基于基因组学技术鉴定奶牛副结核病易感/抗性基因

The purpose of this study is aimed to detect the serum antibody levle of Mycobacterium avium subsp. Paratuberculosis (MAP) with ELISA kit and identify genes affecting susceptibility/resistence to MAP in the Holstein population in Beijing region.1) Based on 50K SNP chip, genome-wide association analysis (GWAS) will be conducted for susceptibility/resistenc to MAP with case-control design to identify genome-wide and chromosome-wide significant SNPs. Through further bioinformation analysis, candidate genes for such trait will be mined and function annotiated; 2) Using RNA-seq，miRNA-seq,CHIP-seq technologies which are based on the next generation high- throughput sequencing technique, jejunum tissue samples will be selected from 9 Holstein cows those were infected with MAP and showed clinical symptom, infected with MAP but no symptom, and health, respectively, with 3 cows in each group, and conducted transcriptome, miRNA, transcript factor and histone modification analysis to identify the significantly expressed genes among 3 groups and regulatory pathways; 3)Corresponding, integrated the above results, we will confirm the genetic effects of such candidate key genes on susceptibility/resistence to MAP in the independant Holstein population, and then investigate the biological functions of the candidates with large genetic effects based on jejunum cell line in order to determine the ture key genes and causal mutations, so that marker-assisted selection program on resistance to MAP will be put into effect for Holstein bulls and cows.

旨在以北京地区中国荷斯坦牛为试验群体，鉴定奶牛副结核病易感/抗性基因，研究内容包括：1）采用ELISA方法检测奶牛血清副结核菌抗体水平，基于牛50K SNP芯片，采用基于case-control设计的全基因组关联分析（GWAS）检测与奶牛血清副结核菌抗体水平病显著关联的SNP位点，通过生物信息学分析，鉴定奶牛副结核病易感/抗性基因并功能注释；2）采用基于第二代高通量测序的RNA-seq、miRNA-seq、CHIP-seq技术，针对副结核菌感染且临床发病、感染但未发病、健康的奶牛个体空肠组织进行转录组、microRNA、转录因子和组蛋白修饰分析，鉴定差异表达基因及调控通路；3）综合上述4个层次的数据分析结果，筛选易感/抗性候选基因并进行大群验证及细胞水平功能分析，最终鉴定奶牛副结核病易感/抗性关键基因和致因突变，为实施奶牛副结核病抗性标记辅助预选青年公牛和母牛提供有效的分子信息。

奶牛副结核病（约翰氏病），是由副结核分支杆菌（MAP）感染导致的慢性肠道疾病，在全世界范围内引起奶业巨大经济损失，为了解该病的遗传机制，本研究从宏观和微观角度开展了系统研究：（1）采集了北京地区10,000头中国荷斯坦母牛血清样本并进行抗体检测，根据ELISA测定结果，采用线性模型、二分类和有序阈模型对副结核病易感性遗传力进行估计，结果为0.0389到0.1069；（2）采用Case-control策略（阳性185头，阴性760头）、基于Illumina Bovine 50 K（54,609 SNPs）和GeneSeek HD（138,893 SNPs）芯片数据进行个体基因分型，利用GRAMMAR-GC和ROADTRIPS软件进行全基因组关联分析，分别检测到15和11个全基因组水平显著SNPs（P < 5×10-5）；（3）对血清和粪便双阳性且临床发病、血清和粪便双阳性但未发病及健康组荷斯坦母牛的空肠组织进行RNA-seq和Small RNA-seq。共鉴定到副结核易感/抗性候选功能基因21个、lncRNAs 10个、miRNAs 28个和 ceRNAs 144个。研究结果为中国荷斯坦牛副结核的抗病育种提供了有价值的基因信息并初步发现了基因/非编码RNA的部分调控机制。