Associaiton Analysis Between MC4R Gen Polymorphism And Body Weight And Body Size Traits Of Hu Sheep

In this study,202 female individuals of the new group G3 of the Hu sheep were used as experimental materials,and the MC4R gene was used as a candidate gene.Based on the gender identification,the genetic structure and diversity of Hu sheep population were discussed in this study by PCR amplification、direct sequencing and sequence analysis technique.In order to screen out the SNPs which had significant effects on body weight traits and body size traits of Hu sheep,we conducted the correlation analysis between SNPs and the two traits respectively and analysis of effects of candidate SNPs on gene transcriptional activity.The aim of this study was to provide theoretical foundation for sheep breeding.1.Sex identification of Hu sheepThe blood samples of the G3 generation of the core group of the Hu sheep were gender-identified.There were 202 female Hu sheep individuals filtered out for subsequent trials whose identification results were consistent with the practical production records.2.Correlation analysis between MC4R gene polymorphism and body weight and body size traits in Hu sheepIn this study,19 SNPs were detected in the core group of G3 generation in Hu sheep.15 loci among them were low polymorphism and the others were moderate polymorphism.g.222 C→T and g.222 C→G were strong linkage status.The genetic parameters of g.306A→G was the highest with rich polymorphism while the g.139 G→A and g.1382 G→C were the lowest with relatively narrow variation range.g.139 G→A and g.706 C→A were out of the Hard-Weinberg equilibrium state(P<0.05),and the remaining 17 SNPs were in the Hard-Weinberg equilibrium state.There are two SNPs in the coding region of MC4R gene which are significantly associated with the adult body height of female sheep.The g.548 C→T can lead the mutation from 112th threonine(the second transmembrane region and the extramembranous region of the third transmembrane region)to methionine while the g.732 position C→G can cause the mutation from 173th isoleucine(the fourth transmembrane region)to methionine.The adult body height(76.09±3.07 cm)of g.706 C→A wild CC-genotype female lake sheep was significantly lower than that of mutant homozygous AA-genotype individuals(79.50±1.91 cm)(P<0.05),but no significant difference with CA-genotype individuals(76.77±2.77 cm)(P>0.05).The adult body height of g.732 C→G wild CC-genotype females(77.57±2.49 cm)was significantly higher than that of mutant heterozygous CG-genotype individuals(76.20±2.88 cm)(P<0.05),which was significantly higher than the mutant homozygous GG-genotype individuals(75.72±3.30 cm)(P<0.01).There was no significant difference in adult height between GG-genotype of female mutant heterozygous individuals and CG-genotype(P>0.05).3.The effect of MC4R candidate functional SNPs on gene transcriptional activityThe results of dual luciferase reporter gene assay showed that the relative fluorescence activity of wild CCCC haplotype of ORF(g.706 C→A,g.732 C→G)was significantly higher than that of mutant AAGGhaplotype and CCGG(P<0.05),in which the g.732 C→G locus has a greater influence on the transcriptional activity of the gene.The relative fluorescence activity and luc2 gene mRNA expression of wild CCGGGG-haplotype of 5’ UTR(g.1337 C→A,g.1352 G→C,g.1382 G→C)were significantly higher than that of mutant AACCCC-haplotype(P<0.05).