孵化温度和性染色体对乌龟性别决定的影响

温度依赖型性别决定（TSD）物种一般认为缺乏性染色体。乌龟为典型TSD物种，在本项目前期已证明乌龟具有Z染色体的基础上，我们提出了乌龟性别决定的调控模型。为验证该模型，本项目拟开展两方面的工作：一是采用限制性酶切简化基因组技术（RAD-seq）筛选性别特异分子标记，即W染色体特异片段；同时以Nanopore测序的雌性乌龟全基因组序列（已完成）为参考基因组，结合RAD-seq雌性和雄性序列，采用染色体商（CQ）计算方法，组装Z、W染色体；二是通过孵化温度置换实验，辅以转录组测序，采用qRT-PCR和荧光免疫技术检测性别决定相关基因在温度敏感期（TSP）的差异表达，筛选响应温度变化的Z染色体雄性性别决定基因，重点关注如DMRT1等基因的表达。本项目将提供TSD龟类具性染色体的直接证据，拓展TSD调控模式的认知，对深入研究TSD的分子机制具有重要的科学意义。

Temperature-dependent sex determination (TSD) species are generally considered to lack sex chromosomes. Reeves' pond turtle is a typical TSD species and the Z chromosome has been identified in our previous studies. Based on the previous data, we have proposed a regulatory model of sex determination for Reeves’ pond turtle. In order to verify this model, the project intends to carry out two aspects of work: firstly, the gender-specific molecular markers (W chromosome-specific fragments) will be identified by using restriction-site associated DNA sequencing (RAD-seq). At the same time, the whole Reeves' pond female genome sequence by nanopore sequencing (has been completed) as a reference genome, will combine with the female and male sequences of RAD-seq, in order to assemble the Z and W chromosomes using chromosome quotient (CQ) method. Secondly, the male sex-determining genes of Z chromosome response to temperature changes will be identified based on the incubation temperature-shift experiments and transcriptome sequencing. The differential expression of sex-related genes will be detected by qRT-PCR and fluorescent immunoassay technology during the temperature-sensitive period (TSP), especially the expression of DMRT1. This study aims to provide the direct evidence of the existence of sex chromosomes in a TSD turtle species, which expands the cognition of TSD regulation patterns and has important scientific significance for the in-depth study of the molecular mechanism of TSD.