The First Comprehensive Genetic Linkage Map of a Marsupial: The Tammar Wallaby (Macropus eugenii)

Corresponding author: Kyall R. Zenger, Faculty of Veterinary Science, The University of Sydney, Camden, NSW 2570, Australia., kzenger{at}camden.usyd.edu.au (E-mail)

Communicating editor: N. JENKINS

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The production of a marsupial genetic linkage map is perhaps one of the most important objectives in marsupial research. This study used a total of 353 informative meioses and 64 genetic markers to construct a framework genetic linkage map for the tammar wallaby (Macropus eugenii). Nearly all markers (93.8%) formed a significant linkage (LOD > 3.0) with at least one other marker, indicating that the majority of the genome had been mapped. In fact, when compared with chiasmata data, >70% (828 cM) of the genome has been covered. Nine linkage groups were identified, with all but one (LG7; X-linked) allocated to the autosomes. These groups ranged in size from 15.7 to 176.5 cM and have an average distance of 16.2 cM between adjacent markers. Of the autosomal linkage groups (LGs), LG2 and LG3 were assigned to chromosome 1 and LG4 localized to chromosome 3 on the basis of physical localization of genes. Significant sex-specific distortions toward reduced female recombination rates were revealed in 22% of comparisons. When comparing the X chromosome data to closely related species it is apparent that they are conserved in both synteny and gene order.

MARSUPIAL and eutherian (placental) mammals diverged 130 million years ago (HOPE et al. 1990 ; KILLIAN et al. 2001 ). The two groups are similar in most of their biology, but have distinctive attributes with regard to reproduction and cytogenetics. For example, marsupials are born at a very early stage of development equivalent to that of the end of embryogenesis in placentals, and marsupial development is completed usually in a pouch attached to a teat (GEMMELL et al. 2002 ). Sex differentiation appears to be much more complex in marsupials than in placentals (PASK and RENFREE 2001 ). While eutherians exhibit a wide range of chromosome numbers (2n = 6–92; WURSTER and BENIRSCHKE 1970 ; SCHMID et al. 1988 ), marsupials are characterized by low numbers, ranging from 2n = 10–32 (HAYMAN 1990 ), and sex-specific recombination rates appear to be contrary to those observed in eutherians (BENNETT et al. 1986 ; SAMOLLOW et al. 2000 ). Given these unique characteristics, marsupials have been regarded as ideal candidates for research encompassing reproductive biology, immunology, developmental studies, and comparative genomics (e.g., SAMOLLOW and GRAVES 1998 ; MILLER and BELOV 2000 ; PASK and GRAVES 2001 ; RENFREE and SHAW 2001 ).

The tammar wallaby (Macropus eugenii) is the Australian marsupial model for genetic mapping studies. Its value arises from a number of characteristics, including: (i) small numbers of chromosomes (2n = 16) that are variable in both length and morphology and include a XX/XY female/male sex-chromosome dimorphism; (ii) a relatively small overall map length of 1172 cM (estimated from chiasmata data); (iii) delayed blastocysts, whereby removing the pouch young stimulates the reactivation of the blastocyst producing multiple offspring annually; (iv) small size (4.5–8.5 kg) relative to other macropodids and ease of breeding in captivity; and (v) subspecies that hybridize together, producing the perfect genetic pedigree system for genetic linkage mapping experiments (SHARP and HAYMAN 1988 ; HINDS et al. 1990 ; MCKENZIE and COOPER 1997 ; RENFREE and SHAW 2000 ).

Genetic mapping progress in M. eugenii to date has proceeded primarily by physical approaches based on in situ hybridization methods (e.g., SAMOLLOW and GRAVES 1998 ; HAWKEN et al. 1999 ; WATERS et al. 2001 ), while linkage mapping in M. eugenii has produced four small linkage groups incorporating a total of nine genetic markers (VAN OORSCHOT and COOPER 1990 ; MCKENZIE et al. 1993 , MCKENZIE et al. 1996 ). Traditionally, research has been directed at the in-depth comparisons of genome structure between metatherian and eutherian gene maps to better understand genome evolution. These differences can then be used as probes to explore the phylogenetic distinction of sex determination, gene expression, and other questions pertaining to the evolution of gene structure and function. However, gene mapping is of interest not only as a comparative tool, but also for its intrinsic value for localizing genes and examining their function. Currently there is great interest in identifying trait loci connected with disease resistance and immune response within overabundant or endangered marsupials (e.g., BUDDLE and YOUNG 2000 ). In addition, the localization of unique marsupial trait loci, which could be of economic importance, has been the focus of recent attention (e.g., GELLIN et al. 2000 ). The production of a M. eugenii genetic linkage map would be the first necessary step to undertake these investigations.

In this article, we report the first comprehensive framework genetic linkage map of a marsupial (M. eugenii) consisting of coding (functional) and anonymous (microsatellites) genetic markers. The impetus for the development of this map has been primarily to develop a system for comparative and quantitative trait mapping. However, the application of this map should not be limited only to these objectives. In essence the map also provides a mechanism for investigating how different genetic control mechanisms evolved and how they function.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Reference pedigrees:
The genetic data used to construct the M. eugenii genetic linkage map were obtained from a total of 353 informative meioses. The majority of these are derived from hybrid phase-known backcrosses from genetically distinct Kangaroo Island (KI) and Garden Island (GI) tammar wallabies bred at Macquarie University (see MCKENZIE et al. 1993 ). There are six different types of crosses (Fig 1), with the majority of informative data being derived from pedigrees 1, 2, and 3.

View larger version (46K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Systematic representation of crosses derived from Kangaroo Island (KI) and Garden Island (GI) animals. Females are presented first in all parental genotypes, e.g., GI–KI and KI–(KI–GI). Pedigree 4 and 5 mothers are the progeny of pedigrees 2 and 3, respectively. Solid symbols represent multiple individuals.

Separate male and female hybrid crosses were used so that independent information regarding male/female recombination rates and allelic transmission could be determined. Four informative hybrid males in pedigree 1 and pedigree 2 produced 105 progeny, while pedigrees 3–6 consisted of 21 informative females who generated 127 offspring. In addition, a number of KI or GI males in the female pedigrees were heterozygous, allowing for extra male informative crosses, which produced an additional 121 meiosis events. These males were originally phase unknown. However, their phase was inferred after careful consideration and examination with the original hybrid male results. Accordingly, all male hybrid-type crosses were combined to produce a total of 226 possible informative meiosis events. All DNA extractions were carried out according to the "salting-out" procedure described in SUNNUCKS and HALES 1996 .

Genetic markers and data integrity:
Sixty-four genetic markers were used for analysis in this study. Seventeen of these have previously been described and tested for genetic linkage (MCKENZIE et al. 1993 , MCKENZIE et al. 1996 ; MCKENZIE 1994 ). These markers include 12 coding genes, 4 anonymous DNA markers, and 1 pseudogene (Table 1). Genotyping of these 17 markers was accomplished via allozyme electrophoresis (MPI and NP) according to RICHARDSON et al. 1986 , isoelectric focusing (TF) according to RIGHETTI 1983 , and Southern hybridization using ³²-P-labeled probes (AR, CASA, DBB, G6PD, HBB, LALBA, LLP, LPL, RNR, pB12, pB15, pB65, pB72, and PGK_9) according to SAMBROOK et al. 1989 . All probes were macropodid specific, except HBB and RNR, which were derived from Dasyurus viverrinus and Xenopus laevis. Although a small number of microsatellite loci have previously been tested (TAYLOR and COOPER 1998 ), they were tested again within this study due to limited numbers of meioses and inconsistencies within these data. The remaining 47 genetic markers are described in Table 1. All but 4 of these genetic markers were based upon anonymous macropodid-specific microsatellite genetic markers. These remaining four loci consisted of a gene-specific microsatellite marker located within interleukin 5 (IL5; HAWKEN et al. 1999 ), a hypoxanthine phosphoribosyltransferase (HPRT)-specific X-linked polymorphism (GenBank accession nos. AF503635 and AF503636) characterized by restriction fragment length polymorphism (RFLP) and two autosomal anonymous DNA polymorphisms identified as pHPRT1 and pHPRT2, which were resolved by single-strand conformation polymorphism (SSCP).

All microsatellite loci and anonymous DNA markers were amplified via PCR in 10-µl reaction volumes containing 100–200 ng of genomic DNA, 2–3 mM MgCl₂, 10 mM Tris-HCL (pH 8.3), 50 mM KCL, 0.1% Triton X-100, 0.1% Tween 20 and NP40, 200 µM each of dTTP, dCTP, and dGTP, 20 µM dATP, 0.05 µl of [³³-P]dATP at 1000 Ci/mmol, 1.0 µM of each primer, and 0.5 units of Taq polymerase (QIAGEN, Chatsworth, CA). PCR amplifications were carried out using an MJ Research (Watertown, MA) PTC100 thermocycler, with an initial 94° denaturation for 3 min, followed by "touchdown" cycles of 94° denaturation for 30 sec, annealing temperatures (60°, 58°, 56°, 54°, 52°, and 50°) for 45 sec, and an extension step of 72° for 1 min. The touchdown annealing temperatures decreased by 2° each cycle, whereby on completion of the last touchdown cycle another 30 cycles were performed at this annealing temperature with a final extension of 72° for 3 min. The amplified microsatellite PCR products were resolved on 6% denaturing polyacrylamide gels and visualized by autoradiography according to TAYLOR et al. 1994 , while PCR-SSCP of pHPRT1 and pHPRT2 was carried out according to SUNNUCKS et al. 2000 .

Preferential PCR amplification of introns 6–8 of the X-linked HPRT gene was accomplished using primers designed over exon/intron boundaries from closely related M. robustus sequences (PIPER et al. 1993 ). PCR conditions are as above with the omission of [³³-P]dATP and the replacement of 20 µM dATP with 200 µM dATP. Characterization of the X-linked HPRT polymorphism was accomplished via RFLP using the restriction enzyme BsrDI.

Even a minute proportion of genotypic errors in a data set can dramatically affect the precision of a linkage map. Several steps were carried out to ensure strict data integrity prior to map construction. This was accomplished by the identification and correction of errant typings by two different approaches. First, random samples of animals from previous typings were duplicated in subsequent analysis to ensure continuity and to allow for quality assessment of newly acquired data, and second, genotype data were inspected for typing inconsistencies via examination of Mendelian segregation patterns against known pedigrees. This second procedure was carried out manually twice at independent times to ensure correct genotyping of the markers. All discordant and non-Mendelian genotypes were either repeated or deleted from the primary data. A further step was carried out following the genetic linkage map construction. This entailed searching for potential errors by looking for dubious double crossovers using the "error detection" command of MAPMAKER version 3.0b with the default setting of 1% a priori probability of error (LANDER et al. 1987 ; LINCOLN and LANDER 1992 ). This procedure was repeated at least three times for each linkage group. Once errors were identified and corrected, the linkage map was rederived.

Segregation distortions, which may be caused by gametic selection or postzygotic selection, have been observed frequently in divergent hybrid backcrosses (e.g., SIRACUSA et al. 1991 ; XU et al. 1997 ; VOGL and XU 2000 ). To test if any of the loci within the hybrid (KI-GI) x KI crosses (excluding F₁ x GI individuals due to insufficient meioses) were expressing segregation distortions, chi-square tests with 100,000 randomizations were calculated (SOKAL and ROHLF 1995 ) using the "Monte Carlo" exact test function of SPSS version 10.0. This application calculated exact segregation heterogeneity for all 64 loci across pedigrees 1, 2, and 3. In addition, to examine if there were sex-specific differences in F₂ allelic inheritance, a Fisher's exact probability (SOKAL and ROHLF 1995 ) was calculated for all loci using SPSS version 10.0. After examination of the loci, the number of informative meioses per locus and the average number of informative meioses across the data set were calculated.

Map construction:
Several strategies were carried out to ensure the most precise genetic linkage maps were obtained. This included the utilization of MAPMAKER version 3.0b (LANDER et al. 1987 ) and CARTHAGENE version 0.4 (SCHIEX and GASPIN 1997 ) linkage analysis programs, which employ different map-ordering algorithms, so that a confident consensus map could be derived. MAPMAKER uses EM algorithms to obtain multipoint maximum-likelihood estimations, while CARTHAGENE combines the multipoint maximum-likelihood criteria with local search techniques. In addition, before map construction commenced, F₁ hybrid individual heterogeneity was calculated for all locus pairs within and across all reference pedigrees of the same sex using two-point linkage analysis results. This was to ensure that all individuals were representative of the collective data within each of the sexes.

Map construction was performed by first grouping the markers together using a stringent minimum LOD score of 4.0 and a maximum recombination fraction of 0.4 as linkage thresholds for both programs. Trial maps were obtained by inspection and by applying the two-point analysis method. Subsequent tests and validation of locus orders were performed using the more powerful maximum-likelihood methods incorporated in both MAPMAKER and CARTHAGENE. The Kosambi map function (KOSAMBI 1944 ) was used to convert recombination fractions to distances within both programs. Markers that did not maintain linkages with the original stringent LOD score threshold were placed within linkage groups only if their most likely position was supported by a LOD score of 3. A linkage map was considered fixed when loci within the linkage map could not be placed within 100:1 odds of their next maximum-likelihood positions. Loci within this range were placed in their maximum-likelihood positions for the final map. All tests were performed over male, female, and pooled reference pedigrees.

Once sex heterogeneity in recombination rates was taken into account, a consensus multipoint map was produced from the pooled data to ascertain a final sex-average genetic linkage map. Confidence intervals for the location of each marker were calculated using the Z_max - 1 criteria of CONNEALLY et al. 1985 utilized in the LINKMAP program of the LINKAGE package (LATHROP and LALOUEL 1988 ). To obtain confidence intervals, the location of each marker locus was varied (all other loci being fixed at their maximum-likelihood locations) to both the proximal and distal sides of its best location until the log₁₀ likelihood value decreased by at least one LOD unit. This method was repeated for each marker to provide an 90–95% confidence limit on each location in the multipoint map. The total multipoint map length and standard error of each linkage group and over the entire map were calculated according to OTT 1999 .

Heterogeneity:
Individual F₁ hybrid recombination heterogeneity within each reference pedigree was calculated using a likelihood ratio test, termed the M-test (OTT 1999 ), and follows a chi-square distribution with (c - 1) degrees of freedom. Sex-specific differences of the intervals, linkage groups, and over the entire map were calculated on the basis of the difference in likelihood values of the combined male/female linkage data with the sex-pooled data using the same number of markers and map order in each. Tests of differences in specific linkage intervals were calculated according to OTT 1999 . Differences in the linkage groups and across the entire map were tested by adding the chi-square values in each representative category, where the degree of freedom equals the number of intervals in each (ARCHIBALD et al. 1995 ). In addition, the ratios of female-to-male map distances (R = X_f/X_m) were calculated for each interval, linkage group, and over the entire map, where X is the map distance.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Genotyping:
With the sexes combined 64 genetic markers were informative for analysis in this study; 59 of these were typed across male pedigrees, while 52 genetic markers were typed over the female pedigrees. The average number (±SE) of informative meioses across the 64 loci was 212 ± 13 for both sexes combined, while the average number for males and females was 139 ± 7 and 104 ± 4, respectively (Table 1).

The integrity of the newly acquired genotypic data was monitored repeatedly throughout this project. Among duplicate typings and with scrutiny of Mendelian inheritance there was a 98.4% concordance with the original data. Most of the discrepancies (95%) were due to genotyping errors, which were remedied once detected. The remaining errors resulted from inconsistencies in the Mendelian inheritance of the markers. Locus T28-1 produced null alleles in 2 of the 21 female hybrids and their offspring, and six microsatellite loci exhibited a total of 11 stepwise mutations of one repeat unit. Following the detection of inconsistent inheritance of alleles, all discordant individuals were removed from the analysis with the exception of the mutations, which were reassigned inheritance where possible.

Analysis of segregation distortions within the sex-pooled data, separate male/female data, and sex-specific allelic inheritance data revealed no significant deviations following Bonferroni correction.

Map construction:
The two-point linkage analysis of the sex-pooled data revealed a total of 60 markers (93.8%) that showed significant linkage with at least one other marker at a LOD score of 3 or greater (except Me2 with LOD score of 2.58), while four markers (G16-2, MPI, TF, and Y148) did not show any linkage with any other markers.

Within the multipoint linkage analysis, both MAPMAKER and CARTHAGENE produced identical linkage groups and map-ordering results. On the basis of the consensus analysis of the sex-pooled data, nine groups were initially established with only locus T19-1 not being placed into a group at the stringent LOD threshold of 4.0. However, the most likely position of this marker was supported with a LOD score of 3.33 when the threshold was reduced. Ultimately, nine linkage groups ranging from 15.7 ± 2.1 cM (linkage group 9) to 176.5 ± 9.8 cM (linkage group 1) were formed based on the two-point and multipoint analysis. These linkage groups almost certainly cover proportions of all seven autosomes and the X chromosome and have a total map length of 828.4 ± 23.2 cM with an average distance between adjacent markers of 16.2 ± 1.8 cM (Fig 2).

View larger version (52K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Systematic representation of the sex-pooled multipoint linkage maps for LG1–9. Confidence limits (Zmax - 1) of maximum-likelihood locations for each locus are indicated in hatched bars. Distance (in centimorgans) of loci relative to first locus are indicated within parentheses as with total group map length (±SE). For comparison, human cytological positions of coding loci within linkage groups are indicated with an asterisk.

The sex-average multipoint linkage maps with approximate Z_max - 1 confidence intervals for each genetic marker are illustrated in Fig 2. Linkage groups 1and 4–9 exhibited comparatively discrete confidence limits attached to each marker's maximum-likelihood location. The minority of markers that exhibited confidence intervals that overlapped considerably with a neighboring marker were Y105–T3-1T and DBB–G12-6 within linkage group 2 and IL5–T28-1 and LPL–Me28 within linkage group 3. In each respective pair both markers have confidence intervals that overlap with each other, probably resulting from their close proximity to each other and the relatively small numbers of informative meioses events observed between each pair. These departures were also observed during validation of the multipoint map gene orders. Consequently, linkage groups 2 and 3 were placed at their most-likelihood gene order position, while all others demonstrated gene orders greater than the 100:1 odds criteria.

Recombination heterogeneity:
No significant deviations were observed between individuals for a particular recombination interval, providing evidence that the F₁ individuals are homogeneous within the sexes for recombination rates.

Sex-specific differences in recombination rates were evaluated for intervals, linkage groups, and over the entire map. Of the 37 intervals, 8 (22%) demonstrated significant sex-specific distortions (P < 0.05), all toward higher male recombination rates (Table 2). When investigating distortions over the linkage groups, five of the eight groups were significant (P < 0.05), with all significant groups incorporating at least 1 significant linkage interval (Table 2). Consequently, to determine if these significant results were caused only by the significant intervals or from an overall distortion across the linkage groups, the significant linkage pairs were removed and the groups reanalyzed. No linkage groups were significant following the removal of the significant linkage intervals, indicating that the trend for higher male recombination occurs at specific intervals and not across the entire linkage group. This was also observed when the overall map length was investigated for sex-specific distortions (Table 2).

Although less informative due to sampling error, the F/M distance ratio was comparable to the likelihood ratio tests. Results indicated a F/M ratio distortion of 0.78 toward a larger male map compared to the female map when incorporating significant intervals. However, after removal of the eight significant sex-skewed intervals, the ratio was 1.01 with both sexes displaying equivalent overall map lengths (Table 2).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Genotyping:
This study describes the first comprehensive framework genetic linkage map for a marsupial, the tammar wallaby (M. eugenii). This map amends and extends previous tammar wallaby linkage data (MCKENZIE et al. 1993 , MCKENZIE et al. 1996 ; TAYLOR and COOPER 1998 ) and contains 64 genetic markers, of which 43 are anonymous microsatellite markers, 14 are type-I coding genes, 6 are anonymous DNA markers, and 1 is a pseudogene.

The integrity of the genotypic data was high with an overall concordance of 98.4% when compared to duplicate typings and examination of Mendelian inheritance. Due to the relatively large amount of genetic divergence between the Kangaroo Island and Garden Island tammar wallaby populations (OLIVER et al. 1979 ; POOLE et al. 1991 ; MCKENZIE and COOPER 1997 ), it was a concern that null alleles may be present. However, examination of the pedigrees revealed only 2 female hybrid individuals and their offspring, which failed to amplify both alleles within locus T28-1. Considering that these null alleles were confined to only 2 of the 21 female hybrids, it is postulated that this is an individual characteristic rather than a hybrid effect.

Segregation distortion:
Segregation distortions have been observed in numerous crosses between genetically divergent genomes (e.g., SIRACUSA et al. 1991 ; XU et al. 1997 ; WHITKUS 1998 ; CHETELAT et al. 2000 ). In this study, backcross progeny genotypic ratios were calculated to detect distortions in gametic proportions. Unfortunately, due to the nature of this backcross experimental design, it is not possible to distinguish between gametic selection and viability selection postfertilization. Consequently, in this study, segregation distortions were investigated to check for non-Mendelian inheritance of alleles and the accountability of these for errors in this mapping project. The segregation distortion analysis of the overall and sex-specific data revealed no skewed distortions from the expected Mendelian ratios. Consequently, the linkage map has not been compromised by segregation distortion arising from gametic selection or by postzygotic epistatic selection originating from the hybrid nature of the cross. This result also confirms the original work of MCKENZIE and COOPER 1997 , who demonstrated hybridization between the two populations with no obvious loss of fitness.

Map construction:
The number of markers within the sex-pooled data that showed significant linkages (LOD > 3.0) with at least one other marker was high (93.8%), suggesting that a large proportion of the genome had been mapped. The four markers (G16-2, MPI, TF, and Y148) that did not show any linkage with any other markers with the exception of Y148 all had relatively low numbers of informative meioses and hence less power. The suggestion that a large proportion of the genome had been covered was strengthened when nine linkage groups of varying sizes were established (Fig 2). On the basis of the tammar wallaby karyotype (n = 8), these linkage groups almost certainly cover large proportions of all seven autosomes and the X chromosome. In fact, when comparing the estimated genome size of 1172 cM with 95% confidence intervals of 1145 and 1199 calculated from chiasmata data (SHARP 1984 ; SHARP and HAYMAN 1988 ) to the total linkage map length of 828.4 ± 23.2 cM calculated in this study, 71% of the total genome has been mapped. Interestingly, the estimated sex-average total map length of M. eugenii is considerably smaller when compared to sex-average human data (3600 cM; OTT 1999 ). Given that tammar wallabies have 15% more DNA content than humans have (HAYMAN and MARTIN 1974 ), it is proposed that this difference reflects an overall lower M. eugenii recombination rate rather than a smaller physical genome size.

Assignment of linkage groups:
All linkage groups excluding linkage group 7 can be placed on the autosomes on the basis of the Mendelian inheritance of markers. Using the inheritance pattern and physical assignment of genes, linkage group 7 is most certainly located on the X chromosome. In fact, all but locus T47-1 has been physically assigned to this chromosome by in situ hybridization or somatic cell hybrids in M. eugenii or closely related species (DONALD and HOPE 1981 ; ROBINS et al. 1984 ; DAWSON and GRAVES 1986 ; SPENCER et al. 1991A , SPENCER et al. 1991B ).

Utilizing loci that have been physically assigned to M. eugenii autosomes, it can be proposed that linkage groups 2 and 3 reside on chromosome 1, while linkage group 4 is located on chromosome 3. The assignment of linkage groups 2 and 3 to chromosome 1 is derived from the in situ hybridization of the DNA gene, which is expected to closely flank the DBB gene (SLADE et al. 1994 ), and from the location of the IL5 gene anchored via fluorescence in situ hybridization (HAWKEN et al. 1999 ). Although another marker (LLP) was located within linkage group 3, which had been physically assigned by in situ hybridization using radiolabeled probes (WESTERMAN et al. 1991 ), it was disregarded because of uncertain elements of its localization. The authors propose that, on the basis of grain density, LLP is located on chromosome 3, but they are unable to show clearly how they distinguished chromosome 3 from chromosome 4. However, they did attempt to distinguish them post-localization by means of exclusion Southern blot mapping to hybrid cells, although this indicated only that LLP was not positioned on chromosome 4. Interestingly, when chromosome 3 was disregarded, the most likely position was on chromosome 1, as in this study. Evidence for the location of linkage group 4 on chromosome 3 is derived from the physical mapping of HBB performed by SINCLAIR and GRAVES 1991 .

X chromosome:
On the basis of Mendelian inheritance and in situ hybridization, five genetic markers (AR, G6PD, RNR, HPRT, and T47-1) have been assigned to the X chromosome (Fig 2). The gene order of these markers is p-{(G6PD, RNR, AR)–HPRT–T47-1}-q, with the most likely orientation being G6PD, RNR, and AR flanking the centromere, HPRT 50% along the length of the long arm, and T47-1 positioned distally on the long arm.

Data presented in this study indicate close linkage between G6PD, RNR, and AR on the basis of recombination rates. Previous work performed by MCKENZIE et al. 1996 suggests that this reduced or absent recombination may be a result of the genes being located closely together in heterochromatic regions flanking the centromere. Based on the in situ hybridization of AR and RNR in M. eugenii, this appears to be correct (ROBINS et al. 1984 ; SPENCER et al. 1991B ). Although G6PD has been tentatively positioned on the distal third of the long arm (SPENCER et al. 1991A ), in situ hybridization results on the closely related species M. robustus (BLAIR 1993 ) and somatic cell hybrid studies performed on M. r. erubescens and M. giganteus (DAWSON and GRAVES 1986 ) contradict this placement and confirm the results in this study that G6PD is positioned near the centromere in close proximity to AR and RNR.

When incorporating all the data together, the linkage group presented for the X chromosome appears to be conserved in both synteny and gene order when compared to closely related species. The location of the G6PD and HPRT genes in M. eugenii within this linkage analysis is synonymous with in situ hybridization investigations in M. robustus (BLAIR 1993 ). In addition, somatic cell hybrid studies performed on M. r. erubescens and M. giganteus (DAWSON and GRAVES 1986 ) indicate that G6PD forms a syntenic group with HPRT and PGK1, with both G6PD and HPRT located in analogous positions as indicated within this study. In fact, PGK1 has also been localized to the same place in M. eugenii via in situ hybridization (COOPER et al. 1994 ).

Recombination heterogeneity:
Sex-specific differences in recombination rates of M. eugenii were evaluated for intervals, linkage groups, and over the entire map. The data revealed significant distortions toward reduced female recombination rates in 8 of the 36 intervals (22%; Table 2). These were localized to specific intervals and were not a uniform characteristic across the linkage groups or across the entire map. In fact, when the 8 significant linkage intervals were removed from the analysis no significant distortions were discovered and the overall female/male map distance ratio was 1:1. This result is not unexpected and is consistent with other eutherian and marsupial linkage investigations (e.g., BENNETT et al. 1986 ; OTT 1999 ; SAMOLLOW et al. 2000 ), which also indicate interval sex-specific distortions.

The reduced rate of female recombination observed within this investigation is contrary to a previous M. eugenii linkage study that used small numbers of loci and individuals (MCKENZIE et al. 1995 ). Their results have since been amended within this investigation, with the disparity being attributable to the limited data in the earlier study. Consequently, there is now strong evidence for reduced female recombination when compared to males in M. eugenii. This sex-specific difference is also consistent in other marsupial linkage studies performed on Sminthopsis crassicaudata and Monodelphis domestica (BENNETT et al. 1986 ; SAMOLLOW et al. 2000 ), but stands in contrast to eutherian studies where crossing over appears to be more frequent within the homogamatic sex (XX). Accordingly, it can be postulated that reduced female recombination rates in marsupials may be of considerable antiquity, since the marsupial species investigated have been separated by as much as 60–80 million years (SPRINGER et al. 1994 ; KIRSCH et al. 1997 ). In addition, it can be suggested that other marsupial species may well show similar differences. Unfortunately, linkage data from more distantly related marsupial families and from distant mammalian groups (i.e., monotremes) have yet to emerge.

Sex differences in recombination rates have been observed in numerous organisms (e.g., mammals, insects, and birds). However, there has been no theoretical consensus regarding the mechanisms controlling these recombination differences. Early theories of HALDANE 1922 and HUXLEY 1928 suggested that recombination tends to be reduced in the heterogametic sex. These views have been scrutinized on many occasions, both because there are some exceptions to the empirical generalizations and because the proposed explanations cannot account for the observed sex differences in many organisms (e.g., TRIVERS 1988 ; BURT et al. 1991 ; OTTO and BARTON 1997 ; BARTON and CHARLESWORTH 1998 ; LENORMAND and OTTO 2000 ). However, these alternate hypotheses may attempt to provide improved explanations for the sex differences, but they all have failed to explain the differences seen within marsupials. In fact, most theorists ignore marsupial data altogether when developing models, either because previously there had been limited studies or because the data did not adhere to their hypotheses. Whichever of these is the case, marsupials must now be considered when evaluating theories regarding recombination differences between the sexes.

	FOOTNOTES

Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession nos. AF503635 and AF503636.
² Present address: The Jackson Laboratory, 600 Main St., Bar Harbor, ME 04609.

	ACKNOWLEDGMENTS

We are especially thankful to Lisa Pope and Craig Moritz for providing details of unpublished microsatellite loci. We are also grateful to Mark Eldridge and the two anonymous referees for reviewing this manuscript and providing much valued advice, Bill Poole for locating the original tammar wallaby hybrid pedigree data buried within archives for more than 10 years, Michael Baxter for compiling and debugging many of the programs used throughout this project, and Professor Trevor Tansley, Dean of Graduate Students, Macquarie University, and Professor D. Yerbury, Vice Chancellor, Macquarie University, for financial support.

Manuscript received April 24, 2002; Accepted for publication June 6, 2002.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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