1. Improve chickpea productivity for
marginal environments in
sub-Saharan Africa and
South Asia

2.  Introduction
 Genomic resources
 Genetic resources
 Trait mapping- QTLs and GWAS
 Molecular breeding for drought tolerance
 Enhancing Capacity of NARS
 Data management and decision support tools
Contents

3. Introduction…

4. Global distribution
of chickpea area
Grown on about 12 m ha across 54 countries
>6 m ha
0.5 to 1.0 m ha
150,000 to 210,000 ha
50,000 to 100,000 ha
10,000 to 40,000 ha

5. Production constraints
Abiotic stresses
1. Drought
2. Heat
3. Salinity
4. Cold
Max. Temperature (0C) Mean Temp
Min. Temperature (0C)
Harvest
0
50
100
150
200
250
300
350
400
450
1 2 3 4 5 6 7 8 9 10 11 12
Month
mm
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
45.0
Sowing
Terminal drought
Precipitation Evaporation
Million(US$)
Totalproduction
Drought
Lowtemperature
Wilt
Ascochytablight
Podborer

6. Constraints in molecular
breeding
 Very few molecular (SSR) markers
 Either no genetic map or maps with low
marker density
 Non-availability of appropriate
germplasm, mapping populations and
phenotyping data
 Non-availability of trait-associated
markers in breeding
 Capacity and skills in molecular breeding

7. Genomic resources...

8. Resource Amount
SSRs ~2,000
DArT arrays 15,360 features
SNPs 9,000
GoldenGate assays 768 SNPs
KASPar assays 2,068 SNPs
Veracode assays 96 SNPs
Summary of marker resources

9. LG1
LG2
LG3 LG4 LG5 LG6
LG7
LG8
Marker Loci: 1,291
Coverage: 845.56 cM
Comprehensive genetic map
TAG 2010, 2011
PLoS One 2011
Plant Biotech J 2012

10. KASPars assays integrated
in transcript map
Markers mapped : 1328
Map distance : 788.6cM
Average number of markers/LG : 166
Average inter-marker distance : 0.59cM
TAG 2010, 2011
PLoS One 2011
Plant Biotech J 2012

11. Towards genome-wide
physical map
Clones CAH library CAE library Old
library
Total
1st 2nd 1st 2nd 1st 2nd
Clones
targeted
29,664 5,376 29,568 5,376 337 773 71,094
(12X )
Clones with
usable data
28,492 5,160 28,272 5,240 319 765 68, 248
Clones in
FPC
18,285 3,502 22,571 3,926 319 765 49,368
Old library, 1st instance are the clones from which BES-SSR
were developed
Old library, 2nd instance are the RGH hybridizing clones
Fingerprinting statistics of different BAC-libraries

12. Clone statistics in contigs:
Total no. clones in 1,174 contigs 46,112
Range of clone in contigs 2 to 3,007
Average no. of clones in each contig 39.27
Genome coverage 8X
Genome represented 615 Mb
Band statistics in clones:
Total no. of bands in clones 318,971
Average no. of bands in clones 271.69
Range of bands in clones 34 to 2,268
Minimum tiling path (MTP):
Total no. of contigs 1,174
No of clones in MTP 4,290
Statistics of physical map
Collaboration: NIPGR, India and UC-Davis, USA

13. Integrating sequence,
physical and genetic maps

14. The chickpea genome

15. Re-sequencing in chickpea
Samples:
- 5 wild species
- 25 landraces
- 60 breeding lines/
released varieties
Or
- 5 wild species
- 57 Desi genotypes
- 28 Kabuli genotypes
Sequencing:
-RAD sequencing (35 Gb)
- 61 genotypes
(40 desi, 16 kabuli, 5 wild)
-WGRS (205 Gb)
- 29 genotypes
(17 desi, 12 kabuli)

16. Genetic resources…

17. Harnessing
alleles from
genebanks
 1,700 genebanks
 By 1997, the world
economy had accrued
annual benefits of ca.
$115 billion from use of
crop wild relatives
 Genomic characterization
of gene bank material is
essential
 Needs to associate allelic
value with phenotype
 Transfer in elite varieties

18. Genetic resources
300
(A) Intra-specific mapping populations:
1. ICCV 283 × ICC 8261 - 289 RILs
2. ICCV 4958 × ICC 1882 - 268 RILs
(B) Chickpea reference set
-300 genotypes
(C) Pre-breeding populations
ICRISAT, Patancheru - 17
ICRISAT, Nairobi - 20
Egerton Uni. Kenya - 5
EIAR, Ethiopia - 2

19. MAGIC populations
Genotypes Remarks
ICC 4958 Drought tolerant genotype found promising in
Ethiopidrought tolerant parent of two mapping populations
ICCV 10 Widely adapted drought tolerant cultivar found promising in
India and Kenya
JAKI 9218 Farmer-preferred cultivar in central and southern India
JG 11 Farmer-preferred cultivar in southern India and also
performing well in Kenya
JG 130 Farmer-preferred cultivars from central India
JG 16 Farmer-preferred cultivar in northern and central India
ICCV 97105 Farmer-preferred elite line identified in Kenya and
Tanzania
ICCV 00108 Farmer-preferred elite line identified in Tanzania
Eight well performing elite chickpea lines
(TLI & TLII)

20. Trait mapping…

21. Experiment of chickpea root
growth in rain out shelter
(ROS)
Crane for lifting root cylinders for
moisture under water stress
determinations
Semi-automated precise
high-throughput phenotyping

22. Phenotyping in rainfed and irrigated
conditions

23. Phenotyping in Sub-Saharan Africa
(Kenya, Ethiopia)

24. Phenotyping in India
(Pantcheru, Kanpur, Bangalore)

25. “QTL- hotspot” on CaLG04
ICC 4958 × ICC1882 Consensus map ICC283 × ICC8261
PVE 58.2%

26. Saturating “QTL- hotspot” region
 701.049 million reads
 59030.7 Mbp
 828 SNPs

27. Genotyping data on
Reference set
Homozygote
KASPar assays
Homozygote
Heterozygote
SSR analysis (CE)
SNPs in candidate genes
Marker data on reference set
 SSR - 35
 DArT - 1157
 SNPs - 651
 Gene based SNPs - 113
Total 1,956
Candidate genes
 Abscisic acid stress and ripening gene (ASR)
 DREB2A
 ERECTA
 Sucrose synthase (SuSy)
 Sucrose phosphate synthase (SPS)
 AKIN (SNF1 related protein kinase)
 Aminoaldehyde dehydrogenase (AMADH)
 Dehydrin (DHN),
 Myb transcription factor

28. Root related traits
Root traits
Traits Seasons
Root length (cm) 3
Root length density (cm cm
-3
)
Root volume (cm
3
) 3
Root dry weight (g) 3
Rooting depth (cm) 3
Root surface area 3
R-T ratio(%) 3
Shoot dry weight (g) 3
Stem dry weight (g) 3
Leaf dry weight (g) 3
Projected area 2
Average diameter 2
Water stress
determination
Exp. of chickpea root
growth in ROS

29. Agronomic traits
Morphological
traits
Yield related traits
Traits SeasonsTraits Seasons
Plant height (cm) 14 Pods/plant 2
Plant width (cm) 7 100 SDW (g) 10
Plant stand 7 Yield (g/m
2
) 3
Apical primary
branch
7 Yield (Kg/ha) 10
Apical secondary
branch
7 Yield per plant 7
Basal primay branch 7 Production 7
Basal secondary
branch
7 Biomass 6
Teritiary branches 7 Biomass/plant 2
Phenological traits Harvest index 6
Days to flowering 13 TDM weight
(g/m
2
)
2
Days to maturity 9 Transpiration efficiency
Seeds per pod 7
13
C 2
Seeds/plant 2 SPAD 2
Field phenotyping under rainfed
and irrigated environments
Heat tolerance phenotyping

30. Δ Carbon on LG4
Yield under rainfed on LG4
.
….
GWAS for ΔC and Yield

31. Root length density
Harvest index
GWAS for RLD and HI

32. GWAS for 100 seed weight

33. Molecular breeding for drought
tolerance…

34.  MABC, MARS and GS
approaches seem to most
promising for crop improvement
 Need to have genomic
resources and cost-effective
genotyping platforms
 Precise phenotyping platforms
required
 Breeders-friendly pipelines and
decision support tools required
for prediction of phenotype
Modern breeding approaches
for enhancing genetic gains

35. Marker-assisted backcross (MABC) method for
introgressing genomic region controlling root and
other drought tolerance related traits
Donors
Cultivars
JG 11 Chefe KAK 2

36. Phenotyping of MABC
products in ROS
BC3F3 lines phenotyped in ROS for
assessing root traits

37. 0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
Yield(kg/ha)
Irrigated
Rainfed
QTL introgression into JG 11
Enhanced root length density
Enhanced yield The Plant Genome, 2013

38. Institution Cross/parents Current status
ICRISAT, India JG 11 × ICC 4958 20 BC3F5 lines
Chefe × ICC 8261 8 BC3F5 lines
KAK2 × ICC 8261 2 BC3F5 lines
ICCV 10 × ICC 4958 22 BC3F5
IIPR, India DCP92-3 × ICC 4958 60BC1F1
KWR 108 × ICC 4958 7 BC1F1
IARI, India Pusa 362 × ICC 4958 170 BC2F1
EIAR, Ethiopia Ejere × ICC 4958 384 BC2F1
Arerti × ICC 4958 27 BC3F4 lines
EU, Kenya ICCV 97105 × ICC 4958 33 BC3F1
ICCV 95423 × ICC 4958 10 BC3F5 lines
MABC for enhancing drought tolerance
in Asia and sub-Saharan Africa

39. MABC status @ IARI
(Indian project)
 4 BC2F1 plants (with >96% genome recovery) were
used in making backcrosses with recurrent parent Pusa
362 to generate BC3F1 (105 seeds)
 BC2F2 seeds also harvested from plants with more
than 96% genome recovery

40. PopulationdevelopmentRecombinationPopulationdevelopment
1st Recombination cycle
2nd Recombination cycle
3rd Recombination cycle
Multilocation phenotyping
Genotyping
Parent 1 × Parent 2
F1
F2
F3
F3:4
F3:5
Single seed descent
282 F3 progenies
282 progenies
Multilocation phenotyping
A B C D E F G H
F1 F1 F1 F1
F1 F1
F1
F2
F3
F3:4
10 plants/family (A-H), 6 sets of 8 families/cross
QTL detection
JG 11 × ICC 04112 JG 130 × ICC 05107
Kenya, Ethiopia and India
Rainfed and irrigated environments
(2010-11)
70 marker 92 markers
QTL analysis completed
Marker-assisted recurrent
selection (MARS)
MARS lines for RC selected
OptiMAS
Indian
project
TLI
Phase II
First RC completed
Second RC in progress

41. ×
JG 130 X ICCV 05107

42. ×
JG 11 X ICCV 04112

43. Exploring genomic selection
in chickpea
 320 elite-breeding lines;>3000 markers

44. GS
ISMU
V2
Raw Reads
Reference
Assemble& Align
Raw Reads
Mine SNPs
Generate Marker
Matrix
Visualizein TABLET
and FLAPJACK
Export in FLAT Files
GDMS
Genotypic
Matrix & QTLs
Lines selected
for further
crossing in
GS
External
Genotyping
Platforms
Called
SNPs
ISMU V2.0
44

45. Enhancing capacity of NARS in
modern breeding…

46. NARS partners practicing
modern breeding
Serah Alice
Paul examining the crossesRobert

47. Musa Jarso
marker analysis for MABC crosses
Mosses oyier
marker analysis for MABC
crosses
4th International Workshop
on
Next generation genomics
and integrated breeding for
crop improvement,
Feb 19-21,2014,
NARS partners practicing
modern breeding

48. Products
 Reference set, pre-breeding populations,
MAGIC lines
 >3000 SSRs, 2068 KASPar, DArT arrays
 High density genetic maps and physical
map
 Draft genome sequence and re-sequencing
of 90 lines
 MABC lines with enhanced drought
tolerance in the genetic background of
JG11, KAK2 and Chefe

49. Summary
 Well characterized reference set and 39 pre-breeding
populations developed
 1200 F8 MAGIC lines developed ready for genotyping and
phenotyping
 Large scale markers (SSRs, SNPs, DArTs) and several
maps developed
 A genome-wide physical map developed (574 Mb) that
contributed to genome sequencing of chickpea
 A most promising “QTL-hotspot” with about 50 markers
for MABC for drought tolerance
 Promising introgression (BC3F5) lines with higher yield in
rainfed conditions
 Next generation of molecular breeders (4 PhD and 8 MSc
students from NARS, >50 researchers) trained
 TLI Phase I and Phase II datasets curated into IBWS

50. ICRISAT, Patancheru, India: Pooran Gaur, Hari Upadhyaya, L Krishnamurthy, Junichi
Kashiwagi, Abhishek Rathore, Trushar Shah, Mahendar Thudi, Manish Roorkiwal, Rachit
Saxena, Ashish Kumar, Murali Mohan, HimaBindu, Shailesh, Pavana, Neha, Serah, Gafoor
UC-Davis, USA: Doug Cook, R Varma Penmetsa
NCGR, Santa Fe, USA: Greg May, Andrew Farmer
Uni Georgia, USA: Scott Jackson; JCVI, USA: Chris Town, Yongli Xiao
DArT Pty Ltd, Australia: Andrzej Killian
Uni Frankfurt, Germany: Peter Winter, Guenter Kahl
Osmania University: PB Kavikishor
NRCPB, New Delhi, India: NK Singh, TR Shrma, R Srinivasan, PK Jain
NIPGR, New Delhi, India: AK Tyagi, Sabhyata Bhatia
IIPR-Kanpur, India: SK Chaturvedi, Aditya Garg, S Datta
IARI, New Delhi, India: Jitendra Kumar, C Bharadwaj, S
MPKV, Rahuri, India: L Mhase, P N Harer, PL Kulwal
JNKVV, Jabalpur, India: A Babbar, N Saini, O Gupta
ARS-Gulburga, India: D Mannur, Jayalaksmi
UAS-Bangalore, India: Sheshashayee, M Udayakumar, KP Vishwanath
Egerton University, Kenya: Paul Kimurto, Richard Mulwa
EIAR, Addis Abba, Ethiopia: Asnake Fikre, Million Eshete
LZARDI, Debre Zeit, Tanzania: Robert Kileo
Many thanks to all contributors

51. Collaborators and partners