Identification of polymorphic, conserved simple sequence repeats (SSRs) in cultivated Brassica species.

The application of simple sequence repeat (SSR) genotyping for the characterization of genetic variation in crop plants has been hindered by ready access to useful primer pairs and potentially limited conservation of the repeat sequences among related species. In this phase of work, we report on the identification and characterization of SSRs that are conserved in Brassica napus L. (rapeseed) and its putative progenitors, B. oleracea L. (cabbage, and related vegetable types) and B. rapa (vegetable and oil types). Approximately 140 clones from a size-fractionated genomic library of B. napus were sequenced, and primer pairs were designed for 21 dinucleotide SSRs. Seventeen primer pairs amplified products in the three species and, among these, 13 detected variation between and within species. Unlike findings on SSR information content in human, no relationship could be established between the number of tandem repeats within the target sequence and heterozygosity. All primer pairs have been designed to work under identical amplification conditions; therefore, single-reaction, multiplex polymerase chain reaction (PCR) with these SSRs is possible. Once moderate numbers of primer pairs are accessible to the user community, SSR genotyping may provide a useful method for the characterization, conservation, and utilization of agricultural crop diversity.

Abundance and characterization of simple-sequence repeats (SSRs) isolated from a size-fractionated genomic library of Brassica napus L. (rapeseed).

A size-fractionated library of Brassica napus L. (rapeseed), composed of 15000 clones, was screened for the presence of GA-, CA-, and GATA-simple-sequence repeats (SSRs). GA-SSRs were four- and five-fold more abundant than CA- and GATA-SSRs, respectively, and present at a frequency of approximately one SSR for every 100 kb of DNA. Following the sequencing of 124 positive clones, primer pairs were designed and evaluated for seven selected SSRs. Products were amplified in an array of individuals of B. napus, B. oleracea and B. rapa, demonstrating that the seven SSRs were conserved among species. Two SSRs were polymorphic. Among 11 accessions, the dinucleotide (GA)-repeat, B.n.9A, yielded 12 fragments, while the tetranucleotide-repeat (GATA), B.n.6A2, revealed two fragments. Automated, fluorescence-based detection of polyacrylamide gels has been employed to simultaneously increase throughput, reduce unit cost, improve analytical resolution, and expedite data acquisition of SSR analysis. Though initial financial investment and technical capabilities may prevent some from directly employing our documented approach, SSR analysis warrants further investigation as a tool in genetic studies for enhancing both the conservation and utilization of genetic resources.