First-Generation Linkage Map of the Gray, Short-Tailed Opossum, … › content › genetics › 166 › 1 › 307.full.pdf · 2004-02-24 · Primate Research Center, San Antonio,

Copyright 2004 by the Genetics Society of America

First-Generation Linkage Map of the Gray, Short-Tailed Opossum,Monodelphis domestica, Reveals Genome-Wide Reduction

in Female Recombination Rates

Paul B. Samollow,*,1 Candace M. Kammerer,† Susan M. Mahaney,* Jennifer L. Schneider,*Scott J. Westenberger,*,2 John L. VandeBerg*,‡ and Edward S. Robinson*,3

*Department of Genetics, Southwest Foundation for Biomedical Research, San Antonio, Texas, 78245-0549, †Department of Human Genetics,Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania 15261 and ‡Southwest National

Primate Research Center, San Antonio, Texas 78245-0549

Manuscript received July 1, 2003Accepted for publication September 22, 2003

ABSTRACTThe gray, short-tailed opossum, Monodelphis domestica, is the most extensively used, laboratory-bred

marsupial resource for basic biologic and biomedical research worldwide. To enhance the research utilityof this species, we are building a linkage map, using both anonymous markers and functional gene loci,that will enable the localization of quantitative trait loci (QTL) and provide comparative informationregarding the evolution of mammalian and other vertebrate genomes. The current map is composed of83 loci distributed among eight autosomal linkage groups and the X chromosome. The autosomal linkagegroups appear to encompass a very large portion of the genome, yet span a sex-average distance of only633.0 cM, making this the most compact linkage map known among vertebrates. Most surprising, themale map is much larger than the female map (884.6 cM vs. 443.1 cM), a pattern contrary to that ineutherian mammals and other vertebrates. The finding of genome-wide reduction in female recombinationin M. domestica, coupled with recombination data from two other, distantly related marsupial species,suggests that reduced female recombination might be a widespread metatherian attribute. We discusspossible explanations for reduced female recombination in marsupials as a consequence of the metatheriancharacteristic of determinate paternal X chromosome inactivation.

METATHERIAN (“marsupial”) mammals have a biomedical models, all of which are eutherian species.However, marsupials and eutherians are more closelylong history in research as models for organismalrelated to one another than to any other vertebrateand cellular physiology, endocrinology, and develop-model species (e.g., birds, amphibians, fishes). Thus,mental patterns and processes and more recently havemarsupials represent a unique midpoint between euthe-taken their place alongside eutherian (“placental”) mam-rian and nonmammalian vertebrate models.mals as serious subjects for genetically oriented biomedi-

As a legacy of their common ancestry, marsupials andcal and evolutionary research (reviewed by Samolloweutherians share genetic mechanisms and molecularand Graves 1998; Graves and Westerman 2002). Thisprocesses that represent fundamental (ancestral) mam-increased interest in the genetic characteristics of mar-malian characteristics. Nevertheless, since their diver-supials reflects the growing utility and importance ofgence from a common ancestor �150–180 million yearscomparative models in biomedical research (Womackago (MYA; Hope et al. 1990; Kumar and Hedges 1998;1997; Graves 1998; Pollock et al. 2000; PostlethwaitWoodburne et al. 2003) eutherian and marsupial mam-et al. 2000) and the broadening recognition of the valuemals have evolved many distinctive morphologic, phys-of the unique phylogenetic position of marsupials iniologic, and genetic variations on these elementalthe mammalian lineage. Marsupials are phylogeneti-mammalian designs. These phylogenetically restrictedcally distinct from the more commonly used mammaliandifferences can be used as comparative tools for examin-ing the underlying molecular and genetic processes thatare common to all mammalian species and thereby help

Sequence data from this article have been deposited with the to reveal how variations in these mechanisms contributeEMBL/GenBank Data Libraries under accession nos. AY369173–

to differences in gene regulation, expression, and func-AY369206.tion.1Corresponding author: Department of Genetics, Southwest Founda-

tion for Biomedical Research, 7620 NW Loop 410, San Antonio, TX Monodelphis domestica (also known as the laboratory78245-0549. E-mail: [email protected] opossum) is a small South American marsupial that has

2Present address: Department of Microbiology, Immunology and Mo- become widely used as a model organism for compara-lecular Genetics, University of California, Los Angeles, CA 90025.tive research on a broad range of topics that are relevant3Present address: Department of Biological Sciences, Macquarie Uni-

versity, Sydney, NSW 2109, Australia. to human development, physiology, and disease suscep-

Genetics 166: 307–329 ( January 2004)

308 P. B. Samollow et al.

tibility (reviewed by VandeBerg 1990; VandeBerg and al. 1986). A cytological examination of meiotic cellsrevealed reduced chiasma frequency in females as com-Robinson 1997). Because of its small size (100–150 g),

rapid growth and maturation (maturity at �5 months), pared to males and restriction of female chiasmata pri-marily to terminal (subtelomeric) regions, suggesting afavorable reproductive characteristics (mean litter size

of approximately eight; up to three litters per year), causal relationship between chiasma characteristics andrecombination rates (Bennett et al. 1986). Soon there-and simple husbandry (rodent cages and commercial

feed; VandeBerg 1999), M. domestica has become the after, Hayman et al. (1988) reported similar cytologicalphenomena in M. domestica (Didelphidae; an Americanmost extensively used laboratory-bred marsupial for ba-

sic biologic and biomedical research worldwide (Samol- family composed of the opossums and woolly opos-sums), and subsequent inheritance studies revealedlow and Graves 1998; and recent database searches).

Examples of recent research involving M. domestica in- drastically reduced recombination among six loci infemales of this species as well (van Oorschot et al.clude: gene and genome evolution, genomic imprinting

(autosomal and X-linked genes), photobiology and 1992b, 1993; Perelygin et al. 1996). The combinedweight of these limited findings prompted guardedDNA repair, molecular characterization of ultraviolet

radiation-induced skin and eye neoplasias, structure and speculation that reduced female recombination mightbe a general phenomenon in marsupials (van Oor-evolution of mammalian antigen receptors, lipoprotein

metabolism and response to dietary fat and cholesterol, schot et al. 1992b, 1993; Hope 1993).Most recently, Zenger et al. (2002) published a com-neurophysiology, normal and regenerative neurodevel-

opment (central and peripheral nervous systems), cra- prehensive linkage map of the tammar wallaby, Mac-ropus eugenii (Macropodidae; an Australian family com-niofacial ontogeny and evolution, skeletal and skeleto-

muscular development, reproductive endocrinology, posed of the kangaroos, wallabies, and related forms),the first of its kind for any marsupial species. Composedand more.

To further expand the potential of this species as a of 60 loci and believed to cover �71% of the M. eugeniigenome, the map shows marked heterogeneity of re-research model, we have undertaken construction of a

linkage map of the M. domestica genome, consisting of combination rates including regions of severely reducedfemale recombination interspersed with intervals inboth anonymous markers and functional gene loci. The

primary objective is to establish a resource that will en- which female and male recombination rates are equiva-able the localization of quantitative trait loci (QTL) that lent. Overall, the female map is �78% the size of thecontribute to normal and abnormal physiologic and male map, and in none of the interlocus intervals doesdevelopmental variation and will provide comparative female recombination exceed that in males.information regarding the evolution of gene synteny The linkage map of M. domestica presented in thisand linkage relationships among distantly related mam- article is composed of 83 loci distributed among eightmalian species. autosomal linkage groups and the X chromosome and

An important outcome of our ongoing linkage analy- represents the most extensive linkage data for any mar-ses has been to extend and refine fundamental ideas supial species. Although the map is still under construc-and speculations regarding sex-specific recombination tion, the available data indicate that recombination isrates in marsupials. Early linkage studies involving small considerably more male biased in M. domestica than innumbers of genes in two distantly related marsupial the tammar wallaby and is by far the most male-skewedspecies yielded the surprising result that meiotic recom- pattern known for any vertebrate species.bination in female marsupials was much lower thanthat in males. This pattern is contrary to the generalmammalian one in which recombination rates are simi- MATERIALS AND METHODSlar between the sexes or are reduced in males: e.g.,

Animal resources and genetic mapping panel: Gray, short-humans (Broman et al. 1998; Kong et al. 2002), dogs tailed opossums (M. domestica) were obtained from the re-(Mellersh et al. 1997; Neff et al. 1999), pigs (Archi- search colony maintained at the Southwest Foundation forbald et al. 1995; Mikawa et al. 1999), cattle (Barendse Biomedical Research (SFBR), San Antonio, Texas. The

“GMBX” mapping panel was established by crossing memberset al. 1997; Kappes et al. 1997), and mice (Dunn andof laboratory stocks that were descended from the most geo-Bennett 1964; Davisson et al. 1989; Copeland et al.graphically separated populations available (�1200 km) at1993). the inception of the study: population 1 (from Exu, in the

Specifically, in the fat-tailed dunnart, Sminthopsis cras- state of Pernambuco, Brazil) and population 3 (Joaima, Minassicaudata (Dasyuridae; an Australian marsupial family Gerais, Brazil). Two wild-captured Pop3 males were crossed

to six pedigreed Pop1 females to produce the F1 generation.of small, mouse-like carnivores), four pairwise geneNineteen F1 offspring of both sexes were crossed to 22 pedi-combinations that exhibited no recombination in fe-greed Pop1 mates to produce the backcross generation com-males exhibited modest to high recombination frequen-posed of 313 progeny in 30 sibships. Insufficient Pop3 animals

cies (rf, 0.16–0.37) in males, and two pairwise combina- were available to produce the reciprocal backcross. Tissue andtions that had minimal recombination in females (rf, blood samples were collected from all of the animals produced

by this crossing scheme with the exception of the six Pop10.06 and 0.12) were unlinked in males (Bennett et

309Linkage Map of the Laboratory Opossum

grandmothers. Thus, the 356-member GMBX panel is com- bridization probes for Southern blots were derived from mar-supial sources: PHL and the anonymous U15557 probes fromposed of 30 backcross families including 313 backcross prog-M. domestica and the HBE probe from the fat-tailed dunnart,eny, all of their parents (19 F1 and 22 Pop1 mates), and theS. crassicaudata. These yielded unique, strong hybridizationtwo Pop3 grandfathers.signals at high stringency, indicative of homology between theSample collection and preparation: Whole blood and vari-probe and the target sequence. NRASLA and NRASLB wereous solid tissues (including liver, brain, kidney, heart, skeletaldetected using a human NRAS genomic probe (Murray et al.muscle, and ear pinna) were collected from lethally anesthe-1983). Although originally reported as homologous to humantized animals under a protocol approved by the SFBR Institu-NRAS (van Oorschot et al. 1991; Perelygin et al. 1994),tional Animal Care and Use Committee. Dissected tissues wereresults from the present study indicate that NRASLA andimmediately frozen on dry ice and stored in small, heavy-duty,NRASLB are unlinked to one another, thus obviating orthologyzipper-type plastic bags at �80�. Blood was allowed to clot forinferences. Preliminary DNA hybridization studies using M.1 hr at ambient temperature and then separated into clot anddomestica-derived HRAS and KRAS genomic probes (data notserum fractions. Both fractions were stored at �80� in 50-�lshown) have failed to clarify the relationships between thesealiquots, which were sealed in plastic tubing “pillows” followingpolymorphic NRAS fragments and other RAS family genes.the method of Cheng and co-workers (Cheng et al. 1986;We propose that both polymorphic fragments be classifiedCheng and VandeBerg 1987). Crude extracts of solid tissuesas NRAS-LIKE loci until their homologies are ascertained byand blood clots were prepared by standard methods (e.g.,sequencing studies.Selander et al. 1971; Samollow et al. 1987) for application

Microsatellite and randomly amplified polymorphic DNAto electrophoretic or isoelectric focusing gels. Serum samples(RAPD) polymorphisms were developed for this study usingfor protease inhibitor (PI) and transferrin (TF) analysis werePCR primers designed from cloned M. domestica DNA se-used directly without additional preparation. For AT3 and C6quences and commercial RAPD primers, respectively (de-analyses, 20-�l serum samples were mixed with 14 �l of 30 mmscribed below).sodium acetate-95 mm (NH4)2SO4 buffer (pH 5.0) containing

All type I protein and DNA polymorphisms exhibited strict0.008 units of neuraminidase and incubated overnight at 4�codominant inheritance, and no unusual segregation patternsprior to use. High-molecular-weight genomic DNA was pre- (i.e., segregation distortions) were detected for these or anypared using routine methods adapted from a variety of of the loci examined in this study. Microsatellite variation wassources. Briefly, frozen tissue (usually liver) was ground to a primarily codominant, although nonamplifying (null) alleles

powder in liquid nitrogen, mixed with a 100 �g/ml proteinase were detected at seven loci (Table 2). In these cases, individu-K solution, and incubated at 55� overnight with gentle rota- als exhibiting dominant phenotypes were included in the ge-tion. The resulting solution was RNase treated (20 mg/ml) for notype data set only if their genotypes could be unambiguously1 hr at 37�, and the DNA was extracted by phenol/chloroform inferred from pedigree data. Specifically, individuals thatphase separation. The DNA was purified by ethanol precipita- could be either homozygous for an amplifiable allele or het-tion and resuspended in 1� Tris-EDTA (TE) and stored at erozygous for that allele and a null allele were excluded from4� (or �80� for archival storage) until used. the data set. Apparent null homozygotes were reamplified and

Polymorphisms—inferred locus homologies and inheri- rescored multiple times to verify the absence of an amplifiabletance: Twelve previously published genetic polymorphisms allele.and 72 newly developed ones were used as genetic markers to RAPD phenotypes encompassed both dominant/recessivegenotype all 356 members of the M. domestica GMBX mapping and codominant patterns of inheritance (Table 2). Domi-panel. The genetic markers included 21 coding (type I) and nant/recessive inheritance occurred at 25 of the 28 RAPD63 anonymous (type II) genetic loci (Tables 1 and 2, respec- loci, with the presence (P) of an amplimer on a gel dominanttively). All polymorphisms were tested for Mendelian inheri- to its absence (A). For loci with this inheritance pattern, indi-tance in a subset of families before being screened in the full viduals with dominant phenotypes were included in the geno-mapping panel. type data set only if their genotypic status (homozygous P/P

Homologies (putative orthologies) of the type I loci to other or heterozygous P/A) could be unambiguously inferred fromvertebrate genes were inferred by a variety of approaches. For pedigree data. For some loci, this criterion excluded entirepolymorphic proteins, criteria included: (1) uniqueness of families (one parent known or suspected of being homozygousloci in mammals [i.e., only a single adenylate kinase (AK1) is P/P) from consideration and thereby reduced the overallknown in mammalian erythrocytes and only one gene encod- informativeness of those loci for mapping purposes. Oneing GAPD and one gene encoding GPT are known to be RAPD locus exhibited codominant variation in amplimer size,expressed in mammals]; (2) immunological cross-reactivity and the two remaining RAPD polymorphisms were essentiallywith antibodies developed for other mammalian species—AT3 restriction fragment length polymorphisms (RFLPs) detected(Bell et al. 1992), C6 (van Oorschot et al. 1993), PI (van by restriction digestion of the RAPD-PCR amplimer. In gen-Oorschot et al. 1992b), and TF (P. B. Samollow and S. M. eral, the RAPD polymorphisms, by dint of their dominantMahaney, unpublished data); (3) characteristic tissue-specific inheritance patterns, were the least informative of the poly-expression and/or coenzyme requirements—ALDOC (brain- morphisms used in this study.specific; Sokolova et al. 1997), DIA1 (predominant erythro- Protein polymorphisms: Protein polymorphisms were de-cytic diaphorase, NADH requirement; P. B. Samollow and tected using electrophoretic and isoelectric focusing methodsS. M. Mahaney, unpublished data), and LDHB (predominant modified from a variety of published sources. Tissues usedheart isoenzyme); and (4) subcellular localizations (P. B. and electrophoretic method/buffer combinations are listedSamollow and S. M. Mahaney, unpublished data)—ACP2 in Table 3. Horizontal starch gel electrophoresis (SGE) was(lysosomal), ACONC (cytosolic), and ME1 (cytosolic). conducted in 12% starch gels (e.g., Shaw and Prasad 1970;

For PCR-based, type I DNA polymorphisms (AMG, GPD, Shaklee and Keenan 1986; Morizot and Schmidt 1990).SP17, and TP53), DNA fragments were generated using prim- Vertical polyacrylamide gel electrophoresis (PAGE) was con-ers derived from cloned M. domestica sequences. Homology ducted using a 10% acrylamide (1:37.5 bis:acrylamide) run-inferences are based on the strong amplification of single ning gel overlaid with a 4% acrylamide (1:37.5 bis:acrylamide)fragments and lack of additional amplification products under stacking gel (e.g., Gahne et al. 1977; Samollow et al. 1987).

Cellulose acetate gel electrophoresis (CAGE) was conductedhigh stringency conditions. With the exception of NRAS, hy-


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atio

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atio

n.

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ils.

dIn

GM

BX

pan

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ly;

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ptM

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and

Mdo

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ited

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ng

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ith

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bp),

and

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)w

ere

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min

ant;

the

four

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had

iden

tica

lm

obili

tyto

alle

leB

(780

bp),

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had

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low

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ase-

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tect

ed).

kT

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les;

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ent;

B,

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15

0-bp

frag

men

ts.


TA

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E3

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in Cellogel (Chemtron, Milan, Italy) cellulose acetate medium extension at 72� (unless otherwise specified, all PCR proce-dures for this study were conducted using ABI 9600 or(e.g., Richardson et al. 1986). Polyacrylamide gel isoelectric

focusing (PAGIEF) was performed in 5% polyacrylamide (1:29 9700 thermocyclers). The amplified M. domestica fragmentcontained a polymorphic Pst I restriction site. This PCR-bis:acrylamide) gels prepared by two methods. For AT3, 0.45-

mm-thick gels were prepared according to the riboflavin pho- RFLP was visualized on 1% agarose gels (1� TE) stainedwith ethidium bromide.topolymerization protocol of Pollitt and Bell (1983). For

PI, 0.30-mm gels containing 10% sucrose (w:v) were produced SP17 : M. domestica SP17 cDNA sequence data (GenBank acces-sion no. AF054290 and M. G. O’Rand, personal communi-by standard TMED/ammonium persulfate polymerization.

Agarose gel isoelectric focusing (AGIEF) was performed in cation) were used to design primers for sequencing theintron 1 region of the M. domestica SP17 gene. The intron0.5-mm, 1.25% isoagarose (FMC BioProducts, Rockland, ME)

gels containing 13.75% sucrose (w:v). 1 boundary was inferred from gene structure informationof human, mouse, and rabbit homologs. PCR primers wereVisualization (“staining”) of 13 protein polymorphisms on

gels was accomplished by a variety of methods modified from designed to amplify an �2400-bp fragment of intron 1,which was partially sequenced (data available on request)published and unpublished sources (Table 3). Details of theseand found to contain a single nucleotide polymorphismprocedures are available online at the Genetics supplemental(SNP) in an HaeIII restriction site. The PCR primers were:information website: http://www.genetics.org/supplemental.forward, 5�-CACTGTATTCCATTTTACTC-3� and reverse,Southern blotting/RFLP analysis: Restriction enzyme di-5�-TTCCTACTTTACATATGAGG-3�. Amplification was ac-gests of M. domestica genomic DNA were electrophoreticallycomplished using touchdown PCR: initial 5-min denatur-separated on 1% agarose gels and then transferred and fixedation step at 95�; 10 cycles of 40-sec denaturation at 94�,to nylon filters (Hybond-N; Amersham Biotech, Piscataway,30-sec annealing beginning at 67� for the first cycle andNJ) by standard methods (e.g., Sambrook et al. 1989). Radiola-decreasing 1� each subsequent cycle, and 150-sec extensionbeled (32P) probes for HBE, NRAS, PHL, and U15557 (de-at 72�; 30 identical cycles of 40 sec at 94�, 30 sec at 57�, andscribed below) were prepared by random priming (Amersham150 sec at 72�; and a final 7-min extension at 72�. TheBiotech oligolabeling kit). Labeled HBE and PHL probes werepolymorphism was analyzed as an HaeIII PCR-RFLP on 1%hybridized to filters for 16 hr at 65�, after which the filtersagarose gels stained with ethidium bromide.were washed three times to a final stringency of 2� SSC/0.1%

TP53 : Published M. domestica TP53 exon 4–11 cDNA sequenceSDS at 65�. Hybridization and wash conditions for NRAS anddata (Kusewitt et al. 1999) and unpublished partial intronU15557 are given by Perelygin et al. (1994, 1996, respec-sequence data (D. F. Kusewitt, personal communication)tively). The hybridization probes used for Southern blot analy-were used to generate sequencing primers for intron re-ses of RFLPs were:gions. Intron 7 was partially sequenced (data available on

HBE : embryonic �-globin genomic DNA fragment, clone pSG- request) and found to contain an SNP in an AluI restriction2H, from the dasyurid marsupial, S. crassicaudata (Hope et site, which was analyzed as a PCR-RFLP pattern visualizedal. 1992), gift of Rory Hope. on 1% agarose gels stained with ethidium bromide. The

NRAS : human NRAS proto-oncogene genomic DNA fragment, PCR primer sequences for amplification of the �850-bp,clone p52c- (Murray et al. 1983) from American Type Cul- intron 7-containing fragment of the TP53 gene were: for-ture Collection (no. 41030). ward, 5�-GCGCCCAATCCTGACTATCAT-3� and reverse, 5�-

PHL : probe generated by PCR amplification from M. domestica AGGAAGGGACAGGTAGAAGGA-3�. PCR amplificationgenomic DNA using primers designed from published M. was accomplished using touchdown PCR: initial 5-min dena-domestica photolyase cDNA sequence data (Kato et al. 1994). turation at 95�; 10 cycles of 30-sec denaturation at 94�, 30-Forward primer was 5�-AAAAAGGGAGGAGCAGAAGGC-3�; sec annealing beginning at 78� for the first cycle and decreas-reverse primer was 5�-GTCATAATGGCGAATGGTAG ing 2� for each subsequent cycle, and 60-sec extension atCAC-3�. The amplified fragment was cloned into pT7Blue(R) 72�; 30 identical cycles of 30 sec at 94�, 30 sec at 58�, andand transformed into NovaBlue competent cells (Novagen, 60 sec at 72�; and a final 5-min extension at 72�.Madison, WI). AMG : M. domestica AMG cDNA sequence data (Hu et al. 1996)

U15557 : anonymous M. domestica genomic DNA fragment clone were used to design primers to amplify a region spanning(Perelygin et al. 1996), GenBank accession no. U15557. 22 bp of exon 5, all of intron 5, and 69 bp of exon 6.

The amplified fragment varied from �430 to �450 bp,Polymerase chain reaction amplimer-restriction fragment indicating an �20-bp insertion/deletion (indel) polymor-

length polymorphisms: PCR was used to generate amplimers phism. The primers were: forward, 5�-TCAAAGCATGATGthat were subsequently subjected to restriction endonuclease CGACAGC-3� and reverse, 5�-CTGAGATAGCACTGGGAdigestion to reveal restriction site polymorphisms (PCR- TGA-3�. Touchdown PCR procedures were identical toRFLPs): those for G6PD except that the initial and final annealing

temperatures were 68� and 58�, respectively. The indel poly-G6PD : PCR primers were designed from published G6PD ge-morphism was visualized on 1-mm thick, 6.5% polyacryl-nomic DNA sequence of the Virginia opossum, Didelphisamide (1:37.5 bis:acrylamide) gels stained with ethidiumvirginiana (Kaslow et al. 1987) and used to amplify anbromide.�2400-bp M. domestica genomic DNA fragment. This frag-

ment was partially sequenced (data available on request), RAPD analysis: RAPD polymorphisms were detected usingarbitrarily primed PCR as described by Williams et al. (1990).and the resulting data were used to design new, M. domestica -

specific PCR primers: forward primer (exon 5), 5�-CAT Briefly, arbitrary 10-base oligonucleotides (Operon RAPD 10-CAAAGAAACTTGCATGAGCCAG-3� and reverse primer Mer kits A and B; Operon Technologies, Alameda, CA) were(exon 8), 5�-ATCTGGAGGAGGTGGTTCTGCATCA-3�. Am- used alone or in pairs to amplify random sequences fromplification was achieved using a “touchdown” PCR proce- genomic DNA. The arbitrary primers used in this study aredure: initial 5-min denaturation step at 95�; 10 cycles of 40- listed in Table 2. RAPD-PCR reaction mixtures consisted ofsec denaturation at 94�, 30-sec annealing beginning at 69� 2.0 mm MgCl2, 0.1 mm each dNTP (dATP, dCTP, dGTP, andfor the first cycle and decreasing 1� each subsequent cycle, dTTP), arbitrary primer(s) (0.4 �m if a single primer was used;and 60-sec extension at 72�; 30 identical cycles of 40 sec at 0.2 �m each if two primers were used), 5 units AmpliTaq

Polymerase (Applied Biosystems, Foster City, CA), 1� PCR94�, 30 sec at 59�, and 60 sec at 72�; and a final 5-min


AmpliTaq PCR buffer, and �25 ng of genomic DNA. Cycle were performed; thus the final genotyping error rate wasslightly lower than that suggested by these random repeatparameters (ABI 480 thermocycler) were: denaturation at 94�

for 2 min, followed by 45 cycles of 1 min at 94�, 1 min at 36�, error rates. Overwhelming agreement of parent-offspring ge-notypes suggests that there were no pedigree errors amongand 2 min at 72�. Resultant amplification products were run

out on 1.4% agarose gels and visualized by ethidium bromide the 356 animals used in the mapping analysis.Map construction: The GMBX panel was constructed bystaining. RAPD banding patterns on gels were scrutinized for-

variation in three categories: (1) PA, presence/absence varia- crossing members of two outbred populations that had fixedgenetic differences at some loci, but shared alleles at othertion of a band at a specific position on the gel, presumed to

reflect priming sequence variation or large insertion/deletion loci. Therefore, the number of informative meioses variedsubstantially across loci (Tables 1 and 2). The average numbervariants that preclude successful amplification; (2) SV, size

variation of a fragment revealed by differences in the position of informative meioses per locus, 150 in females and 177 inmales, was sufficient to assure high levels of support for locusof a band on a gel presumed to reflect small insertion/deletion

variation; and (3) RFLP, length variation of restriction endo- order for most loci.Construction of the M. domestica linkage group maps pro-nuclease-digested RAPD-PCR amplification product.

Microsatellite development and polymorphisms: Detection ceeded in four phases: identifying linked loci, ordering linkedloci, removal of double recombinants, and final map construc-of short tandem repeat sequences (microsatellites) was con-

ducted following the methods outlined in Hillis et al. (1996). tion. We used the computer program Crimap (Green 1990)to perform a series of two-point (pairwise) linkage analysesBriefly, a small-fragment genomic DNA library was constructed

by cloning partially Sau3AI-digested and size-selected (200– and assigned markers to linkage groups. To be included withina linkage group, loci had to be linked to at least one other800 bp) genomic DNA into the pT7 Blue T-Vector (Novagen)

according to the manufacturer’s instructions. The library locus within the group at a LOD 3.00 (1000:1 odds). Wenext ordered the loci within each autosomal linkage group(maintained in Nova Blue cells) was plated, grown, and trans-

ferred to nylon membranes, which were probed at high strin- using the expert system program MULTIMAP (Matise et al.1994), which implements routines of the computer programgency with (CA)15 or (GT)15 probes (Genosys Biotechnologies,

The Woodlands, TX) to detect the presence of dinucleotide Crimap. In MULTIMAP, the two loci with the highest pairwiseheterozygosity are chosen to start the map and additional locirepeat sequences in the plasmid vectors. DNA from strongly

hybridizing clones was isolated and purified (Wizard Plus Mini- are added in order of informativeness. At each step, MULTI-MAP determines a reasonable order for the set of loci, as wellpreps DNA purification system; Promega, Madison, WI) andas validating that this order has a higher likelihood than otherthen used for sequencing analysis. Sequencing of plasmidorders, by inverting groups of three loci and keeping the orderDNA was accomplished either by standard dideoxy sequencingwith the highest likelihood. This process continues until nomethods (e.g., Sambrook et al. 1989) with 35S-labeled dATPmore loci can uniquely be added to the map, on the basis ofusing the Sequenase version 2.0 sequencing kit (Amershama criterion of LOD 2.0 (100:1 odds) as statistical supportBiotech) or by fluorescence-based cycle sequencing methodsfor locus order. After ordering the loci within each linkage(ABI PRISM Big Dye Terminator Cycle Sequencing ready reac-group, we used Simwalk2 to detect possible double recombi-tion kit; Applied Biosystems). For cycle sequencing, PCR reac-nants (Weeks et al. 1995; Sobel et al. 2001). Simwalk2 usestion products were “cleaned” with shrimp alkaline phospha-Markov chain Monte Carlo and simulated annealing algo-tase and exonuclease I (Amersham Biotech) according to therithms to perform these multipoint haplotype analyses andmethod of Nickerson et al. (1998) and sequenced on anreports the overall probability of mistyping, i.e., evidence ofApplied Biosystems (ABI) 377 automated DNA sequencer fol-a double recombinant, at a locus. We removed individuallowing the manufacturer’s instructions. The DNA sequencegenotypes that had �50% likelihood of mistyping. Finally,data were analyzed using Sequencing Analysis Version 3.3MULTIMAP was rerun to obtain estimates of sex-specific or-and Factura Version 2.2 software. Data from the forward andders and distances. Map distances were calculated as centi-reverse reactions were used to determine the base sequencesmorgans (cM) using the Kosambi mapping function (see Ottof the unique (nonrepetitive) regions flanking the repeat1999). With the exception of linkage group 7, which was com-region and the nature and size of the repeat region itself.posed of essentially four recombining loci, we again requiredPCR primer pairs were designed to match unique flankingLOD 2.00 as statistical support for order.sequences, and DNA samples from the smallest subset of com-

Sex-specific differences: We tested for sex-specific heteroge-pletely informative individuals in the GMBX panel (those thatneity in linkage group length by using the likelihood ratiocontained all alleles that contributed to the backcross genera-test to compare the likelihood of each linkage group with sex-tion) were amplified and screened for variation in amplimerspecific recombination vs. the null hypothesis of sex-equallength at each microsatellite locus.recombination. The likelihood ratio test is asymptotically dis-Genotyping and data quality: Genotypes were scored inde-tributed as a chi square with degrees of freedom equal to thependently by two observers. Samples yielding ambiguous scor-number of intervals minus one. We also tested for sex-specificings were rerun and rescored until agreement was reachedheterogeneity of recombination within each interval by com-or it was decided that the sample could not be scored forparing the maximum LOD for linkage when male and femalethe particular marker. In rare cases, wherein an individual’srecombination is equal [Z(�m � �f): the null hypothesis] vs.genotype at a particular microsatellite locus was unambiguousthe maximum LOD obtained with sex-specific recombinationbut could not be reconciled with parental and sibling geno-[Z(�m, �f)] (Ott 1999). The resulting likelihood ratio test,types even after repeated typings (apparent mutations), the 2 � 2 � ln(10) � [Z(�m, �f) � Z(�m � �f)], is asymptoticallyindividual’s genotype was excluded from the mapping analysis.distributed as a chi-square distribution with 1 d.f.The overall quality of the genotyping data was assessed by

random retyping of an average of 17.2% (range, 10.6–23.0%per locus) of previously typed samples. Discrepancy rates were

RESULTSlowest for type I DNA (0.19%) and protein (0.39%) polymor-phisms and somewhat higher for microsatellite (0.82%) and

Linkage map construction: Two-point linkage analysisRAPD polymorphisms (1.0%). The average random repeatusing a linkage criterion of LOD 3.0 identified eightdiscrepancy rate across all loci was 0.77%. All discrepancies

were reconciled, and many nonrandom sample repetitions autosomal linkage groups (LGs 1–8) and an X chromo-


TABLE 4

Linkage groups of M. domestica : number of markers, support values for locus orders, andfemale/male comparisons

Map length (cM)No. of

Linkage No. of markers No. of Log odds Sex F/Mgroup markers ordered intervalsa for order average Female Male ratio P valueb

LG 1 16 14 11 5.2 113.4 51.2 172.9 0.296 2 � 10�7

LG 2c 8 7 4 14.8 56.4 18.9 100.4 0.188 �10�12

LG 3 15 13 11 2.6d 139.6 111.2 194.9 0.571 �10�11

LG 4 13 12 10 5.2 80.7 48.4 105.5 0.458 2 � 10�5

LG 5 6 6 4 8.9 41.6 40.5 46.8 0.865 0.29LG 6 9 8 7 6.0 70.6 56.5 82.9 0.682 0.003LG 7 7 6 3 1.6 64.9 61.4 73.4 0.837 0.19LG 8 7 7 6 13.9 65.8 55.0 107.8 0.510 0.41(LG X) (3) N/A — — — — — — —Totale 81 (3) 73 56 — 633.0 443.1 884.6 0.501 —

a Intervals with nonzero recombination in at least one sex.b Likelihood ratio test of homogeneity of sex-specific recombination fractions across the individual linkage

group (Ott 1999).c Includes locus C7; assumed to cosegregate with locus C6 according to van Oorschot et al. (1993).d Support increases to 3.6 if two loci (TP53 and MdoLR07) are removed.e Autosomal linkage groups only.

somal linkage group (LG X) comprising 83 of the 84 tion for two of these pairs could be attributable to a lownumber of pairwise informative meiosis rather than toloci examined (LDHB failed to link to another locus

with LOD 3.0). Loci were ordered within LGs 1–8 at tight linkage (AT3-Mdo2LR10 with 11 pairwise informa-tive meioses and AMG-Mdo5LR17 with 20 pairwise infor-a minimum criterion of LOD 2.0 (100:1 odds) for

inclusion. Following detection and elimination of im- mative meioses), but the remaining six locus pairs hadbetween 44 and 426 pairwise informative meioses (totalprobable double recombinants, sex-specific locus orders

and recombination distances were recalculated to yield for sexes combined), so tight linkage is the more likelyexplanation in these cases.female- and male-specific recombination maps for LGs

1–8. Multipoint analysis was not pursued for the 3 loci Statistical support for map order is very good; averageLOD is 27.9 across all 56 recombining intervals. Onlyin LG X. Data pertaining to the number of loci, statistical

support levels, and sizes of the sex-average and sex- 3 intervals had multipoint support levels less than LOD3.00. The structure of LG 7 is problematic. This linkagespecific linkage groups obtained from multipoint link-

age analyses are listed in Table 4. Diagrammatic repre- group contains only six loci, two pairs of which exhibitno recombination; thus, the LG 7 map has only 3 interlo-sentations of the eight autosomal linkage group maps

for both sexes, including locus orders, map positions, cus intervals. The overall order for LG 7 is supportedonly at LOD 1.6, although support for 2 of the LG 7and interlocus interval support levels are shown in Fig-

ure 1. Locus C7 was not examined in the present study intervals is strong for the given order (LOD 22.0 andLOD 5.8). We therefore consider the map for LG 7 asbut was placed on the map by inference from the finding

of van Oorschot et al. (1993) that C6 and C7 are provisional with regard to the placement of Mdo7LR27.Map size and sex-specific recombination rates: Twoextremely tightly linked in M. domestica (� � 0.00, Zmax �

31.18). striking features of the M. domestica linkage map areits small overall size and the very large differences inNot all of the 80 loci assigned to autosomal linkage

groups could be unambiguously ordered. Five RAPD recombination rates between sexes. The sex-averagemap size of only 633.0 cM is, to our knowledge, theloci (Mdo1LR04, Mdo2LR06, Mdo3LR08, Mdo4LR12, and

Mdo6LR18 in LGs 1, 2, 3, 4, and 6, respectively) could smallest of any vertebrate species. This small size is duein part to the substantial reduction in recombinationbe placed only within multilocus regions rather than at

specific points on the corresponding linkage group seen in females as compared to males (see below), buteven the larger male map, which spans 884.6 cM, ismap. Three additional RAPD loci (Mdo1LR05, Mdo3-

LR11, and Mdo7LR22 in LGs 1, 3, and 7, respectively) smaller than any other vertebrate linkage map, regard-less of sex.could not be placed on the maps with any confidence

despite linking strongly to one or more loci within their Noninclusion of large portions of the genome doesnot appear to be an adequate explanation for the com-corresponding linkage groups. Eight locus pairs exhib-

ited no recombination in either sex. Lack of recombina- pact linkage map of M. domestica for several reasons.


Figure 1.—Female- and male-specific linkage group maps of M. domestica. The female map is to the left in each linkage group.Locus abbreviations lie between the female and male maps, and locus positions are indicated by their distance in centimorgansfrom the “top” of the linkage group. Boxes enclose nonrecombining loci, which can differ between sexes. Bars to the right ofthe male map indicate the most likely ranges for loci that could not be ordered with high confidence. Numbers to the extremeright of each linkage group indicate the statistical support (LOD) for each interval, given the locus order shown.


Figure 1.—Continued.

The M. domestica karyotype is composed of eight pairs loci has not dramatically altered the size of the map.For example, a series of locus additions that increasedof large autosomes and the X and Y sex chromosomes

(Pathak et al. 1993). The coalescence of 83 genetic the map content from 59 loci to its current 83 loci (a46% increase) increased the overall map size by onlymarkers into eight autosomal linkage groups and one X

chromosomal linkage group in the M. domestica linkage 13%. For all of these reasons we believe that the eightautosomal linkage groups correspond to the eight au-map is consistent with this species karyotype. Failure of

LDHB to link to any of the linkage groups is almost tosomes of this species and that the current linkage mapencompasses a very large proportion of the genome. Ifcertainly due to its lack of variation (only 19 informative

meioses) rather than to its location in an uncharted this is true, M. domestica possesses a generally low rateof meiotic recombination and particularly so in females.portion of the genome. Further, the smallest autosomal

linkage group in the M. domestica map includes six loci, Overall, the male genetic map is twice the size of thefemale map (884.6 vs. 443.1 cM), and male linkageand LG X includes all of the X-linked loci examined.

There are no other unlinked loci, nor are there any group sizes exceed those for each of the correspondingfemale linkage groups (Figure 2). These differences arelocus pairs or triplets that do not link to one of the

nine linkage groups. Moreover, LGs 1–8 were identified statistically significant for the five larger linkage groups(LGs 1–4 and 6), but not for the three smaller ones,during an early phase of mapping using only 46 loci.

Since that time, no newly examined locus has failed which have fewer loci (Table 4). Of the 56 recombiningintervals, 20 (representing 45.9% of the total sex-aver-to link to one of the existing linkage groups. Another

indication that no large, undetected regions of the ge- age map length) show significantly higher male recom-bination, while only two (representing 6.7% of totalnome remain is that the continuing addition of new


the genome, but that the intensity of the differencevaries among chromosomes and chromosomal regions.

DISCUSSION

Sex-average linkage map: The length of the M. domes-tica sex-average linkage map, excluding X-linked loci,is only 633 cM. As argued above (results), the mapappears to represent nearly full coverage of the M. domes-tica genome. However, an estimate of the total linkagemap size must account for undetected regions beyondthe terminal markers of each linkage group and for thelength of the X chromosome. To estimate the lengthof putative undetected telomeric regions, we used thesimple expedient of assuming them to be the same size

Figure 2.—Relative sex-specific linkage group sizes of M. as the average interlocus distance (ILD) of the mappeddomestica. Linkage groups are arranged in descending order loci (e.g., Fishman et al. 2001). Specifically, the averageof male linkage group size, with male linkage groups above ILD was calculated as the total map length divided byand corresponding female linkage groups below. Linkage

the number of recombining intervals: 633/56 � 11.3group sizes (in centimorgans) are indicated above and belowcM. A commonly used alternative method (e.g., Mikawathe male and female bars, respectively. Probabilities are for

tests of male vs. female differences in linkage group size (see et al. 1999; Zenger et al. 2002) uses the total numberfootnote b of Table 4). of linked autosomal loci minus the number of link-

age groups (80 � 8 � 72) as the denominator. Thismore liberal approach yields an average ILD estimate

sex-average map length) exhibit higher female recombi- of 8.79 cM.nation (Table 5). Male recombination appears to be We have no estimate of the length of LG X, buthigher on average over the remaining 34 (nonsignifi- physically the X chromosome is less than half the sizecant) intervals as well, as judged from the summed of the smallest M. domestica autosome (Pathak et al.lengths of these intervals: 345.5 cM in males and 250.3 1993). It seems reasonable to assume that its recombina-cM in females. Only LGs 5 and 6 exhibit no significant tional length would not exceed that of the average ofdifferences in interval lengths between sexes, but, as the three smallest autosomes; thus we use the averagementioned, the overall length of LG 6 is significantly length of the three smallest linkage groups, 54.3 cM, asgreater in males than in females. LG 5, which is the our estimate of LG X length. Combining the lengths ofsmallest with regard to length and number of loci, is LGs 1–8 with the estimate for LG X and 18 linkagethe only linkage group that exhibits no significant be- group ends yields an estimate of 633 54.3 (18 �tween-sex differences either in total length or at the 11.3) � 890.7 cM for the M. domestica sex-average re-interlocus interval level. combinational map. To our knowledge, this is the most

The magnitude of the recombination rate differences compact sex-average linkage map of any vertebrate spe-varies widely among linkage groups, with male linkage cies.group sizes ranging from 1.2 to 5.3 times the size of the The lengths of mammalian sex-average linkage mapscorresponding female linkage groups. Consideration of are highly variable, indicating a broad range of recombi-deviations of sex-specific recombination rates from sex- nation rates among species. Examples range from �1398average rates for each interlocus interval (illustrated in cM for laboratory mice (Rhodes et al. 1998) to �3500Figure 3) suggest that sex differences, while widespread, cM in humans (Kong et al. 2002), cattle (Barendse etare exaggerated in specific chromosomal regions rather al. 1997; Kappes et al. 1997), and sheep (Mikawa et al.than uniform across a chromosome. Interpretation of 1999). However, the length of the mouse linkage mapthese regional and linkage-group level differences is could underestimate the true recombination rate ofhampered by the varying levels of informativeness individual mouse species or strains because it was assem-among the loci studied; some seemingly large differ- bled using combined data from both conspecific crossesences in interlocus distances between the sexes un- and interspecific hybrids (Copeland et al. 1993; Die-doubtedly failed to reach significance because of the low trich et al. 1996). Among maps constructed using strictnumber of informative meioses among the loci involved. conspecific crosses, the 1510-cM dog (Canis familiaris)However, 43 of the 56 interlocus intervals are longer in map (Neff et al. 1999) and 1790-cM rat (Rattus norvegi-males than in females, and the male map significantly cus) map (Bonne et al. 2002) are the smallest, but bothexceeds the female map for almost half (�46%) of its are incomplete. The full-coverage dog and rat mapslength. The overall impression is that male recombina- are estimated to be �2700 and 1989 cM, respectively.

Among other vertebrates, estimated map lengths varytion is greater than female recombination for most of


TABLE 5

Interlocus intervals exhibiting significantly different sex-specific lengths

Map length (cM)

Linkage Sex Chigroup Interval average Female Male square Probabilitya

LG 1 Mdo1L001-Mdo1LR01 11.7 3.3 16.2 6.08 0.014Mdo1LR01-AK1 12.0 2.9 18.8 9.21 0.002Mdo1L019-Mdo1L003 4.7 1.6 7.6 8.05 0.0046Mdo1L003-PI 13.2 5.9 21.6 17.3 0.00003PI- Mdo1L004 25.2 11.7 42.9 17.1 0.00004Mdo1L004-Mdo1L002 3.9 2.2 5.7 5.53 0.0187Mdo1L002-Mdo1L021 4.6 2.1 7.0 6.68 0.0098

LG 2 GPT-Mdo2L023 10.8 1.7 19.3 32.3 1 � 10�8

Mdo2L023-Mdo2L022 19.8 6.3 36.0 58.1 2 � 10�14

Mdo2L022-Mdo2L005 11.2 4.0 18.9 30.0 4 � 10�8

Mdo2L006-C6/C7 14.7 6.9 26.1 21.9 0.000003LG 3 Mdo3L024-AT3 39.0 59.0 27.5 7.64 0.0057

Mdo3L025-Mdo3L008 30.5 8.0 69.2 92.1 9 � 10�22

Mdo3L008-TP53 2.3 0.0 3.8 6.54 0.011TP53-ME1 14.1 2.1 24.5 16.5 0.000049

LG 4 SP17-Mdo4L029 11.2 2.7 22.9 20.3 0.000007Mdo4L029-Mdo4L009 16.7 8.4 23.3 21.2 0.000004Mdo4LR16-Mdo4L028 9.1 3.7 13.7 5.76 0.016Mdo4L028-Mdo4LR14 3.6 7.6 1.2 7.41 0.0065

LG 5 None — — — — —LG 6 None — — — — —LG 7 Mdo7L033-Mdo7L013 37.9 26.1 54.5 6.54 0.0105LG 8 Mdo8L016-Mdo8LR25 5.2 1.3 9.4 7.60 0.0058

Mdo8L025-ACP2 31.4 25.2 68.8 7.51 0.0061Totals 22 intervals 332.8 cM 192.7 cM 538.9 cM

Longer interval is indicated in italic type.a Probability that sex-specific recombination rates for an interval are equal: likelihood ratio test of heterogene-

ity of sex-specific recombination fractions (Ott 1999).

from �1350 cM in rainbow trout (Oncorhyncus mykiss ; (1988) and Gregory (2001)]. Clearly, recombinationrates (centimorgans per megabases) in these two dis-Sakamoto et al. 2000) to 7291 cM in hybrid crosses of

the salamanders Ambystoma mexicanum and A. tigrinum tantly related marsupial species are very low relative tothose of other mammals and of vertebrates in general.(Voss et al. 2001).

The tammar wallaby, M. eugenii, is the only other It is tempting to propose that the short map lengthsof M. domestica and M. eugenii are related to the lowmarsupial for which a comprehensive linkage map has

been published (Zenger et al. 2002). The evolutionary chromosome numbers of these two species. However,an examination of total map lengths (in centimorgans)distance between M. eugenii and M. domestica is �60–70

million years (Kirsch et al. 1997; Springer 1997; and chromosome numbers (n) among the 19 nonmeta-therian vertebrates for which sufficient data are avail-Graves and Westerman 2002), which is roughly com-

parable to that between humans and mice. The M. eu- able, suggests that chromosome number does not ex-plain the majority of the observed variation in mapgenii map length of 828.4 cM includes the X chromo-

some, but is not corrected for putative undetected lengths (Figure 4). Among eutherian mammals, for ex-ample, dogs have more than twice as many chromo-telomeric regions and is estimated to represent only

71% of a total length of 1172 cM. This total map length somes as cats (n � 39 and 19, respectively), but the doglinkage map (�2700 cM) is substantially shorter thanis 31.6% larger than our estimate of the M. domestica

sex-average map, but is still quite small by vertebrate the cat map (�3300 cM). Humans and Syrian hamsters,on the other hand, have nearly identical chromosomestandards. The M. domestica genome (3.6–3.7 pg of the

DNA/haploid genome, estimated by flow cytometry; J. numbers (23 and 22, respectively), but their maplengths (�3600 and �2000 cM, respectively) are widelyDanke, unpublished data) is �6% larger than that of

humans, while that of M. eugenii may be as much as divergent. Even comparing the marsupials, M. eugeniihas fewer chromosomes than M. domestica, while its map18% larger than the human genome [data of Hayman

and Martin (1974), cited by Sharp and Hayman is nearly a third longer. Moreover, regression analyses


Figure 3.—Sex-specificdeviations from the sex-average recombination ratein M. domestica. Intervalnumbers correspond to in-terlocus intervals in Figure1, beginning at the top ofthe linkage group diagram;e.g., interval 3 of LG 1 corre-sponds to AK1–U15557, andinterval 4 of LG 2 corre-sponds to the nonrecombin-ing MdoL005–MdoL006 “in-terval.” The horizontal linerepresents the sex-averagerecombination rate. Over-lapping male and femalesymbols on the horizontalline indicate that recombi-nation occurred in the in-terval, but that the sex-spe-cific rates were identical.Solid squares indicate inter-vals with no recombinationin either sex.

(excluding the clearly anomalous amphibian relation- 0.22 (P � 0.107); eutherians fishes, 0.07 (P � 0.322);and eutherians chicken fishes, 0.16 (P � 0.102).ship) of map length on chromosome number for various

vertebrate groupings reinforce the visual impression However, when the two marsupials were added to theanalyses, all of the corresponding correlations becamethat chromosome number is a poor predictor of map

length. The correlations (r 2) between map length and stronger (r 2 ranged from 0.32 to 0.51) and were statisti-cally significant (P � 0.003–0.012). Because the marsu-chromosome number for the various groups were: eu-

therians alone, 0.12 (P � 0.277); eutherians chicken, pial data were extreme values and provided strong lever-


and the present study shows that ALDOC and TP53 aresyntenic on M. domestica LG 3. These 8 loci are knownto be syntenic on Mmu 11 and Hsa 17q, suggesting thata substantial 8-locus synteny may have been conservedamong M. domestica, mice, and humans. However, LG3 is not entirely homologous to Hsa 17q and Mmu 11,as it also contains loci found on Hsa 1p (NRASLB), Hsa1q (AT3), and Hsa 6q (ME1); these loci are similarlydispersed in the mouse genome. The impression ofmixed conservation and rearrangement relative to eu-therian genomes agrees with extensive physical map-ping results from M. eugenii and a few other marsupialspecies (reviewed by Samollow and Graves 1998). Un-

Figure 4.—Total map length vs. chromosome number infortunately, except for several X- and Y-linked genes,vertebrates. See text for discussion. Data sources are as follows:there is almost no overlap among the type I loci mappedhuman (Broman et al. 1998; Kong et al. 2002), baboon (Rog-

ers et al. 2000), mouse (Rhodes et al. 1998), rat (Bonne et al. in M. domestica and M. eugenii (Samollow and Graves2002), Syrian hamster (Okuizumi et al. 1997), cat (Menotti- 1998; and more recent articles by Delbridge et al. 1999;Raymond et al. 1999), dog (Neff et al. 1999), cattle (average Hawken et al. 1999; Charchar et al. 2000; Waters et al.of estimates of Barendse et al. 1997; Ferretti et al. 1997;

2001), so there is currently no opportunity for syntenyKappes et al. 1997), pig (Mikawa et al. 1999), sheep (Maddoxcomparisons between these two marsupial species.et al. 2001), deer (Slate et al. 2002), horse (Guerin et al.

2003), M. domestica (this study), M. eugenii (Zenger et al. 2002), Sex-specific recombination: The small size of the M.chicken (Groenen et al. 2000), salamander (Voss et al. 2001), domestica linkage map is remarkable in its own right, butchannel catfish (Waldbieser et al. 2001), medaka (Naruse more surprising is the difference in sex-specific recombi-et al. 2000), rainbow trout (mean of estimates derived from

nation rates implied by the female- and male-specificYoung et al. 1998; Sakamoto et al. 2000), Xiphophorus sp. (S.linkage maps. The combined male linkage map (884.6Kazianis, R. S. Nairn, R. B. Walter, D. A. Johnston, J.

Kumar et al., unpublished data), and zebrafish (Shimoda et al. cM) is twice the size of the female map (443.1 cM; the1999). Vertebrate species with substantially incomplete maps female to male recombination ratio, F/M, is 0.50), andwere excluded from this analysis. For consistency with the each of the individual linkage groups is larger in malesother species examined, the published total map lengths for

than in females. This sex difference is extraordinary notrat, deer, horse, and medaka were adjusted for missing telo-only for its magnitude, but also for the direction of themeric regions by adding twice the average interlocus distance

for the particular species map to each of the identified linkage sex-specific recombination bias, which is unlike any-groups. thing observed in other vertebrates except in two distant

marsupial relatives, M. eugenii and S. crassicaudata (fat-tailed dunnart). Unfortunately, linkage data from S.crassicaudata (Bennett et al. 1986) are very limited (six

age points for the regression analyses, the implications loci) and not directly comparable to those from M.of this latter outcome are not clear. In general, however, domestica or M. eugenii. However, interesting compari-there is little support for the concept of a robust relation- sons are possible with the M. eugenii map. The reductionship between map length and chromosome number in female recombination in M. domestica is greater inamong the vertebrate species examined. More data from both extent and magnitude than that seen in M. eugenii.marsupials and other vertebrates with low chromosome The M. eugenii map exhibits significant sex differencesnumbers will be needed to adequately test this hypoth- in only 8 interlocus intervals covering 26.2% of the sex-esis. average autosomal map length (calculated from data of

Interspecific synteny comparisons: Inspection of the Zenger et al. 2002), and the overall F/M ratio is 0.78,linkage group affiliations of the 20 type 1 loci mapped compared with 0.50 in M. domestica. After removal ofin this study is inconclusive with regard to conservation the 8 significant intervals from the M. eugenii map, theof gene synteny with eutherian species. SP17 and TF are F/M ratio increased to a sex-equal 1.01 (Zenger et al.syntenic in M. domestica (LG 4), mice (Mmu 9), and 2002), while in M. domestica, removal of the 20 significanthumans (Hsa 3q), but LG 4 also includes HBE, which intervals yields an F/M ratio of 0.72, a ratio that is moreis not syntenic with SP17 or TF in mice or humans (a skewed than the F/M ratio for the full M. eugenii mapfourth coding locus in LG 4, PHL, has no confirmed with all 8 significant intervals included.homolog in eutherians). C6/C7 and GPT are syntenic Sex-specific differences in meiotic recombination ratein M. domestica (LG 2) and mouse (Mmu 15), but not are common among a broad range of animal and plantin humans (Hsa 5p and Hsa 8q, respectively). A series of species. Haldane (1922) and Huxley (1928) eachtwo-point linkage comparisons reported by Sokolova et noted this general trend and suggested that in speciesal. (1997) suggested that NF1, ERBB2, RARA, HOX2, where the sexes had substantially different recombina-

tion rates, it was the heterogametic sex that had theMPO, PRKCA, and ALDOC are syntenic in M. domestica,


reduced rate. While neither author proposed this situa- nome as a whole. In summary, we have been unableto identify a single unambiguous case of excess maletion to be inviolable, and exceptions are known [e.g.,

flour beetles, Tribolium castaneum (Sokoloff 1964)], recombination, as gauged from genome-wide geneticdata, in any nonmetatherian vertebrate species.this prediction has turned out to be accurate in the

great majority of species in which strong sex-specific The occurrence of reduced female recombination inboth American and Australian marsupial species, whichdifferences in recombination frequency have been doc-

umented on a genome-wide basis (regional sex-specific last shared a common ancestor at least 60 MYA (Kirschet al. 1997; Springer 1997; and discussions by Kirschreversals over short map distances are known in many

species, but these do not affect the genome-wide gener- and Mayer 1998; Springer et al. 1998), tempts specula-tion that reduced female recombination is a generalalization).

Most eutherians examined exhibit substantially higher characteristic that distinguishes metatherian from eu-therian species. With so few mammalian species mapsfemale than male recombination rates, and, among

those with extensive maps, the F/M ratio is almost completed, such speculation might be premature; how-ever, cytologic data from the brushtail possum, Tricho-always �1.0; e.g., 1.56–1.65 in humans (Broman et al.

1998; Kong et al. 2002); 1.36–1.41 in dogs (Mellersh surus vulpecula, (family Phalangeridae) also suggests re-duced female recombination, although less extremeet al. 1997; Neff et al. 1999), and 1.30–1.55 in pigs

(Archibald et al. 1995; Mikawa et al. 1999). In cattle, than that in M. domestica and S. crassicaudata (Haymanand Rodger 1990). The only contrary finding comesthe female map is just slightly larger than the male map:

F/M � 1.03–1.06 (Barendse et al. 1997; Kappes et al. from a cytologic study of the brush-tailed bettong, Betton-gia penicillata (family Potoroidae), in which no obvious1997). Genome-wide female bias in recombination is

also evident in the baboon linkage map (J. Rogers difference in sex-specific chiasma number or distribu-tion was detected (Hayman et al. 1990). Unfortunately,and M. C. Mahaney, personal communication) and in

linkage data from each of six rhesus macaque chromo- linkage data are not available for T. vulpecula or B.penicillata. Thus, reduced female recombination surelysomes examined to date (J. Rogers, personal communi-

cation). A possible exception to this pattern has been does occur in three marsupial species and perhaps afourth. Notwithstanding the data from B. penicillata, itobserved in sheep, in which the F/M ratio � 0.83; but

this ratio must be regarded as provisional because pecu- seems probable that reduced female recombination is avery common, if not universal, marsupial characteristic.liarities of the mating scheme used to produce the sheep

maps could have contributed to an underestimate of What influences recombination rates in marsupials?Marsupial genomes are about the same size as those ofmale map length (Maddox et al. 2001, p. 1285).

The tendency for female map length to exceed that eutherian mammals (Gregory 2001) but are far moreconservative with regard to chromosomal compartmen-of males appears to be greatly exaggerated in fishes:

zebrafish F/M � 2.74 (Singer et al. 2002); catfish F/M � talization (discussed by Samollow and Graves 1998;Graves and Westerman 2002). Whereas eutherian3.18 (Waldbieser et al. 2001); rainbow trout F/M �

3.25 (Sakamoto et al. 2000), but absent in birds. In the chromosome numbers range from 2n � 6 to 2n � 118,those of marsupials range only from 2n � 10 to 2n �only avian map available, that of chicken, F/M � 0.99

(Groenen et al. 1998, 2000), and a cytologic study in 32, and in �90% of marsupial species examined, 2n �14–22. On average, then, marsupials package the samepigeons revealed equal numbers of chiasmata in female

and male meiotic cells (Pigozzi and Solari 1999). Sep- amount of DNA into fewer, larger chromosomes thando their eutherian counterparts. Low chromosomearate female and male maps are not available for am-

phibians, but recombination data for six autosomal loci number reduces the opportunity for genome reshuf-fling via interchromosomal assortment, but this ten-in the frog Rana brevipoda and a pair of loci in its conge-

ner R. japonica indicate that strongly female-biased re- dency can be compensated by an increase in intrachro-mosomal recombination (crossing over). That suchcombination occurs in at least some amphibians (Sum-

ida and Nishioka 2000). Average chiasma counts for compensation does not appear to occur in M. domestica(2n � 18), M. eugenii (2n � 16), or S. crassicaudata (2n �male crested newts (Triturus cristatus) exceed those of

females on all chromosomes (Wallace et al. 1997), 14) suggests that the intergenerational reshuffling ofgenetic information in these marsupials is considerablysuggesting higher male than female recombination

rates (chiasma-based F/M � 0.77) in this species. How- less intense than that in eutherians and other verte-brates and especially so in females.ever, the chiasmata in oocytes are strongly clustered

around centromeres, while those in spermatocytes are The substantial literature on the evolution of sex andrecombination rates (reviewed by Otto and Bartonrestricted to the more distal regions of the chromosome

arms; thus the male and female recombining regions are 1997; Barton and Charlesworth 1998; Burt 2000;Otto and Lenormand 2002; Rice 2002) suggests thatlargely distinct and nonoverlapping. This dimorphism,

together with the lack of genetic mapping data for this the frequency of meiotic recombination can be shapedby natural selection to optimize the extent to whichspecies, inhibits interpretation of these cytological ob-

servations with regard to recombination across the ge- multilocus allelic associations are maintained or obliter-


ated from one generation to the next. Discussions of on the vast majority of X-linked genes that follow thesingle-active-allele pattern). In metatherians, the pat-recombination rate have focused primarily on genetic

interactions and environmental conditions (various tern is decidedly nonrandom; it is always the paternallyderived X that is inactive (paternal XCI or PXI). Thus,forms of epistasis, linkage relationships, environmental

heterogeneity) that act to modify population-average the only X-linked genes expressed in a metatherianfemale’s cells are those derived from her mother;recombination rates. Recent modeling of the evolution

of sex-specific recombination differences (Lenormand X-linked genes derived from her father contribute virtu-ally nothing to her phenotype (as in eutherians, excep-2003) indicates that similar forces can also adjust male

and female recombination rates independently of those tions occur; some paternally derived genes escape fullinactivation, but we focus here on the great majority ofthat determine average rates. If so, the small size of the

M. domestica linkage map and the striking difference paternally derived X-linked genes that are fully si-lenced). This distinction in XCI patterns results in phy-between male and female maps could indicate that the

selective environments that have shaped recombination logenetically restricted differences in the effective con-tributions of mothers and fathers to the expressedrates in this and perhaps other marsupial species differ

at both species- and sex-specific levels from those that genomes of their offspring.Hope (1993) proposed a mechanistic model whereinhave influenced recombination rates in other verte-

brates. the female response to the paternal imprint that causesthe paternally derived X chromosome to be inactiveUnfortunately, we do not know why any particular

species has the recombination rate it does or why marsu- might actually be applied indiscriminately to all pater-nally derived chromosomes. This would render thempials as a group should have reduced recombination.

Nevertheless, it is worth remembering that the species- molecularly distinct from maternally derived chromo-somes and could interfere with and reduce recombina-average rate of recombination can evolve for reasons

unrelated to the forces acting on sex-specific rates (Len- tion in females. Genomic imprinting of selected autoso-mal genes, similar to that seen in eutherians, doesormand 2003), so the low recombination rates observed

in female marsupials need not dictate low male recombi- indeed occur in marsupials (Killian et al. 2000, 2001;O’Neill et al. 2000), but there is no evidence of whole-nation as a correlated response. More likely, low average

recombination in M. domestica (and M. eugenii and per- sale gene silencing or any other form of global paternalimprinting in any marsupial species, so this explana-haps other marsupial species) is a result of pressures

acting to reduce recombination generally, while other tion for reduced female recombination seems unlikely.Alternatively, it has been shown that sex-specific asym-factors acting in sex-specific ways have adjusted male

and female rates within this general, species-average metries in epistatic interactions among autosomal loci(imprinted or not) can lead to divergence of male andrange.

Despite our inability to explain the low average re- female recombination rates (Lenormand 2003). Theexclusive expression of maternally derived X-linkedcombination rate in marsupials, a possible explanation

for reduced female recombination rates could lie in the genes in metatherian females generates an asymmetryof parental contribution to female offspring relative topattern of X-linked dosage compensation that occurs

in metatherian mammals. X chromosome inactivation the situation in eutherian wherein both parents contrib-ute active X-linked genes to their daughters (neither(XCI) is a uniquely mammalian process that results in

the coordinate silencing of the great majority of genes metatherian nor eutherian males contribute X chromo-somes to their sons). Given current understanding ofon one of the two X chromosomes in female somatic

cells during embryogenesis (reviewed by Heard et al. the power of epistatic interactions to alter sex-specificrecombination rates, it seems unlikely that PXI alone1997; Avner and Heard 2001; Boumil and Lee 2001;

Brockdorff 2002). Eutherians and metatherians ex- could favor reduced female recombination in the ab-sence of autosomal imprinting (T. Lenormand and S.hibit differences in several details of the X-inactivation

process (reviewed by VandeBerg et al. 1987; Cooper et Otto, personal communications). However, if benefi-cial epistatic interactions among combinations of allelesal. 1990, 1993; see also Keohane et al. 1998), of which

the most salient is the origin of the inactivated chromo- at linked autosomal loci were dependent on both theparent of origin of the interacting genes (autosomalsome. In eutherian females inactivation is random with

regard to the parental source of the X chromosome. imprinting) and the presence of a maternally imprintedX chromosome, then it might be plausible that PXIAbout half of a female’s cells express genes from the X

chromosome she inherited from her mother (mater- could create a selective environment in which the bene-fit to females of keeping favorable sex-specific epistaticnally derived X) and the other half express the genes

from the X inherited from her father (paternally de- autosomal associations intact would be greater than thedetriment (if any) to male offspring and greater thanrived X). Although no individual cell expresses the ma-

ternally and paternally derived copies of X-linked genes, the pressure to break up such combinations in malemeiosis. To the extent that such multilocus associationsboth contribute to the female’s overall phenotype (some

X-linked genes escape X inactivation, but here we focus occur throughout the genome, this might lead to an


increase in alleles at recombination modifier loci that mapped region or that chiasmata on this chromosomereduce female recombination. Although no empirical are concentrated in the extreme terminal region(s) inor theoretical evidence supports these or any other re- females. As the male LG 2 map is more than five timeslated hypotheses, the unique pattern of paternal XCI in larger (100.4 cM) than the female LG 2 map, lack ofmetatherians may have the potential to explain reduced recombination in female LG 2 is most simply attribut-recombination in females relative to that in males. able to extreme terminal distribution of crossover

Linkage and physical recombination/future research: events, rather than to inadequate marker coverage.Whatever the evolutionary impetus for the disparate Whether these various inflations and compressionsrecombination rates of male and female M. domestica, correspond to telomeric and interstitial regions as pre-the proximate explanation is likely to involve sex-spe- dicted from the data of Hayman et al. (1988) or to thecific differences in the number and distribution of chias- commonly observed exaggeration and diminution (ormata during gametogenesis. The data of Hayman et reversal) of sex-specific recombination differences asso-al. (1988) revealed a clear sexual dimorphism in M. ciated with telomeric and centromeric regions, respec-domestica meiotic cells, with male chiasmata being more tively, in other species [e.g., humans (Broman et al. 1998;numerous and more evenly distributed than female chi- Kong et al. 2002), fishes (Sakamoto et al. 2000; Singerasmata. Males averaged 21.5 chiasmata per meiotic cell, et al. 2002), and even monoecious plants (Lagerkrantzand two-thirds of these were interstitial, suggesting that and Lydiate 1995)] will be known only when the link-crossing over occurs more or less uniformly across the age groups are physically anchored and oriented on thechromosome arms. Females averaged only 13 chiasmata M. domestica chromosomes. As a preliminary step towardper meiosis, and virtually all were terminal or subtermi- this objective, we are isolating M. domestica bacterialnal. The difference in chiasma number should be re- artificial chromosome (BAC) clones (BACPAC Re-flected in a smaller female map, as is seen in the current sources: http://www.chori.org/bacpac) containing ge-linkage data; but it should also proportionally (not abso- netically (linkage) mapped loci for use as probes inlutely) inflate the ends of the female linkage groups fluorescence in situ hybridization mapping. Ultimately,and compress them in intermediate regions, relative to BAC fingerprinting and sequencing combined with thethe male linkage group maps. mapping of linkage map markers (sequence tagged

The small number of loci mapped in several linkage sites) to BAC clones, will enable construction of angroups precludes rigorous analysis, but the larger link- integrated physical and linkage map of the M. domesticaage groups do appear to exhibit terminal inflation in genome. When such an integrated map becomes estab-the female map when the relative proportions of the lished, the relationship between the physical peculiari-individual intervals are compared between the male and ties of recombination in M. domestica and the astound-female maps [relative interval length is defined as (sex- ing sex-specific differences in the genetic map will bespecific interval length)/(sex-specific linkage group clarified.length); data not shown]. For example, the last interval

We thank Lynn Cherry, Nicole Stowell, and Dan Rodriguez forof LG 1 (Mdo1L021–Mdo1L020) on the female map is technical assistance; Jim Bridges and Nicole Stowell for aspects ofproportionally larger than that on the male map. This data management; Debbie Christian, Janice MacRossin, and Jane Van-is also true for the last interval of LG 4, the first three deBerg for their help with animal dissections and sample processing;

and Don Taylor, Gerardo Colon, Susan Collins, and Ernesto Morinintervals of LG 6, and the first interval of LG 3 andfor superb animal care and pedigree record keeping. Thanks also goLG 8. Conversely, compressions of the female map areto Thomas Lenormand, Sarah Otto, and William Rice for helpfulexpected to occur in interstitial regions. Such compres-comments regarding the possible relationship between low female

sions are not obvious, with the possible exception of recombination rates and X chromosome inactivation. This work wasLG 3, but compressions do occur at the ends of several supported in part by grants from the National Institutes of Health

(RR-09919 and RR-14214), the Samuel Roberts Noble Foundation,linkage groups that exhibit inflations at their oppositethe Ellwood Foundation, and the Robert J. Kleberg, Jr. and Helen C.ends, e.g., the first two intervals of LG 4, the last threeKleberg Foundation.intervals of LG 6, and the last interval of LG 8.

Additional support for the idea that localization ofchiasmata to chromosome ends is an important contrib-utor to low female recombination rate can be inferred LITERATURE CITEDfrom female LG 2. It is commonly assumed that each Aivaliotis, M. J., T. Cantu, R. Gilligan and J. L. VandeBerg,meiotic bivalent must undergo at least one non-sister- 1995 Glutathione S-transferase class pi polymorphism in ba-

boons. Biochem. Genet. 33: 35–40.chromatid exchange to ensure proper disjunctionArchibald, A. L., C. S. Haley, J. F. Brown, S. Couperwhite, H. A.(Egel 1995; Roeder 1997; Moore and Orr-Weaver

McQueen et al., 1995 The PiGMaP consortium linkage map of1998). Under this constraint, each linkage group should the pig (Sus scrofa). Mamm. Genome 6: 157–175.

Avner, P., and E. Heard, 2001 X-chromosome inactivation: count-equal or exceed 50 cM if full map coverage has beening, choice and initiation. Nat. Rev. Genet. 2: 59–67.achieved. At 18.9 cM, female LG 2 falls far short of this

Barendse, W., D. Vaiman, S. J. Kemp, Y. Sugimoto, S. M. Armitagemark, suggesting either that a large portion of the LG et al., 1997 A medium-density genetic linkage map of the bovine

genome. Mamm. Genome 8: 21–28.2-bearing chromosome lies outside of the currently


Barton, N. H., and B. Charlesworth, 1998 Why sex and recombi- Graves, J. A., and M. Westerman, 2002 Marsupial genetics andgenomics. Trends Genet. 18: 517–521.nation? Science 281: 1986–1990.

Graves, J. A. M., 1998 Background and overview of comparativeBell, K., H. Arthur, R. A. van Oorschot and J. L. VandeBerg,genomics. ILAR J. 39: 48–65.1992 Antithrombin III (AT3) polymorphism in the marsupial

Green, P., 1990 Documentation of CRIMAP, Version 2.4. WashingtonMonodelphis domestica: identification and genetics. Biochem.University School of Medicine, St. Louis.Genet. 30: 591–601.

Gregory, T. R., 2001 Animal Genome Size Database (http://www.Bennett, J. H., D. L. Hayman and R. M. Hope, 1986 Novel sexgenomesize.com/).differences in linkage values and meiotic chromosome behaviour

Groenen, M. A., R. P. Crooijmans, A. Veenendaal, H. H. Cheng,in a marsupial. Nature 323: 59–60.M. Siwek et al., 1998 A comprehensive microsatellite linkageBonne, A. C., M. G. den Bieman, G. F. Gillissen, L. F. van Zutphenmap of the chicken genome. Genomics 49: 265–274.and H. A. van Lith, 2002 A rat linkage map based on BC �

Groenen, M. A., H. H. Cheng, N. Bumstead, B. F. Benkel, W. E.LEW intercross. Folia Biol. 48: 120–123.Briles et al., 2000 A consensus linkage map of the chickenBoumil, R. M., and J. T. Lee, 2001 Forty years of decoding thegenome. Genome Res. 10: 137–147.silence in X-chromosome inactivation. Hum. Mol. Genet. 10:

Guerin, G., E. Bailey, D. Bernoco, I. Anderson, D. F. Antczak et2225–2232.al., 2003 The second generation of the International EquineBrockdorff, N., 2002 X-chromosome inactivation: closing in onGene Mapping Workshop half-sibling linkage map. Anim. Genet.proteins that bind Xist RNA. Trends Genet. 18: 352–358.34: 161–168.Broman, K. W., J. C. Murray, V. C. Sheffield, R. L. White and J. L.

Haldane, J. B. S., 1922 Sex ratio and unisexual sterility in hybridWeber, 1998 Comprehensive human genetic maps: individualanimals. J. Genet. 12: 101–109.and sex-specific variation in recombination. Am. J. Hum. Genet.

Harris, H., and D. A. Hopkinson, 1976 Handbook of Enzyme Electro-63: 861–869.phoresis in Human Genetics. Elsevier-North Holland, Amsterdam.Burt, A., 2000 Perspective: Sex, recombination, and the efficacy of

Hawken, R. J., P. Maccarone, R. Toder, J. A. Marshall Gravesselection—was Weismann right? Evolution 54: 337–351.and J. F. Maddox, 1999 Isolation and characterization of marsu-Charchar, F. J., M. Svartman and J. A. Graves, 2000 Assignmentpial IL5 genes. Immunogenetics 49: 942–948.of SPROUTY 1 (SPRY1) gene to tammar wallaby chromosome 6

Hayman, D., H. Moore and E. Evans, 1988 Further evidence ofby fluorescence in situ hybridisation. Cytogenet. Cell Genet. 90:novel sex differences in chiasma distribution in marsupials. He-240–241.redity 61: 455–458.Cheng, M. L., and J. L. VandeBerg, 1987 Cryopreservation of eryth-

Hayman, D. L., and P. G. Martin, 1974 Mammalia I: Monotrematarocytes in small aliquots for isozyme electrophoresis. Biochem.and Marsupialia, in Animal Cytogenetics, Vol. 4, edited by B. John.Genet. 25: 535–541.Borntraeger, Berlin.Cheng, M. L., S. C. Woodford, J. L. Hilburn and J. L. VandeBerg,

Hayman, D. L., and J. C. Rodger, 1990 Meiosis in male and female1986 A novel system for storage of sera frozen in small aliquots.Trichosurus vulpecula (Marsupialia). Heredity 64: 251–254.J. Biochem. Biophys. Methods 13: 47–51.

Hayman, D. L., M. J. Smith and J. C. Rodger, 1990 A comparativeClayton, J. W., and D. N. Tretiak, 1972 Amine-citrate buffers forstudy of chiasmata in male and female Bettongia penicillata (Marsu-pH control in starch gel electrophoresis. J. Fish. Res. Bd. Can.pialia). Genetica 83: 45–49.29: 1169–1172.

Heard, E., P. Clerc and P. Avner, 1997 X-chromosome inactivationCooper, D. W., P. G. Johnston, J. L. VandeBerg and E. S. Robinson,in mammals. Annu. Rev. Genet. 31: 571–610.1990 X-chromosome inactivation in marsupials, pp. 269–275 in

Hillis, D. M., B. K. Mable, A. Larson, S. K. Davis and E. A. Zimmer,Mammals From Pouches and Eggs: Genetics, Breeding and Evolution of1996 Nucleic acids IV: sequencing and cloning, pp. 321–381 inMarsupials and Monotremes, edited by J. A. M. Graves, R. Hope andMolecular Systematics, Ed. 2, edited by D. M. Hillis, C. MoritzD. W. Cooper. Commonwealth Scientific and Industrial Researchand B. K. Mable. Sinauer Associates, Sunderland, MA.Organization, Melbourne.

Hope, R., S. Cooper and B. Wainwright, 1990 Globin macromolec-Cooper, D. W., P. G. Johnston, J. M. Watson and J. A. M. Graves,ular sequences in marsupials and monotremes. Aust. J. Zool. 37:1993 X-inactivation in marsupials and monotremes. Semin. 289–313.Dev. Biol. 4: 117–128. Hope, R., H. Bennett, C. Chesson and S. Cooper, 1992 RestrictionCopeland, N. G., N. A. Jenkins, D. J. Gilbert, J. T. Eppig, L. J. fragment length variants in the marsupial Sminthopsis crassicau-Maltais et al., 1993 A genetic linkage map of the mouse: cur- data. Biochem. Genet. 30: 361–370.rent applications and future prospects. Science 262: 57–66. Hope, R. M., 1993 Selected features of marsupial genetics. Genetica

Davisson, M. T., T. H. Roderick and D. P. Doolittle, 1989 Recom- 90: 165–180.bination percentages and chromosomal assignments, pp. 432–505 Hu, C. C., C. Zhang, Q. Qian, O. H. Ryu, J. Moradian-Oldak etin Genetic Variants and Strains of the Laboratory Mouse, edited by al., 1996 Cloning, DNA sequence, and alternative splicing ofM. F. Lyon and A. G. Searle. Oxford University Press, Oxford. opossum amelogenin mRNAs. J. Dent. Res. 75: 1728–1734.

Delbridge, M. L., P. A. Lingenfelter, C. M. Disteche and J. A. Huxley, J. S., 1928 Sexual difference of linkage in GammarusGraves, 1999 The candidate spermatogenesis gene RBMY has chevreuxi. J. Genet. 20: 145–156.a homologue on the human X chromosome. Nat. Genet. 22: Kappes, S. M., J. W. Keele, R. T. Stone, R. A. McGraw, T. S. Sonste-223–224. gard et al., 1997 A second-generation linkage map of the bovine

Dietrich, W. F., J. Miller, R. Steen, M. A. Merchant, D. Damron- genome. Genome Res. 7: 235–249.Boles et al., 1996 A comprehensive genetic map of the mouse Kaslow, D. C., B. R. Migeon, M. G. Persico, M. Zollo, J. L. Vande-genome. Nature 380: 149–152. Berg et al., 1987 Molecular studies of marsupial X chromo-

Dunn, L. C., and D. Bennett, 1964 Sex differences in recombina- somes reveal limited sequence homology of mammalian X-linkedtion of linked genes in animals. Genet. Res. 9: 211–220. genes. Genomics 1: 19–28.

Egel, R., 1995 The synaptonemal complex and the distribution of Kato, T., Jr., T. Todo, H. Ayaki, K. Ishizaki, T. Morita et al., 1994meiotic recombination events. Trends Genet. 11: 206–208. Cloning of a marsupial DNA photolyase gene and the lack of

Ferretti, L., B. G. Urquhart, A. Eggen, I. Olsaker, B. Harlizius et related nucleotide sequences in placental mammals. Nucleical., 1997 Cosmid-derived markers anchoring the bovine genetic Acids Res. 22: 4119–4124.map to the physical map. Mamm. Genome 8: 29–36. Keohane, A. M., J. S. Lavender, L. P. O’Neill and B. M. Turner,

Fishman, L., A. J. Kelly, E. Morgan and J. H. Willis, 2001 A 1998 Histone acetylation and X inactivation. Dev. Genet. 22:genetic map in the Mimulus guttatus species complex reveals 65–73.transmission ratio distortion due to heterospecific interactions. Killian, J. K., J. C. Byrd, J. V. Jirtle, B. L. Munday, M. K. StoskopfGenetics 159: 1701–1716. et al., 2000 M6P/IGF2R imprinting evolution in mammals. Mol.

Gahne, B. R., R. Kumar Juneja and J. Grolmus, 1977 Horizontal Cell 5: 707–716.polyacrylamide gradient gel electrophoresis for the simultaneous Killian, J. K., T. R. Buckley, N. Stewart, B. L. Munday and R. L.phenotyping of transferrin, post-transferrin, albumin and post- Jirtle, 2001 Marsupials and Eutherians reunited: genetic evi-albumin in the blood plasma of cattle. Anim. Blood Groups dence for the Theria hypothesis of mammalian evolution. Mamm.

Genome 12: 513–517.Biochem. Genet. 8: 127–137.


Kirsch, J., F.-J. Lapointe and M. Springer, 1997 DNA-hybridisation Perelygin, A. A., P. B. Samollow and J. L. VandeBerg, 1994 A newHindIII RFLP of NRAS in the laboratory opossum, Monodelphisstudies of marsupials and their implications for metatherian classi-

fication. Aust. J. Zool. 45: 211–280. domestica. Anim. Genet. 25: 432.Perelygin, A. A., P. B. Samollow, L. M. Perelygina, L. M. Cherry,Kirsch, J. A., and G. C. Mayer, 1998 The platypus is not a rodent:

DNA hybridization, amniote phylogeny and the palimpsest the- S. M. Mahaney et al., 1996 A new DNA marker, U15557, is linkedto protease inhibitor and adenylate kinase-1 in the laboratoryory. Philos. Trans. R. Soc. Lond. B Biol. Sci. 353: 1221–1237.

Kong, A., D. F. Gudbjartsson, J. Sainz, G. M. Jonsdottir, S. A. opossum, Monodelphis domestica. Anim. Genet. 27: 113–116.Pigozzi, M. I., and A. J. Solari, 1999 Equal frequencies of recombi-Gudjonsson et al., 2002 A high-resolution recombination map

of the human genome. Nat. Genet. 31: 241–247. nation nodules in both sexes of the pigeon suggest a basic differ-ence with eutherian mammals. Genome 42: 315–321.Kumar, S., and S. B. Hedges, 1998 A molecular timescale for verte-

brate evolution. Nature 392: 917–920. Pollitt, C. C., and K. Bell, 1983 Characterisation of the �1-prote-ase inhibitor system in thoroughbred horse plasma by horizontalKusewitt, D. F., T. E. Sherburn, K. B. Miska, G. B. Tafoya, J. M.

Gale et al., 1999 The p53 tumor suppressor gene of the marsu- two-dimensional (ISO-DALT) electrophoresis. 1. Protein stain-ing. Anim. Blood Groups Biochem. Genet. 14: 83–105.pial Monodelphis domestica : cloning of exons 4–11 and mutations

in exons 5–8 in ultraviolet radiation-induced corneal sarcomas. Pollock, D. D., J. A. Eisen, N. A. Doggett and M. P. Cummings,2000 A case for evolutionary genomics and the comprehensiveCarcinogenesis 20: 963–968.

Lagerkrantz, U., and D. J. Lydiate, 1995 RFLP mapping in Bras- examination of sequence biodiversity. Mol. Biol. Evol. 17: 1776–1788.sica nigra indicates differing recombination rates in male and

female meioses. Genome 38: 255–264. Postlethwait, J. H., I. G. Woods, P. Ngo-Hazelett, Y. L. Yan,P. D. Kelly et al., 2000 Zebrafish comparative genomics and theLenormand, T., 2003 The evolution of sex dimorphism in recombi-

nation. Genetics 163: 811–822. origins of vertebrate chromosomes. Genome Res. 10: 1890–1902.Rhodes, M., R. Straw, S. Fernando, A. Evans, T. Lacey et al.,Maddox, J. F., K. P. Davies, A. M. Crawford, D. J. Hulme, D. Vaiman

et al., 2001 An enhanced linkage map of the sheep genome 1998 A high-resolution microsatellite map of the mouse ge-nome. Genome Res. 8: 531–542.comprising more than 1000 loci. Genome Res. 11: 1275–1289.

Matise, T. C., M. Perlin and A. Chakravarti, 1994 Automated Rice, W. R., 2002 Experimental tests of the adaptive significance ofsexual recombination. Nat. Rev. Genet. 3: 241–251.construction of genetic linkage maps using an expert system

(MultiMap): a human genome linkage map. Nat. Genet. 6: 384– Richardson, B. J., P. R. Baverstock and M. Adams, 1986 AllozymeElectrophoresis: A Handbook for Animal Systematics and Population390.

Mellersh, C. S., A. A. Langston, G. M. Acland, M. A. Fleming, K. Studies. Academic Press, Sydney, Australia.Roeder, G. S., 1997 Meiotic chromosomes: it takes two to tango.Ray et al., 1997 A linkage map of the canine genome. Genomics

46: 326–336. Genes Dev. 11: 2600–2621.Rogers, J., M. C. Mahaney, S. M. Witte, S. Nair, D. Newman et al.,Menotti-Raymond, M., V. A. David, L. A. Lyons, A. A. Schaffer,

J. F. Tomlin et al., 1999 A genetic linkage map of microsatellites 2000 A genetic linkage map of the baboon (Papio hamadryas)genome based on human microsatellite polymorphisms. Geno-in the domestic cat (Felis catus). Genomics 57: 9–23.

Mikawa, S., T. Akita, N. Hisamatsu, Y. Inage, Y. Ito et al., 1999 A mics 67: 237–247.Sakamoto, T., R. G. Danzmann, K. Gharbi, P. Howard, A. Ozakilinkage map of 243 DNA markers in an intercross of Gottingen

miniature and Meishan pigs. Anim. Genet. 30: 407–417. et al., 2000 A microsatellite linkage map of rainbow trout (Oncor-hynchus mykiss) characterized by large sex-specific differences inMoore, D. P., and T. L. Orr-Weaver, 1998 Chromosome segrega-

tion during meiosis: building an unambivalent bivalent. Curr. recombination rates. Genetics 155: 1331–1345.Sambrook, J., E. F. Fritsch and T. Maniatis, 1989 Molecular Clon-Top. Dev. Biol. 37: 263–299.

Morizot, D. C., and M. E. Schmidt, 1990 Starch gel electrophoresis ing: A Laboratory Manual, Ed. 2. Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, NY.and histochemical visualization of proteins, pp. 23–80 in Electro-

phoretic and Isoelectric Focusing Techniques in Fisheries Management, Samollow, P. B., and J. A. M. Graves, 1998 Gene maps of marsupi-als. ILAR J. 39: 203–224.edited by D. H. Whitmore. CRC Press, Boca Raton, FL.

Murray, M. J., J. M. Cunningham, L. F. Parada, F. Dautry, P. Samollow, P. B., A. L. Ford and J. L. VandeBerg, 1987 X-linkedgene expression in the Virginia opossum: differences betweenLebowitz et al., 1983 The HL-60 transforming sequence: a ras

oncogene coexisting with altered myc genes in hematopoietic the paternally derived Gpd and Pgk-A loci. Genetics 115: 185–195.Selander, R. K., M. H. Smith, W. E. Johnson and J. B. Gentry,tumors. Cell 33: 749–757.

Naruse, K., S. Fukamachi, H. Mitani, M. Kondo, T. Matsuoka 1971 Biochemical polymorphism and systematics in the genusPeromyscus. I. Variation in the old field mouse (Peromyscus poliono-et al., 2000 A detailed linkage map of medaka, Oryzias latipes:

comparative genomics and genome evolution. Genetics 154: tus). Stud. Genet. VI. Univ. Texas Publ. 7103: 49–90.Shaklee, J. B., and C. P. Keenan, 1986 A Practical Laboratory Guide1773–1784.

Neff, M. W., K. W. Broman, C. S. Mellersh, K. Ray, G. M. Acland to the Techniques and Methodology of Electrophoresis and Its Applicationto Fish Fillet Identification. CSIRO Marine Laboratories Report 177,et al., 1999 A second-generation genetic linkage map of the

domestic dog, Canis familiaris. Genetics 151: 803–820. Commonwealth Scientific and Industrial Research Organization,Melbourne.Nickerson, D. A., S. L. Taylor, K. M. Weiss, A. G. Clark, R. G.

Hutchinson et al., 1998 DNA sequence diversity in a 9.7-kb Sharp, P. J., and D. L. Hayman, 1988 An examination of the roleof chiasma frequency in the genetic system of marsupials. Hered-region of the human lipoprotein lipase gene. Nat. Genet. 19:

233–240. ity 60 (Pt. 1): 77–85.Shaw, C. R., and R. Prasad, 1970 Starch gel electrophoresis ofOkuizumi, H., T. Ohsumi, N. Sakaki, H. Imoto, Y. Mizuno et al.,

1997 Linkage map of Syrian hamster with restriction landmark enzymes—a compilation of recipes. Biochem. Genet. 4: 297–320.Shimoda, N., E. W. Knapik, J. Ziniti, C. Sim, E. Yamada et al., 1999genomic scanning. Mamm. Genome 8: 121–128.

O’Neill, M. J., R. S. Ingram, P. B. Vrana and S. M. Tilghman, 2000 Zebrafish genetic map with 2000 microsatellite markers. Geno-mics 58: 219–232.Allelic expression of IGF2 in marsupials and birds. Dev. Genes

Evol. 210: 18–20. Singer, A., H. Perlman, Y. Yan, C. Walker, G. Corley-Smith et al.,2002 Sex-specific recombination rates in zebrafish (Danio rerio).Ott, J., 1999 Analysis of Human Genetic Linkage. Johns Hopkins Uni-

versity Press, Baltimore. Genetics 160: 649–657.Slate, J., T. C. Van Stijn, R. M. Anderson, K. M. McEwan, N. J.Otto, S. P., and N. H. Barton, 1997 The evolution of recombina-

tion: removing the limits to natural selection. Genetics 147: 879– Maqbool et al., 2002 A deer (subfamily Cervinae) genetic link-age map and the evolution of ruminant genomes. Genetics 160:906.

Otto, S. P., and T. Lenormand, 2002 Resolving the paradox of sex 1587–1597.Sobel, E., H. Sengul and D. E. Weeks, 2001 Multipoint estimationand recombination. Nat. Rev. Genet. 3: 252–261.

Pathak, S., M. Ronne, N. M. Brown, C. L. Furlong and J. L. Vande- of identity-by-descent probabilities at arbitrary positions amongmarker loci on general pedigrees. Hum. Hered. 52: 121–131.Berg, 1993 A high-resolution banding pattern idiogram of

Monodelphis domestica chromosomes (Marsupialia, Mammalia). Sokoloff, A., 1964 Sex and crossing over in Tribolium castaneum.Genetics 50: 491–496.Cytogenet. Cell Genet. 63: 181–184.


Sokolova, O. V., R. A. van Oorschot and J. L. VandeBerg, 1997 of the South American marsupial Monodelphis domestica. Cyto-genet. Cell Genet. 60: 64–67.Aldolase C polymorphism in the laboratory opossum, Monodelphis

domestica. Anim. Genet. 28: 358–359. van Oorschot, R. A., V. Birmingham, P. A. Porter, C. M. Kammererand J. L. VandeBerg, 1993 Linkage between complement com-Springer, M. S., 1997 Molecular clocks and the timing of the placen-

tal and marsupial radiations in relation to the Cretaceous-Tertiary ponents 6 and 7 and glutamic pyruvate transaminase in the marsu-pial Monodelphis domestica. Biochem. Genet. 31: 215–222.boundary. J. Mamm. Evol. 4: 285–302.

Voss, S. R., J. J. Smith, D. M. Gardiner and D. M. Parichy, 2001Springer, M. S., M. Westerman, J. R. Kavanagh, A. Burk, M. O.Conserved vertebrate chromosome segments in the large sala-Woodburne et al., 1998 The origin of the Australasian marsu-mander genome. Genetics 158: 735–746.pial fauna and the phylogenetic affinities of the enigmatic monito

Waldbieser, G. C., B. G. Bosworth, D. J. Nonneman and W. R.del monte and marsupial mole. Proc. R. Soc. Lond. Ser. B Biol.Wolters, 2001 A microsatellite-based genetic linkage map forSci. 265: 2381–2386.channel catfish, Ictalurus punctatus. Genetics 158: 727–734.Sumida, M., and M. Nishioka, 2000 Sex-linked genes and linkage

Wallace, H., B. M. Wallace and G. M. Badawy, 1997 Lampbrushmaps in amphibians. Comp. Biochem. Physiol. B Biochem. Mol.chromosomes and chiasmata of sex-reversed crested newts. Chro-Biol. 126: 257–270.mosoma 106: 526–533.VandeBerg, J. L., 1990 The gray, short-tailed opossum (Monodelphis

Waters, P. D., P. J. Kirby and J. A. Graves, 2001 Assignment ofdomestica) as a model didelphid for genetic research. Aust. J. Zool.the SMARCF1 gene to tammar wallaby chromosome 5q by fluo-37: 235–247.rescence in situ hybridisation. Cytogenet. Cell Genet. 93: 315–VandeBerg, J. L., 1999 The laboratory opossum (Monodelphis domes-316.tica), pp. 193–209 in UFAW Handbook on the Management of Labora-

Weeks, D. E., E. Sobel, J. R. O’Connell and K. Lange, 1995 Com-tory Animals: Terrestrial Vertebrates, Vol. 1, Ed. 7, edited by T. Pooleputer programs for multilocus haplotyping of general pedigrees.and P. English. Blackwell Science, Oxford.Am. J. Hum. Genet. 56: 1506–1507.VandeBerg, J. L., and E. S. Robinson, 1997 The laboratory opossum

Williams, J. G., A. R. Kubelik, K. J. Livak, J. A. Rafalski and S. V.(Monodelphis domestica) in laboratory research. ILAR J. 38: 4–12.Tingey, 1990 DNA polymorphisms amplified by arbitrary prim-VandeBerg, J. L., E. S. Robinson, P. B. Samollow and P. G. John-ers are useful as genetic markers. Nucleic Acids Res. 18: 6531–ston, 1987 X-linked gene expression and X-chromosome inac-6535.tivation: marsupials, mouse, and man compared. Isozymes Curr.

Williams, R. A. D., 1964 Location of glyceraldehyde-3-phosphateTop. Biol. Med. Res. 15: 225–253. dehydrogenase in starch-gels. Nature 203: 1070–1071.van Oorschot, R., V. Birmingham and J. L. VandeBerg, 1991 Hin- Womack, J. E., 1997 Mapping animal genomes. Adv. Vet. Med. 40:dIII RFLP for NRAS in Monodelphis domestica. Nucleic Acids Res. 157–189.19: 6976. Woodburne, M. O., T. H. Rich and M. S. Springer, 2003 The

van Oorschot, R. A., and J. L. VandeBerg, 1989 Genetic variation evolution of tribospheny and the antiquity of mammalian clades.in adenylate kinase 1, glyceraldehyde-3-phosphate dehydroge- Mol. Phylogenet. Evol. 28: 360–385.nase, and glutamic-pyruvate transaminase in the marsupial Mono- Young, W. P., P. A. Wheeler, V. H. Coryell, P. Keim and G. H.delphis domestica. Biochem. Genet. 27: 761–765. Thorgaard, 1998 A detailed linkage map of rainbow trout

van Oorschot, R. A., S. Williams-Blangero and J. L. VandeBerg, produced using doubled haploids. Genetics 148: 839–850.1992a Genetic diversity of laboratory gray short-tailed opossums Zenger, K. R., L. M. McKenzie and D. W. Cooper, 2002 The first(Monodelphis domestica): effect of newly introduced wild-caught comprehensive genetic linkage map of a marsupial: the tammaranimals. Lab. Anim. Sci. 42: 255–260. wallaby (Macropus eugenii). Genetics 162: 321–330.

van Oorschot, R. A., P. A. Porter, C. M. Kammerer and J. L.VandeBerg, 1992b Severely reduced recombination in females Communicating editor: S. P. Otto

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