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Development of Genomic Resources in Chickpea (Cicer arietinum L.)
Applied Genomics Laboratory,ICRISAT,Patancheru.
Pavana J. Hiremath
Department of Plant Genetics,Osmania University,
Hyderabad.
•The third largest food legume crop, 650 genera , 18,000 species
•Grown over 40 different geographically diverse regions major producers being South Asia.
•Grown by marginal farmers –rainfed crop
Chickpea crop
•Maintenance of soil fertility
•Self‐pollinated, diploid (2x=2n=16), ~740 Mb genome size
•Major source of protein (~20%)
• Two types of seeds‐ Desi (80%)Kabuli (20%)
Kingdom: PlantaeUnranked: EudicotsOrder: FabalesFamily: FabaceaeGenus: CicerSpecies: C. arietinum
>6 m ha0.5 to 1.0 m ha150,000 to 210,000 ha50,000 to 100,000 ha10,000 to 40,000 ha
Chickpea global area ‐ 11.55 million Ha Global annual production ‐ 10.46 million MtAverage productivity‐ 905 Kg/ HaIndia production‐ 894 kg/HaTotal India production: area‐ 7.89 million Ha and production‐ 7.06 Mt
Chickpea production (FAO‐2009)
Major growing states in India•Madhya Pradesh•Uttar Pradesh•Rajasthan•Maharashtra•Andhra Pradesh
India
Australia
Myanmar
PakistanTurkey
Iran
Ethiopia
Canada
Mexico
YearsIndia's
production (Tonnes)
World Production (Tonnes)
% Share
1998‐99 6800000 9429651 72.11
1999‐00 5120000 7963448 64.29
2000‐01 3860000 6911965 55.85
2001‐02 5470000 8292377 65.96
2002‐03 4240000 7127232 59.49
2003‐04 5720000 8435392 67.81
2004‐05 5470000 8515421 64.24
2005‐06 5600000 8510056 65.8
2006‐07 6330000 9685967 65.35
2008‐09 7060000 10460000 67.49
Chickpea production by India against world’s production
Average production share‐ 64.83%
agropedia.iitk.ac.in/?q=content/production‐productivity‐chickpea‐0
Biotic stresses
Abiotic stresses
Terminal drought Salinity
Wilt Pod borer Blight
Production constraints
Total produ
ction
Drought
Low te
mpe
rature
Wilt
Ascochyta
blight
Pod bo
rer
• Development of early maturing varieties for drought escape• Development of varieties with enhanced drought tolerance• Development of wilt, helicoverpa resistant varieties
Pre‐requisites
Molecular breeding approach Transgenic approachConventional breeding approach
Phenotypic markers Transcriptomics‐ESTs‐Candidate genes
•MAS•MABC•Germplasm screening•Population studies
Transformation
Improved or new variety
Genetic markers‐AFLPS‐RFLPs‐RAPDs‐SSRs‐SNPs
Genomic resources available then…!
BAC libraries ‐ 48,796
Genomic survey sequences ‐ 50,853
Protein records ‐ 1,233
DArT features ‐ 5,397
Super‐SAGE tags ‐ 80,238
Isozymes‐ ~50
ESTs ‐ 7,097
COS‐SNPS ‐ 768
SNPs ‐ <100
SSR discovery and validation ‐ 1,655
Objectives of the study
1. Generation of ESTs from drought and salinity stressed libraries of chickpea genotypes‐ ICC 4958, ICC 1882, JG 11 and ICCV 2.
2. Development of chickpea transcriptome assembly.
3. Large‐scale identification of SNPs and SSRs between ICC 4958 and PI 489777
4. Mapping of newly designed SNPs and SSRs on inter‐specific chickpea map.
To develop sequence and marker resource !!
Objective 1: Generation of ESTs from drought andsalinity stressed libraries of chickpeagenotypes
ICC4958 ICC 1882
ICC 4958 ICC 1882ICC 4958 ICC 1882
C S SC
ICC 1882ICC 4958
1. PEG Treatment (1mM); Hydroponic medium
2. Dehydration; Hydroponic 4. Slow drought in field
3. Slow drought in greenhouse
Total RNA extracted
cDNA constructed
ESTs sequenced‐Sanger technology
Roots harvested
Plants grown to 22 days veg. stage
Drought stress imposition
Relative water content – 50%Transpiration ratio‐ 0.1
Drought‐responsive EST libraries
JG 11‐C JG 11‐S ICCV 2‐C ICCV 2‐S
Salinity‐responsive EST libraries
‐ 80 mMNaCl on 22 days old stage‐ Stress applied for 7 days‐ Roots harvested after 7 days of stress‐ RNA isolation‐ cDNA library construction‐ EST sequencing‐ Sanger technology
Alignment
Contig/ Consensus sequence
Singletons
Tentative unique sequences (TUSs)or Unigenes
Derivation of tentative unique sequences (TUSs) using CAP3 program
•To remove any redundant sequences•To represent unique number of genes at specific stage
3,798 4,460
3,132 4,307
315 1,392
1,707
704 547
1,251
945 1,650
2,595 (UG‐II)
3,132 4,3077,439
RS
HQS
Contigs Singletons
Salinity
JG 11
Root tissue
ICCV 2
Root tissue
Total RS
ICC 4958
Root tissue
Drought
PEG Induction
96 960 1,920 3,006 384 1,056 1,922 2,560
96 849 1,822 2,694 365 932 1,808 2,430
5,461
398 2,258
2,656
398 1,994
2,392
763 3,795
4,558 (UG‐I)
5,535
Drought Green House
Drought field
PEG Induction
Drought Green House
Drought field
10,996
RS
HQS
Contigs Singletons
Dehydration Dehydration
ICC 1882
Root tissue
Total RS 5,982
Total RS 5,922
5,461
18,435
1,590 4,814
6,404 (UG‐III)
5,535 3,132 4,307
ICC 4958 ICC 1882 JG 11 ICCV 2
HQS
Contigs Singletons
5,982 5,922 3,798 4,460Total RS
7,09731,32 4,3075,5355,461
25,532
2,431 7,138
9,569 (UG‐IV)
ICC 4958 ICC 1882 JG 11 ICCV 2 Public Domain
HQS
Contigs Singletons
5,982 5,922 3,798 4,460 7,097
Chickpea tentative unigene sequence (TUS) assembly of ESTs
(CAP3)
Clustering and Assembly Program (CAP3)
Average length of high quality ESTs‐ 569 bp
Contig size in terms of number of ESTs aligned in each contig2 EST members ‐ 587 contigs874 EST members‐ 1 contigAverage assembly size‐ 8
Contig assembly features of UG/TUS‐III (6,404 TUSs)
BLASTN analysis of UG/TUS‐III against legumes and model plant species
Percent Identity
Similar across all legume databases‐ 284 (4.4%)
No similarity with legume ESTs‐ 37 (0.5%)
Similar across all plant EST databases analysed‐ 228
82%
97.90%
98.30%
99.10%
97.30%
97.20%
98.10%
98.30%
100%
21.50%
45.80%
45.19%
9.44%
31.12%
53.31%
65.84%
68.83%
0% 20% 40% 60% 80% 100% 120%
Oryza sativa
populus Alba
Arabidopsis thaliana
Vigna radiata
Arachis hypogaea
Lotus japonicus
Glycine max
Medicago truncatula
Total Chickpea Unigenes
Plan
t EST datab
ases
E‐value threshold of ≤1E‐05
BLASTX annotation of UG/TUS‐III (6,404 TUSs) against the UniProt database
4558
4102
1912
1576
1413
2595
2374
1476
1342
1278
6404
5796
2965
2538
2333
9569
8729
4657
4013
3693
0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000
Unigenes
Total Hits
1.00E-05
1.00E-08
1.00E-10
Number of unigenes
Drought responsive unigenes Salinity responsive unigenesDrought and Salinity responsive unigenes Drought, Salinity and Public domain ESTs
E‐value thresholds
367
8
1284
129
301
12 20
305
1140
63113
56 54
371
1528
97 12911
389
111
1048
456
36
1171
876
7733 13 41
17876 43
132
0
200
400
600
800
1000
1200
1400
1600
1800
biologica
l regula
tion
cell a
dhes
ion
cellu
lar pr
oces
s
develo
pmental p
rocess
estab
lishmen
t of lo
caliza
tion
growth
immun
e syste
m process
localiz
ation
metabo
lic pr
ocess
multi-organ
ism pr
oces
s
multicellu
lar organ
ismal
process
reproducti
on
reproducti
ve proce
ss
respo
nse to
stim
ulusce
ll part
enve
lope
extra
cellu
lar re
gion
extra
cellu
lar re
gion part
macromolec
ular co
mplex
membra
ne-enclo
sed lu
menorgan
elle
organell
e part
antio
xidant a
ctivit
ybindin
g
catal
ytic a
ctivit
y
electr
on carri
er ac
tivity
enzy
me reg
ulator a
ctivity
nutrient
reservo
ir activ
ity
signal
transd
ucer
activ
ity
structu
ral m
olecu
le ac
tivity
transc
riptio
n reg
ulator a
ctivity
transla
tion f
actor a
ctivit
y, nucle
ic ac
id binding
transp
orter a
ctivit
y
Biological process Cellular component Molecular function
Num
ber o
f unigene
sFunctional categorization of UG/TUS‐III (6,404 TUSs)
Hydrolases (206)
Transferases (208)Oxidoreductases (183)
Ligases (67)
Isomerases (41)
Lyases (38)
Classification of chickpea UG/TUS‐III according to Enzyme Commission IDs
Sl. No UNIQID Description Lib1(norm) Lib2(norm)
1 Cp_Dr_Contig301 >Q6CQE5|KLULA Protein TAR1 - Kluyveromyces lactis (Yeast) (Candida sphaerica) 228.9 3.6
2 Cp_Dr_Contig312 >P72823|SYNY3 NAD(P)H-quinone oxidoreductase chain 4-2 - Synechocystis sp. (strn PCC 6803) 258.2 45.2
3 Cp_Dr_Contig318 >Q8TGM7|YEAST Uncharacterized protein ART2 - Saccharomyces cerevisiae (Bakers yeast) 131.8 37.9
4 Cp_Dr_Contig332 >Q9SSK5|ARATH MLP-like protein 43 - Arabidopsis thaliana (Mouse-ear cress) 69.6 14.5
5 Cp_Dr_Contig335 >Q9SSK5|ARATH MLP-like protein 43 - Arabidopsis thaliana (Mouse-ear cress) 93.4 30.7
6 Cp_Dr_Contig339 >Q6J163|PINTA Auxin-induced protein 5NG4 - Pinus taeda (Loblolly pine) 45.8 119.2
7 Cp_Dr_Contig341 >Q06931|PEA ABA-responsive protein ABR17 - Pisum sativum (Garden pea) 538.4 791.3
8 Cp_Dr_Contig378 >P04929|PLALO Histidine-rich glycoprotein precursor - Plasmodium lophurae 104.4 25.3
9 Cp_Dr_Contig564 >O22309|MEDSA Isoflavone-7-O-methyltransferase 9 - Medicago sativa (Alfalfa) 0 168
10 Cp_Dr_Contig644 >Q06930|PEA ABA-responsive protein ABR18 - Pisum sativum (Garden pea) 9.2 74.1
11 Cp_Dr_Contig656 >Q8TGM7|YEAST Uncharacterized protein ART2 - Saccharomyces cerevisiae (Bakers yeast) 117.2 272.8
12 Cp_Dr_Contig743 >Q43766|HORVU Non-specific lipid-transfer protein 3 precursor - Hordeum vulgare (Barley) 31.1 126.5
13 Cp_Dr_Contig746 >Q9PLI5|CHLMU Uncharacterized protein TC_0114 - Chlamydia muridarum 536.5 943.1
Identification of differentially expressed drought‐responsive genes using IDEG.6 tool
Clustering of all ESTs‐ CAP3 Contigs/EST count Input to IDEG.6
Drought 763 contigs
Salinity 945 contigs
ICC 4958 ICC 1882
R of Stekel and FalcianiGeneral Chi‐squaredChi‐Sqaured 2x2Fisher‐ExactSteller GoblinAudic and Claverie
Sl. No. UNIQID Description Lib1(norm)
Lib2(norm)
1 Contig1 >A2A6M5|MOUSE Calcium-binding and coiled-coil domain-containing protein 2 - Mus musculus (Mouse) 287.4 4.6
2 Contig2 >Q6CQE5|KLULA Protein TAR1 - Kluyveromyces lactis (Yeast) (Candida sphaerica) 1123.9 285.6
3 Contig4 >Q06931|PEA ABA-responsive protein ABR17 - Pisum sativum (Garden pea) 60.7 162.5
4 Contig72 >Q9PLI5|CHLMU Uncharacterized protein TC_0114 - Chlamydia muridarum 134.1 34.8
5 Contig103 >P48534|PEA L-ascorbate peroxidase, cytosolic - Pisum sativum (Garden pea) 54.3 2.3
6 Contig146 >A2A6M5|MOUSE Calcium-binding and coiled-coil domain-containing protein 2 - Mus musculus (Mouse) 134.1 0
7 Contig147 >Q9FS16|ARATH Extensin-3 precursor - Arabidopsis thaliana (Mouse-ear cress) 67 6.1
8 Contig230 >P27047|PEA Disease resistance response protein DRRG49-C - Pisum sativum (Garden pea) 38.3 146.3
9 Contig294 >A2A6M5|MOUSE Calcium-binding and coiled-coil domain-containing protein 2 - Mus musculus (Mouse) 38.3 0
10 Contig332 >O65740|CAPAN Defensin J1-2 precursor - Capsicum annuum (Bell pepper) 0 41.8
11 Contig385 >Q9SLP4|TULBA Chitinase 1 precursor - Tulipa bakeri (Tulip) 3.2 55.7
12 Contig402 >Q9SX33|ARATH Putative phospholipid-transporting ATPase 9 - Arabidopsis thaliana (Mouse-ear cress) 0 51.1
13 Contig441 >Q06930|PEA ABA-responsive protein ABR18 - Pisum sativum (Garden pea) 35.1 183.4
14 Contig467 >P29062|TOBAC Pathogenesis-related protein PR-4A precursor - Nicotiana tabacum (Common tobacco) 0 62.7
15 Contig636 >Q06930|PEA ABA-responsive protein ABR18 - Pisum sativum (Garden pea) 6.4 139.3
16 Contig727 >Q8TGM7|YEAST Uncharacterized protein ART2 - Saccharomyces cerevisiae (Bakers yeast) 648.1 41.8
17 Contig761 >P93147|GLYEC Cytochrome P450 81E1 - Glycyrrhiza echinata (Licorice) 0 69.7
18 Contig779 >P41512|XENLA DNA topoisomerase 1 - Xenopus laevis (African clawed frog) 0 55.7
19 Contig793 >Q00016|CICAR Isoflavone reductase - Cicer arietinum (Chickpea) (Garbanzo) 0 46.4
20 Contig939 >P36908|CICAR Acidic endochitinase precursor - Cicer arietinum (Chickpea) (Garbanzo) 0 60.4
Identification of differentially expressed salinity responsive genes using IDEG.6
JG 11 ICCV 2
cDNA libraries
ICC 18
82_P
EG_Ind
uction
ICC 49
58_P
EG_Ind
uction
ICC 49
58_D
rought_G
H
ICC 18
82_D
rought_G
H
ICC 49
58_D
ehydration
ICC 18
82_D
ehydration
ICC 18
82_D
rought_Field
ICC 49
58_D
rought_Field
I
IIa
IIb
IIc
IIIa
IIIb
IIIc
IIId
IV
Clusters of con
tigs
JG 11_Salin
ity
ICCV
2_Salinity
Average LinkagePearson’s r: Centered, Unabsolute105 Contigs10 libraries
Clustered correlation map of highly expressed genes in different libraries of chickpea roots using HCE version 2.0 beta web tool
Cluster IV
JG 11 salin
ity
Only contigs with ≥5 reads used (>200)
Contigs with significant changein EST counts‐ IDEG.6
Hierarchical clustering explorer
UG‐III 1,590 contigs from CAP3
Sampling errors
Genuine differences
Total number of sequences examined 9,569Total size of examined sequences (bp) 5,269,104Total number of identified SSRs 3,728Number of SSR containing sequences 2,029Number of sequences containing more than one SSR 581Number of SSRs present in compound formation 1,354Frequency of SSR 1/ 700 bp
Number of mono‐nucleotide repeats 1,793Number of di‐nucleotide repeats 126Number of tri‐nucleotide repeats 110Number of tetra‐nucleotide repeats 7Number of penta‐nucleotide repeats 8Number of hexa‐nucleotide repeats 5
Identification of SSRs using MISA
SSR identification
Distribution of SSRs
Chickpea EST‐SSR primer validationTotal primers designed ‐ 177 Primers synthesized‐ 77 (Class‐I markers: di‐>6, tri‐>5, tetra‐>4, penta>4, hexa‐4)Validated on‐ 24 chickpea genotypesAverage alleles/ marker‐ 4.6Average PIC value‐ 0.43
Mapping population No. of poly markers
ICC 4958 × PI 489777 22
ICC 4958 × ICC 1882 8
ICC 283 × ICC 8261 10
EST derived SNPs and SNP2CAPS
Number of contigs 2,431
Total number of SNPs identified 36,086
Number of contigs containing SNPs 2,047
Contigs with 2‐4 ESTs 1,305
Contigs with ≥5 ESTs 742
Number of SNPs in contigs with 2‐4 ESTs 14,681
Number of SNPs in contigs with ≥5 ESTs 21,405
Number of contigs containing CAPS convertible SNPs 240
Number of identified CAPS 7,884
SNP frequency 1/50bp
Summary of objective‐ I
• 20,162 new chickpea ESTs generated(NCBI: GR390696‐ GR410171 and GR420430‐ GR421115)
• 9,569 chickpea TUSs derived
•Expression patterns of 105 contigs/genes that were up‐ or down‐regulated in response to imposed abiotic stresses
•177 SSR markers designed and 77 validated
•742 genes with SNPs, 240 CAPS convertible SNPs
BMC Genomics (2009) 10:523
OBJECTIVE 2 : Generation of chickpea transcriptome assembly using454/FLX technology
Overview of the 454/FLX sequencing technology
(a) Genomic RNA/cDNA fragmented, ligated
(b) Fragments are bound to beads (c) The emulsion is broken, the DNA
strands are denatured,(d) Smaller beads carrying(e) Scanning electron micrograph(f) The 454 sequencing instrument
consists of
1. a fluidic assembly 2. fiber‐optic slide 3. CCD camera‐based
Generates more than 1,000,000 individual reads with improved Q20 with read length of 250 bases per 10‐hour instrument run
Cost‐ ~ 60$ per megabase base data generated
Total RNA from different tissues/stages/conditions of ICC 4958 was extracted, pooled , normalized and sequenced by 454/FLX technology ( JCVI, USA)
Sequencing of chickpea transcriptome using 454/FLX technology
No. Tissue samples
Stages/stresses
C1 Embryo 2 days embryo
C2 Shoot 3, 4 & 5 days old seedlings
C3 Roots 3, 4 & 5 days old seedlings
C4 Shoot 10 & 20 days old plant
C5 Roots 10 & 20 days old plant
C6 Leaves 25 days old & etiolated leaves
C7 Roots 30 days old plant
C8 Apical meristem
seedling stage & 25 days old plants
C9 Buds and flowers
25 days old plants
C10 Pods 35 days old plants
C11 Roots Drought stressed plants
C12 Roots 60, 80 and 100 mM Salt stressed plants
FLX‐ 454 ReadsTotal numbers of reads : 435,018 Average read length : 216 bpNumber of ESTs with >200 bp : 379,039
Sanger + FLX 454 ESTsNumber of ESTs: 25,532Number of TUSs : 103,215 CaTANumber of Contigs : 46,740Number of Singletons: 56,475
Pool
Normalization by DSN principle
454/FLX technology
List of chickpea tissues used for 454/FLX sequencing
Assembly statistics of chickpea transcriptome
Average length of Roche/454 reads‐ 216 bp Average length of Sanger EST reads‐ 486 bpAverage length of Roche/454 contigs‐ 432 bpAverage length of EST contigs‐ 712 bpAverage length of Roche/454 + EST contigs‐ 459 bp
BLASTN analysis of CaTA against legume and model plant EST databases
Number of TUSs subjected for BLASTX annotation: 103,215 TUSsNumber of TUSs annotated : 49,437 (47.8%), E‐value: ≤1e‐10 Functionally categorised based on GO: 20,634 (19.9%)
Functional categorization of annotated TUSs based on Gene Ontology (GO) description
Biological Process Cellular Localization
Molecular function
Transcription factors Enzyme classification
Classification of TUSs into transcription factors and enzyme classes
Path 1: MtChr5:cmap.cmtv:MtChr5
Path 2: MtChr1:cmap.cmtv:MtChr1
Path 3: MtChr2:cmap.cmtv:MtChr2
OverviewMt1
Region
Details
GBROWSE window showing mapping of chickpea TUSs on Mt 3.0 genome build
http://medtr.comparative‐legumes.org/gb2/gbrowse/3.5.1/
•Exonerate mapping tool (Slater and Birney, 2005),
•Thresholds ‐minimum percent identity of 75maximum intron length of 5,000 bp
•42,141 (40.8%) of the TUSs aligned, intersecting on 14,580 predicted Mt genes (Mt 3.0)
•Predicted 39,281 splice sites in 20,137 of the TUS alignments
•Designed 2,088 intron‐spanning primer sets.
Solexa/Illumina‐ sequencing
• Up to 20 G base of DNA sequence
• Average read length only 36 bp
• ~50 million DNA sequence reads
• Eight channels of sequence
• 345,600 TIFF images
• One Terabyte of data
Cost comparatively 1‐5$ per megabasedata generated
ICC 4958 PI 489777
Total RNA extracted from root tissues of22 days old stage plants , pooled andsequenced by Solexa/Illumina 1Gsequencer (NCGR, USA)
15.6 million short tags(36 bp)
26.3 million short tags(36 bp)
Illumina/Solexa sequencing of chickpea parental genotypes
Summary of objective II
•Generation of 435,018 short reads using 454/FLX technology
•Developed chickpea transcriptome assembly (reference assembly) with 103,215 TUSs
•Predicted physical location for 42,141 (40.8%) TUSs on Mt genome
•2,088 Intron spanning region markers designed
•Generated 15.6 million and 26.3 million Solexa/Illumina short tags for ICC 4958 and PI 489 777, respectively
Plant Bioteh J 2011 pp.1‐10
OBJECTIVE 3: Large‐scale identification and development of SSRs and SNPs
Total number of TUSs examined 103,215Total size of examined sequences (bp) 34,718,996Total number of identified SSRs 26,252Number of SSR containing sequences 23,330Number of sequences containing >1 SSR 2,480Number of SSRs present in compound formation 2,012Mono-nucleotide repeats 24,428Di-nucleotide repeats 743Tri-nucleotide repeats 893Tetra-nucleotide repeats 91Penta-nucleotide repeats 51Hexa-nucleotide repeats 46Primer pair designed 3,172Class-I primer pairs selected 728Primers synthesised and validated 75
Identification of SSRs using MISA from CaTA‐103,215 TUSs
(i) Generation of RNA sequence data from parental genotypes
(ii) Aligning short sequences of both parental genotypes against the reference assembly of chickpea
(iii) Identification of variants
Overall strategy for SNP identification
15.6 million short tags/reads (36 bp) 26.3 million short tags/reads (36 bp)
Total number of variants identified‐ 26,533Total number of InDels‐ 1,847Total number of substitutions‐ 24,686
Solexa/Illumina sequence generation and mapping to reference using Alpheus pipeline
ICC 4958 PI 489777
Frequency difference range
Number of reads in tentative contigs
>500 101‐500 11‐100 3‐10
<0.1 389 751 2,109 158
0.10‐0.19 107 414 2,431 500
0.20‐0.29 17 123 3,856 827
0.30‐0.39 4 47 1,478 992
0.40‐0.49 1 13 746 828
0.50‐0.59 8 18 502 1,442
0.60‐0.69 ‐ 17 297 1,361
0.70‐0.79 ‐ 1 85 374
0.80‐0.89 ‐ ‐ 55 166
0.90‐1.0 ‐ ‐ 40 1,463
SNP identification statistics
Parameters for SNP selection
‐Higher read depth‐High frequency difference range
Sl.No. Population/population_combinationRead depth frequency
No. of SNPs
1 ICC 4958 X ICC 1882, ICC 4958 X PI 489777 and ICC 506 X ICCC 37 ≥3 ≥0.75 and ≤0.25 162 ICC 4958 X ICC 1882, ICC 4958 X PI 489777 ≥3 ≥0.75 and ≤0.25 813 ICC 4958 X P I489777, ICC 506 X ICCC 37 ≥3 ≥0.75 and ≤0.25 1124 ICC 4958 X ICC 1882, ICC 506 X ICCC 37 ≥3 ≥0.75 and ≤0.25 555 ICC 4958 X ICC 1882 ≥10 ≥0.75 and ≤0.25 396 ICC 4958 X PI 489777 ≥10 ≥0.75 and ≤0.25 2,4807 ICC 506 X ICCC 37 ≥10 ≥0.75 and ≤0.25 741
Total no. of SNPs 3,524
SNP selection
Assay design tool to identify high probabilistic SNP by Illumina
ADT scores (0.1‐0.9) for 1,409 non‐redundant SNPs
SNP discovery and validation using KASPar assay
Phule G12ICCV 04112C 214ICC 14402ICC 3946ICCV 10ICC 506‐EBICC 15567ICC 15606ICC 3325WR 315ICC 5135ICC 12492DCP 92‐3JG 74ICC 1431ICC 283JG 62JG 16ICCV 97105ICCV 92318ICCV 05107KAK 2ICCV 95423ICC 4958ICCV 2VijayJG 130JG 11JAKI 9218JG 14VishalPhule G5ICCV 00108ICC 1882ICC 6263ICC 16796ICC 8261ICC 13187ICC 11764ICC 995ICC 3279EjereICC 7554ICC 3137ICC 6877ICC 9895ICC 8855ICC 7571AnnigeriICCV 05530ICC 4593ArertiICC 12037ICCC 37IG 72933PI 489777IG 72953
AfghanistanChileCyprusEthiopiaIndiaIranIsrealMexicoPortugalRussia and CISsTurkeyFormer Soviet Union
Cluster‐II
Cluster‐
IIbCluster‐
IIa
Cluster‐I
Genetic relationship of 58 accessions as revealed by CKAMs using DARwin 5.0
58 chickpea accessions
CKAMs based polymorphims in some segregating populations of chickpea
Summary of objective III
• 3,728 SSR primers, 728 class‐I primers, 75 validated on 5 chickpea genotypes
• Identified 26,533 SNPs between ICC 4958 and PI 489777
• 2,005 SNPs validated by KASPar assay
OBJECTIVE 4: Mapping of newly designed SNPs and SSRs on inter‐specific chickpea map (ICC 4958 × PI 489777)
50 ng
50 ng
50 ng
100 ng
100 ng
100 ng
100 ng
50 ng
150 ng
200 ng
Blanks: PL 1‐ C2, H5, F9, A10PL2‐ D3, G4, B5, C5 ICC 4958 x PI 489777 stock plate
Genomic DNA assessment of 131 recombinant inbred lines (RILs) of inter‐specific mapping population
LG1
CGMM070
CGMM009
CGMM055
CGMM048
LG2
CISR002
CGMM005;CGMM021
CGMM049
CGMM072
CISR090
CGMM032
CaHa61
CGMM024
CGMM011
CGMM006
CGMM071
CGMM045
LG3 LG4 LG5 LG6 LG7 LG8
Gujaria et al. 2010 – chickpea transcript map with 300 loci
CaLG01 CaLG02 CaLG03 CaLG04 CaLG05 CaLG06 CaLG07 CaLG08
CAPsCISRsCKAM/ KASPar assayCOS‐SNPs/Goldengate assayDArTICCeM/ EST‐SSRsLegacy markers (CaM, ISSR, SSR, ICCM)
High density chickpea transcript map with 1, 328 loci
Summary of 1,328 loci transcript map
Marker type
Totalmarkersused
Totalmarkersmapped
Chickpea linkage groups
CaLG01 CaLG02 CaLG03 CaLG04 CaLG05 CaLG06 CaLG07 CaLG08
New marker loci
CKAMs 651 52 81 57 132 90 86 59 68 568
TOGs‐SNPs 317 56 29 16 67 58 56 19 13 371
Published marker loci
GMMs
CGMMs 31 4 10 2 6 3 2 2 2 31
CISRs 15 2 ‐ 2 ‐ 4 4 ‐ 3 15
ICCeMs 15 2 2 2 2 1 1 1 1 12
Legacy markers
H‐series 44 4 7 6 5 7 5 6 4 44
ICCMs 46 3 4 9 10 7 6 5 2 46
CAMs 10 1 ‐ 1 1 2 4 1 ‐ 10
SSRs (Frankfurt University) 93 14 11 16 14 14 10 9 5 93
ISSRs 26 8 8 ‐ 2 2 1 5 ‐ 26
SNaPshot Assay‐based SNPs 79 8 8 18 12 8 8 9 5 79
CAPS 13 ‐ 1 4 2 2 1 ‐ ‐ 10
DArTs 20 1 ‐ 3 2 5 2 5 1 19
RAPDs 4 1 ‐ ‐ ‐ ‐ ‐ 2 1 4
Total number of markers 1,364 156 161 136 255 203 186 124 107 1,328
Total distance (cM) 116.6 92.9 101.8 92.5 95.6 107 112 70.5 788.6
Average inter‐ markerdistance (cM) 0.75 0.58 0.75 0.36 0.471 0.57 0.9 0.66 0.59
Summary statistics of different chickpea linkage maps with 1328 loci map
Gujaria et al. 2010 Thudi et al. 2011Hiremath et al.
2012
Total no. of loci 300 1,291 1,328
Total distance (cM) 766.56 845.56 788.6
Inter‐marker distance 2.55 0.65 0.59
Average no. of markers/ linkage group
~38 ~161 ~166
Chickpea against Medicago Chickpea against Cowpea
Chickpea against Glycine max Chickpea against Lotus
Chickpea vs. Cowpea Lotus Medicago SoybeanCaLG01 6 67 212 173CaLG02 4 31 173 301CaLG03 2 32 268 172CaLG04 14 123 178 374CaLG05 6 54 197 252CaLG06 14 58 218 328CaLG07 5 29 91 77CaLG08 4 44 133 114Total number of matches 55 438 1470 1791
0
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f seq
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Chickpea linkage groups
Cowpea
Lotus
Medicago
G. max
Comparative mapping of 1,064 chickpea loci with related legumes
Plant Biotech J 2012 pp.1‐17
Summary Objective I: ‐ 20,162 ESTs generated, 9,569 chickpea TUSs derived
‐ 177 SSR primer designed , 77 SSRs validated
‐ 240 contigs with 742 SNPs that can be converted to CAPS
‐ Expression patterns of 105 contigs/genes that were up‐ or down‐regulated in responseto imposed abiotic stresses observed
Objective 2: ‐ 435,018 454/FLX reads generated
‐ Chickpea transcriptome assembly developed ‐103,215 TUSs
‐ 2,088 ISR markers designed
Objective 3: ‐ 728 Class I primer designed, 75 SSRs validated
‐ 42 million Solexa/Illumina tags (36 bp) for ICC 4958, PI 489777, Identified 26,533 SNPs
‐ 2005 novel SNPs assayed by KASPar genotyping technology
Objective 4: ‐Chickpea transcript map with 1,328 loci spanning 788.6 cM distance developed
‐Determined syntenic positions of 1,064 mapped loci on 4 legume genomes
Large‐scale transcriptome analysis in chickpea (Cicer arietinum L.), an orphan legume crop of the semi‐aridtropics of Asia and AfricaPavana J. Hiremath, Andrew Farmer, Steven B. Cannon, Jimmy Woodward, HimabinduKudapa, ReetuTuteja, Ashish Kumar, A BhanuPrakash, Benjamin Mulaosmanovic, NehaGujaria, LKrishnamurthy, Pooran M. Gaur, P. B. KaviKishor, TrusharShah, Ramamurthy Srinivasan, Marc Lohse, YongliXiao, Christopher D. Town, Douglas R. Cook, Gregory D. May, Rajeev K. Varshney (2011) Plant Bioteh J, pp.1‐10.A comprehensive resource of drought‐ and salinity‐ responsive ESTs for gene discovery and markerdevelopment in chickpea (Cicer arietinum L.)Varshney, R.K., Hiremath, P.J., Lekha, P., Kashiwagi, J., Balaji, J., Deokar, A.A., Vadez, V., Xiao, Y., Srinivasan, R.,Gaur, P.M., Siddique, K.H.M., Town, C.D. and Hoisington, D.A. (2009). BMC Genomics,10:523.
Development and use of genic molecular markers (GMMs) for construction of a transcript map of chickpea(Cicer arietinum L.)Neha Gujaria, Ashish Kumar, Preeti Dauthal, Anuja Dubey, Pavana Hiremath, A Bhanu Prakash, AndrewFarmer, Mangla Bhide, Trushar Shah, Pooram M. Gaur, Hari D. Upadhyaya, Sabhyata Bhatia, Douglas R.Cook, Greg D. May, and Rajeev K. Varshney. (2011) Theor Appl Genet 122(8):1577‐1589.
Research PublicationsLarge‐scale development of cost‐effective SNP marker assays for diversity assessment and genetic mapping in chickpea and comparative mapping in legumes Pavana J. Hiremath, Ashish Kumar, R. Varma Penmetsa, Andrew Farmer, Jessica A. Schlueter, Siva K. Chamarthi, Adam M. Whaley, Noelia Carrasquilla‐Garcia , Pooran M. Gaur, Hari D. Upadhyaya, Polavarapu B. Kavi Kishor, Trushar M. Shah, Douglas R. Cook, Rajeev K. Varshney (2012) Plant Biotech J, pp.1‐17.
Acknowledgements
•Dr Rajeev K. Varshney (PI), Director‐CEG, ICRISAT•Prof Dr. P. B. Kavikishor (Co‐PI) Osmania University
Acknowledgements
AGLLekha PRaju NLMir ReyazSpurthi NayakAshish KumarHimabindu KudapaMayank KaashyapSarwar AzamRachit SaxenaAnujaNeha GujariaMahendarThudiAbhishek Bohra
Bio‐informatics UnitTrushar ShahVivek ThakurBhanuPrakashSatish PatetiReetu TutejaPradeep
StatisticsAbhishek RathoreRoma
Crop PhysiologyJunichi KashiwagiVincent VadezL KrishnamurthySunitha
JCVI, USAYongli XiaoChristopher Town
NCGR, USAAndrew FarmerJimmy WoodwardGreg May
UC Davis, USADoug CookVerma Pennmetsa
Administration‐AGLMrs Seetha KannanMrs Manjula BaddamPrasad KDV
University of North CarolinaAdam WhaleyJessica Schleuter
Max‐Planck Institute, GermanyMarc Lohse
Learning System UnitDr Rosana MulaPrasad RaoDamodar
Funding ResourceCGIAR‐GCP Bill and Melinda Gates Foundation DBT, India
THANK YOU ALL
