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.     

A PCR-RFLP for KIT associated with tobiano spotting pattern in horses

An MspI polymorphism was identified in intron 13 of the equine homologue of proto-oncogene c-kit (KIT) by comparing DNA sequences from horses with solid coat colour and horses homozygous for the tobiano spotting (To) gene. The allele associated with solid coat colour was designated KM0, while the allele associated with the tobiano pattern created an additional MspI restriction site and was designated KM1. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) studies using DNA from hair follicles demonstrated that all 129 of 129 tobiano patterned horses possessed the KM1 allele. However, three of 104 solid-coloured thoroughbred horses also possessed the KM1 allele. Therefore, while KM1 is strongly associated with the gene for To, the association is not absolute. However, this test appears more efficacious to identify putative homozygotes for To than current biochemical testing methods using albumin (Alb) and vitamin D binding protein (Gc) haplotypes.     

Polymorphism at the porcine Dominant white/KIT locus influence coat colour and peripheral blood cell measures

We have examined the phenotype of different KIT genotypes with regard to coat colour and several blood parameters (erythrocyte numbers and measures, total and differential leucocyte numbers, haematocrit and haemoglobin levels and serum components). The effect of two different iron supplement regimes (one or two iron injections) on the blood parameters was also examined. For a total of 184 cross-bred piglets (different combinations of Hampshire, Landrace and Yorkshire) blood parameters were measured four times during their first month of life, and the KIT genotypes of these and 70 additional cross-bred piglets were determined. Eight different KIT genotypes were identified, which confirms the large allelic diversity at the KIT locus in commercial pig populations. The results showed that pigs with different KIT genotypes differ both in coat colour and in haematological parameters. In general, homozygous Dominant white (I/I) piglets had larger erythrocytes with lower haemoglobin concentration, indicating a mild macrocytic anaemia. The effect of two compared with one iron injection was also most pronounced for the I/I piglets.     

An equine chromosome 3 inversion is associated with the tobiano spotting pattern in German horse breeds

The tobiano white-spotting pattern is one of several known depigmentation phenotypes in horses and is desired by many horse breeders and owners. The tobiano spotting phenotype is inherited as an autosomal dominant trait. Horses that are heterozygous or homozygous for the tobiano allele ( To ) are phenotypically indistinguishable. A SNP associated with To had previously been identified in intron 13 of the equine KIT gene and was used for an indirect gene test. The test was useful in several horse breeds. However, genotyping this sequence variant in the Lewitzer horse breed revealed that 14% of horses with the tobiano pattern did not show the polymorphism in intron 13 and consequently the test was not useful to identify putative homozygotes for To within this breed. Speculations were raised that an independent mutation might cause the tobiano spotting pattern in this breed. Recently, the putative causative mutation for To was described as a large chromosomal inversion on equine chromosome 3. One of the inversion breakpoints is approximately 70 kb downstream of the KIT gene and probably disrupts a regulatory element of the KIT gene. We obtained genotypes for the intron 13 SNP and the chromosomal inversion for 204 tobiano spotted horses and 24 control animals of several breeds. The genotyping data confirmed that the chromosomal inversion was perfectly associated with the To allele in all investigated horses. Therefore, the new test is suitable to discriminate heterozygous To/+ and homozygous To/To horses in the investigated breeds.     

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.     

A possible dominant white gene in Jersey cattle

A white heifer ("Snow") was born in 1991 from coloured registered Jersey parents. She produced six calves sired by coloured Jersey bulls: three white bull calves, two white heifer calves, and one coloured bull calf. One of the white bull calves was mated with 40 Hereford × Friesian yearling heifers (white face, predominantly black body with some white patches). The 38 resulting calves included 16 white and 22 coloured calves. Twelve of the 16 white calves were heifers and four were bulls. Red or black spotting was recorded on some white calves. The results are consistent with an autosomal dominant mutant causing the white phenotype. The mutation appears to have arisen spontaneously in Snow, then passing to her white progeny and white grand-progeny. The white individuals varied from entirely white in a few cases, to most having some residual small areas of red or black pigmentation in patterns not typical of other reported white spotting patterns of cattle.     

Linked markers exclude KIT as the gene responsible for appaloosa coat colour spotting patterns in horses

The appaloosa coat colour pattern of the horse is similar to that caused by the rump-white (Rw) gene in the mouse. In the mouse Rw colour pattern is the result of an inversion involving the proto-oncogene c-kit (KIT). Therefore, we investigated KIT as a candidate gene that encodes the appaloosa coat colour gene (Lp) in horses. KIT plays a critical role in haematopoiesis, gametogenesis, and melanogenesis and encodes a transmembrane tyrosine kinase receptor that belongs to the PDGF/CSF-1/c-KIT receptor subfamily. Half-sib families segregating for Lp were uninformative for a reported polymorphism in KIT. However, KIT is located on horse chromosome 3 close to albumin (ALB), serum carboxylesterase (ES), vitamin D-binding protein (GC) and microsatellite markers ASB23, LEX007, LEX57, and UCDEQ437. Indeed, KIT and ASB23 were localized to ECA3q21-22.1 and 3q22.1-22.3, respectively, by fluorescent in situ hybridization. Family studies were conducted to investigate linkage of Lp to these markers using eight half-sib families in which Appaloosa stallions were mated to solid coloured mares. Linkage of Lp to the chromosome region containing ES, ALB, GC, ASB23, UCDEQ437, LEX57, and LEX007 was investigated by a multipoint linkage analysis using the computer program GENEHUNTER. LOD scores over the interval under investigation ranged from -4.28 to -12.48, with a score of -12.48 at the location for ASB23. Therefore, it was concluded that appaloosa (Lp) is not linked to any of the tested markers on ECA3, and thus Lp is unlikely to be the product of KIT.     

White spotting in the domestic cat (Felis catus) maps near KIT on feline chromosome B1

Five feline-derived microsatellite markers were genotyped in a large pedigree of cats that segregates for ventral white spotting. Both KIT and EDNRB cause similar white spotting phenotypes in other species. Thus, three of the five microsatellite markers chosen were on feline chromosome B1 in close proximity to KIT; the other two markers were on feline chromosome A1 near EDNRB. Pairwise linkage analysis supported linkage of the white spotting with the three chromosome B1 markers but not with the two chromosome A1 markers. This study indicates that KIT, or another gene within the linked region, is a candidate for white spotting in cats. Platelet-derived growth factor alpha (PDGFRA) is also a strong candidate, assuming that the KIT-PDGFRA linkage group, which is conserved in many mammalian species, is also conserved in the cat.     

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.     

Black-ear gene and blood polymorphism in four southern Chinese cattle groups

The gene for black-ear coat colour pattern, commonly found among cattle of tropical origin, was observed in 809 animals of four breeds of local cattle in southern China. Gene frequencies for Tf, Hb and Alb demonstrated that these groups of cattle were quite divergent from Bos taurus. These breeds of cattle are thought to be descended from ancient Chinese cattle. At the same time certain influences observed in coat colour may be derived from Bali cattle.     

A complex structural variant at the KIT locus in cattle with the Pinzgauer spotting pattern

A specific white spotting phenotype, termed finching or line‐backed spotting, is known for all Pinzgauer cattle and occurs occasionally in Tux‐Zillertaler cattle, two Austrian breeds. The so‐called Pinzgauer spotting is inherited as an autosomal incompletely dominant trait. A genome‐wide association study using 27 white spotted and 16 solid‐coloured Tux‐Zillertaler cattle, based on 777k SNP data, revealed a strong signal on chromosome 6 at the KIT locus. Haplotype analyses defined a critical interval of 122 kb downstream of the KIT coding region. Whole‐genome sequencing of a Pinzgauer cattle and comparison to 338 control genomes revealed a complex structural variant consisting of a 9.4‐kb deletion and an inversely inserted duplication of 1.5 kb fused to a 310‐kb duplicated segment from chromosome 4. A diagnostic PCR was developed for straightforward genotyping of carriers for this structural variant (KIT^PINZ) and confirmed that the variant allele was present in all Pinzgauer and most of the white spotted Tux‐Zillertaler cattle. In addition, we detected the variant in all Slovenian Cika, British Gloucester and Spanish Berrenda en negro cattle with similar spotting patterns. Interestingly, the KIT^PINZ variant occurs in some white spotted animals of the Swiss breeds Evolèner and Eringer. The introgression of the KIT^PINZ variant confirms admixture and the reported historical relationship of these short‐headed breeds with Austrian Tux‐Zillertaler and suggests a mutation event, occurring before breed formation.     

Genetic mapping of the belt pattern in Brown Swiss cattle to BTA3

The white belt pattern of Brown Swiss cattle is characterized by a lack of melanocytes in a stretch of skin around the midsection. This pattern is of variable width and sometimes the belt does not fully circle the body. To identify the gene responsible for this colour variation, we performed linkage mapping of the belted locus using six segregating half-sib families including 104 informative meioses for the belted character. The pedigree confirmed a monogenic autosomal dominant inheritance of the belted phenotype in Brown Swiss cattle. We performed a genome scan using 186 microsatellite markers in a subset of 88 animals of the six families. Linkage with the belt phenotype was detected at the telomeric region of BTA3. Fine-mapping and haplotype analysis using 19 additional markers in this region refined the critical region of the belted locus to a 922-kb interval on BTA3. As the corresponding human and mouse chromosome segments contain no obvious candidate gene for this coat colour trait, the mutation causing the belt pattern in the Brown Swiss cattle might help to identify an unknown gene influencing skin pigmentation.     

Colour-sidedness in Gloucester cattle is associated with a complex structural variant impacting regulatory elements downstream of KIT

Colour-sidedness is a striking coat colour pattern found in a number of cattle breeds, typically characterised by a white stripe that extends along the back, head and belly of the animal. This dominant phenotype is caused by two related translocations (Cs₆ and Cs₂₉) that alter a region downstream of the KIT gene. Gloucester cattle are native to the UK and are known for an unusual colour-sided pattern that does not extend to the head. We carried out whole-genome sequencing of two Gloucester bulls as well as colour-sided Irish Moiled, British White and Pustertaler Sprinzen for comparison. We found that the Gloucester cattle also have a complex structural variant downstream of KIT, which overlaps the regions involved in Cs₆ and Cs_29. All three alleles potentially disrupt a number of putative regulatory elements downstream of KIT. These results complement and expand on the recently published work focused on the Pinzgauer breed from Austria, a carrier of the same colour-sided pattern as seen in Gloucester cattle.     

Genetic polymorphisms and antiviral activity in the bovine MX1 gene

Bovine MX1 cDNAs consisting of 2280 bp from 11 animals of five breeds and from a cultured cell line were sequenced and compared with previously reported data. Ten nucleotide substitutions were synonymous mutations, and a single nucleotide substitution at 458 resulted in an amino acid exchange of Ile (ATT) and Met (ATG). A 13-bp deletion-insertion mutation was also found in the 3'-UTR. Based on the nucleotide substitutions found in this study, bovine MX1 cDNA was classified into 11 genotypes. A phylogenetic tree of the 11 genotypes suggested that the genotypes observed in Brahman were a great genetic distance from other genotypes. An 18-bp deletion-insertion variation at position 171 was found to be the result of alternative splicing. The 18-bp deletion-insertion is located at the boundary between exon 3 and intron 3. Permanently transfected 3T3 cell lines expressing bovine MX1 mRNA were established to analyse the antiviral potential against VSVDeltaG*-G infection. Transfected cell clones expressing bovine MX1 mRNA showed a significantly smaller number of cells infected with VSVDeltaG*-G compared with the control cells. These results indicate that the bovine MX1 protein has potent antiviral activity.     

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.     

Indels within promoter and intron 1 of bovine prion protein gene modulate the gene expression levels in the medulla oblongata of two Japanese cattle breeds

Genetic differences which exist in the prion protein gene (PRNP) have been reported to influence susceptibility of humans, sheep and goats to prion diseases. In cattle, however, none of the known coding polymorphisms has a direct effect on bovine spongiform encephalopathy (BSE). It has been reported that 23‐bp insertion/deletion (indel) polymorphisms within the promoter region have a tentative association to BSE susceptibility in German cattle, and a lower number of 24‐bp repeat units in the open reading frame (ORF) was reported to reduce BSE susceptibility in transgenic mice. In this study, because of the hypothesis that bovine PRNP promoter polymorphisms cause changes in PRNP expression, we genotyped PRNP polymorphisms in the promoter and intron 1 using 218 genomic DNA samples from two Japanese cattle breeds. We also analysed the expression levels of prion in 40 animals by quantification of real‐time PCR using mRNAs extracted from the medulla oblongata to study the relationship between PRNP genotypes and PRNP expression. We found a significant correlation between promoter indel polymorphisms and PRNP‐mRNA expression (P_0.0413) and therefore hypothesize that differences in polymorphisms could be one of the causes of differences in PRNP expression levels. We also report a novel difference in PRNP expression (P < 0.0001) between Japanese Black and Japanese Brown cattle breeds. There was no significant difference based on age and sex of the animals.     

SNP-based association mapping of the polled gene in divergent cattle breeds

Naturally, hornless cattle are called polled. Although the POLL locus could be assigned to a c. 1.36‐Mb interval in the centromeric region of BTA1, the underlying genetic basis for the polled trait is still unknown. Here, an association mapping design was set up to refine the candidate region of the polled trait for subsequent high‐throughput sequencing. The case group comprised 101 homozygous polled animals from nine divergent cattle breeds, the majority represented by Galloway, Angus, Fleckvieh and Holstein Friesian. Additionally, this group included some polled individuals of Blonde d’Aquitaine, Charolais, Hereford, Jersey and Limousin breeds. The control group comprised horned Belgian Blue, Fleckvieh, Holstein Friesian and Illyrian Bu?a cattle. A genome‐wide scan using 49 163 SNPs was performed, which revealed one shared homozygous haplotype block consisting of nine neighbouring SNPs in all polled animals. This segment defines a 381‐kb interval on BTA1 that we consider to be the most likely location of the POLL mutation. Our results further demonstrate that the polled‐associated haplotype is also frequent in horned animals included in this study, and thus the haplotype as such cannot be used for population‐wide genetic testing. The actual trait‐associated haplotype may be revealed by using higher‐density SNP arrays. For the final identification of the causal mutation, we suggest high‐throughput sequencing of the entire candidate region, because the identification of functional candidate genes is difficult owing to the lack of a comparable model.     

Evaluation of the genetic variability of 23 bovine microsatellite markers in four Belgian cattle breeds

The polymorphism of 23 microsatellites in the four main cattle breeds in Belgium (Holstein Friesian, Belgian Blue, Belgian Red Pied and East Flemish) was analysed. Heterozygosity, polymorphism information content, the effective number of alleles, exclusion probability and the probability of genotypic identity for two random individuals were calculated for all microsatellites and all breeds. The Belgian Blue breed is generally a little less polymorphic in comparison with the other three breeds. Estimates of the genetic distances between these breeds confirmed the widely accepted proposition that the Belgian Blue is the most genetically distinct of these breeds. The three other breeds are likely to become one population, given current breeding strategies. Exclusion probabilities in parentage control cases are >0·9999 in all four breeds when all 23 microsatellites are used and >0·98 with only the two most polymorphic multiplexes.     

Genetic variation in eight Chinese cattle breeds based on the analysis of microsatellite markers

Genetic variability and genetic relationships were investigated among eight Chinese cattle breeds using 12 microsatellite markers. Three hundred and fifty-two alleles were detected and the average number of alleles per locus ranged from 8.33 ± 1.67 in the Jiaxian breed to 21.33 ± 5.60 in the Qinchuan breed with a mean value of 13.91. The total number of alleles per microsatellite ranged from 21 (INRA005, HEL1) to 40 (HEL13), with a mean of 29.33 per locus. The fixation indices at the 12 loci in the eight breeds were very low with a mean of 0.006. A principal components analysis and the construction of a neighborjoining tree showed that these eight Chinese cattle breeds cluster into three groups i.e. the Yanbian andChineseHolstein, theNanyang and Jiaxian, and the four remaining breeds.This clustering agrees with the origin and geographical distributions of these Chinese breeds.