Supplementary Material for: Effects of Incubation Temperature on the Expression of Sex-Related Genes in the Chinese Pond Turtle, Mauremys reevesii

Despite widespread temperature-dependent sex determination (TSD) in reptiles, it is still unclear how the molecular network responds to temperature variation and drives the sexual fate. Profiling of sex-related genes is the first step in understanding the sex determination system in reptiles. In this study, we cloned the full-length coding sequences of Cyp19a1, Foxl2, Rspo1, Sf1, and Sox9 in an Asian freshwater turtle (Mauremys reevesii) with TSD and identified the expression patterns of these genes and Dmrt1 at different incubation temperatures to understand their roles in urogenital development. Our results showed that Cyp19a1, Foxl2, and Rspo1 were expressed in the adrenal-kidney-gonadal complex at a high level in females, while Sf1 and Dmrt1 were highly expressed in males. In addition, Foxl2 and Rspo1 showed sex-dimorphic expression in the presumed early thermosensitive period (TSP), Dmrt1 was upregulated at the beginning of the presumed TSP, and Sox9 did not show sex-dimorphic expression until the end of the presumed TSP. These results suggest that Foxl2 and Rspo1 are probably upstream genes involved in female sex determination and that Dmrt1 may be a key factor in male sex determination. Therefore, our study provides a solid foundation for further investigations on the molecular mechanism underlying sex determination in M. reevesii.