A genome-wide scan study identifies a single nucleotide substitution in ASIP associated with white versus non-white coat-colour variation in sheep (Ovis aries)

In sheep, coat colour (and pattern) is one of the important traits of great biological, economic and social importance. However, the genetics of sheep coat colour has not yet been fully clarified. We conducted a genome-wide association study of sheep coat colours by genotyping 47 303 single-nucleotide polymorphisms (SNPs) in the Finnsheep population in Finland. We identified 35 SNPs associated with all the coat colours studied, which cover genomic regions encompassing three known pigmentation genes (TYRP1, ASIP and MITF) in sheep. Eighteen of these associations were confirmed in further tests between white versus non-white individuals, but none of the 35 associations were significant in the analysis of only non-white colours. Across the tests, the s66432.1 in ASIP showed significant association (P=4.2 × 10⁻¹¹ for all the colours; P=2.3 × 10⁻¹¹ for white versus non-white colours) with the variation in coat colours and strong linkage disequilibrium with other significant variants surrounding the ASIP gene. The signals detected around the ASIP gene were explained by differences in white versus non-white alleles. Further, a genome scan for selection for white coat pigmentation identified a strong and striking selection signal spanning ASIP. Our study identified the main candidate gene for the coat colour variation between white and non-white as ASIP, an autosomal gene that has been directly implicated in the pathway regulating melanogenesis. Together with ASIP, the two other newly identified genes (TYRP1 and MITF) in the Finnsheep, bordering associated SNPs, represent a new resource for enriching sheep coat-colour genetics and breeding.     

Coat colours in the Massese sheep breed are associated with mutations in the agouti signalling protein (ASIP) and melanocortin 1 receptor (MC1R) genes

Massese is an Italian dairy sheep breed characterized by animals with black skin and horns and black or apparent grey hairs. Owing to the presence of these two coat colour types, this breed can be considered an interesting model to evaluate the effects of coat colour gene polymorphisms on this phenotypic trait. Two main loci have been already shown to affect coat colour in sheep: Agouti and Extension coding for the agouti signalling protein (ASIP) and melanocortin 1 receptor (MC1R) genes, respectively. The Agouti locus is affected by a large duplication including the ASIP gene that may determine the Agouti white and tan allele (A(Wt)). Other disrupting or partially inactivating mutations have been identified in exon 2 (a deletion of 5 bp, D(5); and a deletion of 9 bp, D(9)) and in exon 4 (g.5172T>A, p.C126S) of the ASIP gene. Three missense mutations in the sheep MC1R gene cause the dominant black E(D) allele (p.M73K and p.D121N) and the putative recessive e allele (p.R67C). Here, we analysed these ASIP and MC1R mutations in 161 Massese sheep collected from four flocks. The presence of one duplicated copy allele including the ASIP gene was associated with grey coat colour (P = 9.4E-30). Almost all animals with a duplicated copy allele (37 out of 41) showed uniform apparent grey hair and almost all animals without a duplicated allele (117 out of 120) were completely black. Different forms of duplicated alleles were identified in Massese sheep including, in almost all cases, copies with exon 2 disrupting or partially inactivating mutations making these alleles different from the A(Wt) allele. A few exceptions were observed in the association between ASIP polymorphisms and coat colour: three grey sheep did not carry any duplicated copy allele and four black animals carried a duplicated copy allele. Of the latter four sheep, two carried the E(D) allele of the MC1R gene that may be the cause of their black coat colour. The coat colour of all other black animals may be determined by non-functional ASIP alleles (non-agouti alleles, A(a)) and in a few cases by the E(D) Extension allele. At least three frequent ASIP haplotypes ([D(5):g.5172T], [N:g.5172A] and [D(5):g.5172A]) were detected (organized into six different diplotypes). In conclusion, the results indicated that coat colours in the Massese sheep breed are mainly derived by combining ASIP and MC1R mutations.     

Explicit evidence for a missense mutation in exon 4 of SLC45A2 gene causing the pearl coat dilution in horses

Four loci seem responsible for the dilution of the basic coat colours in horse: Dun (D), Silver Dapple (Z), Champagne (CH) and Cream (C). Apart from the current phenotypes ascribed to these loci, pearl has been described as yet another diluted coat colour in this species. To date, this coat colour seems to segregate only in the Iberian breeds Purebred Spanish horse and Lusitano and has also been described in breeds of Iberian origin, such as Quarter Horses and Paint Horse, where it is referred to as the ‘Barlink Factor’. This phenotype segregates in an autosomal recessive manner and resembles some of the coat colours produced by the champagne CH^CH and cream C^Cr alleles, sometimes being difficult to distinguish among them. The interaction between compound heterozygous for the pearl C^prl and cream C^Cr alleles makes SLC45A2 the most plausible candidate gene for the pearl phenotype in horses. Our results provide documented evidence for the missense variation in exon 4 [SLC45A2:c.985G>A; SLC45A2:p.(Ala329Thr)] as the causative mutation for the pearl coat colour. In addition, it is most likely involved as well in the cremello, perlino and smoky cream like phenotypes associated with the compound C^Cr and C^prl heterozygous genotypes (known as cream pearl in the Purebred Spanish horse breed). The characterization of the pearl mutation allows breeders to identify carriers of the C^prl allele and to select this specific coat colour according to personal preferences, market demands or studbook requirements as well as to verify segregation within particular pedigrees.     

Behavioural reactivity of the Korean native Jindo dog varies with coat colour

This study aimed to compare the behavioural reactivity of Jindo dogs with two different coat colours. Fawn (16 males, 15 females; mean age ± S.D. = 7.2 ± 2.1 years) and white (10 males, 10 females; mean age ± S.D. = 6.9 ± 2.1 years) Jindo dogs were exposed to a set of behavioural tests. All of the dogs were videotaped during the testing period to allow further analysis. The intensity of social, aggressive, fearful, and submissive reactivity and the frequency of urination as a scent-marking behaviour were scored on a scale running from 0 to 4 points. For each dog, each variable was defined as the average of the scores of nine behaviour tests. Then, the behavioural reactivities of Jindo dogs of each coat colour were compared. The results suggested that Jindo dogs of fawn coat colour exhibited a significantly lower intensity of fearful and submissive reactivity than those of white coat colour. In addition, fawn Jindo dogs produced scent-marking behaviour significantly more frequently. The results of the present study may provide useful information for scientific researchers, potential owners and breeders of Jindo dogs.     

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.     

Frequencies of genes for coat colour and horns in Nordic cattle breeds

Gene frequencies of coat colour and horn types were assessed in 22 Nordic cattle breeds in a project aimed at establishing genetic profiles of the breeds under study. The coat colour loci yielding information on genetic variation were: extension, agouti, spotting, brindle, dun dilution and colour sided. The polled locus was assessed for two alleles. A profound variation between breeds was observed in the frequencies of both colour and horn alleles, with the older breeds generally showing greater variation in observed colour, horn types and segregating alleles than the modern breeds. The correspondence between the present genetic distance matrix and previous molecular marker distance matrices was low (r = 0.08 – 0.12). The branching pattern of a neighbour-joining tree disagreed to some extent with the molecular data structure. The current data indicates that 70% of the total genetic variation could be explained by differences between the breeds, suggesting a much greater breed differentiation than typically found at protein and microsatellite loci. The marked differentiation of the cattle breeds and observed disagreements with the results from the previous molecular data in the topology of the phylogenetic trees are most likely a result of selection on phenotypic characters analysed in this study.     

Linkage between genes for coat colour and for blood plasma esterase in dogs

A study on linkage in dogs has been made on the basis of comparable studies in other mammal species. In a breeding experiment one dog was mated to 14 bitches. The dog was heterozygous for the plasma esterase locus (Es-1) and the extension locus (E) for coat colour. The 14 bitches, homozygous for both loci, produced a total of 96 offspring. The recombination distance between the loci is calculated to be 34.4 +/- 4.8 cM. The basis for homology between species for the two loci has been discussed.     

An investigation into the genetic background of coat colour dilution in a Charolais x German Holstein F2 resource population

The molecular background of many loci affecting coat colour inheritance in cattle is still incompletely characterized, although it is known that coat colour results from the joint effects of several loci, e.g. agouti, extension and dilution. Dilution alleles are responsible for a dilution effect on the original coat colour of an individual, which is determined by the agouti and extension loci. Different loci affecting dilution of pigment are suggested in Charolais (Dc) and Simmental (Ds). To enable chromosomal mapping of the Dc mutation, 133 animals from an F2 full-sib resource population generated from a cross of Charolais and German Holstein were scored for the coat colour dilution phenotype. Linkage analysis covering all autosomes revealed a significant linkage of the dilution phenotype with microsatellite markers on bovine chromosome 5. No recombination was observed between marker ETH10 and the Dc locus. Positional and functional information identified the bovine silver homolog (SILV) gene as a candidate for the Dc mutation. Results from comparative sequencing of the SILV gene in individuals with different dilution coat colour phenotypes confirmed the presence of a c.64G>A non-synonymous mutation, which had previously been identified in the Charolais breed. The alleles at this locus were associated with coat colour dilution in this study. However, further investigation of colour inheritance within the F2 resource population indicated that a single diallelic mutation in the SILV gene cannot explain the total observed variation of coat colour dilution.     

Colours of domestication

During the last decade, coat colouration in mammals has been investigated in numerous studies. Most of these studies addressing the genetics of coat colouration were on domesticated animals. In contrast to their wild ancestors, domesticated species are often characterized by a huge allelic variability of coat‐colour‐associated genes. This variability results from artificial selection accepting negative pleiotropic effects linked with certain coat‐colour variants. Recent studies demonstrate that this selection for coat‐colour phenotypes started at the beginning of domestication. Although to date more than 300 genetic loci and more than 150 identified coat‐colour‐associated genes have been discovered, which influence pigmentation in various ways, the genetic pathways influencing coat colouration are still only poorly described. On the one hand, similar coat colourations observed in different species can be the product of a few conserved genes. On the other hand, different genes can be responsible for highly similar coat colourations in different individuals of a species or in different species. Therefore, any phenotypic classification of coat colouration blurs underlying differences in the genetic basis of colour variants. In this review we focus on (i) the underlying causes that have resulted in the observed increase of colour variation in domesticated animals compared to their wild ancestors, and (ii) the current state of knowledge with regard to the molecular mechanisms of colouration, with a special emphasis on when and where the different coat‐colour‐associated genes act.     

Olfactory receptor sequence polymorphism within and between breeds of dogs

Olfactory receptors, to which odorant molecules specifically bind, are encoded by the largest gene family yet identified in the mammalian genome. We investigated additional polymorphism due to the possible existence of multiple alleles dispersed in different dog breeds by carrying out a survey of the sequences of 16 olfactory receptor genes in a sample of 95 dogs of 20 different breeds. The level of polymorphism was high--all genes were found to have allelic variants--leading to amino acid changes and pseudogenization of some alleles in a number of cases. This preliminary study also revealed that some alleles are breed specific (or rare in the dog population), with some representing the major allele in the breeds concerned.     

DLA-DRB1, DQA1, and DQB1 alleles and haplotypes in North American Gray Wolves

The canine major histocompatibility complex contains highly polymorphic genes, many of which are critical in regulating immune response. Since domestic dogs evolved from Gray Wolves (Canis lupus), common DLA class II alleles should exist. Sequencing was used to characterize 175 Gray Wolves for DLA class II alleles, and data from 1856 dogs, covering 85 different breeds of mostly European origin, were available for comparison. Within wolves, 28 new alleles were identified, all occurring in at least 2 individuals. Three DLA-DRB1, 8 DLA-DQA1, and 6 DLA-DQB1 alleles also identified in dogs were present. Twenty-eight haplotypes were identified, of which 2 three-locus haplotypes, and many DLA-DQA1/DQB1 haplotypes, are also found in dogs. The wolves studied had relatively few dog DLA alleles and may therefore represent a remnant population descended from Asian wolves. The single European wolf included carried a haplotype found in both these North American wolves and in many dog breeds. Furthermore, one wolf DQB1 allele has been found in Shih Tzu, a breed of Asian origin. These data suggest that the wolf ancestors of Asian and European dogs may have had different gene pools, currently reflected in the DLA alleles present in dog breeds.     

Melanism and coat colour polymorphism in the Egyptian Wolf Canis lupaster Hemprich & Ehrenberg (Carnivora: Canidae) from Egypt

The Egyptian Wolf Canis lupaster was recently rediscovered as a distinct species on the basis of both morphologic and molecular genetic evidence. Phenotypical variability, including coat colour of this species across its vast, ecologically diverse range is yet to be investigated. In this paper, we present the first record of melanistic individuals of this species and compare their morphological characters and mitochondrial cytochrome b DNA sequences with those of typically coloured Canis lupaster and other closely related canids to verify their identity. We also study pelage polymorphism in a population of this species in the Egyptian Nile Valley and the Nile Delta and define its different colour variants. The typical colour, as well as the rare, very light and reddish coat colours are described. We discuss the possibility that the observed coat colour polymorphism is the result of hybridisation with the domestic dog and their potential adaptive significance.     

Not so colourful after all: eggshell pigments constrain avian eggshell colour space

Birds'' eggshells are renowned for their striking colours and varied patterns. Although often considered exceptionally diverse, we report that avian eggshell coloration, sampled here across the full phylogenetic diversity of birds, occupies only 0.08–0.10% of the avian perceivable colour space. The concentrations of the two known tetrapyrrole eggshell pigments (protoporphyrin and biliverdin) are generally poor predictors of colour, both intra- and interspecifically. Here, we show that the constrained diversity of eggshell coloration can be accurately predicted by colour mixing models based on the relative contribution of both pigments and we demonstrate that the models'' predictions can be improved by accounting for the reflectance of the eggshell''s calcium carbonate matrix. The establishment of these proximate links between pigmentation and colour will enable future tests of hypotheses on the functions of perceived avian eggshell colours that depend on eggshell chemistry. More generally, colour mixing models are not limited to avian eggshell colours but apply to any natural colour. Our approach illustrates how modelling can aid the understanding of constraints on phenotypic diversity.     

On the way to functional agro biodiversity: coat colour gene variability in goats

Functional agro biodiversity defines the exploitation of biodiversity to provide ecosystem services, support sustainable agricultural production and benefit the regional and global environment and the public at large (ELN-FAB, 2009; www.eln_fab.eu). Tracking of animal products back to the breed of origin based on their genetic make-up undoubtedly falls in this category. The aim of this paper was to identify and validate a set of single nucleotide polymorphisms (SNPs) in goat coat colour genes, most of which have not been investigated before, to trace five goat populations of the Italian Alps and their product. Several regions of 28 genes influencing coat colour pathways were amplified in eight animals (two per breed). Sequence comparison revealed 48 SNPs and three INDEL (INsertion DELetion). No breed-specific alleles were detected; however, several SNPs showed an uneven frequency distribution between breeds. In BIO, the genotype frequency distribution of a non-synonymous SNP suggested a possible role of TYRP1 in brown eumelanic goat coat colour. A total of 29 independent SNPs in 20 genes were selected and used to allocate 159 minimally related goat samples using STRUCTURE 2.2 and GeneClass 2 software. STRUCTURE 2.2 assigns 99% of individuals to the correct breed considering the prior information on putative breed of origin for each sample and 81% using only the genotypic data. The three algorithms available in GeneClass 2 performed with nearly equal efficiency, with 86% and 87% correct allocations. All the methods yielded an average probability of assignment >0.92 and a specificity index >0.86. Despite their coat colour variability, individuals belonging to ORO were fully assigned, showing that, in the absence of a breed-specific allele tied to coat colour, the best assignment resulted for the most genetically distinct breed. The lowest rate of correct assignment was observed in Verzaschese (73%), not ascertained in the breed panel used in the SNP discovery phase.     

Genetic heterogeneity and selection signature at the KIT gene in pigs showing different coat colours and patterns

Mutations in the porcine KIT gene (Dominant white locus) have been shown to affect coat colours and colour distribution in pigs. We analysed this gene in several pig breeds and populations (Sicilian black, completely black or with white patches; Cinta Senese; grey local population; Large White; Duroc; Hampshire; Pietrain; wild boar; Meishan) with different coat colours and patterns, genotyping a few polymorphisms. The 21 exons and parts of the intronic regions were sequenced in these pigs and 69 polymorphisms were identified. The grey-roan coat colour observed in a local grey population was completely associated with a 4-bp deletion of intron 18 in a single copy KIT gene, providing evidence that this mutation characterizes the I^d allele described in the early genetic literature. The white patches observed in black Sicilian pigs were not completely associated with the presence of a duplicated KIT allele (I^p), suggesting that genetic heterogeneity is a possible cause of different coat colours in this breed. Selection signature was evident at the KIT gene in two different belted pig breeds, Hampshire and Cinta Senese. The same mutation(s) may cause the belted phenotype in these breeds that originated in the 18th–19th centuries from English pigs (Hampshire) and in Tuscany (Italy) in the 14th century (Cinta Senese). Phylogenetic relationships of 28 inferred KIT haplotypes indicated two clades: one of Asian origin that included Meishan and a few Sicilian black haplotypes and another of European origin.     

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.     

Human pigmentation genes: identification, structure and consequences of polymorphic variation

The synthesis of the visible pigment melanin by the melanocyte cell is the basis of the human pigmentary system, those genes directing the formation, transport and distribution of the specialised melanosome organelle in which melanin accumulates can legitimately be called pigmentation genes. The genes involved in this process have been identified through comparative genomic studies of mouse coat colour mutations and by the molecular characterisation of human hypopigmentary genetic diseases such as OCA1 and OCA2. The melanocyte responds to the peptide hormones alpha-MSH or ACTH through the MC1R G-protein coupled receptor to stimulate melanin production through induced maturation or switching of melanin type. The pheomelanosome, containing the key enzyme of the pathway tyrosinase, produces light red/yellowish melanin, whereas the eumelanosome produces darker melanins via induction of additional TYRP1, TYRP2, SILV enzymes, and the P-protein. Intramelanosomal pH governed by the P-protein may act as a critical determinant of tyrosinase enzyme activity to control the initial step in melanin synthesis or TYRP complex formation to facilitate melanogenesis and melanosomal maturation. The search for genetic variation in these candidate human pigmentation genes in various human populations has revealed high levels of polymorphism in the MC1R locus, with over 30 variant alleles so far identified. Functional correlation of MC1R alleles with skin and hair colour provides evidence that this receptor molecule is a principle component underlying normal human pigment variation.     

Differences in the expression of the ASIP gene are involved in the recessive black coat colour pattern in sheep: evidence from the rare Xalda sheep breed

Here we have tested the hypothesis of association between different levels of agouti signalling peptide (ASIP) mRNA and the recessive black coat colour in the rare Xalda breed of sheep. To deal with this task, we first tested the possible action of both the dominant black extension allele (E(D)) and a 5-bp deletion (X99692:c.100_104del; A(del)) in the ovine ASIP coding sequence on the black coat colour pattern in 188 Xalda individuals. The E(D) allele was not present in the sample and only 11 individuals were homozygous for the A(del)ASIP allele. All Xalda individuals carrying the A(del)/A(del) genotype were phenotypically black. However, most black-coated individuals (109 out of 120) were not homozygous for the 5-bp deletion, thus rejecting the A(del)/A(del) genotype as the sole cause of recessive black coat colour in sheep. Differences in expression of ASIP mRNA were assessed via RT-PCR in 14 black-coated and 10 white-coated Xalda individuals showing different ASIP genotypes (A(wt)/A(wt), A(wt)/A(del) and A(del)/A(del)). Levels of expression in black animals were significantly (P < 0.0001) lower than those assessed for white-coated individuals. However, the ASIP genotype did not influence the ASIP mRNA level of expression. The consistency of these findings with those recently reported in humans is discussed, and the need to isolate the promoter region of ovine ASIP to obtain further evidence for a role of ASIP in recessive black ovine pigmentation is pointed out.     

The complex history of the domestication of rice

BACKGROUND: Rice has been found in archaeological sites dating to 8000 bc, although the date of rice domestication is a matter of continuing debate. Two species of domesticated rice, Oryza sativa (Asian) and Oryza glaberrima (African) are grown globally. Numerous traits separate wild and domesticated rices including changes in: pericarp colour, dormancy, shattering, panicle architecture, tiller number, mating type and number and size of seeds. SCOPE: Genetic studies using diverse methodologies have uncovered a deep population structure within domesticated rice. Two main groups, the indica and japonica subspecies, have been identified with several subpopulations existing within each group. The antiquity of the divide has been estimated at more than 100 000 years ago. This date far precedes domestication, supporting independent domestications of indica and japonica from pre-differentiated pools of the wild ancestor. Crosses between subspecies display sterility and segregate for domestication traits, indicating that different populations are fixed for different networks of alleles conditioning these traits. Numerous domestication QTLs have been identified in crosses between the subspecies and in crosses between wild and domesticated accessions of rice. Many of the QTLs cluster in the same genomic regions, suggesting that a single gene with pleiotropic effects or that closely linked clusters of genes underlie these QTL. Recently, several domestication loci have been cloned from rice, including the gene controlling pericarp colour and two loci for shattering. The distribution and evolutionary history of these genes gives insight into the domestication process and the relationship between the subspecies. CONCLUSIONS: The evolutionary history of rice is complex, but recent work has shed light on the genetics of the transition from wild (O. rufipogon and O. nivara) to domesticated (O. sativa) rice. The types of genes involved and the geographic and genetic distribution of alleles will allow scientists to better understand our ancestors and breed better rice for our descendents.