Development of genome-wide SSR markers in rapeseed by next generation sequencing.

ABSTRACT

Rapeseed (Brassica napus L.) is an important oil crop with a huge genome. This study used next generation sequencing technology to develop SSR markers in rapeseed. A total of 213,876 sequence reads were obtained in 58.8 Mb. For these reads, 21,523 SSRs were recovered from 18,575 microsatellites sequences and 8,964 SSR primer pairs were identified. Di- and mono-nucleotides were the most abundant, accounting for 47.5% and 30.7% of all SSRs, respectively. A total of 8,776 SSRs were designed from contigs and 100 SSR primers were tested for validation of SSR locus amplification. Nearly all (94%) of the markers were found to produce clear amplicons and to be reproducible. For these markers, forty-three SSRs showed polymorphic bands in eight rapeseed accessions. Thirty-four SSRs were then applied to 78 rapeseed accessions from China to evaluate the genetic diversity. Result showed that the allele number varied from two to seven, with a mean value of 3.59. The effective allele number of ranged from 1.14 to 3.25, with an average of 2.09. The average values of observed heterozygosity and expected heterozygosity were 0.54 and 0.49, respectively. The Nei's gene diversity varied from 0.12 to 0.69, with a mean value of 0.48. Resulting of the markers testing showed that the identified genome-wide SSRs were useful in rapeseed genetic studies, including genetic diversity, QTL mapping and marker-assisted selection for breeding.