Association Analysis Of MSTN Gene Polymorphism And Growth Traits At Cattle In Guizhou

This article took Guizhou local yellow cattle(Weining cattle,Guanling cattle,Sinan cattle),Hybrid Guizhou yellow cattle and Angus cattle as the research object.MSTN gene cDNA of Guizhou hybrid yellow cattle was cloned by RT-PCR and analyzed by bioinformatics.MSTN gene polymorphic sites were screened by PCRSSCP method,PCR-RFLP method and direct sequencing method.Plasma MSTN protein concentration was determined by ELISA method and associated with MSTN gene polymorphism in different breeds of cattle.The polymorphic sites of MSTN gene polymorphism related to growth traits were analyzed to provide a theoretical basis for the breeding of MSTN gene in Guizhou high-quality yellow cattle.The results are summarized as follows:1.Cloning and Bioinformatics Analysis of MSTN Gene cDNA of hybrid Guizhou yellow cattleThe cDNA sequence of MSTN gene of hybrid Guizhou yellow cattle was cloned by RT-PCR,and four base mutations were found,of which three were missense mutations: 76 bp A→G mutation in exon 1 caused asparagine to change to aspartic acid,The A→G mutation at 79 bp of exon 1 causes serine to change to glycine,the G→A mutation at 1066 bp in exon 3 causes glutamate to change to lysine;and A→G mutation at 267 bp in exon 2 is a synonymous mutation.The results of bioinformatics analysis showed that the basic physical and chemical properties and structure of MSTN protein of hybrid Guizhou yellow cattle were basically the same as those of ordinary cattle,with a transmembrane structure region and an acidic hydrophilic protein in the signal peptide region,and the theoretical value of protein was about 42.43 k Da.Through gene homology and phylogenetic tree,combined with motif,protein conserved domain and domain analysis,it is concluded that MSTN gene is highly conserved in mammals,but there is genetic diversity.The occurrence of three missense mutations led to changes in the secondary structure and stability of m RNA,and changes in codon preference caused by synonymous mutations.2.Polymorphic Loci and Analysis of MSTN GeneThree polymorphic loci were screened from Guizhou local yellow cattle,hybrid Guizhou yellow cattle and Angus cattle by PCR-SSCP method and direct sequencing method.They were: g.6279107G>C mutation on EXON1 located at 111 th position in CDS region,producing two genotypes GG and GC,Weining cattle with low polymorphism(0.25<PIC<0.5,0.25),and Guanling cattle and Sinan cattle with moderate polymorphism;The g.6281238T>C mutation located on EXON2 was located at position 414 in CDS region,producing three genotypes TT,TC and CC,Weining cattle,Sinan cattle and Angus cattle were moderately polymorphic at this site;The g.6283933 A>C mutation in EXON3,located at 1077 th position in CDS region,and three genotypes AA,CA and CC were produced,Weining cattle,Guanling cattle and Sinan cattle were moderately polymorphic.However,the polymorphic sites in the 5’untranslated region of MSTN gene of four breeds of cattle and a hybrid cattle population were not screened by PCR-RFLP method.None of the three polymorphic loci deviated from the Hardy-Weinberg equilibrium and could provide reasonable genetic information.Linkage disequilibrium analysis showed that there was no strong linkage equilibrium among the three SNPs.Haplotype analysis showed that the three SNPs could construct 6 haplotypes and 13 diplotypes.Bioinformatics analysis showed that three SNPs caused changes in codon preference,secondary structure and stability of m RNA,as well as changes in exon splicing enhancers.3.Association analysis of MSTN gene polymorphism and plasma MSTN protein concentrationThe results of ELISA detection of plasma MSTN protein in Guizhou local yellow cattle and hybrid Guizhou yellow cattle showed that the concentration of MSTN in hybrid cattle was significantly lower than that in Guanling cattle and Sinan cattle.The relationship between three SNPs loci and the concentration of MSTN protein was analyzed,and two polymorphic sites were found to be associated with the concentration of MSTN protein.Among them,the MSTN concentration of Weining cattle was significantly different from that of Guanling cattle and Sinan cattle among different genotypes of g.6279107G>C mutation site,and the MSTN concentration of Weining cattle and Sinan cattle was significantly different among different genotypes of g.6283933A>C mutation site.The correlation between 13 diplotypes produced by 3SNPs loci and MSTN concentration was analyzed.It was found that the MSTN concentration of individuals with H3H5 diplotype was significantly lower than that of H1H5 and H3H4 diplotypes(P<0.05).The plasma MSTN concentration of hybrid Guizhou yellow cattle was lower than that of Guizhou local yellow cattle,which also explained the main reason for the rapid growth of hybrid cattle.The interaction between haplotype H3(GCA)can be used as molecular markers for the breeding of Guizhou yellow cattle.4.Association analysis of polymorphic sites and growth traitsThe MSTN genes of 4 breeds of cattle and a hybrid cattle population were screened out and 3 polymorphic loci were screened for correlation analysis with their growth traits,and only 2 loci were associated with growth traits.In the g.6279107G>C mutation of MSTN gene,the chest circumference,tube circumference and body height of Weining cattle with GG genotype were significantly higher than those with GC genotype(P<0.05).The weight and height of TT type individuals in Weining cattle were significantly higher than those of TC type individuals in the mutation site of MSTN gene g.6281238T>C(P<0.05).The chest girth of TT type and TC type in Angus individuals was significantly higher than that of CC type individuals(P<0.05).The heterozygous TC type and the mutant homozygous CC type of Sinan cattle,and the growth traits of TC type were higher than those of CC type individuals,but the difference was not significant(P>0.05).Hybrid Guizhou yellow cattle only have heterozygous TC type individuals at this locus.It is preliminarily believed that the g.6281238 T>C mutation has the potential to affect the growth traits of Guizhou native cattle,and suggests that the T allele may be related to the growth traits.