Isolation of microsatellite markers for Pelophylax nigromaculata and a tentative application in detecting...

Twenty-seven microsatellite markers were obtained from the black-spotted frog, Pelophylax nigromaculata,using the FIASCO (Fast Isolation by AFLP of Sequences containing repeats) protocol. Genotyping of 60 individuals showed that the number of alleles ranged from 4 to 25 with an average of 12.78 alleles per locus. The observed and expected heterozygosities ranged from 0.137 to 0.956 and 0.594 to 0.948, with averages of 0.531 and 0.809, respectively. Many of these loci were successfully cross-amplified in five other ranid species. The introgression between P. nigromaculata and Pelophylax plancyi was assessed using 5 microsatellite loci on 159 individuals collected from Liaoning, northeast China. Two distinct genetic clusters (pure species) as well as seven unassigned individuals were identified by factorial correspondence analysis (FCA), New Hybrids and Structure. The unassigned individuals, which are likely the product of interspecific hybridization, indicate the usefulness of these microsatellite loci for future studies of hybridization and introgression between these two ranid species.