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Open Access Research article

Analysis of BAC-end sequences (BESs) and development of BES-SSR markers for genetic mapping and hybrid purity assessment in pigeonpea (Cajanus spp.)

Abhishek Bohra12, Anuja Dubey13, Rachit K Saxena12, R Varma Penmetsa4, KN Poornima15, Naresh Kumar16, Andrew D Farmer7, Gudipati Srivani1, Hari D Upadhyaya1, Ragini Gothalwal3, S Ramesh5, Dhiraj Singh6, Kulbhushan Saxena1, PB Kavi Kishor2, Nagendra K Singh8, Christopher D Town9, Gregory D May7, Douglas R Cook4* and Rajeev K Varshney110*

Author affiliations

1 International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru, Hyderabad, 502324, India

2 Department of Genetics, Osmania University, Hyderabad 500007, India

3 Department of Biotechnology and Bioinformatics Centre, Barkatullah University, Bhopal 462026, India

4 Department of Plant Pathology, University of California, Davis, CA 95616, USA

5 Department of Biotechnology, University of Agricultural Sciences (UAS), Bangalore 560065, India

6 Department of Plant Breeding and Genetics, CCS Haryana Agricultural University (CCSHAU), Hisar 125004, India

7 National Center for Genome Resources (NCGR), Santa Fe, N M 87505, USA

8 National Research Center on Plant Biotechnology (NRCPB), New Delhi 110012, India

9 J. Craig Venter Institute (JCVI), Rockville, MD 20850, USA

10 Generation Challenge Programme (GCP), c/o CIMMYT, 06600 Mexico DF, Mexico

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Citation and License

BMC Plant Biology 2011, 11:56  doi:10.1186/1471-2229-11-56

Published: 29 March 2011

Abstract

Background

Pigeonpea [Cajanus cajan (L.) Millsp.] is an important legume crop of rainfed agriculture. Despite of concerted research efforts directed to pigeonpea improvement, stagnated productivity of pigeonpea during last several decades may be accounted to prevalence of various biotic and abiotic constraints and the situation is exacerbated by availability of inadequate genomic resources to undertake any molecular breeding programme for accelerated crop improvement. With the objective of enhancing genomic resources for pigeonpea, this study reports for the first time, large scale development of SSR markers from BAC-end sequences and their subsequent use for genetic mapping and hybridity testing in pigeonpea.

Results

A set of 88,860 BAC (bacterial artificial chromosome)-end sequences (BESs) were generated after constructing two BAC libraries by using HindIII (34,560 clones) and BamHI (34,560 clones) restriction enzymes. Clustering based on sequence identity of BESs yielded a set of >52K non-redundant sequences, comprising 35 Mbp or >4% of the pigeonpea genome. These sequences were analyzed to develop annotation lists and subdivide the BESs into genome fractions (e.g., genes, retroelements, transpons and non-annotated sequences). Parallel analysis of BESs for microsatellites or simple sequence repeats (SSRs) identified 18,149 SSRs, from which a set of 6,212 SSRs were selected for further analysis. A total of 3,072 novel SSR primer pairs were synthesized and tested for length polymorphism on a set of 22 parental genotypes of 13 mapping populations segregating for traits of interest. In total, we identified 842 polymorphic SSR markers that will have utility in pigeonpea improvement. Based on these markers, the first SSR-based genetic map comprising of 239 loci was developed for this previously uncharacterized genome. Utility of developed SSR markers was also demonstrated by identifying a set of 42 markers each for two hybrids (ICPH 2671 and ICPH 2438) for genetic purity assessment in commercial hybrid breeding programme.

Conclusion

In summary, while BAC libraries and BESs should be useful for genomics studies, BES-SSR markers, and the genetic map should be very useful for linking the genetic map with a future physical map as well as for molecular breeding in pigeonpea.