Copyright � 2007 by the Genetics Society of AmericaDOI: 10.1534/genetics.107.074237

Linkage and Segregation Analysis of Black and Brindle Coat Color inDomestic Dogs

Julie A. Kerns,*,1 Edward J. Cargill,†,2 Leigh Anne Clark,† Sophie I. Candille,*Tom G. Berryere,‡ Michael Olivier,§,3 George Lust,§ Rory J. Todhunter,§

Sheila M. Schmutz,‡ Keith E. Murphy† and Gregory S. Barsh*,4

*Departments of Genetics and Pediatrics, Stanford University, Stanford, California 94305, †Department of Pathobiology,College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas 77843,

§College of Veterinary Medicine, Cornell University, Ithaca, New York 14853 and ‡Department of Animaland Poultry Science, University of Saskatchewan, Saskatoon S7N 5A8, Saskatchewan, Canada

Manuscript received April 4, 2007Accepted for publication April 26, 2007

ABSTRACT

Mutations of pigment type switching have provided basic insight into melanocortin physiology andevolutionary adaptation. In all vertebrates that have been studied to date, two key genes, Agouti andMelanocortin 1 receptor (Mc1r), encode a ligand-receptor system that controls the switch between synthesis ofred–yellow pheomelanin vs. black–brown eumelanin. However, in domestic dogs, historical studies based onpedigree and segregation analysis have suggested that the pigment type-switching system is more compli-cated and fundamentally different from other mammals. Using a genomewide linkage scan on a Labrador 3

greyhound cross segregating for black, yellow, and brindle coat colors, we demonstrate that pigment typeswitching is controlled by an additional gene, the K locus. Our results reveal three alleles with a dominanceorder of black (KB) . brindle (kbr) . yellow (ky), whose genetic map position on dog chromosome 16 is distinctfrom the predicted location of other pigmentation genes. Interaction studies reveal that Mc1r is epistatic tovariation at Agouti or K and that the epistatic relationship between Agouti and K depends on the alleles beingtested. These findings suggest a molecular model for a new component of the melanocortin signalingpathway and reveal how coat-color patterns and pigmentary diversity have been shaped by recent selection.

MORPHOLOGIC variation among domestic dogsexemplifies the power of selective breeding to

uncover a diversity of phenotypes from a relatively ho-mogeneous founder population. Major questions posedby this phenomenon are the extent to which widelydifferent phenotypes are caused by previously existinggenetic variation or new mutations and by epistatic in-teractions vs. single loci (reviewed in Falconer 1992;BartonandKeightley2002). Inseveralcases, linecrossesbetween divergent populations, e.g., mice or chickens withhigh or low body weight (Carlborg et al. 2006; Hrbek et al.2006), maize with high or low oil content (Laurie et al.2004), and Drosophila melanogaster with different num-bers of bristles (Dilda and Mackay 2002), have beenused to study selective breeding; for the most part, theseapproaches provide a genome-level view of geneticarchitecture and are particularly useful if little is known

about the underlying cell and molecular biology of thephenotypes, if there are a large number of candidategenes, or if one wishes to make no prior assumptionsabout the number or types of genes involved.

An alternative approach, taken here, is to consider aparticular phenotype that has been subject to selectionand use classical transmission genetics to investigatequestions of allelism and epistasis. This approach is par-ticularly useful for color variation, which often exhibitspatterns of inheritance that are consistent with Mendeliantransmission and for which the underlying biochemicaland molecular genetic pathways have been investigated inlaboratory animals (Searle 1968; Silvers 1979; Jackson

1997). The case of eumelaninic vs. pheomelaninic color-ation is particularly intriguing, since available evidencepoints to a genetic system in domestic dogs that is distinctfrom that known to operate in other mammals (Little

1957).In all mammals that have been studied to date, hair

follicle melanocytes synthesize red–yellow pheomelaninor black–brown eumelanin, depending on the balancebetween two key genes, Agouti and Melanocortin 1 receptor(Mc1r) (Andersson 2003; Klungland and Vage 2003).Agouti encodes a signaling molecule secreted fromspecialized cells in the dermis that acts as an inhibitoryligand for the Mc1r expressed on melanocytes (reviewed

1Present address: Fred Hutchinson Cancer Research Center, Seattle, WA98109.

2Present address: Monsanto, St. Louis, MO 63167.3Present address: Human and Molecular Genetics Center, Medical

College of Wisconsin, Milwaukee, WI 53226.4Corresponding author: Beckman Center B271A, Stanford University

School of Medicine, Stanford, CA 94305.E-mail: gbarsh@cmgm.stanford.edu

Genetics 176: 1679–1689 ( July 2007)

in Barsh 2006; Cone 2006). Mutations that constitu-tively activate the Mc1r cause a uniform black appear-ance, generally inherited in a dominant manner, whilemutations that inactivate the Mc1r cause a uniform redor yellow appearance, generally inherited in a recessivemanner. Conversely, because Agouti protein inhibitsMc1r activity, gain-of-function mutations yield domi-nant inheritance of a yellow coat, while loss-of-functionmutations yield recessive inheritance of a black coat.Much of the classical genetics underlying the aforemen-tioned relationships was summarized in a series of articlesby Sewall Wright (1917a,b,c,d, 1918a,b), when Mc1r wasknown as the Extension locus (because different allelescould extend the amount of yellow vs. black pigment),and loss-of-function Mc1r mutations were known asrecessive yellow (e).

Most dogs with a uniform black appearance, e.g., theNewfoundland, the flat-coated retriever, black Labradorretrievers, or black poodles, exhibit dominant trans-mission of the black color, consistent with mutationsthat constitutively activate the Mc1r. However, pedigreeand segregation analyses carried out by Clarence CookLittle (1957) indicated that dominant black was non-allelic with recessive yellow, leading to the suggestion thatdominant black might be an unusual allele of the Agoutilocus, AS. Recently, we examined a Labrador retriever 3

greyhound backcross with molecular probes for Agouti andMc1r and concluded that neither gene could accountfor the Labrador-derived black variant, which was in-herited in an apparent autosomal dominant mannerand to which we provisionally assigned the symbol K(Kerns et al. 2003).

An additional aspect of coat-color variation in domes-tic dogs that appears distinct from most other mammalsis the phenotype known as brindle, in which stripes ofred–yellow hair alternate with black–brown hair. Brindlestripes form an irregular pattern, typically with a ‘‘V’’shape over the dorsum and an ‘‘S’’ shape over flanks andventrum and are somewhat reminiscent of a dermatologicphenomenon in humans known as lines of Blaschko,thought to be caused by mosaicism of gene expression inkeratinocyte clones ( Jackson 1976; Bolognia et al. 1994;Widelitz et al. 2006). Brindle segregates as a single gene ina variety of dog breeds, such as the boxer, greyhound, andFrench bulldog, and has been thought by some authors tobe caused by variation in Agouti, but by others to be causedby variation in Mc1r (Winge 1950; Little 1957; Willis

1989).To better understand the genetic mechanisms re-

sponsible for coat-color diversity among domestic dogs,we carried out a genomewide linkage scan on pedigreessegregating dominant black, brindle, or both. Our re-sults reveal a single locus with three alleles—yellow (ky),brindle (kbr), and black (KB)—whose genetic map positionis clearly distinct from pigmentation genes known inother mammals. Interactions between alleles of theK locus and those of Agouti and Mc1r uncover a simple

genetic architecture that explains all known eumelanic–pheomelanic variation and helps to reveal how selectionhas shaped morphologic diversity among different breedsof domestic dogs.

MATERIALS AND METHODS

DNA samples and pedigrees: Genomic DNA from blood orcheek swab samples was isolated according to standard pro-cedures. Pedigrees in Figures 1 and 2 were established by threeof us (R. J. Todhunter, G. Lust and M. Olivier) at CornellUniversity to study hip dysplasia; pedigrees in Figure 3 wereascertained by one of us (S. M. Schmutz) as part of a series ofongoing studies on dog coat-color genetics and were donatedby private breeders. In all cases, pedigree relationships wereverified by determining that multiple markers exhibited Men-delian segregation in accord with expectations.

Genotyping, statistical analysis, and genomics: Genotypingfor the minimal screening set I panel of simple sequencelength polymorphism (SSLP) repeat markers described byRichman et al. (2001) was carried out using multiplex PCR aspreviously described (Cargill et al. 2002; Clark et al. 2004).Fluorescently labeled PCR products were separated on theautomated laser fluorescence DNA sequencer ABI377 (Perkin-Elmer, Norwalk, CT), using GENESCAN (version 2.1) frag-ment analysis software, and alleles were identified using theGENOTYPER program (version 2.0; Perkin-Elmer).

Prior to linkage analysis, Mendelian error checking was per-formed. The data were then analyzed under a model of auto-somal dominant inheritance for black vs. nonblack, assumingcomplete penetrance. Two-point linkage analyses were carriedout using the MLINK (to generate LOD scores at differentu-values) and ILINK programs (to maximize LOD) from theLINKAGE 5.1 package (Lathrop and Lalouel 1984). Giventhe small number of animals in the scan, further analysis ofadditional markers was done manually to determine the criticalregion and to infer haplotypes, as depicted in Figures 1–3.

To infer the epistasis relationships among Agouti, Mc1r, andK alleles, we determined the Agouti and Mc1r coding sequenceby sequencing PCR-amplified fragments of genomic DNA.Primer sequences have been described previously (Newton

et al. 2000; Schmutz et al. 2003; Kerns et al. 2004; Berryere

et al. 2005) and are available upon request. We determined Kgenotypes using linkage and pedigree analysis (as describedbelow) and, in some cases, by using additional markers that arein linkage disequilibrium with K locus alleles, which will bedescribed elsewhere. Genotypes for all three loci were de-termined (by resequencing Agouti and Mc1r or by genotypingflanking markers for K as described above) for every individualdepicted in Figures 1–3. This included 35 animals from theCornell Labrador retriever 3 greyhound cross, 10 Afghanhounds, 8 Great Danes, and 10 Staffordshire bull terriers. Atleast 5 animals from each of four additional breeds—Germanshepherd dogs, French bulldogs, boxers, and poodles—werealso genotyped for all three loci as described in Table 3.

The physical location of markers and consideration ofcandidate genes is based on the CanFam1.0 assembly of thedog whole-genome sequence (Lindblad-Toh et al. 2005)available through the UCSC Genome Browser (Karolchik

et al. 2003).

RESULTS

Nomenclature: Historically, Little and others (Little

1957; Willis 1989) recognized that at least two different

1680 J. A. Kerns et al.

genes could give rise to a uniform pheomelanic coat andused the term ‘‘fawn’’ to refer to the phenotype caused bythe ay allele of Agouti, distinguished from a similar pheno-type caused by a loss-of-function Mc1r allele, originallyknown as recessive yellow or e, and now known to representMc1r R306ter. In some cases, differential gene action was in-ferred on the basis of the phenotype, with homozygosityfor Mc1r R306ter giving rise to a clear or diluted yellow color,and the ay allele of Agouti referring to a deeper, often dark-tinged shade of red–yellow. In hindsight, the effects ofAgouti and Mc1r alleles cannot always be distinguished byvirtue of their phenotype; also, the K locus genotype isequally important in determining the balance and distri-bution of pheomelanin vs. eumelanin. To reconcile thehistorical terms with both common usage and moderngenetics, we propose that alleles of the K locus be desi-gnated as yellow (ky), brindle (kbr), and black (KB). A summaryof this nomenclature that relates the historical terms tothose used here and the underlying genetics (describedfurther below) is given in Table 1.

Genomewide linkage scan and fine mapping fordominant black: In a Labrador retriever 3 greyhoundcross that was established at Cornell University to studyhip dysplasia, a subset of kindreds exhibit transmissionof coat-color variation in a pattern that is consistent withinheritance of dominant black as originally suggestedby Little (1957). Black Labrador retrievers crossed toyellow or brindle greyhounds invariably yield black F1

offspring; when an F1 animal is backcrossed to the grey-

hound parent, backcross progeny exhibit 1:1 segrega-tion of black to nonblack.

In previous studies of the EB and GB kindreds (Figure1) from this cross (Kerns et al. 2003), we observed thatvariation at neither Mc1r nor Agouti could account fordominant black (as an allele of the putative K locus);therefore we carried out a genomewide linkage scanof the same kindreds using a dense panel of highlyinformative SSLP markers. For the initial screen of 19animals (Figure 1), 125 of 155 markers from the ‘‘minimalscreening set’’ described by Richman et al. (2001) wereinformative. We analyzed the results by two-point linkageanalysis under a model of dominant inheritance withcomplete penetrance and observed that there were threeloci on chromosomes 4 and 16 (CFA4, CFA16) thatexceeded a LOD score of 2 (Table 2).

The strongest evidence for linkage was obtained onCFA16 with marker FH2155 (Zmax ¼ 3.6 at u ¼ 0). Weused the same marker, FH2155, to analyze four addi-tional kindreds from the Cornell pedigree (Figure 2 anddata not shown) and observed no recombinants be-tween FH2155 and the K locus, yielding a LOD score of6 at u ¼ 0.

To refine the map location, we examined three addi-tional markers surrounding FH2155 that span a dis-tance of �24 Mb—FH3592, REN275L19, and FH2175.In the EB and GB kindreds, recombinant chromosomesin two animals, EB57 and GB17, define a critical regionbetween FH2175 and FH3592 of 23.7 Mb (33.7–57.4).

TABLE 1

Phenotype–genotype relationships for Agouti, K, and Mc1r

Possible genotypes based on this worka

Common namePhenotype andbreed example

Historicalnames (symbols) Agouti K locus Mc1r

Dominant black or‘‘self-colored’’

Uniformly black, can bemodified to brown orby white spotting:Newfoundland, black orbrown Labrador retriever

Dominant black,Agouti-Self (AS)

ay/a, ay/at, ay/a KB/KB, KB/ky,KB/kbr

1/1, 1/R306ter

Recessive yellow Uniformly red–yellow, canbe modified to paleyellow or cream: yellowLabrador retriever,Irish setter, Samoyed

recessive yellow,extension (e)

ay, at, a (allcombinations)

KB, kbr, ky, (allcombinations)

R306ter/R306ter

Fawn Red–tan, can be darktinged: Great Dane,fawn (i.e., yellow) boxer

dominant yellow,golden sable (ay)

ay/a, ay/at, ay/a ky/ky 1/1, 1/R306ter

Black and tan Dobermann pinscher tan points (at) at/a, at/a ky/ky 1/1, 1/R306terBrindle Black- and yellow-colored

stripes: brindle Frenchbulldog, brindle boxer

brindle, partialextension (ebr)

ay/a, ay/at, ay/a kbr/kbr, kbr/ky 1/1, 1/R306ter

Recessive black Uniformly black: blackGerman shepherd dog

recessive black(a) a/a KB, kbr, ky (allcombinations)

1/1, 1/R306ter

Explanations and references for names and symbols are given in the text.a Possible genotypes according to epistasis relationships as described in the text and in Table 3. Only three Agouti alleles are

considered for the sake of simplicity; the aW allele would behave identically to the at allele. Also, the ‘‘1’’ allele at Mc1r is used todesignate any Mc1r allele other than R306ter (also known as recessive yellow or e).

Dog Black and Brindle Genes 1681

An additional marker that lies between FH2175 andFH2155, REN292N24, was informative only for the FBand HB kindreds (Figure 2), but exhibited the samesegregation pattern as FH2175 (3/10 recombinants),which therefore narrows the critical region to a 12-Mbsegment (45.4–57.4, Figure 2B).

This region of the dog genome contains two humanhomology segments, 4q34–4q35 and 8p12, and has beenannotated with .250 genes, mostly from other mam-malian genomes (Figure 2C). Notably, none of thosegenes has been previously implicated in pigmentation,i.e., as a cause of human albinism or a mouse coat-colormutation. Thus, the dog K locus is likely to represent apreviously unappreciated component of the Agouti–Melanocortin pathway.

Allelism of yellow (ky) and brindle: As depicted inFigures 1 and 2, the FB, GB, and HB litters contain onlyblack and yellow animals, whereas the EB litter andseveral of the parents in the Cornell cross are brindle.These observations are consistent either with brindlebeing an intermediate allele of the K locus, recessiveto black (K) and dominant to yellow (ky), or with brindlebeing caused by another gene whose effects are hypo-static to those of the K allele. On the basis of the previous

studies in which brindle 3 brindle crosses often yieldeda mixture of brindle and yellow (but never black)progeny, many dog breeders and geneticists assumedthat brindle is caused by an intermediate allele of theextension locus, ebr, that is dominant to recessive yellow (e orMc1rR306ter) but hypostatic to dominant black (originallyassigned to AS).

To investigate whether allelism or epistasis was morelikely to explain the relationship between brindle andthe K locus, we ascertained several kindreds in whichbrindle and yellow segregated and asked whether FH2155,which cosegregated perfectly with black vs. yellow (Figures1 and 2), might also cosegregate with brindle vs. yellow.As depicted in Figure 3, there was perfect cosegregationbetween FH2155 and brindle vs. yellow in five phase-known meioses (in an Afghan pedigree) and 14 phase-unknown meioses (across a Great Dane and a Staffordshirebull terrier pedigree), corresponding to a LOD score of3.6. We also reexamined all kindreds in the entire Cornellcross and a boxer pedigree (data not shown) and foundthat, in every case, transmission of brindle was consistentwith an intermediate allele of theK locus with the followingdominance relationships: dominant black (KB) . brindle (kbr). yellow (ky).

Figure 1.—Segregation ofCFA16 haplotypes in the EB andGB litters. (A) Haplotypes basedon the four SSLP markers in B areindicated with vertical bars, justabove genotypes for K locus alleles.(As described in the text, brindleand yellow were considered in thesame class, ‘‘nonblack,’’ for analysisof the genome scan; genotypesgiven here for kbr and ky are basedon information presented in Figure3.) Black-colored haplotypes origi-nate from the Labrador retrievergrandparent carrying dominantblack (B53 or Andy); white-coloredhaplotypes originate from the non-black greyhound parent or grand-parent (Esther or Isis). SSLPalleles are numbered arbitrarilyaccordingtoincreasingsize foreachmarker. (B) Physical location ofSSLP markers used in A indicatedinmegabases (Mb) fromthe centro-mere. To the right of each markername is the number of animals re-combinant between that markerand the K allele over the total num-ber of animals that were informa-tive for that marker. Recombinantchromosomes are carried by EB57and GB17 and define a criticalregion for K between FH2175and FH3592. In addition to theK locus genotypes depicted, Agoutiand Mc1r genotypes were deter-mined for every dog as describedin materials and methods.

1682 J. A. Kerns et al.

TABLE 2

Two-point LOD scores for selected markers from genomewide linkage analysis

u

Marker name Chromosome 0.1 0.2 0.3 0.4

FH2309 1 �2.66 �1.163 �0.45 �0.106FH2598 1 �6.48 �3.57 �1.93 �0.81FH2294 1 �2.98 �1.58 �0.82 �0.33CO2.342 2 �3.8717 �1.9691 �0.96843 �0.36165CO2.864 2 �4.57067 �2.36704 �1.19028 �0.45856C02.608 2 �4.57 �2.37 �1.19 �0.459FH2302 3 �2.98431 �1.58146 �0.81699 �0.32619FH2107 3 �3.62 �1.76 �0.822 �0.282FH2531 3 �2.66219 �1.16292 �0.45432 �0.10637AHT128 4 2.11 1.85 1.39 0.774FH2534 4 2.11 1.85 1.39 0.774GLUT4 5 �4.57067 �2.36704 �1.19028 �0.45856CPH18 5 �2.47 �1.17 �0.52 �0.168TAT 5 �2.984 �1.58 �0.82 �0.326FH2119 6 �3.61643 �1.76498 �0.8223 �0.28246CPH3 6 �2.66219 �1.16292 �0.45432 �0.10637FH2396 7 �2.66219 �1.16292 �0.45432 �0.10637CO8.618 8 �3.93855 �2.18352 �1.18496 �0.50228FH2138 8 �4.57067 �2.36704 �1.19028 �0.45856FH2186 9 �2.66 �1.16 �0.454 �0.106FH2537 10 �2.66 �1.16 �0.454 �0.106FH2293 10 �2.00838 �0.85516 �0.35447 �0.11861FH2422 10 �2.54061 �1.38764 �0.74127 �0.30846FH2319 11 �2.66219 �1.16292 �0.45432 �0.10637FH2096 11 �3.49485 �1.9897 �1.10924 �0.48455AHT137 11 �4.57067 �2.36704 �1.19028 �0.45856C12.852 12 �3.49 �1.99 �1.12 �0.484CXX.391 13 �3.49 �1.2 �1.12 �0.485FH2060 14 �5.08122 �2.77528 �1.48253 �0.61692FH2547 14 �5.5 �2.7 �1.56CPH5 15 �2.21849 �0.9691 �0.3786 �0.08864FH2321 15 �2.66219 �1.16292 �0.45432 �0.10637COS15 15 �2.03006 �0.9794 �0.44901 �0.1501AHT139 15 �4.2 �2.4 �1.33 �0.58FH2171 15 �6.48 �3.6 �1.9 �0.81FH2278 15 �2.00897 �0.86147 �0.38114 �0.17759FH2175 16 2.109028 1.84738 1.38556 0.774084FH2155 16 3.0632 2.449 1.754 0.950175AHTK209 20 �2.98431 �1.58146 �0.81699 �0.32619FH2312 21 �3.62 �1.76 �0.822 �0.282FH2233 21 �2.98431 �1.58146 �0.81699 �0.32619FH2538 22 �2.66219 �1.16292 �0.45432 �0.10637REN49F22 22 �2.66 �1.16 �0.454 �0.11FH2079 24 �2.96262 �1.45722 �0.72245 �0.2947FH2261 24 �5.52491 �2.9691 �1.55826 �0.63465C26.733 26 �2.54061 �1.38764 �0.74127 �0.30846REN48E01 26 �2.54061 �1.38764 �0.74127 �0.30846PEZ6 27 �2.03006 �0.9794 �0.44901 �0.1501CXX.176 28 �2.47 �1.17 �0.525 �0.168FH2208 28 �2.66219 �1.16292 �0.45432 �0.10637FH2585 28 �2.66219 �1.16292 �0.45432 �0.10637FH2305 30 �4.57067 �2.36704 �1.19028 �0.45856FH2199 31 �4.57067 �2.36704 �1.19028 �0.45856FH2239 31 �2.54061 �1.38764 �0.74127 �0.30846FH2238 32 �3.61643 �1.76498 �0.8223 �0.28246CPH2 32 �3.61643 �1.76498 �0.8223 �0.28246REN41D20 32 �3.61643 �1.76498 �0.8223 �0.28246

(continued )

Dog Black and Brindle Genes 1683

Epistatic interactions between alleles of the Agouti,K, and Mc1r loci: As indicated above, loss of function forMc1r (recessive yellow, e) causes a yellow coat color thatmay appear very similar or even indistinguishable fromthat caused by homozygosity for yellow (ky). Likewise, lossof function for Agouti (nonagouti, a) causes a black coatcolor that is indistinguishable from that caused byheterozygosity for black (KB). To investigate the epistaticrelationships between K locus alleles and those of Agoutiand Mc1r, we determined the genotype for all three lociin key animals from the pedigrees depicted in Figures1–3 and additional animals described in previous studies(Schmutz et al. 2003; Kerns et al. 2004; Berryere et al.2005). For Agouti, we used the predicted cDNA sequenceto distinguish among the ay, at, and a alleles (Berryere

et al. 2005); for Mc1r, we used the predicted cDNA se-quence to distinguish between the R306ter (e) allele andall others (referred to below as Mc1r1).

The consequent genotype–phenotype relationshipsprovide a coherent view of epistatic interactions. Forexample, the Labrador retrievers Andy (Figure 1A) andA14 (Figure 2A) have a genotype of at/at; KB/KB; e/e ;both animals have a yellow coat, demonstrating that lossof function for Mc1r is epistatic to both the black-and-tan(at) allele of Agouti and the black (KB) allele of the Klocus. In fact, Labrador retrievers are fixed for the black(KB) allele of the K locus and the black-and-tan (at) alleleof the Agouti locus; thus, black Labrador retrieversdemonstrate that the ability of the K locus to produceblack pigment is epistatic to that of the Agouti locus toproduce yellow pigment (because at/at; KB/KB animalsare black rather than black and tan).

Observations for an additional two breeds are partic-ularly demonstrative. Traditionally marked German shep-herd dogs are fixed for the yellow (ky) allele of the K locusand the 1 allele of Mc1r; the difference between blackand black-and-tan German shepherd dogs is determinedsolely by the nonagouti (a) vs. the at allele of Agouti. Thus,the ability of Agouti to prevent production of yellowpigment is epistatic to that of the K locus to allow yellowpigment. (Stated differently, the yellow allele (k�y) of the Klocus can give rise only to yellow pigment in the presenceof a functional Agouti allele.) Finally, Afghan houndswith a K genotype that would ordinarily yield brindle

(kbr/kbr or kbr/ky) may vary at both Agouti (at or ay) and Mc1r(1 or R306ter). In all cases, the interactions between kbr

and Agouti or Mc1r alleles can be predicted on the basis ofwhat happens for ky and for KB. In at/at; kbr/kbr; 1/1

animals, brindling is restricted to the areas of the coat thatwould otherwise be tan (‘‘black and brindle’’); in e/eanimals, brindling is not apparent because Mc1r isepistatic not only to KB but also to kbr.

These relationships, together with specific examplesin which we have directly determined the genotypes forAgouti, Mc1r, and K, are summarized in Table 3, andtheir implications for understanding the underlying bio-chemical pathways are depicted in Figure 4. There areseveral key points. First, the relationship between Mc1rand Agouti in dogs is identical to that which occurs inother mammals where Agouti acts to antagonize mela-nocortin signaling in a manner that is completely de-pendent on a functional receptor. Second, Mc1r is epistaticto all K locus variation, and the K gene product behavessimilarly to Agouti protein in this way; each requires afunctional Mc1r to modulate melanocortin signaling.Finally, the epistatic relationship between Agouti and Kdepends on the alleles being tested: ‘‘black alleles’’ of K areepistatic to ‘‘yellow alleles’’ of Agouti, but ‘‘black alleles’’ ofAgouti are epistatic to ‘‘yellow alleles’’ of K. Thus, the rela-tionship between Agouti and K is fundamentally differentfrom the relationship between Mc1r and either Agouti or K.

These considerations suggest two alternative models(Figure 4). The K gene product may lie geneticallyupstream of Agouti and inhibit its function, either as anegative regulator of Agouti mRNA expression or as apost-translational inhibitor that reduces the levels ofactive Agouti protein at the Mc1r (Figure 4A). Alterna-tively, the K gene product may act directly at the Mc1r tostimulate melanocortin signaling and thereby opposethe action of Agouti protein indirectly (Figure 4B).

DISCUSSION

A general theme of pigmentary genetics for the lastcentury is that patterns of Mendelian variation withinone species frequently display apparent homology tothose in other species. For example, similar segregation

TABLE 2

(Continued)

u

Marker name Chromosome 0.1 0.2 0.3 0.4

AHT133 37 �2.98431 �1.58146 �0.81699 �0.32619FH2532 37 �3.61643 �1.76498 �0.8223 �0.28246FH2587 37 �2.66219 �1.16292 �0.45432 �0.10637

A complete set of the 155 markers used for the linkage scan and the results are available upon request. Thetable shows those markers for which two-point LOD scores between the marker and dominant black were either.2.0 or , �2.0 at u ¼ 0.1. The former category (.2.0) is shown in italics.

1684 J. A. Kerns et al.

and dominance relationships are observed among mice,guinea pigs, rabbits, and cats for the phenotypic seriesfull color . chinchilla . acromelanic . albino, leadingto the suggestion that mutations in the same gene—nowknown as Tyrosinase—are responsible. These types ofobservations, first made by Sewall Wright (1917c) andlater by Clarence Cook Little (1957) and A. G. Searle

(1968), foreshadowed the field of comparative genomics.Indeed, comparison of genome sequences not only clar-ified the evolutionary relationships among mammals(and most other organisms), but also provided the toolsto identify molecular alterations responsible for the Tyros-inase color series in mice (Kwon et al. 1989), cats (Lyons

et al. 2005; Schmidt-Kuntzel et al. 2005), cattle (Schmutz

et al. 2004), and rabbits (Aigner et al. 2000) (although,ironically, not yet in guinea pigs).

Dominant black and brindle in dogs have been curiousand somewhat confusing exceptions to the aforemen-

tioned theme. Historically, the allelic relationships forthe Agouti locus—to which dominant black was assignedas the AS allele—were thought to be opposite to whatpertains in other mammals, where ‘‘yellow alleles’’ aredominant to ‘‘black alleles’’ (Little 1957). In the case ofbrindle, assigned to the Mc1r locus as ebr, epistasis relation-ships were confusing, with ebr epistatic to the ay allele butnot to the AS allele (ay/ay; ebr/ebr animals would be brindlebut AS/AS; ebr/ebr animals would be black).

The work described here resolves this confusion bydemonstrating that both dominant black and brindlingare due to alleles of a previously unappreciated pigmen-tation gene that we have named the K locus. Althoughthe K locus is an apparent exception to the idea that thesame set of molecular tools are used in all mammals, itsrecognition reinforces the general theme that geneticinteractions and pathways for orthologous genes areconserved. Thus, interactions both within and between

Figure 2.—Segregation ofCFA16 haplotypes in the FB andHB litters. (A and B) Symbols areas in Figure 1. Recombinant chro-mosomes are carried by FB27,FB67, and HB27 and indicate thatK must lie centromere distal toREN292N24. In addition to the Klocus genotypes depicted, Agoutiand Mc1r genotypes were deter-mined for every dog as describedin materials and methods. (C)Diagram of the K critical regionfrom REN292N24 to FH3592, indi-cating the location of RefSeq genesin the region (blue) and evolution-arily conserved regions in the hu-man genome. Annotation is basedon the CanFam1.0 dog genome as-sembly (Lindblad-Toh et al. 2005)as displayed by the UCSC GenomeBrowser(Karolchik et al. 2003) us-ing the ‘‘Human Net’’ comparativegenomics track, in which red andyellow indicate sequence similarityto human chromosomes 8 and 4,respectively.

Dog Black and Brindle Genes 1685

Agouti and Mc1r alleles in dogs are identical to thoseobserved in other mammals: ‘‘black’’ Mc1r alleles aredominant to ‘‘yellow’’ Mc1r alleles, ‘‘yellow’’ Agouti allelesare dominant to ‘‘black’’ Agouti alleles, and double mutants

for Mc1r and Agouti always exhibit the phenotype of singleMc1r mutants.

Our original survey of Mc1r variation among domesticdogs (Newton et al. 2000) was motivated by the idea thatdominant black might be due to a gain-of-function Mc1rallele, as described in many other vertebrates (Eizirik

Figure 3.—Segregation of FH2155 al-leles in three kindreds with brindle andyellow. As described in the text, there isperfect cosegregation of FH2155 withbrindle vs. yellow under a model ofdominant inheritance with KB . kbr .ky, corresponding to a LOD score of 3.6.

TABLE 3

Epistasis relationships for Agouti, K, and Mc1r

Genotypea

Agouti K locus Mc1r Phenotypeb Examplec

at/at k/k 1/1 Black and tan German shepherddog

at/at k/k e/e Yellow Afghan houndat/at kbr/kbr 1/1 Black, brindle

pointsStaffordshire bull

terrierat/at kbr/kbr e/e Yellow French bulldogat/at KB/KB 1/1 Black Black Labrador

retrieverat/at KB/KB e/e Yellow Yellow Labrador

retrieveray/ay k/k 1/1 Yellow Boxeray/ay k/k e/e Yellow Afghan hounday/ay kbr/kbr 1/1 Brindle Boxeray/ay kbr/kbr e/e Yellow Afghan hounday/ay KB/KB 1/1 Black Great Daneay/ay KB/KB e/e Yellow Poodle

a Nomenclature is similar to Table 1, with the R306ter alleleof Mc1r indicated as e. For each category, only homozygousgenotypes are shown for the sake of simplicity; more geno-types are possible according to dominance relationships foreach locus as indicated in Table 1.

b These designations refer only to the distribution of eume-lanin and pheomelanin and ignore the effects of modifiersthat affect spotting and/or pigment quality. For example,black and tan in a cocker spaniel homozygous for the b alleleof the Tyrp1 locus would be modified to liver and tan; brindlein a French bulldog carrying an s mutation would appearwhite with brindle spots.

c Examples are based on genotyping studies of dogs fromthe indicated breeds as described in the text (Newton

et al. 2000 or Berryere et al. 2005).

Figure 4.—Models for gene action at the K locus. Bothmodels must account for the observations that (1) the dom-inance order of Agouti is opposite to that of K; (2) Mc1r allelesare epistatic to both Agouti and K locus alleles; (3) ‘‘black al-leles’’ of K are epistatic to ‘‘yellow alleles’’ of Agouti; and (4)‘‘black alleles’’ of Agouti are epistatic to ‘‘yellow alleles’’ ofK. 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 geneproduct acts directly at the Mc1r to stimulate melanocortinsignaling and thereby oppose the action of Agouti protein in-directly.

1686 J. A. Kerns et al.

et al. 2003; Klungland and Vage 2003; Mundy andKelly 2003; Nachman et al. 2003; Rosenblum et al.2004; Hoekstra et al. 2006). Although we and othershave identified a number of Mc1r polymorphisms amongdomestic dogs (Everts et al. 2000; Newton et al. 2000;Schmutz et al. 2003), the only one for which there is anunequivocal effect on function is R306ter, responsible forthe loss-of-function allele originally described as recessiveyellow (e). Given the diversity of coat colors and patternsselected in modern breeds, it is perhaps surprising thatan Mc1r mutation that causes dominant black has notbeen found in dogs. However, a likely explanation is thatvariation at the K locus is relatively old among the canidlineage, since preexisting polymorphism for k vs. K wouldmake it less likely that a new dominant black mutation atMc1r would be noticed.

Because the black (KB) allele is epistatic to variationat Agouti, the yellow (ky) allele probably represents theancestral state; otherwise, the Agouti phenotype (andother aspects of Agouti-induced variation such as white-bellied Agouti and black and tan) would have been crypticin the ancestral population where variation at K firstoccurred. According to this hypothesis, wolf populationsfrom which dogs were domesticated some 15,000–40,000years ago were Agouti colored or a gray modification ofAgouti, similar to the appearance of modern wolves (Vila

et al. 1999; Savolainen et al. 2002). Mutation from ky to KB

is likely to have occurred prior to the origin of modernbreeds several hundred years ago and could even havebeen present in wolves as an adaptive polymorphism priorto domestication. An alternative scenario—positing thatbrindle (kbr) is the ancestral allele, with yellow (ky) and black(KB) as derivative alleles—is less likely, given that thebrindle phenotype is not present in modern canids otherthan domestic dogs.

Superficially, the brindle phenotype in domestic dogsshares some features with tabby striping in domestic cats(Lomax and Robinson 1988). Both involve patches orstripes of eumelanic vs. pheomelanic hairs, and bothrequire the presence of a functional Agouti gene. How-ever, the allelic system for tabby striping is probablyopposite to brindle: the presence of black tabby stripes(tb) is recessive to the absence of such stripes associatedwith the Abyssinian (Ta) allele in cats, while the presenceof black brindle stripes (kbr) is dominant to the absenceof such stripes associated with the yellow (ky) allele indogs. Equally important, the pattern of tabby striping isalternating and regular, consistent with an underlyingpattern based on a Turing-like reaction–diffusion mech-anism (Suzuki et al. 2003; Jiang et al. 2004; Widelitz

et al. 2006). By contrast, the pattern of brindle stripesis irregular and variegated, most consistent with anepigenetic mechanism (discussed further below). Theseconsiderations are consistent with the view based onphylogenetic distribution of color patterns that tabbystriping and brindling have independent evolutionaryhistories (Searle 1968).

The evolutionary history of variation at the K locuswill also have an impact on strategies for its molecularidentification. The 12-Mb region to which K has beenmapped contains �250 genes, many of which mightplausibly be involved in melanocortin signaling (butnone of which are obvious candidates). While additionalpedigree-based linkage analysis could further narrow thecritical interval, a potentially more effective strategy isbased on genetic association. Additional genotyping ofSSLP and SNP markers within the 12-Mb interval shouldreveal whether the K, kbr, and k alleles have specifichaplotypes with which they are associated. If so, compar-ing the length of those haplotypes among unrelatedanimals may delineate a small candidate region. Successof this approach will depend on the degree to which Klocus alleles are identical by descent.

The epistasis relationships between K and Agouti orMc1r may also help to prioritize candidate genes. A func-tional Mc1r is required to ‘‘visualize’’ variation at K andat Agouti; e.g., animals homozygous for the Mc1r R306ter(e) allele are yellow regardless of their genotype at Kor Agouti. Furthermore, a functional Agouti gene is re-quired to ‘‘visualize’’ variation at K. This latter point isespecially apparent from interactions between the black-and-tan (at) and the brindle (kbr) mutations. The at muta-tion affects transcriptional regulation of Agouti codingsequences, limiting their expression to the dorsum orsaddle areas; thus, the tan areas in black-and-tan animals(of genotype at/at; ky/ky; Mc1r1/1) represent locations ofAgouti expression. In at/at; kbr/kbr; Mc1r1/1 animals, theeffects of kbr are restricted to the areas of Agouti ex-pression, producing the phenotype known as ‘‘blackand brindle’’ or ‘‘black with brindle points.’’ Takentogether, these considerations suggest that the K geneproduct functions outside, rather than within, melano-cytes either as a negative regulator of Agouti proteinlevels or as an alternative Mc1r ligand that activatesmelanocortin signaling (Figure 4).

A corollary of this argument is that the stripes in abrindle animal are likely to represent clones of skin cellsthat behave genetically as either KB or ky, in which theirregular and unpredictable distance between stripesreflects a stochastic event that initially ‘‘sets’’ the appar-ent genotype for each clone. The brindle-stripe patternis similar to Blaschko lines in humans, thought to becaused by mosaicism of gene expression in keratinocyteclones (Bolognia et al. 1994; Widelitz et al. 2006).From this perspective, the fascinating pattern caused bythe kbr mutation is most likely explained by an unstableallele—between yellow (ky) and black (KB)—that acquiresone or the other state by chance and then maintains thatstate epigenetically as keratinocytes divide and migrateduring embryonic development. An epigenetic eventacting on keratinocyte clones would also explain why thebrindle pattern in dogs is qualitatively different fromvariegation observed in X-inactivation mosaics or em-bryonic stem cell chimeras, where the relevant cell type

Dog Black and Brindle Genes 1687

is usually a neural-crest-derived melanocyte, as withchimeras involving the albino mutation, or dermal pa-pilla cells, as with chimeras involving Agouti (Mintz

1971a,b; Millar et al. 1995; Wilkie et al. 2002). Thus, alikely candidate for the K gene product is a secretedprotein produced primarily by keratinocytes, but which,like Agouti protein, has a short radius of action.

Molecular identification of the Agouti and Mc1r genesprovided much of the molecular groundwork for un-derstanding the role of melanocortin signaling in avariety of physiologic processes, including regulation ofenergy balance, sexual behavior, and adrenocortical ho-meostasis. Additional studies of the K locus in domesticdogs may allow similar opportunities.

We thank J. Longmire for his support of J.A.K., Elaine Ostrander forhelpful discussions, and Alma Williams for collecting and organizingdata from the Cornell pedigree. We are grateful to the dog breederswho generously submitted DNA samples from their litters and to theDogMap and the National Human Genome Research Institute doggenome project for providing public access to the canine map athttp://www.dogmap.ch/index.html and http://research.nhgri.nih.gov/dog_genome/. This work was supported by funds from theNational Institutes of Health.

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Communicating editor: T. R. Magnuson

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