半滑舌鳎抗病性状的遗传解析及抗病分子育种的基础研究

Half smooth tongue sole (Cynoglossus semilaevis) is an important species of flatfish. Recent years, the problem of disease occurrence, especially the disease caused by Edward's and vickers bacteria, has become a main limiting factors for sustainable development of half smooth tongue sole aquaculture in China. Disease resistance is an important trait in farmed fishes, and the urgent problems to solve at the moment are to create and breed the fish species with excellent disease resistances. Research on molecular mechanism of disease resistance in fish is an important guarantee of new variety breeding. And the finding of disease resistance-related functional genes and molecular markers is an important foundation of researching on molecular mechanism of disease resistance. In view of above, based on the availability of whole genome fine map and high density SNP genetic linkage map in half smooth tongue sole, the present project focuses on identification the main functional genes resistant to main bacterial pathogens by QTL fine mapping, transcriptome and proteome analysis; exploration of the disease resistant genes function through genome editing, RNA inference and epigenetics; determination disease-resistant gene regulatory networks; and revelation the molecular mechanisms of disease resistance in half smooth tongue sole. And then, we will carry out whole genome re-sequencing for the produced families and population to screen disease resistance-related SNP loci, implement genome wide association study (GWAS) and develop genomic selection (GS) breeding theory for half smooth tongue sole. The project will provide a theoretical basis and a technical support for breeding new stock with enhanced disease resistant in fish.

半滑舌鳎是我国鲆鲽鱼类养殖产业的主导品种之一。近年来，各类病害问题日益严重，尤其是由爱德华氏菌、哈维氏菌等病原菌引起的疾病频发，已成为制约半滑舌鳎养殖业可持续发展的瓶颈。抗病力作为鱼类重要的经济性状，其分子机理的解析是抗病育种的重要前提，而抗病功能基因和分子标记的发掘又是进行抗病分子机理解析的重要基础。本项目拟以半滑舌鳎为材料，在已完成的全基因组精细图谱、高密度SNP遗传连锁图谱的基础上，开展抗细菌病相关QTL精细定位，鉴定出抗细菌病相关主效功能基因，采用基因组编辑、表观遗传学等途径阐明抗病基因功能，构建抗病基因调控网络，揭示半滑舌鳎抗病性状的分子机理；进而利用半滑舌鳎抗病和易感家系开展基因组重测序，筛选抗病相关SNP位点，进行全基因组关联分析，建立抗病性状全基因组选择育种的理论基础。本项目成果将为鱼类抗病分子育种奠定理论基础和提供技术支撑，将推动我国鱼类抗病育种学科发展及抗病育种的进程。

半滑舌鳎是我国海水鱼产业技术体系的9大主导品种之一，也是近年来遭受细菌类疾病危害巨大的品种之一，其养殖产业受到病害频发和优良种质匮乏等问题的严重困扰，这些问题成为制约半滑舌鳎养殖业可持续发展的瓶颈。抗病力是鱼类重要的经济性状，其分子机理的解析是抗病育种的重要前提。本项目利用半滑舌鳎基因组和高密度遗传连锁图谱，完成了抗细菌病QTL精细定位，新定位到11个哈维氏弧菌病相关的QTL位点，从基因区间筛选到12个免疫相关基因，对其中的dctn5、stat5bl等基因进行了功能研究。通过同源克隆获得半滑舌鳎CD40、TLR5、TRAFs、perforin等20多个基因，并开展了哈维氏菌感染后的表达模式分析及重组表达蛋白的抑菌实验分析，揭示出这些基因在抵抗细菌感染过程的重要作用。利用转录组学技术，对哈维氏弧菌感染前后的差异基因进行了全面的比较和分析，绘制了半滑舌鳎抗细菌病相关基因调控网络和lncRNA调控网络，筛选到6个半滑舌鳎抗病性状相关基因和7个关键的lncRNA。探索了表观遗传学在鱼类抗病性状中的调控机制，发现抗病力与抗病免疫相关基因的甲基化调控有关，鉴定了类肿瘤坏死因子和类脂多糖结合蛋白的DNA甲基化与抗病性状相关。建立半滑舌鳎家系近500个，开展了抗哈维氏弧菌感染试验，构建了基因组选择参考群体，对抗病家系、不抗病家系、快速生长家系和慢速生长家系等进行了全基因组重测序，绘制了半滑舌鳎抗病性状的全基因组 SNP 变异图谱，解析了抗病性状的基因组变异机制，筛选33个抗病相关SNP位点。发现抗哈维氏弧菌病性状是有多个微效基因控制的数量性状，为基因组选择技术的建立奠定了理论基础，构建了 SNP 基因型和抗病性状关联的 G-P 模型。计算了个体的基因组育种值，建立了半滑舌鳎抗病基因组选择技术，采用该技术培育出抗病新品种“鳎优1号”半滑舌鳎，完成新品种申报和现场评审。本项目发表研究论文29篇，包括SCI论文19篇，中文核心论文10篇，其中在Nature Genetics杂志发表1篇。申报国家发明专利6项，其中已授权4项。本项目解析了半滑舌鳎抗病性状相关的分子机制，成功建立了半滑舌鳎抗病基因组选择育种技术并培育出半滑舌鳎抗病新品种，将海水鱼类抗病育种研究提升至国际领先水平。

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