Copyright � 2009 by the Genetics Society of AmericaDOI: 10.1534/genetics.108.095240

A Domestic cat X Chromosome Linkage Map and the Sex-Linkedorange Locus: Mapping of orange, Multiple

Origins and Epistasis Over nonagouti

Anne Schmidt-Kuntzel,*,†,1 George Nelson,* Victor A. David,‡ Alejandro A. Schaffer,§

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, NationalCancer 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 HumanServices, National Institutes of Health, Bethesda, Maryland 20894, **Faculdade de Biociencias, Pontifica

Universidade Catolica do Rio Grande do Sul, Porto Alegre, RS-900, Brazil, ††Instituto Pro-Carnivoros,Atibaia, 12945-010, Brazil, and ‡‡Nestle Purina PetCare, Saint Louis, Missouri 63102

Manuscript received August 14, 2008Accepted for publication January 8, 2009

ABSTRACT

A comprehensive genetic linkage map of the domestic cat X chromosome was generated with the goal oflocalizing the genomic position of the classic X-linked orange (O) locus. Microsatellite markers with an averagespacing 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 (centimorgansper megabase) was observed along the X chromosome, ranging from a virtual absence of recombinationevents in a region estimated to be .30 Mb to recombination frequencies of 15.7 cM/Mb in a segmentestimated to be ,0.3 Mb. This detailed linkage map was applied to position the X-linked orange gene, placingthis locus on the q arm of the X chromosome, as opposed to a previously reported location on the p arm. Finemapping placed the locus between markers at positions 106 and 116.8 Mb in the current 1.93-coveragesequence assembly of the cat genome. Haplotype analysis revealed potential recombination events that couldreduce the size of the candidate region to 3.5 Mb and suggested multiple origins for the orange phenotype inthe domestic cat. Furthermore, epistasis of orange over nonagouti was demonstrated at the genetic level.

THE domestic cat displays a broad diversity ofphenotypic variation, including an array of colora-

tion patterns resulting from interacting genotypes atmultiple loci.Pigmentationgeneshavebeenidentifiedonthebasisofcomparativegeneticdatasupportedbygeneticlinkage 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-Kuntzel etal.2005; Imes etal.2006);brown locus(chocolate and cinnamon), TYRP1 (Schmidt-Kuntzel

et al. 2005); dilute locus (dilute), MLPH (Ishida et al.2006)]. Other genes involved in domestic cat pigmenta-tion remain unknown, including the X-linked orange (O)locus (Searle 1968; Vella et al. 1999). This locus hasattracted the attention of geneticists for over a century(e.g., Doncaster 1904; Wright 1918). Orange controlsan unknown molecular mechanism that causes thesuppression of black-brownish pigmentation (eumela-nin) in favor of orange-yellowish coloration (pheomela-nin) (Vella et al. 1999). The resulting orange phenotype

is likely caused by the exclusive presence of pheomelanicpigments in the hair shaft (Figure 1A).

Pheomelanic phenotypes have been reported inother species. In mice, cattle, horses, pigs, humans,dogs, bears, rabbits, and chickens, mutations in theMelanocortin 1 receptor (MC1R) have been reported ascausative 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-linkedorange. An X-chromosomal region for the cat orangelocus was previously proposed on the basis of exclusionmapping (Grahn et al. 2005). Only one other mammal,the Syrian hamster (Mesocricetus auratus), has beenreported to have an X-linked pheomelanic phenotype(sex-linked yellow; Robinson 1966). Mapping of Sex-linked yellow in the hamster is presented in an accompa-nying article by Alizadeh et al. (2009, this issue).

Two striking phenotypic variants are seen in thetortoiseshell (mottled orange and nonorange) and thecalico (mosaic pattern of large patches of orange, non-

1Corresponding author: Bldg. 560, Room 11-38, National Cancer In-stitute, Frederick, MD 21702. E-mail: anne.c.sk@gmail.com

Genetics 181: 1415–1425 (April 2009)

orange, and white) cats (Figure 1A). The coloration ofthe nonorange patches is influenced by the geneticbackground at other pigmentation loci, which segregateindependently of the O locus (e.g., the brown locus inLamoreux 1973). The tortoiseshell/calico phenotypesare a consequence of embryonic X inactivation wherebythe alternative expression of orange vs. wild-type alleles indifferent skin patches creates a mosaic color patterningcharacteristic of female cats heterozygous at the O locus(Lyon 1999). Mosaic phenotypes were presented as acompelling argument for the establishment of theLyon hypothesis for X inactivation (Lyon 1961). Dueto the presence of both orange and nonorange color-ation in heterozygous individuals, the orange allele (O)

can be regarded as codominant with the wild-typeallele. The mosaic phenotype is almost exclusive tofemales; rare occurrences in males have been ex-plained by sex chromosome aneuploidy (XXY), chime-rism, mosaicism, and somatic mutations (reviewed inMoran et al. 1984).

An interesting epistatic interaction involving X-linkedorange is mediated by the autosomal white spotting (S)locus. Heterozygous orange females, without the mu-tant white spotting allele, will develop as tortoiseshellwith tiny mottled spots (Figure 1A, f), while heterozy-gous orange females that carry a white spotting alleledevelop as calico cats with much larger patches (Figure1A, e) (Searle 1968; Vella et al. 1999). The large calico

Figure 1.—(A) Phenotypic variation at the orange locus. The top row depicts variation at the orange locus. Genotypes are pre-sented 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 isepistatic 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 depictsthe influence of the white spotting (S) locus on the size and appearance of color patches in female cats that are heterozygous at theO locus. From left to right: (e) heterozygote or homozygote for white spotting allele (S/s, S/S) and (f) wild type at the white spottinglocus (s/s). O/o, orange alleles; A/a, agouti alleles; S/s, white spotting alleles. (B) Phenotypic epistasis of orange pattern overnonagouti. 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 locuswas 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 orangeand nonorange fur parts on an agouti (A/A or A/a) background. It is seen that the pattern is continuous between the patches ofdifferent color. (C) Stylized representation of hairs within the different color patches. The types of hair responsible for a particularfur coloration are represented in a simplified way for each color patch. Of note, hair banding can show variation and some hairsmay have multiple bands. Ca, Cb, and Cc correspond to the phenotypes represented in Ba, Bb, and Bc, respectively. O and o standfor orange and nonorange fur patches. (D) Tufts of hair from orange and nonorange individuals from patterned and backgroundregions of the coat. (a) Orange patterned: hairs are largely uniformly pigmented a dark pheomelanin; note tips of some hairsexhibit narrow light pheomelanic bands. (b) Orange background: background hairs display dark pheomelanic base and lighterpheomelanic band. (c) Nonorange, patterned: hairs are largely uniformly pigmented with eumelanin; see occasional narrowagouti 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.

1416 A. Schmidt-Kuntzel et al.

patches have been inferred to result from a reducednumber of melanoblasts in the skin, allowing for a spa-tial expansion of the clones leading to larger coloredpatches. The white areas in between are caused by anabsence of mature melanocytes (Vella et al. 1999).

Another interesting epistatic situation involves theinteraction of X-linked orange and the agouti locus. Adeletion at the agouti locus changes nonorange agouticoats displaying tabby (which specifies striped,blotched, or spotted coats) or ticked to melanistic catswith a solid coat (Vella et al. 1999; Eizirik et al. 2003). In

nonorange cats, a nonagouti (a/a) homozygote effec-tively masks the tabby pattern (Eizirik et al. 2003), butin orange (O/Y or O/O) cats the tabby pattern is easilyobserved as coat markings of dark and light orangeregardless of the agouti status (Figure 2). In nonagouti(a/a) calico cats this leads to nonorange solid patchesjuxtaposed to orange patches displaying tabby pattern-ing (Figure 1B, c). Epistasis of orange over nonagouti isinvestigated at a genotypic level in this study. Interest-ingly, cats of calico/tortoiseshell coat color carrying atleast one agouti wild-type allele show continuity in the

Figure 1.—Continued.

Cat X-Linkage Map and orange Locus 1417

tabby pattern between orange and nonorange patches(Figure 1B, b) (Vella et al. 1999).

In addition to the O locus, the cat X chromosomecontains at least two loci of biomedical interest: thelocus for feline muscular dystrophy, encoding dystro-phin, (Winand et al. 1994) and another locus implicatedin skin dysplasia, believed to be X-linked in the domesticcat on the basis of breeding information (S. Pflueger,personal communication). The presence of additionalfeline hereditary X-linked diseases corresponding to hu-man X-linked pathologies can be expected on the basisof the fact that X-linked traits are believed to be con-served in eutherian mammals (Ohno 1973). X-linkedtraits in the domestic cat are therefore also expected tobe X-linked in humans, making the study of the domes-tic cat X chromosome of comparative genetic importance.

We report here the generation of a comprehensivegenetic linkage map of microsatellites for the domesticcat X chromosome, which has been applied to the map-ping of the O locus. Special attention has been givento include markers in the pseudoautosomal region(PAR), a segment that displays an autosomal pattern ofinheritance undergoing recombination during malemeiosis with the homologous region on the Y chromo-some. The generation of a dense map (�1 marker/3 Mb) provides an important addition toward the futuremapping of X-linked traits in the domestic cat.

MATERIALS AND METHODS

Animals: Linkage study: The pedigree used for the linkagestudy is a multigeneration pedigree of 287 nonbreed catsgenerated by Nestle Purina PetCare (Eizirik et al. 2003) ofwhich 256 were genotyped. Orange coat color segregates inthis pedigree, which has 109 informative meioses for this trait.Photographs of 55 individuals from this pedigree displayingorange coat color were used to evaluate presence/absence ofpattern in orange coat/patches for the determination of epis-tasis of orange over nonagouti. An additional 88 informativemeioses were obtained from a pedigree of 107 individuals. Aportion of the 107-individual pedigree was generated atMichigan State University for the mapping of spinal muscularatrophy (Fyfe et al. 2006); a subset of the pedigree wasgenerated at the National Institutes of Health (NIH) AnimalCenter for coat color segregation projects of the Laboratoryof Genomic Diversity (LGD). Two individuals utilized in bothsubsets allow for merging into one pedigree for linkageanalysis. DNA was extracted from blood samples with aQIAamp DNA Blood mini kit (QIAGEN, Valencia, CA)following the manufacturer’s protocol.

Figure 2.—Domestic cat X chromosome linkage map. Foreach marker the physical location (pPosition) on the domes-tic cat X chromosome is indicated in megabases (column 2).Positions in the cat were determined using ABCC Get TraceMapping 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 indicatedin centimorgans (column 3). Pairwise genetic distance (gDis-tance in centimorgans) is represented in column 4. LODscores are $3.0, unless otherwise noted [marker pairs thatparticipate in a flip scored at LOD , 3.0 (odds of,1000:1) by CRI-MAP are represented in italics and the lowerof the LOD scores estimated by CRI-MAP and Superlink areshown with superscript footnote numbers]. Recombinationrates (Rec. rate) are indicated in centimorgans per megabase

(column 5). The background color of the recombinationrates is color coded to reflect the extreme variation in the val-ues. *, linkage was determined between markers FCA1434 andFCA1435. However, the PAR1 and the X-specific region arekept 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.

1418 A. Schmidt-Kuntzel et al.

Population study: One hundred eleven unrelated male catswere sampled from a rural environment in Frederick, Mary-land (53 orange, 13 wild type), and an urban setting in PortoAlegre, Rio Grande do Sul, southern Brazil (36 orange, 9 wildtype). Male cats were chosen for the population study as theyare hemizygous for the X chromosome and their haplotypescan be determined with certainty. Sampling of the U.S. cats wasbased on testicular tissue obtained as a by-product of routineneutering procedures, with permission of each pet owner.DNA was extracted from testis samples with a DNeasy bloodand tissue kit (QIAGEN). Samples of the Brazilian cats wereobtained from buccal swabs collected from individuals kept inprivate homes (with permission of owners) and veterinaryclinics, as well as stray animals. Genomic DNA was extractedfrom freshly collected buccal swabs, using a simple salting-outmethod (Abrao et al. 2005).

Marker development: Testing of the previous candidate region:A microsatellite (FCA1446; supplemental Table 1) was de-signed in the candidate region published by Grahn et al.(2005), to examine for linkage to orange in the Nestle Purinapedigree.

Microsatellite linkage map: Ten microsatellites described inprevious publications were used in this study (Menotti-Raymond et al. 1999, 2003a,b). Forty-nine new microsatellitemarkers were genotyped for possible addition to the Xchromosome linkage map (supplemental Table 1). Micro-satellites were selected from the sequence traces of the cat1.93 WGS for inclusion in the X chromosome map, on thebasis of their conserved syntenic position on the dog Xchromosome, following the method described by Ishida

et al. (2006). Following the availability of a cat genomesequence assembly (Pontius et al. 2007), microsatellitemarkers were selected on the basis of their location on thecat 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/cgi-bin/primer3/primer3_www.cgi; Rozen and Skaletsky 2000),including an M13 tail for fluorescent labeling of PCR prod-ucts (Boutin-Ganache et al. 2001).

Fine-mapping of X-linked orange: Thirty-six additional micro-satellite markers were designed for the localization of the Olocus according to the methods described above (see supple-mental Table 2). In addition, a single-nucleotide polymor-phism (SNP) was identified within the gene RAP2C, located inthe candidate region, through exploratory sequencing. Aprimer pair was designed for amplification and genotypingof this SNP (supplemental Table 2).

The physical location on the domestic cat X assembly wasdetermined for each marker. Markers identified from cattraces were localized on the cat X chromosome on the basis ofthe location attributed to the trace using the ABCC tool, ‘‘GetTrace Mapping Info’’ (ABCC trace-centric tools; http://www.abcc.ncifcrf.gov/Genomes/Cat/index.php) or the mi-crosatellite flanking region using the ABCC tool ‘‘GMap asequence to cat’’ (ABCC mapping and browsing tools; Wu andWatanabe 2005; http://www.abcc.ncifcrf.gov/Genomes/Cat/index.php). Markers from previous publications (ob-tained prior to the cat 1.93 WGS) were attributed to sequencetraces with cross-species MegaBLAST (http://www.ncbi.nlm.nih.gov; Zhang et al. 2000), if possible. In the event that amarker could not be localized on the domestic cat assembly, itsposition in the cat was inferred using a comparative genomicsapproach. The locations of the microsatellite markers on thedog and human X chromosomes were obtained by applying aBLAT search (Kent 2002; UCSC genome browser, http://genome.ucsc.edu/) of the trace sequence containing themicrosatellite or of the microsatellite flanking region. The

location on the domestic cat radiation hybrid map wasobtained from the literature for the previously publishedmarkers (Menotti-Raymond et al. 2003b).

Genotyping: Microsatellite markers and SNP: Primers of 95microsatellite markers and one SNP were amplified in theNestle Purina PetCare pedigree (Eizirik et al. 2003) followingtouchdown PCR conditions as in Menotti-Raymond et al.(2005). Following initial mapping of orange, 6 microsatellitemarkers were amplified in a second pedigree of 107 individ-uals segregating for this trait in an attempt to reduce thecandidate region. To establish haplotypes and investigate theirassociation with orange, primers of 25 markers located in theregion of zero recombination (between megabases 106 and116.8 on the X chromosome physical map) were amplified inthe 111 male cats of the population study. PCR products wereanalyzed as in Ishida et al. (2006). PCR product length wasused as a surrogate for actual repeat number when determin-ing allele identity. Inheritance was verified with Pedcheck(O’Connell and Weeks 1998) for genotypes of pedigreeindividuals.

ASIP locus: Primers designed to genotype the 2-bp deletionin ASIP (agouti locus), causative of the melanistic phenotype inthe domestic cat (Eizirik et al. 2003), were modified accord-ing to Boutin-Ganache et al. (2001) and amplified in 55individuals of the Nestle Purina pedigree. Individuals oforange and calico phenotype were selected on the basis ofthe availability of haplotype data and photographs providingunequivocal evaluation of pattern in the orange coat/patches.Tortoiseshell phenotypes were not evaluated due to theinsufficient surface of undisturbed orange coat color. Geno-typing at the agouti locus was determined on the basis of thesize of the fluorescent amplicon as described by Eizirik et al.(2003).

Map construction: Markers with an autosomal inheritancepattern, designed in the pseudoautosomal region 1 (PAR1),were analyzed independently from the markers with an X-specific mode of inheritance due to software limitations. Finallinkage analysis was performed for 51 markers (7 markersinherited in an autosomal manner and 44 X-linked markers).

Two-point LOD scores between all marker pairs werecomputed with Superlink (Fishelson and Geiger 2002,2004) for values of the recombination fraction (u) between 0and 0.4 in steps of 0.01. Two markers were considered linked iftheir peak LOD exceeded 3.0 (Ott 1991). Using this defini-tion, single-linkage clustering determined that the PAR1 andX-specific markers each composed a single linkage group.

A preliminary order was suggested by treating the estimatedKosambi distance (Ott 1991) between markers as a distance,reducing the marker ordering problem to the travelingsalesman problem (TSP) and using CONCORDE (Applegate

et al. 2006) to solve TSP instances. The reduction to TSP isimperfect for linkage data of this type, so some rearrangementto get the optimal order was expected, especially in regionswhere markers are very close to one another in geneticdistance. The order of linked markers was then iterativelytested 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 theycould not be ordered with any confidence or caused instabilityin the flips analysis. For flips that CRI-MAP scored at ,1000:1odds (LOD score ,3.0), Superlink was used to examinesupport for alternative orders. For such low-scoring flips, wereport the lower of the CRI-MAP and Superlink LOD scores.

Using CRI-MAP, the female u was estimated between onepseudoautosomal marker (FCA1434) and one X-specificmarker (FCA1435).

Linkage analysis for the mapping of the orange locus: Formapping orange, LOD scores were computed with Superlink.

Cat X-Linkage Map and orange Locus 1419

Orange was coded as a fully penetrant X chromosome locusfollowing the inheritance pattern described in the Introduc-tion in which (unlike most traits) phenotype completelydetermines genotype at the locus. Linkage to orange wasdetermined for the markers of the X-linkage map, followedby fine mapping with additional microsatellite markers de-signed for that purpose.

Haplotype determination for the orange locus candidateregion: Haplotypes were directly obtained from the genotypingdata for the 111 male individuals collected for the populationstudy. For the individuals from the Nestle Purina PetCarepedigree, 22 haplotypes were inferred by ferret (G. Nelson,unpublished results), an in-house implementation of theexpectation-maximization (EM) algorithm (Excoffier andSlatkin 1995), that was modified for this project to allow inputof monosome (male X) data to improve the accuracy of theinference of the female haplotypes. Because ferret considers allpossible haplotypes consistent with the genotypes, and waslimited to considering �300 million distinct haplotypes in aninference, the analysis was first performed for three overlappingshorter sequences (loci FCA1466–FCA1474, FCA1474–FCA1489, and FCA1482–FCA1498); these haplotypes were thenassembled into larger segments for all 33 loci by an additionalhaplotype inference. Ferret tests haplotype inferences by boot-strap resampling of the genotype data; inferences for the threesegments as well as the ultimate assembly of these segments had.99% bootstrap replicability. Inheritance for the haplotypesegments and final haplotypes was verified with Pedcheck(O’Connell and Weeks 1998).

RESULTS AND DISCUSSION

Two approaches are classically used for mapping ofgenetic loci of interest, a candidate gene approach andgenome scans. The sole candidate gene for orange,MC1R was mapped in the cat in a previous study to anautosome, chromosome E2 (Eizirik et al. 2003). Addi-tionally, a candidate region on the X chromosome wasproposed for the O locus in the vicinity of megabase 46.7(Grahn et al. 2005) on the basis of exclusion analysisutilizing the available X chromosome genetic linkagemaps (Menotti-Raymond et al. 1999, 2003a) contain-ing two linkage groups spanning an estimated 25 cM,along with three unmapped loci. A microsatellite(FCA1446, see supplemental Table 1), was designed inthe proposed candidate region. A demonstrated lack oflinkage between FCA1446 and orange (LOD ¼ �55, u ¼0.01), at a LOD score below the frequently used exclu-sion criterion of�2 (Ott 1991), led to our developmentof a comprehensive linkage map of the X chromosomefor the mapping of orange.

Linkage map: To generate a comprehensive map ofthe cat X chromosome, microsatellites were selected asdescribed in Marker development (materials and meth-

ods). A total of 59 microsatellite markers (supplementalTable 1) were amplified in a multigeneration domesticcat pedigree. Forty-six of the loci (FCA1435–FCA1503;supplemental Table 1) demonstrated an X-linked pat-tern of inheritance.

Thirteen microsatellite markers (FCA1422–FCA1434;supplemental Table 1), selected on the basis of the

homology of their flanking segments to regions locatednear the telomeric region of the p arm of the dog Xchromosome, demonstrated an autosomal inheritancepattern. These markers were determined to be locatedin PAR1 of the X chromosome on the basis of criteriarecommended for the identification of pseudoautoso-mal genes (Burgoyne 1982): (1) failure to map to an auto-some [marker FCA1433 demonstrated lack of linkagewith�500 autosomal markers in a third-generation link-age map of the cat (Menotti-Raymond et al. 2008)], (2)linkage to an X-specific marker [marker FCA1434 linkedto the X-specific marker FCA1435 with a female re-combination fraction estimated at 0.02 (LOD . 85)],and (3) physical mapping to the X chromosome [12 ofthe 13 markers are placed on the cat X chromosome inthe current assembly (Garfield browser 12.2; Pontius

and O’Brien 2007; http://lgd.abcc.ncifcrf.gov/cgi-bin/gbrowse/cat)]. Indirect physical mapping was obtainedby selecting 2 markers, FCA1432 and FCA1434, withinintrons of NLGN4X and TBL1X, respectively, that werepreviously mapped to the pseudoautosomal region ofthe domestic cat radiation hybrid (RH) map (Murphy

et al. 2007). Additionally, sequence traces containingmarker FCA1426 (data not shown) align to homologousregions on the human X and Y chromosomes.

The final genetic recombination map (Figure 2)comprises 46 microsatellite markers (7 pseudoautoso-mal and 39 X-specific markers) spanning the estimated140 Mb of the domestic cat X chromosome (Garfieldbrowser 12.2; Pontius and O’Brien 2007). Eight micro-satellites located in high marker density regions wereexcluded from the map, and 5 others were droppedduring the analysis (see materials and methods).Genetic distances between adjacent loci range from 0 to23.0 cM (Figure 2), and the total genetic length of thedomestic 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 forcomplete coverage of the X chromosome includesdemonstration of linkage between a pseudoautosomal(FCA1434) and an X-specific marker (FCA1435). Themost distal cat marker is located at megabase 139.7,,0.3 Mb from the end of the current cat X sequenceassembly. The total genetic length of the cat X chromo-some is larger than that reported in other mammals[34 cM in the silver fox (covering two-thirds of the Xchromosome; 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 thecow (Ihara et al. 2004), and 195 cM in humans (Matise

et al. 2007)]. Additionally, some of the older maps mayunderestimate the size if they do not cover the entiretyof the X chromosome. Similarly, the recombinationlength of the domestic cat autosomal map is longer thanthat observed in most reported mammalian maps(Menotti-Raymond et al. 2008).

1420 A. Schmidt-Kuntzel et al.

The order of microsatellite markers on the X chro-mosome inferred by genetic linkage data is consistentwith their physical coordinates in the domestic catgenome assembly (Figure 2). Only one difference wasdetected between the genetic linkage and radiationhybrid maps: marker FCA53, which follows an X-linkedinheritance pattern, was mapped to chromosome B1in the first genetic linkage map of the cat (Menotti-Raymond et al. 1999). This first genetic linkage map wasgenerated in a hybrid pedigree between the domesticcat the Asian leopard cat (Prionailurus bengalensis)(Menotti-Raymond et al. 1999). The high incidenceof ‘‘null’’ alleles in hybrid individuals for this locuswas misinterpreted as an autosomal inheritance pat-tern. FCA53 was mapped on cat B2 in the cat RHmap (Menotti-Raymond et al. 2003b); however, itslocation has presently been determined to be on theX chromosome (W. J. Murphy, personal communica-tion). Of note, FCA053 could not be placed on thecat, dog, or human assemblies. For other cat X markersthe colinearity between the domestic cat, dog, andhuman X chromosome coordinates is shown in sup-plemental Figure 1 and confirms the conserved syn-teny of the mammalian X chromosome between dog,human, and cat, as previously reported by Murphy

et al. (1999).Extreme variation of recombination rates was appar-

ent across the X chromosome, ranging from 0 cM/Mb,extending over 34 Mb, to 15.7 cM/Mb localized within300 kb (supplemental Figure 2). Several phenomenahave been suggested to be causative of variation in re-combination frequencies; among these are differencesin GC content, gene density, and location on the chro-mosome (telomeric vs. centromeric; Nachman 2002).No evidence for a correlation between GC contentand recombination frequencies was found utilizing thedomestic cat genome browser (Garfield browser 12.2;Pontius and O’Brien 2007). The centromere, a regionthat is expected to have suppressed recombination(Nachman 2002), is located approximately at position53 Mb of the domestic cat X chromosome (Garfieldbrowser 12.2; Pontius and O’Brien 2007) and is indeedsituated within the region of zero recombination. In-terestingly, the telomeric portion of the q arm alsoexhibits low recombination rates (0 and 0.3 cM/Mb) inthe terminal 10 Mb of the domestic cat X chromosome.This is an unusual observation, as telomeric regions areclassically reported as chromosomal areas undergoingfrequent recombination (Nachman 2002). Suppressionof recombination has also been attributed to chromo-somal rearrangements such as inversions, in whichrecombination occurring in the inverted region in aheterozygous individual leads to nonviable recombi-nants (Griffiths et al. 1996). However, a large inversionis unlikely to account for these observations, becausethere is no ambiguity in marker order associated eitherwith the linkage analysis or from previous cytogenetic

in situ hybridization studies (T. Raudsepp, personalcommunication).

Mapping of the orange locus: Linkage analysis: Themarkers placed on the domestic cat X chromosomelinkage map were utilized to identify linkage to the Olocus in a multigeneration nonbreed domestic cat ped-igree (Eizirik et al. 2003), segregating for orange. Sig-nificant linkage to the O locus was detected for markersFCA1466 and FCA1494, (u ¼ 0, LOD ¼ 19.0 and 29.9,respectively) (Figure 3).

Fine-scale mapping utilizing six additional micro-satellite markers (Figure 3, supplemental Table 2) andone 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 re-gion could not be reduced further despite the additionof 88 informative meioses from an additional pedigreesegregating for orange (Figure 3).

The candidate region for X-linked orange in thedomestic cat corresponds to an interval between 120and 131 Mb in the human X chromosome. Interestinglythe candidate region for Sex-linked yellow in the Syrianhamster was mapped in a nonhomologous region,situated between 46 and 54 Mb on the human Xchromosome (Alizadeh et al. 2009). Additional in situhybridization data provided by Alizadeh et al., utilizingmouse BAC probes from the conserved syntenic posi-tion in the cat orange region, provide further supportthat the orange loci in the Syrian hamster and thedomestic cat are located in nonhomologous regions.However, we cannot rule out the possibility that micro-rearrangements have occurred in either the hamster orthe cat genome that have not been detected by ourmethodologies and that the two orange loci could beorthologs. Alternatively, the two genes could be in-dependent members of the same pathway or encodegene products with similar function.

Haplotype analysis: An attempt was made to reduce thesize of the orange candidate region through identifica-tion of ancestral recombination events within the orangeinterval delimited by the linkage analysis, using ahaplotype-based approach. The logic followed here isthat recombination between an orange and a nonorangehaplotype could ‘‘erode’’ the length of the orange hap-lotype, further refining the size of the orange candidateregion.

Haplotype analysis was performed in a collection of367 cats, including the Nestle Purina pedigree and apopulation sample set of 111 male individuals for whichhaplotypes could be unambi guously determined. In-dividuals in the population survey were sampled fromtwo distant locales, one in southern Brazil and the otherin the eastern United States. The broad geographicscope of the survey, including populations that have notbeen in direct genetic contact for many generations,should increase the probability of sampling distantlyrelated alleles, implying an enhanced likelihood of

Cat X-Linkage Map and orange Locus 1421

spanning ancestral recombination events within a nar-row chromosomal interval. Additional markers (n¼ 22;supplemental Table 2) were designed to increase themarker density in the candidate region.

The haplotypes generated here included 25 micro-satellite markers (FCA1466–FCA1498) located across theregion of zero recombination (supplemental Figure 3).One hundred unique haplotypes were characterized,including 36 haplotypes observed in association with thewild-type or nonorange phenotype (subsequently re-ferred to as nonorange haplotypes) and 64 haplotypesassociated with the orange phenotype (referred to sub-sequently as orange haplotypes; supplemental Figure 3).

To identify alleles that are exclusive or prevalentin orange haplotypes relative to nonorange haplotypes(which would thus be useful for detecting ancestralrecombination events), the alleles were color coded withrespect to their relative frequency of occurrence in thetwo alternative color phenotypes (supplemental Figure3). Three major groups were identified in both the orange(groups I–III) and nonorange (groups 1–3) haplotypes onthe basis of their allele composition. Orange haplotypegroups 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 IIIappears distinct, being represented in only one nonorangehaplotype (hap17; supplemental Figure 3).

Group III (hap43–100; supplemental Figure 3) com-prises 91% of the orange haplotypes and is characterizedby five ‘‘signature’’ alleles (at loci FCA1470, FCA1472,FCA1482, FCA1486, and FCA1494; A–E in supplementalFigure 3), which are found almost exclusively in orangehaplotypes. We identified 6 haplotypes (hap43–48) that,at locus A, exhibited a change from the 194-bp signatureallele observed in the remaining group III haplotypes.The additional introduction of the adjacent 243-bpallele at the locus FCA1466 in hap44 and -48 is suggestiveof a recombinatorial origin of the flanking markeralleles 243 and 168 (see hap19 and -21). At the telomericboundary of the candidate region, 14 haplotypes(hap47–60) exhibited a change at locus E from the268-bp signature allele observed in all other group IIIhaplotypes. Both signature loci, A and E, show littleallelic variation relative to the major nonorange andorange haplotype groups, suggesting a recombination-based vs. mutational origin of the nonsignature alleles.

Figure 3.—Genetic linkage mapping of the orange locus on the cat X chromosome. (A) The majority of the markers of thelinkage 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% recom-bination and transition between 0% and 2 or 4%, respectively). (C) Specific recombination values are represented. Optimal re-combination 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; Nestle 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 col-umn 6 (determined using ABCC Get Trace Mapping Info, ABCC trace-centric tools; http://www.abcc.ncifcrf.gov/Genomes/Cat/index.php).

1422 A. Schmidt-Kuntzel et al.

Signature loci C (FCA1482) (hap59) and D (FCA1486)(hap55–60) display a high level of allele complexity;nonsignature alleles may therefore represent mutationalor gene conversion origins. Ancestral recombinationevents with nonorange haplotypes affecting signature lociA and E would decrease the orange candidate region to3.5 Mb, extending from marker FCA1470 at 109.8 Mb tomarker FCA1494 at 113.3 Mb. Nonetheless, the possi-bility of mutational events at loci A and E cannot beexcluded. The identified region contains no obviouscandidate genes and the annotation of this genomicsegment is still unclear. Within the 3.5 Mb, 4 putativegenes were predicted by the cat genome browser (Gar-field browser 12.2; Pontius and O’Brien 2007) and .20RefSeq genes align to the dog genome (UCSC genomebrowser; www.genome.ucsc.edu).

The six remaining orange haplotypes (groups I and II)contain one (hap37) or none (hap38–42) of the fivesignature alleles mentioned above (supplemental Fig-ure 3). The high degree of similarity observed betweenthe orange haplotypes of groups I and II and thenonorange haplotypes of groups 1 and 2, respectively,suggests the occurrence of additional independentmutational events causative of orange, leading to theorange haplotypes of groups I and II. To identify thenumber of times orange arose in this data set, investiga-tion of variation at the nucleotide level is required oncethe gene responsible for the orange phenotype isidentified.

A noteworthy observation is that each of the orangehaplotype groups is represented in both the Americanand the Brazilian data sets. This could be explained byrecent gene flow between the populations due to ease ofmodern travel or by an old origin of the haplotypegroups, preceding the colonization of domestic cats intoBrazil and the United States, in both cases originatingfrom European gene pools.

Epistasis of orange over nonagouti: We have previouslyshown that the nonagouti mutation in domestic cats iscaused by a 2-bp deletion in exon 2 of the AgoutiSignaling Protein (ASIP) gene, leading to an early frame-shift and premature truncation (Eizirik et al. 2003).Nonorange cats, homozygous for nonagouti (a/a), arecompletely black, similar to laboratory mice that carrynull alleles of agouti. In most mammals, the phenotypicdifference between agouti and nonagouti individuals isthe presence of pheomelanic-banding in all hairs in theformer but not the latter. However, the situation indomestic cats is more complicated due to the presenceof tabby patterns. In nonorange cats, banding of hairs issuppressed in the striped or spotted regions of tabbypattern; these hairs are more uniformly pigmented, andhence ‘‘stand out’’ against the lighter-colored back-ground of banded (agouti) hairs (see hairs in Figure1D, c and d). The overall effect, generated by regions ofdarker largely unbanded hairs juxtaposed with regionsof lighter-banded hairs, creates pattern in the coat. In

orange cats, banding of hairs is also suppressed instriped and spotted regions; however, the hair in theseregions is a dark orange (as opposed to black in wild-type cats), and the light-colored background hairs are adark orange with light-colored orange banding (Figure1C, b and c, and 1D, a and b).

Another particularity of orange cats is that visibility oftabby pattern does not seem to be affected by thegenotype of the agouti locus. While nonagouti, nonorangecats exhibit no pattern (other than occasional faint ‘‘ghostpatterns’’), nonagouti, orange cats do exhibit pattern(Figure 1B). Thus, to investigate this epistatic relationshipbetween orange and nonagouti, we determined agoutigenotypes for orange or calico individuals (n ¼ 55) inour pedigree and confirmed orange status at a genotypiclevel using haplotype data.

In calico cats, tabby pattern was exhibited in orangefur independent of the presence or absence of patternin nonorange patches (Table 1; Figure 1B). In non-orange fur patches, tabby pattern was fully hidden inpresence of the nonagouti (a/a) genotype (Figure 1B)All orange cats exhibited pattern. On the basis of amolecular genetic test for the agouti locus (Eizirik et al.2003), 45 of the 55 individuals with orange coatdemonstrated a wild-type agouti genotype (A/A or A/a),and 10 demonstrated a nonagouti genotype (a/a) (Table1). Thus, genotype status at the agouti locus exhibited noinfluence on the presence or absence of tabby pattern inorange-colored fur, but genotype at ASIP did correlatewith the presence of tabby pattern in nonorange fur asassessed in individuals of calico phenotype (n ¼ 19;Table 1). Independence of tabby pattern from the agoutigenotype in areas of orange coat color, and therefore

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 32 0 0Calico (O/o) 6a 13b 0 0Tortoiseshell (O/o) NA NA NA NANonorange (o/o or o/Y) NA NA NA NATotal 10 45 0 0

Genotypes at the agouti locus (a/a, A/a, or A/A) are repre-sented in columns 2–5, based on the phenotype in the orangefur parts of domestic cats. No cats of orange coat color lackedtabby pattern (columns 4 and 5) and cats with tabby pattern inorange 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 nonorangefur parts.

b All 13 individuals carrying at least one wild-type allele (A/Aor A/a) displayed tabby pattern in the nonorange fur parts.

Cat X-Linkage Map and orange Locus 1423

epistasis of orange over nonagouti, is clearly demonstratedby these results. This observation illustrates the com-plexity of the molecular mechanisms underlying do-mestic cat (and mammalian) coat color, with multipleprocesses interacting to generate different combina-tions of phenotypes.

Summary: We developed a comprehensive geneticlinkage map of the domestic cat X chromosome,including its pseudoautosomal region 1 (46 markersspanning 38 cM in the PAR1 region and 228 cM in the X-linked region). The map was successfully used to definea candidate interval of 10.8 Mb (delineated by markersFCA1464 and RAP2cSNP) for the X-linked orange locus.Additional haplotype analysis suggested multiple ori-gins for orange in the domestic cat and the potential ofhistoric recombination events at the telomeric andcentromeric boundary of our orange candidate region,which would reduce the orange interval to 3.5 Mb.

The linkage map will provide a valuable resource forfuture localization of other X-linked traits. We recom-mend including the markers of the pseudoautosomalregion in whole genome scans seeking loci controllingtraits that have an autosomal inheritance pattern as theinheritance patterns of pseudoautosomal and autoso-mal loci are indistinguishable. The O locus and itsinfluence on the poorly understood pheomelanicpathway are currently unknown. To better understandthe process, as well as the genetic basis for the epistasisof orange over nonagouti, the causative gene for this traitneeds to be identified. A solid genomic basis for this in-depth search has been laid out by this study, which willhopefully foster additional investigation on this trait, itsfunctional implications, and its relevance for mamma-lian pigmentation genetics in general.

We thank Gregory Barsh for discussion and sharing of unpublishedresults, Ana Carolina Garcia Escobar for the collection and extractionof the DNAs obtained from Brazil, the Laboratory of GenomicDiversity core lab for the extraction of the local samples, Joan Pontiusand the Advanced Biomedical Computing Center for their adviceregarding the whole genome sequencing of the cat, Carlos Driscoll forhis suggestion of using microsatellites for haplotype analysis, SolveigPflueger for her advice as a cat breeding specialist, Richa Agarwala andJames Tomlin for assistance with map computation, and John Fyfe(Michigan State University) for making available to this study samplesfrom a feline spinal muscular atrophy pedigree that also segregated fororange. We also acknowledge the photographers who generated theimages used in Figure 1A: Susan Feingold (Fulton County AnimalServices), Joanna Harkin (Alliance for Stray Animals and People), BillHopkins (pet owner), Andrea Thompson (pet owner), AnthonyGriffith (author of Introduction to Genetic Analysis), and Martin Featherand Cristy Bird (feral tortoiseshell cat living on the grounds of a wat inBangkok, Thailand). We also thank Marti Welch (Scientific Publica-tion, Graphics and Media; Advanced Technology Program, ScienceApplications International Corporation, Frederick, MD) for excellentassistance in generating figures and photographs. This publication hasbeen funded in whole or in part with federal funds from the NationalCancer Institute, National Institutes of Health (NIH), under contractno. NO1-CO-12400. The content of this publication does notnecessarily reflect the views or policies of the Department of Healthand Human Services, nor does mention of trade names, commercialproducts, or organizations imply endorsement by the U.S. Govern-

ment. This research is supported in part by the Intramural ResearchProgram of the NIH, National Library of Medicine.

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Communicating editor: L. Siracusa

Cat X-Linkage Map and orange Locus 1425