Considering Transposable Element Diversification in De Novo Annotation Approaches

A major portion of most eukaryotic genomes are transposable elements (TEs). During evolution, TEs have introduced profound changes to genome size, structure, and function. As integral parts of genomes, the dynamic presence of TEs will continue to be a major force in reshaping genomes. Early computational analyses of TEs in genome sequences focused on filtering out "junk" sequences to facilitate gene annotation. When the high abundance and diversity of TEs in eukaryotic genomes were recognized, these early efforts transformed into the systematic genome-wide categorization and classification of TEs. The availability of genomic sequence data reversed the classical genetic approaches to discovering new TE families and superfamilies. Curated TE databases and their accurate annotation of genome sequences in turn facilitated the studies on TEs in a number of frontiers including: (1) TE-mediated changes of genome size and structure, (2) the influence of TEs on genome and gene functions, (3) TE regulation by host, (4) the evolution of TEs and their population dynamics, and (5) genomic scale studies of TE activity. Bioinformatics and genomic approaches have become an integral part of large-scale studies on TEs to extract information with pure in silico analyses or to assist wet lab experimental studies. The current revolution in genome sequencing technology facilitates further progress in the existing frontiers of research and emergence of new initiatives. The rapid generation of large-sequence datasets at record low costs on a routine basis is challenging the computing industry on storage capacity and manipulation speed and the bioinformatics community for improvement in algorithms and their implementations.

An analysis of high-resolution transposable element annotations in Drosophila melanogaster suggests the existence of a global surveillance system against the majority of transposable elements families in the fly.

DNA methylation and epigenetic silencing play important roles in the regulation of transposable elements (TEs) in many eukaryotic genomes. A majority of the maize genome is derived from TEs that can be classified into different orders and families based on their mechanism of transposition and sequence similarity, respectively. TEs themselves are highly methylated and it can be tempting to view them as a single uniform group. However, the analysis of DNA methylation profiles in flanking regions provides evidence for distinct groups of chromatin properties at different TE families. These differences among TE families are reproducible in different tissues and different inbred lines. TE families with varying levels of DNA methylation in flanking regions also show distinct patterns of chromatin accessibility and modifications within the TEs. The differences in the patterns of DNA methylation flanking TE families arise from a combination of non-random insertion preferences of TE families, changes in DNA methylation triggered by the insertion of the TE and subsequent selection pressure. A set of nearly 70,000 TE polymorphisms among four assembled maize genomes were used to monitor the level of DNA methylation at haplotypes with and without the TE insertions. In many cases, TE families with high levels of DNA methylation in flanking sequence are enriched for insertions into highly methylated regions. The majority of the >2,500 TE insertions into unmethylated regions result in changes in DNA methylation in haplotypes with the TE, suggesting the widespread potential for TE insertions to condition altered methylation in conserved regions of the genome. This study highlights the interplay between TEs and the methylome of a major crop species.

Transposable elements (TEs) pervade most eukaryotic genomes. The repetitive nature of TEs complicates the analysis of their expression. Evaluation of the expression of both TE families (using unique and multi-mapping reads) and specific elements (using uniquely mapping reads) in leaf tissue of three maize (Zea mays) inbred lines subjected to heat or cold stress reveals no evidence for genome-wide activation of TEs; however, some specific TE families generate transcripts only in stress conditions. There is substantial variation for which TE families exhibit stress-responsive expression in the different genotypes. In order to understand the factors that drive expression of TEs, we focused on a subset of families in which we could monitor expression of individual elements. The stress-responsive activation of a TE family can often be attributed to a small number of elements in the family that contains regions lacking DNA methylation. Comparisons of the expression of TEs in different genotypes revealed both genetic and epigenetic variation. Many of the specific TEs that are activated in stress in one inbred are not present in the other inbred, explaining the lack of activation. Among the elements that are shared in both genomes but only expressed in one genotype, we found that many exhibit differences in DNA methylation such that the genotype without expression is fully methylated. This study provides insights into the regulation of expression of TEs in normal and stress conditions and highlights the role of chromatin variation between elements in a family or between genotypes for contributing to expression variation. The highly repetitive nature of many TEs complicates the analysis of their expression. Although most TEs are not expressed, some exhibits expression in certain tissues or conditions. We monitored the expression of both TE families (using unique and multi-mapping reads) and specific elements (using uniquely mapping reads) in leaf tissue of three maize (Zea mays) inbred lines subjected to heat or cold stress. While genome-wide activation of TEs did not occur, some TE families generated transcripts only in stress conditions with variation by genotype. To better understand the factors that drive expression of TEs, we focused on a subset of families in which we could monitor expression of individual elements. In most cases, stress-responsive activation of a TE family was attributed to a small number of elements in the family. The elements that contained small regions lacking DNA methylation regions showed enriched expression while fully methylated elements were rarely expressed in control or stress conditions. The cause of varied expression in the different genotypes was due to both genetic and epigenetic variation. Many specific TEs activated by stress in one inbred were not present in the other inbred. Among the elements shared in both genomes, full methylation inhibited expression in one of the genotypes. This study provides insights into the regulation of TE expression in normal and stress conditions and highlights the role of chromatin variation between elements in a family or between genotypes for contributing to expression.

The availability of the sequenced Drosophila melanogaster genome provides an opportunity to study sequence variation between copies within transposable element families. In this study,we analyzed the 624 copies of 22 transposable element (TE) families (14 LTR retrotransposons, five non-LTR retrotransposons, and three transposons). LTR and non-LTR retrotransposons possessed far fewer divergent elements than the transposons,suggesting that the difference depends on the transposition mechanism. However,there was not a continuous range of divergence of the copies in each class,which were either very similar to the canonical elements,or very divergent from them. This sequence homogeneity among TE family copies matches the theoretical models of the dynamics of these repeated sequences. The sequenced Drosophila genome thus appears to be composed of a mixture of TEs that are still active and of ancient relics that have degenerated and the distribution of which along the chromosomes results from natural selection. This clearly demonstrates that the TEs are highly active within the genome,suggesting that the genetic variability of the Drosophila genome is still being renewed by the action of TEs.

Eukaryotic genomes contain large amount of repetitive DNA, most of which is derived from transposable elements (TEs). Progress has been made to develop computational tools for ab initio identification of repeat families, but there is an urgent need to develop tools to automate the annotation of TEs in genome sequences. Here we introduce REPCLASS, a tool that automates the classification of TE sequences. Using control repeat libraries, we show that the program can classify accurately virtually any known TE types. Combining REPCLASS to ab initio repeat finding in the genomes of Caenorhabditis elegans and Drosophila melanogaster allowed us to recover the contrasting TE landscape characteristic of these species. Unexpectedly, REPCLASS also uncovered several novel TE families in both genomes, augmenting the TE repertoire of these model species. When applied to the genomes of distant Caenorhabditis and Drosophila species, the approach revealed a remarkable conservation of TE composition profile within each genus, despite substantial interspecific covariations in genome size and in the number of TEs and TE families. Lastly, we applied REPCLASS to analyze 10 fungal genomes from a wide taxonomic range, most of which have not been analyzed for TE content previously. The results showed that TE diversity varies widely across the fungi “kingdom” and appears to positively correlate with genome size, in particular for DNA transposons. Together, these data validate REPCLASS as a powerful tool to explore the repetitive DNA landscapes of eukaryotes and to shed light onto the evolutionary forces shaping TE diversity and genome architecture.

The genomes of barley and wheat, two of the world's most important crops, are very large and complex due to their high content of repetitive DNA. In order to obtain a whole-genome sequence sample, we performed two runs of 454 (GS20) sequencing on genomic DNA of barley cv. Morex, which yielded approximately 1% of a haploid genome equivalent. Almost 60% of the sequences comprised known transposable element (TE) families, and another 9% represented novel repetitive sequences. We also discovered high amounts of low-complexity DNA and non-genic low-copy DNA. We identified almost 2300 protein coding gene sequences and more than 660 putative conserved non-coding sequences. Comparison of the 454 reads with previously published genomic sequences suggested that TE families are distributed unequally along chromosomes. This was confirmed by in situ hybridizations of selected TEs. A comparison of these data for the barley genome with a large sample of publicly available wheat sequences showed that several TE families that are highly abundant in wheat are absent from the barley genome. This finding implies that the TE composition of their genomes differs dramatically, despite their very similar genome size and their close phylogenetic relationship.

The Drosophila melanogaster genome contains approximately 100 distinct families of transposable elements (TEs). In the euchromatic part of the genome, each family is present in a small number of copies (5-150 copies), with individual copies of TEs often present at very low frequencies in populations. This pattern is likely to reflect a balance between the inflow of TEs by transposition and the removal of TEs by natural selection. The nature of natural selection acting against TEs remains controversial. We provide evidence that selection against chromosome abnormalities caused by ectopic recombination limits the spread of some TEs. We also demonstrate for the first time that some TE families in the Drosophila euchromatin appear to be only marginally affected by purifying selection and contain many copies at high population frequencies. We argue that TEs in these families attain high population frequencies and even reach fixation as a result of low family-wide transposition rates leading to low TE copy numbers and consequently reduced strength of selection acting on individual TE copies. Fixation of TEs in these families should provide an upward pressure on the size of intergenic sequences counterbalancing rapid DNA loss through small deletions. Copy-number-dependent selection on TE families caused by ectopic recombination may also promote diversity among TEs in the Drosophila genome.

Genome size variation in eukaryotic species is largely influenced by repetitive DNA sequences such as transposable elements (TEs), simple repeats, and satellite DNAs (satDNAs), which do not necessarily correlate with organismal complexity. In insects, TEs are crucial to evolutionary processes and are correlated with variations in genome size. In this study, we describe, for the first time, the mobilome and satellitome of Drosophila amaguana, an Ecuadorian Neotropical species with a large, unexplored genome size, to assess the contribution of these repetitive DNA sequences to its genome composition. Using a draft genome assembly of approximately 455.5 Mb, generated from Illumina short-read sequences obtained from 10 wild specimens of D. amaguana collected at the Refugio de Vida Silvestre Pasochoa, we employed a de novo approach to create a manually curated TE library of 737 consensus sequences. We identified 716 novel TE families that had not been previously described, 20 TEs previously characterized in other Drosophila species, and one DNA transposon previously described in the Lepeophtheirus genus. The total TE content in the D. amaguana genome was 21.54%, distributed as follows: 6.35% Helitrons (1 superfamily), 5.13% LTR retrotransposons (5 superfamilies), 3.63% TIRs (9 superfamilies), 3.61% LINEs (7 superfamilies), 1.17% MITEs, 0.94% Maverick, 0.67% PLE, 0.02% SINEs, and 0.01% DIRS. We also identified 11.8% of simple repeats. Additionally, we estimated the satDNA content using Illumina raw reads and identified 16 satDNA families, all unique to the Drosophila genus, which comprise 4.90% of the genome. Overall, our results based on short-read data suggest that the large genome size of D. amaguana may not be the consequence of a high amount of TEs or satDNAs. Instead, its large genome size could be attributed to other factors (e.g., noncoding DNA occupying substantial portions of the genome or a high percentage of duplicated genes) that remain to be determined or explored in future studies using long-reads to overcome short-reads limitations. These findings may currently offer valuable insights into the adaptative and evolutionary processes of the mesophragmatica species group in the Andean forests.

Transposable elements (TEs) constitute the majority of flowering plant DNA, reflecting their tremendous success in subverting, avoiding, and surviving the defenses of their host genomes to ensure their selfish replication. More than 85% of the sequence of the maize genome can be ascribed to past transposition, providing a major contribution to the structure of the genome. Evidence from individual loci has informed our understanding of how transposition has shaped the genome, and a number of individual TE insertions have been causally linked to dramatic phenotypic changes. Genome-wide analyses in maize and other taxa have frequently represented TEs as a relatively homogeneous class of fragmentary relics of past transposition, obscuring their evolutionary history and interaction with their host genome. Using an updated annotation of structurally intact TEs in the maize reference genome, we investigate the family-level dynamics of TEs in maize. Integrating a variety of data, from descriptors of individual TEs like coding capacity, expression, and methylation, as well as similar features of the sequence they inserted into, we model the relationship between attributes of the genomic environment and the survival of TE copies and families. In contrast to the wholesale relegation of all TEs to a single category of junk DNA, these differences reveal a diversity of survival strategies of TE families. Together these generate a rich ecology of the genome, with each TE family representing the evolution of a distinct ecological niche. We conclude that while the impact of transposition is highly family- and context-dependent, a family-level understanding of the ecology of TEs in the genome can refine our ability to predict the role of TEs in generating genetic and phenotypic diversity.

Transposable elements (TEs) constitute the majority of flowering plant DNA, reflecting their tremendous success in subverting, avoiding, and surviving the defenses of their host genomes to ensure their selfish replication. More than 85% of the sequence of the maize genome can be ascribed to past transposition, providing a major contribution to the structure of the genome. Evidence from individual loci has informed our understanding of how transposition has shaped the genome, and a number of individual TE insertions have been causally linked to dramatic phenotypic changes. Genome-wide analyses in maize and other taxa have frequently represented TEs as a relatively homogeneous class of fragmentary relics of past transposition, obscuring their evolutionary history and interaction with their host genome. Using an updated annotation of structurally intact TEs in the maize reference genome, we investigate the family-level dynamics of TEs in maize. Integrating a variety of data, from descriptors of individual TEs like coding capacity, expression, and methylation, as well as similar features of the sequence they inserted into, we model the relationship between attributes of the genomic environment and the survival of TE copies and families. In contrast to the wholesale relegation of all TEs to a single category of junk DNA, these differences reveal a diversity of survival strategies of TE families. Together these generate a rich ecology of the genome, with each TE family representing the evolution of a distinct ecological niche. We conclude that while the impact of transposition is highly family- and context-dependent, a family-level understanding of the ecology of TEs in the genome can refine our ability to predict the role of TEs in generating genetic and phenotypic diversity.

BackgroundGuayule (Parthenium argentatum A. Gray) is a rubber-producing desert shrub native to Mexico and the United States. Guayule represents an alternative to Hevea brasiliensis as a source for commercial natural rubber. The efficient application of modern molecular/genetic tools to guayule improvement requires characterization of its genome.ResultsThe 1.6 Gb guayule genome was sequenced, assembled and annotated. The final 1.5 Gb assembly, while fragmented (N50 = 22 kb), maps > 95% of the shotgun reads and is essentially complete. Approximately 40,000 transcribed, protein encoding genes were annotated on the assembly. Further characterization of this genome revealed 15 families of small, microsatellite-associated, transposable elements (TEs) with unexpected chromosomal distribution profiles. These SaTar (Satellite Targeted) elements, which are non-autonomous Mu-like elements (MULEs), were frequently observed in multimeric linear arrays of unrelated individual elements within which no individual element is interrupted by another. This uniformly non-nested TE multimer architecture has not been previously described in either eukaryotic or prokaryotic genomes. Five families of similarly distributed non-autonomous MULEs (microsatellite associated, modularly assembled) were characterized in the rice genome. Families of TEs with similar structures and distribution profiles were identified in sorghum and citrus.ConclusionThe sequencing and assembly of the guayule genome provides a foundation for application of current crop improvement technologies to this plant. In addition, characterization of this genome revealed SaTar elements with distribution profiles unique among TEs. Satar targeting appears based on an alternative MULE recombination mechanism with the potential to impact gene evolution.

Genome dynamics is an essential process of eukaryote genome evolution. Hybridization and inter-species gene flow result in new interactions among divergent genomes and may reveal genetic incompatibilities having accumulated after the origin of species. Being highly mutagenic and repressed by various epigenetic mechanisms, transposable elements (TEs) are postulated to play a central role in fuelling genome reorganization following inter-genomic conflicts after hybridization. However, we are still far from understanding mechanisms and forces of such genome dynamics. <br> The main aim of this was to test the implication of multiple TE families on genome reorganization, hybridization success and introgression. Four <i>Aegilops</i> allotetraploid species with interconnected genomes belonging to the wheats group, <i>Aegilops crassa</i> (DDMM), <i>Ae. cylindrica</i> (CCDD), <i>Ae. geniculata</i> (MMUU) and <i>Ae. triuncialis</i> (CCUU) that derived from the diploids <i>Ae. caudata</i> (CC), <i>Ae. comosa</i> (MM), <i>Ae. tauschii</i> (DD) and <i>Ae. umbellulata</i> (UU) were used as model. These species have complex genomes with 80% of repetitive elements that have differentially diverged under the influence of TEs and hybridization. <br> As a first step, the TE composition was assessed using high-throughput sequencing in selected model species and TE families were classified as recently active or quiescent. Based on these results, fingerprint assays were designed and evaluated restructuring in 17 active TE families by comparing genome wide restructuring in diploid and derived tetraploid species and highlighted different TE specific evolutionary trajectories following polyploidy. Restructuring was TE-specific and species-specific, but consistently correlated with TE divergence between progenitors. Using this knowledge, levels of restructuring and methylation changes around insertions of nine TE families were assessed in artificial F1 hybrids between the tetraploid species and, again, correlated with TE divergence between parents. Asymmetrical patterns of genome reorganization paralleling patterns of reproductive isolation among tetraploids, together with nonrandom loss and methylation changes in hybrids suggest that certain events are necessary to produce viable plants. In a last study, hybridization and backcrossing was assessed in natural hybrid zones between the <i>Ae. geniculata</i> and <i>Ae. triuncialis</i>, relating important restructuring of which sequences lost was dominant and that three TE families among the six selected ones were differently exchanged. <br> <i>Aegilops</i> as model offered unparalleled opportunities to address the evolutionary trajectories of multiples candidate TE families and reported evidence of important genome reorganization following various independent hybridization, with multiple TE families clearly playing a central role in host genome evolution and revealing that the progenitors divergence between TE families impacts on the reorganization level. This work reported potential proximate and ultimate factors of genome reorganization driven by conflicts between intragenomic parasites.

The high content of repetitive sequences in the genomes of many higher eukaryotes renders the task of annotating them computationally intensive. Presently, the only widely accepted method of searching and annotating transposable elements (TEs) in large genomic sequences is the use of the RepeatMasker program, which identifies new copies of TEs by pairwise sequence comparisons with a library of known TEs. Profile hidden Markov models (HMMs) have been used successfully in discovering distant homologs of known proteins in large protein databases, but this approach has only rarely been applied to known model TE families in genomic DNA. We used a combination of computational approaches to annotate the TEs in the finished genome of Oryza sativa ssp. japonica. In this paper, we discuss the strengths and the weaknesses of the annotation methods used. These approaches included: the default configuration of RepeatMasker using cross_match, an implementation of the Smith-Waterman-Gotoh algorithm; RepeatMasker using WU-BLAST for similarity searching; and the HMMER package, used to search for TEs with profile HMMs. All the results were converted into GFF format and post-processed using a set of Perl scripts. RepeatMasker was used in the case of most TE families. The WU-BLAST implementation of RepeatMasker was found to be manifold faster than cross_match with only a slight loss in sensitivity and was thus used to obtain the final set of data. HMMER was used in the annotation of the Mutator-like element (MULE) superfamily and the miniature inverted-repeat transposable element (MITE) polyphyletic group of families, for which large libraries of elements were available and which could be divided into well-defined families. The HMMER search algorithm was extremely slow for models over 1000 bp in length, so MULE families with members over 1000 bp long were processed with RepeatMasker instead. The main disadvantage of HMMER in this application is that, since it was developed with protein sequences in mind, it does not search the negative DNA strand. With the exception of TE families with essentially palindromic sequences, reverse complement models had to be created and run to compensate for this shortcoming. We conclude that a modification of RepeatMasker to incorporate libraries of profile HMMs in searches could improve the ability to detect degenerated copies of TEs. The Perl scripts and TE sequences used in construction of the RepeatMasker library and the profile HMMs are available upon request.

Global warming is forcing insect populations to move and adapt, triggering adaptive genetic responses. Thermal stress is known to alter gene expression, repressing the transcription of active genes, and inducing others, such as those encoding heat shock proteins. It has also been related to the activation of some specific transposable element (TE) families. However, the actual magnitude of this stress on the whole genome and the factors involved in these genomic changes are still unclear. We studied mRNAs and small RNAs in gonads of two Drosophila subobscura populations, considered a good model to study adaptation to temperature changes. In control conditions, we found that a few genes and TE families were differentially expressed between populations, pointing out their putative involvement in the adaptation of populations to their different environments. Under heat stress, sex-specific changes in gene expression together with a trend toward overexpression, mainly of heat shock response-related genes, were observed. We did not observe large changes of TE expression nor small RNA production due to stress. Only population and sex-specific expression changes of some TE families (mainly retrotransposons), or the amounts of siRNAs and piRNAs, derived from specific TE families were observed, as well as the piRNA production from some piRNA clusters. Changes in small RNA amounts and TE expression could not be clearly correlated, indicating that other factors as chromatin modulation could also be involved. This work provides the first whole transcriptomic study including genes, TEs, and small RNAs after a heat stress in D. subobscura.