Development of EST-SSR Markers in Flowering Chinese Cabbage (Brassica campestris L.) by de novo Transcriptome Assembly

RNA-seq of flowering Chinese cabbage was performed by using Illumina HiSeq2500 platform and generated 295 million clean reads with 36.9 Gb clean bases from 8 different samples. A total of 48,975 unigenes were assembled with an average length of 779 bp, and 83% of them were annotated against public databases with average accuracy of 97%. A collection of 8,165 genic-SSR loci, containing 1-6 nucleotide repeats, were discovered from 48,975 unigenes with 38.17 Mb. The major types of SSR loci were 1-3 nucleotide repeats which accounted for 98.57% of all SSR loci discovered. The proportion of mono-, di- and tri- nucleotide repeats were 14.32%, 41.08% and 43.17%, respectively. Fifty eight repeat motifs with iteration numbers ranging from 5 to 23 were identified. A/T, AG/CT, AT/AT, AC/GT, AAG/CTT and AGG/CCT were the most abundant motifs. The lengths of repeat nucleotide sequences for all SSR loci ranged from 10 to 60 bp. Most of the repeat motifs were below 20 bp, and only 11.49% (938 SSR loci) were 20 bp or above. A total of 676 SSR primers pairs with a potential to produce polymorphism were designed, and 170 primer combinations were randomly selected for amplification test, of which 96 primer pairs amplified unambiguous bands and were highly reproducible, 53 of them were able to be detect polymorphisms in four flowering Chinese cabbage accessions. These results could provide the foundation for development and application of SSR markers in the genetic improvement of flowering Chinese cabbage.