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Abstract

Hair samples of various colors of horses were analyzed for content of both eumelanin and pheomelanin by a procedure using high performance liquid chromatography. The results are in accord with generally accepted genetic hypotheses accounting for the various colors. However, the results support the hypothesis that the chestnut/sorrel group of colors is conditioned by the extension locus, not the brown locus. The results also indicate that the brown locus is a likely contributor to some rare color phenotypes.
Pigment Cell Research
1:410-413 (1988)
Pigment Types
of
Various
Color
Genotypes
of
Horses
D.
PHILLIP SPONENBERG,' SHOSUKE IT0,2 LUDEMAN
A.
ENG,'
AND
KAY
SCHWINK'
'Virginia-Maryland Regional College
of
Veterinary Medicine, VPI&SU, Blacksburg, Virginia
24061,
'Fujita-Gakuen Health University, School
of
Hygiene, Toyoake, Aichi
470-11,
Japan
Hair samples of various colors of horses were analyzed for content of both eumelanin
and pheomelanin by
a
procedure using high performance liquid chromatography. The
results
are
in accord with generally accepted genetic hypotheses accounting for the
various colors. However, the
results
support the hypothesis that the chestnut/sorrel
group
of
colors is conditioned by the extension locus, not the brown locus. The results
also indicate that the brown locus is
a
likely contributor
to
some rare color phenotypes
Key
words: Eumelanin, Pheomelanin, Coat color, Horse
INTRODUCTION
The genetics and biochemistry of the coat colors of
mice and guinea pigs have been documented and de-
scribed by several researchers
(It0
et
al.,
1984;
Russell,
1946;
Silvers,
1979).
The study of coat colors in other
mammalian species is frequently based on the assump-
tion that homologies as
to
locus action occur between
species, but biochemical evidence for most nonrodent
species, including the horse,
is
lacking.
Not all breeds of horses include all possible colors
within their gene pools. Most breeds are
at
least reason-
ably polymorphic for color and the genetic basis of the
various colors is at least basically known (Sponenberg
and Beaver,
1983).
Therefore, appearance of unexpected
colors of foals from parents of known color
is
frequently
used to disallow registration of the foal or as a reason
for bloodtyping of sire, dam, and foal as a check on the
accuracy of parentage. The basic hypotheses of horse
color genetics assume homologies
to
that of rodents, but
they have not been rigorously tested. Some details of
nomenclature and homologies do remain uncertain, and
for that reason we investigated the pigment types of
these as
a
mechanism for establishing or refuting pro-
posed homologies.
The loci reported
to
be active in horses
are
several.
The agouti locus controls the distribution of black vs.
red areas
(A
bay,
a
black) (Adalsteinsson,
1974).
The
brown locus
is
controversial in that some authors doubt
its
existence, whereas others attribute
to
it
the chestnut
and sorrel range
of
colors (Adalsteinsson,
1974;
Evans
1981;
Jones
1982;
Odriozola
1951).
The albino locus is
reported to be active (C-normal Cr-cremello dilution),
and does not act
to
change melanin type but only inten-
sity (Adalsteinsson,
1974).
The dilute locus has poor
homology
to
rodents, since in horses the dominant al-
lele causes dilution, whereas the recessive allele allows
0
1988
Alan
R.
Liss,
Inc.
full pigmentation (D-dun dilution, d-nondilute) (Adal-
steinsson,
1974).
Finally, the extension locus is reported
by some
to
cause the chestnutisorrel group of colors,
instead of the brown locus (E-normal extension, e-re-
striction of eumelenin) (Adalsteinsson,
1974).
The wild
type genotype of horses
is
speculated
to
be
either zebra
dun
(A-B-CCDE-)
or grulla
(aa
B-CC
DE)
depending
on the population decreed
to
be the appropriate wild
horse (Sponenberg,
1983).
MATERIALS
AND
METHODS
Eumelanin and pheomelanin in samples of hair from
various colors of horses were analyzed by the method of
Ito
and Fujita
(1985)
using chemical degradation and
high performance liquid chromatography (HPLC). Ap-
proximately
30
mg hair were homogenized in water
at
a
concentration of
10
mg/ml. For eumelanin estimations
which were performed in duplicate,
200
pl of homoge-
nate
(2
mg hair) were transferred
to
a screwcapped
test-
tube mixed with
800
p1 of
1
M
HzS04
and oxidized with
3%
KMn04. The product, pyrrole-2,
3,
5-tricarboxylic
acid WCA), was analyzed by HPLC with ultraviolet
detection. For pheomelanin estimation,
200
pl of the
homogenate were transferred
to
a screwcapped
test-
tube and hydrolyzed at
130°C
for
24
h with
500
p1
of
57%
hydriodic acid in the presence of HsP02. The prod-
uct,
aminohydroxyphenylalanine
(AHP), was analyzed
by HPLC with electrochemical detection. Contents of
PTCA and AHP of
1
ng roughly correspond to
a
eume-
Address reprint
requests
to
D.P. Sponenberg, Virginia-Maryland
Re-
gional College
of
Veterinary Medicine,
WI&SU,
Blacksburg, VA 24061.
Received
December 7,1987; accepted March 10,1988.
Pigment Types
of
Horses
411
lanin content of
50
ng and
a
pheomelanin content of
5
ng, respectively.
RESULTS
The results indicate that visually black areas of horse
color are eumelanic (blue roan body and mane, grullo
body and mane, bay mane and tail, bay roan mane,
liver chestnut dark tail, chestnut dark tail). Dark red
areas are of mixed melanin
type
(bay body, liver chest-
nut body, red chestnut body). Lighter red
to
yellow
areas are due
to
pheomelanin (blonde sorrel body, red
chestnut body, chestnut mane and body), or
are
of
weak,
indeterminate melanin type (pale buckskin body, blond
sorrel body [pale areas], silver mane of chestnut). Pale
brown areas
are
weakly melanized, but tend
to
be pri-
marily eumelanin (lilac dun body, mane, and tail; yel-
low dun mane).
The results of this analysis are presented in Table
1.
DISCUSSION
Although this study determines the melanin content
of only
a
few representative animals, it nonetheless
provides some useful results. Using melanosome struc-
ture
to
determine melanin type was attempted but
found
to
be ineffective due
to
the size
of
the hairs. Blue
roan and grulla horses (Table
1)
have predominantly
eumelanin. This
fits
in well with the theory that blue
roan and grulla are modifications of black and that both
are conditioned by
au
at
the agouti locus. The bay group
is interesting in that the body color seems
to
be due to
a
mixture of eumelanin and pheomelanin. This is still
in keeping with the theory that bays are due
to
a
domi-
nant
(A)
gene
at
the agouti locus. The low levels of
melanin in the mahogany bay roan body hair probably
are
due
to
having
a
mixture of white hairs in the coat
of this animal. The low levels of melanin in the body
coat
of
the pale buckskin
are
probably due
to
a decrease
in pigment production caused by the
Cr
gene, but num-
bers are
too
few
to
state
this with certainty. The
cc
genotype in mice has been shown
to
drastically reduce
melanin content (Ito et.
al.,
1985).
Intermediate
c
locus
alleles in mice have also been shown to reduce melanin
content (Dunn and Einsele,
1938).
The chestnut group is interesting. This group of colors
is considered
to be
recessive epistatic to the agouti lo-
cus,
so
it is expected that they are
at
the extension
(El
locus (Adalsteinsson,
1974;
Sponenberg and Beaver,
1983).
Earlier genetic interpretation, however, was that
TABLE
1.
Melanin and pheomelanin content
of
various hair coat genotypes
of
horses
ma
AHP~
PTCNAHP Melanin
Genotype Color name Body location ndmg hair ratio' typed
183 40 4.6
E
178 18
E
aaB-CCddE-Rnrn
Blue roan Body
408
18 23
E
E
126 9.0
aaB-CCDdE-
Grulla Body
A-B-CCddE
Bay Tail
-
263 24
11
E
65 349 3.19
M
246
22
11
E
A-B-CCddE
Bay roan Body
Mane
A-B-CCddE- Rnrn
Mahogany bay roan Body
46 88 0.52
Mane
357 15 24
E
4.7 29 0.16
-
84 387 0.22
M
A-B-CCC'ddE
Pale buckskin Body
172
m
0.29
M
203
562 0.36
M
-CCddee
Liver chestnut Body
Tail
263
22
12
E
Body
-CCddee
Blonde sorrel Pale body
2.8 53 0.053
-
Red body
13 358 0.036
P
31
195 0.16
M
76 37 2.1
-
-CCddee
Red chestnut Body
Silver mane
13 725 0.018
P
12 181 0.066
-
Body
Mane
-
130 319 0.41
M
16 256 0.063
P
-CCddee
Chestnut Body
Mane
41
876
0.047
P
Tail
159
-
102 1.6
E
Body
WbbCCC'ddE-
Lilac dun Tail
47 27 1.7
-
Flank
43 18 2.4
-
Mane
63 27 2.3
-
Rump
32 24 1.3
-
A- b
b
CFddE-
Yellow dun Mane
67 27 2.9
-
aPyrrole-2,3,5
tricarboxyic acid,
a
eumelanin indicator. Values exceeding
100
ng/mg are underlined.
bAminohydroxyphenylalanin,
a
pheomelanin indicator. Values exceeding
100
ng/mg are underlined.
'Values above
1.0
or
below
0.1
are underlined, provided that either or both of PTCA and AHP are above
100
ng/mg.
dE, eumelanic: PTCA
>
100
ngkg; PTCNAHP ratio
>
1.0.
P, mainly pheomelanic. AHP
>
100
ng/mg; PTCNAHP ratio
<
0.1.
M, mixed
type:
either or both
of
PTCA and AHP
>
100
ng/mg; PTCNAHP ratio
0.1
to 1.0-weak, indeterminate type
failing to meet the criteria of E,
P,
or M.
Mane
Mane
-
14
-
-
-
-
-CCddee
Liver chestnut Tail
-
412
D.P.
Sponenberg et
al.
the chestnutlsorrel group of colors was caused by a
recessive gene at the brown
(B)
locus (Wright,
1917),
and this
is
still sometimes published
as
the correct
genetic mechanism (Evans,
1981;
Jones,
1982).
If
that
were the case, then chestnut and sorrel horses should
be eumelanic.
If
the extension locus interpretation be
correct, then these colors should
be
pheomelanic. The
biochemical data presented here indicate that the ex-
tension
(El
locus is
a
more probable cause of the chest-
nutlsorrel group than
is
the brown
(B)
locus
(Adalsteinsson,
1974;
Andersson and Sandberg,
1982).
Interaction with the albino
(0
locus and linkage data
with several serum proteins, Roan
(Rn)
and Tobiano
(7')
loci also support the extension locus as responsible for
these colors (Adalsteinsson,
1974;
Andersson and Sand-
berg,
1982;
Sponenberg et. al.,
1984).
The fact remains,
however, that many horses with chestnut color
(ee)
ap-
pear very dark visually, and this seems from our results
to
be due
to
the presence of eumelanin. This visual
interpretation
is
especially true of body color in darker
chestnuts and the dark manes and tails of several
shades of chestnut. This visual suspicion
is
borne out in
the biochemical data since several of these animals
produce both eumelanin and pheomelanin, with the
tails (visually black) of two individuals having predom-
inantly eumelanin. These results suggest that the
ee
genotype in horses does not entirely suppress eume-
lanin production
as
it
seems
to
in other species. The
only examples of low melanin levels in this group are
the blonde sorrel and the silver mane
of
one individual.
These
are
visually pale,
so
may be indicative of de-
creased pigment production.
A
very rare group of horse colors does seem
to
be
homologous
to
the brown based
bb
colors of rodents.
These colors
are
not chestnutlsorrel as postulated in the
past, but are odd colors in which black areas are re-
placed with brown, and for which specific names are
lacking in traditional horse lore (Sponenberg and Bea-
ver,
1983).
This locus has been postulated but not docu-
mented in horses (Odriozola,
19-51],
although
(as
discussed above)
it
has been suspected
to
be the locus
involved in the production of the chestnutlsorrel phe-
notypes.
Two
animals suspected
as
being
bb
were avail-
able for study, but melanin production was
so
weak in
both
as
to
preclude accurate determination of the type
of melanin production. Both of these horses were het-
erozygous for cremello
(C'l
dilution, which may in part
account for the weak melanogenesis of these individu-
als if the data from the pale buckskin
B
horse are
indicative of a real tendency. The
PTCNAHP
ratios in
these horses do indicate primarily eumelanin in the
visually brown
areas
of these animals, and if eumelanin
content
in
bb
horses is reduced
as
it
is
in
bb
mice, then
the data
fit
the expectation of primarily eumelanin in
the brown areas. The
bb
genotype in mice has been
shown'
to
decrease eumelanin
to
112
to
113
that of
BB
animals
(It0
et.
al.,
1984)
If
homologous in horses this
may account for the low levels of eumelanin in these
two animals. The melanin data, therefore, suggest but
do not prove the existence of
bb
horses. One of these
horses was examined ophthalmoscopically and did have
the retinal morphology typical of
bb
animals in other
species,
a
change that has not been found with any
other genotype of horses. These changes include
a
ta-
petal area that was much yellower than that present in
other colors of horses. This yellow had none of the
greenish tint usual for horses, but rather tended to
orange. The nontapetal areas were much lighter orange
in color than is normal for horses, which is probably
due
to
blood showing through the weakly melanized
tissues.
This study was clearly limited
to
a few color types,
but the colors chosen for inclusion were those most
likely
to
support or refute homology
to
rodents. The
attempt
to
characterize melanosome size and shape
proved fruitless due
to
the difficulty of working with
horsehairs, which are rather large in diameter. The
expected actions of various loci can clearly
be
seen
to
be
active in horses. The mixed melanic
types
are
interest-
ing.
It
would be useful to know whether or not these
are heteropolymers or
if
the two melanins exist within
separate melanosomes in the same hair. Without the
morphologic data
it
is
impossible
to
speculate on which
of these alternatives
is
the accurate choice, or whether
both might be active in separate individuals.
The advantage of using the present technique
to
de-
termine melanin content of hair
is
that this technique
chemically quantitates both pheomelanin and eume-
lanin.
As
such it can accurately measure both melanins
in the same sample. The two types, eumelanin and
pheomelanin, can sometimes be confused phenotypi-
cally. The other common method of determining mela-
nin content rests on the detection of pheomelanin by an
electron spin resonance spectroscopy technique (Sealy
et al.,
1982;
Vsevolodov et. al.,
1987).
Whereas some
studies indicate that the character of the
ESR
spectra
can predict mixtures (Sealy et al.,
19821,
others indicate
solely the presence or absence of pheomelanin and could
potentially overlook samples in which mixed melano-
genesis had occurred (Vsevolodov et al.,
1987).
The
ESR
technique also is limited
to
determining the ratio of
pheomelanin
to
eumelanin rather than the absolute
quantities. The
HPLC
technique has the advantage
of
being a direct measurement of both melanins.
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To increase the current understanding of the gene-expression profiles in different skin regions associated with different coat colors and identify key genes for the regulation of color patterns in goats, we used the Illumina RNA-Seq method to compare the skin transcriptomes of the black- and white-coated regions containing hair follicles from the Boer and Macheng Black crossbred goat, which has a black head and a white body. Six cDNA libraries derived from skin samples of the white-coated region (n = 3) and black-coated region (n = 3) were constructed from three full-sib goats. On average, we obtained approximately 76.5 and 73.5 million reads for skin samples from black- and white-coated regions, respectively, of which 75.39% and 76.05% were covered in the genome database. A total of 165 differentially expressed genes (DEGs) were detected between these two color regions, among which 110 were upregulated and 55 were downregulated in the skin samples of white- vs. black-coated regions. The results of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed that some of these DEGs may play an important role in controlling the pigmentation of skin or hair follicles. We identified three key DEGs, i.e., Agouti, DCT, and TYRP1, in the pathway related to melanogenesis in the different skin regions of the crossbred goat. DCT and TYRP1 were downregulated and Agouti was upregulated in the skin of the white-coated region, suggesting a lack of mature melanocytes in this region and that Agouti might play a key developmental role in color-pattern formation. All data sets (Gene Expression Omnibus) are available via public repositories. In addition, MC1R was genotyped in 200 crossbred goats with a black head and neck. Loss-of-function mutations in MC1R as well as homozygosity for the mutant alleles were widely found in this population. The MC1R gene did not seem to play a major role in determining the black head and neck in our crossbred goats. Our study provides insights into the transcriptional regulation of two distinct coat colors, which might serve as a key resource for understanding coat color pigmentation in goats. The region-specific expression of Agouti may be associated with the distribution of pigments across the body in Boer and Macheng Black crossbred goats.
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Farmed mink (Neovison vison) is one of the most important fur-bearing species worldwide, and coat colour is a crucial qualitative characteristic that contributes to the economic value of the fur. To identify additional genes that may play important roles in coat colour regulation, Illumina/Solexa high-throughput sequencing technology was used to catalogue the global gene expression profiles in mink skin with two different coat colours (black and white). RNA-seq analysis indicated that a total of 12,557 genes were differentially expressed in black versus white minks, with 3,530 genes up-regulated and 9,027 genes down-regulated in black minks. Significant differences were not observed in the expression of MC1R and TYR between the two different coat colours, and the expression of ASIP was not detected in the mink skin of either coat colour. The expression levels of KITLG, LEF1, DCT, TYRP1, PMEL, Myo5a, Rab27a and SLC7A11 were validated by qRT-PCR, and the results were consistent with RNA-seq analysis. This study provides several candidate genes that may be associated with the development of two coat colours in mink skin. These results will expand our understanding of the complex molecular mechanisms underlying skin physiology and melanogenesis in mink and will provide a foundation for future studies.
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To characterize the colorization patterns of bovine hairs, the melanin contents were quantitatively assayed and compared among cattle breeds. The total melanin levels measured by spectrophotometric assay (A500) from Jeju Black cattle were significantly lower than those from Holstein or Angus with black coat color but significantly higher than those from Hanwoo with yellow coat color or Angus and Holstein with red coat color (P
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SEARLE (1968) listed in "Comparative Genetics of Coat Colour in Mammals" for horses the following polymorphic loci: A, B, C, D, E,G, R, S (Z) and W. In mice the loci a, b, c, e and d have been identified, localized and characterized on the molocular level. The consequences of these results in mice on colour genetics in horses are discussed. The conclusions of ADALSTEINSSON (1974), ANDERSSON and SANDBERG (1987), that chestnut/ sorrel colours are caused by the recessive genotype at the E-Locus rather than by the B-Locus are in good agreement with molecular genetic results at the respective loci in mice. This good agreement also holds true for allels at the A, B and E Loci which determine black colours as well. Blacks have the genotypes aa B-EE or aa B-Ee and jet blacks - B- Ed-. The correlations to Loci assumed to be responsible for colour dilutions C, D and Z are also of interest. By comparative mapping, the homologies between horses and other mammalian species can be shown. It is concluded that identification, localization and characterization of colour loci in horses is of great interest for horse breeding. Since experimental crosses in horses are too expensive, the informative families necessary for comparative mapping have to be established in herdbook populations. International standardized colour description systems and the complete registration of the colours of horses are necessary for this pulpose. Since genetic defects and reduced resistance against diseases can be caused by pleiotropic effects of colour genes, horse colour genetics is of economic and ethical importance.
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The inheritance of colour in horses had been investigated for many years but since Adalsteinsson's findings on basic and diluted colours, made in the seventies, opinions have changed considerably. Many authors supported this hypothesis by detailed breeding and laboratory experiences. This article contains a review of the most important publications concerning the mode of inheritance of all known horse colours. Some of the papers were published long ago but are still valid. The role of different loci determining horse colour is discussed in relation to other mammals and to the current knowledge on melanogenesis. The paper presents references for 14 loci responsible for horse coat colour.
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Synthetic pheomelanins from enzymic oxidation of the 3,4-dihydroxyphenylalanine (dopa) derivative 5-S-cysteinyldopa have been examined by ESR spectroscopy. These alkalisoluble polymers contain a novel kind of free radical that is spectroscopically distinct from that found in eumelanins. Delocalization of the unpaired electron onto a nitrogen atom and the ability of the radical to chelate complexing metal ions strongly suggest an o-semiquinonimine structure. The synthetic pheomelanin was compared with natural red pigments extracted from human red hair and from red chicken feathers. Spectroscopically, the chicken feather pheomelanin is almost identical to synthetic cysteinyldopa pheomelanin. In contrast, the pigment from red hair has a major spectral component very similar to that found in dopa melanin, with a smaller component corresponding to that found in cysteinyldopa melanin.
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These experiments were chiefly addressed to the question whether the effects of changes at the same locus bear a purely quantitative relation to each other. The wild type and three mutant changes at the c locus have been examined in seven different compounds in combination with black (B) and seven in combination with brown (b). The effects of these changes on the dark granular melanins of the hair have been observed. In combination with black, the reduction in intensity of hair colour by graded steps from full colour (black) to white is accompanied by a parallel graded reduction in the quantity of melanin as measured by weight. The chief tangible factor in this reduction in quantity is the decrease in the size of the pigment granules. Each mutant gene in thec series thus exerts a characteristic effect on granule size. It is probable that the type of melanin molecule which is affected by these mutations is the same in all genotypes. As far as our data go, the changes brought about by mutations at this locus may be described as alterations in the quantity of melanin produced.
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A method for the quantitative analysis of eumelanin and pheomelanin in tissues, e.g., hair and melanoma, is described. The method is simple and rapid because it does not require the isolation of melanins from the tissues. The rationale is that permanganate oxidation of eumelanin yields pyrrole-2,3,5-tricarboxylic acid (PTCA) which may serve as a quantitatively significant indicator of eumelanin, while hydriodic acid hydrolysis of pheomelanin yields aminohydroxyphenylalanine (AHP) as a specific indicator of pheomelanin. The degradation products, PTCA and AHP, can be readily analyzed by high-performance liquid chromatography. Chemical degradations of synthetic melanins, prepared from dopa, 5-S-cysteinyldopa, and their mixtures in various ratios, gave PTCA and AHP in yields that correlated with the dopa/5-S-cysteinyldopa ratio. The ratio as well as the contents of PTCA and AHP reflected well the type of melanogenesis in hair and melanomas. The amounts needed for each degradation were 0.5 mg of melanin, 2 mg of hair, and 5 mg of tissue samples. As many as 20 samples can be analyzed within 3 working days.
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Additional peaks that were known on the esr-spectrograms of red human and reddish-brown Karakul hair to be diagnostic traits of phaeomelanin esr-signal also were found on esr-spectrograms of the tan, but not of black or chocolate brown wool from Icelandic sheep. This tan color is thought to depend on the presence of phaeomelanin and is due to the top dominant allele at the A locus. The two methods of distinguishing between eu- and phaeomelanin-dependent brown colors--esr-spectrometrical and genetical--are in agreement for European as well as for Asiatic breeds. Both light and dark brown Soay fleece samples lacked the additional peaks and are interpreted as eumelanin pigmentation.
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This study examined how various genotypes of coat color in mice and guinea pigs are related to the type and content of melanin and to the levels of free and protein-bound dopa and 5-S-cysteinyldopa in hair. In analysis of black, yellow, and white areas of tortoiseshell guinea pigs, the melanogenesis type was in parallel to the type and content of melanin and was correlated fairly well with the levels of melanin precursors. In mouse hair, substitution of the brown allele (bb) for black (BB) reduced the eumelanin content to 1/2 to 1/3, while it significantly increased the dopa level. The dilution (dd) gene of mice reduced the eumelanin content only slightly, while the gene for pink-eyed dilution (pp) reduced the content of eumelanin and the level of dopa to as much as 1/10. From the eumelanin/pheomelanin ratio, the melanin of brown and dilute brown mice was found to be eumelanic, while the melanin of pink-eyed dilution mice appeared to be a mixed type because of an extremely low content of eumelanin. The levels of bound dopa and 5-S-cysteinyldopa in hair were found to largely reflect the tyrosinase activity.
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A bay roan Brabant Belgian stallion (ERn/ ern) was bred to eight chestnut American Belgian mares (ern/ ern), producing 57 foals. Thirty foals were bay roan, 25 were chestnut, one was bay, and one was chestnut roan. The recombination rate was 0.035 +/- 0.024, indicating fairly close linkage between the roan (Rn) and extension (E) loci.