Molecular characterization of coat color gene in Sahiwal versus Karan Fries bovine

Background Melanocortin-1-receptor gene (MC1R) plays a significant role in signaling cascade of melanin production. In cattle, the coat colors, such as red and black, are an outcome of eumelanin and pheomelanin pigments, respectively. The coat colors have become critical factors in the animal selection process. This study is therefore aimed at the molecular characterization of reddish-brown coat-colored Sahiwal cattle in comparison to the black and white-colored Karan Fries. Results The Sequence length of the MC1R gene was 954 base pairs in Sahiwal cattle. The sequences were examined and submitted to GenBank Acc.No. MG373575 to MG373605. Alignment of both (Sahiwal and Karan Fries) protein sequences by applying ClustalO multiple sequence alignment programs revealed 99.8–96.8% sequence similarity within the bovine. MC1R gene phylogenetic studies were analyzed by MEGA X. The gene MC1R tree, protein confines, and hereditary difference of cattle were derived from Ensemble Asia Cow Genome Browser 97. One unique single-nucleotide polymorphism (c.844C>A) (SNP) was distinguished. Single amino acid changes were detected in the seventh transmembrane structural helix region, with SNP at p.281 T>N of MC1R gene in Karan Fries cattle. Conclusions In this current research, we first distinguished the genomic sequence of the MC1R gene regions that showed evidence of coat variation between Indian indigenous Sahiwal cattle breed correlated with crossbreed Karan Fries. These variations were found in the Melanocortin 1 receptor coding regions of the diverse SNPs. The conclusions of this research provide new insights into understanding the coat color variation in crossbreed compared to the Indian Sahiwal cattle. Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1186/s43141-021-00117-2.


Background
Coat color is a part of the major essential features for identifying the modern breeds of cattle. Each breed has its own specific phenotypic, physiological, hormonal, and metabolic functions to sustain and adapt to diverse agro-geographical and tropical climatic conditions. Coloration is an effective part of variable phenotypical traits in a diversity of animals, and there are several hypotheses for its function, such as camouflage and signaling of diverse detectable progression [1][2][3]. In mammals, coat color has been associated with their production and environmental adaptation. Darwin for the first time stated that a broad range of domestic animals shares multiple phenotypic characteristics, the most evident of which is a wide variety of coat colors [4,5]. In vertebrates, the only character other than variable coat colors that occur The Sequence length of the MC1R gene was 954 base pairs. Coat color phenotypes are useful in the identification of diverse cattle breeds and other livestock. The coat color in animals is dependent on the percentage of eumelanin coating for black-brown pattern [6] to that of the pheomelanin coating for yellow-reddish pattern [7].
The α-melanocyte-stimulating hormone (α-MSH) and the melanocortin-1 receptor (MC1R) have been implicated to execute a pivotal function in regulating the pigment synthesis in several animal species for coat coloration [8,9]. The hormone α-MSH, upon binding with MC1R results in stimulating of adenylate cyclase through the G-protein coupled receptors, consequently leading to high intracellular cAMP levels. Tyrosinase is a well-known rate-limiting enzyme in the melanin pigment synthesis pathway, whose activity is dependent on the prominence of cyclic AMP [10][11][12][13]. Therefore, MC1R performs an essential role in the production of melanin in melanocytes. The variations within the MC1R gene must reveal an effect on the coat color of the diverse animal species like the cow, buffalo, Chinese yakow [14][15][16][17][18] sheep, goat [19,20], pigs, horses [21,22] foxes, dogs [23,24], cats, and mice [25,26].
The MC1R gene encoding for a 7-transmembrane domain [27] contains a single exon spanning over 954 base pairs. Its extension locus is positioned on chromosome 18 in cattle. It is well known that variation in the regulation and expression of the MC1R gene plays a vital role in causing variations in the pigmentation patterns [28]. For instance, differentiation in base coat color of the cattle is due to the hereditary alterations in the MC1R gene, traditionally termed as the extent locus, with alleles coding for black (ED), red (e), and wild-type (E+). Those alternative alleles were identified to observe a dominance model, in which ED > E+ > e. The native population allele (E+) encodes for a receptor that responds to both the α-melanocyte-stimulating hormone (α-MSH) ligand and its competitor, agouti-signaling protein (ASP). The effective ED allele causes a point mutation altering the amino acid from leucine to proline, and promoting the constitutive expression of an active receptor, which acts on most liable eumelanin because of its α-MSH ligand binding mimicry [29].
Sahiwal cattle is a milch breed that mainly existed in the home track of Punjab, Haryana, Uttar Pradesh, and Madhya Pradesh states of India. Its milk yield ranges from 1140 to 3180 kg per lactation. It is remarkably resistant to tick-borne infections, thermotolerant, and perceived for its enhanced protection to internal and external parasites. The color of these animals ranges from lighter reddish-brown to a thicker predominant red occasionally with varying degrees of white patches over the neck and underline. Further, the intensity of the color normally shades in the extremities such as legs, tail, and head. In the current research study, the MCIR gene was amplified from the genomic DNA of Sahiwal cattle breed and compared its sequence with that of Karan Fries breed. In addition, a small cohort of Sahiwal cattle was genotyped for a SNP of the MC1R gene, c.844C>A.

Experimental environment and geographical location
All the experimental cows were housed in Climate Resilient Livestock Research Centre (CRLRC) located in Karnal district, Haryana state, India. The region is positioned at an altitude of 250 ms elevated than the mean sea level (MSL) geographically located at 29°42″ N 79°54″ E, latitude, and longitudinal view, respectively. The location experiences a huge variation in the temperatures measured between summers and winters. While the summers record a maximum temperature of about 46°C, winter temperature drops to as low as 0°C. The mean rainfall from July to August month is around 700 mm.

Experimental design
A total of 30 clinically healthy Sahiwal cattle free from any physical abnormalities were selected in this study. All animals were closely scrutinized and maintained. Blood samples (5-7 ml) were collected from the animals by jugular vein perforation with the help of 18 G needle in K2-EDTA (1 mg/ml concentration) anticoagulant Vacutainer tubes® (BD Biosciences USA) under sterile conditions. The coat colors of the animals were determined by direct visual inspection and recorded at the time of blood specimen collection. Blood samples isolated were cautiously stored at − 20°C for further molecular studies.

Genomic DNA isolation from whole blood samples
The genomic DNA was isolated by standard proteinase K digestion process and phenol, chloroform, and isoamyl alcohol method [30]. To illustrate the quality of the isolated DNA, 200 ng of genomic DNA was loaded on 0.8% agarose gel electrophoresis along with 1 kb marker. Genomic DNA quality and intensity were also investigated by Nanodrop (Shimadzu BioSpec-nano, Japan). We achieved the DNA sample quality ranging between 1.80 and 1.85. An amount of 50-100 ng/μl of genomic DNA was applied as the template for the polymerase chain reaction.

Polymerase chain reaction (PCR)
The primer sequences were designed using the reference gene sequence of Bos taurus cattle (GenBank accession number: NM_174108) and the Primer 3.0 software (Applied Biosystems, USA). To amplify the MC1R sequence of 1280 bp, we employed the following primers set.

MC1R gene PCR product sequencing and analysis
A complete quantity of 50 μl PCR products was sequenced proceeding with 10 μl of the forward and reverse primers of the MC1R gene. All the PCR products were washed in the spin column kit (Sigma, USA) with the maintenance of the ABI3730 DNA analyzer (Applied Biosystems) sequencing service done by Sci-Genom, Kerala, India, Pvt. Ltd. The sequence chromatogram ABI files were visualized and analyzed by using Bio-Edit version 7.2. The MC1R gene Open reading frames were distinguished by using the ORF finder tool by NCBI (http://www.ncbi.nlm.nih.gov/orffinder/) and the nucleotide sequence was interpreted using the translate tool by ExPASy (http://www.expasy.org). The MC1R protein signal peptide prediction was recognized and exhibited by applying SignalP 4.0 server online (http://www.cbs.dtu. dk/services/SignalP/) based on the neural interface instructed on eukaryotes [31]. The bio-physicochemical properties of MC1R proteins were analyzed by applying Protparam server ExPASy [32]. The integrated membrane proteins were analyzed by PROTTER online server (http://wlab.ethz.ch/protter) [33]. MC1R amino acid sequence, secondary structure, and hydrophobic surface accessibility (see Supplementary file) were predicted by NetSurfP-2.0 webserver (http://www.cbs.dtu.dk/services/ NetSurfP/) [34]. The distinguished MC1R protein sequences were associated with the protein data sequence of Bos frontalis, Bos grunniens, Bos taurus, and Bubalus bubalis available at NCBI GenBank database using the pBLAST program.  Ala alanine, Asp aspartic acid, Cys cysteine, Glu glutamic acid, Phe phenyl alanine, Gly glycine, His histidine, Ile iso-leucine, Lys lysine, Leu leucine, Met methionine, Asn asparagine, Pro proline, Gln glutamine, Arg arginine, Ser serine, Thr theronine, Val valine, Trp tryptophan, Tyr tyrosine

Phylogenetic analysis
The MC1R protein sequences of diverse mammalian varieties were regained from the NCBI GenBank database. The phylogenetic tree study was done by MEGA X software by applying the Maximum Parsimony method. This method takes a minimum basic number of mutations into consideration to analyze and elucidate any given series of sequences that are positioned together and does not allow any modification or amendment at every single site. Using this method, the evolutionary records were obtained from all the parsimony sites represented in the parenthesis, in which the rate of duplicate trees, the associated grouped taxa from the bootstrap, distributed (1000 replicates) is presented near to the branches. Limited than fifty percentage of bootstrap replicates are deflated but the branches following to partitions were reproduced [35]. A total of nine amino acid sequences were carried in the study. In the last dataset, a total of 317 positions were covered. The molecular genetic analyses were carried in MEGA X [36].

Results
The MC1R (Fig. 3). The MC1R gene genomic region alignment was related with the other thirteen mammalian species (Fig. 4). Among them, the first internode represents genomic sequence alignment of the cattle and goat that are closely related to the same taxa. The second, third, fourth, fifth, and sixth branch internodes were connected with the pig, horse, dog, and dingo, followed by the seventh branch with cat, respectively. Eight and ninth branches were interconnected with the Chinese hamster and Prairie vole, followed by the ten, eleven, twelve, and thirteen internodes, linked with Ryukyu mouse, mouse, Algerian mouse, Shrew mouse, and rat.
Moreover, the gene MC1R tree of cow was compared with vertebrate species by using Ensemble Asia cow genome browser and we retrieved the MC1R reference sequence (ENSBTAT00000032494) (Fig. 5) Fig. 6. MC1R has seven transmembrane regions. MC1R gene codes the 317 amino acids; the MC1R protein domain was linked to a melanocyte-stimulating hormone receptor, adrenocorticotropic hormone receptor, and G-protein linked receptor, rhodopsin-like receptors. Protein super family domain region of the MC1R is linked with the G-protein linked receptor, rhodopsinlike receptor, serpentine-like receptor, seven transmembrane G-protein receptor, and chemoreceptors. PRO-SITE family was similar to the GPCR, rhodopsin-like 7TM region. In Fig. 6, the MC1R coding domain (shown in the red-shaded area) predominantly represents missense variation and those shown in the greenshaded area represent synonymous variants. In the coding region, the amino acids present at 75th and 109th, position showed loss of a function of the stop codon. Similarly, the amino acids present at 104 and 231 positions of the coding region showed four frame shift variations.
The genetic variations in the genomic region of the MC1R gene are shown in Fig. 7. The full length of the MC1R gene which comprises the 1751 base pairs consists of 5′, 3′ flanking and un-translated regions; the range of the MC1R coding region was 954 base pairs. Most of the variants presented at the coding region and dominantly all were missense variants. Synonymous variants showed lightly in the coding domain. There were less frame shift variations found in the coding region of the MC1R gene in cattle.

Identification of mutation in the melanocortin 1 receptor 7-transmembrane region
Upon comparison of the Karan Fries with that of the zebu Sahiwal cattle, only one inter-breed SNP was identified (c.844C>A) in exonic region of the MC1R gene. One amino acid differences were found in the seven transmembrane helix region of the MC1R protein between Sahiwal and Karan Fries (Fig. 8). The amino-terminal

Discussion
Coat color shows various types of shades in cattle and other mammalian species. MC1R gene plays a key role in regulating the eumelanin and pheomelanin biosynthesis pathways in mammals. Prior to the current study, there was no information available regarding coat color genes in Sahiwal (Bos indicus). Due to an inherited variation in the MC1R gene, which codes for the melanocyte-stimulating hormone (α-MSH) receptor, has been proved to be associated with the coat color of cows [9,37,38] [14]. Red coat coloration is connected with the e locus of Holstein cow [15]. In Korean and Japanese beef cattle ED, MC1R gene has been briefly described in the e loci [39]. Black coat color Japanese cow had no homozygous allele's e/e, and consequently obtained consistent, expressing their black coat color as ED and E+ alleles, which resulted in black pigmentation. In addition, the majority of Korean Hanwoo cattle varieties, which had a coat color changing from yellowish-brown to dark brown, including red skin color, were all homozygous for e/e [40]. Despite, Korean cattle owning a yellowishbrown color were perceived to have gene regularities of the E+/e (0.05) and e/e (0.95) [41]. In (Bubalus bubalis) black coat color of 49 buffaloes, white and grey coat color of 136 swamp buffaloes, 31 hybrid off-springs of river buffalo, and swamp buffaloes, MC1R gene was sequenced. Among these, a total of three SNPs were found. First SNP, c.618 G>C was the same sense mutation and among other SNPs, two were missense mutation c.310G>A and c.384C>T due to differences in the amino acid array p.S104G and p.I128M mentioned already. The black coat color was linked with the SNP104 in buffaloes [17].  [45]. Those were the only extra coding modifications that we recognized in this intron-less gene and they were in the complete phase with the Bos indicus E+ allele (c.296C>T). Discovery of this unique haplotype (TTC) in Bos indicus breed and crossbreed cattle prescribed that nucleotide divergence in MC1R obtained from Bos indicus did not provide to variation in the degree of black in EDE+ heterozygotes [45]. The dominant allele ED, owning a particular amino acid substitution from leucine to proline is reported in bovine E locus [14]. Three leucine amino acid residues were changed to leucine-proline-leucine motifs found in mouse ESO allele. In bovine, proline-leucine-leucine read motifs were found in the ED allele region [14]. We also observed the same kind of results in Karan Fries cross-breed cattle having dominant black color due to the amino acid substitution at 99th position. Moreover, we recognized extensive haplotypes (due to linkage) that stretched for more than 1 Mb either side of MC1R. Each EDE+ having a couple of distinct alleles from the MC1R gene were attributed from the haplotypes of their progenitors to be heterozygous for the CCT/TTC MC1R haplotypes. Inside families, heterozygotes that received the related set of long-range haplotypes revealed a broad change in their degree of black coat color pigmentation; consequently, we hypothesize that it is unlikely that a regulative modification associated with MC1R could explain the observed black and white patch effect.
Coat color might be a complex trait, which is linked to many genes, pathways, or networks. MC1R, a known key determinant of color phenotype in bovine [46]. The transmembrane domain is composed of several polar and non-polar amino acid changes in the transmembrane domain have been described to regulate gene function due to variation of the protein localization and interaction with different molecules [47,48]. Due to SNP changes (c.844C>A, p281T>N) might alter the function and the structure of MC1R protein. A novel Single SNP in Karan Fries cattle (black and white color) breed compared to indigenous Sahiwal cattle (reddish yellow color) due to amino acid changes threonine to asparagine in A 7-TM inner region of the MC1R gene. Because of the protein sequence changes due to the hydroxylation of the amino acid threonine which will involve in the pathway of the pheomelanin synthesis it can cause reddish coat color. Due to the lack of the eumelanin synthesis in the production of black and white color. Several studies reported that in mammals, the pigmentation of the coat color can be affected by 150 to 300 genes reported by [49,50]. Various coat color phenotypes distinguished by the Reggiana and Holstein cattle are recognized in the MC1R gene region, on the eighteenth chromosome. These two breeds showed the intense allele frequency divergence in the MC1R region was observed by [51], which recently reported that the MC1R gene (c.871G>A) SNP can cause the mutation in brown Japanese cattle [52]. Senczuk et al. reported that different grey and non-grey coat color cattle breeds [53]. The MC1R gene variant is associated with black color in Swiss Holstein cattle breed [54]. Recently, Kasprzak et al. described MC1R polymorphism in different coat color cattle breeds of Central Europe [55].

Conclusions
Analysis of Sahiwal MC1R gene revealed that the SNP c.844C>A or p.281 T>N might be the possible reason for its coat color variation from Karan Fries cattle. Further molecular studies need to be conducted to prove this association with the synthesis of different melanins.