Regulatory mutations in TBX3 disrupt asymmetric hair pigmentation that underlies Dun camouflage color in horses

Abstract

Dun is a wild-type coat color in horses characterized by pigment dilution with a striking pattern of dark areas termed primitive markings. Here we show that pigment dilution in Dun horses is due to radially asymmetric deposition of pigment in the growing hair caused by localized expression of the T-box 3 (TBX3) transcription factor in hair follicles, which in turn determines the distribution of hair follicle melanocytes. Most domestic horses are non-dun, a more intensely pigmented phenotype caused by regulatory mutations impairing TBX3 expression in the hair follicle, resulting in a more circumferential distribution of melanocytes and pigment granules in individual hairs. We identified two different alleles (non-dun1 and non-dun2) causing non-dun color. non-dun2 is a recently derived allele, whereas the Dun and non-dun1 alleles are found in ancient horse DNA, demonstrating that this polymorphism predates horse domestication. These findings uncover a new developmental role for T-box genes and new aspects of hair follicle biology and pigmentation.

Full text

15 2 VOLUME 48 | NUMBER 2 | FEBRUARY 2016 Nature GeNetics
The Dun coat color phenotype in horses is characterized by pigmentary
dilution affecting most of the body hair, leaving areas with undi-
luted pigment in a variable pattern, with the most common fea-
ture being a dark dorsal stripe. This stripe and other Dun pattern
elements are termed primitive markings (Fig. 1a, Online Methods
and Supplementary Fig. 1). Most domestic horses, including the
individual used for the genome assembly1, are non-dun, with little or
no pigment dilution and a faint or absent dorsal stripe. The Dun coat
color is presumed to be wild type, as the Przewalski’s horse, a close
relative of the ancestor of domestic horses2,3, exhibits Dun color, as do
other wild equids—the kiang, onager and African wild ass, as well as
the quagga, a now extinct subspecies of plains zebra. The phylogenetic
distribution of the Dun phenotype and the reduced pigment intensity
of Dun horses (Supplementary Fig. 1) suggest that Dun coloring
serves an important camouflage role in equids.
Dun (D) is fully dominant over non-dun (d) (ref. 4). However, the
corresponding phenotypes are sometimes misclassified because some
non-dun horses exhibit faint primitive markings and may appear super-
ficially similar to Dun horses, especially if mutations in other pigment
dilution genes are present4 (Fig. 1a and Supplementary Fig. 2).
Here we show that in horses the TBX3 gene (encoding the T-box
3 transcription factor) is normally expressed in a pattern resulting in
the Dun phenotype and that regulatory mutations specifically impair-
ing TBX3 expression in the hair follicle cause non-dun coat color. In
humans, heterozygosity for loss-of-function mutations in TBX3 causes
a well-recognized pattern of developmental defects, ulnar-mammary
syndrome, with abnormalities in limb, apocrine gland, tooth and
genital development5. Experimental studies of Tbx3 in mice have pro-
vided insight into the mechanism of these abnormalities6,7, but TBX3
has not previously been implicated in pigmentation.
RESULTS
Dun color is caused by asymmetric deposition of hair pigment
Microscopic examination of dilute-colored hairs from the dorsal
hindquarters (croup; Supplementary Fig. 2a) of Dun horses showed
a striking reduction in pigment in a stereotyped, radially asymmetric
pattern (Fig. 1b–e). In sections perpendicular to the hair shaft, pig-
ment granules in dilute hairs from the croup were limited to approx-
imately 25–50% of the cortex (Fig. 1b, left). By contrast, pigment
granules in dorsal stripe hairs from Dun individuals (Supplementary
Fig. 2a) and in both croup and dorsal midline hairs from non-dun
individuals (Fig. 1b and Supplementary Fig. 2a) are more evenly
dispersed throughout the hair cortex. A similar observation was
described by Gremmel8 more than 75 years ago as pigment granule
Regulatory mutations in TBX3 disrupt asymmetric hair
pigmentation that underlies Dun camouflage color in horses
Freyja Imsland1,11, Kelly McGowan2,3,11, Carl-Johan Rubin1, Corneliu Henegar3, Elisabeth Sundström1,
Jonas Berglund1, Doreen Schwochow4,5, Ulla Gustafson4, Páll Imsland6, Kerstin Lindblad-Toh1,7,
Gabriella Lindgren4, Sofia Mikko4, Lee Millon8, Claire Wade7, Mikkel Schubert9, Ludovic Orlando9,
Maria Cecilia T Penedo8, Gregory S Barsh2,3 & Leif Andersson1,4,10
Dun is a wild-type coat color in horses characterized by pigment dilution with a striking pattern of dark areas termed primitive
markings. Here we show that pigment dilution in Dun horses is due to radially asymmetric deposition of pigment in the growing
hair caused by localized expression of the T-box 3 (TBX3) transcription factor in hair follicles, which in turn determines the
distribution of hair follicle melanocytes. Most domestic horses are non-dun, a more intensely pigmented phenotype caused
by regulatory mutations impairing TBX3 expression in the hair follicle, resulting in a more circumferential distribution of
melanocytes and pigment granules in individual hairs. We identified two different alleles (non-dun1 and non-dun2) causing
non-dun color. non-dun2 is a recently derived allele, whereas the Dun and non-dun1 alleles are found in ancient horse DNA,
demonstrating that this polymorphism predates horse domestication. These findings uncover a new developmental role for T-box
genes and new aspects of hair follicle biology and pigmentation.
1Science for Life Laboratory Uppsala, Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden. 2HudsonAlpha Institute for
Biotechnology, Huntsville, Alabama, USA. 3Department of Genetics, Stanford University School of Medicine, Stanford, California, USA. 4Department of Animal
Breeding and Genetics, Swedish University of Agricultural Sciences, Uppsala, Sweden. 5Institut National de la Recherche Agronomique (INRA), AgroParisTech,
Génetique Animale et Biologie Intégrative, Jouy-en-Josas, France. 6Menntaskólinn við Hamrahlíð, Reykjavík, Iceland. 7Broad Institute of Harvard and MIT, Cambridge,
Massachusetts, USA. 8Veterinary Genetics Laboratory, School of Veterinary Medicine, University of California, Davis, Davis, California, USA. 9Centre for GeoGenetics,
Natural History Museum of Denmark, University of Copenhagen, Copenhagen, Denmark. 10Department of Veterinary Integrative Biosciences, College of Veterinary
Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas, USA. 11These authors contributed equally to this work. Correspondence should be
addressed to L.A. (leif.andersson@imbim.uu.se) or G.S.B. (gbarsh@hudsonalpha.org).
Received 6 May; accepted 25 November; published online 21 December 2015; doi:10.1038/ng.3475
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Nature GeNetics VOLUME 48 | NUMBER 2 | FEBRUARY 2016 153
crowding or clumping but has not been otherwise investigated with
regard to the underlying mechanisms.
Asymmetric pigment distribution in dilute hairs was also apparent
in histological sections of skin, with the most intensely pigmented area
lying on the outward-facing side of the hair (Fig. 1c). Furthermore,
examination of longitudinal sections of anagen hair follicles showed
that the asymmetry in pigmentation begins in the hair bulb (Fig. 1d)
and therefore arises during or before melanin synthesis rather than
after pigment deposition.
We also examined pigment distribution in hairs from other equids
(Fig. 1f,g and Supplementary Table 1). Przewalski’s horse exhibits
a Dun phenotype with a dilute coat color and primitive markings,
including a dark dorsal stripe. As in Dun domestic horses,
dilute hairs from Przewalski’s horses exhibit asymmetric pigmen-
tation, whereas dorsal stripe hairs are uniformly pigmented. The
African wild ass, which diverged from the domestic horse more
than 4 million years ago2, also has a Dun phenotype with especially
prominent primitive markings on the legs and asymmetric hair
pigmentation (Fig. 1a,g).
non-dun is caused by noncoding TBX3 mutations
We first mapped the Dun locus to a region on horse chromosome
8 (chr. 8: 18,061,745–18,482,196) using microsatellite markers and
then fine-mapped the locus with a 27-SNP panel to a 200-kb region
containing only one gene, TBX3 (Fig. 2a). In a recent study of the
Gait-keeper mutation in horses9, we used a non-dun and a hetero-
zygous Dun horse for whole-genome resequencing, with each horse
sequenced to about 30× coverage with paired-end reads. A careful
examination of the associated region identified partially aligned
(soft-clipped) and unpaired reads in the Dun individual in an area
where the reads from the non-dun individual aligned well (Fig. 2b),
indicating the presence of a structural variant about 5 kb downstream
of TBX3. PCR amplification and Sanger sequencing showed that the
Dun haplotype has 1,617 bp that is missing from the EquCab2 assem-
bly, which is based on a non-dun horse. The missing sequence con-
sists of nearly contiguous 1,609-bp and 8-bp segments (Fig. 2c). The
Dun-specific sequence shows 86% identity to the correspond-
ing human sequence. Thus, the Dun allele is representative of the
ancestral state, and the deletion present on the sequenced non-dun
chromosomes, as well as in the reference assembly, is derived.
We genotyped a large number of Dun and non-dun horses from
various breeds for the deletion and concluded that it was not present
in the homozygous state in any of the 529 Dun horses, as expected if
it is a causal mutation for the recessive non-dun phenotype (Table 1).
However, only two-thirds of the non-dun horses were homozygous
for the deletion. Careful inspection of a subset of non-dun horses
demonstrated that horses homozygous for the deletion did not show
primitive markings, whereas many horses homozygous or hetero-
zygous for the non-deleted allele did (P = 4.3 × 10−17, Fisher’s exact
test; Fig. 1a, Table 1 and Supplementary Fig. 2). Thus, the non-dun
phenotype is caused by either of two alleles: non-dun1 (d1; associ-
ated with markings) or non-dun2 (d2; not associated with markings,
containing the derived 1.6-kb deletion). As described further below
(Online Methods and Supplementary Fig. 2), the non-dun1 allele has
a weaker effect than the non-dun2 allele with regard to both gross and
microscopic pigmentary phenotypes.
Dun
D/– D/–
D/–
D/–
non-dun
d1/d1
non-dun
d2/d2 d1/d1
d2/d2
Croup
170
**
35
0
2
Flank
Flank
Age (million years)
E. f. caballus
E. zebra, grevyi,
quagga
E. a. asinus
E. kiang
E. h. onager
543210
3
4
5
6
Intensity difference
Dorsal midline
d2/d2
a
D/– d1/d1 d2/d2
b
c
d
e
f
g
E. f.
przewalskii
E. a.
somaliensis
E. f. przewalskii
E. a. somaliensis
Figure 1 Phenotypic characterization. (a) Three horses with different genotypes at the Dun
locus on a similar pigmentary (E/−; a/a) background, including a blue Dun (D/−) horse, a
black horse with primitive markings (d1/d1) and a black horse without primitive markings
(d2/d2). Photographs by Freyja Imsland and Páll Imsland. (b) Cross-sections of hairs from
the croup of the three horses in a. (c) Skin and hair section stained with hematoxylin and
eosin from a Dun horse. (d) Hair sections stained with hematoxylin and eosin from Dun and
non-dun horses. (e) Color intensity differences across the diameter of the hair cortex
(means ± s.e.m.) as shown in the inset for the croup and dorsal midline in each phenotype
(n = 6 Dun and n = 6 non-dun). **P < 0.01 for Dun versus non-dun croup, two-tailed t test.
(f) Cladogram of the Equidae family (based on ref. 10); species with hair histology in g are
shown in bold. (g) Photographs of Przewalski’s horses (Equus ferus przewalskii) and Somali
wild ass (Equus africanus somaliensis) (left) and photomicrographs of transverse sections through
dilute-colored flank hairs (right). Photographs by Waltraut Zimmerman and the St. Louis Zoo.
Scale bars, 35 µm (b), 100 µm (c), 35 µm (d), 10 µm (e) and 50 µm (g).
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15 4 VOLUME 48 | NUMBER 2 | FEBRUARY 2016 Nature GeNetics
ARTICLES
To further explore genetic relationships between the Dun,
non-dun1 and non-dun2 alleles, we identified SNPs from the TBX3
region using resequencing data from 21 modern domestic horses, a
modern Przewalski’s horse and two ancient horses, one ~4,400 years
old from Yakutia and one ~42,700 years old from the Taymyr peninsula,
both of which are in Russia10 (Supplementary Table 2). We designed a
384-SNP panel covering ~375 kb with a SNP density in the core 200-kb
region of approximately 1 SNP/550 bp, including three SNPs within
the region deleted in non-dun2. Genotyping data were combined
with resequencing data (in total, for 71 Dun domestic horses, 152
non-dun domestic horses, 15 Przewalski’s horses and one domestic
horse–Przewalski’s horse hybrid; Supplementar y Table 2) to
analyze genotype-phenotype relationships. Only two SNPs showed
complete concordance with the Dun versus non-dun phenotype
when Przewalski’s horses were included in the genetic analysis
(Fig. 2d and Supplementary Table 3). One of these, SNP1 (G in Dun, T in
non-dun1), is located within the region deleted in non-dun2, 1,067 bp
downstream of the deletion breakpoint at chr. 8: 18,227,267. The
second, SNP2 (G in Dun, A in non-dun1 and non-dun2), is located 362 bp
upstream of the deleted region in non-dun2 at chr. 8: 18,226,905.
We analyzed resequencing data from modern horses, Przewalski’s
horses, the two ancient horses and 13 other equids, for a total of
87 equids (Supplementary Table 2), to comprehensively investigate
potential causal SNPs for non-dun1. This analysis identified three
additional SNPs and a 1-bp indel, all within the deleted region in
non-dun2, showing nearly complete linkage disequilibrium with the
Dun and non-dun1 alleles in domestic horses (Fig. 2e). However,
these four variants could be excluded as causal for the non-dun
phenotype because they were variable in Przewalski’s horses, which
are always Dun.
A total of 1,814 horses of known phenotype, representing more than
45 breeds (including Przewalski’s horses), were genotyped for SNP1
and SNP2 and for the presence of the 1.6-kb deletion. We observed
three common haplotypes: GG (associated with Dun), AT (associated
with non-dun1) and A-del (associated with non-dun2), where
genotypes are given for SNP2 and SNP1 or for SNP2 and the deletion.
These haplotypes showed complete concordance with phenotype
across this diverse set of horses (Table 1 and Supplementary Table 4).
In addition, a rare AG haplotype (associated with Dun) was found in
Estonian native horses, suggesting that SNP2 is not required for the
Dun phenotype. Taken together with the resequencing data (Fig. 2e),
these results indicate that SNP1 is sufficient to cause a non-dun1
phenotype and that the 1.6-kb deletion is the causal mutation for the
non-dun2 phenotype.
Bioinformatic analysis11 predicted binding sites in the region
deleted in non-dun2 for ALX4 and MSX2, two transcription factors
with an established role in hair follicle development12–15. Furthermore,
SNP1 is predicted to affect binding of the CCAT box–binding
transcription factors, NF-Y and NF-I.
We used the pattern of sequence variation in the TBX3 region in
equids to explore the evolutionary history of the Dun and non-dun
haplotypes (Fig. 2e and Supplementary Tables 3 and 5). There was
almost no diversity among non-dun2 chromosomes, as expected for a
recently derived allele (Fig. 2f). In contrast, non-dun1 chromosomes
60
25
20
15
10
5
0
40
–log10 (P value)
–log10 (P value)
20
0
18.0 18.0
18,227,000
115,100,000
18.20 18.2418.22
Mouse
Rabbit
Pig
Orca
Horse
Dog
Elephant
4
0
–4 1,609 bp
Mammalian conservation
hg19 chr. 12 (Mb)
115,104,500
0.3
0.2
0.1
018.15
Nucleotide diversity
18.20
d1
d2
D (Przewalski’s horses)
D (domestic horses)
18.25 18.30
Multiz alignment
D/d2
d2/d2
18,227,500
EquCab2 chr. 8 (Mb) EquCab2 chr. 8 (Mb)
EquCab2 Phenotype Genotype
Deletion
18,226,905
18,227,267 + 328
18,227,267 + 871
18,227,267 + 1,066
18,227,267 + 1,480
18,227,267 + 1,500
d2/d2
d1/d1
d1/d1
D/d1
D/D
D/D
D/D
D/D
D/D
D/D
D/D
D/D
D/D
D/D
D/D
D/D
non-dun
Dun
Dun
Dun
Dun
Dun
Dun
Dun
Dun
Zebra
Zebra
Zebra
Zebra/Dun
?
?
non-dun
Domestic horse
Domestic horse
Domestic horse
Domestic horse (Estonian)
4,400-year-old wild horse
Przewalski’s horse
Przewalski’s horse
EquCab2 chr. 8 (bp)
EquCab2 chr. 8 (Mb)
EquCab2 chr. 8 (Mb)
18.1 18.2 18.3 18.4 18.5 18.1 18.2 18.3 18.4
TBX5TBX3
TBX3
TBX3
TBX3
42,700-year-old wild horse
African wild ass
Domestic donkey
Kiang
Onager
Grévy’s zebra
Mountain zebra
Plains zebra (Burchell’s)
Plains zebra (quagga)
D
d1
d2
a d
b
c
e
f
Figure 2 Genetic analysis. (a) Association
analysis (
χ
2) of the Dun phenotype with SNP
genotypes at chr. 8: 18,061,745–18,482,196.
(b) Read alignments from whole-genome
sequencing of a Dun heterozygote and a non-
dun homozygote. Red borders denote a read
pair where one of the reads is unmapped; blue
segments represent soft-clipped parts of reads
where part of the read cannot be aligned. The
position of the deletion downstream of TBX3 at
chr. 8: 18,227,267–18,227,279 is indicated,
and the extent of sequence conservation is
illustrated using human genome annotation.
(c) Alignment of the Dun, non-dun1 and non-dun2
alleles at the deletion breakpoints, showing how
the deletion breakpoint closer to TBX3 is flanked
by an additional deletion of 8 bp in non-dun2;
there is a 1-bp indel polymorphism between
Dun and non-dun1. (d) SNP association analysis
(−log10 (P value), two-tailed Fisher’s exact
test) between Dun and non-dun1 haplotypes,
including both domestic and Przewalski’s
horses. SNP1 and SNP2 are marked with a black
arrow. Red dots mark candidate SNPs selected
on the basis of allele frequencies of more than
50% in Dun individuals (dominant trait) and less
than 5% in non-dun individuals (erroneous calls)
(Supplementary Table 3). The location of the
deletion is marked with a red arrow in d and f.
(e) Alignment of six sequence polymorphisms
associated with Dun and non-dun1 haplotypes
from domestic horses. SNP1 and SNP2 are
shown in bold. (f) Nucleotide diversity at
polymorphic sites estimated in sliding windows
of 100 SNPs for Dun (D), non-dun1 (d1) and
non-dun2 (d2) chromosomes among domestic
horses and Przewalski’s horses.
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Nature GeNetics VOLUME 48 | NUMBER 2 | FEBRUARY 2016 155
ARTICLES
showed extensive diversity across the entire region (Fig. 2f and
Supplementary Table 5), demonstrating that non-dun1 cannot be
a recently derived allele. Surprisingly, Dun chromosomes in domes-
tic horses were associated with as little nucleotide diversity as the
non-dun2 allele. This relative lack of diversity likely represents either
a founder effect of horse domestication or a subsequent bottleneck,
as Dun chromosomes from Przewalski’s horses showed extensive
nucleotide diversity (Fig. 2f).
Interestingly, the 4,400-year-old ancient horse could be deduced to
be homozygous non-dun1, whereas the 42,700-year-old horse was het-
erozygous Dun/non-dun1. Thus, the divergence of Dun and non-dun1
haplotypes predates domestication. All other equids, including three
species of asses and three species of zebras, could be deduced to be
homozygous Dun on the basis of SNP1 and SNP2 genotypes (Fig. 2e).
Thus, non-dun1 is a derived allele that arose over 40,000 years ago.
Differential gene expression in skin
To gain insight into the mechanism of pigmentary dilution, we gener-
ated and analyzed transcriptome data from the croup skin of seven
Dun and 11 non-dun horses. We identified 57 differentially expressed
genes (Fig. 3a and Supplementary Table 6), including TBX3, which
was downregulated by 1.6-fold in non-dun skin. Quantitative RT-PCR
(RT-qPCR) confirmed downregulation of TBX3 in both non-dun1
(3.5-fold; n = 3) and non-dun2 (2.5-fold; n = 6) homozygotes; notably,
the neighboring gene, TBX5, did not show differential expression
(Supplementary Fig. 3a). The RNA sequencing (RNA-seq) results
also showed upregulation of eight genes with well-established roles in
melanocyte-specific pigment production (TYR, DCT, MC1R, TRPM1,
SLC24A5, MLANA, KIT and OCA2) in non-dun croup skin, consist-
ent with increased pigmentation intensity in non-dun hair (Fig. 1b,d).
ASIP, EDN3 and KITLG are three genes with known regulatory roles
in animal color patterns; of these, only KITLG exhibited differential
expression. We also examined transcriptome data from the dorsal
stripe skin of Dun horses and skin from the corresponding location in
non-dun horses and did not observe differential expression of mela-
nogenic or melanocyte regulatory genes (Supplementary Table 6).
TBX3 is asymmetrically expressed in Dun hair follicles
Immunohistochemistry of sections from Dun croup skin displayed
an unusual pattern of TBX3 expression in hair follicles mirroring the
pattern of pigment deposition (Fig. 3b and Supplementary Fig. 4a).
In sequential sections of anagen hair follicles, TBX3 immunostaining
was localized to a cluster of keratinocytes in the developing hair cortex
(Supplementary Fig. 4a). TBX3 was not expressed in hair cortex kerat-
inocytes from non-dun croup skin (Fig. 3b) nor from the dark dorsal
midline of Dun and non-dun horses (Supplementary Fig. 4b) but
was expressed in the outer cuticular layer of hair follicles in all samples
(Fig. 3b and Supplementary Fig. 4). These observations suggest that
TBX3 operates in a specific subset of hair cortex keratinocytes to inhibit
pigment synthesis in the dilute hair of Dun horses.
We used immunohistochemistry for MITF and KIT, markers of
mature pigment cells16, to investigate the distribution of melanocytes
in croup skin biopsies from Dun and non-dun horses. MITF was
detected in the pigmented epidermis of Dun and non-dun horses
and showed symmetrical immunostaining in hair follicles of non-dun
horses (Fig. 3c and Supplementar y Fig. 5a). In contrast, asymmet-
ric immunostaining of MITF and KIT was observed in hair follicles
from the croup skin of Dun horses, corresponding to the pattern of
pigment deposition (Fig. 3c,d and Supplementary Fig. 5a). Thus,
both the histological and transcriptomic differences between Dun
and non-dun horses arise from differences in melanocyte distribution
within the hair follicle.
In the RNA-seq data, we observed no difference between non-dun
and Dun skin in the expression of MITF, likely owing to the presence
of cells in the dermis whose expression of MITF was not affected by
Dun genotype. By contrast, the RNA-seq data showed a significant
increase in the expression of KIT and, notably, KITLG (encoding KIT
ligand) in non-dun skin in comparison to Dun skin (Fig. 3a), a finding
confirmed by RT-qPCR experiments (Supplementary Fig. 3b).
Previous studies have identified KITLG as a keratinocyte-derived mol-
ecule necessary for melanocyte migration and survival in the skin and
hair follicle17,18. Immunohistochemistry showed that KITLG-positive
cells were uniformly distributed in the hair bulbs of non-dun ani-
mals and in the pigmented epidermis of all animals examined (Fig. 3e
and Supplementary Fig. 5b). However, KITLG showed asymmetric
expression in hair bulb keratinocytes from Dun croup skin, consistent
with the asymmetry of melanocyte distribution and pigment deposi-
tion. Apparent suppression of KITLG expression occurred in a similar
but not identical location to where asymmetric expression of TBX3
occurred in Dun croup skin (Fig. 3e and Supplementary Fig. 5b).
TBX3 and hair follicle symmetry and differentiation
To further investigate the mechanism by which TBX3 affects KITLG
expression and melanocyte localization, we examined the morphology
and differentiation of croup hair follicles in Dun horses in comparison
to those in non-dun horses. In addition to asymmetry in pigment
deposition, we observed that croup hair follicles from Dun horses
exhibited an apparent asymmetry in hair bulb volume. To assess this
difference quantitatively, we measured hair bulb volume in serial hair
follicle sections, using the center of the dermal papilla as a central
boundary, and expressed the extent of asymmetry as the percent dif-
ference in volume between the outward-facing and inward-facing
halves of each hair bulb (Fig. 4a). Croup hair follicles from Dun horses
(n = 4) exhibited significantly greater asymmetry in bulb volume than
those from non-dun horses (n = 4). In contrast, no difference between
Table 1 Association between TBX3 sequence variation and Dun
phenotypes
Genotype frequency for the 1.6-kb deletion
Deletion
PhenotypeaWT/WT WT/del del/del Total
Dun 222 307 0 529
non-dun, dorsal stripe status unknown 108 280 803 1,191
non-dun, with dorsal stripe 28 44 0 72
non-dun, without dorsal stripe 0 3 19 22
Total 358 634 822 1,814
Genotype frequency for causal TBX3 variants
TBX3 genotypes DunbPhenotype
SNP2 SNP1 Deletion Dun non-dun Total
G/G G/G WT/WT D/D152 0 152
A/A G/G WT/WT D/D2 0 2
A/G G/T WT/WT D/d1 66 0 66
A/A G/T WT/WT D/d1 2 0 2
A/G G/− WT/del D/d2 303 0 303
A/A G/− WT/del D/d2 4 0 4
A/A T/T WT/WT d1/d1 0 136 136
A/A T/− WT/del d1/d2 0 327 327
A/A −/− del/del d2/d2 0 822 822
529 1,285 1,814
Animals representing more than 45 breeds were used (Supplementary Table 4).
Del, deletion; WT, wild type.
aOf the 1,285 non-dun horses tested for the deletion, 94 were carefully examined for the
presence of a dorsal stripe. bGenotype inferred from TBX3 genotypes and Dun versus non-dun
phenotype.
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15 6 VOLUME 48 | NUMBER 2 | FEBRUARY 2016 Nature GeNetics
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Dun and non-dun horses was observed for
dorsal midline follicles (Fig. 4a).
To better refine the location of hair bulb
TBX3 expression, we examined its distri-
bution relative to established hair follicle
differentiation markers, including AE13
(cortex, hair cuticle), Ki67 (proliferative
cells), AE15 (medulla, inner root sheath) and
keratin 6 (KRT6; companion cell layer). The
patterns of AE15 and KRT6 immunostain-
ing were very different from that of TBX3
(Supplementary Fig. 6a). TBX3-expressing
cells lay in close proximity to the develop-
ing hair cortex (Fig. 3b and Supplementary Fig. 4a). We observed
no colocalization of TBX3 and AE13 (Supplementary Fig. 6b), but
double immunostaining demonstrated that a subset of Ki67-positive
cells also expressed TBX3 (Fig. 4b). Thus, TBX3 expression occurs
during early differentiation of croup hair follicle keratinocytes but
does not correspond to a known lineage or compartment. Instead,
TBX3 appears to shift the balance between dividing and differentiat-
ing cells, leading to reduced hair bulb volume. Taken together, our
results demonstrate that asymmetric TBX3 expression in hair bulb
keratinocytes from Dun horses leads to asymmetric pigmentation via
altered KITLG expression and that the mutations in non-dun horses
disrupt this mechanism (Fig. 4c).
1,000
800
600
400
200
0
Expression level (normalized counts)
Dun
MITF
SLC7A11
GPR143
ASIP
POMC
EDN3
TAQPEP
TBX3
non-dun
TYR
DCT
MC1R
TRPM1
SLC24A5
MLANA
KIT
OCA2
KITLG
–2 –1 0 1 2
non-dun/Dun log2 (fold change)
D/– TBX3 (green)
Pigment (red)
d1/d1 d2/d2
a b
Dl– MITF (green)
DAPI
(blue)
d2/d2
D/– KIT (green)
DAPI (blue)
D/– KITLG (green)
DAPI (blue)
d2/d2
d2/d2
c
d
e
Figure 3 Differential gene and protein expression
in the croup skin of Dun and non-dun horses.
(a) Transcript levels (normalized gene counts)
plotted as a function of differential expression
(log2-transformed fold change) in Dun (D/−,
n = 7) versus non-dun (d1/d1, n = 3; d2/d2,
n = 6; d1/d2, n = 2) samples. The 57 genes
demonstrating significant (false discovery rate
(FDR) < 0.1) differential expression are shown
in dark yellow (higher expression in Dun) or
red (lower expression in Dun; Online Methods
and Supplementary Table 6); seven additional
pigmentation-related genes that are not
differentially expressed are shown in blue.
(b–e) Immunofluorescence for TBX3 (green) (b),
MITF (green) (c), KIT (green) (d) and KITLG
(green) (e) in sections of anagen hair follicles from
the croup of Dun/−, non-dun1/non-dun1 and non-
dun2/non-dun2 horses. Pigment is pseudocolored
in b (red). Corresponding bright-field images are
on the right in c–e. DAPI staining is in blue in c–e,
and white lines indicate the basement membrane.
Each photomicrograph is representative of at least
two individuals of each genotype. Scale bars,
100 µm (b–e).
Outward
facing
Inward
facing
Percent difference
in area (inward- vs.
outward-facing
hair bulb)
0.2
** TBX3 (green) Ki67 (red) TBX3 (green)
Ki67 (red)
merged
D/– D/– D/–
Croup
Dorsal midline
0.1
0
D/– (n = 4)
d/d (n = 4)
TBX3 in croup
hair bulb cells
KITLG
KITLG
Dun
non-dun
Croup
Melanocyte
migration/
survival
Melanocyte
migration/
survival
TBX3 in croup
hair bulb cells
Dorsal midline
a b
c
Figure 4 Relationship between TBX3
expression and hair follicle symmetry
and differentiation. (a) Left, hair follicle
section stained with hematoxylin and eosin
from Dun croup skin. Black lines outline the
outward-facing and inward-facing halves of
the hair bulb. Right, the percent difference in
area (mean ± s.e.m.) between the outward-
and inward-facing halves of the hair bulb was
calculated for the croup and dorsal midline
from Dun (D/−; n = 4) and non-dun (d/d; n = 4) horses. Bulb area was measured from
at least two serial sections (range of 2–12 sections/follicle) from at least two follicles
(range of 2–7 follicles) per anatomical location. **P = 0.004 for Dun versus non-dun
croup, two-tailed t test. (b) Immunofluorescence for TBX3 (green; left) and Ki67 (red;
center) in a croup hair follicle from a Dun horse. The merged image is on the right; white
lines delineate the basement membrane. Scale bars, 25 µm (a) and 50 µm (b).
(c) Asymmetric expression of TBX3 (green) in hair bulb keratinocytes of Dun croup
hairs impairs the expression of KITLG (red) and melanocyte survival in one-half of
the follicle. Dun croup hairs are asymmetrically pigmented and appear lighter colored
than hairs from the dorsal stripe or from non-dun horses.
npg © 2016 Nature America, Inc. All rights reserved.

Nature GeNetics VOLUME 48 | NUMBER 2 | FEBRUARY 2016 157
ARTICLES
DISCUSSION
The genetics of animal coat color is a longstanding model system
for studying fundamental aspects of gene action and interaction. As
shown here, equine Dun coloration exemplifies a new aspect of pig-
mentary variation with implications for epithelial biology, mamma-
lian evolution and the history of animal domestication.
In contrast to the numerous loss-of-function mutations caus-
ing dilute pigmentation in mice and other vertebrates as a result of
defects in the pigmentary machinery, dilute pigmentation in equids
represents the wild-type state and maintains intact melanogenesis.
The mutations in non-dun horses disrupt this process and, in doing
so, uncover an unexpected mechanism whereby TBX3 regulates
pigment deposition along the radial axis of hair follicles (Fig. 4c).
Regulation of pigment deposition along the longitudinal axis of hair
follicles, controlled by the ASIP gene, underlies color differences
between body regions in many species of mammals. Whereas ASIP
variation affects coat color in horses (Supplementary Fig. 1a), the
longitudinal hair banding mediated by ASIP is largely absent from
equids. Instead, differential expression of TBX3 and its localized
effect on pigment deposition provide an alternative and unexpected
mechanism modulating the appearance of individual hairs in different
regions of the body. From this perspective, TBX3 expression seems
to mark a previously unappreciated hair follicle compartment that is
sensitive to both the microscopic position of cells within individual
follicles (radial asymmetry) and the macroscopic region of the body
(for example, croup versus dorsal stripe).
Hair follicle cells that express TBX3 include keratinocytes of the
cuticle and the hair bulb cortex, but only the latter group is sensitive to
position and controls Dun coloration. These TBX3-expressing cells in
the hair bulb cortex do not correspond to a known cell lineage or differ-
entiation compartment. Instead, they are likely to respond to positional
signals correlated with the direction of hair follicle downgrowth and
bending19, as TBX3 is expressed in the inward-facing half of the hair
follicle where KITLG expression is extinguished and melanocytes are
excluded. However, several observations suggest that the connection
between expression of TBX3 and KITLG in the hair bulb is likely to
be indirect. First, the location of TBX3 immunostaining within the
hair bulb only partially overlap with the location in which KITLG
immunostaining is absent. Second, our histological and morphometric
observations point to altered distributions of hair bulb cell composi-
tion and differentiation rather than just alterations in gene expres-
sion. Finally, existing chromatin immunoprecipitation and sequencing
(ChIP-seq) data for TBX3 do not include potential regulatory regions
for KITLG20. Nonetheless, our data suggest that the most striking effect
of TBX3 expression in hair bulb keratinocytes concerns hair pigmenta-
tion, and, thus, most of the differentially expressed genes we identified
represent differences in melanocyte abundance.
TBX3 is expressed in many cell types and has a critical role during
development5. Our data indicate that the non-dun2 deletion as well as
the non-dun1 allele disrupt the function of a transcriptional enhancer
regulating TBX3 expression in a specific subset of hair bulb kerati-
nocytes during hair growth. This conclusion is based on the clear
difference in TBX3 expression between Dun and non-dun horses,
the high sequence conservation across mammals in the region and
the prediction of transcription factor binding sites affected by the
non-dun mutations. Furthermore, the horse TBX3 mutations appear
to have tissue-specific effects, as neither Dun nor non-dun horses
show any recognized differences in limb development or other
pleiotropic effects outside the hair follicle.
The ability of TBX3 to influence pigmentation by altering hair
bulb keratinocyte differentiation and KITLG expression could
be limited to equids with a last common ancestor present 4.0–4.5
million years ago. Alternatively, a role for TBX3 in hair color may
have a more ancient mammalian origin. Various pigment markings
and patterned coat color dilutions are widely distributed among
Ruminantia (with an origin ~55 million years ago). Animals such
as the wildebeest, eland, okapi, fallow deer and wild goat display
some aspects of coat patterning reminiscent of Dun coloring, and
it will be interesting to investigate the developmental and genetic
basis of these markings. A closely related question within equids has
to do with the potential role of TBX3 in zebra stripes, which could
be viewed as a modification of the Dun phenotype where primitive
markings extend over the entire body and the dilution has become
so pronounced that the hairs are unpigmented. From this perspec-
tive, understanding the spatial control of TBX3 expression in horse
skin, that is, dorsal stripe versus croup, may provide insight into the
etiology of zebra stripes.
The non-dun2 deletion allele has a stronger phenotypic effect than
the non-dun1 allele, exhibits very low nucleotide diversity in flanking
DNA sequences and was not observed outside of domestic horses,
suggesting a relatively recent origin, probably during the last several
thousand years. In contrast, the non-dun1 allele exhibits far greater
nucleotide diversity and is much older. Haplotype comparisons and
breed distribution suggest that the non-dun2 deletion arose on a
non-dun1 chromosome and then increased in frequency, a history
consistent with persistent selection during and after horse domestica-
tion for the non-dun phenotype; in fact, selection against camouflage
color is likely an important reason for changes in coat color during
animal domestication21.
Additional studies of ancient horse DNA should help to more
precisely date the origin of the non-dun1 allele, but its presence in
a ~43,000-year-old sample is consistent with other work inferring
that multiple color morphs existed in predomestic horses22, pointing
toward a hybrid origin of the domestic horse. The major contribu-
tion to horse domestication may have occurred from a population
carrying the non-dun1 allele at high frequency, potentially the same
population as the now extinct tarpan (Equus ferus ferus). Indeed,
the ~43,000-year-old horse from the Taymyr peninsula carr ying
non-dun1 belonged to a population that contributed substantially
to the domestic horse lineage10. Conversely, a predominantly Dun
population, the ancestor of Przewalski’s horse or a close relative, could
have made a minor contribution to domestic horses, as suggested by
the low diversity observed among domestic horse Dun chromosomes.
This idea can be further explored with additional ancient horse DNA
samples spanning a large temporal and geographic range.
URLs. Picard, http://broadinstitute.github.io/picard/; Genome
Analysis Toolkit (GATK) wiki, http://www.broadinstitute.org/gsa/
wiki/index.php/The_Genome_Analysis_Toolkit; ImageJ, http://
imagej.nih.gov/ij/.
METHODS
Methods and any associated references are available in the online
version of the paper.
Accession codes. Data from sequence capture and whole-genome
resequencing have been deposited in GenBank under BioProject
PRJNA277815. Sanger sequences of the non-dun2 deletion have been
submitted to GenBank with accessions KT896508–KT896515.
Note: Any Supplementary Information and Source Data files are available in the
online version of the paper.
npg © 2016 Nature America, Inc. All rights reserved.

15 8 VOLUME 48 | NUMBER 2 | FEBRUARY 2016 Nature GeNetics
ACKNOWLEDGMENTS
We thank the numerous horse owners who provided samples, T. Raudsepp and
I. Randlaht for samples from Estonian native horses, C. Asa and M. Fischer
(St. Louis Zoo) and F. Marshall for providing hair samples from the Somali wild
ass, W. Zimmerman for photographs of Przewalski’s horse, S. Fard, H. Ring and
F. Hallböök for advice on histological characterization, O. Ryder, L. Chemnick and
C. Steiner for delivering DNA extracts from Przewalski’s horses, C. Der Sarkissian
and L. Ermini for assistance in whole-genome resequencing at the Centre for
GeoGenetics, Denmark, and the HudsonAlpha Genomic Services Laboratory for
RNA-seq. This work was supported by grants from the Knut and Alice Wallenberg
foundation (to L.A.) and the US National Institutes of Health (to G.S.B.), as well as
by an Erasmus Mundus fellowship within the framework of the European Graduate
School of Animal Breeding and Genetics (to D.S.). Sequencing was performed
by the SNP&SEQ Technology Platform, supported by Uppsala University and
Hospital, the Science for Life Laboratory and the Swedish Research Council
(80576801 and 70374401).
AUTHOR CONTRIBUTIONS
L.A. led the genetic characterization and G.S.B. led the RNA-seq and
immunohistochemistry studies. F.I., P.I., K.M. and M.C.T.P. did the sampling.
F.I. was responsible for phenotyping and carried out genotyping together with
U.G., L.M. and M.C.T.P. K.M. performed immunohistochemistry analysis. C.H.
performed RNA-seq analysis. C.-J.R., F.I., J.B., M.S. and L.O. were responsible for
genome sequence analysis. E.S. analyzed transcription factor binding sites. D.S.
contributed to TBX3 expression analysis. F.I., L.A., M.C.T.P., K.L.-T., G.L., S.M.
and C.W. took part in the initial mapping of Dun. L.A., F.I., K.M., C.H. and G.S.B.
wrote the manuscript with input from other authors. All authors approved the
manuscript before submission.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Reprints and permissions information is available online at http://www.nature.com/
reprints/index.html.
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ARTICLES
npg © 2016 Nature America, Inc. All rights reserved.

Nature GeNetics
doi:10.1038/ng.3475
ONLINE METHODS
Animals and phenotyping. We obtained extracted DNA, hair or blood
samples collected by authors and collaborators, provided by horse owners,
with permission from the St. Louis Zoo, or archived at the Veterinary Genetics
Laboratory (University of California, Davis), the Animal Genetics Laboratory
(Swedish University of Agricultural Sciences, Sweden) or the Smithsonian
National Museum of Natural History. DNA was extracted from samples fol-
lowing standard methods. DNA from the CITES protected Przewalski’s horse
was transferred from the Zoological Society of San Diego to the Centre for
GeoGenetics to perform high-throughput sequencing analyses under biomate-
rial request BR2013045.
Skin plug biopsies were collected from horses in Iceland by practicing
veterinarians. Local anesthetic was given to the horses before biopsy extrac-
tion. Punch biopsies were taken from the dorsal midline on the hindquarters,
sampling the dorsal stripe if present, and from the croup a few centimeters
lateral to the sampling on the dorsal midline, beyond the area in which a
dorsal stripe is present in Dun horses. The samples from beyond the dorsal
midline are referred to as croup samples. Anatomical locations of sampling
are indicated in Supplementar y Figure 2a.
Dun phenotypes were assigned by visual assessment of live individuals or
photographs; in some instances, Dun phenotypes were assigned on the basis of
owner assessment and/or breed criteria. The Dun coat color is characterized by
specific patterns of intensely pigmented markings on a diluted background. These
specific patterns can be broken down into elements, which can occur in different
combinations. Some elements are rare in general but common in certain breeds.
Other elements, such as the dorsal stripe, are nearly universal. The dorsal stripe
is an intensely pigmented stripe extending from the forehead, along the dorsal
midline all the way down to the tip of the horse’s tail. A non-exhaustive list of
other Dun pattern elements includes ear markings, dark legs, leg barring, shoul-
der crosses, neck or shoulder shadows, dark facial masks, eye shadows, darkly
pigmented areas around vibrissae, dorsal barbs, cobwebbing on the forehead, and
netting on the neck, shoulder and flank (Supplementary Fig. 1b).
non-dun horses carrying the non-dun1 allele often show primitive markings
in the absence of marked color dilution. As the body hair color of non-dun
horses is much darker than that of Dun horses, these markings can be faint.
Furthermore, the base color of the individual horse affects the visibility of
primitive markings. A horse with primarily red body color, such as a chestnut
or a red bay, will show such markings more readily than a horse with prima-
rily black body pigmentation, such as a black bay or black horse, as the visual
intensity of black pigment is greater than that of red.
Dun is dominant to non-dun1 and to non-dun2, that is, Dun/Dun, Dun/
non-dun1 and Dun/non-dun2 horses are phenotypically identical. However,
the effects of the non-dun1 allele are not as strong as those of the non-dun2
allele. Cross-sections of croup hairs from non-dun1/non-dun1 or non-dun1/
non-dun2 horses exhibit some asymmetry in pigment deposition, although
not nearly as great as in Dun/− horses (Fig. 1a,b and Supplementary Fig. 2a).
Likewise, primitive markings in non-dun1/non-dun1 horses are usually more
pronounced than in non-dun1/non-dun2 horses (Supplementary Fig. 2b,c).
Initial SNP association analysis. Twenty-seven SNPs were genotyped across
the region ECA8: 18,061,745–18,482,196 in 366 individuals from 19 breeds,
including Przewalski’s horses. Ninety-six individuals were homozygous Dun,
111 were heterozygous Dun/non-dun and 159 were homozygous non-dun.
Genotyping was performed on the Sequenom platform. Primers and probes
were designed in a multiplex format using SpectroDESIGNER software
(Sequenom). Assay amplification was performed as previously described23.
Statistical evaluation of data was carried out using Haploview24.
Targeted resequencing. Two Dun individuals, an Icelandic horse and a
Quarter Horse, were chosen for massively parallel resequencing of the Dun
region. Both were known to be homozygous for Dun on the basis of offspring
data. Five non-dun horses were also included: an Appaloosa, an Arabian, a
Knabstrupper, a Lipizzaner and a Noriker. NimbleGen Sequence Capture
Arrays (Roche NimbleGen) were designed to target the 200-kb region on
horse chromosome 8 harboring the Dun locus. Targeted resequencing was per-
formed as described25. Subsequent to the discovery of the non-dun2 deletion,
sequence for the deletion was obtained via Sanger sequencing for these seven
individuals. Primer sequences are given in Supplementary Table 7.
Genome resequencing. DNA samples from two Icelandic horses, one Dun
heterozygote and one non-dun homozygote, were prepared for whole-genome
sequencing as part of our previous study on the Gait-keeper mutation9. Initially,
these two samples were aligned to the horse reference assembly (EquCab2.0).
Following the detection of the deletion in non-dun2, we added Sanger-derived
sequence from a Dun chromosome (GenBank, KT896508–KT896515) to the
EquCab2 chromosome 8 sequence for bases chr. 8: 18,227,267–18,227,279,
thereby creating an alternative assembly in silico (EquCab2.0-Dun), which
was used for all subsequent alignment steps.
Whole-genome sequencing was carried out using 2 × 100-bp paired-end
libraries on an Illumina HiSeq instrument at the SciLifeLab Illumina platform,
with insert sizes ranging between approximately 200 and 600 bases, to generate
approximately 6–10× coverage. The reads from both targeted resequencing and
whole-genome sequencing (BioSample accession numbers SAMN03396927–
SAMN03396975; Supplementary Table 2) were mapped to EquCab2.0-Dun
using Burrows-Wheeler Aligner (BWA)2 6. PCR duplicates were removed
using Picard. SNPs and small insertion-deletions were called from mapping
data using the Genome Analysis Toolkit (GATK)27 UnifiedGenotyper com-
mand after having performed realignment around indels. Sequence variants
were collected for EquCab2.0-Dun chromosome 8: 18,120,000–18,322,000.
The variant calls were subjected to recommended filters for SNPs listed on
the GATK wiki page.
Sequences with BioSample accession numbers SAMN04002334–
SAMN04002382 (Supplementary Table 2) had their reads mapped to a seg-
ment of chromosome 8 of the EquCab2.0-Dun hybrid assembly, using the
PALEOMIX pipeline28, omitting mapping quality checks and PCR duplicate fil-
tering. Sequencing reads were first trimmed using AdapterRemoval v.1 (ref. 29).
All mapped reads were then collected and converted from BAM records to
FASTQ reads. Next, the reads were remapped against the full EquCab2.0-
Dun genome using the PALEOMIX pipeline28, filtering for mapping quality
(MAPQ ≥25), removing PCR duplicates and realigning around indels. Paired
mates were kept in pairs if both mates were found among the alignments and
orphaned mates were treated as single-end reads during remapping. Finally,
all hits mapping to the region EquCab2.0-Dun chr. 8: 18,120,000–18,322,000
were collected. This procedure, which first identifies candidate hits against
the target region and then checks whether better alignment exists somewhere
else in the genome, was devised to avoid remapping the whole set of reads
(comprising several billion reads per individual). The software ANNOVAR30
was used to annotate SNPs in relation to Ensembl genes.
SNP genotyping using BeadXpress. A VeraCode GoldenGate assay (Illumina)
was designed targeting 384 SNPs that were called in the resequencing samples.
A total of 200 domestic horses, one Przewalski’s horse and five other equids
were genotyped using the standard protocol. The GoldenGate assays were
read using a BeadXpress Reader (Illumina), and data were analyzed on
GenomeStudio v2011.1 software (Illumina). SNPs located outside the
non-dun2 deletion with a call rate of less than 80% (17 SNPs) or invariable in
typed individuals (one SNP) were excluded from the analysis. The three SNPs
within the deleted region had a call rate of 67–68%, consistent with one-third
of the samples being homozygous for the non-dun2 deletion. All samples used
had call rates of no less than 97% (domestic horses) or 90% (other equids).
In-depth SNP association analysis. Phenotypic association tests assuming
dominant inheritance of the Dun phenotype were carried out with PLINK31
using Fisher’s exact test. SNP genotypes from the GoldenGate assay were
combined with genotypes for those particular SNPs from the resequencing
data. Candidate SNPs were selected on the basis of allele frequencies of more
than 50% in Dun individuals (dominant trait) and less than 5% in non-dun
individuals (erroneous calls) (Supplementary Table 3).
A similar analysis was performed on SNP calls from the resequencing
experiments. Over 5,000 SNPs from the interval chr. 8: 18,119,043–18,321,208
passed quality filtering criteria of quality over 100, genotype quality higher
than 9 and proportion of missing genotypes across samples less than 0.2.
npg © 2016 Nature America, Inc. All rights reserved.

Nature GeNetics doi:10.1038/ng.3475
SNPs at positions 18,276,126, 18,276,338 and 18,277,478 on chromosome 8
showed strong association with the Dun phenotype in the resequencing data.
The SNPs at 18,276,338 and 18,277,478 on chromosome 8 were included in the
GoldenGate assay (Supplementary Table 3). All three SNPs were genotyped via
Sanger sequencing for a family of three recombinant Dun Icelandic horses, which
excluded them as causal. Primer sequences are given in Supplementary Table 7.
Haplotype diversity analysis. To calculate diversity, all screened SNPs from
the GoldenGate assay were phased with SHAPEIT2 (ref. 32; using 100 burn-in
iterations, 100 pruning iterations, 100 main iterations and 1,000 conditioning
states per SNP, effective population size of 100 and recombination parameter
rho of 0.001) and had haplotypes inferred according to genotype at SNP1,
SNP2 and the deletion. Average nucleotide diversity per polymorphic site
was calculated for each of the Dun, non-dun1 and non-dun2 haplotypes as
2 × f1 × f2 × n/(n − 1), where f denotes allele frequency and n the total number
of alleles.
Genotyping of Dun, non-dun1 and non-dun 2 alleles. DNA samples from
horses of various breeds were genotyped for the Dun, non-dun1 and non-dun2
alleles. The non-dun2 deletion was typed by standard agarose or capillary (ABI
3730 DNA Analyzer, Applied Biosystems) electrophoresis for detection of the
amplification length polymorphism. Betaine or DMSO additives were required
for successful PCR amplification. SNP2 was genotyped by pyrosequencing on
PSQ 96MA 2.1 and PyroMark Q96 MD instruments (Qiagen) according to the
manufacturer’s recommendations. SNP1 was genotyped with pyrosequencing
or with a TaqMan allelic discrimination assay on a 7900HT Fast Real-Time
PCR system (Applied Biosystems) according to the manufacturer’s instruc-
tions. Primer sequences are given in Supplementary Table 7.
Expression analysis. Punch biopsies of skin from the croup and dorsal midline
of 18 horses (seven Dun and 11 non-dun) were collected. The samples were
preserved in RNAlater and stored at −80 °C before processing. Total RNA was
isolated from skin biopsies using the RNeasy Fibrous Tissue Mini kit (Qiagen).
RNA quality was assessed with an Agilent Bioanalyzer instrument. All samples
had an RNA integrity number (RIN) greater than 7.0. cDNA libraries were
further prepared for each sample using Illumina’s TruSeq RNA Sample
Preparation Kit v2. Individual libraries were multiplexed, six per lane, and
sequenced as single-end 50-bp reads on an Illumina HiSeq 2000 instrument
at the Genome Sequencing Laboratory of the HudsonAlpha Institute.
RNA-seq reads obtained for each sample were aligned against the horse
reference genome with TopHat2 software (v. 2.0.12), using genomic sequence
and transcript annotations obtained from Ensembl (release 75). Gene
counts were computed from read alignments with GenomicRanges and
GenomicFeatures packages from Bioconductor (release 2.14) and then used
to test for differential gene expression between Dun and non-dun skin samples
with the DESeq2 package from Bioconductor. DESeq2 relies on negative bino-
mial generalized linear models to determine whether the number of counts
for a transcript or gene is significantly different across a range of experimental
conditions. To account for expression changes related to differences in the
base coat color (black, bay or chestnut; Supplementary Fig. 1a) of different
samples rather than the presence of the Dun allele, we included base coat color
as a covariate in the generalized linear model.
RT-qPCR for TBX3, TBX5 and KITLG. Total RNA for RT-qPCR was isolated
from skin biopsies using TRIzol (Invitrogen Life Technologies), purified using
the PureLink RNA Mini kit (Ambion) and treated with DNase I (Invitrogen
Life Technologies) before reverse transcription with Superscript III (Invitrogen
Life Technologies). cDNA was amplified using the LightCycler FastStart DNA
Master Plus SYBR Green I System (Roche Diagnostics). β2-microglobulin
(B2M) was used to compare relative levels of mRNA. Primer sequences are
given in Supplementary Table 7.
Histology and immunofluorescence. Skin samples were fixed in 4% para-
formaldehyde, washed in PBS, dehydrated and embedded in paraffin. Five-
micrometer sections of hair, croup skin and dorsal midline skin were stained
with hematoxylin and eosin. ImageJ was used to measure hair bulb area and
hair color intensity. Color intensity was measured at six points across the
diameter of the hair cortex and averaged for each hair. Hair color intensity
measurements were taken from at least five hairs from each genotype.
Immunofluorescence was carried out with goat antibody to TBX3 (Santa Cruz
Biotechnology, sc-17871), mouse antibody to MITF (Abcam, ab12039), rabbit
antibody to SCF (KITLG; Abcam, ab64677), mouse antibody to AE13 (Abcam,
ab16113), rabbit antibody to Ki67 (Abcam, ab15580), mouse antibody to AE15
(Abcam, ab58755), rabbit antibody to KRT6 (Abcam, ab24646) and rabbit
antibody to KIT (Dako, A4502) after antigen retrieval using Tris-EDTA, pH
9 (TBX3) or 0.01 M citrate buffer, pH 6 (MITF, KITLG, AE13, Ki67, AE15,
KRT6 and KIT) in a pressure cooker. All photomicrographs are representative
of at least two animals of each genotype and anatomical location.
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