Rapid development of novel microsatellite markers from Mauremys reevesii (Testudines: Geoemydidae) using next-generation DNA sequencing technology

Abstract

Mauremys reevesii (Gray, 1831), which belongs to Mauremys of Geoemydidae (Testudines), distributed in China, as well as Japan and Korea. Previous studies have developed several polymorphic microsatellite loci, but most of them were dinucleotide motifs. Here, we developed 15 polynucleotide-repeat microsatellite loci (including di-, tri, tetra-and pentanucleotide motifs) for M. reevesii through Restriction-site Associated DNA tags sequencing (RAD-seq). A total of 987 microsatellite loci with flanking sequences were suitable for setting primers for polymerase chain reactions (PCR). To verify the identified SSRs, 40 primer pairs were selected for PCR detection. In total, 32 primer sets produced strong PCR products matching their expected sizes, in which species amplification tests showed that 15 were polymorphic. And the number of alleles per locus ranged from 3 to 16. The observed and expected heterozygosity per locus varied from 0.3784 to 1.000 and from 0.3995 to 0.9700, respectively. The methodology of microsatellite isolation constructed in this study is not only cost-effective and time-saving in comparison to traditional approaches, but also can be served as useful tools which benefit population genetics studies and conservation management of M. reevesii.