牙鲆性逆转的基因与环境互作分子解析

The olive flounder (Paralichthys olivaceus) is an important maricultured fish in China. As female flounder generally exhibits more rapid growth than the male, its sex control will exert high commercial and practical value. The sex determination and differentiation of flounder is influenced by both genetic and environmental factors, and it is possible to reverse the genotypic female to phenotypic male when reared at high water temperatures. However, the exact mechanism of gene and environment interactions is still unclear. In order to investigate the gene×environment interactions in flounder sex reversal, the different sex phenotypic flounder will be obtained from indoor experiments under different temperatures using double haploid flounders, which were derived by mitosis gynogenesis. And the genotype of each experimental individual will be got by 2b-RAD sequencing. The genome-wide association study (GWAS) based on the interaction between gene and environment will be explored. The genome anatomy of interactions between gene and environment in sex reversal will be performed using GWAS. The quantitative trait nucleotides (QTNs) by environment interaction with large controlling effect on sex reversal in flounder will be identified through GWAS with thousands of single nucleotide polymorphisms (SNPs) and then will be used to predict the phenotype. Finally, the temporal and spatial expression patterns and DNA methylation level of selected QTNs and their corresponding gene will be analyzed to verify their functions and infer the genetic mechanism. The achievements will provide a scientific reference for the study of the genetic mechanisms of fishes with similar sex differentiation characteristics, and will guide the early selection and prediction of sex in flounder breeding practice.

牙鲆是我国重要的海水养殖鱼类，其雌性个体的生长速度显著快于雄性，性别控制有较高的经济及应用价值。牙鲆的性别分化受遗传和环境因子的共同作用，遗传型雌性个体在高温下可以性逆转产生雄性表型，但其遗传与环境的相互作用并不清楚。为了揭示牙鲆性逆转中遗传与环境的互作机制，本研究首先采用有丝分裂雌核发育技术建立遗传型全雌牙鲆群体，然后在不同的温度梯度下观测性逆转表型。利用简化基因组测序技术对实验个体进行标记分型。探索基因与环境互作全基因组关联分析（GWAS）方法，依此对牙鲆性逆转的基因与环境互作进行基因组解析:定位与环境互作的数量性状核苷酸位点（QTN），并据此预测性别表型。对筛选的位点及相应主效基因进行时空表达、甲基化水平分析，以验证其功能并推测其遗传机制。研究成果可为解析类似性别分化特征的鱼类的遗传机制提供科学参考，并指导育种生产过程中牙鲆性别的早期预测和选择。

牙鲆是我国及日韩的重要海水养殖鱼类，其雌性比雄性具有明显的生长优势，培育全雌群体能够提高养殖效益，因而其性别相关研究一直倍受关注。牙鲆性别表型的形成不仅受到遗传因子的调控，还受到外界环境因子特别是温度的影响。尽管可以通过雌核发育技术获得遗传全雌群体，但外源因子如高温诱导会使其变为雄性。故对牙鲆性逆转中遗传与环境的互作机制进行研究，不仅有利于指导性别控制，而且对丰富鱼类性别分化理论具有重要意义。本项目通过诱导和培育获得有丝分裂雌核发育牙鲆群体，获得遗传型XX雌性，通过置于不同水温下培育，获得不同性别表型个体，其雄性比例呈现明显的温度依赖性。利用高通量测序技术对352尾实验个体进行基因组分型，质控后获得约894万SNP标记。利用测序得到的SNP 标记进行基因与环境互作的全基因组关联分析，检测到与性逆转相关的主效QTNs8个，基因环境互作主效候选QTN1个。同时利用基因组SNP标记对有丝分裂雌核发育牙鲆和普通牙鲆群体的遗传变异进行了分析，表明有丝分裂雌核发育群体遗传多样性低于普通群体，但两个群体之间无显著的遗传分化。与普通群体相比，有丝分裂雌核发育群体长连续性纯合片段（ROH）比例较高。通过转录组分析明确了雌核发育伪雄鱼与雌核发育雌鱼及普通雄鱼性腺的基因表达差异，表明伪雄鱼精巢与普通雄鱼精巢的基因表达模式相似，而与雌核发育雌鱼卵巢间存在显著差异。通过对比基因组重测序与转录组数据，本项目还分析了牙鲆雌雄性腺RNA编辑的差异，共检测出742个RNA编辑位点，大部分位于非编码3’UTRs，61个位点位于编码序列中，A-to-G替换是最常见的类型，占牙鲆性腺中已鉴定RNA编辑位点的83.30%。本研究不仅有助于阐释牙鲆性逆转的基因与环境互作遗传机制，也可筛选获得性逆转相关基因型，为牙鲆的选择育种提供重要参考。