SNP Screening Of Somatotropic Axis Gene In Yiling Goat And Functional Stduy On Variant Sites Of MC4R

Goat is one of the extremely major economic animals as a source of meat for humans,and the transition from wild to housing environment leads to a shift in its phenotype towards the direction of human production needs.In this study,we resequenced 35 Yiling goats and downloaded sequencing data from 20 wild goats,and comparative analysis of selection signals about the two populations to obtain somatotropic axis genes and sites subject to positive selection.Furthermore,positive selection site functional study of key gene was conducted,aiming of providing theoretical reference for breeding new breed of Yiling goat.The results of the research were as follows:（1）The selection signal of Yiling goat and wild goats were detected.A total of 775genomic regions subjected to positive selection were obtained,involving 2586 genes.These genes were enriched in 105 pathways,including two growth-related pathways:the development of muscle cells（GO:0055001）and the development of striated muscle cells（GO:0055002）.Furthermore,mining of somatotropic axis genes yielded 76 somatotropic axis genes subject to positive selection.A total of 4116 SNPs subject to positive selection were screened on these genes,among which 10 missense sites were presented on 10 genes（MC4R、GPX1、NPR3、CHRNB1、TIPARP、NFATC3、MC5R、LRRC17、PGM5、SREBF2）.The c.612A>G site on MC4R gene was subjected to the strongest positive selection(F_ST=0.68),and the allele frequencies presented significantly different in the Yiling goat and the wild goat population.（2）Polymorphism analysis of the CDS region of MC4R gene in 425 goats of nine goat populations.A total 15 SNPs were discovered,of which 7 belonged to missense sites（c.110C>A,c.334G>A,c.360T>C,c.502A>G,c.612A>G,c.765C>T,c.906C>A）,and 5were novel SNPs（c.144C>T,c.243C>T,c.334G>A,c.613C>T,c.763G>A）.The frequency of the A allele of c.612A>G was 0.6-1 in the eight domestic goat populations,whereas it was 0 in wild goats.In addition,to further determine the details of the c.612A>G site in other goat breeds around the world.A total of 422 representative goats were collected and aligned,the results demonstrated that A allele of this mutation was specific for domestic goats.（3）The three-dimensional model,physicochemical properties and conservativeness of MC4R were predicted.Sequence comparison of MCRs among 20 species found that c.612A>G existed as G-base type in all species except domestic goat and zebrafish,and all codes for methionine（Met）.The mutation of isoleucine to methionine（I204M）made the residue groups larger,which led to the wider spatial distance betweenα4 andα7,causing a decrease in hydrophobicity coefficient from 2.767 to 2.478.（4）The effect of I204M mutation on MC4R protein function was examined in this study.The results showed that the localization of I204 and M204 receptors on the membrane were normal.When stimulated withα-MSH,M204 receptor was defective in c AMP signaling compared with the I204.Conversely,the level of basal c AMP also significantly lower than that of I204 receptor under stimulation withoutα-MSH（P<0.001）.In the MAPK pathway,the I204 and M204 receptors both had normal p ERK1/2 levels.In summary,abundant SNP variants in the somatotropic axis gene of Yiling goats,the MC4R gene,which controls feeding behavior,was positively selected during domestication,and its allele frequency of the selected c.612A>G site was polarized between domestic and wild goats,affecting function by reducing MC4R-mediated basal c AMP level and weakening signaling in the Gs-c AMP-PKA pathway.