卵形鲳鲹性别特异性标记分离及快捷性别鉴定技术建立

Benefit from its good aquaculture and product performance, Trachinotus ovatus has become a priority species in marine aquaculture of Guangxi, Guangdong, Hainan and other coastal areas in China. However, it has a long sexual maturity period of 7-8 years, and none gender-specific appearance feature can be found throughout its reproductive cycle, which has brought great inconvenience to the breeding and production, and greatly increasing the cost of production, for male and female could not be divided during broodstock conduction and spawning. Thus, development of a simple sex identification technique is an urgent need for industrial of Trachinotus ovatus. But previous effort on screening gender marker was failed because it is difficult to obtain family groups with a clear genetic background. The present study was based on whole genome sequencing result. DNA of parents and DNA pools of female and male offspring of a full-sib family will be used for genome resequencing, to locate and analyze gender-related genome sites, then screening sex-specific genomic sequences to establish SCAR technique for quick sex identification. The technique will be useful in breeding and seedling raising production practices in Trachinotus ovatus, and the study also supplied basis for researches on sex-determining way and sex-determining genes in the species.

卵形鲳鲹具有良好的养殖和商品性能，是广西、广东、海南等沿海地区海水养殖重点品种。但其性成熟周期长，且在其整个生殖周期，雌雄鱼均不具备性别特异的外形特征。进行亲鱼培育和催产的时候无法分雌雄，大大增加了生产成本，也给育种和生产带来巨大不便。因此，发展一种简易的卵形鲳鲹性别鉴定技术是产业发展的迫切需要，但因难以获得背景清晰的家系群体，性别标记筛选失败。本研究拟在全基因组测序的基础上，通过全同胞家系父母本和子代雌雄个体DNA混池的简化基因组测序，定位并解析性别相关位点，筛选性别特异性基因组序列，建立用于快速性别鉴定的SCAR技术，应用于育种和育苗生产实践，也为金鲳鱼性别决定方式和性别决定基因的研究等提供基础。

卵形鲳鲹是广西、广东等沿海地区海水养殖重点品种。但其性成熟周期长，且即使在在其性成熟时期，雌雄鱼均不具备性别特异的外形特征。进行亲鱼培育和催产的时候无法分雌雄，大大增加了生产成本。因此，发展一种基于分子生物学的简易的卵形鲳鲹活体性别鉴定技术是产业发展的迫切需要，而背景清晰的家系群体是发展性别分子标记和鉴定技术的前提。. 本研究首次构建卵形鲳鲹全同胞家系 1个，获得父母本及性别明确的子代个体组织和DNA样本200余份。完成父母本和雌雄子代混池的基因组重测序，SNP 关联分析获得14号染色体上，雌鱼基因组中6个与性别紧密相关的位点，总长度为2.29 Mb；雄鱼基因组中3个与性别紧密相关的位点，总长度为2.31 Mb。解析性别相关基因组位点，发现雌鱼性别相关基因组区域内共注释到基因131个，其中非同义突变SNP位点的基因16个，移码突变基因1个。雄鱼性别相关基因组区域内共注释到基因112个，其中非同义突变SNP位点的基因7个，移码突变基因1个。通过转录组和miRNA测序、分析和荧光定量PCR验证，挖掘性别特异性表达基因，与混池法基因组重测序分析中的性别相关基因进行关联分析获得性别特异性基因4个。通过60个非同义突变位点和4个性别特异基因组的序列验证，发现wnt9b 基因上存在雌雄特异性序列。针对wnt9b 基因上的雌雄特异性序列设计引物，初步建立基于 PCR 技术的卵形鲳鲹性别快速鉴定技术。为卵形鲳鲹育种和育苗生产实践，以及性别决定基因的研究等提供了基础。