Identification Of EDNRB Causative Mutation(s) Affecting The Two-end-black Coat Color In Pigs And Genetic Evolutionary Analysis Of The Porcine MUC4Gene

Coat color is one of breed characteristics in pigs, and two-end-black is one of representative coat color phenotypes in Chinese indigenous pigs. Our previous study demonstrated that EDNRB is a major gene affecting the two-end-black coat color. EDNRB together with other modifier genes like EDN3, KIT and LITLG determine the two-end-black phenotype in Chinese indigenous pigs including Shaziling, Bamaxiang and Tongcheng pigs. The aim of this study was to identify the causal mutation(s) of the EDNRB gene and to dissect the molecular basis of the two-end-black coat color phenotype in pigs.For this purpose,10representative pigs from8Chinese and Western breeds (Shaziling, Bamaxiang, Tongcheng, Rongchang, Erhualian, Min, Duroc, Large white) were used for EDNRB resequceing. A total of341SNPs were detected and52diagnostic SNPs distinguishing two-end-black pigs from non-two-end-black pigs were identified. Subsequently, the52diagnostic SNPs were genotyped in94pigs from the above-mentioned8breeds. A13-kb IBD region shared by all two-end-black pigs was found and24candidate causal mutations were identified in IBD region. The24SNPs were further genotyped in123unrelated pigs from11breeds representing a variety of coat color phenotypes, revealing14candidate causative mutations. These SNPs were analyzed for their conservation among mammals and potential functional elements. The findings indicate that causal variants are most likely regulating mutations in an enhancer region with intron1of EDNRB. The genetic variation at EDNRB diagnostic SNP g.16484A>G was detected in353pigs from diverse breeds. The result showed that all two-end-black and white-belt pigs form Chinese local breeds except Jinhua were homozygous for the mutant allele, indicating that this mutation is most likely the causative site in EDNRB gene affecting the two-end-black and white-belt phenotypes but causative gene in Jinhua pigs could be different. While All pigs having other coat color phenotypes including solid black, solid white, red, brown were homozygous for the wild-type allele. Moreover, we also confirmed that the white-belt phenotype in Hampshire was regulated by KIT not EDNRB.The results provide novel insights into the molecular basis of coat colors in pigs, and enrich our understanding of the pigmentation mechanism in mammals. Furthermore, this study improves our knowledge about the genetic mechanisms of breed characteristics in Chinese pigs, and provides effective molecular genetic makers for Chinese two-end-black pigs breeding. MUC4is a type of membrane anchored glycoprotein and serves as the major constituent of mucus that covers epithelial surfaces of many tissues such as trachea, colon and cervix. MUC4plays important roles in the lubrication and protection of the surface epithelium, cell proliferation and differentiation, immune response, cell adhesion and cancer development. To gain insights into the evolution of the porcine MUC4gene, we surveyed the nucleotide variability and linkage disequilibrium (LD) within this gene in Chinese indigenous breeds and Western commercial breeds.A total of53SNPs covering the MUC4gene were genotyped on5wild boars and307domestic pigs representing11Chinese breeds and3Western breeds. The nucleotide variability, haplotype phylogeny and LD extent of MUC4were analyzed in these breeds. Both Chinese and Western breeds had considerable nucleotide diversity at the MUC4locus. Western pig breeds like Duroc and Large White have comparable nucleotide diversity as many of Chinese breeds, thus artificial selection for lean pork production have not reduced the genetic variability of MUC4in Western commercial breeds. Haplotype phylogeny analyses indicated that MUC4had evolved divergently in Chinese and Western pigs. The dendrogram of genetic differentiation between breeds generally reflected demographic history and geographical distribution of these breeds. LD patterns were unexpectedly similar between Chinese and Western breeds, in which LD usually extended less than20kb. This is different from the presumed high LD extent (more than100kb) in Western commercial breeds. The significant positive Tajima’D, and Fu and Li’s D statistics in a few Chinese and Western breeds implied that MUC4might undergo balancing selection in domestic breeds. Nevertheless, we cautioned that the significant statistics could be upward biased by SNP ascertainment process.Conclusion:Chinese and Western breeds have similar nucleotide diversity but evolve divergently in the MUC4region. Western breeds exhibited unusual low LD extent at the MUC4locus, reflecting the complexity of nucleotide variability of pig genome. The finding suggests that high density (e.g. ISNP/lOkb) markers are required to capture the underlying causal variants at such regions.