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, 2003 Accepted for publication September 22, 2003

ABSTRACT The 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 utility of 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 information regarding the evolution of mammalian and other vertebrate genomes. The current map is composed of 83 loci distributed among eight autosomal linkage groups and the X chromosome. The autosomal linkage groups appear to encompass a very large portion of the genome, yet span a sex-average distance of only 633.0 cM, making this the most compact linkage map known among vertebrates. Most surprising, the male map is much larger than the female map (884.6 cM vs. 443.1 cM), a pattern contrary to that in eutherian mammals and other vertebrates. The finding of genome-wide reduction in female recombination in 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 discuss possible explanations for reduced female recombination in marsupials as a consequence of the metatherian characteristic of determinate paternal X chromosome inactivation.

ETATHERIAN (“marsupial”) mammals have a biomedical models, all of which are eutherian species. M long history in research as models for organismal However, marsupials and eutherians are more closely and cellular physiology, endocrinology, and develop- related to one another than to any other vertebrate mental patterns and processes and more recently have model species (e.g., birds, amphibians, fishes). Thus, taken their place alongside eutherian (“placental”) mam- marsupials represent a unique midpoint between euthe- mals as serious subjects for genetically oriented biomedi- rian and nonmammalian vertebrate models. cal and evolutionary research (reviewed by Samollow As a legacy of their common ancestry, marsupials and and Graves 1998; Graves and Westerman 2002). This eutherians share genetic mechanisms and molecular increased interest in the genetic characteristics of mar- processes that represent fundamental (ancestral) mam- supials reflects the growing utility and importance of malian characteristics. Nevertheless, since their diver- million years 180–150ف comparative models in biomedical research (Womack gence from a common ancestor 1997; Graves 1998; Pollock et al. 2000; Postlethwait ago (MYA; Hope et al. 1990; Kumar and Hedges 1998; et al. 2000) and the broadening recognition of the value Woodburne et al. 2003) eutherian and marsupial mam- of the unique phylogenetic position of marsupials in mals have evolved many distinctive morphologic, phys- the mammalian lineage. Marsupials are phylogeneti- iologic, and genetic variations on these elemental cally distinct from the more commonly used mammalian mammalian designs. These phylogenetically restricted differences can be used as comparative tools for examin- ing the underlying molecular and genetic processes that are 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 contribute EMBL/GenBank Data Libraries under accession nos. AY369173– AY369206. to differences in gene regulation, expression, and func- 1Corresponding author: Department of Genetics, Southwest Founda- tion. tion for Biomedical Research, 7620 NW Loop 410, San Antonio, TX Monodelphis domestica (also known as the laboratory 78245-0549. E-mail: [email protected] opossum) is a small South American marsupial that has 2Present address: Department of Microbiology, Immunology and Mo- lecular Genetics, University of California, Los Angeles, CA 90025. become widely used as a model organism for compara- 3Present address: Department of Biological Sciences, Macquarie Uni- tive research on a broad range of topics that are relevant 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 cells Robinson 1997). Because of its small size (100–150 g), revealed reduced chiasma frequency in females as com- -months), pared to males and restriction of female chiasmata pri 5ف rapid growth and maturation (maturity at favorable reproductive characteristics (mean litter size marily to terminal (subtelomeric) regions, suggesting a of approximately eight; up to three litters per year), causal relationship between chiasma characteristics and and simple husbandry (rodent cages and commercial recombination rates (Bennett et al. 1986). Soon there- feed; VandeBerg 1999), M. domestica has become the after, Hayman et al. (1988) reported similar cytological most extensively used laboratory-bred marsupial for ba- phenomena in M. domestica (Didelphidae; an American sic biologic and biomedical research worldwide (Samol- family composed of the opossums and woolly opos- low and Graves 1998; and recent database searches). sums), and subsequent inheritance studies revealed Examples of recent research involving M. domestica in- drastically reduced recombination among six loci in clude: gene and genome evolution, genomic imprinting females of this species as well (van Oorschot et al. (autosomal and X-linked genes), photobiology and 1992b, 1993; Perelygin et al. 1996). The combined DNA repair, molecular characterization of ultraviolet weight of these limited findings prompted guarded radiation-induced skin and eye neoplasias, structure and speculation that reduced female recombination might evolution of mammalian antigen receptors, lipoprotein be a general phenomenon in marsupials (van Oor- metabolism and response to dietary fat and cholesterol, schot et al. 1992b, 1993; Hope 1993). neurophysiology, normal and regenerative neurodevel- Most recently, Zenger et al. (2002) published a com- opment (central and peripheral nervous systems), cra- prehensive linkage map of the tammar wallaby, Mac- niofacial ontogeny and evolution, skeletal and skeleto- ropus eugenii (Macropodidae; an Australian family com- muscular development, reproductive endocrinology, posed of the kangaroos, wallabies, and related forms), and more. the first of its kind for any marsupial species. Composed of the M. eugenii %71ف To further expand the potential of this species as a of 60 loci and believed to cover research model, we have undertaken construction of a genome, the map shows marked heterogeneity of re- linkage map of the M. domestica genome, consisting of combination rates including regions of severely reduced both anonymous markers and functional gene loci. The female recombination interspersed with intervals in primary objective is to establish a resource that will en- which female and male recombination rates are equiva- the size of the %78ف able the localization of quantitative trait loci (QTL) that lent. Overall, the female map is contribute to normal and abnormal physiologic and male map, and in none of the interlocus intervals does developmental 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 this and linkage relationships among distantly related mam- article is composed of 83 loci distributed among eight malian 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 is rates in marsupials. Early linkage studies involving small considerably more male biased in M. domestica than in numbers of genes in two distantly related marsupial the tammar wallaby and is by far the most male-skewed species yielded the surprising result that meiotic recom- pattern known for any vertebrate species. bination in female marsupials was much lower than that in males. This pattern is contrary to the general mammalian one in which recombination rates are simi- MATERIALS AND METHODS lar 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 for bald et al. 1995; Mikawa et al. 1999), cattle (Barendse Biomedical Research (SFBR), San Antonio, Texas. The et al. 1997; Kappes et al. 1997), and mice (Dunn and “GMBX” mapping panel was established by crossing members of laboratory stocks that were descended from the most geo- ف .Bennett 1964; Davisson et al. 1989; Copeland et al 1993). graphically separated populations available ( 1200 km) at 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, Minas sicaudata (Dasyuridae; an Australian marsupial family Gerais, Brazil). Two wild-captured Pop3 males were crossed of small, mouse-like carnivores), four pairwise gene to six pedigreed Pop1 females to produce the F1 generation. combinations that exhibited no recombination in fe- Nineteen F1 offspring of both sexes were crossed to 22 pedi- males exhibited modest to high recombination frequen- greed Pop1 mates to produce the backcross generation com- 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 and tions that had minimal recombination in females (rf, blood samples were collected from all of the animals produced 0.06 and 0.12) were unlinked in males (Bennett et by this crossing scheme with the exception of the six Pop1 Linkage Map of the Laboratory Opossum 309 grandmothers. Thus, the 356-member GMBX panel is com- bridization probes for Southern blots were derived from mar- posed of 30 backcross families including 313 backcross prog- supial sources: PHL and the anonymous U15557 probes from eny, all of their parents (19 F1 and 22 Pop1 mates), and the M. domestica and the HBE probe from the fat-tailed dunnart, two Pop3 grandfathers. S. crassicaudata. These yielded unique, strong hybridization Sample collection and preparation: Whole blood and vari- signals at high stringency, indicative of homology between the ous solid tissues (including liver, brain, kidney, heart, skeletal probe and the target sequence. NRASLA and NRASLB were muscle, and ear pinna) were collected from lethally anesthe- detected using a human NRAS genomic probe (Murray et al. tized animals under a protocol approved by the SFBR Institu- 1983). Although originally reported as homologous to human tional Animal Care and Use Committee. Dissected tissues were NRAS (van Oorschot et al. 1991; Perelygin et al. 1994), immediately frozen on dry ice and stored in small, heavy-duty, results from the present study indicate that NRASLA and zipper-type plastic bags at Ϫ80Њ. Blood was allowed to clot for NRASLB are unlinked to one another, thus obviating orthology 1 hr at ambient temperature and then separated into clot and inferences. Preliminary DNA hybridization studies using M. serum fractions. Both fractions were stored at Ϫ80Њ in 50-␮l domestica-derived HRAS and KRAS genomic probes (data not aliquots, which were sealed in plastic tubing “pillows” following shown) have failed to clarify the relationships between these the method of Cheng and co-workers (Cheng et al. 1986; polymorphic NRAS fragments and other RAS family genes. Cheng and VandeBerg 1987). Crude extracts of solid tissues We propose that both polymorphic fragments be classified and blood clots were prepared by standard methods (e.g., as NRAS-LIKE loci until their homologies are ascertained by Selander et al. 1971; Samollow et al. 1987) for application sequencing studies. to electrophoretic or isoelectric focusing gels. Serum samples Microsatellite and randomly amplified polymorphic DNA for protease inhibitor (PI) and transferrin (TF) analysis were (RAPD) polymorphisms were developed for this study using used directly without additional preparation. For AT3 and C6 PCR primers designed from cloned M. domestica DNA se- analyses, 20-␮l serum samples were mixed with 14 ␮lof30mm quences and commercial RAPD primers, respectively (de- sodium acetate-95 mm (NH ) SO buffer (pH 5.0) containing scribed below). 4 2 4 All type I protein and DNA polymorphisms exhibited strict 0.008 units of neuraminidase and incubated overnight at 4Њ codominant inheritance, and no unusual segregation patterns prior to use. High-molecular-weight genomic DNA was pre- (i.e., segregation distortions) were detected for these or any pared using routine methods adapted from a variety of of the loci examined in this study. Microsatellite variation was sources. 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 55Њ overnight with gentle rota- als exhibiting dominant phenotypes were included in the ge- tion. The resulting solution was RNase treated (20 mg/ml) for Њ notype data set only if their genotypes could be unambiguously 1hrat37, and the DNA was extracted by phenol/chloroform inferred from pedigree data. Specifically, individuals that phase separation. The DNA was purified by ethanol precipita- could be either homozygous for an amplifiable allele or het- tion and resuspended in 1ϫ Tris-EDTA (TE) and stored at Њ Ϫ Њ erozygous for that allele and a null allele were excluded from 4 (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 amplifiable tance: Twelve previously published genetic polymorphisms allele. and 72 newly developed ones were used as genetic markers to RAPD phenotypes encompassed both dominant/recessive genotype 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 RAPD 63 anonymous (type II) genetic loci (Tables 1 and 2, respec- loci, with the presence (P) of an amplimer on a gel dominant tively). 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 from vertebrate genes were inferred by a variety of approaches. For pedigree data. For some loci, this criterion excluded entire polymorphic proteins, criteria included: (1) uniqueness of families (one parent known or suspected of being homozygous loci in mammals [i.e., only a single adenylate kinase (AK1) is P/P) from consideration and thereby reduced the overall known in mammalian erythrocytes and only one gene encod- informativeness of those loci for mapping purposes. One ing 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 essentially with 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 dominant Mahaney, 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 methods S. M. Mahaney, unpublished data), and LDHB (predominant modified from a variety of published sources. Tissues used heart isoenzyme); and (4) subcellular localizations (P. B. and electrophoretic method/buffer combinations are listed Samollow 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). high stringency conditions. With the exception of NRAS, hy- Cellulose acetate gel electrophoresis (CAGE) was conducted 310 P. B. Samollow et al. nt sizes are lymorphisms follows g mobility classes on letion polymorphism in Informative meioses 90 132 222 104164 45234191 45 149 189 210 209 423 401 115 196 311 c c c c c c A, BA, BA, B 154A, 50 BA, B 217 177 29 102 371 67 5 79 279 146 14 213 19 A, B A, B A, B A, B, C 190 233 423 A, B, C A, B, D A, B, D, F A (7.0 kb), B (4.5 kb)A (7.2 115 kb), B (6.4 kb) 198 115 313 198 313 Alleles in GMBX panel Females Males Combined A (11.5 kb), B (8.5 kb) 153 187 340 j l i a III) A (1600 bp), B (1050/550 bp) 260 201 461 I) A (450 bp), B (300/150 bp) 114 197 311 TABLE 1 I) A (2570 bp), B (2000/570 bp) 107 (X-linked) 107 dIII) dIII) dIII) I) A (11.6 kb), B (8.1 kb) 56 101 157 I) A (13.5 kb), B (9.4 kb) 61 181 242 Pst Alu Hae Pst Hin Taq Hin Hin b d e f g b d d h h k h stocks. , unpublished data. Functional gene and functional gene-related polymorphisms in the GMBX mapping panel M. domestica (1989). Samollow (1993). (1991). VandeBerg . (1992b). . (1992a). andP.B. (1994). and et al. et al et al (1997). et al. -like B SB/RFLP ( -like A SB/RFLP ( . (1996). ras ras -Globin, embryonic SB/RFLP ( Acid phosphatase 2 Protein Transferrin Protein Glyceraldehyde-3-phosphate dehydrogenase Protein Sperm protein 17 PCR/RFLP ( N Aldolase CAmelogeninAntithrombin 3␤ Lactate dehydrogenase BN Protein Protein PCR/INDEL Protein A (450 bp), B (430 bp) 47 123 170 Protease inhibitor Protein Malic enzyme 1 Protein Photolyase SB/RFLP ( Glutamate-pyruvate transaminase Protein Unidentified sequence U15557 SB/RFLP ( Aconitase CAdenylate kinase 1Complement component 6Diaphorase 1Glucose-6-phosphate dehydrogenase Protein Protein PCR/RFLP Protein ( Tumor protein 53 Protein A, B PCR/RFLP ( 115 198 313 et al. et al et al. (1992). Perelygin et al. Unless otherwise indicated (by footnoted reference), polymorphisms were developed by this study. Allele nomenclature for previously described po van Oorschot A. A. van Oorschot van Oorschot van Oorschot Additional alleles known in other Sokolova Bell van Oorschot Perelygin Perelygin a b c d e f g h i j k l LocusACP2 Protein or gene name Polymorphism TF the original source.electrophoretic For or polymorphisms isoelectric focusing developedPCR gels, by fragment, with PCR/RFLP A this indicates indicatingapproximate. study, restriction most Details nomenclature fragment anodally given migrating; length is in for polymorphism text. as DNA in follows: polymorphisms, PCR PCR/INDEL for fragment, indicates and protein insertion SB/RFLP de polymorphisms, indicates letters RFLP on indicate Southern decreasin blot. DNA fragme GAPD SP17 NRASLB ALDOC AMG AT3 HBE LDHB NRASLA PI ME1 PHL GPT U15557 ACONC AK1 C6 DIA1 G6PD TP53 Linkage Map of the Laboratory Opossum 311 ) continued ( Informative meioses e Size (bp) Females Males Combined 176–178 134 234 368 135–272 250 297227–265 547 239 266 505 181–219 249 200195–229 449 104 160 264 ف ف ف d f f f f 5 4 2 127–129 219 150 369 6 172–182 19268 196 171–183 175–201 388 244 206 313 31366 557 519 218–240 119–139 254 291 283 (X-linked) 291 537 2 58 127–14187 149–171 263 131–167 196–220 257 218 2885 175 2814 481 272 176–200 266 538 262–272 560 2207 441 152 217 111–1455 50 437 2219 202 121–133 282 227–255 211 503 234 305 151 516 385 10 11 Alleles range Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј TABLE 2 Ј Ј Ј Ј Ј Ј Primers detected Anonymous locus polymorphisms in the GMBX mapping panel -GCAGTTCCTGCTTAATTTC-3 -GATTGAATGGTTATGGATGTA-3 -ACTATGTGCTAGGCTCTCAG-3 -GAAGCTTCAATGAACAAT-3 -CACAAATACTTTCAGGAGGA-3 -TCCCATATAGTCTTGAACCT-3 -TCTATTAGAATGCCCAGGA-3 -GATGTTTGATAAGTCCTATGTT-3 -TTCCTGGAAGAACTTCTTGA-3 -GAAGGTAGTACCAAATGGAG-3 -GCACTTAGCACAGTGTCTGGC-3 -CTTGAGTCTATTACAAAGCCGTG-3 -TTACTGCCTGCAGCCTGTAG-3 -TCCTCTGAAAGCGTAGCATA-3 -TGCTACTCACTTCTATTGAGTC-3 -TCCTCCTGAGATTGCCATTG-3 -CCAGCCTATCTTAGATGTAG-3 -GATCATAAGATATGGAAATA-3 -CTTCTGCCTTGGAACCAGAG-3 -ACCAGATCTGAAGTCTTAGAAGG-3 -ACTCTTTGTCTCTGAGTCT-3 -GAAGGAAATTAAGAAACAAGTG-3 -GTCCTCCACCCAATAACCTT-3 -ATACGAATTGGAAGACTGCA-3 -ATTCTTCACACAGGAGGACA-3 -CATTGGCAACCTGTCCTCCTG-3 -TGCAGTTTGAGTCTTTCTGG-3 -ATCATTACTCACAATGAGCT-3 -AGAGAGCTGATTCTGAAGTA-3 -ATCTAGGAAGCAAACCCAGGTCT-3 -GTAGGTGGTGGATTCCTGCT-3 -CAGAAATTTGCATACAATCA-3 -TATCTTTGTAGACAGTAGTGAC-3 -TGTCGGCTATGGGAAGGATG-3 -ATTCAGTCCAAGAACCTTG-3 -TACTTTGGACAAGTCACCTA-3 -GATCTCATGGACTTAACTG-3 -CATGTGTCCCCACAGTCTTT-3 -TGTCCAGGGTCAAACATTGTA-3 -GGGTAATCCACAAGTGTCCAA-3 Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј F: 5 F: 5 F: 5 F: 5 F: 5 F: 5 F: 5 F: 5 F: 5 F: 5 F: 5 F: 5 F: 5 F: 5 F: 5 F: 5 F: 5 F: 5 F: 5 F: 5 19 2 10 7 (TA) (GA) 19 (CA) (CA) 2 25 2 2 17 8 c (GA) (CA) (TG) (CG) (TG) (CA) Type 12 16 11 25 27 18 19 19 12 7 21 4 13 5 25 12 32 4 3 M-(TA) M-(CA) M-(TA) M-(CT) M-(CA) M-(CA) M-(GT) M-(GT) M-(CA) M-(GA) M-(CT) M-(GT) M-(CA) M-(TG) M-(GT) M-(GAGACA) M-(CA) M-(GA) M-(GT) M-(GT) b , a Mdo1L002 (AY369179) R: 5 Mdo8L014 (AY369177) R: 5 Mdo1L003 Locus Mdo1L001 Mdo1L019 Mdo1L004 Mdo4L009 MdoXL017 (AY369180)(AY369182)(AY369183) R: 5 Mdo5L010 Mdo5L012 R: 5 R: 5 (AY369188)(AY369189)Mdo1L020 R: 5 R: 5 (AY369173)(AY369181)(AY369174)Mdo2L006 (AY369175) R: 5 (AY369176) R: 5 Mdo5L011 (AY369184) R: 5 R: 5 (AY369187) R: 5 R: 5 (AY369190)(AY369192) R: 5 R: 5 R: 5 Mdo2L005 Mdo3L007 Mdo8L016 Mdo7L013 (AY369178)Mdo3L008 (AY369185) R: 5 R: 5 Mdo8L015 Mdo1L021 (AY369186)(AY369191) R: 5 R: 5 312 P. B. Samollow et al. ) continued ( Informative meioses 65 1115250 176 60 36 112 86 i i i e 1500 1500 2000 Size (bp) Females Males Combined 135–153 272120–158 330158–162 247 602 213 285600–800 267 532 88 480 146 234 Ͼ Ͼ Ͼ d f f f h 9 9 4 76 161–181 218–234 269 285 323 328 592 4 613 92–1088 199 108–154 296 261 495 281 542 75 173–2155 113–149 2487 89–1034 261 274 112–134 245 143–149 283 522 2856 246 188 544 268 491 105–132 226 553 287 414 332 619 4 11 254–286 292 331 623 Alleles range Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј TABLE 2 Ј (Continued) g ) ) 2 (P, A) 900 44 103 147 ) 2 (P, A) 830 25 60 85 ) 2 (P, A) 720 52 83 135 ) ) 2 (P, A) ) 2 (P, A) ) 2 (P, A) ) 2 (P, A) 820 39 166 205 ) 2 (P, A) 610 56 7 63 ) ) 2 (P, A) 380 33 77 110 Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Primers detected -TTCCGAACCC-3 -CAATCGCCGT-3 -GTGATCGCAG-3 -TTCCGAACCC-3 -AGCCAGCGAA-3 -GTGATCGCAG-3 -TCTGTGCTGG-3 -GTAGACCCGT-3 -TTTGCCCGGA-3 -AGGGAACGAG-3 -TCCGCTCTGG-3 -GTCCACACGG-3 Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј -AGCACATGCAGTCCTTTGAA-3 -ACTGGCATGGAATGGGACTA-3 -TGAATGGCTGCTTTACTGAA-3 -GTACTGGGCAACAGTATATTGAG-3 -TCATCAAATGAGGCTCAAAG-3 -TGTGTTCAATGTGGGTCTCAA-3 -TTAAATCTCTGGCAACCACA-3 -TCCTCTACAATAGGAACCTCTTATC-3 -TTGCCTCCTGTCACTTTGGA-3 -CCGAAGGGATCTCAAGTTCA-3 -GGAGGTGATTGGTTCACAATG-3 -CACATTGCCCATTTCCTGAT-3 -TGTGCAGGACTACAGGGCAA-3 -TGCATGACCAAACACCTAA-3 -TTGGCTCTCTGCTTTCCAAT-3 -CCTCTGTGCTTTTCGGCTAT-3 -TTTGTAGGGAGGTAGAAGATAGG-3 -CCTTCAGGGTATAGTATGTTC-3 -TTATGGGATCTGGAATTTGAGG-3 -GAAGGTAATACATCACATGACCACA-3 -TTTCTGCATTGTCCATGTCC-3 -TGGAATACCACCTATTCTCTGC-3 -TGACTCAGACTTAACCCCATCC-3 -ACGGTATGTTCTCTGACCGTCT-3 -CATTCTTCATTTCATGACCAC-3 -CCTTGGGCATGATCAAGTTT-3 -TCCCATTTCTCTTCCACTCTT-3 -GAAAATCTGCCCTTCCATGA-3 Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј F: 5 F: 5 F: 5 F: 5 F: 5 F: 5 F: 5 F: 5 F: 5 F: 5 F: 5 F: 5 F: 5 F: 5 OPB-17 (5 OPA-16 (5 20 6 5 24 14 c (GA) G(GT) (GC) GG(GT) (GT) Type 12 14 29 12 17 26 7 12 15 27 2 13 20 17 M-(GT) M-(GA) M-(GT) M-(CA) M-(GT) M-(GT) R-SV OPA-15 (5 M-(GT) M-(CA) M-(CT) M-(GT) M-(CA) R-PAR-PA OPA-11 (5 OPB-11 (5 M-(CA) M-(CA) M-(CA) R-PA OPB-16 (5 R-PA OPA-10 (5 R-PA OPA-15 (5 R-PA OPA-10 (5 R-PA OPB-14 (5 R-PA OPA-14 (5 R-PA OPB-08 (5 b , a Locus Mdo2L022 Mdo3L024 Mdo3L026 Mdo7L033 (AY369193)Mdo2L023 (AY369194)(AY369196) R: 5 R: 5 Mdo5L030 R: 5 (AY369203)(AY369206)Mdo1LR01 R: 5 R: 5 (AY369195)Mdo3L025 (AY369197)Mdo4L027 (AY369198)(AY369200)(AY369201) R: 5 Mdo6L031 R: 5 R: 5 (AY369205)Mdo8L035 R: 5 R: 5 R: 5 Mdo4L028 Mdo1LR05 Mdo3LR10 Mdo4L029 Mdo8L034 (AY369199)(AY369202)Mdo6L032 (AY369204) R: 5 Mdo1LR02 R: 5 R: 5 Mdo2LR06 Mdo1LR03 Mdo3LR07 Mdo1LR04 Mdo3LR08 Mdo3LR09 Linkage Map of the Laboratory Opossum 313 ed. were estimated resence/absence ف Informative meioses 46 7064 116 88 152 114 174109 288 86 195 , unique numeric locus identifier. Example: j k 003 i i e 1500 2000 Size (bp) Females Males Combined Ͼ Ͻ I fragments I fragments Rsa Mbo d cloned microsatellite standard. Sizes preceded by , linkage group 1 identifier; Alleles range 1L ; larger than or smaller than the nominal size indicated. M. domestica slightly TABLE 2 (Continued) ) 2 (P, A) 1100 31 24 55 ) )) 2 (P, A) 710 2 (P, A) 74 920 81 91 155 80 171 ) ) ) ) 2 (P, A) 1350 136 100 236 ) 2 (P, A) 1450 113 198 311 Monodelphis domestica ) ) 2 (P, A) 920 54 51 105 )) 2 (P, A) ) 2 (P, A) 620 115 197 312 ) ) 2 (P, A) ) 2 (P, A) 405 110 165 275 ) 2 (P, A) 1000 46 58 104 ) 2 (P, A) 1700 115 198 313 )2 ) 2 (P, A) 1000 20 9 29 ) 2 (P, A) 1000 74 105 179 ) 2 (P, A) 550 127 158 285 )2 ) 2 (P, A) 310 84 (X-linked) 84 Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј , Mdo : exhibited additional alleles in animals derived from other populations. Primers detected (assumed 80-bp fragment; not detected). Mdo1L003 ϩ Mdo1L019 -AGGTGACCGT-3 -GGTCCCTGAC-3 -TCGGCGATAG-3 -CAATCGCCGT-3 -TTCCGAACCC-3 -AGCCAGCGAA-3 -AGGGGTCTTG-3 -CAGGCCCTTC-3 -TGCCGAGCTG-3 -GGGTAACGCC-3 -GTTGCGATCC-3 -AGGTGACCGT-3 -GGTCCCTGAC-3 -GTAGACCCGT-3 -GTCCACACGG-3 -GTCCACACGG-3 -TCCGCTCTGG-3 -TGCTCTGCCC-3 -GTCCACACGG-3 -TGCGCCCTTC-3 -GTAGACCCGT-3 -TTTGCCCGGA-3 -AGGGAACGAG-3 -TGATCCCTGG-3 -GTCCACACGG-3 Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј and 150-bp fragments. ϩ OPA-18 (5 OPB-08 (5 OPA-12 (5 OPA-16 (5 OPB-17 (5 OPA-20 (5 OPA-18 (5 Mdo1L001 indicate band positions that are, respectively, Ͻ or , sizes were determined by comparison to a corresponding Ͼ ف c Type R-PA OPB-11 (5 R-PA OPA-06 (5 R-PA OPA-11 (5 R-PA OPB-08 (5 R-PAR-PA OPB-14 (5 OPA-15 (5 R-PAR-PA OPB-06 (5 OPB-08 (5 R-PA OPA-05 (5 R-RFLP OPB-05 (5 R-PA OPB-11 (5 R-PAR-PA OPA-01 (5 R-PA OPA-02 (5 OPB-16 (5 R-PA OPA-09 (5 R-PA OPA-06 (5 R-RFLP OPB-02 (5 R-PA OPB-08 (5 had four detectable alleles: A (800 bp), B (780 bp), and C (600 bp) were codominant; the fourth allele, b, had identical mobility to allele B (780 bp), but : linkage group 7, RAPD locus 27. Linkage group identifiers are temporary; they will be replaced by chromosome identifiers when loci are physically mapp b , a Temporary anonymous locus nomenclature. Example: In GMBX panel only; all loci except Mdo1LR01 Primer oligonucleotide codes are those of Operon Technologies (Alameda, CA) as supplied with their RAPD 10-Mer kitsTwo A alleles; and A, B. 440-bp fragment; B, 290- GenBank accession number is in parentheses. M- indicates microsatellite marker, followed by its major repeat motif. R- indicates RAPD marker. R-SV, RAPD-fragment size variation; R-PA, RAPD-p Except where indicated by Includes null (nonamplifying) allele(s). Base-pair sizes preceded by Two alleles; A, 515-bp fragment; B, 435-bp fragment a b c d e f g h i j k Unique numeric locus identifiers are permanent. Mdo7LR27 variation; R-RFLP, RAPD-restriction fragment length variation. See text for details. from comparison with commercial DNA ladder standards. had very low fluorescence, rendering it recessive to allele B. Locus Mdo3LR11 Mdo4LR12 Mdo4LR13 Mdo7LR27 Mdo6LR21 MdXLR26 Mdo4LR15 Mdo4LR16 Mdo7LR22 Mdo4LR14 Mdo8LR28 Mdo5LR17 Mdo6LR18 Mdo7LR24 Mdo7LR23 Mdo6LR19 Mdo8LR25 Mdo6LR20 314 P. B. Samollow et al. et et al. (1976) c Tretiak Gahne phosphate, . (1992b) (1970) and m . (1993); 7:7:4 d unpublished . (1986) . (1986) . (1986) . (1986) Selander van Oorschot et al . (1995) . (1995) . (1971) . (1971) et al et al et al et al et al Hopkinson et al et al Prasad et al et al (1964) Clayton and NaOH. e gel isoelectric focusing; and Staining reference m Richardson Richardson Shaw Richardson van Oorschot Selander Selander Harris Aivaliotis Aivaliotis Williams Richardson citric acid, titrated to pH 6.0 with maleic acid, pH 7.8; system C of van Oorschot m m citrate, pH 8.0; system 5 of b NaOH. IEF-3: modified from m Tris-0.020 m m system (1986). TB: modified gradient gel system of Tris-0.157 m phosphoric acid; catholyte, 1.0 et al. m a NaOH. IEF-2: modified from (1972); electrode buffer, 0.04 m PAGIEFAGIEF IEF-1 IEF-2 Richardson Tretiak glutamic acid; catholyte, 0.1 m (1970). TC-2: 0.687 and d d (1992); equal parts of pH 5–6 (Pharmacia, Peapack, NJ) and pH 3–10 (SERVA /www.genetics.org/supplemental. et al. Prasad Clayton TABLE 3 Bell Tris-HCl stacking gel, pH 6.8. TM: 0.05 and m glutamic acid; catholyte, 0.1 m Shaw E.C. Tissue Buffer (1983) and phosphate, pH 7.0; system B of m Bell and Protein electrophoresis and isoelectric focusing methods supplemental information website: http:/ Pollitt citrate, pH 7.0; system I of m (1970). PHOS-2: 0.2 Genetics Prasad Tris-0.043 citric acid, titrated to pH 6.0 with N-(3 aminopropyl)-morpholine; gel buffer, 1:9 dilution of electrode buffer. PHOS-1: 0.2 m m and Tris-sulfate running gel, pH 9.0, and single 0.125 m Shaw (1986). IEF-1: modified from Antithrombin 3 — Serum Aldolase C 4.1.2.13 Brain SGE CAAPM 1:9 Adenylate kinase 1 2.7.4.3 Rbc SGE TC-2 Acid phosphatase 2Aconitase C 3.1.3.2 Liver 4.2.1.3 Liver SGE SGE TC-1 CAAPM 1:19 Protease inhibitorTransferrin — — Serum Serum PAGIEF PAGE IEF-3 TB This study Lactate dehydrogenase B 1.1.1.27 Rbc SGE TC-2 Malic enzyme 1 1.1.1.40 Brain SGE CAAPM 1:9 Complement component 6Diaphorase 1 — Serum 1.6.2.2 Rbc SGE PHOS-1 Glyceraldehyde-3-phosphate dehydrogenaseGlutamate-pyruvate transaminase 1.2.1.12 Rbc 2.6.1.2 CAGE Rbc TM CAGE PHOS-2 et al. (1992b); 2:2:1 ratio of pH 4–5 (SERVA):pH 4–5.5 (SERVA):pH 3–10 (SERVA) ampholines; anolyte, 0.3 Methods: SGE, starch gel electrophoresis; PAGE, polyacrylamide gel electrophoresis; CAGE, cellulose acetate gel electrophoresis; AGIEF, agaros Buffer systems: TC-1: 0.155 Visualization of protein polymorphisms was accomplished by methods modified from the published or unpublished sources listed. Details of modified an (1977) using 0.187 a b c AT3 ratio of pH 4–6.5et (Pharmacia):pH 5–6 al. (SERVA):pH 3–10 (SERVA) ampholines; anolyte, 0.04 ALDOC (1972); electrode buffer, 0.04 N-(3 aminopropyl)-morpholine; gel buffer, 1:19 dilution of electrode buffer.pH CAAPM 5.8; 1:9: system citric acid-aminopropyl XIX morpholine, of pH 6.0, modified from Electrophoresis GmbH, Heidelberg, Germany) ampholines; anolyte, 0.04 AK1 al. Richardson procedures are available online at the ACP2 ACONC PI TF (1971). CAAPM 1:19: citric acid-aminopropyl morpholine, pH 6.0, modified from PAGIEF, polyacrylamide gel isoelectric focusing. See text for details. LDHB Locus Protein or gene name number used Method ME1 C6 DIA1 GAPD GPT Linkage Map of the Laboratory Opossum 315 in Cellogel (Chemtron, Milan, Italy) cellulose acetate medium extension at 72Њ (unless otherwise specified, all PCR proce- (e.g., Richardson et al. 1986). Polyacrylamide gel isoelectric dures for this study were conducted using ABI 9600 or focusing (PAGIEF) was performed in 5% polyacrylamide (1:29 9700 thermocyclers). The amplified M. domestica fragment bis:acrylamide) gels prepared by two methods. For AT3, 0.45- contained a polymorphic PstI restriction site. This PCR- mm-thick gels were prepared according to the riboflavin pho- RFLP was visualized on 1% agarose gels (1ϫ TE) stained topolymerization protocol of Pollitt and Bell (1983). For with ethidium bromide. PI, 0.30-mm gels containing 10% sucrose (w:v) were produced SP17: M. domestica SP17 cDNA sequence data (GenBank acces- by standard TMED/ammonium persulfate polymerization. sion no. AF054290 and M. G. O’Rand, personal communi- Agarose gel isoelectric focusing (AGIEF) was performed in cation) were used to design primers for sequencing the 0.5-mm, 1.25% isoagarose (FMC BioProducts, Rockland, ME) intron 1 region of the M. domestica SP17 gene. The intron gels containing 13.75% sucrose (w:v). 1 boundary was inferred from gene structure information Visualization (“staining”) of 13 protein polymorphisms on of human, mouse, and rabbit homologs. PCR primers were ,bp fragment of intron 1-2400ف gels was accomplished by a variety of methods modified from designed to amplify an published and unpublished sources (Table 3). Details of these which was partially sequenced (data available on request) procedures are available online at the Genetics supplemental and found to contain a single nucleotide polymorphism information website: http://www.genetics.org/supplemental. (SNP) in an HaeIII restriction site. The PCR primers were: Southern blotting/RFLP analysis: Restriction enzyme di- forward, 5Ј-CACTGTATTCCATTTTACTC-3Ј and reverse, gests of M. domestica genomic DNA were electrophoretically 5Ј-TTCCTACTTTACATATGAGG-3Ј. Amplification was ac- separated on 1% agarose gels and then transferred and fixed complished using touchdown PCR: initial 5-min denatur- to nylon filters (Hybond-Nϩ; Amersham Biotech, Piscataway, ation step at 95Њ; 10 cycles of 40-sec denaturation at 94Њ, NJ) by standard methods (e.g., Sambrook et al. 1989). Radiola- 30-sec annealing beginning at 67Њ for the first cycle and beled (32P) probes for HBE, NRAS, PHL, and U15557 (de- decreasing 1Њ each subsequent cycle, and 150-sec extension scribed below) were prepared by random priming (Amersham at 72Њ; 30 identical cycles of 40 sec at 94Њ, 30 sec at 57Њ, and Biotech oligolabeling kit). Labeled HBE and PHL probes were 150 sec at 72Њ; and a final 7-min extension at 72Њ.The hybridized to filters for 16 hr at 65Њ, after which the filters polymorphism was analyzed as an HaeIII PCR-RFLP on 1% were washed three times to a final stringency of 2ϫ SSC/0.1% agarose gels stained with ethidium bromide. SDSat65Њ. Hybridization and wash conditions for NRAS and TP53: Published M. domestica TP53 exon 4–11 cDNA sequence U15557 are given by Perelygin et al. (1994, 1996, respec- data (Kusewitt et al. 1999) and unpublished partial intron tively). The hybridization probes used for Southern blot analy- sequence data (D. F. Kusewitt, personal communication) ses of RFLPs were: were used to generate sequencing primers for intron re- 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 restriction 2H, from the dasyurid marsupial, S. crassicaudata (Hope et site, which was analyzed as a PCR-RFLP pattern visualized al. 1992), gift of Rory Hope. on 1% agarose gels stained with ethidium bromide. The ,bp-850ف NRAS: human NRAS proto-oncogene genomic DNA fragment, PCR primer sequences for amplification of the 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, 5Ј-GCGCCCAATCCTGACTATCAT-3Ј and reverse, 5Ј- PHL: probe generated by PCR amplification from M. domestica AGGAAGGGACAGGTAGAAGGA-3Ј. PCR amplification genomic 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 95Њ; 10 cycles of 30-sec denaturation at 94Њ, 30- Forward primer was 5Ј-AAAAAGGGAGGAGCAGAAGGC-3Ј; sec annealing beginning at 78Њ for the first cycle and decreas- reverse primer was 5Ј-GTCATAATGGCGAATGGTAG ing 2Њ for each subsequent cycle, and 60-sec extension at CAC-3Ј. The amplified fragment was cloned into pT7Blue(R) 72Њ; 30 identical cycles of 30 sec at 94Њ, 30 sec at 58Њ, and and transformed into NovaBlue competent cells (Novagen, 60 sec at 72Њ; and a final 5-min extension at 72Њ. 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. ,bp 450ف to 430ف The amplified fragment varied from -bp insertion/deletion (indel) polymor-20ف Polymerase chain reaction amplimer-restriction fragment indicating an length polymorphisms: PCR was used to generate amplimers phism. The primers were: forward, 5Ј-TCAAAGCATGATG that were subsequently subjected to restriction endonuclease CGACAGC-3Ј and reverse, 5Ј-CTGAGATAGCACTGGGA digestion to reveal restriction site polymorphisms (PCR- TGA-3Ј. Touchdown PCR procedures were identical to RFLPs): those for G6PD except that the initial and final annealing Њ Њ G6PD: PCR primers were designed from published G6PD ge- temperatures were 68 and 58 , respectively. The indel poly- nomic DNA sequence of the Virginia opossum, Didelphis morphism was visualized on 1-mm thick, 6.5% polyacryl- virginiana (Kaslow et al. 1987) and used to amplify an amide (1:37.5 bis:acrylamide) gels stained with ethidium .bp M. domestica genomic DNA fragment. This frag- bromide-2400ف ment was partially sequenced (data available on request), RAPD analysis: RAPD polymorphisms were detected using and the resulting data were used to design new, M. domestica- arbitrarily primed PCR as described by Williams et al. (1990). specific PCR primers: forward primer (exon 5), 5Ј-CAT Briefly, arbitrary 10-base oligonucleotides (Operon RAPD 10- CAAAGAAACTTGCATGAGCCAG-3Ј and reverse primer Mer kits A and B; Operon Technologies, Alameda, CA) were (exon 8), 5Ј-ATCTGGAGGAGGTGGTTCTGCATCA-3Ј. Am- used alone or in pairs to amplify random sequences from plification was achieved using a “touchdown” PCR proce- genomic DNA. The arbitrary primers used in this study are dure: initial 5-min denaturation step at 95Њ; 10 cycles of 40- listed in Table 2. RAPD-PCR reaction mixtures consisted of Њ Њ sec denaturation at 94 , 30-sec annealing beginning at 69 2.0 mm MgCl2, 0.1 mm each dNTP (dATP, dCTP, dGTP, and for the first cycle and decreasing 1Њ each subsequent cycle, dTTP), arbitrary primer(s) (0.4 ␮m if a single primer was used; and 60-sec extension at 72Њ; 30 identical cycles of 40 sec at 0.2 ␮m each if two primers were used), 5 units AmpliTaq 94Њ, 30 sec at 59Њ, and 60 sec at 72Њ; and a final 5-min Polymerase (Applied Biosystems, Foster City, CA), 1ϫ PCR 316 P. B. Samollow et al. ng of genomic DNA. Cycle were performed; thus the final genotyping error rate was 25ف AmpliTaq PCR buffer, and parameters (ABI 480 thermocycler) were: denaturation at 94Њ slightly lower than that suggested by these random repeat for 2 min, followed by 45 cycles of 1 min at 94Њ,1minat36Њ, error rates. Overwhelming agreement of parent-offspring ge- and 2 min at 72Њ. Resultant amplification products were run notypes suggests that there were no pedigree errors among out on 1.4% agarose gels and visualized by ethidium bromide the 356 animals used in the mapping analysis. staining. RAPD banding patterns on gels were scrutinized for- Map construction: The GMBX panel was constructed by variation in three categories: (1) PA, presence/absence varia- crossing members of two outbred populations that had fixed tion of a band at a specific position on the gel, presumed to genetic differences at some loci, but shared alleles at other reflect priming sequence variation or large insertion/deletion loci. Therefore, the number of informative meioses varied variants that preclude successful amplification; (2) SV, size substantially across loci (Tables 1 and 2). The average number variation of a fragment revealed by differences in the position of informative meioses per locus, 150 in females and 177 in of a band on a gel presumed to reflect small insertion/deletion males, was sufficient to assure high levels of support for locus variation; and (3) RFLP, length variation of restriction endo- order for most loci. nuclease-digested RAPD-PCR amplification product. Construction of the M. domestica linkage group maps pro- Microsatellite development and polymorphisms: Detection ceeded in four phases: identifying linked loci, ordering linked of short tandem repeat sequences (microsatellites) was con- loci, removal of double recombinants, and final map construc- ducted following the methods outlined in Hillis et al. (1996). tion. We used the computer program Crimap (Green 1990) Briefly, a small-fragment genomic DNA library was constructed to perform a series of two-point (pairwise) linkage analyses by cloning partially Sau3AI-digested and size-selected (200– and assigned markers to linkage groups. To be included within 800 bp) genomic DNA into the pT7 Blue T-Vector (Novagen) a linkage group, loci had to be linked to at least one other according to the manufacturer’s instructions. The library locus within the group at a LOD Ն 3.00 (1000:1 odds). We (maintained in Nova Blue cells) was plated, grown, and trans- next ordered the loci within each autosomal linkage group ferred to nylon membranes, which were probed at high strin- using the expert system program MULTIMAP (Matise et al. gency with (CA)15 or (GT)15 probes (Genosys Biotechnologies, 1994), which implements routines of the computer program The Woodlands, TX) to detect the presence of dinucleotide Crimap. In MULTIMAP, the two loci with the highest pairwise repeat sequences in the plasmid vectors. DNA from strongly heterozygosity are chosen to start the map and additional loci hybridizing clones was isolated and purified (Wizard Plus Mini- are added in order of informativeness. At each step, MULTI- preps DNA purification system; Promega, Madison, WI) and MAP determines a reasonable order for the set of loci, as well then used for sequencing analysis. Sequencing of plasmid as validating that this order has a higher likelihood than other DNA was accomplished either by standard dideoxy sequencing orders, by inverting groups of three loci and keeping the order methods (e.g., Sambrook et al. 1989) with 35S-labeled dATP with the highest likelihood. This process continues until no using the Sequenase version 2.0 sequencing kit (Amersham more loci can uniquely be added to the map, on the basis of Biotech) or by fluorescence-based cycle sequencing methods a criterion of LOD Ն 2.0 (100:1 odds) as statistical support (ABI PRISM Big Dye Terminator Cycle Sequencing ready reac- for locus order. After ordering the loci within each linkage tion kit; Applied Biosystems). For cycle sequencing, PCR reac- group, we used Simwalk2 to detect possible double recombi- tion products were “cleaned” with shrimp alkaline phospha- nants (Weeks et al. 1995; Sobel et al. 2001). Simwalk2 uses tase and exonuclease I (Amersham Biotech) according to the Markov chain Monte Carlo and simulated annealing algo- method of Nickerson et al. (1998) and sequenced on an rithms to perform these multipoint haplotype analyses and Applied Biosystems (ABI) 377 automated DNA sequencer fol- reports the overall probability of mistyping, i.e., evidence of lowing the manufacturer’s instructions. The DNA sequence a double recombinant, at a locus. We removed individual data were analyzed using Sequencing Analysis Version 3.3 genotypes that had Ͼ50% likelihood of mistyping. Finally, and Factura Version 2.2 software. Data from the forward and MULTIMAP was rerun to obtain estimates of sex-specific or- reverse reactions were used to determine the base sequences ders and distances. Map distances were calculated as centi- of the unique (nonrepetitive) regions flanking the repeat morgans (cM) using the Kosambi mapping function (see Ott region and the nature and size of the repeat region itself. 1999). With the exception of linkage group 7, which was com- PCR primer pairs were designed to match unique flanking posed of essentially four recombining loci, we again required sequences, and DNA samples from the smallest subset of com- LOD Ն 2.00 as statistical support for order. pletely informative individuals in the GMBX panel (those that Sex-specific differences: We tested for sex-specific heteroge- contained all alleles that contributed to the backcross genera- neity in linkage group length by using the likelihood ratio tion) were amplified and screened for variation in amplimer test to compare the likelihood of each linkage group with sex- length at each microsatellite locus. specific recombination vs. the null hypothesis of sex-equal Genotyping and data quality: Genotypes were scored inde- recombination. The likelihood ratio test is asymptotically dis- pendently by two observers. Samples yielding ambiguous scor- tributed as a chi square with degrees of freedom equal to the ings were rerun and rescored until agreement was reached number of intervals minus one. We also tested for sex-specific or it was decided that the sample could not be scored for heterogeneity of recombination within each interval by com- the particular marker. In rare cases, wherein an individual’s paring the maximum LOD for linkage when male and female ␪ ϭ␪ genotype at a particular microsatellite locus was unambiguous recombination is equal [Z( m f): the null hypothesis] vs. but could not be reconciled with parental and sibling geno- the maximum LOD obtained with sex-specific recombination ␪ ␪ types even after repeated typings (apparent mutations), the [Z( m, f)] (Ott 1999). The resulting likelihood ratio test, ␹2 ϭ ϫ ϫ ␪ ␪ Ϫ ␪ ϭ␪ individual’s genotype was excluded from the mapping analysis. 2 ln(10) [Z( m, f) Z( m f)], is asymptotically The overall quality of the genotyping data was assessed by distributed as a chi-square distribution with 1 d.f. random retyping of an average of 17.2% (range, 10.6–23.0% per locus) of previously typed samples. Discrepancy rates were lowest for type I DNA (0.19%) and protein (0.39%) polymor- RESULTS phisms and somewhat higher for microsatellite (0.82%) and RAPD polymorphisms (1.0%). The average random repeat Linkage map construction: Two-point linkage analysis discrepancy rate across all loci was 0.77%. All discrepancies using a linkage criterion of LOD Ն 3.0 identified eight were reconciled, and many nonrandom sample repetitions autosomal linkage groups (LGs 1–8) and an X chromo- Linkage Map of the Laboratory Opossum 317

TABLE 4 Linkage groups of M. domestica: number of markers, support values for locus orders, and female/male comparisons

Map length (cM) No. of Linkage No. of markers No. of Log odds Sex F/M group 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 ϫ 10Ϫ7 LG 2c 8 7 4 14.8 56.4 18.9 100.4 0.188 Ͻ10Ϫ12 LG 3 15 13 11 2.6d 139.6 111.2 194.9 0.571 Ͻ10Ϫ11 LG 4 13 12 10 5.2 80.7 48.4 105.5 0.458 2 ϫ 10Ϫ5 LG 5 6 6 4 8.9 41.6 40.5 46.8 0.865 0.29 LG 6 9 8 7 6.0 70.6 56.5 82.9 0.682 0.003 LG 7 7 6 3 1.6 64.9 61.4 73.4 0.837 0.19 LG 8 7 7 6 13.9 65.8 55.0 107.8 0.510 0.41 (LG X) (3) N/A — — — — — — — Totale 81 ϩ (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 low loci examined (LDHB failed to link to another locus number of pairwise informative meiosis rather than to with LOD Ն 3.0). Loci were ordered within LGs 1–8 at tight linkage (AT3-Mdo2LR10 with 11 pairwise informa- a minimum criterion of LOD Ն 2.0 (100:1 odds) for tive meioses and AMG-Mdo5LR17 with 20 pairwise infor- inclusion. Following detection and elimination of im- mative meioses), but the remaining six locus pairs had probable double recombinants, sex-specific locus orders between 44 and 426 pairwise informative meioses (total and recombination distances were recalculated to yield for sexes combined), so tight linkage is the more likely female- and male-specific recombination maps for LGs explanation in these cases. 1–8. Multipoint analysis was not pursued for the 3 loci Statistical support for map order is very good; average in LG X. Data pertaining to the number of loci, statistical LOD is 27.9 across all 56 recombining intervals. Only support levels, and sizes of the sex-average and sex- 3 intervals had multipoint support levels less than LOD specific linkage groups obtained from multipoint link- 3.00. The structure of LG 7 is problematic. This linkage age analyses are listed in Table 4. Diagrammatic repre- group contains only six loci, two pairs of which exhibit sentations of the eight autosomal linkage group maps no recombination; thus, the LG 7 map has only 3 interlo- for both sexes, including locus orders, map positions, cus intervals. The overall order for LG 7 is supported and interlocus interval support levels are shown in Fig- only at LOD 1.6, although support for 2 of the LG 7 ure 1. Locus C7 was not examined in the present study intervals is strong for the given order (LOD 22.0 and but was placed on the map by inference from the finding LOD 5.8). We therefore consider the map for LG 7 as of van Oorschot et al. (1993) that C6 and C7 are provisional with regard to the placement of Mdo7LR27. ␪ϭ ϭ extremely tightly linked in M. domestica ( 0.00, Zmax Map size and sex-specific recombination rates: Two 31.18). striking features of the M. domestica linkage map are Not all of the 80 loci assigned to autosomal linkage its small overall size and the very large differences in groups could be unambiguously ordered. Five RAPD recombination rates between sexes. The sex-average loci (Mdo1LR04, Mdo2LR06, Mdo3LR08, Mdo4LR12, and map size of only 633.0 cM is, to our knowledge, the Mdo6LR18 in LGs 1, 2, 3, 4, and 6, respectively) could smallest of any vertebrate species. This small size is due be placed only within multilocus regions rather than at in part to the substantial reduction in recombination specific points on the corresponding linkage group seen in females as compared to males (see below), but map. Three additional RAPD loci (Mdo1LR05, Mdo3- even the larger male map, which spans 884.6 cM, is LR11, and Mdo7LR22 in LGs 1, 3, and 7, respectively) smaller than any other vertebrate linkage map, regard- could not be placed on the maps with any confidence less of sex. despite linking strongly to one or more loci within their Noninclusion of large portions of the genome does corresponding linkage groups. Eight locus pairs exhib- not appear to be an adequate explanation for the com- ited no recombination in either sex. Lack of recombina- pact linkage map of M. domestica for several reasons. 318 P. B. Samollow et al.

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 centimorgans from the “top” of the linkage group. Boxes enclose nonrecombining loci, which can differ between sexes. Bars to the right of the male map indicate the most likely ranges for loci that could not be ordered with high confidence. Numbers to the extreme right of each linkage group indicate the statistical support (LOD) for each interval, given the locus order shown. Linkage Map of the Laboratory Opossum 319

Figure 1.—Continued.

The M. domestica karyotype is composed of eight pairs loci has not dramatically altered the size of the map. of large autosomes and the X and Y sex chromosomes For example, a series of locus additions that increased (Pathak et al. 1993). The coalescence of 83 genetic the map content from 59 loci to its current 83 loci (a markers into eight autosomal linkage groups and one X 46% increase) increased the overall map size by only chromosomal linkage group in the M. domestica linkage 13%. For all of these reasons we believe that the eight map is consistent with this species karyotype. Failure of autosomal linkage groups correspond to the eight au- LDHB to link to any of the linkage groups is almost tosomes of this species and that the current linkage map certainly due to its lack of variation (only 19 informative encompasses a very large proportion of the genome. If meioses) rather than to its location in an uncharted this is true, M. domestica possesses a generally low rate portion of the genome. Further, the smallest autosomal of meiotic recombination and particularly so in females. linkage group in the M. domestica map includes six loci, Overall, the male genetic map is twice the size of the and LG X includes all of the X-linked loci examined. female map (884.6 vs. 443.1 cM), and male linkage There are no other unlinked loci, nor are there any group sizes exceed those for each of the corresponding locus pairs or triplets that do not link to one of the female linkage groups (Figure 2). These differences are nine linkage groups. Moreover, LGs 1–8 were identified statistically significant for the five larger linkage groups during an early phase of mapping using only 46 loci. (LGs 1–4 and 6), but not for the three smaller ones, Since that time, no newly examined locus has failed which have fewer loci (Table 4). Of the 56 recombining to link to one of the existing linkage groups. Another intervals, 20 (representing 45.9% of the total sex-aver- indication that no large, undetected regions of the ge- age map length) show significantly higher male recom- nome remain is that the continuing addition of new bination, while only two (representing 6.7% of total 320 P. B. Samollow et al.

the genome, but that the intensity of the difference varies 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 map appears to represent nearly full coverage of the M. domes- tica genome. However, an estimate of the total linkage map size must account for undetected regions beyond the terminal markers of each linkage group and for the length of the X chromosome. To estimate the length of putative undetected telomeric regions, we used the simple 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 mapped domestica. Linkage groups are arranged in descending order loci (e.g., Fishman et al. 2001). Specifically, the average of male linkage group size, with male linkage groups above ILD was calculated as the total map length divided by and corresponding female linkage groups below. Linkage ϭ group sizes (in centimorgans) are indicated above and below the number of recombining intervals: 633/56 11.3 the male and female bars, respectively. Probabilities are for cM. A commonly used alternative method (e.g., Mikawa tests of male vs. female differences in linkage group size (see et al. 1999; Zenger et al. 2002) uses the total number footnote b of Table 4). of linked autosomal loci minus the number of link- age groups (80 Ϫ 8 ϭ 72) as the denominator. This more 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, but higher on average over the remaining 34 (nonsignifi- physically the X chromosome is less than half the size cant) 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 of differences in interval lengths between sexes, but, as the three smallest autosomes; thus we use the average mentioned, the overall length of LG 6 is significantly length of the three smallest linkage groups, 54.3 cM, as greater in males than in females. LG 5, which is the our estimate of LG X length. Combining the lengths of smallest with regard to length and number of loci, is LGs 1–8 with the estimate for LG X and 18 linkage the only linkage group that exhibits no significant be- group ends yields an estimate of 633 ϩ 54.3 ϩ (18 ϫ tween-sex differences either in total length or at the 11.3) ϭ 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 maps corresponding female linkage groups. Consideration of are highly variable, indicating a broad range of recombi- 1398ف deviations of sex-specific recombination rates from sex- nation rates among species. Examples range from average rates for each interlocus interval (illustrated in cM for laboratory mice (Rhodes et al. 1998) to Ͼ3500 Figure 3) suggest that sex differences, while widespread, cM in humans (Kong et al. 2002), cattle (Barendse et are 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 map these regional and linkage-group level differences is could underestimate the true recombination rate of hampered 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 crosses ences 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 strict number 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 both of its are incomplete. The full-coverage dog and rat maps (%46ف) exceeds the female map for almost half .and 1989 cM, respectively 2700ف length. The overall impression is that male recombina- are estimated to be tion is greater than female recombination for most of Among other vertebrates, estimated map lengths vary Linkage Map of the Laboratory Opossum 321

TABLE 5 Interlocus intervals exhibiting significantly different sex-specific lengths

Map length (cM) Linkage Sex Chi group Interval average Female Male square Probabilitya LG 1 Mdo1L001-Mdo1LR01 11.7 3.3 16.2 6.08 0.014 Mdo1LR01-AK1 12.0 2.9 18.8 9.21 0.002 Mdo1L019-Mdo1L003 4.7 1.6 7.6 8.05 0.0046 Mdo1L003-PI 13.2 5.9 21.6 17.3 0.00003 PI- Mdo1L004 25.2 11.7 42.9 17.1 0.00004 Mdo1L004-Mdo1L002 3.9 2.2 5.7 5.53 0.0187 Mdo1L002-Mdo1L021 4.6 2.1 7.0 6.68 0.0098 LG 2 GPT-Mdo2L023 10.8 1.7 19.3 32.3 1 ϫ 10Ϫ8 Mdo2L023-Mdo2L022 19.8 6.3 36.0 58.1 2 ϫ 10Ϫ14 Mdo2L022-Mdo2L005 11.2 4.0 18.9 30.0 4 ϫ 10Ϫ8 Mdo2L006-C6/C7 14.7 6.9 26.1 21.9 0.000003 LG 3 Mdo3L024-AT3 39.0 59.0 27.5 7.64 0.0057 Mdo3L025-Mdo3L008 30.5 8.0 69.2 92.1 9 ϫ 10Ϫ22 Mdo3L008-TP53 2.3 0.0 3.8 6.54 0.011 TP53-ME1 14.1 2.1 24.5 16.5 0.000049 LG 4 SP17-Mdo4L029 11.2 2.7 22.9 20.3 0.000007 Mdo4L029-Mdo4L009 16.7 8.4 23.3 21.2 0.000004 Mdo4LR16-Mdo4L028 9.1 3.7 13.7 5.76 0.016 Mdo4L028-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 0.0105 LG 8 Mdo8L016-Mdo8LR25 5.2 1.3 9.4 7.60 0.0058 Mdo8L025-ACP2 31.4 25.2 68.8 7.51 0.0061 Totals 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). cM in rainbow trout (Oncorhyncus mykiss; (1988) and Gregory (2001)]. Clearly, recombination 1350ف from Sakamoto et al. 2000) to 7291 cM in hybrid crosses of rates (centimorgans per megabases) in these two dis- the salamanders Ambystoma mexicanum and A. tigrinum tantly related marsupial species are very low relative to (Voss et al. 2001). those of other mammals and of vertebrates in general. The tammar wallaby, M. eugenii, is the only other It is tempting to propose that the short map lengths marsupial for which a comprehensive linkage map has of M. domestica and M. eugenii are related to the low been published (Zenger et al. 2002). The evolutionary chromosome numbers of these two species. However, (an examination of total map lengths (in centimorgans 70–60ف distance between M. eugenii and M. domestica is million years (Kirsch et al. 1997; Springer 1997; and chromosome numbers (n) among the 19 nonmeta- Graves and Westerman 2002), which is roughly com- therian vertebrates for which sufficient data are avail- parable to that between humans and mice. The M. eu- able, suggests that chromosome number does not ex- genii map length of 828.4 cM includes the X chromo- plain the majority of the observed variation in map some, but is not corrected for putative undetected lengths (Figure 4). Among eutherian mammals, for ex- telomeric regions and is estimated to represent only ample, dogs have more than twice as many chromo- 71% of a total length of 1172 cM. This total map length somes as cats (n ϭ 39 and 19, respectively), but the dog cM) is substantially shorter than 2700ف) is 31.6% larger than our estimate of the M. domestica linkage map ,cM). Humans and Syrian hamsters 3300ف) sex-average map, but is still quite small by vertebrate the cat map standards. The M. domestica genome (3.6–3.7 pg of the on the other hand, have nearly identical chromosome DNA/haploid genome, estimated by flow cytometry; J. numbers (23 and 22, respectively), but their map cM, respectively) are widely 2000ف and 3600ف) larger than that of lengths %6ف Danke, unpublished data) is humans, while that of M. eugenii may be as much as divergent. Even comparing the marsupials, M. eugenii 18% larger than the human genome [data of Hayman has fewer chromosomes than M. domestica, while its map and Martin (1974), cited by Sharp and Hayman is nearly a third longer. Moreover, regression analyses 322 P. B. Samollow et al.

Figure 3.—Sex-specific deviations from the sex- average recombination rate in M. domestica. Interval numbers correspond to in- terlocus intervals in Figure 1, beginning at the top of the linkage group diagram; e.g., interval 3 of LG 1 corre- sponds to AK1–U15557, and interval 4 of LG 2 corre- sponds to the nonrecombin- ing MdoL005–MdoL006 “in- terval.” The horizontal line represents the sex-average recombination rate. Over- lapping male and female symbols on the horizontal line 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 recombination in either sex.

(excluding the clearly anomalous amphibian relation- 0.22 (P ϭ 0.107); eutherians ϩ fishes, 0.07 (P ϭ 0.322); ship) of map length on chromosome number for various and eutherians ϩ chicken ϩ fishes, 0.16 (P ϭ 0.102). vertebrate groupings reinforce the visual impression However, when the two marsupials were added to the that chromosome number is a poor predictor of map analyses, all of the corresponding correlations became length. The correlations (r 2) between map length and stronger (r 2 ranged from 0.32 to 0.51) and were statisti- chromosome number for the various groups were: eu- cally significant (P ϭ 0.003–0.012). Because the marsu- therians alone, 0.12 (P ϭ 0.277); eutherians ϩ chicken, pial data were extreme values and provided strong lever- Linkage Map of the Laboratory Opossum 323

and the present study shows that ALDOC and TP53 are syntenic on M. domestica LG 3. These 8 loci are known to be syntenic on Mmu 11 and Hsa 17q, suggesting that a substantial 8-locus synteny may have been conserved among M. domestica, mice, and humans. However, LG 3 is not entirely homologous to Hsa 17q and Mmu 11, as it also contains loci found on Hsa 1p (NRASLB), Hsa 1q (AT3), and Hsa 6q (ME1); these loci are similarly dispersed in the mouse genome. The impression of mixed conservation and rearrangement relative to eu- therian genomes agrees with extensive physical map- ping results from M. eugenii and a few other marsupial species (reviewed by Samollow and Graves 1998). Un- Figure 4.—Total map length vs. chromosome number in vertebrates. See text for discussion. Data sources are as follows: fortunately, except for several X- and Y-linked genes, human (Broman et al. 1998; Kong et al. 2002), baboon (Rog- there is almost no overlap among the type I loci mapped ers et al. 2000), mouse (Rhodes et al. 1998), rat (Bonne´ et al. in M. domestica and M. eugenii (Samollow and Graves 2002), 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; Kappes et al. 1997), pig (Mikawa et al. 1999), sheep (Maddox 2001), so there is currently no opportunity for synteny et al. 2001), deer (Slate et al. 2002), horse (Gue´rin et al. comparisons between these two marsupial species. 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, but channel 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 Young et al. 1998; Sakamoto et al. 2000), Xiphophorus sp. (S. nation rates implied by the female- and male-specific Kazianis, R. S. Nairn, R. B. Walter, D. A. Johnston, J. linkage maps. The combined male linkage map (884.6 Kumar et al., unpublished data), and zebrafish (Shimoda et al. cM) is twice the size of the female map (443.1 cM; the 1999). Vertebrate species with substantially incomplete maps female to male recombination ratio, F/M, is 0.50), and were excluded from this analysis. For consistency with the each of the individual linkage groups is larger in males other species examined, the published total map lengths for rat, deer, horse, and medaka were adjusted for missing telo- than in females. This sex difference is extraordinary not meric regions by adding twice the average interlocus distance only for its magnitude, but also for the direction of the 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 reduction ship between map length and chromosome number in female recombination in M. domestica is greater in among 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 differences numbers 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 of in this study is inconclusive with regard to conservation the 8 significant intervals from the M. eugenii map, the of 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 significant humans (Hsa 3q), but LG 4 also includes HBE, which intervals yields an F/M ratio of 0.72, a ratio that is more is not syntenic with SP17 orTFin mice or humans (a skewed than the F/M ratio for the full M. eugenii map fourth 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 rate in M. domestica (LG 2) and mouse (Mmu 15), but not are common among a broad range of animal and plant in humans (Hsa 5p and Hsa 8q, respectively). A series of species. Haldane (1922) and Huxley (1928) each two-point linkage comparisons reported by Sokolova et noted this general trend and suggested that in species al. (1997) suggested that NF1, ERBB2, RARA, HOX2, where the sexes had substantially different recombina- MPO, PRKCA, and ALDOC are syntenic in M. domestica, tion rates, it was the heterogametic sex that had the 324 P. B. Samollow et al. reduced rate. While neither author proposed this situa- nome as a whole. In summary, we have been unable tion to be inviolable, and exceptions are known [e.g., to identify a single unambiguous case of excess male flour beetles, Tribolium castaneum (Sokoloff 1964)], recombination, as gauged from genome-wide genetic this prediction has turned out to be accurate in the data, in any nonmetatherian vertebrate species. great majority of species in which strong sex-specific The occurrence of reduced female recombination in differences in recombination frequency have been doc- both American and Australian marsupial species, which umented on a genome-wide basis (regional sex-specific last shared a common ancestor at least 60 MYA (Kirsch reversals over short map distances are known in many et al. 1997; Springer 1997; and discussions by Kirsch species, but these do not affect the genome-wide gener- and Mayer 1998; Springer et al. 1998), tempts specula- alization). tion that reduced female recombination is a general Most eutherians examined exhibit substantially higher characteristic that distinguishes metatherian from eu- female than male recombination rates, and, among therian species. With so few mammalian species maps those with extensive maps, the F/M ratio is almost completed, such speculation might be premature; how- always Ͼ1.0; e.g., 1.56–1.65 in humans (Broman et al. ever, cytologic data from the brushtail possum, Tricho- 1998; Kong et al. 2002); 1.36–1.41 in dogs (Mellersh surus vulpecula, (family Phalangeridae) also suggests re- et al. 1997; Neff et al. 1999), and 1.30–1.55 in pigs duced female recombination, although less extreme (Archibald et al. 1995; Mikawa et al. 1999). In cattle, than that in M. domestica and S. crassicaudata (Hayman the female map is just slightly larger than the male map: and Rodger 1990). The only contrary finding comes F/M ϭ 1.03–1.06 (Barendse et al. 1997; Kappes et al. from a cytologic study of the brush-tailed bettong, Betton- 1997). Genome-wide female bias in recombination is gia penicillata (family Potoroidae), in which no obvious also evident in the baboon linkage map (J. Rogers difference in sex-specific chiasma number or distribu- and M. C. Mahaney, personal communication) and in tion was detected (Hayman et al. 1990). Unfortunately, linkage data from each of six rhesus macaque chromo- linkage data are not available for T. vulpecula or B. somes examined to date (J. Rogers, personal communi- penicillata. Thus, reduced female recombination surely cation). A possible exception to this pattern has been does occur in three marsupial species and perhaps a observed in sheep, in which the F/M ratio ϭ 0.83; but fourth. Notwithstanding the data from B. penicillata,it this ratio must be regarded as provisional because pecu- seems probable that reduced female recombination is a liarities of the mating scheme used to produce the sheep very common, if not universal, marsupial characteristic. maps could have contributed to an underestimate of What influences recombination rates in marsupials? male map length (Maddox et al. 2001, p. 1285). Marsupial genomes are about the same size as those of The tendency for female map length to exceed that eutherian mammals (Gregory 2001) but are far more of males appears to be greatly exaggerated in fishes: conservative with regard to chromosomal compartmen- zebrafish F/M ϭ 2.74 (Singer et al. 2002); catfish F/M ϭ talization (discussed by Samollow and Graves 1998; 3.18 (Waldbieser et al. 2001); rainbow trout F/M ϭ Graves and Westerman 2002). Whereas eutherian 3.25 (Sakamoto et al. 2000), but absent in birds. In the chromosome numbers range from 2n ϭ 6to2n ϭ 118, only avian map available, that of chicken, F/M ϭ 0.99 those of marsupials range only from 2n ϭ 10 to 2n ϭ (Groenen et al. 1998, 2000), and a cytologic study in 32, and in Ͼ90% of marsupial species examined, 2n ϭ pigeons revealed equal numbers of chiasmata in female 14–22. On average, then, marsupials package the same and male meiotic cells (Pigozzi and Solari 1999). Sep- amount of DNA into fewer, larger chromosomes than arate female and male maps are not available for am- do their eutherian counterparts. Low chromosome phibians, but recombination data for six autosomal loci number reduces the opportunity for genome reshuf- in the frog Rana brevipoda and a pair of loci in its conge- fling via interchromosomal assortment, but this ten- ner R. japonica indicate that strongly female-biased re- dency can be compensated by an increase in intrachro- combination occurs in at least some amphibians (Sum- mosomal recombination (crossing over). That such ida and Nishioka 2000). Average chiasma counts for compensation does not appear to occur in M. domestica male crested newts (Triturus cristatus) exceed those of (2n ϭ 18), M. eugenii (2n ϭ 16), or S. crassicaudata (2n ϭ females on all chromosomes (Wallace et al. 1997), 14) suggests that the intergenerational reshuffling of suggesting higher male than female recombination genetic information in these marsupials is considerably rates (chiasma-based F/M ϭ 0.77) in this species. How- less intense than that in eutherians and other verte- ever, the chiasmata in oocytes are strongly clustered brates and especially so in females. around centromeres, while those in spermatocytes are The substantial literature on the evolution of sex and restricted to the more distal regions of the chromosome recombination rates (reviewed by Otto and Barton arms; thus the male and female recombining regions are 1997; Barton and Charlesworth 1998; Burt 2000; largely distinct and nonoverlapping. This dimorphism, Otto and Lenormand 2002; Rice 2002) suggests that together with the lack of genetic mapping data for this the frequency of meiotic recombination can be shaped species, inhibits interpretation of these cytological ob- by natural selection to optimize the extent to which servations with regard to recombination across the ge- multilocus allelic associations are maintained or obliter- Linkage Map of the Laboratory Opossum 325 ated from one generation to the next. Discussions of on the vast majority of X-linked genes that follow the recombination rate have focused primarily on genetic single-active-allele pattern). In metatherians, the pat- interactions and environmental conditions (various tern is decidedly nonrandom; it is always the paternally forms of epistasis, linkage relationships, environmental derived X that is inactive (paternal XCI or PXI). Thus, heterogeneity) that act to modify population-average the only X-linked genes expressed in a metatherian recombination rates. Recent modeling of the evolution female’s cells are those derived from her mother; of sex-specific recombination differences (Lenormand X-linked genes derived from her father contribute virtu- 2003) indicates that similar forces can also adjust male ally nothing to her phenotype (as in eutherians, excep- and female recombination rates independently of those tions occur; some paternally derived genes escape full that determine average rates. If so, the small size of the inactivation, but we focus here on the great majority of M. domestica linkage map and the striking difference paternally derived X-linked genes that are fully si- between male and female maps could indicate that the lenced). This distinction in XCI patterns results in phy- selective environments that have shaped recombination logenetically restricted differences in the effective con- rates in this and perhaps other marsupial species differ tributions of mothers and fathers to the expressed at both species- and sex-specific levels from those that genomes of their offspring. have influenced recombination rates in other verte- Hope (1993) proposed a mechanistic model wherein brates. the female response to the paternal imprint that causes Unfortunately, we do not know why any particular the paternally derived X chromosome to be inactive species has the recombination rate it does or why marsu- might actually be applied indiscriminately to all pater- pials as a group should have reduced recombination. nally derived chromosomes. This would render them Nevertheless, it is worth remembering that the species- molecularly distinct from maternally derived chromo- average rate of recombination can evolve for reasons somes and could interfere with and reduce recombina- unrelated to the forces acting on sex-specific rates (Len- tion in females. Genomic imprinting of selected autoso- ormand 2003), so the low recombination rates observed mal genes, similar to that seen in eutherians, does in female marsupials need not dictate low male recombi- indeed occur in marsupials (Killian et al. 2000, 2001; nation as a correlated response. More likely, low average O’Neill et al. 2000), but there is no evidence of whole- recombination in M. domestica (and M. eugenii and per- sale gene silencing or any other form of global paternal haps other marsupial species) is a result of pressures imprinting in any marsupial species, so this explana- acting to reduce recombination generally, while other tion for reduced female recombination seems unlikely. factors acting in sex-specific ways have adjusted male Alternatively, it has been shown that sex-specific asym- and female rates within this general, species-average metries in epistatic interactions among autosomal loci range. (imprinted or not) can lead to divergence of male and Despite our inability to explain the low average re- female recombination rates (Lenormand 2003). The combination rate in marsupials, a possible explanation exclusive expression of maternally derived X-linked for reduced female recombination rates could lie in the genes in metatherian females generates an asymmetry pattern of X-linked dosage compensation that occurs of parental contribution to female offspring relative to in metatherian mammals. X chromosome inactivation the situation in eutherian wherein both parents contrib- (XCI) is a uniquely mammalian process that results in ute active X-linked genes to their daughters (neither the coordinate silencing of the great majority of genes metatherian nor eutherian males contribute X chromo- on one of the two X chromosomes in female somatic somes to their sons). Given current understanding of cells during embryogenesis (reviewed by Heard et al. the power of epistatic interactions to alter sex-specific 1997; Avner and Heard 2001; Boumil and Lee 2001; recombination rates, it seems unlikely that PXI alone Brockdorff 2002). Eutherians and metatherians ex- could favor reduced female recombination in the ab- hibit differences in several details of the X-inactivation sence of autosomal imprinting (T. Lenormand and S. process (reviewed by VandeBerg et al. 1987; Cooper et Otto, personal communications). However, if benefi- al. 1990, 1993; see also Keohane et al. 1998), of which cial epistatic interactions among combinations of alleles the most salient is the origin of the inactivated chromo- at linked autosomal loci were dependent on both the some. In eutherian females inactivation is random with parent of origin of the interacting genes (autosomal regard to the parental source of the X chromosome. imprinting) and the presence of a maternally imprinted About half of a female’s cells express genes from the X X chromosome, then it might be plausible that PXI chromosome she inherited from her mother (mater- could create a selective environment in which the bene- nally derived X) and the other half express the genes fit to females of keeping favorable sex-specific epistatic from the X inherited from her father (paternally de- autosomal associations intact would be greater than the rived X). Although no individual cell expresses the ma- detriment (if any) to male offspring and greater than ternally and paternally derived copies of X-linked genes, the pressure to break up such combinations in male both contribute to the female’s overall phenotype (some meiosis. To the extent that such multilocus associations X-linked genes escape X inactivation, but here we focus occur throughout the genome, this might lead to an 326 P. B. Samollow et al. increase in alleles at recombination modifier loci that mapped region or that chiasmata on this chromosome reduce female recombination. Although no empirical are concentrated in the extreme terminal region(s) in or theoretical evidence supports these or any other re- females. As the male LG 2 map is more than five times lated hypotheses, the unique pattern of paternal XCI in larger (100.4 cM) than the female LG 2 map, lack of metatherians 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 compressions recombination 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 the cific differences in the number and distribution of chias- commonly observed exaggeration and diminution (or mata 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; Singer asmata. Males averaged 21.5 chiasmata per meiotic cell, et al. 2002), and even monoecious plants (Lagerkrantz and 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 the chromosome arms. Females averaged only 13 chiasmata M. domestica chromosomes. As a preliminary step toward per meiosis, and virtually all were terminal or subtermi- this objective, we are isolating M. domestica bacterial nal. 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 in lutely) 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 the the 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 an groups precludes rigorous analysis, but the larger link- integrated physical and linkage map of the M. domestica age 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 be specific 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 for of LG 1 (Mdo1L021–Mdo1L020) on the female map is technical assistance; Jim Bridges and Nicole Stowell for aspects of proportionally 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; intervals of LG 6, and the first interval of LG 3 and and Don Taylor, Gerardo Colon, Susan Collins, and Ernesto Morin LG 8. Conversely, compressions of the female map are for superb animal care and pedigree record keeping. Thanks also go expected to occur in interstitial regions. Such compres- to Thomas Lenormand, Sarah Otto, and William Rice for helpful 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 was LG 3, but compressions do occur at the ends of several supported in part by grants from the National Institutes of Health linkage groups that exhibit inflations at their opposite (RR-09919 and RR-14214), the Samuel Roberts Noble Foundation, ends, e.g., the first two intervals of LG 4, the last three the Ellwood Foundation, and the Robert J. Kleberg, Jr. and Helen C. intervals of LG 6, and the last interval of LG 8. Kleberg Foundation. Additional support for the idea that localization of chiasmata to chromosome ends is an important contrib- utor to low female recombination rate can be inferred LITERATURE CITED from 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- chromatid exchange to ensure proper disjunction boons. Biochem. Genet. 33: 35–40. 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