白斑狗鱼性别特异标记筛选与性别发育机制的转录组学分析

Northern pike (Esox lucius) is naturally distributed in Irtysh River basin of Xinjiang in China, and now it has become the most important economical fish in Xinjiang Province, and spread over the whole country. Females of northern pike grow significantly faster than males; it has important significance for improving culture efficiency to implement unisexual production through sex control. But the differences of northern pike male and female genomics have not yet been studied, and there were no available sex-specific markers for sex control research; even nothing about gene regulatory network mechanism of sex development of northern pike was known, which have seriously hindered the research on sex control and sex decision of this fish. Simplified genomic sequencing will be performed on female and male individuals of northern pike using RAD-seq technology in this project, and the differences of northern pike male and female genomics will be studied, sex-specific markers of northern pike will be screened. Transcriptome sequencing will be performed on early female and male gonads of northern pike in different developmental stages using RNA-seq technology, and sex differentially expressed genes will be screened. The studied on spatiotemporal expression and functional analysis will be performed on some important sex developmental genes using the methods of quantitative PCR, in situ hybridization and gene knockout, and the gene regulation network mechanism of northern pike sex development will be revealed, the foundation will be laid for further development of sex control and sex decision research of northern pike, and so the work has important scientific significance and application value.

白斑狗鱼自然分布于我国新疆额尔齐斯河流域，现已成为新疆最重要经济鱼类，并向全国推广。白斑狗鱼雌鱼生长明显快于雄鱼，通过性别控制实现单性生产对提高养殖效益有重要意义。但目前对白斑狗鱼雌雄基因组差异尚未研究，没有可用于性别控制研究的特异标记，对白斑狗鱼性别发育的基因调控网络机制更是一无所知，这些严重阻碍着白斑狗鱼性别控制和性别决定机制研究。本项目利用RAD-seq技术对白斑狗鱼雌雄个体进行简化基因组学测序，研究白斑狗鱼雌雄基因组间差异，筛选白斑狗鱼性别特异标记；利用RNA-seq技术对白斑狗鱼早期不同发育阶段的雌雄性腺进行转录组测序，筛选白斑狗鱼性别差异表达基因；利用定量PCR、原位杂交和基因敲除等方法对部分白斑狗鱼重要性别发育基因进行时空表达和功能分析研究，揭示白斑狗鱼性别发育的基因调控网络机制，为进一步开展白斑狗鱼性别控制研究和性别决定机制研究奠定基础，具有重要的科学意义和应用价值。

白斑狗鱼自然分布于我国新疆额尔齐斯河流域，现已成为新疆最重要经济鱼类，并向全国推广。白斑狗鱼雌鱼生长明显快于雄鱼，通过性别控制实现单性生产对提高养殖效益有重要意义。但目前对白斑狗鱼雌雄基因组差异尚未研究，没有可用于性别控制研究的特异标记，对白斑狗鱼性别发育的基因调控网络机制更是一无所知，这些严重阻碍着白斑狗鱼性别控制和性别决定机制研究。本项目利用混池重测序技术和个体重测序技术，对生理性别鉴定的雌雄各15条鱼白斑狗鱼雌雄个体进行二代基因组学测序，进行SNP位点和Indel位点筛选和GWAS分析，研究白斑狗鱼雌雄基因组间的差异，通过PCR验证筛选得到位于24号染色体上的2个特异性非常高的白斑狗鱼性别标记。利用RNA-seq技术对白斑狗鱼孵出后20d、58d、126d、188d和320d等不同发育阶段的雌雄性腺进行精准转录组测序，通过表达定量分析，筛选出大量的白斑狗鱼性别差异表达基因，并进行了差异表达基因GO富集分析、KEGG Pathway 分析和加权基因相关网络分析，为构建白斑狗鱼性别发育的网络调控机制打下良好基础；利用定量PCR技术对FOXL2、Sox9、DmRTA1、StAR和Wnt4等白斑狗鱼重要性别发育基因进行克隆和时空表达分析研究，揭示白斑狗鱼性别发育的基因调控网络机制，为进一步开展白斑狗鱼性别控制研究和性别决定机制研究奠定基础，具有重要的科学意义和应用价值。项目组还建立了循环水斑马鱼养殖系统，初步建立了斑马鱼繁殖和受精卵的显微注射技术，为后续开展斑马鱼和白斑狗鱼性别相关基因的CRISPR/Cas9靶向敲除研究打下基础。