Polygenic sex determination in vertebrates - is there any such thing?

Genetic sex determination (SD) in most vertebrates is controlled by a single master sex gene, which ensures a 1:1 sex ratio. However, more complex systems abound, and several have been ascribed to polygenic SD (PSD), in which many genes at different loci interact to produce the sexual phenotype. Here we examine claims for PSD in vertebrates, finding that most constitute transient states during sex chromosome turnover, or aberrant systems in species hybrids. To avoid confusion about terminology, we propose a consistent nomenclature for genetic SD systems.

Two transcriptionally distinct pathways drive female development in a reptile with both genetic and temperature dependent sex determination.

How temperature determines sex remains unknown. A recent hypothesis proposes that conserved cellular mechanisms (calcium and redox; 'CaRe' status) sense temperature and identify genes and regulatory pathways likely to be involved in driving sexual development. We take advantage of the unique sex determining system of the model organism, Pogona vitticeps, to assess predictions of this hypothesis. P. vitticeps has ZZ male: ZW female sex chromosomes whose influence can be overridden in genetic males by high temperatures, causing male-to-female sex reversal. We compare a developmental transcriptome series of ZWf females and temperature sex reversed ZZf females. We demonstrate that early developmental cascades differ dramatically between genetically driven and thermally driven females, later converging to produce a common outcome (ovaries). We show that genes proposed as regulators of thermosensitive sex determination play a role in temperature sex reversal. Our study greatly advances the search for the mechanisms by which temperature determines sex.

Gene expression of male pathway genes sox9 and amh during early sex differentiation in a reptile departs from the classical amniote model.

BACKGROUND: Sex determination is the process whereby the bipotential embryonic gonads become committed to differentiate into testes or ovaries. In genetic sex determination (GSD), the sex determining trigger is encoded by a gene on the sex chromosomes, which activates a network of downstream genes; in mammals these include SOX9, AMH and DMRT1 in the male pathway, and FOXL2 in the female pathway. Although mammalian and avian GSD systems have been well studied, few data are available for reptilian GSD systems. RESULTS: We conducted an unbiased transcriptome-wide analysis of gonad development throughout differentiation in central bearded dragon (Pogona vitticeps) embryos with GSD. We found that sex differentiation of transcriptomic profiles occurs at a very early stage, before the gonad consolidates as a body distinct from the gonad-kidney complex. The male pathway genes dmrt1 and amh and the female pathway gene foxl2 play a key role in early sex differentiation in P. vitticeps, but the central player of the mammalian male trajectory, sox9, is not differentially expressed in P. vitticeps at the bipotential stage. The most striking difference from GSD systems of other amniotes is the high expression of the male pathway genes amh and sox9 in female gonads during development. We propose that a default male trajectory progresses if not repressed by a W-linked dominant gene that tips the balance of gene expression towards the female trajectory. Further, weighted gene expression correlation network analysis revealed novel candidates for male and female sex differentiation. CONCLUSION: Our data reveal that interpretation of putative mechanisms of GSD in reptiles cannot solely depend on lessons drawn from mammals.

Sex reversal triggers the rapid transition from genetic to temperature-dependent sex.

Sex determination in animals is amazingly plastic. Vertebrates display contrasting strategies ranging from complete genetic control of sex (genotypic sex determination) to environmentally determined sex (for example, temperature-dependent sex determination). Phylogenetic analyses suggest frequent evolutionary transitions between genotypic and temperature-dependent sex determination in environmentally sensitive lineages, including reptiles. These transitions are thought to involve a genotypic system becoming sensitive to temperature, with sex determined by gene-environment interactions. Most mechanistic models of transitions invoke a role for sex reversal. Sex reversal has not yet been demonstrated in nature for any amniote, although it occurs in fish and rarely in amphibians. Here we make the first report of reptile sex reversal in the wild, in the Australian bearded dragon (Pogona vitticeps), and use sex-reversed animals to experimentally induce a rapid transition from genotypic to temperature-dependent sex determination. Controlled mating of normal males to sex-reversed females produces viable and fertile offspring whose phenotypic sex is determined solely by temperature (temperature-dependent sex determination). The W sex chromosome is eliminated from this lineage in the first generation. The instantaneous creation of a lineage of ZZ temperature-sensitive animals reveals a novel, climate-induced pathway for the rapid transition between genetic and temperature-dependent sex determination, and adds to concern about adaptation to rapid global climate change.

Landscape of DNA Methylation on the Marsupial X.

DNA methylation plays a key role in maintaining transcriptional silence on the inactive X chromosome of eutherian mammals. Beyond eutherians, there are limited genome wide data on DNA methylation from other vertebrates. Previous studies of X borne genes in various marsupial models revealed no differential DNA methylation of promoters between the sexes, leading to the conclusion that CpG methylation plays no role in marsupial X-inactivation. Using reduced representation bisulfite sequencing, we generated male and female CpG methylation profiles in four representative vertebrates (mouse, gray short-tailed opossum, platypus, and chicken). A variety of DNA methylation patterns were observed. Platypus and chicken displayed no large-scale differential DNA methylation between the sexes on the autosomes or the sex chromosomes. As expected, a metagene analysis revealed hypermethylation at transcription start sites (TSS) of genes subject to X-inactivation in female mice. This contrasted with the opossum, in which metagene analysis did not detect differential DNA methylation between the sexes at TSSs of genes subject to X-inactivation. However, regions flanking TSSs of these genes were hypomethylated. Our data are the first to demonstrate that, for genes subject to X-inactivation in both eutherian and marsupial mammals, there is a consistent difference between DNA methylation levels at TSSs and immediate flanking regions, which we propose has a silencing effect in both groups.

Independent evolution of transcriptional inactivation on sex chromosomes in birds and mammals.

X chromosome inactivation in eutherian mammals has been thought to be tightly controlled, as expected from a mechanism that compensates for the different dosage of X-borne genes in XX females and XY males. However, many X genes escape inactivation in humans, inactivation of the X in marsupials is partial, and the unrelated sex chromosomes of monotreme mammals have incomplete and gene-specific inactivation of X-linked genes. The bird ZW sex chromosome system represents a third independently evolved amniote sex chromosome system with dosage compensation, albeit partial and gene-specific, via an unknown mechanism (i.e. upregulation of the single Z in females, down regulation of one or both Zs in males, or a combination). We used RNA-fluorescent in situ hybridization (RNA-FISH) to demonstrate, on individual fibroblast cells, inactivation of 11 genes on the chicken Z and 28 genes on the X chromosomes of platypus. Each gene displayed a reproducible frequency of 1Z/1X-active and 2Z/2X-active cells in the homogametic sex. Our results indicate that the probability of inactivation is controlled on a gene-by-gene basis (or small domains) on the chicken Z and platypus X chromosomes. This regulatory mechanism must have been exapted independently to the non-homologous sex chromosomes in birds and mammals in response to an over-expressed Z or X in the homogametic sex, highlighting the universal importance that (at least partial) silencing plays in the evolution on amniote dosage compensation and, therefore, the differentiation of sex chromosomes.

Genome analysis of the platypus reveals unique signatures of evolution.

We present a draft genome sequence of the platypus, Ornithorhynchus anatinus. This monotreme exhibits a fascinating combination of reptilian and mammalian characters. For example, platypuses have a coat of fur adapted to an aquatic lifestyle; platypus females lactate, yet lay eggs; and males are equipped with venom similar to that of reptiles. Analysis of the first monotreme genome aligned these features with genetic innovations. We find that reptile and platypus venom proteins have been co-opted independently from the same gene families; milk protein genes are conserved despite platypuses laying eggs; and immune gene family expansions are directly related to platypus biology. Expansions of protein, non-protein-coding RNA and microRNA families, as well as repeat elements, are identified. Sequencing of this genome now provides a valuable resource for deep mammalian comparative analyses, as well as for monotreme biology and conservation.

Extant and extinct bilby genomes combined with Indigenous knowledge improve conservation of a unique Australian marsupial.

Ninu (greater bilby, Macrotis lagotis) are desert-dwelling, culturally and ecologically important marsupials. In collaboration with Indigenous rangers and conservation managers, we generated the Ninu chromosome-level genome assembly (3.66 Gbp) and genome sequences for the extinct Yallara (lesser bilby, Macrotis leucura). We developed and tested a scat single-nucleotide polymorphism panel to inform current and future conservation actions, undertake ecological assessments and improve our understanding of Ninu genetic diversity in managed and wild populations. We also assessed the beneficial impact of translocations in the metapopulation (N = 363 Ninu). Resequenced genomes (temperate Ninu, 6; semi-arid Ninu, 6; and Yallara, 4) revealed two major population crashes during global cooling events for both species and differences in Ninu genes involved in anatomical and metabolic pathways. Despite their 45-year captive history, Ninu have fewer long runs of homozygosity than other larger mammals, which may be attributable to their boom-bust life history. Here we investigated the unique Ninu biology using 12 tissue transcriptomes revealing expression of all 115 conserved eutherian chorioallantoic placentation genes in the uterus, an XY1Y2 sex chromosome system and olfactory receptor gene expansions. Together, we demonstrate the holistic value of genomics in improving key conservation actions, understanding unique biological traits and developing tools for Indigenous rangers to monitor remote wild populations.

Sequence and gene content of a large fragment of a lizard sex chromosome and evaluation of candidate sex differentiating gene R-spondin 1.

BACKGROUND: Scant genomic information from non-avian reptile sex chromosomes is available, and for only a few lizards, several snakes and one turtle species, and it represents only a small fraction of the total sex chromosome sequences in these species. RESULTS: We report a 352 kb of contiguous sequence from the sex chromosome of a squamate reptile, Pogona vitticeps, with a ZZ/ZW sex microchromosome system. This contig contains five protein coding genes (oprd1, rcc1, znf91, znf131, znf180), and major families of repetitive sequences with a high number of copies of LTR and non-LTR retrotransposons, including the CR1 and Bov-B LINEs. The two genes, oprd1 and rcc1 are part of a homologous syntenic block, which is conserved among amniotes. While oprd1 and rcc1 have no known function in sex determination or differentiation in amniotes, this homologous syntenic block in mammals and chicken also contains R-spondin 1 (rspo1), the ovarian differentiating gene in mammals. In order to explore the probability that rspo1 is sex determining in dragon lizards, genomic BAC and cDNA clones were mapped using fluorescence in situ hybridisation. Their location on an autosomal microchromosome pair, not on the ZW sex microchromosomes, eliminates rspo1 as a candidate sex determining gene in P. vitticeps. CONCLUSION: Our study has characterized the largest contiguous stretch of physically mapped sex chromosome sequence (352 kb) from a ZZ/ZW lizard species. Although this region represents only a small fraction of the sex chromosomes of P. vitticeps, it has revealed several features typically associated with sex chromosomes including the accumulation of large blocks of repetitive sequences.

Truncated jarid2 and kdm6b transcripts are associated with temperature-induced sex reversal during development in a dragon lizard.

Sex determination and differentiation in reptiles are complex. In the model species, Pogona vitticeps, high incubation temperature can cause male to female sex reversal. To elucidate the epigenetic mechanisms of thermolabile sex, we used an unbiased genome-wide assessment of intron retention during sex reversal. The previously implicated chromatin modifiers (jarid2 and kdm6b) were two of three genes to display sex reversal-specific intron retention. In these species, embryonic intron retention resulting in C-terminally truncated jarid2 and kdm6b isoforms consistently occurs at low temperatures. High-temperature sex reversal is uniquely characterized by a high prevalence of N-terminally truncated isoforms of jarid2 and kdm6b, which are not present at low temperatures, or in two other reptiles with temperature-dependent sex determination. This work verifies that chromatin-modifying genes are involved in highly conserved temperature responses and can also be transcribed into isoforms with new sex-determining roles.

A first-generation integrated tammar wallaby map and its use in creating a tammar wallaby first-generation virtual genome map.

BACKGROUND: The limited (2X) coverage of the tammar wallaby (Macropus eugenii) genome sequence dataset currently presents a challenge for assembly and anchoring onto chromosomes. To provide a framework for this assembly, it would be a great advantage to have a dense map of the tammar wallaby genome. However, only limited mapping data are available for this non-model species, comprising a physical map and a linkage map. RESULTS: We combined all available tammar wallaby mapping data to create a tammar wallaby integrated map, using the Location DataBase (LDB) strategy. This first-generation integrated map combines all available information from the second-generation tammar wallaby linkage map with 148 loci, and extensive FISH mapping data for 492 loci, especially for genes likely to be located at the ends of wallaby chromosomes or at evolutionary breakpoints inferred from comparative information. For loci whose positions are only approximately known, their location in the integrated map was refined on the basis of comparative information from opossum (Monodelphis domestica) and human. Interpolation of segments from the opossum and human assemblies into the integrated map enabled the subsequent construction of a tammar wallaby first-generation virtual genome map, which comprises 14336 markers, including 13783 genes recruited from opossum and human assemblies. Both maps are freely available at http://compldb.angis.org.au. CONCLUSIONS: The first-generation integrated map and the first-generation virtual genome map provide a backbone for the chromosome assembly of the tammar wallaby genome sequence. For example, 78% of the 10257 gene-scaffolds in the Ensembl annotation of the tammar wallaby genome sequence (including 10522 protein-coding genes) can now be given a chromosome location in the tammar wallaby virtual genome map.

A second-generation anchored genetic linkage map of the tammar wallaby (Macropus eugenii).

BACKGROUND: The tammar wallaby, Macropus eugenii, a small kangaroo used for decades for studies of reproduction and metabolism, is the model Australian marsupial for genome sequencing and genetic investigations. The production of a more comprehensive cytogenetically-anchored genetic linkage map will significantly contribute to the deciphering of the tammar wallaby genome. It has great value as a resource to identify novel genes and for comparative studies, and is vital for the ongoing genome sequence assembly and gene ordering in this species. RESULTS: A second-generation anchored tammar wallaby genetic linkage map has been constructed based on a total of 148 loci. The linkage map contains the original 64 loci included in the first-generation map, plus an additional 84 microsatellite loci that were chosen specifically to increase coverage and assist with the anchoring and orientation of linkage groups to chromosomes. These additional loci were derived from (a) sequenced BAC clones that had been previously mapped to tammar wallaby chromosomes by fluorescence in situ hybridization (FISH), (b) End sequence from BACs subsequently FISH-mapped to tammar wallaby chromosomes, and (c) tammar wallaby genes orthologous to opossum genes predicted to fill gaps in the tammar wallaby linkage map as well as three X-linked markers from a published study. Based on these 148 loci, eight linkage groups were formed. These linkage groups were assigned (via FISH-mapped markers) to all seven autosomes and the X chromosome. The sex-pooled map size is 1402.4 cM, which is estimated to provide 82.6% total coverage of the genome, with an average interval distance of 10.9 cM between adjacent markers. The overall ratio of female/male map length is 0.84, which is comparable to the ratio of 0.78 obtained for the first-generation map. CONCLUSIONS: Construction of this second-generation genetic linkage map is a significant step towards complete coverage of the tammar wallaby genome and considerably extends that of the first-generation map. It will be a valuable resource for ongoing tammar wallaby genetic research and assembling the genome sequence. The sex-pooled map is available online at http://compldb.angis.org.au/.

Evolutionary transitions between mechanisms of sex determination in vertebrates.

Sex in many organisms is a dichotomous phenotype--individuals are either male or female. The molecular pathways underlying sex determination are governed by the genetic contribution of parents to the zygote, the environment in which the zygote develops or interaction of the two, depending on the species. Systems in which multiple interacting influences or a continuously varying influence (such as temperature) determines a dichotomous outcome have at least one threshold. We show that when sex is viewed as a threshold trait, evolution in that threshold can permit novel transitions between genotypic and temperature-dependent sex determination (TSD) and remarkably, between male (XX/XY) and female (ZZ/ZW) heterogamety. Transitions are possible without substantive genotypic innovation of novel sex-determining mutations or transpositions, so that the master sex gene and sex chromosome pair can be retained in ZW-XY transitions. We also show that evolution in the threshold can explain all observed patterns in vertebrate TSD, when coupled with evolution in embryonic survivorship limits.

Non-homologous sex chromosomes of birds and snakes share repetitive sequences.

Snake sex chromosomes provided Susumo Ohno with the material on which he based his theory of how sex chromosomes differentiate from autosomal pairs. Like birds, snakes have a ZZ male/ZW female sex chromosome system, in which the snake Z is a macrochromosome much the same size as the bird Z. However, the gene content shows clearly that the snake and bird Z chromosomes are completely non-homologous. The molecular aspect of W chromosome degeneration in snakes remains largely unexplored. We used comparative genomic hybridization to identify the female-specific region of the W chromosome in representative species of Australian snakes. Using this approach, we show that an increasingly complex suite of repeats accompanies the evolution of W chromosome heteromorphy. In particular, we found that while the python Liasis fuscus exhibits no sex-specific repeats and indeed, no cytologically recognizable sex-specific region, the colubrid Stegonotus cucullatus shows a large domain on the short arm of the W chromosome that consists of female-specific repeats, and the large W of Notechis scutatus is composed almost entirely of repetitive sequences, including Bkm and 18S rDNA-related elements. FISH mapping of both simple and complex probes shows patterns of repeat amplification concordant with the size of the female-specific region in each species examined. Mapping of intronic sequences of genes that are sex-linked in both birds (DMRT1) and snakes (CTNNB1) reveals massive amplification in discrete domains on the W chromosome of the elapid N. scutatus. Using chicken W chromosome paint, we demonstrate that repetitive sequences are shared between the sex chromosomes of birds and derived snakes. This could be explained by ancestral but as yet undetected shared synteny of bird and snake sex chromosomes or may indicate functional homology of the repeats and suggests that degeneration is a convergent property of sex chromosome evolution. We also establish that synteny of snake Z-linked genes has been conserved for at least 166 million years and that the snake Z consists of two conserved blocks derived from the same ancestral vertebrate chromosome.

The evolution of epigenetic regulators CTCF and BORIS/CTCFL in amniotes.

CTCF is an essential, ubiquitously expressed DNA-binding protein responsible for insulator function, nuclear architecture, and transcriptional control within vertebrates. The gene CTCF was proposed to have duplicated in early mammals, giving rise to a paralogue called "brother of regulator of imprinted sites" (BORIS or CTCFL) with DNA binding capabilities similar to CTCF, but testis-specific expression in humans and mice. CTCF and BORIS have opposite regulatory effects on human cancer-testis genes, the anti-apoptotic BAG1 gene, the insulin-like growth factor 2/H19 imprint control region (IGF2/H19 ICR), and show mutually exclusive expression in humans and mice, suggesting that they are antagonistic epigenetic regulators. We discovered orthologues of BORIS in at least two reptilian species and found traces of its sequence in the chicken genome, implying that the duplication giving rise to BORIS occurred much earlier than previously thought. We analysed the expression of CTCF and BORIS in a range of amniotes by conventional and quantitative PCR. BORIS, as well as CTCF, was found widely expressed in monotremes (platypus) and reptiles (bearded dragon), suggesting redundancy or cooperation between these genes in a common amniote ancestor. However, we discovered that BORIS expression was gonad-specific in marsupials (tammar wallaby) and eutherians (cattle), implying that a functional change occurred in BORIS during the early evolution of therian mammals. Since therians show imprinting of IGF2 but other vertebrate taxa do not, we speculate that CTCF and BORIS evolved specialised functions along with the evolution of imprinting at this and other loci, coinciding with the restriction of BORIS expression to the germline and potential antagonism with CTCF.

The status of dosage compensation in the multiple X chromosomes of the platypus.

Dosage compensation has been thought to be a ubiquitous property of sex chromosomes that are represented differently in males and females. The expression of most X-borne genes is equalized between XX females and XY males in therian mammals (marsupials and "placentals") by inactivating one X chromosome in female somatic cells. However, compensation seems not to be strictly required to equalize the expression of most Z-borne genes between ZZ male and ZW female birds. Whether dosage compensation operates in the third mammal lineage, the egg-laying monotremes, is of considerable interest, since the platypus has a complex sex chromosome system in which five X and five Y chromosomes share considerable genetic homology with the chicken ZW sex chromosome pair, but not with therian XY chromosomes. The assignment of genes to four platypus X chromosomes allowed us to examine X dosage compensation in this unique species. Quantitative PCR showed a range of compensation, but SNP analysis of several X-borne genes showed that both alleles are transcribed in a heterozygous female. Transcription of 14 BACs representing 19 X-borne genes was examined by RNA-FISH in female and male fibroblasts. An autosomal control gene was expressed from both alleles in nearly all nuclei, and four pseudoautosomal BACs were usually expressed from both alleles in male as well as female nuclei, showing that their Y loci are active. However, nine X-specific BACs were usually transcribed from only one allele. This suggests that while some genes on the platypus X are not dosage compensated, other genes do show some form of compensation via stochastic transcriptional inhibition, perhaps representing an ancestral system that evolved to be more tightly controlled in placental mammals such as human and mouse.

Unravelling the evolutionary origins of X chromosome inactivation in mammals: insights from marsupials and monotremes.

Determining the evolutionary origin of X inactivation mechanisms in mammals requires knowledge of features of X inactivation across all three major mammal lineages; monotremes, marsupials and eutherians. In the past, research into X inactivation in marsupials and monotremes lagged far behind the major advances made in understanding the mechanisms of X inactivation in human and mouse. Fragmentary knowledge of the genic content and sequence of marsupial and monotreme X chromosomes has been alleviated by the recent release of genome sequences for two marsupials and one monotreme. This has lead to a number of important findings, among which is the absence of XIST in marsupials and monotremes, and the surprising finding that X-borne genes in platypus are subject to stochastic transcriptional inhibition rather than whole chromosome inactivation. Availability of sequence data, and new techniques for studying expression and chromatin modification, now make rapid advance possible.

The ZW sex microchromosomes of an Australian dragon lizard share no homology with those of other reptiles or birds.

Reptiles show a diverse array of sex chromosomal systems but, remarkably, the Z sex chromosomes of chicken are homologous to the ZW sex chromosomes of a species of gecko, Gekko hokouensis, suggesting an ancient but common origin. This is in contrast to the ZW sex chromosomes of snakes and a species of soft-shelled turtle, Pelodiscus sinensis, which are nonhomologous to those of chicken or each other and appear to have been independently derived. In this paper, we determine what homology, if any, the sex chromosomes of the Australian dragon lizard Pogona vitticeps shares with those of snake and chicken by mapping the dragon homologs of five snake Z chromosome genes (WAC, KLF6, TAX1BP1, RAB5A, and CTNNB1) and five chicken Z chromosome genes (ATP5A1, GHR, DMRT1, CHD1, and APTX) to chromosomes in the dragon. The dragon homologs of snake and chicken sex chromosome genes map to chromosomes 6 and chromosome 2, respectively, in the dragon and that DMRT1, the bird sex-determining gene, is not located on the sex chromosomes of P. vitticeps. Indeed, our data show that the dragon homolog to the chicken Z chromosome is likely to be wholly contained within chromosome 2 in P. vitticeps, which suggests that the sex-determining factor in P. vitticeps is not the sex-determining gene of chicken. Homology between chicken Z chromosome and G. hokouensis ZW chromosome pairs has been interpreted as retention of ancient ZW sex chromosomes in which case the nonhomologous sex chromosomes of snake and dragons would be independently derived. Our data add another case of independently derived sex chromosomes in a squamate reptile, which makes retention of ancient sex chromosome homology in the squamates less plausible. Alternatively, the conservation between the bird Z chromosome and the G. hokouensis ZW chromosomes pairs is coincidental, may be an example of convergent evolution, its status as the Z chromosome having been independently derived in birds and G. hokouensis.

In the platypus a meiotic chain of ten sex chromosomes shares genes with the bird Z and mammal X chromosomes.

Two centuries after the duck-billed platypus was discovered, monotreme chromosome systems remain deeply puzzling. Karyotypes of males, or of both sexes, were claimed to contain several unpaired chromosomes (including the X chromosome) that form a multi-chromosomal chain at meiosis. Such meiotic chains exist in plants and insects but are rare in vertebrates. How the platypus chromosome system works to determine sex and produce balanced gametes has been controversial for decades. Here we demonstrate that platypus have five male-specific chromosomes (Y chromosomes) and five chromosomes present in one copy in males and two copies in females (X chromosomes). These ten chromosomes form a multivalent chain at male meiosis, adopting an alternating pattern to segregate into XXXXX-bearing and YYYYY-bearing sperm. Which, if any, of these sex chromosomes bears one or more sex-determining genes remains unknown. The largest X chromosome, with homology to the human X chromosome, lies at one end of the chain, and a chromosome with homology to the bird Z chromosome lies near the other end. This suggests an evolutionary link between mammal and bird sex chromosome systems, which were previously thought to have evolved independently.

Retrotransposon silencing by DNA methylation can drive mammalian genomic imprinting.

Among mammals, only eutherians and marsupials are viviparous and have genomic imprinting that leads to parent-of-origin-specific differential gene expression. We used comparative analysis to investigate the origin of genomic imprinting in mammals. PEG10 (paternally expressed 10) is a retrotransposon-derived imprinted gene that has an essential role for the formation of the placenta of the mouse. Here, we show that an orthologue of PEG10 exists in another therian mammal, the marsupial tammar wallaby (Macropus eugenii), but not in a prototherian mammal, the egg-laying platypus (Ornithorhynchus anatinus), suggesting its close relationship to the origin of placentation in therian mammals. We have discovered a hitherto missing link of the imprinting mechanism between eutherians and marsupials because tammar PEG10 is the first example of a differentially methylated region (DMR) associated with genomic imprinting in marsupials. Surprisingly, the marsupial DMR was strictly limited to the 5' region of PEG10, unlike the eutherian DMR, which covers the promoter regions of both PEG10 and the adjacent imprinted gene SGCE. These results not only demonstrate a common origin of the DMR-associated imprinting mechanism in therian mammals but provide the first demonstration that DMR-associated genomic imprinting in eutherians can originate from the repression of exogenous DNA sequences and/or retrotransposons by DNA methylation.