The genetics of brown coat color and white spotting in domestic yaks (Bos grunniens)

Domestic yaks (Bos grunniens) exhibit two major coat color variations: a brown vs. wild‐type black pigmentation and a white spotting vs. wild‐type solid color pattern. The genetic basis for these variations in color and distribution remains largely unknown and may be complicated by a breeding history involving hybridization between yaks and cattle. Here, we investigated 92 domestic yaks from China using a candidate gene approach. Sequence variations in MC1R, PMEL and TYRP1 were surveyed in brown yaks; TYRP1 was unassociated with the coloration and excluded. Recessive mutations from MC1R, or p.Gln34*, p.Met73Leu and possibly p.Arg142Pro, are reported in bovids for the first time and accounted for approximately 40% of the brown yaks in this study. The remaining 60% of brown individuals correlated with a cattle‐derived deletion mutation from PMEL (p.Leu18del) in a dominant manner. Degrees of white spotting found in yaks vary from color sidedness and white face, to completely white. After examining the candidate gene KIT, we suggest that color‐sided and all‐white yaks are caused by the serial translations of KIT (Cs₆ or Cs₂₉) as reported for cattle. The white‐faced phenotype in yaks is associated with the KIT haplotype S^wf. All KIT mutations underlying the serial phenotypes of white spotting in yaks are identical to those in cattle, indicating that cattle are the likely source of white spotting in yaks. Our results reveal the complex genetic origins of domestic yak coat color as either native in yaks through evolution and domestication or as introduced from cattle through interspecific hybridization.     

Molecular genetics of coat colour variations in White Galloway and White Park cattle

White Galloway cattle exhibit three different white coat colour phenotypes, that is, well marked, strongly marked and mismarked. However, mating of individuals with the preferred well or strongly marked phenotype also results in offspring with the undesired mismarked and/or even fully black coat colour. To elucidate the genetic background of the coat colour variations in White Galloway cattle, we analysed four coat colour relevant genes: mast/stem cell growth factor receptor (KIT), KIT ligand (KITLG), melanocortin 1 receptor (MC1R) and tyrosinase (TYR). Here, we show that the coat colour variations in White Galloway cattle and White Park cattle are caused by a KIT gene (chromosome 6) duplication and aberrant insertion on chromosome 29 (Cs₂₉) as recently described for colour‐sided Belgian Blue. Homozygous (Cs₂₉/Cs₂₉) White Galloway cattle and White Park cattle exhibit the mismarked phenotype, whereas heterozygous (Cs₂₉/wt₂₉) individuals are either well or strongly marked. In contrast, fully black individuals are characterised by the wild‐type chromosome 29. As known for other cattle breeds, mutations in the MC1R gene determine the red colouring. Our data suggest that the white coat colour variations in White Galloway cattle and White Park cattle are caused by a dose‐dependent effect based on the ploidy of aberrant insertions and inheritance of the KIT gene on chromosome 29.     

Whole genome sequencing reveals a novel deletion variant in the KIT gene in horses with white spotted coat colour phenotypes

White spotting phenotypes in horses can range in severity from the common white markings up to completely white horses. EDNRB, KIT, MITF, PAX3 and TRPM1 represent known candidate genes for such phenotypes in horses. For the present study, we re‐investigated a large horse family segregating a variable white spotting phenotype, for which conventional Sanger sequencing of the candidate genes’ individual exons had failed to reveal the causative variant. We obtained whole genome sequence data from an affected horse and specifically searched for structural variants in the known candidate genes. This analysis revealed a heterozygous ~1.9‐kb deletion spanning exons 10–13 of the KIT gene (chr3:77,740,239_77,742,136del1898insTATAT). In continuity with previously named equine KIT variants we propose to designate the newly identified deletion variant W22. We had access to 21 horses carrying the W22 allele. Four of them were compound heterozygous W20/W22 and had a completely white phenotype. Our data suggest that W22 represents a true null allele of the KIT gene, whereas the previously identified W20 leads to a partial loss of function. These findings will enable more precise genetic testing for depigmentation phenotypes in horses.     

Interaction of MC1R and PMEL alleles on solid coat colors in Highland cattle

Six solid colors occur in Highland cattle: black, dun, silver dun and red, yellow, and white. These six coat colors are explained by a non‐epistatic interaction of the genotypes at the MC1R and PMEL genes. A three base pair deletion in the PMEL gene leading to the deletion of a leucine from the signal peptide is observed in dilute‐colored Highland cattle (c.50_52delTTC, p.Leu18del). The mutant PMEL allele acts in a semi‐dominant manner. Dun Galloway cattle also have one copy of the deletion allele, and silver dun Galloway cattle have two copies. The presence of two adjacent leucine residues at the site of this deletion is highly conserved in human, horse, mouse and chicken as well as in cattle with undiluted coat colors. Highland and Galloway cattle thus exhibit a similar dose‐dependent dilution effect based on the number of PMEL :c.50_51delTTC alleles, as Charolais cattle with PMEL :c.64G>A alleles. The PMEL :c.64G>A allele was not found in Highland or Galloway cattle.     

Analysis of ROH patterns in the Noriker horse breed reveals signatures of selection for coat color and body size

Overlapping runs of homozygosity (ROH islands) shared by the majority of a population are hypothesized to be the result of selection around a target locus. In this study we investigated the impact of selection for coat color within the Noriker horse on autozygosity and ROH patterns. We analyzed overlapping homozygous regions (ROH islands) for gene content in fragments shared by more than 50% of horses. Long‐term assortative mating of chestnut horses and the small effective population size of leopard spotted and tobiano horses resulted in higher mean genome‐wide ROH coverage (S_ROH) within the range of 237.4–284.2 Mb, whereas for bay, black and roan horses, where rotation mating is commonly applied, lower autozygosity (S_ROH from 176.4–180.0 Mb) was determined. We identified seven common ROH islands considering all Noriker horses from our dataset. Specific islands were documented for chestnut, leopard spotted, roan and bay horses. The ROH islands contained, among others, genes associated with body size (ZFAT, LASP1 and LCORL/NCAPG), coat color (MC1R in chestnut and the factor PATN1 in leopard spotted horses) and morphogenesis (HOXB cluster in all color strains except leopard spotted horses). This study demonstrates that within a closed population sharing the same founders and ancestors, selection on a single phenotypic trait, in this case coat color, can result in genetic fragmentation affecting levels of autozygosity and distribution of ROH islands and enclosed gene content.     

Locus‐specific ancestry to detect recent response to selection in admixed Swiss Fleckvieh cattle

Identification of selection signatures is one of the current endeavors of evolutionary genetics. Admixed populations may be used to infer post‐admixture selection. We calculated local ancestry for Swiss Fleckvieh, a composite of Simmental (SI) and Red Holstein Friesian (RHF), to infer such signals. Illumina Bovine SNP50 BeadChip data for 300 admixed, 88 SI and 97 RHF bulls were used. The average RHF ancestry across the whole genome was 0.70. To identify regions with high deviation from average, we considered two significance thresholds, based on a permutation test and extreme deviation from normal distribution. Regions on chromosomes 13 (46.3–47.3 Mb) and 18 (18.7–25.9 Mb) passed both thresholds in the direction of increased SI. Extended haplotype homozygosity within (iHS) and between (Rsb) populations was calculated to explore additional patterns of pre‐ and post‐admixture selection signals. The Rsb score of admixed and SI was significant in a wide region of chromosome 18 (6.6–24.6 Mb) overlapped with one area of strong local ancestry deviation. FTO, with pleiotropic effect on milk and fertility, NOD2 on dairy and NKD1 and SALL1 on fertility traits are located there. Genetic differentiation of RHF and SI (F_st), an alternative indicator of pre‐admixture selection in pure populations, was calculated. No considerable overlap of peaks of local ancestry deviations and F_st was observed. We found two regions with significant signatures of post‐admixture selection in this very young composite, applying comparatively stringent significance thresholds. The signals cover relatively large genomic areas and did not allow pinpointing of the gene(s) responsible for the apparent shift in ancestry proportions.     

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

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

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.     

A novel MLPH variant in dogs with coat colour dilution

Coat colour dilution may be the result of altered melanosome transport in melanocytes. Loss‐of‐function variants in the melanophilin gene (MLPH) cause a recessively inherited form of coat colour dilution in many mammalian and avian species including the dog. MLPH corresponds to the D locus in many domestic animals, and recessive alleles at this locus are frequently denoted with d. In this study, we investigated dilute coloured Chow Chows whose coat colour could not be explained by their genotype at the previously known MLPH:c.–22G>A variant. Whole genome sequencing of such a dilute Chow Chow revealed another variant in the MLPH gene: MLPH:c.705G>C. We propose to designate the corresponding mutant alleles at these two variants d¹ and d². We performed an association study in a cohort of 15 dilute and 28 non‐dilute Chow Chows. The dilute dogs were all either compound heterozygous d¹/d² or homozygous d²/d², whereas the non‐dilute dogs carried at least one wildtype allele D. The d² allele did not occur in 417 dogs from diverse other breeds. However, when we genotyped a Sloughi family, in which a dilute coloured puppy had been born out of non‐dilute parents, we again observed perfect co‐segregation of the newly discovered d² allele with coat colour dilution. Finally, we identified a blue Thai Ridgeback with the d¹/d² genotype. Thus, our data identify the MLPH:c.705G>C as a variant explaining a second canine dilution allele. Although relatively rare overall, this d² allele is segregating in at least three dog breeds, Chow Chows, Sloughis and Thai Ridgebacks.     

A candidate gene association study for nine economically important traits in Italian Holstein cattle

We genotyped 58 single nucleotide polymorphisms (SNPs) in 25 candidate genes in about 800 Italian Holstein sires. Fifty‐six (minor allele frequency >0.02) were used to evaluate their association with single traits: milk yield (MY), milk fat yield (FY), milk protein yield (PY), milk fat percentage (FP), milk protein percentage (PP), milk somatic cell count (MSCC); and complex indexes: longevity, fertility and productivity–functionality type (PFT), using deregressed proofs, after adjustment for familial relatedness. Thirty‐two SNPs were significantly associated (proportion of false positives <0.05) with different traits: 16 with MSCC, 15 with PY, 14 with MY, 12 with PFT, eight with longevity, eight with FY, eight with PP, five with FP and two with fertility. In particular, a SNP in the promoter region of the PRLR gene was associated with eight of nine traits. DGAT1 polymorphisms were highly associated with FP and FY. Casein gene markers were associated with several traits, confirming the role of the casein gene cluster in affecting milk yield, milk quality and health traits. Other SNPs in genes located on chromosome 6 were associated with PY, PP, PFT, MY (PPARGC1A) and MSCC (KIT). This latter association may suggest a biological link between the degree of piebaldism in Holstein and immunological functions affecting somatic cell count and mastitis resistance. Other significant SNPs were in the ACACA, CRH, CXCR1, FASN, GH1, LEP, LGB (also known as PAEP), MFGE8, SRC, TG, THRSP and TPH1 genes. These results provide information that can complement QTL mapping and genome‐wide association studies in Holstein.     

Increasing the accuracy of genomic prediction in pure‐bred Limousin beef cattle by including cross‐bred Limousin data and accounting for an F94L variant in MSTN

Explicitly fitting effects for major genes or QTL that account for a large percentage of variation in a whole genomic prediction model may increase prediction accuracy. This study compared approaches to account for a major effect of an F94L variant in the MSTN gene within the genomic prediction using bovine whole‐genomic SNP markers. Among the beef cattle breeds, Limousin have been known to have an F94L variant that is not present in Angus. The reference population in this study consisted of 3060 beef cattle including pure‐bred Limousin (PL), cross‐bred Limousin with Angus (LF) and pure‐bred Angus, genotyped using a BovineSNP50 BeadChip and directly for the MSTN‐F94L variant. We compared prediction accuracies in PL animals using the three datasets from only the PL population, admixed PL and LF (AL) or multibreed analysis using all of the PL, LF and Angus (MB) population according to four‐fold cross‐validation after K‐means clustering. The MSTN‐F94L variant was the most strongly associated with five traits (birth weight, calving ease direct, milk, weaning weight and yield grade) among the 13 measured traits in PL and AL populations. Fitting the MSTN‐F94L variant as a random effect, the genomic prediction accuracies for birth weight increased by 2.7% in PL, by 2.2% in AL and by 3.2% in MB. Prediction accuracies for five traits increased in the MB analysis. Fitting MSTN‐F94L as a fixed effect in PL, AL and MB analyses resulted in increased prediction accuracy in PL for eight traits. Prediction accuracies can be improved by including a causal variant in genomic evaluation compared with simply using whole‐genome SNP markers. Fitting the causal variant as a fixed effect along with markers fitted as random effects resulted in greater prediction accuracies for most traits. Causal variants should be genotyped along with SNP markers.     

Novel variants in the KIT and PAX3 genes in horses with white‐spotted coat colour phenotypes

Variants in the EDNRB, KIT, MITF, PAX3 and TRPM1 genes are known to cause white spotting phenotypes in horses, which can range from the common white markings up to completely white horses. In this study, we investigated these candidate genes in 169 horses with white spotting phenotypes not explained by the previously described variants. We identified a novel missense variant, PAX3:p.Pro32Arg, in Appaloosa horses with a splashed white phenotype in addition to their leopard complex spotting patterns. We also found three novel variants in the KIT gene. The splice site variant c.1346+1G>A occurred in a Swiss Warmblood horse with a pronounced depigmentation phenotype. The missense variant p.Tyr441Cys was present in several part‐bred Arabians with sabino‐like depigmentation phenotypes. Finally, we provide evidence suggesting that the common and widely distributed KIT:p.Arg682His variant has a very subtle white‐increasing effect, which is much less pronounced than the effect of the other described KIT variants. We termed the new KIT variants W18–W20 to provide a simple and unambiguous nomenclature for future genetic testing applications.     

Detection of genetic variants affecting cattle behaviour and their impact on milk production: a genome‐wide association study

Behaviour traits of cattle have been reported to affect important production traits, such as meat quality and milk performance as well as reproduction and health. Genetic predisposition is, together with environmental stimuli, undoubtedly involved in the development of behaviour phenotypes. Underlying molecular mechanisms affecting behaviour in general and behaviour and productions traits in particular still have to be studied in detail. Therefore, we performed a genome‐wide association study in an F₂ Charolais × German Holstein cross‐breed population to identify genetic variants that affect behaviour‐related traits assessed in an open‐field and novel‐object test and analysed their putative impact on milk performance. Of 37 201 tested single nucleotide polymorphism (SNPs), four showed a genome‐wide and 37 a chromosome‐wide significant association with behaviour traits assessed in both tests. Nine of the SNPs that were associated with behaviour traits likewise showed a nominal significant association with milk performance traits. On chromosomes 14 and 29, six SNPs were identified to be associated with exploratory behaviour and inactivity during the novel‐object test as well as with milk yield traits. Least squares means for behaviour and milk performance traits for these SNPs revealed that genotypes associated with higher inactivity and less exploratory behaviour promote higher milk yields. Whether these results are due to molecular mechanisms simultaneously affecting behaviour and milk performance or due to a behaviour predisposition, which causes indirect effects on milk performance by influencing individual reactivity, needs further investigation.     

Whole‐genome sequencing reveals a large deletion in the MITF gene in horses with white spotted coat colour and increased risk of deafness

White spotting phenotypes in horses are highly valued in some breeds. They are quite variable and may range from the common white markings up to completely white horses. EDNRB, KIT, MITF, PAX3 and TRPM1 represent known candidate genes for white spotting phenotypes in horses. For the present study, we investigated an American Paint Horse family segregating a phenotype involving white spotting and blue eyes. Six of eight horses with the white‐spotting phenotype were deaf. We obtained whole‐genome sequence data from an affected horse and specifically searched for structural variants in the known candidate genes. This analysis revealed a heterozygous ~63‐kb deletion spanning exons 6–9 of the MITF gene (chr16:21 503 211–21 566 617). We confirmed the breakpoints of the deletion by PCR and Sanger sequencing. PCR‐based genotyping revealed that all eight available affected horses from the family carried the deletion. The finding of an MITF variant fits well with the syndromic phenotype involving both depigmentation and an increased risk for deafness and corresponds to human Waardenburg syndrome type 2A. Our findings will enable more precise genetic testing for depigmentation phenotypes in horses.     

Genome‐wide association study for body weight in cattle populations from Siberia

Body weight is a complex trait in cattle associated with commonly used commercial breeding measurements related to growth. Although many quantitative trait loci (QTL) for body weight have been identified in cattle so far, searching for genetic determinants in different breeds or environments is promising. Therefore, we carried out a genome‐wide association study (GWAS) in two cattle populations from the Russian Federation (Siberian region) using the GGP HD150K array containing 139 376 single nucleotide polymorphism (SNP) markers. Association tests for 107 550 SNPs left after filtering revealed five statistically significant SNPs on BTA5, considering a false discovery rate of less than 0.05. The chromosomal region containing these five SNPs contains the CCND2 gene, which was previously associated with average daily weight gain and body mass index in US beef cattle populations and in humans respectively. Our study is the first GWAS for body weight in beef cattle populations from the Russian Federation. The results provided here suggest that, despite the existence of breed‐ and species‐specific QTL, the genetic architecture of body weight could be evolutionarily conserved in mammals.     

A novel KIT deletion variant in a German Riding Pony with white‐spotting coat colour phenotype

White spotting phenotypes in horses may be caused by developmental alterations impairing melanoblast differentiation, survival, migration and/or proliferation. Candidate genes for white‐spotting phenotypes in horses include EDNRB, KIT, MITF, PAX3 and TRPM1. We investigated a German Riding Pony with a sabino‐like phenotype involving extensive white spots on the body together with large white markings on the head and almost completely white legs. We obtained whole genome sequence data from this horse. The analysis revealed a heterozygous 1273‐bp deletion spanning parts of intron 2 and exon 3 of the equine KIT gene (Chr3: 79 579 925–79 581 197). We confirmed the breakpoints of the deletion by PCR and Sanger sequencing. Knowledge of the functional impact of similar KIT variants in horses and other species suggests that this deletion represents a plausible candidate causative variant for the white‐spotting phenotype. We propose the designation W28 for the mutant allele.     

Thymus vulgaris alleviates UVB irradiation induced skin damage via inhibition of MAPK/AP‐1 and activation of Nrf2‐ARE antioxidant system

Solar ultraviolet (UV) radiation‐induced reactive oxidative species is mainly responsible for the development of photoageing. Rosmarinic acid was one of the main bioactive components detected in Thymus vulgaris (TV) we extracted. In this study, UVB‐induced skin damages have been shown to be ameliorated by treatment with TV in hairless mice (HR‐1) skin, demonstrated by decreased matrix metalloproteinases (MMPs) and increased collagen production. However, the underlying molecular mechanism on which TV acted was unclear. We examined the photoprotective effects of TV against UVB and elucidated the molecular mechanism in normal human dermal fibroblasts. Thymus vulgaris remarkably prevented the UVB‐induced reactive oxygen species and lactate dehydrogenase. Dose‐dependent increase in glutathione, NAD(P)H: quinone oxidoreductase1 and heme oxygenase‐1, by TV was confirmed by increased nuclear accumulation of Nrf2. Furthermore, 5‐Methoxyindole‐2‐carboxylic acid was introduced as a specific inhibitor of dihydrolipoyl dehydrogenase (DLD). We demonstrated that Nrf2 expression was regulated by DLD, which was a tricarboxylic acid cycle‐associated protein that decreased after UVB exposure. Besides, TV significantly diminished UVB induced phosphorylation of mitogen activated protein kinases pathway, containing extracellular signal‐regulated kinase, Jun N‐terminal kinase and p38, which consequently reduced phosphorylated c‐fos and c‐jun. Our results suggest that TV is a potential botanical agent for use against UV radiation‐induced oxidative stress mediated skin damages.