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

SMDSusceptible 0

20

40

60

80

100

120

Dis

ea

se

sc

ore

Parents and selected susceptible PRILs

SMD FW

0

5

10

15

20

25

30

35

40

45

Dis

ea

se

sc

ore

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 1.75x 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