Development Of SSR Markers Based On Transcriptome Sequences In Brassica Species

This study detected SSR in the Brassica napus sequences of transcriptome genome, and analyzed their basic characteristics. Next, we developed the corresponding functional SSR markers. Finally, we used part of SSR markers for genetic diversity analysis and clustering analysis in 127 Brassica species so as to assess the value of these markers. The result as below.(1) We obtained 189,116 Unigenes from Barssica transcriptome which have been published. We searched SSR in these sequences and detected 68,212 qualified SSRs. The occurrence frequency of SSR in Unigenes was 36.1%. We detected 101,856 SSR loci of the whole B. napus transcriptome, the frequency of which was 53.86%. The average distribution distance of the transcriptome SSR was 1.25kb. In addition, the main SSR repeat types in B. napus transcriptome were mononucleotide, dinucleotide and trinucleotide. Where the largest number of repeat unit was mononucleotide, accounting for 89.39% of all detected SSRs, followed by dinucleotide and trinucleotide, the proportion of which were 5.22% and 3.88%, respectively. B. napus transcriptome sequences included 468 kinds of SSR repeat units. In single-nucleotide repeat unit, A/T and C/G were dominant, accounting for 68.3% and 21.09% in all SSR sequences, respectively, followed by dinucleotide repeat units:AG/CT and GA/TC, accounting for 2.06% and 1.94%, respectively.(2) The 68,212 transcriptome sequences containing SSRs were used as queries to perform BLAST2GO analysis against the Arabidopsis thaliana genome sequence database. The study showed that these genes containing SSRs were mainly involved in cellular process, metabolic process, response to stimulus, biolocical regulation.(3) We designed 58 pairs of SSR primers, and randomly selected 100 pairs of SSR primers to test the polymorphism level using the genomic DNA of 127 Brassica species. The results showed that a total of 58 pairs of SSR primers exhibited polymorphism with the amplified 120 clear and repeatable bands. The polymorphism percentage of SSR primers was 76.47%. Brassica genetic diversity indices varied from 0.1588 to 0.6931 with an average of 0.536. Nei gene diversity ranged from 0.0715 to 0.5, the average of which was 0.3626. In this study, the PIC values of 58 pairs SSR primers ranged between 0.2714 and 0.6988, the average of which was 0.7, which indicated that our selected SSR primers were relatively abundant in polymorphism.(4) We used NTSYS software to perform genetic diversity analysis of 127 Brassica species, and the genetic distance of Brassica species ranged from 0.03 to 1.18. When the genetic distance was at the point of 0.66,127 Brassica species were divided into eight categories. The result of clustering analysis was consistent with the specie types. It is suggested that the development of genetic markers can be used for genetic diversity analysis effectively.This study analyzed the characteristics of SSR located in the coding region and developed 58 pairs functional primers. Through validation of SSR primers, we thought that the developed SSR primers can be better used to the study of rapeseed species. It will provide more choices of markers for Brassica researchers.