Towards utilization of genome sequence information for pigeonpea improvement By ICAR institutes, SAUs and ICRISAT

 A major source of protein to about 20% of the world population (Thu et al., 2003)  An abundant source of minerals and vitamins (Saxena et al., 2002)  Most versatile food legume with diversified uses such as food, feed, fodder and fuel  It is hardy, widely adaptable crop with better tolerance to drought and high temperature  Belongs to family Leguminosae with chromosome no. 2n=22 and genome size of ~833 Mbp Pigeonpea (Cajanus cajan L. Millsp)

Climate change!

Pigeonpea – production trends (last five decades) Unfortunately, no increase has been witnessed in its productivity (yield kg ha-1), which in the past five decades has remained stagnant at around 700 kg ha-1

Some constraints in pigeonpea production Sterility mosaic disease (SMD) Fusarium wilt (FW)

A route developed and taken by breeders: From germplasm to variety/hybrid Germplasm Superior variety

Genomics-assisted breeding: Predicting the phenotype Genotype Gene(s) Trait/QTL Phenotype Transcriptomics Proteomics Metabolomics TILLING EcoTILLING EST Sequencing Genome Sequencing Map-based Cloning Genetic Mapping Physical Mapping Genetic Mapping Association Mapping QTL Mapping Trait Correlations Genetic Resources Improved germplasm Trends Pl Science 2005; Trends Biotech 2006

A variety of approaches (cars) MAS: MARKER-ASSISTED SELECTION - Plants are selected for one or more (up to 8-10) alleles MABC: MARKER-ASSISTED BACKCROSSING –One or more (up to 6-8) donor alleles are transferred to an elite line MARS: MARKER-ASSISTED RECURRENT SELECTION –Selection for several (up to 20-30) mapped QTLs relies on index (genetic) values computed for each individual based on its haplotype at target QTLs GWS: GENOME-WIDE SELECTION –Selection of genome-wide several loci that confer tolerance/resistance/ superiority to traits of interest using GEBVs based on genome-wide marker profiling

Example of development of a submergence tolerant version of Swarna, a widely grown variety, in 2½ years Marker-assisted backcrossing IR : tolerant Swarna-Sub1 Swarna: Non-tolerant Sub1 Target gene selection Recombinant selection Recombinant selection Background selection Background selection BC2 or BC3 X Courtesy of David Mackill, IRRI

New Sub1 lines (in yellow) and recurrent parents (in white) after 17 days submergence in field at IRRI, 2007DS Samba Samba-Sub1 IR64-Sub1 IR49830 (Sub1) IR64 IR42 IR64 IR64-Sub1 Samba-Sub1 IR49830 (Sub1) Samba IR64 IR64-Sub1 IR49830 (Sub1) IR42 IR64-Sub1 IR64 IR49830 (Sub1) IR42 Samba IR42 Samba Courtesy of David Mackill, IRRI

Swarna-Sub1 in U.P. (Faizabad area) Courtesy of David Mackill, IRRI, The Philippines

Challenges in genomics- assisted crop improvement  Narrow genetic base in the primary gene pool  Very few molecular (SSR) markers  Non-availability of appropriate germplasm such as mapping populations  Intraspecific genetic map with low marker density  Non-availability of trait-associated markers in breeding  Issues of costs and expertise in molecular breeding

Germplasm Superior variety

Developing infrastructures and sign posts for providing directions (Indo-US AKI, CGIAR-GCP, US-NSF)

ResourcePigeonpea SSRs29,000 SNPs35,000 GoldenGate768 SNPs KASPar assays1,616 SNPs DArT arrays15,360 Sanger ESTs~20, /FLX reads496,705 TUSs21,432 Illumina reads (million reads) >160 (14 parents) Gene/transcriptomic/ SNP resources

CMS and mt genome sequencing of pigeonpea Production of A- line seeds Production of hybrid seeds for commercial crop Commercial pigeonpea hybrids production  ICPA 2039, ICPB 2039, ICPH 2433 & ICPW 29 sequenced using 454 technology

From Orphan crop- genomic resources rich crop

Phylogenetic analysis of Cajanus spp. using KASPar assays Cluster-I Cluster-II Cluster-III

How to use this genome information…

Objectives  Molecular mapping of resistance to biotic and abiotic stresses - Mapping populations available - Genotyping and phenotyping - Marker trait association for resistance to FW, SMD and Rf  Enhancing the genetic base of pigeonpea genepool by developing multi-parents populations - MAGIC population (2000 lines) developed using 8 parents - NAM population (50 crosses-1000 lines) with 50 parents - High density genotyping or genotyping by sequencing of 3000 lines - Phenotyping of MAGIC and NAM populations (each population at least in 3 environments) - Marker trait association analysis for traits of interest

 Genome wide association studies based on re- sequencing and phenotyping of germplasm set - Germplasm set of lines assembled - Genotyping-by-sequencing of the germplasm set - Precise phenotyping of the germplasm set by different partners - Fine mapping of traits of interest for breeders  Bioinformatics analysis to improve the quality of draft genome - Two genome assemblies need to be merged - Defining a consensus genes set - Breeders-friendly genome databases

 Validation and characterization of 1213 disease resistance genes - Genetic mapping of disease resistance genes - Association of genes with disease resistance traits - Functional validation of selected set of candidate genes - Mining of superior allleles/haplotypes for disease resistance  Validation and characterization of ca. 200 abiotic stress tolerance genes - Genetic mapping of abiotic stress tolerance genes - Association of genes with abiotic stress tolerance traits - Functional validation of selected set of candidate genes - Mining of superior allleles/haplotypes for abiotic stress tolerance genes

Possible outcomes  Superior breeding lines for traits of interest with enhanced genetic diversity  Molecular markers associated with resistance to biotic stresses and tolerance to abiotic stresses  Alleles and haplotype information available on germplasm set so that breeders can use informative lines  Set of well characterized disease resistance and abiotic stress tolerance genes Breeder-friendly genome database of pigeonpea

Possible partners NRCPB, New Delhi NBPGR, New Delhi IIPR, Kanpur IARI, New Delhi Uni Agril Sciences- Bangalore Banaras Hindu University ANGRAU- Hyderabad