Genetic variation of ASIP and MC1R in past and present sheep of Gotland

Abstract: Ovine black coat colour is determined by the MC1R (Melanocortin 1 receptor) and ASIP (Agouti signalling peptide) genes at the Agouti and Extension loci. The black phenotype is caused by production of eumelanin by melanocytes, whereas yellow-tan or light phenotype is the result of phaeomelanin synthesis. The ovine MC1R gene has two known alleles: the wildtype (E+) and the dominant black (ED) alleles. Two missense mutations (c.218T>A and c.361G>A) constitute ED. The presence of a third allele, the e allele, is proposed and believed to give rise to phaeomelanic phenotype. There are many alleles in the ASIP gene, the two most important are the dominant white (Awt) and the most recessive black (Aa). Two mutations are alleged to cause recessive black phenotype: a missense mutation g.5172T>A and a nonsense mutation, a five base pairs (bp) deletion (D5). However, not all black sheep, lacking the dominant black allele, are homozygous for at least one of the above mentioned ASIP mutations. A third mutation, a nine bp deletion (D9), is identified but its complete phenotypic influence is unknown. The aim of this study was to compare the presence of ED and the ASIP mutations in past and present sheep native to the Swedish island of Gotland in the Baltic Sea, to gain knowledge regarding the coat colour phenotype in these sheep. Fragments of the genes mentioned above concerning parts of interest were sequenced from present Gute Sheep and Gotland Pelt Sheep (breeds native to Gotland) using Sanger sequencing and from ovine remains contextually dated to the Medieval Times and the Iron Age found on Stora Karlsö, Gotland, (as well as some sheep remains from the Swedish mainland) using pyrosequencing. All present sheep possessed the E+E+ genotype. The three black sheep included in this study were homozygous for at least one of g.5172T>A or D5. Eighty-two grey present sheep possessed several diplotypes, but none was homozygous for g.5172T>A or D5. The ancient individuals possessed TT of c.218 and GG of c.361G>A. Two ancient individuals possessed the A of c.361G>A of ED, indicating that this mutation was indeed present in medieval sheep of Gotland and in mainland sheep of the 7th century. While some of the ancient sheep possessed the wildtype genotype of D5, no genotype could be determined for g.5172T>A. The full genetic background for recessive black coat colour could not be elucidated since some black sheep, that lack ED, are not always homozygous for D5 and/or g.5172TT>A.