MC1R Gene Variants and Their Relationship with Coat Color in South American Camelids

In domestic camelids, fleece color is an essential characteristic because it defines the direction of production. Variants were determined in the MC1R gene that showed a relationship with coat color in alpacas and llamas at the level of the coding region. This report sequenced the MC1R gene from 290 alpacas (142 white, 84 black, 50 brown, and 14 light fawn), five brown llamas, nine vicuñas, and three guanacos to analyze the association between coat color and the MC1R gene among South American camelids. A total of nineteen polymorphisms were identified. Seven polymorphisms were significant; three of them were of nonsynonymous type (c.82A > G, c.376G > A, and c.901C > T), two were of synonymous type (c.126 T > C and c.933G > A), one was in the promoter region (−42C > G), and one was in the 3′ UTR (+5T > C). More polymorphisms were found in domestic camelids than in wild camelids. Besides polymorphism, the association of polymorphisms might cause white and dark pigmentation in the fleece of South American camelids. In addition, the MC1R protein would answer the pigmentation in alpacas.


Introduction
Mammals have distinct colors, which are determined by the biochemical function, distribution, and availability of pheomelanin and eumelanin pigments.Pheomelanin gives red and yellow color, while eumelanin gives black and brown phenotypes [1].Te discovery and analysis of candidate genes associated with coat color led to a better understanding of pigmentation mechanisms [2].More than 150 genes associated with color have been identifed with or without epistatic interactions [3].In this sense, the extension locus encodes the melanocortin one receptor (MC1R) expressed in the hair follicle and skin melanocytes.Melanocyte-stimulating hormone activates the melanocytic activity of MC1R, and mutations at some single-nucleotide polymorphism (SNP) loci have been shown to alter mammalian coat color in guinea pigs [4] and the chestnut horse [5].On the other hand, the mutation of the MC1R gene has been linked to diferent colors in pigs [6,7], cattle [8,9], sheep [10,11], goats [12], dogs [13,14], chicken [15,16], and foxes [17,18].In those cases, a functional mutation increased to give a black/dark color, and lack of function usually generated a light coat color.
Some studies in alpacas identifed mutations in the MC1R gene that determine light phenotypes, but without being conclusive [19][20][21], and in the case of dromedaries, it is indicated that a polymorphism (c.901C > T) is typical of light-coated animals [2,22].
Tis study aimed at analyzing the association between dark coat color and the candidate gene MC1R among South American camelids.

Animal Material.
Tree hundred seven unrelated camelids were studied for MC1R gene sequencing; 290 were alpacas (142 white, 84 black, 50 brown, and 14 light fawn), fve brown llamas, nine vicuñas, and three guanacos.Animals were from three diferent sites in the Cusco region, two of them with signifcant recognition for the quality of the animals (Oquemarca in Phinaya and Chaupi Wasi in Maranganí) and the La Raya Research Center, UNSAAC, as well as samples from the Puno region (Huaycho and Corani).All the sites are located more than 4000 meters above sea level.Te samples were obtained by venipuncture from the jugular vein into EDTA vacutainer tubes.Blood was frozen at −20 °C until handling at the La Raya Research Center from Universidad Nacional de San Antonio Abad del Cusco.

DNA Extraction.
Deoxyribonucleic acid (DNA) was extracted from the whole blood of all sampled animals using the PureLink Genomic Mini Kit (Qiagen) according to the manufacturer's protocol.

Primer Design and Amplifcation.
Polymerase chain reaction (PCR) primers to amplify the coding region and 5′ and 3′ regions were designed with the Ion AmpliSeq 6.0.1 designer for Ion Torrent new generation (Life Technologies, https://www.ampliseq.com)from the VicPac3.1 reference sequence.A total of 5 amplicons ranging from 275 bp to 335 bp covered 100% of the target sequence (Table 1).Te primers were supplied in two pools, each at a concentration of 100 nM (Life Technologies, Carlsbad, CA, USA).

Sequencing of Candidate Genes.
Te sequencing library was prepared with the Ion AmpliSeq Library Kit (version 2.0) according to the manufacturer's protocols for afected individual CSA110.03.Te amplifed library was diluted to 10 pM, and 25 μL was used for template preparation according to the Ion PGM Template OT2 200 Kit protocol using the Ion PGM Template OT2 200 kit.Te clonally amplifed library was enriched on Ion OneTouch ES.Sequencing was performed on an Ion Torrent bulk sequencing machine using the Ion PGM Sequencing 200 v2 kit and an Ion 540 chip.Using Torrent Suite (version 3.6.56201)variant call format (VCF) fles were uploaded to Ion Reporter V4.0 (https://ionreporter. lifetechnologies.com/ir/)for variant annotation.

Variant Analysis.
Te frst run of 168 samples was performed on Ion Torrent Variant Caller (TVC).Tose variants were combined using Genome Analysis Tool Kit (GATK), and the resulting VCF fle had 20 variants.Variant calling was then rerun with 344 samples using the list of variants generated in the frst pass as hotspots for the variants.Tis procedure allowed us to detect variants and spots with the reference allele.Te fnal matrix with all samples had 19 variants.Te MC1R gene sequences were deposited in GenBank with accession numbers MT789859-MT790208.Te sequences were aligned using the software Bosque 2.0.2 [23].Two software programs, DNAsp 6.0 [24] and Network v.10.1.0.0.(Fluxus Technology Ltd.), were employed to construct a haplotype network [25].
2.6.Statistical Analysis.Te Beagle 4.1 software was used to perform the imputation of nongenotyped tags; the resulting fle was processed with PLINK v1.90b4.7 using MAF flters of 0.05, MIND 0.5, HWE 0.001, and GENO 0.2.Minor allele frequency criteria, Hardy-Weinberg equilibrium criteria, and missing genotype criteria fltered 19 variants.Finally, the PLINK association test for feece color was used.Variants were analyzed with a P value adjusted by the Holm-Bonferroni method.

Phenotypic Characterization.
Te phenotypic characteristics of alpacas with feeces ranging from white to black, llamas with brown and white coats, and vicuñas and guanacos, some of the colors analyzed, are presented in Figures 1 and 1S.

Genetic Analysis.
A total of 1238 bp corresponding to the alpaca MC1R gene, including the 5′ and 3′ UTR regions, were determined from 307 animals (Tables 1S and 2S).Seventeen SNP polymorphisms and two indels were also identifed.Six were silent mutations, seven were nonsense polymorphisms, and one was in the 5′ region; there were also a four-base pair deletion, another one-base pair deletion, a fve-base pair insertion, and two polymorphisms in the 3′ UTR (Table 2 and Figure 2S).Five of the nineteen polymorphisms were unreported and are presented in Table 2. Te comparison between genotypes and phenotypes to determine the association with coat color is shown in Table 3.
Strong associations with coat color were found only in eight polymorphisms (Table 3) from genotyping data (Table 3S): three involved amino acid change (c.82A > G, c. 376G > A, and c. 901C > T), two involved synonymous changes (c.126 T > C and c. 933G > A), one located in the 5′ region (5′c.−42C> G), and two in the 3′ UTR (c.+5T > C and +170 G> C; Tables 3 and  4S with more details of the analyses performed).Forty-one haplotypes were identifed (Table 5S) with a haplotypic diversity of 0.9523.Likewise, MC1R gene sequence translation revealed an amino acid size of 318 amino acids.

Discussion
Te present study of the MC1R gene in South American camelids allowed a broader overview since a complete genome was used.In this sense, amplifying a large region of the MC1R gene was possible, providing, at the same time, a better understanding of the expression of this gene with the actual color of animals.Tus, the results of the present study are consistent with those of previous reports [19-21, 26, 28, 30].

2
Te Scientifc World Journal Although eight signifcant polymorphisms are reported (Table 3), three are of nonsynonymous type (c.82A < G, c.376G > A, and c.901C > T that caused amino acid change p.T28A, p.G126S, and p.R301C, respectively), two are of synonymous type (c.126T > G and c.933G> A), one polymorphism is in the 5′ region (−42C > G), and two polymorphisms are in the 3′ region (+8T > C and +170G > C).Te three polymorphisms that were of nonsynonymous type have been responsible for the diference in tones in alpaca feece, interacting in combination with each other for diferent color phenotypes where the combination of genotypes G82/C126/ G − 42(5′) and genotypes T901/A933/C + 5/C + 170 and C901T/G933A/T + 8C/G + 170C has a better association for white feece color (Table 2).In the case of brown feece alpacas, the majority were heterozygous and homozygous for the total set of polymorphisms, and the three genotypes marked in white alpacas were not signifcant.It is also signifcant that the A376 polymorphism was found in 89.58% of white animals.Tus, this polymorphism could be used as a marker for color.In some species, there are several alleles at MC1R [29].Te authors in [19,30] indicated that a combination of mutations at the MC1R locus could cause eumelanin and pheomelanin synthesis in alpacas.Tis assessment was corroborated in the present study.
Te c.82A > G SNP genotypes were signifcantly associated with the color phenotypes analyzed.Some residues in this domain are critical in the normal functioning of the protein [31].Te authors in [20,21] mentioned that there are a more signifcant number of alpacas with the A82G genotype than in the present study; they found a signifcant population of white alpacas with the G82 genotype.Tis fnding could be due to the population analyzed and a more signifcant number of animals studied.
Although synonymous, the c.126T > C polymorphism was shown to play a signifcant role in feece pigmentation in statistical analysis.Tis SNP is likely linked to a promoter mutation [32] and may be a good predictor for pheomelanic [20] and eumelanic animals.All the primers were using the reference (GCF_000164845.3).Vicugna_pacos 3.1.Primary assembly.AMPL: amplicon; Fw: forward strand; Rv: reverse strand.Te Scientifc World Journal 3 Te c.376G > A polymorphism occurring at codon 126 was also shown to play a role in alpaca feece color in statistical analysis, whereas it could serve as a marker for color.Tis codon is in the middle portion of the third transmembrane fragment [31], where it alters the structure of the protein and afects its ability to function [33].
Although this polymorphism was mentioned in [20,21,29], all found no association with this polymorphism; this could be again due to the number of animals analyzed and the origin of alpacas and llamas.In this study, nonrelated animals were used, which may provide a better understanding of this polymorphism.Polymorphisms were signifcant for white, black, and brown coat colors in alpacas.Some of them are found at the level of the 5′ and 3′ UTRs, indicating that these regions are also important for gene characterization studies and their role with coat color. 4 Te Scientifc World Journal Te c.901C > T polymorphism was the one with the highest statistical value in the present study, where white alpacas were found with the TT and CT genotypes (58.78% and 36.73%,respectively) and black and brown alpacas were found with CC and CT genotypes, with the majority being the C901 genotype.Te same situation was also determined in llamas, guanacos, and vicuñas analyzed where all the animals were of the CC genotype.Tese results difer from those found by the authors in [21,34] who did not see white alpacas with the TT genotype and only found heterozygous white alpacas.Te authors in [20,29] mentioned genotypes similar to those reported in the present study.In the case of dromedaries, it has been reported that the polymorphism located at position 901 is responsible for white pigmentation in a dominant manner because heterozygosity of the T allele at c901C > T is sufcient for a white phenotype to occur [25].Te c.901C > T polymorphism occurs at the C-terminal end of the protein and might afect the structure of C-terminal MC1R [35,36].Evaluating the C901 genotype in the two wild camelids could indicate that it is wild type or ancestral and its role in the presentation of feece shades in domestic camelids.
Another polymorphism found in the coding region and located at the C-terminal end, which was signifcant to the statistical analysis, was the c.933G > A synonymous polymorphism.Tis SNP was found in a highly signifcant domain concerning the structural integrity and function of the receptor [35,37].Tis polymorphism was found in Peruvian alpacas [16] and domestic and wild camelids [26].However, they do not mention its signifcance for association studies with coat color.
Finally, it was identifed that polymorphisms present at the level of the 5′ region (−42C > G) and in the 3′ UTR (+5T > C, +170G> C) were signifcantly associated with coat color in alpacas; Tis fact was revealing on this little region and afected gene expression changes.In addition, its efect should be analyzed systematically.Mutations afecting promoter regions and specifc transcription factors have increased melanin synthesis [38]; 3′ untranslated regions (3′ UTRs) contain a critical class of noncoding variants that might impact posttranscriptional and translational processes [39].Although one of these polymorphisms (3′+5T > C) was reported in [19,34], it was not signifcant compared to the results of the present study.
When polymorphisms present in domestic and wild camelids were analyzed, domestic species presented a higher number of polymorphisms than the case of vicuñas and guanacos.Tese results are consistent with those of [26], which indicated that there is higher variability in domestic camelids apparently caused by the efects of the selection to which they were subjected.
In conclusion, from many animals (307), 19 polymorphisms were identifed and presented in Peruvian alpacas, llamas, guanacos, and vicuñas.In addition, an indepth study of this gene by studying 5′ and 3′ UTRs demonstrated their role in the coat of these camelids.Only 14 polymorphisms have been reported in this gene in alpacas [20,26].Te present study adds four new polymorphisms that might also participate in expressing eumelanic and pheomelanic animals.Furthermore, the present study has identifed a combination of polymorphisms that might play an essential role in feece shade, as well as the case of heterozygosity and homozygosity, and not a single polymorphism as the cause of feece color in alpacas.However, they act together to defne feece color.S1: information from the animals used in this study, with signifcant polymorphisms in statistical analysis for the trait analyzed.Table S2: information from animals used in this study, with polymorphisms found in the MC1R gene that were not signifcant in the statistical analysis.Table S3: distribution of genotypes for polymorphisms among diferent color phenotypes.Table S4:

Supplementary Materials
Te Scientifc World Journal statistical analyses used in the population studied.Table S5: frequency of haplotypes in the population studied.(Supplementary Materials)

Figure S1 :
FigureS1: some coat color in alpacas and llamas.FigureS2: structural organization of the MC1R gene with identifed polymorphisms.TableS1: information from the animals used in this study, with signifcant polymorphisms in statistical analysis for the trait analyzed.TableS2: information from animals used in this study, with polymorphisms found in the MC1R gene that were not signifcant in the statistical analysis.TableS3: distribution of genotypes for polymorphisms among diferent color phenotypes.TableS4: FigureS1: some coat color in alpacas and llamas.FigureS2: structural organization of the MC1R gene with identifed polymorphisms.TableS1: information from the animals used in this study, with signifcant polymorphisms in statistical analysis for the trait analyzed.TableS2: information from animals used in this study, with polymorphisms found in the MC1R gene that were not signifcant in the statistical analysis.TableS3: distribution of genotypes for polymorphisms among diferent color phenotypes.TableS4:

Table 1 :
Primers used to amplify the coding region of the MC1R gene and 5′ and 3′ regions.
SNP: single-nucleotide polymorphism; Disp.: displacement; del: deletion; ins: insertion.Five new polymorphisms contributed to the study of the MC1R gene.