Study On The Relationship Between Melanocortin Receptor 1(MC1R) Gene And Color Phenotype In Pigs And Its Variation Of 5'UTR

Coat color is a kind of available genetic marker. It plays an important role in determining crossing combination, breed purity and phylogenesis. Melanocortin Receptor 1(MCIR) Gene is a candidate gene for coat color heredity. Sequences of pig MCIR gene were aligned with Bioedit software. Two mutations, CCC insertion at 894 site(nt894insCCC)and G1197A, were found in Landrace (AY365253) and Yorkshire (AY365255), whereas Kijas reported that there are CC insertion at 894 site. C1318T and G1554A mutations were found in partial Duroc individuals, For instance, AY916524 hasn't C1318T and AY916523 hasn't G1554A, however Kijas reported the two mutations existed in all Duroc individuals. Four primers were designed with pig sequence(AF326520) from GenBank, which used to amplificate the coding region of MCIR gene. The mutations of coding region were investigated with PCR-RFLP and PCR-SSCP. Result revealed that all the individuals in Landance and Yorkshire have CC insertion at 894 site and present G1197A mutation, and all of the individuals in Duroc present C1318T and G1554A mutations. No heterozygote was found at these mutation sites, nt894insCC causes a frameshift mutation that introduces a premature stop at codon 56 and makesG1197A become nonsense.It could be inferred that nt894insCC might be associated with the white color in Landrace and Yorkshire breed. C1318T and G1554A mutations in Duroc resulting in Ala164Val and Ala243Thr respectively might be loss of function mutations and give rise to the ratio of phaeomelanin/eumelanin associating with the recessive red color. To study the relationship between MCIR gene Polymorphisms and pig coat color further, Polymorphisms screening of 5'UTR has also been done. According to AF326520, one pair of primers was designed to amplificate 5'UTR sequence. Two individuals from every breeds were selected to be sequenced. G668C was found in Duroc individuals. PCR-RFLP analysis showed that all Duroc individuals had G668C mutation and no heterozygote was found. G 668C in 5'UTR perhaps acts on both transcriptional and post-transcriptional level and influencs MCIR expression. So it could be inferred that G668C, C1318T and G1554A might be associated with the recessive red color of Duroc. Finally, we investigated the genetic distance for 5'UTR of different species, Primers were designed according to cattle sequence AF445641 for amplificating 5'UTR of goat and sheep with different coat color. After sequencing, 11 mutations, T86G, G104A, 127G(deletion), C129T, 233-236GGAC(deletion), G296A, C353T, G544A, C582T, A583G, C594A were found between goat and sheep. 3 Single Nucleotide Polymorphisms(SNPS), G296A, A544G, T129C were found in Suffolk while aligning with Small Tailed Han Sheep, Mongol Sheep and Tibet Sheep. No Variations were found in goat. Molecular phylogenetie trees were made with the thirteen 5'UTR sequences including Ovis aries, Capra hircus and Sus serofa sequence obtained by sequencing, and Capra hircus, Bos taurus, Equus caballus, Homo sapiens, Mus musculus, Mammuthus primigenius, Canis familiaris, Takifugu rubripes, Gallus gallus, Danio rerio, Oreochromis mossambicus obtained from genbank. Results showed that Mus musculus, Homo sapiens, Mammuthus primigenius and Sus scrofa were clustered into the first group, Ovis aries and Bos taurus were clustered into the second group, and Canis familiaris, Equus caballus, Gallus gallus, Oreochromis mossambicus, Danio rerio and Takifugu rubripes were clustered into the other groups of themselves. The results got here was identical with animal taxonomy.