Deep ESTs (+/-) genome sequence

Primary focus of NSF project

Generation Challenge Program

INDO-US Agricultural Knowledge Initiative

SALIACEAE: Populus (poplar)

Figure modified from Gepts P et al (2005) Plant Physiol. 137:1228-1235

Comparative Genomics and Evolution of the Papilionoid Legumes

Focus on legume species at key nodes in legume evolution.

1. Develop detailed comparative genetic maps based on orthologous markers.

•Define genome segments intact since the last common ancestor ~60 MYA

2. 10X BAC libraries and 50K BAC end sequences for each species.

•SSR marker resource; gene discovery; inventory of heterochromatin•Genetically-mapped BAC clones as seed points for physical map development

3. Clone, characterize and map all Resistance Gene Homologs (RGHs).

•Sequence genome regions containing legume RGH clusters and singletons•Infer evolutionary history of genes and gene clusters•Tools for molecular breeding

Outcomes (Evolution of genome structure and gene diversity)

1. Comparative maps to model species allows inference of gene content in genetic intervals of less-well characterized legume species.

2. SSR markers: rapidly diverging sequences are useful for basic and applied genetic studies, especially in the conext of the narrow domesticated germplasm characteristic of crop legumes.

3. BAC clones and BAC-SSRs linked to genetic maps in each species. Provides seed points for physical maps and sequencing projects. Anchors ~50% of gene space in each species.

4. Genetically mapped and sequenced NBS-LRR R gene clusters. Ancestry inferred from synteny and phylogenetics. Genetic markers as tools for marker assisted selection.

5. Comprehensive knowledge of NBS-LRR Resistance gene evolution across the Fabaceae

•Identify ancient stable vs recently evolved fast changing genes.•Nature of selection and placement relative to speciation (e.g.,

radiation, gene conversion, birth-death, recombination, diversifying/purifying selection, etc.)

Development of Orthologous Genetic Markers

Status:

•Set of 1,369 orthologous amplicons developed.

•Allele discovery in mapping parents of 4N peanut, 2N peanut, chickpea, pigeonpea, common bean, cowpea and lupin.

•Diversity analysis in germplasm collections.

•Develop Illumina SNP assay.

Goal:

Genetic mapping:

•Detailed genetic map documenting common descent from a 60 MYA ancestor.

Population genetics:•Characterize genetic diversity and define domestication history in cultivated genomes.

EGO72K

homology clusters

(a) CALTL

Lotus

A17A20DZA

JI15JI399JI1194

JI281L. filicaulisiL. japonicus

PI209332Evans TC1966

VC3890BATJalo

At1g563400.05 substitutions/site

50

100100

100100

100

100

100

100

100

Mt

Ps

Gm

Vr

Pv

At

0.5Kb

0.1 Kb

Gm-EST

Mt-EST

Mt-genomic

Lj-EST

Crop SpeciesGenotype (major features)

# of orthologous genes with high quality sequence

# of sequence

assemblies

# of singletons

total high quality

sequence (Mbp)

# of cross-genotype

alignments

# of SNPs (predicted)

# of genes with SNPs

(predicted)

# of SNPs (validated)

# of genes with SNPs (validated)

PigeonpeaCajanus cajan cultivar ICP28 (BAC library; parent of mapping RILs)

1206 919 399 0.915

Cajanus scarabaeoides ICPW 94 (parent of mapping RILs)

1130 774 509 0.849

ChickpeaCicer arietinum ICC4958 (parent of mapping RILs)

1237 985 336 0.949C. arietinum FLIP_84-92C

Cicer reticulatum PI 489777 (parent of mapping RILs)

1132 934 270 0.895C. reticulatum PI-599072

Common Bean

Phaseolus vulgaris cultivar JALO (parent of mapping RILs)

1125 983 232 0.903 Pv - DOR364

Phaseolus vulgaris cultivar BAT93 (parent of mapping RILs)

1022 912 159 0.794 Pv - G19833

CowpeaVigna unguiculata 9405C (BAC library; derived from 524B)

1209 914 379 0.873

Vigna unguiculata 524B (parent of mapping RILs)

962 654 396 0.689 Vu "wild" CG003

Vigna unguiculata UCR430 (parent of mapping RILs)

1015 788 292 0.740

Peanut 2nArachis duranensis Dur 2 (parent of F2 mapping population)

786 568 300 0.519 A. magna 30097 A. ipaensis 30076

Arachis duranensis Dur 25 (parent of F2 mapping population)

793 550 288 0.531A. duranensis K7988

A. stenosperma V10309

Peanut 4nArachis hypogea cultivar TifRunner (BAC library; parent of mapping F2s)

766 384 471 0.437

3 additional tetraploid accessions

LupinLupinus angustifolius cultivar Tanjil (BAC library)

1001 752 325 0.803

LentilLens culinaris cultivar redberry (mapping parent; Anthracnose suscp)

in progress

72912406

PCR, topTA cloning, colony picking, sequencing

Ongoing sequencing

827 73614131

749 5525239 473/623*

218/646*

4029

1241 5573956 157

5131 379691 529

1801 / 3815 *

619

3082

888 5945039

Pigeon pea

0

5

10

15

20

25

30

35

40

45

0 1 2 3 4 5 6 7 8 91011121314151617181920212223242526272829303132333435363738394041424344454649505153555657596263646566676870737578818386889091

101104107118119124142156247248259

# Curated SNPs/analyzed KB

Count

Defining the Resistance Gene Content of Crop legumes

Goals:

• Understand the evolution of this large and diverse gene family.

• Develop tools for marker-assisted resistance breeding.

>2,400 distinct NBS-LRR genes cloned to date

24 additional clones from

winners

12 clones from all

BAC end sequencing

SSR and SNP markers

544 degenerate primer combinations (1X105)

250 amplicon mixtures

250 amplicon mixtures30-50% with RGH~600 unique homologs

50-90 lineages at 90%

~10 Mbp unique DNA (Cc, Ca, Vu)~35 Mbp unique DNA (4N peanut)

>1.4 Mbp (Cc, Ca, Vu)>2 Mbp (4N peanut)

130 SSRs/Mbp

HIC Fingerprinting

PCR and agarose gel

TopoTA clone, plate and pick

Sequencing (7K)

Phylogenetic analysis and probe design

Hybridization and rearray

LentilChickpeaCowpeaCommon beanPigeonpeaLupinPeanut 4NCercis

Pat AY747579Pat AY747574

Pat AY747586Pat AY747585

Pat AY747584Peanut1 C05

Peanut7 C07Peanut6 C06

Peanut10 A03Peanut9 H11rc

Pat AY747558Pat AY747530

Pat AY747542

Peanut6 D04Peanut1 C04rc

Peanut6 G10Peanut6 D01rc

Pat AY747548rcPat AY747529rc

Pat AY747556rcPat AY747532rc

Pat AY747560rcPeanut1 H11

Peanut6 C02rcPeanut6 B07rc

Peanut6 B11Peanut6 B10rc

Peanut1 F04rcPeanut6 C07rc

Peanut6 C08Peanut1 C07rc

Peanut10 A06rcPeanut6 C03rc

Pat AY747553

Pat AY747535Peanut10 B07

Peanut9 H08rcPeanut1 D06

Peanut7 C06rcPeanut2 A10

Peanut6 C10Peanut2 A11

Peanut6 C11rc

Extreme and recent RGA radiation: the case of cultivated peanutPat AY747579Pat AY747574

Pat AY747586Pat AY747585

Pat AY747584Peanut1 C05

Peanut7 C07Peanut6 C06

Peanut10 A03Peanut9 H11rc

Pat AY747558Pat AY747530

Pat AY747542Peanut6 D04

Peanut1 C04rcPeanut6 G10

Peanut6 D01rcPat AY747548rcPat AY747529rc

Pat AY747556rcPat AY747532rc

Pat AY747560rcPeanut1 H11

Peanut6 C02rcPeanut6 B07rc

Peanut6 B11Peanut6 B10rc

Peanut1 F04rcPeanut6 C07rc

Peanut6 C08Peanut1 C07rcPeanut10 A06rcPeanut6 C03rc

Pat AY747553Pat AY747535

Peanut10 B07Peanut9 H08rc

Peanut1 D06Peanut7 C06rc

Peanut2 A10Peanut6 C10

Peanut2 A11Peanut6 C11rc

Pat AY747538Peanut6 D05rc

Pat AY747393Pat AY747394

Pat AY747392Peanut2 H02rc

Peanut2 H09rctirAC143338.1

tirAC143338.2Peanut2 G11

Pat AY747379Pat AY747375

Pat AY747381Pat AY747383Pat AY747385

Pat AY747351Pat AY747431

Peanut7 B03rcPeanut1 F11rc

Peanut1 D08rcPat AY747360

Peanut2 C07Peanut2 G12Peanut2 H05rc

Peanut9 A06rcPeanut9 E01rcPeanut9 D10rc

Peanut9 H03Peanut2 C09rc

Peanut2 H07rcPeanut2 H06Peanut9 B10rc

Peanut9 A01rcPeanut9 B02tirAC125480.1

Peanut9 H02rcPeanut9 E08rc

Peanut11 C08tirAY372412

tirAC135415.1Pat AY747559Pat AY747533

Pat AY747544Pat AY747550rc

Pat AY747516rcPat AY747536rc

Pat AY747539Pat AY747545

Pat AY747573Pat AY747363Peanut4 A03rcPat AY747368Pat AY747353Pat AY747425rc

Pat AY747427rcPat AY747370

Pat AY747333Pat AY747345

Pat AY747371Pat AY747337

Pat AY747344Pat AY747541

Pat AY747364Peanut12 F11rcPeanut13 F04rc

Peanut13 A03rcPeanut12 H04rc

Peanut12 H01rcPat AY747526

Pat AY747401Pat AY747415rc

Pat AY747578Pat AY747412rc

Pat AY747332Pat AY747334

Pat AY747421rcPat AY747365

Pat AY747391Pat AY747537

Pat AY747508rcPeanut10 D10rc

Peanut11 G03rcPeanut12 E11rcPat AY747429

Pat AY747432Pat AY747402

Pat AY747339Pat AY747378

Pat AY747388Pat AY747330

Pat AY747555rcPat AY747426rcPat AY747359

Pat AY747356Pat AY747366

Pat AY747404Pat AY747377

Pat AY747403Pat AY747350

Pat AY747346Pat AY747430

Pat AY747525rcPat AY747527rc

Pat AY747552Pat AY747551rc

Pat AY747587Pat AY747534rc

Pat AY747376Peanut4 D11rc

Peanut6 G08Pat AY747540Pat AY747511rcPat AY747546

Pat AY747367Peanut6 H09rc

Pat AY747399Pat AY747417

Pat AY747422Pat AY747369

Pat AY747352Pat AY747389

Pat AY747424Pat AY747372

Pat AY747354tirAC127167.1

tirAC127167.3tirAC127167.2tirAC127167.5

tirAC127167.6tirAC127167.4

Pat AY747380Pat AY747390

Pat AY747382tirAC144658.1.1

tirAC144658.2.1tirAC144658.4.1

tirAC144658.3.2Peanut3 D09Peanut3 B07rcPeanut3 B06rcPeanut1 A01rc

Peanut3 B09rcPeanut3 D04rc

Peanut3 A07rcPeanut3 A08

Peanut3 B01Peanut1 A03rc

Peanut3 B08Peanut3 C05

Peanut3 A01Peanut3 D10

Peanut1 A10rcPeanut3 C03rc

Peanut3 A04rcPeanut3 D08

Peanut3 A05rcPeanut3 C09rc

Peanut3 C08Peanut3 A11

Peanut3 A03rcPeanut1 A06

Peanut3 A10Peanut1 B01

Peanut3 C07Peanut1 A08

Peanut1 B03Peanut3 C12

Peanut3 B04rcPeanut3 B12

Peanut3 A02rcPeanut3 C06rcPeanut3 D01rc

Peanut3 A09rcPeanut3 D02rc

Peanut3 C01rcPeanut2 E06

Peanut3 A12Peanut3 B11

Peanut10 G01Peanut10 E07rc

Peanut10 D09rcPeanut11 B01rc

Peanut10 G11Peanut7 H06

Peanut7 H07Peanut4 A08rc

Pat AY747396Peanut4 A04rcPeanut7 H08rcPeanut5 D05

tirAC144658.1.2tirAC144658.2.2

tirAC144658.3.1tirAC144658.5tirAC144658.4.2

tirAC143338.3Pat AY747342Peanut12 E03rc

Peanut13 B02rcPat AY747340

Peanut11 G05rcPeanut11 F11rc

Peanut12 C12rcPeanut11 G08rc

Peanut12 D10rcPeanut12 H11rc

Peanut11 E01tirAC130653.3

tirAC145513.3tirAC144645.1

tirAC145513.2tirAC145513.1

tirAC145513.4tirAC144345.1

Pat AY747400Pat AY747374Pat AY747338

Pat AY747331Pat AY747358

Pat AY747410Pat AY747362

Pat AY747373Pat AY747397

Pat AY747428tirAC121233.1

tirAC144344.2tirAC140067.1

tirAC123574.1tirAC126790.6

tirAC126790.4tirAC126790.5

tirAC126790.1tirAC144344.1

tirAC126790.2tirAC126790.3tirAC126790.7

tirAC127020.1tirAC127020.2

tirAC134824.1tirAC134824.2

tirAC127020.3tirAC135396.6

tirAC135396.7tirAC135396.2

tirAC135396.3tirAC135396.1

tirAC135396.5tirAC127020.5

tirAC138199.1tirAC127020.4

tirAC126790.8tirAC135396.4

tirAC130808.1tirAC124959.3

tirAC124959.4tirAC124959.1

Peanut4 D12rctirAC124959.2

Pat AY747395Pat AY747420rc

Pat AY747343Pat AY747423

Pat AY747386Pat AY747387

Pat AY747413Pat AY747348

tirAC138015.1tirAC138015.2

tirAC123975.1Pat AY747411rc

Pat AY747406rcPat AY747405rc

Pat AY747416rcPat AY747414rc

Pat AY747408rctirAC137825.1tirAC144473.2

tirAC144473.3tirAC144473.1

tirAC146563.1tirAC138526.1

tirAC122722.1tirAC144502.3

tirAC135160.2tirAC144502.1

tirAC135160.1tirAC144502.2

tirAC144502.4Peanut2 G10

Peanut2 H10MtCC5fg23230.05 changes

NJ

Mt-Peanut TIR NJ ......................... .................. ..... ... .. ...... ................ ................... ...

...

.................. .. .......... ............. ...... .. ....................... .... ........................ ........... ..... .. ............... .... ........... ... ............ . .. .................. ... ..... ...... ......... ..

cv. Florunner

cv. Tifrunner

Saskatoon - lentil (Aschochyta, Sally Vail)Tuskegee, USDA, Brazil - peanut (Guohao He, Corley Holbrook, David Bertioli)Kolkotta - Vigna radiataW Australia - lupinICRISAT - chickpeaCIAT - common bean (Aschochyta, Nayibe Garzon)India AKI - pigeonpeaDavis - cowpea (CpMV).

Linking - Resistance genes, markers and phenotypes

NCBI submission+ metadata

+ accession #’s

CAP3 clustering

BLASTN vs Mitochondrial

Chloroplast data set

95% similarity with 200 bp overlap

BLASTN vs Nuclear rRNA

BLASTN vs Chloroplast

Mitochondrial data set

Ribosomaldata set

Non-redundant Genomic BAC clusters

and singletons

5557 legume seqs,4 complete legume seqs

31 complete plant seqs, 236 partial legume seqs

PseudoContigs : Data reductionDefragmentation

25 Arabidopsis, Ca and Vuribsomal annotated seqs, augmented with clusters

1e-20

1e-20

1e-20

Gene identification

Gene annotation

SSR discovery, selection &

primer pipeline

Mt genome relationships

Repeat and mobile DNA

analysis

•Screen against repeat database containing nucleotide repeats and mobile DNA elements

•BLASTN vs TIGR transcript assemblies

•BLASTX vs NCBI-NR

•Interpro

•GO Biological Process

•GO Molecular Function

•ICRISAT pipeline containing SSR-IT and Primer3.

•BLASTN vs Mt genome.

BAC-linked Simple Sequence Repeat Markers

1344 Chickpea

1152 Cowpea

50K 2N Peanut

100K Pigeonpea

10K lupin

0

5

10

15

20

25

30

Relative SSR frequency

CsSat Mobile ANO+EST NSGenome Fraction

SSR rate in varous BES fractions

Nature of "genic" BES-SSRs

no annotation

other

metabolism

transport

protein modification

electron transport

DNA metabolic process

transcription

response to stimulus

proteolysis

signal transduction

protein biosynthesis

RNA metabolic process

34% of the SSRs currently used as genetic markers are derived from gene-containing BAC end sequences, with GO biological process annotations.

SSR rate in genome fractions.

Evolution NSFDoug Cook UC-DavisGeorge Bruening UC-DavisGuohao He Tuskegee UniversityMei Yuan Tuskegee UniversityDeka Allie Tuskegee UniversityLimin Gong Tuskegee UniversityGreg May NCGRAndrew Farmer NCGRVarma Penmetsa UC-DavisBirinchi Sarma UC-Davis (BOYSCAST, BHU, Varanasi)Bhuvaneshwar Patil UC-Davis (DBT, UAS-Dharwad)Ben Rosen UC-DavisNoelia Garcia UC-DavisBullo Mamo UC-Davis (Ethiopia)Jimmy Woodward UC-DavisJinliang Gao UC-DavisSally Vail University of SaskatchewanNayibe Garzon Columbia/IITARhonda Foley CSIRO, WAKaram Singh CSIRO, WAFrancisco Goes daSilva UC-DavisAnand Surendrarao UC-Davis (Chennai)Rakesh Chahota UC-Davis (Borlaug Fellow, India)Zack Chestnut UC-DavisCecelia Osorio UC-DavisSubhojit Datta UC-Davis (IIPR, Kanpur, ICAR Fellow)Esperanza Martinez UC-Davis

Applied Genomics and Breeding GCPDavid Hoisington ICRISATRajeev Varshney ICRISAT-GCP

Doug Cook UC-DavisMatthew Blair CIAT

Jeff Ehlers UC-RiversideTim Close UC-Riverside

Carmen de Vicente GCPSpurthi Nayak ICRISAT

Andrew Paterson University of GeorgiaDavid Bertioli EMBRAPA

AKI Pigeonpea Genomics initiativeDoug Cook UC-Davis

NK Singh NRCPBRajeev Varshney ICRISAT

Subhojit Datta IIPR, Kanpur M Kurivanaschetty UAS, Dharwad

KB Wanjari UA, Akola MN Singh BHU, Varanasi

Chickpea and Groundnut initiativesSubhra Chakraborty NIPGR

Sabhyata Bhatia NIPGRVidya Gupta NCL Pune

Rajeev Varshney ICIRSATHari Upadhyaya ICRISAT

Doug Cook UC-DavisFred Muehlbauer USDA-ARS Pullman

US National Science Foundation Plant Genome Program; CGIAR Generation Challenge Program; Bill and Melinda Gates Foundation; INDO-US Science and Technology Forum; INDO-US Agricultural Knowledge Initiative; Indian Council for Agricultural Research; Indian Department of Biotechnology;

USDA-Foreign Agricultural Service; US Agency for International Development