Unexpectedly high allelic diversity at the KIT locus causing dominant white color in the domestic pig.

Mutations in KIT encoding the mast/stem cell growth factor receptor (MGF) are responsible for coat color variation in domestic pigs. The dominant white phenotype is caused by two mutations, a gene duplication and a splice mutation in one of the copies leading to skipping of exon 17. Here we applied minisequencing and pyrosequencing for quantitative analysis of the number of copies with the splice form. An unexpectedly high genetic diversity was revealed in white pigs. We found four different KIT alleles in a small sample of eight Large White females used as founder animals in a wild boar intercross. A similar number of KIT alleles was found in commercial populations of white Landrace and Large White pigs. We provide evidence for at least two new KIT alleles in pigs, both with a triplication of the gene. The results imply that KIT alleles with the duplication are genetically unstable and new alleles are most likely generated by unequal crossing over. This study provides an improved method for genotyping the complicated Dominant white/KIT locus in pigs. The results also suggest that some alleles may be associated with negative pleiotropic effects on other traits.     

白色基因座(I)在中国地方猪种毛色遗传中的作用研究

通过利用PCR—RFLP和PCR—SSCP技术对中国地方猪种KIT基因内含子17、18的序列进行多态性分析。结果表明：内含子17上的替换突变(G→A)发生于毛色为白色的个体——白色五指山猪、大白猪、长白猪上,其基因型(AB型)频率分别为1、1和0．8;其他中国地方猪种的此基因型频率均为0。内含子18上的缺失突变(AGTT)也同样发生在上述3个猪种的白色个体中,其基因型(AA型)频率分别为1、1和0．93;而且同样在其他的地方品种中其基因型频率均为0。这充分证明KIT基因对于猪的白毛色有重要的调控作用,而且I基因座对于其他的经典遗传基因座有上位作用。另一方面,中国地方猪种荣昌猪虽然在表型上与引入猪种大白猪、长白猪相似(白毛色),但是在KIT基因上发生的突变完全不同,推测它们分别属于不同的毛色遗传体系。     

Seven novel KIT mutations in horses with white coat colour phenotypes

White coat colour in horses is inherited as a monogenic autosomal dominant trait showing a variable expression of coat depigmentation. Mutations in the KIT gene have previously been shown to cause white coat colour phenotypes in pigs, mice and humans. We recently also demonstrated that four independent mutations in the equine KIT gene are responsible for the dominant white coat colour phenotype in various horse breeds. We have now analysed additional horse families segregating for white coat colour phenotypes and report seven new KIT mutations in independent Thoroughbred, Icelandic Horse, German Holstein, Quarter Horse and South German Draft Horse families. In four of the seven families, only one single white horse, presumably representing the founder for each of the four respective mutations, was available for genotyping. The newly reported mutations comprise two frameshift mutations (c.1126_1129delGAAC; c.2193delG), two missense mutations (c.856G>A; c.1789G>A) and three splice site mutations (c.338-1G>C; c.2222-1G>A; c.2684+1G>A). White phenotypes in horses show a remarkable allelic heterogeneity. In fact, a higher number of alleles are molecularly characterized at the equine KIT gene than for any other known gene in livestock species.     

Identification of the e allele at the Extension locus (MC1R) in Brazilian Creole sheep and its role in wool color variation

The melanocortin 1 receptor (MC1R) gene has been described as responsible for the black color in some breeds of sheep, but little is known about its function in many colored breeds, particularly those with a wide range of pigmentation phenotypes. The Brazilian Creole is a local breed of sheep from southern Brazil that has a wide variety of wool colors. We examined the MC1R gene (Extension locus) to search for the e allele and determine its role in controlling wool color variation in this breed. One hundred and twenty-five animals, covering the most common Creole sheep phenotypes (black, brown, dark gray, light gray, and white), were sequenced to detect the mutations p.M73K and p.D121N. Besides these two mutations, three other synonymous sites (429, 600, and 725) were found. The dominant allele (E(D): p.73K, and p.121N) was found only in colored animals, whereas the recessive allele (E(+): p.73M, and p.121D) was homozygous only in white individuals. We concluded that MC1R is involved in the control of wool color in Brazilian Creole sheep, particularly the dark phenotypes, although a second gene may be involved in the expression of the white phenotype in this breed.     

Allelic heterogeneity at the equine KIT locus in dominant white (W) horses

White coat color has been a highly valued trait in horses for at least 2,000 years. Dominant white (W) is one of several known depigmentation phenotypes in horses. It shows considerable phenotypic variation, ranging from ~50% depigmented areas up to a completely white coat. In the horse, the four depigmentation phenotypes roan, sabino, tobiano, and dominant white were independently mapped to a chromosomal region on ECA 3 harboring the KIT gene. KIT plays an important role in melanoblast survival during embryonic development. We determined the sequence and genomic organization of the ~82 kb equine KIT gene. A mutation analysis of all 21 KIT exons in white Franches-Montagnes Horses revealed a nonsense mutation in exon 15 (c.2151C>G, p.Y717X). We analyzed the KIT exons in horses characterized as dominant white from other populations and found three additional candidate causative mutations. Three almost completely white Arabians carried a different nonsense mutation in exon 4 (c.706A>T, p.K236X). Six Camarillo White Horses had a missense mutation in exon 12 (c.1805C>T, p.A602V), and five white Thoroughbreds had yet another missense mutation in exon 13 (c.1960G>A, p.G654R). Our results indicate that the dominant white color in Franches-Montagnes Horses is caused by a nonsense mutation in the KIT gene and that multiple independent mutations within this gene appear to be responsible for dominant white in several other modern horse populations.     

The gene for dominant white color in the pig is closely linked to ALB and PDGRFRA on chromosome 8.

White is a widespread coat color among domestic pig breeds and is controlled by an autosomal dominant gene I. The segregation of this gene was analyzed in a reference pedigree for gene mapping developed by crossing the European wild pig and a Large White domestic breed. The gene for dominant white color was shown to be closely linked to the genes for albumin (ALB) and platelet-derived growth factor receptor alpha (PDGFRA) on chromosome 8. An unexpected phenotype with patches of colored and white coat was observed among the F1 and F2 animals. The segregation data indicated that the phenotype was controlled by a third allele, denoted patch (Ip), most likely transmitted by one of the Large White founder animals. It is shown that the ALB, PDGFRA, I linkage group shares homologies with parts of mouse chromosome 5, human chromosome 4, and horse linkage group II, all of which contain dominant genes for white or white spotting. Candidate genes for the dominant white and patch mutations in the pig are proposed on the basis on these linkage homologies and the recent molecular definition of the dominant white spotting (W) and patch (Ph) mutations in the mouse.     

Two cysteine substitutions in the MC1R generate the blue variant of the Arctic fox (Alopex lagopus) and prevent expression of the white winter coat

We have characterized two mutations in the MC1R gene of the blue variant of the arctic fox (Alopex lagopus) that both incorporate a novel cysteine residue into the receptor. A family study in farmed arctic foxes verified that the dominant expression of the blue color phenotype cosegregates completely with the allele harboring these two mutations. Additionally to the altered pigment synthesis, the blue fox allele suppresses the seasonal change in coat color found in the native arctic fox. Consequently, these findings suggest that the MC1R/agouti regulatory system is involved in the seasonal changes of coat color found in arctic fox.     

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.     

Reverse genetic screen for loss‐of‐function mutations uncovers a frameshifting deletion in the melanophilin gene accountable for a distinctive coat color in Belgian Blue cattle

In the course of a reverse genetic screen in the Belgian Blue cattle breed, we uncovered a 10‐bp deletion (c.87_96del) in the first coding exon of the melanophilin gene (MLPH), which introduces a premature stop codon (p.Glu32Aspfs*1) in the same exon, truncating 94% of the protein. Recessive damaging mutations in the MLPH gene are well known to cause skin, hair, coat or plumage color dilution phenotypes in numerous species, including human, mice, dog, cat, mink, rabbit, chicken and quail. Large‐scale array genotyping undertaken to identify p.Glu32Aspfs*1 homozygous mutant animals revealed a mutation frequency of 5% in the breed and allowed for the identification of 10 homozygous mutants. As expression of a colored coat requires at least one wild‐type allele at the co‐dominant Roan locus encoded by the KIT ligand gene (KITLG), homozygous mutants for p.Ala227Asp corresponding with the missense mutation were excluded. The six remaining colored calves displayed a distinctive dilution phenotype as anticipated. This new coat color was named ‘cool gray’. It is the first damaging mutation in the MLPH gene described in cattle and extends the already long list of species with diluted color due to recessive mutations in MLPH and broadens the color palette of gray in this breed.     

Two variants in the KIT gene as candidate causative mutations for a dominant white and a white spotting phenotype in the donkey

White spotting phenotypes have been intensively studied in horses, and although similar phenotypes occur in the donkey, little is known about the molecular genetics underlying these patterns in donkeys. White spotting in donkeys can range from only a few white areas to almost complete depigmentation and is characterised by a loss of pigmentation usually progressing from a white spot in the hip area. Completely white‐born donkeys are rare, and the phenotype is characterised by the complete absence of pigment resulting in pink skin and a white coat. A dominant mode of inheritance has been demonstrated for spotting in donkeys. Although the mode of inheritance for the completely white phenotype in donkeys is not clear, the phenotype shows similarities to dominant white in horses. As variants in the KIT gene are known to cause a range of white phenotypes in the horse, we investigated the KIT gene as a potential candidate gene for two phenotypes in the donkey, white spotting and white. A mutation analysis of all 21 KIT exons identified a missense variant in exon 4 (c.662A>C; p.Tyr221Ser) present only in a white‐born donkey. A second variant affecting a splice donor site (c.1978+2T>A) was found exclusively in donkeys with white spotting. Both variants were absent in 24 solid‐coloured controls. To the authors’ knowledge, this is the first study investigating genetic mechanisms underlying white phenotypes in donkeys. Our results suggest that two independent KIT alleles are probably responsible for white spotting and white in donkeys.     

Single nucleotide polymorphism analysis on melanocortin receptor 1 (MC1R) of Chinese native pig

Thecoatcolorisanimportantcharacteristicofapigbreed,andcanbeclassifiedintomanytypes.Thecolorvariationsareeitherduetothedistributionofmelanocytesintheskinortotheabilityofmelano-cytestoproducemelaningranules.Thesynthesisofmelaninoriginatedfromtheformationofneuralcrest-derivedcells,whichhavetwomigrationroutes[1,2].Themelanocytesaretheramificationswhiletheneuralcrest-derivedcellsmigrateviadorsalroute[3].Andtheyprovidethefactoryformelaninsynthesis.Al-thoughtherearemanykindsofpigmentsinvertebralanim…     

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.     

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     

Single nucleotide polymorphism analysis on melanocortin receptor 1 (<Emphasis Type="Italic">MC1R</Emphasis>) of Chinese native pig

Melanocortin receptor 1 (MC1R) gene, one of the important candidate genes for coat color trait, was used to analyze the single nucleotide polymorphism (SNP) in Chinese native pig breeds by PCR-single strand conformation polymorphism (PCR-SSCP). The study had also taken 3 imported pig breeds as control. The results showed that the three mutations G284A, T309C and T364C found in Chinese native pigs were consistent to the mutation found in the European Large Black individuals. However, 68CC or C492T and G728A were only found in the imported individuals, which were obviously different from the Chinese native pigs. Accordingly, we presumed that the coat colors of Chinese native pigs belonged to dominant black color system, which was completely distinct to that of imported pig breeds. Thus it was implied thatMC1R gene was not the principal factor affecting the coat color differences of Chinese native pig breeds, but could be used to trace the molecular evolution of pig breeds.     

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).     

利用混合样本池法对鸡显性白羽基因PMEL17突变位点的检测

显性白羽基因座是影响鸡羽色形成的重要基因座位之一, 该基因座上的显性等位基因I 会抑制黑色素合成, 从而使携带该基因的个体全身羽毛呈现白色。目前已确认鸡显性白羽基因座编码PMEL17蛋白: 是一种黑素细胞特异性蛋白, 在黑素细胞的分化与成熟中起到重要作用, 并证明PMEL17基因的突变与显性白羽的形成有关。文章利用混合样本池建立了一种低成本、高效率, 并能在大规模群体中检测PMEL17基因突变的方法, 称为PCR产物混合样本池法。该方法的基本步骤如下: 首先, 提取个体基因组DNA, 并设计相关引物对每一个体单独进行PCR扩增; 其次, 将PCR产物等比例混合, 10个样品混在一个池中; 然后, 将PCR产物混合池样品于非变性聚丙烯酰胺凝胶上进行电泳; 最后, 待电泳结束后进行银染, 根据凝胶上所显条带判定是否存在突变体。此外, 文章还将这种方法与传统基因组DNA混合样本池法进行了比较试验, 并利用该方法对试验鸡群显性白羽基因PMEL17突变进行检测, 证实该方法具有较高准确度。