Agouti sequence polymorphisms in coyotes, wolves and dogs suggest hybridization

Domestic dogs have been shown to have multiple alleles of the Agouti Signal Peptide (ASIP) in exon 4 and we wished to determine the level of polymorphism in the common wild canids of Canada, wolves and coyotes, in comparison. All Canadian coyotes and most wolves have banded hairs. The ASIP coding sequence of the wolf did not vary from the domestic dog but one variant was detected in exon 4 of coyotes that did not alter the arginine at this position. Two other differences were found in the sequence flanking exon 4 of coyotes compared with the 45 dogs and 1 wolf. The coyotes also demonstrated a relatively common polymorphism in the 3' UTR sequence that could be used for population studies. One of the ASIP alleles (R96C) in domestic dogs causes a solid black coat color in homozygotes. Although some wolves are melanistic, this phenotype does not appear to be caused by this same mutation. However, one wolf, potentially a dog-wolf hybrid or descendant thereof, was heterozygous for this allele. Likewise 2 coyotes, potentially dog-coyote or wolf-coyote hybrid descendants, were heterozygous for the several polymorphisms in and flanking exon 4. We could conclude that these were coyote-dog hybrids because both were heterozygous for 2 mutations causing fawn coat color in dogs.     

Sequence analysis of three pigmentation genes in the Newfoundland population of Canis latrans links the Golden Retriever Mc1r variant to white coat color in coyotes

Three genes, Mc1r, Agouti, and CBD103, interact in a type-switching process that controls much of the pigmentation variation observed in mammals. A deletion in the CBD103 gene is responsible for dominant black color in dogs, while the white-phased black bear (“spirit bear”) of British Columbia, Canada, is the lightest documented color variant caused by a mutation in Mc1r. Rare all-white animals have recently been discovered in a new northeastern population of the coyote in insular Newfoundland and Labrador, Canada. To investigate the causative gene and mutation of white coat in coyotes, we sequenced the three type-switching genes in white and dark-phased animals from Newfoundland. The only sequence variants unambiguously associated with white color were in Mc1r, and one of these variants causes the amino acid variant R306Ter, a premature stop codon also linked to coat color in Golden Retrievers and other dogs with yellow/red coats. The allele carrying R306Ter in coyotes matches that in the Golden Retriever at other variable amino acid sites and hence may have originated in these dogs. Coyotes experienced introgression with wolves and dogs as they colonized northeastern North America, and coyote/Golden Retriever interactions have been observed in Newfoundland. We speculate that natural selection, with or without a founder effect, may contribute to the observed frequency of white coyotes in Newfoundland, as it has contributed to the high frequency of white bears, and of a domestic dog-derived CBD allele in gray wolves.     

Chocolate coated cats: TYRP1 mutations for brown color in domestic cats

Brown coat color phenotypes caused by mutations in tyrosinase-related protein-1 (TYRP1) are recognized in many mammals. Brown variations are also recognized in the domestic cat, but the causative mutations are unknown. In cats, Brown, B, has a suggested allelic series, B > b > b^l. The B allele is normal wild-type black coloration. Cats with the brown variation genotypes, bb or bb^l, are supposedly phenotypically chocolate (aka chestnut) and the light brown genotype, b^lb^l, are supposedly phenotypically cinnamon (aka red). The complete coding sequence of feline TYRP1 and a portion of the 5′ UTR was analyzed by direct sequencing of genomic DNA of wild-type and brown color variant cats. Sixteen single nucleotide polymorphisms (SNPs) were identified. Eight SNPs were in the coding regions, six are silent mutations. Two exon 2 on mutations cause amino acid changes. The C to T nonsense mutation at position 298 causes an arginine at amino acid 100 to be replaced by the opal (UGA) stop codon. This mutation is consistent with the cinnamon phenotype and is the putative light brown, b^l, mutation. An intron 6 mutation that potentially disrupts the exon 6 downstream splice-donor recognition site is associated with the chocolate phenotype and is the putative brown, b, mutation. The allelic series was confirmed by segregation and sequence analyses. Three microsatellite makers had significant linkage to the brown phenotype and two for the TYRP1 mutations in a 60-member pedigree. These mutations could be used to identify carriers of brown phenotypes in the domestic cat.     

The brown coat colour of Coppernecked goats is associated with a non‐synonymous variant at the TYRP1 locus on chromosome 8

The recent development of a goat SNP genotyping microarray enables genome‐wide association studies in this important livestock species. We investigated the genetic basis of the black and brown coat colour in Valais Blacknecked and Coppernecked goats. A genome‐wide association analysis using goat SNP50 BeadChip genotypes of 22 cases and 23 controls allowed us to map the locus for the brown coat colour to goat chromosome 8. The TYRP1 gene is located within the associated chromosomal region, and TYRP1 variants cause similar coat colour phenotypes in different species. We thus considered TYRP1 as a strong positional and functional candidate. We resequenced the caprine TYRP1 gene by Sanger and Illumina sequencing and identified two non‐synonymous variants, p.Ile478Thr and p.Gly496Asp, that might have a functional impact on the TYRP1 protein. However, based on the obtained pedigree and genotype data, the brown coat colour in these goats is not due to a single recessive loss‐of‐function allele. Surprisingly, the genotype distribution and the pedigree data suggest that the ⁴⁹⁶Asp allele might possibly act in a dominant manner. The ⁴⁹⁶Asp allele was present in 77 of 81 investigated Coppernecked goats and did not occur in black goats. This strongly suggests heterogeneity underlying the brown coat colour in Coppernecked goats. Functional experiments or targeted matings will be required to verify the unexpected preliminary findings.     

TYRP1 is associated with dun coat colour in Dexter cattle or how now brown cow?

Tyrosinase related protein 1 (TYRP1), which is involved in the coat colour pathway, was mapped to BTA8 between microsatellites BL1080 and BM4006, using a microsatellite in intron 5 of TYRP1. The complete coding sequence of bovine TYRP1 was determined from cDNA derived from skin biopsies of cattle with various colours. Sequence data from exons 2-8 from cattle with diluted phenotypes was compared with that from non-diluted phenotypes. In addition, full-sib families of beef cattle generated by embryo transfer and half-sib families from traditional matings in which coat colour was segregating were used to correlate TYRP1 sequence variants with dilute coat colours. Two non-conservative amino acid changes were detected in Simmental, Charolais and Galloway cattle but these polymorphisms were not associated with diluted shades of black or red, nor with the dun coat colour of Galloway cattle or the taupe brown colour of Braunvieh and Brown Swiss cattle. However, in Dexter cattle all 25 cattle with a dun brown coat colour were homozygous for a H424Y change. One Dexter that was also homozygous Y434 was red because of an "E+/E+" genotype at MC1R which lead to the production of only phaeomelanin. None of the 70 remaining black or red Dexter cattle were homozygous for Y434. This tyrosine mutation was not found in any of the 121 cattle of other breeds that were examined.     

Molecular variation in pigmentation genes contributing to coat colour in native Korean Hanwoo cattle

Pigmentation genes such as TYR (tyrosinase), TYRP1 (tyrosinase-related protein 1), DCT (previously TYRP2, or tyrosinase-related protein 2), ASIP (agouti) and MC1R (melanocortin receptor 1) play a major role in cattle coat colour. To understand the genotypic profile underlying coat colour in native Korean Hanwoo cattle and Angus black cattle, portions of the above-mentioned genes were amplified. Sequence analysis revealed variation in the TYRP1 (exon 5) and MC1R genes. Restriction enzyme analysis of these two genes could distinguish between different colours of Hanwoo cattle. Quantitative estimates of melanin and eumelanin in hair from three different-coloured Hanwoo phenotypes and Angus black showed significant differences at the breed and phenotypic levels. Finally, sequence variants in MC1R were associated with total melanin and eumelanin in breeds as well as in Hanwoo phenotypes.     

Melanocortin 1 receptor variation in the domestic dog

The melanocortin 1 receptor (Mc1r) is encoded by the Extension locus in many different mammals, where a loss-of-function causes exclusive production of red/yellow pheomelanin, and a constitutively activating mutation causes exclusive production of black/brown eumelanin. In the domestic dog, breeds with a wild-type E allele, e.g., the Doberman, can produce either pigment type, whereas breeds with the e allele, e.g., the Golden Retriever, produce exclusively yellow pigment. However, a black coat color in the Newfoundland and similar breeds is thought to be caused by an unusual allele of Agouti, which encodes the physiologic ligand for the Mc1r. Here we report that the predicted dog Mc1r is 317 residues in length and 96% identical to the fox Mc1r. Comparison of the Doberman, Newfoundland, Black Labrador, Yellow Labrador, Flat-coated Retriever, Irish Setter, and Golden Retriever revealed six sequence variants, of which two, S90G and R306ter, partially correlated with a black/brown coat and red/yellow coat, respectively. R306ter was found in the Yellow Labrador, Golden Retriever, and Irish Setter; the latter two had identical haplotypes but differed from the Yellow Labrador at three positions other than R306ter. In a larger survey of 194 dogs and 19 breeds, R306ter and a red/yellow coat were completely concordant except for the Red Chow. These results indicate that the e allele is caused by a common Mc1r loss-of-function mutation that either reoccurred or was subject to gene conversion during recent evolutionary history, and suggest that the allelic and locus relationships for dog coat color genes may be more analogous to those found in other mammals than previously thought.     

Tyrosinase and tyrosinase related protein 1 alleles specify domestic cat coat color phenotypes of the albino and brown loci

The genes encoding enzymes of the tyrosinase family are strong candidates for coat color variation in mammals. To investigate their influence in domestic cat coat color, we determined the complete nucleotide coding sequence of the domestic cat genes tyrosinase (TYR)--a plausible candidate gene for the albino (C) locus, and tyrosinase related protein 1 (TYRP1)--a candidate gene for the brown (B) locus. Sequence variants between individuals exhibiting variation in pigmentation were submitted to association studies. In TYR, two nonsynonymous substitutions encoding TYR-G301R and TYR-G227W were associated with the siamese and burmese phenotypes of the albino locus, respectively. TYRP1 was mapped on chromosome D4 within 5 cM of a highly polymorphic microsatellite, previously found to be fixed in a cat breed selected for the chocolate (b) allele of the B locus, which reinforced TYRP1 as a candidate gene for the B locus in the domestic cat. Two DNA polymorphisms, one leading to a TYRP1-A3G substitution in the signal peptide and another to an in-frame insertion TYRP1-421ins17/18 caused by a donor splice site mutation in intron 6, were associated with the chocolate (b) allele. A premature UAG stop codon at position 100 of TYRP1 was associated with a second allele of the B locus, cinnamon (b(l)). The results provide very strong evidence that the specific nucleotide variants of feline TYR (chromosome D1) are causative of the siamese (c(s)) and burmese (c(b)) alleles of the albino locus, as well as nucleotide variants of TYRP1 (chromosome D4) as specifying the chocolate (b) and cinnamon (b(l)) alleles of the B locus.     

Exclusion of melanocortin-1 receptor (mc1r) and agouti as candidates for dominant black in dogs

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.     

Melanocortin receptor 1 (MC1R) mutations and coat color in pigs.

The melanocortin receptor 1 (MC1R) plays a central role in regulation of eumelanin (black/brown) and phaeomelanin (red/yellow) synthesis within the mammalian melanocyte and is encoded by the classical Extension (E) coat color locus. Sequence analysis of MC1R from seven porcine breeds revealed a total of four allelic variants corresponding to five different E alleles. The European wild boar possessed a unique MC1R allele that we believe is required for the expression of a wild-type coat color. Two different MC1R alleles were associated with the dominant black color in pigs. MC1R*2 was found in European Large Black and Chinese Meishan pigs and exhibited two missense mutations compared with the wild-type sequence. Comparative data strongly suggest that one of these, L99P, may form a constitutively active receptor. MC1R*3 was associated with the black color in the Hampshire breed and involved a single missense mutation D121N. This same MC1R variant was also associated with EP, which results in black spots on a white or red background. Two different missense mutations were identified in recessive red (e/e) animals. One of these, A240T, occurs at a highly conserved position, making it a strong candidate for disruption of receptor function.     

Identification of two TYRP1 loss‐of‐function alleles in Valais Red sheep

The Valais Red sheep breed is a local breed of the Swiss canton Valais. Although the breed is characterised by its brown colour, black animals occasionally occur and the objective of this study was to identify the causative genetic variants responsible for the obvious difference. A GWAS using high‐density SNP data to compare 51 brown and 38 black sheep showed a strong signal on chromosome 2 at the TYRP1 locus. Haplotype analyses revealed three different brown‐associated alleles. The WGS of three sheep revealed four protein‐changing variants within the TYRP1 gene. Three of these variants were associated with the recessively inherited brown coat colour. This includes the known missense variant TYRP1:c.869G>T designated as b^Soay and two novel loss‐of‐function variants. We propose to designate the frame‐shift variant TYRP1:c.86_87delGA as b^VS1 and the nonsense variant TYRP1:c.1066C>T as b^VS2. Interestingly, the b^VS1 allele occurs only in local breeds of Switzerland whereas the b^VS2 allele seems to be more widespread across Europe.     

The genetics of brown coat color and white spotting in domestic yaks (Bos grunniens)

Domestic yaks (Bos grunniens) exhibit two major coat color variations: a brown vs. wild‐type black pigmentation and a white spotting vs. wild‐type solid color pattern. The genetic basis for these variations in color and distribution remains largely unknown and may be complicated by a breeding history involving hybridization between yaks and cattle. Here, we investigated 92 domestic yaks from China using a candidate gene approach. Sequence variations in MC1R, PMEL and TYRP1 were surveyed in brown yaks; TYRP1 was unassociated with the coloration and excluded. Recessive mutations from MC1R, or p.Gln34*, p.Met73Leu and possibly p.Arg142Pro, are reported in bovids for the first time and accounted for approximately 40% of the brown yaks in this study. The remaining 60% of brown individuals correlated with a cattle‐derived deletion mutation from PMEL (p.Leu18del) in a dominant manner. Degrees of white spotting found in yaks vary from color sidedness and white face, to completely white. After examining the candidate gene KIT, we suggest that color‐sided and all‐white yaks are caused by the serial translations of KIT (Cs₆ or Cs₂₉) as reported for cattle. The white‐faced phenotype in yaks is associated with the KIT haplotype S^wf. All KIT mutations underlying the serial phenotypes of white spotting in yaks are identical to those in cattle, indicating that cattle are the likely source of white spotting in yaks. Our results reveal the complex genetic origins of domestic yak coat color as either native in yaks through evolution and domestication or as introduced from cattle through interspecific hybridization.     

Mutations in the agouti (ASIP), the extension (MC1R), and the brown (TYRP1) loci and their association to coat color phenotypes in horses (Equus caballus)

Coat color genetics, when successfully adapted and applied to different mammalian species, provides a good demonstration of the powerful concept of comparative genetics. Using cross-species techniques, we have cloned, sequenced, and characterized equine melanocortin-1-receptor (MC1R) and agouti-signaling-protein (ASIP), and completed a partial sequence of tyrosinase-related protein 1 (TYRP1). The coding sequences and parts of the flanking regions of those genes were systematically analyzed in 40 horses and mutations typed in a total of 120 horses. Our panel represented 22 different horse breeds, including 11 different coat colors of Equus caballus. The comparison of a 1721-bp genomic fragment of MC1R among the 11 coat color phenotypes revealed no sequence difference apart from the known chestnut allele (C901T). In particular, no dominant black (E ^D) mutation was found. In a 4994-bp genomic fragment covering the three putative exons, two introns and parts of the 5′- and 3′-UTRs of ASIP, two intronic base substitutions (SNP-A845G and C2374A), a point mutation in the 3′-UTRs (A4734G), and an 11-bp deletion in exon 2 (ADEx2) were detected. The deletion was found to be homozygous and completely associated with horse recessive black coat color (A ^a /A ^a ) in 24 black horses out of 9 different breeds from our panel. The frameshift initiated by ADEx2 is believed to alter the regular coding sequence, acting as a loss-of-function ASIP mutation. In TYRP1 a base substitution was detected in exon 2 (C189T), causing a threonine to methionine change of yet unknown function, and an SNP (A1188G) was found in intron 2. Received: 22 November 2000 / Accepted: 07 February 2001     

A premature stop codon in the TYRP1 gene is associated with brown coat colour in the European rabbit (Oryctolagus cuniculus)

Classical genetic studies in European rabbits (Oryctolagus cuniculus) suggested the presence of two alleles at the brown coat colour locus: a wild‐type B allele that gives dense black pigment throughout the coat and a recessive b allele that in the homozygous condition (b/b genotype) produces brown rabbits that are unable to develop black pigmentation. In several other species, this locus is determined by mutations in the tyrosinase‐related protein 1 (TYRP1) gene, encoding a melanocyte enzyme needed for the production of dark eumelanin. In this study, we investigated the rabbit TYRP1 gene as a strong candidate for the rabbit brown coat colour locus. A total of 3846 bp of the TYRP1 gene were sequenced in eight rabbits of different breeds and identified 23 single nucleotide polymorphisms (SNPs; 12 in intronic regions, five in exons and six in the 3′‐untranslated region) and an insertion/deletion of 13 bp, in the 3′‐untranslated region, organised in a few haplotypes. A mutation in exon 2 (g.41360196G>A) leads to a premature stop codon at position 190 of the deduced amino acid sequence (p.Trp190ter). Therefore, translation predicts a truncated TYRP1 protein lacking almost completely the tyrosinase domain. Genotyping 203 rabbits of 32 different breeds identified this mutation only in brown Havana rabbits. Its potential functional relevance in disrupting the TYRP1 protein and its presence only in brown animals strongly argue for this non‐sense mutation being a causative mutation for the recessive b allele at the brown locus in Oryctolagus cuniculus.     

Canine TCOF1; cloning, chromosome assignment and genetic analysis in dogs with different head types

We describe the construction of a dog embryonic head/neck cDNA library and the isolation of the dog homolog of the Treacher Collins Syndrome gene, TCOF1. The protein shows a similar three-domain structure to that described for human TCOF1, but the dog gene lacks exon 10 and contains two exons not present in the human sequence. In addition, exon 19 is differentially spliced in the dog. How these structural differences relate to TCOF1 phosphorylation is discussed. Isolation of a genomic clone allowed the exon/intron boundaries to be characterized and the dog TCOF1 gene to be mapped to CF Chr 4q31, a region syntenic to human Chr 5. Genetic analysis of DNA of dogs from 13 different breeds identified nine DNA sequence variants, three of which gave rise to amino acid substitutions. Grouping dogs according to head type showed that a C396T variant, leading to a Pro117Ser substitution, is associated with skull/face shape in our dog panel. The numbers are small, but the association between the T allele and brachycephaly, broad skull/short face, was highly significant (p= 0.000024). The short period of time during which the domestic dog breeds have been established suggests that this mutation has arisen only once in the history of dog domestication. Received: 12 January 2001 / Accepted: 1 April 2001     

Allelic Combinations of Promoter and Exon 2 in <Emphasis Type="Italic">DQB1</Emphasis> in Dogs and Wolves

Polymorphism of PBRs of the major histocompatibility complex (MHC) genes is well recognized, but the polymorphism also extends to proximal promoter regions. Examining DQB1 variability in dogs and wolves, we identified 7 promoter variants and 13 exon 2 alleles among 89 dogs, including a previously unknown DQB1 exon 2 allele, and 8 promoter variants and 9 exon 2 alleles among 85 wolves. As expected from previous studies and from a close chromosomal location, strong linkage disequilibrium was demonstrated in both wolves and dogs by having significantly fewer promoter/exon 2 combinations than expected from simulations of randomized data sets. Interestingly, we noticed weaker haplotypic associations in dogs than in wolves. Dogs had twice as many promoter/exon 2 combinations as wolves and an almost 2-fold difference in the number of exon 2 alleles per promoter variant. This difference was not caused by an admixture of breeds in our group of dogs because the high ratio of observed to expected number of haplotypes persisted within a single dog breed, the German Shepherd. Ewens-Watterson tests indicated that both the promoter and exon 2 are under the balancing selection, and both regions appear to be more recently derived in the dog than in the wolf. Hence, although reasons for the differences are unknown, they may relate to altered selection pressure on patterns of expression. Deviations from normal MHC expression patterns have been associated with autoimmune diseases, which occur frequently in several dog breeds. Further knowledge about these deviations may help us understand the source of such diseases.     

Coat color determination by miR-137 mediated down-regulation of microphthalmia-associated transcription factor in a mouse model

Coat color is a key economic trait in wool-producing species. Color development and pigmentation are controlled by complex mechanisms in animals. Here, we report the first production of an altered coat color by overexpression of miR-137 in transgenic mice. Transgenic mice overexpressing miR-137 developed a range of coat color changes from dark black to light color. Molecular analyses of the transgenic mice showed decreased expression of the major target gene termed MITF and its downstream genes, including TYR, TYRP1, and TYRP2. We also showed that melanogenesis altered by miR-137 is distinct from that affected by UV radiation in transgenic mice. Our study provides the first mouse model for the study of coat color controlled by miRNAs in animals and may have important applications in wool production.