Introduction
Chickpea (Cicer arietinum L.) is a self-pollinated cool-season grain legume, cultivated on 13.2 million ha globally with a total production of 11.6 million metric tonnes (FAOSTAT, 2012). Chickpea is an important source of protein in the diets of the poor in the semi-arid tropics and West Asia and North Africa regions and is particularly important in vegetarian diets. Vast collections of chickpea germplasm are maintained at the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), India, and the International Center for Agricultural Research in the Dry Areas (ICARDA), Syria. The former contains 17,258 accessions (135 wild and 17,123 cultivated), while the latter houses 12,647 accessions (304 wild and 12,343 cultivated).
With the availability of large numbers of genomic simple sequence repeats (SSRs) in chickpea, it is now possible to conduct extensive molecular diversity studies in chickpea to identify diverse germplasm with beneficial traits for use in crop improvement programmes. Although a large number of SSR markers have been developed in recent years, inter-laboratory variations have made comparisons of results difficult. In the framework of the CGIAR Generation Challenge Program, the ICRISAT in India and the ICARDA in Syria assessed the genetic diversity of 3,000 chickpea accessions using 35 SSR markers distributed throughout the genome. Based on genotyping data obtained with the 35 SSR markers, a reference genotyping kit was developed to allow the international community to better integrate other genetic diversity studies.
Materials and methods
For genotyping, 35 SSR markers were chosen from published and unpublished data from other colleagues (Hüttel et al., Reference Hüttel, Winter, Weising, Choumane, Weigand and Kahl1999; Winter et al., Reference Winter, Pfaff, Udupa, Hüttel, Sharma, Sahi, Arrequin-Espinoza, Weigand, Muehlbauer and Kahl1999; Sethy et al., Reference Sethy, Shokeen, Edwards and Bhatia2006; Table S1, available online). The markers were selected based on quality criteria, genome coverage and polymorphism information content. Furthermore, the 35 selected markers are distributed across all the eight linkage groups, with a range of two to six markers per linkage group. Polymerase chain reaction (PCR) was carried out in a 5 μl volume containing 10 ng of DNA, 1 × buffer, 200 μM of dNTP, 2.5 mM MgCl2, 1–5 pmol of forward (fluorescence dye labelled) and reverse primers, and 0.2 U of Taq DNA polymerase. PCR was carried out using Perkin Elmer 384-well Thermal Cyclers (Applied Biosystems, Foster City, CA, USA) using a touchdown PCR (65–60, 60–55 and 55–45°C) at corresponding annealing temperatures (Table S1, available online). The amplified DNA fragments were separated and detected using an ABI 3700 sequencer (Applied Biosystems, Foster City, CA, USA). Allele size data were base-called using the Genotyper version 3.1 software (Applied Biosystems, Foster City, CA, USA) and were subsequently analysed using an in-house-constructed program ‘AlleloBin’ and alleles were binned as per the corresponding SSR length.
Results and discussion
A set of nine chickpea accessions representing the largest genetic diversity was chosen based on phenotyping data. After analysing the allele sizes for these genotypes, three pools comprising three genotypes each were created (C1: ICC 14 446, ICC 15 996, and ICC 15 994; C2: ICC 11 265, ICC 6537, and ICCV 2; and C3: Annigeri, ICC 13 454, and ICC 4366). For each marker, the nine genotypes and three mix controls were amplified and analysed on an ABI 3700 genetic analyser (Applied Biosystems, Foster City, CA, USA). Allele sizes obtained in control pools are given in Table 1. The allelic patterns of control samples for all the 35 markers can be found at http://www.icrisat.org/gt-bt/chickpea/varshney-2013. Distinct alleles amplified in an individual genotype with each marker as well as control pools are given with the called allele size in Table S2 (available online).
Table 1 Allele sizes obtained for each simple sequence repeat marker of the kit for reference accessions
a C1: ICC 14 446, ICC 15 996, and ICC 15 994; C2: ICC 11 265, ICC 6537, and ICCV 2; C3: Annigeri, ICC 13 454, and ICC 4366.
The developed SSR marker kit of chickpea is recommended for use in assessing the diversity in chickpea germplasm. Using the kit, other germplasm collections can be more accurately compared with that of the global composite, reference and mini core collections.
Supplementary material
To view supplementary material for this article, please visit http://dx.doi.org/10.1017/S1479262114000392
Acknowledgements
This work was carried out under the CGIAR Generation Challenge Program and was undertaken as part of the CGIAR Research Program on Grain Legumes. ICRISAT and ICARDA are members of the CGIAR Consortium.
