Journal of Heredity 2003:94(1)
© 2003 The American Genetic Association 94:75-79
Exclusion of Melanocortin-1 Receptor (Mc1r) and Agouti as Candidates for Dominant Black in Dogs
From the Departments of Pediatrics (Barsh) and Genetics (Kerns, Olivier, and Barsh), Stanford University School of Medicine, Stanford, CA 94305-5323, and the College of Veterinary Medicine, Cornell University, Ithaca, NY 14853 (Lust). M. Olivier is currently at the Human and Molecular Genetics Center, Medical College of Wisconsin, Milwaukee, WI 53226.
Address correspondence to Greg Barsh, Stanford University, Beckman Center B271A, Stanford, CA 94305-5323, or e-mail: gbarsh{at}cmgm.stanford.edu.
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The domestic dog exhibits a variety of coat colors that encompass a wide range of variation among different breeds. Very little is known about the molecular biology of dog pigmentation; current understanding is based mostly on traditional breeding experiments, which in some cases have suggested genetic interactions that are different from those reported in other mammals. We have examined the molecular genetics of dominant black, a uniform coat color characteristic of black Labrador retrievers or Newfoundlands that has been proposed to be caused by either variation in the melanocortin-1 receptor gene (Mc1r) or by variation in the Agouti gene (A). We identified several coding polymorphisms within Mc1r and several simple sequence repeat polymorphisms closely linked to A, and examined their inheritance in a Labrador retriever x greyhound cross that segregates dominant black. No single Mc1r allele was found consistently in animals carrying dominant black, and neither Mc1r nor A cosegregated with dominant black. These results refine our understanding of mammalian coat color inheritance and suggest that dominant black coat color in dogs is caused by a gene not previously implicated in pigment type switching.
The study of coat color variation in mammals has resulted in the identification of many genes required for melanocyte development, migration, and regulation. A variety of pigmentation patterns are created by differential production of two types of pigment: pheomelanin, which is usually yellow or red, and eumelanin, which is usually black or brown. Individual melanocytes can switch between the synthesis of eumelanin and pheomelanin in response to a paracrine signaling molecule, Agouti protein, and a seven transmembrane domain receptor expressed by hair follicle melanocytes, melanocortin-1 receptor (Mc1r).
Mc1r is coupled to adenylate cyclase and its activation causes accumulation of cAMP and promotes eumelanin instead of pheomelanin synthesis. Agouti protein inhibits Mc1r activation, and production of Agouti protein therefore causes hair follicle melanocytes to produce pheomelanin instead of eumelanin (Lu et al. 1994; Ollmann et al. 1998). In mice, gain-of-function Agouti mutations or loss-of-function Mc1r mutations give rise to animals with pheomelanic coats, whereas loss-of-function Agouti mutations or gain-of-function Mc1r mutations give rise to animals with eumelanic coats. Mc1r mutations are epistatic to Agouti mutations; thus mice carrying gain-of-function mutations in both Agouti and Mc1r produce eumelanic coats, while those carrying loss-of-function mutations in both Agouti and Mc1r produce pheomelanic coats (reviewed in Silvers 1979).
Gain-of-function Mc1r mutations have also been described in cattle, sheep, foxes, and pigs, and produce a uniform black coat color transmitted in a dominant manner (Kijas et al. 1998; Klungland et al. 1995; V
ge et al. 1997, 1999). Dominant black also exists in the domestic dog, but its genetic basis is unclear. Based on pedigree and segregation studies carried out several decades before Agouti and Mc1r were identified at the molecular level, C. C. Little proposed that dominant black was one of a series of Agouti alleles, named As, along with ay (sable/tan) and at (tan points), with a dominance hierarchy of As > ay > at. Little (1957) also proposed that production of different pigment types was controlled by allelic variation at the Extension (E) locus, in which four alleles, Em (mask), E (wild type), ebr (brindle), and e (red-yellow), exhibited a dominance hierarchy of Em > E > ebr > e, and in general were epistatic to Agouti alleles (an exception was the proposal that AS would be epistatic to ebr) (Little 1957).
Previous work from our laboratory demonstrated that Mc1r is the Extension gene and that a nonsense mutation, R306ter, was responsible for the e mutation in a number of red or pale-colored dogs including yellow Labrador retrievers, Irish setters, golden retrievers, and Samoyeds. We also identified an Mc1r polymorphism that was a candidate for dominant black, S90G, because it was found in Newfoundlands, flat-coated retrievers, and black Labrador retrievers, but not in golden retrievers or Irish setters; however, the absence of pedigree analysis prevented a more direct test for allelism (Newton et al. 2000).
Here we report a more extensive study of the genetics of pigment type switching in dogs using multiple molecular markers for both Mc1r and Agouti to carry out a haplotype analysis of a large Labrador retriever x greyhound cross. No single Mc1r haplotype or variant was found consistently in animals carrying dominant black, and neither Mc1r nor A cosegregated with dominant black. These results refine our understanding of mammalian coat color inheritance and suggest that dominant black coat color in dogs is caused by a gene not previously implicated in pigment type switching.
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Results and Discussion |
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In our initial study of Mc1r variation in dogs, we determined the complete coding sequence in black and yellow Labrador retrievers, Newfoundlands, golden retrievers, Irish setters, flat-coated retrievers, and Doberman pinschers. Each of these breeds is thought to be fixed for dominant black except for the Doberman. A pale or pheomelanin-colored coat in, for example, yellow Labrador retrievers, golden retrievers, and Irish setters, is caused by homozygosity for the Mc1r R306ter allele, with a genotype referred to as AS/AS e/e in the original notation used by Little. Thus the presence of dominant black in pedigrees becomes apparent when an animal of genotype AS/AS E/E (black Labrador retrievers, Newfoundlands, flat-coated retrievers) or AS/AS e/e (yellow Labrador retrievers, golden retrievers, or Irish setters) is crossed to an animal of genotype ay/ay E/E (e.g., a tan Basenji) or at/at E/E (e.g., a black-and-tan Doberman), yielding litters where all progeny are completely black.
These criteria fit those of a cross established at Cornell by Lust and colleagues to study hip dysplasia, in which black or yellow Labrador retrievers were crossed to greyhounds and their progeny backcrossed to a greyhound parent (Todhunter et al. 1999). Greyhounds can be fawn, yellow, red, brindle, or masked; Little (1957) suggested that the uniform pheomelanic appearance (fawn, yellow, or red) could be caused by either Agouti (ay/ay) or Extension, that is, Mc1r (e/e); however, as described below, we did not find the Mc1r R306ter allele in any of the greyhound samples we examined.
Informative kindreds from this cross are depicted in Figure 1, in which a total of 18 F1 progeny were produced from three sets of Labrador retriever x greyhound matings; all progeny are completely black. In two of the crosses, the Labrador retriever parent was yellow and homozygous for the Mc1r R306ter allele, yielding F1 black offspring whose genotype in the original notation is AS/ay E/e (Figure 1B,C). A single black F1 animal from each mating was then backcrossed to a greyhound (sometimes from a different pedigree), yielding a total of 21 BC1 progeny, of which 10 were completely black and 11 were nonblack. These observations conform to Mendelian expectations for a locus that controls uniform black coloration in which the Labrador retriever parents are homozygous for a "black" allele and the greyhound parents are homozygous for a "nonblack" allele, with the Labrador retriever allele dominant to the greyhound allele, and the Labrador retriever allele hypostatic to the Mc1r R306ter allele.
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Coat color phenotypes of nonblack progeny in the pedigrees depicted in Figure 1 include yellow, red, reddish-brown, and brindle. We cannot exclude the possibility that some of these animals carried a dominant black allele whose effects are not apparent. For example, the gene or genes responsible for brindling may be epistatic to those responsible for dominant black; also, mutations that impair but do not abolish eumelanin synthesis, such as Tyrp1, yield a brown coat color that may be difficult to distinguish from a reddish-brown pheomelanic coat color (Schmutz et al. 2002). Thus, for purposes of evaluating specific loci as candidates, we treated the dominant black allele inherited from Labrador retrievers as potentially nonpenetrant in greyhound backcross progeny, and considered genotypes of the backcross progeny only for black animals.
Like most seven transmembrane receptors, the Mc1r coding sequence is contained within a single exon. We polymerase chain reaction (PCR) amplified a fragment of genomic DNA that contained this exon and determined the complete Mc1r coding sequence for all black animals and all parental animals except one of the greyhound parents (Isis, Figure 1B). We identified four single-nucleotide variants that alter protein coding sequence, S90G (AGC
GGC), A105T (GCCACC), M264V (ATGGTG), and R306ter (CGATGA); all but the M264V variant were identified in our previous survey. As described above, we had previously considered S90G as a candidate for dominant black because it was found in Newfoundlands, flat-coated retrievers, and Labrador retrievers, but not in golden retrievers or Irish setters. However, of the 16 black animals whose genotypes we determined, 10 did not carry S90G, including 1 black Labrador retriever (B53, Figure 1A) and 1 yellow Labrador retriever (A14, Figure 1C), therefore the S90G variant probably has no functional significance.
As a more rigorous approach to determine if a specific Mc1r allele might cause dominant black, we first used pedigree information to determine Mc1r haplotypes, then asked whether black backcross progeny had inherited a consistent Mc1r allele from their Labrador retriever grandparent. The four coding sequence variants were found in seven different haplotype combinations (Table 1). In each kindred we found one or more animals that failed to inherit any Mc1r allele from their Labrador retriever grandparent: EB67 (Figure 1A), GB27 and GB47 (Figure 1B), and FB47 (Figure 1C). Thus dominant transmission of a uniform black coat color cannot be explained by an Mc1r allele in any of the kindreds.
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We also determined the complete coding sequence for Agouti in 14 animals (Figure 2); however, no sequence variants were identified (data not shown). To analyze inheritance of different Agouti alleles, we identified three microsatellite markers contained on the same bacterial artificial chromosome clone as the dog Agouti gene, genotyped a large series of animals (Figure 2), then, as for Mc1r, asked whether black backcross progeny had inherited a consistent Agouti allele from their Labrador retriever grandparent. Alleles for the three microsatellite markers were found in five different haplotypes (Table 2). In one of the kindreds, FX16 x Ester, none of the four black backcross progeny inherited an Agouti allele from their Labrador retriever grandparent, Andy (Figure 2). In the second kindred, Ester x B53, two of three black backcross progeny, DB27 and DB47, failed to inherit an Agouti allele from their Labrador retriever grandparent, B53 (Figure 2). Thus dominant transmission of a uniform black coat color cannot be explained by an Agouti allele.
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Our findings provide compelling evidence to exclude Agouti and Mc1r as the genes responsible for dominant black in the Cornell Labrador retriever x greyhound cross. In mice, three additional classic mutations affect pigment type switching: Ragged, mahogany, and mahoganoid; Ragged and mahogany have been identified at the molecular level (and are now known as Sox18 and Attractin, respectively) (Gunn et al. 1999; Pennisi et al. 2000). However, each of these genes has nonpigmentary effects, and therefore is unlikely to be responsible for dominant black. Assuming that dominant black is caused by a single locus, the Cornell cross should provide sufficient resolution to identify that locus by linkage using a genomewide panel of microsatellite markers. Molecular identification of dog dominant black will bring unique insight to the biochemistry of pigment type switching and Agouti-melanocortin signaling, since the same gene is apparently not represented in other model organisms.
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Materials and Methods |
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DNA Samples
Frozen leukocytes from a pedigree involving a cross between Labrador retrievers and greyhounds were kindly provided by George Lust and Michael Olivier from the Cornell Veterinary School, Ithaca, NY. DNA was isolated using a standard low-salt/phenol/chloroform DNA extraction protocol. Pedigree structures depicted in Figures 1 and 2 were verified by demonstrating that multiple unlinked microsatellite markers in addition to Agouti and Mc1r exhibited Mendelian segregation in accordance with expectations.
Mc1r Sequence Analysis
The entire coding sequence of Mc1r was amplified using the following primers: (F: 5'-GGTCATTGCTGAGCTGACAC-3') and (R: 5'-GAGATGCTGTCCAGTAGTCTCCC-3') and the following conditions: 40 cycles of 30 sec denaturing at 94°C, 1 min annealing at 60°C, and a 2-min extension at 72°C (Newton et al. 2000). The 1.3 kb amplicon was purified using Qiagen column purification and directly sequenced using ABI technology. The sequences were analyzed using Polyphred to detect polymorphisms.
Agouti Sequence Analysis
A bacterial artificial chromosome clone that contains the dog Agouti locus was isolated and sequenced; details will be presented elsewhere. We identified three microsatellites within the BAC sequence as follows:
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Acknowledgments |
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We thank Sheila Schmutz for useful discussion and Jon Longmire for his generous support of J.A.K. This paper was delivered at the Advances in Canine and Feline Genomics symposium, St. Louis, MO, May 16–19, 2002.
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Footnotes |
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Corresponding Editor: Urs Giger
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