Mutations in the agouti (ASIP), the extension (MC1R), and the brown
(TYRP1) loci and their association to coat color phenotypes in horses
Laboratoire de Ge´ne´tique biochimique et de Cytoge´ne´tique, De´partement de Ge´ne´tique animale, INRA Centre de Recherche de Jouy, 78352
Jouy-en-Josas cedex, France
Station de Ge´ne´tique quantitative et applique´e, De´partement de Ge´ne´tique animale, INRA Centre de Recherche de Jouy, 78352
Jouy-en-Josas cedex, France
Received: 22 November 2000 / Accepted: 07 February 2001
Abstract. Coat color genetics, when successfully adapted and ap-
plied to different mammalian species, provides a good demonstra-
tion of the powerful concept of comparative genetics. Using cross-
species techniques, we have cloned, sequenced, and characterized
equine melanocortin-1-receptor (MC1R) and agouti-signaling-
protein (ASIP), and completed a partial sequence of tyrosinase-
related protein 1 (TYRP1).
The coding sequences and parts of the flanking regions of
those genes were systematically analyzed in 40 horses and muta-
tions typed in a total of 120 horses. Our panel represented 22
different horse breeds, including 11 different coat colors of Equus
caballus. The comparison of a 1721-bp genomic fragment of
MC1R among the 11 coat color phenotypes revealed no sequence
difference apart from the known chestnut allele (C901T). In par-
ticular, no dominant black (E
) mutation was found.
In a 4994-bp genomic fragment covering the three putative
exons, two introns and parts of the 5Ј- and 3Ј-UTRs of ASIP, two
intronic base substitutions (SNP-A845G and C2374A), a point
mutation in the 3Ј-UTRs (A4734G), and an 11-bp deletion in exon
2 (ADEx2) were detected. The deletion was found to be homozy-
gous and completely associated with horse recessive black coat
) in 24 black horses out of 9 different breeds from our
panel. The frameshift initiated by ADEx2 is believed to alter the
regular coding sequence, acting as a loss-of-function ASIP muta-
tion. In TYRP1 a base substitution was detected in exon 2 (C189T),
causing a threonine to methionine change of yet unknown func-
tion, and an SNP (A1188G) was found in intron 2.
Mammalian coat and skin color seems to be determined by a small
number of genes shared among different species (Jackson et al.
1994; Barsh 1996; Newton et al. 2000). These genes can be clas-
sified into two main groups: those acting on the melanocyte—its
development, differentiation, proliferation, and migration; and
those acting directly on pigment synthesis. Variation in coat and
skin colors is, therefore, likely to be understood as the effect of
modified genes causing changes to either the melanocyte or the
pigment synthesis or its combinations (detailed in Searl 1968;
Melanocortin-1-receptor (MC1R), encoded by the Extension
(E) locus, and its peptide antagonist agouti-signaling-protein
(ASIP), encoded by the Agouti (A) locus, control the relative
amounts of melanin pigments in mammals (Lu et al. 1994; Sir-
acusa, 1994). ASIP acts as an antagonist of MC1R by nullifying
the action of ␣-melanocyte-stimulating hormone (␣-MSH). Loss-
of-function of MC1R results in yellow pigment (pheomelanin),
whereas gain-of-function of MC1R or loss-of-function of ASIP
seems to result in the production of black pigment—eumelanin
(reviewed in Barsh 1996).
Tyrosinase-related protein 1 (TYRP1) coded by the Brown lo-
cus is believed to be a melanosomal membrane protein. Its enzy-
matic function is thought to represent 5,6-dihydroxyindole-2-
carboxylic acid (DHICA) oxidase (Kwon 1993; Jackson et al.
1994; Sturm et al. 1995; Lee et al. 1996). Apart from the original
brown mouse mutation (Zdarsky et al. 1990), alterations in TYRP1
are known to be involved in progressive greying of mice (Johnson
and Jackson 1992; Javerzat and Jackson 1998). In the horse, re-
duced levels of TYRP1 mRNA were found in grey horses com-
pared with solid colored horses (Rieder et al., 2000). TYRP1 is
believed to be involved in the synthesis of an intermediate “choco-
late” melanin, represented in horses of a dark chestnut, liver chest-
nut, silver or seal brown phenotype.
The successful molecular definition of coat color mutations in
different mammals (Jackson 1994; Klungland et al. 1995; Joerg et
al. 1996; Marklund et al. 1996, 1999; Moller et al. 1996; Vage et
al. 1997, 1999; Kijas et al. 1998; Rana et al. 1999; Newton et al.
2000) and the homology among involved genes enhances the gen-
eral concept of comparative genetics between species (Rudolph et
al. 1992; Hayes 1995; Raudsepp et al. 1996; Caetano et al. 1999;
Santschi et al. 1998; Godard et al. 2000), and its application to
color determination in particular.
In Equus caballus, one can roughly distinguish between the
black, bay, chestnut, and chocolate coat color “families” (detailed
in Wagoner 1978; Evans et al. 1990; Adalsteinsson and Thorkels-
son 1991; Sponenberg 1996).
Horse breeds usually display a huge variety of distinct coat
color patterns. Nevertheless, some of them are known for their
particular coat color, indicating homozygozity for this character.
Me´rens and Friesian horses, for example, are thought to be all
black, except for a low frequency of the chestnut allele (E
sulting occasionally in chestnut-colored horses when homozygous.
Solid black is quite a rare coat color in most horse breeds and
seems to be essentially recessive (A
), although some authors
mention cases of dominant inheritance—E
(Dreux 1966; Sponen-
berg and Weise 1997). The mutation leading to the chestnut allele
) is a single base substitution in MC1R (Marklund et al. 1996).
In the present study, we report for the first time the complete
coding sequence and the genomic structure of equine MC1R, ASIP,
and TYRP1 loci. We provide molecular evidence for a recessive
segregation of horse black coat color (A
) and show that black
horses are homozygous for a deletion in the Agouti locus. We
Correspondence to: G. Guérin; E-mail: email@example.com
Mammalian Genome 12, 450–455 (2001).
© Springer-Verlag New York Inc. 2001