Download A Domestic cat X Chromosome Linkage Map and the Sex

Copyright Ó 2009 by the Genetics Society of America
DOI: 10.1534/genetics.108.095240
A Domestic cat X Chromosome Linkage Map and the Sex-Linked
orange Locus: Mapping of orange, Multiple
Origins and Epistasis Over nonagouti
Anne Schmidt-Küntzel,*,†,1 George Nelson,* Victor A. David,‡ Alejandro A. Schäffer,§
Eduardo Eizirik,‡,**,†† Melody E. Roelke,* James S. Kehler,‡ Steven S. Hannah,‡‡
Stephen J. O’Brien‡ and Marilyn Menotti-Raymond‡
*Laboratory of Genomic Diversity, Science Applications International Corporation and ‡Laboratory of Genomic Diversity, National
Cancer Institute, Frederick, Maryland 21702, †Genetics Department, George Washington University, Washington, DC 20037,
§
National Center for Biotechnology Information, National Library of Medicine and Department of Health and Human
Services, National Institutes of Health, Bethesda, Maryland 20894, **Faculdade de Biociências, Pontifica
Universidade Católica do Rio Grande do Sul, Porto Alegre, RS-900, Brazil, ††Instituto Pró-Carnivoros,
Atibaia, 12945-010, Brazil, and ‡‡Nestlé Purina PetCare, Saint Louis, Missouri 63102
Manuscript received August 14, 2008
Accepted for publication January 8, 2009
ABSTRACT
A comprehensive genetic linkage map of the domestic cat X chromosome was generated with the goal of
localizing the genomic position of the classic X-linked orange (O) locus. Microsatellite markers with an average
spacing of 3 Mb were selected from sequence traces of the cat 1.93 whole genome sequence (WGS),
including the pseudoautosomal region 1 (PAR1). Extreme variation in recombination rates (centimorgans
per megabase) was observed along the X chromosome, ranging from a virtual absence of recombination
events in a region estimated to be .30 Mb to recombination frequencies of 15.7 cM/Mb in a segment
estimated to be ,0.3 Mb. This detailed linkage map was applied to position the X-linked orange gene, placing
this locus on the q arm of the X chromosome, as opposed to a previously reported location on the p arm. Fine
mapping placed the locus between markers at positions 106 and 116.8 Mb in the current 1.93-coverage
sequence assembly of the cat genome. Haplotype analysis revealed potential recombination events that could
reduce the size of the candidate region to 3.5 Mb and suggested multiple origins for the orange phenotype in
the domestic cat. Furthermore, epistasis of orange over nonagouti was demonstrated at the genetic level.
T
HE domestic cat displays a broad diversity of
phenotypic variation, including an array of coloration patterns resulting from interacting genotypes at
multiple loci. Pigmentation genes have been identified on
the basis of comparative genetic data supported by genetic
linkage or association studies [agouti locus (melanism),
ASIP (Eizirik et al. 2003); albino locus (siamese, burmese,
and albino), TYR (O’Brien et al. 1986; Lyons et al. 2005;
Schmidt-Küntzel et al. 2005; Imes et al. 2006); brown locus
(chocolate and cinnamon), TYRP1 (Schmidt-Küntzel
et al. 2005); dilute locus (dilute), MLPH (Ishida et al.
2006)]. Other genes involved in domestic cat pigmentation remain unknown, including the X-linked orange (O)
locus (Searle 1968; Vella et al. 1999). This locus has
attracted the attention of geneticists for over a century
(e.g., Doncaster 1904; Wright 1918). Orange controls
an unknown molecular mechanism that causes the
suppression of black-brownish pigmentation (eumelanin) in favor of orange-yellowish coloration (pheomelanin) (Vella et al. 1999). The resulting orange phenotype
1
Corresponding author: Bldg. 560, Room 11-38, National Cancer Institute, Frederick, MD 21702. E-mail: [email protected]
Genetics 181: 1415–1425 (April 2009)
is likely caused by the exclusive presence of pheomelanic
pigments in the hair shaft (Figure 1A).
Pheomelanic phenotypes have been reported in
other species. In mice, cattle, horses, pigs, humans,
dogs, bears, rabbits, and chickens, mutations in the
Melanocortin 1 receptor (MC1R) have been reported as
causative of red/yellow/white hair or plumage (Robbins
et al. 1993; Klungland et al. 1995; Marklund et al. 1996;
Kijas et al. 1998; Rees et al. 1999; Newton et al. 2000;
Ritland et al. 2001; Kerje et al. 2003; Fontanesi et al.
2006). However, the autosomal location of MC1R,
mapped to chromosome E2 in the domestic cat (Eizirik
et al. 2003), eliminates this gene as causal for X-linked
orange. An X-chromosomal region for the cat orange
locus was previously proposed on the basis of exclusion
mapping (Grahn et al. 2005). Only one other mammal,
the Syrian hamster (Mesocricetus auratus), has been
reported to have an X-linked pheomelanic phenotype
(sex-linked yellow; Robinson 1966). Mapping of Sexlinked yellow in the hamster is presented in an accompanying article by Alizadeh et al. (2009, this issue).
Two striking phenotypic variants are seen in the
tortoiseshell (mottled orange and nonorange) and the
calico (mosaic pattern of large patches of orange, non-
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A. Schmidt-Küntzel et al.
Figure 1.—(A) Phenotypic variation at the orange locus. The top row depicts variation at the orange locus. Genotypes are presented for a female with representative coat color. From left to right: (a) nonorange, wild-type agouti; (b) nonorange, nonagouti;
(c) orange heterozygote, nonagouti; (d) orange homozygote. No phenotype for the agouti locus is given for cat ‘‘d’’ as orange is
epistatic over nonagouti. A female O/O, A/A or O/O, A/a would be indistinguishable from an O/O, a/a cat. The bottom row depicts
the influence of the white spotting (S) locus on the size and appearance of color patches in female cats that are heterozygous at the
O locus. From left to right: (e) heterozygote or homozygote for white spotting allele (S/s, S/S) and (f) wild type at the white spotting
locus (s/s). O/o, orange alleles; A/a, agouti alleles; S/s, white spotting alleles. (B) Phenotypic epistasis of orange pattern over
nonagouti. Orange and agouti genotypes are indicated under each picture representing phenotypic variation at the orange locus.
(a) Skin patch from a domestic cat of orange coat color demonstrating pattern and for which the genotype at the agouti locus
was determined to be nonagouti (O/O, a/a or O/Y, a/a). (b) Skin patch from a calico cat (O/o) demonstrating pattern in orange
and nonorange fur parts on an agouti (A/A or A/a) background. It is seen that the pattern is continuous between the patches of
different color. (C) Stylized representation of hairs within the different color patches. The types of hair responsible for a particular
fur coloration are represented in a simplified way for each color patch. Of note, hair banding can show variation and some hairs
may have multiple bands. Ca, Cb, and Cc correspond to the phenotypes represented in Ba, Bb, and Bc, respectively. O and o stand
for orange and nonorange fur patches. (D) Tufts of hair from orange and nonorange individuals from patterned and background
regions of the coat. (a) Orange patterned: hairs are largely uniformly pigmented a dark pheomelanin; note tips of some hairs
exhibit narrow light pheomelanic bands. (b) Orange background: background hairs display dark pheomelanic base and lighter
pheomelanic band. (c) Nonorange, patterned: hairs are largely uniformly pigmented with eumelanin; see occasional narrow
agouti band at tips. (d) Nonorange, background: background hairs display eumelanic base, broad pheomelanic agouti band,
eumelanic tips. Note that hairs can display multiple pheomelanic agouti bands.
orange, and white) cats (Figure 1A). The coloration of
the nonorange patches is influenced by the genetic
background at other pigmentation loci, which segregate
independently of the O locus (e.g., the brown locus in
Lamoreux 1973). The tortoiseshell/calico phenotypes
are a consequence of embryonic X inactivation whereby
the alternative expression of orange vs. wild-type alleles in
different skin patches creates a mosaic color patterning
characteristic of female cats heterozygous at the O locus
(Lyon 1999). Mosaic phenotypes were presented as a
compelling argument for the establishment of the
Lyon hypothesis for X inactivation (Lyon 1961). Due
to the presence of both orange and nonorange coloration in heterozygous individuals, the orange allele (O)
can be regarded as codominant with the wild-type
allele. The mosaic phenotype is almost exclusive to
females; rare occurrences in males have been explained by sex chromosome aneuploidy (XXY), chimerism, mosaicism, and somatic mutations (reviewed in
Moran et al. 1984).
An interesting epistatic interaction involving X-linked
orange is mediated by the autosomal white spotting (S)
locus. Heterozygous orange females, without the mutant white spotting allele, will develop as tortoiseshell
with tiny mottled spots (Figure 1A, f), while heterozygous orange females that carry a white spotting allele
develop as calico cats with much larger patches (Figure
1A, e) (Searle 1968; Vella et al. 1999). The large calico
Cat X-Linkage Map and orange Locus
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Figure 1.—Continued.
patches have been inferred to result from a reduced
number of melanoblasts in the skin, allowing for a spatial expansion of the clones leading to larger colored
patches. The white areas in between are caused by an
absence of mature melanocytes (Vella et al. 1999).
Another interesting epistatic situation involves the
interaction of X-linked orange and the agouti locus. A
deletion at the agouti locus changes nonorange agouti
coats displaying tabby (which specifies striped,
blotched, or spotted coats) or ticked to melanistic cats
with a solid coat (Vella et al. 1999; Eizirik et al. 2003). In
nonorange cats, a nonagouti (a/a) homozygote effectively masks the tabby pattern (Eizirik et al. 2003), but
in orange (O/Y or O/O) cats the tabby pattern is easily
observed as coat markings of dark and light orange
regardless of the agouti status (Figure 2). In nonagouti
(a/a) calico cats this leads to nonorange solid patches
juxtaposed to orange patches displaying tabby patterning (Figure 1B, c). Epistasis of orange over nonagouti is
investigated at a genotypic level in this study. Interestingly, cats of calico/tortoiseshell coat color carrying at
least one agouti wild-type allele show continuity in the
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A. Schmidt-Küntzel et al.
tabby pattern between orange and nonorange patches
(Figure 1B, b) (Vella et al. 1999).
In addition to the O locus, the cat X chromosome
contains at least two loci of biomedical interest: the
locus for feline muscular dystrophy, encoding dystrophin, (Winand et al. 1994) and another locus implicated
in skin dysplasia, believed to be X-linked in the domestic
cat on the basis of breeding information (S. Pflueger,
personal communication). The presence of additional
feline hereditary X-linked diseases corresponding to human X-linked pathologies can be expected on the basis
of the fact that X-linked traits are believed to be conserved in eutherian mammals (Ohno 1973). X-linked
traits in the domestic cat are therefore also expected to
be X-linked in humans, making the study of the domestic cat X chromosome of comparative genetic importance.
We report here the generation of a comprehensive
genetic linkage map of microsatellites for the domestic
cat X chromosome, which has been applied to the mapping of the O locus. Special attention has been given
to include markers in the pseudoautosomal region
(PAR), a segment that displays an autosomal pattern of
inheritance undergoing recombination during male
meiosis with the homologous region on the Y chromosome. The generation of a dense map (1 marker/
3 Mb) provides an important addition toward the future
mapping of X-linked traits in the domestic cat.
MATERIALS AND METHODS
Figure 2.—Domestic cat X chromosome linkage map. For
each marker the physical location (pPosition) on the domestic cat X chromosome is indicated in megabases (column 2).
Positions in the cat were determined using ABCC Get Trace
Mapping Info, ABCC trace-centric tools (http://www.abcc.
ncifcrf.gov/Genomes/Cat/index.php). The genetic location
(gPosition) within the domestic cat linkage map is indicated
in centimorgans (column 3). Pairwise genetic distance (gDistance in centimorgans) is represented in column 4. LOD
scores are $3.0, unless otherwise noted [marker pairs that
participate in a flip scored at LOD , 3.0 (odds of
,1000:1) by CRI-MAP are represented in italics and the lower
of the LOD scores estimated by CRI-MAP and Superlink are
shown with superscript footnote numbers]. Recombination
rates (Rec. rate) are indicated in centimorgans per megabase
Animals: Linkage study: The pedigree used for the linkage
study is a multigeneration pedigree of 287 nonbreed cats
generated by Nestlé Purina PetCare (Eizirik et al. 2003) of
which 256 were genotyped. Orange coat color segregates in
this pedigree, which has 109 informative meioses for this trait.
Photographs of 55 individuals from this pedigree displaying
orange coat color were used to evaluate presence/absence of
pattern in orange coat/patches for the determination of epistasis of orange over nonagouti. An additional 88 informative
meioses were obtained from a pedigree of 107 individuals. A
portion of the 107-individual pedigree was generated at
Michigan State University for the mapping of spinal muscular
atrophy (Fyfe et al. 2006); a subset of the pedigree was
generated at the National Institutes of Health (NIH) Animal
Center for coat color segregation projects of the Laboratory
of Genomic Diversity (LGD). Two individuals utilized in both
subsets allow for merging into one pedigree for linkage
analysis. DNA was extracted from blood samples with a
QIAamp DNA Blood mini kit (QIAGEN, Valencia, CA)
following the manufacturer’s protocol.
(column 5). The background color of the recombination
rates is color coded to reflect the extreme variation in the values. *, linkage was determined between markers FCA1434 and
FCA1435. However, the PAR1 and the X-specific region are
kept separate since recombination mechanisms are different
(PAR1 being able to recombine with the Y chromosome).
1
, LOD ¼ 1.58; 2, LOD ¼ 0.17; 3, LOD ¼ 1.21; 4, LOD ¼ 1.19;
5
, LOD ¼ 1.56.
Cat X-Linkage Map and orange Locus
Population study: One hundred eleven unrelated male cats
were sampled from a rural environment in Frederick, Maryland (53 orange, 13 wild type), and an urban setting in Porto
Alegre, Rio Grande do Sul, southern Brazil (36 orange, 9 wild
type). Male cats were chosen for the population study as they
are hemizygous for the X chromosome and their haplotypes
can be determined with certainty. Sampling of the U.S. cats was
based on testicular tissue obtained as a by-product of routine
neutering procedures, with permission of each pet owner.
DNA was extracted from testis samples with a DNeasy blood
and tissue kit (QIAGEN). Samples of the Brazilian cats were
obtained from buccal swabs collected from individuals kept in
private homes (with permission of owners) and veterinary
clinics, as well as stray animals. Genomic DNA was extracted
from freshly collected buccal swabs, using a simple salting-out
method (Abrão et al. 2005).
Marker development: Testing of the previous candidate region:
A microsatellite (FCA1446; supplemental Table 1) was designed in the candidate region published by Grahn et al.
(2005), to examine for linkage to orange in the Nestlé Purina
pedigree.
Microsatellite linkage map: Ten microsatellites described in
previous publications were used in this study (MenottiRaymond et al. 1999, 2003a,b). Forty-nine new microsatellite
markers were genotyped for possible addition to the X
chromosome linkage map (supplemental Table 1). Microsatellites were selected from the sequence traces of the cat
1.93 WGS for inclusion in the X chromosome map, on the
basis of their conserved syntenic position on the dog X
chromosome, following the method described by Ishida
et al. (2006). Following the availability of a cat genome
sequence assembly (Pontius et al. 2007), microsatellite
markers were selected on the basis of their location on the
cat X chromosome, using the algorithm ABCC Retrieve STRs
(ABCC STR-centric tools, http://www.abcc.ncifcrf.gov/
Genomes/Cat/index.php). Primers (supplemental Table 1)
were designed with Primer 3 (http://frodo.wi.mit.edu/cgibin/primer3/primer3_www.cgi; Rozen and Skaletsky 2000),
including an M13 tail for fluorescent labeling of PCR products (Boutin-Ganache et al. 2001).
Fine-mapping of X-linked orange: Thirty-six additional microsatellite markers were designed for the localization of the O
locus according to the methods described above (see supplemental Table 2). In addition, a single-nucleotide polymorphism (SNP) was identified within the gene RAP2C, located in
the candidate region, through exploratory sequencing. A
primer pair was designed for amplification and genotyping
of this SNP (supplemental Table 2).
The physical location on the domestic cat X assembly was
determined for each marker. Markers identified from cat
traces were localized on the cat X chromosome on the basis of
the location attributed to the trace using the ABCC tool, ‘‘Get
Trace Mapping Info’’ (ABCC trace-centric tools; http://
www.abcc.ncifcrf.gov/Genomes/Cat/index.php) or the microsatellite flanking region using the ABCC tool ‘‘GMap a
sequence to cat’’ (ABCC mapping and browsing tools; Wu and
Watanabe 2005; http://www.abcc.ncifcrf.gov/Genomes/
Cat/index.php). Markers from previous publications (obtained prior to the cat 1.93 WGS) were attributed to sequence
traces with cross-species MegaBLAST (http://www.ncbi.nlm.
nih.gov; Zhang et al. 2000), if possible. In the event that a
marker could not be localized on the domestic cat assembly, its
position in the cat was inferred using a comparative genomics
approach. The locations of the microsatellite markers on the
dog and human X chromosomes were obtained by applying a
BLAT search (Kent 2002; UCSC genome browser, http://
genome.ucsc.edu/) of the trace sequence containing the
microsatellite or of the microsatellite flanking region. The
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location on the domestic cat radiation hybrid map was
obtained from the literature for the previously published
markers (Menotti-Raymond et al. 2003b).
Genotyping: Microsatellite markers and SNP: Primers of 95
microsatellite markers and one SNP were amplified in the
Nestlé Purina PetCare pedigree (Eizirik et al. 2003) following
touchdown PCR conditions as in Menotti-Raymond et al.
(2005). Following initial mapping of orange, 6 microsatellite
markers were amplified in a second pedigree of 107 individuals segregating for this trait in an attempt to reduce the
candidate region. To establish haplotypes and investigate their
association with orange, primers of 25 markers located in the
region of zero recombination (between megabases 106 and
116.8 on the X chromosome physical map) were amplified in
the 111 male cats of the population study. PCR products were
analyzed as in Ishida et al. (2006). PCR product length was
used as a surrogate for actual repeat number when determining allele identity. Inheritance was verified with Pedcheck
(O’Connell and Weeks 1998) for genotypes of pedigree
individuals.
ASIP locus: Primers designed to genotype the 2-bp deletion
in ASIP (agouti locus), causative of the melanistic phenotype in
the domestic cat (Eizirik et al. 2003), were modified according to Boutin-Ganache et al. (2001) and amplified in 55
individuals of the Nestlé Purina pedigree. Individuals of
orange and calico phenotype were selected on the basis of
the availability of haplotype data and photographs providing
unequivocal evaluation of pattern in the orange coat/patches.
Tortoiseshell phenotypes were not evaluated due to the
insufficient surface of undisturbed orange coat color. Genotyping at the agouti locus was determined on the basis of the
size of the fluorescent amplicon as described by Eizirik et al.
(2003).
Map construction: Markers with an autosomal inheritance
pattern, designed in the pseudoautosomal region 1 (PAR1),
were analyzed independently from the markers with an Xspecific mode of inheritance due to software limitations. Final
linkage analysis was performed for 51 markers (7 markers
inherited in an autosomal manner and 44 X-linked markers).
Two-point LOD scores between all marker pairs were
computed with Superlink (Fishelson and Geiger 2002,
2004) for values of the recombination fraction (u) between 0
and 0.4 in steps of 0.01. Two markers were considered linked if
their peak LOD exceeded 3.0 (Ott 1991). Using this definition, single-linkage clustering determined that the PAR1 and
X-specific markers each composed a single linkage group.
A preliminary order was suggested by treating the estimated
Kosambi distance (Ott 1991) between markers as a distance,
reducing the marker ordering problem to the traveling
salesman problem (TSP) and using CONCORDE (Applegate
et al. 2006) to solve TSP instances. The reduction to TSP is
imperfect for linkage data of this type, so some rearrangement
to get the optimal order was expected, especially in regions
where markers are very close to one another in genetic
distance. The order of linked markers was then iteratively
tested and modified with the CRI-MAP version 2.4 (Lander
and Green 1987) flips option until the order stabilized.
During this process, five markers were dropped because they
could not be ordered with any confidence or caused instability
in the flips analysis. For flips that CRI-MAP scored at ,1000:1
odds (LOD score ,3.0), Superlink was used to examine
support for alternative orders. For such low-scoring flips, we
report the lower of the CRI-MAP and Superlink LOD scores.
Using CRI-MAP, the female u was estimated between one
pseudoautosomal marker (FCA1434) and one X-specific
marker (FCA1435).
Linkage analysis for the mapping of the orange locus: For
mapping orange, LOD scores were computed with Superlink.
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A. Schmidt-Küntzel et al.
Orange was coded as a fully penetrant X chromosome locus
following the inheritance pattern described in the Introduction in which (unlike most traits) phenotype completely
determines genotype at the locus. Linkage to orange was
determined for the markers of the X-linkage map, followed
by fine mapping with additional microsatellite markers designed for that purpose.
Haplotype determination for the orange locus candidate
region: Haplotypes were directly obtained from the genotyping
data for the 111 male individuals collected for the population
study. For the individuals from the Nestlé Purina PetCare
pedigree, 22 haplotypes were inferred by ferret (G. Nelson,
unpublished results), an in-house implementation of the
expectation-maximization (EM) algorithm (Excoffier and
Slatkin 1995), that was modified for this project to allow input
of monosome (male X) data to improve the accuracy of the
inference of the female haplotypes. Because ferret considers all
possible haplotypes consistent with the genotypes, and was
limited to considering 300 million distinct haplotypes in an
inference, the analysis was first performed for three overlapping
shorter sequences (loci FCA1466–FCA1474, FCA1474–
FCA1489, and FCA1482–FCA1498); these haplotypes were then
assembled into larger segments for all 33 loci by an additional
haplotype inference. Ferret tests haplotype inferences by bootstrap resampling of the genotype data; inferences for the three
segments as well as the ultimate assembly of these segments had
.99% bootstrap replicability. Inheritance for the haplotype
segments and final haplotypes was verified with Pedcheck
(O’Connell and Weeks 1998).
RESULTS AND DISCUSSION
Two approaches are classically used for mapping of
genetic loci of interest, a candidate gene approach and
genome scans. The sole candidate gene for orange,
MC1R was mapped in the cat in a previous study to an
autosome, chromosome E2 (Eizirik et al. 2003). Additionally, a candidate region on the X chromosome was
proposed for the O locus in the vicinity of megabase 46.7
(Grahn et al. 2005) on the basis of exclusion analysis
utilizing the available X chromosome genetic linkage
maps (Menotti-Raymond et al. 1999, 2003a) containing two linkage groups spanning an estimated 25 cM,
along with three unmapped loci. A microsatellite
(FCA1446, see supplemental Table 1), was designed in
the proposed candidate region. A demonstrated lack of
linkage between FCA1446 and orange (LOD ¼ 55, u ¼
0.01), at a LOD score below the frequently used exclusion criterion of 2 (Ott 1991), led to our development
of a comprehensive linkage map of the X chromosome
for the mapping of orange.
Linkage map: To generate a comprehensive map of
the cat X chromosome, microsatellites were selected as
described in Marker development (materials and methods). A total of 59 microsatellite markers (supplemental
Table 1) were amplified in a multigeneration domestic
cat pedigree. Forty-six of the loci (FCA1435–FCA1503;
supplemental Table 1) demonstrated an X-linked pattern of inheritance.
Thirteen microsatellite markers (FCA1422–FCA1434;
supplemental Table 1), selected on the basis of the
homology of their flanking segments to regions located
near the telomeric region of the p arm of the dog X
chromosome, demonstrated an autosomal inheritance
pattern. These markers were determined to be located
in PAR1 of the X chromosome on the basis of criteria
recommended for the identification of pseudoautosomal genes (Burgoyne 1982): (1) failure to map to an autosome [marker FCA1433 demonstrated lack of linkage
with 500 autosomal markers in a third-generation linkage map of the cat (Menotti-Raymond et al. 2008)], (2)
linkage to an X-specific marker [marker FCA1434 linked
to the X-specific marker FCA1435 with a female recombination fraction estimated at 0.02 (LOD . 85)],
and (3) physical mapping to the X chromosome [12 of
the 13 markers are placed on the cat X chromosome in
the current assembly (Garfield browser 12.2; Pontius
and O’Brien 2007; http://lgd.abcc.ncifcrf.gov/cgi-bin/
gbrowse/cat)]. Indirect physical mapping was obtained
by selecting 2 markers, FCA1432 and FCA1434, within
introns of NLGN4X and TBL1X, respectively, that were
previously mapped to the pseudoautosomal region of
the domestic cat radiation hybrid (RH) map (Murphy
et al. 2007). Additionally, sequence traces containing
marker FCA1426 (data not shown) align to homologous
regions on the human X and Y chromosomes.
The final genetic recombination map (Figure 2)
comprises 46 microsatellite markers (7 pseudoautosomal and 39 X-specific markers) spanning the estimated
140 Mb of the domestic cat X chromosome (Garfield
browser 12.2; Pontius and O’Brien 2007). Eight microsatellites located in high marker density regions were
excluded from the map, and 5 others were dropped
during the analysis (see materials and methods).
Genetic distances between adjacent loci range from 0 to
23.0 cM (Figure 2), and the total genetic length of the
domestic cat X chromosome was estimated to be 268 cM
(38 cM for PAR1, 2 cM between FCA1434 and FCA1435,
and 228 cM for the X-specific region). Evidence for
complete coverage of the X chromosome includes
demonstration of linkage between a pseudoautosomal
(FCA1434) and an X-specific marker (FCA1435). The
most distal cat marker is located at megabase 139.7,
,0.3 Mb from the end of the current cat X sequence
assembly. The total genetic length of the cat X chromosome is larger than that reported in other mammals
[34 cM in the silver fox (covering two-thirds of the X
chromosome; Kukekova et al. 2007), 65 cM in the horse
(Swinburne et al. 2006), 71 cM in the mouse (Shifman
et al. 2006), 78 cM in the rat (Bihoreau et al. 2001),
121 cM in the sheep (Maddox et al. 2001), 147 cM in the
cow (Ihara et al. 2004), and 195 cM in humans (Matise
et al. 2007)]. Additionally, some of the older maps may
underestimate the size if they do not cover the entirety
of the X chromosome. Similarly, the recombination
length of the domestic cat autosomal map is longer than
that observed in most reported mammalian maps
(Menotti-Raymond et al. 2008).
Cat X-Linkage Map and orange Locus
The order of microsatellite markers on the X chromosome inferred by genetic linkage data is consistent
with their physical coordinates in the domestic cat
genome assembly (Figure 2). Only one difference was
detected between the genetic linkage and radiation
hybrid maps: marker FCA53, which follows an X-linked
inheritance pattern, was mapped to chromosome B1
in the first genetic linkage map of the cat (MenottiRaymond et al. 1999). This first genetic linkage map was
generated in a hybrid pedigree between the domestic
cat the Asian leopard cat (Prionailurus bengalensis)
(Menotti-Raymond et al. 1999). The high incidence
of ‘‘null’’ alleles in hybrid individuals for this locus
was misinterpreted as an autosomal inheritance pattern. FCA53 was mapped on cat B2 in the cat RH
map (Menotti-Raymond et al. 2003b); however, its
location has presently been determined to be on the
X chromosome (W. J. Murphy, personal communication). Of note, FCA053 could not be placed on the
cat, dog, or human assemblies. For other cat X markers
the colinearity between the domestic cat, dog, and
human X chromosome coordinates is shown in supplemental Figure 1 and confirms the conserved synteny of the mammalian X chromosome between dog,
human, and cat, as previously reported by Murphy
et al. (1999).
Extreme variation of recombination rates was apparent across the X chromosome, ranging from 0 cM/Mb,
extending over 34 Mb, to 15.7 cM/Mb localized within
300 kb (supplemental Figure 2). Several phenomena
have been suggested to be causative of variation in recombination frequencies; among these are differences
in GC content, gene density, and location on the chromosome (telomeric vs. centromeric; Nachman 2002).
No evidence for a correlation between GC content
and recombination frequencies was found utilizing the
domestic cat genome browser (Garfield browser 12.2;
Pontius and O’Brien 2007). The centromere, a region
that is expected to have suppressed recombination
(Nachman 2002), is located approximately at position
53 Mb of the domestic cat X chromosome (Garfield
browser 12.2; Pontius and O’Brien 2007) and is indeed
situated within the region of zero recombination. Interestingly, the telomeric portion of the q arm also
exhibits low recombination rates (0 and 0.3 cM/Mb) in
the terminal 10 Mb of the domestic cat X chromosome.
This is an unusual observation, as telomeric regions are
classically reported as chromosomal areas undergoing
frequent recombination (Nachman 2002). Suppression
of recombination has also been attributed to chromosomal rearrangements such as inversions, in which
recombination occurring in the inverted region in a
heterozygous individual leads to nonviable recombinants (Griffiths et al. 1996). However, a large inversion
is unlikely to account for these observations, because
there is no ambiguity in marker order associated either
with the linkage analysis or from previous cytogenetic
1421
in situ hybridization studies (T. Raudsepp, personal
communication).
Mapping of the orange locus: Linkage analysis: The
markers placed on the domestic cat X chromosome
linkage map were utilized to identify linkage to the O
locus in a multigeneration nonbreed domestic cat pedigree (Eizirik et al. 2003), segregating for orange. Significant linkage to the O locus was detected for markers
FCA1466 and FCA1494, (u ¼ 0, LOD ¼ 19.0 and 29.9,
respectively) (Figure 3).
Fine-scale mapping utilizing six additional microsatellite markers (Figure 3, supplemental Table 2) and
one SNP reduced the candidate region to 10.8 Mb,
between markers FCA1464 (106.0 Mb) and RAP2cSNP
(116.8 Mb). The extent of the zero-recombination region could not be reduced further despite the addition
of 88 informative meioses from an additional pedigree
segregating for orange (Figure 3).
The candidate region for X-linked orange in the
domestic cat corresponds to an interval between 120
and 131 Mb in the human X chromosome. Interestingly
the candidate region for Sex-linked yellow in the Syrian
hamster was mapped in a nonhomologous region,
situated between 46 and 54 Mb on the human X
chromosome (Alizadeh et al. 2009). Additional in situ
hybridization data provided by Alizadeh et al., utilizing
mouse BAC probes from the conserved syntenic position in the cat orange region, provide further support
that the orange loci in the Syrian hamster and the
domestic cat are located in nonhomologous regions.
However, we cannot rule out the possibility that microrearrangements have occurred in either the hamster or
the cat genome that have not been detected by our
methodologies and that the two orange loci could be
orthologs. Alternatively, the two genes could be independent members of the same pathway or encode
gene products with similar function.
Haplotype analysis: An attempt was made to reduce the
size of the orange candidate region through identification of ancestral recombination events within the orange
interval delimited by the linkage analysis, using a
haplotype-based approach. The logic followed here is
that recombination between an orange and a nonorange
haplotype could ‘‘erode’’ the length of the orange haplotype, further refining the size of the orange candidate
region.
Haplotype analysis was performed in a collection of
367 cats, including the Nestlé Purina pedigree and a
population sample set of 111 male individuals for which
haplotypes could be unambi guously determined. Individuals in the population survey were sampled from
two distant locales, one in southern Brazil and the other
in the eastern United States. The broad geographic
scope of the survey, including populations that have not
been in direct genetic contact for many generations,
should increase the probability of sampling distantly
related alleles, implying an enhanced likelihood of
1422
A. Schmidt-Küntzel et al.
Figure 3.—Genetic linkage mapping of the orange locus on the cat X chromosome. (A) The majority of the markers of the
linkage map are represented on the domestic cat X chromosome. (B) Markers used for fine mapping (FCA1462–FCA1500)
are represented on the magnified chromosome section. Solid areas and hatched areas represent the candidate region (0% recombination and transition between 0% and 2 or 4%, respectively). (C) Specific recombination values are represented. Optimal recombination fractions (u) between the markers and the O locus are shown in columns 3 and 5. LOD scores at the optimal u (LOD)
are represented in columns 2 and 4. Values are given for pedigree 1 (columns 2 and 3; Nestlé Purina PetCare) and for pedigree 2
(columns 4 and 5; spinal muscular atrophy and LGD combined) when available. The position of each locus is represented in column 6 (determined using ABCC Get Trace Mapping Info, ABCC trace-centric tools; http://www.abcc.ncifcrf.gov/Genomes/Cat/
index.php).
spanning ancestral recombination events within a narrow chromosomal interval. Additional markers (n ¼ 22;
supplemental Table 2) were designed to increase the
marker density in the candidate region.
The haplotypes generated here included 25 microsatellite markers (FCA1466–FCA1498) located across the
region of zero recombination (supplemental Figure 3).
One hundred unique haplotypes were characterized,
including 36 haplotypes observed in association with the
wild-type or nonorange phenotype (subsequently referred to as nonorange haplotypes) and 64 haplotypes
associated with the orange phenotype (referred to subsequently as orange haplotypes; supplemental Figure 3).
To identify alleles that are exclusive or prevalent
in orange haplotypes relative to nonorange haplotypes
(which would thus be useful for detecting ancestral
recombination events), the alleles were color coded with
respect to their relative frequency of occurrence in the
two alternative color phenotypes (supplemental Figure
3). Three major groups were identified in both the orange
(groups I–III) and nonorange (groups 1–3) haplotypes on
the basis of their allele composition. Orange haplotype
groups I and II are more similar to the nonorange groups
1 and 2, respectively, than to any other haplotype group
(see supplemental Figure 3). Haplotype group III
appears distinct, being represented in only one nonorange
haplotype (hap17; supplemental Figure 3).
Group III (hap43–100; supplemental Figure 3) comprises 91% of the orange haplotypes and is characterized
by five ‘‘signature’’ alleles (at loci FCA1470, FCA1472,
FCA1482, FCA1486, and FCA1494; A–E in supplemental
Figure 3), which are found almost exclusively in orange
haplotypes. We identified 6 haplotypes (hap43–48) that,
at locus A, exhibited a change from the 194-bp signature
allele observed in the remaining group III haplotypes.
The additional introduction of the adjacent 243-bp
allele at the locus FCA1466 in hap44 and -48 is suggestive
of a recombinatorial origin of the flanking marker
alleles 243 and 168 (see hap19 and -21). At the telomeric
boundary of the candidate region, 14 haplotypes
(hap47–60) exhibited a change at locus E from the
268-bp signature allele observed in all other group III
haplotypes. Both signature loci, A and E, show little
allelic variation relative to the major nonorange and
orange haplotype groups, suggesting a recombinationbased vs. mutational origin of the nonsignature alleles.
Cat X-Linkage Map and orange Locus
Signature loci C (FCA1482) (hap59) and D (FCA1486)
(hap55–60) display a high level of allele complexity;
nonsignature alleles may therefore represent mutational
or gene conversion origins. Ancestral recombination
events with nonorange haplotypes affecting signature loci
A and E would decrease the orange candidate region to
3.5 Mb, extending from marker FCA1470 at 109.8 Mb to
marker FCA1494 at 113.3 Mb. Nonetheless, the possibility of mutational events at loci A and E cannot be
excluded. The identified region contains no obvious
candidate genes and the annotation of this genomic
segment is still unclear. Within the 3.5 Mb, 4 putative
genes were predicted by the cat genome browser (Garfield browser 12.2; Pontius and O’Brien 2007) and .20
RefSeq genes align to the dog genome (UCSC genome
browser; www.genome.ucsc.edu).
The six remaining orange haplotypes (groups I and II)
contain one (hap37) or none (hap38–42) of the five
signature alleles mentioned above (supplemental Figure 3). The high degree of similarity observed between
the orange haplotypes of groups I and II and the
nonorange haplotypes of groups 1 and 2, respectively,
suggests the occurrence of additional independent
mutational events causative of orange, leading to the
orange haplotypes of groups I and II. To identify the
number of times orange arose in this data set, investigation of variation at the nucleotide level is required once
the gene responsible for the orange phenotype is
identified.
A noteworthy observation is that each of the orange
haplotype groups is represented in both the American
and the Brazilian data sets. This could be explained by
recent gene flow between the populations due to ease of
modern travel or by an old origin of the haplotype
groups, preceding the colonization of domestic cats into
Brazil and the United States, in both cases originating
from European gene pools.
Epistasis of orange over nonagouti: We have previously
shown that the nonagouti mutation in domestic cats is
caused by a 2-bp deletion in exon 2 of the Agouti
Signaling Protein (ASIP) gene, leading to an early frameshift and premature truncation (Eizirik et al. 2003).
Nonorange cats, homozygous for nonagouti (a/a), are
completely black, similar to laboratory mice that carry
null alleles of agouti. In most mammals, the phenotypic
difference between agouti and nonagouti individuals is
the presence of pheomelanic-banding in all hairs in the
former but not the latter. However, the situation in
domestic cats is more complicated due to the presence
of tabby patterns. In nonorange cats, banding of hairs is
suppressed in the striped or spotted regions of tabby
pattern; these hairs are more uniformly pigmented, and
hence ‘‘stand out’’ against the lighter-colored background of banded (agouti) hairs (see hairs in Figure
1D, c and d). The overall effect, generated by regions of
darker largely unbanded hairs juxtaposed with regions
of lighter-banded hairs, creates pattern in the coat. In
1423
TABLE 1
Interaction of orange with tabby pattern and nonagouti
Tabby pattern in orange
Yes
No
Agouti genotype
Phenotype
a/a A/A or A/a a/a A/A or o/a
Orange (O/O or O/Y)
4
Calico (O/o)
6a
Tortoiseshell (O/o)
NA
Nonorange (o/o or o/Y) NA
Total
10
32
13b
NA
NA
45
0
0
NA
NA
0
0
0
NA
NA
0
Genotypes at the agouti locus (a/a, A/a, or A/A) are represented in columns 2–5, based on the phenotype in the orange
fur parts of domestic cats. No cats of orange coat color lacked
tabby pattern (columns 4 and 5) and cats with tabby pattern in
orange were of both agouti and nonagouti genotype.
a
All 6 individuals homozygous for the agouti deletion
(a/a) displayed an absence of pattern in the nonorange
fur parts.
b
All 13 individuals carrying at least one wild-type allele (A/A
or A/a) displayed tabby pattern in the nonorange fur parts.
orange cats, banding of hairs is also suppressed in
striped and spotted regions; however, the hair in these
regions is a dark orange (as opposed to black in wildtype cats), and the light-colored background hairs are a
dark orange with light-colored orange banding (Figure
1C, b and c, and 1D, a and b).
Another particularity of orange cats is that visibility of
tabby pattern does not seem to be affected by the
genotype of the agouti locus. While nonagouti, nonorange
cats exhibit no pattern (other than occasional faint ‘‘ghost
patterns’’), nonagouti, orange cats do exhibit pattern
(Figure 1B). Thus, to investigate this epistatic relationship
between orange and nonagouti, we determined agouti
genotypes for orange or calico individuals (n ¼ 55) in
our pedigree and confirmed orange status at a genotypic
level using haplotype data.
In calico cats, tabby pattern was exhibited in orange
fur independent of the presence or absence of pattern
in nonorange patches (Table 1; Figure 1B). In nonorange fur patches, tabby pattern was fully hidden in
presence of the nonagouti (a/a) genotype (Figure 1B)
All orange cats exhibited pattern. On the basis of a
molecular genetic test for the agouti locus (Eizirik et al.
2003), 45 of the 55 individuals with orange coat
demonstrated a wild-type agouti genotype (A/A or A/a),
and 10 demonstrated a nonagouti genotype (a/a) (Table
1). Thus, genotype status at the agouti locus exhibited no
influence on the presence or absence of tabby pattern in
orange-colored fur, but genotype at ASIP did correlate
with the presence of tabby pattern in nonorange fur as
assessed in individuals of calico phenotype (n ¼ 19;
Table 1). Independence of tabby pattern from the agouti
genotype in areas of orange coat color, and therefore
1424
A. Schmidt-Küntzel et al.
epistasis of orange over nonagouti, is clearly demonstrated
by these results. This observation illustrates the complexity of the molecular mechanisms underlying domestic cat (and mammalian) coat color, with multiple
processes interacting to generate different combinations of phenotypes.
Summary: We developed a comprehensive genetic
linkage map of the domestic cat X chromosome,
including its pseudoautosomal region 1 (46 markers
spanning 38 cM in the PAR1 region and 228 cM in the Xlinked region). The map was successfully used to define
a candidate interval of 10.8 Mb (delineated by markers
FCA1464 and RAP2cSNP) for the X-linked orange locus.
Additional haplotype analysis suggested multiple origins for orange in the domestic cat and the potential of
historic recombination events at the telomeric and
centromeric boundary of our orange candidate region,
which would reduce the orange interval to 3.5 Mb.
The linkage map will provide a valuable resource for
future localization of other X-linked traits. We recommend including the markers of the pseudoautosomal
region in whole genome scans seeking loci controlling
traits that have an autosomal inheritance pattern as the
inheritance patterns of pseudoautosomal and autosomal loci are indistinguishable. The O locus and its
influence on the poorly understood pheomelanic
pathway are currently unknown. To better understand
the process, as well as the genetic basis for the epistasis
of orange over nonagouti, the causative gene for this trait
needs to be identified. A solid genomic basis for this indepth search has been laid out by this study, which will
hopefully foster additional investigation on this trait, its
functional implications, and its relevance for mammalian pigmentation genetics in general.
We thank Gregory Barsh for discussion and sharing of unpublished
results, Ana Carolina Garcia Escobar for the collection and extraction
of the DNAs obtained from Brazil, the Laboratory of Genomic
Diversity core lab for the extraction of the local samples, Joan Pontius
and the Advanced Biomedical Computing Center for their advice
regarding the whole genome sequencing of the cat, Carlos Driscoll for
his suggestion of using microsatellites for haplotype analysis, Solveig
Pflueger for her advice as a cat breeding specialist, Richa Agarwala and
James Tomlin for assistance with map computation, and John Fyfe
(Michigan State University) for making available to this study samples
from a feline spinal muscular atrophy pedigree that also segregated for
orange. We also acknowledge the photographers who generated the
images used in Figure 1A: Susan Feingold (Fulton County Animal
Services), Joanna Harkin (Alliance for Stray Animals and People), Bill
Hopkins (pet owner), Andrea Thompson (pet owner), Anthony
Griffith (author of Introduction to Genetic Analysis), and Martin Feather
and Cristy Bird (feral tortoiseshell cat living on the grounds of a wat in
Bangkok, Thailand). We also thank Marti Welch (Scientific Publication, Graphics and Media; Advanced Technology Program, Science
Applications International Corporation, Frederick, MD) for excellent
assistance in generating figures and photographs. This publication has
been funded in whole or in part with federal funds from the National
Cancer Institute, National Institutes of Health (NIH), under contract
no. NO1-CO-12400. The content of this publication does not
necessarily reflect the views or policies of the Department of Health
and Human Services, nor does mention of trade names, commercial
products, or organizations imply endorsement by the U.S. Govern-
ment. This research is supported in part by the Intramural Research
Program of the NIH, National Library of Medicine.
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