Transposable Element Insertion Sites Research Articles

Ongoing transposition in cell culture reveals the phylogeny of diverse Drosophila S2 sublines.

Cultured cells are widely used in molecular biology despite poor understanding of how cell line genomes change in vitro over time. Previous work has shown that Drosophila cultured cells have a higher transposable element content than whole flies, but whether this increase in transposable element content resulted from an initial burst of transposition during cell line establishment or ongoing transposition in cell culture remains unclear. Here, we sequenced the genomes of 25 sublines of Drosophila S2 cells and show that transposable element insertions provide abundant markers for the phylogenetic reconstruction of diverse sublines in a model animal cell culture system. DNA copy number evolution across S2 sublines revealed dramatically different patterns of genome organization that support the overall evolutionary history reconstructed using transposable element insertions. Analysis of transposable element insertion site occupancy and ancestral states support a model of ongoing transposition dominated by episodic activity of a small number of retrotransposon families. Our work demonstrates that substantial genome evolution occurs during long-term Drosophila cell culture, which may impact the reproducibility of experiments that do not control for subline identity.

Genome-Wide Screening of Transposable Elements in the Whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae), Revealed Insertions with Potential Insecticide Resistance Implications

Simple SummaryTransposable elements (TEs) are mobile DNA sequences hosted in the genomes of various organisms. These elements have the ability to mediate regulatory changes, which can result in changes in gene expression. Bemisia tabaci is an important agricultural pest that has been linked to several cases of insecticide resistance. In this study, we conducted a genome-wide screening of TEs in the B. tabaci genome using bioinformatics tools. Results revealed a total of 1,292,393 TE copies clustered into 4872 lineages. The TE insertion site analysis revealed 94 insertions within or near defensome genes.Transposable elements (TEs) are genetically mobile units that move from one site to another within a genome. These units can mediate regulatory changes that can result in massive changes in genes expression. In fact, a precise identification of TEs can allow the detection of the mechanisms involving these elements in gene regulation and genome evolution. In the present study, a genome-wide analysis of the Hemipteran pest Bemisia tabaci was conducted using bioinformatics tools to identify, annotate and estimate the age of TEs, in addition to their insertion sites, within or near of the defensome genes involved in insecticide resistance. Overall, 1,292,393 TE copies were identified in the B. tabaci genome grouped into 4872 lineages. A total of 699 lineages were found to belong to Class I of TEs, 1348 belong to Class II, and 2825 were uncategorized and form the largest part of TEs (28.81%). The TE age estimation revealed that the oldest TEs invasion happened 14 million years ago (MYA) and the most recent occurred 0.2 MYA with the insertion of Class II TE elements. The analysis of TE insertion sites in defensome genes revealed 94 insertions. Six of these TE insertions were found within or near previously identified differentially expressed insecticide resistance genes. These insertions may have a potential role in the observed insecticide resistance in these pests.

TEAseq-based identification of 35,696 Dissociation insertional mutations facilitates functional genomic studies in maize

In plants, transposable element (TE)-triggered mutants are important resources for functional genomic studies. However, conventional approaches for genome-wide identification of TE insertion sites are costly and laborious. This study developed a novel, rapid, and high-throughput TE insertion site identification workflow based on next-generation sequencing and named it Transposable Element Amplicon Sequencing (TEAseq). Using TEAseq, we systemically profiled the Dissociation (Ds) insertion sites in 1606 independent Ds insertional mutants in advanced backcross generation using K17 as background. The Ac-containing individuals were excluded for getting rid of the potential somatic insertions. We characterized 35,696 germinal Ds insertions tagging 10,323 genes, representing approximately 23.3% of the total genes in the maize genome. The insertion sites were presented in chromosomal hotspots around the ancestral Ds loci, and insertions occurred preferentially in gene body regions. Furthermore, we mapped a loss-of-function AGL2 gene using bulked segregant RNA-sequencing assay and proved that AGL2 is essential for seed development. We additionally established an open-access database named MEILAM for easy access to Ds insertional mutations. Overall, our results have provided an efficient workflow for TE insertion identification and rich sequence-indexed mutant resources for maize functional genomic studies.

A Transposon Story: From TE Content to TE Dynamic Invasion of Drosophila Genomes Using the Single-Molecule Sequencing Technology from Oxford Nanopore.

Transposable elements (TEs) are the main components of genomes. However, due to their repetitive nature, they are very difficult to study using data obtained with short-read sequencing technologies. Here, we describe an efficient pipeline to accurately recover TE insertion (TEI) sites and sequences from long reads obtained by Oxford Nanopore Technology (ONT) sequencing. With this pipeline, we could precisely describe the landscapes of the most recent TEIs in wild-type strains of Drosophila melanogaster and Drosophila simulans. Their comparison suggests that this subset of TE sequences is more similar than previously thought in these two species. The chromosome assemblies obtained using this pipeline also allowed recovering piRNA cluster sequences, which was impossible using short-read sequencing. Finally, we used our pipeline to analyze ONT sequencing data from a D. melanogaster unstable line in which LTR transposition was derepressed for 73 successive generations. We could rely on single reads to identify new insertions with intact target site duplications. Moreover, the detailed analysis of TEIs in the wild-type strains and the unstable line did not support the trap model claiming that piRNA clusters are hotspots of TE insertions.

Transposon insertion profiling by sequencing (TIPseq) for mapping LINE-1 insertions in the human genome

BackgroundTransposable elements make up a significant portion of the human genome. Accurately locating these mobile DNAs is vital to understand their role as a source of structural variation and somatic mutation. To this end, laboratories have developed strategies to selectively amplify or otherwise enrich transposable element insertion sites in genomic DNA.ResultsHere we describe a technique, Transposon Insertion Profiling by sequencing (TIPseq), to map Long INterspersed Element 1 (LINE-1, L1) retrotransposon insertions in the human genome. This method uses vectorette PCR to amplify species-specific L1 (L1PA1) insertion sites followed by paired-end Illumina sequencing. In addition to providing a step-by-step molecular biology protocol, we offer users a guide to our pipeline for data analysis, TIPseqHunter. Our recent studies in pancreatic and ovarian cancer demonstrate the ability of TIPseq to identify invariant (fixed), polymorphic (inherited variants), as well as somatically-acquired L1 insertions that distinguish cancer genomes from a patient’s constitutional make-up.ConclusionsTIPseq provides an approach for amplifying evolutionarily young, active transposable element insertion sites from genomic DNA. Our rationale and variations on this protocol may be useful to those mapping L1 and other mobile elements in complex genomes.

Abstract P3-04-10: Analysis of somatic transposable element insertions in a breast cancer genome

Abstract Introduction: More than 50% of the human genome consists of transposable elements (TE). However most TEs are inactive and remain relatively stable among different human populations. However, some classes of retrotransposons consisting of Alu, SVA, ERV and L1s are active and believe to be involved in cancer. Commonly used bioinformatics pipelines of whole genome sequencing (WGS) data do not currently analyze for somatic TE insertions, and maybe missing an important class of somatic mutations. As a proof-of-concept, we analyzed the HCC1143 triple-negative breast cancer cell line along with a matched normal control to analyze for the presence of somatic TEs. Methods: WGS data of the HCC1143 cell line along with the matched lymphoblastoid control cell line, HCC1143BL, were downloaded from the NCI GDC Commons. We used discordant read-pair (only one paired-end read matches to the reference genome) and split-read (part of a read matches to the reference) analysis to detect novel somatic TE insertion sites in genes known to play important roles in cancer proliferation. Results: Most TE insertions are heterogenous in nature such that they insert only in a small subset of the cells. Therefore by comparing the number of split reads and discordant reads to the total number of reads the heterogenity of the insertion can be inserted. The discordant and split read analysis revealed about 30 high confidence hits. A subset of the high confidence hits were validated experimentally using PCR amplification and gel electrophoresis. About 85% of the hits were successfully validated. A subset of the validated hits known to be relevant to breast cancer have been shown in Table 1. Table 1 - Validated somatic TE insertion locations in HCC1143 cell lineGeneFunctionChromosome numberStart positionStop position% heterogeneity in tumor line = (split_reads + discordant_reads)*2 / total_reads% heterogeneity in blood line = (split_reads + discordant_reads)*2 / total_readsPBRM1Chromatin remodeling352662648526635501001.1GLIS3Overexpressed in TNBC/involved in Wnt pathway942369794237291402.5XPR1Receptor of murine leukemia viruses1180857399180857714253.2CSMD1Tumor suppressor gene84452610445304323.520TMEM181Involved in tamoxifen response61590532671590538254.710 Discussion: Using the approach described above, we were able to detect somatic TEs within several genes known to be involved in cancer. PBRM1, a gene involved in chromatin remodeling, was found to have several exons replaced with a TE insert. Other genes such as CSMD1 is a known tumor suppressor gene, and TMEM181 which is involved in tamoxifen response. These results suggest that TE insertions may be involved in cancer biology. Therefore, TE insert prediction can serve as useful biomarkers for early detection and have precision medicine implications in the treatment of breast cancer. Analyses in additional breast cancer patient genomes is currently underway. Citation Format: Paul R, Solzak JP, Chen Y-H, Hancock BA, Radovich M. Analysis of somatic transposable element insertions in a breast cancer genome [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-04-10.

The distribution of transposable elements (TEs) in the promoter regions of moso bamboo genes and its influence on downstream genes

Transposable elements are abundant in the promoter regions of moso bamboo genome and influence the expression of downstream genes. As important components of animal and plant genomes, transposable elements (TEs) can shape host genomes and regulate gene expression. In the present study, TEs distributed in the promoter regions of moso bamboo genome were systematically investigated with stringy parameters using RepeatMasker. Approximately 85.7% of the promoter regions were anchored into the TE sequences. Among TE families, three types of TEs are preferentially inserted into the promoter regions: hAT-like transposons, miniature inverted-repeat elements (MITEs), and short interspersed elements (SINEs). The TE insertion sites in promoter regions were amplified by PCR. One site (TE-20) exhibited insertion polymorphism. The expression of downstream gene PH01003704G0280 was three to five times higher with the absence of TE-20 than with the presence of it. On the basis of previous studies it was hypothesized that TEs distributed in the promoter regions of four homologs of floral pathway integrators (FPIs) might be responsible for the observed low expression level. To test the hypothesis, four promoter sequences with TE insertions and TE deletions were inserted upstream of the open reading frame of the β-glucuronidase (GUS) gene and green fluorescent protein (GFP) reporter genes. The expression level of downstream genes was higher with TE deletions than with TE insertions. These results show that the TEs are abundant in the promoter regions and influence the expression of downstream genes in moso bamboo genome.

EpiTEome: Simultaneous detection of transposable element insertion sites and their DNA methylation levels

The genome-wide investigation of DNA methylation levels has been limited to reference transposable element positions. The methylation analysis of non-reference and mobile transposable elements has only recently been performed, but required both genome resequencing and MethylC-seq datasets. We have created epiTEome, a program that detects both new transposable element insertion sites and their methylation states from a single MethylC-seq dataset. EpiTEome outperforms other split-read insertion site detection programs, even while functioning on bisulfite-converted reads. EpiTEome characterizes the previously discarded fraction of DNA methylation at sites of new insertions, enabling future investigation into the epigenetic regulation of non-reference and transposed elements.

RelocaTE2: a high resolution transposable element insertion site mapping tool for population resequencing.

BackgroundTransposable element (TE) polymorphisms are important components of population genetic variation. The functional impacts of TEs in gene regulation and generating genetic diversity have been observed in multiple species, but the frequency and magnitude of TE variation is under appreciated. Inexpensive and deep sequencing technology has made it affordable to apply population genetic methods to whole genomes with methods that identify single nucleotide and insertion/deletion polymorphisms. However, identifying TE polymorphisms, particularly transposition events or non-reference insertion sites can be challenging due to the repetitive nature of these sequences, which hamper both the sensitivity and specificity of analysis tools.MethodsWe have developed the tool RelocaTE2 for identification of TE insertion sites at high sensitivity and specificity. RelocaTE2 searches for known TE sequences in whole genome sequencing reads from second generation sequencing platforms such as Illumina. These sequence reads are used as seeds to pinpoint chromosome locations where TEs have transposed. RelocaTE2 detects target site duplication (TSD) of TE insertions allowing it to report TE polymorphism loci with single base pair precision.Results and DiscussionThe performance of RelocaTE2 is evaluated using both simulated and real sequence data. RelocaTE2 demonstrate high level of sensitivity and specificity, particularly when the sequence coverage is not shallow. In comparison to other tools tested, RelocaTE2 achieves the best balance between sensitivity and specificity. In particular, RelocaTE2 performs best in prediction of TSDs for TE insertions. Even in highly repetitive regions, such as those tested on rice chromosome 4, RelocaTE2 is able to report up to 95% of simulated TE insertions with less than 0.1% false positive rate using 10-fold genome coverage resequencing data. RelocaTE2 provides a robust solution to identify TE insertion sites and can be incorporated into analysis workflows in support of describing the complete genotype from light coverage genome sequencing.

Population and clinical genetics of human transposable elements in the (post) genomic era.

ABSTRACTRecent technological developments—in genomics, bioinformatics and high-throughput experimental techniques—are providing opportunities to study ongoing human transposable element (TE) activity at an unprecedented level of detail. It is now possible to characterize genome-wide collections of TE insertion sites for multiple human individuals, within and between populations, and for a variety of tissue types. Comparison of TE insertion site profiles between individuals captures the germline activity of TEs and reveals insertion site variants that segregate as polymorphisms among human populations, whereas comparison among tissue types ascertains somatic TE activity that generates cellular heterogeneity. In this review, we provide an overview of these new technologies and explore their implications for population and clinical genetic studies of human TEs. We cover both recent published results on human TE insertion activity as well as the prospects for future TE studies related to human evolution and health.

Benchmarking computational tools for polymorphic transposable element detection.

Transposable elements (TEs) are an important source of human genetic variation with demonstrable effects on phenotype. Recently, a number of computational methods for the detection of polymorphic TE (polyTE) insertion sites from next-generation sequence data have been developed. The use of such tools will become increasingly important as the pace of human genome sequencing accelerates. For this report, we performed a comparative benchmarking and validation analysis of polyTE detection tools in an effort to inform their selection and use by the TE research community. We analyzed a core set of seven tools with respect to ease of use and accessibility, polyTE detection performance and runtime parameters. An experimentally validated set of 893 human polyTE insertions was used for this purpose, along with a series of simulated data sets that allowed us to assess the impact of sequence coverage on tool performance. The recently developed tool MELT showed the best overall performance followed by Mobster and then RetroSeq. PolyTE detection tools can best detect Alu insertion events in the human genome with reduced reliability for L1 insertions and substantially lowered performance for SVA insertions. We also show evidence that different polyTE detection tools are complementary with respect to their ability to detect a complete set of insertion events. Accordingly, a combined approach, coupled with manual inspection of individual results, may yield the best overall performance. In addition to the benchmarking results, we also provide notes on tool installation and usage as well as suggestions for future polyTE detection algorithm development.

Jitterbug: somatic and germline transposon insertion detection at single-nucleotide resolution.

BackgroundTransposable elements are major players in genome evolution. Transposon insertion polymorphisms can translate into phenotypic differences in plants and animals and are linked to different diseases including human cancer, making their characterization highly relevant to the study of genome evolution and genetic diseases.ResultsHere we present Jitterbug, a novel tool that identifies transposable element insertion sites at single-nucleotide resolution based on the pairedend mapping and clipped-read signatures produced by NGS alignments. Jitterbug can be easily integrated into existing NGS analysis pipelines, using the standard BAM format produced by frequently applied alignment tools (e.g. bwa, bowtie2), with no need to realign reads to a set of consensus transposon sequences. Jitterbug is highly sensitive and able to recall transposon insertions with a very high specificity, as demonstrated by benchmarks in the human and Arabidopsis genomes, and validation using long PacBio reads. In addition, Jitterbug estimates the zygosity of transposon insertions with high accuracy and can also identify somatic insertions.ConclusionsWe demonstrate that Jitterbug can identify mosaic somatic transposon movement using sequenced tumor-normal sample pairs and allows for estimating the cancer cell fraction of clones containing a somatic TE insertion. We suggest that the independent methods we use to evaluate performance are a step towards creating a gold standard dataset for benchmarking structural variant prediction tools.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1975-5) contains supplementary material, which is available to authorized users.

Detection of Transposable Element Insertion Site Polymorphisms by Sequence-Specific Amplification Polymorphism (SSAP)

Transposable elements represent a major part of any eukaryotic genomes. Notably in plants they can account for more than 80% of the whole genomic sequence (such as in maize). Due to their mobility across the genome, they can act as mutagens but can also be considered as an important source of genetic diversity. It has been shown that they may be activated following various stresses, and it has been assumed that they may contribute to genome evolution and adaptation. Molecular methods have thus been proposed to allow identification of new transposition events, or more generally to tag transposable element insertion site polymorphisms. Sequence-Specific Amplification Polymorphism (SSAP) is a high throughput method derived from AFLP, which has been first tested on the barley genome (Waugh et al., 1997). Its efficiency in tagging TEs in comparison to AFLP is based on the use of specific primers anchored in the TE sequences of interest, requiring the TEs under survey to be previously characterized. SSAP can thus be used to identify any genomic reorganization in the vicinity of TE insertion sites, and still represents an efficient approach to analyse evolutionary dynamics of TEs.

Genomic dynamics of transposable elements in the western clawed frog (Silurana tropicalis).

Transposable elements (TEs) are repetitive DNA sequences that can make new copies of themselves that are inserted elsewhere in a host genome. The abundance and distributions of TEs vary considerably among phylogenetically diverse hosts. With the aim of exploring the basis of this variation, we evaluated correlations between several genomic variables and the presence of TEs and non-TE repeats in the complete genome sequence of the Western clawed frog (Silurana tropicalis). This analysis reveals patterns of TE insertion consistent with gene disruption but not with the insertional preference model. Analysis of non-TE repeats recovered unique features of their genome-wide distribution when compared with TE repeats, including no strong correlation with exons and a particularly strong negative correlation with GC content. We also collected polymorphism data from 25 TE insertion sites in 19 wild-caught S. tropicalis individuals. DNA transposon insertions were fixed at eight of nine sites and at a high frequency at one of nine, whereas insertions of long terminal repeat (LTR) and non-LTR retrotransposons were fixed at only 4 of 16 sites and at low frequency at 12 of 16. A maximum likelihood model failed to attribute these differences in insertion frequencies to variation in selection pressure on different classes of TE, opening the possibility that other phenomena such as variation in rates of replication or duration of residence in the genome could play a role. Taken together, these results identify factors that sculpt heterogeneity in TE distribution in S. tropicalis and illustrate that genomic dynamics differ markedly among TE classes and between TE and non-TE repeats.

Allopolyploidy has a moderate impact on restructuring at three contrasting transposable element insertion sites in resynthesized Brassica napus allotetraploids

The role played by whole-genome duplication (WGD) in evolution and adaptation is particularly well illustrated in allopolyploids, where WGD is concomitant with interspecific hybridization. This 'Genome Shock', usually accompanied by structural and functional modifications, has been associated with the activation of transposable elements (TEs). However, the impact of allopolyploidy on TEs has been studied in only a few polyploid species, and not in Brassica, which has been marked by recurrent polyploidy events. Here, we developed sequence-specific amplification polymorphism (SSAP) markers for three contrasting TEs, and compared profiles between resynthesized Brassica napus allotetraploids and their diploid Brassica progenitors. To evaluate restructuring at TE insertion sites, we scored changes in SSAP profiles and analysed a large set of differentially amplified SSAP bands. No massive structural changes associated with the three TEs surveyed were detected. However, several transposition events, specific to the youngest TE originating from the B. oleracea genome, were identified. Our study supports the hypothesis that TE responses to allopolyploidy are highly specific. The changes observed in SSAP profiles lead us to hypothesize that they may partly result from changes in DNA methylation, questioning the role of epigenetics during the formation of a new allopolyploid genome.

Transposing from the Laboratory to the Classroom to Generate Authentic Research Experiences for Undergraduates

Large lecture classes and standardized laboratory exercises are characteristic of introductory biology courses. Previous research has found that these courses do not adequately convey the process of scientific research and the excitement of discovery. Here we propose a model that provides beginning biology students with an inquiry-based, active learning laboratory experience. The Dynamic Genome course replicates a modern research laboratory focused on eukaryotic transposable elements where beginning undergraduates learn key genetics concepts, experimental design, and molecular biological skills. Here we report on two key features of the course, a didactic module and the capstone original research project. The module is a modified version of a published experiment where students experience how virtual transposable elements from rice (Oryza sativa) are assayed for function in transgenic Arabidopsis thaliana. As part of the module, students analyze the phenotypes and genotypes of transgenic plants to determine the requirements for transposition. After mastering the skills and concepts, students participate in an authentic research project where they use computational analysis and PCR to detect transposable element insertion site polymorphism in a panel of diverse maize strains. As a consequence of their engagement in this course, students report large gains in their ability to understand the nature of research and demonstrate that they can apply that knowledge to independent research projects.

A transposable element insertion within ZmGE2 gene is associated with increase in embryo to endosperm ratio in maize

Most of the maize kernel oil is located in the embryo while the majority of starch is located in the endosperm. Maize kernel composition and value are affected significantly by the ratio of the embryo size to the endosperm size; however, the genetic regulation of embryo to endosperm ratio (EER) in maize is unknown. Here we identified ZmGE2 gene, which encodes a cytochrome p450 protein, as a gene associated with EER variation in maize. We first expressed rice Giant Embryo (GE) gene driven by oleosin promoter in maize and detected a 23.2 % reduction in EER in transgenic seeds, demonstrating the existence of evolutionarily conserved mechanisms for EER determination in rice and maize. We next identified maize GE2, a homolog of rice GE sharing 70 % identity in amino sequence, as a candidate based on the similar expression pattern and co-localization with a previously detected QTL for EER. Followed by linkage and association mapping, a 247-bp transposable element (TE) insertion in 3'-untranslated region of ZmGE2 gene was identified to be associated with increase in EER and kernel oil content. Expression level of the favorable ZmGE2 allele containing the 247-bp TE insertion was strongly reduced. In addition, the 247-bp TE insertion site was a selection target during the artificial long-term selection for the high EER trait in a high oil population. This is the first report that demonstrates an association of ZmGE2 with EER variation in maize and identifies ZmGE2 gene as a promising target for manipulation of EER and grain composition by either transgenic approach or molecular breeding in maize.

Sequencing of Pooled DNA Samples (Pool-Seq) Uncovers Complex Dynamics of Transposable Element Insertions in Drosophila melanogaster

Transposable elements (TEs) are mobile genetic elements that parasitize genomes by semi-autonomously increasing their own copy number within the host genome. While TEs are important for genome evolution, appropriate methods for performing unbiased genome-wide surveys of TE variation in natural populations have been lacking. Here, we describe a novel and cost-effective approach for estimating population frequencies of TE insertions using paired-end Illumina reads from a pooled population sample. Importantly, the method treats insertions present in and absent from the reference genome identically, allowing unbiased TE population frequency estimates. We apply this method to data from a natural Drosophila melanogaster population from Portugal. Consistent with previous reports, we show that low recombining genomic regions harbor more TE insertions and maintain insertions at higher frequencies than do high recombining regions. We conservatively estimate that there are almost twice as many “novel” TE insertion sites as sites known from the reference sequence in our population sample (6,824 novel versus 3,639 reference sites, with on average a 31-fold coverage per insertion site). Different families of transposable elements show large differences in their insertion densities and population frequencies. Our analyses suggest that the history of TE activity significantly contributes to this pattern, with recently active families segregating at lower frequencies than those active in the more distant past. Finally, using our high-resolution TE abundance measurements, we identified 13 candidate positively selected TE insertions based on their high population frequencies and on low Tajima's D values in their neighborhoods.

A proposal for the reference-based annotation of de novo transposable element insertions

Understanding the causes and consequences of transposable element (TE) activity in the genomic era requires sophisticated bioinformatics approaches to accurately identify individual insertion sites. Next-generation sequencing technology now makes it possible to rapidly identify new TE insertions using resequencing data, opening up new possibilities to study the nature of TE-induced mutation and the target site preferences of different TE families. While the identification of new TE insertion sites is seemingly a simple task, the mechanisms of transposition present unique challenges for the annotation of de novo transposable element insertions mapped to a reference genome. Here I discuss these challenges and propose a framework for the annotation of de novo TE insertions that accommodates known mechanisms of TE insertion and established coordinate systems for genome annotation.

Massive alterations of the methylation patterns around DNA transposons in the first four generations of a newly formed wheat allohexaploid

Rapid and reproducible genomic changes can be induced during the early stages of the life of nascent allopolyploid species. In a previous study, it was shown that following allopolyploidization, cytosine methylation changes can affect up to 11% of the wheat genome. However, the methylation patterns around transposable elements (TEs) were never studied in detail. We used transposon methylation display (TMD) to assess the methylation patterns of CCGG sites flanking three TE families (Balduin, Apollo, and Thalos) in the first four generations of a newly formed wheat allohexaploid. In addition, transposon display (TD), using a methylation-insensitive restriction enzyme, was applied to search for genomic rearrangements at the TE insertion sites. We observed that up to 54% of CCGG sites flanking the three TE families showed changes in methylation patterns in the first four generations of a newly formed wheat allohexaploid, where hypermethylation was predominant. Over 70% of the changes in TMD patterns occurred in the first two generations of the newly formed allohexaploid. Furthermore, analysis of 555 TE insertion sites by TD and 18 cases by site-specific PCR revealed a full additive pattern in the allohexaploid, an indication for lack of massive rearrangements. These data indicate that following allopolyplodization, DNA-TE insertion sites can undergo a significantly high level of methylation changes compared with methylation changes of other genomic sequences.