全基因组关联分析鉴别白色杜洛克×二花脸F_2资源家系中猪阴囊疝易感基因位点的鉴定

Genome-Wide Association Study Reveals Candidate Susceptibility Loci for Pig Scrotal Hernia Using Both F_2 Intercross and Outbred Populations

【目的】通过基于单倍型的病例-对照全基因组关联分析(GWAS)鉴别白色杜洛克×二花脸F2资源家系中影响猪阴囊疝的易感位点及位置功能候选基因,并在远缘纯种猪阴囊疝三联体核心家系(Trio)群体中进行重复验证。【方法】利用Illumina porcine 60K SNP芯片对1 020头白色杜洛克×二花脸F2资源家系阴囊疝群体(包含19个阴囊疝患病个体)进行扫描获取基因型。通过Plink v1.07软件对基因型数据进行质量控制;剔除个体基因型检出率<90%的个体,剔除SNP位点检出率<90%、哈迪温伯格平衡卡方检验P≤10-3和最小等位基因频率<0.05及性染色体上和无法定位的SNP位点。质控合格的SNP位点用PHASEBOOK构建出F2资源家系中每个个体的单倍型,并采用隐马尔可夫模型将其归类到数目预先定义的祖先单倍型中。使用基于广义线性混合模型的GLASCOW软件进行利用祖先单倍型的病例-对照全基因组关联分析。采用保守的Bonferroni校正方法得到基因组显著水平和染色体显著水平的阈值。对F2资源家系中达到染色体显著水平的易感SNP位点在包含237个患病个体的远缘纯种猪阴囊疝三联体核心家系(Trio)群体中进行同样的质控,并利用Haploview软件对质控合格的SNP位点分别展开基于单点和基于单倍型的传递不平衡分析(TDT),进行重复验证。【结果】白色杜洛克×二花脸F2资源家系中全部个体的38 033个SNP标记通过质控。在GWAS分析中,共发现108个达染色体显著水平(P<2.63×10-5)的猪阴囊疝关联SNP位点,分别位于染色体2、8和17上,最强相关的SNP位点位于17号染色体上。在远缘三联体核心家系群中,724个个体的96个SNP位点通过质控。对质控合格的SNP位点进行基于单点的TDT分析,结果发现5个显著相关的SNP位点得到重复验证(P<0.05),其中2号染色体上有1个SNP位点和17号染色体上有4个SNP位点,分别位于IQGAP2,CHMP4B,SERINC3,ZNF334等4个基因内或其上下游。基于单倍型的TDT验证分析发现两个易感单倍型框,其中与基于单点TDT验证分析有重合的仅有SSC17上CHMP4B内及下游的两个易感位点。结合疝气发生机制及TDT分析结果,推测IQGAP2和CHMP4B可能是影响猪阴囊疝发生的两个重要的位置功能候选基因。【结论】本研究利用基于小样本的GWAS分析和较大样本群验证分析,在SSC2和SSC17上分别鉴别1个和4个阴囊疝易感位点,在SSC17上鉴别到两个易感单倍型。易感位点附近的2个基因IQGAP2和CHMP4B可能是影响猪阴囊疝发生的位置功能候选基因,值得进一步研究。

【Objective】Isolation of susceptible loci and positional candidate genes associated with pig scrotal hernia through case-control genome-wide association study（GWAS） based on haplotypes in a F2 White Duroc × Erhualian intercross and it＇s replication in outbred trio families.【Method】Phenotypic data and Illumina porcine 60 K SNP genotypes were collected from 1020 individuals in the White Duroc × Erhualian intercross（containing 19 affected pigs） and outbred trio families（containing 237 scrotal hernia pigs）. Quality control was carried out using PLINK v1.07 for each population, separately. Samples of low（90%） call rate were removed. SNP markers were removed if they had genotype-missing rates 0.1, or Hardy-Weinberg P≤10-3（based on Chi-squared test） or minor allele frequencies（MAF） 0.05, and SNPs on sex chromosomes or with unknown positions were also excluded. PHASEBOOK package was used to infer haplotypes from the genotyped individuals and to assign them to K ancestral haplotype clusters by hidden monto carlo markov models. The software GLASCOW based on generalized linear mixed models was used to carry out GWAS for pig scrotal hernia in the F2 intercross by the ancestral haplotype and significant SNPs were chosen to perform transmission disequilibrium test（TDT） based on both single SNP markers and haplotypes in the outbred population（containing 237 affected pigs） by Haploview. 【Result】After quality control, all samples in F2 population and 724 samples in outbred populations passed the filter and a final set of 38 033 SNPs and 96 SNPs were selected for GWAS and TDT analysis. In total, 108 SNPs with Bonferroni chromosome-wise significance（P2.63×10-5） were identified to be associated with scrotal hernia in F2 intercross, and located on SSC2, SSC8 and SSC17, respectively. The most promising SNP was located on SSC17. Five significant SNPs, located in on nearby IQGAP2,CHMP4 B,SERINC3,ZNF334 genes, were replicated（located on SSC2 and SSC17） in outbred trio families by TDT analysis. TDT analysis based on haplotype revealed that two haplotypes contribute to risk of scrotal hernia, one of which was overlapped with two significant SNP on SSC17 in the TDT analysis based on single marker. IQGAP2 and CHMP4 B genes close to the SNPs most associated with pig scrotal hernia were selected as positional candidate genes according to its biological and physiological functions.【Conclusion】A GWAS with small samples was conducted and variation and replication test was performed in a larger populations, which revealed 5 susceptible loci for pig scrotal hernia both in the F2 intercross and the outbred populations. IQGAP2 and CHMP4 B may play a role in the risk of scrotal hernia, and deeper analysis should be carried out in the future study.