Upload
others
View
0
Download
0
Embed Size (px)
Citation preview
Specific research activities within WGIN –how science can identify new traits and the tools to exploit them
Kim Hammond-Kosack
Wheat Pathogenesis ProgrammePlant-Pathogen InteractionsDivision
23rd June 2004
The Defra WGIN Core ProjectAims:To Underpin Wheat Improvement by Plant
BreedersApproaches:1. Characterisation and provision of genetic resources2. Genetic mapping and marker development3. Trait identification 4. Identification and generation of novel variation in
key traits : using non-GM approaches5. Central storage of grain from field trials6. Liaison and communication
Genetic mapping and marker development
• Establish a reference UK mapping population
Avalon Cadenza
Avalon x Cadenza -
204 double haploid lines
• Switch to ‘within the gene’ molecular markers
> 500,000 wheat ESTs available
Trait identification 1. Nitrogen utilisation and economy (NUE)
2. Plant architecture traits to lower disease pressure
Septoria leaf blotch Fusarium ear blight
3. Grain quality • Field trial at RRes, 0,100, 200, 350 kg nitrogen,31 cultivars, UK(20), Fra (5), Ger (5), Poland (1)
• Desktop scoping study NUE (U of Notts, RRes, ADAS)
WGIN cultivar list for Year 1 field trial
HerewardFlandersEnormELS 02-30EinsteinCappelle-DesprezCaphornCadenzaBeaverBatisAvalonArche
PetrusPBIS 00/77=PrivilegParagon (Spring)Opus MonopolMerciaMaris Widgeon MalaccaLynxIsengrain
ZytaXi19SparkSolsticeSoissonsSokratesScorpion 25RibandRialto
Blue = public molecular data availableGreen = Broadbalk long term exp RRes
Underlined – parent of public DH mapping population
Year 1: Field trial at Rothamsted Research
3 replicates, 4 N treatments, plot size 3 m x 14 m
Split N application
kg N/ha Mar Apr May0100 50 50 0200 50 100 50350* 50 250 50
Tillering GS31/32 GS37
* PGR applications very important to prevent lodging
Year 1: Field trial at Rothamsted Research
Sowing date: Last week November
Emergence: scored December
Residual soil N – deep soil cores taken Feb
Established protocols to measure the variousplant architecture traits
GS 31, 39, 65 and 92 determined for each genotype / treatment
Peter Barraclough, Dimah Habash,Darren Lovell, Caroline Shepherd
Exploitation of diploid wheat
Why ? A rich source of additional gene diversity Ability to test the function of individual alleles
Species: T. monococcum (AA genome)
AA x BB
AABB x DD
AABBDD
T. urartuT. monAA
+ mycotoxins
Novel resistance to problematic pathogens
Septoria leaf blotch
Tapesia eyespot
Fusarium ear blightPolymyxa graminis- Soil borne cerealmosaic virus
Infected roots
T. monococcum collection – three sources
Vavilov Institute, St. Petersburg, Russia
24 accessions all land races, 17 accessions collected prior to 194019 countriesall previously shown to exhibit resistance to multiple pathogens and insect pests in Russia
Sort Number MDR VIR nomber Variety Origin Country Year1 MDR 24 K-105 flavescens, hornemannii Chechen-Ingushetia 19042 MDR 25 K-8365 flavescens, macedonicum Crimea, Ukraine 19233 MDR 26 K-8555 macedonicum, symphaeropolitanum Crimea, Ukraine 19234 MDR 27 K-18105 macedonicum Azerbaijan 19275 MDR 28 K-20399 flavescens Germany 19276 MDR 29 K-20491 flavescens Spain 19277 MDR 30 K-20589 monococcum Spain 19278 MDR 31 K-20994 vulgare, macedonicum Turkey 19279 MDR 32 K-21308 vulgare Italy 1927
10 MDR 33 K-23032 vulgare Yugoslavia 192811 MDR 34 K-23653 hornemannii Armenia 192812 MDR 35 K-25968 vulgare Austria 193013 MDR 36 K-29603 flavescens, monococcum Czechoslovakia 193214 MDR 37 K-30086 macedonicum Armenia 193415 MDR 38 K-30090 monococcum Armenia 193416 MDR 39 K-31683 hornemannii Georgia 193417 MDR 40 K-38079 macedonicum Bulgaria 194018 MDR 41 K-39417 nigricultum, flavescens Albania 195019 MDR 42 K-39471 macedonicum Balkans region 195020 MDR 43 K-39722 vulgare Greece 195021 MDR 44 K-45024 hornemannii Turkey 196522 MDR 45 K-45927 vulgare Denmark 197023 MDR 46 K-46748 macedonicum, vulgare Romania 197024 MDR 47 K-46752 macedonicum Hungary 1970
* also 96 samples from National Small Grains Collection, Aberdeen, USA and 3 from JIC, Norwich will be included in assessment
Description of T. monococcum accessions from VIRNote: Each accession is a land-race NOT a pure line
T. monococcum collection
3 accessions from John Innes Centre, UK
1. EMS mutagenised population of 600 linesavailable (V97031)
2. Can be regenerated in vitro3. Transformable by Agrobacterium - mediated
method ( H. Jones, RRes, 2003)
96 samples from National Small Grains Collection, Aberdeen, USA
Complete collection n = 123
Comparison of ear morphology
T. monococcumPI 119422
T.aestivumGabo
Resistance to Septoria leaf blotchScreening for resistance to Septoria leaf blotch –the No1 disease of wheat
crops in the UK
12
34 5
Field experiment at RRes (2003-2004) to explore reaction of 24 Triticum monococcum samples to
natural infection by Septoria tritici
Septoria disease progress – Jan 04
Good natural winter epidemic
Hexaploids
Claire R lesionsExcept R lesionsSpark R lesionsRiband S lesionsConsort S lesions
All 24 accession No lesionsDiploids
Disease measurements based on thermal time
T. monococcumK-20589
T. aestivumcv. Consort
Verification of Septoria diseaseon Triticum monococcum plants
Diagnostic PCR for Septoria triticiβ-Tubulin Cytochrome B
f Tm Ta f Tm Ta
Feb 2004
Pycnidia(Trypan blue
staining)
infected leaves
Septoria leaf blotch symptoms on hexaploid bread wheatcv. “Consort”, 5th May 2004
All Triticum monococcum plants (n = 3000+ plants) – no Septoria leaf blotch disease symptoms !
Glasshouse screening of T. monococcum
Host Plants
3 x T. mon; 2 x T. ave
Pathogen
5 x Isolates derived from T. monococcum3 x Isolates derived from Cv. Consort
3 x Isolates derived from Cv. Claire
Both hexaploid cultivars were heavily infected by all Septoria
isolates
Introgression of gene diversity from diploid wheat
Interspecies sexual crosses
T. aestivumAABBDD
T. monococcumAA
X
fertile F1 hybridAABBDD
deploy cytogenetics to select for 42 chromosomes
Overview of diploid wheat plant inoculation results
Septoria leaf blotch – most accessionsextremely resistant
Tapesia eyespot
Fusarium ear blight
Soil borne cereal mosaic virus – both resistantand susceptible accessions
vector Polymyxa graminis – only susceptible
Expected variation among accessions
SBWMV/Polymyxa
Tapesiaeyespot
Fusariumear blight
Ideal accessions for mutagenesis
Identification and generation of novel variationin key traits
Two demonstration PCR TILLINGprojects involving hexaploid bread wheat and diploid T. monococcum
Steven Henikoff and Luca Comai (Seattle, USA)
Targeting Induced Local Lesions IN Genomics
Originally developed for the model plant - Arabidopsis
PCR TILLING Technique to identify gene variants
P1 P2Gene of interest
PCR primers 1000bp apart
PCR product lines 1- 7
PCR product line 8
Pool of PCRproducts(1000 bp)
Single bp mis-matches cut in heteroduplex DNA by Cel1 enzyme
denaturingelectrophoresis
Wild-type allele
Variant allele
Assess lines carrying variant allele for novel phenotypes
Application of PCR TILLING technique
- chemically mutagenised populations
ethyl methane sulphonate (EMS) causes GC to AT base pair changes
- Diverse germplasm collections (EcoTilling)
Compare the extent of gene diversitybetween diploid and hexaploid wheat
Demonstration PCR TILLING project No 1
Rht3 gene variants - wild-type phenotype extreme dwarf
rht Rht1 Rht2 Rht1Rht2
Rht3 Rht2Rht3
EMS mutagenised - Mercia Rht3 line- Cadenza
Demonstration PCR TILLING project No 2
Global plant defence signalling regulatorsin both cereal and non-cereal species
PATHOGEN RECOGNITION
Plant cell signalling
A MULTI-COMPONENTRESISTANCE RESPONSE
3 COMPONENTS TO INDUCIBLE PLANT DEFENCE
Overall PCR Tilling Objective for wheat
Correlate using the diploid accessionsspecific gene sequences with specific phenotypes (trait types)
Example: the rar1 gene – required for disease resistanceto multiple pathogens
R1 R2 R3
rar1 (null)
Fully Susceptible
stop
R1 R2 R3
rar1 (weak)
Moderately R
stop
R1 R2 R3
rar1 (elite)
Fully Resistant
The Defra WGIN Core ProjectAims:To Underpin Wheat Improvement by Plant
BreedersApproaches:1. Characterisation and provision of genetic resources2. Genetic mapping and ‘within gene’ marker
development3. Trait identification 4. Identification and generation of novel variation in
key traits : using non-GM approaches- chemical mutagenesis- diverse diploid and hexaploidaccessions
- PCR Tilling technique5. Central storage of grain from field trials6. Liaison and communication
http://www.wgin.org.uk/
Wheat Genetic Improvement NetworkMany thanks to ….
Rothamsted ResearchPPI Division CPI DivisionHai-Chun JingDmitry Kornyukhin*Kostya KanyukaDarren Lovell
Peter ShewryAndy PhillipsKatie Tearall
Funded byJohn Innes CentreJohn SnapeRobert KoebnerLeodie AlbertChristian Rogers
Defra *Rothamsted InternationalFellows Programme