data obtained from wild population samples or from plants scattered over the entire species' range are informative about many aspects of the evolutionary process, including modes of speciation and changes in effective population size. We present new DNA sequence data on eight nuclear loci from population samples of Solanum habrochaites and S. arcanum (formerly part of S. peruvianum) that complement our previous data on S. chilense and S. peruvianum. Intriguingly, levels of molecular divergence between the four taxa do not fit expectations from published phylogenetic analyses, but rather indicate near-equal differentiation in all pairwise comparisons. Our emerging results underscore the importance of population subdivision that will be quantified in various ways. Moreover, the joint impact of underlying population structure and the actual sampling scheme on summary statistics of the allele frequency spectrum that are generally used to test for selection is complex, implying that attention to sampling and its genealogical consequences should have high priority. Generally, our results highlight the importance of using an appropriate demographic model in future efforts to disentangle effects of natural selection from those due to demographic history.


SOL 2009 97


SOL 200998

POTATO GENETIC RESOURCES IN INDIA

J. Gopal, Vinod Kumar and S.K. Pandey

Central Potato Research Institute, Shimla-171 001, India

Potato is believed to be introduced in India in 17th century from Europe, and now it is the third most important crop of the country. The introduced long day adapted potato varieties were not suitable for cultivation under sub-tropical short day conditions prevalent in the plains of India. Thus potato breeding programmes were initiated to develop varieties suited to the different agro-climatic conditions of the country. To start with genetic variability prevalent in the country was collected. These cultivars represented some of the earliest introductions or their clonal variants, but proved to be too meager for the breeding purpose. Thus import of potato germplasm was initiated and now the national repository of potato germplasm maintained at the Central Potato Research Institute has more than 3500 accessions belonging to cultivated as well as more than 120 wild species. This collection is being maintained in field as well as in vitro gene banks, and the wild species are conserved mainly as true seeds. Till date about 1,200 tuberosum accessions have been conserved in vitro on MS medium supplemented with 2% sucrose and 4% sorbitol. The plantlets are incubated under 16 h photoperiod at 6-8oC. These conditions induce slow growth and help to avoid frequent sub-culturing. This collection is regularly checked for their freedom from viruses and viroids. Infected accessions are freed of viruses through meristem culture combined with thermo- and chemotherapies. Minitubers of new germplasm accessions received in vitro are produced for adding these accessions to field for evaluations and utilization. The collections has been screened for all important biotic and abiotic stresses including late blight, viruses, stem necrosis, bacterial wilt, cyst nematodes, frost and heat tolerance. The collection has been evaluated for adaptability under varying thermo- photoperiods, as well as for physiological and biochemical characters including those important to the processing industry. The accessions have been also characterized morphologically for characters important for their identification as well as for use in the hybridization programmes. To identify good general combiners among the germplasm accessions found promising for various characters, combining ability studies have been conducted. Pre-breeding of Andigena and wild species for developing clones suitable for their utilization in the breeding has also been undertaken. A number of improved clones have been developed and registered at the national level. The databases in electronic form have been developed and inventories on different groups of germplasm have been published from time to time. The information is made available to the breeders, and the utilization of the parental lines from this national repository has led to the development and release of as many as 47 potato varieties out which five varieties have been developed specifically for processing into chips and/or French fries. The present germplasm collection in India though modest, is the best in South Asia.

THE HISTORY OF EGGPLANT DOMESTICATION: PHYLOGEOGRAPHIC RELATIONSHIPS AMONG CANDIDATE PROGENITORS AND ASIAN HEIRLOOM VARIETIES

Rachel Meyer and Amy Litt

Eggplants appear to be products of an unusual domestication syndrome in which ethnobotanical uses have shifted from medicinal to culinary. Hundreds of heirloom cultivars maintained by communities throughout Asia differ from each other regarding gastrononomic and health-beneficial attributes. We are using a collection of 100 heirloom eggplant cultivars and wild relatives to study changes in regulation of the phenolic pathway (which yields many of the desirable compounds) that may be correlated with domestication and subsequent selection. To understand how these changes have occurred historically, we must identify the closest wild relatives, an issue that has been in dispute. To elucidate the relationships among eggplant and its wild relatives we have explored variability in several nuclear loci, as well as population genetic techniques such as AFLP and SNP analysis. Phylogenetic analysis using the Internal Transcribed Spacer (ITS) has revealed eggplants forming two separate clades. This raises the possibility that either eggplant or its putative progenitor are the product of an ancient hybridization among two members of Section Melongena, or that it may have been domesticated twice. However, AFLP and SNP data do not show two distinct clades, in contrast to the ITS data. These data are being used to address the relationship of the putative progenitor to domesticated eggplants, as well as to select cultivars for studies of gene regulation, phytochemistry, and ethno botany.


SOL 2009 99


SOL 2009100

HP Singh

ICAR, New Delhi, India

TBD

SESSION X RNAI AND EMERGING AREAS

101


SOL 2009102

TRANSCRIPTOME NETWORK ANALYSIS OF RIPENING TOMATO FRUIT SUGGESTS CROSS-TALK BETWEEN DEVELOPMENTAL AND ENVIRONMENTAL REGULATORY ELEMENTS

1 1,B 1 1 1,2Rob Alba ,A, Benjamin J. Cole , Paul P. Debbie , Lukas A. Mueller , and James J. Giovannoni

1 4Boyce Thompson Institute for Plant Research, Cornell University Campus, Ithaca, NY 14853, USA; USDA AAgricultural Research Service, Plant, Soil, and Nutrition Laboratory, Ithaca, NY 14853; Current address:

BMonsanto Company, 800 N. Lindbergh Blvd, St. Louis, MO 63167, USA; Current address: The Salk Institute for Biological Studies, La Jolla, CA 92037, USA.

Fruit transcriptome dynamics was investigated via time-series expression profiling using the TOM1 cDNA microarray and three ripening mutants (rin, nor, hp-1). 2975 unigenes are differentially expressed in wild-type fruit; lesions in LeMADS-RIN and NOR alter the expression of 89% of these unigenes. Eighty-eight of these unigenes were computationally mapped to the available partial tomato genome sequence, and appear evenly distributed within and among chromosomes. Gene correlation analysis predicts a network with scale-free topology and identifies 13 co-expression modules. Five prominent expression modules (containing 894, 587, 141, 134, 113 unigenes, respectively) are correlated with RIN and NOR transcript accumulation, C2H4 biosynthesis, and carotenoid accumulation. Putative drivers of these ripening-related expression modules were identified and 20 have homology to transcription factors. LeMADS-RIN and a putative WRKY-30 are central network nodes and mutation of LeMADS-RIN virtually eliminates network connectivity during ripening. Other key drivers of this co-expression network include a 26S proteosome regulatory subunit, a cysteine protease, histone H2B, a protein kinase, and ribosomal protein P1a. GT2, CONSTANS, and COP1 are highly correlated with these putative network drivers, and the analysis suggests a direct link between COP1 and LeMADS-RIN. Ripening-related expression of ~200 genes, including LeMADS-RIN, ACS4, and ERF2, is delayed in fruit containing the HIGH-PIGMENT1 lesion indicating a role in ripening beyond pigmentation. The HIGH-PIGMENT1 lesion represses C2H4 biosynthesis in fruit, perhaps by delaying expression of ACS4. These results suggest cross-talk between developmental and environmental regulators of ripening, confirming known interactions and suggesting novel regulators for functional analysis.


SOL 2009 103

SCENT, COLOR OR BOTH: PETUNIA'S PERSPECTIVE

Vainstein A., Spitzer B., Moyal Ben Zvi M., Marhevka, E.

The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, The Hebrew University of Jerusalem, P.O.Box 12, Rehovot 76100, Israel

Flower fragrance is a composite character determined by secondary metabolites of diverse biosynthetic origins. Together with other traits, such as flower color, it is used by plants to lure pollinators and seed dispersers, thereby ensuring plant survival. To enable the exploration of metabolic fluxes and the identification of genes that perform key functions in the production of phenylpropanoids and terpenoids—representing two major metabolic pathways—we employed various high-throughput approaches in conjunction with transgenics in both plants and yeast.

Expression in petunia flowers of the transcriptional regulator Pap1 (production of anthocyanin pigment 1) allowed us to genetically engineer flowers (rose and petunia) with an up to 10-fold increase in scent production/emission, as well as in pigmentation. Remarkably, incorporation of increased substrate into these lines led to a continuous strong increase in aroma production, both during the day and at night, abolishing the natural nocturnal rhythms of volatile production that are otherwise observed in flowers.

Using virus-induced gene silencing (VIGS) for large-scale identification of floral scent genes, we identified and then isolated and characterized a flower-specific MYB-like regulatory genes in petunia named EMISSION OF BENZENOIDS (EOBs). Silencing of EOBII resulted in a reduced level of phenylpropanoid volatile production by flowers. Target genes of EOBII that are involved in the production of phenylpropanoid volatiles and whose expression was reduced in flowers with suppressed expression of EOBII were identified. Based on gene-expression profiling, the levels of phenylpropanoid-pathway intermediates, and the coordinated wide-ranging effect of EOBII on the production of floral volatiles but not on that of anthocyanin, a central regulatory role for EOBII in the biosynthesis of phenylpropanoid volatiles is suggested.


SOL 2009104

THE MECHANISM OF RNAI SUPPRESSION BY MYMIV-AC2 PROTEIN

Sumona Karjee, Vikash Kumar, and Sunil Kumar Mukherjee

Plant Mol. Biol. Lab., ICGEB, New Delhi 110067

The geminiviruses have emerged as very destructive pathogens of crops in recent years. The AC2 molecules encoded by the viruses belonging to the genus begomoviridae have shown pathogenic characteristics and behave as the suppressors of RNAi. To understand the mechanism of RNAi-suppression of these molecules, we have used the AC2 proteins encoded by the model geminiviruses, namely, Mungbean yellow mosaic india virus (MYMIV) and Tomato leaf curl virus (ToLCV) as these viruses impact a lot on the agricultural economy. We also developed several assays to detect the in-planta RNAi suppression activities of the proteins of interest.

The C-terminal 16 amino acid residues of AC2 showed transactivation property but these were dispensable for the RNAi suppression activity as evidenced in the suppression reversal assay. The recombinant protein, either with or without the transactivation domain, showed oligomerization characteristics, and its loss resulted in the failure of RNAi suppression. Unlike other RNAi suppressors, the recombinant AC2 did not show any affinity of binding with any form of RNA as evident from the EMSA assays. However, AC2 interacted with a few host RNAi factors, namely RDR6 and AGO1 but not with DICER. In an in-vitro dicing reaction, AC2 did not have any effect on the amount of diced product. But the slicing characteristics of AGO1 were lost when AC2 was added exogenously in an in-vitro slicing reaction. All of these data together reflect the novel functions of AC2 as the suppressor of RNAi.

FUNCTION AND EVOLUTION OF THE TPP RIBOSWITCH IN THE PLANT KINGDOM

Asaph Aharoni

Department of Plant Sciences, Weizmann Institute of Science, Rehovot 76100, Israel

Riboswitches are natural RNA sensors that mediate gene regulation via their capacity to bind small molecules. We have identified a novel post-transcriptional feedback mechanism that employs a riboswitch to modulate alternative splicing in the 3'-UTR of plant thiamin pyrophosphate (TPP) biosynthesis genes. Once cellular TPP levels rise, metabolite sensing by the riboswitch directs an increase in an unstable RNA splicing product and consequently TPP levels drop. Alternative splicing in the 3'-UTR of TPP biosynthesis genes occurs already in the ancient lycophytes, suggesting that this mechanism has been active since vascular plants emerged 400 million years ago. We demonstrate that recombinant riboswitches can act autonomously in transgenic plants, thus paving the way for their exploitation in mapping metabolite concentrations and designing novel approaches for regulation of RNA splicing in living plants.


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SOL 2009106

DEVELOPMENT FOR MASSIVE MIRACULIN PRODUCTION USING TRANSGENIC TOMATO FRUITS IN PLANT FACTORY

1 1 1 1 1 1 1Kato K. , Hiwasa-Tanase K. , Hirai T. , Kim Y-W. , Yano M. , Narendra D. , Fukukawa G. , 1 1 1,3 1 1 2 3 1

Kikkawa N. , Kurokawa N. , Ichikawa T. , Mizoguchi T. , Fukuda N. , Kakuta H. , Takane K. , Ezura H.

1 2Graduate School of Life and Environmental Sciences, University of Tsukuba; Plant Ecochemicals Research

Center, 3Inplanta Innovations Inc.

Miraculin is a taste-modifying protein isolated from miracle fruit (Richadella dulcifica) in West Africa. Miraculin is able to turn a sour taste into a sweet taste. Therefore miraculin has a great potential as an alternative low-calorie sweetener for diabetic and dietetic purposes. Recently, we have succeeded in producing transgenic tomato (line 56B, upright type, cv. Moneymaker) expressing miraculin protein. In this study, we tried to further improve the transgenic tomato for massive miraculin production in plant factory. The miraculin gene expression of line 56B was genetically stable in tomato as a result of advancing the generation to T6. Some useful constructions, in which native miraculin 3' non-coding genomic sequence was used as a terminator, or selection makers were eliminated, were newly used in order to develop the transgenic lines accumulating more miraculin. Moreover we tried to produce suitable tomato architecture for plant factory using breeding and transgenic techniques. The line 56B was crossed with a dwarf tomato (cv. Micro-Tom), and the generation was promoted to F6. And various genes, which are related with miniaturization of plant size or floral regulation, were transformed into tomato, and useful characters for plant factory were investigated. We also tried to develop a new cultivation system for tomato in plant factory. The yield of line 56B using this system was higher than that cultivated under field condition in Japan because the system is better in space utility and time management. These works were supported by the Ministry of Economy, Trade, and Industry of Japan (METI).

SYSTEMS BIOLOGY OF TRANSGENIC SOLANACEAE WITH ALTERED CAROTENOID CONTENTS REVEAL NOVEL AND UNINTENDED EFFECTS

1 1 1 2 2 3 3 4 Scossa F. , Diretto G. , Tavazza R. , Schauer N. , Fernie A. , Matas A. , Rose J. , Giovannoni J. and

1Giuliano G.

1Ente per le Nuove Tecnologie, l'Energia e l'Ambiente, Rome, Italy; Max-Planck Institute of Plant Molecular

3Physiology, Golm, Germany; Department of Plant Biology, 228 Plant Science Building, Cornell University, Ithaca, 4NY 14853; Boyce Thompson Institute for Plant Research, Cornell University Campus, Ithaca, New York, 14853

We performed a global profiling at transcriptional, metabolomic and post-harvest level on several tomato carotenoid mutants and transgenic fruits overexpressing lycopene beta-cyclase (LCY-b), beta-carotene hydroxylase (CHY) or both transgenes. In addition, a multilevel characterization has also been carried out on transgenic potato tubers expressing simultaneously three genes of bacterial origin leading to the accumulation of beta-carotene. Gene expression profiles, integrated with high-throughput metabolomic analyses, revealed unintended transcript-metabolite correlations and shed light on novel co-regulatory dynamics which emerged in berry ripening or tuber aging during post-harvest storage. Networks of biological interactions and correlations have been employed to visualize and predict the consequences of metabolic engineering approaches on the whole plant metabolism. Large-scale network analysis proved to be a valuable approach for the future and rational design of new biofortified crops.


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SOL 2009108

RNA INTERFERENCE FOR THE CONTROL OF A FUNGAL PATHOGEN (FUSARIUM OXYSPORUM) IN TOMATO

M. V. Rajam, Neeru Singh and Madhu Khatri

Plant Polyamine and Transgenic Research Lab, Department of Genetics, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021

RNA interference (RNAi) approach is novel and has proven potential for the control of viral infections and pests in plants. However, there are no attempts for the prevention of fungal diseases by RNAi. Therefore, we have been exploring RNAi strategy for engineering fungal resistance in tomato. Our RNAi strategy involves the plant RNAi-mediated silencing of vital genes of fungal pathogens, particularly polyamine biosynthesis genes like ornithine decarboxylase (ODC) for the control of fungal diseases as polyamines (putrescine, spermidine and spermine) are absolutely essential for growth and development of fungi. As a prelude to these investigations, initially we had undertaken in vitro work to silence ODC gene of Aspergillus nidulans by using siRNA. Five different concentrations of siRNA (5-25 nM) and an unrelated siRNA were tested for their effect on germinating spores by adding them in the culture medium and incubating for 72 h. This has resulted in specific silencing effects leading to significant reduction in fungal germ tube length, mycelial growth, sporulation, target mRNA titers and cellular polyamine levels in the fungal ODC-siRNA treated samples, whereas no adverse effects were observed in unrelated siRNA treated and untreated control samples. Further, we have also developed transgenic tomato plants expressing dsRNA of ODC gene of Fusarium oxysporum, and preliminary results showed that such transgenic plants exhibited increased resistance to Fusarium wilt. These results suggest that RNAi approach would be a promising alternative for the control of fungal pathogens and polyamine pathway would be a novel target for this approach.

SATELLITE SESSION I POTATO

109


SOL 2009110

ASSEMBLY ISSUES IN GENOME SEQUENCING OF HETEROZYGOUS PLANTS

Christian Bachem, Theo Borm, Jan de Boer, Erwin Datema, Roeland van Ham and Richard Visser

Laboratory of Plant Breeding, Dept. of Plant Sciences, Wageningen-UR, Wageningen, The Netherlands

The PGSC started in 2006 with the aim of completing the potato genome sequence by 2010 using a BAC-by-BAC, chromosome by chromosome approach. The basis for this project was a BAC library and physical map from the diploid potato genotype RH89-039-16 (RH). This clone was the male parent of a crossing population (SH x RH) that had a high density AFLP genetic marker map with around 10K markers. With the rapid advancement of genome sequencing technologies, the consortium decided to adopt whole genome shotgun (WGS) sequencing as a faster way to achieve the compete genome. To achieve a rapid high quality assembly an additional homozygous potato genotype (DM1-3516) was included for sequencing using next generation sequencing technology. Nevertheless, the WGS sequencing of the RH genotype was also initiated with the aim of overcoming the assembly problems associated with a heterozygous genome. Whole Genome Profiling was also done in RH to aid both the sequence anchoring and the assembly scaffolding. Progress and challenges toward these goals will be discussed.

WHOLE GENOME SHOTGUN SEQUENCING OF DOUBLED MONOPLOID POTATO

Sanwen, Huang

Institute of vegetables and Flowers Chinese Academy of Agricultural Sciences, China

Potato (Solanum tuberosum L) genome sequencing has been initiated four years ago by the mutilateral Potato Genome Sequencing Consortium (PGSC). However, the overall progress of this project is very slow because of some technical limitations such as the inherent problems of BAC-by-BAC strategy and the heterozygosity of the sequenced clone RH89-039-16. Moreover, the next generation sequencing technologies have brought about new opportunities to de novo sequencing of genomes. To accelerate the potato genome sequencing, we adopted a whole genome shotgun strategy to sequence a doubled monoploid (DM) potato genome using the next generation sequencing technologies. We have finished 48.5 Gb sequences representing 58.8x coverage of potato genome using Illumina technology. A preliminary assembly showed that 90% the genome was covered, and 22,474 gene models were predicted. More Illumina GA reads with different insert size are being sequenced, and the assembly will be improved further. On the basis of the sequenced sequences, SSRs, DArTs and SNPs are being developed to construct a high saturated genetic map for DM to facilitate the whole genome sequencing. In addition, the end sequences of 10x fosmid and 10x BAC library are being sequenced by PGSC partners. The high quality draft potato genome sequence will be released in later 2009.


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SOL 2009112

ANCHORING THE DRAFT POTATO GENOME TO A DIPLOID GENETIC MAP

Glenn Bryan and The Potato Genome Sequencing Consortium

SCRI , Invergowrie, Dundee, DD2 5DA, United Kingdom

Potato, the world's most important vegetable crop, is being sequenced by the multi-national Potato Genome Sequencing Consortium (PGSC, see www.potatogenome.net). A major focus is the generation of a whole genome shotgun (WGS) sequence of a homozygous 'doubled monohaploid' (DM) clone (DM1-3 516R44, referred to as DM) to accompany the effort directed at the heterozygous RH89-039-16 clone. The DM sequencing effort lacks a genome wide physical clone map, and so a key issue is genetically anchoring the assembled genome scaffolds. A segregating backcross population has been established between the DM clone and a heterozygous Solanum goniocalyx clone as the recurrent parent. Mapping is being performed using a variety of marker technologies. Scaffolds, in combination with extensive transcriptome data, have been used to identify simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers, these markers currently being analysed by several partners within the consortium, dependent on expertise and resource availability. Diversity Array Technology (DArT) is also being used, and marker sequences will provide more sequence-tagged anchors to the genetic map. At the same time in silico approaches are being developed. For example it is possible to putatively assign many genome scaffolds to chromosomal locations by virtue of sequence homologies between scaffolds and sequence data from BAC clones in the RH physical map. The integrated sequence and genetic map will form an important resource for linking to all future genetic mapping efforts by the potato community.

QTL META-ANALYSIS FOR LATE BLIGHT RESISTANCE IN POTATO INCLUDING THE TWO NOVEL RESISTANCE SOURCES SOLANUM SPARSIPILUM AND S. SPEGAZZINII

Véronique, Lefebvre, Sarah, Danan, Patrick, Signoret, Jean-Batiste, Veyrieras

INRA, France

The resurgence of late blight epidemics in potato (Solanum tuberosum) is due to the apparition of new Phytophthora infestans strains, resistant to chemicals and overcoming deployed R-genes. The alternative exploitation of polygenic resistance controlled by QTLs requires to inventory resistance sources and to get a better insight of the genetic architecture and diversity of quantitative resistance to late blight in potato. To find out novel resistance sources to late blight in the wild germplasm for potato breeding, we examined the polygenic resistance of Solanum sparsipilum and Solanum spegazzinii by a QTL analysis (Danan et al., 2009, TAG). The assessment of stem and foliage resistances made possible to identify 30 QTLs including a large-effect QTL region on chromosome X detected in both potato wild species. The mapping of literature-derived anchor markers suggested colinearities with published late blight QTLs or R-genes. We integrated the QTL results in a meta-analysis for late blight resistance in potato together with the data of 19 other published studies. It consisted in constructing a consensus map of potato, on which we projected late blight resistance meta-QTLs. Results highlighted some well-conserved QTLs in the potato related species. The relationships of late blight resistance meta-QTLs with R-genes and maturity QTLs were examined.

[This work was supported by grants from the European BIOEXPLOIT project FOOD CT2005-513959.]


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SOL 2009114

IDENTIFICATION OF NEW SOURCES OF RESISTANCE TO POTATO LATE BLIGHT WITHIN SOLANUM MICHOACANUM AND S. RUIZ-CEBALLOSII

Jadwiga Śliwka, Henryka Jakuczun, Iwona Wasilewicz-Flis, Agnieszka Hara, Konrad Dębski, Ewa Zimnoch-Guzowska

Plant Breeding and Acclimatization Institute, Młochów Research Centre, Platanowa 19, 05-831 Młochów, Poland

Breeding potatoes resistant to such fast-evolving pathogen as Phytophthora infestans (Mont.) de Bary is a challenge. There is constant need for novel resistance sources that can be applied in breeding programs. In the presented work, we focus on two wild species: Solanum michoacanum and S. ruiz-ceballosii. The first one is a 1 EBN (Endosperm Balance Number) species and a natural hybrid of S. pinnatisectum and S. bulbocastanum, both well known for their late blight resistance. Due to its EBN, S. michoacanum cannot be crossed with cultivated potato. Therefore, to characterize the genetics of its resistance, we developed a segregating population from the intraspecific cross between resistant and susceptible clones. S. ruiz-ceballosii is also known as S. sparsipilum and it is a 2 EBN species. Resistant clone of this species was directly crossed to dihaploid of the cultivar Balbina to obtain a second population segregating for resistance to P. infestans. Both populations were tested in laboratory detached leaflet and tuber assays and in both cases the progenies segregated into two distinct classes of resistant and susceptible individuals. It indicated that single R genes underlie the resistance to P. infestans in our two new sources. We plan to map both genes using sequence specific and Diversity Array Technology (DArT) markers. Furthermore, the resistance of S. michoacanum will be introduced in S. tuberosum gene pool via somatic hybridization in collaboration with Dr. Thieme from Institute of Agricultural Crops, Germany.

EXPERIMENTAL GENE ANNOTATION: PAINTING ACTIVE EXONS IN THE POTATO GENOME

Kare Lehman Nielson

Aalborg University, Section of Biotechnology, Sohngaardsholmsvej 4, Denmark

TBD


SOL 2009 115


SOL 2009116

COMPARATIVE TRANSCRIPTOME PROFILING OF DIPLOID POTATO CLONES WITH VARIATION IN MATURATION TIMING

Helen Tai, David De Koeyer, Claudia Goyer, Agnes Murphy

Agriculture and Agri-Food Canada

Potato maturation timing is an important agronomic trait that is regulated by both endogenous and exogenous controls. Cultivation of potato germplasm in temperate regions requires adaptation to long-days. In this study we compare the transcriptomes of diploid potato clones, 12120-03 (early maturing) and 07506-01 (late maturing). Developing leaflets 1-1.5 cm in length was sampled from three replicates of the two clones 11 weeks after planting. Gene expression analysis was done using the 44K 60-mer POCI arrays. A t-test was used to identify 3639 genes showing significant differences in gene expression at a significance of p<0.01. Functional annotation analysis was done using GenMapper. 07506-01 has increased expression of protein biosynthesis and photosynthesis activities over clone 12120-03. These results are consistent with clone 07506-01 maintaining metabolic functions at 11 weeks where early maturing clone 12120-02 is entering senescence. Analysis of individual genes show that blue light photoreceptor cryptochrome 1b is expressed higher in 12120-02 indicating that differences in light responses may play a role in early maturation in potato. Interestingly, the CONSTANS-interacting protein 5, which is involved in flower timing, is expressed higher in late maturing 07506-01. These data indicate that day length sensing mechanisms may differ between 12120-03 and 07506-01 potatoes.

POTATO WART: A BIOTECHNOLOGICAL APPROACH FOR EFFICIENT EXPLOITATION OF NATURAL RESISTANCE

1 2 2 3 4 5Agim Ballvora , Jens Lübeck , Josef Strahwald , Eckhart Tacke , Hans-Reinhard Hofferbert Kerstin Flath , 1 1 1Elmon Schmelzer , Merle Noschinski , Christiane Gebhardt

1 2Max-Planck Institute for Plant Breeding Research, Carl von Linné Weg 10, 50829 Cologne, Germany; Saka 3

Pflanzenzucht GbR, Zuchtstation Windeby, 24340 Windeby/Eckernförde, Germany; Bioplant GmbH, 29574 4 5

Ebstorf, Germany; Böhm-Nordkartoffel Agrarproduktion GbR, 29574 Ebstorf, Germany; Julius Kühn Institute 14532 Kleinmachnow, Germany

Wart caused by the obligate biotrophic fungus Synchytrium endobioticum is increasingly becoming a threat to potato cultivation in temperate climates. Sporangia produced during sexual reproduction of the fungus are able to survive in soil for at least two decades causing a long term soil contamination problem. The only ways of controlling the disease are quarantine and phyto-sanitary measurements, and genetic resistance. Two populations segregating for resistance to paths.1, 2, 6, 18 of S. endobioticum were obtained by crossing res. with suscep. tetraploid parents. Resistance was evaluated after infecting 25 tuber-eyes from each progeny with a specific pathotype. The resistance data show a complex pattern of inheritance of genetic resistance to path.1, 2, 6, 18. To identify loci conferring resistance to S. endobioticum bulked segregant analysis was combined with molecular marker technologies. Resistant and susceptible DNA pools were constructed based on phenotypic means for resistance. The pools were screened with SSR, ISSR and RAPD markers. Three resistance loci were identified by means of linked markers: one for resistance to path 1; a second for resistance to path18 and a third for resistance to paths.2, 6, 18. Cytological analysis of infected potato shoot tissues revealed that cell division is induced around the site of sporangia development, leading to wart formation. This is the first report on genetic analysis of resistance to four paths 1, 2, 6, 18 of S. endobioticum in potato.


SOL 2009 117

SATELLITE SESSION II COFFEE

118

G. V. Krishna Rau

Coffee Board, Bangalore, India

OVERVIEW OF INDIAN INDUSTRY VIS-À-VIS WORLD COFFEE: CHALLENGES AND OPPORTUNITIES

TBD


SOL 2009 119


SOL 2009120

INDIAN INITIATIVE ON COFFEE GENOMICS: A BRIEF OVERVIEW

1 2 3 4 5Ramesh K. Aggarwal , H.L. Sreenath , M. UdayKumar , K. Veluthambi , G.A. Ravishankar , 2 2

N. Suryapraksh , Jayarama

1 2Centre for Cellular & Molecular Biology (CSIR), Uppal Road, Hyderabad 500007, India; Central Coffee 3Research Institute, Coffee Board, Chikmagalur, India; University of Agricultural Sciences, GKVK, Bangalore,

4 5India; Madurai Kamaraj University, Madurai, India; Central Food Technological Research Institute, Mysore, India.

Coffee is an economically important plantation tree crop belonging to the genus Coffea that comprise two main cultivated species (tetraploid C. arabica L. and diploid C. canephora Pierre ex Froehner) and ~100 diploid wild species. There is a continuous need for genetic improvement of coffee, which unfortunately is severely constrained owing to inherently slow pace of tree breeding using conventional methods compounded with a general lack of genetic markers, screening and selection tools. Inspite of these limitations, commendable success has been achieved in evolving new coffee varieties, from onerous conventional breeding efforts worldwide. However, the new challenges posed by biotic and abiotic stress factors, increasing quality concerns from consumer sector and scarcity of plantation labour needs to be addressed quickly for sustaining the world coffee industry. The situation warrants recourse to newer, easy and efficient practical alternatives/ technologies that can surmount the above problems and provide acceleration, reliability and directionality to the breeding efforts. To this end, a 'Multi-institutional National Network on Coffee' was initiated in early 2000 with support from the Department of Biotechnology, India. The major emphasis of this first effort was to create a knowledge base, and genomic resources, in the form of:

Characterization of the available coffee genepool for overall diversity, as well as, resistance sources for abiotic/biotic stresses, development of coffee-specific DNA/genetic markers, mapping populations, molecular linkage maps, EST pool, understanding the genetic-biochemical-physiological basis of drought tolerance and caffeine biosynthesis, and development of protocols/genetic constructs as prelude to transgenesis based improvement of coffee. The talk will briefly outline the scope and successes of these efforts made in India for the last few years.

SEQUENCING THE COFFEE GENOME: OVERALL STRATEGY AND PROGRESS

Philippe Lashermes

UMR RPB (CIRAD, IRD, Um2), Centre IRD de Montpellier, BP 64501, F-34394, Montpellier, France

The International Coffee Genomics Network (ICGN) is a worldwide network of scientists from universities, research institutes and industry within the coffee producing and coffee consuming countries (http://www.coffeegenome.org/). ICGN's ultimate goal is to sequence the coffee genome and decipher through international partnerships the genetic and molecular bases of important biological traits in coffee tree species that are relevant to growers, processors, and consumers. As part of ICGN efforts, French institutions (Genoscope-CEA, IRD and CIRAD) are combining their scientific resources to sequence and analyze the entire genome of Coffea canephora Pierre. The canephora genome consists of 11 chromosomes, is about 710 Mb in size and constitutes a reference genome for Coffea. The overall strategy based on a combination of sequencing technologies as well as the progress of the project will be described.


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SOL 2009122

MOLECULAR ANALYSIS OF DROUGHT TOLERANCE MECHANISMS IN COFFEE PLANTS

1, 2 2 2 2 2Pierre Marraccini , Luciana P. Freire , Natalia G. Vieira , Gabriel S. C. Alves , Felipe Vinecky , 3 1 4 2Humberto J O Ramos , Thierry Leroy , Gustavo C. Rodrigues , Alan C. Andrade

1 2CIRAD UMR DAP, Montpellier, France; Embrapa Recursos Genéticos e Biotecnologia, LGM-NTBio, Brasília- 3 4

DF, Brazil; IAPAR, LBI-AMG, Londrina-PR, Brazil; Embrapa Cerrados, Planaltina-DF, Brazil

Drought stress significantly affects coffee yield, productivity and quality. Thus, the development of molecular tools for rapid generation of drought-tolerant coffee varieties is amongst the priorities of the Brazilian Research Program on Coffee Genomics. The goal of this study was to investigate the molecular mechanisms involved in drought-stress responses in coffee plants, by different approaches. The first strategy used EST data generated by the Brazilian Coffee EST project in order to identify candidate genes (CGs) by an in silico analysis (electronic Northern-blot). Differential expression of those CGs was confirmed in leaves and roots from drought-tolerant and susceptible clones of C. canephora var. Conilon grown with and without water restriction by quantitative PCR experiments. The second approach was based on screening macroarrays spotted with coffee ESTs which were hybridized separately with leaf cDNA probes of the same coffee clones. Finally, 2D gel electrophoresis was also performed to select proteins displaying differential accumulation in leaves. These proteins were analyzed by MALDI-TOF-MS/MS leading to the identification of a new set of CGs mainly involved in the photosynthesis process. Results concerning the CGs identification by these different approaches will be presented and discussed. Currently, the natural genetic variation of some selected CGs among different coffee genotypes is also under investigation.

FUNCTIONAL GENOMICS FOR THE MAJOR COFFEE-PEST INTERACTIONS IN COLOMBIA

Alvaro Gaitan

Colombian National Coffee Research Center (CENICAFE), Chinchiná, Caldas, Colombia, co-sponsored by the Colombian Ministry of Agriculture.

Using a combination of differential display techniques such as subtractive libraries, comparison of full-length cDNA libraries and microarrays, we have undertaken the task of identifying candidate genes and metabolic pathways that can be involved in the differential response of genotypes against the Coffee Leaf Rust (CLR) and the Coffee Berry Borer (CBB), currently the two most limiting pests for Colombian coffee production. Timor Hybrid accession 1343 still displays complete resistance under strong inoculum pressure after 26 years of continuous coffee rust pressure in the country, and a few genotypes of the Colombian Germplasm Collection show a negative effect on the life cycle of Hypothenemus hampei. A better understanding of plant host responses at the molecular level is essential to progress towards significant and durable resistance to be used in integrated control measures, either through breeding or by alternative applications. For CLR, a set of 17 genes have been repeatedly obtained from subtractive libraries as differentially expressed after rust attack. Six of them have no known homologous in other plant genomes. For CBB, 83% of the cDNAs from the genotype C. liberica have orthologous sequences present in un-challenged tissues of the species C. arabica and C. canephora. From a set of 217 unigenes, at least 20 corresponded to orthologous genes in other genera involved in stress responses. For both pests, spotted cDNA microarrays were hybridized containing a 36,480 probe set from normalized libraries from C. arabica and C. liberica, representing 21,373 unigenes. Under CLR challenges 1,600 genes were detected as differentially expressed, as well as 2,500 for CBB infestations. Although defense involved genes were identified under Hemileia vastatrix attack, changes related to cell wall and cytoskeleton metabolism, activities of sugar transferases and invertases, and protein degradation were more frequent, indicating plant metabolism adjustments to fungal nutrition. Genes present along the jasmonic acid pathway were notorious under CBB attack. Differential expressions of candidate genes have been validated through quantitative RT-PCR. Better experimental designs using a more complete gene set printed on oligoarrays are being carried out, as well as attempts to achieve a transient expression system in coffee to either over-express or silence candidate genes in vivo and complete the evidence for the role these pathways play in pathogen and insect defense.


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SOL 2009124

TOWARDS THE FIRST DRAFT GENOME SEQUENCE OF COFFEA CANEPHORA

1 2 3 4 5Alexandre de Kochko , Victor Albert , Alan C. Andrade , Giovanni Giuliano Giorgio Graziosi , 6 7 8 9

Robert Henry , Ray Ming , Chifumi Nagai , David Sankoff

1 2 UMR DIAPC, IRD - BP34394, Montpellier Cedex 5, France; Department of Biological Sciences, University at

3Buffalo (SUNY), Buffalo NY, USA; Laboratory of Molecular Genetics (LGM-NTBio), Embrapa, Recursos

4Genéticos e Biotecnologia, C.P. 02372, 70770-900 Brasília-DF, Brazil; ENEA Casaccia Research Center- Post Bag 5026, 00123 S.M. di Galeria Rome, Italy; Dipartimento di Scienze della Vita, Università di Trieste P.le Valmaura 9

6 734143 Trieste, Italy; Southern Cross University, PO Box 157, Lismore NSW 2480, Australia; Department of Plant 8

Biology, University of Illinois at Urbana Champaign, Urbana, IL 61801, USA; Hawaii Agriculture Research 9

Center, Kunia, HI 96759, USA; Department of Mathematics and Statistics University of Ottawa 585 King Edward Avenue Ottawa, Canada K1N 6N5

During the last ICGN (http://www.coffeegenome.org/) meeting at the Plant and Animal Genome conference in San Diego in January 2009 and following previous discussions at the 5th SOL Genome Workshop in Cologne (Germany), an international consortium was set up with the goal to produce a draft sequence of the Coffea canephora (Robusta) genome. Eight laboratories from 6 different countries are now part of the consortium and several other laboratories are committed to collaborate.

Institution Name Country Status

EMBRAPA Alan C. ANDRADE Brazil Member

University at Buffalo Victor ALBERT USA Member

ENEA Giovanni GIULIANO Gaetano Italy MemberPERROTTA

Institut de Recherche Alexandre de KOCHKO France Memberpour le Développement Romain GUYOT (IRD) UMR DIAPC Marie COUDERC

Università di Trieste Giorgio GRAZIOSI Italy MemberAlberto PALLAVICINI

Southern Cross University Robert HENRY Australia Member

University of Illinois at Ray MING USA MemberUrbana Champaign

Hawaii Agriculture Chifumi NAGAI USA MemberResearch Center Ming-Li WANG

University of Ottawa David SANKOFF Canada Member

University of Arizona Steve ROUNSLEY USA Collaborator

University of Florida Brab BARBAZUK USA Collaborator

Institut National de Sylvain SANTONI France CollaboratorRecherche Gregory CARRIERAgronomique (INRA) Loïc LECUNFF

Institut de Recherche Diana FERNANDEZ France Collaboratorpour le Développement (IRD) UMR RPB

Instituto de Investigacao Maria do Céu SILVA Portugal CollaboratorCientifica Tropical (IICT) Centro de Investigacao das Ferrugens do Cafeeiro (CIFC)

Coffea canephora is a diploid species with a genome size of about 695 Mb (2C value = 1,44 pg/ nucleus). The sequencing of the genome is being carried out with deep sequencing technologies: 454 pyrosequencing by Roche and Illumina (Solexa). To avoid problems of allelic polymorphism inherent to C. canephora due to its allogamous condition, the genotype we chose is a double haploid (DH200-94) produced by IRD and kept in its tropical green house in Montpellier, France. DNA was extracted from purified nuclei by INRA in Montpellier and sent to the University of Illinois where the library was constructed. Sequencing is being carried out at the University of Illinois at Urbana-Champaign and at the Italian National Agency for New Technologies, Energy and the Environment for the 454 part, and at Southern Cross University, Australia, for the Illumina technology. Assembly and initial annotations will be done at the University of Arizona and the University of Florida. Sequencing of a Coffea plant will provide a new perspective compared to the dicotyledonous genomes currently available (Arabidopsis, grape, papaya, poplar) in that Coffea is a member of the Asterid clade whereas the others are all Rosids. We expect to get a sufficiently assembled draft genome to allow an assessment of the general organization of the genome, to permit comparisons with existing sequenced genomes and to lead to better understanding of Coffea genome evolution. Identification of the majority of genes should provide insight into specific metabolic and developmental pathways. There will be a dramatic increase in the quantity of genetic markers, permitting the establishment of more dense genetic maps for C. arabica. Identification of transposable elements and analysis of their distribution will be also greatly facilitated. We hope to demonstrate that with relatively low investment but great involvement of the scientific community and the sharing of means and will; valuable advances in knowledge are within reach.


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SOL 2009126

ADVANCES IN C. ARABICA PHYSICAL MAPPING

1 2 1 1 1 2 2Moncada P .; Goicoechea J.L. ; Maldonado C.E. ; Tovar E. ; Rivera L. ; Kudrna D. ; Wing R.

1 2National Center of Coffee Research CENICAFE. A. A. 2427. Manizales, Caldas, Colombia; Arizona Genomics

institute, Department of Plant Sciences, 303 Forbes Building, Tucson, AZ 85721-0036, USA

We construct a bacterial artificial chromosome (BAC library) from Coffea arabica cv Caturra with the aim of developing a physical map of this important crop. The BAC library contains 114,816 clones archived in 384 well plates. Estimated clone average insert size (120 kb) and representation, provides a library coverage of 9.5 haploid genome equivalents. The library contains low chloroplast contamination (1 %). BAC clones were fingerprinted using the SNaP shot technique and end sequenced. BAC fingerprints were assembled with the FPC software, resulting in 2087 contigs and BAC end sequences (BES) allowed the incorporation of 17,310 "in silico" markers, mainly derived from EST from C. arabica and C. canephora. These sequences also were screened with the Repeat Scout for "de novo" identification of repeats, resulting in 400 putative families, which are currently under characterization and will be also conveniently incorporated to the physical map. BES also allowed to align the FPC contigs from the coffee map to the current available sequence from tomato, using SyMAP. Based on this result BAC from the minimum tiling path from two regions with high synteny to chromosomes 2 and 8 from tomato are being sequenced with GS-FLX Titanium sequencing platform. Additionally we are carrying out hybridization experiments with probes from SSR and EST genetically mapped to connect the genetic and physical maps of this species. This map will allow us to find genes of interest for the coffee breeding program.

DEVELOPMENT AND ANALYSIS OF AN EST DATABANK OF COFFEA ARABICA

Gianluca De Moro, Martina Modonut, Elisa Asquini, Patrizia Tornincasa, Alberto Pallavicini, Giorgio Graziosi

Department of Life Sciences, University of Trieste, Trieste, Italy

Messenger RNA was extracted from leaves, stem, root, flowers and cherries at several developmental stages of Coffea arabica v. Catuai. Following synthesis of cDNA, the majority of the ESTs underwent pyrosequencing while a proportion of the cDNA underwent classical Sanger sequencing. We obtained 148,701 sequences by pyrosequencing with an average length of 212 bp and 12,959 sequences with an average length of 543 bp by the Sanger method. Both types of sequences were assembled together though the MIRA program to create longer contigs of presumptive genes. This approach allowed for the development of a data base of 23,844 contigs having an average length of 450 bp.

Next step was the annotation through the software Annot8r which is based on the search of similarities in the databases KEGG (Kyoto Encyclopedia of Genes and Genomes), GO (Gene Ontology), EC (Enzyme Commission). KEGG allowed for the annotation of 3174 sequences, GO allowed for the identification of the function of 1982 contigs and the Enzyme Database allowed for the attribution of possible enzymatic activity to 3275 contigs. As some contigs appeared in more than one database, the total number of annotated contigs was 5774, about 25% of the total of our contigs. We also used the FrameDP software to identify potential protein and, out of the 20,555 hypothetical proteins obtained, more than 70% had a positive confirmation by Interproscan.

All the data and sequences of the 161,660 ESTs will be publicly available at the website www.coffeeDNA.net.


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BIOINFORMATICS ANALYSIS OF COFFEA ARABICA ESTS

1,2 2 2 2 2Jorge M.C. Mondego , Ramon O. Vidal , Marcelo F. Carazzolle , Lucas P. Parizzi , Gustavo G.L. Costa , 2 3 4 1 5Eric Keiji , Mirian T.S. Eira , Luiz G. E. Vieira , Carlos A. Colombo , Alan C. Andrade ,

2Gonçalo A.G. Pereira

1Centro de Pesquisa e Desenvolvimento em Recursos Genéticos Vegetais, Instituto Agronômico de Campinas,

213001-970, Campinas - SP, Brazil; Laboratório de Genômica e Expressão – Departamento de Genética, Evolução e Bioagentes, Instituto de Biologia, Universidade Estadual de Campinas, 13083-970, Campinas - SP, Brazil; 3Empresa Brasileira de Pesquisa Agropecuária, Embrapa Café. Av. W3 Norte Final. 70770-901, Brasilia - DF,

4Brazil; Laboratório de Biotecnologia Vegetal (LBI), Instituto Agronômico do Paraná, 86001-970, Londrina, PR, 5Brazil; Laboratório de Genética Molecular (LGM-NTBio), Embrapa Recursos Genéticos e Biotecnologia, Pq.

Estação Biológica, Final W5 Norte, Plano Piloto. 70770-917, Brasilia- DF, Brazil

Coffee is an important agricultural commodity produced in more than 60 countries. It generates a turnover of US$10-12 billion per year, ranking as second on international trade exchanges. Coffea arabica represent about 70 % of the total coffee market. Due to its world economical importance, a Brazilian consortium composed by a series of academic and scientific institutions made an effort in sequencing ESTs from C. arabica. A total of 187,412 ESTs derived from 32 cDNA libraries of diverse tissues were assembled in 32,007 clusters (15,656 contigs and 16,351 singlets). These clusters were evaluated in structural and functional annotation. We verified that C. arabica has a similar GC content in comparison to Arabidopsis and other dicotyledons. In addition, the annotation of protein families indicates that C. arabica has less retrotransposons and heat shock proteins than C. canephora, other economical important Coffea species. We also present an annotation of Coffea specific gene families and a tentative evaluation of C. arabica metabolic pathways. We believe that these data can provide insights into Coffee development and aid in Coffee biotechnology.Financial Support: FAPESP, Consórcio Brasileiro de Pesquisa e Desenvolvimento do Café and CNPq

SATELLITE SESSION III TOBACCO

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TOBACCO: AN IDEAL PLANT FOR BIOTECHNOLOGICAL RESEARCH

V. Krishnamurthy, T.G.K. Murthy and K. Sarala

Central Tobacco Research Institute, Rajahmundry- 533 105, India

Tobacco, a commercial crop, cultivated in fifteen States in India producing 700 M kg cured leaf from an area of 0.40 M ha. India produces a wide spectrum of tobacco types including Virginia, bidi, burley, oriental, chewing, hookah, cheroot, cigar wrapper/ filler etc. India exports 225 M kg of tobacco and its products to about 100 countries earning foreign exchange to the tune of INR 338 billions. It is the lifeline for 6 million farmers and supports 30 million workers. Out of the 69 species of genus Nicotiana, only two species viz., N. tabacum and N. rustica are cultivated in the country. N. rustica, being cold-loving is cultivated in north India while N. tabacum is grown through out the country. Tobacco has the chromosomal no 2n=48. Nicotine is the principal alkaloid for which the crop is commercially cultivated for smoking, chewing and sniffing. Besides its commercial utility, tobacco is valued and extensively used during the course of evolution of the present genetic engineering and biotechnology. Since the entire plant can easily be regenerated from a single cell, it is extensively used in basic research in tissue culture. Availability of different ploidy levels in tobacco makes it a potent tool in cytogenetic research. Tobacco is an ideal plant for genetic transformation. In biotechnology, tobacco is often used as a model plant for the testing of new procedures and in research on the function of specific genes. It may be possible to change the genetic make up of tobacco plant so that it would produce valuable chemicals and drugs useful to man. The plants would then be harvested and processed for these useful chemicals and drug molecules. Hence, molecular farming is another area where tobacco plant is advantageously exploited. In India, Central Tobacco Research Institute (CTRI), Rajahmundry working under the aegis of Indian Council of Agricultural Research (ICAR), Ministry of Agriculture, Govt. of India is involved in research and developmental activities of different types of tobacco. At CTRI, 2,535 germplasm lines collected from different parts of the world are maintained for conducting basic and applied research. Biotechnology work at CTRI is mainly focussed on development of stress resistant transgenic (Bt) lines, somaclones tolerant to viruses (leaf curl/ CMV), molecular mapping of tobacco traits like nicotine, solanesol and TSNA, DNA fingerprinting of varieties/ germplasm lines, tagging of disease resistant genes (Fusarium), development of tobacco specific markers, micro-propagation of elite lines, embryo rescue of inter-specific hybrids, etc.

The paper reviews the practical utility of tobacco plant as a model system for biological research and experiences of CTRI. The paper also enlists certain critical areas of research where tobacco has the potential to be used as research tool.

GENETIC STRUCTURE ANALYSIS OF FCV TOBACCO POPULATION FOR BREEDING BENEFITS

1 1 1 1Ganesh CT *, Saiprasad GVS , Navin K Sharma , Ambika Prasad Upadhyay ,

2 2Sheshshayee MS , Mohan Raju B and Udayakumar M

1. 2. ITC R&D Centre, SP Biotech Park, Hyderabad, INDIA; Department of Crop Physiology, UAS, GKVK Campus, Bangalore, INDIA

DNA marker association studies for traits largely depend on the genetic nature of the population used for mapping. An understanding of genetic structure of populations assists in inferring nature of variations in population and thus aid in designing further studies that suits the dynamics of variability in population. Our work was aimed to analyze genetic structure of tobacco (Nicotiana tabacum L.) population of 144 FCV genotypes using multilocus genotyping approach with mapped simple sequence repeat (SSR) markers. Twenty five unlinked SSR markers delineated all the 144 genotypes revealing a total of 85 alleles with an average of 3.4 alleles per locus. Contrasting allelic frequencies were observed in most of the loci studied. Bayesian method of inference employed genotyping data to derive genetic structure information on the population. High Fst measures in the subpopulations (0.44, 0.09 and 0.29 at population level K=3) indicated high inbreeding coefficient or heterozygote deficit signifying a structured population. Cluster analysis using Neighbor-Joining method and evolutionary dissimilarity estimate using DARwin also supported the finding of strong structure in the population. These findings were found highly useful in designing appropriate mapping strategies in our work. Results suggest that tobacco might have experienced a series of selection followed by high inbreeding for several decades leading to a genetic bottleneck and limited divergence during its commercial cultivation.


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IN VITRO INDUCTION OF HAPLOIDS AND REGENERATION OF FERTILE DOUBLED HAPLOIDS IN FCV TOBACCO THROUGH ANDROGENESIS

Salej Sood, Samresh Dwivedi, T Venkata Reddy and Navin Sharma

ITC R&D Centre, Spark Tower, SP Biotech Park Pvt Ltd Survey No; 230-243, Turkapally Village, Shamirpet RR District, Andhra Pradesh-500078, India

Haploid breeding followed by hybrid sorting is the shortest route of breeding new varieties. Production of doubled haploid plants through androgenesis in FCV tobacco (Nicotiana tabacum L.) is a promising and convenient alternative to conventional selfing techniques for the generation of pure lines for breeding programs. This process comprises three main steps: induction of haploids, identification of haploids and duplication of the haploid genome. In the present study five F1 hybrids were used as base material for production of haploids using anther culture. Anther response varied from 13.28 to 39.80% and 18.6 to 40.4% in colchicine treatment I and II, respectively in stage 2 buds. Chromosome count confirmed haploid (24) and doubled haploids (48) by studying meiosis in PMC's of anther derived plants. Doubled haploids developed from F1 plant anthers are fixed recombinants due to crossing over, segregation and independent assortment which might be desirable for yield or quality components. Thirty (30) doubled haploid lines were recovered in in vitro colchicine treatment where average chromosome doubling was 27% in comparison to 4.45% doubling with 24 doubled haploid lines in in vivo treatment. The results demonstrate more efficient plant breeding methodology for exploitation of gametic embryogenesis

FUNCTIONAL ANALYSIS OF CAMV35S, DUPLICATED CAMV35S AND ROLC PROMOTER BY USING GUS (Β- GLUCURONIDASE) ASSAY IN TOBACCO

Akhilesh Kumar, Rekha Kansal and K.R. Koundal

National Research Centre on Plant Biotechnology; IARI-Pusa New Delhi 110012

An important consideration in transgenic research is the choice of promoter for regulating the expression of a cloned foreign gene. Efficient expression of foreign genes in transformed plants requires that they are placed under control of suitable promoter that is stable and active in plant cells. Appropriate regulation of transcription in higher plants requires specific cis elements in the regulatory regions of genes and their corresponding trans-acting proteins. We have compared the level of GUS gene expression, when cloned with three different promoters' i.e. single 35S promoter, double 35S promoter and rolC (Agrobacterium rhizogenes) by using pBI series of binary vectors in tobacco using Agrobacterium method of genetic transformation. Analysis of the cauliflower mosaic virus (CaMV) 35S promoter conferred constitutive expression whereas rolC-promoter driven expression was mainly seen in glandular cells in transgenic tobacco plants. Duplicated cauliflower mosaic virus 35S promoter showed transcriptional activity approximately tenfold higher than that of the single 35S promoter since it consist of tandem duplication of 250 base pairs of upstream sequences, which acts as a strong enhancer of heterologous promoters. The significance of these results is desirable for higher expression of foreign genes. Such study related to transgenic research will be very useful in managing the biotic stress.


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TOBACCO MONOSOMIC LINES AS A MOLECULAR GENETICS TOOL

Carla Ceoloni, Carlo Pozzi, Andrea Gennaro, Nicolas Lugon-Moulin, Vincent Robert, Martin Ganal, Paolo Donini Philip Morris Intl. Switzerland

The aim of this work was to link the tobacco genetic and cytogenetic maps and to ensure the propagation of the full set of tobacco monosomics as a valuable genetic resource. This was achieved by: a) the selection of monosomic lines (i.e., 2n=47 instead of 48) and b) the attribution of linkage groups (LG) to specific chromosomes using monosomic lines. A collection of 24 monosomic lines was propagated and cytologically monitored for ploidy state. For propagation, each of the lines was crossed with the non-monosomic Red Russian (RR) background. Further crossing was performed with Hicks Broad Leaf (HBL) tobacco type, which is one of the parents of the genetic map produced by Philip Morris International. F1 plants were produced and both 2n=47 plants (monosomics) and 2n=48 plants (controls) were identified. The genetic material was then used to assign single sequence repeat (SSR) markers to specific chromosomes. To date, 17 of the monosomic lines have been successfully crossed with HBL. For nine of these lines, a clear attribution of molecular markers to specific chromosomes was possible: in four cases, a single LG was shown to be associated with a specific chromosome and in five cases two LG were shown to be associated with a specific chromosome. The attribution of LG to specific chromosomes allows us to link the tobacco genetic and cytogenetic maps. This is instrumental to further integration with the physical map being developed by Philip Morris International Res. & Dev.

INVOLVEMENT OF TRANSPORTERS AND CHANNELS IN TRANSLOCATION AND REDISTRIBUTION OF POTASSIUM IN NICOTIANA TABACUM

Deva Kumari, Mahavishnan K, Gurumurthy D S, Ambika Prasad Upadhyay and Navin Sharma

ITC R&D centre, SP biotech Park, Spark Towers, Survey nos 230-243,Turkapally village, Shameerpet -500 078, RR district, Andhra Pradesh

Potassium (K+) is an essential macronutrient in plant system, regulating many cellular activities like osmoregulation, turgor maintenance, stomatal function and membrane potential. It is also involved in cellular metabolism, activating various enzymes required for plant growth and development. Multiple mechanisms are responsible for K+ uptake from soil solution, which comprise of transporters (high affinity uptake) and channels (low affinity uptake). Potassium uptake and transport is regulated by differential expression of K+ transporters and channels. To verify our hypothesis "differential expression of Potassium transporters and channels lead to the differences in the distribution of Potassium in plant organs ", in the present study differential accumulation of K+ in Nicotiana tabacum and correlation of gene expression profile with the K+ content was observed. Potassium content in various organs was estimated using Atomic Absorption Spectrophotometry (AAS). The results show that leaf midrib accumulate more K+, translocated from stem, when compared to leaf lamina. Gene expression analysis was performed for transporter genes, HAK, NtTPK1, NtKC1, and NKT2, involved in K+ translocation from leaf midrib to leaf lamina, using quantitative Real Time PCR. The results indicate decreased expression of K+ outward rectifier, NtTPK 1, in midrib. Inward rectifiers, NtKC 1 and HAK 1, show correlation with K+ accumulation in leaf lamina. Additionally, application of Thiourea may facilitate the redistribution of K+ between midrib and lamina.


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ROLE OF GLUTATHIONE AS A SIGNALING MOLECULE IN PLANT DEFENSE

Srijani Ghanta, Dipto Bhattacharyya, Ragini Sinha and Sharmila Chattopadhyay*

Plant Biotechnology Laboratory, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata 700 032, India

Glutathione, a ubiquitous non-protein thiol, plays a significant role in protecting the plants from environmental stresses. In this study, transgenic tobacco (Nicotiana tabacum), overexpressing γ-ECS, the rate-limiting enzyme of the glutathione biosynthetic pathway, was raised upto the T1 generation. Transgenesis T1 plants were confirmed by PCR, RT-PCR and Southern blot analysis. Further, a subtracted cDNA library has been constructed using wild-type tobacco, as driver and transgenic tobacco as tester. 250 upregulated ESTs were obtained from the transgenic plants as compared to the wild-type plants. After successful sequencing, Blastn analysis of these upregulated clones was performed. The high level of PR1a expression was noted along with other stress related genes. Taken together, a significant role of glutathione in plant defense has been discussed.

TOBACCO BY2 CELLS AS A TOOL FOR FUNCTIONAL VALIDATION OF GENES INVOLVED IN HORMONE BIOSYNTHESIS/SIGNALING FROM HETEROLOGOUS SYSTEMS

1Saraswati Nayar, Rita Sharma and Sanjay Kapoor

Interdisciplinary Center for Plant Genomics and Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi-110021, India1Present address: Department of Plant Pathology, University of California, Davis, CA 95616, USA

Ectopic expression of a rice MADS box gene has been found to result in overall dwarfing, and early flowering phenotype in transgenic rice. Since cell division is considerably reduced in the transgenic lines, aberrant cytokinin metabolism was thought to be a suspect. To test this hypothesis tobacco BY2 cells were stably transformed with the rice MADS box gene. Ectopic expression of the rice MADS box genes resulted in overall enlargement of the BY-2 nucleus resulting in increased nucleus area/cell size ratio, which could be a direct effect of altered cytokinin levels affecting cell cycle. In a previous study Miyazawa et al., 1999, 2002 had shown that higher cytokinin concentration results in amyloplast development in BY2 cells which otherwise have plastids in the form of proplastids. Furthermore, excess of auxin was shown to inhibit the same. The transgenic BY2 lines expressing the OsMADS gene also exhibited higher accumulation of amyloplasts indicating high endogenous levels of cytokinin in the transgenic cells. The results obtained from these transgenic BY2 studies have given headway to the plausible role of this transcription factor in cytokinin biosynthesis or signaling pathway in Rice.


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ROLE OF CMV-2B PROTEIN IN REGULATING MIR164 DEPENDENT DEVELOPMENTAL PROCESSES IN TOBACCO

Priyanka Singh, Vikas Koundal and Shelly Praveen*

Advanced Centre for Plant Virology, Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi.

Tobacco plants infected with Cucumber mosaic virus developed symptoms like stunted growth, initiation of auxiliary buds, lateral root growth, aberrant leaf shape, early senescence etc. The exact regulatory mechanisms for development of these aberrations during viral infection are not known. Here we demonstrated that CMV suppressor 2b protein when over express in tobacco plants develops viral like symptoms. In vitro expression of 2b and its activity in binding micro RNA showed its affinity towards miR164. Recent studies have shown that miR164 controls a group of transcription factors belonging to NAC family (NAC1, CUC1/2), which regulate various transcripts involved in developmental processes and also cell death during senescence. We relate our results by showing regulation of miR164 by CMV 2b which finally leads to virus like symptoms during viral infection.

SATELLITE SESSION IV BRINJAL

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VARIETAL DEVELOPMENT IN EGGPLANT BY COMBINING CLASSICAL AND BIOTECHNOLOGICAL APPROACHES

1 2 3 4 5 6 7Rotino GL , Toppino L , Mennella G , Valè GP , Lo Scalzo R , Lanteri S , Acciarri N

1 2CRA-ORL, Unità di Ricerca per l'Orticoltura, Montanaso Lombardo (LO), Italy; CRA-ORT, Pontecagnano (SA), 3 4 5

Italy ; CRA-GPG, Fiorenzuola d'Arda (PC), Italy; CRA-IAA, Milano, Italy; DIVAPRA, Genetica Agraria, 6

Università di Torino, Grugliasco (TO); CRA-ORA, Monsampolo del Tronto (AP), Italy

Breeding programs, aimed at the development of eggplant cultivar displaying innovative traits into the eggplant have been set up in the middle of 1990's. A sexual interspecific hybrid was obtained with S. sodomaeum (syn. S. linneanum) carrying tolerance to verticillium wilt, drought and salt. Tetraploid somatic hybrids were obtained by electrofusion of eggplant and S. aethiopicum gr. gilo or S. integrifolium protoplasts, both species being a source of resistance to Fusarium and bacterial wilts, and dihaploids were obtained through anther culture. Phenotypic, biochemical and molecular analyses demonstrated that partial genetic recombination occurred between eggplant and allied species genomes. Advanced introgression lines were obtained through several cycles of backcrosses and selection, followed by selfing and/or anther culture to obtain pure lines currently employed to release commercial F1 hybrids. The introgressed Fusarium resistance trait is controlled by the dominant Rfo-sa1 locus, and tightly linked codominant CAPS markers were developed. Biochemical analyses showed high differences between eggplant and its allied species in chlorogenic acid, total polyphenol, glycoalkaloids, anthocyanins and lipid fatty acids contents, PPO activity and antioxidant potential. Agrobacterium-mediated transformation enabled to obtain eggplant lines and hybrids resistant to the insect Colorado Potato Beetle or displaying parthenocarpic fruit development, further transgenesis approaches are under development to engineered useful traits. Functional and structural genomic researches to identify genes involved in the resistance to the wilting fungi and to develop a dense intraspecific genetic map are in progress.

EGGPLANT BREEDING DEVELOPMENTS IN SOUTHEAST ASIA

S.J. de Hoop

East-West Seed, Philippines

Before the early eighties little work had been done in Southeast Asia on variety improvement of local vegetable crops, including eggplant. In the late seventies and early eighties the first local hybrid field corn programs were established; the first commercial vegetable crop breeding programs were initiated slightly later in the early to mid-eighties. East-West Seed (EW) played a pioneering role to transform a traditional trading oriented market into a quality seed market. In terms of acreage in SE Asia, eggplant at about 120,000 ha is significantly smaller and about similar in size to tomato and much smaller than hot pepper. In terms of seed value it is, however, less than half of the total value of the tomato seed market. For this reason, and because of fragmentation of the eggplant markets, this crop has generally received a much lower breeding priority than the 2 other major Solanaceous crops. For EW it has always been an important program as a result of which we have established a market leading position. Breeding has focused on the major types in SE Asia: long purple in the Philippines, long violet in Indonesia and miscellaneous other types throughout the region. The main breeding objectives have included improvement of yield, disease resistance, particularly bacterial wilt, shipping and shelf-life qualities, and plant habit.

The first commercially successful hybrid eggplant hybrid variety 'Jackpot' was introduced in 1987, followed by the first successful hybrid introductions in Thailand and Indonesia in 1989 and 1992. Hybrid penetration has gradually increased to about 65-70% with EW continuing to maintain a leading position in this market.


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THE INDIAN BRINJAL CORE COLLECTION USING GEOGRAPHICAL AND MORPHOLOGICAL DESCRIPTORS

KK. Gangopadhyay, R.K. Mahajan, S.K. Sharma, Gunjeet Kumar, S.K. Yadav, S.K. Mishra andSunil Archak

National Bureau of Plant Genetic Resources, Pusa Campus, New Delhi-110 012

Brinjal, an important Solanaceae vegetable, is a good source of minerals and phenolic constituents and having medicinal properties including anti carcinogenic activity. It is available round the year at affordable prices to poor consumers when prices of many other vegetable crops rise dramatically in the hot-wet season. India, being the primary centre of origin, possesses wide diversity for growth habit, leaf blade lobing, calyx color, fruit size, and shape, color, and color distribution. NBPGR, being the nodal organization in the country for over all management of plant genetic resources (PGR) of agrihorticultural crops, conserves large number of brinjal germplasm collected from different regions of the country. Core collection is a representative of entire collection i.e. the available germplasm in the country, with minimum repetitiveness and capturing the maximum diversity. A core collection of 181 accessions (10% of entire collections) was fixed in advance. The entire collections (1798 accessions), mostly indigenous, were classified in 15 groups on the basis of regions within the country/continents(using geographical data) and numbers of accessions from the groups were selected on the basis of the diversity. Allelic evenness (H') and allelic richness are the most commonly used parameters for measuring the Shannon-Wiener Diversity Index (H') derived from 14 qualitative descriptors whereas the number of accessions from each group was selected on the basis of Principal Components Score Strategy (PCSS) derived from 14 quantitative descriptors. Analysis of frequency distribution indicated homogeneity of distribution between the entire collection and core set for all the descriptors except fruit colour distribution and fruit shape. However, the rare alleles (very strong leaf blade lobing, violet leaf color, greenish white corolla color, and 'LI' shaped fruit curvature) in the entire collection were captured in the core collection. The results showed that the variability in the entire collection is well represented in the core collection. The core set is presently under validation testing at multi-location and will be subsequently subjected to molecular characterization.

GENOME FUNCTIONAL STUDIES IN FUSARIUM RESISTANT EGGPLANT INOCULATED WITH FUSARIUM OXYSPORUM F. SP. MELONGENAE AND/OR VERTICILLIUM DAHLIAE

1 1 1 1 2 1 Toppino L. , Barbierato V. , Rinaldi P. , Caponetto G. ., Vale' G.P. , Rotino G.L.

1 2CRA-ORL,Unità di Ricerca per l'Orticoltura, Montanaso Lombardo, Italy; CRA-GPG, Fiorenzuola d'Arda (PC),

Italy

The locus Rfo-sa1 conferring resistance to Fusarium oxysporum f.sp. melongenae was introgressed from the species S. aethiopicum and S. integrifolium into cultivated eggplant through protoplasts electrofusion. Identification of differentially expressed genes involved in the plant-pathogen interactions between Fusarium and/or Verticillium dahliae and the new eggplant advancend introgressed lines was started. The resistant lines were inoculated with Fusarium, Verticillium and both fungi together, while roots dipping in water were used as mock inoculation. Three libraries were created by PCR-select between the mRNA extracted from roots of inoculated and mock inoculated plantlets. A total of 1000 transcripts for each library were cloned and validated through colony Northern analysis. Up to now, 800 cDNAs were sequenced and subjected to Blast analyses employing the NCBI, SGN and MiBASE databases. All the analysed sequences were grouped into three major categories: clones aligned with sequences of known function, clones aligned with sequences of unknown roles and clones with no alignment with sequences in the database. The clones belonging to the group with known function were assigned to the following metabolic groups: primary metabolism and photosynthesis; DNA replication, regulation and expression; translation; protein synthesis, degradation and modification; protein transport and translocation/membrane associated; cell wall, division and cytoskeleton; defence mechanism/secondary metabolism; stress induced proteins. Considering the assigned categories, we evaluated and compared the different expression profiles distinctive of each fungal inoculation alone, and also of the mixed inoculation. Of particular interest will be genes differently associated with response to pathogen among the three types of inoculations.


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EXPRESSION OF E.COLI OTSB-A OPERON IN CHLOROPLASTS FOR ENHANCED TOLERANCE TO DROUGHT AND SALINITY STRESSES IN EGGPLANT (SOLANUM MELONGENA)

1,2 1,3 1 2 1Geeta Prakash , Ajay Singh , Viswanathan Chinnusamy , S.K. Sawhney and K.C. Bansal

1National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute, New Delhi-110012,

2 3India; Department of Botany, University of Delhi, New Delhi-110007; Department of Plant Pathology, University of Kentucky, USA

Abiotic stress responses in plants lead to a wide variety of biochemical and physiological changes including the accumulation of various organic compounds of low molecular weights, collectively known as compatible solutes, or osmolytes. Transgenic approaches - nuclear transformation and plastid transformation have opened up new opportunities to improve tolerance to abiotic stresses by incorporating genes involved in stress tolerance into agronomically superior crops. Plastid transformation is a powerful tool to obtain crop plants with improved properties and to study fundamental aspects of plastid function. Since there are many copies of plastid DNA per plant cell, proteins encoded by plastid transgenes can be expressed at a very high level. Here, we report the introduction of E. coli OtsB-A operon in the plastid genome of eggplant for enhanced tolerance to salt and water stress. The operon was incorporated into the plastid DNA using particle bombardment and constitutively expressed in Solanum melongena cv. Pusa Shyamla. Molecular analysis by PCR, RT-PCR and Southern blotting revealed the site- specific integration of the transgene and expression into the chloroplast genome. The transplastomic eggplant plants when subjected to salt and water stress showed high level of tolerance as compared to the wild type plant. The results revealed that enhanced accumulation of trehalose in chloroplasts provides increased tolerance against abiotic stresses in plants and hence, this strategy could be used for the improvement of high yield stress sensitive crops.

INSECT RESISTANT CROPS ARE AN INTEGRAL PART OF IPM PROGRAMS: BT BRINJAL

Srinivas Parimi and Usha B. Zehr

Mahyco, Jalna - 431 203, India

Brinjal (Eggplant), Solanum melongena, is one of the most important vegetable crops of India and is being cultivated in an area of 0.51 million ha with a production of 8.2 million tons. India is one of the largest producers of brinjal in the world. Brinjal is attacked by more than 30 arthropod pests of which brinjal fruit and shoot borer (FSB), Leucinodes orbonalis (Guenee) is recognized as the most serious and destructive pest (causing upto 90% yield losses). Other important pests include fruit borer, stem borer, sucking pests and hadda beetles. Pest management in brinjal relies on insecticide applications besides other practices following the principles of Integrated Pest Management (IPM). The constraints in available pest control methods places an increased emphasis on the search for alternative methods and the insect resistant Bt Brinjal is a viable option. Bt Brinjal, developed by Mahyco, can be effectively used to manage economically important pests such as fruit and shoot borer, fruit borer and stem borer. Management of insect pests by inserting Bt genes into crops is considered as a host plant resistance mechanism, an integral part of IPM. The sustainability of Bt technology depends on development and implementation of insect resistance management strategies. This presentation will discuss briefly on available techniques to manage FSB, management constraints, Bt Brinjal technology, insect resistance management (IRM) and integration of Bt Brinjal in the existing FSB management systems.


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DEVELOPMENT OF BT BRINJAL AND THE REGULATORY PROCESS

Usha Barwale Zehr

Mahyco Research Center, Dawalwadi, India

Brinjal crop is affected heavily with several insect pests, Fruit and shoot borer being the most damaging. Conventional sources of tolerance are not available as a result the farmers undertake pesticide applications to control the pest pressure. The number of pesticide applications range from 25-80 per cropping season. Bt technology offers an built-in protection to control this pest. Bt brinjal was developed at Mahyco and transferred to different types of brinjal which are preferred by different regions. A full regulatory package has also been generated as per Government of India guidelines for Biotechnology products. The field trials conducted till date demonstrates the benefits of the technology at farm level, farmer level, and consumer level plus has positive impact on the environment. The regulatory studies also establish the safety of the product. Data from the studies till date will be discussed.

BT EGGPLANT (OR BRINJAL) FOR THE PHILIPPINES: A SUCCESSFUL CASE OF SOUTH-SOUTH PARTNERSHIP

Desiree M. Hautea

University of the Philippines Los Baños, Regional Coordinator, ABSPII-SEAsia College, Laguna 4031, The Philippines

In many developing countries like the Philippines concerns on food security, poverty alleviation and environmental sustainability are putting more pressure on people and institutions to find alternative ways to achieve higher agricultural productivity. Recognizing that modern biotechnology offers unique opportunities to help address production and productivity constraints, the Philippines became one of the early adopters of genetically modified (or biotech) crops. However, adoption of biotech crop in the country has been limited only to Bt corn due to lack of access to proprietary gene technology for use in other crops of extreme importance to resource-poor farmers. In 2003, a South-South partnership between a private institution in India (MAHYCO) and a public institution in the Philippines (UPLB) was created to assist the product development and commercialization of Bt eggplant in the Philippines. Eggplant is one of the most important vegetable crops produced and consumed in the Philippines. Like India, the production of marketable eggplant is compromised due to extreme infestation of the insect commonly known as the eggplant fruit and shoot borer (Leucinodes orbonalis Guenee) or EFSB. Since then, product development of Bt eggplant for commercial release in the Philippines has progressed tremendously. This presentation will provide an update of the progress of the Bt eggplant product development in Philippines and describe the elements that make this South-South partnership a success.


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SATELLITE SESSION V PEPPER

148

CHARACTERIZATION OF PUTATIVE CAPSAICIN SYNTHASE PROMOTER ACTIVITY

June-Sik, Kim, Minkyu, Park, Dong Ju, Lee, Byung-Dong, Kim

Dept. of Plant Science and Center for Plant Molecular Genetics and Breeding Research, Seoul National University.

Capsaicin is a very important secondary metabolite that is unique to Capsicum. Capsaicin biosynthesis is regulated developmentally and environmentally in the placenta of hot pepper. To investigate regulation of capsaicin biosynthesis, the promoter (1,537 bp) of pepper capsaicin synthase (CS) was fused to GUS and introduced into Arabidopsis thaliana (Col-0) via A. tumefaciens to produce CSPRO: GUS transgenic plants. The CS was specifically expressed in the placenta tissue of immature green fruit. However, the transgenic Arabidopsis showed ectopic GUS expressions in the leaves, flowers and roots, but not in the stems. The CSPRO activity was relatively high under light conditions and was induced by both heat shock and wounding, as CS transcripts were increased by wounding. Exogenous capsaicin caused strong suppression of the CSPRO activity in transgenic Arabidopsis, as demonstrated by suppression of CS expression in the placenta after capsaicin treatment. Furthermore, the differential expression levels of Kas, Pal and pAmt, which are associated with the capsaicinoid biosynthetic pathway, were also suppressed in the placenta by capsaicin treatment. These results support that capsaicin, a feedback inhibitor, plays a pivotal role in regulating gene expression which is involved in the biosynthesis of capsaicinoids.


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SOL 2009150

THE GENETIC CONTROL OF FLOWERING TIME AND SHOOT ARCHITECTURE IN PEPPER

Oded Cohen, Dar Jeifetz and Ilan Paran

Institute of Plant Sciences, The Volcani Center, Agricultural Research Organization, Israel

Pepper develops 8-20 leaves in the primary shoot that terminates in a single flower. Further development continues after bifurcation of the primary stem into two sympodial shoots, each consisting a flower and two leaves. In order to identify the key regulators of shoot architecture and flowering time in pepper, we performed EMS mutagenesis and identified several mutants that are altered in these traits. One mutant, late flowering2 (lfl2) is characterized by late flowering, an increase of leaf-to-flower ratio in the sympodial shoot and vegetative growth in the floral meristem. lfl2 was found to be controlled by the pepper ortholog of JOINTLESS that suppresses vegetative growth in the inflorescence meristem of tomato. Pepper JOINTLESS (Ca-JOINTLESS) functions as promoter of flowering in both the primary and sympodial shoots, and it suppresses vegetative growth in the floral meristem. The second mutant is characterized by inhibition of lateral branching in the primary stem, late flowering, an increase of leaf-to-flower ratio in the sympodial shoot and suppression of sympodial development. This mutation was found to be controlled by the pepper ortholog of BLIND that controls the formation of lateral meristems in tomato. While the main function of BLIND orthologs is conserved in pepper and tomato, minor effects of the genes are species-specific. The comparative analysis of homologous mutants affecting shoot architecture in both pepper and tomato allows to define common and unique functions of these genes that contribute to the differentiation of shoot development in these closely related species.

VALUABLE MOLECULAR MARKERS ASSOCIATED WITH SEVERAL IMPORTANT TRAITS FOR BREEDING COMMERCIAL F1 VARIETY OF CHILLI PEPPER IN KOREA

Jundae Lee, Won Phil Lee, Jung-Heon Han, Jae Wahng Do and Jae Bok Yoon

R&D Unit, Pepper & Breeding Institute, Seodun-dong 103-2, Suwon 441-853, Korea

Pepper (Capsicum annuum L.) is one of the most important money making vegetables in Korea. Most of the commercial varieties are F1 hybrids produced by male sterility including GMS and CGMS. Pepper production, however, has been seriously damaged by several diseases including Phytophthora blight, anthracnose and some viral diseases (CMV, TMV and PepMoV). It is that reasons why we need to develop molecular markers associated with male sterility and disease resistance for increasing pepper breeding efficiency. To develop molecular marker, we usually used BSA-AFLP method in a trait-related near-isogenic line or F2 segregating population. Then, most of the AFLP markers developed were converted into the codominant STS markers (CAPS and SCAR) by using internal sequencing and/or genome walking analysis. Up to now, we have developed several molecular markers; it is the four different GMS linked markers that MS1-SCAR, MS3-CAPS, MSK-CAPS and MSP-CAPS. The PR-CAPS and OPP13-CAPS markers are linked to the Rf locus in CGMS. The PRM-CAPS marker is linked to the major locus resistant to Phytophthora root rot. The AR1-CAPS and AR2-SCAR markers are linked to the major locus resistant to anthracnose caused by Colletotrichum acutatum and C. capsici, respectively. The CMR1-CAPS marker is linked to the resistance gene to CMVfny isolate. The TMR2-CAPS, TMR3-SCAR and TMR4-CAPS are allele-specific markers linked to the L locus resistant to TMV. The PVR4-SCAR marker is linked to the Pvr4 gene resistant to PepMoV. All the markers we developed are now using for practical breeding program of F1 hybrids in Korea.


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SOL 2009152

COMPARATIVE GENOMIC ANALYSIS BETWEEN PEPPER AND TOMATO CONFIRMED GENETIC SYNTENY BUT REVEALED THE GENOME DIFFERENTIATION BY ACCUMULATION OF LTR RETROTRANSPOSONS AND UNIQUE REPEAT SEQUENCES IN PEPPER GENOME

Minkyu Park, SungHwan Jo, Jin-Kyung Kwon, Jong Wha Ahn, Ik-Hyun Bae, Cheol-Goo Hur, Byund-Dong Kim, Doil Choi

Seoul National University

Among Solanaceae, pepper is an evolutionarily close species with tomato but has 3 times larger genome than that of tomato. Though the well conserved gene synteny and repertoire between the two species have been reported, the causes of genome size differentiation are to be elucidated. In this retrospective study, we investigated the cause of pepper genome expansion by comparing pepper gene-rich regions with their orthologous tomato sequences and analyzing heterochromatic sequences. For the purpose, a total of 985,237 bp pepper BAC sequences harboring gene-rich regions were generated and compared with their orthologous tomato BAC sequences corresponding to total 490,745 bp. In addition, two pepper BAC sequences harboring heterochromatin regions were analyzed in detail. The comparative analysis of the orthologous sequences revealed that pepper gene-rich regions are highly syntenic but twice larger than that of tomato due to multiple insertions of LTR retrotransposons. Analysis of heterochromatic sequence revealed that, besides LTR retrotransposons, long-unit tandem repeats (LUTRs) that have about 3.3 Kbp or 5 Kbp of unit length constitute the pepper heterochromatic regions. The distributions of those LTR retrotransposons and LUTRs in the pepper genome were identified by Fluorescence In Situ Hybridization (FISH) analysis. Our findings indicate that the pepper genome had been expanded its size by massive accumulation of the LTR retrotransposons and the LUTRs.

DIVERSE PIN-II PROTEINASE INHIBITOR GENES FROM CAPSICUM ANNUUM LINN. ASSOCIATE WITH ENDOGENOUS AND DEFENSE RELATED ROLES

Mishra M, Tamhane VA, Khandelwal N, Kulkarni MJ, Gupta VS and Giri AP

Plant Molecular Biology Unit, Division of Biochemical sciences, National Chemical Laboratory, Pune - 411008

Plant proteinase inhibitors (PIs) are proteins that interact with gut proteases of Lepidopteran insects and suppress their proteolytic activity. Potato type-II (Pin2) PIs having large structural and functional diversity have been reported from various crop plants of Solanaceae. Pin-II PI genes and precursor proteins are composed of multiple inhibitory repeat domains (IRDs), their number varying between 1 and 8; connected by protease sensitive 5 AA linker regions. A typical IRD (55 AA residue) is characterized by presence of 8 cysteines and an active site either for trypsin or chymotrypsin. Pin-II PIs have demonstrated influence in plant defense against herbivores pests and also have an important endogenous role in planta. Twenty one novel genes of the Pin-II type with 1- to 4-IRD types were isolated from fruit and stem tissues of Capsicum annuum L. Deduced amino acid sequences of the CanPIs showed 2 to 20% sequence variation pronounced close to the reactive site loop. CanPIs distinctly separate from full length Pin-II type PIs from other solanaceae members. Expression patterns of CanPIs in the fruit and stem tissues of mature C. annuum plants were shown to vary qualitatively and quantitatively. CanPI expression was differentially up-regulated upon wounding and insect attack, thus supporting their endogenous and defense roles. To elucidate the functional significance of eminent CanPI diversity, representatives of each type were expressed in Pichia pastoris and the recombinant CanPI (rCanPIs) proteins were analyzed in vitro and in vivo for their inhibitory activity against H. armigera gut proteinases.


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SOL 2009154

PHY-P5, A MAJOR BROAD-SPECTRUM RESISTANCE QTL CONFERRING RESISTANCE TO PHYTOPHTHORA IN PEPPER

Stéphanie Mallard, Sophie Ewert, Anne Blattes, Véronique Lefebvre

INRA, UR 1052 GAFL Génétique et Amélioration des Fruits et Légumes, Bp94,84140 Montfavet, France.

Blight caused by the pathogen Phytophthora spp. (Oomycete) is one of the most damaging diseases of Solanaceae crops. Contrary to monogenic resistances that are easily overcome by virulent strains, polygenic resistances generally confer a durable control of disease severity. Despite the potential importance of polygenic disease resistances, little is known about their molecular bases in plants and the mechanisms of action of the genes controlling them. A major-effect QTL region that confers resistance towards P. capsici was detected on pepper chromosome P5 from 5 partially resistant parents and with 11 different isolates. We were not able to determine whether this QTL region, that we called Phy-P5, corresponds to the same QTL in all populations because of the lack of common markers between maps. Moreover, comparative mapping suggested colinearities between Phy-P5 and resistance QTLs to P. infestans on potato chromosome IV and on tomato chromosome T4. To determine whether Phy-P5 is a broad-spectrum resistance QTL to Phytophthora and to identify candidate genes, we anchored the pepper genomic region harbouring Phy-P5 (i) within the published pepper maps, (ii) with the other members of the Solanaceae family and (iii) with the model plants by developing bridge markers. Up to now, our results showed that Phy-P5 was conserved across 5 genetic backgrounds in pepper and controlled 4 P. capsici isolates. We also determined the colinear region of Phy-P5 in tomato, potato and Arabidopsis making a step forward the research of functional orthologs of Phy-P5 in those species

SATELLITE SESSION VII SECONDARY METABOLISM

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SOL 2009156

METABOLIC ENGINEERING OF TOMATO AROMA: THE SOUNDS OF SILENT METABOLISM

Efraim Lewinsohn

Department of Vegetable Crops, Newe Ya'ar Research Center, Agricultural Research Organization, P.O. Box 1021, Ramat Yishay, 30095 Israel

Plants invest a great deal of resources in synthesizing and accumulating natural products. Still, there is growing evidence indicating that many biosynthetic enzymes leading to the accumulation of plant natural products are often active in cells that not necessary produce a given metabolite. In attempts to divert the existing terpenoid pathway to the formation of pigments, to the formation of aroma volatiles in tomato fruit, we have uncovered occult sets of enzymes that are able to further metabolize the novel substrates made available in the transgenic plants. Thus, enzymes without any apparent endogenous substrate or function were found, suggesting that tomato fruits have a reservoir of metabolic capabilities that normally remains hidden, concealed or unused. It could be that such concealed enzyme activities had important biological roles in the tomato ancestors and have not been fully eliminated through selection and evolution. Also, it could be that such activities possess important biochemical but yet unknown roles and coincidentally are able to accept novel substrates. We have coined the term "silent metabolism" to describe occult metabolic capacities present or induced in plants. A few examples illustrating such concealed metabolism in tomato, as well as its possible repercussion in plant metabolic engineering will be presented.

POTATO TUBER QUALITY TRAITS AND SECONDARY METABOLISM

Wayne Morris, Mark Taylor

Scottish Crop Research Institute, UK

Secondary metabolites impact on many aspects of potato tuber nutritional and sensory quality. Research at SCRI has focussed on tuber isoprenoid metabolites particularly carotenoids. Transgenic experiments have clearly illustrated the potential for enhancing potato tuber carotenoid content. However, equally clear is that the further development of the potato tuber as a platform for carotenoid biosynthesis requires a greater understanding of this pathway in terms of its regulation and storage organelle capacity. Thus we have applied a genetic approach combined with the use of the 44K element POCI microarray to give us new insights into how the pathway is regulated. The use of the POCI array has also enabled us to investigate the molecular basis of differences in isoprenoid -copaenelevels within potato germplasm. For example the sesquiterpene volatile is a significant flavour volatile in some types of cooked potato tuber. Gene expression comparisons between different germplasm enabled us to rapidly -copaene synthase gene. Taste trials of potatoes, transgenicallyidentify the -copaene to-copaene, will reveal the contribution of modified to produce overall potato flavour. As the nutritional value of flavonoids such as anthocyanins becomes more apparent, tuber flavonoid metabolism is also attracting more research interest. From comparative transcript profiling of potato germplasm differentiated in tuber anthocyanin content several novel candidate genes have been identified.


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SOL 2009158

ASHWAGANDHA WITHANOMICS: LEADS IN WITHANOLIDE BIOSYNTHESIS, NOVEL CHEMOTYPES AND THEIR METABOLIC CONNOTATIONS IN WITHANIA SOMNIFERA

1 2Rajender S. Sangwan and Rakesh Tuli

1 2Central Institute of Medicinal and Aromatic Plants (CSIR), Lucknow-226015, India; National Botanical Research

Institute (CSIR), Lucknow-226001, India

Ashwagandha (Withania somnifera, Solanaceae) is one of the most reputed medicinal plant of Indian systems of medicine (Ayurveda, Sidha, Unani) used in 100 therapeutic and nutraceutical formulations. Its pharmacological (Rasayana) description like physiological and metabolic restoration, anti-arthritic, cognitive function improvement in geriatric states, recovery from neurodegenerative disorders have inspired its phytochemical and pharmacological investigations. However, understanding of biosynthesis of its unique secondary metabolome (withanolides) and chemo-genomic diversity as chemotypes has been meagre.

Our recent investigations on different aspects of withanolide biosynthesis have led to several conceptual developments on 'withanomics'. These include (i) withanolides are the characteristic secondary metabolites, rather than alkaloids, (ii) DOXP (non-mevalonate) pathway of isoprenogenesis contributes significantly to withanogenesis, (iii) root and shoot inherently possess independent complete metabolic pathway for de novo biosynthesis of withanolides (iv) a novel withanolidal cross-talk operates between shoot and roots (v) development of several novel chemotypes including a phytochemical hybrid of Ashwagandha, (vi) identification and characterization of some of the enzymes and genes related to withanolide metabolism in the plant and their putative function in physiological performance/defense, (vii) metabolic connotation of phytochemical diversity via position-specific hydroxylations (cyt P450), glycosylations (UGTs) and acylations (like withaferin A acetyl Co A acetyltrasferase, WAAT), (viii) development of a working metabolic model of origin of withanogenesis in relation to ubiquitous sterol and brassinolide pathway and putative functional overtones of the biosynthetic relationships, (ix) ability of some withanolides being selectively facile to interact with thiol-reagents due to their characteristic hydroxyl- and epoxy configurations-a putative bio-mimic of their reported therapeutic effects via covalent binding with cysteine residues of several regulatory proteins.

In our opinion, with these dimensions of diversity as withanolide chemotypes and with the presence of other novel classes of secondary metabolites like calystegines, withanamides biosynthesized by the plant, it fits as a model plant for secondary metabolism, joining the food and nutrition model counterparts (potato, tomato) in the Solanaceae. Details on these results and leads would be discussed in the presentation.

MEDICINAL PLANTS OF SOLANACEAE: GROWING SIGNIFICANCE IN HEALTHCARE SINCE ANTIQUITY

Janardan Singh

Central Institute of Medicinal and Aromatic Plants, Lucknow – 226015, India

Solanaceae family occupies an exalted position amongst the Angiosperms, in view of their multiferous uses as a food, vegetable, beverage, ornamental and valuable medicinal products. It is one of the largest families and its species are found all over the world. In India, it is well represented by many species. The earliest record of use of solanaceous drugs can be traced back to ancient Hindu, Greek, Persian, Egyptian, Chinese and other medicines. Species of Solanum viz. S. violaceum and S. virginianum are the important medicinal plants of Ayurvedic and Unani medicines. Their roots are a constituent of drug “Dasmula – Ten roots”, often used in Ayurvedic formulations. In Unani medicine, many plants of family are extensively utilized such as: Atropa belladonna, Capsicum spp, Datura metel and Hyoscymus niger etc. Lycium barbatum, L. chinense, Physochlaina infundibularis, Datura metel and D. innoxia are some of the plants used in Chinese medicines. Hyoscyamus albus has been used in European, Arabian and Persian medicines for ophthalmia, gout and rheumatism. The true “Mandrake” (Mandragora officinarum) has been prized as an aphrodisiac in Roman and Greek medicine. Recently, potential of solanaceous raw drugs has been increasingly realized and various plants yielding more secondary metabolites have been identified. However, some of the wild species have also been exploited for important secondary metabolites. These include: Scopolia japonica, S. cornifolia, Atropa baetica and Datura querefolia etc. Their presence constitutes an interesting chemo-taxonomic study. A specific group of secondary metabolites “Withanolides” have evinced keen research interest in plants like Withania somnifera (Ashwagandha), a plant of Indian origin. Looking forward, use of solanaceous plants globally, the potentialities of a number of other plants used in various traditional or ethno medicines are yet to be explored for human health.


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SOL 2009160

SECONDARY METABOLISM DURING ACTIVE ANTHOCYANIN DEGRADATION IN BRUNFELSIA CALYCINA FLOWERS

1 1 1 1 1 1Michal Oren-Shamir , Ayelet Bar-Akiva , Rinat Ovadia , Raya Leiberman , Liat Shahar , Hinanit Koltai , 2 2 3 3 4Ilana Rogachev , Asaph Aharoni , Einat Bar , Efraim Lewinsohn , David Weiss

1Department of Ornamental Horticulture, Agriculture Research Institute, The Volcani Center, Bet Dagan, 50250, 2

P.O Box 6, Israel; Department of Plant Sciences, Weizmann Institute of Science, Rehovot 76100, Israel; 3Department of Vegetable Crops, Agriculture Research Institute, Newe Ya'ar Research Center, P.O. Box 1021,

4Ramat Yishay, 30095; Plant Sciences and Genetics in Agriculture, Faculty of Agricultural, Food, and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel

Brunfelcia calycina flowers change color from purple to white due to active anthocyanin degradation, parallel to increase in fragrance and petal size. An extensive characterization of the events taking place in Brunfelsia flowers is presented. Anthocyanin characterization was performed using UPLC-QTOF-MS/MS. Volatiles emitted were identified by HS-SPME-GC-MS. Accumulated proteins were identified by 2D gels electrophoresis. Transcription profiles were characterized by cross-species hybridization of Brunfelsia cDNAs to potato cDNA microarrays. Identification of accumulated metabolites was performed by UPLC-QTOF-MS non-targeted metabolite analysis. Results include characterization of the nine main anthocyanins in Brunfelsia flowers. In addition we identified 146 upregulated genes, 19 volatiles, seven proteins, and 17 metabolites that increased during anthocyanin degradation. A multi level analysis suggests induction of the shikimate pathway. This pathway is the most probable source for the phenolic acids, which in turn are precursors of both the benzenoid and lignin production pathways. However, since these phenolics are also constituents of Brunfelsia anthocyanins, the degradation of anthocyanins may contribute to their increase in concentration in the flower petals. In an attempt to reveal the catabolic process of anthocyanins in Brunfelsia, several genes that may be candidates for anthocyanin degradation have been isolated and will be tested in a transient transformation system in petunia flowers. The knowledge obtained from this study is valuable for future studies regarding the degradation of anthocyanins, formation of volatiles, and the network of secondary metabolism in Brunfelsia and related species such as petunia.

REGULATION OF GLYCOALKALOIDS SYNTHESIS IN POTATO

Idit Ginzberg*, Jim Tokuhisa†, Richard Veilleux†, Thippeswamy Muddarangappa, Ufuk Demirel, Edna Fogelman, Dana Kadosh

*Institute of Plant Sciences, ARO, the Volcani Center, Bet Dagan 50250, Israel †Department of Horticulture, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, USA

The potato steroidal glycoalkaloids (SGAs) are important components of plant resistance against pests and pathogens. The two major SGAs in cultivated potato (Solanum tuberosum) are α-chaconine and α-solanine that exhibit strong cellular lytic properties and inhibit acetylcholinesterase activity, and can be toxic to humans at high levels. SGA composition and level are genetically determined; however unfavorable growth conditions and inappropriate post-harvest management increase their levels in tubers of “safe” cultivars. Deciphering how stress conditions regulate SGA gene expression would enable genetic manipulation of new cultivars with limited accumulation of SGAs under unfavorable growth conditions. SGA biosynthesis is derived from the isoprenoid pathway, however only sketchy details are available on SGAs specific genes. Previous results indicated of an association between high SGA levels in leaves and tubers and high expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase-1 (hmg1) and squalene synthase-1 (ss1) genes in potato genotypes that exhibit various levels of SGA content. Sequence comparison of hmg1 and ss1 from the cultivar Desiree (low SGA producer) and the wild type S. chacoense Bitter (high SGA producer) indicated of genotype specific amino acid substitutions that may affect protein efficiency in metabolism. To isolate genes and precursors of SGA biosynthesis, the expression of its pathway was modified by transforming plants of Desiree with hmg1 and ss1 genes of S. chacoense Bitter. Real-time PCR and HPLC techniques were used to verify induction of the biosynthetic pathway, and cDNA-AFLP was used to isolate SGA putative sequences with altered expression by comparing transgenic and non-transgenic plants.


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SOL 2009162

SYSTEMATIC INVESTIGATION OF CAROTENOID ACCUMULATION IN TRANSGENIC POTATO TUBERS: THE LONG AND WINDING ROAD TO THE “GOLDEN” PHENOTYPE

Gianfranco Diretto, Salim Al-Babili, Raffaela Tavazza, Federico Scossa, Velia Papacchioli, Melania Migliore, Peter Beyer, Giovanni Giuliano

Italian Agency for New Technologies, Energy and Environment. Roma, Italy

Vitamin A deficiency (VAD) is a public health problem in a large number of countries, especially in Africa and South-East Asia. To alleviate this problem, enhancement of the β-carotene (provitamin A) content of the major staple crops (wheat, rice, maize or potato) is desirable. Previously, we engineered transgenic “Golden” potato tubers overexpressing 3 bacterial genes for β-carotene synthesis (CrtB, CrtI and CrtY) and accumulating the highest amount of this provitamin in the four aforementioned crops. In this work, we report a systematic characterization of transgenic plants carrying different combinations of the three genes under the control of the 35S and Pat promoters. When expressed in leaves, Crt genes negatively affect leaf chlorophyll and carotenoid content in an additive way. In tubers, total carotenoids increase in all transgene combinations. Contrary to “Golden” rice and canola, all three genes are needed for α- and β-carotene accumulation. Real Time experiments show a complex network of alterations in endogenous carotenoid gene expression in tubers. Clustering and pathway network analysis shine light on the perturbation of leaf and tuber carotenogenesis by different transgene combinations. Modulation of “late” transcripts and of PSY1 shows a strong correlation with tuber β-carotene levels. Total transcription of the carotenoid pathway shows a strong increase in tubers expressing the BI transgenes, while is relatively unaltered in “Golden” (YBI) tubers.

ASSESSMENT OF GENETIC AND ENVIRONMENTAL FACTORS INVOLVED IN TOMATO FLAVOR

1 2 3 2 1 1 1Carli P , Arima S , Fogliano V , Tardella L , Frusciante L , Barone A , Ercolano M. R.

1Department of Soil, Plant, Environmental and Animal Production Sciences, University of Naples “Federico II”, 2Via Università 100, 80055 Portici (Italy); Department of Statistical Sciences, University of Rome “La Sapienza”,

3P.le Aldo Moro 5, 00185 Roma, (Italy); Department of Food Science, University of Naples “Federico II” Via Università 133, 80055 Portici (Italy)

Commercial market is becoming more demanding about external appearance nutritional and organoleptic characteristics of tomato fruits. Flavor is a very complex trait that is affected by numerous genetic components and non genetic factors, not all of which are known or well understood. A complex mixture of sugars, acids, amino acids, minerals and volatile compounds contribute to the characteristic flavor of fresh tomato fruits. The development of high-throughput data-collection techniques allows the simultaneous analysis of several traits and helps to determine how and when these interact with each other. The aim of this work was to assess flavor diversity in six Italian heirlooms growth in three different environmental conditions and to identify important components affecting flavor. In particular, the six heirlooms were grown in three areas located in the southern Campania and characterized for biochemical and sensory traits. Sensory attributes intensity showed marked differences in the three environments tested. Considering single sample data, peculiar traits of each typology were also evidenced. Principal component analysis was carried out on the biochemical and sensorial traits for to describe relations between the different attributes as well as to reduce the data set to important components. A clear discrimination of tomato heirlooms in the three different fields, based on the first three factors, was evidenced.


SOL 2009 163

SATELLITE SESSION VIII VIRUSES

164

POTATO VIRUS Y SYMPTOMS IN POTATO TUBERS GROWN IN DIFFERENT ENVIRONMENTS

1 2 3 4Whitworth , J.L., Hamm, P.B. , Gray, S.M. , Crosslin, J.M.

1 2USDA-ARS, 1693 S 2700 W, Aberdeen, Idaho, 83210, USA; Oregon State University, Hermiston, Oregon, USA;

3 4USDA-ARS, Ithaca, New York, USA; USDA-ARS, Prosser, Washington, USA

Strains of Potato virus Y (PVY) known to cause tuber necrotic ringspot disease (PTNRD) have been detected in recent surveys and screening in the USA, Canada, and Mexico. Many diagnostic tests are available for detecting and differentiating PVY strains, but more than one test must be used due to differences in genomic composition. PVY has traditionally been divided into PVY, PVYN, PVYC based upon reaction in tobacco with N strains producing veinal necrosis, while O strains only produce a leaf mottle or mosaic. The same isolates have been designated as different strains depending on whether molecular data (sequencing) is used or serological data is used. Isolates identified as subcategories of N (N-Wi or NTN) can either show no tobacco veinal necrosis and may or may not produce tuber necrosis. A few O strains have also been shown to produce tuber necrosis. At this point, a tuber bioassay remains a definitive test for PTNRD. In order to better characterize PVY strains, this study was done under different environmental conditions using the same potato varieties and PVY isolates.


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BIOLOGICAL AND MOLECULAR PROPERTIES OF POTATO VIRUS S (PVS) AND THE RESPONSE OF SELECTED POTATO GENOTYPES TO PVS INFECTION

1 2Hanu R. Pappu and Jonathan Whitworth

1 2Department of Plant Pathology, Washington State University, Pullman, WA 99164, USA; USDA-ARS, Aberdeen, ID, USA.

Potato virus S (PVS) belongs to the genus Carlavirus, family Flexiviridae. Two strains of PVS were reported: ordinary and Andean. PVS was reported to cause yield losses in some parts of the world. In the US, most potato cultivars show mild or no symptoms to PVS infection. Late blight, caused by Phytophthora infestans, is an extremely devastating disease of potato worldwide. Potato cv. Defender is the first and only commercially released potato cultivar with tubers and leaves that survive late blight. Severe symptoms including severe leaf mottling, stunting and malformation of plants were displayed in cv. Defender. Out of all the known potato viruses, PVS was the only potato virus consistently found in association with these symptomatic plants. Tubers from these PVS-positive, symptomatic Defender plants were grown under controlled, greenhouse conditions and tuber-borne transmission of the virus was demonstrated based on the development of symptoms identical to those observed in the field and confirmation of the presence of PVS by ELISA and RT-PCR. PVS usually induces inconspicuous symptoms in potato cultivars such as Russet Burbank, Norkotah, Gemstar, Ranger Russet and 6LS. Our recent studies further showed that LBR potato clone 4106 (also known as A95053-61) infected with PVS also resulted in similar severe symptoms. To better understand this phenomenon and to characterize PVS at biological and molecular levels, the complete nucleotide sequence of the PVS isolates from cv. Defender and LBR4106 were determined. Host response studies of PVS showed that Defender and other late blight resistant potatoes appear to be susceptible to PVS infection. Screening of LBR clones and the pedigree of LBR clones might provide important clues regarding the possible genetic linkage between late blight resistance and virus susceptibility.

PRESENT STATUS OF CHRONIC AND EMERGING TOSPOVIRUSES AFFECTING SOLANACEOUS CROPS IN INDIA

R. K.

Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi-110012, India

Tospoviruses (Family Bunyaviridae) are fast emerging as serious pathogens negatively impacting the cultivation of several field and horticultural crops in India. Of the 20 tospovirus species recorded world wide, Capsicum chlorosis virus (CaCV) on capsicum, Groundnut bud necrosis virus (GBNV) on groundnut, Iris yellow spot virus (IYSV) on onion, Peanut yellow spot virus (PYSV) on groundnut and Watermelon bud necrosis virus (WBNV) on watermelon have been reported from India. Of the Indian tospoviruses, GBNV, WBNV and CaCV have been recorded on Solanaceous crops and are serologically indistinguishable. Complete genome properties are studied only for GBNV. WBNV and CaCV have not been adequately characterized and are recognized based on nucleocapsid protein (N) gene characteristics. Intensive cultivation, introduction of susceptible genotypes, favorable environmental conditions for thrips populations, change in genetic makeup of the virus through mutation and genomic reassortments are some important factors for emergence of tospoviruses.

Jain


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SOL 2009168

VIRAL DERIVED RNAI SUPPRESSORS: ROLE IN PATHOGENESIS AND SYNERGISM

1 1 1 2Shelly Praveen , Priyanka Singh ,Vikas Koundal and Satendra K.Mangruthia

1 2Advanced Center for Plant Virology, Division of Plant Pathology, IARI, New Delhi; Plant Pathology Section, Directorate of Rice Research, Hyderabad

RNA silencing is a novel gene regulatory mechanism that operates at the level of RNA. Upon activation, this mechanism can degrade viral and transgene RNAs in a sequence-specific manner. In plants and insects, RNA interference (RNAi) is the main responder against viruses and shapes the basis of antiviral immunity. Viruses counter this defense by expressing viral suppressors of RNAi. Transgenic expression of viral suppressors of RNAi produces viral like symtoms, presumably by altering miRNA pathway. To investigate the role of RNAi suppressors in altering miRNAs levels and subsequently developing viral like symptoms, three distinct viral suppressors i.e. Cucumoviral-2b, Potyviral-HcPro and Geminiviral-AC4 have been characterized and their involvement in altering five different miRNAs responsible for hormone signaling, flower and plant development were analyzed in transgenic Nicotiana tabacum. Their role in synergistic behaviour for establishment of heterologous virus was also studied.

HIGH-THROUGHPUT MUTATION BREEDING IN SOLANACEA FOR VIRUS RESISTANCE

1 2Manash Chatterjee and Abdelhafid Bendahmane

1 2Bench Bio Private Ltd., C/O Jai Research Foundation, NH8, Valvada, Valsad, Gujarat- 396108, India; INRA-CNRS, UMR1165, Unité de Recherche en Génomique Végétale, 2 rue Gaston Crémieux, F-91057 Evry, France

The challenge of the next decade will be the speed at which DNA sequence databases are analysed and candidate genes of agronomic importance selected and tested for function. The bottleneck in this strategy is the lack of high throughput (HTP) tools to test the function of specific genes in a cellular context. Transgenic (GM) method is one route to validate the gene function and to also carry out crop improvement. However, this is not available in all crops. Moreover, GM foods face tremendous difficulties in public acceptance; the technology is expensive and laborious in most species, and most importantly frequently limits 'freedom to operate'. Additionally, the GM method cannot keep pace with the speed at which candidate genes for important traits are identified and prioritised for functional evaluation. A recently developed tool, TILLING (Targeting Induced Local Lesions IN Genomes), offers an alternative way to improve crops without transgenics. TILLING combines traditional chemical mutagenesis with sensitive molecular screenings to discover induced point mutations in genes controlling important traits whose sequence is known. By means of TILLING new genotypes with potentially high agronomic value can be isolated and directly commercialized. Several natural resistance genes against Potyviruses, (which form the largest genus of plant virus) from distinct crops, were shown to encode defective forms of eIF4E; for example, pvr2 for resistance against Potato virus Y (PVY) and Tobacco etch virus (TEV) in pepper, mo1 for resistance to Lettuce mosaic virus (LMV), sbm1 for resistance to Pea seed-borne mosaic virus (PSbMV), and pot-1 for resistance to PVY and TEV in Tomato. In all cases, resistance results from a small number of amino acid changes in the proteins encoded by the recessives resistance alleles that harbour point mutations. We will present our data to demonstrate that TILLING can be used successfully to create Potyvirus resistant plants.


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VIRUS DIAGNOSIS AND DEVELOPMENT OF MAKERS FOR RESISTANCE TO VIRUSES IN CHILLI (CAPSICUM ANNUUM)

M. Krishna Reddy, K. Madhavi Reddy and K.V. Ravishanker

Indian Institute of Horticultural Research, Hessaraghatta Lake PO, Bangalore-560089, [email protected].

Peppers (Capsicum sop.) both hot and sweet are important and remunerative vegetables cultivated all over India, ranking first in the world with an annual production of 0.6 million dry chilli from an area of 0.8 m.ha. Virus diseases cause serious losses and became the most limiting factor affecting pepper production in Andhra Pradesh, Karnataka, Tamil Nadu, Maharashtra, and Rajasthan. Several viruses have been reported on chilli, The most important viruses, which have emerged during the last two decades, are Alfamo, Begomo, Cucumo, Ilar, Poty and Tospoviruses. Characterization and identification of viruses is a prerequisite for developing effective management of viral diseases. In India, diagnosis was mainly based on symptomatology, which is not reliable. Several plant viruses produce very similar symptoms but are unrelated and strains of the same virus can produce very different symptoms. Moreover, mixed infections of unrelated viruses are very common in chilli. Detection of viruses in plant material, vectors or natural reservoirs is essential to ensure safe and sustainable agriculture. During the last two decades immunoassays particularly ELISA using polyclonal and monoclonal antibodies become widely accepted as valid tool in the diagnosis and detection of plant viruses. Immunoassays have now been developed for nearly all groups of plant viruses. New molecular detection techniques such as nucleic acid hybridization, blotting techniques using radio and novel non-radioactive labeled probes, variants of PCR, and real-time monitoring of amplicons or quantitative PCR have been used for diagnosis of viruses. This paper highlights the current diagnostic methods available for Chilli viruses and their utilization in resistance identification and marker development against chilli viruses.

CHILLI LEAF CURL DISEASE IS CAUSED BY INTERACTION AMONG DIVERSE BEGOMOVIRUSES IN INDIA

S. Chakraborty, A. K. Singh, B. Chattopadhyay, N. Kushwaha, R. Vinothkumar, S. Basu

Molecular Virology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi – 110067, India

Leaf curl disease of chilli (ChiLCD) is a devastating disease to chilli (Capsicum annuum) in India. We have cloned full-length genomes of DNA-A, DNA-B and satellite DNA-βs of 25 chilli-infecting begomovirus isolates from several major chilli growing regions of the country. Based on phylogenetic analyses of the DNA-As, we have identified association of nine distinct species of begomoviruses viz., Chilli leaf curl Multan virus (ChiLCV-Mul), Chilli leaf curl India virus (ChiLCV-In), Pepper leaf curl Bangladesh virus (PepLCBDV), Tomato leaf curl Joydebpur virus (ToLCJoV), Tomato leaf curl New Delhi virus, Tomato leaf curl Gujarat virus (ToLCGV), Tomato leaf curl Karnataka virus including two new species reported in this study. Out of these, four begomovirus species viz., Chilli leaf curl Multan virus, Chilli leaf curl India virus, Pepper leaf curl Bangladesh virus and Tomato leaf curl Joydebpur virus are the predominant ones associated with ChiLCD throughout the country, not being restricted to any particular geographical area. Occurrence of DNA-B and satellite DNA-βs were also found to be associated with the disease in some cases. Mixed infection of genomic components of ChiLCV-Mul & ToLCNDV (in New Delhi), ToLCJoV & PepLCBDV (in Ghazipur), and ToLCJoV & ToLCGV (in Kolkata) were found to be associated with this disease. Synergistic interaction among chilli begomoviruses has lead to occasional breakdown of host resistance of hitherto known resistant cultivars. Molecular diversity of these chilli-infecting begomoviruses and mechanism of synergistic interaction leading to severe leaf curl disease will be presented.


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SOL 2009172

VALIDATION OF TOLCV RESISTANCE CAPS MARKER FOR TY2 IN THE PARENTAL LINES OF TOMATO

Ashish Kumar, Sanjeev Kumar, Major Singh, Mathura Rai and D. Datta

Indian Institute of Vegetable Research, Varanasi

Tomato leaf curl virus (ToLCV) disease is a serious threat for tomato cultivation in the tropics and subtropics. The disease can be managed by evolving cultivars with pyramided ToLCV resistance genes through the use of molecular markers. These markers must be validated before their use in gene pyramiding programme. The Ty2 CAPS marker developed by Peter Hanson and co-workers was validated in the parental lines. The experimental material comprised H24 (Ty2), DVRT-1, DVRT-2, H86, Fla478 and backcross lines of H24/H86. PCR amplification was performed in a 25 µl reaction mixture containing, 20 ng genomic DNA, 2.5mM Mgcl2, 75 µM dNTPs, 10 x assay buffer, 0.1µM of each primer. PCR product amplified by Ty2 gene marker was digested with Taq I. Digested product was resolved through electrophoresis using 1X TAE buffer in 2.6% agarose gel. Upon PCR amplification of the lines DVRT-1, DVRT-2, H24, H86, Fla478 and Punjab Chhuhara, monomorphic band was obtained. PCR amplification product was digested with TaqI which generated two kinds of fragments. The resistant amplicon (~200bp) was detected in H24, DVRT-1, DVRT-2 and surprisingly in Punjab Chuhara as well. H86 carried the susceptibility allele (~350bp). The backcross of DVRT-1/H24 and DVRT-2/H24 carried the resistance allele whereas the backcross of H86/H24 segregated in the ratio of 1:1 for H24 type allele. Though Punjab Chhuhara is highly susceptible to ToLCV but it also showed resistant type marker allele. This kind of marker is not diagnostic for Ty2 gene and can be used for detecting Ty2 gene in few known parents only.

IDENTIFICATION AND MOLECULAR MAPPING OF TOMATO LEAF CURL NEW DELHI VIRUS (TOLCNDV) RESISTANT GENE(S) IN TOMATO (SOLANUM LYCOPERSICUM L.)

1 1 2 2 3 1 2M. Prasad , N. K Rai , M. K Reddy , K.V. Ravishankar , S. Chakraborty , P. P. Sahu and A.T. Sadashiva

1 2 National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi-67, INDIA; Indian Institute of

3Horticultural Research, Hessaraghatta Lake Post, Bangalore-560 089; School of Life Sciences, Jawaharlal Nehru University, New Delhi-67

The cultivated tomato, Solanum lycopersicum L., is the second most consumed vegetable worldwide and a well studied crop species in terms of genetics, genomics, and breeding. Tomato Leaf Curl Disease (ToLCD) causes important yield loss in tomato crops all over the world and despite serious efforts, till date there are no immune commercial varieties or F1 hybrids available. Genes controlling resistance to TYLCV have been identified and introgressed from several wild tomato species , including: Solanum pimpinel l i fol ium, S. peruvianum (Ty-5), S. chilense (Ty-1, Ty-3 and Ty-4), and S. habrochaites (Ty-2). In this study, we present the results of a research aimed at the identification of PCR-based markers [Random Amplified Polymorphic DNA, Simple Sequence Repeats, Resistance Gene Analogues, Inter Simple Sequence Repeat and Cleaved Amplified Polymorphic Sequence] linked to gene(s) confer resistance to ToLCNDV. To this purpose, bulked segregant analysis (BSA) was applied to a BC1F1 population segregating for the ToLCNDV resistance gene and derived from a cross between a ToLCNDV tolerant accession Solanum habrochaites LA-1777 and susceptible line 15SBSB. Genetic analysis indicated that three partial dominant genes confer resistance to ToLCNDV in the accession LA-1777. By using the BSA method, we have identified one PCR-based molecular marker linked with ToLCNDV and mapped at long arm of chromosome 11 of tomato at a distance of 5.9 cM from the ToLCNDV gene. The identified marker has been validated in a set of 19 different accessions of tomato for effective use in marker assisted breeding (MAB).


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ADDENDUM to the ABSTRACT BOOK

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GENE COEXPRESSION ANALYSIS OF TOMATO AND DEDUCED FUNCTION OF ZINC-FINGER PROTEIN GENE ASSOCIATED WITH FLAVONOID BIOSYNTHESESIS

1,2 1 1 1 3 1Soichi Ozaki , Yoshiyuki Ogata , Kunihiro Suda , Atsushi Kurabayashi , Ttsuya Suzuki , Yoko Iijima ,

1 1Daisuke Shibata , Koh Aoki

1 2Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu, 292-0818 Japan. Grad. School of Life Sci., Tohoku Univ. 3Chiba Prefectural Agric. Forestry Res. Center. Contact: [email protected]

In recent years, gene-to-gene and gene-to-metabolite coexpression analyses are regarded as potentially powerful approaches to infer functions of unknown genes. In Arabidopsis research, these strategies have successfully uncovered functions of genes involved in glucosinolate and flavonoid (e.g., Tohge et al., 2005 Plant J.). Here we report results of gene-to-gene coexpression analysis using Affymetrix Tomato Genechip array and experimental verification of gene function inferred from it.

We collected a gene-expression dataset from various tissues of Solanum lycopersicum cv. Micro-Tom by 71 hybridization experiments. Although we were aware of the small size and bias in experimental conditions of the Micro-Tom transcriptome dataset, we attempted to extract biologically relevant coexpression clusters using a novel algorithm based on the strength of correlation and density of connectivity. By this method, more than 200 coexpression clusters were identified, which contains clusters associated with biological processes such as flavonoid biosynthesis, proteinase inhibition, and jasmonate signaling. The analysis also revealed that many of the natural antisense transcripts showed high correlation with their sense counterparts. We then focused on an analysis of a zinc finger protein gene that was coexpressed with genes of flavonoid pathway enzymes. By using transgenic Micro-Tom, we demonstrated that the overexpression of zinc-finger gene is accompanied by the upregulation of early flavonoid pathway genes and enhanced accumulation of flavonol glycosides and tricaffeoylquinic acid. This result verifies the usefulness of coexpression analysis in inferring function of unknown genes of tomato. We acknowledge the support from Research and Development Program for New Bio-industry Initiatives.


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ASSEMBLY AND ANNOTATION OF TOMATO GENOME BAC SEQUENCES FOR THE IDENTIFICATION OF AGRICULTURALLY IMPORTANT GENES

A.K.Mahato, Sheetal Arora, Aditi Mathur, Vivek Dogra, Archana Singh, Jitendra K.Pal ,Manju Singh, Shubhra Barwa, Awadhesh Pandit, K.Gaikwad, T.Mohapatra, T.R.Sharma, N.K.Singh

National Research Centre on Plant Biotechnology, IARI, Pusa, New Delhi – 110012Email:[email protected]

Tomato is an important member of the Solanaceae family for studying fruit biology and in terms of economic value as a prime vegetable crop. Gene Prediction and annotation is one of the first and most important steps in understanding the genome biology of a species once it has been sequenced. Gene annotation is the process of attaching biological information to sequences which includes both functional as well as structural annotation. The analysis of Tomato genome is providing good framework of biological information. The shotgun sequence reads of 27 tomato BAC clones generated obtained from the MegaBACE-4000 sequencing machine were assembled using PHRED, PHRAP and CONSED software package. Out of these 16 BACS were submitted in Phase II and Phase III in GenBank. High quality phase III sequences were generated by using different genome finishing strategies like Transposons sequencing, custom primer and PCR amplification of the regions followed by sequencing. As on 26th March 2009 a total 645 finished BACs of 12 chromosomes were downloaded from FTP site of SOL genomics network (SGN; http://www.sgn.cornell.edu/) for annotation. The sequences were downloaded in FASTA format in local server of NRCPB and used for gene prediction by using FGENESH software.(www.softberry.com). We predicted 11019 genes in all the BACs. Of these genes 5541 were in forward stand and 5478 were in reverse strand. The protein sequences of all the genes were used for annotation using NR database of NCBI BLAST x. Many agriculturally important genes were identified in these BACs. The results obtained on tomato BAC genome assembly, annotated will be presented.


SOL 2009 177

DEVELOPMENT OF UNIGENE DERIVED MICROSATELLITE MARKERS IN INDIAN GINSENG WITHANIA SOMNIFERA

Shivam Mishra, Sahabjada, Shefali Pal, Shefali Singh, AK Shasany & Tripta Jhang

Genetic Resources and Biotechnology Division, CIMAP,(CSIR), Kukrail Picnic Spot Road, P.O. CIMAP, Lucknow 226015.

Withania somnifera (Ashwagandha) an important medicinal plant is in high demand for its wide spectrum of steroidal lactones (Withanolides and Withaferrin). It is used in more than 200 Ayurvedic, Siddha, Unani and other herbal formulations in India indicated as antiarthritic, anticancer, immunomodulatory, aphrodisisiac, diuretic, restorative and rejuvenative pharmacological activities. Mainly two types of ecotypes Kashmiri and Nagori with very distinct chemotypes is reported from India. Till date there is no report of microsatellite markers from this highly valuable medicinal plant which is required crucially for molecular breeding for high withanolide content. In total 300 sequences were generated from leaf and root specific cDNA libraries. These sequences were assembled by CAP3 assembler (http://www.genome.clemson.edu/cgi-.bin/cugi_cap3) into 22 unigene comprising 18521bp sequence. 75 SSRs were identified using MISA (http://www.pgrc.ipk-gatersleben.de/misa). 51 SSR were class II type. 24 primer-pairs were designed exclusively from trinucleotides, pentanucleotides and hexanucleotides. These were validated on the 59 accessions of W.somnifera including 47 tall,red berry Kashmiri type and 12 short, yellow berry, Nagori type. One accession each from Lycopericum esculentum and Capsicum annum were also screened to test cross-transferability. 82% of primer pairs were functional. EST sequencing is under way to increase microsatellite marker resource. This will help to generate markers for genetic characterization, development of genetic map and mapping of economic metabolic traits in Withania somnifera.


SOL 2009178

PHYSICAL MAPPING OF THE LONG ARM OF TOMATO CHROMOSOME 5

Manju Singh, Shubhra Barwa, Archana Singh, Awadhesh Pandit, Jitendra K. Pal, Ajay K. Mahato, Vivek Dogra, Sheetal Arora, Aditi Mathur , K. Gaikwad, T.R. Sharma, T. Mohapatra and N.K. Singh

National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute, Pusa Campus, New Delhi

The NRCPB, New Delhi has been assigned the sequencing of region between 73-119cM on the long arm of tomato chromosome 5. Initially total of 72 probable seed BACs were received from the Cornell University, USA, which were anchored by 13 genetic markers located at 9 different genetic positions in this region. However, after confirmatory tests using BAC FISH, it was found that three of these markers actually belonged to other tomato chromosomes. Physical mapping by end walking using STC approach was followed to construct extension tiling path from the seed BAC nucleation points. BAC end sequences available on SGN site were used in BLAST search against the complete sequence/end sequence contigs of the seed BACs to identify extension BACs and determine the size of overlapping regions. Minimum 5 Kb overlap with seed BACs anchored to a genetic markers was taken as the standard. For further validation of the identified extension BACs, additional 3 pairs of primers were designed from the regions of overlap at different sequence intervals and used to amplify the putative extension BAC. The basis of selection was positive validation with all the three primers and larger BAC size as estimated by PFGE following Not1 digestion. For BAC purity testing, DNA was isolated from 8 separate colonies of each identified BAC and fingerprint analysis was done after digestion with Hind-III restriction endonuclease. BACs showing uniform fingerprints for the eight colonies were taken as pure BACs. A total of 21 extension BACs with insert size ranging from 40 kbp to 170 kbp and end sequence overlap with seed BACs ranging from 8 kbp to 58 kbp were selected, tested for purity and used for sub-cloning and sequencing. Presently there are total 31 BACs (10 seed BACs and 21 Extension BACs) in the minimum tiling path of the long arm of chromosome 5 at different stages of sequencing and finishing.


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HIGH QUALITY SHORTGUN LIBRARIES FOR THE SEQUENCING OF LONG ARM OF CHROMOSOME 5 OF TOMATO( LYCOPERSICUM ESCULENTUM,VAR. HEINZ 1706)

Archana Singh, Manju Singh, Shubhra Barwa, Awadhesh Pandit, Jitendra K. Pal, Ajay K. Mahato, Vivek Dogra, Sheetal Arora, Aditi Mathur , K. Giakwad, T.R.Sharma, T. Mohapatra, N.K. Singh

National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute, Pusa Campus, New Delhi

The International consortium of tomato genome sequencing project assigned the sequencing of chromosome 5 to India. National Research Centre on Plant Biotechnology (NRCPB), IARI, New Delhi, has the responsibility to sequence the long arm of tomato chromosome 5 (5Mb) of the region spanning between 73-119cM, through our internal arrangements. Information for genetic maps, FPC, seed BAC libraries and BAC end sequences available on SOL web site (http://www.sgn.cornell.edu) enabled us to identify 10 Seed BACs anchored to corresponding genetic markers and 21 extension BACs for our region. Out of these 31 BACs, total 27 shotgun libraries have been constructed and high quality sequences have been generated. Shotgun cloning is a stepwise procedure including, fragmentation of large BAC DNA into 2-5 kb sized smaller fragments using Argon gas at 30 psi pressure for 7-8 seconds for 10 µg of DNA by nebulization, extraction of these smaller sized fragments from agarose gel against Hind III size marker (Elution), gap filling/ polishing /end repairs of these fragments and ligation with cloning vector (Sma I digested PUC19), followed by electro - transformation into E.coli. The whole process leads to picking of desired clones by blue and white colony selection. Plasmids isolated from white colonies were sequenced and assembled to provide full length sequences of the BAC clones. Completion of high quality tomato Genome sequence is going to provide a valuable tool for forward genetic approach to map, tag and finally clone these genes and revalidate their functions by making transgenics. Also, give information for comparative genomics of related solanaceae family members.


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SEQUENCE GAP CLOSURE STRATEGIES FOR BAC CLONES FROM THE LONG ARM OF TOMATO CHROMOSOME 5

Jitendra K. Pal, Awadhesh Pandit, Archana Singh, Manju Singh, Ajay K. Mahato, Shubhra Barwa, Vivek Dogra, Sheetal Arora, Aditi Mathur, K. Gaikwad, T.R. Sharma, T. Mohapatra and N.K. Singh.

National Research Centre on Plant Biotechnology, IARI, New Delhi 110012, India

The goal of the international tomato genome sequencing project at NRCPB, New Delhi is to produce a high quality finished sequence of 5 Mb gene rich euchromatin region of long arm of chromosome 5. At our centre we apply four main strategies for sequence gap closure (i) custom primer walking (ii) sequencing of transposon insert library of linker clones (iii) alternate chemistry (Templiphi Sequence Resolver Kit, GE Healthcare, UK) and (iv) PCR amplification of gap region for sequencing. First strategy utilizes the automated primer picking program in the Consed Sequence editor for on the existing linker clones that span a gap. The main advantage of primer walking is that it allows closure of small gaps with a minimum number of sequencing reactions. For transposon insertions we currently use an in vitro GPS-1 Genome Priming System (Bio Labs Inc, USA) for generating a population of clones with randomly interspersed primer-binding sites. Unique priming sites on the both ends of the transprimer element, allow DNA sequence to be obtained from both strands of the target DNA at the position of the insertion. In the third strategy we use TempliPhi Sequence Resolver Kit to produce exceptional sequencing results from difficult templates such as those with high GC content and secondary structures and resolve most of the sequencing stops or gaps and problem of sequencing difficult templates. The data generated by this kit are very high in quality and found very effective in filling of gaps between contigs. Fourth strategy is, using PCR primer for gap closure in this we design four sets of PCR primers of that unfinished region and carry out PCR cycling with normal PCR reagents with Taq Polymerase, after if amplification appears we do sequencing of that amplified product with ET terminator chemistry. This method is very useful where linker clone is not available between gaps. We have used various combinations of these four strategies to increase our output finished sequences.


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TOMATO MITOGEN ACTIVATED PROTEIN KINASES REGULATE THE EXPRESSION OF EXTRACELLULAR INVERTASE LIN6 IN RESPONSE TO STRESS RELATED STIMULI

A A B,C ATae Kyung Hyun , Anja Hoffmann , Alok K. Sinha ,Thomas Roitsch

AJulius-von-Sachs-Institut fuer Biowissenschaften, Universitaet Wuerzburg, Julius-von-Sachs-Platz 2, 97082 BWuerzburg, Germany, National Institute of Plant Genome Research, PB No. 10531, Aruna Asaf Ali Road,

New Delhi – 110067, India

Activation of mitogen-activated protein kinases (MAPKs) is a common reaction of plant cells in defense-related signal transduction pathways. Since the downstream events after the activation of MAPKs are largely unknown in plants, the role of MAPKs in the co-ordinate regulation of defence reactions and primary carbon metabolism by stress related stimuli have been analyzed in tomato. Thus, the relationship between mitogen activated protein kinase, LpMPK2 and LpMPK3 and extracellular invertase Lin6, as the key enzyme of an apoplasmic phloem unloading pathway, has been analyzed. It was observed that the mRNAs of LpMPK3 and Lin6 are sequentially induced by the same set of stress related stimuli while LpMPK2 transcripts are constitutively expressed. In a gain of function approach, a His-tagged version of LpMPK2 and a HA-tagged version of LpMPK3 were transiently and functionally expressed in leaves of transgenic tobacco plants expressing the ß-glucuronidase reporter gene under control of the Lin6 promoter via agro-infection. Immunokinase MAPK assays showed that a set of four different stimuli, wounding, a fungal elicitor derived from Fusarium oxysporum lycopersici, the endogenous plant derived elicitor PGA, and salt stress results in the fast and transient activation of both LpMPK2 and LpMPK3 within 3 to 12 minutes. The induction of the Lin6 promoter, as revealed by an increase in ß-glucuronidase activity after 24 hours, was dependent both on the expression and activation of both LpMPK2 and LpMPK3. These data suggest that the induction of extracellular invertase Lin6 by stress related stimuli requires LpMPK2 and LpMPK3 and thus demonstrate that MAPK signaling might be involved the regulation of primary carbon metabolism in general and sink metabolism in particular.


SOL 2009182

DEEP TRANSCRIPTOME SEQUENCING AND SNP DETECTION IN S. LYCOPERSICUM AND S. PENNELLII.

José M Jiménez-Gómez1, Seisuke Kimura1, Neelima Sinha1 and Julin N Maloof1

1. Section of Plant Biology, University of California Davis, Davis, CA 95616, USA.

Wild tomato species are native to diverse habitats in South America and show great morphological and ecological diversity that has proven useful in breeding programs. However, relatively little is known about nucleotide diversity between tomato species. High throughput sequencing (HTS) technologies allow for rapid acquisition of significant amounts of sequence data. Application of HTS to wild species could help revolutionize the identification and characterization of natural alleles of ecological and breeding interest.

With this objective in mind, we are deeply sequencing the transcriptomes of the S. lycopersicum and S. pennellii strains used as parents of a publicly available, well characterized NIL population. Samples were obtained by pooling RNA from different tissues and developmental stages, which were converted to cDNA and normalized to aid detection of rare transcripts.

We are in the process of producing enough sequence data to assemble the transcriptomes by combining de novo and reference based assembly. In addition to other resources, we are developing a dense and accurate set of polymorphisms for genotyping the above-mentioned NIL population. As a proof-of-principle we mined ~5K SNPs and indels from ~620 kb of ESTs found in the SGN databases. The SNP rates per gene region and estimations of the likelihood of the genotypes at each SNP defined in our preliminary study will be used in a Bayesian model to estimate SNP probabilities in the upcoming HTS data.

A RECESSIVE RESISTANT GENE AGAINST CHILLI VEINAL MOTTLE POTYVIRUS (CHIVMV) IN CHILLI (CAPSICUM ANNUUM L.) CORRESPONDS TO THE EUKARYOTIC INITIATION FACTOR 4E (ELF4E)

K. Madhavi Reddy, M. Krishna Reddy and K.V. Ravishankar

Indian Institute of Horticultural Research, Hessaraghatta Lake Post, Bangalore – 560 089

1The pvr1 locus identified in chilli, conferring recessive resistance against strains of Chilli veinal mottle virus (ChiVMV), corresponds to a eukaryotic initiation factor 4E (elF4E) gene. RT-PCR amplification of ChiVMV susceptible and resistant chilli cultivars, using an elF4E ORF primer, resulted DNA fragment of 0.8kb from all the cultivars used. Further, to characterize the ORF of elF4E contributing to chilli veinal mottle virus resistance in chilli genotypes, DNA fragment was cloned into pTZ56 vector. At least three independent positive clones were sequenced from both ends to PCR product of chilli resistant line, IHR3291. Nucleotide and amino acid sequence alignment were produced using the clustal algorithm. The sequence has resulted an ORF of elF4E with 687 nucleotides and 228 amino acids. Nucleotide comparison of IHR3291 elF4E sequence with other potyvirus resistance elF4E has indicated 51.8 to 68% with pea elF4E, 86.6 to 87.6% identity with tomato elF4E and 99.1 to 100% with pepper elF4E. Comparative sequence analysis of nucleotide and protein sequence of elF4E from C. annuum shown that IHR3291 sequence has 98.8 to 100% nucleotide and 97.3 to 100% protein identity, whereas with tomato elF4E it has 86.6 to 87.6% nucleotide and 83.7 to 86.1% protein identity. The comparative sequence analysis of IHR3291

+ 1 1 2nucleotide sequence with the elF4E sequences of Pvr1 , pvr , pvr1 and pvr1 has revealed single nucleotide polymorphism. The A151G and C196A substitutions in pvr1 were not present in IHR3291. The T200A, A218C and

1 2A319G substitutions present in pvr1 and pvr1 were present in IHR3291 sequence. The substitutions C203A that is

+ 2 2present in Pvr1 and pvr1 , substitution A325G present in pvr1 were absent in IHR3291. From this analysis it

1indicates that pvr1 is present in IHR3291 which holding resistance to chilli veinal mottle virus. However, the substitution T236G as present in IHR3291 sequence did not result in susceptibility to ChiVMV, indicating this nucleotide change does not contribute to virus susceptibility as revealed with ChiVMV.


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SOL 2009184

EXPLORING THE TRANSCRIPTOME OF LEAF AND ROOT OF WITHANIA SOMNIFERA (ASHWGANDHA) USING EXPRESSED SEQUENCE TAGS.

1,2 1 2 2Neha G. Wasnik , Kalaiselvi Senthil , Yu-Jin Kim , Deok-Chun Yang

1Department of Biochemistry, Biotechnology and Bioinformatics, Avinashilingam University for Women,

2Coimbatore, 641043, India. ; Korean Ginseng Center for Most Valuable Products and Ginseng Genetic Resource Bank, Kyung Hee University, Yongin 449-701, Korea.

Ashwgandha (Withania somnifera, Dunal) is a plant of repute in the Indian Systems of Traditional Medicine and a source of various withanolides. A cDNA library constructed from samples of the 2-months old, in vitro cultured leaves and roots, which generated 1047 leaf cDNA and 1034 root cDNA clones representing 48.5% and 61.5% unique sequences. The ESTs from leaf and root grouped into 239 and 230 clusters representing 22.8% and 22.2% of total sequences. Of these, about 70% encoded proteins found similar (E-value?10-14) to characterized or annotated proteins from the NCBI non-redundant database and diverse molecular functions and biological processes based on gene ontology (GO) classification. We identified genes with potential role in photosynthesis (cytochrome p-450), pathogenesis (arginine decarboxylase, chitinase) and withanolide biosynthesis (squalene epoxidase, CDP-ME kinase). Highly expressed transcripts, with a particularly high abundance of cytochrome p450 (0.85% in leaf) were noticed. W. somnifera is a source of multifarious and beneficial alkaloids referred as withanolides. High levels of withanolides accumulate in mature leaves and roots. Since, the knowledge for synthesis and presence of some of these important biochemical constituent is limited, identification of the genes involved in two different pathways of secondary metabolite synthesis (MVA and MEP), in different tissue will be requisite for articulation of withanolide biosynthesis. This investigation aimed at elucidating the differential gene expression in two vital sites where withanolides essentially found and leaf and root trascriptomes were comparatively analyzed.

ISOLATION AND EXPRESSION ANALYSIS OF A PUTATIVE SLSTP (SOLANUM LYCOPERSICUM STOLON TIP PROTEIN) GENE FROM 40-DAY OLD ROOTS OF TOMATO (SOLANUM LYCOPERSICUM L.)

Sethuraman Sivapriya, Nagarajan Kalidhasan, Kanneganti Vydehi and Aditya K. Gupta

Dept of Plant Biotechnology, School of Biotechnology, Madurai Kamaraj University, Madurai – 625021.

Plant root development requires a coordinated expression of many tissue and/or stage specific genes apart from the expression of housekeeping genes. Studying genome – wide changes at specific developmental time points would reveal the genetic network that control development of roots including processes like root hair development, lateral root initiation, stolonisation that determine root architecture. Differential expression studies have revealed the expression of specific genes in stolon tips of potato and similar sequences are found in its non-tuberizing relatives including tomato. In this study, we report the isolation and expression analysis of the gene encoding a Stolon tip protein (SlSTP) from 40 day old roots of tomato.Screening of Suppression Subtractive Hybridization (SSH) libraries (generated using two distinct stages of root development - 8 and 40 day old tomato roots) resulted in ESTs (Expressed Sequence Tags) specific for the developmental stages. After validating them by RT-PCR, a candidate EST (EX149669) coding for a stolon tip protein (SlSTP) was chosen for full length completion and characterization studies. Expression profile studies have revealed that it is specifically expressed during later stages in root development. This was also found to be expressing at varying levels in the reproductive tissues (buds, open flower, post pollinated flower, and premature fruit). The sequence was completed using 5' and 3' RACE and a full length sequence of 1.3 Kb was obtained. The functional characterization of this gene for its role in root development is in progress using over expression and silencing studies. This would be a valuable addition to our understanding of the genetic regulation of root development.


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SOL 2009186

DEVELOPMENT OF SCAR MARKERS FOR FRUIT AND SHOOT BORER RESISTANCE IN BRINJAL Makesh Sekar, Asish Ghosh , Pugalendhi Lakshmanan , Veeraragavathatham Desikathatahachari

Department of Vegetable crops, Horticultural College and Research Institute Tamil Nadu Agricultural University, Coimbatore – 641 Email : [email protected]

Brinjal, Solanum melongena L., is one of the important vegetable crops grown in India with an estimated area of 0.51 million ha with an annual production of 8.45 million tonnes (FAO, 2007). Brinjal cultivation is severely limited by the fruit and shoot borer (FSB) (Leucinodes orbonalis Guen.) damage with yield losses ranging from 50-70%. The affected fruits loose their market value. The control of the insect by chemical pesticides is highly ineffective as the larvae of the FSB would have caused the damage to the shoot and fruit by the time the symptoms were visually observed .The only way to control the insect is to kill the larvae before it enters the shoot and fruit. Extensive and repeated use of the insecticides to control this pest results in large amount of pesticide residues and increases the cost of cultivation to a great extent. The best way to overcome the FSB problem is to impart host resistance. However, the cultivated brinjal is not having any such resistance germplasm. Hence, an attempt was made to explore the resistance in other species of Solanum. In this screening the bitter fruited Solanum viarum was identified as immune (complete resistance) to FSB. We tried to impart this resistance to cultivated brinjal by Interspecific crossing between EP65 (popular brinjal cultivar otherwise known as Palur 1) and Solanum viarum followed by backcross breeding to transfer the resistance gene pool. Some of the BC3F6 homozygous progenies showed high level of resistance along with low bitterness in fruits. In order to track the resistance gene source, molecular profiling through RAPD was performed with Solanum viarum, backcross progenies and on resistance and susceptible bulks, which resulted in identification of a marker that is tightly linked with FSB resistance. This RAPD marker was converted into Sequence Characterized Amplified Region (SCAR) marker OPZ-7407 and validated.

The BLAST Analysis of the SCAR Fragment (OPZ-7407) showed a match with an intron and attempts are under way to identify the flanking exon regions by using Thermal Asymmetric Interlaced PCR (TAIL- PCR).The application of the SCAR marker in developing FSB resistance is discussed.

USE OF MOLECULAR MARKERS IN THE BREEDING OF TOMATOES RESISTANT TO DISEASES CAUSED BY FUSARIUM OXYSPORUM F. SP. LYCOPERSICI AND PSEUDOMONAS SYRINGAE PV. TOMATO.

1 2 1 1M. Staniaszek , W. Marczewski , E. U. Kozik , H. Habdas

1 Research Institute of Vegetable Crops, Konstytucji 3 maja 1/3, 96-100 Skierniewice, Poland; 2 Plant Breeding and Acclimatization Institute, Platanowa 19, 05-831 Młochów, Poland

Classical selection methods involving the inoculation of plants with the relevant pathogens in breeding directed to obtain disease resistant varieties are generally used. Such techniques are time-consuming, costly and, of necessity, repetitive. An alternative to biological tests is to be found in molecular methods, especially those based on PCR amplification. In studies undertaken at the Research Institute of Vegetables Crops in Skierniewice, two markers were identified. The first marker, CAPS TAO1902, is linked to the I-2 gene, which confers resistance to Fusarium oxysporum f. sp. lycopersici race 2 in tomato. The second amplicon, RP487GAAA1500, is linked to the Pto gene, conferring resistance to Pseudomonas syringae p.v. tomato. Usefulness of the both markers for MAS of Polish tomato varieties, breeding lines and F1 hybrids are presented and discussed.


SOL 2009 187

POSTERS

188

SESSION I STRUCTURAL GENOMICS

189


SOL 2009190

POPULATION STRUCTURE AND LINKAGE DISEQUILIBRIUM PATTERNS IN POTATO

Björn B. D'hoop, Paul L.C., Keizer , Caroline, Marques-Castro

Wageningen University, Wagningen, The Netherlands.

We investigated population structure in potato germplasm using a collection of 430 tetraploid potato cultivars, progenitor and breeder's clones, representing worldwide commercial potato germplasm. Genetic variation was analysed with 3364 AFLP™ markers from 41 primer combinations and 653 alleles from 53 microsatellite loci. Phenotypic data have been collected by breeders during consecutive years of clonal selection and commercial testing on a variable number of locations and during a variable number of years. Population structure was inferred with three different approaches: Bayesian, distance-based hierarchical clustering and principal coordinates analysis. All three methods identified weak, but significant population structure, which repeatedly coincided with different market segments (Fresh consumption, Processing industry, Starch) as well as different breeding periods (Ancient = pre-1950 and modern breeding). Decay of genome-wide LD below r2=0.1 was observed beyond on average 5cM throughout the twelve chromosomes. LD patterns were analysed in greater detail, removing noise caused by parent-specific variations in genetic recombination, and population structure (within-group LD patterns). The molecular variation could be related to particular phenotypic traits. The most discriminative markers with respect to population structure were identified. Additionally, we will elaborate on association mapping results with respect to those population-informative markers.

POTATO CBP20 GENE POSSESSES CONSERVED U12 INTRON AND ITS 5TH INTRON CONTAINS SIGNATURES OF LINE1 RETROTRANSPOSON

Pieczynski M., Karlowski W., Bielewicz D., Dolata J., Jarmolowski A., Szweykowska-Kulinska Z

Department of Gene Expression, Institute of Molecular Biology and BiotechnologyAdam Mickiewicz University, Poznañ, Poland

Cap binding complex (CBC) consist of two proteins: CBP20 and CBP80. CBC binds to the cap structure at the 5' end of all mRNAs and is known to stimulate, at least in animals, splicing of the first intron. In the A. thaliana genome CBP20 gene contains seven introns. Intron nr 4 is of U12 type. We analyzed, both experimentally (potato, tobacco) and using bioinformatics tools, CBP20 genes from different plant species. We found the presence of seven introns within the CBP20 gene and U12 intron in all plant species studied – from mosses to dicots. Moreover the U12 intron position is highly conserved – it is always present as intron nr 4. The length of CBP20 U12 intron varies considerably in different plants. The comparison of the CBP20 gene structure revealed that the exons upstream of the U12 intron are highly conserved in length in all analyzed plants. It may suggest the role of the U12 intron in regulation of CBP20 expression. In the case of potato intron nr5 is extremely long (~6050bp) and we found there signatures of LINE1 retrotransposon. Interestingly intron nr5 in tobacco CBP20 gene does not contain any remnants of LINE1 retrotransposon suggesting that retrotransposon invasion occurred after the evolutionary divergence leading to tobacco and potato species.


SOL 2009 191


SOL 2009192

CURRENT SEQUENCING PROGRESS OF SOLANUM LYCOPERSICUM CHROMOSOME 4

1 1 2 2 2 2Rosa Lopez-Cobollo , Giulia Bonciani , Daniel Buchan , James Abbott , Rosalind Cutts , Sarah Butcher , 3 4 4 4 4Jane Rogers , Helen Beasley , Carol Churcher , Sean Humphray , Clare Riddle and Mapping Core Group ,

4 4Karen McLaren and Finishing Team 46 , Stuart McLaren and Pre-finishing Team 58 , Christine Lloyd and

4 4 4 4 5 6 7QC Team 57 , Karen Oliver , Matt Jones , Carol Scott , Graham Seymour , Glenn Bryan , Lukas Mueller ,

7 8 9 10 10 11 11Jim Giovannoni , Klaus Mayer , Stephen Stack , Hans de Jong , Dora Szinay , Shusei Sato , Satoshi Tabata

1and Gerard J. Bishop

1 2 Division of Biology, Imperial College London, Exhibition Road, London SW7 2AZ, UK; . Bioinformatics Support 7 3Service, Imperial College London, Exhibition Road, London SW 2AZ, UK; The Genome Analysis Centre (TGAC).

4Norwich Research Park, Colney, Norwich, NR4 7UH, UK; Wellcome Trust Sanger Institute, Wellcome Trust 5

Genome Campus, Hinxton, Cambridge, CB10 1SA, UK; University of Nottingham, School of Biosciences, Division 6

of Plant Sciences, Sutton Bonington Campus, Loughborough, Leics LE12 5RD UK; The Scottish Crop Research 7

Institute, Invergowrie, Dundee, DD2 5DA, Scotland, UK; Department of Plant Breeding and Genetics. Cornell 8

University, 251 Emerson. Hall, Ithaca NY 14853, USA; Institute for Bioinformatics (IBI) at National Research 9Centre for Environment and Health, Neuherberg, Germany; Department of Biology, Colorado State University,

10Fort Collins, Colorado 80523, USA; University and Research Centre – Plant Sciences Group Laboratory of 11Genetics, 6703 BD Wageningen, Netherlands; Department of Plant Gene Research, Kazusa DNA Research

2-6-7Institute, Kazusa-kamatari, Kisarazu, Chiba 292-0818 JAPAN

The estimated 19Mb of gene space on Tomato (Solanum lycopersicum) chromosome 4 is being sequenced in the UK as part of the International Tomato Genome Sequencing Project(http://www.sgn.cornell.edu/help/about/tomato_sequencing.html). A tile path of minimally overlapping BAC clones was selected using both fingerprint and BAC end sequence (BES) information. The sequenced BAC clones have been placed in an AGP file that gives the user the accession numbers and assembly information needed to construct the pseudo-molecule at the end of the sequencing project. All data has been released into the public domain throughout the process (http://www.ncbi.nlm.nih.gov/HTGS/index.html). The sequencing activities were transferred to Imperial College. Verification of Chr4 location of 64 sequenced BACs has been carried out by IL mapping. 9 BACs were confirmed to be on Chr4, 55 were placed on Chr0 17 of which are definitely not on Chr4. To identify further Chr4 BACs 3D DNA superpools have been screened using the Chr4 markers that have not been identified in the current sequenced contigs. Extension of BACs sequence contigs has also been performed using the BES and Fosmid end sequences (FES). A total of 18 BACs and 3 Fosmids have been identified by these methods and a further two BACs were moved from Chr0 to Chr4 by IL mapping. We are also contributing to the next generation sequencing initiative and in the process of generating a ~7-8kb paired end read using SOLID3 technology. Current progress of these results will be presented.

INDIAN CONTRIBUTION TOWARDS SEQUENCING THE TOMATO GENOME

1 1 1 1 1 1 1 1S. Mathur , S. Vyas , A.U. Solanke , R. Kumar , V. Gupta , A.K. Sharma , P. Khurana , J.P. Khurana , 1 2 2 2 2 2 2 2 2A.K. Tyagi , M. Singh , A. Singh , A. Pandit , J.K. Pal , A.K. Mahto , V. Dogra , K. Gaikwad , T.R. Sharma ,

2 2 3 3 T. Mohapatra , N.K. Singh , Sarita, P. Chowdhury , D. Chattopadhyay

1IITGS, Inter-disciplinary Centre for Plant Genomics, Department of Plant Molecular Biology, University of Delhi

2South Campus, Benito Juarez Road, New Delhi-110021, INDIA; IITGS, National Research Centre on Plant

3Biotechnology, Indian Agricultural Research Institute, New Delhi-110012, INDIA; IITGS, National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi-110067, INDIA

A consortium of ten countries has joined hands to sequence the genome of the model Solanaceae plant, Solanum lycopersicum cv. Heinz 1706. The Indian contribution towards this effort is aimed to generate ~12 Mb sequence from the gene-dense euchromatic region of chromosome 5 using BAC shot-gun sequencing. Presently, 95 BAC clones from chromosome 5, mapped using Introgression Lines (provided by Dr. Dani Zamir), are in the sequencing pipeline and 62 GenBank accessions have been obtained. In all, more than ten sequence contigs, covering nearly 10 Mb have been generated for chromosome 5 (www.genomeindia.org and www.nrcpb.org). Efforts are on to find new extension BACs to fill the gaps. In addition, 67 BACs have been mapped on the 12 tomato chromosomes by CAPS markers generated using BACs that contained ORFs /unique sequences at their ends. Annotation of finished BACs has revealed several genes of interest and a gene density of one gene per 5 kb sequence.

To boost the sequencing of the tomato genome, a next generation whole genome sequencing platforms has also been initiated internationally. India has contributed 5X 454 sequencing coverage in collaboration with The Netherlands and the results are available to the sequencing community.

As a participating member of the International Tomato Annotation Group, we are performing functional annotation on the proteins predicted on sequenced BACs by performing BLAST analysis against 5 protein databases.


SOL 2009 193


SOL 2009194

THE ITALIAN CONTRIBUTION TO THE SEQUENCING OF THE TOMATO GENOME

1 1 1 1 1 2 2Pietrella M. , Falcone G. , Fantini E. , Gonzalez, M. , Rosato, V. , Ercolano M.R , Barone A. , Chiusano 2 2 3 3 3 3 4 4M.L. , Frusciante L. , Facella P. , Lopez L. , D'addiego L. , Perrotta G. , Vezzi A. , Valle G. and

1Giuliano G.

1 2ENEA, Casaccia Research Center, Via Anguillarese 301, 00123 Roma, Italy; Department of Soil, Plant,

Environmental and Animal Production Sciences, University of Naples "Federico II", Via Università 100, 80055 3 4

Portici, Italy; ENEA, Trisaia Research Center, S.S. Ionica - Km 419.5, 75026 Rotondella (MT), Italy; CRIBI Biotechnology Centre and Department of Biology, Univ. of Padova, via U. Bassi 58/B, 35131 Padova, Italy

The international tomato sequencing project has started in 2004, based on a BAC-by-BAC strategy (http://sgn.cornell.edu/about/tomato_sequencing.pl). Italy is sequencing chromosome 12 and providing mapping and bioinformatic tools to the international effort. Seed BACs are selected for the presence of genetic markers and validated via genetic (Introgression Line) and cytogenetic (FISH) mapping, in collaboration with the De Jong lab (Wageningen). To date 43% of the projected euchromatic portion of chromosome 12 has been completed. Additionally, 54 BACs have been mapped “de novo” on the various chromosomes to provide new starting points for sequencing and 12 already sequenced BACs have been reassigned from “Chromosome 0” to the various chromosomes. A bioinformatic platform has been built to provide a preliminary annotation of the genome. An effort to obtain a draft WGS sequence with Next Generation sequencing has been launched in late 2008 in collaboration with other international partners. This strategy is based on 454 (Titanium) and SOLiD shotgun and Paired End runs, as well as 454 and SOLiD RNA seq, and is aimed at obtaining a high quality, annotated assembly by the end of 2009.

KOREAN EFFORTS ON TOMATO CHROMOSOME 2 GENOME SEQUENCING

Sung-Hwan Jo, Bo-Ra Kang, Doil Choil

Korea Research Institute of Bioscience & Biotechnology (KRIBB)

The genome of tomato (Solanum lycopersicum L.) is being sequenced by an international consortium of 10 countries as part of the project “International Solanaceae Genome Project (SOL): Systems Approach to Diversity and Adaptation”. The goal of the consortium is to sequence the approximately 220 Mb of euchromatin that may contain the majority of gene coding sequences and to use it as a reference genome for the other solanaceous species. The Korean SOL is aiming to complete sequencing of about 22 Mb euchromatic region of chromosome 2 using BAC by BAC approach. From the initial sequencing stage, 60 BAC clones, which are anchored on genetic markers, were selected as “seed BACs”. Further BAC extension from the “seed BACs” has been performed by utilization of BAC-end sequence and fosmid-end sequence database validating their location with FISH and IL mapping. Currently, 23 Mb of unique sequence was determined from 194 BACs using conventional Sanger method and pool of 85 BACs and 95 fosmids using 454 FLX titanium platform. Among them, 184 BACs (14.7Mb) were mapped on chromosome 2 and assembled in 29 super-contigs in order. An annotation effort is also underway by collaboration with International Tomato Annotation Group. To complete the chromosome 2 sequencing project, we are applying next generation sequencing platforms and comparative genomic analysis


SOL 2009 195

SESSION II BIOTIC STRESSES

196

INFECTIVITY OF A NEW BEGOMOVIRUS: TOMATO LEAF CURL AURANGABAD VIRUS (TOLCAUV)

VB Singh , Neha T, Haq QMI, S Richa , Jyothsana P, Archna K, VG Malathi

Plant Virology Unit, Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi-110012

A new begomovirus was cloned from tomato and Parthenium showing severe leaf curl symptoms through rolling circle amplification. DNA-A components showed 86% similarity with Tomato leaf curl Karnataka virus (Accession number: AY754812) and DNA β showed 100% similarity with Papaya leaf curl beta satellite (Accession number: DQ118862). As per the taxonomic criteria of 89% sequence identity in DNA-A, the present virus is designated as Tomato leaf curl Aurangabad virus (ToLCAuV). To investigate the infectivity of this virus partial tandem repeat (PTR) constructs of Tomato leaf curl Aurangabad Virus-A (ToLCAUV-A) and satellite DNA β were made in pBin 19 and agroinoculation was done on Nicotiana benthamiana and tomato seedlings. ToLCAuV could infect and cause symptoms on N. benthamiana, but could not cause any symptom on tomato. The symptoms were severe and took 21 days to develop. DNA-A alone is more infectious and percentage of infection was high (29%). The percentage infectivity (16%) was less in those plants, which was inoculated with both DNA-A and DNA- β. In PCR in both the DNA-A alone and DNA-A and DNA- β inoculated plants, only DNA-A was amplified. In Southern hybridization, high levels of viral replicative form were seen in both DNA-A alone and A+β inoculated plants.


SOL 2009 197


SOL 2009198

INFECTIVITY OF TOMATO LEAF CURL BANGALORE VIRUS

Neha T, Veer B.S., Haq.Q.M.I., Jyothsana P., Richs S., Archana K., V.G.Malathi


Begomoviruses are a group of plant viruses that have circular single-stranded DNA genome of 2.7 kb of length, which is encapsidated within twinned para-isomeric particles and are transmitted by whitefly (Bemisia tabaci).The genomic component of a virus infecting tomato plants from Bangalore were cloned in the vector pUC18 through RCA and sequenced. The nucleotide sequence of DNA A component of the present isolate showed 95% identity with [ToLCBV ( DQ8526232)] and hence it is designated as an isolate of Tomato leaf curl Bangalore virus (ToLCBV).The satellite DNA β showed 92% identity with Tomato leaf curl Bangalore beta satellite[ToLCBV-β (AY428768)]. Partial tandem repeat (PTR) constructs of DNA-A and DNA-β were made and mobilized into Agrobacterium tumefaciens EHA-105 for inoculation. Tomato (cultivar - Pusa Ruby) and Nicotiana benthamiana seedlings were inoculated and were maintained at 28°C, 80% humidity and 8-9000 lux. Hundred percent infectivity was found in the case of DNA-A alone and DNA-A+ β inoculated plants. Severe symptoms in the form of leaf curling, yellowing were observed in tomato seedlings 15-18 days post inoculation. The presence of replicative forms of viral DNA was confirmed by Southern blotting. Differential transmission of A and A + β components by whitefly was examined.

CLONING AND INFECTIVITY OF TOMATO BEGOMOVIRUSES THROUGH ROLLING CIRCLE AMPLICATION (RCA) OF THE GENOME

Haq.Q.M.I., Neha T., Jyothsna P., Veer B.S., Richa S., Archana K., V.G.Malathi

Plant Virology Unit, Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi-110012, INDIA

During our survey in 2008-09, typical symptoms of leaf curl, yellow mottling, vein clearing and enation were seen on cultivated tomato crops in and around Pune (Maharashtra), Bangalore (Karnataka) and in Kalyani (West Bengal). In order to identify the virus, cloning of genomic components were attempted which is reported here. The total DNA was extracted by Gem-CTAB method (Rouhibakhsh et al., 2008) and Rolling Circle Amplification (RCA) was performed (Haible et al., 2006). These rolling circle amplicons were restricted with BamHI, XbaI and HindIII and full length 2.7kb, 1.3kb linearised products were cloned in pUC18. Totally 32 different tomato samples were tested for RCA, out of which 25 samples were found positive for 2.7kb (DNA A & DNA B) and 1.3kb (DNA β) products, which were cloned. Based on 89% sequence identity in DNA component, the clones were identified as, two DNA A, one DNA B clones of ToLCNDV; two DNA A clones of ToLCBV; one DNA A, one DNA β clone of ToLCGV; one DNA A and one DNA β clone of ToLCJV. Nucleotide sequence analysis of one of the tomato samples showed less than 89% identity with other tomato begomoviruses, and therefore designated as a new virus and named as Tomato leaf curl Aurangabad virus (ToLCAuV). Severe symptoms were expressed on tomato seedlings after biolistic delivery of RCA of respective clones 15 - 40 DPI.


SOL 2009 199


SOL 2009200

EXPRESSION OF S-ADENOSYL METHIONINE DECARBOXYLASE GENE UNDER STRESS INDUCIBLE PROMOTER IN TRANSGENIC TOMATO CONFERS ENHANCED RESISTANCE TO FUNGAL PATHOGENS AND MULTIPLE ABIOTIC STRESSES

M. V. Rajam and Pranjal Hazarika

Department of Genetics, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India

The role of polyamines (putrescine, spermidine and spermine) in stress tolerance has been implicated in recent findings. However, the inducible over-expression of stress-related genes can be more desirable than their constitutive expression to avoid unnecessary burden on the plant machinery. Therefore, the present study was undertaken to express human S-adenosyl methionine decarboxylase (SAMDC) gene (a key polyamine biosynthesis gene) under stress-inducible phenylalanine ammonia-lyase (PAL) promoter in transgenic tomato for engineering tolerance to both biotic and abiotic stresses. Several tomato tranformants were generated via Agrobacterium tumefaciens-mediated tranformation, and the validation of transgene integration and expression was done by PCR/Southern analysis and RT-PCR analysis, respectively. Polyamine levels were found to be not only enhanced on induction by the pathogens, Fusarium oxysporum and Alternaria solani but readjustments via inter-conversion among the three fractions (free, conjugated and bound) of polyamines were also evident. The generated transgenic plants were normal in phenotype and showed enhanced tolerance to multiple abiotic stresses such as salinity, drought, cold and high temperature as well as enhanced resistance against two important fungal pathogens of tomato, F. oxysporum and A. solani.

USE OF MOLECULAR MARKERS IN THE BREEDING OF TOMATOES RESISTANT TO DISEASES CAUSED BY FUSARIUM OXYSPORUM F. SP. LYCOPERSICI AND PSEUDOMONAS SYRINGAE PV. TOMATO

1 2 1 1M. Staniaszek , W. Marczewski , E. U. Kozik , H. Habdas

1 2 Research Institute of Vegetable Crops, Konstytucji; 3 maja 1/3, 96-100 Skierniewice, Poland; Plant Breeding and

Acclimatization Institute, Platanowa 19, 05-831, Młochów, Poland

Classical selection methods involving the inoculation of plants with the relevant pathogens in breeding directed to obtain disease resistant varieties are generally used. Such techniques are time-consuming, costly and, of necessity, repetitive. An alternative to biological tests is to be found in molecular methods, especially those based on PCR amplification. In studies undertaken at the Research Institute of Vegetables Crops in Skierniewice, two markers were identified. The first marker, CAPS TAO1902, is linked to the I-2 gene, which confers resistance to Fusarium oxysporum f. sp. lycopersici race 2 in tomato. The second amplicon, RP487GAAA1500, is linked to the Pto gene, conferring resistance to Pseudomonas syringae p.v. tomato. Usefulness of the both markers for MAS of Polish tomato varieties, breeding lines and F1 hybrids are presented and discussed.


SOL 2009 201


SOL 2009202

HIGHER ACCUMULATION OF PROTEINASE INHIBITORS IN FLOWERS THAN LEAVES AND FRUITS AS A POSSIBLE BASIS FOR DIFFERENTIAL FEEDING PREFERENCE OF HELICOVERPA ARMIGERA ON TOMATO (LYCOPERSICON ESCULENTUM MILL,) CV. DHANASHREE

Mrunal S. Damle, Ashok P. Giri, Mohini N. Sainani, Vidya S. Gupta

National Chemical Laboratory, Pune, India

Tomato (Lycopersicon esculentum, Mill; cultivar- Dhanashree) proteinase inhibitors (PIs) were tested for their trypsin inhibitory (TI) and Helicoverpa armigera gut proteinases inhibitory (HGPI) activity in different organs of the tomato plants. Analysis of TI and HGPI distribution in various parts of the plant showed that flowers accumulated about 300 and 1000 times higher levels of TI while 700 and 400 times higher levels of HGPI as compared to those in leaves and fruits, respectively. Field observation that H. armigera larvae infest leaves and fruits but not the flowers could be at least partially attributed to the protective role-played by the higher levels of PIs in the flower tissue. Tomato PIs inhibited about 50–80% HGP activity of H. armigera larvae feeding on various host plants including tomato, of larvae exposed to non-host plant PIs and of various larval instars. Tomato PIs were found to be highly stable to insect proteinases wherein incubation of inhibitor with HGP even for 3 h at optimum conditions did not affect inhibitory activity. Bioassay using H. armigera larvae fed on artificial diet containing tomato PIs revealed adverse effect on larval growth, pupae development, adult formation and fecundity.

IDENTIFICATION AND CHARACTERIZATION OF DIFFERENTIALLY EXPRESSED GENES INDUCED BY TOMATO LEAF CURL NEW DELHI VIRUS INFECTION IN TOLERANT CULTIVAR OF TOMATO ( L.)

1 1 2 3 3Pranav Pankaj Sahu , Neeraj K Rai , Supriyo Chakraborty , Major Singh , HC Prasanna and

1Manoj Prasad

1 2 National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi-67, INDIA; School of Life 3

Sciences, Jawaharlal Nehru University, New Delhi-67; Indian Institute of Vegetable Research, Gandhinagar (Naria), Varanasi-221 005

Tomato leaf curl virus infection causes significant yield loss in tomato. In our present study we used suppression subtractive hybridization (SSH) technique to identify the genes involved in Tomato leaf curl New Delhi virus (ToLCNDV) tolerance and validated the obtained results through Reverse Northern (RN) and quantitative real time PCR (qRT-PCR). The library was made from H-88-78-1 (a ToLCNDV tolerant cultivar) between ToLCNDV inoculated and Agrobacterium inoculated mock plants of this cultivar at 21 days post inoculation (dpi). A total of 106 non redundant transcripts were identified which were subsequently classified in 12 different categories according to there putative functions. The plant response against ToLCNDV infection was complex, representing major transcripts involved in defense signaling, hormonal response and proteolysis pathway at different time points of infection. By RN technique we identified the differential expression pattern of 106 transcripts out of which 34 transcripts were upregulated (>2.5 fold induction). Among upregulated transcripts, 8 showed more than four fold induction post ToLCNDV infection. qRT-PCR analysis was carried out to have a comparative expression profiling of these 8 transcripts between susceptible cv. Punjab Chhuhara and the tolerant cv. H-88-78-1, upon ToLCNDV infection. The expression patterns of these transcripts showed a significant increase of differential expression in tolerant cultivar mostly after 14 and 21 dpi in comparison to susceptible cultivar as analyzed by qRT-PCR. The direct and indirect relationship of identified transcripts with ToLCNDV tolerance mechanism is discussed.

SOLANUM HABROCHAITIS


SOL 2009 203


SOL 2009204

GENECHIP PROFILING OF TRANSCRIPTIONAL RESPONSES TO EARLY BLIGHT (ALTERNARIA SOLANI ) TOMATO (SOLANUM LYCOPERSICUM)

1 2 2 2 1Priti Upadhyay , Ashutosh Rai , Rajesh Kumar , Major Singh , Brajesh Sinha , 2 2

Prabhash Chandra Singh , Mathura Rai

1Department of Genetics and Plant Breeding, Institute of Agricultural Sciences, Banaras Hindu University,

2Varanasi-221005; Crop Improvement Division, Indian Institute of Vegetable Research, Jakhini (Shahanshahpur), Varanasi-221305

Early blight of tomato caused by Alternaria solani is one of the most destructive foliar diseases in many tomato production areas worldwide. The objective of this study was to identify differentially expressed genes in response to early blight disease through microarray analysis. Cultivated lines in tomato have showed very less resistance against early blight and that too are influenced by the environment. Fourteen tomato genotypes, representing two species of tomato (S. lycopersicum, S. habrochaites) were screened for resistance against early blight disease. Evaluations were conducted in growth chamber for disease severity and host resistance of the plants. EC-520061 (S. habrochaites) was found highly resistant against infection and CO-3 (S. lycopersicum) was found to be susceptible. To identify transcripts involved in defense response, gene expression profiling was done for above two tomato genotypes for control and inoculated conditions with a GeneChip microarray containing probe sets for approximately 10209 probes. The samples were collected from the infected plants after 24hr of infection at the stage of increased appressoria formation. Significant variation in responses between susceptible and resistant genotypes to Alternaria solani infection was found which include genes from various pathways like response (PR P2 and pr p23) to biotic stimulus and response to fungus at p-values less than 0.05. These genes may be involved in the resistant response by catalyzing the hydrolysis of chitin and chitodextrins. Real- Time PCR analysis is being progressed for Significant up and down regulated genes. These results will provide new clues to the molecular mechanisms that underlie plant resistance against fungal resistance and will be useful in the genetic improvement of early blight resistance in tomato.

VALIDATION OF TOLCV RESISTANCE CAPS MARKER FOR Ty2 IN THE PARENTAL LINES OF TOMATO

Ashish Kumar, Sanjeev Kumar, Major Singh, Mathura Rai and D. Datta

Indian Institute of Vegetable Research, Varanasi

Tomato leaf curl virus (ToLCV) disease is a serious threat for tomato cultivation in the tropics and subtropics. The disease can be managed by evolving cultivars with pyramided ToLCV resistance genes through the use of molecular markers. These markers must be validated before their use in gene pyramiding programme. The Ty2 CAPS marker developed by Peter Hanson and co-workers was validated in the parental lines. The experimental material comprised H24 (Ty2), DVRT-1, DVRT-2, H86, Fla478 and backcross lines of H24/H86. PCR amplification was performed in a 25 µl reaction mixture containing, 20 ng genomic DNA, 2.5mM MgCl2, 75 µM dNTPs, 10 x assay buffer, 0.1µM of each primer. PCR product amplified by Ty2 gene marker was digested with Taq I. Digested product was resolved through electrophoresis using 1X TAE buffer in 2.6% agarose gel.

Upon PCR amplification of the lines DVRT-1, DVRT-2, H24, H86, Fla478 and Punjab Chhuhara, monomorphic band was obtained. PCR amplification product was digested with TaqI which generated two kinds of fragments. The resistant amplicon (~200bp) was detected in H24, DVRT-1, DVRT-2 and surprisingly in Punjab Chuhara as well. H86 carried the susceptibility allele (~350bp). The backcross of DVRT-1/H24 and DVRT-2/H24 carried the resistance allele whereas the backcross of H86/H24 segregated in the ratio of 1:1 for H24 type allele. Though Punjab Chhuhara is highly susceptible to ToLCV but it also showed resistant type marker allele. This kind of marker is not diagnostic for Ty2 gene and can be used for detecting Ty2 gene in few known parents only.


SOL 2009 205


SOL 2009206

MICROARRAY ANALYSIS OF THE EARLY BLIGHT (ALTERNARIA SOLANI) RESPONSE IN TOMATO (SOLANUM LYCOPERSICUM)

1 2 2 2 1Priti Upadhyay , Ashutosh Rai , Major Singh , Rajesh Kumar , Brajesh Sinha , 2 2

Prabhash Chandra Singh , Mathura Rai

1Department of Genetics and Plant Breeding, Institute of Agricultural Sciences, Banaras Hindu University,

2Varanasi-22100; Crop Improvement Division, Indian Institute of Vegetable Research, Jakhini (Shahanshahpur), Varanasi-221305

Early blight of tomato caused by Alternaria solani is one of the most destructive foliar diseases in many tomato production areas worldwide. Development of resistant cultivars is the most economical and sustainable control measure of early blight. The objective of this study was to identify differentially expressed genes in response to early blight disease through microarray analysis. Resistance source in cultivated lines are less and that too are influenced by the environment. Fourteen tomato genotypes, representing two species of tomato (S. Lycopersicum, S. habrochaites) were screened for resistance against early blight disease. Evaluations were conducted in growth chamber for disease severity and host resistance of the plants. EC-520061 (S. habrochaites) was found highly resistant against infection and CO-3 (S. lycopersicum) was found to be susceptible. To identify transcripts involved in defense response, gene expression profiling was done for above two tomato genotypes (resistant and susceptible to early blight) for control and inoculated conditions, with a GeneChip microarray with probe sets for approximately 10209 probes. The samples were collected from the infected plants after 24hr of infection at the stage of increased appressoria formation. Significant variation in responses between susceptible and resistant genotypes to Alternaria solani infection was found which include genes from various pathways like response (PR P2 and pr p23) to biotic stimulus and response to fungus at p-values less than 0.05. These genes may be involved in the resistant response by catalyzing the hydrolysis of chitin and chitodextrins. Significant up and down regulated genes were selected for further validation with Real- Time PCR analysis. These results will provide new clues to the molecular mechanisms that underlie plant resistance against fungal resistance and will be useful in the genetic improvement of early blight resistance in tomato.

QTL META-ANALYSIS FOR LATE BLIGHT RESISTANCE IN POTATO INCLUDING THE TWO NOVEL RESISTANCE SOURCES SOLANUM SPARSIPILUM AND S. SPEGAZZINII

1 1 1 2Véronique Lefebvre , Sarah Danan , Patrick Signoret , Jean-Baptiste Veyrieras

1 2INRA, UR 1052 GAFL Génétique et Amélioration des Fruits et Légumes, BP94, 84140 Montfavet, France; INRA,

UMR UMR 320 Génétique Végétale, Ferme du Moulon, 91190 Gif-sur-Yvette, France.

The resurgence of late blight epidemics in potato (Solanum tuberosum) is due to the apparition of new Phytophthora infestans strains, resistant to chemicals and overcoming deployed R-genes. The alternative exploitation of polygenic resistance controlled by QTLs requires to inventory resistance sources and to get a better insight of the genetic architecture and diversity of quantitative resistance to late blight in potato. To find out novel resistance sources to late blight in the wild germplasm for potato breeding, we examined the polygenic resistance of Solanum sparsipilum and Solanum spegazzinii by a QTL analysis (Danan et al., 2009, TAG). The assessment of stem and foliage resistances made possible to identify 30 QTLs including a large-effect QTL region on chromosome X detected in both potato wild species. The mapping of literature-derived anchor markers suggested colinearities with published late blight QTLs or R-genes. We integrated the QTL results in a meta-analysis for late blight resistance in potato together with the data of 19 other published studies. It consisted in constructing a consensus map of potato, on which we projected late blight resistance meta-QTLs. Results highlighted some well-conserved QTLs in the potato related species. The relationships of late blight resistance meta-QTLs with R-genes and maturity QTLs were examined.

[This work was supported by grants from the European BIOEXPLOIT project FOOD CT2005-513959.]


SOL 2009 207

SESSION III ABIOTIC STRESSES

208

DEVELOPMENT OF TRANSGENIC TOMATO (VAR. KASHI VISHESH) LINES USING BCZAT12 GENE FOR ABIOTIC STRESS RESISTANCE

1 1 2 1 1Avinash Chandra Rai , Major Singh , Kavita Shah , Sanjeev Kumar and Mathura Rai

1Division of Vegetable Improvement, Indian Institute of Vegetable Research, P.O. Jakhini- Shahanshahpur, Varanasi 2

221 305 India; Department of Zoology (Biochemistry), M.M.V., Banaras Hindu University, Varanasi 221 005 India

Tomato (Solanum lycopersicum) is considered to be a model plant for studying many of the biological processes, including biotic and abiotic stress resistance. Response of tomato under various stresses is controlled by an array of regulatory genes that create distinct stress response regulons. In contrast to many signaling genes and regulatory elements, which are generally specific to a particular stress, zinc finger protein ZAT12 responds to a range of abiotic stresses. Conventional breeding methods are proved to be difficult as well as time taking in developing genotypes with enhanced tolerance to abiotic stresses. In recent times, genetic transformation of the plant species is gaining importance for developing novel phenotypes and significant achievements have been made in isolation and cloning of abiotic stress response elements, viz., DREB-1, ZAT12, etc. An efficient regeneration system was developed for tomato Var. Kashi Vishesh (H-86) involving cotyledon and hypocotyl explants, and MS medium with BAP (0.44-4.44 µM) and kinetin (2.32 µM). Later, the BcZAT12 gene on the binary plasmid pBinAR was used for Agrobacterium-mediated gene transformation to develop transgenic tomato lines. The expression cassette included abiotic stress inducible lea1 promoter, nptII selection marker, and nos terminator. A total of 16 transgenic plants were obtained from 10 independent transformation events; the selection media included cefotoxime (500 mg l-1) and kanamycin (150 mg l-1). The transformation event was detected using PCR with BcZAT12 and nptII specific primers. The tests for copy number and level of BcZAT12 expression is under progress. The putatively transgenic lines were transferred to a glass house after acclimation and seeds are being collected for further evaluation. The parameters for regeneration and genetic transformation are discussed herein.


SOL 2009 209


SOL 2009210

BIOTECHNOLOGICAL APPROACHES TO HARNESS HEAT TOLERANCE IN TOMATO (SOLANUM LYCOPERSICUM L.)

S.V.R.Reddy., P. Mosconi, A. Mazzucato

Department of Agrobiology and Agrochemistry, Laboratory of Vegetable Biotechnology, University of Tuscia, Via S.C. de Lellis s.n.c., 01100 Viterbo (Italy)

The negative effect of high temperature is becoming a major problem because of predicted increase of 2°C of earth surface temperature by 2050. Exposure to high temperatures causes reduced yields in tomato (Solanum lycopersicum L.). The level of membrane fatty acids in leaves, carbohydrate and proline content of anthers plays important role in acquiring heat tolerance in tomato. We aimed to silence tomato omega 3 fatty acid desaturase (LeFAD7) using a dsDNA construct under the CaMV 35S promoter and to increase carbohydrate and proline supply to the developing anthers during heat stress by expressing anther sugar transporter (STP), acid invertase (LIN6) and Proline transporter (LePROT1) genes under the control of heat shock protein (HSP) 18.2 promoter. We isolated partial coding sequences of both sense and antisense fragments of LeFAD7 with primers designed on AY157317 and cloned into PBI121 vector with GUS fragment as a loop-forming structure in silencing mechanism. Open reading frames of STP, LIN6 and LePROT1 genes were isolated with the primers designed on the sequences of AF506005 for LIN6, AF014808.1 for LePROT1 and TC211922 for STP. Isolated open reading frames of three genes were cloned into pGind01 vector under the control of HSP 18.2 promoter and kanamycin as selectable marker. Microtom tomato was transformed with Agrobacterium containing vectors for respective genes. Transformed plants were confirmed by PCR and their acquired tolerance towards heat stress was assessed by analysis of fatty acids content, level of anthers carbohydrate and proline content and pollen and fruit fertility.

OVER-EXPRESSION OF GENES INVOLVED IN GLYOXALASE PATHWAY IN TOMATO CONFERS SALT TOLERANCE

Patrani Madhulatha, Riffat John, Sneh Latha Singla-Pareek, S. K. Sopory, M. V. Rajam

Department of Genetics, University of Delhi South Campus, New Delhi 110021, India

Tomato is affected by a variety of abiotic stresses, including salinity, drought and extreme temperatures, which cause a significant reduction in tomato yield and quality. Therefore, the development of tomato varieties for abiotic stress tolerance is extremely important. Metabolic engineering is one of the important strategies for developing transgenic crops for abiotic stress tolerance, and glyoxalase pathway is one such important target for engineering crops for abiotic stress tolerance. Glyoxalase I (GlyI) and glyoxalase II (GlyII) are important enzymes in glyoxalase pathway, which are involved in glutathione based detoxification of methylglyoxyl. Thus, the present study was undertaken to develop abiotic stress tolerant transgenic tomato plants by engineering glyoxalase pathway. Several putative tomato transformants were generated using a binary vector that harbors key genes of glyoxalase pathway, GlyI and GlyII, and hpt plant selection marker gene. The presence of transgene in tomato transformants was shown by PCR analysis using primers specific to GlyI and hpt genes. Preliminary abiotic stress tolerance assays, based on the bleaching of chlorophyll in leaf discs floated on salt (NaCl 200 mM) amended medium revealed that these transgenic plants exhibited increased salt tolerance. These transgenic plants are subjected for further molecular characterization and multiple abiotic stress tolerance assays. Besides, to understand the molecular basis of glyoxalase pathway mediated abiotic stress tolerance, the expression profile of the important genes involved in abiotic stress tolerance is also being studied. These preliminary results indicate that glyoxalase pathway may be an important target for creating abiotic stress tolerant crop plants.


SOL 2009 211


SOL 2009212

TRANSGENIC TOMATO PLANTS EXPRESSING OSISAP1 FROM RICE CONFERS TOLERANCE TO DROUGHT AND SALT STRESS

Amolkumar Solanke, Akhilesh Tyagi, Arun Sharma

Plant Molecular Biology, University of Delhi South Campus, New Delhi 110021

Tomato is sensitive to almost all types of abiotic stresses. For improving stress tolerance in an Indian tomato cultivar Pusa Ruby, OSISAP1 (Oryza sativa stress associated protein) from rice was utilized, which encodes for protein containing A20/AN1 zinc finger domains. Expression of this gene provided tolerance to cold, dehydration and salt stress in transgenic tobacco plants. Transgenic lines were generated using Agrobacterium-mediated transformation and transgenic nature of plants was confirmed by PCR and Southern analysis. Out of 11 lines confirmed for the presence of transgene, fruiting was observed only in 5 lines. Expression level of OSISAP1 transcript was checked by semi-quantitative PCR and northern analysis. Responses to cold, drought and salt stresses were analyzed in nine-day-old seedlings in T2 generation. Plants from all five transgenic lines performed better in salt and drought stress with improvement up to 57% and 30% in fresh weight, respectively, over control plants. No significant improvement was observed under cold stress in transgenic plants. To check the redundant nature of native SAP family genes, expression of SAP genes from tomato was checked in transgenic lines. Expression of some other stress related genes was also checked. It was observed that expression of few stress related genes was altered in OSISAP1 expressing plants. In conclusion, expression of rice OSISAP1 provides tolerance under drought and salt stress condition in transgenic tomato.

DEVELOPMENT OF DROUGHT TOLERANT TRANSGENIC TOMATO LINES USING ATDREB1A GENE

1,2 1 1 2 1Govind Kumar Rai , Neha Prakash Rai , Major Singh , Sushma Rathaur , Sanjeev Kumar and 1

Mathura Rai

1 2Indian Institute of Vegetable Research, P.O. Box 5002, PO-BHU, Varanasi - 221005; Department of Biochemistry,

Banaras Hindu University, Varanasi -221005

Plants resist drought stress by modifying water uptake and loss, accumulating compatible solutes, modifying the properties of cell walls, and synthesizing protective proteins to tolerate or repair cell damage. Many transcription factors have been identified that provide enhanced plant tolerance to drought stress. Among them, a group of genes, called DREB/CBF are considered as 'master switches', since DREB/CBF proteins (Dehydration Responsive Element Binding or C-repeat Binding Factors) bind to the cis-acting DRE/C-repeat sequence. The DRE/C-repeat is a 5-bp core sequence of CCGAC, present in the promoter region of many downstream water deficit stress-inducible genes and cold-regulated (COR) genes. Heterologous constitutive over-expression of DREB1/CBF genes in plants like Arabidopsis, Brassica napus, Rice, Chrysanthemum etc. conferred strong constitutive expression of the stress-inducible genes, leading to an increased tolerance to drought. In an attempt to improve drought tolerance of tomato (var. H-86), a gene construct containing AtDREB1A/CBF3 cDNA driven by water deficit stresses-inducible promoter rd29A along with nos terminator, and nptII selection marker in T-DNA of binary vector pCambia 2300, cloned in Agrobacterium tumefaciens (GV3101) was used to develop transgenic tomato lines. Parameters of Agrobacterium-mediated tomato transformation were standardized to get high transformation efficiency, minimize tissue necrosis and Agrobacterium over-growth during the selection of transformed explants. A total of 96 putative transgenic were obtained from 50 independent transformation events. The selection of transformants was made on the MS medium containing 200 mg l-1 kanamycin and 500 mg l-1 cephotaxime. PCR amplification with AtDREB1A and nptII specific primers indicates that and 61 putative transformants contain copies of AtDREB1A and nptII gene. Confirmed transgenic lines are multiplied and maintained in vitro for further confirmation and assay. The T0 plants were also transferred in glass house for further evaluation. T1 seeds are being collected from selfed fruits.


SOL 2009 213


SOL 2009214

MICROARRAY ANALYSIS FOR BIOCHEMICAL AND MOLECULAR MECHANISMS OF HEAT TOLERANCE IN TOMATO (SOLANUM LYCOPERSICUM)

2 1 2 1 2Ashutosh Rai , Upama Mishra , Major Singh , Rama Shankar Dubey , Rajesh Kumar , 2 2

Rajiv Kumar , Mathura Rai

1 2Department of Biochemistry, Faculty of Science, Banaras Hindu University, Varanasi-221005; Crop Improvement

Division, Indian Institute of Vegetable Research, Jakhini (Shahanshahpur), Varanasi-221305

The physiological aspects of plant responses to high temperature are well studied. The present constrain is now to find the genetic and molecular pathways that modulates this response and the actual factors that determine relative sensitivities. Among the abiotic stresses, heat stress is the most difficult to model experimentally. One heat tolerant (PS-1) and one susceptible line (H-24) were selected on the basis of three successive year morphological, cytological, physiological and biochemical data. These two lines were grown in glass house under regime of 24°C/20°C (day/night) temperature and 14 h photoperiod with photon flux density of 300 µmol m-2s-1 and 75% relative humidity and at the stage of 50% flowering RNA has been isolated for microarray analysis. Hybridization percentage for probe hybridization was 64.6% to 80.8% for all 12 chips. Analysis of heat-regulated transcripts revealed structured responses of genes associated with cellular and biochemical activities in metabolism, energy, transcription, protein synthesis, defense, cell rescue, transport facilitation, and signal transduction pathways. Further validation with Real Time PCR analysis is being progress. 100 cDNA sequences also have been submitted to NCBI db ESTs. These results were then compared with the growing databases containing transcript and metabolite profiles during episodes. Further analyses will be focused on a comparison of gene expression changes under different stresses in an attempt to define which subsets of the responding genes identified stress-type specific reactions, as opposed to those that signified common responses to drought and early blight.

PHYSIOLOGICAL AND BIOCHEMICAL PARAMETERS FOR IDENTIFICATION OF DROUGHT TOLERANT LINES IN TOMATO (SOLANUM LYCOPERSICUM)

1 2 2 2 1 2Upama Mishra , Ashutosh Rai , Major Singh , Rajesh Kumar , Hausila Pandey , Mathura Rai

1 2Department of Biochemistry, Faculty of Science, Banaras Hindu University, Varanasi-221005; Crop Improvement Division, Indian Institute of Vegetable Research, Jakhini (Shahanshahpur), Varanasi-221305

To understand the adaptability of four different genotypes of tomato to drought stress, we analyzed physiological and biochemical parameters including Relative water content (RWC), net photosynthetic rate, proline, lipid peroxidation, hydrogen peroxide (H2O2) level, catalase (CAT) and superoxide dismutase (SOD), in tomato leaves subjected to moderate (7days) and severe (14 days) drought stress during flowering stage. RWC was used as measure of tolerance level in plant tissues. Under severe drought EC-520071 showed maximum RWC followed by EC-520061 compared to control plant. CO-3 showed minimum RWC, under mild (7 days) and severe drought (14 days). Genotypes EC-520061 and EC-520071 showed maximum photosynthesis under moderate and severe drought. Whereas CO-3 showed minimum net photosynthetic rate under moderate and severe drought conditions followed by DVRT-1. EC-520061 and EC-520071 showed high level of proline, SOD and CAT compared with CO-3 and DVRT-1. EC-520061 and EC-520071 also exhibited low level of lipid peroxidation and H2O2. Based on the combined investigation into the physiological and biochemical properties it was found that genotype EC-520061 and EC-520071 were drought tolerant compared to CO-3 and DVRT-1.


SOL 2009 215

SESSION IV FUCTIONAL GENOMICS

216

TOMATO TILLING MUTANTS IN EXPANSIN1 GENE SHOW ALTERATION IN SOFTENING AND CELL WALL METABOLISM DURING RIPENING

1 3 3 1Silvia, Minoia , Jacqueline, Vigouroux , Bernard, Quéméner , Giuseppina, Mosca , Marie Françoise, 3 2 2 1 3

Devaux , Florence, Piron , Abdelhafid, Bendahmane , Filomena, Carriero , Marc, Lahaye

1 2 3 Metapontum Agrobios, 75010 Metaponto (MT) Italy; URGV/ INRA, 91057 Evry; BIA, INRA, Nantes, France

Tomato fruit ripening involves the expression of several types of enzymes and proteins targeted at the cell wall disassembly. Expansin1 gene (SlExp1) codes for the major expansin protein involved in fruit softening. This protein controls non-covalent linkages at the hemicellulose-cellulose interface and regulates cell wall polysaccharides metabolism. To date, no non-GMO tomato genotypes modulated on expansin are available to realize controlled texture hybrids. We identified five new SlExp1 allelic variants by TILLING screening of two mutant collections generated in the M82 and Red Setter genetic background. Two of the five mutant lines bear mutations within the gene region coding for the active site of the protein. The nucleotide substitutions led to a nonsense and a missense mutation that affected more strongly the protein function than the three other point mutations mutants. Mechanical, histological and biochemical analyses were performed on the pericarp tissue of the two nonsense and missense Exp1 mutant lines at different ripening stages. Significant differences were observed in the pericarp tissue module of deformation during compression, in cell size distribution and in cell wall polysaccharides composition with respect to the control lines. The results are interpreted with regard to the impact the mutations on pericarp tissue construction and cell wall disassembly in relation to mechanical properties.


SOL 2009 217


SOL 2009218

FUNCTIONAL CHARACTERIZATION OF A CHAPERONE PROTEIN DNAJ-LIKE FOR S. LYCOPERSICUM AND S. PENNELLII, ASSOCIATED WITH CHANGES IN SUGARS, AMINOACIDS, AND CAROTENOIDS CONTENTS IN TOMATO FRUIT

Bermudez, Luisa; de Godoy, Fabiana; Carrari, Fernando; Rossi, Magdalena

Departamento de Botânica, Instituto de Biociências, USP, Brasil.

Tomato (Solanum lycopersicum) is an important food crop worldwide and represents an excellent model plant for genomic research and metabolism of fruit. Numerous wild related species show great morphological and ecological diversity that has proven useful in breeding programs because of its valuable genetic variability including nutritional and industrial quality traits. It has been described a list of 9 candidate genes co-localizing with a subset of 27 QML and 4 YAL in chromosome 4 (segment 4I). One of these candidate genes identified is a Chaperone protein dnaJ-like. This gene could be involved in the metabolite changes observed because of the known functions of this protein family, that includes protein folding, assembly and disassembly, translocation of proteins into organelles and recently association with the differentiation of proplastids. By means of a comprehensive and comparative approach in this work we present a functional characterization of this gene for S. pennelli and S. lycopersicum. Both alleles were complete cloned and sequenced. Results will present amino acids polymorphisms, differential promoter motifs, differences in expression profile data and promoter transient expression. Moreover, silencing and sub-cellular localization experiments are in progress. Results presented here represent a novel characterization of a Chaperone protein dnaJ-like in tomato that could act as a regulatory factor explaining important metabolite changes in fruit.

SLSTP CODING FOR STOLON TIP PROTEIN IS SPECIFICALLY EXPRESSED DURING LATER STAGES IN ROOT DEVELOPMENT AND IN REPRODUCTIVE TISSUES OF TOMATO (SOLANUM LYCOPERSICUM L.)

Sivapriya, Sethuraman, Kalidhasan, Nagarajan, Kanneganti Vydehi ,Aditya K. Gupta

Dept of Plant Biotechnology, School of Biotechnology, Madurai Kamaraj University, Madurai - 625 021

Plant root development requires a coordinated expression of many tissue and/or stage specific genes apart from the expression of housekeeping genes. Though several genes are characterized for their role in root development, much remains unknown about the temporal regulation of root development. We sought to identify genes expressed in specific stages of root development in tomato (Solanum lycopersicum L.) which is a typical representative for most crop species than Arabidopsis in which most root development studies have been reported so far. Suppression subtractive hybridization (SSH) libraries were generated using two distinct stages of root development - 8 day old tomato roots (driver) and 40 day old tomato roots (tester). Screening of the libraries and sequencing of the confirmed clones has generated ESTs (Expressed Sequence Tags) specific for the developmental stage. After validating them by RT-PCR, a candidate gene specifically expressed during late root development and coding for stolon tip protein was chosen for full length completion and characterization studies. The sequence was completed using 5' and 3' RACE and a full length sequence of 1.3 Kb was obtained. The functional characterization of this gene would be possible by raising transgenics for over expression and silencing. This would in turn, improve our understanding of the genetic regulation of root development.


SOL 2009 219


SOL 2009220

UNDERSTANDING THE COMPLEXITY OF FRUIT RIPENING BY TRANSCRIPTOME ANALYSIS OF RIN MUTANT FRUIT AND IN SILICO ANALYSIS OF PROMOTERS OF DIFFERENTIALLY REGULATED GENES

Rahul Kumar, Sanjay Kapoor, Akhilesh K. Tyagi and Arun K. Sharma

Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi, 110021

Tomato (Solanum lycopersicum) is widely used as model to study fruit ripening in plants. A MADS-box transcription factor, LeMADS-RIN, controls fruit ripening as indicated by ripening inhibitor (rin) mutation resulting in non-ripening phenotype of fruit. A complete understanding of rin mutation and its effect on fruit transcriptome during ripening is not clear. In this study, Affymetrix GeneChip® Tomato Genome Arrays have been used to investigate the influence of rin mutation on fruit transcriptome during ripening. Gene expression data of the pericarp of wild type and rin mutant fruits from three different ripening stages were analyzed to identify RIN regulated genes. A total of 1,802 genes were found to be differentially expressed between wild type and rin mutant fruits. Since it is known that LeMADS-RIN gene binds to the CArG box of LeACS2 promoter, 309 available putative promoter sequences of differentially expressed genes were searched for presence of CArG box. The study revealed that 206 promoter sequences harbor CArG box while rest of the promoter sequences lacked CArG box suggesting an indirect regulation by LeMADS-RIN for CArG box deficient genes.

ALTERATIONS IN CAROTENOID CONTENTS DURING FRUIT DEVELOPMENT IN TRANSGENIC TOMATO OVEREXPRESSING SPERMIDINE SYNTHASE GENE

3 3 2 2 2Mohamed Hichem Neily , Chiaki Matsukura , Stéphane Bernillon , Mickaël Maucourt , Annick Moing , 2 3 1Dominique Rolin , Takaya Moriguchi , Hiroshi Ezura

1 2Grad Sch Life Environ Sci, Univ Tsukuba; UMR619 Biologie du Fruit; Centre INRA de Bordeaux, France; 3NAFRO-NIFTS.

Carotenoids are lipid-soluble pigments produced strictly by photosynthetic organisms, well known by their valuable benefits for human health in the prevention of numerous diseases. In our study, the content of carotenoids in mature tomato fruits of homozygous lines overexpressing apple spermidine synthase MdSPDS1 under the control of CaMV 35S promoter was evaluated by HPLC with diode array detector. The analysis of the total carotenoids revealed that transgenic fruits exhibited significantly increased content of lycopene (1.5-2.0 folds) compared with the wild type fruit particularly in the pericarp-columella tissue. To investigate the relationship between carotenoid content and gene expression, key genes of carotenoid metabolic pathway, encoding Psy-1, Pds, Dxs, Lcy-e and Lcy-b were checked by real time PCR. Since ethylene is known to play critical role during ripening and carotenoid accumulation, ethylene production of fruits ripened on the vine at different stages of fruit development was also measured and the results have showed a clear differences between transgenic and wild type fruit. These results lend credence to the idea that elevated polyamines might modulate carotenoid synthesis in tomato fruits.


SOL 2009 221


SOL 2009222

FUNCTIONAL CHARACTERIZATION OF PROLYL OLIGOPEPTIDASE: A GENE DIFFERENTIALLY EXPRESSED IN THE EARLY DEVELOPMENTAL STAGES OF TWO COFFEE CULTIVARS

Ratnesh Sing, Beth Irikura, Chifumi Nagai, Henrik H. Albert, Monto Kumagai, Robert E Paull, Ming-Li Wang

University of Hawaii at Manoa, Department of Molecular Biosciences and Bioengineering

Tall-Mokka (MA2) and Kona-Typica (KO34) cultivars of Coffee (Coffea arabica L.) are renowned for their high quality beans and distinct flavors. MA2 has smaller organs than KO34, including leaves, fruits and seeds. In an attempt to identify genes responsible for organ size control, a gene encoding prolyl oligopeptidase (CaPOP) was found to be upregulated during the early developmental stages of MA2 in comparison to KO34. POP, a serine endopeptidase that hydrolyzes proline bonds in small peptides (<30 aa) has been widely studied in animals but largely ignored in plants. Ubiquitous presence of POP in all domains of life from archaea to green plants and mammals suggests its conserved and important role in all organisms. To understand its role in plants, the POP genes from MA2 and KO34 were cloned and sequenced. Sequence analysis identified two POP variants, arbitrarily named CaPOP1 and CaPOP2, each in both cultivars. Primarily, CaPOP1 differs from CaPOP2 in containing two deletions (158 nt and 720 nt, respectively) in its promoter region. For functional characterization of CaPOP, we have generated several expression constructs to study the effect of overexpression, promoter function and subcellular localization, in transgenic Arabidopsis thaliana. The transformants are currently being analyzed. Furthermore, we are assessing the effects of TDNA insertion mutation of AtPOP in Arabidopsis. This study will increase our understanding of the role of POP in plant growth and development.

THE POTATO GENOME SEQUENCING INITIATIVEThe Potato Genome Sequencing Consortium

Sanjeev Kumar Sharma (on behalf of PGSC), Programme of Genetics

Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, Scotland, United Kingdom; [email protected]

Potato is the world's most important vegetable crop, and a key member of the Solanaceae. The 840-Mb genome of potato is being sequenced by the multi-national Potato Genome Sequencing Consortium (PGSC, see www.potatogenome.net). Initially the sequencing effort employed a chromosome by chromosome, BAC by BAC sequencing strategy of the diploid RH89-039-16 clone, the male parent of the potato Ultra High Density map. Using a whole genome shotgun approach the PGSC has also generated a draft sequence of a completely homozygous 'doubled monoploid' clone (DM1-3 516R44) of S. tuberosum group Phureja, as well as the RH89-039-16 clone. Progress towards generation of draft sequences of the two genotypes has been rapid, with genome coverage greater than 70X produced using a combination of next generation sequencing technologies and conventional Sanger sequencing. Genome assembly is well underway, and resources developed include fosmid and additional BAC libraries, improved physical maps, and an anchored genetic reference map, onto which genome scaffolds are mapped using a range of marker technologies. This integrated genome data will form an important resource for linking to all future genetic mapping efforts by the potato community. The timely release of the potato genome will give the entire Solanaceae research community an early opportunity to exploit the genome sequence for fundamental and applied biological studies and various other widespread applications in potato genetics. The potato genome sequence will serve to accelerate potato improvement and help to meet the challenges facing food production in the 21st century.


SOL 2009 223

SESSION V METABOLOMICS AND PROTEOMICS

224

INFLUENCE OF DNA DAMAGE BINDING PROTEIN ON TOMATO FRUIT PROTEOME

Rakesh Kumar, SJS Rama Devi, Rameshwar Sharma and Yellamaraju Sreelakshmi

Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad-500 046, India

Biofortification is one of the most efficient means to provide essential nutrients to human diets, particularly in developing countries. Lycopene, which is one of the major nutraceuticals targeted for biofortification is present in high quantity in naturally occurring tomato high pigment1 mutant. Gene mapping and cloning studies have identified that high pigment phenotype is caused by a mutation in the DAMAGED DNA BINDING PROTEIN 1 gene (Sl-DDB1). Physiological studies have suggested that the hp1 gene product is a negative regulator of phototropic signal transduction pathway. DDB1 is also a part of the CDD complex (composed of DDB1, DET1 and COP10) that mediates the effect of light and cytokinin activity, thus affecting both cell size and plastid multiplication. It is expected that hp1 mutation may shift temporal patterns of proteins regulating pigment accumulation during fruit ripening. The present work was undertaken to explore the proteome of wild type and hp1 fruit tissue at three developmental stages (mature green, breaker and red ripe). The differentially expressed proteins from hp1 mutant were identified by MALDI-TOF/TOF. The up-regulated proteins in mature green fruits of the mutant belonged to photosynthesis, energy and nitrogen metabolism indicating the diversity in proteome shift. Heat shock proteins and photosynthetic proteins were found to be up-regulated in the red ripe fruit of the mutant. In contrast, a protein regulating cell wall metabolism was found to be down regulated. The results obtained in this study highlight that high pigment locus regulates a variety of metabolic pathways during fruit development in tomato.


SOL 2009 225


SOL 2009226

TOWARDS THE IDENTIFICATION OF GENES INVOLVED IN VITAMIN C ACCUMULATION THROUGH A COMBINATION OF METABOLOMIC, TRANSCRIPTOMIC, AND REVERSE GENETICS APPROACHES

1 1 2 1 1Viviana Lima , Mariana López , Antonio Granell , Victoriano Valpuesta and Miguel A. Botella

1Departamento de Biología Molecular y Bioquímica, Universidad de Málaga, 29071 Málaga, Spain;

2Instituto de Biología Molecular y Celular de Plantas, Consejo Superior de Investigaciones Científicas,

Universidad Politécnica de Valencia, 46022 Valencia, Spain

Tomato species constitute one of the most popular and extensively consumed vegetable crops in the world and represent an important dietary source of nutrients, vitamins and antioxidants. Vitamin C (L-ascorbic acid) is an important antioxidant in plants and animals and an enzyme cofactor in various hydroxylation reactions. Because primates have lost the capacity for vitamin C biosynthesis they are dependent on fruits and vegetables to obtain it, being tomato a major source. Despite advances on the elucidation of vitamin C biosynthesis and recycling pathways in plants, the regulatory mechanisms leading to the modulation of vitamin C content is still elusive. We have combined “omics” approaches together with a reverse genetic approach in order to identify genes involved in vitamin C accumulation in tomato fruits. A RIL population generated from an inter-specific cross between Solanum Lycopersicum x Solanum pimpinellifolium was analyzed for vitamin C content and the expression profile of those RILs displaying extreme contents were analyzed using the TOM2 microarray. The list of candidates genes obtained belonged to different functional categories such as transcription, energy production and conversion, signal transduction, and others. The possible involvement of several candidates genes selected in the regulation of vitamin C is being explored using a visually traceable VIGS system.

IMPROVEMENT OF FRUIT CHARACTERISTICS AND EXPRESSION PROFILE OF RIPENING GENES IN TOMATO TRANSGENICS OVER-EXPRESSING POLYAMINE BIOSYNTHESIS GENES

Patrani Madhulatha, Roopali Pandey, Anuj Chaudhary, Ramakrishna Pal, Manchikatla Rajam

Department of Genetics, University of Delhi South Campus, New Delhi 110021, India

The naturally occurring polyamines (PAs) - putrescine (Put), spermidine (Spd) and spermine (Spm) have been implicated in several plant developmental processes, including fruit development. The present study was aimed to improve fruit characteristics in tomato by engineering polyamine metabolism in fruits. Tomato transgenics over-expressing PA biosynthesis genes viz., mouse ornithine decarboxylase (ODC), oat arginine decarboxylase (ADC) and human S-adenosylmethionine decarboxylase (SAMDC), under the control of fruit-specific promoter (2A11) were developed. These transgenics have exhibited significant increase in polyamine levels and decrease/no change in ethylene production in fruits with a delay in fruit ripening, longer shelf-life of fruits, increased lycopene content and improved post-harvest qualities with respect to processing. The transgene expression and the improved fruit characteristics were stably inherited into the subsequent generations (T3/T4). To understand the molecular basis of improved fruit traits, the expression profile of all the polyamine biosynthesis genes and also various genes involved in fruit ripening is being studied in transgenic and wild-type tomato fruits by semi-quantitative RT-PCR, at different stages of fruit development. Preliminary results showed that there was no difference in the expression profile of ODC, SPDSYN, E8 and ACC synthase (ACC4) genes in fruits of the tomato transgenic ODC line (LeODC27), when compared to control fruits. However, an enhanced expression profile of SAMDC, polygalacturonase (PG) and β-galactosidase (TBG1) genes was observed in transgenic fruits as compared to control fruits. These results suggest that the improved fruit traits in transgenic lines may be due to the up-regulation of some fruit-specific genes.


SOL 2009 227


SOL 2009228

RELATING POTATO FLESH COLOUR TO A METABOLOMICS DATA SET

Animesh Acharjee, Chris Maliepaard

UR Plant Breeding, Wageningen, The Netherlands

We use a mapping population of potato to relate tuber flesh colour traits to LC-MS profiles. In such an analysis the number of is much larger than the sample size. Due to this, traditional statistical regression methods cannot be applied. One of the ways to handle this situation is to apply “regularization”. We applied different regression methods and compared their properties and results. Flesh colour of the harvested tubers, enzymatic discoloration of grated potato flesh exposed to air after five minutes and three hours were collected. We used different regression methods like univariate regression, stepwise multivariate regression, ridge regression, lasso, elastic net, principal component regression, partial least squares regression, sparse partial least squares regression, support vector regression and random forest regression and compared these methods by the R2 statistic of the fitted versus observed phenotypic values and by the mean squared error of prediction. We compared the rank orders of the regression coefficients of the LC-MS peaks in the different methods. Support vector regression outperformed the other methods in terms of the R2 value. Flesh colour and enzymatic discoloration of the grated material could be predicted by a small subset of the variables. Some of these variables are found in all the traits and some of these variables are likely to be related to the same compounds since they show high correlations and have the same retention time.

SESSION VI BIOINFORMATICS AND COMPUTATIONAL BIOLOGY

229


SOL 2009230

SGN DATABASE: QTL ANALYSIS, VISUALIZATION AND THE LINK TO GENOMES

Isaak Y Tecle, Naama Menda, Robert Buels and Lukas Mueller

Boyce Thompson Institute for Plant Research, Cornell University, Tower Rd, Ithaca, NY 14853, USA

Quantitative trait loci (QTL) analysis is one of the genetic approaches employed to understand the genetic basis underlying complex traits, of which most traits of economic importance fall into. At the Sol Genomics Network (SGN; http://sgn.cornell.edu), we are developing web technologies for online QTL analysis, visualization and sharing of the outcomes with the wider research community. Specifically, we are: (1) developing user-friendly web interfaces for researchers to submit their raw phenotype and genotype data from their QTL studies; (2) designing database schema for storing the raw data; (3) implementing R/QTL statistical software (http://www.rqtl.org) for performing on-the-fly QTL analysis; (4) designing interfaces for online visualization of QTLs on a genetic map; and (5) making the relevant cross-links between related SGN datasets. With our QTL analysis tool one can identify peak and flanking markers for QTLs of traits of interest. And since the QTL mapping output is integrated with other SGN datasets (eg. Marker, BACs, and Unigenes), and analysis tools such as the Comparative Map Viewer (http://sgn.cornell.edu/cview/) users can compare QTL regions of interest to corresponding regions in genetic maps of the same or different Solanaceae species at SGN. As the tomato genome sequencing advances, we envision the possibility for researchers to identify candidate BAC sequences or loci on the tomato physical map, which can be suggestive of candidate genes for a trait of interest.

Furthermore at SGN, images, quantitative phenotype and genotype data, publications, and genetic maps generated by QTL studies are displayed and available for download. Currently, data from three F2 and two backcross population QTL studies on fruit morphology traits (18 – 46 traits per population) is available at the SGN website for viewing at population, accession, and trait levels. Traits are described using ontology terms. Phenotype data is presented in tabular and graphical formats such as frequency distributions with basic descriptive statistics. Mapping data showing location of parental alleles on individual offspring accession genetic maps is also available.

We invite researchers to use the QTL analysis tool and share their research output with the Solanaceae research community.

SGN a public database hosted at Boyce Thomson Institute, Cornell University, and funded by USDA CSREES and NSF.

AB INITIO MODELING OF DNA BINDING WITH ONE FINGER (DOF) TRANSCRIPTION FACTOR PROTEIN OF SORGHUM BICOLOR BY ITERATIVE TASSER SIMULATIONS

1 2 3Vinay Kumar Singh , Hariom Kushwaha and Dinesh Yadav

1 2School of Biotechnology,Banaras Hindu University,Varanasi-221005; Institute of Microbial Technology, Sector

339-A, Chandigarh - 160036 ; Department of Biotechnology, D.D.U Gorakhpur University, Gorakhpur-273009

Prediction of protein structure from amino-acid sequences has been one of the most challenging problems in computational structural biology for many years. Historically, protein structure prediction was classified into three categories namely (i) comparative modeling (ii) threading and (iii) ab initio folding. The first two approaches build protein models by aligning query sequences onto solved template structures. When close templates are identified, high-resolution models could be built by the template-based methods. If templates are absent from the Protein Data Bank (PDB) library, the models need to be built from scratch, i.e. ab initio folding. This is the most difficult category of protein-structure prediction. An attempt has been made to reveal the 3-D structure of one of the DNA binding with One finger (DOF) Transcription factor protein sequence (Accession number ACO53859) (sbdof-1) using TASSER (threading/assembly/refinement) method for the ab initio modeling. The Secondary structure predicted based on Confidence score (C-score) demonstrates a strong correlation with the real quality of the final models. C-score is a confidence score for estimating the quality of predicted models by I-TASSER. It is calculated based on the significance of threading template alignments and the convergence parameters of the structure assembly simulations. C-score of the SbDof-1 protein is typically in the range of [-5, 2]. A total of top 10 templates were used for the structural modeling and 10 proteins in PDB which were structurally closest to the first model (identified by TM-align) were elucidated. The five best models for the structure of sbdof-1 were predicted using TASSER method.


SOL 2009 231


SOL 2009232

IN SILICO PREDICTION OF DNA BINDING WITH ONE FINGER (DOF) TRANSCRIPTION FACTOR GENES IN SOLANUM TUBEROSUM AND LYCOPERSICON ESCULENTUM

1 2 3Vinay Kumar Singh , Hariom Kushwaha and Dinesh Yadav

1 2School of Biotechnology,Banaras Hindu University,Varanasi-221005; Institute of Microbial Technology, Sector

339-A, Chandigarh - 160036 ; Department of Biotechnology, D.D.U Gorakhpur University, Gorakhpur-273009

Plant gene expression involves classes of transcription factors that have specifically evolved to regulate plant-specific genes and/or to mediate a variety of plant-specific signals. The DOF (DNA binding with One Finger) family is a typical example of Zinc-finger transcription factors involved with multifarious diverse roles exclusively in plants extensively like the expression of genes associated with carbon assimilation, phytochrome signalling, seed maturation and germination, the auxin response, the salicylic acid response, involved with the function of the stomata guard cells, photoperiodic flowering and biosynthesis of glucosinolates etc. In cereals DOF genes have been studied in maize, barley, wheat and rice. There exists great diversity of DOF genes in cereals with 30 and 24 different DOF genes being reported in rice and barley respectively. An attempt has been made to study the genome-wide comparative analysis of the rice and Arabidopsis DOF gene families. The availability of complete genome sequence provides an opportunity to reveal the existing diversity of DOF genes in a particular plant species.An attempt has been made to investigate the diversity of DOF genes in two of the economically important crops namely Solanum tuberosum and Lycopersicon esculentum using various bioinformatic tools. An extensive annotations at both family and gene level has been attempted. An extensive homology search for DOF like genes were attempted from Database of GenBank, EMBL and DDBJ sequences from EST divisions, whole-genome shotgun reads (wgs) and nucleotide collection (nt/nr) databases. Based on all the homologous sequences the classification was done using multiple sequence alignment and phylogenetic analysis tool (clustalw, MEGA).It was predicted that there exist nearly 21 and 23 C2C2-like DOF genes in Solanum tuberosum and Lycopersicon esculentum respectively. Further gene prediction and protein functional analyses of the predicted DOF genes were done using bioinformatics tools namely Genescan, fgenesh and Interproscan tool.

SESSION VII DEVELOPMENT AND SIGNALING

233


SOL 2009234

AN INSIGHT INTO AUXIN CONCENTRATION GRADIENT CONTROLLING PATTERN DEVELOPMENT IN TOMATO

1 2 3 1Nongmaithem Sapana Devi , Min Raj Dhakal , Maria Ivanchenko , Yellamaraju Sreelakshmi and 1

Rameshwar Sharma

1 2School of Life Sciences, University of Hyderabad, Hyderabad- 5000 46, India; Department of Botany, Tribhuvan

3University, Biratnagar, Nepal; Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, USA

Auxin plays an important role in every aspect of plant growth and development. Earlier studies have elucidated the role and overall distribution pattern of auxin in Arabidopsis but little is known in tomato. In the present study, auxin concentration gradient and its activities during the development of tomato flower and fruits were analyzed in transgenic lines having auxin sensitive reporter construct (DR5::GUS). In flower, expression of DR5::GUS was found to be confined to pollen maturation stages, particularly pollen before pre-anthesis. Following pollination, GUS expression was observed at style and later in the ovary, suggesting that auxin in pollen may be responsible for the formation of pollen tube. In developing embryos, no detectable GUS expression was observed until the globular stage. After the globular stage, GUS expression was restricted to the proximal and distal parts of the heart-shaped embryo and the same was observed till late torpedo stage. In mature embryos till seedling stages, auxin accumulation was found to be more prominent and confined to root tips. The observation of GUS expression in parenchymatous cells in the primary xylem of floral stalk from the day of pollination till fruit ripening indicated auxin transport in later stages of fruit development. To validate DR5 activity, crosses were performed with DR5::GUS line (pollen donor) and polycotyledon (poc) mutant having enhanced Polar Auxin Transport (PAT) activity. The positive POC:DR5::GUS lines showed increased GUS expression than DR5::GUS lines indicating higher DR5 activity due to enhanced PAT of poc mutant. These studies will give an insight to the overall auxin gradient and its subsequent role in different developmental stages of various organs in tomato.


SOL 2009 235

ELUCIDATION OF THE FUNCTION OF PHYB1 IN LYCOPERSICON ESCULENTUM USING THE REVERSE GENETIC TECHNIQUE TILLING

Vineeta Singh Chauhan, Soni Gupta, Vijee Mohan, Yellamaraju Sreelakshmi, Rameshwar Sharma

Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad- 500046, India

Light plays a vital role in regulating plant growth and development and the perception of light is accomplished by plants due to the presence of photoreceptors. Till date, three classes of photoreceptors, Phytochromes, Cryptochromes and Phototropins have been identified in plants. Phytochrome is the principal photoreceptor in plants that detects the amount of red (600-700nm) and far-red (700-800nm) light available to plants. Five different phytochrome genes are known in tomato (PHYA, PHYB, PHYC, putative PHYD and PHYE). In view of the large number of phytochrome genes in tomato, there is a need for more type-specific phytochrome mutants to enable the physiological roles of different phytochromes to be elucidated. Since PHYB has a critical role in photomorphogenic response to red light, one of the PHYB genes (PHYB1) has been taken in this study. We have targeted the N-Terminal region of the PHYB1 gene, which has the PAS and the GAF domain, having a critical role in light reception. Previous reports suggest that mutations in this region could lead to drastic phenotypes which may even be insensitive to light. The reverse genetics approach, TILLING, which combines mutagenesis with high throughput mutation discovery, has been used for screening allelic series of mutations in an EMS (60 mM) mutagenized population of 3000 lines of L.esculentum cv Arka Vikas. DNA was pooled eight fold in a 2 dimensional manner and screened for point mutations in the PHYB1 gene by using CEL1 endonuclease, which, cleaves at the site of mismatch in a sequence. Two putative mutant lines of PHYB1 were detected and the individual lines were confirmed by screening the corresponding plate of the 2-D pool. To increase the number of mutations detected another population treated at a dose of 120 mM EMS is being screened. A unique nucleotide polymorphism has been detected in one of the lines using the Ecotilling strategy, for characterization of natural variations in various accessions of tomato. The array of mutants obtained by screening induced and natural variations will help in further studying the role of PHYB1 gene on growth and development of the plant.


SOL 2009236

ENGINEERED POLYAMINE ACCUMULATION IN TAPETAL TISSUE RESULTS IN MALE STERILITY IN TRANSGENIC TOMATO PLANTS

M. V. Rajam, Soumen Nandy and V. Rajendher

Department of Genetics, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India

The polyamines (putrescine, spermidine and spermine) have been implicated in a variety of plant developmental processes, including the induction of floral organ differentiation and regulation of male sterility. Thus, the present study was undertaken to induce male sterility by engineering polyamine metabolism in tomato through the expression of key genes involved in polyamine biosynthesis, arginine decarboxylase (ADC) and S-Adenosylmethionine decarboxylase (SAMDC) genes under the control of tapetum-specific TA29 promoter. Several putative transgenic plants have been generated by Agrobacterium using binary vectors harboring oat ADC (antisense) or human SAMDC gene, and NPT-II plant selection marker gene. Transgenic status of these transformants was demonstrated by PCR and Southern blot hybridization. RT-PCR analysis revealed the expression of transgenes in tapetum tissue, which resulted in polyamine accumulation. Although transgenic plants were phenotypically indistinguishable from wild-type plants, they exhibited varying degree of male sterility, ranging from partial to complete sterility. The flower buds in these plants were drooping downwards and showed burning symptoms at the pedicel and the buds dried up. Complete male sterile lines showed the aborted and distorted pollen in comparison to the round and fully developed pollen in wild-type anthers. Microscopic and SEM examination of anthers showed the selective degradation of tapetum with no microspore development in male sterile lines. When male sterile lines were crossed with pollen from wild-type plants, they showed normal fruit and seed setting, indicating that they are female fertile. These results suggest that polyamine biosynthesis genes would be novel targets for inducing male sterility in plants.

MINING CHEMICALLY INDUCED MUTANTS IN TOMATO TO UNRAVEL THE FUNCTIONS OF PHYTOCHROME A

Sherinmol Thomas, Soni Gupta, Yellamaraju Sreelakshmi, Rameshwar Sharma

Department of Plant Sciences, School of Life sciences, University of Hyderabad, Hyderabad -500046, India

Light is one of the most complex and variable environmental factors to which the growing plant is exposed. Plants employ an array of photoreceptors to detect and respond to a broad spectrum of light. Phytochromes are a family of red/far-red light-absorbing photoreceptors that control plant developmental and metabolic processes in response to changes in light. Unique properties like strong photocontrol to its transcript level, protein stability and ability to mediate photomorphogenic responses to continuous far red light makes Phytochrome A, a molecule of interest among the members of Phytochrome family. Phytochrome A consists of an N-terminal photosensory region with three conserved domains (termed P2 or PAS domain, P3 or GAF domain, and P4 or PHY domain) and a C-terminal regulatory histidine kinase or histidine kinase-related domain (HKRD). The isolation of missense alleles of Phytochrome A have provided valuable insight into the molecular basis of phytochrome signaling. Most of the known loss of function alleles fall within the PAS domain and other photosensory region. Isolation of mutant alleles for Phyochrome A would be valuable for improving crop yield, as it has important roles in shade detection and Fruit ripening. In our study we have used TILLING (Targeting Induced Local Lesion In Genome), which is a reverse genetic non transgenic approach for the discovery of induced mutations. Two EMS mutagenised populations of tomato (cv. Arka Vikas and cv. M82) were screened for mutation in regions of Phytochrome A gene which is important for light perception and signal transduction and few mutations were detected. The role and significance of the mutants would be presented.


SOL 2009 237


SOL 2009238

MOLECULAR AND GENETIC MAPPING OF SHORT ROOT MUTANT (SHR), A RECESSIVE GENE INVOLVED IN ENHANCED NITRIC OXIDE LEVEL IN TOMATO (SOLANUM LYCOPERSICUM)

Reddaiah B, Osman Basha P, Sakthivel K, Sangeeta Negi, Yellamaraju Sreelakshmi and Rameshwar Sharma

Department of plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad- 500 046, India

Nitric oxide (NO) plays a pivotal role in controlling diverse functions in various cell types in animals. However its mode of action in regulation of development process in plants is still not known. Identification of mutants defective in nitric oxide signaling and synthesis pathway can help us to understand its role(s) in different phases of plant development. We identified a NO overproducing mutant with short root mutant (shr) phenotype from the gamma irradiated mutant population of Ailsa craig cv (L.esculentum). Both etiolated and light grown seedlings of mutant exhibited short root phenotype and the root length was almost five times shorter than the wild type. During the vegetative growth shr mutant plants showed sluggish growth, stunted appearance, smaller leaves and flowers and were less fertile than wild type. Two mapping populations were developed from the crosses between shr with L. pimpinellifolium and L. pennelli. Genetic analysis of long and short root phenotype in the segregating F2 population of shr X L. pimpinellifolium, shr X L. pennelli and shr X Ailsa craig was found to be in 3:1 ratio, indicating that shr phenotype is controlled by a single recessive genetic locus. SSR and Indel markers were used to screen the mapping populations in combination with bulk segregant analysis, to identify markers that are linked to the shr locus. The shr locus was then genetically mapped on to chromosome 9 to a region of 2.2 cM and 5.4 cM distance from markers SSR19 and SSR110 respectively. The genetic map developed from this study can be used to clone the gene by map based cloning method.

ROLE OF SIGNALOSOME IN PLANT DEVELOPMENT

Chaitanya CH, Soni Gupta, Vijee Mohan, Rameshwar Sharma, Yellamaraju Sreelakshmi

Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad- 500046, India Plant development is regulated by the precise manipulation of protein levels involving a multitude of proteolytic pathways, ubiquitination being the major pathway. Accumulating evidence indicates that signalosome functions at the interface of signal transduction and protein degradation where it interacts with a repertoire of E3 ligases for the rapid turn over of targeted proteins. One of the central regulatory molecules regulating signalosome function is COP1. Constitutive photomorphogenic1 (COP1), one of the E3 ligases in plants repress light signaling by targeting photoreceptors and downstream transcription factors for ubiquitination and degradation. A single copy of COP1 gene in tomato encodes for COP1 protein comprising a RING finger, coiled-coil and WD40 domains. It is believed that WD-40 region is the key domain in fishing the targeted proteins for degradation. Studies on LeCOP1LIKE gene in tomato fruit ripening suggest that manipulation of COP1 level enhance the nutritional quality of tomato fruit. Given the central role of COP1, availability of an allelic series of mutants of COP1 gene would be valuable to examine the structure function relation of COP1 and the interacting partners of COP1 and signalosome. To achieve this we have adopted the TILLING (Target Induced Local Lesions in Genomes) strategy to screen for point mutations in the region coding for the WD 40 domain of COP1. In parallel the natural variations existing within the different accessions of tomato are also being screened through Eco - TILLING. The current status in identifying COP1 mutants in the TILLING and Eco-TIILING populations of our model organism tomato would be presented.


SOL 2009 239


SOL 2009240

ENDOGENOUS AND EXOGENOUS GIBBERELLINS PLAY A PIVOTAL ROLE IN THE DEVELOPMENT OF IN VITRO CULTURED PARTHENOCARPIC OVARIES AT PRE-ANTHESIS STAGES

Venkataramireddy Sanampudi, Picarella Maurizio, Luigi Selleri, Andrea Mazzucato

Department of Agrobiology and Agrochemistry, University of Tuscia, Via S.C. de Lellis s.n.c., 01100

The parthenocarpic growth of the ovary is a useful trait for the breeders. Its exploitation needs a comprehensive understanding of the underlying physiological events. Objective: (i) to set up a protocol for the early identification and the selection of tomato parthenocarpic mutants in environmental conditions “masking” their expression; (ii) to gain new clues on the hormonal interactions acting during the first stages of ovary growth. Methodology: the supply of NAA and GA3, alone or in combination with the respective inhibitors (TIBA or PAC) were tested on the in vitro growth of tomato flowers harvested at pre-anthesis stages. For objective (i) plants of the parthenocarpic fruit (pat) mutant and the corresponding (near isogenic) normal counterpart were used. For the second (ii), the flowers were picked up from a segregating F2 population derived from a cross between a pat mutant line and a line harbouring an auxin reporter gene, where the GUS gene was expressed under the control of an auxin-inducible promoter. Results: due to its unbalanced hormonal content, the pat mutant was clearly distinguishable from the WT. A greater development of pat ovaries has been observed without additional hormones in the medium; the presence of GA determined a further swelling of pat ovaries but also of the WT ones. GUS assay on WT and pat ovaries cultured in vitro gave new insights on the relationship between gibberellins and auxins, indicating a pivotal role for GA in the mutant phenotype.

A CYTOKININ INDUCED ETHYLENE UNDERPRODUCER MUTANT OF TOMATO

Suresh Kumar Gupta, Shaktivel K, Reddaiah B, Santisree P, Sonal, Yellamaraju Sreelakshmi, and Rameshwar Sharma


Ethylene is one of the gaseous signaling molecules which has a major role in developmental responses such as germination; root, shoot and flower development; stress, defense, fruit ripening and senescence. One powerful approach to decipher plant hormone signaling pathways is isolation of mutants defective in hormone signaling and synthesis. Among cytokinins, kinetin is one endogenous factor that modulates ethylene biosynthesis. Tomato seedlings grown on kinetin rich medium exhibit ethylene induced triple response resulting in inhibition of root and hypocotyl elongation and also exaggerated curvature of apical hook. A tomato mutant, kinetin resistant (kin-27), defective in above ethylene response was isolated from an EMS mutagenized population. kin-27 mutant was phenotypically different from the wild type (Ailsa Craig) plant with respect to plant architecture with bushy appearance, pale green color and short internodes. The mutant fruits were also pale green in color and were slow to progress to the red ripe stage (70 days post anthesis). The mutant fruits also showed extended shelf life after harvesting at red ripe stage. Gas chromatographic analysis revealed that kin-27 fruits produced less ethylene at all stages of fruit development in comparison to WT. Segregation and inheritance analysis confirmed the recessive nature of the mutant and was controlled by a monogenic recessive locus. The mapping of the mutant locus is in progress by analyzing F2 population of the mutant crossed withwild relatives, L. pimpinellifolium and L. pennellii.


SOL 2009 241


SOL 2009242

THE ROLE OF CAROTENOID CLEAVAGE PRODUCTS IN TUBER APICAL MERISTEM ACTIVATION AND DEACTIVATION PROCESSES

1 1 1 2Raymond Campbell , Wayne L. Morris , Laurence J.M. Ducreux , Jenny A Morris , 1 2 2 2 1

Mariaconcetta Scandura , Gavin Ramsay , Glenn J. Bryan , Pete E Hedley , Mark A. Taylor

1 2Plant Products and Food Quality; Genetics, Scottish Crop Research Institute, Invergowrie, Dundee, UK.

The life cycle of the potato tuber includes organogenesis, tuber development, dormancy and sprouting. This developmental sequence requires the co-ordinated control of a complex set of physiological processes and metabolic pathways and impacts directly on many economically important traits such as tuber dormancy and tuber size distribution. Thus although an understanding of meristem activation and deactivation processes is fundamental in the drive for increased tuber quality and crop productivity, there are currently gaps in our knowledge about these processes. Although many of the classic plant growth regulators have been shown to influence the tuber life-cycle, roles for carotenoid cleavage products other than abscisic acid have not been considered. Recently it has been elegantly demonstrated that carotenoid-derived strigolactones have an inhibitory effect on the outgrowth of axillary meristems in species such as Arabidopsis, rice and pea. A key step in strigolactone formation is the action of carotenoid cleavage dioxygenases. Comparison of gene expression profiles between potato germplasm that exhibit different degrees of tuber dormancy revealed variation in the expression level of a gene encoding a carotenoid cleavage dioxygenase. A transgenic approach was used to investigate the function of this gene. Transgenic lines were developed in which the carotenoid cleavage dioxygenase was down-regulated using both tuber-specific and constitutive promoters. Dramatic effects on the tuber apical meristem were observed, consistent with there being a delayed deactivation of the tuber apical meristem at the onset of tuber formation when dormancy is established. We are currently investigating whether this is due to perturbed apocarotenoid metabolism.

EXPRESSION ANALYSIS OF CAROTENOID BIOSYNTHESIS PATHWAY GENES IN TOMATO (SOLANUM LYCOPERSICUM)

Ravi Rajwanshi, Sangram K. Lenka, Shuchi Smita, Amit Katiyar, Kailash C. Bansal

National Research Center on Plant Biotechnology, Indian Agricultural Research Institute, New Delhi-110012, India

Tomato (Solanum lycopersicum) is an appropriate model system for studying the fruit ripening process because of the drastic color changes that occurs during different stages of fruit development. Carotenoids play an important role in biosynthesis of vitamin A, abscisic acid (ABA), and their ability to act as an excellent source of natural antioxidants for plants and animals. These reports motivated us to take up the objective of developing high lycopene containing tomato for human consumption. To elucidate the global regulatory changes in fruit transcriptome, expression profiling was carried out in two tomato genotypes, VRT-32-1 (Low lycopene) and EC-521086 (High lycopene). Expression level of different set of genes involved in carotenoid biosynthetic pathway at different stages of fruit maturity i.e. (mature green to red ripe) was analyzed. Global transcriptome profiling revealed that many of the genes of carotenoid biosynthesis pathway were differentially expressed and some of these potential candidate genes such as 1-deoxyxylulose-5-phosphate synthase (DXS), Isopentyl pyrophosphate isomerase (IPI), ζ-carotene desaturase (ZDS) and β-carotene hydroxylase (CrtR-b1 and CrtR-b2) were induced in tomato genotype having higher lycopene content in comparison to genotype having lower lycopene at various stages of fruit development during ripening. Quantitative PCR analysis validated the expression level of selected candidate genes of carotenoid biosynthesis pathway as observed in Affymetrix tomato genome array expression analysis. We presume the induction of carotenoid biosynthesis pathway genes might be responsible for higher lycopene content in the genotype EC-521086. Functional validation of potential candidate genes identified in this study might lead to development of tomato genotypes having higher antioxidative property.


SOL 2009 243


SOL 2009244

A DOMINANT MUTATION IN THE PHOTOTROPIN1 HOMOLOGUE OF TOMATO IMPAIRS PHOTOTROPIN 1 AND PHOTOTROPIN 2 SIGNALING

1 1 1 2 2Sulabha Sharma , Eros Kharshiing , Ankanagari Srinivas , Kazunori Zikihara , Satoru Tokutomi , 1 1 1Rajendra K Behera , Reddaiah Bodanapu , Rameshwar Sharma ,

1 2School of Life Sciences, University of Hyderabad, Hyderabad-500046, India; Department of Biological Science, Graduate School of Science, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan.1

In higher plants, blue light phototropism is primarily controlled by the phototropins, which also mediate stomatal movements and chloroplast relocation. These photoresponses are mediated by two phototropins, Phot1 and Phot2. Phot1 mediates responses with higher sensitivity than Phot2, and Phot2 specifically mediates chloroplast avoidance and dark positioning response. By screening under continuous blue light mutant we selected a Non phototropic shoot (Nps1) mutant which was found to be defective in low as well as high fluence phototropic curvature. It also lacks any detectable accumulation movement of chloroplasts. Genetic analyses show that the mutant locus is dominant negative in nature. The mutant protein accumulates at a reduced level relative to wild type in dark grown seedlings and lacks blue light induced autophosphorylation. The mutant harbors a single G1484 to A transition in the phototropin1 homologue resulting in an arginine to histidine in a highly conserved A?α helix of the translated gene product. Molecular modeling indicates a possible alteration of the interaction between A?α helix and Jα helix in the mutant Phot1 that may reduce stability and photoreactivity resulting in loss of phototropic activity in the mutant. The results indicate that a conserved arginine residue in the A?α helix region of PHOT1 plays a critical role in phot1 mediated signal transduction in tomato.

THE TOMATO MUTANT ERB (ENHANCED ROOT BRANCHING) HAS ALTERED AUXIN AND ETHYLENE RESPONSES

1,2 2 1Sangeeta Negi , Rameshwar Sharma and Gloria Muday

1 2Department of Biology; Wake Forest University; Winston-Salem, NC 27109; USA; School of Life Sciences; University of Hyderabad; Hyderabad-500 046; India

Lateral and adventitious roots form by initiation of cell division in pericycle cells of the primary root or hypocotyl, respectively. To better understand the molecular events that drive this process in tomato, we have isolated a gamma-irradiation induced mutant named erb (enhanced root branching) with 1.5-fold and 3-fold increases in lateral and adventitious roots, respectively, relative to its wild-type parent Ailsa Craig (AC). We have begun to characterize the hormonal responses that may drive enhanced root formation in erb. Auxin has long been known to positively regulate lateral and adventitious root formation, while a negative role for ethylene in lateral root formation has recently been uncovered. Exogenous indole-3-acetic acid (IAA) stimulates lateral root formation in AC, while in the erb mutant, similar doses of IAA inhibit lateral root formation. Additionally, the erb mutant has reduced acropetal and basipetal auxin transport in roots and enhanced transport in hypocotyls suggesting that this mutant is defective in long distance auxin transport or signaling. We also examined the ethylene responses in erb, and find that it is less sensitive to the inhibition of lateral root formation and triple response by 1-aminocyclopropane carboxylic acid (ACC), the ethylene precursor, compared to the wild type. We are using a candidate gene approach to ask if this mutant has a defect in genes implicated in auxin and ethylene signaling and transport. Characterization of the erb mutant should provide insight into the molecular mechanisms that modulate cross talk between auxin and ethylene in root development in tomato.


SOL 2009 245

SESSION VIII MOLECULAR BREEDING AND GENETIC ENHANCEMENT

246

ESTABLISHMENT OF GENETIC TOOLS FOR TOMATO FUNCTIONAL GENOMICS

1 1 1 1 1Tohru Ariizumi , Erika Asamizu , Takashi Saito , Tsuyoshi Mizoguchi , Naoya Fukuda , 1 2 2 3 3 2Chiaki Matsukura , Atsushi Kurabayashi , Kunihiro Suda , Ayako Suzuki , Kentaro Yano , Koh Aoki ,

1Hiroshi Ezura

1 2 3University of Tsukuba, Gene Research Center; Kazusa DNA Research Institute, School of Agriculture, Meiji University

Tomato is one of the most developing bio-resource materials attracting vast ranges of scientists who are interested in genomics, genetics, pathology, molecular and physiology. Tomato is also a benefit crop material for scientists who want to characterize its unique feature such as fruit development where other model plants (Arabidopsis, rice, legume, etc) do not produce in their life cycles. As a part of National BioResourse Project (NBRP) launched in Japan, a total of 3,620 mutagenesis lines of dwarf variety Micro-Tom, consisting of 2,078 and 1,542 lines were generated by EMS-mutagenesis and gamma-irradiation, respectively. Their M3 seeds are now available forscientists who want to use these materials upon request to NBRP

(http://tomato.nbrp.jp/indexEn.html). From our mutagenesis lines, a number of different varieties of mutants defective in organ development have been isolated and some mutants are being under characterization. Here we present construction of Micro-Tom bacterial artificial chromosome (BAC) library consisting of 50,000 clones.


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EXPRESSIVITY OF PS AND PS-2 GENES CONTROLLING FUNCTIONAL MALE STERILITY IN TOMATO

Elzbieta U. Kozik, Marzena Nowakowska

Research Institute of Vegetable Crops, 96-100 Skierniewice, Poland

The objectives of the study was to analyze the phenotypic variation in expression of male-sterility and morphological characteristics (growth habit, inflorescence length, number of flowers per inflorescence, fruit size) in 15 tomato lines with ps and ps-2 genes, and to discuss their level of sterility. Over an eight-year period (2001-2008) twelve ps and three ps-2 male sterile lines were evaluated in greenhouse conditions. Expressivity of sterility was determined by the number of seeds per seeded fruit. The lines differed for most of the morphological traits and in their tendency toward higher or lower sterility level. For most lines high fruit number was correlated with both low percentage of seeded fruit and low number of selfed seed. Based on the yield of selfed seed over an eight year period all the studied lines were divided into three categories: 1/ five lines with increasing level of sterility, 2/ six lines with varying sterility, and 3/ four lines with uniform and stable sterility. All three ps-2 lines had the highest level of sterility expressed by no selfed seed, while the ps lines differentiated significantly in number of selfed seed through the eight-year study.

FINE MAPPING OF THE MALE STERILITY GENE, MS1035 IN TOMATO

Hee-jin Jeong, Jin Kyung Kwon, Jung Hwan Bae, Sunghwan Cho, Hak sun Choi, Doil Choi, Byoung-Cheorl Kang

Dept. of Plant Science, College of Agricultural and Life Science, Seoul National University

The male sterile accession 2-517 contains ms1035, which shows abnormal flower development such as small flower, pale color of anther, protrusion of stigma. Flowers of 2-517 accession are pollenless and have empty pollen mother cells in the locules. It is reported that this trait is located on chromosome 2 between two RFLP markers per-2.3 and CT38. Genetic analysis showed that the ms1035 mutant phenotype segregated as a single recessive trait confirming the previous study. For fine mapping of ms1035, we took advantage of sequence information of Tomato chromosome 2, which is being sequenced by Korean efforts. Eight BAC clones corresponding to 67cM to 72.5cM on Chromosome 2 were selected to pinpoint the ms1035 gene. This BAC contig contains 109 genes in 758,417 base pairs. For fine-mapping, a total of 480 F2 plants were phenotyped and tested for cosegregation with SNP markers derived from the BAC contig. We developed a high-resolution linkage map around the ms1035 locus and narrowed the candidate region to about 79 kb. This region contains 6 predicted genes and we are currently testing for candidate genes for ms1035


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SOL 2009250

MUTANT ALLELES OF CHROMOPLAST SPECIFIC PHYTOENE SYNTHASE 1 GENE IN TOMATO

Hanjabam Mickey, Soni Gupta, Reddaiah B, Yellamaraju Sreelakshmi, Rameshwar Sharma


Carotenoids are one of the important natural pigments which play an important role in human health and nutrition. The dominance of red color in tomato fruits is due to lycopene, which occupies the major portion of the carotenoid pool. Carotenoids are synthesized through isoprenoid biosynthetic pathway in which phytoene synthase (Psy) plays the key role of conversion of GGPP to phytoene. In tomato PSY is encoded by two different genes, Psy1 (chromoplast) and Psy2 (chloroplast). Since Psy1 controls the total carotenoid pool in tomato fruit, the functional analysis of this gene will be of much importance. One of the tomato mutants, 'yellow flesh' was found to be lacking all the carotenoids in the ripened fruit, due to a natural single point mutation in the intronic region. The objective of the present work is to isolate induced mutants for Psy1 gene through TILLING (Targeting Induced Local Lesions In Genomes), which is a reverse genetic strategy for the identification of point mutations in specific genes of interest .High throughput screening of EMS mutagenized populations of two cultivars 'Arka Vikas' and 'M-82, having ~3000 lines each, led to the identification of a putative mutant for Psy1 gene by TILLING. The screening of fruit phenotype of the above populations in field led to the identification of another mutant line having low carotenoids conferring a yellow colored flesh to fruits. The mutant lines identified by both forward and reverse genetic screening will help in understanding the role of Psy1 in carotenoid biosynthetic pathway.

DEVELOPMENT OF SNP MARKERS USING HRM ANALYSIS AND CONSTRUCTION OF INTER-SPECIFIC LINKAGE MAP IN CAPSICUM

Jin-Kee Jung, Byoung-Cheorl Kang

Department of Plant Science, CALS, Seoul National University, Seoul 151-921

Single nucleotide polymorphism (SNP) is sequence variation by the difference of a single nucleotide in the genome and the most abundant in genomes of animal and plant. For discovery of SNP markers, 440 conserved ortholog set II (COSII) markers were amplified and sequenced. Two hundred three COSII markers were found to have SNPs between C. annuum “RNaky” and C. chinense “PI159234”. Intron-Based Polymorphic (IBP) was also developed using intron flanking segments. To detect SNPs, high resolution melting analysis (HRM) was performed. This method is the most inexpensive, simple, and rapid because, it requires only conventional PCR plus double strand specific fluorescence dye instead of expensive labeled probes. Up to now, we developed SNP markers which include 160 intron based polymorphic (IBP) markers and 140 COSII markers. These markers were placed in an interspecific pepper linkage map. The entire genetic map covered 2,998.9 cM with an average density of 8.3 cM.


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SOL 2009252

A RECESSIVE ALLELE OF ZEAXANTHIN EPOXIDASE (ZEP), INVOLVED IN THE ACCUMULATION OF HEALTH-BENEFICIAL ZEAXANTHIN AND ORANGE FLESH COLOUR IN POTATO TUBERS, DIFFERS FROM OTHER ZEP ALLELES BY THE PRESENCE OF AN INTRONIC NON-LTR RETROTRANSPOSON

Anne-Marie A. Wolters, Jan G.A.M.L. Uitdewilligen, Bjorn A. Kloosterman, Ronald C.B. Hutten, Richard G.F. Visser, Herman J. van Eck

Laboratory of Plant Breeding, Wageningen University, P.O. Box 386, 6700 AJ Wageningen, The Netherlands

We have investigated the genetics and molecular biology of orange flesh colour in potato. To this end the natural diversity in three genes of the carotenoid pathway was assessed by SNP analyses. Association analysis was performed between the SNP haplotypes and flesh colour phenotypes in diploid and tetraploid potato genotypes. We observed that among eleven beta-carotene hydroxylase 2 (CHY2) alleles only one dominant allele has a major effect, changing white into yellow flesh colour. In contrast, none of the lycopene epsilon cyclase (LCYe) alleles seemed to have a large effect on flesh colour. Analysis of nine zeaxanthin epoxidase (ZEP) alleles showed that all (diploid) genotypes with orange tuber flesh were homozygous for one specific ZEP allele. This ZEP allele showed a reduced level of expression. An epistatic interaction was observed between the CHY2 and ZEP genes. Only genotypes combining presence of the dominant CHY2 allele with homozygosity for the recessive ZEP allele produced orange-fleshed tubers that accumulated large amounts of zeaxanthin. The complete genomic sequence of the recessive ZEP allele, including the promoter, was determined, and compared with the sequence of other ZEP alleles. The most striking difference was the presence of a non-LTR retrotransposon sequence in intron 1 of the recessive ZEP allele, which was absent in all other ZEP alleles investigated. We hypothesise that the presence of this large sequence in intron 1 caused the lower expression level, resulting in reduced ZEP activity and accumulation of zeaxanthin.

A WIDE TOMATO GENOME SCANNING FOR R GENE MARKERS DEVELOPMENT

1 1 1 2 2 1 1Sanseverino W ,Carli P , Ferriello F , Van De Peer Y Rombauts, S , Frusciante L , Ercolano M.R .

1Department of Soil, Plant, Environmental and Animal Production Sciences, University of Naples “Federico II”,

2Via Università 100, 80055 Portici, Italy; VIB Department of Plant Systems Biology, Ghent University, Technologiepark 927, 9052 Gent, Belgium

Plant disease resistance genes (R-Genes) are an important class of genes from which a subset are well characterized at the molecular level. These genes play a key role in the recognition of the products of avirulence (Avr) genes from pathogens and in the activation of plant defence responses. In the Solanaceae family, 29 R-genes have been isolated and well studied. Starting with this genes pool, we were able to predict 257 putative R genes in tomato genome. In silico information were used to characterize some R regions by SNPs markers.

Such markers have been employed to detect R genes polymorphisms among closely related individuals within species (e.g. between elite cultivars) or between S. lycopersicum and closely related species. A SNaPshot multiple single nucleotide interrogation technique was adapted to identify simultaneously SNPs in tomato resistance genes. To facilitate detection of several polymorphisms in a single run, the length of the extension primers was adjusted to a distinct size by addition of a poly (dA) code to their 5' site. In conclusion, we developed a rapid, accurate, and thoroughly validated method to discover new sequences putative for resistance function in tomato genome. Results have been used to perform simultaneous genotyping of several polymorphisms in different resistance pathogen genes. This assay provides a useful tool to identify simultaneously more polymorphisms in multiple genes and to screening large populations.


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SOL 2009254

DEVELOPMENT OF A MUTANT POPULATION IN TOMATO AND HIGH THROUGHPUT PHENOTYPE DATA COLLECTION BY USING PDA

Reddaiah B, Raju Naik, Zakir Hussain, Vineeta Singh Chauhan, Osman Basha P, Vijee Mohan, Hanjabam Micky, Sherinmol Thomas, Chaitanya Ch., Suresh Kumar Gupta, Sapana N, Alka Kumari, Yellamaraju Sreelakshmi and Rameshwar Sharma

Department of plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad- 500 046, India

The present work aims to create a core collection of mutants in one of the representatives of Solanaceous species, tomato (Lycopersicon esculentum) Arka Vikas, which is a table variety in India, has been selected for this purpose. Two EMS (60 mM and 120 mM) mutagenized population were grown and a total of 4,000 and 3,000 seeds from M2 lines were collected, respectively, and systematically catalogued. High throughput phenotypic characterization of the M2 populations were done by tagging the individual plants with barcode labels which was scanned with the help of an inbuilt barcode scanner in portable palm top Personal Digital Assistant (PDA). PDA in concert with, PHENOME, specially designed data collection software, aided fast and hassle free documentation of the phenotypic traits of hundreds of plants in field. The data recorded in the field was then transferred to a master PC in lab where a central phenotype database of the populations is being maintained. The phenotypic variations in the populations were categorized and consisted of alterations mainly in plant architecture; leaf morphology and color; flower morphology and color; and fruit shape, size and color. The genomic DNA from M2 lines is currently serving as a resource for screening induced point mutations by a reverse genetics approach TILLING. The above mutant resource in tomato with a detailed phenotype database will add to functional genomics study of various genes similar to the model plant, Arabidopsis.

ELUCIDATION OF LE-ACS-2 MUTANT ALLELES IN TOMATO BY REVERSE GENETICS APPROACH, TILLING

Soni Gupta, Rajeswari Srinivasan, Vijee Mohan, Yellamaraju Sreelakshmi, Rameshwar Sharma

Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad – 500046, INDIA

In climacteric fruits such as tomato, ethylene is a key player and affects the transcription and translation of many ripening related genes. Till now no naturally occurring / induced mutant alleles for ethylene biosynthetic enzymes have been reported, which could have given a better insight of its role in ripening. The enzyme, 1-aminocyclopropane -1-carboxylic acid synthase (ACS), is one of the key enzymes of the ethylene biosynthetic pathway and out of the eight genes of the ACS identified in tomato; LeACS-2 has been shown to be responsible for the production of ethylene during climacteric phase of fruit ripening. Site directed as well as random mutagenesis have revealed the important amino acid residues regulating its enzyme activity. The current work envisages screening a mutagenized population for an allelic series of mutants for LeACS-2 by TILLING, in tomato. High throughput screening of eight folds pooled EMS mutagenized populations of Solanum lycopersicon cv Arka Vikas mediated by CEL I has led to the identification of some ACS-2 mutant alleles. The point mutations in these lines were confirmed by screening complementary 2-dimensionally arrayed plates and then by sequencing. Frequency of ACS-2 mutations in 60 mM EMS population was found to be 1 / 700Kbp. To obtain a wider spectrum of allelic series of mutants in this gene, 120 mM EMS treated population is being currently screened. Single nucleotide polymorphisms in the third exon of the ACS-2 gene were also observed in naturally occurring accessions by EcoTILLING. These allelic series of mutants obtained for the gene will provide useful resource for understanding its role in fruit ripening and also in developing varieties with longer shelf life.


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SOL 2009256

EXPLOITING GENETIC VARIATION FOR ELEVATED MINERAL CONCENTRATIONS IN POTATOES

1, 2 1 1 2Nithya Subramanian , Gavin Ramsay , Philip White , and Martin Broadley

1 2Scottish Crop Research Institute, Invergowrie, Dundee, Scotland – DD2 5DA; Division of Plant Sciences, University of Nottingham, Loughborough, LE12 5RD, UK

At least 22 minerals are required for the well-being of humans and these can be supplied by a balanced diet. Mineral deficiencies are common in both developing and developed countries and the enhancement of mineral elements in food crops represents a possible strategy to increase the human dietary mineral intake. To determine the prospects for this approach in potatoes (Solanum tuberosum L.), a diverse set of cultivated potato germplasm including the Phureja-Tuberosum Core Collection and the Neotuberosum population (derived from Andean tetraploids) were grown in randomized block replicated field trials. The mineral concentrations were determined using Inductively Coupled Plasma Mass Spectrometry on freeze-dried tuber samples. Minerals measured included those of human dietary significance such as iron, zinc, copper, calcium, magnesium and manganese in addition to others of more importance to the plant such as phosphorus, potassium and sulphur. Significant mineral variation was found between diploid Phureja and tetraploid Tuberosum clones. Neotuberosum clones showed a greater variation for most minerals and have a broader genetic background. We are using an F1 tetraploid mapping population, Stirling x 12601ab1, to study the genetic basis for this variation. Statistical analysis showed a significant variation between clones for tuber mineral concentrations and the QTL analysis for this population is currently underway. QTL mapping and identification of genes underlying mineral traits will enable marker-assisted breeding to enrich potatoes with essential dietary minerals.

ASCORBIC ACID AND POLYPHENOLS ACCUMULATION IN CALLUS CULTURE OF TOMATO INTROGRESSION LINES

M. Minutolo, A. Di Matteo, P. Chiaiese, E. Filippone, A. Errico

University of Naples - Dept of Soil, Plant, Environmental and Animal Production, Portici (NA), Italy

Antioxidants such as ascorbic acid (AsA) and polyphenols (Phe) have often been studied for their cancer-preventive and immune system empowering activity. Plant production of such antioxidant metabolites is quite low and not constant over life cycle. Our aim was to assess the production of AsA and Phe in tomato callus culture. In particularly, we have focused our attention on leaf and petiole explants of three introgression lines (IL7-3, IL10-1 and IL12-4) of Solanum pennellii into S. lycopersicum cv. M82 background expressing QTLs for fruit AsA concentration. Callus cultures were successfully obtained from plants growth in vitro and frequencies of callus and antioxidants content were scored at 0, 15, 30, 45 and 60 days of in vitro culture. Callus induction and proliferation were explants and genotype dependent. At stationary phase of callus growth the Phe content was found to be higher than the initial stages in all genotypes and explants assayed. On the contrary, total AsA accumulation decreased during callus growth. Thus, callus cultures may be used as system to enhance Phe synthesis and accumulation as model for investigating genetic mechanisms controlling antioxidant accumulation. Furthermore, efforts will focus on changes in gene expression leading to antioxidant accumulation through transcriptomic approach.


SOL 2009 257


SOL 2009258

IDENTIFICATION OF MARKERS LINKED TO TOMATO YELLOW LEAF CURL VIRUS RESISTANCE IN TOMATO (SOLANUM LYCOPERSICUM)

D.P. Sinha, Major Singh, Sanjeev Kumar, H.C. Prasanna, D. Dutta and Mathura Rai

Indian Institute of Vegetable Research, Post Box No. 01, P.O. Jhakhini (Shahanshahpur), Varanasi 221 305 (India)

Tomato yellow leaf curl disease caused by yellow leaf curl virus (TYLCV) is one of the most severe problems to tomato cultivation in India and in many temperate areas of the world. Typical symptoms of this disease are yellowing and curling of the leaf margins with distinguishable venal chlorosis. This disease is systemic and spreads to the other tomato plants through whitefly (Bemesia tabaci) in a persistent manner. Genetic resistance seems to be the best long-term strategy to control the damage caused by this devastating viral disease. Generally, the resistant genes available in wild taxa pose difficulty in introgression to cultivated species we focused on the cultivated species of tomato for resistance. A breeding line, H-88-78-2 originating from Solanum hirsutam expressing the resistant phenotype was crossed to a highly susceptible female parent, Punjab Chhuhara. The line H-88-78-2 also showed excellent resistance of whitefly inoculations. Hybrid plants from the cross H-88-78-2 and Punjab Chhuhara were selfed to develop F2 segregating population. This segregating population was analyzed using 42 SSR primers spanning the entire tomato genome. All the 42 SSR primers gave polymorphic products between the two parents during the screening; however, only the primers SSR179, SSR193 and TG324 produced polymorphism suitable to assess in segregating populations. The further studied are in progress to assess the efficiency of the identified markers in mapping of the TYLCV resistance genes.

STUDIES ON POPULATION STRUCTURE IN S. PIMPINELLIFOLIUM GERMPLASM AS A PREREQUISITE FOR ASSOCIATION MAPPING

E.S.Rao, Kadirvel Palchamy and Andreas Ebert

AVRDC, Taiwan

The currant tomato (Solanum pimpinellifolium) is a close wild species of cultivated tomato which is easily crossable and a source of genes for biotic and abiotic stress tolerance, nutritional quality and yield traits. Traditionally, linkage mapping has been a valuable tool for discovering marker/trait associations useful for crop improvement, but an alternative new approach showing much promise is association mapping especially when we have a diverse germplasm panel. Association mapping has the potential to play a central role in identifying genes encoding complex traits, but the possibility of spurious associations due to population structure has restricted the utility of these methods in the past. Recently the development of statistical techniques has removed much of the problem with spurious associations, or false positives in association mapping, allowing this technique to be used in a wide range of studies in crop plants. Hence as a prerequisite for association mapping studies, an effort has been made to understand the population structure in the S.pimpinellifolium germplasm. A germplasm consisting of 192 accessions from regions including 12 countries of South and North Americas have been assembled for the study. Population structure inferred from SSR data at 46 loci chosen to ensure coverage of the entire genome and statistically analysed using the STRUCTURE 2.2 software (Falush et al., 2003; Pritchard et al., 2000) will be presented and prospects for association mapping shall be discussed.


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SOL 2009260

PRELUSIVE SURVEY OF TOMATO LINES WITH MICROSATELLITE MARKERS FOR EFFECTIVE MAPPING STUDIES

Smita Singh, Prabash Chandra Singh, Major Singh, Rajesh Kumar, and Mathura Rai

Crop Improvement Division, Indian Institute of Vegetable Research, Jakhini (Shahanshahpur), Varanasi-221305

Genetic markers have been employed in several diversity analyses and construction of maps in tomato. Among these microsatellites markers (codominant) are valuable source of information. This study surveys the usage of SSR markers for mapping populations.

These lines were chosen as morphologically contrasting wild and cultivated parental pairs to produce segregating populations to be used in mapping QTL for disease resistance and fruit quality traits in tomato crop. viz, (Co-3 X EC-520061, Co-3 X NCEBR-4, Punjab Chuara X H-88-78-1, Punjab Chhuhara X EC 538408, BL1208 X VRT-32, Sel-7 X PDT-3-1, H-86 X EC 538380, Punjab Chhuhara X H-86 ). Polymorphic survey was done using five hundred SSR primers, in contrasting parents, yielded 10-12% polymorphism depending on the cross. Potentially useful bands were characterized as having mobility between 80-1500 bp. Putative marker loci were identified as bands that were bright and reproducible and used for further analysis. This represented on average 6.8% recovery of potentially useful bands (loci) from the survey. From the above results, 82 bands (loci) were applied in F2 population derived from Co-3 X EC-520061 cross which segregated in a predictable manner represented 4.2% recovery of useable marker loci. Further to know the genetic relation between the parental pairs the SSR data were analyzed in UPGMA clustering, NT Sys Software.

We found, out of eight crosses only three Co-3 x EC-520061, Pb.Ch X EC-538408, Pb.ChXH-88-78-1, showed large molecular variation whereas other crosses showed narrow variation between themselves.

The results of the present study could be attributed to the fact that selection of mapping population for its utilization in QTL association studies would be beneficial with significant morphological and molecular variations between the respective crosses. Overall, a standard marker array and reference lines used in this study will provide powerful set of tools for genetic and molecular studies in tomato lines.

SESSION IX BIODIVERSITY

261


SOL 2009262

CHROMOPLAST SPECIFIC LYCOPENE BETA CYCLASE GENE PROMOTER SHOWS HYPERVARIABILITY IN TOMATO

Vijee Mohan, Yellamaraju Sreelakshmi, Rameshwar Sharma


Conversion of lycopene to β-carotene in tomato is mediated by a chloroplast specific lycopene β-cyclase enzyme. A paralog of this gene exists in tomato and it has the same function but its expression is specific to the chromoplast hence, called chromoplast specific lycopene beta cyclase gene (B-gene). In most of the tomato varieties lycopene content in fruits is at least ten times more than the β-carotene content. The orange colored high beta-carotene containing fruits of tomato mutant 'Beta' was found to be due to the increased expression of B-gene resulting from changes in its promoter region. In the present study in order to find out the promoter variations which might have happened during evolution, the promoter regions from different wild relatives of Solanum lycopersicum were amplified and sequenced. Alignment of the sequences showed high variation between different Solanum species. A screening of 615 tomato accessions based on amplicon length further revealed the occurrence of several natural variations in the promoter region of B-gene. Sequencing and BLASTn search in NCBI database for the promoter region of B-gene in one of the accessions, LA0348 revealed the presence of a probable Nup (Nuclear localized plastid) DNA fragment of around 250bp from rps4 (ribosomal protein small subunit 4) gene of the tomato chloroplast genome. But in all the above mentioned cases the exon size was found to be the same. These results imply that the promoter region of B-gene might be a hot spot for variations.

A COMPARATIVE STUDY ON ONTOGENIC EXPRESSION OF ANTIOXIDANTS AND SECONDARY METABOLITES IN IN VITRO AND IN VIVO GROWN LEAVES OF WITHANIA SOMNIFERA

Nathiya. S, Savitha. C, Santhi. N and Kalaiselvi Senthil

Department of Biochemistry, Biotechnology and Bioinformatics, Avinashilingam University for Women, Coimbatore. Withania somnifera (WS), also known as ashwagandha, Indian ginseng and winter cherry, has been an important herb in the Ayurvedic and indigenous medical systems for over 3000 years. The objective of the study is to estimate the antioxidant activity and secondary metabolites present in both in vitro and in vivo grown leaves of Withania somnifera at different time period. The estimated antioxidant enzymes were superoxide dismutase, catalase, glutathione peroxidase and glutathione-s-transferase. The secondary metabolites from leaves were extracted with increasing and decreasing polarity of solvents and analysed by thin layer chromatography using chloroform: methanol (9:1) as solvent system. The chromatogram was developed using 10% sulphuric acid. It was found that ethyl acetate extract of decreasing polarity contains all the metabolites present in the leaves. The level of enzymatic antioxidants were found to be present in increased quantity at third month compared to first and second month leaves of in vitro maintained plants, whereas there is no significant change in the level of enzymatic antioxidants in the leaves of in vivo grown plants. Search for the concerned active compounds has led to the identification of several withanolides, alkaloids, flavonoids, steroids, phenolics, saponins and quinones from leaves of Withania somnifera. These findings have high significance in the pharmacological industry.


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SOL 2009264

GENETIC DIVERSITY IN CAPSICUM GERMPLASM AS REVEALED BY MICROSATELLITE MARKERS

Ved Prakash Rai, Sanjay Kumar, Neeraj Dwivedi, Rajesh Kumar*, Major Singh and Mathura Rai

Indian Institute of Vegetable Research, PB. No. 01, PO. Jakhini (Shahanshahpur) Varanasi 221 305

The present study on genetic diversity was conducted with 48 diverse genotypes, selected from four species of Capsicum based on morphological variability with respect to plant type, fruit shape, disease resistance and pungency, using simple sequence repeats (SSR) and random amplified microsatellite polymorphism (RAMPO) markers during 2008-09 at IIVR, Varanasi. A total of 102 SSR primers were screened among six diverse genotypes initially for the search of polymorphism, out of which 26 SSR primers were found to be polymorphic and further used in all the 48 genotypes for diversity analysis. Twenty six SSR primers amplified a total of 94 alleles, varying from 2 (CAMS-142) to 5 alleles (CAMS-348) with polymorphism information content (PIC) ranging between 0.28 (CAMS-844) to 0.92 (CAMS-846). Dendrogram generated through UPGMA based cluster analysis by NTSys pc 2.1 software, grouped the genotypes in to four clusters. Clusters I consisted of three, cluster II of six, cluster III of one and cluster IV consisted of 38 genotypes, respectively. Pepper leaf curl virus resistant genotype GKC-29 separated alone from the cultivated genotypes at coefficient of 0.39, indicating reason for non-crossabiity with other genotypes (as attempted several times in the past). In another approach, RAMPO, firstly RAPD reactions were performed and then PCR products were taken as template for ISSR (inter simple sequence repeats) analysis. Twenty primer combinations (4 RAPD primers and 5 ISSR primers) based on greater polymorphism were tested for their ability to generate RAMPO banding patterns from 48 genotypes. Among 180 generated bands, 140 were found polymorphic (77 %). The number of markers varied from 2 (OPL-19 x BV 35) to 8 (OPJ-17 x BV-17) with a mean of seven bands per primer combination. UPGMA based cluster analysis grouped the genotypes into three major clusters. Genotypes Bhut Jolokia, Tripura collection, COO-304, BS-35, IC-383072 were separated from the others. The present study portrays the efficiency of SSRs and RAMPO procedures for the genetic diversity study in chilli genotypes.

SESSION X RNAI AND EMERGING AREAS

265


SOL 2009266

CONTROL OF HELICOVERPA ARMIGERA LARVAL GROWTH AND DEVELOPMENT BY RNAI STRATEGY

Manoj Goel, Maneesh Kumar, Mamta Kaushik, R Srujana, M V Rajam

University of Delhi, south campus, New Delhi-110021

RNA interference has been proved as novel and potential approach for control of diseases and insect pests. This involves the introduction of hairpin dsRNA in transgenic plants targeting vital genes of insects. For present study, acaetylcholinesterase (AChE), Juvenile hormone acid methyltransferase (JHAMT) and Cathepsin L (HarCL) genes, which play key role in growth and development of insects were identified as potential RNAi targets for protection of crop plants against insect. We studied the effect of siRNA on larval development by selective targeting of AChE gene of Helicoverpa armigera, along with other genes. Different concentrations of siRNA were fed to H. armigera larvae through artificial diet, which resulted in mortality, retarded larval growth and pupal malformation, and low fecundity against control ones. No adverse effect was seen on larval growth by unrelated siRNA, which indicates the specificity of AChE-siRNA effect. The effect of gene silencing was observed both at transcription and protein level. Our studies demonstrate that siRNA can readily be taken up along with the artificial diet and also reveal the role of AChE in growth and development of insect larvae. Besides, the efficacy of siRNAs specific to JHAMT and HarCL genes on growth and development of H. armigera larvae is also being examined. Further, RNAi constructs harbouring insect target genes were prepared and are being used to develop insect resistant transgenic plants of tomato, brinjal and cotton.

RNA SILENCING SUPPRESSORS ENCODED BY BETA SATELLITE OF TOMATO BEGOMOVIRUSES

Richa S., Haq.Q.M.I., Neha T., V.B. Singh, P. Jyothsana, Archana K., V.G. Malathi


Virus encoded RNA silencing suppressors (RSSs) are the key components evolved by the viruses to counter RNA silencing defence of plants. Whitefly transmitted begomoviruses. Infecting tomato crop code for three different proteins, ORF AC4, ORF AC2 in DNA-A component and ORF βC1 in satellite DNA β which are predicted to function as silencing suppressors. In the present study suppressor function of ORF βC1 of two satellite DNA β as Tomato leaf curl Bangalore beta satellite (Bang βc1) and cotton leaf curl multan beta satellite (Gang βC1) are examined. The ORF βC1of both satellites was cloned in the plant expression vector PBI 121 and mobilized into Agrobacterium tumefaciens strain EHA105. Agroinfilteration of transgenic GFP silenced Nicotiana tabaccum xanthi done with the cells having 35S-GangβC1 and 35S-BangβC1 resulted in expression. It was comparable with activity of another well established suppressor, ORF-AC2 of Mungbean yellow mosaic India virus


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SOL 2009268

SILENCING OF SUPPRESSORS: A MICRORNA APPROACH

1 1 1Vikas Koundal , Suneha Upadhyay , Shelly Praveen

1Advance Centre for Plant Virology, Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi 110012, India

RNA silencing is a sequence-specific defense mechanism of plants against invading viruses. As a counter defense, viruses evolved silencing suppressors to interfere with host silencing. Most of these proteins bind 21 to 24 nucleotide long double stranded RNA (miRNA, siRNA) molecules which have a key function in RNAi.The biogenesis of mature miRNA is not affected if few nucleotides are changed in the mature miRNA sequence. Thus, by replacing natural mature miRNA sequences in the miRNA precursors with the virus specific sequences, artificial miRNAs (amiRNAs) can be generated. Hence, for functional gene analysis, amiRNAs can be designed to target any gene of interest. In the present study, amiRNAs targeting two different RNAi silencing suppressors i.e. AC4 of Tomato leaf curl New Delhi virus and another suppressor NSs of Groundnut bud necrosis virus were designed using siRNA design tools. These amiRNAs were cloned in the backbone of Arabidopsis miRNA159a precursor and subcloned into an Agrobacterium binary vector. Transgenic N. tabacum lines expressing amiRNA-AC4 were challenge inoculated with viruliferous whiteflies and ToLCV resistance was evaluated by quantifying AC4 transcript using qPCR. Similarly GBNV infected cowpea plants were agro-infiltrated with amiRNA-NSs and down regulation of GBNV was assayed using ELISA. Both qPCR and ELISA data shows that both the designed amiRNAs confers down regulation of viruses. The BLAST analysis showed that designed siRNAs had 100 percentage homology with other related viruses, suggesting its possibility to silence these viruses also.

SATELLITE SESSION I POTATO

269


SOL 2009270

POTATO CYST NEMATODE: NEW LEGISLATION, NEW TECHNOLOGY

Reid, A.

Scottish Agricultural Science Agency, Roddinglaw Road, Edinburgh, EH12 9FJ, UK.

In July 2010 the new European Union Potato Cyst Nematode directive comes into force affecting fields scheduled for planting from 2011. The result of this new legislation will be to increase sampling rates approximately fourfold throughout Europe. Currently samples are examined at SASA by visual observation of the float material for PCN cysts which are subsequently identified as Globodera palida or G. rostochiensis based on a range of morphological characteristics. All of the soil samples sent to SASA have to be processed in a very short period of time prior to the beginning of the planting season and due to the significant rise in the number of samples that will be require examination it will be impractical to continue using visual examination to fulfill our obligation for PCN detection. Therefore a new, high throughput real-time PCR detection method has been developed for use directly on float material to enable SASA to process the large numbers of samples in a timeous nature.

GENETIC ANALYSIS OF RESISTANCE TO COLD-INDUCED SWEETENING IN POTATO TUBERS

1,2 1 1 2 1Sagar Datir , Martin L. Shaw , Kathryn Wright , Hayley J. Ridgway , Jeanne M.E.Jacobs , 1,2

Anthony J. Conner

1 2New Zealand Institute for Plant & Food Research; Private Bag 4704, Christchurch, New Zealand; Faculty of

Agriculture and Life Sciences, Private Bag 84, Lincoln University 7647, Canterbury, New Zealand

Cold-induced sweetening (CIS) is an important quality trait in potato breeding programmes worldwide. Post-harvest storage of potato tubers at low temperature causes accumulation of reducing sugars which limits the processing quality. The acceptability of such potatoes for processing into French fries and chips is dependent on the colour of the final product which is directly related to the sugar and dry matter content in the tuber. To facilitate the breeding of resistance to CIS we are investigating the genetic basis of this trait. A tetraploid mapping population of over 300 progeny was established from a cross derived from Crop20 (a breeding line with very high resistance to CIS) and Karaka (a cultivar with high susceptibility to CIS). The effects on processing quality were determined immediately after harvest and after one and four months of cold storage at 6°C (with and without ten days of reconditioning at 18°C). Phenotypic traits such as dry matter, reducing sugar and starch content are being measured in all progeny. Cold storage resulted in segregation for unacceptable chip colour scores although reconditioning improved the processing quality of some lines. The inheritance of these quality related traits will be presented.


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WHOLE GENOME PROFILING OF THE DIPLOID POTATO CLONE RH89-039-16

1 1 1 1 2 2Edwin van der Vossen , Richard Feron , Jan van Oeveren , Kim Jansen , Jan de Boer , Christian Bachem , 3 1 1Bert Bogden , Marcel Prins , Michiel van Eijk

1 2 3 Keygene N.V.; Potato Genome Sequencing Consortium; Amplicon Express

KeyGene has developed Whole Genome Profiling (WGP), a new cost effective method to construct high quality sequence-based physical maps. Using the Illumina Genome Analyzer II, WGP maps are constructed by sequence-based fingerprinting of a BAC library. BAC clones are ordered into contigs using sequence-based anchor points forming a whole genome physical map. Here we report on the WGP of the diploid potato clone R89-39-16, thereby generating a sequence-based scaffold for assembly of the potato whole genome shotgun (WGS) sequence that has been generated within the international Potato Genome Sequencing Consortium (PGSC). A total of 86,016 BAC clones (11.4 genome equivalents), 55,296 from an RE based library (RHPOTKEY) and 30,720 from a random sheared library (RHPOTLUC), with average insert sizes of 120kb and 90 kb, were analyzed. The WGP map was assembled using an improved version of FPC software (Keygene N.V.), capable of processing sequence-based BAC fingerprint (WGP) data instead of fragment mobility information as used in the original FPC software (Soderlund et al., 1997: Comput. Appl. Biosci. 13: 523-535). Using relatively conservative FPC settings, a total of 57,890 BAC clones were assembled into 3459 contigs (~ 8x effective coverage), with an N50 contig size of 30 BAC clones and 465 Kbp. The calculated genome coverage of 1125 Mbp approximates 1.3x the potato genome size (840 Mbp), reflecting the heterozygous nature of the diploid potato clone RH89-39-16. This potato WGP map will facilitate assembly of the potato WGS sequences that have been generated within the international PGSC.

SATELLITE SESSION II COFFEE

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IDENTIFICATION OF GENES DISPLAYING DIFFERENTIAL EXPRESSION IN LEAVES FROM COFFEE PLANTS (COFFEA CANEPHORA AND C. ARABICA) GROWN UNDER DIFFERENT WATER STRESS CONDITIONS

1, 2 2 2 2 2Pierre Marraccini , Gabriel S. C. Alves , Felipe Vinecky , Luciana P. Freire , Natalia G. Vieira , 3 4 2 2Humberto J O Ramos , Gustavo C. Rodrigues , Carlos Bloch Jr. , Alan C. Andrade

1 2CIRAD UMR DAP, Montpellie; France; Embrapa Recursos Genéticos e Biotecnologia; LGM-NTBio, Brasília- 3 4DF, Brazil; IAPAR, LBI-AMG, Londrina-PR, Brazil; Embrapa Cerrados, Planaltina-DF, Brazil

With the aim of establishing breeding programs based on marker-assisted selection for drought tolerance on coffee, the present work had as a goal, the identification of candidate genes for this trait. The data was obtained by comparing gene expression and protein profile of different genetic materials (tolerant vs. susceptible) as well as, under different conditions of water supply (irrigated vs. non-irrigated). In this work, the genetic materials studied were the Conillon clones 22 and 14 (Coffea canephora) and the cultivars Rubi and Iapar 59 (C. arabica). The applied water stress (PD=-3, 0 MPa) to the conillon plants was achieved under green-house conditions and, in the case of Arabica, adult plants cultivated under field conditions were used. In the later case, leaves were collected during day and night, and the most pronounced observed water-stress was of PD=-1, 7 MPa. After selection of candidate genes by different strategies, the expression was confirmed by qPCR analysis. The data obtained indicated that several genes displayed decreased expression upon water stress and usually these were encoding-genes of proteins involved in photosynthesis. On the other hand, the applied water stress on coffee plants also induced a set of genes such as RD29, DREBA and NAC, which have already been described in literature as genes involved in plant responses to drought. Results also confirmed previously observed differential expression of a Dehydrin encoding-gene upon water stress. In addition, this study revealed the importance of other factors controlling the expression of these genes, such as the circadian clock and the age.

FRAMEWORK MOLECULAR LINKAGE MAP OF ROBUSTA COFFEE (COFFEA CANEPHORA): FIRST GENERATION MAP DEVELOPED USING PSEUDO-TESTCROSS STRATEGY WITH RAPD, AFLP, G-SSRS AND EST-SSRS MARKERS

Ramesh K. Aggarwal, Prasad Suresh Hendre, A. V. Rao

Centre for Cellular and Molecular Biology, Uppal Road, Tarnaka, Hyderabad- 500 007, India

We describe for the first time, a linkage map of cultivated diploid coffee C. canephora (2n= 22), developed using RAPD, AFLP and SSR markers by applying pseudo testcross strategy. A trait specific (drought tolerance) mapping population comprising 175 plants, was developed by crossing CxR hybrid (C. congensis X C. canephora) and a local selection Kagganahalla (C. canephora). This population was used to generate segregation data using different DNA markers; which comprised 282 RAPDs, 181 AFLPs and 97 novel SSR specific segregating alleles. Of these, 110 marker alleles exhibited distorted segregation ratio (P<0.01, 19.64% of all polymorphic) and thus excluded from the data. Subsequently, 374 of the polymorphic/segregating alleles (about 68% of total) could be mapped using JoinMap 3.0 package. The resulting map comprised eleven major linkage groups (LGs) having 17 to 48 markers, and five minor LGs (with 2 to 12 markers). Further, the 30 accessory/floating markers on the 11 major LGs could also be mapped on eight accessory LGs. Overall, the combined (integrated) robusta map spanned a total length of ~1230 cM with average marker spacing of 3.6 cM. Similarly, the individual parental maps of CxR and Kagganahalla were generated which were comparable with the combined map. The genome coverage estimate indicated near complete coverage for the integrated map. To our understanding, this is the first molecular linkage map of cultivated robusta coffee where large numbers of SSRs have been mapped, using the pseudo testcross strategy. Further, the map is expected to be of immediate utility for undertaking QTL analysis, followed by MAS in near future.


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IDENTIFICATION, CHARACTERIZATION AND UTILIZATION OF EST-DERIVED GENIC MICROSATELLITE MARKERS FOR GENOME ANALYSES OF COFFEE AND RELATED SPECIES

Ramesh K. Aggarwal*, R. Prasoona, Prasad S. Hendre, A. V. Rao

Centre for Cellular and Molecular Biology (CSIR), Uppal Road, Tarnaka, Hyderabad-500007, India

Analysis of variation at DNA level using various DNA-marker approaches has become the key for modern genetics studies. Despite the apparent advantages, only few such markers have been developed/ described for coffee, which is an important beverage and plantation crop belonging to genus Coffea (family Rubiaceae); moreover, these efforts have generally been limited to genomic microsatellites. Genic microsatellites or EST-SSRs represent one other class of microsatellite markers that are: derived from expressed sequence tags (ESTs), inexpensive to develop utilizing available transcriptome resources, and often a putative function can be assigned to them. In this study, we attempted identification and development of genic microsatellite markers using 2553 coffee ESTs (461 from the public domain and 2092 in-house generated ESTs). These ESTs were searched for SSRs using MISA –search module followed by stackPACK clustering that revealed a total of 425 microsatellites in 331 (13.5%) non-redundant ESTs/consensus sequences suggesting an approximate frequency of 1 SSR/2.16 kb of the analysed coffee transcriptome. Identified microsatellites mainly comprised of di-/tri-nucleotide repeats, of which repeat motifs AG and AAG were the most abundant. A total of 224 primer pairs could be designed from the non-redundant SSR-positive ESTs for possible use as potential genic markers. About 10% of these primer pairs were tested and 18 could be validated as usable markers. Sixteen of these markers revealed moderate to high polymorphism information content (PIC) across 23 genotypes of C. arabica and C. canephora, while 2 markers were found to be monomorphic. In general, the new markers showed broad cross-species/genera transferability (also confirmed by cloning and sequencing of the amplified alleles) when tested across 14 Coffea and 4 Psilanthus species. Thus, the study provides an insight about the frequency and distribution of SSRs in coffee transcriptome, and also demonstrates the successful development of genic-SSRs. It is expected that the 200+ potential markers developed in the study would greatly help DNA markers based genetic studies on cultivated coffee, as well as, related taxa that constitute the important secondary genepool for coffee improvement.

DEVELOPMENT OF NEW GENOMIC MICROSATELLITE MARKERS FROM ROBUSTA COFFEE (COFFEA CANEPHORA PIERRE EX A. FROEHNER) SHOWING BROAD CROSS-SPECIES TRANSFERABILITY AND UTILITY IN GENETIC STUDIES

Prasad Suresh Hendre, Regur Phanindranath, Ramesh K. Aggarwal

Centre for Cellular and Molecular Biology (CCMB), Uppal Road, Tarnaka, Hyderabad- 500 007, Andhra Pradesh, India

Species-specific microsatellite markers are the most desired for genetic studies and to harness the potential of MAS based breeding for genetic improvement. Limited availability of such markers for coffee, one of the most important beverage tree crop, warrants newer efforts to develop additional microsatellite markers that can be effectively deployed in genetic analysis and improvement programs of coffee. In this study, 44 new coffee-specific SSR markers were successfully developed using the conventional approach of screening/mining of the genomic library albeit with low efficiency. A small-insert partial genomic library (made in pMOS plasmid vector) of Coffea canephora was probed for various SSR motifs by Southern hybridization. Characterization of the putative repeats positive clones by sequencing revealed a very high abundance of DNRs (1/15 Kb) over TNRs (1/406 kb). The relative frequencies of different DNRs were found as AT>>AG>AC, whereas among TNRs, AGC was the most abundant repeat. The SSR positive sequences were used to design 58 primer pairs of which 44 pairs could be validated as single locus markers using a panel of arabica and robusta genotypes. The analysis revealed an average of 3.3 and 3.78 alleles and 0.49 and 0.62 PIC per marker for the tested arabicas and robustas, respectively. The markers were tested for Hardy-Weinberg equilibrium, linkage dis-equilibrium, and were successfully used to ascertain generic diversity/affinities in the tested germplasm (cultivated as well as species). Nine markers could be mapped on robusta linkage map. Importantly, the markers showed ~92% transferability across related species/genera of coffee. The characterization/validation studies thus show that the new markers are highly informative. These can be used studies on: genetic diversity in coffee germplasm, individualization/bar-coding for germplasm protection, linkage mapping, taxonomic studies; and possibly also as conserved orthologous sets across secondary genepool of coffee.


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EPIC-PCR REVEALS HIGH LEVEL OF POLYMORPHISM IN THE THIRD INTRON OF N- METHYLTANSFERASES INVOLVED IN CAFFEINE BIOSYNTHESIS IN COFFEA SP

P.S.Simmi, P.Giridhar, Avinash Kumar and G.A.Ravishankar

Plant Cell Biotechnology Department, Central Food Technological Research Institute, Mysore- 570 020

S-Adenosylmethionine dependant N-methyl transferases (NMTs) are involved in the sequential methylation of Xanthosine at the N7, N3 and N1 locations to produce caffeine in Coffee. Previous work on caffeine biosynthesis in Coffea arabica has led to the cloning and characterization of seven different NMTs namely CaXMT1, CmXRS1, CaMXMT1, CaMXMT2, CTS1, CaDXMT1 and CCS1, each having different Km value and different substrate preferences. Apart from these a few genes with no known substrate specificity in vitro, have also been mentioned. These NMTs share a high level of similarity in both protein and nucleic acid sequence indicating that they have evolved via gene duplication events. To further strengthen this postulation we isolated the genomic sequences of the second NMT- theobromine synthase, from C. canephora using homology based PCR methods. Seven genomic clones were obtained, two of which appear to be pseudogenes due to the presence of premature stop codons in the exonic regions. Global alignment of the coding region of the rest five clones indicates closer match to the theobromine synthase 'CaMXMT1' cDNA of C. arabica. Interestingly, all these genomic clones have striking similarity in the coding regions but differ mainly in the size and sequence of the third intron, the reasons for which is not known. In order to identify more such genomic variations we used Exon-Primed Intron Crossing PCR (EPIC-PCR) using primers annealing to conserved sequences of the third and fourth exon. EPIC-PCR was carried out with genomic DNA of thirteen different species of Coffea including C. arabica and C. canephora. At least fifty five polymorphic loci were identified among the thirteen species by separation on 5.5% polyacrylamide gel, indicating high complexity in the evolution of these genes. The polymorphic loci were cloned and sequenced to identify the levels of diversity. A probable path for the evolution of the NMTs has been described based on the diversity between the intronic sequences.

IDENTIFICATION OF ROOT AND ASSOCIATED PHYSIOLOGICAL TRAITS USING MORPHOLOGICAL CHARACTERIZATION AND RAPD MARKERS IN COFFEA CANEPHORA PIERRE EX. FROEHNER ACCESSIONS

1 2 2 2 2Mallikarjun G. Awati , Anand, C. G. , Venkataramanan, D. , Renuka Swamy, N. S. , D'souza, G. F. ,

3 3Udaya Kumar, M and Jayarama

1 2Regional Coffee Research Station, Thandigudi - 624 216, Dindigul District,Tamil Nadu, India; Central Coffee 3Research Institute, C R S (Post) - 577 117, Chikmagalur District, Karnataka, India ; Department of Crop

Physiology, University of Agricultural Sciences, GKVK, Bangalore-560 065, Karnataka, India

A study was conducted using 31 robusta accessions from Central Coffee Research Institute (CCRI), Karnataka, India with an objective to discriminate the robusta accessions for root traits and to estimate their genetic relatedness. Eighteen 10mer RAPD (Random Amplified Polymorphic DNA) primers were used to amplify DNA by the polymerase chain reaction (PCR) and thirty morphological and physiological traits were used to identify the high and low root biomass traits in robusta coffee. Eighteen 10mer RAPD (Random Amplified Polymorphic DNA) primers generated total 256 fragments, out of which 157 bands were polymorphic in nature. Based on morphological and physiological traits and genetic (RAPD) markers cluster (dendrogram) three and two dimensional scaling analyses were performed. Considerable genetic variability was found in cluster analyses based on morphological and physiological traits for root traits. Cluster analyses of morphological and physiological characters revealed a maximum linkage distance of 520 SED units. Where as, a maximum of 138 SED units were noticed in dendrogram based on RAPD markers. The RAPD markers revealed a genetically low variation among the robusta accessions even though a large variation was noticed in the morphological and physiological traits. Comparison of both dissimilarity matrices generated by RAPD markers and physiological and morphological traits revealed a significant (p<0.05) correlation (r=0.418) between the clustering obtained with molecular and morphological characters in robusta accessions. It is evident that the correlation between RAPD markers and morpho-physiological characteristics exists in robusta coffee.


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MARKER ASSISTED SELECTION IN COFFEE (COFFEA ARABICA L.)

1 2 1 2 3 1Sundaresha , M.H Divya , B. Muniswamy , B.T. Hanumantha , N. Surya Prakash , H.L Sreenath , 3 2 3 4

S.S. Bhat , C. B. Prakasan , Jayarama and P. Lashermes

1 2Coffee Biotechnology Centre, Coffee Board, Mysore – 570 006, India; Coffee Research Sub-Station, Coffee Board,

3Chettalli, Kodagu, Karnataka, India; Central Coffee Research Institute, Coffee Board, CRS - 577 117, India; 4Institut de Recherche pour le Développement (IRD), BP 64501-34394 Montpellier Cedex 5, France

Coffee leaf rust (CLR) popularly known as orange rust caused by the obligate parasitic fungus Hemileia vastatrix is a major disease of concern and an important limiting factor for arabica coffee (Coffea arabica L) production in almost all growing countries, including India. Resistance to coffee leaf rust is reported to be determined by at least nine resistance genes, SH1 to SH9, either singly or in combination; the corresponding virulence factors in the pathogen are v1 to v9. Of these resistance factors, SH1, SH2, SH4 and SH5 are identified in the tetraploid species C. arabica. SH6, SH7, SH8 and SH9, have been introgressed to C. arabica from the diploid species C. canephora, while SH3 is introgrssed from another diploid species C. liberica. The genes introgressed from the diploid species are found to be more durable and provided longer-lasting protection to arabica under field conditions, as compared to the genes identified in tetraploid C. arabica itself. Hence, accumulating resistance genes introgressed from diploid coffee species in one genotype (gene pyramiding) or using them in composite varieties is expected to provide durable CLR resistance to arabica coffee. Availability of molecular markers linked to SH genes and other genes that influence resistance to CLR could be extremely useful for marker-assisted breeding for CLR resistance.

In early coffee breeding programmes of India, S. 26, a spontaneous hybrid of C. arabica and C. liberica was used as main source of CLR resistance and the country's most popular cultivar S.795 (Selection 3) was evolved. The populations of S.795 carry SH3 CLR resistance factor introgressed from C. liberica. The main objective of the present study was to validate two sequence-characterized DNA markers (Sat244 and BA-124-12K-f) reported to be closely linked to SH3 gene and application of these markers for maintenance breeding of S.795 through marker-assisted selection.

A random survey was undertaken in S.795 populations established as early as 1950s to 1970s. Around 150 plants manifesting different levels of CLR build up, plant vigour and berry yield were marked and used for SCAR assays. Both the markers gave clear amplification profiles that could distinguish the presence or absence of SH3 gene. Based on the marker data, SH3-positive and SH3-negative plants were identified and characterized for CLR resistance pattern by screening with CLR race I & VIII. In general, the marker data correlated well with the expected CLR resistance/susceptibility and plant vigour observed in the field. Backed with this positive correlation between the presence of SCAR markers, presence of SH3 gene and CLR resistance in the random populations, SCAR analysis has been taken up for selecting desirable plants of S.795 cultivar. Seed gardens will be established from these elite plants by clonal propagation. This is the first report on successful application of marker-assisted selection for CLR resistance and has potential implications for durable CLR resistance breeding.

SRAP MARKER: A PROMISING APPROACH FOR MOLECULAR GENETIC ANALYSIS IN COFFEE

1 1 1 1 2 1M. K. Mishra , Asha Bhat , N. Suresh , S. Satheesh Kumar , Anil Kumar , N. SuryaPrakash , 2 1

M. Jagadishan , Jayarama

1Central Coffee Research Institute, Coffee Research Station, Chikmagalur District- 577117, Karnataka, India;

2Regional Coffee Research Station, Thandigudi – 624 216, Dindigul Dist, Tamil Nadu, India.

The major constraint that limits the arabica coffee (Coffea arabica) breeding is the selection of parental lines and identification of hybrids at an early stage of plant growth based on morphological traits. This is because most of the commercial arabica cultivars are morphologically indistinguishable from each other. Uniformity of morphological traits is due to the narrow genetic origin and self fertility in C. arabica. Compared to the morphological and biochemical markers, molecular markers are more precise and reliable to discriminate closely related species and cultivars and therefore widely used in marker assisted breeding programs. Among the various molecular markers, a simple marker technique called sequence–related amplified polymorphism (SRAP) is used extensively in recent years for estimating genetic diversity and phenetic relationships in many crop plant species. In the present study, the scope and potential of SRAP marker approach was tested for analyzing hybrid status of selected F1 hybrids of C. arabica. In all, 115 F1 hybrids belonging to 10 different hybrid combinations were screened with 80 SRAP primers. Among the primers tested 34 primers were found highly polymorphic and generated consistent amplification products and therefore selected for further analysis. Based on the amplification profile in parents and hybrids, these 34 SRAP primers could be classified in to eight different groups. Clear differences in banding pattern were obtained between the parental lines using specific primer combinations. Comparison of SRAP banding pattern of the parents and their respective F1 hybrids established the hybrid status in most of the combinations. Molecular analysis of reciprocal crosses revealed that F1 hybrids are more closer to maternal parent in the banding pattern than the paternal parent. The results obtained in the present study inveterate the effectiveness of SRAP markers in discriminating parental genotypes and hybrids by distinctive fingerprints. Therefore the SRAP marker is a promising approach for molecular genetic analysis and marker assisted selection (MAS) in coffee.


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TRANSGENIC ARABICA COFFEE (COFFEA ARABICA L CV CAUVERY) PLANTS WITH RICE CHITINASE (CHI11) GENE

1 2H.L. Sreenath and K. Veluthambi

1 2Coffee Biotechnology Centre, Coffee Board, Manasagangothri, Mysore - 570 006, India; School of Biotechnology, Madurai Kamaraj University, Madurai - 625 021, India

Coffea arabica popularly known as arabica coffee and Coffea canephora called as robusta coffee are the two main species under commercial cultivation contributing 70% and 30% to the total global coffee production, respectively. Arabica cultivars produce coffee with better cup quality coffee, but they are highly susceptible to several pests and diseases. C. canephora has been the main source of disease and pest resistance for breeding improved C. arabica cultivars. The coffee leaf rust (Hemileia vastatrix) is a great threat for arabica coffee production in India due to favourable climatic factors leading to crop losses up to 70%. Timor Hybrid (HDT), a natural hybrid arabica type plant originating from a spontaneous interspecific cross between C. arabica and C. canephora, is used extensively as the main source of resistance to pests and diseases, including coffee leaf rust. Coffea arabica cv. Cauvery (Catimor) derived by crossing HDT with the high yielding arabica cultivar Caturra was released for cultivation in India about 2 decades back. Cauvery is a semi dwarf cultivar with high yielding potential. After performing well in the field initially, the cultivar became highly susceptible to CLR due to development of new races.

Coffee varietal improvement has so far relied on conventional breeding procedures. However it is a lengthy procedure and a minimum of 25 years are required after hybridization to develop a new variety. Recent progress in genetic transformation technology has provided powerful tools for more efficient breeding of newer varieties of arabica coffee with desirable agronomic and commercial traits. We made efforts to transform Cauvery variety using rice chitinase (chi11) gene to provide resistance for fungal pathogens. Chitinases, representing a group of pathogenesis-related (PR) proteins, exhibit antifungal activity owing to their ability to degrade chitin in the fungal cell wall. Embryogenic calli of Cauvery were co-cultivated with Agrobacterium tumefaciens EHA 105 strain containing the binary plasmid pKVD6 carrying the rice chitinase gene. The T-DNA region of the pKVD6 contained rice chitinase gene driven by Nos promoter, hph (hpt) gene and gus-intron genes driven separately by 35S promoter. Transgenic calli were selected in the presence of 100 mg/l hygromycin. Somatic embryos were regenerated from the selected calli and germinated into plantlets on modified MS medium. Histochemical GUS assay was used to monitor transformation and regeneration process. Six transgenic plants are hardened and grown in soil under containment conditions. The oldest plant is in the soil for slightly more than 2 years. The transgenic nature of the plants was confirmed by PCR, Southern and Northern analyses. Southern analysis revealed integration of single copy of T-DNA in all the plants. Northern analysis confirmed the expression of chi11 gene in the transgenic Cauvery plants. The plants growing in potted soil are showing the normal semi-dwarf morphology of donor Cauvery cultivar. Bioassay studies are being done on transgenic plants using CLR spores.

FIELD PERFORMANCE OF COFFEA ARABICA PLANTS PROPAGATED THROUGH SOMATIC EMBRYOGENESIS

1 1 2B. Muniswamy , H.L Sreenath and Jayarama

1 2Coffee Biotechnology Centre, Coffee Board, Mysore – 570 006, India; Central Coffee Research Institute, Coffee

Board, CRS - 577 117, Chickmagalur Dist. Karnataka

Coffee is an important plantation crop in India and many countries are involved for improvement of this crop. For last three decades there is tremendous progress in coffee biotechnology. The major milestones are the in vitro multiplication of elite materials of coffee. In India, we are successful in vitro multiplication of elite materials via somatic embryogenesis and plant regeneration. In the present study, field performance of tissue cultured plants of the selected F1 hybrid plants of S.2800 3/1 (Bourbon X Hybrido de Timor (HDT)), S.2790 10/1 (HDT X Taferikela) and S.2794 7/1 (HDT X Geisha) genotypes were studied along with their seedling progenies under different agro-climatic conditions. Tissue cultured plants and seedlings of these genotypes were established in five different coffee growing areas in southern states of Karnataka and Tamilnadu. For comparison, same stage of TC plants and seedlings were planted in the field for studied both vegetative and fertility characters. Morphologically, tissue cultured plants of S.2800 3/1 showed more vigorous, uniform and higher yield than seedlings followed by S.2790 10/1 and S.2794 7/1 genotypes. These plants produced more number of longer primaries and bigger leaf size. However, significant variation among the plant materials for stem girth, number of primaries and fruit yield per plant was recorded in TC plants of S.2800 3/1. The fruit yield, bean size and bean grade of TC plants is found to be significant as compared to the seedlings. The result obtained from this data shows that, tissue cultured plants can be produced from the F1 hybrid populations to maintain the true-to-type with the normal agronomic performance from the somatic embryos due to less variation recorded in some characters. These plants are high yielding and possess resistance to various diseases and pests as compared to traditional varieties. The results obtained are discussed.


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DIFFERENTIAL EXPRESSION OF MICRORNA AND THEIR ROLE IN REGULATION OF AUXIN RESPONSE IN COFFEA ARABICA AND COFFEA CANEPHORA

Shibin Mohanan, Parvatam Giridhar, Ravishankar G, Arun Chandrashekar

Department of Plant Cell Biotechnology, Central Food Technological Research Institute (C.F.T.R.I.),Mysore, Karnataka-570020

MicroRNA are non-coding RNA ~22 nucleotide. Several hundred miRNA classes have been identified from different plant species using both experimental and computational methods. Many of them mediate gene expression in both plant and animals through various mechanisms such as blocking of transcription, sequestration of target mRNA, and DNA methylation. There are miRNA in plants conserved across species and many of these are known to regulate the levels of different hormones. The differences between Coffea arabica and Coffea canephora have yet to be characterized at the molecular level hence we have initiated an investigation into the role of micro RNAS in coffee. Our results indicate that the levels of RNA coding for proteins involved in auxin signaling pathways were differentially expressed in the leaf, endosperm and suspension culture commensurate with the level of specific miRNAs. Based on our result, we hypothesize that auxin signaling may be different between the two species of coffee.

DEVELOPMENT AND CHARACTERIZATION OF DROUGHT SPECIFIC ESTS IN COFFEE (COFFEA CANEPHORA)

1 1 1 2 1 1Jeyaraman R , Raveendra GM , Rama N , Venkataramanan D , Nataraja KN and Udayakumar M

1 2Department of Crop Physiology, University of Agricultural Sciences, GKVK, Bangalore 560 065, India; Central Coffee Research Institute, Coffee Board, Balehonnur, India.

Coffee, the most popular non-alcoholic beverage consumed by 40% of the world population, is an important agricultural commodity produced from coffee tree. The genus Coffea (family Rubiaceae) comprises >100 species of which Coffea arabica (arabica_coffee) and C. canephora (robusta_coffee) are cultivated commercially. Despite its social and economic importance, coffee tree has not received full attention with respect to crop improvement. Drought, an important abiotic stress limiting plant productivity, affects coffee tree growth and productivity and hence there is a need to improve drought tolerance. In this context, we developed and characterized drought specific cDNA library to identify novel drought genes in C. canephora.

Drought was created at whole plant level by withholding irrigation and different levels of stress were imposed by varying soil field capacity, ranging from 60 to 30% using gravimetric approaches to simulate field conditions. Drought cDNA library was developed from the leaf tissue and subjected for analysis. Sequencing and annotation (homology analysis) of cDNA library indicated the expression of diverse genes, which were classified into different categories based on their putative functions viz., cellular transport, protein fate and activity regulation, transcription, cell communication, rescue and defense. Approximately 9% of the expressed genes are related to transcription activity and 36% of the genes expressed are unknown. Expression analysis indicated stress responsive nature of the ESTs and some of the relevant gene identified are APE2; HSP70; DEAD box RNA helicase (RH30); Early Responsive to Dehydration (ERD1); DNA binding SUMO-protein ligase (SIZ1) etc. A few candidate genes are being validated.


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IDENTIFICATION AND VALIDATION OF MOLECULAR MARKERS LINKED TO ROOT AND OTHER ASSOCIATED PHYSIOLOGICAL TRAITS IN COFFEA CANEPHORA

1 2 1 1 2Devaraja Achar A.M., Mallikarjuna G. Awati, Madhura J.N., Sheshshayee M., Venkataramanan D.,

1 1Prasad, T.G and Udayakumar M

1Department of Crop Physiology, University of Agricultural Sciences, Gandhi Krishi Vignana Kendra, Bangalore 2560 065, Karnataka, India; Central Coffee Research Institute, Coffee Research Station, Balehonnur, Karnataka,

India

Coffee is one of the important beverage crops and also the second important commodity in the global trade. India stands second and contributes to 25% of Asia's coffee production. It is predominantly grown in the peninsular India, where growth is constrained by water availability during rain free periods. Despite ample genetic variation for many root related parameters, genetic improvement in coffee using conventional selection has been difficult. Therefore, major emphasis is to identify and validate molecular markers linked to root and other associated physiological traits in Coffea canephora.

A mapping population (L1 valley x S.3334) was developed and phenotyped for several morphological and physiological characters viz., root traits, total dry matter, root to shoot ratio, cumulative water transpired, and water use efficiency. Parental lines were genotyped with 41 RAPD and 9 SSR primers, which generated 70 and 9 informative loci, respectively, which was further characterized in the mapping progeny. Marker trait association by Single marker analysis revealed that 29 traits were associated with 42 markers. Thirteen RAPD markers associated for various root traits, while only two microsatellite markers linked to root length and root to shoot ratio. Few of the markers were associated with positively related traits - OPP51800 (LAD & shoot dry weight), OPZ101030 (MTR and gs) and OPK20350 (Ci and NAR). Further two RAPD markers, (root length - OPS1850) and (root dry weight - OPL11200) were converted into sequence characterized amplified region (SCAR) markers (SCARL1 and SCARD1) and validated in the same mapping population.

DEVELOPMENT OF SSR MARKERS FOR COFFEE

1 2 1 1 1 1Devaraja Achar A.M., Jeena D., Madhura J.N., Sheshshayee M., Nataraja Karaba N., Prasad, 1

T.G and Udayakumar M

1Department of Crop Physiology, University of Agricultural Sciences, Gandhi Krishi Vignana Kendra, Bangalore 2560 065, Karnataka, India; Department of Genetics and Plant Breeding, University of Agricultural Sciences,

Gandhi Krishi Vignana Kendra, Bangalore 560 065, Karnataka, India

Although impressive progress has been achieved through conventional breeding, the potential of genetic improvement for coffee production is still large. In view of the time-consuming process in coffee breeding, modern molecular technologies like DNA markers have a great potential to assist and accelerate breeding programmes. Since there are only a few SSR markers reported in coffee genome, the major aim was to develop and characterize a comprehensive set of SSR markers for coffee (Coffea canephora), which could be used to tag genes and quantitative trait loci controlling traits of agronomic interest.

Pre-cloning enrichment (selective hybridization) strategy was followed to fish out microsatellites from coffee genome. From this strategy, 1368 microsatellite enriched colonies were screened and 534 clones were sequenced and annotated for the presence of microsatellite repeat regions. Based on the annotation results, primers were designed for the 345 sequences containing microsatellite repeat regions. The enriched library resulted in 25 % of microsatellite containing clones. Initially 100 primers were designed, custom synthesized and standardized to obtain locus specific amplification. These SSRs were validated in nine related diploid cultivated and distant related Coffea species. These markers were also characterized in the F1 population, where only two primers (CPCM 11 and CPCM 13) showed polymorphism.

Genic-SSRs from EST database were developed by annotating 13,175 Coffea canephora unigene sequences for the presence of microsatellite repeats. SSR primers were designed for 368 sequences and 100 Coffea canephora EST sequence (CCES) primers are being standardized for locus specific amplification.


SOL 2009 287

SATELLITE SESSION IV BRINJAL

288

MOLECULAR CLONING AND CHARACTERIZATION OF POLYPHENOL OXIDASE GENES FROM EGGPLANT (SOLANUM MELONGENA)

1 2 1Santoshkumar M Shetty , Arun Chandrashekar , Yeldur P. Venkatesh

1 2Department of Biochemistry &Nutrition; Department of Plant Cell Biotechnology, Central Food Technological Research Institute,Mysore-570020,Karnataka ,India

Polyphenol oxidase (PPO), a copper metalloprotein present in many members of Solanaceae, is responsible for the enzymatic browning of plant tissues upon wounding; it exists as a multigene family in tomato and potato. Information on structural and functional aspects of eggplant PPO gene(s) is lacking. A full-length PPO gene (gcSOMELPPO4, GenBank accession GQ246219.1) was cloned using degenerate primers, revealed high degree of similarity to potato and tomato PPO genes. The deduced amino acid sequence reveals a 90-aa chloroplast transit peptide, a 510-aa mature peptide and highly conserved copper-binding regions . RNA-ligation mediated rapid amplification of cDNA ends technique was adapted to identify the 5’ and 3’ untranslated regions of gcSOMELPPO4 gene using gene-specific primers. Two distinct 5’ partial clones of PPO genes with conspicuous differences in the coding sequences of the 5’ end were obtained indicating multigene expression in the fruit. The two cDNA clones contain a transcription start site approximately 40 bp upstream of start codon. The eggplant PPO gene is expressed as a GST-fusion protein in E. coli TB1. Semi-quantitative RT-PCR has provided useful information on spatiotemporal expression of different PPO genes of eggplant.


SOL 2009 289

SATELLITE SESSION V PEPPER

290

GENETIC ANALYSIS OF PHENYLALANINE AMMONIA LYASE ACTIVITY IN CHILLI (CAPSICUM ANNUUM L.)

Sathiyamurthy Appachi, Veeraragavathatham Desikathathachari, Pugalendhi Lakshmanan

Horticultural College and Research Institute, Tamil Nadu Agricultural University

Chilli (Capsicum annuum L.) is one of the important vegetable cultivated for its pungency in most part of the country. Pungency is mainly due to the capsaicin present in the fruit. Accumulation of capsaicin occurs over relatively short period during the later stages of fruit development. Phenylalanine ammonia-lyase is the enzyme that catalyses the first step in the pathway using l-phenylalanine -coumaric, as the substrate. Later this will transform to cinnamic acid, caffeic and ferulic acids. Ferulic acid will be converted to vanillin and vanillylamine and finally capsaicin will be synthesized. A study was conducted at Horticultural College and Research Institute, Tamil Nadu Agricultural University, Coimbatore to assess the genetic nature of the Phenylalanine Ammonia Lyase (PAL) activity at 15, 30 and 45 days after flowering on capsaicin synthesis under the different genetic background of the chilli. The results revealed that at 15 days after flowering, the genetic parameters D, H1, H2 and h2 were significant. The F and E were non-significant. Among the ratios of the genetic parameters, KD/KR value was more than unity. h2/H2 was more than unity and the ratio of (H1/D)1/2 was less than unity. Narrow sense heritability was 19.23 per cent. All the components viz., D, F, H1, H2, h2 and E recorded significant values for PAL activity at 30 days after flowering. The ratios of genetic parameters revealed that mean degree of dominance was more than unity. The ratio of H2/4H1 was not equal to 0.250. The KD/KR ratio was less than unity and the number of gene groups controlling this character was found to be more than one. The heritability in narrow sense was 41.88 per cent. At 45 days after flowering all the genetic parameters except E were non-significant. The ratios of genetic parameters revealed that one group of gene controlling this trait. The KD/KR ratio was more than unity and H2/4H1 was not equal to than 0.250. Key words: Chilli, Capsicum annuum, genetic analysis, phenylalanine ammonia lyase


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SOL 2009292

A SINGLE DOMINANT GENE CONTROLS CMV RESISTANCE IN PEPPER (CAPSICUM ANNUUM L.)

Hoang Ngoc Huy, Hee-Bum Yang, Jin-Kyung Kwon, Sung-hwan Jo, Doil Choi, Byoung-Cheorl Kang

Department of Plant Science, CALS, Seoul National University, Seoul, 151-921, Korea.

Cucumber mosaic virus (CMV) is one of the most destructive viruses in Solanaceae crops. C. annuum 'Bukang' is a commercial cultivar known to contain a single dominant gene resistant to CMV, including CMVKorean and CMVFNY strains. We designated the name Cmr1 in 'Bukang' to this resistant gene. Analysis of cellular localization using CMV-GFP showed that 'Bukang' inhibits systemic movement of virus from mesophyll cell layer to mesophyll cells. Mapping study and FISH analysis revealed the Cmr1 gene is located at the upper end of LG2 near TG31A. A total of three markers were developed by comparative genetic mapping study. One intron based marker was developed using a pepper EST sequence homologous to Tm-1. We also developed two additional SNP markers using tomato BAC sequence which is located syntenic position to Cmr1. This result could be used for fine mapping and cloning the Cmr1 gene.

MOLECULAR DETECTION AND EXPRESSION OF COAT PROTEIN GENE OF LEAF CURL VIRUS IN PEPPER (CAPSICUM ANNUM)

1 1 2 1 1D.P. Sinha , Major Singh Sangeeta Saxena , Sanjeev Kumar and Mathura Rai

1Indian Institute of Vegetable Research, Post Box No. 01, P.O. Jhakhini (Shahanshahpur), Varanasi 221 305 2(India); Department of Biotechnology, Babasaheb Bhimrao Ambedkar University, Lucknow

Chilli (Capsicum annuum) is one of the most diverse vegetable species and is considered to be a high value crop. Chilli is susceptible to various pathogens including viruses, which cause heavy production losses. Leaf curl disease of chilli caused by pepper leaf curl virus (PepLCV) the most destructive in terms of incidence and yield and economic losses. Epidemics of PepLCV can result in upto 100% yield loss under severe epiphytotic conditions. Curling and puckering of leaves and stunted growth of the plants are some of the typical symptoms of the PepLCV infection. The first step towards minimizing the effects of PepLCV includes efficient detection of the virus. For a reliable detection of PepLCV through PCR, coat protein specific primer pairs were designed and ~1 kb amplification product specific to coat protein gene was amplified. Cloning of this amplification product in an expression vector resulted in high-level expression of a 28 kD protein product. Coat protein gene product is responsible for viral capsid formation and is important for vector specificity for efficient whitefly-mediated transmission. The purified protein can be used for development of coat protein specific antisera leading to development of an efficient detection system for this very important viral pathogen of pepper. Serological based assay methods offer an option for rapid detection of virus. The cloning and expression of AVI gene in a prokaryotic system will be discussed herein.


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SOL 2009294

VALIDATION OF MOLECULAR MARKERS ASSOCIATED WITH PUNGENCY IN CHILLI (CAPSICUM ANNUUM L.)

1 1 2 1 1Neeraj Dwivedi , Rajesh Kumar *, R.K. Singh , Major Singh , Mathura Rai

1Division of Crop Improvement and Biotechnology, Indian Institute of Vegetable Research, Post Box 5002, 2Varanasi, India; Biochemistry Department, Banaras Hindu University, Varanasi-221005

Capsaicin is a unique alkaloid of the plant kingdom restricted to the genus Capsicum. Capsaicin, the pungency factor, is a bioactive molecule of food and medicinal importance. During recent years gene specific marker development has proved as a potential tool for MAS. Once an association has been identified, validations studies on independent populations are necessary to ensure that the association is valid throughout the population and could also be use to develop more linked marker.A set of 7 earlier reported specific markers for pungency were screened to validate the reported markers. One of each SNP, CAPS, PAP-SSR and four SCAR makers were validated in our two contrasting breeding lines for pungency. Only three earlier reported markers PAP (plastid lipid associated protein) -SSR and two SCAR markers; UBC145 and UBC106 were found polymorphic. The PAP-SSR marker amplified loci of 150 bp in non-pungent line whereas 112 bp fragments produced in highly pungent line which showed distinct polymorphism with difference of approx 40 bp. PAP-SSR has also been validated in to 20 different accessions (pungent and non-pungent) of chili and found that this marker was able to distinguish pungent and non pungent lines.

The SCAR marker UBC145 amplified a monomorphic 600 bp band for the two parents. After digesting this product by Hha1 as reported, a fragment of 450 bp appeared in pungent line and was absent in non-pungent and SCAR UBC106 marker amplified 1,500 bp band in non-pungent which is absent in pungent line. These three specific markers will be highly useful for our ongoing linkage analysis of genes associated to pungency in our breeding population.

SCREENING OF PEPPER ACCESSIONS FOR RESISTANCE AGAINST TWO THRIPS SPECIES (FRANKLINIELLA OCCIDENTALIS AND THRIPS PARVISPINUS)

1,2 1 3 2 1Awang Maharijaya , Greet Steenhuis-Broers , Asep Harpenas , Agus Purwito , Ben Vosman 1

and Roeland E. Voorrips ,

1 2Plant Research International. P.O. Box 16, 6700 AA Wageningen, the Netherlands; Bogor Agricultural University.

3Jalan Raya Darmaga 16680 Bogor, Indonesia; East-West Seed, P.O. Box 1, 41181 Campaka, Purwakarta, Indonesia

Thrips are damaging pests in pepper worldwide. They can cause damage directly by feeding on leaves, fruits or flowers, and also indirectly by transferring viruses, especially Tomato Spotted Wilt Virus (TSWV). Although thrips are among the most damaging pests in pepper, until now there is no commercial variety with a useful level of resistance to thrips. This is at least partly due to the lack of knowledge of pepper germplasm with sufficient resistance to thrips species, of QTLs and/or genes for resistance, and of information about resistance mechanisms to thrips in pepper. We have carried out research aimed at identifying genetic variation for thrips resistance in pepper in order to identify pepper accessions covering a wide range of thrips resistance, and at developing practical and reliable screening methods for thrips resistance. Thirty-two pepper accessions from four species of pepper (Capsicum annuum, C. baccatum, C. chinense and C. frutescens) and two species of thrips (Frankliniella occidentalis and Thrips parvispinus) were used in this study. Several screening methods were tested, using both choice (screenhouse, greenhouse) and non-choice situations (leaf disc, detached leaf and cutting). Screening methods were compared and correlation among these methods was investigated. We observed large variation for resistance to thrips in Capsicum that might be exploited in breeding programs. Our result also indicated that both the leaf disc test and the detached leaf test can be used as screening methods for thrips resistance in pepper.


SOL 2009 295

SATELLITE SESSION VI TOMATO

296

WHOLE GENOME PROFILING OF SOLANUM LYCOPERSICUM HEINZ 1706

1 1 1 1 1Marco van Schriek , Richard Feron , Jan van Oeveren , Peter de Heer , Lorena DaPonte , 1 1 2 2 3Saskia Jacobs-Oomen , Mike Cariaso , Rene Klein Lankhorst , Roeland van Ham , Bert Bogden ,

1 1Marcel Prins , Michiel van Eijk

1 2 3Keygene N.V.; CBSG; Amplicon

The Whole Genome Profiling (WGP) method enables the de novo construction of a complete high quality physical map. This sequence-based physical map forms the framework to construct a complete genome sequence assembly of plant and animal genomes in a fast and cost-effective manner. For the WGP tomato project, the Heinz1706 enzyme libraries MboI, HindIII, EcoRI and the Heinz1706 random sheared library were applied (2X, 2X, 2X, 4X genome coverage respectively). The resulting tomato physical map is 2521 contigs in size and contains over 52000 BACs. The N50 contig size is larger than 563 Kbp and the calculated genome coverage is 947 Mbp. The tomato WGP map will serve as a scaffold to anchor and order the sequence contigs to yield a draft sequence of the tomato genome by the International SequencingConsortium SOL.


SOL 2009 297

SATELLITE SESSION VII SECONDARY METABOLISM

298

PURIFICATION AND PHYSICO-KINETIC CHARACTERIZATION OF A Β-GLUCOSIDASE FROM WITHANIA SOMNIFERA (ASHWAGANDHA) LEAF

1 1 2 1Siddhartha K. Mishra ,*Neelam S. Sangwan , Rakesh Tuli and Rajender S. Sangwan

1 2Central Institute of Medicinal and Aromatic Plants (CSIR), Lucknow-226015, India; National Botanical Research Institute (CSIR), Lucknow-226001, India

Withania somnifera (Solanaceae) contains withanolides and their glycosides (withanosides) for recruitment in specialized physiological functions or situations. Withanolides may be produced by their de novo synthesis and/or remobilization from pre-stored form like glycosylation in the vacuoles through glucosidic cleavage by glucosidase. This secondary metabolomic scenario of phyto-pharmaceutically important withanolides and their gluco-conjugates prompted us to examine the -glucosidase of the plant. Therefore, a β-glucosidase was homogeneity (SDS-PAGE) purified through a sequence of gel filtration and ion-exchange column chromatography and characterized for its physical and kinetic characterisitics. The major properties of the enzyme included an acidic pH optima (4.8), alkaline pI (8.7), meso-thermostabity, monomeric structure with subunit molecular weight of about 50 kDa, high affinity for substrate (Km) for pNPG (0.19 mM), high (105,263 M-1.s-1) catalytic efficiency (Kcat/Km). The mesostable enzyme had a stringent substrate specificity restricted to only β-linked gluco-conjugate. The enzyme is optimally active at 40 ºC with 12.4 kCal Mol-1 activation energy and was highly sensitive to D-gluconic acid lactone inhibition (94% at 1 mM) with an apparent Ki 0.21 mM. The enzyme could catalyze transglucosylation of geraniol with pNPG as glucosyl donor but not with cellobiose and could produce un-characterized aglycones from the butanol fraction (small molecule glycosides) of Withania somnifera. The catalytic diversity of substrates and specificity of linkage suggest its potential role in generating aglycones from b-linkage glycosides like withanosides and sitoindosides in the plant.


SOL 2009 299


SOL 2009300

STUDIES ON PURIFICATION OF WITHANOLIDE GLYCOSIDES FROM ROOTS OF WITHANIA SOMNIFERA AND ITS CONVERSION TO AGLYCONES BY ACID HYDROLYSIS

D.Pradeepa, A.Geetha, and Kalaiselvi Senthil

Department of Biochemistry,Biotechnology and Bioinformatics, Avinashilingam University for Women, Coimbatore -641043

Molecular and pharmacological investigations of Withania somnifera have elucidated numerous pharmacological activities of ashwaganda with specific secondary metabolites collectively termed as withanolides includes withanolide glycosides and withanolide aglycones. Withanolide glycosides are metabolized by the intestinal micro flora to withanolide aglycone and absorbed more effectively than the withanolide glycosides. But under diseased condition the intestinal micro flora gets altered thereby no conversion. Thus withanolide aglycones are mostly preferred. In the present study an attempt has been made to purify withanolide glycosides which are present at a higher concentration and to analyze the withanolide aglycones obtained by acid hydrolysis of withanolide glycosides. Powdered roots of Withania somnifera was extracted with 50% ethanol exhaustively 3-4 times and the pooled extracts were found to contain sugars on analysis by thin layer chromatography. The chloroform fraction of 50% ethanol extract was found to contain less sugar and showed the glycoside contents in it with more spots when compared to the 50% ethanol extract. The concentrated chloroform fraction was subjected to silica gel column chromatography followed by elution with different ratios of chloroform and ethyl acetate in the order of increasing polarity resulted in a fraction containing single spot corresponding to Rf value 0.2.The purified compound obtained was acid hydrolyzed using hydrochloric acid and glacial acetic acid at 80ºC for 12 hours resulted in conversion of the spot with Rf value 0.2 to 0.43, 0.45, 0.54 and 0.64 respectively. Thus the present study indicates that the withanolide glycosides of Withania somnifera roots can be purified and converted using mild acids to more biologically potential aglycone.

A COMPARATIVE STUDY ON ONTOGENIC EXPRESSION OF ANTIOXIDANTS AND SECONDARY METABOLITES IN IN VITRO AND IN VIVO GROWN LEAVES OF WITHANIA SOMNIFERA

Nathiya. S, Savitha. C, Santhi. N and Kalaiselvi Senthil

Department of Biochemistry, Biotechnology and Bioinformatics, Avinashilingam University for Women, Coimbatore.

Withania somnifera (WS), also known as ashwagandha, Indian ginseng and winter cherry, has been an important herb in the Ayurvedic and indigenous medical systems for over 3000 years. The objective of the study is to estimate the antioxidant activity and secondary metabolites present in both in vitro and in vivo grown leaves of Withania somnifera at different time period. The estimated antioxidant enzymes were superoxide dismutase, catalase, glutathione peroxidase and glutathione-s-transferase. The secondary metabolites from leaves were extracted with increasing and decreasing polarity of solvents and analysed by thin layer chromatography using chloroform: methanol (9:1) as solvent system. The chromatogram was developed using 10% sulphuric acid. It was found that ethyl acetate extract of decreasing polarity contains all the metabolites present in the leaves. The level of enzymatic antioxidants were found to be present in increased quantity at third month compared to first and second month leaves of in vitro maintained plants, whereas there is no significant change in the level of enzymatic antioxidants in the leaves of in vivo grown plants. Search for the concerned active compounds has led to the identification of several withanolides, alkaloids, flavonoids, steroids, phenolics, saponins and quinones from leaves of Withania somnifera. These findings have high significance in the pharmacological industry.


SOL 2009 301


SOL 2009302

MICROPROPAGATION AND IN VITRO FLOWERING IN WITHANIA SOMNIFERA

Viji.MO, Kalaiselvi Senthil and R .Parvatham

Department of Biochemistry, Biotechnology and Bioinformatics, Avinashilingam University for Women, Coimbatore -641043

Effective micropropagation, in vitro flowering, and in vitro fruiting protocol were studied in Withania somnifera, an antitumor medicinal plant using axillary bud as explants. Maximum number of multiple shoots was achieved in four weeks when the shoot tips of germinated seedlings were inoculated in MS basal medium supplemented a combination of.2mg/l TDZ and 1mg/l BAP .Murashige and Skoog's medium (MS) supplemented with kinetin(1mg/l) and BAP(.1mg/l) found optimum for elongation of shoots. The multiple shoots obtained were found to form tiny green floral buds after 4–6 weeks of inoculation in MS medium supplemented with Kinetin and (2.0 mg/ l) BAP (0.5 mg/ l). The elongated shoots of Withania somnifera from MS medium supplemented with Kinetin and (2.0 mg/ l) BAP (0.5 mg/ l) were transferred to MS0 medium (no hormones supplemented in it).The flowering was observed within 3 months. In autogamy, the pollen number was high. The present study describes in vitro flowering system to overcome problems associated with flower growth and development as well as fruit and seed production in vitro. In addition a shortened time period to obtain in vitro flowering and fruit set would be an added advantage in accomplishing in vitro pollination for rapid production of desired hybrids from rare stocks during off seasons too.

IN SILICO ANALYSIS OF CYCLOARTENOL SYNTHASE GENE, A KEY METABOLIC DIVERSIFICATION STEP OF THE PENTACYCLIC TRITERPENOID PATHWAY TO SEEK ITS ROLE IN WITHANOLIDE BIOSYNTHESIS IN INDIAN MEDICINAL PLANT ASHWAGANDHA (WITHANIA SOMNIFERA) DUNAL

Asha and Neelam S. Sangwan

Central Institute of Medicinal and Aromatic plants (CIMAP-CSIR), PO CIMAP, Lucknow-226015

Withania somnifera Dunal of the Solanaceae family is a highly reputed plant of Indian traditional system of medicines used in various preparations for the treatment of diseases like leprosy, nervous disorders, rheumatism and to combat stress. The medicinal properties of ashwagandha are attributed to withanolides which are steroidal lactone of triterpenoid ancestry. Though, the plant is of immense importance, withanolides biosynthetic pathway, and the enzymes and genes involved are not well understood so far. It is hypothesized that the triterpenoid pathway through the cyclization of oxidosqualene leads to the withanolides biosynthetic routes. Thus, we analyzed the sequences of cycloartenol synthase, the most intriguing biochemical junction/diversification in secondary metabolism where multi-direction highly conserved gene based cyclization of 2, 3 oxido-squalene singly or in combination might create cyclized product(s) that further diversify to a myriad of phytochemicals. We planned to work on WS-cas gene in Withania somnifera with aim to clone and characterize the gene and analyze its role in the metabolomic, physiological and ecological status of the plant. In-silico analysis using various bioinformatics tools revealed that cas is a 750-760 amino acid long protein having conserved sites 'DCTAE motif' MWCHCR and repeated 'QW motifs' which are important for the activity. Solanaceae cas exhibited its uniqueness when compared with other gene sequences from Arabidopsis thaliana, Medicago truncatula and other plants by falling in distinct and distant cluster. Conserved domain based primers were utilized for amplification of partial cas gene fragment from Withania somnifera. Expression analysis along with the levels of phytomolecules-sterols and withanolides is likely to reveal the dependence of the plant in terms of their interactions and biogenetic priorities vis-à-vis their functions in W. somnifera.


SOL 2009 303

SATELLITE SESSION VIII VIRUSES

304

PHYTOHORMONAL RESPONSES TO PLANT DEVELOPMENT DURING VIRAL INFECTION

1 2 1 1 1Vipin Permar , Anil K. Mishra , Vikas Koundal , Priyanka Singh and Shelly Praveen

1 2Advanced Center for Plant Virology,Indian Agricultural Research institute, New Delhi; Quality control laboratory, Delhi Jal Board, New Delhi.

Plant developmental processes are synergistically regulated by multiple hormones. These processes are greatly affected during viral infection. Over the past years, viral suppressor proteins have been validated to play crucial role in affecting plant gene regulation through non-coding regulatory RNAs (microRNAs). Recent research has witnessed the identification of intersecting role of viral suppressor proteins, miRNA pathway and phyto-hormones responses, which improved over understanding of altered hormonal requirement for plant development during infection. In present study, exogenous hormones were applied on tomato and tobacco explants infected with virus for their regeneration. A relatively higher doses of auxin and cytokinins hormones are required to regenerate tomato and tobacco explants infected with Tomato leaf curl virus (ToLCV) as compared to Cucumber mosaic virus (CMV).This might be due to presence of multiple suppressor proteins in case of ToLCV (AC4, AC2 & C1 ) as compared to single suppressor protein in CMV (2b). Furthermore northern analysis indicated altered levels of five different miRNAs in infected tissues as compared to normal tissues of tomato and tobacco.


SOL 2009 305


SOL 2009306

ROLE OF CMV-2B PROTEIN IN REGULATING MIR164 DEPENDENT DEVELOPMENTAL PROCESSES IN TOBACCO

Priyanka Singh, Vikas Koundal and Shelly Praveen

Advanced Centre for Plant Virology, Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi.

Tobacco plants infected with Cucumber mosaic virus developed symptoms like stunted growth, initiation of auxiliary buds, lateral root growth, aberrant leaf shape, early senescence etc. The exact regulatory mechanisms for development of these aberrations during viral infection are not known. Here we demonstrated that CMV suppressor 2b protein when over express in tobacco plants develops viral like symptoms. In vitro expression of 2b and its activity in binding micro RNA showed its affinity towards miR164. Recent studies have shown that miR164 controls a group of transcription factors belonging to NAC family (NAC1, CUC1/2), which regulate various transcripts involved in developmental processes and also cell death during senescence. We relate our results by showing regulation of miR164 by CMV 2b which finally leads to virus like symptoms during viral infection.

SCREENING OF TLCV RESISTANCE THROUGH VARIOUS WHITEFLY MEDIATED INOCULATION TECHNIQUE IN TOMATO

1 1 1 2 1Ramesh Kumar Singh , Nagendra Rai , Major Singh , Satya Narayan Singh and Mathura Rai

1Indian Institute of Vegetable Research, Post Box No. 01, P.O. Jakhini (Shahanshahpur),Varanasi-221 305, U. P., 2India; Udai Pratap Autonomous College, Varanasi-221001

Tomato leaf curl virus (TLCV), a whitefly (Bamicia tabaci) transmitted Gemini virus caused upto 99% losses and a serious challenges for tomato producers throughout the world. The infected plants are stunted, shortened internodes with proliferation of lateral branches. Leaves are rolled both in upward and inwards having small chlorotic leaflets and curled lamina between the veins, flower shedding and small sized fruit causing complete destruction of plants.Screening is the most important step for getting confirmed resistance sources for any disease and this process usually practiced in natural & artificial conditions. Therefore, four cultivars (Punjab Chhuhara, DVRT-2, H-86 & H-24 ) belonging Solanum lycopersicum and six accessions of different wild species namely S. Pimpinellifolium (EC-521080), S. chmielewskii (EC-520049), S. ceraseforme (EC-528372), S. habrochaites (EC-520061), S. chilense (WIR-5032) and S. peruvianum (WIR-3957) were crossed in line x tester mating design at Indian Institute of Vegetable Research, Varanasi which is situated at 82.520E longitude, 25.100N latitude and an altitude of 128.93m above mean sea level (MSL) with annual rainfall of 1270 mm, having a soil PH in the range of 6.5 to 7. Twenty four F1's along with their parents were subjected to screen in natural (field condition) and two different artificial (mass & cage) inoculations for two consecutive years (2006 &2007). The disease was scored on 0-4 scale at 30 days and 15 days intervals in natural and artificial screening, respectively. The percent disease incidence (PDI) & coefficient infection (CI) were calculated to study of disease severity in all the F1's with their parents. The highly resistant and susceptible parent was further subjected 60 SSR primers to confirm the hybrid purity of F1 against TLCV.In screening techniques used for testing against TLCV, the cage inoculation technique was found most efficient and effective to identify the resistance / susceptible F1's/ parents. Among the parents, all the cultivated parents were showed susceptible to TLCV, while five accessions (EC-521080, EC-520049, EC-528372, WIR-5032 and WIR-3957) and one accession (EC-520061) belonging to wild species exhibited moderately and highly resistant, respectively. The F1's derived from accession EC-520061 showed highly resistant (Punjab Chhuhara x EC-520061) to resistant (DVRT-2 x EC-520061, H-86 x EC-520061, H-24 x EC-520061) whereas other F1's were found moderately susceptible to highly susceptible. PCR amplification with SSR markers showed maximum polymorphism in S. habrochaites accession than other accession of wild species. In hybrid purity test through SSR polymorphic primers, it was observed that highly resistant F1 derived from S. habrochaites showed true hybrid as phenotypically resistant like S. habrochaites to TLCV.


SOL 2009 307


SOL 2009308

REAL-TIME PCR FOR THE QUANTIFICATION OF TOMATO LEAF CURL VIRUS IN TOMATO PLANTS AND ITS VECTOR BEMISIA TABACI

Jyothsna P, Archana Kumari, V.G. Malathi

Advanced centre for Plant Virology, Division of Plant Pathology, IARI, New Delhi

Tomato leaf curl viruses (Geminiviridae) are group pf important pathogens which affect tomato production all over the world. Although diagnostic protocols are available for its detection in plants and its vector Bemisia tabaci, suitable tools for estimating and comparing viral inoculum in plants and insect tissues are needed.

In the present study, we report quantification of Tomato leaf curl Bangalore virus (ToLCBV) in both tomato plant and whitefly extracts by SYBR Green I based real-time PCR. The Ct values obtained against the known concentrations of serially diluted DNA of ToLCBV clone was used to construct standard curve. For whitefly, the fluorescence measured was directly related to the amount of DNA amplified by using specific primers standardized to the amount of DNA present in each sample using selected endogenous Bemisia gene as an internal reference. A linear relationship was obtained between virus concentration and cycle threshold (Ct) value. The viral DNA levels were relatively obtained by analyzing the samples at regular intervals.

The much lower detection limits reported here in our study make it a valuable tool particularly when early diagnosis of pre-symptomatic tissues is necessary to help reduce spread of infection.

BREEDING FOR BEGOMOVIRUS RESISTANCE IN TOMATO (SOLANUM LYCOPERSICUM L.)

A.T.Sadashiva, M. Krishna Reddy, K.V. Ravishankar, T.H. Singh, B. Manjunath, P. Swarnalatha, J. Deepa, K. Sudarshini and R. Pushpalatha

Division of Vegetable Crops, Indian Institute of Horticultural Research, Hessaraghatta Lake Post, Bangalore-560 089, Karnataka, INDIA

Tomato (Solanum lycopersicum L.) is one of the most important vegetable crops cultivated in India. Tomato Leaf Curl disease caused by begomoviruses is the major production constraint causing considerable yield losses. Adoption of tomato leaf curl virus (ToLCV) resistant varieties is the most practical way to combat this disease. A systematic screening was undertaken at Indian Institute of Horticultural Research, Bangalore to identify stable sources of resistance to ToLCV. Eight lines viz; IIHR-1943 (S. peruvianum), IIHR-1970 (S. peruvianum) IIHR-2101 (S. habrochaites f glabratum LA 1777), IIHR-2195, IIHR-2202, IIHR-2403, IIHR-2611 and IIHR-2321 (TLCVR-1) were found resistant to ToLCBV under screen house conditions. IIHR-2101 was also found to be resistant to ToLCNDV, when screened under field conditions in Delhi. Two F3 mapping populations derived from the crosses viz; IIHR-2611 x IIHR-1334 and IIHR-2403 x IIHR-1334 have been advanced further. Of the 427 BC1F2 plants of the cross 15SBSB x IIHR-2101, 123 plants were immune to ToLCBV. The individual plant BC1F2-26-9 was found to be high yielding with better fruit quality attributes. Of the 49 BC1F3 progeny of the same cross, 18 plants were immune to ToLCBV. Resistance specific primers Ty-1, Ty-2 and Ty-3 were employed to screen tomato genotypes IIHR-2195, IIHR-2101, IIHR-2403, IIHR-2321, IIHR-1970, SH1, IIHR-2611, 15SBSB and Pusa Ruby. IIHR-1970 showed heterozygous bands (500bp and 450bp) for Ty-1, whereas others showed susceptible band of 450bp. While screening for Ty-2 marker, IIHR-2195 and IIHR-2101 showed homozygous resistance band of 900bp. IIHR-1970 and SH1 showed heterozygous bands (800bp and 900bp). Whereas, others showed susceptible band of 800bp for Ty-2. IIHR-2101 showed 640bp when screened for Ty-3 (FLUW) whereas SH-I showed heterozygous band of 640bp and 480bp. Others showed susceptible band of 480bp for Ty-3. IIHR-1970 showed resistance band of 480bp when screened with Ty-3a (P6-25), while others showed susceptible band of 320bp. Resistance specific primer Ty-1 was also employed to screen BC1F3 (of 15 SB SB x IIHR-2101) lines (BC1F3-93 & BC1F3-15) which showed phenotypic resistance. After restriction digestion with Taq1 enzyme. For Ty-1 all the progenies of BC1F3-93 and BC1F3-15 showed susceptible band of 450bp. For Ty-2 also these progenies showed susceptible band of 800bp. Where as BC1F2-26-9 (of 15 SB SB & IIHR-2101) showed unique banding pattern with an extra band between the resistant 900 band and susceptible 800band. For Ty-3 (BC1F3-93 lines and BC1F3-15 lines showed susceptible band of 480bp whereas IIHR-2101 showed resistance band of 640bp.


SOL 2009 309

LIST OF AUTHORS AND PARTICIPANTS

310

A

Aarti Gupta [email protected] IndiaAashima Nijhawan [email protected] IndiaAditi Mathur [email protected] IndiaAditya Gupta [email protected] IndiaAgim Ballvora [email protected] GermanyAgustin Alberto Lopez Pedrosa [email protected] SpainAjay Kumar Mahato [email protected] IndiaAkhilesh Tyagi [email protected] IndiaAkhilesh Kumar [email protected] IndiaAlain Tissier [email protected] FranceAlan Andrade [email protected] BrazilAlexander Vainstein [email protected] Israel Alexander Paul Reid [email protected] UKAlexandrede Kochko [email protected] FranceAlka Kumari [email protected] IndiaAlok Sinha [email protected] IndiaAlvaro Gaitan [email protected] ColombiaAmar Kumar [email protected] IndiaAmeya Mahajan [email protected] IndiaAmit Katiyar [email protected] IndiaAmolkumar Solanke [email protected] IndiaAna-Rosa Ballester Frutos [email protected] NetherlandsAnimesh Acharjee [email protected] NetherlandsAniruddha Sane [email protected] IndiaAnithakumari Arabikothanuru Muniyappa [email protected] NetherlandsAnjan Banerjee [email protected] IndiaAnna Czerednik [email protected] NetherlandsAnne-marie Wolters [email protected] NetherlandsAntonio Di Matteo [email protected] ItalyAntonio Granell [email protected] SpainAnuradha Pujar [email protected] USAArchana Kumari [email protected] IndiaArchana singh [email protected] IndiaArnaud G.Bovy [email protected] NetherlandsArrvapalli Veerabhadra Roa [email protected] IndiaArun Sharma [email protected] IndiaArun Joshi [email protected] IndiaAsaph Aharoni [email protected] IsraelAsha [email protected] IndiaAshok P Giri [email protected] IndiaAshwin Kashikar [email protected] IndiaAsis Hallab [email protected] GermanyAsis Datta IndiaAsma Aouini [email protected] JapanAutar Krishen Mattoo [email protected] USAAvantika Bhaskar [email protected] IndiaAvital Adato [email protected] IsraelAvtar Handa [email protected] USAAwadhesh Pandit [email protected] India

B

B. Muniswamy IndiaBasavarajaiah D [email protected] IndiaBen Vosman [email protected] NetherlandsBjörn D'hoop [email protected] NetherlandsByoung-Cheorl Kang [email protected] Korea (south)Byung-Dong Kim [email protected] Korea (south)


SOL 2009 311


SOL 2009312

C

Carl Jones [email protected] USACarlo Pozzi [email protected] SwitzerlandCees Schuit [email protected] NetherlandsCelestina mariani [email protected] NetherlandsChaitanya Charakana [email protected] IndiaChangbao Li [email protected] ChinaChinna Babu Muvva [email protected] IndiaChristain Chevalier [email protected] FranceChristian Bachem [email protected] NetherlandsChristophe Rothan [email protected] FranceChuanyou Li [email protected] China

D

Dani Zamir IsraelDavid Francis [email protected] USADebashis Rana [email protected] IndiaDebasis Chattopadhyay [email protected] IndiaDesiree Menancio Hautea [email protected] PhilippinesDeva Kumari [email protected] IndiaDibendu Datta [email protected] IndiaDoil Choi [email protected] Korea (south)

E

EA Siddiq IndiaEfraim Lewinsohn [email protected] Israel Elangovan M [email protected] IndiaElzbieta Kozik [email protected] PolandErika Asamizu [email protected] Japan

Ffrancois dorlhac de borne [email protected] FranceFrédéric Moquet [email protected] France

G

Ganesh Chowthi Thimmegowda [email protected] IndiaGeeta Prakash [email protected] IndiaGerald Freymark g [email protected] NetherlandsGerard Bishop [email protected] United KingdomGianfranco Diretto [email protected] ItalyGil Shalev [email protected] Graziosi [email protected] ItalyGiovanni Giuliano [email protected] ItalyGitanjali Yadav [email protected] IndiaG. V. Krishna Rau India Giuseppe leonardo rotino [email protected] ItalyGlenn Bryan [email protected] United KingdomG. D. Shankaranarayana [email protected] IndiaGurumurthy

H

H. P. Singh [email protected] IndiaH. L Sreenath IndiaHanjabam Mickey [email protected] IndiaHanna Habdas [email protected] PolandHannele Lindqvist-Kreuze [email protected] PeruHanumantha Rao PAPPU [email protected] USAHarsh Chauhan [email protected] IndiaHelen Tai [email protected] CanadaHenk Huits [email protected] NetherlandsHenk Hilhorst [email protected] NetherlandsHiroshi Ezura [email protected] Japan

I

Idit Ginzberg [email protected] IsraelIlan Paran [email protected] IsraelIsaak Yosief Tecle [email protected] Netherlands

J

J. Karihaloo IndiaJacobus de Wit [email protected] NetherlandJadwiga Sliwka [email protected] PolandJae Bok Yoon [email protected] Korea (south)Jai Gopal [email protected] IndiaJames Giovannoni [email protected] USAJan Uitdewilligen [email protected] NetherlandsJayarama IndiaJanardhan Singh [email protected] IndiaJavaregowda Nagaraju Jawahar Lal Karihaloo [email protected] IndiaJesus Abad Martin [email protected] SpainJianfeng Ren [email protected] ChinaJitendra Khurana [email protected] IndiaJitendra Kumar Pal [email protected] IndiaJohn Lindbo [email protected] USAJonathan Whitworth jonathan.whitworthoars.usda.gov USAJorge Mondego [email protected] BrazilJose M Jimenez-Gomez [email protected] USAJoyce Marie Van Eck [email protected] USAJulin Maloof [email protected] USAJundae Lee [email protected] Korea (south)Jung-Heon Han [email protected] Korea (south)Junji Kimbara [email protected] JapanJyothsna P [email protected] India

K

K. Rebekah Prasoona [email protected] IndiaKailash Chander Bansal [email protected] Indiakalidhasan Nagarajan [email protected] IndiaKåre Lehmann Nielsen [email protected] DenmarkKaren Gjesing Welinder [email protected] DenmarkKazuhiro Kato [email protected] JapanKoh Aoki [email protected] JapanKoji Sakamoto [email protected] JapanKoteswararao Yadav DVVR [email protected] IndiaKrishna Kumar Gangopadhyay [email protected] India

L

Laia Fito [email protected] SpainLalji Singh IndiaLuisa Fernanda Bermudez [email protected] BrazilLukas Mueller [email protected] USA

M

M. Udayakumar [email protected] IndiaM. K. Mishra IndiaMajor Singh [email protected] IndiaMakarand Keshaorao Pande [email protected] IndiaMakesh Sekar [email protected] IndiaMalathi Ganesh Varagur [email protected] IndiaMallikarjun G. Awati IndiaManash Chatterjee [email protected] IndiaManasi Mishra [email protected] IndiaManchikatla Rajam [email protected] IndiaManju Singh [email protected] IndiaManoj Prasad [email protected] IndiaManoj Goel [email protected] IndiaManupriya [email protected] IndiaMarcin Pieczynski [email protected] Poland


SOL 2009 313


SOL 2009314

Marco Van Schriek [email protected] NetherlandsMaria Del Pilar Moncada [email protected] ChinaMaria Raffaella Ercolano [email protected] ItalyMark Taylor [email protected] UKMartin Ganal [email protected] GermanyMathura Rai [email protected] IndiaMeenakshi Munshi India Michal Oren-Shamir [email protected] Israel"Miguel " Botella [email protected] SoainMinkyu Park [email protected] Korea (south)Miroslawa Staniaszek [email protected] PolandMK Bhan India Mohamed Hichem Neily [email protected] JapanMonika Bansal [email protected] IndiaMyrna Q Sevilla [email protected] USA

N

N. Surya Prakash Rao IndiaNaama Menda [email protected] USANagendra Kumar Singh [email protected] IndiaNandkumar Sheshrao Kunchge [email protected] IndiaNar Singh Chauhan [email protected] IndiaNavin Sharma [email protected] IndiaNeeraj Kr Rai [email protected] IndiaNeeta Shailesh Madan [email protected] India Neha Tewari [email protected] IndiaNeha Gajendra Wasnik [email protected] IndiaNicolas Sierro [email protected] SwitzerlandNiranjan Chakraborty [email protected] IndiaNikolai Ivanov [email protected] SwitzerlandNithya Subramanian [email protected] UKNongmaithem Sapana Devi [email protected] IndiaNurit Firon [email protected] Israel

O

Osman Basha [email protected] India

P

Paramjit Khurana [email protected] IndiaParul Chowdhury [email protected] IndiaPawan Kumar Saraswat [email protected] IndiaPieter Van Poppel [email protected] NetherlandsPieter Martijn Eggink [email protected] NetherlandsPinky Agarwal [email protected] IndiaPooja Sharma [email protected] IndiaPradeep Kumar Jain [email protected] IndiaPradeep Kumar Burma [email protected] IndiaPrakash Chand Sharma [email protected] IndiaPranav Pankaj Sahu [email protected] IndiaPranjal Yadava [email protected] IndiaPravendra Nath [email protected] IndiaPriyanka Deveshwar [email protected] India

Q

Qazi Mohd Imranul Haq [email protected] India

R

R. Phanindranath [email protected] IndiaR. P. Sharma [email protected] IndiaRachel Meyer [email protected] USARahul Kumar [email protected] IndiaRajender Singh Sangwan [email protected] IndiaRajesh Kumar [email protected] IndiaRakesh Upadhyay [email protected] IndiaRakesh Kumar [email protected] IndiaRamesh K. Aggarwal [email protected] India


SOL 2009 315

Rameshwar Sharma [email protected] IndiaRanjita Sinha [email protected] IndiaRatnesh Singh [email protected] USARavi Rajawanshi [email protected] IndiaReddaiah Bodanapu [email protected] IndiaRené Klein Lankhorst [email protected] NetherlandsRenu Swarup [email protected] India Richa Shukla [email protected] IndiaRob Alba [email protected] USARobert Hall [email protected] NetherlandsRobert Buels [email protected] USARoeland van Ham [email protected] NetherlandsRosa Lopez-Cobollo [email protected] UKRuud de Maagdruud [email protected] Netherlands

S

S. Jayasinha [email protected] IndiaSagar Datir [email protected] New ZealandSalej Sood [email protected] IndiaSaloni Mathur [email protected] IndiaSameer Srivastava [email protected] IndiaSamresh Dwivedi [email protected] IndiaSangeeta Negi [email protected] Indiasanjay kapoor [email protected] IndiaSanjeev Sharma [email protected] UKSanjeev Kumar [email protected] IndiaSanjit Aradhye [email protected] IndiaSantanu Acharya [email protected] IndiaSantoshkumar Shetty [email protected] IndiaSanwen Huang [email protected] ChinaSarita [email protected] IndiaSathiyamurthy Appachi [email protected] IndiaSaurabh Raghuvanshi [email protected] IndiaShailendra Vyas [email protected] IndiaSharmila Chattopadhyay [email protected] IndiaSheetal Arora [email protected] IndiaShelly Praveen [email protected] IndiaSherinmol Thomas [email protected] IndiaShibin Mohanan [email protected] IndiaShubhra Barwa [email protected] IndiaShuchi Smita [email protected] IndiaShyam Kumar Sharma [email protected] IndiaSiddhartha Kumar Mishra [email protected] IndiaSilvia Minoia [email protected] ItalySimon Jam De Hoop [email protected] ThailandSimone T Earle Barrett [email protected] IndiaSivapriya Sethuraman [email protected] IndiaSmriti Shridhar [email protected] IndiaSneha Bhogale [email protected] IndiaSoni Gupta [email protected] IndiaSoung-Woo Park [email protected] Korea (south)Sreelakshmi Yellamaraju [email protected] IndiaSreenivasa Rao Eguru [email protected] IndiaStephen Stack [email protected] USASubhra Chakraborty [email protected] IndiaSudhir K. Sopory [email protected] IndiaSulabha Sharma [email protected] IndiaSuneha Upadhyay [email protected] IndiaSunghwan Jo [email protected] Korea (south)Sunil Mukherjee [email protected] India


SOL 2009316

Sunil Archak [email protected] IndiaSuping Zhou [email protected] USASupriya Chakraborty [email protected] IndiaSuresh Kumar Gupta [email protected] IndiaSwatismita Dhar [email protected] India

T

Teresa Mosquera-Vasquez [email protected] ColombiaTeresa Maria Montoro Ponsoda [email protected] SpainThikra Dawood [email protected] NetherlandsThomas Städler [email protected] SwitzerlandTilak Sharma [email protected] IndiaTohru Ariizumi [email protected] JapanTomasz Golas [email protected] NetherlandsTorben Jahrmann [email protected] SpainTrilochan Mohapatra [email protected] IndiaTripta Jhang [email protected] India

U

Uri Krieger [email protected] IsraelUsha Vijayraghavan

V

V Babu Rajendra Prasad [email protected] IndiaVaijayanti Tamhane [email protected] IndiaVaraprasad Kodeboyina [email protected] IndiaVeer Bahadur Singh [email protected] IndiaVeeranki Krishnamurthy [email protected] IndiaVenkata Reddy Thamalampudi [email protected] IndiaVenkatarami Reddy Sanampudi [email protected] ItalyVéronique LEFEBVRE [email protected] FranceVidya Gupta [email protected] IndiaVijee Mohan [email protected] IndiaVikas Kaondal [email protected] IndiaVinay Baranwal [email protected] IndiaVinay Kumar Singh [email protected] IndiaVineeta Singh Chauhan [email protected] IndiaVipen K Sawhnwy [email protected] CanadaVipin Permar [email protected] IndiaVivek Dogra [email protected] India

W

Wencai Yang [email protected] ChinaWilco Ligterink [email protected] NetherlandsWon - Hee Kang [email protected] Korea (south)

X

Xavier BERTHET [email protected] Spain

AUTHOR INDEX

317


SOL 2009318

Abbott, James Acciarri, NAcharjee, Animesh Adato, Avital Aggarwal, Ramesh K. Agrawal, Lalit Aharoni, Asaph Ahn, Jong Wha Akiva, Ayelet Bar-Alabadí, David Alba, Rob Albert, Victor Albert, Henrik H. Althan, Levia Alves, Gabriel S. C. An, Song-Ji Anacleria, Milena Anand, C. G.Anderson, Lorinda K. Andrade, Alan C. Angenent, Gerco C.Aoki, Koh Aouini, Asma Appachi, Sathiyamurthy Archak, SunilAriizumi, Tohru Arima, SAsamizu, Erika AshaAsquini, Elisa Awati, Mallikarjun G.Awati, Mallikarjuna G.

17814021462120, 261, 262, 26336, 5462, 105, 1601521607910212420848122, 260435526527122, 124, 128, 2608189, 2335327714289, 23316353, 89, 233289127265272

A

Babili, Salim AlBabu, B.Sarath Bachem, Christian Bae, Ik-Hyun Bae, Jung Hwan Ballester, ARBallesteros, Carolina Ballvora, Agim Bambarely, Aureliano Bansal, Kailash C.Bansal, K.C. Bar, Einat Barbierato, V.Barone, A.Barry, Cornelius Basu, S. Batiste, JeanBeasley, Helen Behera, Rajendra K Bendahmane, A.Bernard, QuéménerBernillon, Stéphane Beyer, Peter Bhat, S.S. Bhat, Asha Bhattacharyya, DiptoBielewicz, D.Bielewicz, D.Bishop, Gerard J. Bita, C.Blattes, Anne Bloch Jr., Carlos Blok, Vivian C. Bodanapu, Reddaiah Boer, Jan de Bogden, Bert Bonciani, Giulia Borm, Theo Botella, Miguel A. Bovy, AGBradeen, J.M. Broadley, Martin Broers, Greet Steenhuis-Bryan, Glenn Buchan, Daniel Buell, C. Robin Buels, Robert Bum Yang, HeeBusscher, Marco Butcher, Sarah Byung, Dong

1629451, 110, 25815223556351177122914416014355, 163, 1807617111317823047, 169, 209, 79203207162266267136491771784415426041230110, 258258, 28317811047, 21256, 6337242281112, 178, 2281786971, 21627881178149

B

C, Savitha.Campbell, Raymond Caponetto, G.Carazzolle, Marcelo F. Cariaso, Mike Carli, P Caroline, Carriero, FilomenaCastanedo, Itziar Castro, MarquesCeoloni, Carla Chaitanya, RameshwarChaitanya , Ch.Chakrabarti, S.K. Chakraborty, S.Chakraborty, Niranjan Chakraborty, Subhra Chakraborty, S. Chakraborty, Supriyo Chandrashekar, Arun Chapman, Sean Chatterjee, Manash Chattopadhyay, Debasis Chattopadhyay, Sharmila Chattopadhyay, B. Chattopadhyay, D. Chaudhary, Anuj Chauhan, Vineeta SinghChauhan, Vineeta Singh Chen, Jia Cheorl Kang, Byoung-Chiaiese, P. Chinnusamy, Viswanathan Chitwood, Daniel Chiusano, M.L.Cho, Sunghwan Choi, Doil Choi, Hak sun Chowdhury, Sarita, P. Christian, ChevalierChung, MiYoung Churcher, Carol Cobollo, Rosa LopezCock, Peter Cohen, OdedCole, A, Benjamin J. Colombo, Carlos A. Conner, Anthony J.Costa, Gustavo G.L. Crosslin, J.M.Cutts, Rosalind Czerednik, Anna Czyzewska, D.

287228143128283163, 239176203471761342252403717136, 5436, 54173189270, 2754116974136171179213240221922782431447718023538, 152, 235, 278, 1812351795776178178411501021282571281651788149

C

18018811319328311025736, 54172, 1915628310211429555161277272, 2732036922017624310716226615117949, 177149

D'addiego, L.Damle, Mrunal S. Danan, Danan, Sarah DaPonte, Lorena Datema, Erwin Datir, Sagar Datta, Asis Datta, D. de, Vos CHde Heer, Peter Debbie, Paul P. Dębski, Konrad Deepa, J. Delledonne, Massim Demirel, Ufuk Desikathathachari, Veeraragavathatham Devaraja Achar, A.M.Devaux , Marie FrançoiseDeynze, Allen Van Dhakal, Min RajD'hoop, Björn B. Di Matteo, A. Diretto, G.Diretto, Gianfranco Divya, M.H Do, Jae WahngDogra, V. Dolata, J.Dong , Ju

D


SOL 2009 319

Donini, Paolo Douches, Dave Droege, Marcus Dubey, Rama Shankar Ducreux, Laurence J.M. Durstewitz, Gregor Dutta, D. Dwivedi, Samresh Dwivedi, Neeraj

13469322002289188, 244132250, 280

E., Ebert, Andreas Eijk, Michiel van Eira, Mirian T.S. Ercolano, M. R.Errico, A. Ewert, Sophie Ezura, Hiroshi

103245258, 283128163, 180, 23924315453, 80, 89, 106, 207, 233

E

Fabiana, de GodoyFacella, P.Falcone, G.Fantini, E.Fatima, Tahira Fernando, CarrariFernie, Alisdair Fernie, A.Feron, Richard Ferriello , F Filiault, Daniele Filippone, E. Firon, Nurit Flath, Kerstin Flis, Iwona WasilewiczFloras, K.Fogelman, EdnaFogliano, VFrancis, David M. Frank, Gil Freire, Luciana P. Frusciante, Luigi Frusciante, LFukuda, Naoya Fukuda, N.Fukukawa, G.

20418052, 18052, 1806020479107258, 28323977243481171144916116369, 9248122, 26055163, 180, 23989, 233106106

F

G. F., D'souzaGaikwad, Ambika Baldev Gaikwad, K. Gaitan, Alvaro Ganal, Martin Ganesh, CTGangopadhyay, KK. Gebhardt, Christiane Geetha, A.Gennaro, AndreaGhanta, Srijani Ghosh, Rajgourab Ghosh, Sudip Ginzberg, Idit Giorno, M.Giovannoni, James Giovannoni, James J. Giovannoni, J.Giovannoni, Jim Giri, APGiri, Ashok P. Giridhar, P.Giridhar, Parvatam Giuliano, G.Giuliano, Giovanni Goel, Manoj Goicoechea, J.L.Gomez, Aureliano BombarelyGómez, José M Jiménez Gómez, Gustavo Gonzalez, M.Gopal, J.

G2659517912391, 1341311421172861341363636161447610210717815318826427052, 107, 180124, 1622521266631, 773518098

Gosselin, Joseph Goyer, Claudia Grandillo, SGranell, AGranell, Antonio Gray, S.M.Graziosi, Giorgio Grover, Atul Gupta, Aditya K.Gupta, D.K. Gupta, V. Gupta, Vidya S. Gupta, Soni Gupta, Suresh Kumar Gupta , VSGurumurthy, D SGuzowska, Ewa Zimnoch

71116565679, 212165124, 1277020573179188221, 223, 225, 236, 241227, 240153135114

Habdas, H. Hall, Robert D. Ham, Roeland van Hamilton, John Hamm, P.B.Han, Jung-Heon Handa, Avtar K. Hanumantha, B.T. Haq., Q.M.I.Hara, Agnieszka Harpenas, Asep Hautea, Desiree M. Hazarika , Pranjal Headland, Lauren Hedley, Pete E Hendre, Prasad Suresh Henk, Hilhorst Hennig, J.Henry, Robert Hirai, T.Hiwasa, Tanase K.Hofferbert, Hans-Reinhard Hoop, S.J. de Huang, Sanwen Hueso, Leandro Humphray, Sean Hur, Cheol-Goo Hussain, Zakir Hutten, Ronald C.B.hwan Jo, Sung

18756,6326, 1106916515160, 86266183, 184, 185, 25311428114718677228261, 262, 263584912410610611714169, 1117917830, 152240238278

H

Ichikawa, T. Irikura, Beth Ivanchenko, Maria

106208220

I

Jacobs, Jeanne M.E.Jagadishan, M. Jain, R. K. Jakuczun, Henryka Jansen, Kim Jarmołowski, A.Jayarama, Jeena, D.Jeifetz, Dar Jeong, Hee-jinJeyaraman , RJo, SungHwanJo, Yeong deuk Jo, Sung-Hwan Jones, John T. Jong, Walter De Jong, Hans de Jorgensen, MJun, ZhangJung, Jin-Kee Jyothsana, P.

25726716711425849, 177120, 265, 266, 2692731502352711523018141691786168237183, 184, 185, 253

J


SOL 2009320

K., ArchanaKadosh, Dana Kakuta, H. Kalia, S. Kalidhasan, Kang, Byoung-Cheorl Kang, Bo-Ra Kansal, Rekha Kapoor, Sanjay Karihaloo, J. L. Karjee, Sumona Karlowski, W.Karp, Peter Katiyar, Amit Kato, K.Kaur , Manjeet Kaushik, Mamta Keiji, Eric Keizer Khandelwal, NKharshiing, Eros Vasil Kharshiing, Eros Khatri, Madhu Khurana, P. Khurana, J.P. Kikkawa, N. Kim, June-Sik,Kim, Byund-DongKim, Jungeun Kim, Y-W. Kim, Kimura, Seisuke Kloosterman, Bjorn Kloosterman, Bjorn A. Kochko, Alexandre de Koeda, Sota Koenig, Daniel Koeyer, David De Koltai, Hinanit Koundal, K.R. Koundal, Vikas Kozik, Elzbieta UKozik, E. U. Kreuze, Hannele LindqvistKrieger, Uri Krishna Reddy, M. Krishnamurthy, V. Kudrna, D.Kulińska, Z.SzweykowskaKulkarni, MJKumagai, Monto Kumar, Amar Kumar, Rajesh

Kumar, Sanjeev

Kumar, Vinod Kumar, Vikash Kumar, Akhilesh Kumar, Gunjeet Kumar, Ashish Kumar, R. Kumar, Rajiv Kumar, Rahul Kumar, Rakesh Kumar, Sanjay Kumar, Maneesh Kumar, Avinash Kumar, S. Satheesh Kumar, Anil Kumari, Anitha Kumari, Deva Kumari, Alka Kumari, Archana Kurabayashi, Atsushi Kurokawa, N.Kushwaha, Hariom Kwon, Jin KyungKwon, Jin-Kyung

253, 183, 184, 1851611069520530, 43, 235, 237181133137, 2069510417766229106952521281761538323010872, 17972, 1791061491523010614931, 77512381244377116160133138, 168, 254, 291, 2922341878790295130126 49 , 177 ,1532084188 , 190 , 192 ,200, 201 , 246 , 250 , 280

88, 172, 191 , 195 , 199 , 244 , 279

98104133142172,19117920020621125025226426726745135240294233106 , 171217 , 21823543 , 152 , 278

Lahaye, Marc, Lakshmanan, PugalendhiLankhorst, Rene Klein Lanteri, SLashermes, Philippe Lazaro, Esperanza Lee, JundaeLee, Won PhilLee, JeMin LeeLefebvre, Véronique Leiberman, Raya Lenka, Sangram K. Leo, WillemsLeroy, Thierry Lewinsohn, Efraim Li, Jinjie Lima, Viviana Linden, Gerard van der Lippman, Zach B. Litt, Amy Lloyd, Christine and QC TeamLo Scalzo, RLokossou, Anoma Lopez, L.López, Mariana Lübeck, Jens Luerssen, Hartmut Lugon-Moulin, Nicolas Luisa, Bermudez

K L203277283140121 , 2667915115176149113 , 154, 19316022958122156 , 160432124590991781404018021211791134204

Maagd, Ruud de Madhulatha, Patrani Madhura, J.N.Magdalena, RossiMahajan, R.K. Maharijaya, Awang Mahavishnan , KMahto, A.K. Malathi, V.G.

Maldonado , C.E.Maliepaard, ChrisMallard, Stéphanie Maloof, Julin N Mangruthia, Satendra K.Manjunath, B. Marczewski, W.Marhevka, Mariani, C. Marraccini, Pierre Martí, Cristina Martin, CMatas, A.Mathur, S. Matsukura, Chiaki Matt , JonesMatteo, Antonio Di Mattoo, Autar K. Maucourt, Mickaël Maurizio, Picarella Mayer, Klaus Mazzucato, Andrea McLaren, Stuart and Pre-finishing TeamMcLaren , Karen and Finishing TeamMenda, Ron Caspi, Naama Menda, Naama Mennella, GMerino, Carlos Meyer, Rachel Mickey, Hanjabam Migliore, Melania Mills, Adri Ming, Ray Minkyu, Minoia, Silvia Minutolo, M. Mishra, S.K. Mishra, MMishra, Upama Mishra, M. K. Mishra, Siddhartha K. Mishra, Anil K.

81197 , 213272 , 27320414228113517939 , 183 , 184 , 185 , 253 , 294

12621415431 , 7716829549 , 18710344122 , 260795610772 , 17953 , 80 , 89 , 207,233 178556080 , 207226178196 , 2261781786671 , 2161409799236 , 24016271124149203243142153200 , 201267285291

M


SOL 2009 321

Mizoguchi, Tsuyoshi MO, Viji.Modonut, Martina Mohan, Raju BMohan, Vijee Mohanan, Shibin Mohapatra, T. Moing, Annick Molthoff, JMoncada, PMondego, Jorge M.C. Moriguchi, Takaya Moro, Gianluca De Morris, Wayne LMorris, Jenny A Mosca, GiuseppinaMosconi, P. Moyal, Ben Zvi M.Muday, Gloria Muddarangappa, ThippeswamyMueller, Lukas Mukherjee, Sunil Kumar Munetaka, Hosokawa Muniswamy, B. Murphy, Agnes Murthy, T.G.K. Mustafiz, Ananda

89 , 106 , 233288127131221, 225 , 240,241,24827017980 , 2075612612880 , 207127157 , 22822820319610323116166, 71 , 102 , 178 , 21610443266 , 26911613046

N, Santhi.Nagai, ChifumiNagai, Chifumi Nagarajan, Naik, Raju Nandy, SoumenNarendra, D.Nataraja , KNNataraja Karaba, N.Nath, Pravendra Nathiya, S.Nayar, SaraswatiNegi, Sangeeta Neha TNeily, Mohamed Hichem Ngoc Huy, Hoang Nielson, Kare Lehman Nongmaithem , Sapana DeviNoorullah, KahnNoschinski, Merle Nowakowska, Marzena

2872081242052402221062712738224913783, 231 , 224183 , 184 , 185 , 25380 , 20727811522058117234

N

Oeveren, Jan van Okabe, Yoshihiro Oliver, Karen Olson, Douglas J.H. Oomen, Saskia JacobsOphir, Ron Osman , Basha POvadia, Rinat

258 , 283891786428348224 , 240160

O

P, Jyothsna Pal, J.K.Pal, Ramakrishna Palchamy, Kadirvel Pallavicini, Alberto Pandey, RoopaliPandey, S.K. Pandey, Hausila Pandit, A.Pandravada, S.R.Papacchioli, Velia Pappu, Hanu R. Paramás, Ana Ma González Paran, Ilan Pareek, Sneh Lata SinglaParimi, Srinivas Parizzi, Lucas P. Park, MinkyuPark, Jongsun

29417921324512721337 , 98201179941621666315046 , 19714512815230

P

Parvatham, R .PatrickPaul, L.C.Paull, Robert E Pereira, Gonçalo A.G. Permar, Vipin Perrotta, G.Pezzotti, Mario Phanindranath, Regur Phillips, S. AustinPhillips, Mark S. Pieczynski, M.Pietrella, M.Piron, Florence Polley, Andreas Posé, David Pozzi, Carlo Pradeepa, D.Prakasan, C. B. Prakash, Geeta Prasad, ManojPrasad, M. Prasad , T.GPrasanna, HC Prasoona, R. Praveen, ShellyPressman, Etan Prins, Marcel Pujar, Anuradha Purwito, Agus Pushpalatha, R. Pylypenko, Liliya

28811317620812829118055263374149 , 17718079, 2039147134286266144189173272 , 273189 , 244262292, 138 , 168 , 254 , 29148258 , 28366 , 7128129541

Rai, AshutoshRai, Mathura

Rai, N. K Rai, Neeraj K Rai, Avinash Chandra Rai, Govind KumarRai, Neha Prakash Rai, Ved Prakash Rai, Nagendra Rajam, M. V. Rajam, Manchikatla Rajendher, V.Rajwanshi, Ravi Rama , NRaman, K.V. Rambla, Jose Luis Ramos, Humberto J O Ramsay, Gavin Rao, E.S.Rao, A. V. Rashid, KazmiRathaur, Sushma Rau, G. V. Krishna Raveendra, GMRavishankar, G.A. Ravishankar, K.V. Ravishankar, GRay , Soham Reddaiah, BReddy, T VenkataReddy, M. KReddy, M. Krishna Reddy, K. Madhavi Reddy, S.V.R.Renuka Swamy, N. S.Richa , S.Riddle, Clare Ridgway, Hayley J. Rietman, Hendrik Riffat , JohnRinaldi, P.Rivera, L.Robert, Vincent Rodrigues, Gustavo C.Rogachev, Ilana Rogers, Jane

190, 192, 200, 20188 , 172 , 191, 192, 195,199,200, 201, 244,246,250,279,280,293

173189195199199250293108, 186, 197, 222, 2522132222292713779122 ,260228 , 242245261 , 26258199119271120 , 264170, 173 , 29527073224 , 227 , 236,240132173170170196265183 , 184 , 185 , 253 17825740197143126134122, 260160178

R


SOL 2009322

Rolin, Dominique Rombauts, SRosato, V.Rose, J.Ross, Andrew R.S. Rotino, GLRoyer, Suzanne M.

80, 20723918010764140 , 14327

S, Nathiya. Sacco, Adriana Sadashiva, A.T. Sahu, Pranav Pankaj Sainani, Mohini N. Saiprasad, GVSSaito, Takeshi Sakthivel , KSanampudi, Venkataramireddy Sane, Aniruddha P. Sangwan, Rajender S. Sangwan, Neelam S. Sankoff, David Sanseverino, WSanthi, N.Santisree, Parankusam Sapana, NSarah, Sarala, K. Sato, Shusei Sauve, Roger Savitha, CSawhney, Vipen K. Sawhney, S.K. Saxena, Sangeeta Scandura, Mariaconcetta Schauer, N.Schmelzer, Elmon Schriek, Marco van Scossa, FedericoScossa, F.Scott, Carol Selleri, Luigi Semel, Yaniv Senthil, Kalaiselvi Sethuraman, Seymour, Graham Shah, Kavita Shahar, LiatShaked, Rachel Shaktivel, KShamir, Michal OrenSharma, PC Sharma, A.K. Sharma, T.R. Sharma, Rameshwar

Sharma, S.K.Sharma, Navin K Sharma, Navin Sharma, Rita Sharma, Arun Sharma, Sanjeev Kumar Sharma, Sulabha Shaw, Martin L. Shearer, Lindsay A. Shekhar, Shubhendu Shen, Huolin Sheoran, Inder S. Sheshshayee, MSShetty, Santoshkumar M Shridhar, SmritiShukla, Vijaya Signoret, Patrick Simmi, P.S.Singh, PriyankaSingh, VBSingh, Major

28755173 , 295173 , 18918813189 , 23322422682158 , 285285 , 28912423924983 , 227240113130178652496414427922810711728 , 28316210717822685249 , 286 , 287 , 288205178195160482271607072 ,179 , 206 73 , 17983 , 211,220 , 221, 223, 224, 225, 227, 230, 231,236,240, 241,248

94 , 142131132 , 13513719820923025727549264131, 272 , 2732757460113, 193264168, 29218388 ,172 , 189 , 190 , 191,192,195,199,200,201,244,246,250,279,280,293

Singh, Lalji Singh, B.P. Singh, Sonika Singh, HP Singh, Neeru Singh, Priyanka Singh, Ajay Singh, Janardan Singh, A. K. Singh, M. Singh, A. Singh, N.K. Singh, Prabhash Chandra Singh, Ratnesh Singh, Vinay Kumar Singh, Smita Singh, Prabash Chandra Singh, V.B. Singh, R.K. Singh, Priyanka Singh, Ramesh Kumar Singh, Satya Narayan Singh, T.H. Sinha, Neelima Sinha, Ragini Sinha, Brajesh Sinha, D.P. Sivapriya, SJS , Rama DeviŚliwka, Jadwiga Smita, Shuchi Sobolev, Anatoli Solanke, A.U. Solanke, Amolkumar SonalSong, Wonho Sood, SalejSopory, Sudhir K.Sopory, S. K. Spitzer, B.Sreenath, H.L. Srinivas, Ankanagari Srinivasan, Rajeswari Srujana, RStack, Stephen M. Städler, Thomas Staniaszek, M. Stensballe, Allan Stephan, Wolfgang Strahwald, Josef Subramanian, Nithya Suda, Kunihiro Sudarshini, K. SundareshaSuresh, N. Surya Prakash, N. Suzuki, Ayako Swarnalatha, P. Swarup, Renu Szinay, Dora

253795100108138144159171179179179190 , 192208217 , 21824624625328029129329329531 ,77136190, 192244, 279205211114229601791982273013246197103120, 266, 268,26923024125227 , 178971876197117242233295266267120, 266,26723329529178

Tabata, Satoshi Tacke, Eckhart Tai, Helen Takane, K.Tamhane, VATardella, LTavazza, R.Tavazza, Raffaela Taylor, Mark Tecle, Isaak Tellier, Aurelien Thomas, Sherinmol Tikunov, YMTikunov, Yury M. Tissier, Alain Tiwari, Shilesh Tokuhisa, Jim Tokutomi, Satoru

S

178117116106153163107162157 ,22866,71, 21697223, 24056637895161230

T


SOL 2009 323

Toppino, LTornincasa, Patrizia Tovar, E.Tuli, Rakesh Tyagi, Akhilesh K.

140 , 143127126158 , 285206, 72 , 179 , 198

Udaya Kumar, M

Uitdewilligen, Jan G.A.M.L. Umashanker, R Upadhyay, Ambika PrasadUpadhyay, Rakesh Kumar Upadhyay, Priti Upadhyay, Suneha

120, 131 , 265 ,271, 272,273

23896131, 13582190 , 192254

U

Vainstein, A.Vale, G.P.Valpuesta, Victoriano Van De , Peer YVan Eck, Herman J. Van Ham, Roeland Varaprasad, K.S.Vasquez, Teresa MosqueraVeer, B.S.Veilleux, Richard Veluthambi, K. Venkataramanan , DVéroniqueVeyrieras, Jean-Baptiste Vezzi, A.Vidal, Ramon O. Vieira, Natalia G.Vieira, Luiz G. E. Vigouroux, JacquelineVinecky, Felipe Vinothkumar, R. Visser, Richard Voorrips, Roeland E. Vos, Ric C.H. de Vosman, Ben Vossen, Edwin van der Vrebalov, Julia Vriezen, W.Vyas, S. Vydehi , Kanneganti

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V

Wang, Yuanyuan Wang, Ming-Li Wang , Miqia Weiss, David Welinder, KG.White, Philip Whitworth, J.L.Whitworth, Jonathan Wilco, LigterinkWing, R.Wolters, Anne-Marie A. Wright, Kathryn Wu, Tingting

9220840160612421651665812623825792

W

Yadav, Gitanjali Yadav, S.K. Yadav, Dinesh Yamazaki, Yukiko Yang, Wencai Yano, KentaroYano , M.Yeldur, P. VenkateshYellamaraju, Sreelakshmi

Yoon, Joonseon Yoon, Jae Bok York, Tom

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3815171

Zamir, Dani Zanor, Maria Ines Zehr, Usha B. ZhonghuaZhou, Suping Zikihara, Kazunori

Z33,85,9079145, 1466865230


SOL 2009324

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Documents

Expression Analysis of Carotenoid Biosynthesis Genes in Carrot (Daucus Carota) Using Real Time Quantitative PCR