Development and application of polymorphic microsatellite markers in Saxifraga genus / 中国中药杂志

ABSTRACT

In order to develop genomic-SSR markers for species of Saxifraga genus, a mixed plant genomic DNA sample was sequenced based on high-throughput Illumina MiSeq platform. According to genomic sequencing data, SSR loci were identified with MISA software, and then primers were designed with Primer 3 software. A total of 120 pairs of primers were randomly synthesized and amplified in genomic DNA of a few plant samples. Those primers who have yielded polymorphic bands and were considered easy to amplify were identified. After that, transferability of these primers was evaluated, and phylogenetic relationship of 25 species of Saxifraga genus was analyzed with UPGMA (unweighted pair group method analysis). In our results, 587 256 sequences containing SSRs were identified from a total of 1 881 979 combined read pairs obtained in genomic sequencing. Primers were designated to amplify SSRs containing two to six nucleotide repeat units, screened in a small portion of species. Finally, 17 pairs of primers which have produced abundant of polymorphic bands with little problem were amplified in 25 species of Saxifraga genus. A total of 2 687 polymorphic bands were obtained, the average polymorphic rate was 158 bands per pairs of primers. The transferability rate was ranging from 88.0% to 100% across 25 species of Saxifraga. In phylogenetic analysis, the clustering of 25 species based on 17 pairs of SSR primers was different from morphological classification. Our analysis has provided molecular data for genetic relationship of Saxifraga genus, and the transferable and polymorphic SSRs have provided information for genetic diversity research.