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

Abstract

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.

Figure 1.—

Segregation of CFA16 haplotypes in the EB and GB litters. (A) Haplotypes based on the four SSLP markers in B are indicated with vertical bars, just above genotypes for K locus alleles. (As described in the text, brindle and yellow were considered in the same class, “nonblack,” for analysis of the genome scan; genotypes given here for k^br and k^y are based on information presented in Figure 3.) Black-colored haplotypes originate from the Labrador retriever grandparent carrying dominant black (B53 or Andy); white-colored haplotypes originate from the nonblack greyhound parent or grandparent (Esther or Isis). SSLP alleles are numbered arbitrarily according to increasing size for each marker. (B) Physical location of SSLP markers used in A indicated in megabases (Mb) from the centromere. To the right of each marker name is the number of animals recombinant between that marker and the K allele over the total number of animals that were informative for that marker. Recombinant chromosomes are carried by EB57 and GB17 and define a critical region for K between FH2175 and FH3592. In addition to the K locus genotypes depicted, Agouti and Mc1r genotypes were determined for every dog as described in materials and methods.

Figure 2.—

Segregation of CFA16 haplotypes in the FB and HB litters. (A and B) Symbols are as in Figure 1. Recombinant chromosomes are carried by FB27, FB67, and HB27 and indicate that K must lie centromere distal to REN292N24. In addition to the K locus genotypes depicted, Agouti and Mc1r genotypes were determined for every dog as described in materials and methods. (C) Diagram of the K critical region from REN292N24 to FH3592, indicating the location of RefSeq genes in the region (blue) and evolutionarily conserved regions in the human genome. Annotation is based on the CanFam1.0 dog genome assembly (Lindblad-Toh et al. 2005) as displayed by the UCSC Genome Browser (Karolchik et al. 2003) using the “Human Net” comparative genomics track, in which red and yellow indicate sequence similarity to human chromosomes 8 and 4, respectively.

Figure 3.—

Segregation of FH2155 alleles in three kindreds with brindle and yellow. As described in the text, there is perfect cosegregation of FH2155 with brindle vs. yellow under a model of dominant inheritance with K^B > k^br > k^y, corresponding to a LOD score of 3.6.

Figure 4.—

Models for gene action at the K locus. Both models must account for the observations that (1) the dominance order of Agouti is opposite to that of K; (2) Mc1r alleles are epistatic to both Agouti and K locus alleles; (3) “black alleles” of K are epistatic to “yellow alleles” of Agouti; and (4) “black alleles” of Agouti are epistatic to “yellow alleles” of K. These observations are consistent with a model in which (A) the K gene product functions to inhibit Agouti function, but are also consistent with a model in which (B) the K gene product acts directly at the Mc1r to stimulate melanocortin signaling and thereby oppose the action of Agouti protein indirectly.