An improved method for identifying the genotypic sex in Rana dybowskii

Screening for sex-specific markers via PCR is a common method for identifying the genotypic sex of amphibians. Previously, we employed TRAP‒PCR and polyacrylamide gel electrophoresis to effectively amplify two male-specific markers (222 bp and 261 bp) in Rana dybowskii. However, polyacrylamide gels contain hazardous substances that endanger human health and pollute the environment. Agarose gel is superior to polyacrylamide gel and contains no toxic chemicals, making it suitable for the contamination-free detection of PCR products. To obtain more primers that amplify male-specific bands in R. dybowskii DNA and yield amplification products that can be detected via agarose gel electrophoresis, seven pairs of primers were designed to amplify new male-specific markers that should be within the parameters of two previously identified male-specific bands (222 bp and 261 bp sequences). Among the primers tested, the 222–1 F/222-1R primer pair amplified a 192 bp male-specific fragment and yielded the greatest clarity, with bright, easily visualized bands. To evaluate its effectiveness for the identification of the genotypic sex of R. dybowskii, this primer pair was used to amplify a male-specific DNA marker in three R. dybowskii populations from Xinglong, Huadian and Dandong. The results showed that the primer pair 222–1 F/222-1R amplified a stable male-specific marker (192 bp) in male individuals from three populations. In conclusion, our findings suggest that the revised method is a dependable, efficient, and nontoxic strategy.