Improve chickpea productivity for marginal environments in sub-Saharan Africa and

South Asia

Introduction

Genomic resources

Genetic resources

Trait mapping- QTLs and GWAS

Molecular breeding for drought tolerance

Enhancing Capacity of NARS

Data management and decision support tools

Contents

Introduction…

Global distribution of chickpea area

Grown on about 12 m ha across 54 countries

>6 m ha 0.5 to 1.0 m ha 150,000 to 210,000 ha 50,000 to 100,000 ha 10,000 to 40,000 ha

Production constraints Abiotic stresses 1. Drought 2. Heat 3. Salinity 4. Cold

Max. Temperature (0C) Mean Temp Min. Temperature (0C)

Harvest

0 50

100 150 200 250 300 350 400 450

1 2 3 4 5 6 7 8 9 10 11 12 Month

mm

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0

Sowing

Terminal drought

Precipitation Evaporation

Mill

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(US$

)

Tota

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Dro

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Low

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Wilt

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Pod

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Constraints in molecular breeding

Very few molecular (SSR) markers

Either no genetic map or maps with low marker density

Non-availability of appropriate germplasm, mapping populations and phenotyping data

Non-availability of trait-associated markers in breeding

Capacity and skills in molecular breeding

Genomic resources...

Resource Amount SSRs ~2,000 DArT arrays 15,360 features SNPs 9,000 GoldenGate assays 768 SNPs KASPar assays 2,068 SNPs Veracode assays 96 SNPs

Summary of marker resources

LG1 LG2

LG3 LG4 LG5 LG6 LG7

LG8

Marker Loci: 1,291 Coverage: 845.56 cM

Comprehensive genetic map

TAG 2010, 2011 PLoS One 2011 Plant Biotech J 2012

KASPars assays integrated in transcript map

Markers mapped : 1328 Map distance : 788.6cM Average number of markers/LG : 166 Average inter-marker distance : 0.59cM

TAG 2010, 2011 PLoS One 2011 Plant Biotech J 2012

Towards genome-wide physical map

Clones CAH library CAE library Old library

Total

1st 2nd 1st 2nd 1st 2nd

Clones targeted

29,664 5,376 29,568 5,376 337 773 71,094 (12X )

Clones with usable data

28,492 5,160 28,272 5,240 319 765 68, 248

Clones in FPC

18,285 3,502 22,571 3,926 319 765 49,368

Old library, 1st instance are the clones from which BES-SSR were developed

Old library, 2nd instance are the RGH hybridizing clones

Fingerprinting statistics of different BAC-libraries

Clone statistics in contigs: Total no. clones in 1,174 contigs 46,112 Range of clone in contigs 2 to 3,007 Average no. of clones in each contig 39.27 Genome coverage 8X Genome represented 615 Mb Band statistics in clones: Total no. of bands in clones 318,971 Average no. of bands in clones 271.69 Range of bands in clones 34 to 2,268 Minimum tiling path (MTP): Total no. of contigs 1,174 No of clones in MTP 4,290

Statistics of physical map Collaboration: NIPGR, India and UC-Davis, USA

Integrating sequence, physical and genetic maps

The chickpea genome

Re-sequencing in chickpea Samples: - 5 wild species - 25 landraces - 60 breeding lines/ released varieties Or - 5 wild species - 57 Desi genotypes - 28 Kabuli genotypes

Sequencing: -RAD sequencing (35 Gb) - 61 genotypes (40 desi, 16 kabuli, 5 wild) -WGRS (205 Gb) - 29 genotypes (17 desi, 12 kabuli)

Genetic resources…

Harnessing alleles from genebanks 1,700 genebanks

By 1997, the world economy had accrued annual benefits of ca. $115 billion from use of crop wild relatives

Genomic characterization of gene bank material is essential

Needs to associate allelic value with phenotype

Transfer in elite varieties

Genetic resources

300

(A) Intra-specific mapping populations: 1. ICCV 283 × ICC 8261 - 289 RILs 2. ICCV 4958 × ICC 1882 - 268 RILs

(B) Chickpea reference set -300 genotypes

(C) Pre-breeding populations ICRISAT, Patancheru - 17

ICRISAT, Nairobi - 20

Egerton Uni. Kenya - 5

EIAR, Ethiopia - 2

MAGIC populations

Genotypes Remarks ICC 4958 Drought tolerant genotype found promising in

Ethiopidrought tolerant parent of two mapping populations

ICCV 10 Widely adapted drought tolerant cultivar found promising in India and Kenya

JAKI 9218 Farmer-preferred cultivar in central and southern India

JG 11 Farmer-preferred cultivar in southern India and also performing well in Kenya

JG 130 Farmer-preferred cultivars from central India

JG 16 Farmer-preferred cultivar in northern and central India

ICCV 97105 Farmer-preferred elite line identified in Kenya and Tanzania

ICCV 00108 Farmer-preferred elite line identified in Tanzania

Eight well performing elite chickpea lines (TLI & TLII)

Trait mapping…

Experiment of chickpea root growth in rain out shelter

(ROS)

Crane for lifting root cylinders for moisture under water stress

determinations

Semi-automated precise high-throughput phenotyping

Phenotyping in rainfed and irrigated conditions

Phenotyping in Sub-Saharan Africa (Kenya, Ethiopia)

Phenotyping in India (Pantcheru, Kanpur, Bangalore)

“QTL- hotspot” on CaLG04

ICC 4958 × ICC1882 Consensus map ICC283 × ICC8261

PVE 58.2%

Saturating “QTL- hotspot” region 701.049 million reads 59030.7 Mbp 828 SNPs

Genotyping data on Reference set

Homozygote

KASPar assays

Homozygote

Heterozygote

SSR analysis (CE)

SNPs in candidate genes

Marker data on reference set

SSR - 35 DArT - 1157 SNPs - 651 Gene based SNPs - 113 Total 1,956

Candidate genes Abscisic acid stress and ripening gene (ASR) DREB2A ERECTA Sucrose synthase (SuSy) Sucrose phosphate synthase (SPS) AKIN (SNF1 related protein kinase) Aminoaldehyde dehydrogenase (AMADH) Dehydrin (DHN), Myb transcription factor

Root related traits Root traits Traits Seasons Root length (cm) 3 Root length density (cm cm-3) Root volume (cm3) 3 Root dry weight (g) 3 Rooting depth (cm) 3 Root surface area 3 R-T ratio(%) 3 Shoot dry weight (g) 3 Stem dry weight (g) 3 Leaf dry weight (g) 3 Projected area 2 Average diameter 2

Water stress determination

Exp. of chickpea root growth in ROS

Agronomic traits Morphological traits

Yield related traits

Traits Seasons Traits Seasons Plant height (cm) 14 Pods/plant 2 Plant width (cm) 7 100 SDW (g) 10 Plant stand 7 Yield (g/m2) 3 Apical primary branch

7 Yield (Kg/ha) 10

Apical secondary branch

7 Yield per plant 7

Basal primay branch 7 Production 7 Basal secondary branch

7 Biomass 6

Teritiary branches 7 Biomass/plant 2 Phenological traits Harvest index 6 Days to flowering 13 TDM weight

(g/m2) 2

Days to maturity 9 Transpiration efficiency Seeds per pod 7 13C 2 Seeds/plant 2 SPAD 2

Field phenotyping under rainfed and irrigated environments

Heat tolerance phenotyping

Δ Carbon on LG4

Yield under rainfed on LG4

.

….

GWAS for ΔC and Yield

Root length density

Harvest index

GWAS for RLD and HI

GWAS for 100 seed weight

Molecular breeding for drought tolerance…

MABC, MARS and GS approaches seem to most

promising for crop improvement

Need to have genomic resources and cost-effective

genotyping platforms

Precise phenotyping platforms required

Breeders-friendly pipelines and decision support tools required for prediction of phenotype

Modern breeding approaches for enhancing genetic gains

Marker-assisted backcross (MABC) method for introgressing genomic region controlling root and

other drought tolerance related traits Donors

Cultivars

JG 11 Chefe KAK 2

Phenotyping of MABC products in ROS

BC3F3 lines phenotyped in ROS for assessing root traits

0

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Yield

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Irrigated

Rainfed

QTL introgression into JG 11

Enhanced root length density

Enhanced yield The Plant Genome, 2013

Institution Cross/parents Current status ICRISAT, India JG 11 × ICC 4958 20 BC3F5 lines

Chefe × ICC 8261 8 BC3F5 lines KAK2 × ICC 8261 2 BC3F5 lines ICCV 10 × ICC 4958 22 BC3F5

IIPR, India DCP92-3 × ICC 4958 60BC1F1 KWR 108 × ICC 4958 7 BC1F1

IARI, India Pusa 362 × ICC 4958 170 BC2F1 EIAR, Ethiopia Ejere × ICC 4958 384 BC2F1

Arerti × ICC 4958 27 BC3F4 lines EU, Kenya ICCV 97105 × ICC 4958 33 BC3F1

ICCV 95423 × ICC 4958 10 BC3F5 lines

MABC for enhancing drought tolerance in Asia and sub-Saharan Africa

MABC status @ IARI (Indian project) 4 BC2F1 plants (with >96% genome recovery) were

used in making backcrosses with recurrent parent Pusa 362 to generate BC3F1 (105 seeds)

BC2F2 seeds also harvested from plants with more than 96% genome recovery

Popu

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1st Recombination cycle

2nd Recombination cycle

3rd Recombination cycle

Multilocation phenotyping

Genotyping

Parent 1 × Parent 2

F1

F2

F3

F3:4

F3:5

Single seed descent

282 F3 progenies

282 progenies

Multilocation phenotyping

A B C D E F G H

F1 F1 F1 F1

F1 F1

F1

F2

F3

F3:4

10 plants/family (A-H), 6 sets of 8 families/cross

QTL detection

JG 11 × ICC 04112 JG 130 × ICC 05107

Kenya, Ethiopia and India Rainfed and irrigated environments

(2010-11)

70 marker 92 markers

QTL analysis completed

Marker-assisted recurrent selection (MARS)

MARS lines for RC selected

OptiMAS

Indian project

TLI Phase II

First RC completed

Second RC in progress

×

JG 130 X ICCV 05107

×

JG 11 X ICCV 04112

Exploring genomic selection in chickpea

320 elite-breeding lines;>3000 markers

GS

ISMU V2

Raw Reads

Reference

Assemble & Align Raw Reads Mine SNPs Generate Marker Matrix Visualize in TABLET and FLAPJACK Export in FLAT Files

GDMS Genotypic

Matrix & QTLs

Lines selected for further crossing in

GS

External Genotyping Platforms

Called SNPs

ISMU V2.0

44

Enhancing capacity of NARS in modern breeding…

NARS partners practicing modern breeding

Serah Alice

Paul examining the crosses Robert

Musa Jarso marker analysis for MABC crosses

Mosses oyier marker analysis for MABC crosses

4th International Workshop on

Next generation genomics and integrated breeding for

crop improvement, Feb 19-21,2014,

NARS partners practicing modern breeding

Products Reference set, pre-breeding populations,

MAGIC lines >3000 SSRs, 2068 KASPar, DArT arrays High density genetic maps and physical

map Draft genome sequence and re-sequencing

of 90 lines MABC lines with enhanced drought

tolerance in the genetic background of JG11, KAK2 and Chefe

Summary Well characterized reference set and 39 pre-breeding

populations developed

1200 F8 MAGIC lines developed ready for genotyping and phenotyping

Large scale markers (SSRs, SNPs, DArTs) and several maps developed

A genome-wide physical map developed (574 Mb) that contributed to genome sequencing of chickpea

A most promising “QTL-hotspot” with about 50 markers for MABC for drought tolerance

Promising introgression (BC3F5) lines with higher yield in rainfed conditions

Next generation of molecular breeders (4 PhD and 8 MSc students from NARS, >50 researchers) trained

TLI Phase I and Phase II datasets curated into IBWS

ICRISAT, Patancheru, India: Pooran Gaur, Hari Upadhyaya, L Krishnamurthy, Junichi Kashiwagi, Abhishek Rathore, Trushar Shah, Mahendar Thudi, Manish Roorkiwal, Rachit Saxena, Ashish Kumar, Murali Mohan, HimaBindu, Shailesh, Pavana, Neha, Serah, Gafoor UC-Davis, USA: Doug Cook, R Varma Penmetsa NCGR, Santa Fe, USA: Greg May, Andrew Farmer Uni Georgia, USA: Scott Jackson; JCVI, USA: Chris Town, Yongli Xiao DArT Pty Ltd, Australia: Andrzej Killian Uni Frankfurt, Germany: Peter Winter, Guenter Kahl Osmania University: PB Kavikishor NRCPB, New Delhi, India: NK Singh, TR Shrma, R Srinivasan, PK Jain NIPGR, New Delhi, India: AK Tyagi, Sabhyata Bhatia IIPR-Kanpur, India: SK Chaturvedi, Aditya Garg, S Datta IARI, New Delhi, India: Jitendra Kumar, C Bharadwaj, S MPKV, Rahuri, India: L Mhase, P N Harer, PL Kulwal JNKVV, Jabalpur, India: A Babbar, N Saini, O Gupta ARS-Gulburga, India: D Mannur, Jayalaksmi UAS-Bangalore, India: Sheshashayee, M Udayakumar, KP Vishwanath Egerton University, Kenya: Paul Kimurto, Richard Mulwa EIAR, Addis Abba, Ethiopia: Asnake Fikre, Million Eshete LZARDI, Debre Zeit, Tanzania: Robert Kileo

Many thanks to all contributors

Collaborators and partners