Nagendra K. Singh NRC on Plant Biotechnology Indian Agricultural Research Institute, New Delhi Pigeonpea Genomics Initiative

Status of Plant Genome Sequencing Sequencing of 16 Plants in Progress (10 completed + 6 about to be finished) Arabidopsis, Rice, Poplar, Medicago, Sorghum, Papaya, Cassava, Cucumber, Tomato, Potato, Maize, Soybean, Citrus, Grape, Banana, Wheat Arabidopsis and Rice with high quality BAC by BAC sequence data

INDIAN INITIATIVE FOR RICE GENOME SEQUENCING International Rice Genome Sequencing Project

INTERNATIONAL TOMATO GENOME SEQENCING CONSORTIUM Based on Song-Bin Chang’s Ph. D. Thesis 2004

Indo-US AKI Pigeonpea Genomics Initiative from Orphan Legume to Draft Genome Sequence

Productivity (hg/ha) World-Food grains FAOSTAT, 2010 Year Productivity (hg/ha) Cereals Pulses

 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.  Plays an important role in sustaining soil productivity by fixing atmospheric nitrogen. Pigeonpea  Pigeonpea (Cajanus cajan (L.) Millsp.) belongs to f amily Fabaceae with chromosome no. 2n=22 and genome size of 853 Mbp

Area, Production and Productivity YearArea (Mha) Production (MT) Productivity (Kg/ha) World India o India produces about 75% of the global output of pigeonpea. o Very low average productivity (800 kg/ha) as compared to it’s potential (2000 kg/ha) (Ali and Kumar, 2005). (FAOSTAT 2010)

Constraints to High Productivity  Growing traditional landraces on large area  Non-availability of quality seeds of improved varieties  Inferior plant type with low harvest index  Long crop duration (5-9 months)  Wilt, SMD, Water logging, Pod borer  Poor agronomic practices

First meeting of Pigeonpea Consortium on 10 th Nov 2006 at NRCPB, New Delhi Objectives: 1.100,000 ESTs and genic-SSR /SNP markers 2. Genomic SSR markers 3. Mutant lines and mapping populations as resource for gene discovery 4.High density molecular linkage map as a reference map 5.Markers and genes for important agronomic traits 6. Pigeonpea genome informatics platform 7. Sequencing gene-rich BAC clones of pigeonpea

Impact of Indo-US AKI: Pigeonpea Genomics Initiative

Sequencing of Pigeonpea Genome

1 st Draft of Pigeonpea Genome Sequence Submitted to NCBI GenBank, July 2011

Pigeonpea Genome- Repeat Elements

Pigeonpea Genome- Gene Content

Pigeonpea Genome- Comparison with Soybean 152 homologs of genes for abiotic stress tolerance 56 genes for heat shock proteins (HSP), 32 genes for glutathione-S-transferase (GST), 28 genes for trehalose-6-phosphate synthase (TPS), 8 genes for glutamine synthase (GS), 7 genes for water channel protein aquaporins several transcription factors e.g. DREB, NAC and MYB genes

Circular Map of Synteny between 11 Pigeonpea 20 Soybean Chromosomes Based on 512 Single Copy Genes Pigeonpea Genome- Comparison with Soybean

Pigeonpea Genome-miRNA

Pigeonpea Genome- Improved Assembly

Pigeonpea Genome- Development of Genic-SSR and SNP markers by mRNA Sequencing

M M 50bp 100bp 150bp Agarose gel (4.0 %) showing allelic variation among 30 genotypes of pigeonpea and related wild species with genic-SSR marker ASSR-277

C. cajan cultivars Wild species Asha GTR 9 HDMO4-1 H JA 4 PCMF 39-1 PCMF 40 PCMF 43-7 GT288A PS 971 PS 956 Pusa 9 Kudarat ICPA 2089A ICPR 2438 UPAS 120 TTB 7 Pusa Dwarf Bahar Maruti Pusa 992 GTR 11 R. aurea C. platycarpus(1) C. platycarpus(2) C. cajanifolius C. lineatus C. scricea R. bracteata C. albicans Ib Ia II I Ia 2 Ia 1 IIb IIa Similarity coefficient Dendrogram showing phylogenetic relationship of 30 genotypes of Cajanus cajan and related wild species based on 20 genic-SSR markers

Pigeonpea Genome- High density linkage map based on 366 genic-SNP and 24 genic-SSR markers

Pigeonpea Genome- SSR Mining

M HASSR-283 HASSR-284 HASSR-285 M HASSR-127 HASSR-128 HASSR-129 Pigeonpea Genome- HASSR Markers

Ideotype: Set of features delineating the shape, size, canopy and external structure of the plant Plant height Number of primary and secondary branches Number and length of internodes Size, shape and position of leaves and reproductive organs Options for Enhancing Pigeonpea Productivity 1.Hybrids 2.Ideotype Application of Genic SSR/SNP Markers in QTL Mapping

1.Development of molecular linkage map of pigeonpea 2.Mapping of genes/QTLs for traits involved in plant ideotype and maturity Application of Genic SSR/SNP Markers in QTL Mapping

Outline of work Pusa Dwarf/ HDM04-1 F1F1 F1F1 F2F2 F2F2 F 2:3 Marker polymorphism Genotyping Linkage map Phenotyping QTL mapping Cross

Mapping Population: ♀ Pusa Dwarf X ♂ HDM04-1 Trait Pusa Dwarf HDM04-1 Plant height (cm)88118 No. of primary branches/plant 205 No. of pods/plant12024 Days to Flowering10665 Days to Maturity Growth habitDeterminateIndeterminate Plant Material Pusa Dwarf HDM04-1

A. Genic-SSR Total 927 genic SSR markers, 772 developed from 454 TSA contigs and 155 from Sanger ESTs under Indo-US AKI project were used. B. Genomic-SSR 45 genomic SSR markers from literature ( Odeny et al., 2007, 2009) Additional 40 SSR markers were designed from public BAC end sequence database at NCBI BatchPrimer 3 software (You et al., 2008) Markers

C. Intron length Polymorphism (ILP) Markers A total of 60 ILP primers were designed using Medicago genome as subject species genome by ConservedPrimers 2.0 software. (

D. Single Nucleotide Polymorphism Assay:  SNPs were identified by aligning 15,511 common large TSA contigs between the two varieties.  1536-plex and 768-plex Illumina GoldenGate assays were designed and latter was used for genotyping of F2 population  Two pools of RNA from varieties namely Asha and UPAS120 were sequenced by 454-FLX sequencing and TSA contigs were used for in silico SNP identification (Indo- US AKI project).

GoldenGate Genotyping OPA

Segregation analysis:  All markers were tested for goodness of fit by chi-square test. Linkage analysis:  Linkage analysis of segregating markers was done by Mapdisto software ( at LOD = 3http://mapdisto.free.fr/MapDisto/ QTL analysis:  Statistical analysis of Phenotypic data was performed SPSS software version 10.0  QTL analysis was done by QTL Network software version 2.1 (Yang et al. 2008)

Marker typeNo. tested No. amplified Polymorphic No (%) Marker Source/ Reference Genic-SSR (EST- 454 seq.) (75.5%)28 (4.8%)Indo-US AKI, NRCPB Genic-SSR (EST-Sanger seq.) (20%)0Indo-US AKI, NRCPB GENOMIC-SSR (Genomic library) 4532 (71.1%)0Odeny et al., (2007, 2009) GENOMIC-SSR (BAC end sequences) 4027(67.7%)0NCBI GSS database ILP (454 TSA contigs) 6051(85%)0NPTC, NRCPB TOTAL (67.5%)28 Markers Used for Parental Polymorphism Survey

M A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B M Parental polymorphism survey with ASSR markers (1-24), 21-ASSR1486 (polymorphic) L-100bp DNA ladder, A-Pusa Dwarf, B-HDM Polymorphism survey with genic-SSR markers

Genotyping of F 2 with SSR Markers L P 1 P 2 Genotyping of F 2 population with ASSR8 in 4% metaphor agarose gel L- 100bp DNA ladder, F 2 genotypes, P 1 - Pusa Dwarf, P 2 - HDM

ASSR L L Parental polymorphism survey for ASSR markers in 8% PAGE L-50bp DNA ladder, 1- Pusa Dwarf, 2-HDM04-1 Parental Polymorphism survey on PAGE 150bp 200bp 250bp

Genotyping on PAGE Genotyping of ASSR206 on 8% polyacrylamide gels. M-100bp ladder, P1- Pusa Dwarf, P2- HDM04-1, 1-22 F 2 genotypes M P1 P

F 2 Progeny

Homogeneous F 3 Families

Segregating F 3 Families

F 3 Recombinants

Phenotyping of F 2 and F 2:3 (20 plants/lines) 1.Plant height 2.Number of primary branches per plant 3.Number of pods per plant 4.Days to flowering 5.Days to maturity 6.Number of secondary branches 7.Pod bearing length 8.No. of seeds per pod 9.Growth habit (determinate/indetrminate)

cm Frequency Distribution of Plant Height and No. of Primary Branches P1P2 P1 P2 P1

Frequency Distribution for No. of Pods Pod numbers P1 P2 P1

Frequency Distribution for Days to Flowering and Days to Maturity P2 P1 P2 P1 P2 P1 P2

Trait Pusa Dwarf HDM0 4-1 F2F2 F3F3 RangeMeanSDCVRangeMeanSDCV Plant height No. of pri. branches No. of pods Days to flowering Days to maturity Descriptive Statistics of Five Traits for the Parents and Mapping Populations of Pusa Dwarf and HDM04-1

QTL Map for All Traits

QTLs with additive and dominance epistatic effects for number of primary branches per plant in F 2:3 population from Pusa Dwarf/HDM04-1 ▬ Interaction between QTLs with epistatic and main effects. ● QTLs with only additive effect ▀ QTLs with only dominant effect ▀ QTLs with no dominance effect

Summary and Conclusions  Draft of 511 Mb of pigeonpea genome sequence assembled, 47,004 genes, 437 HASSR markers  Deep coverage TSA assembly of 43,324 genes, 550 genic-SSR and 2,304 genic-SNP GoldenGate assays  Intra-species reference map of 366 genic-SNP and SSR markers  1,363 markers screened to find 135 polymorphic (9.9%) markersbetween Pusa Dwarf and HDM04-1 (28 SSR and 107 SNP)  Linkage map of 136 loci, cM, average interval 7.77 cM.  2 QTLs for plant height, qPH3, qPH5 ( 28.2, 28.1% PEV)  3 QTLs for primary branches, qPB3, qPB5, qPB9 (23.4, 11.1, 2.6% PEV)  2 QTLs for number of pods, qPD3, qPD5, (16.4, 18.7% PEV)  2 QTLs for days to flowering, qFL3, qFL5 (52.3, 8.8% PEV)  3 QTLs for days to maturity, qMT3.1, qMT3.2, qMT5 (23.4, 4.4,15.6% PEV)  Significant epistasis of qPB3 with qPB5 and qPB9 (3.5% PEV).  Co-located of QTLs in two genomic regions on LG3 and LG5 with pleiotropic effect  Useful for MAS of semi-dwarf short duration pigeonpea varieties

Acknowledgements ICAR for funding support under Indo- US AKI and NPTC Projects Doug Cook, UC Davis, Chris Town, JCVI and Rajeev Varshney, ICRISAT for quality files of BAC end sequences

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