This document outlines plans for several activities under the TL-I Phase II GCP work plan for objective 4 on chickpea. It details 7 activities including characterization of diversity, development of mapping populations, QTL mapping, marker discovery, phenotyping, marker-assisted backcrossing for drought tolerance, and collaboration with various international institutions. The collaborators will work to develop genomic resources, identify drought tolerance genes and loci, validate QTLs, and generate improved chickpea lines with enhanced drought tolerance.

1. TL-I Phase II GCP
WORK PLAN
for
Objective 4- chickpea

2. Collaborating Scientists
and Institutions GCP
 ICRISAT, India- Rajeev Varshney, Pooran Gaur, HC Sharma,
L Krishnamurthy, Nalini Mallikarjuna, Mahendar Thudi,
Siva K. Chamarthi
 ICRISAT, Kenya- NVPR Ganga Rao, Said N. Silim
 Egerton University, Kenya - Paul Kimurto, Richard Mulwa
 Ethiopian Institute of Agricultural Research (EIAR), Ethiopia-
Asnake Fikre
 Indian Institute of Pulse Research (IIPR) Kanpur, India-
Sushil K Chaturvedi, SD Mahapatra,Aditya Pratap
 University of California-Davis, USA- Doug Cook, Ming Cheng Luo
 DArT Pty Ltd., Yaramulla, Australia- Andrzej Killian

3. Activity 1 Activity 2 Activity 3 Activity 4 Activity 5 Activity 6 Activity 7
Reference Enriched BAC end Interspecific Intraspecific
Elite lines
collection libraries sequences population population 2007
Trait mapping Trait mapping
& Ethiopia
India, Kenya
Phenotyping
GCP
MARS
SSRs
MABC
X
Phase I
DArTs
Major effect Introgression
Minor effect QTLs F3
25-50 Genetic QTLs
Genotyping
contrast lines SNPs maps BC3F2
Phenotypic data, Marker/ BAC end sequences, Marker genotyping/ QTLs,QTLs-associated markers
Harnessing
Diversity
Biotic stress
Data managemnet
resources
Capacity Building
Abiotic stress
Genomic
resistance
resistance
Molecular
breeding

4. Activity 1
GCP
Characterization
of diversity
- extensive phenotyping
Pre-breeding population
MAGIC population
Harnessing diversity

5. Activity 1 Activity 2 Activity 3 Activity 4 Activity 5 Activity 6 Activity 7
Reference Enriched BAC end Interspecific Intraspecific
Elite lines
collection libraries sequences population population 2007
Trait mapping Trait mapping
& Ethiopia
India, Kenya
Phenotyping
GCP
MARS
SSRs
MABC
X
Phase I
DArTs
Major effect Introgression
Minor effect QTLs F3
25-50 Genetic QTLs
Genotyping
contrast lines SNPs maps BC3F2
Phenotypic data, Marker/ BAC end sequences, Marker genotyping/ QTLs,QTLs-associated markers
2010
Pre-
breed.
populations
(GCP central registry &
Data management
MAGIC
populations
Phase II
phenotyping
Subset of
ICGGC)
MAGIC lines
phenotyping
SUPERIOR LINES
Phenotypic data, Markergenotyping data 2014

6. Scheme for MAGIC populations
GCP
Parental line Remarks
ICC 4958 Drought-tolerant genotype found to be promising in Ethiopia,
Kenya and India; drought-tolerant parent of two mapping
populations
ICCV 10 Widely adapted drought-tolerant cultivar found to be
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
Cavanagh et al. 2008

7. Timeline for development of
MAGIC population GCP
Activity Timeline
Make 28 one-way crosses Oct 2009–Feb 2010
Confirm hybrids by genotyping and make 14 four-way Mar 2010–Jun 2010
crosses
Confirm hybrids by genotyping and make 7 eight-way Jun 2010–Sep 2010
crosses
Grow F1s Oct 2011–Feb 2011
Grow F2 (n=500 minimum) Mar 2011– Jun 2011
• MAGIC lines with enhanced drought tolerance
Grow F4 (SSD) Jun 2011–Sep 2011
• Fine resolution mapping population
Grow F5 (SSD) Oct 2011– Feb 2012
Grow F6 (SSD) Mar 2012–Jun 2012
Grow F7 (SSD) Jun 2012– Sep 2012
Grow F8 progenies for seed bulking Oct 2012– Feb 2013
Genotyping of selected MAGIC populations Jan 2013–June 2013
Selection of subset of MAGIC lines based on June 2013- Sept 2013
SNP genotyping/haplotype data

8. Activity 2 in TLI
Large scale markers and GCP
high-density genetic map
LG 8
Total marker loci - 1821
Inter-marker distance- 2.48cM
LG 2 LG 7
LG 1 LG 3 LG 4 LG 5 LG 6

9. High-throughput & cost-
effective marker genotyping GCP
Integrated with Molecular Breeding Platform for
facilitating molecular breeding in high-throughput and
cost-effective manner e.g.
- genotyping of MAGIC population
- genotyping of AB- population
- genotyping of MABC population
- genotyping of MARS population

10. Diagnostic markers for
accelerating molecular breeding
GCP
Marker needs to be
Polymorphic in D x R
Linkage group 5
- 0 genotypes tested
3
harboring hot spot
- 50% success
for drought related QTLs
in TL-I (Act 4)
- 4 markers
Candidate QTLs
Genetic map
CaM0856
ICCM0249
TAA170
STMS11
GA24
Physical map
Additional markers BACs

11. Activity 1 Activity 2 Activity 3 Activity 4 Activity 5 Activity 6 Activity 7
Reference Enriched BAC end Interspecific Intraspecific
Elite lines
collection libraries sequences population population 2007
Trait mapping Trait mapping
& Ethiopia
India, Kenya
Phenotyping
GCP
MARS
SSRs
MABC
X
Phase I
DArTs
Major effect Introgression
Minor effect QTLs F3
25-50 Genetic QTLs
Genotyping
contrast lines SNPs maps BC3F2
Phenotypic data, Marker/ BAC end sequences, Marker genotyping/ QTLs,QTLs-associated markers
2010
SNP resources
& genotyping
platform
Pre-
breed.
populations High density
genetic maps
(GCP central registry &
Data management
MAGIC
populations
BAC contigs
Phase II
phenotyping
sequencing
NGS
Subset of
ICGGC)
MAGIC lines
phenotyping
SSR
markers
SUPERIOR LINES
Phenotypic data, Marker/ BAC end sequences 2014

12. Phase I- Activity:
Marker discovery for insect resistance
GCP
Natural field conditions Detached leaf assay
- No natural variation in cultivated germplasm
- QTL discovery based on interspecific mapping
population didn’t yield appropriate QTLs

13. Scheme for AB-QTL approach
GCP
JG11 × PI 489777 Nov 2010 - Mar 2011
(Cultivated) (Wild)
F1 Jun 2011 - Oct 2011
JG 11 ×
BC1F1 Nov 2011 - Mar 2012
JG 11 ×
BC2F1 Jun 2012 – Oct 2012
×
BC2F2 (Genotyping) Nov 2012 - Mar 2013
QTL Analysis ×
BC2F2:3 (Phenotyping) Jun 2013 – Oct 2013
Identification and charecterisation of ABCLs Nov 2013 - Mar 2013

14. Activity 1 Activity 2 Activity 3 Activity 4 Activity 5 Activity 6 Activity 7
Reference Enriched BAC end Interspecific Intraspecific
Elite lines
collection libraries sequences population population 2007
Trait mapping Trait mapping
& Ethiopia
India, Kenya
Phenotyping
GCP
MARS
SSRs
MABC
X
Phase I
DArTs
Major effect Introgression
Minor effect QTLs F3
25-50 Genetic QTLs
Genotyping
contrast lines SNPs maps BC3F2
Phenotypic data, Marker/ BAC end sequences, Marker genotyping/ QTLs,QTLs-associated markers
2010
SNP resources JG 11 × PI 489777
& genotyping
platform
Expectations
Pre-
breed.
AB-QTL app
- Candidate markers and QTLs
populations High density
Validation
genetic maps
(GCP central registry &
of QTLs
- ABCLs with enhanced resistance to insect resistance
Data management
MAGIC
populations
BAC contigs
Phase II
- Validation of insect resistance QTL
phenotyping
sequencing
NGS
Subset of
ICGGC)
MAGIC lines
ABCLs
phenotyping
SSR
markers AB-QTLs
SUPERIOR LINES
Phenotypic data, Marker/ BAC end sequences, QTLs, QTLs-associated markers 2014

15. Phase I- Activity 4:
Large scale drought phenotyping
GCP
- 000 cylinder
2
(1.2 m long, 20 cm dm)
- Root traits
- Yield
- Harvest index
- CID
- OID
WUE/ TE phenotyping needs to be undertaken

16. Activity 1 Activity 2 Activity 3 Activity 4 Activity 5 Activity 6 Activity 7
Reference Enriched BAC end Interspecific Intraspecific
Elite lines
collection libraries sequences population population 2007
Trait mapping Trait mapping
& Ethiopia
India, Kenya
Phenotyping
GCP
MARS
SSRs
MABC
X
Phase I
DArTs
Major effect Introgression
Minor effect QTLs F3
25-50 Genetic QTLs
Genotyping
contrast lines SNPs maps BC3F2
Phenotypic data, Marker/ BAC end sequences, Marker genotyping/ QTLs,QTLs-associated markers
2010
SNP resources JG 11 × PI 489777
& genotyping
platform
Pre-
AB-QTL app
breed.
populations
WUE, TE
Phenotyping
High density
Validation
genetic maps
(GCP central registry &
of QTLs
Data management
MAGIC
populations
BAC contigs
Phase II
phenotyping
sequencing
NGS
Subset of Identification of
ICGGC)
MAGIC lines superior lines for
ABCLs
phenotyping
SSR drought tolerance
markers AB-QTLs
SUPERIOR LINES
Phenotypic data, Marker/ BAC end sequences, QTLs, QTLs-associated markers 2014

17. Expected outputs
GCP
1. TE phenotyping reference collection and
ICC 4958 × ICC 1882 mapping population
2. Metadata analysis on all set of phenotyping
data- best phenotyping methodology
3. Superior lines with enhanced drought tolerance
4. Validation of drought QTL in elite chickpea
lines

18. TL-I Activity 6
Crosses:
MABC for Donors for root traits
3 Cultivars x 2
drought tolerance GCP
Donors
BC1: Cultivar x F1
↓
BC1F1
BC 2: Cultivar x BC1F1
↓
BC2F1
Subjected to foreground and background selection
BC3: Cultivar x BC2F1 Cultivars
As in BC 2
↓
BC3F1
Selected heterozygous plants for QTL-linked markers
and over 90% genome of the recurrent parent JG 11
↓
BC3F2
2009-2010 Select homozygous plants for QTL-linked markers
↓
BC3F3
Seed multiplication Chefe KAK 2
↓
Multilocation evaluation BC3F4 lines

19. MABC for drought tolerance
by NARS partners GCP
Selection of cultivars
ICCV 97105: Farmer-preferred desi chickpea elite line identified in
Kenya and Tanzania through farmer-participatory varietal selection
(FPVS) trials conducted in phase I of TLII. This is being proposed
for release in Kenya and Tanzania.
Ejere: Farmer-preferred kabuli chickpea cultivar identified in
Ethiopia through FPVS trials conducted in phase I of TLII.
DCP-92-3: Most-popular desi chickpea cultivar in north-eastern
India
ICC 4958: Well known drought-tolerant genotype with deep and
vigorous root system. It is a drought tolerant parent of two
mapping populations for root traits. It has also performed well in
Ethiopia, Kenya and India

20. MABC by NARS partners
GCP
Egerton University, DZARC–Debre IIPR, Kanpur,
Njoro, Kenya Ziet, Ethiopia India
ICCV 97105 × ICC Ejere × ICC 4958 DCP 92-3 × ICC
4958 4958
Make cross Oct 2009–Mar Oct 2009–Mar Nov 2009 – Mar
2010 2010 2010
Grow F1 and make BC1 Apr 2010–Aug 10 Apr 2010–Aug Jun 2010–Oct
2010 2010
Grow BC1F1, foreground and Oct 2010–Mar Oct 2010–Mar Nov 2010–Mar
background selection, make BC2 2011 2011 2011
Grow BC2F1, foreground and Apr 2011–Aug 11 Apr 2011–Aug Jun 2011–Oct
background selection, make BC3 2011 2011
Grow BC3F1, foreground and Oct 2011–Mar 12 Oct 2011–Mar Nov 2011–Mar
background selection, self selected 2012 2012
plants
Grow BC3F2, select homozygous Apr 2012–Aug Apr 2012– Aug Jun 2012–Oct
plants for QTL-linked markers, 2012 2012 2012
self selected plants
Grow BC3F3 progenies for seed Oct 2012–Mar Oct 2012–Mar Nov 2012–Mar
multiplication 2013 2013 2013
Multilocation evaluation of BC3F4 Apr 2013 –Aug Apr 2013–Aug Nov 2013–Mar
progenies 2013 2013 2014

21. TL-I Activity 6
MARS for drought tolerance GCP
Five good-by-good crosses involving cultivars from Africa and Asia
Set 1 (2 crosses) Set-2 (3 crosses)
2008/09 Genotyping of F3 plants F2 grown
F4 (Seed mutiplication) Genotyping of F3 plants
Multilocation evaluation F4 (Seed mutiplication)
of F5 progenies and
QTL analysis
Selection and inter-crossing Multilocation evaluation
of F3s of F5 progenies and
2009/10 QTL analysis

22. Development of MARS
populations continued GCP
Parent 1 × Parent 2
Population development F1
F2 Single seed
descent
F3 282 F3 progenies Genotyping
F3:4 282 progenies
F3:5
QTL detection
Multilocation phenotyping
10 plants/family (A-H), 6 sets of 8 families/cross
Recombination
1 st Recombination cycle A B C D E F G H
2nd Recombination cycle F1 F1 F1 F1
3rd Recombination cycle F1 F1
F1
F2
development
F3
Population
F3:4
Multilocation phenotyping

23. Activity 1 Activity 2 Activity 3 Activity 4 Activity 5 Activity 6 Activity 7
Reference Enriched BAC end Interspecific Intraspecific
Elite lines
collection libraries sequences population population 2007
Trait mapping Trait mapping
& Ethiopia
India, Kenya
Phenotyping
GCP
MARS
SSRs
MABC
X
Phase I
DArTs
Major effect Introgression
Minor effect QTLs F3
25-50 Genetic QTLs
Genotyping
contrast lines SNPs maps BC3F2
Phenotypic data, Marker/ BAC end sequences, Marker genotyping/ QTLs,QTLs-associated markers
phenotyping
Multilocation
2010
SNP resources JG 11 × PI 489777
F5
& genotyping
(NARS)
MABC
platform
Pre-
AB-QTL app
breed.
populations
WUE, TE
Phenotyping
cycles
Recombination
High density
Validation
genetic maps
(GCP central registry &
of QTLs BC3F4 Molecular
Data management
MAGIC
populations
BAC contigs Breeding
Phase II
phenotyping
Multilocation
phenotyping
Platform
sequencing
NGS
phenotyping
Multilocation
Subset of Selection of
ICGGC)
MAGIC lines phenotyping
ABCLs
phenotyping
SSR methodology(ies) BC3F2
markers AB-QTLs
SUPERIOR LINES
Phenotypic data, Marker/ BAC end sequences, QTLs, QTLs-associated markers, Next generation breeders trained 2014

24. New Activity- trait package
GCP
Genotyping
MBP
Development Markers for
of populations Marker screening FW resistance
for root trait QTL
- Act 5.2 of and FW resistance CMBN
TL II associated markers Govt of India
(TL I)
MBP
Phenotyping

25. Activity 6: Capacity-building
GCP
Sub-activities:
Strengthening infrastructure in collaboration with
Activity 3 of Objective 5
Organization of a modern breeding workshop for TL-I &
TL-II breeders of chickpea
Six MSc and three PhD students trained in chickpea
genomics and breeding activities

26. Activity 1 Activity 2 Activity 3 Activity 4 Activity 5 Activity 6 Activity 7
Reference Enriched BAC end Interspecific Intraspecific
Elite lines
collection libraries sequences population population 2007
Trait mapping Trait mapping
& Ethiopia
India, Kenya
Phenotyping
GCP
MARS
SSRs
MABC
X
Phase I
DArTs
Major effect Introgression
Minor effect QTLs F3
25-50 Genetic QTLs
Genotyping
contrast lines SNPs maps BC3F2
Phenotypic data, Marker/ BAC end sequences, Marker genotyping/ QTLs,QTLs-associated markers
phenotyping
Multilocation
2010
SNP resources JG 11 × PI 489777
F5
& genotyping
(NARS)
MABC
platform
Pre-
AB-QTL app
Infrastructure
Strengthening
breed.
populations
WUE, TE
Phenotyping
cycles
Recombination
High density
Validation
genetic maps
(GCP central registry &
of QTLs BC3F4
Data management
MAGIC
populations
BAC contigs
Phase II
phenotyping
Multilocation
phenotyping
studentship
sequencing
NGS
phenotyping
Multilocation
PhD
Subset of Selection of
ICGGC)
MAGIC lines phenotyping
ABCLs
phenotyping
SSR methodology(ies) BC3F2
markers AB-QTLs
SUPERIOR LINES
Phenotypic data, Marker/ BAC end sequences, QTLs, QTLs-associated markers, Next generation breeders trained 2014

27. Data storage & Management
GCP
 Molecular Breeding Platform
 Databases within ICRISAT (Activity 2/ Obj 5)
 GCP Central Registry
 International Chickpea Genetics & Genomics
Consortium (ICGGC)

28. Activity 1 Activity 2 Activity 3 Activity 4 Activity 5 Activity 6 Activity 7
Reference Enriched BAC end Interspecific Intraspecific
Elite lines
collection libraries sequences population population 2007
Trait mapping Trait mapping
& Ethiopia
India, Kenya
Phenotyping
GCP
MARS
SSRs
MABC
X
Phase I
DArTs
Major effect Introgression
Minor effect QTLs F3
25-50 Genetic QTLs
Genotyping
contrast lines SNPs maps BC3F2
Phenotypic data, Marker/ BAC end sequences, Marker genotyping/ QTLs,QTLs-associated markers
phenotyping
Multilocation
2010
SNP resources JG 11 × PI 489777
F5
& genotyping
(NARS)
MABC
platform
Pre-
AB-QTL app
Infrastructure
Strengthening
breed.
populations
WUE, TE
Phenotyping
cycles
Recombination
High density
Validation
genetic maps
(GCP central registry &
of QTLs BC3F4
Data management
MAGIC
populations
BAC contigs
Phase II
phenotyping
Multilocation
phenotyping
studentship
sequencing
NGS
phenotyping
Multilocation
PhD
Subset of Selection of
ICGGC)
MAGIC lines phenotyping
ABCLs
phenotyping
SSR methodology(ies) BC3F2
markers AB-QTLs
SUPERIOR LINES
Phenotypic data, Marker/ BAC end sequences, QTLs, QTLs-associated markers, Next generation breeders trained 2014

29. In summary…
GCP
 Phase II proposal is based on achievements of
Phase I of TLI and requirements of TL II and
heavily interacted with Mol Breed Platform
 All activities are downstream application oriented
 Flexible- open for more suggestions from TL II
 Diagnostic markers for drought and insect res.
 Superior lines (MAGIC, ABCLs, MABCs, MARS)
for drought tolerance and insect resistance
 Mol breeding being undertaken by NARS

30. GCP