马MC1R基因多态位点与毛色相关性分析

Correlation Analysis between the Polymorphism Loci of MC1R Gene and Coat Color in Horses

旨在研究马MC1R基因多态位点与马毛色的关联性,为马的毛色育种提供重要的理论参考依据。本研究采集了10种不同毛色共132匹马的颈静脉血,提取基因组并应用PCR技术克隆MC1R基因编码序列,通过限制性长度多态性(RFLP)分析和测序分析,对马MC1R基因多态性与毛色进行关联分析,并预测了SNPs对其编码蛋白结构的影响。构建和分析了MC1R基因的系统发育树。结果发现,马MC1R基因存在3个多态位点,其中c.G102A为同义突变,c.C248T和c.G250A为错义突变。c.C248T造成MC1R蛋白的第83个氨基酸由极性亲水丝氨酸变为非极性疏水苯丙氨酸,预测突变后该位点氨基酸与第三跨膜区的第127个氨基酸(丝氨酸)之间的氢键消失而改变了MC1R的蛋白构象,该突变导致MC1R基因出现EE、Ee和ee 3种基因型。错义突变c.G250A导致其翻译的氨基酸由天冬氨酸变为天冬酰胺,该位点突变后与邻近氨基酸及跨膜域作用力复杂,推测比只出现c.C248T突变对MC1R蛋白功能的影响要小,该位点突变导致出现基因型ee^a。综合NCBI中的马MC1R基因型数据(共571匹)与其毛色性状进行相关性分析发现,在骝毛马和黑毛马群体中存在EE和Ee两种基因型,青毛马群体中存在EE、Ee、ee和ee^a基因型,栗毛中存在ee和ee^a两种基因型。黑毛和骝毛马的基因型分布差异极显著(P<0.01),呈现Hardy-Weinberg不平衡,等位基因E为优势等位基因;在栗色马中未检测到E等位基因,e为优势等位基因。MC1R基因系统发育树分析显示,EE基因型个体与家马、普氏野马相似性最高。本研究结果显示,马MC1R多态位点产生的不同基因型与马的骝毛、黑毛、栗毛及以这3种毛色为基础毛色的花马毛色存在相关性,骝毛马与黑毛马中不存在基因型ee,栗毛马中则不存在等位基因E。为应用基因型对马的毛色进行定向选择育种奠定了基础。

The study of the association between horse MC1R gene polymorphic loci and coat colors would provide important theoretical reference for coat color breeding of horses.In this study,the CDS of MC1R gene from 132 horses with 10 different coat colors was obtained by cloning and Polymerase Chain Reaction（PCR）,Restriction Fragment Length Polymorphsm（RFLP）and sequencing were implemented to analyze the correlation between MC1R gene polymorphism andcoat color,the effect of SNPs on its coding protein structure was predicted,and the MC1R gene NJ phylogenetic tree was constructed and analyzed.The results showed that 3 SNPs loci were found in horse MC1R gene,including one synonymous mutation（c.G102 A）and two missense mutations（c.C248 T,c.G250 A）.The c.C248 Tcaused change of the 83 rd amino acids of MC1R protein from polar hydrophilic serine to nonpolar hydrophobic phenylalanine,predicting that the hydrogen bond between amino acid at this locus and 127 th amino acid（serine）at the third transmembrane domain would disappear and the conformation of MC1R protein would be changed.This mutation resulted in 3 genotypes of EE,Ee and ee in MC1R gene.The missense mutation c.G250 Acaused the change of amino acid from aspartic acid to asparagine.The mutation site had more complex interaction with adjacent amino acids and transmembrane domains.It was speculated that c.G250 A and c.C248 T had less effect on the MC1R protein function than only the c.C248 T mutation,which resulted in the emergence of genotype eea.Based on the association analysis of the horse MC1R genotype data（a total of 571 individuals）published by NCBI with their coat color traits,we found that there were two genotypes of EE and Ee in the bay and black horse populations,four genotypes of EE,Ee,ee and eeain gray horse population,and two genotypes of ee and eeain the chestnut horse population.The distribution of genotypes were extremely significantly different（P〈0.01）in black and bay horse populations,showing Hardy-Weinberg disequilibrium,and the allele E was the dominant one.However,allele E was not detected in chestnut horses and e was the dominant allele.The result of phylogenetic tree showed that the individuals with EE genotype had the highest similarity to horse and Przewalskii.These results indicate that the different genotypes produced by the MC1R polymorphic loci are related to the bay,black,chestnut coat colors and the colorful hair color of horses.There was no ee genotype in bay and black horses and no allele E in chestnut horses.This study lay the foundation for the application of genotypes in the directional selection breeding of horse coat color.