Association between polymorphism in the melanocortin 1 receptor gene

and E locus plumage color phenotype

S. G. Dávila,1 M. G. Gil, P. Resino-Talaván, and J. L. Campo

Departamento de Mejora Genética Animal, Instituto Nacional de Investigación y Tecnología,

Agraria y Alimentaria, Apartado 8111, 28080 Madrid, Spain

ABSTRACT The purpose of this study was to investi-
gate the effect of the melanocortin 1 receptor (MC1R)
gene on plumage color in chickens. The gene was se-
quenced in 77 males and 77 females from 13 Spanish
breeds, carrying 6 different alleles in the E locus (E*E,
E*R, E*WH, E*N, E*B, E*BC), a recessive wheaten
(yellowish-white) tester line (E*Y), and a White Leg-
horn population (heterozygous E*E). A total of 11 sig-
nificant SNP were detected. Nine of them were nonsyn-
the Birchen Leonesa, had the G274A polymorphism.
Eleven haplotypes were made with 7 of the significant
SNP. The distribution of these haplotypes in the differ-
ent alleles of the E locus showed that each haplotype
was predominantly associated to one allele. The num-
ber of haplotypes was greatest for the Black Menorca,
Birchen Leonesa, and Blue Andaluza breeds, whereas
the Quail Castellana and Red-barred Vasca breeds were
monomorphic. Our results suggested that the Glu92Lys

Downloaded from http://ps.oxfordjournals.org/ by guest on May 6, 2014

onymous (T212C, G274A, G376A, T398AC, G409A,
A427G, C637T, A644C, and G646A, corresponding to
amino acid changes Met72Thr, Glu92Lys, Val126Ile,
Leu133GlnPro, Ala137Thr, Thr143Ala, Arg213Cys,
His215Pro, and Val216Ile), and 2 were synonymous
(C69T and C834T). With respect to the significant
SNP, 7 had an allelic frequency of 0.5 or greater for some
of the alleles at the E locus. These results indicated a
significant correlation between MC1R polymorphism
and the presence of different alleles at the E locus. All
the populations carrying the E*E or E*R alleles, except
Key words: melanocortin 1 receptor, single nucleotide polymorphism, E locus, plumage color, chicken
mutation may be responsible of the activation of the
receptor for eumelanin production, being necessary but
not sufficient to express the extended black phenotype.
They also suggested that the Arg213Cys mutation may
be the cause of the loss or the decrease of function of
the receptor to produce eumelanin, and the Ala137Thr
mutation may be a candidate to attenuate the Glu-
92Lys effect. The observed co-segregation of the E lo-
cus alleles and polymorphisms in MC1R confirms that
the E locus is equivalent to MC1R.
2014 Poultry Science 93:1089–1096
http://dx.doi.org/10.3382/ps.2013-03611

INTRODUCTION (yellowish-white; Smyth, 1990; Crittenden et al., 1996).

In chickens, the process of domestication from a sin-
gle ancestor, the Red Jungle Fowl (Gallus gallus), has
created a wide variety of plumage colors. The study
and knowledge of genes involved in plumage color have
increased considerably, because these genes could pro-
vide genetic markers useful for the identification of
breeds. There are several genes that control the plum-
age color of chickens. One of the main genes is the
E locus with several different alleles, including (in or-
der of dominance): E*E, extended black; E*R, birch-
en; E*WH, dominant wheaten; E*N, wild type; E*B,
brown; E*BC, buttercup; and E*Y, recessive wheaten
©2014 Poultry Science Association Inc.
Received September 9, 2013.
Accepted February 8, 2014.
1 Corresponding author: sgdavila@inia.es
These alleles affect the distribution of the 2 melanin
pigments (eumelanin and phaeomelanin) in feathers,
and are important for accurate down color sexing.
The E locus was originally mapped on chromosome 1
by linkage analysis (Smyth and Ponce de León, 1992;
Carefoot, 1993).
Molecular studies in several mammalian species have
revealed that the extension locus (similar to the E locus
in birds) encodes the melanocortin 1 receptor (MC1R;
Robbins et al., 1993; Klungland et al., 1995; Kijas et
al., 1998; Väge et al., 1999). The MC1R is a 7-trans-
membrane receptor expressed on melanocytes (Lu et
al., 1994). This receptor influences the induction of the
enzyme tyrosinase, the enzyme responsible for the syn-
thesis of eumelanin and phaeomelanin pigments (Hear-
ing and Tsukamoto, 1991). The activation of the MC1R
receptor results in induction of tyrosinase and increased
eumelanin synthesis (Robbins et al., 1993; Schiöth et

1089
1090 Dávila et al.

al., 1999; Schiöth, 2001). Usually, mutations in the gene
encoding MC1R present in mammals carrying domi-
nant alleles of the extension locus result in an active
receptor associated with black color, whereas mutations
that cause loss in receptor function are associated with
recessive alleles and red-yellow color.
In chickens, the E locus has also been shown to cor-
respond to the MC1R gene (Takeuchi et al., 1996a,b;
Okimoto et al., 1999; Kerje et al., 2003), although it
is not clear which mutations are associated with each
phenotype. The physical and the genetic mapping of
the MC1R gene have been examined by Sazanov et al.
(1998), Okimoto et al. (1999), and Kerje et al. (2003),
and this locus has been assigned to chromosome 11. In
chickens, MC1R encodes a transmembrane protein that
shares 64% identity with its counterpart in mammals.
In populations that carry the E*E allele, Takeuchi et
al. (1996a,b) found the same substitution in MC1R
that has been reported in mouse with black color, and
it seems that this mutation (Glu92Lys) leads an ac-
were associated with different phenotypes. Liu et al.
(2010), analyzing levels of expression of MC1R in a
cross between chickens of black and white plumage col-
or, observed that MC1R expression levels only were sig-
nificantly different at 56 d of age but not at other ages.
This study aimed to investigate the effect of MC1R
sequence on plumage color in chickens. The molecular
characterization of the gene encoding MC1R was ana-
lyzed in Spanish breeds of chickens carrying 6 different
alleles in the E locus (E*E, E*R, E*WH, E*N, E*B,
E*BC), a recessive wheaten (yellowish-white) tester
line (E*Y), and a White Leghorn population that is
heterozygous E*E.
MATERIALS AND METHODS
Chickens
A total of 130 chickens (5 male and 5 female by
population) were randomly selected from 11 Spanish

Downloaded from http://ps.oxfordjournals.org/ by guest on May 6, 2014

tive receptor to produce eumelanin. Kerje et al. (2003) chicken breeds (Campo, 1998), a recessive wheaten
observed that this same mutation may be necessary to (yellowish-white) tester line (E*Y; Smyth, 1990), and a
determine the extended black and the buttercup phe- White Leghorn population (Campo and Jurado, 1982).
notypes in chickens, in addition to another different Twenty-four additional chickens (6 males and 6 females
mutation in each one. Ling et al. (2003) characterized by population) were randomly selected from the Black
6 polymorphic variants of the MC1R derived from dif- Castellana and the Red Villafranquina breeds. The
ferent alleles of the E locus (E*E, E*R, E*WH, E*N, Spanish breeds carried different alleles at the E locus
E*B, E*Y ) and observed that 3 of these variants (E*E, (Table 1), whereas the White Leghorn was heterozy-
E*R, and E*B) share the mutation Glu92Lys, this mu- gous E*E and carried an additional allele in this locus
tation leading to receptor activity independent of the (Campo and Alonso, 2000). The Spanish breeds in-
bound ligand. The E*WH allele does not correspond cluded a synthetic population (Quail Castellana), origi-
to a nonfunctional MC1R receptor (Ling et al., 2003), nated from an F2 cross between the Black Castellana
which is different from what is known in mammals. and the Buff Prat breeds followed by 3 generations of
Tixier-Boichard et al. (2006) found 8 polymorphic selection for eumelanins in feather down color (Campo,
sites corresponding with 4 haplotypes in the coding re- 1991), after which all chicks were completely black with
gion of MC1R, in lines with different plumage color a brown face. All these chicken breeds are maintained
(black, yellow, red, and white). Studies of genetic varia- in a conservation program of genetic resources started
tion of the MC1R gene associated with different plum- in 1975, located at the experimental station of El Encín
age color have also been reported in Chinese breeds. (Madrid, Spain), and they are used for quality produc-
Yang et al. (2008) and Guo et al. (2010) observed abun- tion in free range systems. Pictures of all these popula-
dant polymorphisms in the MC1R gene, some of which tions can be found at http://www.inia.es. The effective
population size ranged from 66 to 310 (Campo et al.,
2002), higher than the minimum size (50) required to
Table 1. Description of the plumage color of the 13 Spanish avoid inbreeding depression in the short term.
breeds of chickens
Gene DNA Isolation
Breed symbol Name
Blood samples were collected by brachial venipunc-
Black Castellana E*E Extended black
Black Menorca E*E Extended black
ture aseptically into tubes using EDTA as anticoagu-
White-faced Spanish E*E Extended black lant. Blood samples were stored at −20°C. Deoxyribo-
Black-barred Andaluza E*E Extended black nucleic acid was extracted from 40 μL of blood using
Blue Andaluza E*E Extended black
Birchen Leonesa E*R Birchen
resuspension buffer (0.1 M Tris-HCl, 0.01 M NaCl, 0.1
Blue Leonesa E*R Birchen M EDTA, pH 8), lysis buffer (0.1 M Tris-HCl, 0.1 M
Buff Prat E*WH Dominant wheaten EDTA, 0.01 NaCl, 1% SDS pH 8), 12.5 µL of proteinase
Red-barred Vasca E*WH Dominant wheaten
Quail Castellana E*WH Dominant wheaten
K (20 mg/mL), 2 mL of 5 M NaCl, 6 mL isopropanol,
Black-breasted Red Andaluza E*N Wild type and resuspension in Tris-EDTA. The DNA was quanti-
Red Villafranquina E*B Brown fied spectrophotometrically, and the concentration was
White Prat E*BC Buttercup
adjusted to 60 ng/μL.
 MELANOCORTIN 1 RECEPTOR AND E LOCUS
Amplification and Sequencing of MC1R
The 1,056-bp fragment containing the 945-bp coding
region of MC1R was amplified. The primers used to
amplify and to sequence the coding region of the MC1R
gene were designed with Primer3 software (Rozen and
Skaletsky, 2000) on the basis of the available nucleotide
sequence of the Red Jungle Fowl (Ling et al., 2003). A
pair of PCR primers: forward (MC1R-F1: 5′-TTTG-
TAGGTGCTGCA GTTGTG-3′) and reverse (MC1R-
R: 5′-CCATCCATCCATCCTCCTGTCTGT-3′) were
used to amplify the MC1R gene. The PCR products
were obtained in a total volume of 10 µL reaction, with
1 µL of DNA (60 ng/µL), 1 µM of forward and reverse
primers, 1.5 mM MgCl2, 200 µM deoxynucleoside tri-
phosphate, 5% DMSO (dimethyl sulfoxide), 1 M beta-
ine, and 0.02 U/µL Taq polymerase. The amplification
involved first denaturation at 94°C for 3 min, 30 cycles
of denaturation at 94°C for 1 min, annealing at primer-
specific temperature 58°C for 1 min, and extension at
72°C for 1 min, followed by final extension at 72°C for
10 min. The PCR amplification was detected by 1%
agarose gels stained with 0.25 µg/mL of ethidium bro-
mide.
The PCR products were purified using Ilustra Exo-
Star 1-step (General Electric Company) and sequenced
with BigDye Terminator v3.1 (Secugen S.L.). Three se-
quencing primers, forward (MC1R-F1), reverse (MC1R-
R), and a second forward primer (MC1R-F2: 5′-GGAC-
CGCTACATCACCATCT-3′), were used to cover the
entire MC1R coding region.
Analysis of MC1R
Sequences were edited with the CHROMAS 2.33
software (Technelysium Pty Ltd., South Brisbane, Aus-
tralia) to identify heterozygous individuals. Then, they
were aligned using the MEGA4 package (Tamura et al.,
2007). The SAS statistical package, FREQ procedure,
was used to calculate the allele and genotype frequen-
cies for data analysis with the chi-squared test (SAS
Institute Inc., Cary, NC). The null hypothesis for the
chi-squared test was that the frequency distribution of
the 3 genotypes for each SNP was not associated with
the alleles at the E locus. The polymorphism infor-
mation content was estimated using FSTAT software
(Goudet, 2001). Estimated haplotypes were obtained
by the Bayesian statistic method implemented in the
PHASE 2.1 software (Stephens et al., 2001; Stephens
and Scheet, 2005).
RESULTS
SNP in the MC1R Gene
A total of 12 SNP were detected in the coding region
of MC1R across all populations (Table 2). The number
of animals for each allele at the E locus is included in
this table (for the E*WH allele, there were 2 pieces of
1091
missing data for the A427G SNP). Distribution of ge-
notypic frequencies for each SNP is included in Table
2. Results were significant for 11 SNP (at the 0.1%
level for 10 SNP). Nine were nonsynonymous (T212C,
G274A, G376A, T398AC, G409A, A427G, C637T,
A644C, and G646A) corresponding to amino acid
changes: Met72Thr, Glu92Lys, Val126Ile, Leu133Gl-
nPro, Ala137Thr, Thr143Ala, Arg213Cys, His215Pro,
and Val216Ile), and 2 were synonymous (C69T and
C834T). All the SNP were biallelic, except the SNP at
position T398AC, which had 3 nucleotides segregating.
The linkage between the 2 polymorphisms at positions
C69T and T212C is different in the E*B allele than in
the other alleles that segregate those polymorphisms.
With respect to the significant SNP, 7 had an allelic
frequency of 0.5 or greater for some of the alleles at the
E locus (Table 3). A low level (<0.2) of polymorphism
was found for 2 SNP (T398AC and G409A), and a me-
dium level (0.2–0.5) of polymorphism was found for the
remaining 5 SNP. The significant SNP with allelic fre-
Downloaded from http://ps.oxfordjournals.org/ by guest on May 6, 2014
quency of 0.5 or greater (the former 7 plus the T212C
and G376A) observed for each population are also indi-
cated in Table 3. A medium level of polymorphism was
found for these 2 additional SNP. All the populations
carrying the E*E or E*R alleles, except the Birchen Le-
onesa, had the G274A polymorphism (Black Castella-
na, Black Menorca, White-faced Spanish, Black-barred
Andaluza, Blue Leonesa, Blue Andaluza, and White
Leghorn). Three of these populations (Black Castel-
lana, White-faced Spanish, and Blue Andaluza) had 1
or 2 additional SNP.
MC1R Haplotypes
The MC1R haplotypes (Table 4) were made in the
coding region of the MC1R gene with 7 of the signifi-
cant SNP (C69T, T212C, G274A, T398AC, G409A,
A427G, and C637T). There were 11 haplotypes, with
frequency of 0.5 or greater in at least one population,
compared with the haplotype of the Red Jungle Fowl
(H0).
The frequency of haplotypes of the MC1R gene in the
different alleles of the E locus are indicated in Table
5. The number of haplotypes was maximum for the
E*E allele and minimum for the E*N and E*Y alleles.
The H1 haplotype was predominantly associated with
the E*E and E*R alleles, and it was not found in the
E*WH, E*N, E*B, and E*Y alleles. The H3 and H6
haplotypes were exclusively associated with the E*E
and E*R alleles, respectively, whereas the H8 and H9
haplotypes were exclusively associated with the E*B al-
lele. Finally, the H0, H7, H9, H10, and H11 haplotypes
were predominantly associated with the E*N, E*WH,
E*B, E*BC, and E*Y alleles, respectively.
The frequency of haplotypes of the MC1R gene in the
13 Spanish breeds, the tester line, and the White Leg-
horn population are also indicated in Table 5. The num-
ber of haplotypes was greatest for the Black Menorca,
Birchen Leonesa, and Blue Andaluza breeds, whereas
1092 Dávila et al.
Table 2. Genotypic frequencies and chi-squares (χ2), with the degrees of freedom and P-value, for each SNP1 of the MC1R gene in
the different alleles of the E locus, as compared as the E*N allele of the Red Jungle Fowl
SNP Genotype E*E E*R E*WH E*N E*B E*BC E*Y Item

C69T CC 0.57 0.45 1.00 1.00 0.00 0.90 1.00 χ2 = 95.6

CT 0.27 0.45 0.00 0.00 0.08 0.00 0.00 df = 12
TT 0.16 0.10 0.00 0.00 0.92 0.10 0.00 P < 0.001
T212C TT 0.57 0.45 1.00 1.00 0.92 0.90 1.00 χ2 = 43.1
TC 0.27 0.45 0.00 0.00 0.08 0.00 0.00 df = 12
CC 0.16 0.10 0.00 0.00 0.00 0.10 0.00 P < 0.001
G274A GG 0.02 0.15 1.00 1.00 0.00 0.60 1.00 χ2 = 137.0
GA 0.24 0.30 0.00 0.00 0.00 0.10 0.00 df = 12
AA 0.74 0.55 0.00 0.00 1.00 0.30 0.00 P < 0.001
G376A GG 0.37 0.55 1.00 1.00 0.92 1.00 1.00 χ2 = 63.7
GA 0.40 0.40 0.00 0.00 0.08 0.00 0.00 df = 12
AA 0.23 0.05 0.00 0.00 0.00 0.00 0.00 P < 0.001
T398AC TT 0.82 0.60 0.97 1.00 1.00 0.20 1.00 χ2 = 145.1
TA/TC 0.18 0.25 0.03 0.00 0.00 0.50 0.00 df = 24
AA/CC 0.00 0.15 0.00 0.00 0.00 0.30 0.00 P < 0.001
G409A GG 1.00 1.00 1.00 1.00 0.17 1.00 1.00 χ2 = 126.6
GA 0.00 0.00 0.00 0.00 0.08 0.00 0.00 df = 12
AA 0.00 0.00 0.00 0.00 0.75 0.00 0.00 P < 0.001
A427G AA 0.98 1.00 0.32 1.00 1.00 0.30 1.00 χ2 = 97.6
AG 0.02 0.00 0.00 0.00 0.00 0.00 0.00 df = 12
GG 0.00 0.00 0.68 0.00 0.00 0.70 0.00 P < 0.001

Downloaded from http://ps.oxfordjournals.org/ by guest on May 6, 2014

C637T CC 0.60 0.25 1.00 0.80 0.00 0.90 0.00 χ2 = 103.1
TC 0.27 0.60 0.00 0.20 0.08 0.00 0.50 df = 12
TT 0.13 0.15 0.00 0.00 0.92 0.10 0.50 P < 0.001
A644C AA 1.00 1.00 1.00 1.00 0.84 1.00 1.00 χ2 = 23.9
AC 0.00 0.00 0.00 0.00 0.08 0.00 0.00 df = 12
CC 0.00 0.00 0.00 0.00 0.08 0.00 0.00 P = 0.021
G646A GG 1.00 0.70 1.00 1.00 1.00 1.00 1.00 χ2 = 29.4
GA 0.00 0.30 0.00 0.00 0.00 0.00 0.00 df = 6
AA 0.00 0.00 0.00 0.00 0.00 0.00 0.00 P < 0.001
C663T CC 0.92 1.00 1.00 1.00 1.00 1.00 1.00 χ2 = 7.7
CT 0.06 0.00 0.00 0.00 0.00 0.00 0.00 df = 12
TT 0.02 0.00 0.00 0.00 0.00 0.00 0.00 P = 0.811
C834T CC 0.55 0.70 0.93 1.00 1.00 0.10 1.00 χ2 = 49.8
CT 0.32 0.30 0.07 0.00 0.00 0.60 0.00 df = 12
TT 0.13 0.00 0.00 0.00 0.00 0.30 0.00 P < 0.001
Number of birds 52 20 28 10 12 10 10  
1Each SNP is associated with a given position in the protein: Asn23Asn, Met72Thr, Glu92Lys, Val126Ile, Leu133GlnPro, Ala137Thr, Thr143Ala,
Arg213Cys, His215Pro, Val216Ile, Ser221Pro, Asn278Asn, respectively.

the Quail Castellana and Red-barred Vasca breeds were
monomorphic. The 3 breeds with extended black phe-
notype (Black Castellana, Black Menorca, and White-
Faced Spanish) were associated predominantly with the
H1 haplotype. The other 2 breeds with black pigment
(Black-barred Andaluza and Birchen Leonesa) showed
mainly the H4 and H5 haplotypes, respectively, where-
as the 2 breeds with blue pigment (Blue Andaluza and
Blue Leonesa) were mainly associated with the H4 and
H1 haplotypes, respectively. The Black-breasted Red
Andaluza and the Buff Prat showed mainly the H0 and
H7 haplotypes, respectively, whereas these haplotypes
were fixed in the Quail Castellana and the Red-barred
Vasca. The most frequent haplotypes observed in the
2 remaining Spanish breeds (Red Villafranquina and
White Prat) were the H9 and H10. Finally, the tester
line and the White Leghorn population were associated
with the H11 and H1 haplotypes, respectively.
DISCUSSION
In this study of the E locus at molecular level, in
relation with the influence of the alleles at the E locus
in the structure of the MC1R, we observed consider-
able genetic diversity at the MC1R locus: 12 SNP were
detected, 11 being significant and 9 nonsynonymous
(suggesting that selection has taken place on MC1R
genotypes). Selection on MC1R alleles has been shown
across galliform species by Nadeau et al. (2007). In the
current work, the E locus is defined according to the
breed phenotype, but some similar phenotypes, par-
ticularly the black ones, may be associated with differ-
ent SNP and exhibit variable frequencies of haplotypes.
The MC1R should be affected by mutations associated
with the individual alleles, if the MC1R gene encodes
the E locus. Our data confirm this association with
the 7 alleles at the E locus (E*E, E*R, E*WH, E*N,
E*B, E*BC, E*Y). They also suggest that the plum-
age color traits are affected by the SNP variation in
the MC1R gene. In agreement with this fact, Takeuchi
et al. (1996b) found some variants of the MC1R gene
associated with the E locus, proposing that they might
be linked with feather pigmentation, whereas Okimoto
et al. (1999) and Ellett and Okimoto (2000) indicated
a close linkage between MC1R polymorphism and the
E locus.
 MELANOCORTIN 1 RECEPTOR AND E LOCUS 1093
Table 3. Single nucleotide polymorphisms1 with allelic frequency of 0.5 or greater (in brackets) for the alleles of the E locus, and the 13 Spanish breeds, the tester line, and the Takeuchi et al. (1996a,b) found the Glu92Lys mu-
tation in chickens that were homozygous for the E*E

Asn (0.70)

Asn (0.60)
C834T allele, and Kerje et al. (2003) found that the Glu92Lys
—
—
—

—
—
—

—
—
—

—
—
—
—
—
substitution is the most likely causative mutation for
the E*E allele. The Glu92Lys mutation appears mainly
in our birds carrying the E*E allele, and consequently

Cys (0.96)

Cys (0.75)
this mutation may be responsible of the activation of
C637T

the receptor for eumelanin production. Additionally,

—
—
—
—
—

—
—
—

—
—
—
—
—
—
—
the Met72Thr and the Arg213Cys mutations were ob-

SNP is associated with a given position in the protein: Asn23Asn, Met72Thr, Glu92Lys, Val126Ile, Leu133GlnPro, Ala137Thr, Thr143Ala, Arg213Cys, Asn278Asn.
served by Takeuchi et al. (1996a,b) and Kerje et al.
(2003) in birds carrying the E*E allele. These previ-

Ala (1.00)
Ala (0.68)
Ala (1.00)
Ala (1.00)
ous results suggested that the mutation Glu92Lys was
A427G

—
—
—
—
—

—
—
—

—
—
—

—
—
not solely associated with the black phenotype. Our
results suggest that the Glu92Lys sequence is neces-
sary but not sufficient to express the extended black
Thr (0.79)
phenotype, because we find this mutation in the Red
G409A

Villafranquina breed associated with other SNP. The

—
—
—
—
—

—
—
—

—
—
—

—
—
—
—
—

Glu92Lys sequence can be modified or not by the bar-

ring (Black-barred Andaluza), blue (Blue Andaluza),
or dominant white (White Leghorn) genes, all breeds

Downloaded from http://ps.oxfordjournals.org/ by guest on May 6, 2014

Pro (0.55)
Gln (0.60)

carrying the Glu92Lys mutation without or with other

T398AC

additional sequences. This mutation had the highest

—
—
—
—
—

—
—
—
—

—
—
—
—
—

polymorphism information content, which is favorable

for the genetic improvement of relevant traits.
Okimoto et al. (1999), Ellett and Okimoto (2000),
Tixier-Boichard et al. (2000), and Ling et al. (2003)
Ile (0.60)

Ile (0.70)
G376A

found the Glu92Lys mutation in breeds with the E*E

—

—
—
—
—

—
—
—

—
—
—
—
—

allele, but also in those carrying E*R. Similarly, this

mutation was predominant in our E*R birds. Ellett and
Okimoto (2000) found that the E*R allele had the Glu-
(0.86)
(0.90)
(0.80)
(0.90)
(0.90)

Lys (1.00)
(0.60)
(0.80)
(0.70)

Lys (0.90)

92Lys mutation depending on the breed. They found

G274A

—
—

—
—
—
—
—

that the E*R allele without Glu92Lys mutation was

Lys
Lys
Lys
Lys
Lys
Lys
Lys
Lys

carrying the Leu133Gln substitution. Our results sug-

gesting that the birchen phenotype has at least 2 differ-
ent SNP associated with it. These 2 alleles may give 2
Thr (0.60)

different phenotypes (black breasted or black breasted

T212C

—
—
—
—

—
—

—
—
—

—
—

—
—
—
—
—
—

with lacing in white or gold), traditionally assigned to

the effect of the Columbian restriction gene (Smyth,
1990). The Leu133Gln substitution was not predomi-
3This is also the genotype of the Red Jungle Fowl reference genome.

nant in our study for the E*R birds, although almost

Asn (0.60)

Asn (0.96)

all the E*R birds that were heterozygous for the SNP
C69T

—
—
—
—

—
—

—
—

—
—

—
—
—
—
—
—

associated with this substitution were heterozygous for

the SNP associated with the Glu92Lys substitution,
by previous studies in Mendelian genetics.

and all the E*R birds that did not have the Glu92Lys
substitution were homozygous for the SNP associated
with the Leu133Gln polymorphism. This may indicate
Black-breasted Red Andaluza

that Leu133Gln substitution, located in a highly con-

served region at the third transmembrane domain of
Black-barred Andaluza

MC1R, could be involved in the production of a consti-

White-faced Spanish

Red Villafranquina

tutively active receptor associated with the E*R allele.

Red-barred Vasca
Black Castellana

Quail Castellana
Birchen Leonesa
White Leghorn
Black Menorca

Blue Andaluza

Our results did not find the Glu92Lys mutation in

White Leghorn population

Blue Leonesa

White Prat

chickens carrying the E*N allele, this mutation being

Population

Tester line
Buff Prat

necessary to maintain the receptor in an active confor-

mation (Robbins et al., 1993), in agreement with the
results reported by Takeuchi et al. (1996b), Ling et al.
 

(2003), and Tixier-Boichard et al. (2006). On the oth-

2Identified

er hand, Takeuchi et al. (1996b), Ellett and Okimoto

E locus2

1Each
E*WH

(2000), and Kerje et al. (2003) found the Arg213Cys

E*BC
E*N3
E*R

E*Y
E*B
E*E

polymorphism in E*N birds. This substitution was not

 
 
 
 
 
 

 
 

 
 
 
1094 Dávila et al.
Table 4. Haplotypes (H1–H11) for 7 SNP1 in the MC1R gene (with frequency of 0.5 or greater in at
least one population), as compared as the H0 haplotype of the Red Jungle Fowl
H C69T T212C G274A T398AC G409A A427G C637T

H0 C T G T G A C
Asn Met Glu Leu Ala Thr Arg
H1 * * A * * * *
Lys
H2 * * A * * * T
Lys Cys
H3 T C A * * * *
Asn Thr Lys
H4 T C A * * * T
Asn Thr Lys Cys
H5 * * * A * * *
Gln
H6 * * * A * * T
Gln Cys
H7 * * * * * G *
Ala
H8 T * A * * * T
Asn Lys Cys
H9 T * A * A * T
Asn Lys Thr Cys
H10 * * * C * G *

Downloaded from http://ps.oxfordjournals.org/ by guest on May 6, 2014

Pro Ala
H11 * * * * * * T
Cys
1Each SNP is associated with a given position in the protein: Asn23Asn, Met72Thr, Glu92Lys, Leu133GlnPro,
Ala137Thr, Thr143Ala, Arg213Cys, respectively.
*Indicates that the nucleotide and the amino acid were identical to that of H0.

found in our study for the E*N allele, and thus it is
expected to have only minor phenotypic effect.
Ellett and Okimoto (2000) found the Thr143Ala mu-
tations in a breed with the E*N allele and in wheaten
breeds (E*WH and E*Y). In agreement with this fact,
our results indicate that the Quail Castellana breed
is E*N and H0 haplotype, even though this breed is
carrying the E*WH allele at the E locus (Campo and
Orozco, 1986). However, we found the Thr143Ala SNP
in birds carrying the E*WH allele, but this mutation
was not found in those with the E*Y allele, suggest-
ing that dominant and recessive wheaten could differ
in their sequence and their degree of Mendelian domi-
nance. Besides, we found the Arg213Cys in birds car-
rying the E*Y allele, but this mutation was not found
in birds carrying the E*WH allele, suggesting again the
difference between dominant and recessive wheaten in-
dicated above. Previous work did not support the Ar-
g213Cys variant as being associated with the E*Y al-
lele. As indicated before, Takeuchi et al. (1996b), Ellett
and Okimoto (2000), and Kerje et al. (2003) identified
this mutation in E*N lines and Red Jungle Fowl acces-

Table 5. Haplotypes of the MC1R gene (with frequency of 0.5 or greater in at least 1 population) in the different alleles of the E
locus, and the 13 Spanish breeds, the tester line, and the White Leghorn population
E locus1 Population H0 H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11

E*E   0.02 0.55 0.02 0.07 0.22 0.08 — 0.01 — — — 0.03

  Black Castellana — 0.50 — 0.16 0.25 0.08 — — — — — —
  Black Menorca 0.01 0.52 0.11 0.11 0.14 0.03 — — — — — —
  White-faced Spanish 0.02 0.82 — 0.03 — 0.08 — — — — — —
  Black-barred Andaluza 0.09 0.40 0.01 0.01 0.49 — — — — — — —
  Blue Andaluza 0.03 0.09 — 0.10 0.45 0.26 — 0.03 — — — —
  White Leghorn — 0.85 — — — — — — — — — 0.15
E*R   0.03 0.37 — — 0.32 0.15 0.13 — — — — —
  Birchen Leonesa — 0.25 — — 0.20 0.30 0.25 — — — — —
  Blue Leonesa 0.05 0.50 — — 0.45 — — — — — — —
E*WH   0.32 — — — — — — 0.66 — — 0.02 —
  Buff Prat — — — — — — — 0.95 — — 0.05 —
  Red-barred Vasca — — — — — — — 1.00 — — — —
  Quail Castellana 1.00 — — — — — — — — — — —
E*N2 Black-breasted Red 0.90 — — — — — — — — — — 0.10
Andaluza
E*B Red Villafranquina — — 0.01 — 0.01 — — — 0.14 0.77 — —
E*BC White Prat — 0.20 — — 0.05 — — 0.15 — — 0.45 —
E*Y Tester line 0.25 — — — — — — — — — — 0.75
1Identified by previous studies in Mendelian genetics.
2The haplotype of the Red Jungle Fowl reference genome is H0.
 MELANOCORTIN 1 RECEPTOR AND E LOCUS
sions, although the Red Jungle Fowl utilized by Ling
et al. (2003) did not have the Arg213Cys mutation.
Takeuchi et al. (1996b) and Ellett and Okimoto (2000)
did not observe the Arg213Cys mutation in E*Y birds,
although they used as carriers of the E*Y allele the
Nagoya, the Buff Menorca, and the Rhode Island Red
breeds, instead of the recessive tester line utilized in our
study. Although, Ellett and Okimoto (2000) associated
the Thr143Ala mutation with restriction of black pig-
ment in the feathers of wheaten birds, it is necessary
to bear in mind that other nonextended black alleles
in the E locus also produce a restriction in eumelanin.
On the other hand, it may be speculated that the Arg-
213Cys mutation of the E*Y birds may be the cause of
the loss of function of the receptor to produce eumela-
nin, although the decrease in functional MC1R may be
regulatory, most likely transcriptional regulation.
In agreement with the results of Okimoto et al.
(1999), Ellett and Okimoto (2000), Ling et al. (2003),
and Tixier-Boichard et al. (2006) for the E*B allele, we
found fixation of the Glu92Lys mutation in E*B birds.
Ellett and Okimoto (2000) indicated that the E*B se-
quence also had the His215Pro substitution. The last
result has not been obtained in our experiment. The
His215Pro appeared only at low frequency in our E*B
birds; they instead had the Ala137Thr mutation, sug-
gesting that Ala137Thr may be a candidate to attenu-
ate the Glu92Lys mutation, producing a brown instead
of a black bird, because Ala137Thr is specific to the
Red Villafranquina breed. It could be of interest that
Ellett and Okimoto (2000) used the E*B tester line
(Smyth, 1990), whereas the origin of our E*B allele was
the Red Villafranquina breed.
Kerje et al. (2003) found that the E*BC allele shared
the Glu92Lys and Met71Thr substitutions with the
E*E allele. They also found a third nonsynonymous
mutation (His215Pro) associated with the E*BC allele.
In our study, we found another 3 different mutations
(Leu133Pro, Thr143Ala, and Arg213Cys) instead of
those reported by these authors.
The Ala137Thr mutation appears only in the H9
haplotype. This haplotype (the only new one) is that of
the Red Villafranquina breed, and it has not been pre-
viously described, although Guo et al. (2010) included
the Ala137Thr mutation in their study. New functional
data would be very interesting on the Red Villafran-
quina breed to better characterize this haplotype. The
distribution of MC1R haplotypes showed that each of
them was predominantly associated with 1 allele at the
E locus (exclusively for the H9 and the E*B allele).
With respect to the number of haplotypes found in our
study for MC1R, some breeds (Black-barred Andaluza,
Black Menorca, Birchen Leonesa, and Blue Andaluza)
were more polymorphic that others for the MC1R hap-
lotypes.
The correspondences found in our study between
the SNP in each breed and each allele showed that the
Black Castellana, Black Menorca, White-faced Spanish,
Black Barred Andaluza, and Blue Leonesa are associ-
1095
ated with the E*E allele, whereas the Birchen Leonesa
is associated with E*R and the Black-breasted Red An-
daluza and the Quail Castellana are associated with
E*N. Finally, the Buff Prat and the Red-barred Vasca
are associated with E*WH, the Red Villafranquina
with E*B, the White Prat with E*BC, and the tester
line with E*Y. These correspondences are in agreement
with the results obtained by Mendelian genetic analy-
ses in previous work by our department, with the ex-
ception of the Blue Leonesa and the Quail Castellana,
whose sequences were associated with E*E and E*N,
instead of E*R and E*WH, respectively.
This work investigated the different mutations re-
sponsible for the respective phenotype by sequencing of
the coding region of the MC1R gene from 15 different
populations displaying different plumage color, increas-
ing the number of breeds and phenotypes analyzed for
the E locus alleles. Our results, based on a large sample
of sequenced individuals, indicate a significant correla-
tion between MC1R polymorphism and the presence
Downloaded from http://ps.oxfordjournals.org/ by guest on May 6, 2014
of different alleles at the E locus. This co-segregation
of the E locus alleles and polymorphisms in the MC1R
gene suggests that the E locus is equivalent to MC1R.
The MC1R locus alone cannot explain all the variation
in pigmentation, but it is necessary to know the geno-
types at this locus to monitor feather color in a breed.
On the other hand, the expression of MC1R can be dif-
ferent in chick down, juvenile, postjuvenile, and adult
plumage colors.
REFERENCES
Campo, J. L. 1991. Use of the sex-linked barring (B) gene for chick
sexing on an eumelanotic columbian background. Poult. Sci.
70:1469–1473.
Campo, J. L. 1998. Conservation and genetical study of Spanish
chicken breeds. Pages 155–158 in Proc. 6th World Congr. Genet.
Appl. Livestock Prod., Armidale, Australia. Univ. New England,
Armidale, New South Wales, Australia.
Campo, J. L., and M. A. Alonso. 2000. Segregation of hypostatic
plumage colour genes in five strains of White Leghorn. CD in
Proc. XXI World’s Poultry Congress, Montreal, Canada.
Campo, J. L., M. G. Gil, and S. G. Dávila. 2002. El programa de
conservación de las razas españolas de gallinas. Pages 183–191 in
Proc. V Congreso Sociedad Española Recursos Genéticos Ani-
males, Madrid, Spain. P y Punto, Madrid, Spain.
Campo, J. L., and J. J. Jurado. 1982. Evaluation of multiple trait
selection in strains of layers. Pages 869–874 in Proc. 2nd World
Congr. Genet. Appl. Livest. Prod., Madrid, Spain. Editorial Gar-
si, Madrid, Spain.
Campo, J. L., and F. Orozco. 1986. Genetic basis of the Melanotic
Prat phenotype. Br. Poult. Sci. 27:361–367.
Carefoot, W. C. 1993. Further studies of linkage and mappings of
the loci of genes in group 3 on chromosome 1 of the domestic
fowl. Br. Poult. Sci. 34:205–209.
Crittenden, L. B., J. J. Bitgood, D. W. Burt, F. A. Ponce de Leon,
and M. Tixier-Boichard. 1996. Nomenclature for naming loci, al-
leles, linkage groups and chromosomes to be used in poultry ge-
nome publications and databases. Genet. Sel. Evol. 28:289–297.
Ellett, A. E., and R. Okimoto. 2000. Melanocortin 1-receptor
(MC1R-R) gene polymorphisms associated with chickens E locus
alleles. University Student Journal Inquiry 1:37–41.
Goudet, J. 2001. FSTAT, a program to estimate and test gene diver-
sities and fixation indices, version 2.9.3. Accessed Oct. 15, 2012.
http://www2.unil.ch/popgen/softwares/fstat.htm.
1096 Dávila et al.
Guo, X. L., X. L. Li, Y. Li, Z. L. Gu, C. S. Zheng, Z. H. Wei, J.
S. Wang, R. Y. Zhou, L. H. Li, and H. Q. Zheng. 2010. Genetic
variation of chicken MC1R gene in different plumage colour pop-
ulations. Br. Poult. Sci. 51:734–739.
Hearing, V. J., and K. Tsukamoto. 1991. Enzymatic control of pig-
mentation in mammals. FASEB J. 5:2902–2909.
Kerje, S., J. Lind, K. Schütz, P. Jensen, and L. Andersson. 2003.
Melanoortin 1-receptor (MC1R) mutations are associated with
plumage colour in chicken. Anim. Genet. 34:241–248.
Kijas, J. M. H., R. Wales, A. Törnsten, P. Chardon, M. Moller, and
L. Andersson. 1998. Melanocortin receptor 1 (MC1R) mutations
and coat color in pigs. Genetics 150:1177–1185.
Klungland, H., D. I. Väge, L. Gomez-Raya, S. Adalsteinsson, and S.
Lien. 1995. The role of melanocyte-stimulating hormone (MSH)
receptor in bovine coat color determination. Mamm. Genome
6:636–639.
Ling, M. K., M. C. Lagerström, R. Fredriksson, R. Okimoto, N. I.
Mundy, S. Takeuchi, and H. B. Schiöth. 2003. Association of
feather colour with constitutively active melanocortin 1 receptors
in chicken. Eur. J. Biochem. 270:1441–1449.
Liu, W. B., S. R. Chen, J. X. Zheng, L. J. Qu, G. Y. Xu, and N.
Yang. 2010. Developmental phenotypic-genotypic associations of
tyrosinase and melanortin 1 receptor genes with changing profiles
in chickens plumage pigmentation. Poult. Sci. 89:1110–1114.
Lu, D., D. Willard, I. R. Patel, S. Kadwell, L. Overton, T. Kost,
M. Luther, W. Chen, R. P. Woychik, W. O. Wilkison, and R. D.
Cone. 1994. Agouti protein is an antagonist of the melanocyte-
stimulating-hormone receptor. Nature 371:799–802.
Nadeau, N. J., T. Burke, and N. I. Mundy. 2007. Evolution of an
avian pigmentation gene correlates with a measure of sexual se-
lection. Proc. Biol. Sci. 274:1807–1813.
Okimoto, R., J. T. Stie, S. Takeuchi, W. S. Payne, and D. W. Salter.
1999. Mapping the melanocortin 1-receptor (MC1R-R) gene and
association of MC1-R polymorphisms with E locus phenotypes.
Poult. Sci. 78 (Suppl. 1):60. (Abstr.)
Robbins, L. S., J. H. Nadeau, K. R. Johnson, M. A. Kelly, and L.
Roselli-Rehfuss. 1993. Pigmentation phenotypes of variant exten-
sion locus alleles result from point mutations that alter MSH
receptor function. Cell 72:827–834.
Rozen, S., and H. Skaletsky. 2000. Primer3 on the WWW for gen-
eral users and for biologist programmers. Methods Mol. Biol.
132:365–386.
Sazanov, A., J. Masabanda, D. Ewald, S. Takeuchi, M. Tixier-Boi-
chard, J. Buitkamp, and R. Fries. 1998. Evolutionarily conserved
telomeric location of BBC1 and MC1R on a microchromosome
questions the identity of MC1R and a pigmentation locus on
chromosome 1 in chicken. Chromosome Res. 6:651–654.
Schiöth, H. B. 2001. The physiological role of melanocortin recep-
tors. Vitam. Horm. 63:195–232.
Schiöth, H. B., S. R. Phillips, R. Rudzish, M. A. Birch-Machin,
J. E. S. Wikberg, and J. L. Rees. 1999. Loss of function muta-
tions of the human melanocortin 1 receptor are common and
are associated with red hair. Biochem. Biophys. Res. Commun.
260:488–491.
Smyth, J. R., Jr. 1990. Genetics of plumage, skin and eye pigmenta-
tion in chickens. Pages 118–224 in Poultry Breeding and Genet-
ics. R. D. Crawford, ed. Elsevier, Amsterdam, the Netherlands.
Smyth, J. R., Jr., and F. A. Ponce de León. 1992. Linkage relation-
ship between the pea comb (P) and extended black (E) loci of the
chicken. Poult. Sci. 71:208–210.
Stephens, M., and P. Scheet. 2005. Accounting for decay of linkage
disequilibrium in haplotype inference and missing data imputa-
tion. Am. J. Hum. Genet. 76:449–462.
Stephens, M., N. J. Smith, and P. Donnelly. 2001. A new statistical
method for haplotype reconstruction from population data. Am.
J. Hum. Genet. 68:978–989.
Takeuchi, S., H. Suzuki, S. Hirose, M. Yabuuchi, C. Sato, H. Yama-
moto, and S. Takahashi. 1996a. Molecular cloning and sequence
analysis of the chick melanocortin 1-receptor gene. Biochim.
Biophys. Acta 1306:122–126.
Takeuchi, S., H. Suzuki, M. Yabuuchi, and S. Takahashi. 1996b.
A possible involvement of melanocortin 1-receptor in regulating
Downloaded from http://ps.oxfordjournals.org/ by guest on May 6, 2014
feather color pigmentation in the chicken. Biochim. Biophys.
Acta 1308:164–168.
Tamura, K., J. Dudley, M. Nei, and S. Kumar. 2007. MEGA4: Mo-
lecular Evolutionary Genetics Analysis (MEGA) software version
4.0. Mol. Biol. Evol. 24:1596–1599.
Tixier-Boichard, M., J. L. Coville, and G. Coquerelle. 2000. Poly-
morphism of the MC1R gene and feather color in the chicken:
Between breeds diversity and within family analysis. Page 342 in
Proc. 27th International Conference an Animal Genetics (ISAG),
MN. ISAG, Minneapolis, MN.
Tixier-Boichard, M., R. Faugeras, J. L. Coville, C. M. Chang, and
G. Coquerelle. 2006. Genotyping test for feather colour genes in
chickens. 2006. CD in XII European Poultry Conference, Verona,
Italy.
Väge, D. I., H. Klungland, D. Lu, and R. D. Cone. 1999. Molecular
and pharmacological characterization of dominant black coat co-
lour in sheep. Mamm. Genome 10:39–43.
Yang, Z. Q., Z. R. Zhang, M. Xu, and Q. Zhu. 2008. Study on
association of melanocortin 1-receptor (MC1R) mutations with
melanin trait in Chinese domestic chickens. Res. J. Anim. Sci.
2:45–49.