Rachel J. O'Neill

Undermethylation associated with retroelement activation and chromosome remodelling in an interspecific mammalian hybrid

Genetic models, predict that genomic rearrangement in hybrids can facilitate reproductive isolation and the formation of new species by preventing gene flow between the parent species and hybrid (sunflowers are an example). The mechanism underlying hybridization-induced chromosome remodelling is as yet unknown, although mobile element activity has been shown to be involved in DNA rearrangement in some dysgenic Drosophila hybrids,. It has been proposed that DNA methylation evolved as a means of repressing the movement of mobile elements (the host defence model,). If such a protective mechanism were to fail, mobile elements could be activated, and could cause major and rapid genome alterations,. Here we demonstrate the occurrence of genome-wide undermethylation, retroviral element amplification and chromosome remodelling in an interspecific mammalian hybrid (Macropus eugenii × Wallabia bicolor). Atypically extended centromeres of Macropus eugenii derived autosomes in the hybrid were composed primarily of an unmethylated, amplified retroviral element not detectable in either parent species. These results, taken with the observation of deficient methylation and de novo chromosome change in other mammalian hybrids, indicate that the failure of DNA methylation and subsequent mobile-element activity in hybrids could facilitate rapid karyotypic evolution.

Genomic Instability within Centromeres of Interspecific Marsupial Hybrids

Several lines of evidence suggest that, within a lineage, particular genomic regions are subject to instability that can lead to specific types of chromosome rearrangements important in species incompatibility. Within family Macropodidae (kangaroos, wallabies, bettongs, and potoroos), which exhibit recent and extensive karyotypic evolution, rearrangements involve chiefly the centromere. We propose that centromeres are the primary target for destabilization in cases of genomic instability, such as interspecific hybridization, and participate in the formation of novel chromosome rearrangements. Here we use standard cytological staining, cross-species chromosome painting, DNA probe analyses, and scanning electron microscopy to examine four interspecific macropodid hybrids (Macropus rufogriseus × Macropus agilis). The parental complements share the same centric fusions relative to the presumed macropodid ancestral karyotype, but can be differentiated on the basis of heterochromatic content, M. rufogriseus having larger centromeres with large C-banding positive regions. All hybrids exhibited the same pattern of chromosomal instability and remodeling specifically within the centromeres derived from the maternal (M. rufogriseus) complement. This instability included amplification of a satellite repeat and a transposable element, changes in chromatin structure, and de novo whole-arm rearrangements. We discuss possible reasons and mechanisms for the centromeric instability and remodeling observed in all four macropodid hybrids.

Abundant Human DNA Contamination Identified in Non-Primate Genome Databases

During routine screens of the NCBI databases using human repetitive elements we discovered an unlikely level of nucleotide identity across a broad range of phyla. To ascertain whether databases containing DNA sequences, genome assemblies and trace archive reads were contaminated with human sequences, we performed an in depth search for sequences of human origin in non-human species. Using a primate specific SINE, AluY, we screened 2,749 non-primate public databases from NCBI, Ensembl, JGI, and UCSC and have found 492 to be contaminated with human sequence. These represent species ranging from bacteria (B. cereus) to plants (Z. mays) to fish (D. rerio) with examples found from most phyla. The identification of such extensive contamination of human sequence across databases and sequence types warrants caution among the sequencing community in future sequencing efforts, such as human re-sequencing. We discuss issues this may raise as well as present data that gives insight as to how this may be occurring.

Genome-Wide Reprogramming in Hybrids of Somatic Cells and Embryonic Stem Cells

Recent experiments demonstrate that somatic nuclei can be reprogrammed to a pluripotent state when fused to ESCs. The resulting hybrids are pluripotent as judged by developmental assays, but detailed analyses of the underlying molecular‐genetic control of reprogrammed transcription in such hybrids are required to better understand fusion‐mediated reprogramming. We produced hybrids of mouse ESCs and fibroblasts that, although nearly tetraploid, exhibit characteristics of normal ESCs, including apparent immortality in culture, ESC‐like colony morphology, and pluripotency. Comprehensive analysis of the mouse embryonic fibroblast/ESC hybrid transcriptome revealed global patterns of gene expression reminiscent of ESCs. However, combined analysis of variance and hierarchical clustering analyses revealed at least seven distinct classes of differentially regulated genes in comparisons of hybrids, ESCs, and somatic cells. The largest class includes somatic genes that are silenced in hybrids and ESCs, but a smaller class includes genes that are expressed at nearly equivalent levels in hybrids and ESCs that contain many genes implicated in pluripotency and chromatin function. Reprogrammed genes are distributed throughout the genome. Reprogramming events include both transcriptional silencing and activation of genes residing on chromosomes of somatic origin. Somatic/ESC hybrid cell lines resemble their pre‐fusion ESC partners in terms of behavior in culture and pluripotency. However, they contain unique expression profiles that are similar but not identical to normal ESCs. ESC fusion‐mediated reprogramming provides a tractable system for the investigation of mechanisms of reprogramming.

Ancient and continuing Darwinian selection on insulin-like growth factor II in placental fishes

Despite abundant examples of both adaptation at the level of phenotype and Darwinian selection at the level of genes, correlations between these two processes are notoriously difficult to identify. Positive Darwinian selection on genes is most easily discerned in cases of genetic conflict, when antagonistic evolutionary processes such as a Red Queen race drive the rate of nonsynonymous substitution above the neutral mutation rate. Genomic imprinting in mammals is thought to be the product of antagonistic evolution coincident with evolution of the placenta, but imprinted loci lack evidence of positive selection likely because of the ancient origin of viviparity in mammals. To determine whether genetic conflict is a general feature of adaptation to placental reproduction, we performed comparative evolutionary analyses of the insulin-like growth factor II (IGF2) gene in teleost fishes. Our analysis included several members of the order Cyprinodontiformes, in which livebearing and placentation have evolved several times independently. We found that IGF2 is subject to positive Darwinian selection coincident with the evolution of placentation in fishes, with particularly strong selection among lineages that have evolved placentation recently. Positive selection is also detected along ancient lineages of placental livebearing fishes, suggesting that selection on IGF2 function is ongoing in placental species. Our observations provide a rare example of natural selection acting in synchrony at the phenotypic and molecular level. These results also constitute the first direct evidence of parent–offspring conflict driving gene evolution.

Chromosome heterozygosity and de novo chromosome rearrangements in mammalian interspecies hybrids

Closely related mammalian species frequently differ in chromosome number and morphology. The source of this variation and the role it plays in speciation has never been clarified. It is clear from recent research that chromosomal speciation can occur very rapidly (Garagna et al. 1997), and one possibility is that it is engendered by hybridization (Reiseberg et al. 1995), possibly by the derepression of transposable elements (O’Neill et al. 1998). Studies of chromosomal rearrangements in hybrids to date have relied on traditional cytological techniques like G-banding, which lack the resolution to detect small rearrangements. Crossspecies chromosome painting allows the identification of smallscale interchromosomal rearrangements not visible by traditional, non-fluorescent cytogenetic techniques. Marsupials are an ideal study group because of their low diploid numbers and the availability of many interspecific hybrids. Chromosome paints isolated from sorted and microdissected Macropus eugenii chromosomes and chromosome arms (Toder et al., 1997a, 1997b) were used to identify de novorearrangements in several hybrids (Table 1) between kangaroo (marsupial Family Macropodidae) species. The parent species include some in the genera Petrogale(rock wallabies), Macropus (tammar and agile wallabies), and Wallabia (swamp wallaby). BE-1, a hybrid betweenW. bicolorandM. eugenii,represents the most divergent cross used in this study [parent species diverged 7 MYrBP (Kirsch et al., 1997)]. M. eugenii(Meu) paints have been used to identify the homologous regions in the chromosomes of W. bicolor (Wbi) (Toder et al. 1997b). The Meuchromosomes of the hybrid show obvious centromeric expansions that include amplified transposable elements (O’Neill et al. 1998). Painting revealed two interchromosal rearrangements, including a de novotranslocation of a segment of chromosomal material homologous to Meu 2 to the proximal portion of Meu7q in 100% of the cells examined (Fig. 1a, 2). Chromosome painting of ten individuals from two genetically divergent populations of M. eugeniidid not reveal a rearrangement of this type within the parental species (R. O’Neill, unpublished). In addition, examination of hundreds of individuals over several years has revealed no karyotypic polymorphisms within this species. TheMeuX Chromosome (Chr) has also been greatly modified in this hybrid. A very large centromeric expansion is evident (O’Neill et al. 1998; Fig. 1b, 2), but is not hybridized by the same transposable element located in the expanded centromeres of the Meu autosomes (O’Neill et al. 1998). In addition, the nucleolus organizer region (NOR) detected by hybridization with an rDNA probe appears to be more terminal than previously described in the Meuparent (Toder et al. 1997a), and has MeuXp heterochromatin on the proximal as well as the distal side (Fig. 1b, 2). This suggests either an inversion of Xp in the hybrid, or transposition and amplification of heterochromatic sequences proximal to the NOR, or both. Additionally, the normal cross-hybridization of the Meu Y Chr paint to Xp that is observed in M. eugeniiis not present in the hybrid (Toder et al. 1997a). Meupaints were used to identify the homologous regions in the chromosomes of the agile wallaby ( M. agilis; R. Glass, personal communication), whose genome displays no interchromosomal rearrangement relative to Meuchromosomes. The two parental species both have 2n4 16 but differ in that the X Chr ofMag is acrocentric (Rofe 1979). Painting the chromosomes of the M. agiis × M. eugenii(Mag × Meu) female hybrid AE-1 identified at least fivede novorearrangements (Fig. 2), all in 100% of cells examined. Segments homologous to MeuChr 1 were found in one 2p, in Xp and the distal portion of Meu Xq, and the proximal section ofMag Xq. The other 2p shows homologies to Meu 6. The hybrid LP is the product of a cross between two Petrogale species,Petrogale inornata× P. assimilis.Chromosome homologies withMeuhave been established by G-banding (Eldridge et al. 1988, 1990) and cross-species painting with Meu chromosomespecific paints (O’Neill et al. 1999) for both parental species. This hybrid is a cross that occurs in a natural hybrid zone between P. assimilis (Pas) and P. Inornata (Pin), within which females are fertile, an observation based on introgression studies of mtDNA (Bee and Close 1993). Chromosome painting with Meu probes showed a total of fivede novorearrangement in 100% of the cells examined from the hybrid (Fig. 1c, 2). Three rearrangements involved Meu Chr 1. TheMeu Chr 1 paint hybridized to the pericentric region of one Chr 2 and to one member each of Chr 3 and 4, replacing theP. assimilisshort arm in each case. The centric region of one member of the hybrid Chr 1 pair now shows hybridization to sequences common to the M uY and Xp. TheMeu 7 paint hybridized to the centric region of the other Chr 2. These results demonstrate the frequent occurrence of d novo chromosome rearrangements in the genomes of three independent marsupial hybrids between kangaroo species of disparate taxa. The involvement of Chr 2 and 7 in rearrangements in both the Petro-

Maternal methyl supplemented diets and effects on offspring health

Women seeking to become pregnant and pregnant women are currently advised to consume high amounts of folic acid and other methyl donors to prevent neural tube defects in their offspring. These diets can alter methylation patterns of several biomolecules, including nucleic acids, and histone proteins. Limited animal model data suggests that developmental exposure to these maternal methyl supplemented (MS) diets leads to beneficial epimutations. However, other rodent and humans studies have yielded opposing findings with such diets leading to promiscuous epimutations that are likely associated with negative health outcomes. Conflict exists to whether these maternal diets are preventative or exacerbate the risk for Autism Spectrum Disorders (ASD) in children. This review will discuss the findings to date on the potential beneficial and aversive effects of maternal MS diets. We will also consider how other factors might influence the effects of MS diets. Current data suggest that there is cause for concern as maternal MS diets may lead to epimutations that underpin various diseases, including neurobehavioral disorders. Further studies are needed to explore the comprehensive effects maternal MS diets have on the offspring epigenome and subsequent overall health.

Two different high throughput sequencing approaches identify thousands of de novo genomic markers for the genetically depleted Bornean elephant.

High throughput sequencing technologies are being applied to an increasing number of model species with a high-quality reference genome. The application and analyses of whole-genome sequence data in non-model species with no prior genomic information are currently under way. Recent sequencing technologies provide new opportunities for gathering genomic data in natural populations, laying the empirical foundation for future research in the field of conservation and population genomics. Here we present the case study of the Bornean elephant, which is the most endangered subspecies of Asian elephant and exhibits very low genetic diversity. We used two different sequencing platforms, the Roche 454 FLX (shotgun) and Illumina, GAIIx (Restriction site associated DNA, RAD) to evaluate the feasibility of the two methodologies for the discovery of de novo markers (single nucleotide polymorphism, SNPs and microsatellites) using low coverage data. Approximately, 6,683 (shotgun) and 14,724 (RAD) SNPs were detected within our elephant sequence dataset. Genotyping of a representative sample of 194 SNPs resulted in a SNP validation rate of ∼ 83 to 94% and 17% of the loci were polymorphic with a low diversity (H o = 0.057). Different numbers of microsatellites were identified through shotgun (27,226) and RAD (868) techniques. Out of all di-, tri-, and tetra-microsatellite loci, 1,706 loci had sufficient flanking regions (shotgun) while only 7 were found with RAD. All microsatellites were monomorphic in the Bornean but polymorphic in another elephant subspecies. Despite using different sample sizes, and the well known differences in the two platforms used regarding sequence length and throughput, the two approaches showed high validation rate. The approaches used here for marker development in a threatened species demonstrate the utility of high throughput sequencing technologies as a starting point for the development of genomic tools in a non-model species and in particular for a species with low genetic diversity.

Comparative genome mapping of the deer mouse (Peromyscus maniculatus) reveals greater similarity to rat (Rattus norvegicus) than to the lab mouse (Mus musculus)

BackgroundDeer mice (Peromyscus maniculatus) and congeneric species are the most common North American mammals. They represent an emerging system for the genetic analyses of the physiological and behavioral bases of habitat adaptation. Phylogenetic evidence suggests a much more ancient divergence of Peromyscus from laboratory mice (Mus) and rats (Rattus) than that separating latter two. Nevertheless, early karyotypic analyses of the three groups suggest Peromyscus to be exhibit greater similarities with Rattus than with Mus.ResultsComparative linkage mapping of an estimated 35% of the deer mouse genome was done with respect to the Rattus and Mus genomes. We particularly focused on regions that span synteny breakpoint regions between the rat and mouse genomes. The linkage analysis revealed the Peromyscus genome to have a higher degree of synteny and gene order conservation with the Rattus genome.ConclusionThese data suggest that: 1. the Rattus and Peromyscus genomes more closely represent ancestral Muroid and rodent genomes than that of Mus. 2. the high level of genome rearrangement observed in Muroid rodents is especially pronounced in Mus. 3. evolution of genome organization can operate independently of more commonly assayed measures of genetic change (e.g. SNP frequency).

Cytogenetic and Molecular Evaluation of Centromere-Associated DNA Sequences From a Marsupial (Macropodidae: Macropus rufogriseus) X Chromosome

The constitution of the centromeric portions of the sex chromosomes of the red-necked wallaby, Macropus rufogriseus (family Macropodidae, subfamily Macropodinae), was investigated to develop an overview of the sequence composition of centromeres in a marsupial genome that harbors large amounts of centric and pericentric heterochromatin. The large, C-band-positive centromeric region of the X chromosome was microdissected and the isolated DNA was microcloned. Further sequence and cytogenetic analyses of three representative clones show that all chromosomes in this species carry a 178-bp satellite sequence containing a CENP-B DNA binding domain (CENP-B box) shown herein to selectively bind marsupial CENP-B protein. Two other repeats isolated in this study localize specifically to the sex chromosomes yet differ in copy number and intrachromosomal distribution. Immunocytohistochemistry assays with anti-CENP-E, anti-CREST, anti-CENP-B, and anti-trimethyl-H3K9 antibodies defined a restricted point localization of the outer kinetochore at the functional centromere within an enlarged pericentric and heterochromatic region. The distribution of these repeated sequences within the karyotype of this species, coupled with the apparent high copy number of these sequences, indicates a capacity for retention of large amounts of centromere-associated DNA in the genome of M. rufogriseus.