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Development of chickpea EST-SSR markers and analysis of allelic variation across related species

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Abstract

Despite chickpea being the third important grain legume, there is a limited availability of genomic resources, especially of the expressed sequence tag (EST)-based markers. In this study, we generated 822 chickpea ESTs from immature seeds as well as exploited 1,309 ESTs from the chickpea database, thus utilizing a total of 2,131 EST sequences for development of functional EST-SSR markers. Two hundred and forty-six simple sequence repeat (SSR) motifs were identified from which 183 primer pairs were designed and 60 validated as functional markers. Genetic diversity analysis across 30 chickpea accessions revealed ten markers to be polymorphic producing a total of 29 alleles and an observed heterozygosity average of 0.16 thereby exhibiting low levels of intra-specific polymorphism. However, the markers exhibited high cross-species transferability ranging from 68.3 to 96.6% across the six annual Cicer species and from 29.4 to 61.7% across the seven legume genera. Sequence analysis of size variant amplicons from various species revealed that size polymorphism was due to multiple events such as copy number variation, point mutations and insertions/deletions in the microsatellite repeat as well as in the flanking regions. Interestingly, a wide prevalence of crossability-group-specific sequence variations were observed among Cicer species that were phylogenetically informative. The neighbor joining dendrogram clearly separated the chickpea cultivars from the wild Cicer and validated the proximity of C. judaicum with C. pinnatifidum. Hence, this study for the first time provides an insight into the distribution of SSRs in the chickpea transcribed regions and also demonstrates the development and utilization of genic-SSRs. In addition to proving their suitability for genetic diversity analysis, their high rates of transferability also proved their potential for comparative genomic studies and for following gene introgressions and evolution in wild species, which constitute the valuable secondary genepool in chickpea.

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Acknowledgments

We are grateful to the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) India, National Bureau for Plant Genetic Resources (NBPGR) India, Maharana Pratap Agriculture University (MPAU) India, and Australian Medicago Genetic Resource Centre, SARDI, Australia for providing us the seed material of different accessions of cultivated chickpea, wild Cicer species and other legumes. Financial assistance for this work was provided by National Institute for Plant Genome Research (NIPGR), New Delhi, India and also the Department of Biotechnology (DBT), Government of India by means of a project grant. The fellowship provided to SC by University Grants Commission (UGC), India and to NK and BS by Council for Scientific and Research (CSIR), India is gratefully acknowledged.

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  1. Niroj Kumar Sethy

    Present address: Defence Institute of Physiology and Allied Sciences (DIPAS), Defence Research and Development Organization (DRDO), Timarpur, Delhi, 110 054, India

Authors and Affiliations

  1. National Institute of Plant Genome Research, Post Box Number 10531, Aruna Asaf Ali Marg, Jawaharlal Nehru University Campus, New Delhi, 110067, India

    Shalu Choudhary, Bhumika Shokeen & Sabhyata Bhatia

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  1. Shalu Choudhary
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  2. Niroj Kumar Sethy
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Correspondence to Sabhyata Bhatia.

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Choudhary, S., Sethy, N.K., Shokeen, B. et al. Development of chickpea EST-SSR markers and analysis of allelic variation across related species. Theor Appl Genet 118, 591–608 (2009). https://doi.org/10.1007/s00122-008-0923-z

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