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 <i>Cyp19a1</i>,<i> Foxl2</i>,<i> Rspo1</i>, <i>Sf1</i>, and <i>Sox9</i> in an Asian freshwater turtle (<i>Mauremys reevesii</i>) with TSD and identified the expression patterns of these genes and <i>Dmrt1</i> at different incubation temperatures to understand their roles in urogenital development. Our results showed that <i>Cyp19a1, Foxl2, </i>and <i>Rspo1</i> were expressed in the adrenal-kidney-gonadal complex at a high level in females, while <i>Sf1</i> and<i> Dmrt1</i> were highly expressed in males. In addition, <i>Foxl2</i> and <i>Rspo1</i> showed sex-dimorphic expression in the presumed early thermosensitive period (TSP),<i> Dmrt1 </i>was upregulated at the beginning of the presumed TSP, and <i>Sox9 </i>did not show sex-dimorphic expression until the end of the presumed TSP<i>. </i>These results suggest that <i>Foxl2</i> and <i>Rspo1</i> are probably upstream genes involved in female sex determination and that <i>Dmrt1</i> 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 <i>M. reevesii</i>.