Study On Screening Of Candidate Coat Color Genes And Polymorphisms In Yaks

The coat color of animals is one of the important characteristics of breeds,and as an available genetic marker,it can be used for breed identification and crossbreeding selection,etc.The abundance of coat color types in domestic yaks provides a good model for the genetic regulation of coat color in livestock.Although studies on yak coat color have been reported,the genetic basis of yak coat color is still not fully resolved.The analysis of the genetic basis of yak coat color is helpful to clarify the adaptive evolution of yak and the evolution of yak coat color.On the other hand,it is also helpful to the protection of yak varieties.In this study,healthy black,white and brown yaks around Qinghai Lake were selected as the research objects.The skin hair follicle structure and pigment distribution of yaks with three different coat colors were compared and analyzed by using hematoxylin-eosin(H&E)staining technology;The transcriptome of yak skin was sequenced by Illumina Hi Seq TM2500 sequencing platform to screen the differentially expressed genes related to different coat colors;Then,immunofluorescence and Western blot were used to locate and quantitatively analyze the corresponding proteins of candidate genes screened by transcriptome sequencing;Finally,the SNP sites of coat color related genes screened by transcriptome sequencing were analyzed.My research are as follows.(1)H&E staining showed that a large number of melanin granules were distributed in the epidermal basal layer and around the hair follicles in black and brown yak skin tissues,while no melanin granules were found in the epidermal basal layer and around the hair follicles in white yak skin tissues.(2)A total of 57.57 Gb Clean Data were obtained by sequencing the transcriptomes of three different coat color yak skin tissues,with Q30 above 93%.There were 426 differentially expressed genes between white and black yak skin tissues,including 19 genes related to coat color,246 differentially expressed genes between white and brown yak skin tissues,including 16 genes related to coat color,and 4230 differentially expressed genes between black and brown yak skin tissues,including 138 genes related to coat color.KEGG enrichment analysis showed that white and black yaks differentially expressed genes were mainly enriched in the drug metabolism-cytochrome P450 pathway,and also in the tyrosine metabolism pathway.white and brown yak differential genes were mainly enriched in the tyrosine metabolism pathway(TYRP1,TYR,ALDH3A1 and AOX4).The differential genes between black yak and brown yak are mainly concentrated in sphingolipid metabolism pathway,unsaturated fatty acid biosynthesis and arachidonic acid metabolism pathway.The Venn diagram showed that there were 9 significant genes in the three groups of yaks,and only SOX10 was associated with coat color.Meanwhile,the expression of SOX10 in brown yaks was significantly higher than that in black and white yaks(P < 0.05),and it was speculated that SOX10 was associated with hair color formation in brown yaks.(3)Immunofluorescence staining showed that SOX10 protein was expressed in the basal layer of the epidermis and around the hair follicles in black and brown yak skin tissues,but no SOX10 protein expression was detected in the basal layer of the epidermis and around the hair follicles in white yaks.Moreover,SOX10-positive cells in black yak skin tissue co-localized with the value-added marker KI67,indicating that SOX10-positive cells were active in black yaks,but no co-localized cells were detected in brown and white yaks;TYRP1 protein expression was detected in the basal layer of the epidermis and around hair follicles in black yak skin tissue.In brown yak skin tissues,TYRP1 protein expression was detected only in the basal layer of the epidermis.The expression of TYRP1 protein was not detected in white yak skin tissues.Western blot results showed that SOX10 protein was significantly higher in black yak skin tissues than in brown yak and white yak(P < 0.01),and SOX10 protein was significantly higher in brown yak skin tissues than in white yak(P < 0.01).The TYRP1 protein of black yak skin tissue was highly significantly higher than that of brown yak and white yak(P < 0.01),and the difference between TYRP1 protein of brown yak skin tissue and white yak was not significant.(4)Transcriptome sequencing was used to analyze the SNP loci in the skin groups of yaks of three different coat colors,and the results showed that the SNP loci of the eight genes related to coat color screened in the white yak were located in the non-coding region,while the SNP loci of the genes PMEL,ERBB3 and PAICS screened in the brown group caused amino acid sequence alterations.The PMEL coding region 107 bp C>T made serine No.36 a leucine.The PMEL coding region1835 bp A>G made alanine No.612 a glutamic.The ERBB3 coding region 3253 bp A>G made methionine No.1085 a valine.The PAICS coding region 2233 bp A>G made proline No.745 a serine.According to the above research results,it was concluded that the white and brown yaks have different regulatory mechanisms for coat color formation;inhibition of tyrosine metabolic pathway may be a key factor in white coat color formation;TYRP1,a marker of late melanocyte differentiation,was not detected to be expressed in white yaks;the white phenotype in white yaks may be due to the lack of fully differentiated melanocytes;screening of brown yaks for One SNP site in SOX10 was a synonymous mutation,SNP(1245 C > T);two SNP sites in PMEL were missense mutations,SNP1(107 C > T)and SNP2(1835 C > A);one SNP site in ERBB3 was a missense mutation,SNP(3253 A > G);one SNP site in PAICS was a missense mutation SNP(2233 C > T).