Polymorphism of the Melanocortin 1 Receptor (MC1R) Gene and Its Role in Determining the Coat Colour of Central European Cattle Breeds

animals

Article Polymorphism of the Melanocortin 1 Receptor (MC1R) Gene and its Role in Determining the Coat Colour of Central European Cattle Breeds

Karolina Kasprzak-Filipek 1 , Wioletta Sawicka-Zugaj 1,*, Zygmunt Litwi ´nczuk 1, Witold Chabuz 1,Ruta¯ Šveistiene˙ 2 and Josef Bulla 3

1 Sub-Department of Cattle Breeding and Genetic Resources Conservation, Institute of Animal Breeding and Biodiversity Conservation, University of Life Sciences in Lublin, Akademicka 13, 20-950 Lublin, Poland; karolina.kasprzak-ﬁ[email protected] (K.K.-F.); [email protected] (Z.L.); [email protected] (W.C.) 2 Animal Science Institute, Lithuanian University of Health Sciences, A. Mickeviciaus 9, LT 44307 Kaunas, Lithuania; [email protected] 3 Department of Animal Physiology, Slovak University of Agriculture in Nitra, A. Hlinku 2, 94976 Nitra, Nitriansky Kraj, Slovakia; [email protected] * Correspondence: [email protected]; Tel.: +48-81-445-60-92

Received: 17 September 2020; Accepted: 12 October 2020; Published: 14 October 2020

Simple Summary: Animal coat colour has been the subject of numerous studies for many years. While most phenotypic features of animals result from the interaction of genetic and environmental factors, coat colour is considered to be almost exclusively genetically determined. Differences in coat colour underscore the distinct character of a given breed or group of animals, testify to its uniqueness, and sometimes serve as selection criteria. Observations of changes in cattle coat colour are an important source of information used to track domestication processes and discover how animals were selected for breeding. One of the genes responsible for variation in coat colour is the melanocortin receptor (MC1R) gene locus, which controls the production of black and red pigments that determine basic colours. Other interacting genes also influence cattle phenotypes. In view of the complexity of the genetic factors influencing cattle coat colour, this study investigated the genetic basis of different coat colours of Central European cattle breeds.

Abstract: There are many genes responsible for the appearance of diﬀerent coat colours, among which the melanocortin 1 receptor gene (MC1R) plays an important role. The aim of the study was to characterize genetic variation in Central European cattle breeds based on polymorphism of the MC1R gene and factors determining their coat colour. The study was conducted on 290 individuals of the following breeds: Polish White-Backed (PW), Lithuanian White-Backed (LW), Polish Red (PR), Lithuanian Red (LR), Carpathian Brown (CB), Ukrainian Grey (UG), and Slovak Pinzgau (SP). Polymorphism at the MC1R gene locus was analysed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using two restriction enzymes: Cfr10I and SsiI. The proportions of alleles and genotypes in the MC1R locus indicates a strong relationship between polymorphism and the coat colour of cattle: The ED allele proved to be characteristic for the breeds with a white-backed coat (PW and LW), while the dominant allele in the red breeds (PR and LR) was E+. It is noteworthy that coat colour in the SP population was determined only by the recessive e allele, which resulted in the formation of a separate clade in the phylogenetic tree.

Keywords: MC1R; coat colour determination; Central European cattle breeds

Animals 2020, 10, 1878; doi:10.3390/ani10101878 www.mdpi.com/journal/animals Animals 2020, 10, 1878 2 of 11

1. Introduction A characteristic feature of most phenotypic traits of animals is that they are the result of the interaction of genetic and environmental factors. The coat colour of mammals depends primarily on the proportions of individual pigments: Pheomelanin (red pigment) and eumelanin (determining brown or black colour) [1,2]. The proportion of one of the two pigments is limited by tyrosinase, an enzyme that limits their synthesis. A low tyrosinase level leads to the production of pheomelanin, while increased tyrosinase production results in the production of eumelanin [3]. Two loci are primarily responsible for regulating the level of pigments produced by melanocytes: E (Extension) and A (Agouti) [4]. These loci exert an epistatic eﬀect in various mammalian species. Alleles dominant at the E locus determine the colour black (eumelanin production), while recessive alleles, due to pheomelanin synthesis, cause the appearance of a colour from red to pale yellow. Mutations occurring at the Agouti locus have the opposite eﬀect: Dominant alleles determine pheomelanin production, while recessive ones are usually associated with eumelanin synthesis and black colour [5]. The E locus encodes the melanocortin 1 receptor (MC1R), which binds to melanocyte-stimulating hormone (α-MSH), which induces eumelanin synthesis [6,7]. The A locus, which encodes the protein ASIP (agouti-signalling protein), by acting antagonistically to the E locus causes blockage of α-MSH receptor interaction, resulting in the production of pheomelanin instead of eumelanin [8,9]. Mutation at the MC1R gene locus has been the subject of many studies on various species of mammals, such as humans [10], pigs [11], felids [12], mice [13], sheep [14], dogs [15], foxes [16], bears [17], horses [18] and cattle [3,19,20], where the occurrence of a functional mutation has been associated with black (or dark) coat colour, while a lack of mutation at the MC1R locus has resulted in red, yellow or white colours. The MC1R gene in cattle is located on chromosome 18 [3]. According to research in cattle conducted by Adalsteinsson et al. [21], the colours black, brown and red are determined by the occurrence of alleles from the E and A loci. Three alleles have been distinguished at the E locus in order to clarify the mechanism of inheritance of coat colour in cattle: The dominant black ED allele, the recessive red e allele, and the E+ allele, which enables phenotypic expression of the A locus alleles. Individuals with the E+ allele probably have the A+ allele responsible for the colour brown or the recessive allele a determining the colour black at the A locus. The order of allele dominance is usually described as ED > E+ > e. Analyses carried out by Klungland et al. [3] have shown that information obtained by sequencing or other methods e.g., restriction fragment length polymorphism (RFLP) can be used to identify three alleles at the MC1R gene locus. The aim of the study was to characterise genetic variation in Central European cattle breeds based on polymorphism of the MC1R gene and factors determining their coat colour.

2. Materials and Methods

2.1. Study Area, Data Collection and Characteristics of Populations Selected for Research The animals came from private farms located in Poland, Lithuania, Ukraine and Slovakia. The study was conducted on 290 individuals belonging to seven Central European cattle breeds kept on 57 farms. The animals were not related, which was veriﬁed on the basis of breeding documentation and pedigree data (to two generations back). Hair bulbs were used as biological material. Polish White-Backed cattle (PW) were raised in eastern Poland, in a temperate climate. The animals had black sides and a continuous stripe running from the muzzle, which was dark, along the entire body (Figure1). A total of 50 animals from 10 farms were selected for the study. Animals 2020, 10, 1878 3 of 11 Animals 2020, 10, x 3 of 11 Animals 2020, 10, x 3 of 11

Figure 1. Polish White-Backed (PW) female. Figure 1. Polish White-Backed (PW) female. Lithuanian White-Backed cattle (LW) were raised in central Lithuania, in a temperate climate. Lithuanian White-Backed cattle (LW) were raised in central Lithuania, in a temperate climate. The animalsanimals hadhad black black sides sides and and a continuousa continuous stripe stripe running running from from the muzzle,the muzzle, which which was dark, was alongdark, The animals had black sides and a continuous stripe running from the muzzle, which was dark, thealong entire the entire body (Figurebody (Figure2). A total 2). A of total 50 animals of 50 an fromimals 10 from farms 10 werefarms selected were selected for the for study. the study. along the entire body (Figure 2). A total of 50 animals from 10 farms were selected for the study.

Figure 2. Lithuanian White-Backed (LW) females. Figure 2. LithuanianLithuanian White-Backed White-Backed (LW) females. Polish Red cattle (PR) were raised in Southern Poland, in a temperate climate. The animals had Polish Red cattle (PR) were raised in Southern Poland, in a temperate climate. The animals had a solid red coat and a dark muzzle (Figure 3). A total of 50 animals from 10 farms were selected for a solid red coat and a dark muzzle (Figure 33).). AA totaltotal ofof 5050 animalsanimals fromfrom 1010 farmsfarms werewere selectedselected forfor the study. the study.

Figure 3. Polish Red (PR) female with calf. Figure 3. PolishPolish Red (PR) female with calf. Lithuanian Red cattle (LR) were present in central Lithuania, in a temperate climate. The Lithuanian RedRed cattlecattle (LR) (LR) were were present present in central in central Lithuania, Lithuania, in a temperate in a temperate climate. climate. The animals The animals had a solid red coat and a dark muzzle (Figure 4). A total of 50 animals from 10 farms were animalshad a solid had red a solid coat andred acoat dark and muzzle a dark (Figure muzzle4). (Figure A total of4). 50 A animalstotal of from50 animals 10 farms from were 10 selectedfarms were for selected for the study. selectedthe study. for the study. Animals 2020, 10, 1878 4 of 11 Animals 2020, 10, x 4 of 11 AnimalsAnimals 20202020,, 1010,, xx 44 ofof 1111

Figure 4. LithuanianLithuanian Red Red (LR) female. FigureFigure 4.4. LithuanianLithuanian RedRed (LR)(LR) female.female. Carpathian Brown Brown cattle (CB) were raised in Western Ukraine (Zakarpattia), in a transitional CarpathianCarpathian BrownBrown cattlecattle (CB)(CB) werewere raisedraised inin WesternWestern UkraineUkraine (Zakarpattia),(Zakarpattia), inin aa transitionaltransitional temperatetemperate climateclimate (inﬂuenced(influenced by by continental continental climate). climate). The The animals animals had had a solid a solid coat coat colour, colour, from from dark temperatetemperate climateclimate (influenced(influenced byby continentalcontinental climclimate).ate). TheThe animalsanimals hadhad aa solidsolid coatcoat colour,colour, fromfrom darkbrown brown to light to grey,light withgrey, a with characteristic a characteristic white ringwhite around ring around the nostrils the nostrils (Figure 5(Figure). A total 5). of A 22 total animals of 22 darkdark brownbrown toto lightlight grey,grey, withwith aa characteristiccharacteristic whitewhite ringring aroundaround thethe nostrilsnostrils (Figure(Figure 5).5). AA totaltotal ofof 2222 animalsfrom 5 farms from were5 farms selected were selected for the study. for the study. animalsanimals fromfrom 55 farmsfarms werewere selectedselected forfor thethe study.study.

Figure 5. Carpathian Brown (CB) female. FigureFigure 5. 5. CarpathianCarpathian BrownBrown (CB)(CB) female.female. Ukrainian Grey cattle (UG) were raised in Eastern Ukraine (steppe region), in a continental UkrainianUkrainian GreyGreyGrey cattle cattlecattle (UG) (UG)(UG) were werewere raised raisedraised in Eastern inin EasternEastern Ukraine UkraineUkraine (steppe (steppe(steppe region), region),region), in a continental inin aa continentalcontinental climate climate (hot summer and freezing winter). The animals had a solid grey or light grey coat colour, climate(hotclimate summer (hot(hot summersummer and freezing andand winter).freezingfreezing Thewinter).winter). animals TheThe had animalsanimals a solid hadhad grey aa solid orsolid light greygrey grey oror coat lightlight colour, greygrey coat oftencoat colour,colour, with a often with a darker shade on the neck, chest or abdomen (Figure 6). A total of 18 animals from five oftendarkeroften withwith shade aa darkerdarker on the shadeshade neck, onon chest thethe or neck,neck, abdomen chestchest oror (Figure abdomenabdomen6). A (Figure(Figure total of 6).6). 18 AA animals totaltotal ofof from 1818 animalsanimals ﬁve farms fromfrom were fivefive farms were selected for the study. farmsselectedfarms werewere for selectedselected the study. forfor thethe study.study.

Figure 6. Ukrainian Grey (UG) male. FigureFigure 6. 6. UkrainianUkrainian GreyGrey (UG)(UG) male.male. Slovak Pinzgau cattle (SP) were raised in Northern Slovakia, in a cool temperate climate. The SlovakSlovak PinzgauPinzgau Pinzgau cattlecattle cattle (SP)(SP) (SP) werewere were raisedraised raised inin inNorthernNorthern Northern Slovakia,Slovakia, Slovakia, inin aa in coolcool a cool temperatetemperate temperate climate.climate. climate. TheThe animals had a red coat with a white stripe running from the withers and crossing the tail, udder and animalsTheanimals animals hadhad a hada redred a coatcoat red coatwithwith with aa whitewhite a white stripestripe stripe runningrunning running fromfrom from thethe witherswithers the withers andand crossing andcrossing crossing thethe tail,tail, the udderudder tail, udder andand underbelly, and a solid red head (Figure 7). A total of 50 animals from 7 farms were selected for the underbelly,andunderbelly, underbelly, andand andaa solidsolid a solid redred headhead red head (Figure(Figure (Figure 7).7). AA7). totaltotal A total ofof 5050 of animalsanimals 50 animals fromfrom from 77 farmsfarms 7 farms werewere were selectedselected selected forfor forthethe study. study.thestudy. study. Animals 2020, 10, 1878 5 of 11 Animals 2020, 10, x 5 of 11

Figure 7. Slovak Pinzgau (SP) female.

2.2. Molecular Molecular Identification Identification The genomic DNA of cattle was isolated using a ready-made commercial kit for nucleic acid isolation from biological traces (Sherlock AX AX A&A Biotechnology, Gdynia, Poland) according to the procedure provided by thethe manufacturer.manufacturer. Polymera Polymerasese chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to analyse polymorphisms at the MC1R locus in the seven Central European cattle breeds. Primers given given by by Klungland Klungland et etal. al. [3] [3we] werere used used to assess to assess polymorphism polymorphism at the at MC1R the MC1R gene locus.gene locus.The first The primer firstprimer pair was pair 5 was′-GTGCCTGGAGGTGTCCATC-3 50-GTGCCTGGAGGTGTCCATC-3′ (forward0 (forward primer) primer) and 5and′-GAAGTTCTTGAAGATGCAGCC-3 50-GAAGTTCTTGAAGATGCAGCC-3′ (reverse0 (reverse primer), primer), and andthe thesecond second primer primer pair pair was 5′0-CAAGAACCGCAACCTGCACT-3-CAAGAACCGCAACCTGCACT-3′ 0 (forward(forward primer) andand 550-GCCTGGGTGGCCAGGACA-3′-GCCTGGGTGGCCAGGACA-30′ (reverse primer). PCR-RFLP was used to amplify the MC1R gene fragment. The amplifiedamplified fragments were digested with Thermo Scientific Scientific FastDigest™ FastDigest™ (Thermo Fisher Scientific,Scientific, Waltham,Waltham, MA, MA, USA) USA) restriction restriction enzymes enzymesCfr10I Cfr10Iand SsiIand. TheSsiI. digestion The digestion reaction reaction was carried was carriedout at 37 out◦C. at 37 °C. The fragmentsfragments obtained obtained from from the PCR-RFLPthe PCR-RFLP reaction reaction were subjected were subjected to electrophoretic to electrophoretic separation separationon a 2% agarose on a 2% gel agarose stained gel with stained 0.01% with ethidium 0.01% bromideethidium (EtBr). bromide Molecular (EtBr). Molecular size markers size (Thermomarkers (ThermoScientific GeneRulerScientific GeneRuler 100bp Plus 100bp DNA, Plus Thermo DNA, Fisher Thermo Scientific) Fisher were Scientific) used to trackwere theused electrophoresis to track the processelectrophoresis and assess process the length and assess of the the fragments. length of the fragments.

2.3. Statistical Analysis 2.3. Statistical Analysis To visualize the genetic diversity of the analysedanalysed cattle breeds, the results were subjected to statistical analysis in POPGENE v. 3.2. statistical software. The The following following indicators indicators were were estimated: estimated: Frequency of alleles and genotypes, observed heterozygosity (HO) and genetic distances according Frequency of alleles and genotypes, observed heterozygosity (HO) and genetic distances according to Neito Nei [22]. [22 The]. The obtained obtained values values of ofgenetic genetic distances distances were were used used to to create create a a phylogenetic phylogenetic tree using UPGMA (unweighted pair group method with arithmetic mean)mean) method.method. 3. Results 3. Results Identiﬁcation of genotypes at the MC1R locus was carried out in two stages. Restriction digestion Identification of genotypes at the MC1R locus was carried out in two stages. Restriction of the PCR product (739 bp) with the Cfr10I enzyme resulted in genetic variants ED/e and E+/e, where digestion of the PCR product (739 bp) with the Cfr10I enzyme resulted in genetic variants ED/e and the lengths of the fragments were 739 bp, 531 bp and 208 bp; ee with a length of 739 bp; and ED/ED, E+/e, where the lengths of the fragments were 739 bp, 531 bp and 208 bp; ee with a length of 739 bp; ED/E+ and E+/E+, with fragments of 531 bp and 208 bp (Figure8). and ED/ED, ED/E+ and E+/E+, with fragments of 531 bp and 208 bp (Figure 8). In the second stage of the analysis, the ampliﬁcation resulted in a 130 bp product. Following restriction digestion of the PCR product with the SsiI enzyme, the following genotypes were obtained: ED/E+ and ED/e, with fragments of 130 bp, 97 bp and 33 bp; and E+/E+,E+/e and e/e, where cleavage of the products did not take place (Figure9).

Figure 8. Electrophoretic separation of DNA fragments at the melanocortin 1 receptor (MC1R) gene locus following digestion with restriction enzyme Cfr10I: lane 1: DNA ladder, lane 2: PCR (polymerase chain reaction product—739 bp), Identyfied genotypes: lanes 8–10,12,13,16,17,19,20: Animals 2020, 10, x 5 of 11

Figure 7. Slovak Pinzgau (SP) female.

2.2. Molecular Identification The genomic DNA of cattle was isolated using a ready-made commercial kit for nucleic acid isolation from biological traces (Sherlock AX A&A Biotechnology, Gdynia, Poland) according to the procedure provided by the manufacturer. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to analyse polymorphisms at the MC1R locus in the seven Central European cattle breeds. Primers given by Klungland et al. [3] were used to assess polymorphism at the MC1R gene locus. The first primer pair was 5′-GTGCCTGGAGGTGTCCATC-3′ (forward primer) and 5′-GAAGTTCTTGAAGATGCAGCC-3′ (reverse primer), and the second primer pair was 5′-CAAGAACCGCAACCTGCACT-3′ (forward primer) and 5′-GCCTGGGTGGCCAGGACA-3′ (reverse primer). PCR-RFLP was used to amplify the MC1R gene fragment. The amplified fragments were digested with Thermo Scientific FastDigest™ (Thermo Fisher Scientific, Waltham, MA, USA) restriction enzymes Cfr10I and SsiI. The digestion reaction was carried out at 37 °C. The fragments obtained from the PCR-RFLP reaction were subjected to electrophoretic separation on a 2% agarose gel stained with 0.01% ethidium bromide (EtBr). Molecular size markers (Thermo Scientific GeneRuler 100bp Plus DNA, Thermo Fisher Scientific) were used to track the electrophoresis process and assess the length of the fragments.

2.3. Statistical Analysis To visualize the genetic diversity of the analysed cattle breeds, the results were subjected to statistical analysis in POPGENE v. 3.2. statistical software. The following indicators were estimated: Frequency of alleles and genotypes, observed heterozygosity (HO) and genetic distances according to Nei [22]. The obtained values of genetic distances were used to create a phylogenetic tree using UPGMA (unweighted pair group method with arithmetic mean) method.

3. Results Identification of genotypes at the MC1R locus was carried out in two stages. Restriction digestion of the PCR product (739 bp) with the Cfr10I enzyme resulted in genetic variants ED/e and EAnimals+/e, where2020, 10 the, 1878 lengths of the fragments were 739 bp, 531 bp and 208 bp; ee with a length of 7396 ofbp; 11 and ED/ED, ED/E+ and E+/E+, with fragments of 531 bp and 208 bp (Figure 8).

Animals 2020, 10, x 6 of 11

ED/e, E+/e (739 bp, 531 bp, 208 bp), lanes 3,5: ee (739 bp), lanes 4,6,7,11,14,15,18: ED/ED, ED/E+, E+/E+ (531 bp, 208 bp).

FigureIn the 8.second Electrophoretic stage of separation the analysis, of DNA the amplificationfragme fragmentsnts at the resulted melanocortin in a 130 1 receptor bp product. ( MC1R )Following gene restrictionlocus following followingdigestion digestion digestionof the with PCRwith restriction productrestriction enzyme with enzyme Cfr10Ithe SsiI:Cfr10I lane enzyme, 1:: lane DNA 1: ladder, theDNA following lane ladder, 2: PCR lanegenotypes (polymerase 2: PCR were D + D + + + obtained:(polymerasechain E reaction/E and chain product—739 E reaction/e, with bp), prodfragments Identyﬁeduct—739 of genotypes: bp),130 bp,Identyfied 97 lanes bp and 8–10,12,13,16,17,19,20:genotype 33 bp;s: lanesand E8–10,12,13,16,17,19,20:/E ED, E/e,/e E and+/e (739 e/e, bp, where cleavage531 bp,of the 208 bp),products lanes 3,5:did eenot (739 take bp), place lanes (Figure 4,6,7,11,14,15,18: 9). ED/ED, ED/E+,E+/E+ (531 bp, 208 bp).

Figure 9. Electrophoretic separation of DNA fragme fragmentsnts at the melanocortin 1 receptor ( MC1R) gene locus following digestion with restriction enzyme SsiI: lane 1: DNA DNA ladder, ladder, lane 2: PCR (polymerase chain reaction product—130product—130 bp), bp), lanes lanes 3–9,12,16,17: 3–9,12,16,17: E DE/DE/E+,E+, EDD//ee (130(130 bp, bp, 97 97 bp, bp, 33 33 bp), bp), lane lane 10,11,13–15, 10,11,13– 15,18–20: 18–20: E+ /E+/E,E+,+ E/e,+/e, e /e/ee (130 (130 bp). bp).

D D + + The analyses performed at thethe MC1RMC1R genegene locuslocus identifiedidentified fivefive genotypes, genotypes, E ED/e,/e, EED//EE +,,E E+/e,/e, + + D E+/E/E+ andand e/e, e/e, determined determined by by the the occurrence occurrence of of three three alleles: The The dominant E D allele,allele, the the recessive recessive e + allele, and the wild type E+ allele.allele. The The tables tables present present the the percenta percentagesges of of individuals individuals in in each each breed breed for which the genotypes were identified identified at the MC1R locus (Table (Table1 1)) andand thethe frequenciesfrequencies ofof allelesalleles atat thethe MC1R locus for the breeds (Table2 2).).

Table 1. Observed heterozygosity (Ho) values and percentage shares of individuals with identiﬁed Table 1. Observed heterozygosity (Ho) values and percentage shares of individuals with identified genotype at the melanocortin 1 receptor gene (MC1R) locus (%). genotype at the melanocortin 1 receptor gene (MC1R) locus (%).

Heterozygosity Genotype in MC1R Locus Breed Heterozygosity Genotype in MC1R Locus Breed Observed (Ho) D +/ D + D D + + Observed (Ho) E E/eD/e E Ee+/e E /ED/E+ E /EED/EDE /E E+/E+ e/e e/e LW LW1.00 1.00 84%84% 16%16% PW PW0.98 0.98 28%28% 6%6% 64%64% 2% 2% PR PR0.20 0.20 20%20% 80% 80% LR 0.12 12% 88% LR 0.12 12% 88% CB 1.00 100% CB UG1.00 1.00 100%100% UG SP1.00 0.00 100% 100% SPNotation: LW—Lithuanian0.00 White-Backed, PW—Polish White-Backed, PR—Polish Red, LR—Lithuanian Red,100% Notation:CB—Carpathian LW—Lithuanian Brown, UG—Ukrainian White-Backed, Grey, SP—Slovak PW—Polish Pinzgau. White-Backed, PR—Polish Red, LR—Lithuanian Red, CB—Carpathian Brown, UG—Ukrainian Grey, SP—Slovak Pinzgau. Table 2. Frequency of alleles at the melanocortin receptor gene (MC1R) locus for the breeds tested. Table 2. Frequency of alleles at the melanocortin receptor gene (MC1R) locus for the breeds tested. Allele Frequency in Breeds Overall Alleles LW PWAllele PR Frequency LR in Breeds CB UG SP FrequencyOverall Alleles ED LW0.50 PW 0.46 PR - LR -CB -UG -SP -Frequency 0.17 EDE + 0.500.08 0.46 0.37 - 0.90 - 0.94 - 0.50 - 0.50- - 0.460.17 E+ e 0.080.42 0.37 0.17 0.90 0.10 0.94 0.06 0.50 0.50 0.50 0.50 - 1.00 0.370.46 Notation:e LW—Lithuanian0.42 0.17 White-Backed, 0.10 PW—Polish0.06 White-Backed,0.50 0.50 PR—Polish 1.00 Red, LR—Lithuanian0.37 Red, CB—Carpathian Brown, UG—Ukrainian Grey, SP—Slovak Pinzgau. Notation: LW—Lithuanian White-Backed, PW—Polish White-Backed, PR—Polish Red, LR—Lithuanian Red, CB—Carpathian Brown, UG—Ukrainian Grey, SP—Slovak Pinzgau. Coat colour in cattle is determined by a number of genes, among which an important role is attributedCoat colour to the MC1Rin cattlegene. is determined Our research by a shows number that of assessment genes, among of polymorphism which an important at the MC1Rrole is attributed to the MC1R gene. Our research shows that assessment of polymorphism at the MC1R gene loci makes it possible to partially explain determination of coat colour, and at the same time is an important source of information in the analysis of the genetic diversity of Central European cattle breeds. The phylogenetic tree (Figure 10), based on estimated values of distances and genetic Animals 2020, 10, 1878 7 of 11

Animals 2020, 10, x 7 of 11 gene loci makes it possible to partially explain determination of coat colour, and at the same time issimilarities an important according source ofto informationNei [22], shows in the that analysis the Central of the European genetic diversity cattle breeds of Central included European in the cattlestudy breeds. formed The three phylogenetic distinct clades, tree (Figure according 10), basedto their on estimatedcoat colour values characteristics. of distances The and first genetic clade similaritiesincluded both according breeds to representing Nei [22], shows the thatWhite-Backed the Central coat European type (with cattle black breeds sides), included i.e., inPW the and study LW. formedWhite-Backed three distinct cattle clades, are derived according from to theirprimitive coat colourcattle characteristics.originally present The in first north-western clade included Europe. both breedsCattle representingwith this coat the colour White-Backed became coatwidespread type (with in blackthe Baltic sides), Sea i.e., region PW and together LW. White-Backed with German cattlesettlers are derivedduring the from Middle primitive Ages cattle [23,24]. originally The presentsecond inclade north-western comprised Europe.two groups Cattle of with single-colour this coat colourbreeds—red-coated became widespread breeds in the(PR Balticand SeaLR) regionand grey together and withbrown German cattle settlers (UG and during CB). the These Middle are AgesCentral-Eastern [23,24]. The European second clade breeds. comprised PR and LR two are groups derived of single-colourfrom small, wild breeds—red-coated short-horned cattle breeds living (PRin Eastern and LR) Europe. and grey The and spread brown of cattlethese (UGanimals and is CB). associated These arewith Central-Eastern the migration of European Slavic peoples breeds. in PRthe and early LR are16th derived century from [25]. small, The wildUG and short-horned CB breeds, cattle which living were in Easternin one Europe.group, had The spreada common of theselink—Podolian animals is associated cattle, which with were themigration used in their of Slavic creation. peoples UG inis a the breed early derived 16th century from Podolian [25]. The UGGrey andSteppe CB breeds, cattle kept which in wereRomania, in one Hungary, group, had Italy a commonand Bulgaria link—Podolian in the 19th cattle,century which [26]. wereThe CB used breed in theirwas creation.probably UG created is a breedthrough derived many from years Podolian of cross-breeding Grey Steppe of cattlelocal brown kept in breeds Romania, with Hungary, imported ItalyAlpine and and Bulgaria Podolian in the breeds 19th century [27]. SP [26 cattle]. The formed CB breed a completely was probably separate created cl throughade, which many reflects years ofthe cross-breedingdistinct differences of local in brown both the breeds colour with and imported location Alpine of the and breed Podolian relative breeds to the [27 others.]. SP cattle This formedbreed is aincluded completely among separate Alpine clade, (Central which European) reflects the cattle distinct [28] di. Accordingfferences in to both the estimated the colour genetic and location distances, of thethe breed highest relative genetic to similarity the others. was This between breed is the included CB and amongUG breeds, Alpine while (Central the highest European) genetic cattle diversity [28]. Accordingestimated to on the the estimated basis of po geneticlymorphism distances, analysis the highest at the MC1R genetic gene similarity locus was was found between between the CB the and LR UGand breeds, SP breeds. while the highest genetic diversity estimated on the basis of polymorphism analysis at the MC1R gene locus was found between the LR and SP breeds.

Figure 10. Dendrogram of genetic similarity for seven Central European cattle breeds according to the UPGMAFigure 10. (unweighted Dendrogram pair of group genetic method similarity with arithmeticfor seven Central mean) method,European based cattle on breeds genetic according similarity to calculatedthe UPGMA according (unweighted to Nei’s pair formula group [22 method]. Notation: with LW—Lithuanian arithmetic mean) White-Backed, method, based PW—Polish on genetic White-Backed,similarity calculated PR—Polish according Red, LR—Lithuanian to Nei’s form Red,ula [22]. CB—Carpathian Notation: LW—Lithuanian Brown, UG—Ukrainian White-Backed, Grey, SP—SlovakPW—Polish Pinzgau. White-Backed, PR—Polish Red, LR—Lithuanian Red, CB—Carpathian Brown, UG—Ukrainian Grey, SP—Slovak Pinzgau. 4. Discussion 4. DiscussionThe dominant ED allele was identified in the PW and LW breeds. Given the characteristic black and white colour of the White-Backed individuals included in the study, the results obtained are consistent The dominant ED allele was identified in the PW and LW breeds. Given the characteristic black D withand those white reported colour of by the Russo White-Backed et al. [29], who individual identifieds included the E allelein the in study, the Italian the results Holstein–Friesian obtained are and Black Pied Valdostana breeds; by Rouzaud et al. [20], who found the ED allele to be characteristic consistent with those reported by Russo et al. [29], who identified the ED allele in the Italian of the French Holstein–Friesian breed; and by other authors [19,30–32]. Holstein–Friesian and Black Pied Valdostana breeds; by Rouzaud et al. [20], who found the ED allele + to beThe characteristic wild type E ofallele the French was present Holstein–F withriesian varying breed; frequency and by in other six analysed authors breeds,[19,30–32]. but was not identified in the SP breed (Table2). Importantly, individuals belonging to di fferent Central European The wild type E+ allele was present with varying frequency in six analysed breeds, but was not cattleidentified breeds, in and the thus SP dibreedffering (Table in coat 2). colour Importantly, (from the light-greyindividuals UG belonging to the red to PR different and LR breeds), Central + wereEuropean found tocattle have breeds, the E andallele. thus According differing to in Klungland coat colour et (from al. [3], the the light-grey wild type UG receptor to the encoded red PR byand the E+ allele is probably inhibited, so that the dominant A locus inhibits the MC1R locus effect, resulting LR breeds), were found to have the E+ allele. According to Klungland et al. [3], the wild type receptor in different coat colours in cattle. Similar results have been reported by other authors [20,29,33], encoded by the E+ allele is probably inhibited, so that the dominant A locus inhibits the MC1R locus effect, resulting in different coat colours in cattle. Similar results have been reported by other authors [20,29,33], who found that the E+ allele determined differences in coat colour. Rouzaud et al. [20] identified the E+ allele in individuals of the Gascon breed with a grey coat, in Normande cattle with a Animals 2020, 10, 1878 8 of 11 who found that the E+ allele determined differences in coat colour. Rouzaud et al. [20] identified the E+ allele in individuals of the Gascon breed with a grey coat, in Normande cattle with a brown and white coat, and in brown Aubrac individuals. A study by Lee et al. [33] analysing brown, yellow-brown, and black individuals of Korean and Japanese breeds, also identified individuals with the wild type E+ allele. Similarly, in a study by Russo et al. [29], the E+ allele was identified among specimens of numerous breeds with a variety of coat colours: Modenese, Jersey, Simmental, Grigio Alpina, Piedmontese, Chianina, Romagnola, Marchigiana, Swedish Red and White, and Danish Red. On the other hand, Brenig et al. [34] found that individuals with the E+E+ genotype at the MC1R locus had a red coat colour. The occurrence of the e allele was recorded among individuals of all breeds tested, but it is significant that all SP individuals had the e/e genotype at the MC1R locus. In the study by Russo et al. [29], of the many European (mainly Italian) breeds analysed, only Reggiana individuals had the e/e genotype. Rouzaud et al. [20] found that the coats of individuals with e/e genotypes were very light (Blonde d’Aquitaine), cream-and-white (Charolais), red (Limousin), and dark mahogany (Salers). According to the assumptions of Klungland et al. [3], all individuals with e/e genotypes have a red coat, whereas the results of the authors’ analyses and our own research may indicate a modifying effect of various genes, including the A locus (agouti), on the expression of the MC1R gene, thereby causing varied coat colour in different breeds of cattle. Research by Putra et al. [35] has demonstrated that the environmental conditions in which animals are raised may also have a modifying effect on coat colour, and indirectly on the proportions of alleles at the MC1R gene locus. The estimated allele frequencies among Indonesian Pesisir cattle were 0 for ED, 0.93 for E+ and 0.07 for e. Two genotypes were identified among the individuals studied: E+E+ and E+e. The genotypes EDED,EDE+,EDe and ee were not observed. The proportions of the alleles obtained by the authors may be the result of a selective environmental effect, with the light coat of the Indonesian Pesisir cattle (determined by the E+ and e alleles at the MC1R locus) enabling survival in tropical conditions. The estimated frequency of alleles and genotypes at the MC1R locus and the observed variations in coat colour in the Central European cattle breeds may also be due to their geographic distribution. In a study by Niemi et al. [36], which assessed polymorphism at the MC1R locus in native Scandinavian breeds, the phenotypes were strongly influenced by the environment of the region where the animals were kept. High proportions of heterozygotes (from 26% to 57%) were observed among individuals of the native Scandinavian breeds, with both uniform and multi-coloured coats. In contrast, the level of heterozygosity at the MC1R gene locus in Western, Central and Southern European breeds was much lower (from 0% to 16%). In our research, the average percentage of heterozygous individuals among all analysed Central European cattle breeds was high, amounting to 53%. The high heterozygosity levels at the MC1R gene locus for the PW, LW, UG and CB breeds are likely due to the population bottleneck effect. Previous studies have shown that the current structure of these cattle populations is due to a sudden decline in the number of individuals and its subsequent restoration from the group of surviving individuals [37,38]. According to Mei et al. [39], analysis of polymorphism at the loci of various genes associated with milk traits, meat quality or coat colour (including the MC1R gene locus) makes it possible to track domestication processes and identify the characteristics of a given breed. The authors’ research on individuals of Chinese cattle breeds offers a new view of evolutionary history and the features of domestication of Chinese cattle. Ludwig et al. [40] also state that coat colour is a feature that can be used to track the domestication processes. Differences in coat colour underscore their distinct character and testify to the uniqueness of a given breed or group of animals, sometimes serving as a selection criterion [41]. In addition, a comparative analysis by Niemi et al. [36] of ancient DNA of cattle from Finland and contemporary Scandinavian cattle showed that the frequency of MC1R alleles exhibits changes over time that are similar to those found in studies of mitochondrial DNA and Y chromosome haplotypes. Animals 2020, 10, 1878 9 of 11

At the locus of the MC1R gene as well as the other two markers there was a signiﬁcant change in genotypes in Scandinavian cattle from the late Iron Age to the Middle Ages, followed by a period of slower change, continuing up to the present day.

5. Conclusions The results of analyses of polymorphism at the MC1R gene locus contribute to knowledge of the genetic variation between Central European cattle breeds, their phylogenetic relationships, and the characteristics of a given breed. The proportions of alleles and genotypes at the MC1R locus indicate a strong relationship between polymorphism and the coat colour of cattle. The ED allele was characteristic of the white-backed breeds (PW—0.46 and LW—0.50) but was not present in the other breeds analysed. In the PR and LR breeds, the wild-type allele E+ had the largest share (0.90 and 0.94, respectively). The alleles were evenly distributed in the CB and UG breeds, and all individuals were heterozygous (E+/e). The e allele occurred in all breeds, with the homozygous e/e genotype found only in the SP breed (100% of the studied population). Given the complexity of the genetic mechanisms responsible for the appearance of varied coat colours, such as the characteristic coat of the White-Backed breeds and the SP breed or the uniform coat colours found in the red breeds and the CB and UG breeds, research should be expanded to include analysis of polymorphism at other loci associated with coat colour in cattle.

Author Contributions: Conceptualization, K.K.-F., Z.L., W.S.-Z. and W.C.; methodology, K.K.-F., Z.L., W.S.-Z, W.C., R.Š. and J.B.; validation, K.K.-F., Z.L., W.S.-Z, W.C., R.Š. and J.B.; formal analysis, Z.L., R.Š. and J.B.; investigation, K.K.-F.; resources, W.C.; data curation, K.K.-F.; writing—original draft preparation, K.K.-F.; writing—review and editing, W.S.-Z.; visualization, K.K.-F., Z.L., W.S.-Z.; supervision, K.K.-F., Z.L., W.S.-Z. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Conﬂicts of Interest: The authors declare no conﬂict of interest.

References

1. Hearing, V.J.; Tsukamoto, K. Enzymatic control of pigmentation in mammals. FASEB J. 1991, 5, 2902–2909. [CrossRef][PubMed] 2. Jackson, I.J. Colour-coded switches. Nature 1993, 362, 587–588. [CrossRef][PubMed] 3. Klungland, H.; Vage, D.I.; Gomez-Raya, L.; Adalsteinsson, S.; Lien, S. The role of melanocyte-stimulating hormone (MSH) receptor in bovine coat color determination. Mamm. Genome 1995, 6, 636–639. [CrossRef] [PubMed] 4. Searle, A.G. Comparative Genetics of Coat Colour in Mammals; Logos Press Ltd.: London, UK, 1968. 5. Fontanesi, L.; Beretti, F.; Riggio, V.; Dall’Olio, S.; González, E.G.; Finocchiaro, R.; Davoli, R.; Russo, V.; Portolano, B. Missense and nonsense mutations in melanocortin 1 receptor (MC1R) gene of diﬀerent goat breeds: Association with red and black coat colour phenotypes but with unexpected evidences. BMC Genet. 2009, 10, 47. [CrossRef] 6. Chhajlani, V.; Wikberg, J.E.S. Molecular cloning and expression of the human melanocyte stimulating hormone receptor cDNA. FEBS Lett. 1992, 309, 417–420. [CrossRef] 7. Mountjoy, K.G.; Robbins, L.S.; Mortrud, M.T.; Cone, R.D. The cloning of a family of genes that encode the melanocortin receptors. Science 1992, 257, 1248–1251. [CrossRef][PubMed] 8. Lu, D.; Willard, D.; Patel, I.R.; Kadwell, S.; Overton, L.; Kost, T.; Luther, M.; Chen, W.; Woychik, R.P.; Wilkinson, W.O.; et al. Agouti protein is an antagonist of the melanocyte-stimulating-hormone receptor. Nature 1994, 371, 799–802. [CrossRef] 9. Ollmann, M.M.; Lamoreux, M.L.; Wilson, B.D.; Barsh, G.S. Interaction of Agouti protein with the melanocortin 1 receptor in vitro and in vivo. Genes Dev. 1998, 12, 316–330. [CrossRef] 10. Valverde, P.; Healy, E.; Jackson, I.; Rees, J.; Thody, A. Variants of the melanocyte-stimulating hormone receptor gene are associated with red hair and fair skin in humans. Nat. Genet. 1995, 11, 328–330. [CrossRef] 11. Kijas, J.M.H.; Wales, R.; Törnsten, A.; Chardon, P.; Moller, M.; Andersson, L. Melanocortin receptor 1 (MC1R) mutations and coat color in pigs. Genetics 1998, 150, 1177–1185. Animals 2020, 10, 1878 10 of 11

12. Eizirik, E.; Yuhki, N.; Johnson, W.E.; Menotti-Raymond, M.; Hannah, S.S.; O’Brien, S.J. Molecular genetics and evolution of melanism in the cat family. Curr. Biol. 2003, 13, 448–453. [CrossRef] 13. Robbins, L.; Nadeau, J.; Johnson, K.R.; Kelly, M.; Roselli-Rehfuss, L.; Baack, E. Pigmentation phenotypes of variant extension locus alleles result from point mutations that alter MSH receptor function. Cell 1993, 72, 827–834. [CrossRef] 14. Våge, D.I.; Klungland, H.; Dongsi, L.; Cone, R.D. Molecular and pharmacological characterization of dominant black coat color in sheep. Mamm. Genome 1999, 10, 39–43. [CrossRef] 15. Everts, R.E.; Rothuizen, J.; Van Oost, B.A. Identification of a premature stop codon in the melanocyte-stimulating hormone receptor gene (MC1R) in Labrador and Golden retrievers with yellow coat colour. Anim. Genet. 2000, 31, 194–199. [CrossRef] 16. Våge, D.I.; Lu, D.; Klungland, H.; Lien, S.; Adalsteinsson, S.; Cone, R.D. A non-epistatic interaction of agouti and extension in the fox, Vulpes vulpes. Nat. Genet. 1997, 15, 311–315. [CrossRef][PubMed] 17. Ritland, K.; Newton, C.; Marshall, H.D. Inheritance and population structure of the white-phased “Kermode” black bear. Curr. Biol. 2001, 11, 1468–1472. [CrossRef] 18. Marklund, L.; Moller, M.; Sandberg, K.; Andersson, L. A missense mutation in the gene for melanocyte-stimulating hormone receptor (MC1R) is associated with the chestnut coat color in horses. Mamm. Genome 1996, 7, 895–899. [CrossRef][PubMed] 19. Joerg, H.; Fries, H.; Meijerink, E.; Stranzinger, G. Red coat color in Holstein cattle is associated with a deletion in the MSHR gene. Mamm. Genome 1996, 7, 317–318. [CrossRef] 20. Rouzaud, F.; Martin, J.; Gallet, P.F.; Delourme, D.; Goulemont-Leger, V.; Amigues, Y.; Ménissier, F.; Levéziel, H.; Julien, R.; Oulmouden, A. A first genotyping assay of French cattle breeds based on a new allele of the extension gene encoding the melanocortin-1 receptor (MC1R). Genet. Sel. Evol. 2000, 32, 511–520. [CrossRef] 21. Adalsteinsson, S.; Bjarnadottir, S.; Vage, D.; Jonmundsson, J. Brown coat color in Icelandic cattle produced by the loci Extension and Agouti. J. Hered. 1994, 86, 395–398. [CrossRef] 22. Nei, M. Genetic distance between populations. Am. Nat. 1972, 106, 283–292. [CrossRef] 23. Litwińczuk,Z.; Chabuz, W.; Stanek, P.; Sawicka, W. Genetic potential and reproductive performance of Whitebacks—Polish native breed of cows. Arch. Tierz. 2006, 49, 289–296. 24. Litwińczuk,Z.; Stanek, P.; Chabuz, W.; Jankowski, P. Whitebacks—the native cattle of the Polesie region. Teka Comm. Prot. Form. Nat. Environ. 2004, 1, 130–138. 25. Szarek, J.; Adamczyk, K.; Felenczak, A. Polish Red Cattle breeding: past and present. Anim. Genet. Resour. Inf. 2004, 35, 21–35. [CrossRef] 26. Lorenzo, P.D.; Lancioni, H.; Ceccobelli, S.; Colli, L.; Cardinali, I.; Karsli, T.; Capodiferro, M.R.; Sahin, E.; Ferretti, L.; Marsan, P.A.; et al. Mitochondrial DNA variants of Podolian cattle breeds testify for a dual maternal origin. PLoS ONE 2018, 13, 1–12. [CrossRef] 27. Ruban, S.Y.; Prijma, S.V. Animal genetic resources of Ukraine: Current status and perspectives. J. Vet. Med. Biotechnol. Biosaf. 2015, 1, 23–31. 28. Alderson, L. The categorisation of types and breeds of cattle in Europe. Arch. Zootec. 1992, 41, 4. 29. Russo, V.; Fontanesi, L.; Scotti, E.; Tazzoli, M.; Dall’Olio, S.; Davoli, R. Analysis of melanocortin 1 receptor (MC1R) gene polymorphisms in some cattle breeds: Their usefulness and application for breed traceability and authentication of Parmigiano Reggiano cheese. Ital. J. Anim. Sci. 2007, 6, 257–272. [CrossRef] 30. Maudet, C.; Taberlet, P. Holstein’s milk detection in cheeses inferred from melanocortin receptor 1 (MC1R) gene polymorphism. J. Dairy Sci. 2002, 85, 707–715. [CrossRef] 31. Crepaldi, P.; Marilli, M.; Gorni, C.; Meggiolaro, D.; Cicogna, M.; Renieri, C. Preliminary study on MC1R polymorphism in some cattle breeds raised in Italy. Ital. J. Anim. Sci. 2003, 2, 13–15. [CrossRef] 32. Rolando, A.; Di Stasio, L. MC1R gene analysis applied to breed traceability of beef. Ital. J. Anim. Sci. 2006, 5, 87–91. [CrossRef] 33. Lee, S.S.; Yang, Y.H.; Kang, S.Y.; Oh, W.Y.; Yang, B.S.; Ko, S.B.; Kim, K.I. Comparison of the genotypes and frequencies of MSH receptor (MC1R) gene in Korean Cattle, Cheju Native Black Cattle, Japanese Black and Japanese Brown Cattle. Korean J. Anim. Sci. 2000, 42, 253–260. 34. Brenig, B.; Beck, J.; Floren, C.; Bornemann-Kolatzki, K.; Wiedemann, I.; Hennecke, S.; Swalve, H.; Schütz, E. Molecular genetics of coat colour variations in White Galloway and White Park cattle. Anim. Genet. 2013, 44, 450–453. [CrossRef][PubMed] Animals 2020, 10, 1878 11 of 11

35. Putra, D.E.; Paul, R.C.; Thu, L.N.A.; Okuda, Y.; Ibi, T.; Kunieda, T. Genetic characterization of Indonesian Pesisir cattle using mitochondrial DNA and Y-chromosomal haplotypes and loci associated with economical traits and coat color. J. Anim. Genet. 2018, 46, 17–23. [CrossRef] 36. Niemi, M.; Sajantila, A.; Vilkki, J. Temporal variation in coat colour (genotypes) supports major changes in the Nordic cattle population after Iron Age. Anim. Genet. 2016, 47, 495–498. [CrossRef] 37. Kasprzak-Filipek, K.; Sawicka-Zugaj, W.; Litwi´nczuk,Z.; Chabuz, W.; Šveistiene,˙ R.; Bulla, J. Assessment of the genetic structure of Central European cattle breeds based on functional gene polymorphism. Glob. Ecol. Conserv. 2019, 17.[CrossRef] 38. Sawicka-Zugaj, W.; Chabuz, W.; Litwi´nczuk,Z.; Kasprzak-Filipek, K. Evaluation of reproductive performance and genetic variation in bulls of the Polish White-Backed breed. Reprod. Domest. Anim. 2018, 53, 157–162. [CrossRef] 39. Mei, C.; Wang, H.; Liao, Q.; Wang, L.; Cheng, G.; Wang, H.; Zhao, C.; Zhao, S.; Song, J.; Guang, X.; et al. Genetic architecture and selection of Chinese cattle revealed by whole genome resequencing. Mol. Biol. Evol. 2018, 35, 688–699. [CrossRef] 40. Ludwig, A.; Pruvost, M.; Reissmann, M.; Benecke, N.; Brockmann, G.A.; Castaños, P.; Cieslak, M.; Lippold, S.; Llorente, L.; Malaspinas, A.S.; et al. Coat color variation at the beginning of Horse domestication. Science 2009, 324, 485. [CrossRef] 41. Felius, M.; Beerling, M.L.; Buchanan, D.S.; Theunissen, B.; Koolmees, P.A.; Lenstra, J.A. On the history of cattle genetic resources. Diversity 2014, 6, 705–750. [CrossRef]

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