Journal of Heredity, 2016, 214–219
doi:10.1093/jhered/esw007
Original Article
Advance Access publication February 16, 2016
Original Article
The MC1R and ASIP Coat Color Loci May Impact
Behavior in the Horse
Lauren N. Jacobs, Elizabeth A. Staiger, Julia D. Albright, and
Samantha A. Brooks
From the Department of Animal Science, Cornell University, Morrison Hall, Ithaca, NY 14853 (Jacobs and Staiger);
Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, 2407 River
Drive, Knoxville, TN 37996 (Albright); Department of Animal Science, University of Florida, PO Box 110910, Gainesville,
FL 32611 (Brooks).
Address correspondence to Samantha Brooks at the address above, or e-mail: [email protected].
Received September 11, 2015; First decision October 15, 2015; Accepted January 27, 2016.
Corresponding editor: Elaine Ostrander
Abstract
Shared signaling pathways utilized by melanocytes and neurons result in pleiotropic traits of
coat color and behavior in many mammalian species. For example, in humans polymorphisms at
MC1R cause red hair, increased heat sensitivity, and lower pain tolerance. In deer mice, rats, and
foxes, ASIP polymorphisms causing black coat color lead to more docile demeanors and reduced
activity. Horse (Equus caballus) base coat color is primarily determined by polymorphisms at the
Melanocortin-1 Receptor (MC1R) and Agouti Signaling Protein (ASIP) loci, creating a black, bay,
or chestnut coat. Our goal was to investigate correlations between genetic loci for coat color and
temperament traits in the horse. We genotyped a total of 215 North American Tennessee Walking
Horses for the 2 most common alleles at the MC1R (E/e) and ASIP (A/a) loci using previously
published PCR and RFLP methods. The horses had a mean age of 10.5 years and comprised 83
geldings, 25 stallions, and 107 mares. To assess behavior, we adapted a previously published
survey for handlers to score horses from 1 to 9 on 20 questions related to specific aspects of
temperament. We utilized principle component analysis to combine the individual survey scores
into 4 factors of variation in temperament phenotype. A factor component detailing self-reliance
correlated with genotypes at the ASIP locus; black mares (aa) were more independent than bay
mares (A_) (P = 0.0063). These findings illuminate a promising and novel animal model for future
study of neuroendocrine mechanisms in complex behavioral phenotypes.
Subject areas: Gene action, regulation and transmission, Quantitative genetics and Mendelian inheritance
Keywords: ASIP, equine, MC1R, pigmentation, temperament
Genes controlling coat color dictate the quantity and distribution
of melanin pigments in the skin and hair. In many mammalian species, these same genes often have pleiotropic effects on behavioral
phenotypes. For example, alterations of the stress response are
noted in colorphase foxes (Keeler et al. 1968; Keeler et al. 1970;
Kukekova et al. 2012) and agouti-variant deer mice (Harris et al.
2001). Aggressiveness is associated with red coat color versus black
Cocker Spaniel dogs (Pérez-Guisado et al. 2006). Additionally, a fear
reaction specific to Icelandic horses with the silver coat color was
recently described (Brunberg et al. 2013). Across diverse species,
individuals with loss-of-function ASIP variants tend to be calmer
and have a lesser stress or fear response. The 2 genes primarily
© The American Genetic Association. 2016. All rights reserved. For permissions, please e-mail: [email protected]
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responsible for determination of base coat color in the horse (Equus
caballus) are the Melanocortin-1 Receptor (MC1R) and its antagonist, Agouti-Signaling Protein (ASIP) (Rieder et al. 2001; Bailey and
Brooks 2013, Table 1; Figure 1). Notated as the “extension” (E/e)
locus, MC1R is epistatic to ASIP, or the “agouti” (A/a) locus.
Called chestnut, red or sorrel in horses, a recessively inherited
red coat results from a single base substitution in MC1R, leading to loss-of-function in the receptor and a coat bearing little to
no black eumelanin that might obscure the underlying red/brown
pheomelanin (Rieder et al. 2001). MC1R loss-of-function mutations
in humans also lead to red hair color, as well as increased sensitivity to the sun, thermal discomfort, and a reduced pain tolerance
threshold (Mogil et al. 2003; Liem et al. 2005). Mogil et al. (2003)
specifically observed that, in comparison to their darker-pigmented
counterparts, red-haired women had a greater analgesic response to
certain opioid drugs.
The second gene influencing base coat color in the horse is ASIP,
a paracrine signaling protein that acts as a ligand antagonist for
the MC1R receptor by competing with α-melanocyte-stimulating
hormone (α-MSH) for binding (Rieder et al. 2001). In the horse,
a loss-of-function mutation in ASIP, a recessive 11 base pair (bp)
deletion resulting in a frameshift, causes the uniform production of
eumelanin across the body and the appearance of a black coat in
the homozygote (Rieder et al. 2001). Bay base coat color results if
at least one of the ASIP alleles is the functional wild-type. There is
evidence for a relationship between stress and the ASIP gene in deer
mice. There is increased reactivity to stressors in black-colored individuals, and the black color in deer mice is due to a loss-of-function
ASIP mutation similar to black horses (Harris et al. 2001). There
is also a well-documented relationship between ASIP alleles and
food intake; those with functional variants of the gene often become
obese (Harris et al. 2001), though an obesity phenotype was not
examined in the current study.
To date, the only studies assessing the relationship between coat
color and behavior phenotypes in the horse have examined just
one specific coat color pattern, Silver. However, the Silver locus is
also responsible for ocular abnormalities and blindness, confounding measures of startle behavior using visual stimuli (Ramsey et al.
1999; Brunberg et al. 2013). This study aimed to determine the
relationship between the presence of alleles of the genes for base
coat color and behavioral characteristics in the horse. Results of this
research should provide people involved with horses—owners, riders, trainers, etc.—a greater understanding or foresight in choosing
and working with horses of a particular color.
Table 1. Genotype and corresponding base coat color phenotype
in the horse
Materials and Methods
Horse Sampling
MC1R genotype
ASIP genotype
AA
Aa
aa
EE
Ee
ee
Bay
Bay
Black
Bay
Bay
Black
Chestnut
Chestnut
Chestnut
A total of 276 Tennessee Walking Horses (TWH) were sampled in
conjunction with another study (Staiger 2015). The TWH breed was
chosen for this study due to its popularity for use in trail/pleasure riding and show competition, and the TWH also possesses many coat
colors providing a variety of MC1R and ASIP genotypes. Owners
and trainers of horses either volunteered for participation in the
Figure 1. Photos illustrating the variations of base coat colors in the Tennessee Walking Horse. Examples of a dark chestnut (a), light chestnut (b), black (c), and
bay (d) horse.
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216
study and mailed in required materials or were visited in person by
lab staff at private farms or at horse shows to collect the necessary
data. Sampled horses included in the study resided in the continental
United States or in Alberta and Quebec, Canada. Data were collected
from September 2011 through August 2014. Approximately 50–100
hair bulbs were pulled from the underside of the tail or from the
mane of individuals to extract DNA. Pedigrees, registration information, and photographs were collected from each horse for identification purposes.
A behavior survey (Staiger 2015) was adapted from Momozawa
et al. (2003 and 2005), containing 20 questions referring to temperament traits with answer scores rated on a scale from one (1) to nine
(9) (Supplementary Table S1). Answers to the survey questions quantified the quality of a given horse’s temperament. Those who filled out
the survey relayed how long they had known the horse, their exact
relationship with the horse (e.g. owner, trainer, or barn manager) and
their amount of exposure and familiarity to the animal’s behavior.
Two candidate genes, those for Melanocortin-1 Receptor (MC1R)
and Agouti-Signaling Protein (ASIP), were chosen for their conservation in different species and potential to impact behavior in the horse.
A behavior survey (Staiger 2015) was used to subjectively assess the
behavior of horses. Such a methodology was previously utilized successfully in the horse (Momozawa et al. 2003; 2005), fox (Kukekova
et al. 2012), and dog (Hsu and Serpell 2003). Seaman et al. (2002)
validated a behavior survey as mirroring the results of physical handling and novel object tests like those of Wolff et al. (1997) and Visser
et al. (2001) in determining temperament in the horse.
In total, 215 horses contributed to the temperament scores after
filtering for incomplete behavior surveys and relatedness. The horses
sampled had a mean age of 10.5 years, ranging from 1 to 32 years.
The samples consisted of an equal gender ratio with 83 geldings and
25 stallions, for a total of 108 males, and 107 mares or females.
MC1R and ASIP Genotyping
DNA was extracted from 5 to 15 collected hair bulbs per horse by 2
methods: a kit-based procedure for alcohol precipitation or a crude
lysis technique. The kit-based hair prep protocol was modified from
the Gentra PUREGENETM DNA Isolation Kit (Cook et al. 2010).
The crude lysis hair preparation followed the method outlined by
Locke et al. (2002). Samples were genotyped at the coat color gene
loci following the restriction fragment length polymorphism (RFLP)
methods at MC1R (Marklund et al. 1996; Royo et al. 2008) and polymerase chain reaction (PCR) techniques at ASIP (Royo et al. 2008).
DNA was amplified by PCR using FastStart Taq DNA Polymerase
and included all reagents per the manufacturer’s recommended
conditions (Roche Diagnostics), with the primers obtained from
Marklund et al. (1996) and Rieder et al. (2001), based on the assembly of the equine genome, EquCab2.0 (Table 2). Thermocycling
on an Eppendorf Mastercycler® EP Gradient or Thermo Electron
Corporation Px2 was also according to the manufacturers’ recommendations with respective annealing temperatures presented in
Table 2 and run a total of 40 cycles for both amplicons.
Genotypes for the MC1R locus were determined from a RFLP
test using Taqα1 (1.0 U per reaction; New England Biolabs, Inc.)
incubated at 65°C overnight. The resulting MC1R products were
visualized by electrophoresis following standard conditions on a 1%
agarose gel (Omnipur Agarose; EMD Chemicals, Inc.). The presence
of 150 and 200 base pair (bp) fragments indicate genotype homozygous recessive (ee), a single 350 bp fragment (not digested) indicates
a homozygous dominant (EE) horse, while all 3 bands (150, 200,
Table 2. Polymerase chain reaction (PCR) primers and annealing
temperatures for MC1R and ASIP
Gene
Annealing
temperature
Primer sequences (Forward and Reverse)
ASIP
58°C
MC1R
63°C
Forward: 5′ CTT TTG TCT CTC TTT
GAA GCA TTG 3′
Reverse: 5′ GAG AAG TCC AAG GCC
TAC CTT G 3′
Forward: 5′ CCT CGG GCT GAC CAC
CAA CCA GAC GGG GCC 3′
Reverse: 5′ CCA TGG AGC CGC AGA
TGA GCA CAT 3′
and 350 bp) represents the heterozygote (Figure 2). The ASIP products were visualized by electrophoresis following standard conditions on a 4% agarose gel (Omnipur Agarose, EMD Chemicals, Inc.).
Genotyping at the ASIP locus was called via the presence of a 91-bp
fragment indicating genotype homozygous recessive (aa); a 102-bp
fragment indicates a homozygous dominant (AA), and a heterozygote (Aa) had both bands (91 and 102 bp) (Figure 2). Genotypes
from the MC1R and ASIP loci determined the base coat colors of the
horses sampled (Table 1, Bailey and Brooks 2013).
Statistical Analysis
Statistical analyses were performed using the software JMP Pro 10
(SAS Institute, Inc., Cary, NC). To analyze the temperament trait
data, principle component analysis (PCA) was conducted on the
scores of the 20-question survey. The multivariate method of PCA
on covariances was employed and principle components (PCs) were
retained for analysis based on Peres-Neto et al. (2005) recommendations. PCs were connected to the original behavioral qualities based
on their eigenvalues (Hatcher 1994). Factor analysis using prior
communality and Varimax rotation for orthogonal transformation
was carried out on the significant PCs to correct for human-derived
non-normal distribution of the behavior survey scores, yielding factor components (FCs).
A standard least squares multiple regression was used to model
the dependence of the behavior traits on genetic base coat color,
gender (mare, gelding, or stallion), and age. This method controlled
for the effects of age, gender, genotype, and their interactions with
the behavior traits to explain the best set of covariances to use in
analysis of variance (ANOVA). Owner relationship and observation
frequency were also examined, but found to be not statistically significant and therefore not included in the model. ANOVA was used
to detect significant differences between the FCs and genetic base
coat color by gender. Statistical significance was set at any P value
equal to or below 0.05.
Data Availability
Genotypes, behavior scores, and sampling information for all
horses used in these analyses are available in a table submitted to
Dryad.
Results
Allele Frequencies
The 215 TWH were successfully genotyped at the MC1R and ASIP
loci. Genotypes were more evenly distributed at the MC1R locus
than at ASIP. Base coat color phenotypes of the horses and allele
Journal of Heredity, 2016, Vol. 107, No. 3
217
Figure 2. MC1R (a) and ASIP (b) genotyping gels showing the ladder (L), as well as the non-template control (NTC) in the ASIP gel. Homozygous recessive,
homozygous dominant, and heterozygous conditions are genotyped from left to right.
Table 3. Distribution of MC1R and ASIP genotypes and base coat
color phenotypes of the 215 Tennessee Walking Horses
MC1R genotype
ASIP genotype
AA
Aa
aa
EE
Ee
Ee
0
6
31
0
21
75
3
17
62
frequencies were determined based on the genotypes (Table 3). At
MC1R, the allele frequencies were 0.395 for E and 0.605 for e; at
ASIP, A had a frequency of 0.116 compared to a with 0.884.
Coat Color and Temperament
PCs 1 through 4 were retained for analysis as they accounted
for 34.1, 9.27, 7.80, and 5.78% of the data variance, respectively. Four FCs were derived from the significant PCs retained
for analysis, accounting for 16.9, 23.5, 8.59, and 7.94% of the
data variance. The temperament traits that described each FC
were determined by graphing the eigenvectors of the surveyed
temperament traits against the FC scores (Supplementary Figure
S1). Factor 1 reflected overall trainability, with positive FC1 scores
indicating a less trainable horse versus negative scores defining a
more trainable horse. Factor 2 described neophobia, with negative
scores correlated to a fearful or flighty horse and positive scores a
calmer animal. Negative FC3 scores explained a less friendly, more
competitive horse, while positive scores a friendlier, less competitive horse among peers. Factor 4 strongly loaded with self-reliance, indicating that positively scoring horses were restless when
left alone and negatively scoring individuals more at ease when
solitary.
The results of the standard least squares multiple regression
showed significant values delineating a dependence of behavior on
genotype at the ASIP locus and gender for FC1 and FC4 (P = 0.0449
and P = 0.0383, respectively) and with age for FC2 and FC3
(P = 0.0010 and P = 0.0471, respectively). No significant association
was identified with the MC1R locus or combined genotype effects
for ASIP and MC1R in this sample population. Analyses of variance
indicated black mares (aa) had statistically significantly lower FC4
scores than bay mares (A_) (P = 0.0063), reflecting a lower behavior
survey score or more self-reliant horse that is at ease when alone
(Figure 3).
Figure 3. Mean Factor 4 “self-reliance” scores in mares are significantly
different between the dominant and recessive ASIP genotypes.
Discussion
There are 2 genes that determine base coat color in the horse that
are anecdotally reported to affect behavior: MC1R and ASIP. Our
candidate gene approach using a behavior survey found an association between ASIP genotype and temperament that may influence how humans interact with horses. A black mare (aa as defined
by ASIP) is more self-reliant and perhaps preferable in a solitary
situation than a bay-colored horse (A_). This is reflected in foxes,
where recessive alleles at ASIP correlated to smaller adrenal size and
thus less stress response-related hormone (adrenaline or epinephrine) production (Keeler et al. 1968). Similar effects were described
in deer mice and rats (Harris et al. 2001), and analogous results
were obtained in the English Cocker Spaniel dog (Podberscek and
Serpell 1997; Pérez-Guisado et al. 2006). In a loss-of-function mutation at ASIP, α-MSH binds to melanocortin receptors which inhibit
stress-induced glucocorticoid release, decreasing the stress response
(Harris et al. 2001). When α-MSH binds, it also plays a part in dopamine pathways, increasing feeding and grooming behaviors, while
the protein product of ASIP binds antagonistically (Roseberry et al.
2015). Implications of this finding include considerations for change
in the treatment of horses and potential human applications. Similar
human hair color genetics may show the same correlations with
behavior and physiology.
Journal of Heredity, 2016, Vol. 107, No. 3
218
Age correlated positively with FC2 and negatively with FC3,
indicating that as a horse aged it exhibited a less neophobic behavior
phenotype and a more competitive nature, respectively. The effects of
genotype and age could not be separated in this study, but the results
seen reflected those of Baragli et al. (2014).
Further study in this area is warranted. Ideally, the research sample should be expanded, a more objective measure of behavior developed, and additional coat color genes analyzed. Distributions of
gene alleles and behavior scores were skewed in the TWH (Table 3),
and this bias may have affected our results. The TWH in this study
had a high a allele frequency, likely due to coat color selection following the success of a black-colored World Grand Champion of the
breed from 1946 to 1965, though there was selection for chestnut
(ee as defined by MC1R) for the same reason at a later time in breed
development in the 1980s–1990s (Womack 1994). Line breeding
and selection of TWH may have also affected behavioral tendencies, as a calm disposition is desired both innately and requisitely
by the breed association (Tennessee Walking Horse Breeders’ and
Exhibitors’ Association 2011), limiting the temperament variation
we expected. A revised behavior survey or another type of behavioral assessment can be performed for a more objective measure of
temperament; these assessments include arena tests, handling tests,
and novel object tests (Wolff et al. 1997) or measuring heart rate
(Visser et al. 2002). Other gene loci that encode coat color could
influence behavior in the horse. One study (Brunberg et al. 2013)
examined the gene for silver coat color (PMEL17) and a novel object
test, which may have been a more objective phenotype than the present study, yet did not consider the impact of the genes for base coat
color. A genome wide association study (GWAS) might be a first step
in future studies to find other such significant relationships, by analyzing the allele variation within many genes influencing coat color
and behavior.
From the current study, the genes governing base coat color in
the horse seem to influence behavior. Historically chestnut horses
were considered excitable, but horse breeds, coat color, and their
associated behavior may have changed since that proposition was
originally made. We found no significant relationship with behavior at the MC1R locus, which codes for chestnut coat color, though
detection of such an effect may have been hampered by the low frequency of the e allele in this population. The correlation between the
recessive ASIP allele and a more independent temperament will help
guide horse choice and care, as well as improve our understanding
of pigmentation and behavioral pathways.
Supplementary Material
Supplementary material can be found at http://www.jhered.oxfordjournals.org/.
Funding
This work was supported by the Cornell University College of
Agriculture and Life Sciences Dextra Undergraduate Research
Endowment Fund.
Acknowledgements
We thank the Brooks Equine Genetics Lab, Dr. Heather J. Huson and the
Odyssey DNA lab, as well as the Cornell University Department of Animal
Science for their equipment and assistance in carrying out this research. We
also thank all the horses and people who participated in this study.
Data Availability
Data deposited at Dryad: http://dx.doi.org/doi:10.5061/dryad.3q111
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