Open Journal of Genetics, 2013, 3, 195-200 OJGen
http://dx.doi.org/10.4236/ojgen.2013.33022 Published Online September 2013 (http://www.scirp.org/journal/ojgen/)
Novel intron 2 polymorphism in the melanophilin gene is in
Hardy-Weinberg equilibrium and is not associated with
coat color in goats
Mufliat A. Adefenwa1,2, Brilliant O. Agaviezor1,3, Sunday O. Peters1,4, Matthew Wheto5,
Oludotun J. Ekundayo5, Moses Okpeku6, Bola O. Oboh2, Khalid O. Adekoya2, Christian O. N. Ikeobi5,
Marcos De Donato1, Bolaji N. Thomas7, Ikhide G. Imumorin1*
1Department of Animal Science, Cornell University, Ithaca, USA
2Department of Cell Biology and Genetics, University of Lagos, Lagos, Nigeria
3Department of Animal Science and Fisheries, University of Port Harcourt, Port Harcourt, Nigeria
4Department of Animal Science, Berry College, Mount Berry, USA
5Department of Animal Breeding and Genetics, University of Agriculture, Abeokuta, Nigeria
6Department of Livestock Production, Niger Delta University, Amassomma, Nigeria
7Department of Biomedical Sciences, Rochester Institute of Technology, Rochester, USA
Received 25 January 2013; revised 28 February 2013; accepted 15 March 2013
Copyright © 2013 Mufliat A. Adefenwa et al. This is an open access article distributed under the Creative Commons Attribution Li-
cense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Pigmentation plays important adaptation and physio-
logical efficiency roles in animals. In the sequence of a
648 bp fragment representing intron 1, exon 2, and
part of intron 2 of the MLPH mammalian pigmenta-
tion gene, we identified a novel g.469C > G mutation
in intron 2, and genotyped it in 266 Nigerian goats
using PCR-RFLP analysis. The C allele had frequen-
cies of 0.9625, 0.9804 and 0.97405 in West African
Dwarf (WAD), Sahel (SH) and Red Sokoto (RS)
breeds, respectively. The G allele was the highest in
WAD (0.0375), followed by RS (0.0259 5), and then SH
(0.0196). Overall low FIS and FST and high Nm val-
ues demonstrate little differentiation within and
among the goat breeds at this intronic locus. This
g.469C > G polymorphism in MLPH gene is the first
in any goat breed and also first in Nigerian goats. Our
results suggest that this intronic SNP locus is main-
tained at Hardy-Weinberg equilibrium (P < 0.05) and
the lack of association of this SNP with coat color may
indicate its neutrality in goats.
Keywords: Coat Color; Goats; Melanophilin; Nigeria;
Intron 2; SNP; PCR-RFLP
1. INTRODUCTION
Several coat colors and color patterns are common
among mammals and have evolved through natural and
sexual selection [1]. In many mammals, pigmentation
primarily plays a role in protection against ultraviolet
radiation but may also help in immune system function
[1]. The pigment granules, eumelanin and pheomelanin,
are produced in melanosomes, membrane bound organ-
elles present in melanocytes which are found in the basal
skin layer [2]. Melanosomes gradually develop melanin
as they mature and are translocated from the perinuclear
region to the periphery (dendrites) of the melanocytes
where they are then transferred to neighboring keratino-
cytes in the middle and upper layers of the skin. Skin and
hair colour of many mammals are determined by the
form, contents, transfer and accumulation of melano-
somes in keratinocytes. More deeply-pigmented skins
usually contain a larger and higher number of melano-
somes when compared to light-pigmented skins [3].
Mammalian pigmentation is controlled by several
genes, the products of which form the melanosomal par-
ticles or are involved in their maturation and dispersion.
Many researchers have reported the movement of me-
lanosomes in melanocytes, with defects leading to the
three types of Griscelli syndrome in humans, the ashen,
leaden and dilute phenotype in mice, and the dilute coat
colour phenotype in several animals. They further re-
ported that they are controlled by three major proteins,
which include: myosin VA (MYO5A), Rab27A and
melanophilin (MLPH) [1,4,5]. This interaction between
melanophilin and myosin Va is strengthened by the
presence of a melanocyte-specific exon F in the tail
*Corresponding author.

M. A. Adefenwa et al. / Open Journal of Genetics 3 (2013) 195-200 196
domain of myosin-Va [4,6,7].
Mutations in melanophilin gene have been associated
with coat color dilution in some mammals. Philipp et al.
[8] reported strong associations of single nucleotide
polymorphisms in exon 2 of MLPH and color in dilute
Doberman pinschers, Beagles, and Large munsterlanders
and in exon 7 in dilute German pinschers. Zhou et al. [9]
also found a missense mutation of g.115844A > G in
exon 10 in goat melanophilin gene and inferred from
their results that allele G might be responsible for the tan
color observed in some of the goat breeds in China.
There are no published studies on melanophilin gene in
Nigerian indigenous goats. The three major goat breeds
in Nigeria exhibit specific coat colors except for occa-
sional within breed color variation. The West African
Dwarf is usually black; the Sahel goat is commonly
white while the Red Sokoto is mostly red. In this study,
we identified a novel intronic SNP in the caprine
melanophilin gene and investigated the association of
this polymorphism with coat color and its differentiation
among Nigerian goat populations.
2. MATERIALS AND METHODS
2.1. Animals and DNA Samples
Nigeria is located in West Africa on the Gulf of Guinea
(latitude 10˚00'N, longitude 8˚00'E) with a total area of
923,768 km2. Nigeria is bounded by Niger, Benin and
Cameroon Republics on the North, West and East, re-
spectively. The sample was made up of 266 goats of the
three major breeds of goats in Nigeria; WAD (n = 80),
RS (n = 135) and SH (n = 51) collected from farms and
markets across Nigeria, according to the geographical
distribution of the breeds published by Blench [10] with
locations shown in Figure 1. Blood samples were col-
lected by jugular venipuncture from individual animals
and genomic DNA from collected blood samples were
isolated using ZymoBeadTM Genomic DNA kit (Zymo
Research Corporation, Irvine, CA, USA) following the
manufacturer’s instructions. Quantification of DNA yield
and assessment of quality were done using a Nanodrop
ND-100 UV/Vis Spectrophotometer (Nanodrop Tech-
nologies, Inc., Wilmington, DE, USA).
2.2. Isolation and Sequencing of Melanophilin
Gene Fragments
Using information from Feng et al. [11], a pair of PCR
primers (F: 5’-CGTGGGTTCCCTTATTTTGAC-3’ and
R: 5’-ATCCTGGCTTCTGGGTGTTG-3’) was synthe-
sized to amplify a 648 bp fragment spanning intron 1,
exon 2 and part of intron 2. PCR amplifications were
carried out in a C1000TM Thermal Cycler (Biorad, Her-
cules, CA, USA) in a total reaction volume of 20 µL
containing approximately 50 ng DNA, 10 pmol of each
primer in AccuPowerTM PCR Premix (Bioneer Corpora-
tion, Alameda, CA, USA). The PCR cycling conditions
are as follows: denaturation at 95˚C for 4 min, followed
by 35 amplification cycles of denaturation at 94˚C for 30
Figure 1. Sites of sampling collection.

M. A. Adefenwa et al. / Open Journal of Genetics 3 (2013) 195-200 197
s, annealing at 56˚C for 30S, extension at 72˚C for 3 min,
followed by an extended elongation at 72˚C for 10 min.
PCR products were detected on 1.5% agarose gel in-
cluding ethidium bromide, and photographed under UV
light. Sequencing of the amplified fragments was carried
out using the same PCR primers with the Applied Bio-
systems Automated 3730 DNA Analyzer (Applied Bio-
sytems, Carlsbad, CA, USA) using Big Dye Terminator
chemistry and AmpliTaq-FS DNA Polymerase.
2.3. Genotyping of Goat MLPH Gene by
PCR-RFLP
In order to identify SNPs of MLPH gene in Nigerian
goats, sequences derived from 20 animals were aligned
using ClustalW program
(http://www.ebi.ac.uk/Tools/clustalw/). The transversion
469C > G, according to EU316218, was detected. This
SNP was analyzed by PCR-RFLP in a total of 266 goats.
HinP1l restriction enzyme (Fermentas Life Sciences,
Glen Burnie, MD, USA), recognizing the palindromic
tetranucleotide sequence G↓CGC was used for restriction
enzyme digestion according to NEBcutter V2.0. Aliquots
of 15 µL PCR products, including 2 µL 10X buffer with
BSA, were digested with 1 µL (10U) HinP1l for 16 h at
37˚C. Thermal inactivation of the restriction enzyme was
thereafter done by incubation at 65˚C for 20 min. The
digested products were detected by electrophoresis on
2.0% agarose gel including 0.5 µg/mL of ethidium bro-
mide.
2.4. Statistical Analysis
Genotypic frequencies, allelic frequencies and Hardy-W-
einberg equilibrium (HWE) test were performed using
POPGENE32 software (version 1.32). Genetic diversity
of each breed, population and genetic differentiation
among different breeds and populations including
Wright’s fixation indices, and Shannon Indices were ob-
tained using the same software. Comparisons of allelic
frequencies among breeds were done using chi-square
analysis. All tests were assessed at a significance level of
5%.
2.5. In-Silico Analyses
In-silico functional analyses were obtained using Splice
Site Prediction by Neural Network and Splicing Regula-
tion Online Graphical Engine (Sroogle).
3. RESULTS
3.1. Goat MLPH Gene Sequences and SNP
Identification
The PCR primers produced a 648 bp fragment (Figure 2),
spanning intron 1, exon 2 and part of intron 2 (GenBank
EU316218). The transversion g.469C > G was identified
in intron 2 from aligning sequences obtained from 20
animals. This mutation was analyzed in a larger number
of animals of the three Nigerian goat breeds using
PCR-RFLP method. Digestion of the PCR products with
HinP1l yielded two fragments (532 and 116 bp) for C
allele and one fragment (648 bp) for G allele. Genotype
CC, CG and GG, therefore, demonstrated two, three and
one band respectively (Figure 3).
Figure 2. Amplified fragments of the Melanophilin gene. The
first lane from the left contains the DNA marker. W = Well
(points that PCR products were loaded) casted on gel. B=
Bands of amplified DNA fragments.
Figure 3. Result of PCR-RFLP by Hinp1I. Digestion of the
PCR products with Hinp1I yielded two fragments (532 and 116
bp) for C allele and one fragment (648 bp) for G allele. Geno-
types CC, CG and GG, therefore, demonstrated two, three and
one band respectively M refers to DNA Marker. Lane 1 illus-
trates genotype GG with one band of 648 bp, lanes 2, 3, 6, 7, 8,
9 and 11 illustrate genotype CC with two bands of 116 bp and
532 bp, and lanes 10 and 12 illustrate genotype CG three bands
of 116 bp, 532 bp and 648 bp. Lanes 4 and 5 contain DNA
marker.

M. A. Adefenwa et al. / Open Journal of Genetics 3 (2013) 195-200 198
3.2. The Distribution of g.469C > G in Nigerian
Goat Breeds
Genotypic frequencies, allelic frequencies and the pro-
bability of departure from HWE of the different goat
breeds are shown in Table 1. Allele C had frequencies of
0.9741, 0.9804 and 0.9625 in RS, SH and WAD goats,
respectively. WAD had the highest frequency (0.0375) of
allele G, followed by RS (0.0259) and then SH (0.0196).
Also, homozygous GG was found only in the RS breed.
The chi-square test based on the allelic frequencies re-
vealed no significant differences between the breeds for
g.469C > G (Table 2). Both WAD and SH breeds appear
to be in HWE for g.469C > G (P < 0.05). The deficit of
homozygote GG individuals in SH and WAD probably
accounts for the deviation from HWE in the RS breed.
Overall, our results suggest that the Nigerian goat popu-
lation is in HWE with respect to this locus (P < 0.05).
3.3. Genetic Diversity and Differentiation of
g.469C > G in Different Nigerian Goat
Breeds
Ta b le s 3 and 4 show the genetic diversity and differen-
tiation of g.469C > G in Nigerian goat breeds. The ob-
served heterozygosity ranged from 0.0370 (RS) to
0.0750 WAD. The Levene and Nei’s expected heterozy-
gosity ranged from 0.0388 (SH) to 0.0726 (WAD) and
0.0384 (SH) to 0.0722 (WAD), respectively. The two
kinds of expected heterozygosity had similar values, with
little difference in observed heterozygosity. There was
however, a somewhat large difference in the observed
heterozygosity from the two kinds of expected het-
erozygosity in RS. Only RS goats (red coat color)
showed a positive value of Wright’s fixation index also
indicating an excess of homozygotes in this breed. The
relative magnitude of genetic variation of g.469C > G for
each breed is WAD >RS >SH based on the Shannon In-
dex value. All three goat breeds exhibited low genetic
diversity at g.469C > G. Ta b l e 4 shows overall low FIS
and FST values and high Nm value.
3.4. Functional In-Silico Analysis
Functional in-silico analysis of the sequenced portion of
the gene using Splice Site Prediction by Neural Network
shows that one of the Splice donor site starts at site 103
and ends at 117 and the other starts at 237 and ends at
251 with scores 0.96 and 0.99, respectively. The acceptor
sites were found to start at 89 and 363 and ended at 129
and 403 respectively with 0.93 and 0.69 scores, re-
spectively. The point mutation g.469C > G was found in
a regulator with 0.0354 score according to the Voelker
down dataset as implemented in Sroogle.
4. DISCUSSION
We have analyzed this novel g.469G > C intronic mu-
tation for possible association with coat color because it
has been found that mutations in intronic regions can
alter gene expression by affecting regulation, impairing
protein synthesis and causing aberrant splicing [12-14].
In-silico functional analysis of this mutation shows that
this mutation is in a splicing regulatory sequence and can
therefore potentially affect splicing of the caprine me-
lanophilin mRNA.
The positive value of Wright’s fixation index observed
in RS breed is probably due to its distribution. This breed
is the most common and most widespread breed of goat
in Nigeria. Its use for high quality leather has had
important positive consequences on the distribution and
propagation of this breed [10]. The relatively low degree
of genetic variation for the SH breed can be attributed to
its restricted geographical distribution. This breed is
Table 1. Genotype and gene frequencies of g.469C > G in Nigerian goat Breeds.
Genotypic Frequencies Allelic Frequencies
Goat Breed Sample Size
CC CG GG C G
HWc
Red Sokoto 135 0.956 0.037 0.007 0.974 0.026 0.001
Sahel 51 0.961 0.039 0.000 0.980 0.020 0.920
West African Dwarf 80 0.925 0.075 0.000 0.963 0.038 0.751
Overall 266 0.947 0.049 0.004 0.972 0.028 0.064
cProbability of departure from Hardy-Weinberg equilibrium.
Table 2. χ2 and P values for differences among the three Nigerian goat breeds based on allelic frequencies.
Red Sokoto Sahel West African Dwarf
Red Sokoto - 0.125 (P = 0.724) 0.459 (P = 0.498)
Sahel - 0.674 (P = 0.412)
West African Dwarf -

M. A. Adefenwa et al. / Open Journal of Genetics 3 (2013) 195-200 199
Table 3. The heterozygosis of g.469C > G in different Nigerian goat breeds.
Observed Value Expected** Values
Breed* Sample Size
Hom Het Hom Het
Nei’s H*** Ne Fis I
RS 135 0.963 0.037 0.949 0.051 0.051 1.053 0.267 0.120
SH 51 0.961 0.039 0.961 0.039 0.038 1.040 −0.020 0.097
WAD 80 0.925 0.075 0.927 0.073 0.072 1.078 −0.039 0.160
Overall 266 0.951 0.049 0.945 0.055 0.055 1.058 0.108 0.128
*Breed: RS = Red Sokoto; SH = Sahel; WAD = West African Dwarf. **Expected homozygosity and heterozygosity were computed using Levene (1949);
***Nei’s 1973 expected heterozygosity; Ne = effective number of alleles [Kimura and Crow (1964)]; I = Shannon’s information index [(Lewontin (1972)]; Fis =
Wright’s fixation index.
Table 4. Summary of F-Statistics and Gene Flow [Nei (1987)]
of g.469C > G between Nigerian goat breeds.
Fis Fit Fst Nm
0.061 0.063 0.002 122.323
found mostly along the northern border of Nigeria,
particularly in Borno State [10].
Heterozygosity denotes the frequency of heterozygosis
at the tested site (g.469C > G) in the populations and
represents an appropriate index for population genetic
variation. In this study, the values of expected heterozy-
gosity were all less than 0.5 within the 3 goat breeds.
This shows that genetic diversity was deficient at this site.
The effective number of alleles and Shannon’s Informa-
tion Indices showed similar trends as expected het-
erozygosity in the 3 goat breeds. This relatively low ge-
netic diversity at the g.469C > G locus may be due to the
directional selection for coat color. The Veterinary Ser-
vice in Sokoto Province once castrated 219,688 non-red
male goats in 5 years to replace the non-red skins with
the more valuable red in the local markets [10]. The
overall low FIS and FST values and high Nm value ob-
served in this study showed that there is little or no dif-
ferentiation within and among the breeds based on
g.469C > G. The lower genetic diversity of this mutation
site contrasts with the higher genome diversity based on
microsatellite DNA of goat breeds in Nigeria [15,16].
From this study, the g.469C > G mutation does not
seem to be associated with coat color based on the allele
and genotypic frequencies in the different breeds. This
contrasts with the findings of Zhou et al. [9] who found
an association between coat color and the missense mu-
tation g.11584A > G in exon 10 of caprine MLPH gene.
The G allele was mostly found in Chengdu Ma and Nan-
jiang Brown goat (including three strains), in which ho-
mozygote GG was only found. They inferred that allele
G might be a candidate site for the dilute coat color (tan)
found in Nanjiang Brown goat and Chengdu Ma goat. Li
et al. [17] also found an association between nine com-
pletely linked SNPs and dilute coat color (tan) of
Chengdu Ma goat.
A single base pair deletion in exon 2 of MLPH tran-
scripts that introduces a stop codon 11 amino acids
downstream, resulting in the truncation of the bulk of the
MLPH protein, was also identified by Ishida et al. [18].
They found this homozygous variant in 97 unrelated di-
lute cats representing 26 cat breeds and random-bred cats,
along with 89 unrelated wild-type cats representing 29
breeds and random bred cats. They identified a single
haplotype in dilute cats, suggesting that a single mutation
event in MLPH gave rise to dilute in domestic cats. In
chickens also, Vaez et al. [19] identified a strong asso-
ciation between a mutation in exon 1 of the MLPH gene
and the diluted pigmentation phenotype in Lavender
chickens. The g.469C > G mutation was, however, not
reported in the earlier mentioned studies.
In summary, this novel SNP is maintained in HWE
and is not associated with coat color in Nigerian goats.
Further studies to test for additional variants in MLPH
gene are needed to understand if other molecular differ-
ences in MLPH affect coat color in these and other
populations.
5. ACKNOWLEDGEMENTS
Financial support from College of Agriculture and Life Sciences, Cor-
nell University, Ithaca, NY is gratefully acknowledged.
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