Linkage and segregation analysis of black and brindle coat color in domestic dogs.

Mutations of pigment type switching have provided basic insight into melanocortin physiology and evolutionary adaptation. In all vertebrates that have been studied to date, two key genes, Agouti and Melanocortin 1 receptor (Mc1r), encode a ligand-receptor system that controls the switch between synthesis of red-yellow pheomelanin vs. black-brown eumelanin. However, in domestic dogs, historical studies based on pedigree and segregation analysis have suggested that the pigment type-switching system is more complicated and fundamentally different from other mammals. Using a genomewide linkage scan on a Labrador x greyhound cross segregating for black, yellow, and brindle coat colors, we demonstrate that pigment type switching is controlled by an additional gene, the K locus. Our results reveal three alleles with a dominance order of black (K(B)) > brindle (k(br)) > yellow (k(y)), whose genetic map position on dog chromosome 16 is distinct from the predicted location of other pigmentation genes. Interaction studies reveal that Mc1r is epistatic to variation at Agouti or K and that the epistatic relationship between Agouti and K depends on the alleles being tested. These findings suggest a molecular model for a new component of the melanocortin signaling pathway and reveal how coat-color patterns and pigmentary diversity have been shaped by recent selection.

The genetics of cream coat color in dogs.

Cream dogs of several breeds require a genotype of e/e at MC1R based on 27 individuals in this study. All Akita, Caucasian Mountain Dogs, German Shepherd Dogs, Miniature Schnauzer, and Puli with this genotype are cream, suggesting they are fixed at a second locus which causes the phaeomelanin pigmentation caused by this genotype to be diluted or pale. Conversely, although all Chinese Shar-Pei and Poodles that were cream had an e/e genotype at MC1R, not all dogs with this genotype are cream. Today, many Golden Retrievers and Labrador Retrievers with an e/e genotype are cream instead of the traditional yellow to golden color seen in the past. The second gene in these breeds must have multiple alleles, only one of which causes phaeomelanin pigment to be diluted or pale. Tyrosinase (TYR) and solute carrier family 45, member 2 (SLC45A2) have been shown to cause cream coat color in other species and were therefore investigated in dogs as candidate genes for this second locus. Although polymorphisms were detected in cDNA sequence from TYR and SLC45A2, no polymorphism was consistently associated with cream dogs or cosegregated with cream coat color in any of the families used in this study. A microsatellite was detected in a published BAC sequence (GenBank no. AAEX01017083) in intron 2 and was used to map SLC45A2 to CFA4.

Association of an Agouti allele with fawn or sable coat color in domestic dogs.

The type of pigment synthesized in mammalian hair, yellow-red pheomelanin or black-brown eumelanin, depends on the interaction between Agouti protein and the Melanocortin 1 receptor. Although the genetics of pigmentation is broadly conserved across most mammalian species, pigment type-switching in domestic dogs is unusual because a yellow-tan coat with variable amounts of dark hair is thought to be caused by an allele of the Agouti locus referred to as fawn or sable (a(y)). In a large survey covering thirty seven breeds, we identified an Agouti allele with two missense alterations, A82S and R83H, which was present (heterozygous or homozygous) in 41 dogs (22 breeds) with a fawn or sable coat, but was absent from 16 dogs (8 breeds) with a black-and-tan or tricolor phenotype. In an additional 33 dogs (14 breeds) with a eumelanic coat, 8 (German Shepherd Dogs, Groenendaels, Schipperkes, or Shetland Sheepdogs) were homozygous for a previously reported mutation, non-agouti R96C; the remainder are likely to have carried dominant black, which is independent of and epistatic to Agouti. This work resolves some of the complexity in dog coat color genetics and provides diagnostic opportunities and practical guidelines for breeders.

Characterization of the dog Agouti gene and a nonagoutimutation in German Shepherd Dogs.

The interaction between two genes, Agouti and Melanocortin-1 receptor ( Mc1r), produces diverse pigment patterns in mammals by regulating the type, amount, and distribution pattern of the two pigment types found in mammalian hair: eumelanin (brown/black) and pheomelanin (yellow/red). In domestic dogs ( Canis familiaris), there is a tremendous variation in coat color patterns between and within breeds; however, previous studies suggest that the molecular genetics of pigment-type switching in dogs may differ from that of other mammals. Here we report the identification and characterization of the Agouti gene from domestic dogs, predicted to encode a 131-amino-acid secreted protein 98% identical to the fox homolog, and which maps to chromosome CFA24 in a region of conserved linkage. Comparative analysis of the Doberman Pinscher Agouti cDNA, the fox cDNA, and 180 kb of Doberman Pinscher genomic DNA suggests that, as with laboratory mice, different pigment-type-switching patterns in the canine family are controlled by alternative usage of different promoters and untranslated first exons. A small survey of Labrador Retrievers, Greyhounds, Australian Shepherds, and German Shepherd Dogs did not uncover any polymorphisms, but we identified a single nucleotide variant in black German Shepherd Dogs predicted to cause an Arg-to-Cys substitution at codon 96, which is likely to account for recessive inheritance of a uniform black coat.

A form of albinism in cattle is caused by a tyrosinase frameshift mutation.

We used PCR amplification of cDNA prepared from skin biopsies to determine the full-length protein-coding sequence of tyrosinase ( TYR) in cattle of several coat colors. An insertion of a cytosine was detected in an albino Braunvieh calf, which resulted in a frameshift which caused a premature stop codon at residue 316. This insertion was found in the homozygous state in this calf and the genomic DNA of two related albino calves. All six parents of these calves were heterozygous for this insertion. However, an albino Holstein calf did not have this insertion, nor was any other mutation detected in the partial TYR sequence obtained from the genomic DNA available. Diagnostic genotyping tests were developed to detect this mutation in Braunvieh cattle.

TYRP1 and MC1R genotypes and their effects on coat color in dogs.

We used PCR amplification of cDNA prepared from skin biopsies to determine the nearly full-length, protein-coding sequence of dog TYRP1, and to define sequence variants potentially responsible for the B locus. One common variant contained a premature stop codon in exon 5 (Q331ter), and the other deleted a proline residue in exon 5 (345delP). A third variant in exon 2 (S41C) occurred less frequently. We genotyped 43 brown (including brown and white) and 34 black (including tricolor, black-and-tan, and black and white) dogs. All 43 of the brown group carried two or more of these sequence variants likely to interfere with TYRP1 function, whereas 0 of 34 in the black group carried two or more of these variants (10 carried one variant). We also genotyped 13 black-nosed and 10 brown-nosed dogs whose coat color was described as red, yellow, gold, apricot, or orange (including various degrees of white). All these dogs were homozygous for a R306X MC1R variant shown to be associated with these coat color phenotypes. The black or brown nose correlated perfectly with the absence or presence of the same three TYRP1 variants described above. TYRP1 was linkage mapped to dog chromosome 11, with a SNP in exon 7.