This document summarizes a study that used a combination of whole genome sequencing and bulk segregant analysis to identify candidate genes for fusarium wilt and sterility mosaic disease resistance in pigeonpea. Researchers developed resistant and susceptible bulks from a mapping population and sequenced these along with a resistant parent. They identified over 35,000 SNPs between the bulks and found four candidate genes on two chromosomes associated with initiating defense mechanisms against fungal and viral diseases. This approach combined fast genetic mapping with genome sequencing to efficiently identify targeted genomic regions controlling important traits.

1. Inclusive Market-Oriented Development (IMOD) –
our approach to bringing prosperity in the drylands.
ICRISAT is a member of the CGIAR Consortium.
QTL-Seq: WGS combined with BSA
Fast forward genetic mapping combined with whole genome sequencing (WGS) and bulked segregant analysis (BSA)
approach was used to identify the candidate genes for Fusarium wilt (FW) and sterility mosaic disease (SMD) resistance in pigeonepa. To
map the targeted genomic regions, F7 RILs developed by crossing ICPL 20096 (R) × ICP 332 (S) and segregating for FW and SMD
resistance were phenotyped at two different locations in India. Based on the phenotyping, 16 RILs in each category were selected for
development of resistant (R-Bulk) and susceptible bulks (S-Bulk). These two bulks along with resistant parent (ICPL 20096) were re-
sequenced and generated ~19GB of 250 bp pair-end data with ~15× genome coverage. WGS data generated from R- and S- Bulks were
aligned with resistant parent. As a result a total of 35,877 SNPs with SNP index of ≥3 were identified. Out of 35,877 SNPs only 4,139 (11.54%)
SNPs were found homozygous. Based on the SNP index (0 for R-Bulk and 1 for S-Bulk) and SNP substitution effect three significant SNPs
including two on CcLG07 and one on CcLG11 affects a total of four candidate genes. Functional annotation of these four genes indicated
their role to initiate defence mechanism against fungal and viral diseases.
Abstract
Phenotyping of mapping population
Vikas K. Singh1, Aamir W. Khan1, Hiroki Takagi2, Rachit K. Saxena1, Vinay Kumar1, Mamta Sharma1, C.V. Sameer Kumar1,
Pallavi Sinha1, Annapurna Chitikineni1, Suyash Patil1, Anuradha Ghanta3, K. N. Yamini3, Swathi Parupalli1, S. Muniswamy4,
P. S. Dharmaraj4, Ryohei Terauchi2, Rajeev K. Varshney1,*
1International Crop Research Institute for the Semi-Arid Tropics (ICRISAT), Hyderabad, India; 2Iwate Biotechnology Research Center, Kitakami, Iwate, Japan;
3Agricultural Research Station (ARS)-Tandur, Acharya N G Ranga Agricultural University (ANGRAU), Hyderabad, India; 4Agricultural Research Station (ARS)-
Gulbarga, University of Agricultural Sciences (UAS), Raichur, Karnataka, India
*Address for correspondence: r.k.varshney@cgiar.org
Acknowledgements
Fast forward genetic mapping provides candidate genes
for resistance to fusarium wilt and sterility mosaic disease
in pigeonpea
Summary
SMD
Susceptible 0
20
40
60
80
100
120
Diseasescore
Parents and selected susceptible PRILs
SMD FW
0
5
10
15
20
25
30
35
40
45
Diseasescore
Parents and selected resistant PRILs
SMD FW
(b)
-1
-0.8
-0.6
-0.4
-0.2
0
0.2
0.4
0.6
0.8
1
1.55 1.6 1.65 1.7 1.75
x 10000000
Delta SNP U95 L95 U99 L99
Genotypes Number of
reads
Number of pair
read mapped
Coverage
at 1×
x
Coverage
Average
depth
ICPL 20096 19069648 1585188 88.98 15.29 14.44
R-Bulk (RB) 18140041 1779932 88.33 15.85 12.30
S-Bulk (RB) 16958742 1338885 89.15 12.88 14.76
Linkage
group
Position
(bp)
R- parent
base
R- bulk
base
Read
depth
SNP
Index
S- bulk
base
Read
depth
SNP
Index
CcLG02 26551810 T T 7 0 A 7 1
CcLG07 16064896 G G 8 0 C 8 1
CcLG07 18411642 G G 11 0 A 9 1
CcLG08 354473 G G 10 0 C 7 1
CcLG10 7815091 G G 9 0 A 7 1
CcLG11 19958148 A A 9 0 C 10 1
CcLG11 34310320 C C 7 0 A 7 1
SNPs identified between resistant and susceptible bulks
Projections of the null distribution
of delta SNP index for respective
depths
Calculating the delta SNP index
Simulating the SNP index for the
number of individuals in the bulks
Excluding the positions if reads
aligned are < 7 and SNP index in both
the samples < 0.3
Calculating the SNP index with
respect to resistant parent
Calling of the SNPs through GATK
Aligning the RP, RB and SB to the
reference
Financial support from United States Agency for International
Development (USAID) is gratefully acknowledged. This work has been
undertaken as part of the CGIAR Research Program on Grain
Legumes.
Sequencing of parents and bulks
Delta SNP index plot of CcLG07
Gene function
Pigeonpea Genome
This is the one of the successful example of application of pigeonpea genome
sequence information for identification of targeted genomic regions for FW and SMD
resistance combined with whole genome sequencing (WGS) with bulk segregant
analysis (BSA). This is fast forward genetic mapping approach for identification of
candidate genomic regions for target traits in comparison to the classical method of
QTL mapping, which is time consuming and labor intensive process.
SNP index between R- and S- Bulk