候选基因法筛选团头鲂抗病SNPs标记及其验证和应用研究

With the development of large-scale farming and culture dense of the blunt snout bream (Megalobrama amblycephala), which is one of the most economic and endemic freshwater fish in China, the incidence of eruptive epidemic disease shows a sharp increase. It has caused the significant mortality in the blunt snout bream aquaculture. Currently, the researches related to disease-resistance of blunt snout bream are rare deficient. So,it is very urgent to develop new species or strains with enhanced disease-resistance in blunt snout bream using some potential and fast breeding approaches.In present study, five functional genes, including bactericidal/permeability-increasing protein, natural resistance associated macrophage protein, major histocompatibility complex II, hepcidin and toll-like receptor 4 genes are selected as candidate genes with relevance to disease-resistant trait. Single nucleotide polymorhphisms (SNPs) for five genes are identified using sequence analysis. By analyzing the association between SNPs or haplotype markers and resistance of disease, the molecular markers tightly linked to important disease-resistant trait are further verified. Furthermore, the method of optimal identification of the alleles and genotypes of SNPs is established. In order to further confirm the genetic effect of SNPs or haplotype markers, a Mendelian segregating population with the closer genetic background is generated from a crossing between heterozygous parents which are screened on the basis of molecular markers. Then the relation among genotype, phenotype and gene expression is analyzed in the segregating population. Meanwhile, a molecular breeding strategy for pyramiding multiple genes into blunt snout bream will be established through the evaluation of genetic effect and breeding efficiency in the different combinations of genes. In a word, the study of molecular marker assisted breeding related disease-resistant trait in blunt snout bream will promote breeding efficiency and shorten the breeding cycle as well as may provide valuable reference for other fish in molecular marker assisted breeding.

团头鲂是我国重要的淡水养殖鱼类，随着养殖规模和密度的增加，暴发性疾病呈直线型上升，对其养殖造成巨大影响。但团头鲂抗病育种相关研究工作严重缺乏，因此开发快速可靠的育种技术，加快培育抗病新品种（系）十分迫切。本项目选择5个功能基因（杀菌/渗透性增强蛋白、天然抗性相关巨噬蛋白、主要组织相容性复合体II、抗菌肽和Toll样受体4基因）作为团头鲂抗病候选基因，通过分析SNPs和单倍型标记与抗病力的关联性，筛选到与团头鲂抗病性状密切相关的标记；建立最适的SNPs等位基因检测方法；根据标记选择杂合亲本获得孟德尔分离群体，利用家系分析子代基因型-表型-基因表达之间的关系，进一步验证SNPs功能；确定基因不同组合的遗传效应，评估其育种效率，建立团头鲂分子设计育种的多基因聚合育种技术平台。开展团头鲂抗病性状的分子标记辅助育种研究，将大大提高育种效率，缩短育种周期，也为其它鱼类分子标记辅助育种提供参考和借鉴。

团头鲂是我国重要的淡水养殖鱼类，随着养殖规模和密度的增加，暴发性疾病呈直线型上升，对其养殖造成巨大影响。但团头鲂抗病育种相关研究工作严重缺乏，因此开发快速可靠的育种技术，加快培育抗病新品种（系）十分迫切。本项目选择与天然免疫相关的重要功能基因（BPI、TLR4、Nramp、Tollip、TLR7和LITAF）作为团头鲂抗病性状的候选基因，期望筛选到与团头鲂抗病密切相关的标记。首先，利用同源克隆和RACE技术，已成功获得团头鲂候选基因（BPI、TLR4、Nramp、LITAF）全序列和候选基因（Tollip、TLR7）的部分序列，已提交GenBank数据库保存。运用RT-qPCR技术，发现BPI、TLR4、Nramp基因在不同组织中呈组成型表达，但在头肾、脾脏等免疫组织中是高表达的。注射病原菌刺激物脂多糖（LPS）后，BPI、TLR4、Nramp基因mRNA水平显著上升，随着时间推移mRNA水平逐渐降低，再回落至正常水平。这证明本项目选定的候选基因在团头鲂天然免疫反应中起着重要作用，作为抗病候选基因是准确可行的。在4个候选基因（BPI、TLR4、Nramp、LITAF）上，在抗病和感病团头鲂中未筛查到多态标记，这可能与抗病和感病团头鲂个体数少有关。此外，项目组还开展了团头鲂免疫相关miRNAs筛选和鉴定研究。利用高通量测序技术，对经LPS刺激（实验组）和正常（对照组）团头鲂的两个小RNA文库进行深度测序，共获得218个conserved miRNAs和106个novel miRNAs。从文库中筛选出113个明显差异表达的miRNAs，其中63个miRNAs表达上调、50个miRNAs表达下调。利用RT-qPCR验证随机选取的12个差异表达miRNAs，其结果与高通量测序结果相一致。靶基因预测及GO、KEGG分析结果显示，差异表达miRNAs的与免疫相关的靶基因有75个，其中包括本项目选定的候选基因TLR4、Nramp、LITAF、Tollip和TLR7基因。本项目鉴定的团头鲂天然免疫相关基因及miRNAs，为确定团头鲂miRNAs功能及其与靶基因的相互调控关系，为诠释团头鲂免疫防御分子调控机理和疾病防治新对策奠定基础。