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A Genetic Linkage Map of Microsatellites

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Genomics 57, 9–23 (1999)Article ID geno.1999.5743, available online at http://www.idealibrary.com on

in the Domestic Cat (Felis catus)

Marilyn Menotti-Raymond,*,1 Victor A. David,* Leslie A. Lyons,* Alejandro A. Schaffer,†James F. Tomlin,‡ Michelle K. Hutton,§ and Stephen J. O’Brien*

*Laboratory of Genomic Diversity, NCI–FCRDC, Frederick, Maryland 21702; †NHGRI/IDRB and ‡CIT/CBEL/BIMAS,National Institutes of Health, Bethesda, Maryland 20892; and §PE AgGen, Inc., Davis, California 95616

Received September 18, 1998; accepted January 6, 1999

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Of the nonprimate mammalian species with devel-ping comparative gene maps, the feline gene mapFelis catus, Order Carnivora, 2N 5 38) displays theighest level of syntenic conservation with humans,ith as few as 10 translocation exchanges discriminat-

ng the human and feline genome organization. Toxtend this model, a genetic linkage map of microsat-llite loci in the feline genome has been constructedncluding 246 autosomal and 7 X-linked loci. Two hun-red thirty-five dinucleotide (dC z dA)n z (dG z dT)n and8 tetranucleotide repeat loci were identified andenotyped in a two-family, 108-member multigenera-ion interspecies backcross pedigree between the do-estic cat (F. catus) and the Asian leopard cat (Prio-ailurus bengalensis). Two hundred twenty-nine lociere linked to at least one other marker with a lod

core >3.0, identifying 34 linkage groups. Representa-ive markers from each linkage group were assignedo specific cat chromosomes by somatic cell hybridnalysis, resulting in chromosomal assignments to 16f the 19 feline chromosomes. Genome coverage spanspproximately 2900 cM, and we estimate a geneticength for the sex-averaged map as 3300 cM. The mapas an average intragroup intermarker spacing of 11M and provides a valuable resource for mapping phe-otypic variation in the species and relating ito gene maps of other mammals, includinguman. © 1999 Academic Press

INTRODUCTION

Humankind has long valued its association with theat. It is estimated that cats were domesticated approx-mately 7000 years ago, with the oldest fossil recordsmplicating coexistence with humans originating from

Sequence data from this article have been deposited with theMBL/GenBank Data Libraries under Accession Nos. AF130472–F130701.

1 To whom correspondence should be addressed at Laboratory ofenomic Diversity, National Cancer Institute, Building 560, Room1–38, Fort Detrick, Frederick, MD 21702-1201. Telephone: (301)46-1299. Fax: (301) 846-6327. E-mail: [email protected].

9

ered domestic cat breeds recognized in the Unitedtates (Cat Fancy Association, Manasquan, NJ), some00 disorders that demonstrate Mendelian patterns ofnheritance have been identified and characterizedNicholas et al., 1998; Migaki, 1982; Robinson, 1977).

any of these pathologies are analogous to humannherited disorders, including autosomal dominantolycystic kidney disease (Biller et al., 1996), retinaltrophy (Narfstrom, 1983), primary hyperoxaluriaype 2 (Danpure et al., 1989), glycogen storage diseaseype IV (Fyfe et al., 1992), and hypothyroidism (Ta-ase et al., 1991). Genes associated with some of theseeline disorders have been genetically mapped andharacterized including loci for glycogen branching en-yme, arylsulfatase B, and lipoprotein lipase (Gilbert etl., 1988; Fyfe et al., 1992; Jackson et al., 1992; Ginz-nger et al., 1996), and corrective gene therapy strate-ies have even been examined for some disorders,ncluding feline arylsulfatase B deficiency and

ucopolysaccharidosis Type VI (Gasper et al., 1984;eters et al., 1991; Byers et al., 1997). Nonetheless,enes associated with the majority of feline disordersave yet to be identified.The value of animal models has been demonstrated

epeatedly with hundreds of characterized mouse mu-ations. Model animal systems serve to elucidate mo-ecular mechanisms underlying pathology and ulti-

ately provide useful candidates for drug and geneherapy interventions. For many human hereditaryisorders, multiple animal models from diverse evolu-ionary backgrounds may be required to characterizeolecular pathologies fully. For instance, murine mod-

ls that can be genetically engineered may prove inad-quate for analysis of quantitative characters, as gen-rations of inbreeding may have eliminated geneticiversity at crucial modifying loci. For rare disordersnd those for which candidate gene approaches haveeen exhausted, spontaneous models can offer a hope-ul resource for genomic mapping.

Efficient genetic analysis benefits from integratedenetic linkage maps containing Type I coding genes

0888-7543/99 $30.00Copyright © 1999 by Academic Press

All rights of reproduction in any form reserved.

and Type II polymorphic markers (Lyons et al., 1997;O

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10 MENOTTI-RAYMOND ET AL.

’Brien et al., 1993).The current status of the domestic cat map includesphysical map of 105 Type I loci generated principallyy use of a rodent 3 cat somatic cell hybrid panel anduorescence in situ hybridization (FISH) (O’Brien andash, 1982; Gilbert et al., 1988; O’Brien et al., 1997a;opez et al., 1996; Yuhki and O’Brien, 1988). Remark-ble conservation of synteny has been observed be-ween the human and the feline physical mapsO’Brien et al., 1997b), based on cytogenetic homologyf feline vs human G-band chromosome preparationsNash and O’Brien, 1982) and reciprocal chromosomeainting patterns (Zoo-FISH) of flow-sorted singleetaphase chromosome libraries used as a fluorescent

robe for in situ hybridization (O’Brien et al., 1997b;ienberg et al., 1997; Rettenberger et al., 1995).In addition to high-resolution human and mouse mi-

rosatellite maps (Dib et al., 1996; Dietrich et al.,994), recent microsatellite maps for diverse speciesuch as rat, cattle, pig, goat, dog, and zebrafish haveeen constructed (Jacob et al., 1995; Barendse et al.,994; Bishop et al., 1994; Archibald et al., 1995;aiman et al., 1996; Knapik et al., 1998; Mellersh et al.,997). Microsatellite markers are highly polymorphicTautz, 1989), abundant, and near randomly distrib-ted in eukaryotic genomes (Weber and May, 1989;autz and Renz, 1984). The genome maps of microsat-llites have provided a valuable mapping resource fordentification of genes associated with hereditary dis-ase (Hearne et al., 1992).We report here a genetic linkage map of microsatel-

ite loci in the domestic cat, composed of 246 autosomalnd 7 X-linked markers. Of these loci, 229 are linked tot least one other marker in a total of 34 linkageroups. Physical assignments have been made to 16 ofhe 19 feline chromosomes by use of a rodent 3 catomatic cell hybrid panel. An average intragroup in-ermarker distance of 11 cM was observed. The lengthf the felid map is estimated at 3300 cM. This geneticinkage map in the domestic cat will provide a frame-ork for future mapping efforts and a valuable re-

ource for linkage mapping in disease pedigrees.

MATERIALS AND METHODS

Pedigree. Mendelian inheritance was analyzed in a 108-memberultigeneration interspecies pedigree between the domestic cat (Fe-

is catus) and the Asian leopard cat (Prionailurus bengalensis) (Fig.). We utilized an interspecies pedigree to maximize the number ofoding differences in Type I loci that could be utilized in the con-truction of a linkage map of Type I markers. An in-depth descriptionf the generation of the pedigree is in Lyons et al. (submitted forublication). The samples consisted of two pedigrees, one family withdomestic cats, 4 F1 females, 2 leopard cat males, and 12 backcross

rogeny. A second pedigree was composed of 18 domestic cats, 11eopard cats, 14 F1 hybrids, 34 BXD individuals (progeny of an F1

emale backcrossed to a domestic cat male), and 7 BXL (progeny of an1 female backcrossed to a leopard cat male) progeny. Domestic cats

11) were of mixed breed obtained from a commercial laboratoryupplier (Liberty Labs, Waverly, NY) and 13 were of the Egyptianau domestic cat breed from Dr. Gregory and Elizabeth Kent (Lot-

onca City, OK), and Judy Sugden (EEYAA Cats, Blaisdell, CA).DNA was extracted from leukocytes or fibroblast tissue culture cell

ines established for each animal (Modi and O’Brien, 1988) usingtandard proteinase K/phenol/chloroform extraction protocols (Sam-rook et al., 1989).

Genomic library. Construction and screening of a genomic li-rary for CA/GT repeats have been described (Dietrich et al., 1992;enotti-Raymond and O’Brien, 1995). Genomic DNA from a female

omestic cat (F. catus), LGD accession No. Fca-081, was digestedith MboI (Life Technologies). Products were size-fractionated on a% NuSieve GTG agarose gel (FMC Bioproducts), and fragments insize range of 250–500 bp were subcloned into the alkaline phos-

hatase treated BamHI site of M13mp19 (Life Technologies). Li-rary efficient Escherichia coli (Electromax DH10B cells, Life Tech-ologies) were transformed with ligation product by electroporationsing a Cell-Porator Electroporation System I (Life Technologies)nd plated on 23 YT medium (Sambrook et al., 1989) at a density ofpproximately 5000 plaques/150-mm culture plate. Two separatecreens were performed for CA/GT repeat recombinant clones andne screen using a tetranucleotide repeat oligonucleotide cocktail. Inll, approximately 30 million basepairs of genomic DNA wascreened for dinucleotide and tetranucleotide repeat loci. Plaque liftsere made in duplicate to U-V Duralon nylon membranes (Strat-gene) and hybridized overnight with end-labeled (CA)15 and (GT)15

ligonucleotides (T4 polynucleotide kinase, Life Technologies;g-32P]ATP, 5000 Ci/mmol, New England Nuclear) at 65°C in Churchnd Gilbert hybridization solution (Church and Gilbert, 1984). Fil-ers were washed once in 23 SSC/1% SDS at room temperature andwo times at 65°C in 0.13 SSC/0.1% SDS for approximately 3 min orntil a single membrane registered between 1000 and 2000 countser minute on a handheld monitor. X-OMAT AR film was exposed tohe filters at 280°C overnight with an intensifying screen. Positivelaques were selected and plated at low density for a secondarycreen. Plaques that generated duplicate positive signals were grownn 3 ml of 23 YT broth. DNA was isolated from the supernatantsing a Qiagen M13 mini kit (Qiagen) and sequenced using a Prismeady Reaction Dye Primer Cycle Sequencing Kit (PE Applied Bio-ystems Inc.) and an ABI (PE Applied Biosystems, Inc.) 373 DNAenetic Analyzer apparatus. New sequences were screened against

he sequences already in our data collection using an applicationrogram based on FASTA (Version 1.6) (Pearson and Lipman, 1988)o identify duplicates. Primer pairs were designed in unique se-uence flanking the microsatellite using PRIMER (Version 0.5, Lin-oln, Daly, and Lander, Whitehead Institute for Biomedical Re-earch, Cambridge, MA). All primer pairs were designed for uniformmplification conditions and a Tm of 60°C. Amplification products forndividual loci were initially electrophoresed in 2% agarose gels toxamine product size and quality. Primer pairs for 13 tetranucle-tide repeat loci were designed using similar methods (using differ-nt cat DNA) and characterized at PE AgGen, Inc. (Davis, CA).omestic cat sequences containing microsatellite sequences thatere used for primer design have been submitted to GenBank.

Genotyping. PCR amplifications of individual microsatellite lociDavid and Menotti-Raymond, 1998) were performed in 10-ml reac-ion volumes containing 13 Perkin–Elmer PCR buffer II containing0 mM Tris–HCl (pH 8.3), 50 mM KCl (PE Applied Biosystems Inc.),.0 mM MgCl2; 250 mM each of the four deoxyribonucleoside 59-riphosphates (dATP, dCTP, dGTP, and dTTP) (Pharmacia), 0.4nits AmpliTaq DNA polymerase (Perkin–Elmer Cetus, Norwalk,T), 4.0 pmol each of forward and reverse primer (Life Technologies,aithersburg, MD, and PE Applied Biosystems Inc.), and 50 ng ofenomic DNA. The primer listed as the reverse primer in Table 1 wasabeled with a fluorescent dye phosphoramidite (6-FAM, TET, HEX).CR amplification was performed in a Perkin–Elmer Model 9600hermocycler using the following set of conditions: one cycle of 3 mint 93°C, 10 cycles of 94°C for 15 s, 55°C for 15 s, 72°C for 30 s, 20ycles of 89°C for 15 s, 55°C for 15 s, 72°C for 30 s, and one cycle of2°C for 30 min. PCR amplifications were performed for five lociTable 1) using AmpliTaq gold (PE Applied Biosystems) to reduce

artifactual bands; the cycling parameters were altered to include asmcBdDadmlapwsSettt(stidau“tr

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ingle initial amplification cycle of 94°C for 10 min following theanufacturer’s suggestions. PCR products were diluted as empiri-

ally established for each locus with sterile deionized water (Qualityiological) and pooled in sets of five or six loci. Two microliters ofiluted pooled product was mixed with 4 ml of a gel loading buffer/NA standard mixture composed of a 6:1:1 ratio of deionized form-mide (Sigma):ABI Prism Genescan-350 TAMRA internal lane stan-ard:ABI Genescan loading buffer. Samples were denatured for 3in at 94°C and snap-cooled on ice. Two microliters of sample was

oaded per lane and electrophoresed in 6% denaturing polyacryl-mide gels in an ABI Model 373A Automated DNA Sequencer Ap-aratus for 3.5 h at 2500 V, 40 mA, 25 W. Allele sizes were analyzedith ABI Genescan (Version 1.2.2-1) and Genotyper (Version 1.1)

oftware applications using the local Southern method (Elder andouthern, 1987) to generate a best-fit curve from the size standardslectrophoresed in each lane. Genotypes were checked for consis-ency with Mendelian rules of inheritance using a software applica-ion tailored for the pedigree (Microsoft FoxPro 2.6 for the Macin-osh) and by the software application UNKNOWN of FASTLINK 4.0Cottingham et al., 1993), and genotypes that exhibited any incon-istency were reexamined and regenotyped if necessary. Amplifica-ion product from a standard reference animal was electrophoresedn the multiple gels necessary to type the pedigree to correct for sizeifferences of PCR products between gels. Over the 253 loci, anverage of 13%, or 8 alleles/locus, of the backcross animals werengenotyped or were intentionally left out of the data set due to thehybrid null” phenomenon described under Results. Genotypes forhe entire data set are available at our laboratory Web site (http://ex.nci.nih.gov/RESEARCH/basic/lgd/lgdpage.htm).

Map construction and linkage analysis. The initial lod scorecreening was performed with the MLINK program of FASTLINK.0P (Cottingham et al., 1993; Schaffer et al., 1994). Two loci wereonsidered linked if they achieved a two-locus lod score of 3.0 orreater (Ott, 1991). Two loci that did not have a lod score above 3.0irectly were included in the same linkage group indirectly as aesult of high lod scores with other loci. The ILINK program ofASTLINK 4.0P was used to find exact optimal lod scores andecombination fractions between each pair of loci deemed to be in theame linkage group.To find an order in each linkage group FIRSTORD (Curtis, 1994)

nd CRI-MAP (version 2.4) (Lander and Green, 1987) were used.IRSTORD is a quick method to obtain plausible orders for large

inkage groups. For each linkage group of size 10 or less, the ALLption in CRI-MAP was used to try all orders directly. For the groupsf size larger than 10, FIRSTORD was used to find an initial ordernd CRI-MAP was used with the FLIPS7 option until the ordertabilized. The FLIPS7 option considers all orders that differ fromhe input order in any permutation of seven consecutive loci. For allroups the set of orders that are at all consistent with the two-pointod scores is small, if one coalesces those loci with no recombinationetween them into one locus. Therefore, we feel that the restrictiono FLIPS7 did not miss consideration of any plausible orders. Al-hough the sets of plausible orders are small, CRI-MAP was almostever able to select a single order that is better than all other ordersy 1.0 lod units or more. The CRI-MAP uncertainty is due to insuf-cient informative meioses in these pedigrees to distinguish amonghe plausible alternatives.

In the MLINK computations, equal allele frequencies were used atach locus due to the fact that different allele frequencies werebserved in the two species. We tested the robustness of this decisiony repeating the calculations in the large 22-locus group on A1 usingn average of equal allele frequencies and allele frequencies from theample of all domestic breed founder cats. The map order for thisroup came out identical with negligible changes in distances. Theutcome of this allele frequency test is consistent with the outcome ofhe test of CRI-MAP to compute lod scores; both show that theeioses with ungenotyped parent cats (where allele frequencies aresed and which are ignored by CRI-MAP) have little effect on the lodcore.

oint linkage analysis runs were performed in parallel (Gupta et al.,995) on an SGI multiprocessor on top of the p4 system (Butler andusk, 1994). Programs were written to assemble automatically theedigree and locus files, automatically set up the next run, clean uphe intermediate results files, find the high lod scores, and assemblehe linkage groups. This combination of programs allowed us to setp automatically a batch queueing system to use multiple worksta-ions to handle batches of thousands of small runs. The results weretored in a SYBASE database, and an HTML interface was writteno query the results database.

Physical mapping of microsatellite loci. Loci were physicallyapped with the use of a 39-cell line, rodent 3 cat somatic cellybrid panel previously used to map over 105 Type I markers in theomestic cat (O’Brien and Nash, 1982; O’Brien et al., 1997a). Indi-idual loci were PCR amplified from 100 ng of DNA from each celline and genotyped as described above with the exception that Am-liTaq gold (Perkin–Elmer Cetus) was utilized in the amplificationso decrease potential artifacts amplifying from the rodent back-round. Each hybrid line was scored for the presence or absence ofocus-specific amplification products. Cell line genotype profiles werehecked for concordancy and discordancy with all other known mark-rs typed in the hybrid lines including chromosomes, isozymes, andther genes. x2 values were calculated from a 2 3 2 contingency tablehere marginal frequencies were used to estimate expected values.ssignment of a locus to a chromosome was based on a P value0.001 for two distinct loci in a linkage group to the same chromo-

ome.

Heterozygosity. Heterozygosity estimates were derived from ge-otype assessment of 10 unrelated outbred domestic cats, 8 unre-

ated leopard cats, and 9 unrelated Egyptian Mau domestic cats as

h 5 2n~1 2 (x i2!/2n 2 1),

here ~Xi) is an estimate of allele frequency Xi, and n is the numberf individuals sampled (Nei and Roychoudhury, 1974). Heterozygos-ty estimates are reported for the unrelated outbred domestic catubset (Table 1).

RESULTS

ibrary Construction and Primer Screening

Two small insert libraries of genomic DNA from aemale domestic cat were screened with radiolabeledCA)15 and (GT)15 oligonucleotides. A subsequentcreening was performed using a tetranucleotide re-eat oligonucleotide mixture. Five hundred seventy-wo dinucleotide repeat and 32 tetranucleotide repeatrimary recombinant clones were selected for a second-ry screen. Positive recombinant clones were se-uenced, and primer pairs were designed for 360 dinu-leotide, 8 tetranucleotide, and 2 pentanucleotideepeat loci to amplify products of between 100 and 300p under a standard set of conditions. Recombinantshat proved inappropriate for primer design included5 dinucleotide and 24 tetranucleotide loci (16%) thatither failed the secondary screen or were false posi-ives, 50 (8%) duplicates, 47 (8%) with flanking se-uence too short or inappropriate for primer design,nd 38 (6%) where the insert was too large for a singleass sequence.Based on a screen of approximately 25,600 recombi-

ant clones in our first library with an average insert

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ize of 550 bp, we estimate the frequency of the CA/GTinucleotide motif with 12 or greater repeat units aspproximately 1 in 40 kb in the cat genome (25,600FU 3 550 bp/339 CA/GT recombinant clones), a fre-uency that is about equivalent to that observed inther mammalian genomes, including human (Webernd May, 1989) and the giant panda (Menotti-Ray-ond, unpublished results). The average number of

inucleotide repeat motifs/locus in the sequenced re-ombinant clones was 21. The 370 primer pairs were

Description of 253 Domes

Note. All loci are dinucleotide repeat (dC z dA)n z (dG z dT)n unranges are reported for the domestic cat (mixed breed). Commsharp electropherogram signal, no artifactual bands; 3, someAmpliTaq Gold (PE Applied Biosystems) gave best results. 1 mwere “null” in all leopard cats; T, tetranucleotide repeat locus.not mapped; Informative meioses: parent in which meiosis occu

mplified in genomic DNA of three unrelated individ-als to check for a product of anticipated size, to eval-ate product quality, and to detect allele variation.ltimately, 130 primer pairs (35%) were dropped at

his stage: 46 primer pairs (12%) failed to generate aroduct under the standard set of conditions, 69 (18%)enerated a product with too many artifactual bands orn uninterpretable product, and 15 (4%) were not poly-orphic. Primer pairs for 13 tetranucleotide repeat

oci, isolated (using similar methods from a different

Cat Microsatellite Loci

s indicated in the final column. Heterozygosity and allele sizets: PCR product quality (as observed in the domestic cat); 4,ifactual bands; 2, increased number of artifactual bands; G,, 1.5 mM, 3 mM: final PCR MgCl2 concentrations; N, loci that, tetranucleotide repeat loci isolated by PE AgGen. NA, locusd is heterozygous at both loci examined for two-point linkage.

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at) and characterized at PE AgGen, Inc. (M.K.H.),ere also genotyped and mapped.

enotyping

Two hundred fifty-three loci listed in Table 1 wereenotyped in a 108-member multigeneration pedigreeetween the domestic cat and the Asian leopard catFig. 1). Amplifications were performed using fluores-ently tagged primer pairs; PCR products were dilutednd pooled in sets of five or six independent loci andlectrophoresed in an ABI 373A DNA analyzer. Wedopted a conservative multiplex strategy as large sizeifferences were often observed between domestic catnd leopard cat alleles for a particular locus.A small proportion of loci (N 5 12; 5%) failed to

enerate a product in the leopard cat. This phenome-on of “null” alleles occurs when lack of conservation of

he primer sequence results in failed amplificationCallen et al., 1993). The null alleles could be clearlyracked through the pedigree, exhibiting Mendeliannheritance patterns. A second set of loci (N 5 26; 10%)mplified well in either species independently, butreferentially amplified the domestic cat allele in theybrid background. Inheritance of either the domesticat or the null leopard cat allele from the F1 hybridemale could generally be scored in backcross individ-als. However, in matings where the F1 female and sirexhibited alleles of the same size, homozygous back-ross individuals exhibiting the shared allele size wereot scored, as maternal inheritance could not be deter-ined. Two hundred thirty-three (92%) of 253 markersere polymorphic in the leopard cat. Table 1 presentsrimer sequences, locus heterozygosities, and alleleize ranges observed in the domestic cat for the loci

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hat were incorporated into the map. The averageumber of informative meioses was 87. Average locuseterozygosities were high for both species, with 0.74bserved for the mixed breed domestic cat, 0.77 for theeopard cat, and 0.68 for the Egyptian Mau cat breed.eopard cat PCR products were frequently smallerhan the domestic cat homologue, with an averageedian allele size of 6 bp smaller observed in the

eopard cat.

inkage Analysis

A total of 253 markers within the pedigrees could becored with confidence and exhibited Mendelian inher-tance (Table 1, Fig. 2). Of these loci, 229 markers could

e linked to at least one other marker with a LOD of.0, identifying 34 linkage groups. Two hundred forty-ix loci were autosomally inherited, and 7 exhibited-linked patterns of inheritance. An average intra-roup intermarker distance of 11 cM was observed.he greatest intermarker distance observed was (chro-osome B2) 27.6 cM; 96% of the intermarker distancesere less than 25 cM. The linkage groups were physi-

ally mapped to a chromosome using a rodent 3 catomatic cell hybrid panel with a minimum of 2 markersnchoring each group (http://rex.nci.nih.gov/REEARCH/basic/lgd/lgdpage.htm). A total of 94 lociere physically assigned to a cat chromosome, result-

ng in chromosomal assignments to 16 of the 19 feline

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hromosomes. Additionally, 21 of the remaining 24ingleton loci were physically mapped to a cat chromo-ome using the rodent 3 cat somatic cell hybrid panel.hree singleton loci could not be physically mappedith confidence: FCA164, FCA625, and F141.Figure 2 presents a diagram of the sex-averagedap. The major linkage group on B4 was physicallyapped by one marker, by linkage to several Type Iarkers (data not shown), which were physicallyapped previously to chromosome B4 (O’Brien andash, 1982; O’Brien et al., 1997), and by radiation-ybrid analysis (Murphy et al., 1999). Our criteria tossign physically a linkage group to a particular

hromosome were based on achieving high statisticaloncordance (P # 0.001) of the marker retention inhe somatic cell hybrid panel for at least two markersssigned to a linkage group. For four linkage groups,nly one marker exhibited high concordance for aarticular cat chromosome. Assignment of theseinkage groups is considered as tentative: FCA223–CA344 were assigned to chromosome B3; FCA070 –CA589 were assigned to chromosome C1; FCA466 –CA628 were assigned to chromosome D2, and a

our-member group was assigned to A2 includingCA309, FCA058, FCA531, and FCA665. X-linkageas inferred by Mendelian transmission. Two single-

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on markers, FCA476 and FCA031, were tentativelyapped to E3 (P 5 0.012 and P 5 0.015, respectively)

ecause there was additional evidence showing thathey belong on E3. The genotype profile for these locias discordant with assignment to any other chro-osome. Additionally, one of the markers (FCA476)as tightly linked to a single-nucleotide Type I locusolymorphism (LOD 4.0, 10.4 cM) (M.M.R. et al., inreparation), whose human counterpart maps tohromosome 16p, a region homologous to cat chromo-ome E3 (Wienberg et al., 1997; O’Brien et al.,997b). Table 2 presents a summary of the distribu-ion of markers for each chromosome.

The sum of all measured intermarker distancesquals 2040 cM. To approximate the genetic length ofhe feline map, we used a conservative intermarkeristance of 14.4 cM, the average of all intermarkeristances 5 cM or greater in our calculations. As weere able to detect many linkages above the LOD 3.0

hreshold between loci that are .10 cM apart, we wereonfident that almost all of the loci at the ends ofinkage groups and the singleton loci are at least 5 cMway from the nearest locus in our set. We estimate theenetic length of the present map at 2900 cM as theum of all measured intragroup intermarker linkageistances (2040 cM), the distance between the terminalarkers on each chromosome to the telomere (14.4 3

9 3 2 5 547 cM), and the intervals between each of 22ingletons and a member of a linkage group (22 34.4 5 317 cM).

DISCUSSION

We have constructed a first-generation linkage mapf microsatellite loci in the domestic cat containing 253icrosatellite loci mapped to 16 of the 19 cat chromo-

omes. The estimated 8 million years of evolutionaryistance between the two species (Johnson and

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’Brien, 1997) of the pedigrees generates issues asso-iated with the map we wish to address. It is possiblehat small inversions and other chromosomal rear-angements between domestic cat and leopard cathromosomes may suppress recombination and resultn a shorter map or some ambiguity in marker order.

FIG. 2. Linkage map of the domestic cat (Felis catus). Chromoson the same horizontal line did not demonstrate recombination; –,arkers whose order is supported by a lod score of greater than 2.0

omatic cell hybrid panel (P # 0.001) are underlined; markers thonfidence (0.005 . P . 0.001). All distances are sex-averaged and cith respect to one another or with respect to the chromosome arms

One cytogenetic rearrangement documented betweenhe domestic cat and the leopard cat (Modi and O’Brien,988; Wurster-Hill and Centerwall, 1982) includes a peri-entric inversion in chromosome D2 that spans approxi-ately 10% of its cytogenetic length. A second chromo-

omal rearrangement between domestic cat F1 and the

assignment is indicated on the top of each linkage group. Markersicates regions where markers could not be ordered with confidence.e connected with heavy lines. Markers mapped in the rodent 3 catare underlined and italicized were physically mapped with lowerulated using Kosambi’s map function. The orientation of the groupsnot known with any degree of confidence.

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omologous leapord cat E4 would suppress recombina-ion in interspecies hybrids, precluding recombinationapping for this chromosome in the interspecies hybrid

anel. The failure to identify any markers on chromo-ome F1 is incidental to this phenomenon. We antici-ated identifying a group of loci that exhibited no recom-

FIG. 2—

ination and were physically assigned to F1 with theomatic cell hybrid panel. Two regions of ambiguity inarker order may reflect chromosomal rearrangements

n the two species including the loci FCA340, FCA124,nd FCA105 on A2 and FCA044, FCA051, and FCA210n B4. Marker order inconsistencies between the linkage

ntinued
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md(ecmprelostalaor1

hetolhlbco

leopard cat exhibiting an average median allele size 6b

odipgg

cc3lDwtdfaelmam

wcmpbbbuBusr

lCcatcvrF

A

TABLE 2

c


ap derived here and that derived from a 5000-rad ra-iation hybrid analysis of domestic cat chromosomesMurphy et al. 1999) may reflect small inversions andxchanges that occurred since divergence of the leopardat and domestic cat from a common ancestor. Lack ofarker assignments to E2 and D4 might best be ex-

lained by the small size of these chromosomes, whichepresent less than 6% of the genome length, based on anstimation of its proportion of the cytogenetic mapength. Another small chromosome E3 is represented bynly two singletons. Chromosome B2 is underrepre-ented in the map based on its cytogenetic length, whilehe three chromosomes A1, F2, and D3 are disproportion-tely represented relative to their cytogenetic mapength. The X chromosome is sparsely represented, annomaly of microsatellite library screens observed inther mammalian species, including human, mouse, andat (Dib et al., 1996; Dietrich et al., 1994; Jacob et al.,995).Average locus heterozygosities were equivalently

igh in both outbred domestic cat and leopard cats,xhibiting levels of 0.74 (Table 1) and 0.78, respec-ively. This is interesting in light of the present debatef ascertainment bias phenomenon, which reports thatibrary screening procedures utilizing high-stringencyybridization protocols for microsatellite isolation se-

ect for loci in the target species with the highest num-er of microsatellite repeat motifs, leading to high lo-us heterozygosities (Ellegren et al., 1995). We didbserve smaller alleles in the leopard cat, with the

Distribution of Markers for Each Chromosome

ChromosomeGenetic length

(cM)Total number

of loci

Fraction ofcytogenetic

mapa

A1 331 40 0.096A2 217 25 0.070A3 106 15 0.056B1 207 25 0.077B2 39b 5 0.058B3 99 12 0.056B4 154 15 0.054C1 212 20 0.086C2 135 17 0.058D1 94 13 0.048D2 105 16 0.040D3 135 18 0.040D4 90c 0 0.037E1 54 9 0.037E2 73c 0 0.030E3 56c 2 0.023F1 71c 0 0.029F2 125 13 0.029X 27 7 0.050

a Based on physical length measurements (W. Nash, pers. comm.).b Estimated length for B2 based on cytogenetic map length is 142

M.c Estimated lengths based on the cytogenetic map.

p smaller than the domestic cat.An average locus heterozygosity over the marker set

f 0.68 observed in nine unrelated Egyptian Mau, aomestic cat breed that contributed to part of the ped-gree, suggests that the marker set will be adequatelyolymorphic within cat breeds to be informative inene mapping exercises within cat breeds that segre-ate for inherited disease pathology.The cover of the genetic map is estimated at 2900

M, including 2040 cM observed in linkage groups, 547M between terminal markers and the telomeres, and17 cM between 22 singletons and a member of ainkage group. Three small chromosomes, F1, E2, and4 were unrepresented, and chromosomes E3 and B2ere underrepresented, leaving a small uncertainty as

o their contribution to overall map length. The linkageistance of these chromosomes was estimated (393 cM)rom their measured physical cytogenetic distance rel-tive to other chromosomes with multiple linked mark-rs by calibrating a regression of physical cytogeneticength with measured linkage distance of densely

apped chromosomes (Table 2). We therefore estimategenetic length for the sex-averaged map of the do-estic cat as 3300 cM.In the future, developing resources in our laboratoryill be utilized to expand the cat map. Additional mi-

rosatellite markers under development will beapped in the recently generated radiation hybrid

anel of the cat (Murphy et al., 1999), which will alsoe utilized to examine microsatellite order in a nonhy-rid background. A large-insert domestic cat PAC li-rary (Beck and Yuhki, unpublished results) will betilized to isolate markers from the underrepresented2 or unrepresented chromosomes F1, E2, and D4sing Type 1 PCR products as probes for chromosome-pecific clones or to supply additional markers in largeecombination intervals.

ACKNOWLEDGMENTS

We acknowledge the technical support of Heather Huffer, Car-os Driscoll, Janice Martenson, Kristy Zimmerman, Jenniferhristopherson, and Stanley Cevario in sequencing recombinantlones, genotyping, and physical mapping. Melody Roelke-Parkernd Mary Thompson were instrumental in generation of the in-erspecies pedigree and generation and maintenance of tissueulture cell lines, respectively. We thank Gary Smythers for de-elopment of a FASTA application for identification of duplicateecombinant sequences and Deborah Lomb for optimization of theoxPro software application.

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a genetic linkage map of microsatellites in the...

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