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. 2019 Feb 14;97(4):1578–1585. doi: 10.1093/jas/skz071

Synergy between MC1R and ASIP for coat color in horses (Equus caballus)1

Songyang Shang 1,#, Yan Yu 1,#, Yuxin Zhao 1,#, Wanyi Dang 1,#, Junpeng Zhang 1, Xia Qin 1, David M Irwin 2, Qin Wang 3, Fei Liu 4, Zhenshan Wang 5, Shuyi Zhang 1, Zhe Wang 1,✉
PMCID: PMC6447268  PMID: 30785190

Abstract

Through domestication and human selection, horses have acquired various coat colors, including seven phenotypes: black, brown, dark bay, bay, chestnut, white, and gray. Here we determined the genotypes for melanocortin-1 receptor (MC1R) and agouti signaling protein (ASIP) in 709 horses from 15 breeds. We found that the EEEE genotype frequency at MC1R decreased from dark to light colors (black = 64.5%, brown = 67.5%, dark bay = 47.0%, bay = 16.5%, and chestnut = 0.0%), whereas the AAAA genotype frequency at ASIP increased as coat color lightened (black = 0.0%, brown = 22.9%, dark bay = 69.2%, and bay = 83.0%). When combined genotypes at MC1R and ASIP were examined, different advantage genotype combinations were found for each color: black EEEE–AaAa (64.5%), brown EEEE–AAAa (47.0%), dark bay EEEE–AAAA, and EEEe–AAAA (36.2% and 33.0%, totally 69.2%), bay EEEe–AAAA (69.6%), and chestnut EeEe–AAAA (62.6%). The χ2 test showed that the phenotypes of horse coat colors were significantly related with the genotypes of MC1R and ASIP (p < 0.001). Furthermore, in contrast to a previous study where AaAa was only found in black, chestnut, and gray horses, we also found this allele in brown, dark bay, bay, and white horses. These results indicated that MC1R and ASIP may synergistically affect the levels of melanin in equine coat colors and that additional genes are likely involved in regulating coat colors, especially for white and gray colors. Our research provides new data for further studies on the synergetic actions of MC1R and ASIP in coat color of horses.

Keywords: ASIP, coat color, horse, MC1R, polymorphism

INTRODUCTION

Presently, there are more than 400 distinct horse breeds and various coat colors due to human selective action. Among them, black, bay, chestnut, and gray are common colors. Gray horses that are born with original coat color but then gradually lose hair pigmentation with age (Rosengren Pielberg et al., 2008). The bay coat color phenotype contains three subphenotypes, brown, dark bay, and bay, from dark to light. White is a relatively rare coat color, which is due to a complete coat of white hair and a largely or fully unpigmented skin (Haase et al., 2007). Two strongest candidate genes that influence the coat color in horses are melanocortin-1 receptor (MC1R) and agouti signaling protein (ASIP), both of which are in the same signaling pathway (Cone, 2006; Hoekstra et al., 2006; Campagna et al., 2017; Sponenberg and Bellone, 2017).

MC1R protein located on the surface of melanocytes and activated by melanocyte stimulating hormone (MSH) leading to the production of eumelanin, which is the wild-type allele EE (Chen et al., 2017). The MC1R C901T mutation generates a recessive allele (Ee) that leads to the production of only pheomelanin within melanocytes (Neves et al., 2017). The eumelanin is a black pigment, whereas pheomelanin is a red/yellow pigment (Jackson, 1994). Melanocytes that are homozygous for the recessive mutation (EeEe) at MC1R cannot be activated by MSH, leading to the production of only pheomelanin (Suzuki et al., 1996; Neves et al., 2017).

ASIP encodes the agouti signaling protein (wild-type allele AA), an antagonist to MSH that can block the function of MC1R by inhibiting eumelanin production in horse body melanocytes (Lu et al., 1994). An 11-bp deletion mutation at base 192 in exon 2 of ASIP (allele Aa) lead to loss of agouti signaling protein function, yielding a black phenotypic in the horse (Rieder et al., 2001). Combination of specific genotypes at MC1R and ASIP result in three basic phenotypes: black (EEE−–AaAa), bay (EEE−–AAA−), and chestnut (EeEe–AAA− or EeEe–AaAa) (Neves et al., 2017). The superscript dash character (−) in the E− and A− alleles means any genotype at that gene.

Although a statistically significant association between MC1R and coat color has been found, this was based on a limited number of horses (n = 120) and only three colors were investigated (Rieder et al., 2001). Another statistical analysis of coat colors based on 153,778 horses generated a hypothesis that MC1R genotypes have a direct effect on eumelanin levels, but indicated that other genes are involved in this process (Sakamoto et al., 2017). While the genotype distribution of MC1R has been studied, the distribution of ASIP genotypes in horses with different coat colors remains unclear and how the combination of the two important genes correlate with the coat color has not been studied.

In this study, we determined the MC1R and ASIP genotypes in over 700 horses with seven different coat colors and examined the frequencies of each genotype for both gene combinations in all phenotypes to characterize genotype distribution to coat colors in horses.

MATERIALS AND METHODS

Identifying the Phenotype of Equine Coat Colors

Horse coat colors were classified according to previous studies (Rieder, 2009; Neves et al., 2017; Sponenberg and Bellone, 2017; Figure 1a). Briefly, gray horses are born with an original coat color (such as black, bay, or chestnut) but gradually loose hair pigmentation at ages (6 to 8 yr) but maintained the dark skin. Some gray horses do not loose hair pigmentation but change to darker phenotype by decreasing white and increasing the black or brown hairs. The other six colors are from dark to light, black, brown, dark bay, bay, chestnut, and white. The black color is common in horse breeds such as Freiesian and Percheron, which have black hairs on every part of their body, although some have white makings. Most horses with black color will fade to a brownish color when regularly exposed to the sunlight. Horses with brown coat color have black pigment in the mane, tail and legs, and have a nearly-black phenotype of their whole body, but reddish or tan pigments around their eyes, muzzle, and abdomen between the elbow and stifle. The dark bay horse coat color is a black mane, tail and legs, and very dark brown or reddish hair at the head, neck, back and hip, but with brown or reddish pigment at the belly. The bay horse coat color, the most common coat color, has a brown or reddish body, and a black mane, legs, and tail. Chestnut horses are reddish with no black color on their entire body including the mane. White horses are relatively rare and differ from gray horses as they are born with white hair and remain white for life.

Figure 1.

Figure 1.

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Horse coat colors and variation in the MC1R and ASIP genes. (a) Typical examples of horse coat colors investigated in this study. (b) Sanger sequence maps of variations in MC1R. Missense mutations in MC1R are indicated in the red boxes. (c) Sanger sequence maps of variations in ASIP. Position of the 11 bp deletion in ASIP was indicated by a red box (presence of sequence) and arrows (deleted sequence).

Sample Collection and DNA Extraction

Hair roots from a total of 709 adult horses were collected from 15 breeds (566 Thoroughbreds, 65 Mongolians, 22 Warmbloods, 14 Arabians, 12 Akhal-tekes, nine American standardbreds, five Draft horses, four Friesians, three Ponies, two Budyonnies, two Polos, two Orlov trotters, one Andalusian, one Clydesdale, and one Shetland) and represented all seven coat colors. Photos and legitimate registration information of all the horses from the China Horse Industry Association were used to confirm coat colors. A list of the coat color, breeds, and quantity of collected horses was shown in Table 1. Genomic DNA was extracted from hair roots using the TIANamp Genomic DNA kit (Tlangen Biotech, China) according to manufacturer’s manual with the exception that the lysis time was reduced from 1 h to 10 min. The concentration of the extracted DNA was estimated by measuring the OD260 using a NanoDrop 2000 (Thermofisher Scientific). Each DNA sample was diluted to 20 to 50 ng/μL to prepare for PCR amplification.

Table 1.

Breeds and quantity of different coat color horses used in this study

Coat color Breeds and quantity Total
Black 17 Mongolians, 8 Thoroughbreds, 4 Friesians, 1 Arabian, 1 Warmblood 31
Brown 79 Thoroughbreds, 3 Warmbloods, 1 Akhal-teke 83
Dark bay 179 Thoroughbreds, 3 Warmbloods, 2 Akhal-tekes, 1 American standardbred 185
Bay 146 Thoroughbreds, 5 American standardbreds, 4 Warmbloods, 4 Akhal-tekes, 2 Arabians, 2 Budyonnies, 1 Pony 164
Chestnut 101 Thoroughbreds, 9 Warmbloods, 5 Draft horses, 3 Akhal-tekes, 3 Arabians, 2 American standardbreds 123
White 48 Mongolian, 4 Thoroughbreds, 2 Akhal-tekes, 1 Andalusian 55
Gray 49 Thoroughbreds, 8 Arabians, 2 Warmbloods, 2 Polos, 2 Ponies, 2 Orlov trotter, 1 Shetlands, 1 American standardbred, 1 Clydesdale 68
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PCR Amplification

To determine the identities of the genotypes at the MC1R (i.e., E−E−) and ASIP (i.e., A−A−) genes in each horse, primers for amplification of polymorphic gene sequences were designed based on the horse genome EquCab 3.0 using the software Primer premier v5.0 (Lalitha, 2000). The primer sequences for MC1R were MC1R-forward 5′-TGACCACCAACCAGACGGA-3′, MC1R-reverse 5′-CGAGACAGAGCAGGACAGC-3′, and for ASIP were ASIP-forward 5′-GGCTCTGAGAAATGGAGGGTAG-3′, ASIP-reverse 5′-ATATCCTTAACGCTTCCCC AAC-3′. PCR reactions (20 μL) containing 20 to 50 ng genomic DNA, 0.5 μM of each primer, 10 μL 2× EasyTaq PCR Supermix (Transgen Biotech, China) and the appropriate amount of ddH2O. Condition for MC1R PCR was an initial incubation and enzyme activation at 95 °C for 5 min, followed by 30 cycles of denaturation at 95 °C for 30 s, primer annealing at 59 °C for 30 s and extension of 72 °C for 45 s, and final extension at 72 °C for 10 min. The PCR condition for ASIP was same as for MC1R, except that the primer annealing temperature was 61 °C.

Sanger Sequencing, Genotyping, and Statistical Analysis

PCR products were firstly checked by 1% agarose gels and then directly sequenced by the Sanger method on a 3730xl DNA Analyzer (Applied Biosystems) and genotypes (E−E− and A−A−) at MC1R and ASIP were deduced from the sequences. The MC1R PCR fragment amplified here included the mutation position C901T that had previously been used to genotype MC1R in horses (Marklund et al., 1996). The presence of homozygous cytosines at mutation position C901T was genotyped as EEEE, with heterozygous thymine and cytosine being genotyped as EEEe, and homozygous thymine genotyped as EeEe. Similarly, the PCR amplified ASIP fragment contained the 11 bp deletion in exon 2 and was used to genotype ASIP in horses (Rieder et al., 2001). Fragments homozygous for the deletion were genotyped as AaAa, heterozygous for the deletion was genotype as AAAa, and homozygous for nondeletion was genotyped as AAAA. The frequencies of each genotype (E−E− or A−A−) and each genotype combination (E−E−–A−A−) were calculated for each coat color. To reveal the potential association between genotypes of MC1R and ASIP and phenotypes of the horse coat colors, a χ2 test for independence was performed on R v3.52 (R Foundation for Statistical Computing, Vienna, Austria; https://www.R-project.org; accessed February 26, 2019) using the equation of x2=T[∑(Oij2/RiCj)−1]. The results of the test were shown in Supplementary Figure S1 (Cox and Lewis, 1966; Krajewski and Matthews, 2010).

RESULTS

Typical examples of the investigated coat colors are shown in Figure 1a. PCR products containing the mutation positions in MC1R (501 bp) and ASIP (1,006 bp) were successful amplified and sequenced in 709 horses allowing identification of the genotypes (E−E− and A−A−) for both MC1R and ASIP (Figure 1b and c). A summary of the genotypes at MC1R and ASIP, and genotype frequencies for the seven horse coat colors are shown in Figure 2 and Table 2.

Figure 2.

Figure 2.

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Genotype frequencies for MC1R (a) and ASIP (b) in seven tested coat colors.

Table 2.

Numbers of horses and frequencies of MC1R–ASIP genotype combinations in the seven coat colors

Coat color* E E E E E E E e E e E e
A a A a A A A a A A A A A a A a A A A a A A A A A a A a A A A a A A A A
Black (31) 64.5% (20) 0 0 35.5% (11) 0 0 0 0 0
Brown (83) 2.4% (2) 47.0% (39) 18.1% (15) 9.6% (8) 18.1% (15) 4.8% (4) 0 0 0
Dark bay (185) 1.6% (3) 9.2% (17) 36.2% (67) 1.1% (2) 18.9% (35) 33.0% (61) 0 0 0
Bay (164) 1.2% (2) 1.8% (3) 13.4% (22) 0.6% (1) 13.4% (22) 69.6% (114) 0 0 0
Chestnut (123) 0 0 0 0 0 0 2.4% (3) 35.0% (43) 62.6% (77)
White (55) 0 0 0 9.1% (5) 21.8% (12) 7.3% (4) 7.3% (4) 20.0% (11) 34.5% (19)
Gray (68) 4.4% (3) 17.6% (12) 8.8% (6) 1.5% (1) 25.0% (17) 11.8% (8) 5.9% (4) 7.4% (5) 17.6% (12)
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*The number of horses with each coat color and genotype combination is in parentheses.

M1CR Genotypes and Horse Coat Colors

The frequency of the EEEE genotype at MC1R generally decreased as coat color changed from dark (black) to light (white) colors, in contrast to the frequency of the EEEe genotype that mainly increased from black to bay color (Figure 2a). Furthermore, the EEEe genotype was the advantage genotype observed in bay color horses (83.5%), dark bay horses had the genotypes EEEE and EEEe at similar frequencies (47.0% and 53.0%, respectively), while most black (64.5%) and brown (67.5%) colored horses had the EEEE genotype (Figure 2a). The changes in the frequencies of the three genotypes (EeEe, EEEe, and EEEE) were in keeping with coat color changes from light to dark (chestnut, bay, dark bay, brown, and black). A majority (63.6%) of the white horses possess the EeEe genotype at MC1R, with none of them having the EEEE genotype (Figure 2a). All chestnut colored horses had the EeEe genotype (Figure 2a), as found in a previous study (Marklund et al., 1996). No advantage genotype (EEEE, EEEe or EeEe) was found in the gray horses. The χ2 test results show that the phenotypes of horse coat colors were significantly related with the genotypes of MC1R (p < 0.001, Table 3).

Table 3.

The genotype distribution of MC1R and ASIP gene

Gene Genotype Phenotype class χ2 value
Black Brown Dark bay Bay Chestnut White Gray
MC1R E E 51 139 272 191 0 20 68 χ2 = 445.64 P < 0.01
E e 11 27 98 137 246 50 68
E E E E 20 56 87 27 0 0 21 χ2 = 672.82 P < 0.01
E E E e 11 27 98 137 0 20 26
E e E e 0 0 0 0 123 35 21
ASIP A a 62 74 62 31 49 41 42 χ2 = 286.74 P < 0.01
A A 0 92 308 297 197 69 94
A a A a 31 10 5 3 3 9 8 χ2 = 406.63 P < 0.01
A A A a 0 54 52 25 43 23 26
A A A A 0 19 128 136 77 23 34
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ASIP Genotypes and Horse Coat Colors

Unlike previous reports that the AaAa genotype at ASIP was only in black, chestnut, and gray colored horses, we found the AaAa genotype in horses of all seven coat colors, with even 16.4% of the white colored horse being homozygous for the 11 bp deletion (Figure 2b). From dark (brown) to light (bay) colors, the frequency of the AAAa genotype gradually decreased, while the AAAA genotype increased in these coat colors (Figure 2b). The advantage genotype found in brown horse was AAAa (65.1%), while in the dark bay, bay, and chestnut horse the advantage genotype changed to AAAA (ranging from 62.6% to 83.0%). The AAAA and AAAa genotypes had the same frequencies (41.8% and 41.8%, respectively) in white colored horses, with gray colored horses having a slightly greater frequency of the AAAA genotype than the AAAa genotype (50.0% and 38.2%), respectively. As previously reported (Rieder et al., 2001), all horses with black color coats were AaAa. The χ2 test results show that the phenotypes of horse coat colors were significantly related with the genotypes of ASIP (p < 0.001, Table 3).

Genotype Combinations and Horse Coat Colors

When the combined genotypes at MC1R and ASIP were considered together, advantage genotype combinations for each coat color were identified (Table 2). Horses with black color coats only had two genotype combinations, with most having EEEE–AaAa (64.5%) and remaining being EEEe–AaAa (35.5%). For brown color horses, 83.2% of the horses were found to have one of three genotype combinations, EEEE–AAAa (47.0%), EEEE–AAAA (18.1%), or EEEe–AAAa (18.1%) (Table 2). Similarly, three genotype combinations, EEEE–AAAA (36.2%), EEEe–AAAA (33.0%), and EEEe–AAAa (18.9%), were found in 88.1% of the dark bay colored horses (Table 2). While for bay colored horses, a single-genotype combination, EEEe–AAAA, was found in a majority (69.6%) of these horses, and the addition of two other genotypes, EEEE–AAAA (13.4%) and EEEe–AAAa (13.4%), explained 96.4% of these bay colored horses (Table 2). Only three genotype combinations were found in chestnut colored horses, the majority of which were EeEe–AAAA (62.6%) and the remaining being EeEe–AAAa (35.0%) and EeEe–AaAa (2.4%) (Table 2). For white colored horse, 85.4% had one of the four following genotype combinations, EeEe–AAAA (34.5%), EEEe–AAAa (21.8%), EeEe–AAAa (20.0%), and EEEe–AaAa (9.1%, Table 2). In contrast to the other coat colors, all nine genotype combinations were found in the gray colored horses; however, most of them (72.0%) were EEEe–AAAa (25.0%), EEEE–AAAa (17.6%), EeEe–AAAA (17.6%), or EEEe–AAAA (11.8%, Table 2).

DISCUSSION

MC1R has a demonstrated role in hair and coat color variation in humans and in a number of other mammalian species (Healy et al., 2001; Römpler et al., 2006; Candille et al., 2007; Dreger and Schmutz, 2010; Våge et al., 2014). In previous research, the reddish chestnut horse coat color is due to the EeEe genotype, while horses with the genotypes EEEE and EEEe present with darker coat colors (black and brown) (Marklund et al., 1996). Our results show that the advantage genotype at MC1R change with coat color in horses, through a spectrum from dark to light, i.e., black (EEEE), brown (EEEE), dark bay (EEEE and EEEe), bay (EEEe), chestnut (EeEe), and white (EeEe). In black horses, no EeEe genotypes were found, and similarly, in white no EEEE genotypes at MC1R were found. These results suggest that MC1R affects horse coat color through the melanogenesis process, with the EE allele enhancing melanogenesis and the Ee allele inhibiting it. Previous research on MC1R and coat color variation in the Arabian dromedary camel had indicated that the EEEE genotype was associated with the dark phenotype (black, brown, and light brown), whereas the EEEe and EeEe genotypes were associated with light coat colors (e.g., white) (Almathen et al., 2018). While a minority of the tested horses does not conform to this expectation, this might be due to other genes participating in the eumelanin process or genes that regulate MC1R expression in these horses. In general, our results demonstrate that EEEE genotype is associated with dark coat color phenotypes, including black, brown, dark bay, and bay, whereas EEEe genotype is associated with the light phenotypes.

Most of the horses with dark bay, bay, and chestnut coat colors examined here possessed the AAAA genotype at ASIP, with only the brown horse having AAAa as an advantage genotype. Meanwhile, we found that the genotype of brown hoses at last has one allele EE or Aa. This observation might account for the deeper coat color in brown horses. The distribution of the advantage genotypes with coat color suggests that the Aa allele is associated with dark phenotypes (e.g., black and brown), whereas the AA allele is associated with lighter phenotypes (e.g., bay and chestnut). This pattern is consistent with ASIP acting through MSH antagonism to inhibit eumelanin production (Lu et al., 1994).

The 11 bp deletion mutation in exon 2 (the Aa allele) of ASIP leads to loss of function for the agouti signaling protein and thus should cause dark phenotypes in the horse (Rieder et al., 2001). Previous studies have reported that the AaAa genotype only occurs in black, chestnut, and gray-colored horses (Rieder et al., 2001); however, in this study, we found that the AaAa genotype is also found in brown, dark bay, bay, and even white-colored horses with frequencies between 1.8% and 16.4%. These horses with the AaAa genotype did not show a darker color than other horses with AAA− genotype of the same coat color. These results suggest that another gene(s) might take the place of ASIP to block melanin production promoted by MC1R. Based on the hypothesis that all black or gray (which were born black) horses were AaAa at ASIP (Rieder et al., 2001), researchers had speculated on the coat colors of ancient horses by analyzing DNA from fossils. These analyses lead to the conclusion that black horse had appeared in the Copper Age (Ludwig et al., 2009), but our new findings might question this.

When the genotypes at both MC1R and ASIP were considered, the advantage genotype combinations for coat colors across black to chestnut were: black (EEEE–AaAa), brown (EEEE–AAAa), dark bay (EEEE–AAAA and EEEe–AAAA), bay (EEEe–AAAA), and chestnut (EeEe–AAAA). The differences in the advantage genotype combinations correlate with color deepness. The variations of the advantage genotype combinations (from EEEE–AaAa to EeEe–AAAA) with the darkness of the coat colors (from dark to light colors) indicate that MC1R and ASIP synergistically regulate the production of melanin for horse coat color. All nine genotype combinations were found in the gray horse, perhaps due to their various original coat colors.

Among nine possible genotype combinations for MC1R and ASIP, 97.6% to 100% black and chestnut horses were represented by only two of them, 83.2% to 96.4% of the brown, dark bay, and bay horses had three of them, and 72.0% to 85.4% of the white and gray horses had four of them (Table 2). These results indicate that coat color in horses is dominated by these two genes, but that other genes and loci also participated with the rare genotypes to generate coat colors (Metallinos et al., 1998; Rosengren Pielberg et al., 2008; Holl et al., 2010; Hauswirth et al., 2012, 2013).

Through statistical analyses of MC1R and ASIP genotypes in seven coat color phenotypes in horses, we found that these two genes synergistically and advantaged influence the amount of melanin in black, brown, dark bay, bay, and chestnut horses, but also found that some other genes likely participate in generating the white and gray coat colors. Our results provide a molecular basis for further research on the mechanism of horse and other mammalian coat colors.

Conflict of interest statement. None declared.

Supplementary Material

Supplementary Figure S1
Click here for additional data file. (288.5KB, docx)

Footnotes

1

This work was supported by grants from the Ministry of Science and Technology of the People’s Republic of China (The National Key Research and Development Program, grant nos. 2016YFD0500300 and 2016YFC1200100), the National Natural Science Foundation of China (grant nos. 31672274 and 31570382).

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