Analysis of Frequency and Functional Annotation of SSRs in ESTs of Cole Crops

Aims: In silico analysis of publicly available set of expressed sequence tagged sites of Brassica oleracea (B. oleracea var. botrytis, B. oleracea var. capitata, B. oleracea var. italica and B. oleracea var. gemmifera) was carried out so as to remove redundancy. The assembled sequences were then used to detect SSR motifs present among the EST sequences.

Study Design: Detection and annotation of SSRs in EST sequences.

Place and Duration of Study: Department of Biotechnology, Dr Y. S. Parmar University of Horticulture and Forestry, Nauni, Solan-173230, Himachal Pradesh, India, between July 2009 and August 2010.

Methodology: EST sequences were assembled into a non-redundant set using EGassembler program. The assembled sequences were analysed for SSR motifs. The detected EST-SSRs were then subjected to sequence identification and annotation.

Results: After sequence assembly, 34.12% reduction of data redundancy was obtained. 107 SSRs were identified using SSRIT and they were analysed for their distribution in terms of frequency, density, composition and distribution. Maximum frequency was exhibited by dinucleotide SSR motifs i.e. 68.22%. The detected EST-SSRs were annotated for their putative function using BLASTx program of NCBI. 82% SSR-ESTs were found to show a significant match with protein databases. The 100 SSR-ESTs were also analysed for significant functional domains using PROSITE and 83% sequences were found to code for functional domains. Functional domain markers (FDM) predict the functional property of markers having predicted protein domains Primer sequences were designed for some of these EST – SSRs by making use of PRIMER3 software and they were validated for their usefulness on a set of 20 genotypes of Brassica oleracea var. botrytis. The primers generated high level of genetic polymorphism which could be used for genetic characterization of the genotypes used.

Conclusion: Thus it can be concluded that these detected SSRs can be used to design primers that have functional role and will also facilitate studies on genetic diversity, variability, genome analysis and evolutionary relationships among these cole crops of family Brassicaceae.