The variation observed in the coat colour of the F2 and Backcross
individuals of this Charolais × Holstein population provides an
opportunity to investigate effects and the mode of inheritance of the
Charolais dilution locus. Based on the phenotypic data (Table 1), it is clear that the Dc and the Extension loci are mainly responsible for the variation in coat colour observed. As multiple alleles at the Extension (MC1R) locus were segregating in this population, the effect of the Dc locus
on both types of backgrounds was confirmed by the observation of a
complete or partial dilution affecting individuals with ED-, E+e or ee MC1R genotype. The pale colour observed in individuals with E+e MC1R genotype demonstrates the dilution of pigments produced by both Agouti-responsive (E+-) and non-responsive (ee) melanocytes. This consistency of the effect across MC1R genotypes
was also supported by the results of the genome scan, in which the same
region on chromosome 5 showed linkage with the three dilution-related
traits analyzed. In addition, the additive effects estimated for
Quantitative-Black and Quantitative-Red had similar size. The dominance
effect was very small relative to the additive effect; therefore a
single copy of the Dc allele originating from the Charolais is sufficient to dilute either eumelanin or phaeomelanin. Heterozygous individuals, Dc/dc+, are generally of intermediate phenotype (light-grey or light-red) and two copies of the Dc allele
are required to produce a complete dilution of the original pigment
(white phenotype). These results are consistent with the inheritance of
the Charolais dilution locus described in the literature [17]. The data also support the assumption of alternative fixed alleles at the Dc locus
in the founder lines, on which the regression analysis was based.
Therefore, the power of detection of this locus was maximised, which is
reflected in the high significance of the associations identified on
chromosome 5.
The location of the major gene associated with the
Quantitative-Dilution, Quantitative-Black and Quantitative-Red traits
is coincident with the position reported in a linkage study of
eumelanin dilution (black pigment) in a Holstein × Charolais F2
population [18].
In the region of bovine chromosome 5 flanked by markers ETH10 and
DIK5248, there are several metabolic candidate genes directly related
to pigmentation pathways (ErbB3, SILV), and members of gene families where at least one member is suggested to have an effect on pigmentation (BLOC1S1, RAB5b, DCTN2 and MYO1A). Among these, the SILV gene
is the only one with an established function in the melanocyte and
therefore is the strongest candidate. This gene is between 56.407 and
56.415 Mb in the latest version of the bovine genome sequence assembly
(Build 3.1) [21], according to which, ETH10 is located at 55.333 Mb [21].
It codes for a pre-melanosomal matrix protein (PMEL17) necessary for
the formation of the fibril matrix upon which melanin intermediates are
deposited late in melanosome maturation [14]. Mutations in the SILV gene are known to cause diluted phenotypes in mice [22], horse [23] and dog [24],
although in these species the effect is to block the production of
eumelanin without effects on phaeomelanin. In chicken, allelic
variations in this gene also block the production of black pigment in
the plumage leading to the Smoky, Dun, and Dominant white colour variants [25].
In cattle, a Charolais-specific allele has previously been reported in exon 1 of the bovine SILV gene [19].
This mutation is a G>A substitution that results in a change from
glycine to arginine within the N-terminal signal sequence of the PMEL17
protein. Among thirteen breeds tested, the A allele was only identified
in pure-breed Charolais individuals or Charolais crosses [19].
This mutation was genotyped across the individuals of the resource
population scored for coat colour (F2, CB1 and HB1). The observed
distribution of genotypes within the three genetic background groups
supports the hypothesis of fixation of alleles in the founder lines,
with the A allele only present in Charolais founders. To test the
association between the SILV c.64A>G genotypes and the
diluted phenotype, the genotype of this mutation was included as a
fixed effect in the regression model fitted to the
Quantitative-Dilution, Quantitative-Black and Quantitative-Red traits.
For all traits, inclusion of this variant resulted in the disappearance
of the highly significant linkage associations, suggesting that the Dc locus is either due to or in strong linkage disequilibrium with the SILV c.64A>G mutation.
Some discrepancies between SILV c.64A>G and the phenotype were observed however, which draws into question whether SILV c.64A>G is the causative mutation underlying the Dc dilution effect, as other authors have suggested [18]. For these discordant animals, the CHROMPIC analysis including the SILV c.64A>G mutation and the Dc locus
(presumed genotypes based on phenotypes) did not suggest genotyping
errors for the tested mutation, however, most of these animals appeared
as double recombinants at the Dc locus. The probability of a
genuine double recombination event in such a small chromosomal interval
is very low, and to detect several such double recombinants in the
number of animals examined here would be very unlikely. Hence, apart
from possible phenotype-genotype mismatches, these double recombination
events are more likely to be the result of either phenotypic
mis-scoring or the effect of other loci influencing coat colour.
Possible mis-scoring may be explained by difficulties in distinguishing
between the partially and the completely diluted phenotypes (especially
Light-Grey/Light-Red against Off-White) or in scoring some individuals
showing a non-homogenous dilution along the body (e.g. darker head than
body).
Under the possibility of another locus or loci affecting the coat
colour variation in this population, the results of the analysis of
Grey-Intensity may help to interpret the minor gene effects revealed by
the primary analysis. The proximal region of chromosome 28 was the only
significant effect at the suggestive level for the diluted-related
traits and for the Grey-Intensity trait, which suggests that these
significant associations could result from the true quantitative nature
of coat colour intensity within and between the phenotype classes. This
locus could, therefore, be considered as a candidate for the genetic
background effects that underlie subtle variations in colour, and that
in certain cases could lead to discordance between colour score and the
SILV locus genotype (e.g. this could explain discrepancies
such as AG animals that were scored as White). A colour-associated
gene, LYST (lysosomal trafficking regulator), maps to the proximal end
of chromosome 28 [26]. Mutations in this gene are responsible for Chediak-Higashi syndrome 1 in human and mouse (beige mutant). This disorder has been reported in Japanese black cattle [27]
and is characterized by prolonged bleeding time and, more relevantly
for this paper, a light coat colour. Our results indicate that allelic
variation at this gene, possibly not associated with illness, could
underlie the different shades of colours observed in the partially
diluted colour categories by acting as a modifier of the Dc locus.
Increased marker density in this chromosomal region would be required
before an epistatic analysis between this locus and the Dc locus could be conducted.
Other genetic effects may be the result of the interaction of the
causal mutation of the Charolais dilution phenotype and other mutations
in the SILV gene. For instance, the AG individual with
Dark-Red phenotype rather than the expected Light-Red may be explained
if another mutation rescued the dilution effect due to the SILV c.64A>G mutation as seems to be the case with the Smoky phenotype in chickens, which in addition to the 9-bp deletion in exon 10 of the SILV gene associated with the Dominant white phenotype, also have an additional deletion in exon 6 that partially restores pigment production [25].
Based on the CHROMPIC analysis, only four discordant animals showed a putative single recombination event between the SILV c.64A>G and the Dc locus,
however, these discrepancies could not be conclusively confirmed as the
phenotype of these individuals was intermediate between pale
(Light-Grey/Light-Red) and Off-White. Based on the lack of convincing
recombinants between the SILV c.64A>G mutation and the Dc locus, this allelic variant of the SILV gene
cannot be ruled out as the causal mutation of the Charolais dilution
phenotype. The effect of this locus on the phenotype is supported by
the loss of significance in the regression analysis when this mutation
is included as a fixed effect in the model. However, this does not
exclude the possibility of a different mutation tightly linked to SILV c.64A>G being the Dc causal mutation, although we and others [18] have not found other mutations associated with coat colour in the coding region of the SILV gene.
The interaction between the SILV gene and pigment type appears to be complicated. The pigment-specificity of mutations in the SILV gene observed in other species [22-25] is in agreement with the critical role reported for this protein in eumelanosomes but not in phaeomelanosomes [28] and the suppression of PMEL17 expression seen in murine phaeomelanosomes [29,30]. However, recent work in Highland cattle reported a 3-bp deletion in exon 1 of the bovine SILV gene associated with the dilution of both red and black pigments [16]. This finding, and the likely association of the SILV gene and the Dc locus,
which affects both pigments, are intriguing and may suggest that the
role of PMEL17 differs between species. This is plausible as the
genuine function of the SILV gene product in pigmentation is not completely understood [14] and the biological basis of pigmentation may vary with species. Mutations in the SILV gene that have only been shown to affect eumelanin background are located in the c-terminal sequence of the SILV gene and affect the transmembrane or cytoplasmatic domains of the protein [22-25].
It is possible that mutations closer to the N-terminal end (such as
exon 1, where both cattle mutations are found) could lead to more
general interference with pigment production. Exon 1 codes for the
signal peptide sequence of the protein [14], which is thought to determine the entry of PMEL17 into the secretory pathway prior to its processing and cleavage [31].
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