María Pilar García Guerreiro

A genome-wide survey of genetic instability by transposition in Drosophila hybrids.

Hybridization between species is a genomic instability factor involved in increasing mutation rate and new chromosomal rearrangements. Evidence of a relationship between interspecific hybridization and transposable element mobilization has been reported in different organisms, but most studies are usually performed with particular TEs and do not discuss the real effect of hybridization on the whole genome. We have therefore studied whole genome instability of Drosophila interspecific hybrids, looking for the presence of new AFLP markers in hybrids. A high percentage (27–90%) of the instability markers detected corresponds to TEs belonging to classes I and II. Moreover, three transposable elements (Osvaldo, Helena and Galileo) representative of different families, showed an overall increase of transposition rate in hybrids compared to parental species. This research confirms the hypothesis that hybridization induces genomic instability by transposition bursts and suggests that genomic stress by transposition could contribute to a relaxation of mechanisms controlling TEs in the Drosophila genome.

Distribution of the transposable elements bilbo and gypsy in original and colonizing populations of Drosophila subobscura

BackgroundTransposable elements (TEs) constitute a substantial amount of all eukaryotic genomes. They induce an important proportion of deleterious mutations by insertion into genes or gene regulatory regions. However, their mutational capabilities are not always adverse but can contribute to the genetic diversity and evolution of organisms. Knowledge of their distribution and activity in the genomes of populations under different environmental and demographic regimes, is important to understand their role in species evolution. In this work we study the chromosomal distribution of two TEs, gypsy and bilbo, in original and colonizing populations of Drosophila subobscura to reveal the putative effect of colonization on their insertion profile.ResultsChromosomal frequency distribution of two TEs in one original and three colonizing populations of D. subobscura, is different. Whereas the original population shows a low insertion frequency in most TE sites, colonizing populations have a mixture of high (frequency ≥ 10%) and low insertion sites for both TEs. Most highly occupied sites are coincident among colonizing populations and some of them are correlated to chromosomal arrangements. Comparisons of TE copy number between the X chromosome and autosomes show that gypsy occupancy seems to be controlled by negative selection, but bilbo one does not.ConclusionThese results are in accordance that TEs in Drosophila subobscura colonizing populations are submitted to a founder effect followed by genetic drift as a consequence of colonization. This would explain the high insertion frequencies of bilbo and gypsy in coincident sites of colonizing populations. High occupancy sites would represent insertion events prior to colonization. Sites of low frequency would be insertions that occurred after colonization and/or copies from the original population whose frequency is decreasing in colonizing populations. This work is a pioneer attempt to explain the chromosomal distribution of TEs in a colonizing species with high inversion polymorphism to reveal the putative effect of arrangements in TE insertion profiles. In general no associations between arrangements and TE have been found, except in a few cases where the association is very strong. Alternatively, founder drift effects, seem to play a leading role in TE genome distribution in colonizing populations.

Changes in the chromosomal insertion pattern of the copia element during the process of making chromosomes homozygous in Drosophila melanogaster

In situ hybridization on polytene chromosomes of Drosophila melanogaster was used to compare the insertion patterns of copia and mdgl transposable elements on chromosome 2 in male gametes sampled by two different methods: (i) by crossing the males tested with females from a highly inbred line with known copia and mdgl insertion profiles; (ii) by crossing the same males with females from a marked strain, and analysing the resulting homozygous chromosomes. Crossing of the males with the inbred line led to homogeneous insertion profiles for both the copia and mdgl elements in larvae, thus giving an accurate estimation of the patterns in the two gamete classes of each male. Crossing with the marked strain led, however, to heterogeneity in insertion patterns of the copia transposable element, while no significant polymorphism was observed for mdgl. The use of balancer chromosomes is thus not an adequate way of inferring transposable element insertion patterns of Drosophila males, at least for the copia element. This technique could, however, be powerful for investigating the control of movements of this element.

Adaptation of the AFLP technique as a new tool to detect genetic instability and transposition in interspecific hybrids

An adapted amplified fragment length polymorphism (AFLP) protocol is presented for detection of hybrid instability in the genome of interspecific hybrids between Drosophila buzzatii and D. koepferae species. Analyses of 15 AFLP instability markers (new bands detected in hybrids) show that up to 81% are the result of transposable element (TE) activity. Twenty TEs associated with AFLP instability markers have been detected by this method in backcross hybrids and segmental hybrids, demonstrating its validity in detecting transposition events occurring during the hybridization process. New insertions of Helena TE have been observed in the hybrid genome after hybridization of the TGTCG22 instability marker by FISH. The AFLP marker technique proved to be an efficient method that improves upon traditional and bioinformatic tools previously used to detect TE mobilization. This newly adapted AFLP protocol may also be applied to a large number of organisms outside the Drosophila genus, making it of interest to evolutionary and population genetic researchers working with species where the knowledge of the genome is scarce.

Changes of Osvaldo expression patterns in germline of male hybrids between the species Drosophila buzzatii and Drosophila koepferae.

Hybridization between different genomes is a source of genomic instability, sometimes associated with transposable element (TE) mobilization. Previous work showed that hybridization between the species Drosophila buzzatii and Drosophila koepferae induced mobilization of different (TEs), the Osvaldo retrotransposon being the most unstable. However, we ignore the mechanisms involved in this transposition release in interspecific hybrids. In order to disentangle the mechanisms involved in this process, we performed Osvaldo expression studies in somatic and germinal tissues from hybrids and parental species. There was a trend towards increased Osvaldo expression in the somatic tissues of hybrid females and males, which was always significant in males compared to the parental species D. buzzatii but, not in females compared to maternal species D. koepferae. There were massive changes of Osvaldo expression in the testes, which varied depending on the hybrid generation and family. Moreover, Osvaldo hybridization signals, restricted to the apical and primary spermatocyte regions in parents, occupied broader region in the hybrids. In ovaries, there were no significant differences in Osvaldo expression rates between hybrids and the maternal species D. koepferae. The transcript location was restricted to ovarian nurse cells in both parents and hybrids, undetectable in some hybrids. This research highlights first, the existence of putative complex deregulation mechanisms different between sexes and cell types and second, disruption of Osvaldo activity particularly evident in testes from sterile hybrid males. Deeper studies of the total transcriptome in hybrids and parental species are necessary to gain a better knowledge of the TE deregulation pathways in hybrids.Hybridization between different genomes is a source of genomic instability, sometimes associated with transposable element (TE) mobilization. Previous work showed that hybridization between the species Drosophila buzzatii and Drosophila koepferae induced mobilization of different (TEs), the Osvaldo retrotransposon being the most unstable. However, we ignore the mechanisms involved in this transposition release in interspecific hybrids. In order to disentangle the mechanisms involved in this process, we performed Osvaldo expression studies in somatic and germinal tissues from hybrids and parental species. There was a trend towards increased Osvaldo expression in the somatic tissues of hybrid females and males, which was always significant in males compared to the parental species D. buzzatii but, not in females compared to maternal species D. koepferae. There were massive changes of Osvaldo expression in the testes, which varied depending on the hybrid generation and family. Moreover, Osvaldo hybridization signals, restricted to the apical and primary spermatocyte regions in parents, occupied broader region in the hybrids. In ovaries, there were no significant differences in Osvaldo expression rates between hybrids and the maternal species D. koepferae. The transcript location was restricted to ovarian nurse cells in both parents and hybrids, undetectable in some hybrids. This research highlights first, the existence of putative complex deregulation mechanisms different between sexes and cell types and second, disruption of Osvaldo activity particularly evident in testes from sterile hybrid males. Deeper studies of the total transcriptome in hybrids and parental species are necessary to gain a better knowledge of the TE deregulation pathways in hybrids.

The Evolutionary History of Drosophila buzzatii. XXXVI. Molecular Structural Analysis of Osvaldo Retrotransposon Insertions in Colonizing Populations Unveils Drift Effects in Founder Events

Previous work on transposable element distribution in colonizing populations of Drosophila buzzatii revealed a high frequency of occupancy in several chromosomal sites. Two explanatory hypotheses were advanced: the founder hypothesis, by which founder genetic drift was responsible, and the unstable hypothesis that assigns this unusual distribution to bursts of transposition toward some chromosomal sites. Here, we study the molecular structure of three euchromatic Osvaldo clones isolated from sites occupied at high (A4 and B9) and low frequency (B4) in colonizing populations, to test these hypotheses. Large insertions, duplications, and indels in the Osvaldo coding region and LTR were detected in the A4 clone and a truncated Osvaldo with many substitutions was found in the B9 clone. These altered sequences indicate that the two copies of this retroelement are precolonization insertions. Interestingly, the LTR of the A4 clone and the reverse transcriptase region of B9 show identical sequences in all colonizing populations indicating, most probably, that they are identical by descent. Moreover, Osvaldo is inserted at the same nucleotide site in all colonizing populations. On the other hand an almost identical LTR sequence, except by 1 base deletion, was found in the B4 clone compared to the canonical active Osvaldo element. These results suggest that Osvaldo copies in highly occupied sites are, most probably, identical by descent and strongly favor the founder hypothesis. On the other hand, low-insertion-frequency sites could represent recent transposition events. This work emphasizes the importance of molecular population studies to disentangle the effects of genetic drift and transposition in colonization.

Expression of the Retrotransposon Helena Reveals a Complex Pattern of TE Deregulation in Drosophila Hybrids.

Transposable elements (TEs), repeated mobile sequences, are ubiquitous in the eukaryotic kingdom. Their mobilizing capacity confers on them a high mutagenic potential, which must be strongly regulated to guarantee genome stability. In the Drosophila germline, a small RNA-mediated silencing system, the piRNA (Piwi-interacting RNA) pathway, is the main responsible TE regulating mechanism, but some stressful conditions can destabilize it. For instance, during interspecific hybridization, genomic stress caused by the shock of two different genomes can lead, in both animals and plants, to higher transposition rates. A recent study in D. buzatii—D. koepferae hybrids detected mobilization of 28 TEs, yet little is known about the molecular mechanisms explaining this transposition release. We have characterized one of the mobilized TEs, the retrotransposon Helena, and used quantitative expression to assess whether its high transposition rates in hybrids are preceded by increased expression. We have also localized Helena expression in the gonads to see if cellular expression patterns have changed in the hybrids. To give more insight into changes in TE regulation in hybrids, we analysed Helena-specific piRNA populations of hybrids and parental species. Helena expression is not globally altered in somatic tissues, but male and female gonads have different patterns of deregulation. In testes, Helena is repressed in F1, increasing then its expression up to parental values. This is linked with a mislocation of Helena transcripts along with an increase of their specific piRNA levels. Ovaries have additive levels of Helena expression, but the ping-pong cycle efficiency seems to be reduced in F1 hybrids. This could be at the origin of new Helena insertions in hybrids, which would be transmitted to F1 hybrid female progeny.

Osvaldo and Isis retrotransposons as markers of the Drosophila buzzatii colonisation in Australia

BackgroundTransposable elements (TEs) constitute an important source of genetic variability owing to their jumping and regulatory properties, and are considered to drive species evolution. Several factors that are able to induce TE transposition in genomes have been documented (for example environmental stress and inter- and intra-specific crosses) but in many instances the reasons for TE mobilisation have yet to be elucidated. Colonising populations constitute an ideal model for studying TE behaviour and distribution as they are exposed to different environmental and new demographic conditions. In this study, the distribution of two TEs, Osvaldo and Isis, was examined in two colonising populations of D. buzzatii from Australia. Comparing Osvaldo copy numbers between Australian and Old World (reported in previous studies) colonisations provides a valuable tool for elucidating the colonisation process and the effect of new conditions encountered by colonisers on TEs.ResultsThe chromosomal distributions of Osvaldo and Isis retrotransposons in two colonising populations of D. buzzatii from Australia revealed sites of high insertion frequency (>10%) and low frequency sites. Comparisons between Osvaldo insertion profiles in colonising populations from the Old World and Australia demonstrate a tendency towards a higher number of highly occupied sites with higher insertion frequency in the Old World than in Australian populations. Tests concerning selection against deleterious TE insertions indicate that Isis is more controlled by purifying selection than Osvaldo. The distribution of both elements on chromosomal arms follows a Poisson distribution and there are non-significant positive correlations between highly occupied sites and chromosomal inversions.ConclusionsThe occupancy profile of Osvaldo and Isis retrotransposons is characterised by the existence of high and low insertion frequency sites in the populations. These results demonstrate that Australian D. buzzatii populations were subjected to a founder effect during the colonisation process. Moreover, there are more sites with high insertion frequency in the Old World colonisation than in the Australian colonisation, indicating a probable stronger bottleneck effect in Australia. The results suggest that selection does not seem to play a major role, compared to demography, in the distribution of transposable elements in the Australian populations.

Drosophila Females Undergo Genome Expansion after Interspecific Hybridization.

Genome size (or C-value) can present a wide range of values among eukaryotes. This variation has been attributed to differences in the amplification and deletion of different noncoding repetitive sequences, particularly transposable elements (TEs). TEs can be activated under different stress conditions such as interspecific hybridization events, as described for several species of animals and plants. These massive transposition episodes can lead to considerable genome expansions that could ultimately be involved in hybrid speciation processes. Here, we describe the effects of hybridization and introgression on genome size of Drosophila hybrids. We measured the genome size of two close Drosophila species, Drosophila buzzatii and Drosophila koepferae, their F1 offspring and the offspring from three generations of backcrossed hybrids; where mobilization of up to 28 different TEs was previously detected. We show that hybrid females indeed present a genome expansion, especially in the first backcross, which could likely be explained by transposition events. Hybrid males, which exhibit more variable C-values among individuals of the same generation, do not present an increased genome size. Thus, we demonstrate that the impact of hybridization on genome size can be detected through flow cytometry and is sex-dependent.

Genomic analysis of European Drosophila melanogaster populations on a dense spatial scale reveals longitudinal population structure and continent-wide selection

Abstract Genetic variation is the fuel of evolution, with standing genetic variation especially important for short-term evolution and local adaptation. To date, studies of spatio-temporal patterns of genetic variation in natural populations have been challenging, as comprehensive sampling is logistically difficult, and sequencing of entire populations costly. Here, we address these issues using a collaborative approach, sequencing 48 pooled population samples from 32 locations, and perform the first continent-wide genomic analysis of genetic variation in European Drosophila melanogaster. Our analyses uncover longitudinal population structure, provide evidence for continent-wide selective sweeps, identify candidate genes for local climate adaptation, and document clines in chromosomal inversion and transposable element frequencies. We also characterise variation among populations in the composition of the fly microbiome, and identify five new DNA viruses in our samples.Genetic variation is the fuel of evolution. However, analyzing evolutionary dynamics in natural populations is challenging, sequencing of entire populations remains costly and comprehensive sampling logistically difficult. To tackle this issue and to define relevant spatial and temporal scales of variation, we have founded the European Drosophila Population Genomics Consortium (DrosEU). Here we present the first analysis of 48 D. melanogaster population samples collected across Europe in 2014. Our analysis uncovers novel patterns of variation at multiple levels: genome-wide neutral SNPs, mtDNA haplotypes, inversions, and TEs showing previously cryptic longitudinal population structure; signatures of selective sweeps shared among populations; presumably adaptive clines in inversions; and geographic variation in TEs. Additionally, we document highly variable microbiota and identify several new Drosophila viruses. Our study reveals novel aspects of the population biology of D. melanogaster and illustrates the power of extensive sampling and pooled sequencing of populations on a continent-wide scale.Genetic variation is the fuel of evolution. However, analyzing dynamics of evolutionary change in natural populations is challenging, genome sequencing of entire populations remains costly and comprehensive sample collection logistically challenging. To tackle this issue and to define relevant spatial and temporal scales of variation for a population genetic model system, the fruit fly Drosophila melanogaster, we have founded the European Drosophila Population Genomics Consortium (DrosEU). Our principal objective is to employ the strengths of this collaborative consortium to extensively sample and sequence natural populations on a continent-wide scale and across distinct timescales. Here we present the first analysis of the first DrosEU pool-sequencing dataset, consisting of 48 population samples collected across the European continent in 2014. The analysis of this comprehensive dataset uncovers novel patterns of variation at multiple levels: genome-wide neutral SNPs, mtDNA haplotypes, inversions and TEs that exhibit previously cryptic longitudinal population structure across the European continent; signatures of selective sweeps shared among the majority of European populations; presumably adaptive clines in inversions; and geographic variation in TEs. Additionally, we document highly variable microbiota among European fruit fly populations and identify several new Drosophila viruses. Our study reveals novel aspects of the population biology of D. melanogaster and illustrates the power of extensive sampling and pooled sequencing of natural populations on a continent-wide scale.