安义瓦灰鸡“灰羽”形成的分子机理解析

Plumage color is one of the important varietal characteristics in chicken breeds and molecular genetic studies on plumage color have important implications for the origin, evolution genetic, resource conservation and product quality control in the domestic fowl. Anyi tile-grey chicken is a unique breed with a grey plumage color within all Chinese indigenous chicken breeds, and up to now, the molecular mechanism for causing the grey pigmentation is still unknown. Based on the above, the objective of this study is to dissolve the molecular mechanism of grey plumage coloration in Anyi tile-grey chicken via genetic and genomics approach. Firstly, we herein would perform a genome wide association analysis (GWAS) to mapping the grey loci via genotyping a F2 resource population intercrossed by Ningdu yellow chicken breed and Anyi tile-grey chicken breed with the Affymetrix chicken 600k high density single nucleotide polymorphism(SNP) array. Meanwhile, we would detect the selection signature for the grey feather coloration by using a selective sweep method followed by an Identical-by-descent (IBD) mapping on the top selected region related to the grey phenotype, and so then we would refine the interval with a major effect on the grey pigmentation through combining above results. Finally, the major gene and causative mutation for the grey plumage color would be identified by a combined analysis including the positional candidate cloning and functional verification. The findings will not only greatly enrich our realization of the resource characteristics of the Anyi tile-grey chicken and deepen understanding of the genetic architecture of plumage color, but also facilitate to improve the purification of the Anyi tile-grey chicken breed and breeding procedure on the grey plumage color.

羽色是鸡的重要品种特征之一，其分子遗传研究对于探讨禽类起源及演化遗传、资源保护和产品质量控制等方面都具有重要意义。安义瓦灰鸡是我国唯一以“灰羽”为特征的珍稀地方鸡种，但截止目前，其灰羽形成的分子机理未知。基于此，本研究拟采用遗传学和基因组学研究手段，利用宁都黄鸡和安义瓦灰鸡为亲本的自交F2资源群体，开展基于鸡全基因组600K SNP芯片的全基因组关联分析，同时利用不同羽色鸡品种芯片数据开展安义瓦灰鸡“灰羽”选择性清除分析、染色体共享区段（IBD）分析，共同精细定位安义瓦灰鸡“灰羽”基因座位；最终通过位置候选克隆和功能验证分析鉴别到安义瓦灰鸡的灰羽基因及其因果突变位点，进而科学地阐述安义瓦灰鸡“灰羽”形成的分子机理。预期研究结果不仅将丰富人们对安义瓦灰鸡种质资源特性的认识、加深对家鸡羽色形成的分子机理的了解，还将有利于安义瓦灰鸡纯种的提纯以及以安义瓦灰鸡为亲本之一培育新品系时羽色的选育改良。

为解析安义瓦灰鸡“灰羽”表型形成的分子机制，利用安义瓦灰鸡和宁都黄鸡为亲本构建了F1代正反交群体，F1群体中灰羽个体在两种性别之间分布无差异，初步推定常染色体上存在一个控制灰羽的位点，该位点具有稀释黑色素的作用。.采用RNA-seq技术对 F1代群体中具有纯黑羽、灰羽和黄羽表型的各3个个体的新生羽毛毛囊组织进行转录组测序，共发现黑羽、灰羽和黄羽个体中特异表达基因分别为720个、351个和333个，与黑羽组相比，灰羽组有68个基因显著上调表达，57个基因显著下调表达，其中与羽色形成相关的功能基因有9个。采用GO和KEGG对上述DEGs进行功能和通路富集分析，发现一条由TYR、WNT16、WNT5B和EDNRB2等基因主导的黑色素生成通路与“灰羽”的形成显著相关。.利用鸡Affymetrix 600k SNP芯片和国产55K芯片，对包含安义瓦灰鸡在内的江西省9个地方鸡种开展了遗传多样性和种群间的遗传关系分析，结果显示安义瓦灰鸡和宁都黄鸡、广丰白耳黄鸡及修水黄羽乌鸡等群体遗传关系最近。基于Fst和LSBL两种选择信号检测分析，结合基因注释、单倍型构建成功鉴别到1号染色体上SNP AX-75499197（位于SOX5基因）和Z染色体上SNP AX-77270872两个标记的临近区域。为精确定位“灰羽”基因，对安义瓦灰鸡、宁都黄鸡、白耳黄鸡和修水黄羽乌鸡进行了平均深度约~18X的全基因组重测序，开展了基于Fst和π ratio的选择效应检测和基于case-control模型的全基因组关联分析，最终筛选到显著与“灰羽”形成相关的候选基因，分别为1号染色体上的SOX5基因和位于11号染色体上MC1R、TCF25、TUBB3、DEF8、DBNDD1和GAS8基因，结合MC1R基因突变检测表明灰羽很可能是在黑色素形成基础上受到稀释位点作用而形成，为进一步鉴定瓦灰鸡“灰羽”因果基因和因果突变奠定基础。