A genome-wide scan study identifies a single nucleotide substitution in MC1R gene associated with white coat colour in fallow deer (Dama dama)

Background The coat colour of fallow deer is highly variable and even white animals can regularly be observed in game farming and in the wild. Affected animals do not show complete albinism but rather some residual pigmentation resembling a very pale beige dilution of coat colour. The eyes and claws of the animals are pigmented. To facilitate the conservation and management of such animals, it would be helpful to know the responsible gene and causative variant. We collected 102 samples from 22 white animals and from 80 animals with wildtype coat colour. The samples came from 12 different wild flocks or game conservations located in different regions of Germany, at the border to Luxembourg and in Poland. The genomes of one white hind and her brown calf were sequenced. Results Based on a list of colour genes of the International Federation of Pigment Cell Societies (http://www.ifpcs.org/albinism/), a variant in the MC1R gene (NM_174108.2:c.143 T > C) resulting in an amino acid exchange from leucine to proline at position 48 of the MC1R receptor protein (NP_776533.1:p.L48P) was identified as a likely cause of coat colour dilution. A gene test revealed that all animals of the white phenotype were of genotype CC whereas all pigmented animals were of genotype TT or TC. The study showed that 14% of the pigmented (brown or dark pigmented) animals carried the white allele. Conclusions A genome-wide scan study led to a molecular test to determine the coat colour of fallow deer. Identification of the MC1R gene provides a deeper insight into the mechanism of dilution. The gene marker is now available for the conservation of white fallow deer in wild and farmed animals. Supplementary Information The online version contains supplementary material available at 10.1186/s12863-020-00950-3.


Background
White coat colour or dilution are commonly found within fallow deer in game farming and in the wild. It is important for the management of the white animals to identify the responsible gene variant and develop a gene marker. This is the only way to make informed statements about the distribution of the white gene allele in a population. However, up to now, no scientific articles have been available on the colouring of fallow deer and nothing was known about the genes that are responsible for the white coat colour in this species.
A list of 256 genes involved in the white colour or dilution is available from the International Federation of Pigment Cell Societies (http://www.ifpcs.org/albinism/). The most important proteins are formed in melanocytes where they are involved in pigmentation on five independent levels: melanocyte development and migration, melanosome biogenesis, melanosome transport, biosynthesis of melanin and control of melanogenesis.
In addition to colour inheritance in cattle [17], information is also available on sheep [18], goats [19] and buffalos [20]. Recently, we characterized a single nucleotide substitution in the TYR gene associated with white coat colour in a red deer (Cervus elaphus) population [21]. While variants in tyrosinase are commonly associated with oculocutaneous albinism type 1, an amino acid exchange at position 291 in TYR was found to be associated with coat colour dilution in this population.
Nothing is known about colour inheritance in Dama dama. Although so far only a few genes seem to be associated with the whitening of cattle, there is still a wide range of candidate genes to be considered in the search for the genetic cause of the whitening of fallow deer.
The aim of the present study was therefore to identify the causative variant for coat colour dilution in the fallow deer and to develop a genetic marker to facilitate the conservation of white animals.

Results
Whole genome sequencing of a white hind (Fig. 1) and her brown calf was performed to reveal the causative variant of colour dilution in fallow deer.
Sequencing of the hind and her calf resulted in a coverage of 9.48 and 9.68 fold, respectively. A total of 26.18 and 26.71 gigabases were sequenced. 85.58 and 85.69% of these sequences could be mapped to the bovine genome, respectively. Around 11 million SNPs were identified.
After variant calling and annotation, 12,751 SNPs were extracted as a subset of SNPs based on a list of colour genes detected in mice, human and zebrafish (International Federation of Pigment Cell Societies; http:// www.ifpcs.org/albinism/). Three thousand nine hundred fifty-three of them were non-synonymous (ns) SNPs that covered 149 genes. They were located in ASIP (2 ns + 5 synonymous [s] SNPs), DCT (21 ns + 33 s SNPs), EDNRB (6 ns + 15 s SNPs), KIT (18 ns + 85 s SNPs), MC1R (11 ns + 49 s SNPs), TYR (20 ns + 45 s SNPs) and TYRP1 (25 ns + 46 s SNPs). Synonymous SNPs were excluded from further processing. Following the hypothesis of a recessive inheritance of the white colour, we expected the genotype of the white hind to be homozygous for the white allele and the brown calf to be heterozygous. All genes and SNPs that did not correspond to this assumption were filtered out.
After filtering, sixteen genes with nineteen nonsynonymous SNPs were left and confirmed by Sanger sequencing (Table 1). For each of these SNPs a PCR system was established to test the association of the gene variant with the phenotypes of one additional white and one additional brown individual of the population. Four SNPs showed the expected genotype-phenotype association and were tested on further white (n = 3) and brown (n = 3) individuals. Only the genotypes at one SNP in the MC1R gene were associated to the phenotype in these individuals. Results of the phenotypegenotype association are presented in Supplementary  Table 1. Subsequently all animals (n = 102) were tested for the SNP at the MC1R gene revealing a 100% match between genotype and phenotype (Fisher's exact test: Table 2).
Eleven out of 80 wildtype or dark pigmented individuals carried the TC genotype (14%). Any other wildtype or dark pigmented individual were homozygous for the T-allele. Any of the white individuals was homozygous for the C-allele. The MC1R SNP (c.143 T > C) is predicted to result in an amino acid substitution from leucine to proline (p.L48P).  The MC1R gene is involved in a huge network of colouring genes (for overview see [25]), and thus associated with a broad spectrum of colour variation in human [26], mouse [27] and several other mammalian and bird species, such as horses [28], foxes [29], dogs [30], rabbits [31], chicken [32], alpacas [33], buffalos [20], sheep [34,35], goats [36], and cattle [37,38]. Variants were described together with the prevention of the white winter coat in foxes [39] and increased pigmentation in reindeer [40] and other species (overview by [41]). However, besides oculocutaneous albinism type 2 in humans [26], associations of MC1R variants with white coat colour are rare. They were found in black bears [42], white leghorn chicken [43], martens [44], mice [45], Huskies [46] and the Arabian camel [47]. MC1R has never been associated with colour variation or dilution in cervids.
MC1R is a seven-pass transmembrane G protein [26] coupled receptor that is especially located on the surface of melanocytes. MC1R is activated by the α-melanocyte stimulating hormone (α-MSH) and competitively inhibited by the agouti signalling protein (ASIP). Activation stimulates an adenylate cyclase and increases the amount of cAMP, activating the transcription of enzymes involved in eumelanin production [26], e.g. TYRP1 and TYR, the key enzymes in melanin biosynthesis [2,48]. The loss of function of MC1R because of sequence variation affects the ability to generate cAMP and leads to minimal production of eumelanin in melanocytes. Variation within the transmembrane helices can result in loss of function. The variation which is responsible for white coat colour in fallow deer was detected at nucleotide 143 (c.143 T > C) that leads to an amino acid exchange from leucine to proline (p.L48P) in the present study. This substitution is located within the helix structure of the first transmembrane motif, where several non-synonymous variants have been described in humans (V38M, S41F, V51A) [49], I40T [50], and V60L [51] (for overview see [26]). These variants resulted in reduced cell surface expression of MC1R as a consequence of retention in the endoplasmic reticulum (V38M, S41F, V51A) and/or with a decreased coupling to adenylate cyclase (V60L). Although the variant of the fallow deer has never been described before, it is closely related to the above-described human variants. While leucine, the wild-type amino acid is a typical component of α-helices, introduction of a proline residue into similar membrane-bound proteins was shown to alter the gross secondary structure from αhelix to ß-sheet-like [52], which could be detrimental to the structure. Because of its very rigid structure, which bends the main chain of the protein in a characteristic way, proline is a well-known breaker of secondary structures [53,54]. In contrast to proline, leucine is found with above-average frequency in helix structures and is very rarely replaced by other amino acids, an indication of the important structural function this amino acid occupies there [54]. Further studies are necessary to prove the functional significance of the p.L48P variant in white fallow deer, especially in populations from other parts of the world.

Conclusion
The genomic sequencing of a white hind and her brown calf led to the identification of a non-synonymous variant with exchange of a leucine residue at position 48 of the melanocortin 1 receptor by proline as a likely cause of dilution of the coat colour. This variant was detected using a list of colour genes of the International Federation of Pigment Cell Societies (http://www.ifpcs.org/albinism/). Genetic testing confirmed the expected genotypes in all 22 white and 80 brown animals from 12 different locations examined. The study showed that 14% of the brown animals carry the white allele. This genetic test provides a simple and reliable way of conservation and management for the white animals.

Fallow deer
Samples of fallow deer were collected from 12 locations. Four locations were hunting grounds and eight locations were game parks or game farms ( Table 3). The different locations contributed between 1 and 29 animals, 21.6% of which had a white coat colour. Samples were taken from existing antlers and frozen tissue samples provided either by official game parks or by those authorised to practise hunting. No animals were killed specifically for the study. No live animals were sampled and no dropping antlers were sought or collected for the study.

The phenotype
The white fallow deer were not albinos, but the coat colour was diluted. There were no noticeable differences in the degree of dilution. The eyes and claws were normally pigmented or slightly lightened. Apart from the coat and eye colour, the white animals did not differ from the brown animals in size, weight or habitus.

Sample collection
Samples from pigmented (normal brown and dark pigmented, n = 80) and white (n = 22) fallow deer were collected over the 2017/2018 seasons. Two female animals (one white adult hind with its brown calf) were available for Next Generation Sequencing. All samples were accompanied with information about age, weight, colour, and hunting ground. Samples from antlers were taken as drill core samples from the base and stored dry at ambient temperature. Tissue samples from meat were frozen at − 20°C until use.

DNA extraction
Genomic DNA was extracted from tissue samples and antler drill cores with the Instant Virus RNA Kit (Analytik Jena, Germany). Antler drill cores (0.1 to 0.3 g) were treated in a bead mill (MM200, Retsch, Germany) at a frequency of 25 Hz for 2 min. Tissue samples were suspended in 450 μl of lysis buffer and subsequently treated in the same way as the antler drill cores. All following steps were performed according to the manufacturer's instructions. The extracted DNA was eluted with 60 μl of RNAse-free water.
DNA concentration was measured photometrically with the Nanodrop 2000 spectrophotometer (Thermofisher, All programs used in further processing of raw reads were embedded in python scripts to connect the different steps and programs. In a first step raw sequences were converted from a base call file (bcl) to fastq files and mixed probes were demultiplexed through the program bcl2fastq Conversion Software from Illumina (http://emea.support.illumina.com/downloads/bcl2fastq_conversion_soft-ware_184.html?langsel=/de/). Because a Cervus elaphus reference genome was not available at the time of sequencing, resulting reads were aligned to the reference sequence of the bovine genome (UMD 3.1 [55]) using the BWA-MEM algorithm (https://arxiv.org/abs/1303.3 997). After processing of data, single files were merged and converted from the SAM to the BAM format with SAMtools 56 . Duplicated reads were marked by the PICA RDtools command MarkDuplicates (https://github.com/ broadinstitute/picard/).

Variant calling, annotation and identification of candidate variants
To identify single nucleotide polymorphisms (SNPs) in the annotated reads of the two sequenced fallow deer samples, we used the mpileup algorhithm implemented in SAMtools [56]. With the filter algorithm from PICA RDtools (https://github.com/broadinstitute/picard/) called variants were filtered by excluding all SNPs within 3 base pairs of an INDEL and with lower QUAL score.
For the functional annotation of each called SNP we adapted the VariantEffectPredictor (VEP) from Ensemble [57].
Furthermore, we extracted a subset of SNPs based on a list of colour genes detected in mice, humans and zebrafish (International Federation of Pigment Cell Societies; http://www.ifpcs.org/albinism/). The resulting VEP annotated files containing only genomic regions coding for coat colour were checked on the basis of a recessive genetic inheritance model for non-synonymous impacts of the variants.

Validation of candidate SNPs
SNPs detected by NGS were validated by Sanger sequencing (LGC Genomics, Germany). For this purpose, regions including the candidate SNPs were PCR amplified and sequenced. PCR primers were designed from the NGS data in combination with data from the Bos taurus reference genome.
Additional file 1: Supplementary Table S1. Genotype-phenotype associations in white and brown fallow deer for all tested single nucleotide variants.