The 96 plex and 48plex SNPs assays could successfully characterize reference sets of chickpea and pigeonpea respectively.

These SNPs can easily be employed in molecular breeding programmes where only few markers are required for

genotyping of selected set of genotypes.

BeadXpress Reader at Genotyping Services

Laboratory, ICRISAT

Roorkiwal M1, Sawargaonkar SL1, Thudi M1, Saxena RK1, Upadhyaya HD1,

Varshney RK1,2,*

Single nucleotide polymorphisms (SNPs) have become an ideal marker system due to their greater abundance in the

plant genomes. In cases like marker-assisted selection (MAS) where only few markers are required for genotyping a set of potential

lines, a cost-effective SNP genotyping platform is required. In this scenario GoldenGate assays based on VeraCode technology

using Illumina BeadXpress platform seems to be a cost-effective genotyping method. In the case of chickpea and pigeonpea,

>10,000 SNPs have been identified using next-generation sequencing (NGS) technologies in earlier studies. Based on high

polymorphism information content and assay designing tool (ADT) scores, two sets of SNPs one each for chickpea (96 SNPs) and

pigeonpea (48 SNPs) were designed for custom Oligo Pool Assays (OPAs) for Illumina BeadXpress genotyping platform. These

OPAs were used to genotype reference sets (ca. 300 genotypes each) of both the legumes. All SNPs were successfully scored in

the reference set, with less than 1% missing data in case of chickpea and around 1-2% missing data in pigeonpea. The mean

polymorphism information content (PIC) of SNP markers in case of chickpea was 0.31 and 0.32 in pigeonpea. No unique pattern of

grouping in chickpea reference set was observed based on neighbour joining tree constructed. However, in the case of pigeonpea

two major groups are observed with SNPs data. Our results indicate that Illumina BeadXpress based system provides a cost

effective SNP genotyping method for selected set of potential SNP markers and can easily be used for diversity studies.

Financial support from Department of Biotechnology, Government of India and CGIAR Generation Challenge Programme (GCP) is

gratefully acknowledged.

Abstract

Acknowledgements

Summary

Highly polymorphic SNPs

Utility of designed SNP plex in breeding applications

Mapping population Population Traits No. of SNPs

Chickpea (96-plex)

ICC 283 × ICC 8261 RIL Root traits 57

ICC 3137 × ICC 72933 RIL Helicoverpa resistance 57

ICC 4958 × ICC 1882 RIL Root traits 56

DCP 92-3 × ICC 4958 MABC Drought tolerance 48

ICC 6362 × ICC 1431 RIL Salinity tolerance 44

Pigeonpea (48-plex)

ICPL 20096 × ICPL 332 RIL Fusarium wilt 28

ICP 8863 × ICC 20097 RIL Sterility mosaic disease 14

Designed 96 and 48 OPAs for chickpea and pigeonpea respectively are being

used for diversity analysis, trait mapping and marker-assisted selection

SNP genotyping using Illumina BeadXpress for

germplasm diversity studies in chickpea and

pigeonpea

0

5

10

15

20

25

30

35

40

45

No

. o

f S

NP

s

PIC value range

0

5

10

15

20

25

30

35

40

45

No

. o

f S

NP

s

PIC value range

Chickpea-96 SNPs Pigeonpea-48 SNPs

SNP SSR

SNP SSR

Genetic structure analysis of chickpea reference set

Genetic structure analysis of pigeonpea reference set

Allele calling using GenomeStudio

Data Analysis Software

SNP genotyping using BeadXpress Reader

• 96-plex (chickpea) and 48-plex (pigeonpea) OPAs for SNP

assays was designed based on polymorphism among the

cultivated chickpea and pigeonpea genotypes and high

ADT scores

• 288 and 256 genotypes of chickpea and pigeonpea

reference set respectively were genotyped using the OPAs

• All 96 and 48 SNPs gave clear genotyping and are highly

polymorphic across their respective reference set

• The success rate of genotyping is high 98% in chickpea

and 90% in pigeonpea

• SNPs in both species could effectively characterize the

reference sets compared to SSR markers

1International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Hyderabad, India; 2Generation Challenge Programme (GCP), c/o CIMMYT, Mexico DF, Mexico;

*Address for correspondence: r.k.varshney@cgiar.org

B: Breeding line

L: Landrace

W: Wild relative