Identification Of Growth-related Genes Based On Multi-association Analysis And Their Potential Gene Function Verification In Pig

Domestic pig is one of the earliest domesticated animals.It is the primary source for human meat consumption.Growth trait is a complex trait which controlled by many genes,for which conventional method is difficult to identify major genes responsible.Genome-wide association study(GWAS)is considered as an efficient way to identify candidate gene or causative mutation of complex traits.So in this study,GWAS was used to identify the SNPs correlated to pig growth,which neiboring genes were analyzed by further function assay.The thesis was studied in detail as below:(1)GWAS for growth trait using high-density SNP chip in male Duroc population(GWAS)Based on a 796 male Duroc population,GWAS was performed to identify the SNP associated with growth trait(D100,FCR and ADG).The results revealed that 11 genome-wide significant SNPs and 162 chromosome-wide significant SNPs.Gene ontology analysis identified a list of 14 candidate genes close to significant SNPs,with growth-related functions:six for D100(WT1,FBX03,DOCK7,PPP3CA,GPAT3 and NKX6-1),seven for FCR(MAP2,TBX15,IVL,ARL15,CPS1 VWC2L and VAV3),and one for ADG(COL27A1).Gene functional analysis indicated most of the candidate genes are involved in muscle,fat,bone or nervous system development,nutrient absorption,and metabolism.Additionally,we found haplotype blocks composed of suggestive SNPs located in the growth trait-related QTL and further narrowed down the ranges,of which haplogype block located in SSC10 decreased by?60Mb.(2)Analysis of genetic effect of GPAT3 gene on pig growth trait(post-GWAS)Above GWAS result revealed the SNP ASGA0040372,located at upstream of GPAT3,associated with growth trait.Based on this,pig complete GPAT3 mRNA were obtained,containing 14 exons and three alternative splice variants by cloning and RACE strategy.The obtained five SNPs(including ASGA0040372)were used to confirm their genetic effects on pig growth by association study in another 289 Duroc pig population.The results indicated that three SNPs located at beginning of pig GPAT3 gene are high association with average daily gain(ADG)or days to 100 KG traits(P-value<0.05).Subsequent analysis suggested the promoter region of GPAT3 gene from-950 to-500bp exhibited the highest activity followed by the region from-500 to-229bp,-1262 to-950bp and-1424 to-950,and the ASGA0040372 play a significant effect on the promoter activity.From the above,GPAT3 gene is significant correlated to pig growth rate and significant effect of ASGA0040372 on GPAT3 promoter activity was observed.(3)Negative effect of ARL15 gene on pig feed conversion ratio(post-GWAS)The complete mRNA of pig ARL15 was first obtained,and eight SNPs(including 4 novel SNPs)was obtained in Duroc pig population.The results of Association analysis indicated that SNPs and haplotypes is significantly associated to FCR trait(p<0.01).Lentivirus-mediated ARL15-overexpressed 3T3-L1 cell model was established to uncover the biological function of ARL15.Combined with RNA-seq result,we observed that ARL15 could increase the expression and secretion of Adiponectin(Adipoq)and upregulation of Adipoq controlled by ARL15 is independent of PPARg signal pathway.Furthermore,enhancement of genes involved in energy metabolism pathway(PPARg,AP2,GLUT4,DGAT2,Acly,Acaca etc.)was observed with the treatment of high-dose ARL15.Combining our result with the biological function of Adipoq,it could be concluded that ARL15 enhanced the energy consumption in vivo by increasing the Adipoq expression,and further decreased the feed conversion efficiency and influenced the pig growth.Taken together,this study identified the SNPs and genes correlated to growth traits based on GWAS.Replication of association analysis and wet-lab experiment were performed to study the function of GPAT3 and ARL15,which provide a sufficient evidence for GWAS result.