泥鳅杂交三倍体染色体组稳定性及其表观遗传机制解析

Rencently, studies on triploid fish had become hot in fish genetic thremmatology for the sterility and fast-growing. The triploid could be obtained (100%) by the hybridation between natural tetraploid and diploid, while it is difficult to induce tetraploid. Therefore, how to obtain a new kind triploid by hybridization between natural diploid and tetraploid, has become a bottleneck and difficulty for the industrialization of triploid fishto prepared a new kind of triploid by the hybridation between natural tetraploid and diploid. In our past 8 years studies on the origin and formation mechanism of the unique natural tetraploid loaches in the Yangtze River valley, it has been approved that the natural tetraploid loach wasgenetic tetraploid (4n=100), producing normal diploid (2n) gametes in both male and female. Preliminary researches had also been taken on the cultivation of triploid by hybridization between natural diploid and tetraploid. However, the genetic stability and epigenetic mechanism has not been reported yet. In this study, hybrid triploid loach was analyzed using molecular cytogenetic and molecular biology method for the researches of chromosome formation, configuration, meiosis process, gonadal development and the epigenetic mechanism, hoping to provide evidence for the stability of chromosome formation of the hybrid triploid and explore a effective and feasible way for the industrialization of the triploid fish.

三倍体鱼类以其育性差、生长快等特性而成为鱼类遗传育种研究的热点。四倍体和二倍体杂交理论上可获得100%的三倍体，人工诱导四倍体难度大。因此，利用天然四倍体与二倍体杂交制备1种新型的杂交三倍体是解决某些鱼类三倍体产业化的瓶颈与难点。本课题组在近8年对我国长江流域特有的天然四倍体泥鳅的起源及形成机制进行了系统研究，证实了天然四倍体泥鳅是遗传四倍体(4n=100)、雌雄均能产生正常的2n配子。并对天然四倍体与二倍体杂交获得的杂交三倍体泥鳅的培育进行了初步研究。但关于杂交三倍体泥鳅的遗传稳定性及表观遗传机制迄今国内外尚无报道。本项目以杂交三倍体泥鳅及其亲本为研究对象，采用分子细胞遗传学及分子生物学分析的方法，对杂交三倍体泥鳅的染色体组成、构型、减数分裂行为、性腺发育及其表观遗传机制进行解析。旨在为杂交三倍体泥鳅的染色体组稳定性提供遗传学证据，为我国三倍体鱼类的产业化探索高效易行的新途径奠定基础。

三倍体鱼类以其育性差、生长快等特性而成为鱼类遗传育种研究的热点。四倍体和二倍体杂交理论上可获得100%的三倍体。本项目利用天然四倍体泥鳅与二倍体泥鳅杂交制备了1种新型的杂交三倍体泥鳅。并以育性及杂种优势为切入点，从细胞及分子水平对杂交三倍体泥鳅的染色体组稳定性及表观遗传学机制进行了系统的研究。结果表明，杂交三倍体泥鳅出现大量能存活的非整三倍体个体，而且核型表现出多态性。在卵母细胞（精母细胞）减数分裂的终变期有些细胞可观察到1-5个三价体。精母细胞染色体经银染、CMA3及荧光原位杂交等发现在三个不同的单价体端部有信号，表明同源染色体联会的失败。性腺组织学观察发现虽然少数杂交三倍体个体无论雌雄均能达到性成熟，但产生的卵子和精子较少，其杂交后代无存活个体。针对亲本及其子代性腺组织进行了高通量转录组测序，通过对测序数据的拼接，共获得62821个Genes和178962个Transcripts。以斑马鱼、墨西哥脂鲤、虹鳟等基因组为参考序列，进行基因功能的注释。筛选性腺发育、配子形成和激素调控相关基因（Dmc1，hormad1，cyp19a，dmrt1，FOXL2等），根据基因在亲子代间的表达量变化模式，从分子生物学角度分析了在杂交三倍体泥鳅的育性机制。另外，本项目还建立了泥鳅甲基化敏感扩增多态性(MSAP)方法的反应体系，并利用该方法对不同倍性及亲子代泥鳅鳍基因组DNA甲基化差异进行了分析，不同倍性泥鳅DNA甲基化水平表现出随着倍性增加而减少的倍性效应，DNA甲基化模式也发生了变化。四倍体与二倍体正反杂交F1代的甲基化水平均介于双亲之间，正（反）杂交F1代的甲基化模式也经过了重新调整。不同位点DNA甲基化的增强或减弱对杂交三倍体泥鳅杂种优势均可能发挥作用。本项目研究结果为杂交三倍体泥鳅的染色体组稳定性提供了遗传学证据，同时也为我国三倍体鱼类的产业化探索高效易行的新途径奠定了基础。