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.

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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 approximately 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 approximately 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.