Repetitive sequence characterization and development of SSR and CMS-gene-specific markers in the Brassica mitochondrial genomes

In the Brassica genus, relatively little is known about repetitive sequences in the mitochondrial genomes, and few mitochondrial molecular markers are available. In this study, repetitive sequences in the mitochondrial genomes of six Brassica species (Brassica rapa subsp. campestris, B. oleracea, B. nigra, B. napus (pol and nap), B. juncea and B. carinata) were analyzed. The distribution and characteristics of simple sequence repeats (SSRs), transposons, tandem repeat sequences, and inverted repeat sequences were assessed. A total of 148 SSRs were detected, with a density ranging from one SSR per 15.4 kbp to one SSR per 11.1 kbp per Brassica species. The most commonly identified SSRs were mononucleotide repeats. Many transposons, tandem repeats, and inverted repeats were also detected, with the most repeats in B. oleracea of all the species. Forty-eight bands amplified from 31 polymorphic SSR primers designed from the six Brassica mitochondrial genome sequences were screened across 92 Brassica accessions. Of these 48, 13 clear and reproducible bands (from 12 rimer pairs) could be used to distinguish between mitochondria from different species. Twelve polymorphic CMS (cytoplasmic male sterility)-gene-specific primers were developed from CMS-associated genes in the B. napus mitochondrial genome, and five primers could be used to distinguish mitochondria from different species, providing alternative markers for the identification of different Brassica mitochondrial types. Our results suggest that these SSR markers and CMS-gene-specific markers can be widely applied to characterize Brassica mitochondrial genomes. Further investigation of CMS-gene-associated markers may facilitate identification and utilization of CMS cytoplasm for future breeding approaches.