Polymorphism Loci Of MC1R Gene And Coat Color In Horses

Coat color is an important biological character of horses.It is one of the important references for variety identification and individual identification,and it is related to use,nutrition,health,disease,character and behavior of horses.There are many genes affecting the coat color of horses,of which the melanocortin receptor 1(Melanocortin 1 Receptor,MC1R)is an important function gene that involved in regulation of horse hair color.In this study,we extracted DNA from the jugular vein blood of 132 horses,and classified the coat color.By cloning of the coding sequence of horse MC1R gene through Polymerase Chain Reaction(PCR),analysis of Restriction Fragment Length Polymorphsm(RFLP)and sequencing,we analysed the correlation between MC1R gene polymorphism and coat color,and predicted the effect of SNPs on its coding protein structure.The main achievements of the studies were as following:1.Study on Polymorphism of Exon of MC1R Gene in Horses.The exons of horse MC1R gene were cloned and sequenced,and 3 SNPs were identified,of which c.G102A was a synonymous mutation,c.C248T and c.G250A were missense mutations.c.C248T mutation produced alleles e and c.G250A generated allele ea.In all the samples,there were 63 genotype Ee,31 genotype EE,35 genotype ee,and 3 genotype eea.The Bay,Black,and Bay with Skewbald horses contained the genotype EE and Ee.The genotypes of Gray and White horses were distributed among the three EE,Ee and ee.The Chestnut or the Chestnut with Skewbald horses had ee genotype only.Piebald horses only had the EE genotype;and Roan horses only had the Ee genotype.It can be determined that the parents of Chestnut all carry the MC1R allele e inevitablely.We analyzed the genotypes and allele frequencies of c.C248T loci of exon of MC1R gene.The results showed that the distribution of genotypes were extremely significant difference(X2-test,P<0.01)in black and bay horses.And there was the Hardy-Weinberg imbalance in the heredity,and allele E was the dominant one.E allele was not detected in Chestnut horse,and e was the dominant allele.The genotype of c.C248T locus of Gray horse was in Hardy-Weinberg equilibrium.The analysis of all the color of coats showed that all genotypes of the locus were in the Hardy-Weinberg balance and the allele frequencies were equal.2.The structure prediction of MC1R protein produced by missense mutation.The c.C248T missense mutation in the MC1R gene exon of horse resulted in the 83rd amino acid of MC1R protein changed from polar to non-polar amino acid,the hydrophobic interaction force of which also be changed,and the hydrogen bond between it and the 127th serine which was in the third transmembrane region disappeared,so that the hydrogen bonds acting on them were distributed only in the 1st transmembrane domain.It was speculated that the mutation may lead to change the distance between the 1st transmembrane domain and the 3rd transmembrane region,which affected the whole structure of MC1R protein.In addition,the weakening of maintenance force of the α-helix in that place may cause the structure to be looser than before,and the α-helix in the anterior of the mutation segment may change.This may partially stick out from the the cell membrane and enter the cell,which may affect the function of the MC1R protein.On the basis of the presence of c.C248T in the horse MC1R gene,the missense mutation occured at 250 bp of the CDS,causing the 84th amino acid of the MC1R to change from the negatively charged aspartic acid to the polar uncharged asparagine.After this mutation occured,three new hydrogen bonds acted on the 56th amino acid in the 1st transmembrane region and the 294th amino acid in the 7th transmembrane region,respectively,and the ion binds force at this site also changed after that.Compared with the wild type,after the pro.D84N mutation occured,the number of hydrogen bonds were increased between the 83rd,84th amino acids and surrounding amino acids,and the interaction between amino acids was complex.Compared with the only mutation of pro.S83F,the hydrogen bond reappeared which acted on the 127th serine in the 3 rd transmembrane region and increased the interaction between the 1st and the 7th transmembrane region.We speculated that with the influence of three new hydrogen bonds,mutation at that site may weaken the effect of the overall structure of the MC1R protein when there was only pro.S83F mutation.