Microsatellites in Brassica unigenes: relative abundance, marker design, and use in comparative physical mapping and genome analysis.

Microsatellites present in the transcribed regions of the genome have the potential to reveal functional diversity. Unigene sequence databases are the sources of such genic microsatellites with unique flanking sequences and genomic locations even in complex polyploids. The present study was designed to assay the unigenes of Brassica napus and B. rapa for various microsatellite repeats, and to design markers and use them in comparative genome analysis and study of evolution. The average frequency of microsatellites in Brassica unigenes was one in every 7.25 kb of sequence, as compared with one in every 8.57 kb of sequence in Arabidopsis thaliana. Trinucleotide motifs coding for serine and the dinucleotide motif GA were most abundant. We designed 2374 and 347 unigene-based microsatellite (UGMS) markers including 541 and 58 class I types in B. napus and B. rapa, respectively, and evaluated their use across diverse species and genera. Most of these markers (93.3%) gave successful amplification of target microsatellite motifs, which was confirmed by sequencing. Interspecific polymorphism between B. napus and B. rapa detected in silico for the UGMS markers was 4.16 times higher in 5' untranslated regions than in coding sequences. Physical anchoring of Brassica UGMS markers on the A. thaliana genome indicated their significance in studying the evolutionary history of A. thaliana genomic duplications in relation to speciation. Comparative physical mapping identified 85% of Brassica unigenes as single copy and gave clues for the presence of conserved primordial gene order. Complex chromosomal rearrangements such as inversions, tandem and segmental duplications, and insertions/deletions were evident between A. thaliana and B. rapa genomes. The results obtained have encouraging implications for the use of UGMS markers in comparative genome analysis and for understanding evolutionary complexities in the family Brassicaceae.