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We report here the isolation and sequencing of 10 Y-specific tammar wallaby (Macropus eugenii) BAC clones, revealing five hitherto undescribed tammar wallaby Y genes (in addition to the five genes already described) and several pseudogenes. Some genes on the wallaby Y display testis-specific expression, but most have low widespread expression. All have partners on the tammar X, along with homologs on the human X. Nonsynonymous and synonymous substitution ratios for nine of the tammar XY gene pairs indicate that they are each under purifying selection. All 10 were also identified as being on the Y in Tasmanian devil (Sarcophilus harrisii; a distantly related Australian marsupial); however, seven have been lost from the human Y. Maximum likelihood phylogenetic analyses of the wallaby YX genes, with respective homologs from other vertebrate representatives, revealed that three marsupial Y genes (HCFC1X/Y, MECP2X/Y, and HUWE1X/Y) were members of the ancestral therian pseudoautosomal region (PAR) at the time of the marsupial/eutherian split; three XY pairs (SOX3/SRY, RBMX/Y, and ATRX/Y) were isolated from each other before the marsupial/eutherian split, and the remaining three (RPL10X/Y, PHF6X/Y, and UBA1/UBE1Y) have a more complex evolutionary history. Thus, the small marsupial Y chromosome is surprisingly rich in ancient genes that are retained in at least Australian marsupials and evolved from testis–brain expressed genes on the X.

The Y chromosome is perhaps the most interesting element of the mammalian genome but comparative analysis of the Y chromosome has been impeded by the difficulty of assembling a shotgun sequence of the Y. BAC-based sequencing has been successful for the human and chimpanzee Y but is difficult to do efficiently for an atypical mammalian model species (Skaletsky et al. 2003, Kuroki et al. 2006). We show how Y-specific sub-libraries can be efficiently constructed using DNA amplified from microdissected or flow-sorted Y chromosomes. A Bacterial Artificial Chromosome (BAC) library was constructed from the model marsupial, the tammar wallaby (Macropus eugenii). We screened this library for Y chromosome-derived BAC clones using DNA from both a microdissected Y chromosome and a flow-sorted Y chromosome in order to create a Y chromosome-specific sub-library. We expected that the tammar wallaby Y chromosome should detect ∼100 clones from the 2.2 times redundant library. The microdissected Y DNA detected 85 clones, 82% of which mapped to the Y chromosome and the flow-sorted Y DNA detected 71 clones, 48% of which mapped to the Y chromosome. Overall, this represented a ∼330-fold enrichment for Y chromosome clones. This presents an ideal method for the creation of highly enriched chromosome-specific sub-libraries suitable for BAC-based sequencing of the Y chromosome of any mammalian species.

The X and Y chromosomes of humans and other mammals both have very atypical gene contents. The degenerate Y bears only a handful of genes that are specialized for male sex and reproduction. Now it seems that the X over-represents genes controlling reproductive traits and intelligence. This is hard to explain in terms of function but makes excellent sense in terms of evolution. Comparisons between the gene content of the X and Y in humans, distantly related mammals, and other vertebrates, define the evolutionary past of our sex chromosomes and suggest how special selective forces act on the X and Y.

During male sexual development in reptiles, birds, and mammals, anti-Mullerian hormone (AMH) induces the regression of the Mullerian ducts that normally form the primordia of the female reproductive tract. Whereas Mullerian duct regression occurs during fetal development in eutherian mammals, in marsupial mammals this process occurs after birth. To investigate AMH in a marsupial, we isolated an orthologue from the tammar wallaby (Macropus eugenii) and characterized its expression in the testes and ovaries during development. The wallaby AMH gene is highly conserved with the eutherian orthologues that have been studied, particularly within the encoded C-terminal mature domain. The N-terminus of marsupial AMH is divergent and larger than that of eutherian species. It is located on chromosome 3/4, consistent with its autosomal localization in other species. The wallaby 5' regulatory region, like eutherian AMH genes, contains binding sites for SF1, SOX9, and GATA factors but also contains a putative SRY-binding site. AMH expression in the developing testis begins at the time of seminiferous cord formation at 2 days post partum, and Mullerian duct regression begins shortly afterward. In the developing testis, AMH is localized in the cytoplasm of the Sertoli cells but is lost by adulthood. In the developing ovary, there is no detectable AMH expression, but in adults it is produced by the granulosa cells of primary and secondary follicles. It is not detectable in atretic follicles. Collectively, these studies suggest that AMH expression has been conserved during mammalian evolution and is intimately linked to upstream sex determination mechanisms.

Microsatellites were identified from three fully sequenced Y chromosome-specific bacterial artificial chromosome clones from the tammar wallaby, Macropus eugenii. Ten microsatellites were genotyped in male tammar wallabies. Four loci were polymorphic with between two and six alleles per locus. Eleven different haplotypes were identified from 22 male tammar wallabies. No amplifications were obtained from female samples. Each microsatellite was also shown to amplify reliably in at least one other macropod species. These markers may therefore prove useful as some of the first male-specific genetic markers for marsupials, with potential application to studies of male-biased dispersal and mating systems.

Comparative molecular cytogenetics provides a powerful tool for deciphering the evolutionary history of vertebrate sex chromosomes. We have adapted cell culture and molecular cytogenetic techniques to study the sex chromosomes of many exotic mammals, birds, and reptiles. Here we describe differential chromosome banding and staining techniques that distinguish sex chromosomes in species with no morphologically distinct XY or ZW chromosome pairs. We describe a method to isolate, identify, and map genomic BAC clones from the Y chromosome, and we also identify strategies for isolating candidate sex chromosome genes.

Comparative molecular cytogenetics provides a powerful tool for deciphering the evolutionary history of vertebrate sex chromosomes. We have adapted cell culture and molecular cytogenetic techniques to study the sex chromosomes of many exotic mammals, birds, and reptiles. Here we describe differential chromosome banding and staining techniques that distinguish sex chromosomes in species with no morphologically distinct XY or ZW chromosome pairs. We describe a method to isolate, identify, and map genomic BAC clones from the Y chromosome, and we also identify strategies for isolating candidate sex chromosome genes.

Marsupials occupy a phylogenetic middle ground that is very valuable in genome comparisons of mammal and other vertebrate species. For this reason, whole genome sequencing is being undertaken for two distantly related marsupial species, including the model kangaroo species Macropus eugenii (the tammar wallaby). As a first step towards the molecular characterization of the tammar genome, we present a detailed description of the tammar karyotype, report the development of a set of molecular anchor markers and summarize the comparative mapping data for this species.

All mammals have an XX female:XY male sex chromosome system. The X and Y are thought to have evolved from a homologous pair of autosomes by a process of Y degradation. This process left a small, heterochromatic and gene-poor Y, and a relatively unchanged X chromosome (Ohno, 1967). The origin of the human sex chromosomes has been explored by comparing their gene content with that of sex chromosomes from distantly related mammals. Previous work has defined conserved (XCR, shared with the X of marsupials and monotremes) and added regions of the human X (XAR, homologous to marsupial and monotreme autosomal regions, represented by chromosome 5p in the tammar wallaby) (Watson et al., 1991; Spencer et al., 1991). As a degraded X, the Y also consists of conserved (YCR) and added (YAR) regions (Graves, 1995; Waters et al., 2001). We cloned and characterized the tammar wallaby homologue of TMSB4X (GenBank accession number AY341342), a gene with a copy on both the X and Y in human, and mapped it by fluorescent in situ hybridisation. No signals were found over the X or Y chromosome, but clear signals were apparent on chromosome 5p. This location is consistent with the hypothesis that the region of human Xp that includes TMSB4X was recently added (XAR), and that the human Y is largely derived from the same added region.

During male sexual development in reptiles, birds, and mammals, anti-Müllerian hormone (AMH) induces the regression of the Müllerian ducts that normally form the primordia of the female reproductive tract. Whereas Müllerian duct regression occurs during fetal development in eutherian mammals, in marsupial mammals this process occurs after birth. To investigate AMH in a marsupial, we isolated an orthologue from the tammar wallaby (Macropus eugenii) and characterized its expression in the testes and ovaries during development. The wallaby AMH gene is highly conserved with the eutherian orthologues that have been studied, particularly within the encoded C-terminal mature domain. The N-terminus of marsupial AMH is divergent and larger than that of eutherian species. It is located on chromosome 3/4, consistent with its autosomal localization in other species. The wallaby 5' regulatory region, like eutherian AMH genes, contains binding sites for SF1, SOX9, and GATA factors but also contains a putative SRY-binding site. AMH expression in the developing testis begins at the time of seminiferous cord formation at 2 days post partum, and Müllerian duct regression begins shortly afterward. In the developing testis, AMH is localized in the cytoplasm of the Sertoli cells but is lost by adulthood. In the developing ovary, there is no detectable AMH expression, but in adults it is produced by the granulosa cells of primary and secondary follicles. It is not detectable in atretic follicles. Collectively, these studies suggest that AMH expression has been conserved during mammalian evolution and is intimately linked to upstream sex determination mechanisms.

With the accelerating recognition of the power of comparative genomics, there is now enormous interest in sequencing the genomes of a broad range of species. Marsupials diverged at an important evolutionary time. The model Australian marsupial, the tammar wallaby (Macropus eugenii), has long been a resource for biological and genetic studies of marsupials, and the availability of a bacterial artificial chromosome (BAC) library will be a valuable resource in these studies. A tammar wallaby BAC library was constructed using pRazorBAC vector. It contains 55 296 clones with an average insert size of 108 kb, representing 2.2 times coverage of the wallaby genome (based on an estimated 2.7 × 109 bp haploid genome size). The library was arrayed in 384-well plates, and spotted in duplicate onto nylon membranes. Screening these membranes has yielded clones containing 34 single-copy genes distributed over the genome, while it failed for only one gene. Each probe isolated 1–12 BAC clones and, to date, no chimeric clones have been found. This BAC library will constitute an invaluable resource for creating physical maps, positional cloning of genes and other sequences in the tammar wallaby, as well as comparative mapping studies in mammals.