Genome sequence of an Australian kangaroo, Macropus eugenii, provides insight into the evolution of mammalian reproduction and development

Abstract

Background: We present the genome sequence of the tammar wallaby, Macropus eugenii, which is a member of the kangaroo family and the first representative of the iconic hopping mammals that symbolize Australia to be sequenced. The tammar has many unusual biological characteristics, including the longest period of embryonic diapause of any mammal, extremely synchronized seasonal breeding and prolonged and sophisticated lactation within a well-defined pouch. Like other marsupials, it gives birth to highly altricial young, and has a small number of very large chromosomes, making it a valuable model for genomics, reproduction and development.

Results: The genome has been sequenced to 2 × coverage using Sanger sequencing, enhanced with additional next generation sequencing and the integration of extensive physical and linkage maps to build the genome assembly. We also sequenced the tammar transcriptome across many tissues and developmental time points. Our analyses of these data shed light on mammalian reproduction, development and genome evolution: there is innovation in reproductive and lactational genes, rapid evolution of germ cell genes, and incomplete, locus-specific X inactivation. We also observe novel retrotransposons and a highly rearranged major histocompatibility complex, with many class I genes located outside the complex. Novel microRNAs in the tammar HOX clusters uncover new potential mammalian HOX regulatory elements.

Conclusions: Analyses of these resources enhance our understanding of marsupial gene evolution, identify marsupial-specific conserved non-coding elements and critical genes across a range of biological systems, including reproduction, development and immunity, and provide new insight into marsupial and mammalian biology and genome evolution.

Figure 1

Phylogeny of the marsupials. Phylogenetic relationships of the orders of Marsupialia. Top: the placement of the contemporary continents of South America and Australia within Gondwanaland and the split of the American and Australian marsupials. Relative divergence in millions of years shown to the left in the context of geological periods. The relationship of the Macropodide within the Australian marsupial phylogeny shown is in purple with estimated divergence dates in millions of years [5,162,163]. Representative species from each clade are illustrated. Inset: phylogeny of the genus Macropus within the Macropodidae showing the placement of the model species M. eugenii (purple) based on [59]. Outgroup species are Thylogale thetis and Petrogale xanthopus.

Figure 2

Homology of tammar regions to the human karyotype, and location of major histocompatibility complex, classical class I genes and olfactory receptor gene. Colored blocks represent the syntenic blocks with human chromosomes as shown in the key. A map of the locations of the tammar major histocompatibility complex (MHC) is shown on the right-hand side of each chromosome. The rearranged MHCs are on chromosome 2 and clusters of MHC class I genes (red) near the telomeric regions of chromosomes 1, 4, 5, 6, and 7. MHC class II genes are shown in blue, olfactory receptors are shown in orange and Kangaroo endogenous retroviral elements found within these clusters are shown in green. The location of the conserved mammalian OR gene clusters in the tammar genome are shown on the left-hand side of each chromosome. OR genes are found on every chromosome, except for chromosome 6 but including the X. The location of the OR gene clusters (numbers) are shown, and their approximate size is represented by lines of different thickness.

Figure 3

Classification of novel tammar genes. Summary of protein domains contained within translated novel ESTs isolated from the tammar transcriptomes. A large proportion of unique genes contain receptor or transcriptional regulator domains. The next largest classes of unique ESTs were immune genes, whey acidic protein and lipid domain containing genes. These findings suggest a rapid diversification of genes associated with immune function and lactation in the tammar.

Figure 4

A survey of both conserved and novel small RNAs in the tammar genome. (a) Size ranges of the major classes of small RNAs. The x-axis shows number of reads mapped to the tammar genome while the size of the read in nucleotides is on the y-axis. Boxes denote each major class analyzed in the tammar. Classes targeted for sequencing and full annotation include the miRNAs (18 to 22 nucleotides), the piRNAs (28 to 32 nucleotides) and the newly discovered crasiRNAs (35 to 45 nucleotides). (b) Five tammar miRNA libraries (brain, liver, fibroblast, ovary and testis) were pooled and mapped to the tammar genome. miRNAs with a complete overlap with miRBase entries mapped to the tammar genome were considered conserved and annotated according to species. Heat map showing the frequency of conserved mirBase entries per tissue and per species as identified in the tammar. A high degree of overlap (that is, conservation) was observed between tammar and human for fibroblast and testis, but a relatively low degree of overlap was observed for the brain. (c) The complex tammar centromere. Genome browser view of chromatin immunoprecipitation-sequencing (ChIP-Seq) for DNA bound by the centromere-specific histone CENP-A mapped to a centromeric contig (top, blue). Nucleotide position on the contig is shown on the x-axis and depth of reads shown on the y-axis. Tracks illustrated: MACs peak (model-based analyses of Chip-Seq (black); locations for mapped reads of crasiRNAs (red); location of annotated centromere sequences (in this example, the centromeric LINE L6; purple); modeler repeat prediction track (green). crasiRNAs co-localize to DNA found in CENP-A-containing nucleosomes and are enriched in regions containing known centromere sequences.

Figure 5

Comparative map of X and Y chromosomes. Comparison of X/Y shared gene locations on the tammar wallaby, grey short-tailed opossum and human X chromosomes. Blue represents the X conserved region, which is common to all therian X chromosomes. Green represents the X added region, which is on the X in eutherian mammals, but autosomal in marsupial mammals. Ten genes have been identified on the short arm of the tammar Y chromosome, all with a partner on the X, and an orthologue on the Tasmanian devil Y. In contrast, only four genes on the human Y have a partner on the conserved region of the X.

Figure 6

Desert hedgehog phylogeny. A phylogenetic tree showing the relationship of the SHH, IHH, DHH, and fish desert-like genes. Each group is composed of representatives from mammalian and non-mammalian species. The mammalian DHH group (green) clusters tightly and forms a separate linage to the fish DHH-like genes (red), which are no more closely related to DHH than they are to vertebrate IHH (yellow) and SHH (blue). Hs, human; Tt, dolphin; Xt, Xenopus; Gag, chicken; Mum, mouse; Me, tammar.

Figure 7

HOX genes in the tammar. mVISTA comparison of partial HOXC cluster highlights conserved HOX genes and non-coding RNAs between human and tammar. In the coding regions, HOXC11 and HOXC10 are highly conserved between human and tammar. In the intergenic regions, some conserved regions shown are non-coding RNAs (long non-coding RNA such as HOTAIR, and miRNAs such as mir-196) or unknown motifs participating in gene expression and regulation. The percentage of identities (50 to 100%) (Vertical axis) is displayed in the coordinates of the genomic sequence (horizontal axis).

Figure 8

Olfaction in the tammar. (a) The olfactory apparatus of the tammar showing the pattern of vomeronasal receptor projections to the accessory olfactory bulb with the VN2 receptor cells (expressing Goα) projecting to all parts of the vomeronasal nerve layer (which may also be the case for the VN1 receptor cells (expressing Giα2). This projection pattern may reflect an intermediate type to the 'segregated type' and the 'uniform type' so far described. AOB, accessory olfactory bulb; GL, glomerular layer; GRL, granule cell layer; MOB, main olfactory bulb; MTL, mitral tufted cell layer; VNL, vomeronasal nerve layer; VNO, vomeronasal organ; VN1R and VN2R, vomeronasal receptors 1 and 2. (b) Olfactory receptor (OR) gene family in the tammar. The families of the OR gene repertoire. Neighbor joining tree of 456 full-length functional OR genes was rooted with opossum adrenergic β receptor. Only a few OR gene families (14, 51 and 52) have members that are most closely related to each other, whilst most other families have a high degree of relatedness to other families.