An SSR kit to study genetic diversity in chickpea (Cicer arietinum L.)
Team involved: ICRISAT: Rajeev Varshney, Dave Hoisington, Hari Upadhyaya, SL Dwivedi, Jayashree Balaji , Subash Chandra ICARDA: Sripada Udupa, Bonnie Furman, Wafa Coumane, Michael Baum For more information, please contact Rajeev Varshney / Dave Hoisington

Background information

In the frame of the Generation Challenge Program, an assessment of the overall genetic diversity of 3000 chickpea accessions was assessed through the use of SSR markers, distributed throughout the genome. This task was shared between two institutes: ICRISAT and ICARDA, Aleppo, Syria. The genetic diversity study provides an overview on population structure as well as the allelic diversity that can be associated with the phenotypic trait. In addition, based on genotyping data obtained with 35 SSR markers, an effort has been made to provide the international community with a genotyping kit that could be used as a reference kit to compare with other genetic diversity studies in the future.

SSR Marker Kit

A set of 9 chickpea accessions (Table 1) representing the largest genetic diversity was chosen based on phenotyping data. After analysing the genotyping data for these genotypes in term of allelic sizes, these genotypes were pooled into 3 groups, each group comprising three genotypes (C1, C2 and C3). It is proposed to compare the allelic composition of these controls (genotype pools) with other genetic diversity studies.

Methodology

For genotyping the composite collection of 3,000 chickpea genotypes (Upadhyaya et al. 2006), SSR markers were chosen from published and unpublished data from other colleagues (Table 2, Hüttel et al. 1999, Winter et al. 1999, Sethy et al. 2003). PCR for SSR markers was carried out in a total reaction volume of 5 µl containing 10 ng of DNA; 1X buffer; 200µM dNTP; 2.5 mM MgCl2; 1- 5 pmols forward (fluorescence dye labelled) and reverse primers, 0.2 U of Taq polymerase. PCR were performed with an Perkin Elmer Thermalcycler 384-wells. PCR cycling involved touch down PCR (65°C -60°C, 60°C -55°C, 55°C -45°C) at corresponding annealing temperature (Table 2, Buhariwalla et al. 2005). The amplified DNA fragments were separated and detected with fluorescence detection on the ABI 3700 sequencer (see Buhariwalla et al. 2005). Allele size data was based called using the Genotyper version 3.1 software (ABI) and subsequently the data was analysed using in-house constructed programme “AlleloBin” and alleles were binned as per the corresponding (expected) SSR length.

Features

Details on the primer pairs used are given in Table 2. Allele size obtained by diagnostics alleles observed in the reference accessions as well as in control pools are provided in Table 3. Allelic patterns of control samples (Table 1) are provided for each marker. For each marker, the 9 genotypes and the 3 mix controls were amplified and analyzed on ABI 3700 genetic analyzer. Distinct alleles amplified in an individual genotype with each of the markers are provided with the called allele size.

Acknowledgements

We acknowledge the support of K Eshwar, Seetha Kannan, VP Prasanth in generating and analysing the genotyping data. The study was performed under the Generation Challenge Program SP1 Chickpea genotyping project and SP5 Marker kit project.

References

Buhariwalla HK, et al. (2005). BMC Plant Biology 5: 16
Hüttel B, et al. (1999) Genome 42: 210- 217
Sethy NK et al. (2003). Mol. Ecol. Notes 3:428- 430.
Upadhyaya HD et al. (2006). Plant Genet. Resou. 4: 13-19
Winter P et al. (1999). Mol. Gen. Genet. 262: 90-101