Specificities and Dynamics of Transposable Elements in Land Plants.

Transposable elements (TEs) are important components of most plant genomes. These mobile repetitive sequences are highly diverse in terms of abundance, structure, transposition mechanisms, activity and insertion specificities across plant species. This review will survey the different mechanisms that may explain the variability of TE patterns in land plants, highlighting the tight connection between TE dynamics and host genome specificities, and their co-evolution to face and adapt to a changing environment. We present the current TE classification in land plants, and describe the different levels of genetic and epigenetic controls originating from the plant, the TE itself, or external environmental factors. Such overlapping mechanisms of TE regulation might be responsible for the high diversity and dynamics of plant TEs observed in nature.

Parental transposable element loads influence their dynamics in young Nicotiana hybrids and allotetraploids.

The genomic shock hypothesis suggests that allopolyploidy is associated with genome changes driven by transposable elements, as a response to imbalances between parental insertion loads. To explore this hypothesis, we compared three allotetraploids, Nicotiana arentsii, N. rustica and N. tabacum, which arose over comparable time frames from hybridisation between increasingly divergent diploid species. We used sequence-specific amplification polymorphism (SSAP) to compare the dynamics of six transposable elements in these allopolyploids, their diploid progenitors and in corresponding synthetic hybrids. We show that element-specific dynamics in young Nicotiana allopolyploids reflect their dynamics in diploid progenitors. Transposable element mobilisation is not concomitant with immediate genome merger, but occurs within the first generations of allopolyploid formation. In natural allopolyploids, such mobilisations correlate with imbalances in the repeat profile of the parental species, which increases with their genetic divergence. Other restructuring leading to locus loss is immediate, nonrandom and targeted at specific subgenomes, independently of cross orientation. The correlation between transposable element mobilisation in allopolyploids and quantitative imbalances in parental transposable element loads supports the genome shock hypothesis proposed by McClintock.

Elucidating the major hidden genomic components of the A, C, and AC genomes and their influence on Brassica evolution.

Decoding complete genome sequences is prerequisite for comprehensive genomics studies. However, the currently available reference genome sequences of Brassica rapa (A genome), B. oleracea (C) and B. napus (AC) cover 391, 540, and 850 Mbp and represent 80.6, 85.7, and 75.2% of the estimated genome size, respectively, while remained are hidden or unassembled due to highly repetitive nature of these genome components. Here, we performed the first comprehensive genome-wide analysis using low-coverage whole-genome sequences to explore the hidden genome components based on characterization of major repeat families in the B. rapa and B. oleracea genomes. Our analysis revealed 10 major repeats (MRs) including a new family comprising about 18.8, 10.8, and 11.5% of the A, C and AC genomes, respectively. Nevertheless, these 10 MRs represented less than 0.7% of each assembled reference genome. Genomic survey and molecular cytogenetic analyses validates our insilico analysis and also pointed to diversity, differential distribution, and evolutionary dynamics in the three Brassica species. Overall, our work elucidates hidden portions of three Brassica genomes, thus providing a resource for understanding the complete genome structures. Furthermore, we observed that asymmetrical accumulation of the major repeats might be a cause of diversification between the A and C genomes.

LTR-retrotransposons in plants: Engines of evolution.

LTR retrotransposons are the most abundant group of transposable elements (TEs) in plants. These elements can fall inside or close to genes, and therefore influence their expression and evolution. This review aims to examine how LTR retrotransposons, especially Ty1-copia elements, mediate gene regulation and evolution. Various stimuli, including polyploidization and biotic and abiotic elicitors, result in the transcription and movement of these retrotransposons, and can facilitate adaptation. The presence of cis-regulatory motifs in the LTRs are central to their stress-mediated responses and are shared with host stress-responsive genes, showing a complex evolutionary history in which TEs provide new regulatory units to genes. The presence of retrotransposon remnants in genes that are necessary for normal gene function, demonstrates the importance of exaptation and co-option, and is also a consequence of the abundance of these elements in plant genomes. Furthermore, insertions of LTR retrotransposons in and around genes provide potential for alternative splicing, epigenetic control, transduction, duplication and recombination. These characteristics can become an active part of the evolution of gene families as in the case of resistance genes (R-genes). The character of TEs as exclusively selfish is now being re-evaluated. Since genome-wide reprogramming via TEs is a long evolutionary process, the changes we can examine are case-specific and their fitness advantage may not be evident until TE-derived motifs and domains have been completely co-opted and fixed. Nevertheless, the presence of LTR retrotransposons inside genes and as part of gene promoter regions is consistent with their roles as engines of plant genome evolution.

Ty1-copia elements reveal diverse insertion sites linked to polymorphisms among flax (Linum usitatissimum L.) accessions.

BACKGROUND: Initial characterization of the flax genome showed that Ty1-copia retrotransposons are abundant, with several members being recently inserted, and in close association with genes. Recent insertions indicate a potential for ongoing transpositional activity that can create genomic diversity among accessions, cultivars or varieties. The polymorphisms generated constitute a good source of molecular markers that may be associated with phenotype if the insertions alter gene activity. Flax, where accessions are bred mainly for seed nutritional properties or for fibers, constitutes a good model for studying the relationship of transpositional activity with diversification and breeding. In this study, we estimated copy number and used a type of transposon display known as Sequence-Specific Amplification Polymorphisms (SSAPs), to characterize six families of Ty1-copia elements across 14 flax accessions. Polymorphic insertion sites were sequenced to find insertions that could potentially alter gene expression, and a preliminary test was performed with selected genes bearing transposable element (TE) insertions. RESULTS: Quantification of six families of Ty1-copia elements indicated different abundances among TE families and between flax accessions, which suggested diverse transpositional histories. SSAPs showed a high level of polymorphism in most of the evaluated retrotransposon families, with a trend towards higher levels of polymorphism in low-copy number families. Ty1-copia insertion polymorphisms among cultivars allowed a general distinction between oil and fiber types, and between spring and winter types, demonstrating their utility in diversity studies. Characterization of polymorphic insertions revealed an overwhelming association with genes, with insertions disrupting exons, introns or within 1 kb of coding regions. A preliminary test on the potential transcriptional disruption by TEs of four selected genes evaluated in three different tissues, showed one case of significant impact of the insertion on gene expression. CONCLUSIONS: We demonstrated that specific Ty1-copia families have been active since breeding commenced in flax. The retrotransposon-derived polymorphism can be used to separate flax types, and the close association of many insertions with genes defines a good source of potential mutations that could be associated with phenotypic changes, resulting in diversification processes.

Highly efficient gene tagging in the bryophyte Physcomitrella patens using the tobacco (Nicotiana tabacum) Tnt1 retrotransposon.

Because of its highly efficient homologous recombination, the moss Physcomitrella patens is a model organism particularly suited for reverse genetics, but this inherent characteristic limits forward genetic approaches. Here, we show that the tobacco (Nicotiana tabacum) retrotransposon Tnt1 efficiently transposes in P. patens, being the first retrotransposon from a vascular plant reported to transpose in a bryophyte. Tnt1 has a remarkable preference for insertion into genic regions, which makes it particularly suited for gene mutation. In order to stabilize Tnt1 insertions and make it easier to select for insertional mutants, we have developed a two-component system where a mini-Tnt1 with a retrotransposition selectable marker can only transpose when Tnt1 proteins are co-expressed from a separate expression unit. We present a new tool with which to produce insertional mutants in P. patens in a rapid and straightforward manner that complements the existing molecular and genetic toolkit for this model species.

LTR retrotransposons, handy hitchhikers of plant regulation and stress response.

LTR retrotransposons are major components of plant genomes. They are regulated by a diverse array of external stresses and tissue culture conditions, displaying finely tuned responses to these stimuli, mostly in the form of upregulation. Second to stress conditions and tissue culture, meristems are also permissive for LTR retrotransposon expression, suggesting that a dedifferentiated cell status may represent a frequent activating condition. LTR regions are highly plastic and contain regulatory motifs similar to those of cellular genes. The activation of LTR retrotransposons results from interplay between the release of epigenetic silencing and the recruitment by LTRs of specific regulatory factors. Despite the role of LTR retrotransposons in driving plant genome diversification, convincing evidence for major mobilizations of LTR retrotransposons remains much rarer than observations of massive bursts of transcriptional upregulation. Current evidence suggests that LTR retrotransposon expression may be involved in host functional plasticity, acting as dispersed regulatory modules able to redirect stress stimuli to adjacent plant genes. This may be of crucial importance for plants that cannot escape stress, and have evolved complex and highly coordinated responses to external challenges. This article is part of a Special Issue entitled: Stress as a fundamental theme in cell plasticity.

Different tobacco retrotransposons are specifically modulated by the elicitor cryptogein and reactive oxygen species.

Interactions of plant retrotransposons with different steps of biotic and abiotic stress-associated signaling cascades are still poorly understood. We perform here a finely tuned comparison of four tobacco retrotransposons (Tnt1, Tnt2, Queenti, and Tto1) responses to the plant elicitor cryptogein. We demonstrate that basal transcript levels in cell suspensions and plant leaves as well as the activation during the steps of defense signaling events are specific to each retrotransposon. Using antisense NtrbohD lines, we show that NtrbohD-dependent reactive oxygen species (ROS) production might act as negative regulator of retrotransposon activation.

Detecting epigenetic effects of transposable elements in plants.

Transposable elements (TE) represent a major fraction of eukaryotic genomes and play many roles in plant epigenetics. In this chapter, we describe the use of Sequence-Specific Amplified Polymorphism (SSAP) as a reliable Transposon Display technique applicable for use in many plant species. We also discuss the interpretation of SSAP data and associated risks. This technique has potential to allow rapid screening of plant populations, especially in nonmodel or wild species.

Diploidization and genome size change in allopolyploids is associated with differential dynamics of low- and high-copy sequences.

Recent advances have highlighted the ubiquity of whole-genome duplication (polyploidy) in angiosperms, although subsequent genome size change and diploidization (returning to a diploid-like condition) are poorly understood. An excellent system to assess these processes is provided by Nicotiana section Repandae, which arose via allopolyploidy (approximately 5 million years ago) involving relatives of Nicotiana sylvestris and Nicotiana obtusifolia. Subsequent speciation in Repandae has resulted in allotetraploids with divergent genome sizes, including Nicotiana repanda and Nicotiana nudicaulis studied here, which have an estimated 23.6% genome expansion and 19.2% genome contraction from the early polyploid, respectively. Graph-based clustering of next-generation sequence data enabled assessment of the global genome composition of these allotetraploids and their diploid progenitors. Unexpectedly, in both allotetraploids, over 85% of sequence clusters (repetitive DNA families) had a lower abundance than predicted from their diploid relatives; a trend seen particularly in low-copy repeats. The loss of high-copy sequences predominantly accounts for the genome downsizing in N. nudicaulis. In contrast, N. repanda shows expansion of clusters already inherited in high copy number (mostly chromovirus-like Ty3/Gypsy retroelements and some low-complexity sequences), leading to much of the genome upsizing predicted. We suggest that the differential dynamics of low- and high-copy sequences reveal two genomic processes that occur subsequent to allopolyploidy. The loss of low-copy sequences, common to both allopolyploids, may reflect genome diploidization, a process that also involves loss of duplicate copies of genes and upstream regulators. In contrast, genome size divergence between allopolyploids is manifested through differential accumulation and/or deletion of high-copy-number sequences.

Next generation sequencing analysis reveals a relationship between rDNA unit diversity and locus number in Nicotiana diploids.

BACKGROUND: Tandemly arranged nuclear ribosomal DNA (rDNA), encoding 18S, 5.8S and 26S ribosomal RNA (rRNA), exhibit concerted evolution, a pattern thought to result from the homogenisation of rDNA arrays. However rDNA homogeneity at the single nucleotide polymorphism (SNP) level has not been detailed in organisms with more than a few hundred copies of the rDNA unit. Here we study rDNA complexity in species with arrays consisting of thousands of units. METHODS: We examined homogeneity of genic (18S) and non-coding internally transcribed spacer (ITS1) regions of rDNA using Roche 454 and/or Illumina platforms in four angiosperm species, Nicotiana sylvestris, N. tomentosiformis, N. otophora and N. kawakamii. We compared the data with Southern blot hybridisation revealing the structure of intergenic spacer (IGS) sequences and with the number and distribution of rDNA loci. RESULTS AND CONCLUSIONS: In all four species the intragenomic homogeneity of the 18S gene was high; a single ribotype makes up over 90% of the genes. However greater variation was observed in the ITS1 region, particularly in species with two or more rDNA loci, where >55% of rDNA units were a single ribotype, with the second most abundant variant accounted for >18% of units. IGS heterogeneity was high in all species. The increased number of ribotypes in ITS1 compared with 18S sequences may reflect rounds of incomplete homogenisation with strong selection for functional genic regions and relaxed selection on ITS1 variants. The relationship between the number of ITS1 ribotypes and the number of rDNA loci leads us to propose that rDNA evolution and complexity is influenced by locus number and/or amplification of orphaned rDNA units at new chromosomal locations.

Differential dynamics of transposable elements during long-term diploidization of Nicotiana section Repandae (Solanaceae) allopolyploid genomes.

Evidence accumulated over the last decade has shown that allopolyploid genomes may undergo drastic reorganization. However, timing and mechanisms of structural diploidization over evolutionary timescales are still poorly known. As transposable elements (TEs) represent major and labile components of plant genomes, they likely play a pivotal role in fuelling genome changes leading to long-term diploidization. Here, we exploit the 4.5 MY old allopolyploid Nicotiana section Repandae to investigate the impact of TEs on the evolutionary dynamics of genomes. Sequence-specific amplified polymorphisms (SSAP) on seven TEs with expected contrasted dynamics were used to survey genome-wide TE insertion polymorphisms. Comparisons of TE insertions in the four allopolyploid species and descendents of the diploid species most closely related to their actual progenitors revealed that the polyploids showed considerable departure from predicted additivity of the diploids. Large numbers of new SSAP bands were observed in polyploids for two TEs, but restructuring for most TE families involved substantial loss of fragments relative to the genome of the diploid representing the paternal progenitor, which could be due to changes in allopolyploids, diploid progenitor lineages or both. The majority of non-additive bands were shared by all polyploid species, suggesting that significant restructuring occurred early after the allopolyploid event that gave rise to their common ancestor. Furthermore, several gains and losses of SSAP fragments were restricted to N. repanda, suggesting a unique evolutionary trajectory. This pattern of diploidization in TE genome fractions supports the hypothesis that TEs are central to long-term genome turnover and depends on both TE and the polyploid lineage considered.

Maize genetic diversity and association mapping using transposable element insertion polymorphisms.

Transposable elements are the major component of the maize genome and presumably highly polymorphic yet they have not been used in population genetics and association analyses. Using the Transposon Display method, we isolated and converted into PCR-based markers 33 Miniature Inverted Repeat Transposable Elements (MITE) polymorphic insertions. These polymorphisms were genotyped on a population-based sample of 26 American landraces for a total of 322 plants. Genetic diversity was high and partitioned within and among landraces. The genetic groups identified using Bayesian clustering were in agreement with published data based on SNPs and SSRs, indicating that MITE polymorphisms reflect maize genetic history. To explore the contribution of MITEs to phenotypic variation, we undertook an association mapping approach in a panel of 367 maize lines phenotyped for 26 traits. We found a highly significant association between the marker ZmV1-9, on chromosome 1, and male flowering time. The variance explained by this association is consistent with a flowering delay of +123 degree-days. This MITE insertion is located at only 289 nucleotides from the 3' end of a Cytochrome P450-like gene, a region that was never identified in previous association mapping or QTL surveys. Interestingly, we found (i) a non-synonymous mutation located in the exon 2 of the gene in strong linkage disequilibrium with the MITE polymorphism, and (ii) a perfect sequence homology between the MITE sequence and a maize siRNA that could therefore potentially interfere with the expression of the Cytochrome P450-like gene. Those two observations among others offer exciting perspectives to validate functionally the role of this region on phenotypic variation.

Next generation sequencing reveals genome downsizing in allotetraploid Nicotiana tabacum, predominantly through the elimination of paternally derived repetitive DNAs.

We used next generation sequencing to characterize and compare the genomes of the recently derived allotetraploid, Nicotiana tabacum (<200,000 years old), with its diploid progenitors, Nicotiana sylvestris (maternal, S-genome donor), and Nicotiana tomentosiformis (paternal, T-genome donor). Analysis of 14,634 repetitive DNA sequences in the genomes of the progenitor species and N. tabacum reveal all major types of retroelements found in angiosperms (genome proportions range between 17-22.5% and 2.3-3.5% for Ty3-gypsy elements and Ty1-copia elements, respectively). The diploid N. sylvestris genome exhibits evidence of recent bursts of sequence amplification and/or homogenization, whereas the genome of N. tomentosiformis lacks this signature and has considerably fewer homogenous repeats. In the derived allotetraploid N. tabacum, there is evidence of genome downsizing and sequences loss across most repeat types. This is particularly evident amongst the Ty3-gypsy retroelements in which all families identified are underrepresented in N. tabacum, as is 35S ribosomal DNA. Analysis of all repetitive DNA sequences indicates the T-genome of N. tabacum has experienced greater sequence loss than the S-genome, revealing preferential loss of paternally derived repetitive DNAs at a genome-wide level. Thus, the three genomes of N. sylvestris, N. tomentosiformis, and N. tabacum have experienced different evolutionary trajectories, with genomes that are dynamic, stable, and downsized, respectively.

Impact of transposable elements on the organization and function of allopolyploid genomes.

Transposable elements (TEs) represent an important fraction of plant genomes and are likely to play a pivotal role in fuelling genome reorganization and functional changes following allopolyploidization. Various processes associated with allopolyploidy (i.e. genetic redundancy, bottlenecks during the formation of allopolyploids or genome shock following genome merging) may allow accumulation of TE insertions. Our objective in carrying out a survey of the literature and a comparative analysis across different allopolyploid systems is to shed light on the structural, epigenetic and functional modifications driven by TEs during allopolyploidization and subsequent diploidization. The available evidence indicates that TE proliferation in the short or the long term after allopolyploidization may be restricted to a few TEs, in specific polyploid systems. By contrast, data indicate major structural changes in the TE genome fraction immediately after allopolyploidization, mainly through losses of TE sequences as a result of recombination. Emerging evidence also suggests that TEs are targeted by substantial epigenetic changes, which may impact gene expression and genome stability. Furthermore, TEs may directly or indirectly support the evolution of new functionalities in allopolyploids during diploidization. All data stress allopolyploidization as a shock associated with drastic genome reorganization. Mechanisms controlling TEs during allopolyploidization as well as their impact on diploidization are discussed.

Potential impact of stress activated retrotransposons on genome evolution in a marine diatom.

BACKGROUND: Transposable elements (TEs) are mobile DNA sequences present in the genomes of most organisms. They have been extensively studied in animals, fungi, and plants, and have been shown to have important functions in genome dynamics and species evolution. Recent genomic data can now enlarge the identification and study of TEs to other branches of the eukaryotic tree of life. Diatoms, which belong to the heterokont group, are unicellular eukaryotic algae responsible for around 40% of marine primary productivity. The genomes of a centric diatom, Thalassiosira pseudonana, and a pennate diatom, Phaeodactylum tricornutum, that likely diverged around 90 Mya, have recently become available. RESULTS: In the present work, we establish that LTR retrotransposons (LTR-RTs) are the most abundant TEs inhabiting these genomes, with a much higher presence in the P. tricornutum genome. We show that the LTR-RTs found in diatoms form two new phylogenetic lineages that appear to be diatom specific and are also found in environmental samples taken from different oceans. Comparative expression analysis in P. tricornutum cells cultured under 16 different conditions demonstrate high levels of transcriptional activity of LTR retrotransposons in response to nitrate limitation and upon exposure to diatom-derived reactive aldehydes, which are known to induce stress responses and cell death. Regulatory aspects of P. tricornutum retrotransposon transcription also include the occurrence of nitrate limitation sensitive cis-regulatory components within LTR elements and cytosine methylation dynamics. Differential insertion patterns in different P. tricornutum accessions isolated from around the world infer the role of LTR-RTs in generating intraspecific genetic variability. CONCLUSION: Based on these findings we propose that LTR-RTs may have been important for promoting genome rearrangements in diatoms.

Rapid structural and epigenetic reorganization near transposable elements in hybrid and allopolyploid genomes in Spartina.

*Transposable elements (TE) induce structural and epigenetic alterations in their host genome, with major evolutionary implications. These alterations are examined here in the context of allopolyploid speciation, on the recently formed invasive species Spartina anglica, which represents an excellent model to contrast plant genome dynamics following hybridization and genome doubling in natural conditions. *Methyl-sensitive transposon display was used to investigate the structural and epigenetic dynamics of TE insertion sites for several elements, and to contrast it with comparable genome-wide methyl-sensitive amplified polymorphism analyses. *While no transposition burst was detected, we found evidence of major structural and CpG methylation changes in the vicinity of TE insertions accompanying hybridization, and to a lesser extent, genome doubling. Genomic alteration appeared preferentially in the maternal subgenome, and the environment of TEs was specifically affected by large maternal-specific methylation changes, demonstrating that TEs fuel epigenetic alterations at the merging of diverged genomes. *Such genome changes indicate that nuclear incompatibilities in Spartina trigger immediate alterations, which are TE-specific with an important epigenetic component. Since most of this reorganization is conserved after genome doubling that produced a fertile invasive species, TEs certainly play a central role in the shock-induced dynamics of the genome during allopolyploid speciation.

LTR-retrotransposons Tnt1 and T135 markers reveal genetic diversity and evolutionary relationships of domesticated peppers.

Plant genetic resources often constitute the foundation of successful breeding programs. Pepper (Capsicum annuum L.) is one of the most economically important and diversely utilized Solanaceous crop species worldwide, but less studied compared to tomato and potato. We developed and used molecular markers based on two copia-type retrotransposons, Tnt1 and T135, in a set of Capsicum species and wild relatives from diverse geographical origins. Results showed that Tnt1 and T135 insertion polymorphisms are very useful for studying genetic diversity and relationships within and among pepper species. Clusters of accessions correspond to cultivar types based on fruit shape, pungency, geographic origin and pedigree. Genetic diversity values, normally reflective of past transposition activity and population dynamics, showed positive correlation with the average number of insertions per accession. Similar evolutionary relationships are observed to that inferred by previous karyosystematics studies. These observations support the possibility that retrotransposons have contributed to genome inflation during Capsicum evolution.

Contrasting evolutionary patterns and target specificities among three Tourist-like MITE families in the maize genome.

Miniature inverted-repeat transposable elements (MITEs) are short, non autonomous DNA elements that are widespread and abundant in plant genomes. The high sequence and size conservation observed in many MITE families suggest that they have spread recently throughout their respective host genomes. Here we present a maize genome wide analysis of three Tourist-like MITE families, mPIF, and two previously uncharacterized families, ZmV1 and Zead8. We undertook a bioinformatic analysis of MITE insertion sites, developed methyl-sensitive transposon display (M-STD) assays to estimate the associated level of CpG methylation at MITE flanking regions, and conducted a population genetics approach to investigate MITE patterns of expansion. Our results reveal that the three MITE families insert into genomic regions that present specific molecular features: they are preferentially AT rich, present low level of cytosine methylation as compared to the LTR retrotransposon Grande, and target site duplications are flanked by large and conserved palindromic sequences. Moreover, the analysis of MITE distances from predicted genes shows that 73% of 263 copies are inserted at less than 5 kb from the nearest predicted gene, and copies from Zead8 family are significantly more abundant upstream of genes. By employing a population genetic approach we identified contrasting patterns of expansion among the three MITE families. All elements seem to have inserted roughly 1 million years ago but ZmV1 and Zead8 families present evidences for activity of several master copies within the last 0.4 Mya.

Distribution dynamics of the Tnt1 retrotransposon in tobacco.

Retrotransposons contribute significantly to the size, organization and genetic diversity of plant genomes. Although many retrotransposon families have been reported in plants, to this day, the tobacco Tnt1 retrotransposon remains one of the few elements for which active transposition has been shown. Demonstration that Tnt1 activation can be induced by stress has lent support to the hypothesis that, under adverse conditions, transposition can be an important source of genetic variability. Here, we compared the insertion site preference of a collection of newly transposed and pre-existing Tnt1 copies identified in plants regenerated from protoplasts or tissue culture. We find that newly transposed Tnt1 copies are targeted within or close to host gene coding sequences and that the distribution of pre-existing insertions does not vary significantly from this trend. Therefore, in spite of their potential to disrupt neighboring genes, insertions within or near CDS are not preferentially removed with age. Elimination of Tnt1 insertions within or near coding sequences may be relaxed due to the polyploid nature of the tobacco genome. Tnt1 insertions within or near CDS are thus better tolerated and can putatively contribute to the diversification of tobacco gene function.