Towards utilization of genome sequence information forpigeonpea 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

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 inpigeonpea 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

TranscriptomicsProteomicsMetabolomicsTILLINGEcoTILLING

EST SequencingGenome SequencingMap-based Cloning

Genetic MappingPhysical Mapping

Genetic MappingAssociation MappingQTL MappingTrait 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 backcrossingIR49830-7:tolerant

Swarna-Sub1

Swarna:Non-tolerant

Sub1• Target gene selectionTarget gene selection• Recombinant selectionRecombinant selection• Background selectionBackground selection

BC2or 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

New Sub1 lines (in yellow) and recurrent parents (in white) after 17 days submergence in field at IRRI, 2007DS

Samba

Samba-Sub1

Samba-Sub1

IR64-Sub1IR49830 (Sub1)IR6

4IR42 IR6

4IR64-Sub1

Samba-Sub1

IR49830 (Sub1)

Samba

IR64

IR64-Sub1

IR49830 (Sub1)

IR42

IR64-Sub1

IR64

IR49830 (Sub1)

IR49830 (Sub1)

IR42

Samba

IR42

Samba

Courtesy of David Mackill, IRRI

Swarna-Sub1 in U.P. (Faizabad area)

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)

Resource Pigeonpea

SSRs 29,000

SNPs 35,000

GoldenGate 768 SNPs

KASPar assays 1,616 SNPs

DArT arrays 15,360

Sanger ESTs ~20,000

454 /FLX reads 496,705

TUSs 21,432

Illumina reads(million reads)

>160 (14 parents)

Gene/transcriptomic/ SNP resources

CMS and mt genome sequencing of pigeonpeaProduction

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 300-500 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