Male heterogametic sex determination in Rana dybowskii based on sex-linked molecular markers

Identifying the mechanism for sex determination in amphibians is challenging.Very little is known about sex determination mechanisms of Rana dybowskii,a species of importance to evolutionary and conservation biology.We screened for sex-linked molecular markers in R.dybowskii in China using target region amplification polymorphism with 2 fixed primers against the sequences of Dmrt1.We found 2 male-linked molecular markers in R.dybowskii,which were 222 bp and 261 bp long.The detection rates of 222 bp marker in males form Xinglong,Huadian,and Dandong were 93.79%,69.64%,and 13.64%,respectively,while the rate in females from Huadian was 27.50%.Besides,the detection rates of 261 bp marker in the above 3 regions were only observed in males at the rate of 93.79%,87.50%,and 32.73%,respectively.The inheritance patterns of sex-linked molecular markers showed that the 2 sex-linked molecular markers were heterozygous.Compared to the XY-male parent,progeny from XX-pseudo-male parent possessed lower sex reversal ratio at the same rearing temperature,and the proportion of female froglets from an XX-pseudo-male parent was more than 95%at low rearing temperature(15°C).Our findings suggest that R.dybowskii displays male heterogamety,and the 2 sex-linked molecular markers may have a guiding significance for the protection and utilization of R.dybowskii.