Detection of active transposable elements in Arabidopsis thaliana using Oxford Nanopore Sequencing technology.

BACKGROUND: Transposables elements (TEs) contribute to both structural and functional dynamics of most eukaryotic genomes. Because of their propensity to densely populate plant and animal genomes, the precise estimation of the impact of transposition on genomic diversity has been considered as one of the main challenges of today's genomics. The recent development of NGS (next generation sequencing) technologies has open new perspectives in population genomics by providing new methods for high throughput detection of Transposable Elements-associated Structural Variants (TEASV). However, these have relied on Illumina platform that generates short reads (up to 350 nucleotides). This limitation in size of sequence reads can cause high false discovery rate (FDR) and therefore limit the power of detection of TEASVs, especially in the case of large, complex genomes. The newest sequencing technologies, such as Oxford Nanopore Technologies (ONT) can generate kilobases-long reads thus representing a promising tool for TEASV detection in plant and animals. RESULTS: We present the results of a pilot experiment for TEASV detection on the model plant species Arabidopsis thaliana using ONT sequencing and show that it can be used efficiently to detect TE movements. We generated a ~0.8X genome coverage of a met1-derived epigenetic recombinant inbred line (epiRIL) using a MinIon device with R7 chemistry. We were able to detect nine new copies of the LTR-retrotransposon Evadé (EVD). We also evidenced the activity of the DNA transposon CACTA, CAC1. CONCLUSIONS: Even at a low sequence coverage (0.8X), ONT sequencing allowed us to reliably detect several TE insertions in Arabidopsis thaliana genome. The long read length allowed a precise and un-ambiguous mapping of the structural variations caused by the activity of TEs. This suggests that the trade-off between read length and genome coverage for TEASV detection may be in favor of the former. Should the technology be further improved both in terms of lower error rate and operation costs, it could be efficiently used in diversity studies at population level.

Epigenetic imbalance and the floral developmental abnormality of the in vitro-regenerated oil palm Elaeis guineensis.

BACKGROUND: The large-scale clonal propagation of oil palm (Elaeis guineensis) is being stalled by the occurrence of the mantled somaclonal variation. Indeed, this abnormality which presents a homeotic-like conversion of male floral organs into carpelloid structures, hampers oil production since the supernumerary female organs are either sterile or produce fruits with poor oil yields. SCOPE: In the last 15 years, the prevailing point of view on the origin of the mantled floral phenotype has evolved from a random mutation event triggered by in vitro culture to a hormone-dependent dysfunction of gene regulation processes. In this review, we retrace the history of the research on the mantled variation in the light of the parallel advances made in the understanding of plant development regulation in model systems and more specifically in the role of epigenetic mechanisms. An overview of the current state of oil palm genomic and transcriptomic resources, which are key to any comparison with model organisms, is given. We show that, while displaying original characteristics, the mantled phenotype of oil palm is morphologically, and possibly molecularly, related to MADS-box genes mutants described in model plants. We also discuss the occurrence of comparable floral phenotypes in other palm species. CONCLUSIONS: Beyond its primary interest in the search for discriminating markers against an economically crippling phenotype, the study of the mantled abnormality also provides a unique opportunity to investigate the regulation of reproductive development in a perennial tropical palm. On the basis of recent results, we propose that future efforts should concentrate on the epigenetic regulation targeting MADS-box genes and transposable elements of oil palm, since both types of sequences are most likely to be involved in the mantled variant phenotype.

Construction and characterization of a knock-down RNA interference line of OsNRPD1 in rice (Oryza sativa ssp japonica cv Nipponbare).

In plants, RNA-directed DNA methylation (RdDM) is a silencing mechanism relying on the production of 24-nt small interfering RNAs (siRNAs) by RNA POLYMERASE IV (Pol IV) to trigger methylation and inactivation of transposable elements (TEs). We present the construction and characterization of osnrpd1, a knock-down RNA interference line of OsNRPD1 gene that encodes the largest subunit of Pol IV in rice (Oryza sativa ssp japonica cv Nipponbare). We show that osnrpd1 displays a lower accumulation of OsNRPD1 transcripts, associated with an overall reduction of 24-nt siRNAs and DNA methylation level in all three contexts, CG, CHG and CHH. We uncovered new insertions of known active TEs, the LTR retrotransposons Tos17 and Lullaby and the long interspersed nuclear element-type retrotransposon Karma. However, we did not observe any clear developmental phenotype, contrary to what was expected for a mutant severely affected in RdDM. In addition, despite the presence of many putatively functional TEs in the rice genome, we found no evidence of in planta global reactivation of transposition. This knock-down of OsNRPD1 likely led to a weakly affected line, with no effect on development and a limited effect on transposition. We discuss the possibility that a knock-out mutation of OsNRPD1 would cause sterility in rice. This article is part of a discussion meeting issue 'Crossroads between transposons and gene regulation'.

Retrotranspositional landscape of Asian rice revealed by 3000 genomes.

The recent release of genomic sequences for 3000 rice varieties provides access to the genetic diversity at species level for this crop. We take advantage of this resource to unravel some features of the retrotranspositional landscape of rice. We develop software TRACKPOSON specifically for the detection of transposable elements insertion polymorphisms (TIPs) from large datasets. We apply this tool to 32 families of retrotransposons and identify more than 50,000 TIPs in the 3000 rice genomes. Most polymorphisms are found at very low frequency, suggesting that they may have occurred recently in agro. A genome-wide association study shows that these activations in rice may be triggered by external stimuli, rather than by the alteration of genetic factors involved in transposable element silencing pathways. Finally, the TIPs dataset is used to trace the origin of rice domestication. Our results suggest that rice originated from three distinct domestication events.

DNA methylation and expression of the EgDEF1 gene and neighboring retrotransposons in mantled somaclonal variants of oil palm.

The mantled floral phenotype of oil palm (Elaeis guineensis) affects somatic embryogenesis-derived individuals and is morphologically similar to mutants defective in the B-class MADS-box genes. This somaclonal variation has been previously demonstrated to be associated to a significant deficit in genome-wide DNA methylation. In order to elucidate the possible role of DNA methylation in the transcriptional regulation of EgDEF1, the APETALA3 ortholog of oil palm, we studied this epigenetic mark within the gene in parallel with transcript accumulation in both normal and mantled developing inflorescences. We also examined the methylation and expression of two neighboring retrotransposons that might interfere with EgDEF1 regulation. We show that the EgDEF1 gene is essentially unmethylated and that its methylation pattern does not change with the floral phenotype whereas expression is dramatically different, ruling out a direct implication of DNA methylation in the regulation of this gene. Also, we find that both the gypsy element inserted within an intron of the EgDEF1 gene and the copia element located upstream from the promoter are heavily methylated and show little or no expression. Interestingly, we identify a shorter, alternative transcript produced by EgDEF1 and characterize its accumulation with respect to its full-length counterpart. We demonstrate that, depending on the floral phenotype, the respective proportions of these two transcripts change differently during inflorescence development. We discuss the possible phenotypical consequences of this alternative splicing and the new questions it raises in the search for the molecular mechanisms underlying the mantled phenotype in the oil palm.