大黄鱼性别决定区域的定位及结构、功能研究

Large yellow croaker ( Larimichthys crocea, LYC ) is one of most important marine culture fish species in China. All-female culture of LYC has high economic value for the growth of females is faster than that of males both in wild and in farm. The techniques for producing all-female fries of LYC have been established. However, the innovations in sex-control techniquesare are still "black- box" mainpulations for the lack of knowledge on genetic basis of sex determination in LYC. Therefore, the major objectives of this project are to map sex determination region (SDR) of LYC and further anylize the genomic structure of SDR; to find the candidate sex-determination genes in SDR and annotate them initially. We will use the recent genomic tools to achieve the objectives, which includes the following steps: i) resequencing genome of the females and the males; ii) identifying of sex specific sequence with whole genome in-silico subtractive hybridization; iii) mapping QTL for sex with linkage analysis and linkage disequilibrium analysis with SNP markers called from the genome-resequencing data; iv) physically mapping SDR with sex cosegregated marker; v) analyzing the genome structure of SDR and discovering the genes and the control elements in SDR; vi) gene functional analysis with both in-silico and in-lab tools. The expected achievements of this project include the sex specific sequence, the linkage and physical position of the sex determination region, the genomic structure of SDR, and some candidate sex-determination genes for further analysis in LYC. These expected achievements will serve as the basis for finding the major sex-determination gene, for further investigation on genes and environment interaction during sex determination, for developing the sex specific molecular marker, and for developing innovative methods for sex controlling in LYC.

针对大黄鱼性别决定遗传基础相关知识匮乏的问题，本项目拟利用基因组学新技术，定位大黄鱼性别决定区域（SDR），并分析SDR的结构与功能特征，探讨大黄鱼性别决定的遗传机理。主要研究内容包括：1）通过重测序构建大黄鱼雌雄基因组数据库，再利用全基因组电子消减杂交筛查性染色体特异序列；2）通过连锁分析与连锁不平衡分析精细定位性别连锁位点，进而通过电子工具物理定位SDR；3）分析SRD的基因组结构特征，识别区域内的基因与调控位点；4）综合采用电子工具与实验室方法对SDR区间的基因进行功能预测、分析与验证，发现大黄鱼候选性别决定基因。本项目首次系统开展大黄鱼性别决定的遗传分析，预期成果将为阐明大黄鱼性别决定分子机制奠定基础，为开发高效大黄鱼性别控制技术提供科学依据，还将填补石首鱼科鱼类性别决定遗传基础相关知识缺口，为研究鱼类性别决定进化提供重要资料。

本项目利用基因组学技术，对大黄鱼性别决定区域（SDR）进行了连锁与物理定位，并分析了SDR的结构与功能特征。主要研究内容和结果如下：（1）雌雄大黄鱼基因组重测序数据的比对。结果显示，大黄鱼两种性别的基因组大小没有显著性差异，无大片段性染色体特异片段。（2）性别决定位点的连锁定位。QTL定位分析发现，决定大黄鱼性别的位点只有1个，位于9号连锁群30 cM - 38 cM之间。通过关联分析，将SDR进一步定位在该区域2 Mb范围内。（3）物理定位。通过序列比对将17个性别紧密的连锁的SNP标记定位在参考基因组的相应位置上。构建了1个雄性大黄鱼BAC文库，筛查了含Dmrt1基因的BAC克隆，对中期染色体作BAC-FISH，结果有且仅有1对阳性信号，表明SDR性别间差异小且无大片段重复。（4）SDR基因组结构特征分析。SDR基因扫描结果显示，该区域分布着20个基因，具有较高的基因密度。重测序数据深度分析结果表明，SDR未见大范围测序深度扩大，提示大黄鱼SDR不具有大范围重复序列累积，与其他已报道鱼类的SDR有明显区别。（5）基因功能分析。功能注释结果显示，SDR区域内的Dmrt1、Dmrt3和piwi-样蛋白等基因与性别决定与分化过程相关。基于同源性分析、基因型与性别相关性分析初步推断，Dmrt1基因是大黄鱼性别决定关键基因的候选基因。转录组和RT-PCR分析结果显示，Dmrt1基因在精巢中特异表达，且时间表达谱与精巢分化过程相符合，支持了这一结论。此外，本项目还克隆并验证了2个大黄鱼生殖细胞标志基因，并初步研究了大黄鱼生殖细胞的起源与迁移。本项目的研究结果为开发大黄鱼性别分子标记，深入开展大黄鱼性别决定与分化机制的研究、性别控制技术的优化升级奠定了重要基础。