TYRP1 and MC1R genotypes and their eects on coat color in dogs
Sheila M. Schmutz, Tom G. Berryere, Angela D. Gold®nch
Department of Animal and Poultry Science, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5A8
Received: 27 November 2001 / Accepted: 20 March 2002
Abstract. We used PCRampli®cation of cDNA prepared
from skin biopsies to determine the nearly full-length, protein-
coding sequence of dog TYRP1, and to de®ne sequence vari-
ants potentially responsible for the B locus. One common
variant contained a premature stop codon in exon 5 (Q331ter),
and the other deleted a proline residue in exon 5 (345delP). A
third variant in exon 2 (S41C) occurred less frequently. We
genotyped 43 brown (including brown and white) and 34 black
(including tricolor, black-and-tan, and black and white) dogs.
All 43 of the brown group carried two or more of these se-
quence variants likely to interfere with TYRP1 function,
whereas 0 of 34 in the black group carried two or more of these
variants (10 carried one variant). We also genotyped 13 black-
nosed and 10 brown-nosed dogs whose coat color was de-
scribed as red, yellow, gold, apricot, or orange (including
various degrees of white). All these dogs were homozygous for
a R306X MC1R variant shown to be associated with these
color phenotypes. The black or brown nose correlated
perfectly with the absence or presence of the same three
TYRP1 variants described above. TYRP1 was linkage mapped
to dog chromosome 11, with a SNP in exon 7.
Tyrosinase-related protein 1 (TYRP1) is a protein within the
melanocyte that alters the color of the skin and hair of animals
Some mutations within this gene have been
identi®ed in mice that cause brown coloration (Zdarsky et al.
1990; Bell et al. 1995; Javerzat and Jackson 1998). This gene
has also been referred to as the B locus (Jackson 1988). Winge
(1950) and Little (1957) both described one locus as being
responsible for the brown versus black coat color in dogs and
another for black versus red or yellow coats. Their interpre-
tations dier somewhat, in that Little (1957) lumped liver and
brown together as a single coat color, whereas Winge (1950)
interpreted liver as a shade of red with brown caused by an
allele at the other locus. Unfortunately, their descriptions re-
verse the use of E and B for these two loci, but since Little
(1957) is more widely quoted, we will follow his nomenclature.
Recently, Newton et al. (2000) described a premature stop
codon in the dog MC1R gene that was present in most dogs
with red or yellow coat color that they examined. All dogs with
either black or brown coat colors were lacking this premature
stop codon or were heterozygous for it. This follows the pre-
diction, by both Little (1957) and Winge (1950), that black is
dominant to red in most dogs.
We studied the alternative proposals of Winge (1950) and
Little (1957) regarding brown and/or liver coat colors in the
light of recent molecular ®ndings. Because TYRP1 was shown
to be the gene for brown in mice (Zdarsky et al. 1990; Bell et al.
1995; Javerzat and Jackson 1998), we sequenced TYRP1 in the
dog for the ®rst time. We also extended the data presented on
MC1R (Newton et al. 2000) to additional breeds and studied
the interaction of MC1R with TYRP1.
Materials and methods
DNA was obtained from several dogs, by venipuncture, by
one of several collaborating veterinarians. Some DNA samples were
also obtained from dog owners who used cheek swab brushes (Epi-
centre, Madison, WI) and mailed these back to our lab. We found the
chance of obtaining nonsheared DNA was better when these were
returned after drying or after rotation of the swab in the extraction
buer contained in the eppendorf tubes supplied by the manufacturer.
Skin samples were obtained under local anesthesia from four dogs by a
punch biopsy: two large Munsterlanders and two Labrador Retrievers.
Skin samples were also obtained from a German Longhair and a
German Shorthair during required surgeries with general anesthetic.
Coat color was recorded by the veterinarian and/or owner or by us,
if we took the sample. Nose color was also recorded in most cases.
DNA and microsatellite genotypes from the DogMap families
(Lingaas et al. 2001) were used to map TYRP1.
Because of our ongoing interest in coat color of
cattle and dogs, we designed PCRprimers to most TYRP1 exons from
human TYRP1 sequence (GenBank AF001295) (Table 1). However,
some primers were designed from goat sequence (ex8R) (GenBank
AF136926) or ultimately dog sequence, as described later. Determi-
nation of exon boundaries was based on Box et al. (1998). Although
these primers gave the correct product based on Blast and later con-
®rmation with dog cDNA sequence, we suggest designing dog-speci®c
primers for further research.
cDNA prepared from skin biopsies was sequenced with Ex2F and
Ex8Rprimers (Table 1). Exon 1 and the end of exon 8 are noncoding;
therefore, similarity between dog and species for which sequence was
available did not seem to be high enough to design primers in these
regions that would yield a dog product.
Table 1. Primer pairs designed for amplication of TYRP1 exons from genomic
DNA and their respective annealing temperatures.
Primer Sequence Temperature (°C)
Ex2F TTGTTCTTCACTCTTGCTTAAAGC 55
Ex3F GTCAGGAGAAATCTTCTGGACTTAAG 58
Ex4F GCAGGAAATGTTGCAGGAT 53
Ex5F TGCAGGCACCGAGGATGGGCCAATTA 57
Ex6F TCTAGGTTACAGTGACCCCACG 57
Ex7F GATATATCCACATTTCCATTGG 53
Mammalian Genome 13, 380±387 (2002).
Correspondence to: S. M. Schmutz; e-mail: firstname.lastname@example.org