Genetic variations associated with six-white-point coat pigmentation in Diannan small-ear pigs

Domestication of the pig has been accompanied with retention of a tremendous amount of phenotypic variation regarding growth, behavior, and coat color. The diverse domestic pig breeds thus provide valuable animal models for genetic studies on phenotypic variation1,2,3. Coat color was potentially exposed to artificial selection at an ancient time, dating back to about 5,000 years ago4. Variation in coat color may have important implications for pig breed development and conservation. Distinct coat colors have evolved in different pig breeds and may even have been used as visual characteristic to distinguish between breeds5. The most common coat colors found in domestic pigs include black, brown, white, red, spotted, belted, and two-end-black, which are distinct from the camouflage coat color of wild boars4,6,7,8,9. The genetic mechanisms underlying variation in coat color is an interesting topic. Six-white-point (SWP) is a common kind of coat color in pigs that and is characterized by a white coat on the four feet, the head, and the end of tail but with the remaining coat being black. The SWP phenotype is observed in many well-known European and East Asian domestic pigs, including the Berkshire pigs from Europe10, as well as Diannan small-ear (DSE), Tibetan, Longlin and Anqing six-white-point pigs in China11. The SWP coat color in the Berkshire pigs was found to be influenced by the melanocortin 1 receptor encoding (MC1R) gene, with this phenotype regarded as a special presentation of spotted color, as it is usually observed with black spots on white or red backgrounds5. In Berkshire pigs, two mutations in the MC1R gene are likely involved in formation of the SWP coat color pattern. The D124N MC1R allele probably causes a complete black body color4. SWP in the Berkshire is derived from the MC1R D124N allele through a 2-bp insertion in the coding region that leads to a coding frameshift and a premature stop codon. The background skin expresses the 2-bp inserted MC1R premature transcript, while the black-spotted area expresses a transcript without the 2-bp insertion, possibly due to somatic reversion events10. Additional to the MC1R locus, further evidence indicated that variation in the KITLG gene may also influence the coat color of Berkshire pigs12. Due to the independent domestication of pigs in Europe and East Asia13,14, black coat color has putatively evolved through different genetic mutations in the coding sequence of the MC1R gene in East Asia (L102P) and Europe (D124N)4,15. Thus how the SWP phenotype evolved in East Asian domestic pigs, where these animals have a MC1R allele containing the L102P substitution remains mysterious. In this study, we aimed to screen for genomic variants underlying the SWP phenotype using whole genome resequencing of DSE pigs from Yunnan province, China. DSE pigs have varying coat colors, including whole body black (denoted as “black” below) and SWP. Here we show that the SWP phenotype in DSE pigs is not attributed to variation in the MC1R gene, but rather it is associated with variations in multiple genes with differing phenotypic contributions.