基于GWAS的猪肉品质性状候选基因研究

发布时间：2018-04-29 00:18

本文选题：猪 + 全基因组关联分析　；参考：《中国农业大学》2016年博士论文

【摘要】：骨骼肌由肌纤维、脂肪、结缔组织和神经血管等构成,其中肌纤维性状和肌内脂肪是影响肌肉品质的主要因素。本研究利用大白猪×民猪F2设计资源群体,进行了肌纤维性状和部分肉品质相关性状遗传参数估计,并利用高密度SNP芯片,对猪肌纤维性状及肌内脂肪进行全基因组关联分析,对与肌纤维面积及肌内脂肪相关的显著SNP位点深入研究,初步筛选候选主效基因。1、用MTDFREML方法对肌纤维性状及部分肉质性状进行遗传参数初步估计,肌纤维直径、肌纤维密度、肌纤维面积、中间型肌纤维比例、红肌纤维比例、白肌纤维比例的遗传力分别为0.39、0.46、0.36、0.58、0.69、0.65,肌肉中的pH24、水份含量、滴水损失、肌内脂肪、肉色L值、大理石纹的遗传力分别为0.50、0.51、0.71、0.52、0.35和0.57,以上遗传力均超过0.3,属于高遗传力(h20.3)性状;肌内脂肪与肌纤维密度、pH24、L值、大理石纹中-高程度正遗传相关(rA=0.38～0.91),与肌纤维面积、肌纤维直径、白肌纤维比例、中间型肌纤维比例、水份等性状呈中-高程度负遗传相关(rA=-0.46-0.74)。2、采用全同胞-半同胞混合家系设计,应用混合线性模型,开展肌纤维性状全基因组关联研究(GWAS),将肌纤维直径、肌纤维面积、肌纤维密度显著相关的SNPs位点定位在猪3、4、5、6、7、14号染色体上。针对肌纤维面积性状GWAS显著SNP位点附近已注释基因(Sirt2、NFKBIB、KATNB1、ACP1)在不同日龄民猪、大白猪中荧光定量PCR实验发现,Sirt2基因和ACP1基因具有表达差异显著性。因此,Sirt2和ACP1被确定为肌纤维面积的候选主效基因。3、对肌内脂肪性状进行全基因组关联研究(GWAS),与肌内脂肪显著相关的35个SNPs定位于12号染色体上30个。对肌内脂肪GWAS显著SNP位点H3GA0022758和ALGA0119023附近的一个肌内脂肪性状候选基因MAP2K4基因进行了深入研究,克隆该基因的cDNA,获得cDNA全长3324 bp,包含了846 bp的ORF区,120bp的5'-UTR区和2358 bp的3'-UTR区,该基因编码的蛋白序列包含了281个氨基酸序列：针对MAP2K4基因在大白猪的不同组织中进行荧光定量PCR,该基因在多个组织中广泛表达,在肌肉组织中表达量较高。在不同日龄大白猪、民猪应用荧光定量PCR检测MAP2K4基因mRNA表达量,结果显示,120d民猪MAP2K4基因表达量显著高于大白猪(P0.05)。在北京黑猪群体中对MAP2K4基因中启动子区筛选SNPs位点,发现Chrl2:59330365处AG突变位点与肌内脂肪性状显著关联。对肌内脂肪最高显著点与第二显著点区段包含的10个已有注释的基因(USP43、DHRS7C、GSG1L2、GLP2R、RCVRN、GAS7、 MYH1、ADPRM、TMEM220和MYH2),以肌内脂肪全基因组关联分析最显著SNP位点(MARC0017000)分型,在F2资源群体AA、GG、AG型的个体中进行mRNA表达量检测,检测结果显示,MYH2基因不同基因型的mRNA具有表达差异显著性,综合候选基因的功能注释及试验结果,可将MAP2K4和MYH2基因作为候选基因进行下一步研究。
[Abstract]:Skeletal muscle is composed of muscle fiber, fat, connective tissue and nerve and blood vessel, among which muscle fiber and intramuscular fat are the main factors affecting muscle quality. In this study, the genetic parameters of muscle fiber traits and some meat quality traits were estimated by using F _ 2 design resource population, and the whole genome association analysis of pig muscle fiber traits and intramuscular fat was carried out by using high-density SNP microarray. The significant SNP loci related to muscle fiber area and intramuscular fat were studied, and candidate major gene. 1. The genetic parameters of muscle fiber and some meat traits were estimated by MTDFREML method. The diameter of muscle fiber and density of muscle fiber were estimated. The heritability of muscle fiber area, intermediate muscle fiber ratio, red muscle fiber ratio and white muscle fiber ratio were 0.39 ~ 0.46 ~ 0.36 ~ 0.36 ~ 0.68 ~ 0.68 ~ 0.69 ~ 0.65 respectively. The pH of muscle was 24, water content, drip loss, intramuscular fat, meat color L value. The heritability of marbling was 0.50 / 0.51 / 0.71 / 0.52 / 0.35 and 0.57 respectively, and the heritability of the above was more than 0.3, which belonged to the high heritability (H20.3), the density of intramuscular fat and muscle fiber (pH 24g / L), the positive genetic correlation between middle and high degree of marbling was 0.380.91, and it was related to the area of muscle fiber, the diameter of muscle fiber, the density of muscle fiber and the density of muscle fiber. The proportion of white muscle fiber, the proportion of intermediate muscle fiber and moisture were negatively correlated with middle and high degree of negative genetic correlation. The whole sib / half sib mixed family was designed and the mixed linear model was used. A genome-wide association study of muscle fiber traits was carried out. The SNPs loci of muscle fiber diameter, muscle fiber area and muscle fiber density were located on chromosome 77,14. For the muscle fiber area trait GWAS significant SNP locus, the annotated gene (Sirt2) NFKBIBN KATNB1ACP1) was found to have significant difference in the expression of sirt2 gene and ACP1 gene in different day old pigs and large white pigs by fluorescence quantitative PCR. Therefore, Sirt2 and ACP1 were identified as candidate major genes for muscle fiber area. GWASA was used to study the whole genome association of intramuscular fat traits. 35 SNPs, which were significantly related to intramuscular fat, were located on chromosome 12 30. In this paper, a candidate gene MAP2K4 gene for intramuscular fat traits near H3GA0022758 and ALGA0119023, which is the significant SNP locus of intramuscular fat GWAS, was studied. The cDNA of this gene was cloned. The length of cDNA was 3324 BP, including the 5'-UTR region of 846bp ORF region and the 3'-UTR region of 2358 BP. The protein sequence encoded by this gene contains 281 amino acid sequences: the MAP2K4 gene is widely expressed in many tissues and is highly expressed in muscle tissues for fluorescent quantification of MAP2K4 gene in different tissues of large white pigs. Fluorescence quantitative PCR was used to detect the mRNA expression of MAP2K4 gene in different day old big white pigs. The results showed that the expression of MAP2K4 gene in domestic pigs was significantly higher than that in large white pigs at 120 days. In Beijing Black Pig population, the promoter region of MAP2K4 gene was screened for SNPs locus. It was found that AG mutation at Chrl2:59330365 site was significantly associated with intramuscular fat traits. Among the 10 annotated genes USP43, DHRS7, GSG1L2, GLP2RN, MYH1, ADPRM, TMEM220 and MYH2, the most significant SNP loci in intramuscular fat genome association analysis were found to be MARC0017000. The expression of mRNA was detected in individuals of F _ 2 resource population. The results showed that the mRNA of different genotypes of MYH _ 2 gene had significant difference. The functional annotation of candidate gene and the results of the experiment were synthesised. MAP2K4 and MYH2 genes can be used as candidate genes for further study.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S828

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