猪背膘厚主效基因捕获及变异序列构建

As we all know, most Chinese indigenous pig breeds (fat type) own more thicker backfat (>4 cm) than Western commercial pig breeds (meat type) (<2 cm). The mechanism of inheritance for backfat thickness is very complex. A genome-wide association study (GWAS) has been applied to detect the SNPs associated with backfat thickness in a Large White × Minzhu F2 design population. Our previous results indicated that eight SNPs in a 1.8 Mb region on SSC7 showed genome-wide significant association with backfat thickness. Twenty-four annotated genes were contained in this region. According to the pre-existing results, the aim of present project is to capture the major gene for backfat thickness throughout the study in DNA and RNA level. For DNA level, haplotype sharing analysis and selective sweep analysis will be applied together to refine the region from previous GWAS result. For RNA level, Those genes that are not expressed or with low level in backfat tissues in both Minzhu and Large White will be eliminated. Significantly tests will be done for all retained genes express level at 60, 120, 150, 180 and 200 d between Minzhu and Large White. Synthesizing the obove information, the major gene of backfat thickness will be captured in the significant region from GWAS. Several individuals of Minzhu with H+ and Large White with H- will be selected as QQ and qq genotypes, respectively, by haplotype association analysis. The Agilent SureSelect Target Enrichment will be applied to obtain the whole sequences of major gene in genomes of the H+ Minzhu and H- Large White individuals, respectively. Variations will be detected in the whole region of major gene between Minzhu and Large Whilte and the variation seqences of the two pig breeds will be constructed. The success of this project will provide experience for capturing the major gene from complex traits GWAS results and promote the advance of molecular breeding for backfat thickness in pig.

中外猪种背膘厚的巨大差异有着极其复杂的分子遗传机制。申请人前期在构建的大白猪×民猪F2设计资源群体中，运用全基因组关联研究（GWAS）方法，检测发现7号染色体上存在8个与背膘厚性状显著关联的SNP位点，区间跨度1.8Mb，包含了24个已知基因。本项目旨在及时利用前期研究的成果，通过DNA水平、RNA表达水平开展背膘厚性状主效基因捕获。拟利用单倍型分析、选择性清除分析获得最小显著区间，通过民猪、大白猪GWAS区间内所有基因表达规律及表达差异研究缩减候选基因数量，结合生物信息学综合分析捕获主效基因。以主效基因所在单倍型开展关联分析，以H+民猪、H-大白猪分别作为QQ、qq型个体，开展主效基因全基因序列片段靶向捕获及重测序，获得两猪种间变异位点，构建出猪种间变异位点序列。本项目的实施不但为动物复杂性状GWAS后捕获主效基因方法研究方面有重要理论意义，且为以降低背膘厚为目的的分子选育提供理论基础。

背膘厚性状一直都是猪育种的重要目标性状之一，经济价值巨大。多年的研究表明，中外猪种背膘厚的巨大差异有着极其复杂的分子遗传机制。本课题利用大白猪×民猪F2代资源群体，对其应用Illumina Porcine SNP 60K BeadChip基因分型，之后对猪的6-7肋性状进行了全基因组关联分析（Genome-wide Association Study, GWAS），发现了7号染色体上31.23~47.41 Mb区间内共35个全基因组水平显著关联SNP位点。通过单倍型分析、选择性清除分析方法对背膘厚GWAS显著区间进一步分析，将显著区间缩小至0.50 Mb（34.76~35.33 Mb）区段。对该区域内的六个已注释的基因（HMGA1、GRM4、NUDT3、RPS10、SPDEF和PACSIN1）在大白猪、民猪和二花脸背膘组织荧光定量PCR分析，发现只有HMGA1基因在中外猪种间有显著性的表达差异（P< 0.05）。因此，HMGA1被确定为猪背膘厚的主效候选基因。对小鼠3T3-L1前体脂肪细胞系HMGA1基因进行了RNAi试验，对转染了siRNA的细胞和阴性对照细胞诱导分化后进行荧光定量试验，结果显示干扰后，HMGA1、C/EBP-β和PPARγ基因的mRNA表达量都显著下调（P< 0.05）。此结果说明了HMGA1在3T3-L1前体脂肪细胞分化为脂肪细胞的过程中发挥重要作用。比对大白猪×民猪F2代资源群体中的祖代民猪和大白猪全基因组重测序数据发现，在HMGA1基因启动子及外显子区域共发现19个SNP位点。在北京黑猪和杜洛克猪群体中的多态性与背膘厚关联研究，未发现与背膘厚性状显著关联SNP位点。因此，虽然本研究确定了HMGA1基因为背膘厚性状首选候选主效基因，但其QTN还需进一步深入探索研究，本研究为深入挖掘猪背膘厚主效基因及探索背膘沉积调控机制奠定了研究基础。