Presence of the dominant extension allele E(D) in red and mosaic cattle

The melanocyte-stimulating hormone receptor (MC1-R) is a central regulator of mammalian coat colour, encoded by the extension locus. In cattle, the dominant extension allele E(D) is associated with the production of black pigment in coloured areas. Genotyping of the MC1-R gene in a bull with mosaic expression of red vs. black pigment verified the existence of the E(D) allele, in spite of the fact that the majority of the animal is red coloured. No further mutations were found within the E(D) variant of the MC1-R gene, which was inherited from a completely red mother (genotype E(D)/e).     

The role of melanocyte-stimulating hormone (MSH) receptor in bovine coat color determination

The melanocyte-stimulating hormone (MSH) receptor has a major function in the regulation of black (eumelanin) versus red (phaeomelanin) pigment synthesis within melanocytes. We report three alleles of the MSH-receptor gene found in cattle. A point mutation in the dominant allele E ^D gives black coat color, whereas a frameshift mutation, producing a prematurely terminated receptor, in homozygous e/e animals, produces red coat color. The wild-type allele E ⁺ produces a variety of colors, reflecting the possibilities for regulating the normal receptor. Microsatellite analysis, RFLP studies, and coat color information were used to localize the MSH-receptor to bovine Chromosome (Chr) 18.     

Molecular and pharmacological characterization of dominant black coat color in sheep

Dominant black coat color in sheep is predicted to be caused by an allele E ^D at the extension locus. Recent studies have shown that this gene encodes the melanocyte stimulating hormone receptor (MC1-R). In mouse and fox, naturally occurring mutations in the coding region of MC1-R produce a constitutively activated receptor that switches the synthesis from phaeomelanin to eumelanin within the melanocyte, explaining the black coat color observed phenotypically. In the sheep, we have identified a Met→Lys mutation in position 73 (M73K) together with a Asp → Asn change at position 121 (D121N) showing complete cosegregation with dominant black coat color in a family lineage. Only the M73K mutation showed constitutive activation when introduced into the corresponding mouse receptor (mMC1-R) for pharmacological analysis; however, the position corresponding to D121 in the mouse receptor is required for high affinity ligand binding. The pharmacological profile of the M73K change is unique compared to the constitutively active E92K mutation in the sombre mouse and C123R mutation in the Alaska silver fox, indicating that the M73K change activates the receptor via a mechanism distinct from these previously characterized mutations. Received: 18 September 1997 / Accepted: 14 October 1998     

Temporal variation in coat colour (genotypes) supports major changes in the Nordic cattle population after Iron Age

Variation in coat colour genotypes of archaeological cattle samples from Finland was studied by sequencing 69 base pairs of the extension locus (melanocortin 1‐receptor, MC1R) targeting both a transition and a deletion defining the three main alleles, such as dominant black (E^D), wild type (E⁺) and recessive red (e). The 69‐bp MC1R sequence was successfully analysed from 23 ancient (1000–1800 AD) samples. All three main alleles and genotype combinations were detected with allele frequencies of 0.26, 0.17 and 0.57 for E^D, E⁺ and e respectively. Recessive red and dominant black alleles were detected in both sexes. According to the best of our knowledge, this is the first ancient DNA study defining all three main MC1R alleles. Observed MC1R alleles are in agreement with calculated phenotype frequencies from historical sources. The division of ancient Finnish cattle population into modern Finnish breeds with settled colours was dated to the 20th century. From the existing genotyped populations in Europe (43 breeds, n = 2360), the closest match to ancient MC1R genotype frequencies was with the Norwegian native multicoloured breeds. In combined published genotype data of ancient (n = 147) and genotypes and phenotypes of modern Nordic cattle (n = 738), MC1R allele frequencies showed temporal changes similar to neutral mitochondrial DNA and Y‐chromosomal haplotypes analysed earlier. All three markers indicate major change in genotypes in Nordic cattle from the Late Iron Age to the Medieval period followed by slower change through the historical periods until the present.     

牛黑素皮质素受体1(MC1R)基因与毛色表形的研究

牛MC1R基因不仅与毛色有关, 而且与牛乳中乳蛋白的含量有关。利用PCR-RFLP和DNA测序技术分析了中国荷斯坦黑白花牛, 中国荷斯坦红白花牛, 鲁西黄牛和渤海黑牛共4个品种的MC1R基因。共检测出3种等位基因(E^D, E⁺, e)。中国荷斯坦黑白花牛主要是E^D和E⁺等位基因(E^D=0.12、E⁴=0.80); 渤海黑牛也主要是E^D和E⁺等位基因(E^D=0.52、E⁺=0.47); 中国荷斯坦红白花牛和鲁西黄牛大多为e等位基因(e=0.95)。中国荷斯坦红白花牛和鲁西黄牛还存在E⁺/e基因型。由此推测E^D和E⁺等位基因导致黑色素合成。另外发现牛MC1R基因编码区725处存在一重要的SNP(单核苷酸多态性)。     

Non‐synonymous SNPs in MC1R gene are associated with the extended black variant in domestic ducks (Anas platyrhynchos)

In this study, we performed a sequence characterization of the duck melanocortin 1 receptor (alpha‐melanocyte stimulating hormone receptor) (MC1R) gene to analyze the relationship between MC1R polymorphism and the extended black variant in domestic ducks based on the extended black (E) and non‐extended black (e⁺) allele hypothesis of the duck MC1R gene. Both c.52G>A and c.376G>A substitutions are highly associated with the duck extended black variant (P < 0.01), but the novel c.52G>A substitution is more of a fit for the allele hypothesis of the duck MC1R gene.     

Dominant Red Coat Color in Holstein Cattle Is Associated with a Missense Mutation in the Coatomer Protein Complex,Subunit Alpha (COPA) Gene

Coat color in Holstein dairy cattle is primarily controlled by the melanocortin 1 receptor (MC1R) gene, a central determinant of black (eumelanin) vs. red/brown pheomelanin synthesis across animal species. The major MC1R alleles in Holsteins are Dominant Black (MC1R^D) and Recessive Red (MC1R^e). A novel form of dominant red coat color was first observed in an animal born in 1980. The mutation underlying this phenotype was named Dominant Red and is epistatic to the constitutively activated MC1R^D. Here we show that a missense mutation in the coatomer protein complex, subunit alpha (COPA), a gene with previously no known role in pigmentation synthesis, is completely associated with Dominant Red in Holstein dairy cattle. The mutation results in an arginine to cysteine substitution at an amino acid residue completely conserved across eukaryotes. Despite this high level of conservation we show that both heterozygotes and homozygotes are healthy and viable. Analysis of hair pigment composition shows that the Dominant Red phenotype is similar to the MC1R Recessive Red phenotype, although less effective at reducing eumelanin synthesis. RNA-seq data similarly show that Dominant Red animals achieve predominantly pheomelanin synthesis by downregulating genes normally required for eumelanin synthesis. COPA is a component of the coat protein I seven subunit complex that is involved with retrograde and cis-Golgi intracellular coated vesicle transport of both protein and RNA cargo. This suggests that Dominant Red may be caused by aberrant MC1R protein or mRNA trafficking within the highly compartmentalized melanocyte, mimicking the effect of the Recessive Red loss of function MC1R allele.     

A first genotyping assay of French cattle breeds based on a new allele of the extension gene encoding the melanocortin-1 receptor (Mc1r)

The seven transmembrane domain melanocortin-1 receptor (Mc1r) encoded by the coat color extension gene (E) plays a key role in the signaling pathway of melanin synthesis. Upon the binding of agonist (melanocortin hormone, α-MSH) or antagonist (Agouti protein) ligands, the melanosomal synthesis of eumelanin and/or phaeomelanin pigments is stimulated or inhibited, respectively. Different alleles of the extension gene were cloned from unrelated animals belonging to French cattle breeds and sequenced. The wild type E allele was mainly present in Normande cattle, the dominant E^D allele in animals with black color (i.e. Holstein), whereas the recessive e allele was identified in homozygous animals exhibiting a more or less strong red coat color (Blonde d''Aquitaine, Charolaise, Limousine and Salers). A new allele, named E¹, was found in either homozygous (E¹/E¹) or heterozygous (E¹/E) individuals in Aubrac and Gasconne breeds. This allele displayed a 4 amino acid duplication (12 nucleotides) located within the third cytoplasmic loop of the receptor, a region known to interact with G proteins. A first genotyping assay of the main French cattle breeds is described based on these four extension alleles.     

A missense mutation in the gene for melanocyte-stimulating hormone receptor (MCIR) is associated with the chestnut coat color in horses

The melanocyte-stimulating hormone receptor gene (MC1R) is the major candidate gene for the chestnut coat color in horses since it is assumed to be controlled by an allele at the extension locus. MC1R sequences were PCR amplified from chestnut (e/e) and non-chestnut (E/−) horses. A single-strand conformation polymorphism was found that showed a complete association to the chestnut coat color among 144 horses representing 12 breeds. Sequence analysis revealed a single missense mutation (83Ser → Phe) in the MC1R allele associated with the chestnut color. The substitution occurs in the second transmembrane region, which apparently plays a key role in the molecule since substitutions associated with coat color variants in mice and cattle as well as red hair and fair skin in humans are found in this part of the molecule. We propose that the now reported mutation is likely to be the causative mutation for the chestnut coat color. The polymorphism can be detected with a simple PCR-RFLP test, since the mutation creates a TaqI restriction site in the chestnut allele. Received: 20 May 1996 / Accepted: 31 July 1996     

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.     

New Mechanism of Melanosomes Transport into Hair of Lambs of Different Breeds and Genotypes

By light microscopic investigation of skin and wool specimens of newborn lambs, we discovered a previously unknown mechanism for melanosomes transport in the process of dermal papilla melanocytes regular mitosis and migration into the hair shaft. This mechanism plays a great role in hair pigmentation especially in dominant (E^D/E^D) and recessive (A^a/A^a) black lambs of all investigated breeds. The rate of pigment cell mitosis, proliferation, and migration differs greatly in lambs of investigated color genotypes. In black genotypes the rate of melanocyte mitosis is very high and is approximately the same as in the hair bulb matrix cells, whereas in brown and red genotypes this rate is much lower. Melanocyte mitosis in the light red and tan groups was not found.     

Identification of a major locus interacting with MC1R and modifying black coat color in an F2 Nellore-Angus population

Background

In cattle, base color is assumed to depend on the enzymatic activity specified by the MC1R locus, i.e. the extension locus, with alleles coding for black (E ^D ), red (e), and wild-type (E ⁺ ). In most mammals, these alleles are presumed to follow the dominance model of E ^D  > E ⁺  > e, although exceptions are found. In Bos indicus x Bos taurus F₂ cattle, some E ^D E ⁺ heterozygotes are discordant with the dominance series for MC1R and display various degrees of red pigmentation on an otherwise predicted black background. The objective of this study was to identify loci that modify black coat color in these individuals.

Results

Reddening was classified with a subjective scoring system. Interval analyses identified chromosome-wide suggestive (P < 0.05) and significant (P < 0.01) QTL on bovine chromosomes (BTA) 4 and 5, although these were not confirmed using single-marker association or Bayesian methods. Evidence of a major locus (F = 114.61) that affects reddening was detected between 60 and 73 Mb on BTA 6 (Btau4.0 build), and at 72 Mb by single-marker association and Bayesian methods. The posterior mean of the genetic variance for this region accounted for 43.75% of the genetic variation in reddening. This region coincided with a cluster of tyrosine kinase receptor genes (PDGFRA, KIT and KDR). Fitting SNP haplotypes for a 1 Mb interval that contained all three genes and centered on KIT accounted for the majority of the variation attributed to this major locus, which suggests that one of these genes or associated regulatory elements, is responsible for the majority of variation in degree of reddening.

Conclusions

Recombinants in a 5 Mb region surrounding the cluster of tyrosine kinase receptor genes implicated PDGFRA as the strongest positional candidate gene. A higher density marker panel and functional analyses will be required to validate the role of PDGFRA or other regulatory variants and their interaction with MC1R for the modification of black coat color in Bos indicus influenced cattle.     

Mapping and characterization of the dominant black colour locus in sheep

Dominant black pigment synthesis in sheep is caused by alterations of the melanocortin-1 receptor (MC1-R) coding sequence. Using five bovine microsatellite markers we have mapped the sheep MC1-R gene to chromosome 14, corresponding to the location in other mammalian species. The existence of two independent mutations, both causing an amino acid substitution, in distantly related breeds of sheep, support the hypothesis that the observed black pigment synthesis is caused by a mutual effect of the two mutations. As similar mutations are found separately at both locations in dominant black variants of other animal species, it is also possible that any of the two mutations could be sufficient for a partial pigment switch.     

MOLECULAR AND PHARMACOLOGICAL CHARACTERISATION OF THE MSH-R ALLELES IN SWISS CATTLE BREEDS

ABSTRACT

The coat colour in mammals is determined by the relative amounts of eumelanin (black/brown) and phaeomelanin (red/yellow), produced in melanocytes, which are controlled by melanocyte stimulating hormone receptor (MSH-R). Melanocyte stimulating hormone receptor is activated by α-melanocyte-stimulating hormone (α-MSH). Stimulated MSH-R activates adenylyl cyclase (AC), thereby increasing the amount of cyclic AMP in the cell, which activates the enzyme tyrosinase resulting in eumelanin synthesis. In this study the complete coding sequences of five alleles of the MSH-R gene found in Holstein, Red Holstein, Simmental, and Brown Swiss cattle were cloned into a mammalian expression vector and transfected into human embryonic kidney (HEK) 293 cells. The expressed receptors were analyzed for their ability to increase intracellular cAMP in response to stimulation by α-MSH. The recessive red allele (e) found in Red Holstein and Simmental and the dominant black allele (E^D) found in Holstein were unresponsive to a wide range of α-MSH concentrations. Two alleles from Brown Swiss (E^d1, E^d2) and one allele found in the Simmental breed (e^f) responded to stimulation by α-MSH in a dose-dependent manner. When compared to E^d1 and E^d2, the cells transfected with the e^f MSH-R allele, however, reached the corresponding intracellular cAMP concentrations at a 10-fold higher concentration of α-MSH. In conjunction with the mode of inheritance of coat colour, the results indicate that the e MSH-R allele is a non-functional receptor, E^D is constitutively activated receptor, and E^d1 and E^d2 are hormonally activated receptors. The delay in e^f MSH-R response may explain the similarity between the e and e^f phenotypes.     

Pigmentation in Black-boned sheep (Ovis aries): association with polymorphism of the MC1R gene

Variations in vertebrate skin and hair color are due to varied amounts of eumelanin (brown/black) and phaeomelanin (red/yellow) produced by the melanocytes. The melanocortin 1 receptor (MC1R) is a regulator of eumelanin and phaeomelanin production in the melanocytes, and MC1R mutations causing coat color changes are known in many vertebrates. We have sequenced the entire coding region of the MC1R gene in Black-boned, Nanping indigenous and Romney Marsh sheep populations and found two silent mutation sites of A12G and G144C, respectively. PCR-RFLP of G144C showed that frequency of allele G in Black-boned, Nanping indigenous and Romney Marsh sheep was 0.818, 0.894 and 0, respectively. Sheep with GG genotype had significantly higher (P < 0.05) tyrosinase activity than sheep with CC genotype in the all investigated samples. Moreover, there was significant effect of MC1R genotype on coat color, suggesting that MC1R gene could affect coat color but not black traits. There would be merit in further studies using molecular techniques to elucidate the cause of black traits in these Black-boned sheep.     

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     

Sequence characterization of the melanocortin 1 receptor (MC1R) gene in sheep with different coat colours and identification of the putative e allele at the ovine Extension locus

Sequence of the melanocortin 1 receptor (MC1R) gene (the Extension locus) was obtained from a panel of 73 animals belonging to 9 Italian sheep breeds or populations (Appenninica, Bergamasca, Comisana, Cornigliese-like, Delle Langhe, Massese, Merinizzata Italiana, Sarda and Valle del Belice) with different coat colours. Evaluation of the identified polymorphisms on this phenotype was reported with in silico predictions and comparative approaches within and across breeds and across species. Five novel single nucleotide polymorphisms (SNPs), organized in three haplotypes, were detected. Another haplotype, including the two missense mutations already described for the E^D allele, was identified in few Massese sheep. One SNP (c.199C > T) caused a predicted amino acid substitution (p.R67C) in a highly conserved position of the first intracellular loop of the MC1R protein. The same substitution causes recessive pheomelanism in other species. We propose that the p.67C allele represents the recessive e allele at the ovine Extension series that was, so far, not completely recognized in sheep by classical genetic studies. This polymorphism was analysed in a total of 388 sheep of the 9 investigated breeds. The p.67C allele was identified only in the Valle del Belice breed (allele frequency of 21.3% in 176 analysed animals of this breed) in which the presence of epistatic white-determining loci might mask, at least in part, its effects. Confirming the effect of this novel allele on coat colour will lead to new perspectives on the composition of specialized coloured sheep lines.     

A missense mutation in melanocortin 1 receptor is associated with the red coat colour in donkeys

The seven donkey breeds recognised by the French studbook are characterised by few coat colours: black, bay and grey. Normand bay donkeys seldom give birth to red foals, a colour more commonly seen and recognised in American miniature donkeys. Red resembles the equine chestnut colour, previously attributed to a mutation in the melanocortin 1 receptor gene (MC1R). We used a panel of 124 donkeys to identify a recessive missense c.629T>C variant in MC1R that showed a perfect association with the red coat colour. This variant leads to a methionine to threonine substitution at position 210 in the protein. We showed that methionine 210 is highly conserved among vertebrate melanocortin receptors. Previous in silico and in vitro analyses predicted this residue to lie within a functional site. Our in vivo results emphasised the pivotal role played by this residue, the alteration of which yielded a phenotype fully compatible with a loss of function of MC1R. We thus propose to name the c.629T>C allele in donkeys the e allele, which further enlarges the panel of recessive MC1R loss‐of‐function alleles described in animals and humans.