Isolation and characterization of novel microsatellite loci in the wrinkled frog

The wrinkled frog, Rana rugosa, is a species complex that inhabits plains and mountains near freshwater bodies throughout East Asia, encompassing China, Korea, Japan, and the Russian Primorye region. Although extensive efforts are required to estimate cryptic diversity in the R. rugosa complex, no specifically designed microsatellite loci are available. Here, novel and polymorphic microsatellites were isolated based on the construction of a microsatellite-enriched library and characterized using R. rugosa specimens collected on the Korean Peninsula. A total of 72 primer sets were designed from approximately 400 positive clones, and 22 were validated as being reliably amplified and polymorphic. Overall, high genetic variability was observed (mean number of alleles per locus = 22.23; mean observed and expected heterozygosities = 0.770 and 0.816, respectively) from a total of 60 individuals sampled from two geographically isolated localities. In the two sites analyzed, an extremely low level of relatedness was inferred from the estimation of pairwise relatedness, and no evidence of a genetic bottleneck was detected. The two sites showed a high level of genetic differentiation, suggesting a clear signature of isolation following colonization. With high statistical power in parentage and sibship exclusion, these microsatellite loci will be suitable for the identification of cryptic diversity and population structure as well as the recognition of individuals in social interaction and captive breeding practice.