东湖杂交羊产羔性状的GWAS分析及候选基因GRID 2的验证

GWAS Analysis of Lambing Traits in East Friesian and Hu Crossbred Sheep and Verification of Candidate Gene GRID 2

旨在研究影响东湖杂交羊产羔性状的关键基因并挑选关键基因的变异位点进行验证,以期为东湖杂交奶绵羊多羔性状的选育提供分子标记。本研究从西北农林科技大学金昌奶绵羊试验示范基地采集身体健康无疾病的东湖杂交二代奶绵羊168只进行全基因组重测序,GEMMA和SnpEff软件进行全基因组关联分析,KOBAS 3.0数据库对注释的基因进行富集分析,LDBlockShow软件对显著性最强SNPs周边的连锁区域进行连锁不平衡分析。使用竞争性等位基因特异性PCR(kompetitive allele-specific PCR,KASP)技术对GRID 2基因进行SNP的分型检测,统计该基因两个多态位点的遗传多样性参数,并将其与产羔性状进行关联分析。结果显示,全基因组关联分析确定了6号染色上1163个达到显著性水平的SNPs位点,通过对候选位点的注释,确定与产羔性状相关的候选基因BMPR 1B、PDLIM5、PDHA2、UNC 5C、GRID2、NFKB1、TSPAN5、STPG2、PPP 3CA。同时结合已有报道和最强关联区域的注释,选择GRID 2上的两个SNPs(g.35685218A>G和g.35685499C>T)作为候选位点。通过统计学分析发现,本研究中涉及的两个位点均处于P>0.05的哈代-温伯格平衡状态,表明这两个位点均没有受到过强选择,并且这两个位点与东湖杂交羊的产羔性状有显著关联,两个位点的野生型均处于优势状态,且两个位点在胎次增加时均值也增加。综上所述,GRID 2基因2个SNPs对东湖杂交奶绵羊产羔性状有显著影响,可以作为分子标记辅助选择的候选位点。本研究为提高东湖杂交奶绵羊产羔率,促进奶绵羊的遗传育种进展提供了坚实的理论依据。

The experiment aimed to study the key genes affecting lambing traits in East Friesian and Hu crossbred sheep and select the mutation loci of the key genes for validation,to provide molecular markers for the selection and breeding of multiple lambing traits in East Friesian and Hu crossbred sheep.In this study,168 healthy and disease-free East Friesian and Hu crossbred sheep were collected from the Jinchang Milk Sheep Experimental Demonstration Base of Northwest Agriculture and Forestry University for whole-genome resequencing,genome-wide association study was performed by GEMMA and SnpEff softwares,enrichment analysis of annotated genes was conducted by KOBAS 3.0 database,and analysis of the chain region around the most significant SNPs with LDBlockShow software.Linkage disequilibrium analysis was performed on the linkage regions around the SNPs by LDBlockShow.SNPs were typed in the GRID 2 gene using kompetitive allele-specific PCR(KASP),and the genetic diversity parameters of the two polymorphic loci of the gene were counted and analyzed in association with lambing traits.The results showed that genome-wide association analysis identified 1163 SNP loci at the significance level on chromosome 6,and candidate genes BMPR 1B,PDLIM5,PDHA2,UNC 5C,GRID2,NFKB1,TSPAN5,STPG 2,and PPP 3CA were identified as associated with lambing traits through the annotation of the candidate loci.The two SNPs on GRID 2(g.35685218A>G and g.35685499C>T)were also selected as candidate loci by combining the existing reports and annotations of the most strongly associated regions.Statistical analysis revealed that both loci involved in this study were in Hardy-Weinberg equilibrium at P>0.05,indicating that neither locus had been subjected to over-selection and that both loci were significantly associated with lambing traits in East Friesian and Hu crossbred sheep,with wild-type being dominant at both loci and the means of the two loci increasing with the increasing parities.In summary,the two SNPs in the GRID 2 gene have significant effects on lambing number traits in East Friesian and Hu crossbred sheep and can be used as candidate loci for molecular marker-assisted selection.This study provides a solid theoretical basis for improving the lambing rate of East Friesian and Hu crossbred sheep and promoting the progress of genetic breeding of milk sheep.