Variations in the Coding Region of the Agouti Signaling Protein Gene Do Not Explain Agouti/Non-agouti Phenotypes in Macaques

Agouti is a common pigmentation phenotype in mammals including primates. Mutations in the agouti signaling protein gene (ASIP) are known to result in non-agouti black hairs in laboratory mice. It is still unclear whether sequence variation in ASIP is linked with the agouti/non-agouti phenotypes in macaques (Genus Macaca). To address this issue, we have determined and compared nucleotide sequences of protein coding region of ASIP in 18 macaque species and have identified 16 different sequences of the ASIP. Macaca nemestrina, which showed yellow agouti hairs, shared an identical amino acid sequence of ASIP with several non-agouti species. No sequence changes were found in functionally important sites of ASIP in the macaques showing non-agouti dark hair color. These results indicated that the variation in the protein coding region of ASIP did not explain the non-agouti dark coat color in the macaques. Upstream regulatory regions of ASIP and other genes participating in pigmentation system remain to be investigated for the hair color variation in the macaques.     

Mutations in ASIP and MC1R: dominant black and recessive black alleles segregate in native Swedish sheep populations

By studying genes associated with coat colour, we can understand the role of these genes in pigmentation but also gain insight into selection history. North European short‐tailed sheep, including Swedish breeds, have variation in their coat colour, making them good models to expand current knowledge of mutations associated with coat colour in sheep. We studied ASIP and MC1R, two genes with known roles in pigmentation, and their association with black coat colour. We did this by sequencing the coding regions of ASIP in 149 animals and MC1R in 129 animals from seven native Swedish sheep breeds in individuals with black, white or grey fleece. Previously known mutations in ASIP [recessive black allele: g.100_105del (D₅) and/or g.5172T>A] were associated with black coat colour in Klövsjö and Roslag sheep breeds and mutations in both ASIP and MC1R (dominant black allele: c.218T>A and/or c.361G>A) were associated with black coat colour in Swedish Finewool. In Gotland, Gute, Värmland and Helsinge sheep breeds, coat colour inheritance was more complex: only 11 of 16 individuals with black fleece had genotypes that could explain their black colour. These breeds have grey individuals in their populations, and grey is believed to be a result of mutations and allelic copy number variation within the ASIP duplication, which could be a possible explanation for the lack of a clear inheritance pattern in these breeds. Finally, we found a novel missense mutation in MC1R (c.452G>A) in Gotland, Gute and Värmland sheep and evidence of a duplication of MC1R in Gotland sheep.     

Identification of single nucleotide polymorphisms in the agouti signaling protein (ASIP) gene in some goat breeds in tropical and temperate climates

The agouti-signaling protein (ASIP) plays a major role in mammalian pigmentation as an antagonist to melanocortin-1 receptor gene to stimulate pheomelanin synthesis, a major pigment conferring mammalian coat color. We sequenced a 352 bp fragment of ASIP gene spanning part of exon 2 and part of intron 2 in 215 animals representing six goat breeds from Nigeria and the United States: West African Dwarf, predominantly black; Red Sokoto, mostly red; and Sahel, mostly white from Nigeria; black and white Alpine, brown and white Spanish and white Saanen from the US. Twenty haplotypes from nine mutations representing three intronic, one silent and five missense (p.S19R, p.N35K, p.L36V, p.M42L and p.L45W) mutations were identified in Nigerian goats. Approximately 89 % of Nigerian goats carry haplotype 1 (TGCCATCCG) which seems to be the wild type configuration of mutations in this region of the gene. Although we found no association between these polymorphisms in the ASIP gene and coat color in Nigerian goats, in-silico functional analysis predicts putative deleterious functional impact of the p.L45W mutation on the basic amino-terminal domain of ASIP. In the American goats, two intronic mutations, g.293G>A and g.327C>A, were identified in the Alpine breed, although the g.293G>A mutation is common to American and Nigerian goat populations. All Sannen and Sahel goats in this study belong to haplotypes 1 of both populations which seem to be the wild-type composite ASIP haplotype. Overall, there was no clear association of this portion of the ASIP gene interrogated in this study with coat color variation. Therefore, additional genomic analyses of promoter sequence, the entire coding and non-coding regions of the ASIP gene will be required to obtain a definite conclusion.     

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 deletion spanning the promoter and first exon of the hair cycle-specific ASIP transcript isoform in black and tan rabbits

Black and tan animals have tan-coloured ventral body surfaces separated by sharp boundaries from black-coloured dorsal body surfaces. In the a^t mouse mutant, a retroviral 6 kb insertion located in the hair cycle-specific promoter of the murine Asip gene encoding agouti signalling protein causes the black and tan phenotype. In rabbits, three ASIP alleles are thought to exist, including an a^t allele causing a black and tan coat colour that closely resembles the mouse black and tan phenotype. The goal of our study was to identify the functional genetic variant causing the rabbit a^t allele. We performed a WGS-based comparative analysis of the ASIP gene in one black and tan and three wt agouti-coloured rabbits. The analysis identified 75 a^t-associated variants including an 11 kb deletion. The deletion is located in the region of the hair cycle-specific ASIP promoter and thus in a region homologous to the site of the retroviral insertion causing the a^t allele in mice. We observed perfect association of the genotypes at this deletion with the coat colour phenotype in 49 rabbits. The comparative analysis and the previous knowledge about the regulation of ASIP expression suggest that the 11 kb deletion is the most likely causative variant for the black and tan phenotype in rabbits.     

How the Leopard Hides Its Spots: ASIP Mutations and Melanism in Wild Cats

The occurrence of melanism (darkening of the background coloration) is documented in 13 felid species, in some cases reaching high frequencies at the population level. Recent analyses have indicated that it arose multiple times in the Felidae, with three different species exhibiting unique mutations associated with this trait. The causative mutations in the remaining species have so far not been identified, precluding a broader assessment of the evolutionary dynamics of melanism in the Felidae. Among these, the leopard (Panthera pardus) is a particularly important target for research, given the iconic status of the ‘black panther’ and the extremely high frequency of melanism observed in some Asian populations. Another felid species from the same region, the Asian golden cat (Pardofelis temminckii), also exhibits frequent records of melanism in some areas. We have sequenced the coding region of the Agouti Signaling Protein (ASIP) gene in multiple leopard and Asian golden cat individuals, and identified distinct mutations strongly associated with melanism in each of them. The single nucleotide polymorphism (SNP) detected among the P. pardus individuals was caused by a nonsense mutation predicted to completely ablate ASIP function. A different SNP was identified in P. temminckii, causing a predicted amino acid change that should also induce loss of function. Our results reveal two additional cases of species-specific mutations implicated in melanism in the Felidae, and indicate that ASIP mutations may play an important role in naturally-occurring coloration polymorphism.     

Evolution of a pigmentation gene,the melanocortin-1 receptor,in primates

The melanocortin-1 receptor (MC1R) forms a critical switch in the production of orange/red pheomelanin and black/brown eumelanin pigments during hair development in mammals. The molecular evolution of the melanocortin-1 receptor gene was investigated in a broad range of primate species, including several groups with large differences in distribution of orange/red and black hairs. Primate MC1R has been subject to purifying selection throughout most of its evolution, with small changes in selective constraint being detected early in primate evolution. In contrast to the situation in humans and domestic mammals, many intraspecific and intrageneric differences in primate coat color cannot be attributed to changes in the MC1R coding sequence. Nevertheless, important changes in the biochemical function of MC1R are suggested by mutations in sites of known functional importance, particularly in New World monkeys and lemurs. The evolution of the MC1R in lion tamarins is anomalous, with a combination of a high nonsynonymous to synonymous substitution rate (dN/dS) ratio, deletions, and substitutions.     

Is Brightest Best? Testing the Hamilton-Zuk Hypothesis in Mandrills

Although many primates exhibit striking coloration, including brightly colored pelage and bare areas of skin, our understanding of the function and evolution of these traits pales in the face of knowledge about color in other taxa. However, recent years have seen an increase in the number of studies of individual variation in primate color and evidence is accumulating that these traits can act as important signals to conspecifics. Mandrills are arguably the most colorful of all primates. Here, we review what we have discovered about the signal function of coloration in male and female mandrills from our long-term studies of a semi-free-ranging colony in Franceville, Gabon and test the predictions of the Hamilton-Zuk hypothesis—that bright coloration is condition dependent, and that only individuals of superior quality will be able to express color fully—in this species. We compare measures of facial coloration in both sexes with parasite load (using fecal analysis over 1 annual cycle), immune status (hematological parameters), neutral genetic diversity (microsatellite heterozygosity), and major histocompatability (MHC) genotype to examine whether red coloration acts as an honest signal of individual quality in mandrills. We found that red coloration was unrelated to parasitism and hematological parameters. Red was also unrelated to genome-wide heterozygosity and MHC diversity, although specific MHC genotypes were significantly related to red. The healthy, provisioned nature of the colony and problems associated with observational, correlational studies restrict interpretation of our data, and it would be premature to draw conclusions as to whether color signals individual quality in mandrills. We conclude with some suggestions for future studies on the signal content of color in mandrills and other primates.     

The primate palette: The evolution of primate coloration

Flip through The Pictorial Guide to the Living Primates¹ and you will notice a striking yet generally underappreciated aspect of primate biology: primates are extremely colorful. Primate skin and pelage coloration were highlighted examples in Darwin's² original discussions of sexual selection but, surprisingly, the topic has received little research attention since. Here we summarize the patterns of color variation observed across the primate order and examine the selective forces that might drive and maintain this aspect of primate phenotypic diversity. We discuss how primate color patterns might be adaptive for physiological function, crypsis, and communication. We also briefly summarize what is known about the genetic basis of primate pigmentation and argue that understanding the proximate mechanisms of primate coloration will be essential, not only for understanding the evolutionary forces shaping phenotypic variation, but also for clarifying primate taxonomies and conservation priorities.     

Differential expression of MC1R gene in Liaoning Cashmere goats with different coat colors

Melanocyte stimulating hormone receptor (MC1R) has been known as a regulator of eumelanin and phaeomelanin production in the melanocytes, and MC1R mutations causing coat color changes are known in many vertebrates; however, there are no research reports about the differentially expression of MC1R gene and its coding protein in Cashmere goats with different coat color. We examined the presence of MC1R distribution and MC1R protein and gene expression in the white Cashmere goats and black Cashmere goats, respectively; q-PCR, Western blot and immunhistochemical analysis showed that the expression of the MC1R gene in the black Cashmere goats was 3.39 fold more than the white ones (p?<?0.01), and Cashmere goats with black genotype had significantly higher (2.03, p?<?0.01) MC1R protein expression than white genotype in the all investigated samples. Moreover, all Cashmere goats with different coat color available for immunhistochemical analysis showed either lower (white Cashmere goats) or higher (black Cashmere goats) expression of the MC1R protein; these findings suggested that it had a relationship between the MC1R and the coat color of Cashmere goats. That could lay the foundation for the further research of the MC1R and coat color controllability regulation of the Cashmere goats.     

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.     

The effect of methyl supplements during pregnancy on the phenotypic modification of offspring agouti coat color in rats

The effect of methyl supplements to the diet of pregnant homozygous (AAHH) female rats with agouti coat color mated with homozygous (aahh) males on the phenotypic modification of the coat color of their heterozygous offspring (AaHh) has been studied. Comparative morphological analysis of the main parameters of hair that determine coat color, including the total length of hairs of different types and the length of the upper black (eumelanin) and light (pheomelanin) parts of awn hairs has been performed. The pattern of pigment granule distribution among hair layers has been analyzed. The melanin content of the hair has been determined using electron spin resonance (ESR). Although all offspring have a typical agouti coat color (alternating black and light portions of hair), 39% of them have a darker coat color than control and other experimental rats have. The main differences between the offspring with darkened and standard coat colors are accounted for by the ratio between the eumelanin and pheomelanin portions of awn hairs. In darkened offspring, this ratio is significantly higher than in control rats. The possible mechanisms of the phenotypic modification of agouti coat color in experimental animals are discussed.     

Inheritance of seed coat color genes in <Emphasis Type="Italic">Brassica napus</Emphasis> (L.) and tagging the genes using SRAP,SCAR and SNP molecular markers

Seed coat color inheritance in Brassica napus was studied in F₁, F₂, F₃ and backcross progenies from crosses of five black seeded varieties/lines to three pure breeding yellow seeded lines. Maternal inheritance was observed for seed coat color in B. napus, but a pollen effect was also found when yellow seeded lines were used as the female parent. Seed coat color segregated from black to dark brown, light brown, dark yellow, light yellow, and yellow. Seed coat color was found to be controlled by three genes, the first two genes were responsible for black/brown seed coat color and the third gene was responsible for dark/light yellow seed coat color in B. napus. All three seed coat color alleles were dominant over yellow color alleles at all three loci. Sequence related amplified polymorphism (SRAP) was used for the development of molecular markers co-segregating with the seed coat color genes. A SRAP marker (SA12BG18388) tightly linked to one of the black/brown seed coat color genes was identified in the F₂ and backcross populations. This marker was found to be anchored on linkage group A9/N9 of the A-genome of B. napus. This SRAP marker was converted into sequence-characterized amplification region (SCAR) markers using chromosome-walking technology. A second SRAP marker (SA7BG29245), very close to another black/brown seed coat color gene, was identified from a high density genetic map developed in our laboratory using primer walking from an anchoring marker. The marker was located on linkage group C3/N13 of the C-genome of B. napus. This marker also co-segregated with the black/brown seed coat color gene in B. rapa. Based on the sequence information of the flanking sequences, 24 single nucleotide polymorphisms (SNPs) were identified between the yellow seeded and black/brown seeded lines. SNP detection and genotyping clearly differentiated the black/brown seeded plants from dark/light/yellow-seeded plants and also differentiated between homozygous (Y2Y2) and heterozygous (Y2y2) black/brown seeded plants. A total of 768 SRAP primer pair combinations were screened in dark/light yellow seed coat color plants and a close marker (DC1GA27197) linked to the dark/light yellow seed coat color gene was developed. These three markers linked to the three different yellow seed coat color genes in B. napus can be used to screen for yellow seeded lines in canola/rapeseed breeding programs.     

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     

TBX3 and ASIP genotypes reveal discrepancies in officially recorded coat colors of Hucul horses

Although only a few specific pigmentation types are allowed within the Hucul horse registry, accurate determination of particular coat colors can be uncertain due to the presence of variation in color shades and segregation of multiple dun dilution variants. Herein, we genotyped the previously identified polymorphisms within two coat color loci TBX3 (T-box 3) and ASIP (Agouti Signaling Protein) in 462 Hucul individuals and compared the genotype predicted phenotypes with observed pigmentation types provided in the Polish Horse Breeders Association database. We identified disagreement between the predicted and recorded coat color in 157 horses (34%). The most common error was misclassification of horses with the nd1/nd1 and nd1/nd2 genotypes, what may be related with the occurrence of some ‘intermediate’ dilution phenotypes in such individuals. We have also proven that the frequency of the dominant dun dilution allele (D) (0.30) is higher than previously predicted by available studbooks. The D allele(s) is easily ‘hidden’ in various phenotypic groups including dark bay and black, therefore we hypothesized that the dun dilution effect itself is not as strongly epistatic in the Hucul horse as described in other horse breeds. This may be the result of an additional genetic modifier suppressing D allele phenotypic effect.     

Adaptive change in protective coloration in adult striated shieldbugs Graphosoma lineatum (Heteroptera: Pentatomidae): test of detectability of two colour forms by avian predators

1. Protective coloration in insects may be aposematic or cryptic, and some species change defensive strategy between instars. In Sweden, the adult striated shieldbugs Graphosoma lineatum (Heteroptera: Pentatomidae) undergo a seasonal colour change from pale brown and black striation in the pre‐hibernating adults, to red and black striation in the same post‐hibernating individuals. To the human eye the pre‐hibernating adults appear cryptic against the withered late summer vegetation, whereas the red and black post‐hibernating adults appear aposematic. This suggests a possibility of a functional colour change. However, what is cryptic to the human eye is not necessarily cryptic to a potential predator. 2. Therefore we tested the effect of coloration in adult G. lineatum on their detectability for avian predators. Great tits (Parus major) were trained to eat sunflower seeds hidden inside the emptied exoskeletons of pale or red G. lineatum. Then the detection time for both colour forms was measured in a dry vegetation environment. 3. The birds required a longer time to find the pale form of G. lineatum than the red one. The pale form appears more cryptic on withered late summer vegetation than the red form, not only to the human eye but also to avian predators. The result supports the idea that the adult individuals of G. lineatum undergo a functional change from a cryptic protective coloration to an aposematic one.