Chickpea is an important food legume crop, for the semi-arid regions including Indian subcontinent. Its productivity is adversely affected by various biotic and abiotic stresses. 7 Jan 2013

1. Financial support from Department of Biotechnology, Government of India and CGIAR Generation Challenge Programme (GCP) is
gratefully acknowledged.
Manish Roorkiwal1,2, Spurthi Nayak1,3, Mahendar Thudi1, Hari Upadhyaya1,
Rajeev Varshney1,4,*, Prakash Sharma2
1International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Hyderabad, India;
2Guru Gobind Singh Indraprastha University, New Delhi, India; 3University of Florida, Florida, USA;
4CGIAR Generation Challenge Programme, c/o CIMMYT, Mexico DF, Mexico;
*Address for correspondence: r.k.varshney@cgiar.org
Chickpea is an important food legume crop, for the semi-arid regions including Indian subcontinent. Its productivity is
adversely affected by various biotic and abiotic stresses. Identification of candidate genes that are associated with
abiotic stress response will help breeding efforts aiming to enhance its productivity. With this objective, eleven abiotic stress
responsive candidate genes were selected on the basis of prior knowledge acquired from mutational, biochemical and linkage analysis
of the traits. These 11 genes were subjected to allele specific sequencing across chickpea reference set comprising of 300 genotypes
including 211 mini-core collection genotypes. In total, 1.3Mbp sequence data was generated. Multiple sequence alignment revealed 79
SNPs and 41 indels in ten candidate genes while, CAP2 gene was found to be conserved across all genotypes. Among ten candidate
genes, maximum number of SNPs (34) were observed in ASR gene, amongst which 22 transitions, 11 transversions and one tri-allelic
SNP. Nucleotide diversity varied from 0.0004 to 0.0029, while PIC values ranged from 0.01 (AKIN gene) to 0.43 (CAP2 promoter).
Association analysis using these candidate genes identified 6 SNPs in ASR, 3 SNPs in Dehydrin and 3 SNPs in DREB associated with
different traits like 100-seed weight, delta carbon, plant height, root surface area, root dry weight, pods per plant and yield. Identified
marker trait association after further validation may be useful for enhancing drought tolerance in chickpea through molecular breeding.
Abstract
Gene sequence alignment to identify SNPsBackground
ICC1882
ICC2210
ICC2990
ICC6263
ICC6811
ICC7184
ICC8195
ICC8261
ICC8621
ICC9848
ICC11378
ICC12328
ICC12537
ICC13816
ICC14402
Transition (C-T)
‘ATG’ deletion
b.
ICC9848 TATGGGACCCACAATACAC---GTGG
ICC11378 TATGGGACCTACAATACACATGGTGGICC9848
ICC11378
a.
GURU GOBIND SINGH
INDRAPRASTHA UNIVERSITY
Allelic diversity and association analysis for candidate abiotic
stress responsive genes with drought tolerance in chickpea
 Chickpea (Cicer arietinum L. 2n=16) is an important food legume
crop, which ranks third worldwide as a food legume crop
 India is the largest producer as well as largest importer of
chickpea; Yield and productivity are adversely affected by various
biotic and abiotic stresses
 Drought alone causes 40-50% reduction in the chickpea yield
globally
 A holistic approach, combining genomics with breeding and
physiology, termed as genomics-assisted breeding (Varshney et al.
2005 Trends Plant Sci 10:621-630) should be used
 Identification of abiotic stress responsive genes and determination
of genetic diversity among the mini core collection using SNPs
identified in these genes will lead to identification of superior
alleles of the gene present in chickpea germplasm
Sequence quality check using
DNA Baser V3.01 to compare the
SNP (C/T) and deletion of ATG
Candidate gene AKIN AMADH ASR CAP2 CAP2
promoter
DHN DREB ERECTA
_7
ERECTA
_8
Myb SPS
Genotypes with
successful sequences
208 209 193 227 137 198 191 79 147 200 236
Sequence length (bp) 772 932 621 367 629 381 776 921 1189 335 312
No. of Indels 2 3 2 0 0 7 23 1 0 2 1
Indel frequency 1/386.0 1/310.67 1/310.6 0 0 1/54.4 1/33.7 1/921.0 0 1/167.5 1/312.0
No. of SNPs 2 13 34* 0 1 7 14 13 20 6 3
Transition 2 6 22 0 0 5 8 9 10 1 2
Transversion 0 7 13 0 1 2 6 4 10 5 1
SNP frequency 1/386.0 1/71.7 1/18.3 0 1/629.0 1/54.4 1/55.4 1/70.9 1/69.5 1/55.8 1/104.0
Nucleotide Diversity
(Pi)
0.0004 0.002 0.0014 0 0 0.0022 0.0011 0.0029 0.0029 0.002 0.0011
Average PIC of SNP 0.01 0.04 0.1 0 0.43 0.17 0.14 0.27 0.1 0.04 0.01
No. of Haplotypes 3 9 4 1 2 6 33 4 3 6 4
Haplotype Diversity 0.019 0.326 0.833 0 0.438 0.426 0.879 0.372 0.324 0.256 0.034
PIC of Haplotypes 0.019 0.324 0.829 0 0.436 0.424 0.874 0.367 0.322 0.255 0.034
Sequence diversity analysis for biotic stress responsive genes
Methodology
Gene
sequences
EST
sequences
SNP
Identification
Allele diversity
analysis
Primer design
PCR
Amplification
Sequencing of
purified PCR products
Contigs showing
match with target
gene
Contigs
Confirmation
of gene
Chickpea
Reference set/
mini core
Gene
Amplification
Amplicons
sequencing
Gene
Amplification
Sequence similarity
based identification
and confirmation of
candidate genes in
chickpea
SNP identification
and allele
diversity analysis
in chickpea mini
core collection
Asia
M East
E Africa
Euro Asia
Europe
N Africa
N America
NE Africa
NW Africa
S America
SE Africa
W Africa
Unknown
Haplotype network of SPS gene Salient features
1. Eleven abiotic stress responsive candidate genes were
identified in chickpea based on sequence similarity
approach.
2. SNPs were identified for these eleven genes across 300
(211) accessions of chickpea reference set &/or mini
core collection
3. A total of 79 SNPs and 41 indels were identified in ten
candidate genes while, CAP2 gene was found to be
conserved across all genotypes
4. Diversity analysis reveals that ASR gene was most
variable with 34 SNPs amongst which 22 transitions, 11
transversions and one tri-allelic
5. PIC values ranged from 0.01 (AKIN gene) to 0.43 (CAP2
promoter)
Acknowledgements