鸡蛋用性状相关的CNVs筛选及其影响鸡产蛋的机理研究

Copy number variations(CNVs),a new kind of genomic structure variations that seems to be at least as important as SNPs(Single Nucleotide Polymorphisms),is recently discoveried in the genomics of many species.It is found in human that CNVs are related with many muti-factors diseases and height.The objectives of this proposal are to screen CNVs that are associated with chicken egg performances by using association analysis based on the initial map of CNVs in the chicken just published and a population of specialized male lines breeding now with characteristics of recessive white plumage color and dwarf body size, and to study the mechanisms on how the CNVs affecting chicken egg-laying. The research will be divided into two parts,i.e.① to obtain 2 to 3 candidate CNVs loci that are related with chicken egg performances,the quantitative PCR will be used firstly to detect the copy numbers of different individual in the population at the CNVs loci selected from the initial map and then genotyping of different individual will be conducted by using segregatin analysis integrating the pedigree information of individuals and at last the association analysis will be operated between the genotypes identified and the egg performances. And ② some resources families including the individuals with different genotypes at different candidate loci will be constrcucted and the tissues from different organs that are involved into the egg formation and laying will be collected at first,and then the high-throught cDNA expression profiling sequencing will be used to know the differented expression genes in different tissues of the individuals with different genotypes at each candidate CNVs loci,and finally,the Pathway enrichment analysis will be conducted,based on the differentiated expression genes, to find the gene regulating pathways that the candidate CNVs loci affecting the chicken egg-laying.The results of this project will be helpful for knowing whether the CNVs will affect the quantitative traits or not and the mechnisms on how the CNVs affecting chicken egg-laying,providing new kind of melocular marker for the breeding of quality layer.

拷贝数变异（CNVs）是新发现的一种与单核苷酸多态（SNPs）同等重要的基因组结构变异，人类中研究发现CNVs与许多多因子疾病及身高等表型相关。本研究拟利用已发表的鸡CNVs初级图谱,筛选与鸡蛋用性状相关的CNVs位点并研究它们影响鸡产蛋的机理。研究分两部分进行：① 以隐性白羽矮脚欣华鸡专门化父本为材料，采用qPCR法鉴定CNVs位点的拷贝数、利用分离分析法结合系谱信息确定个体CNVs位点基因型，然后利用群体蛋用性能记录进行关联分析筛选出2-3个影响鸡蛋用性状的候选CNVs位点。② 构建资源家系繁殖在候选CNVs位点具有不同基因型的个体，采集与产蛋相关的器官组织样，通过表达谱测序了解不同基因型个体组织中的差异表达基因，并通过Pathway富集分析构建CNVs影响鸡产蛋的基因调控通路。研究结果将有助于了解CNVs是否影响数量性状及CNVs影响鸡产蛋的机理，并能为优质蛋鸡育种提供新的分子标记。

近年来，通过高通量测序和基因芯片的方法，基因组中大量的结构变异被发现，本研究尝试将拷贝数变异运用到蛋鸡数量性状的选育中去。前期根据别人已发表文献中关于鸡基因组中CNV的报道，结合鸡的数量性状基因座数据库，挑选了17个候选CNVRs，在每一个CNVR上设计两条特异探针进行拷贝数的检测。实验材料为湖北欣华鸡E系，每只欣华鸡都有明确的产蛋记录及40周龄的蛋品质记录。拷贝数检测结果显示有三个位点存在拷贝数变异。关联分析的结果显示位点14B的拷贝数能够关联上开产日龄（p=0.0086）和250天产蛋量(p=0.036)；位点05A的拷贝数能够关联上蛋黄重（p=0.04）。对鸡的各组织进行采集及定量分析，发现位于位点05A下游的TMEM86A基因，在卵泡的膜细胞与颗粒细胞的等级前与排卵前都存在表达差异，推测其可能与卵黄的沉积有关。另外根据位点14拷贝数的不同对Pcdhα基因簇的表达进行分析，发现在垂体中高拷贝组（拷贝数大于2.5）的Pcdhα基因的表达量显著高于拷贝缺失组（p=0.042）。位点14B位于Pcdhα基因簇的内含子中，然而Pcdhα基因簇的结构和功能在鸡上鲜有报道，在已知的多个可变外显子的上下游设计正反向引物，并随机组合进行PCR扩增，我们发现了6个新可变外显子，将该基因簇的可变外显子在4个品种的鸡（欣华鸡，茶花鸡，吐鲁番斗鸡，藏鸡）中进行扩增鉴定，发现大部分的鸡仅拥有部分的可变外显子，个别可变外显子（V5,V9）存在品种差异。鉴于Pcdhα基因簇参与神经元之间树突与轴突的连接等功能，而高拷贝的个体有更多的可变外显子可供表达，神经发育更完善，我们猜测完善的神经系统可以更好地调节神经肽类的激素的分泌，促使蛋鸡开产日龄的提前和产蛋数的提高。