【作者】 王振；

【导师】 潘玉春；

【作者基本信息】 上海交通大学 ， 生物医学工程， 2015， 博士

【摘要】 太湖流域地方品种猪具有繁殖力高、抗病力强、耐粗饲、性情温驯、肉质鲜美、杂种优势等许多优良经济性状,尤其繁殖力在所有猪种中几乎最高。阐明这些复杂性状（complex traits）的分子遗传机制（molecular genetic mechanism）是当前猪遗传育种工作者的重要研究课题。随着研究的不断深入、技术的不断进步以及研究方法的不断更新,单纯依靠传统的单个或少数几个位点（基因）的研究显然已经不能满足研究需要,而从基因组层面（genome-wide）进行整体研究、揭示上述重要经济性状的遗传机制、实现遗传改良的要求已变得越来越迫切。然而令人遗憾的是,迄今为止,只有少量研究针对某一个或几个太湖地方品种进行了全基因组范围遗传变异的研究,且实验样本较少。而全面系统的比较6个太湖品种（包括梅山猪、二花脸猪、米猪、枫泾猪、沙乌头猪和嘉兴黑猪）,在全基因组范围内对其遗传变异进行普查的研究工作目前尚未见报道;并且这些种质特性的遗传机制研究更是远未达到令人满意的地步,其遗传机制尚不清楚;此外,对于这些品种、类群等的划分多依赖于表型、产地、分布而缺乏充分的分子遗传依据。据此,本研究拟开展以下工作:（1）对太湖流域地方猪群体的基因组进行遗传变异（SNP、Indel和CNV等）检测;（2）全基因组范围内的遗传标记在保种等生产实践上的应用,即利用分子标记进行群体遗传多样性和群体结构研究;（3）综合考虑QTL、KEGG和GO等基因注释库信息,对上述基因组遗传变异的功能进行初步预测分析;（4）全基因组范围内的SNP遗传标记在揭示遗传机制上的应用,即结合选择信号分析对太湖流域地方猪群体繁殖力高、抗病能力强、性格温顺等优良性状遗传基础进行研究。具体研究内容和结果如下:一、全基因组遗传变异检测:利用本实验室构建的简化基因组测序平台——GGRS,对太湖流域6个品种,共计252头地方猪进行了全基因组范围内遗传变异的检测。共检测到105,550个SNP、122,632个Indel、31,444个Insertion、44,056个Deletion和455个CNV。与当前dbSNP数据库比对的分析,发现其中有29,575个为新的SNP,这表明太湖流域地方品种存在一定程度的遗传特异性。进一步分析表明,SNP在单个染色体上的分布相对均一,但在不同染色体上数量和密度的分布存在一定差异,其MAF的分布偏向中低值。这些结果将有利于对太湖地方品猪种遗传基础的认识,同时也丰富了中国地方品猪种的分子标记数据库。二、遗传多样性及群体结构分析:品种或类群的划分,直接关乎一个资源群体的保护力度和重视程度,因此合理的品种划分才能更好地实现对当前种质资源的有效保护和执行国家对其有效利用的战略。然而,当前品种和类群的划分多依赖于表型、产地、分布而缺乏充分的分子遗传依据。为此,本研究应用全基因组范围内的遗传标记,对太湖流域地方猪种群体进行了遗传多样性和群体结构的分析。首先对反映群体内遗传多样性的三个重要指标:等位基因的丰度（allelic richness,A_R）、标记多态性的比例（the proportion of polymorphic markers,P_N）、期望杂合度（expected heterozygosity,H_E）进行了分析、计算。结果显示:米猪基因丰度值最高（A_R=1.736）,而枫泾表现最低（A_R=1.481）;P_N范围为0.946（长白）到0.999（二花脸）;长白猪杂合度最高（H_E=0.375）,而米猪最低（H_E=0.343）。其次对群体间的遗传多样性进行了评估。结果显示,二花脸和米猪之间的群体分化指数(F_ST)最小,低于小梅山和中梅山之间的分化程度;PCA、STRUCTURE和NJ-tree等分析均显示各个品种（包括小梅山和中梅山）之间能够清晰地进行聚类区分。以上结果表明,当前太湖流域地方品种划分整体而言是合理的,但需要进行局部微调,若把小梅山和中梅山当作两个品种而非两个类群进行保种或许更加合理。三、遗传变异功能预测分析:为了进一步探究各类变异的初步功能,我们利用当前已有的基因注释库（如NCBI、QTL、KEGG和GO等）对这些变异进行了初步的功能注释。研究结果显示:各类变异位于基因上的比例在27.5%³9.5%之间,位于QTL上的比例在25%左右。基因富集分析结果表明,不同类型的变异在不同的通路上发生了富集,SNP主要与脂肪相关,Indel更多的与疾病相关,Insertion和Deletion与内吞作用相关,而CNV与神经功能相关。此外,我们还发现含有高密度变异的QTL主要集中在胴体与肉质性状、健康性状。这些研究结果,将有利于我们对太湖流域地方猪基因组遗传变异的分布特点及其功能的了解,为进一步深入地研究其功能提供参考。四、选择信号分析:经历了长期的人工或者自然选择,太湖流域地方品种猪群体形成了今天繁殖力高、抗病力强等优良性状,这些选择作用很可能在其基因组上留下了印迹。有鉴于此,我们采用群体内（REHH）和群体间（XPEHH）两种选择信号检测方法,对太湖流域地方品种猪群体进行了全基因组选择信号的检测分析。研究结果显示:在太湖群体基因组上分别发现282（REHH法）和112（XPEHH法）个选择信号候选区域,对应基因组长度为159.78 Mb和62.29 Mb,占基因组总长的6.15%和2.40%。进一步对这些选择信号候选区域进行了基因或QTL映射分析,发现许多基因或QTL与繁殖（比如TLR9基因）、毛色（比如KIT基因）、脂肪代谢（如CPT1A和MAML3基因）等表型相关。此外,通过对选择信号候选区域内基因的富集分析,发现了多个显著的基因富集通路,其中多数与疾病相关,如哮喘、免疫性甲状腺病等。因此,选择信号候选区内的基因可以作为与猪繁殖、抗病等性状相关的基因集,用于证实这些基因功能的后续研究。更多还原

【Abstract】 Taihu pig breeds have many outstanding economic traits,such as high prolificacy,high disease resistance,high resistance to crude feed,docile temperament and heterosis,especially they are the most prolific breeds of swine in the world.Revealing the molecular genetic mechanism of these complex trarits is an important issue for the genetic study.With the development of genetic study and biotechnologies as well as upgarating of study methods,the requirement to reveal the genetic mechanism of these complex traits in the genome level rathter than in the single or several genes level is becoming more intense.However,up to date,only few studies performed genetic variation analysis for Taihu pig breeds on the genome-wide.Moreover,these studies only contained few samples of one or several Taihu pig breeds.To our knowledge,the original survey of a genome-wide of genetic makers for all the six Taihu pig breeds has not yet been reported.What’s more,the genetic mechanism for their outstanding traits is still unclear.Additionally,the current classification of these breeds are mainly based on their characteristics of phenotype(such as body or ear size)or performance(reproductive or meat quality traits)(Resource 2011)as well as their locations.However,it has not been convinced from the terms of genetic characteristics.Therefore,this study will do the researches as follows:(1)Surveying the genome-wide of genetic variations for all the six Chinese Taihu pig breeds.(2)Applying the detected genetic markers for production practices such as breed conservation,etc,namely conducting the genetic diversity and population structure analysis to provide suggestions for breeds classification and future conservation.(3)Predicting their preliminary functions.(4)Applying the detected genetic markers for studies in revealing the genetic mechanism,namely performing the selection signature analysis to uncover the genetic mechansim of outstanding traits such as high prolificacy,high disease resistance for Taihu pig breeds.The results are as follows:(1)Genome variants detection.We detected genetic variants of 252 pigs related to the six breeds(Meishan,Erhualian,Mi,Fengjing,Shawutou and Jiaxing Black)in the Taihu Lake region using GGRS.In total,105,550 SNP,122,632 Indels,31,444 Insertions,44,056 Deletions and 455 CNVs(copy number variants)were identified in the genomes of the pigs.Among those SNPs,29,575(28%)were novel based on compariations with a database of known SNPs,it implied the specific of genetic variants in the Taihhu pig breeds.Further analyses indicated that SNPs were distributed in a relatively uniform fashion in the same chromosome,but its density for each chromosome were distributed in a non-uniform fashion(p < 0.001),and the distribution of the observed SNPs is skewed toward an intermediate frequency.These results are helpful to know the genome information of the Taihu pig breeds and to enrich the databse of genetic markers in the Chinese domestic pig breeds.(2)Genetic diversity and population structure analysis.The breed or sub-type classification of a population can bring a large effect for its protection and conservation.The requirement for effectively protecting and utilizing the genetic resource is to rationally classify them into the correct breed.The current classification of these breeds is mainly based on their characteristics of phenotype or performances as well as their locations.However,it has not been convinced from the terms of genetic characteristics.Therefore,we investigated the genetic diversity and population structure of the six Taihu pig breeds using whole-genome SNP data.Firstly,the allelic richness(AR),proportion of polymorphic markers(PN)and expected heterozygosity(HE)were used to investigate the genome-wide genetic variability within these six breeds.As a result,Mi and Duroc displayed the highest and least AR value compared with the other populations,PN value ranged from 0.946(Landrace)to 0.999(Erhualian),and HE value ranged from 0.343(Mi)to 0.375(Landrace).Secondly,the genetic differentiation shown by the fixation index(FST),principal component analysis(PCA),neighborjoining tree(NJ-tree)and STRUCTURE analysis were performed to investigate interpopulation genetic variability in the six Taihu pig breeds.The result of FST value indicated that the Middle Meishan and Small Meishan breeds exhibited a moderate level of genetic differentiation(FST = 0.099)that was greater than that of the Erhualian and Mi breeds(FST =0.055),which are now regarded as two breeds.What’s more,NJ-tree,PCA and STRUCTURE analysis showed that all the population could be well distinguished from each other(including the Middle Meishan and Small Meishan breeds).In conclusion,we confirmed the rationality of the current breed classification of the Chinese Taihu pig breeds,and we suggested that the Middle Meishan and Small Meishan breeds were separated into two breeds for conservation.(3)Genetic variants functional predicting analysis.We performed functional annotation analysis for these detected genetic variants based on the gene annotation databases(such as NCBI,QTL,KEGG,GO,etc).The results showed that the proportion of these variants in genetic regions ranged from 27.5% to 39.5%,and around 25% of them were in QTLs.The enrichment analysis for genes containing large-effect mutations for each type of variant suggested that they are enriched in different GOs and pathways,which indicated that the different types of variants may have different functions in the genome.SNPs were mainly related to adipose tissue.Indels were more related to disease.Both insertions and deletions were related to endocytosis,and CNVs were more related to nerve function.Moreover,we found the majority of QTLs with high density of variants were related to meat and carcass quality and health traits.Those findings can contribute to reveal Taihu pig breeds’ genetic variants distribution characteristics and functions and also provide valuable references for further studying their functions.(4)Selection signature analysis.Taihu pig breeds have experienced natural and artificial selection over a very long time and those factors might contribute to their current outstanding traits such as high prolificacy,high disease resistance.Therefore, in this study we used both Intra-population(REHH)and Inter-population(XPEHH)methods to detect genomic regions which might be under the process of selection for Taihu pig breeds.As a result,we found 282(REHH)and 112(XPEHH)selection signature candidate regions,corresponding to 159.78 Mb(6.15%)and 62.29 Mb(2.40%)of genomic regions.We found many genes or QTLs under the selection candidate regions were related to reproductive traits(such as TLR9 gene),coat color(such as KIT gene)and fat metabolism(such as CPT1 A gene and MAML3 gene).We further performed gene enrichments analysis for genes under selection candidate regions and found that most of the significant pathways were related to diseases such as autoimmune thyroid and asthma disease.Those findings were consistent with Taihu pig breeds’ traits characteristics of high prolificacy and high disease resistance.Therefore,the genes under selection candidate regions could be took as candidate genes set related to reproductive or disease resistance traits for future convincing.更多还原