Supplementary Material for: Thermal Response of Epigenetic Genes Informs Turtle Sex Determination with and without Sex Chromosomes

Vertebrate sexual fate can be established by environmental cues (e.g., temperature-dependent sex determination, TSD) or by genetic content (genotypic sex determination, GSD). While methylation is implicated in TSD, the influence of broader epigenetic processes in sexual development remains obscure. Here, we investigated for the first time the embryonic gonadal expression of the genome-wide epigenetic machinery in turtles, including genes and noncoding RNAs (ncRNAs) involved in DNA/histone acetylation, methylation, ubiquitination, phosphorylation, and RNAi. This machinery was active and differentially thermosensitive in TSD versus GSD (ZZ/ZW) turtles. Methylation and histone acetylation genes responded the strongest. The results suggest these working hypotheses: (i) TSD might be mediated by epigenetically controlled hormonal pathways (via acetylation, methylation, and ncRNAs), or by (ii) hormonally controlled epigenetic processes, and (iii) key epigenetic events prior to the canonical thermosensitive period may explain differences between TSD and GSD. Novel epigenetic candidate regulators other than methylation were identified, including previously unknown ncRNAs that could potentially mediate gonadogenesis. These findings illuminate the molecular ecology of reptilian sex determination and permitted hypothesis building to help guide future functional studies on the epigenetic transduction of external cues in TSD versus GSD systems.

脊椎动物的性别决定可通过环境信号（例如温度依赖型性别决定（temperature-dependent sex determination, TSD））或遗传物质（基因型性别决定（genotypic sex determination, GSD））得以确立。尽管甲基化已被证实与TSD存在关联，但更广范围的表观遗传过程对性别发育的影响仍不明确。本研究首次针对龟类开展了全基因组表观遗传相关分子系统的胚胎期性腺表达谱分析，涵盖参与DNA/组蛋白乙酰化、甲基化、泛素化、磷酸化以及RNA干扰（RNA interference, RNAi）的基因与非编码RNA（noncoding RNAs, ncRNAs）。该分子系统在TSD与ZZ/ZW型GSD龟类中均呈活跃状态，且表现出差异化的温度敏感性，其中甲基化相关基因与组蛋白乙酰化相关基因的响应最为显著。本研究结果提出以下待验证假说：（1）TSD或可通过表观遗传调控的激素通路（经由乙酰化、甲基化及ncRNAs实现）介导，或是（2）通过激素调控的表观遗传过程实现，（3）经典温度敏感窗口期之前的关键表观遗传事件或可解释TSD与GSD系统间的差异。研究还鉴定出除甲基化之外的新型表观遗传候选调控因子，包括此前未被报道的、可能参与性腺发育调控的ncRNAs。本研究结果阐明了爬行动物性别决定的分子生态调控机制，并为后续假说构建提供了依据，有助于指导未来针对TSD与GSD系统中外源信号表观遗传转导的功能研究。