Single Nucleotide Polymorphism Of MEF2B, RIPK2, CAMKMT Genes And Associations With Growth Traits In Ujumqin Sheep

Our previous GWAS results revealed that, 3 SNPs were associated with post-weaning gain: s58995.1 in MEF2 B gene, OAR9₉1721507.1 in RIPK2 gene, OAR3₈4073899.1 in CAMKMT gene. In 343 Ujumqin sheep experiment group, 30 DNA samples with a fixed concentration were selected to construct the DNA pool, primers were designed to extend all the exons and 1000 bp of upstream and downstream regulate regions. PCR products were tested and the objective strap were sequenced. The sequence peak maps were analyzed by DNAMAN and Chromas2 softwares,Sequenom Mass-array technology was used to genotype the detected SNPs including the SNP obtained by previous GWAS.Haploview was used to construct haplotypes and analyze linkage disequilibrium of the polymorphic loci.Association analysis between the SNPs in the candidate genes and the growth traits were performed using SPSS 22.0 software.1. DNA pool sequencing results One SNP（rs417014745 A>G） was detected in the 3’regulatory regions of MEF2 B gene.In RIPK2 gene,a synonymous mutation rs160673968A>G was detected in exon4,a missen variation rs162034551T>C was in exon7, rs419478383C>T was a synonymous mutation in exon 10,in the upstream regions g.5182G>C was detected.5 SNPs were identified in CAMKMT gene: rs405040355C>A in the 5′-UTR, rs411221065G>A in exon5, rs162300069A>Gin exon8, rs404399494G>A and rs418513655C>G in 3′-UTR.2. Association analysis between single nucleotide polymorphism with growth traits in sheep rs417014745A>G was extremely significantly associated with body weight, chest girth in 4-month age and chest girth in（P<0.01）,it was significantly associated with body weight in 6-month age（P<0.05）. on rs160673968A>G site, individuals with genotype AA/AG were significantly higher than those with GG genotype on body weight, chest girth in 4-month age and body height, chest girth in 6-month age（P<0.05）. On rs162034551T>C site, individuals with CC genotype were significantly higher on body weight, body height, chest girth in 4-month age and chest width（P<0.05） in 6- month age than individuals with TT and TC genotypes, individuals with CC genotype were extremely significantly different from individuals with TT and TC genotypes on body length in 4-month age（P<0.01） and body height, body length in 6-month age（P<0.01）. Individuals with GG genotype on g.5182G>C showed significant difference with individuals with the other two genotypes on body weight and chest girth in 4-month age（P<0.05）, individuals with each genotype on g.5182G>C had significant difference on chest girth in 6-month age（P<0.05）. Individuals with either of the 3 genotypes on g.4456C>T had significant difference on body weight in 4-month age（P<0.05）. Association analysis suggested rs411221065G>A was significantly associated with chest girth in 6-month age（P<0.05）. rs162300069A>G exhibited significant correlation with body weight, chest girth in 4-month age and body weight, chest girth,chest width in 6-month age（P<0.05）. rs404399494G>A was highly associated with body weight and chest girth in 4-month age（P<0.05）, rs418513655C>G was extremely significantly associated with body weight, chest girth in 4-month age and chest girth in 6-month age（P<0.01）. rs420220919 A>G was significantly associated with body weight in 4-month age（P<0.05）.3. Association of combined genotypes with growth traits in sheep Linkage disequilibrium analysis suggested rs160673968A>G and rs162034551T>C were strongly linked in RIPK2 gene, rs162300069A>G and rs420220919 A>G were strongly linked in CAMKMT gene. According to the linkage disequilibrium analysis results, we combined the genotypes of rs160673968A>G- rs162034551T>C and analyzed the associations between the combined genotypes and sheep growth traits.The results suggested 6 genotype combinations were constructed, most of the individuals were with AATT combined genotype.Individuals with GGCC combined genotype were significantly different from individuals with AATT genotype on body weight, body height, body length, chest girth in 4-month age and body height, body length and chest width in 6-month age（P<0.05）.Individuals with GGCCcombined genotyp were superior than sheep with only GG /CC genotype on body weight in 4-month age（P<0.05） and body height in 6-month age（P<0.05）.7 combined genotypes were constructed in rs162300069A>G- rs420220919 A>G of CAMKMT gene,most of the individuals were with GGAA genotype.Association analysis of rs162300069A>G- rs420220919 A>G combined effect showed sheep with GGAA combined genotype were significantly higher than the AAAA individuals on body weight, chest girth in 4-month age（P<0.05） and body weight, chest girth in 6-month age（P<0.05）.4. Bioinformatics analysis The bioinformatics analysis of rs162034551T>C indicated:the minimum structure free energy of m RNA was changed from-435.80 kcal/mol to-436.90 kcal/mol,which enhanced the structural stability. The secondary structure of the protein did not change, the proportion of alpha helix was 36.25%, the proportion of the extended chain was 14.87%, and the ratio of beta folding was 8.74%, and the proportion of random coil was 40.15% The bioinformatics analysis of rs162300069A>G indicated: the minimum structure free energy of m RNA was changed from-294.50kcal/molto-289.4kcal/mol, which decreased structural stability.The secondary structure of the protein did not change, the proportion of alpha helix was 49.38%, the proportion of the extended chain was 18.12%, and the ratio of beta folding was 9.69%, and the proportion of random coil was 22.81%...