Transposable element insertions (TEIs) are an important source of genomic innovation by contributing to plant adaptation, speciation, and the production of new varieties. The often large, complex plant genomes make identifying TEIs from short reads difficult and expensive. Moreover, rare somatic insertions that reflect mobilome dynamics are difficult to track using short reads. To address these challenges, we combined Cas9-targeted Nanopore sequencing (CANS) with the novel pipeline NanoCasTE to trace both genetically inherited and somatic TEIs in plants. We performed CANS of the EVADÉ ( EVD ) retrotransposon in wild-type Arabidopsis thaliana and rapidly obtained up to 40x sequence coverage. Analysis of hemizygous T-DNA insertion sites and genetically inherited insertions of the EVD transposon in the ddm1 genome uncovered the crucial role of DNA methylation in shaping EVD insertion preference. We also investigated somatic transposition events of the ONSEN transposon family, finding that genes that are downregulated during heat stress are preferentially targeted by ONSEN s. Finally, we detected hypomethylation of novel somatic insertions for two ONSEN s. CANS and NanoCasTE are effective tools for detecting TEIs and exploring mobilome organization in plants in response to stress and in different genetic backgrounds, as well as screening T-DNA insertion mutants and transgenic plants.

Long-read data is a great tool to discover new active transposable elements (TEs). However, no ready-to-use tools were available to gather this information from low coverage ONT datasets. Here, we developed a novel pipeline, nanotei, that allows detection of TE-contained structural variants, including individual TE transpositions. We exploited this pipeline to identify TE insertion in the Arabidopsis thaliana genome. Using nanotei, we identified tens of TE copies, including ones for the well-characterized ONSEN retrotransposon family that were hidden in genome assembly gaps. The results demonstrate that some TEs are inaccessible for analysis with the current A. thaliana (TAIR10.1) genome assembly. We further explored the mobilome of the ddm1 mutant with elevated TE activity. Nanotei captured all TEs previously known to be active in ddm1 and also identified transposition of non-autonomous TEs. Of them, one non-autonomous TE derived from (AT5TE33540) belongs to TR-GAG retrotransposons with a single open reading frame (ORF) encoding the GAG protein. These results provide the first direct evidence that TR-GAGs and other non-autonomous LTR retrotransposons can transpose in the plant genome, albeit in the absence of most of the encoded proteins. In summary, nanotei is a useful tool to detect active TEs and their insertions in plant genomes using low-coverage data from Nanopore genome sequencing.

The centromere is a unique part of the chromosome combining a conserved function with an extreme variability in its DNA sequence. Most of our knowledge about the functional centromere organization is obtained from species with small and medium genome/chromosome sizes while the progress in plants with big genomes and large chromosomes is lagging behind. Here, we studied the genomic organization of the functional centromere in Allium fistulosum and A. cepa, both species with a large genome (13 Gb and 16 Gb/1C, 2n = 2x = 16) and large-sized chromosomes. Using low-depth DNA sequencing for these two species and previously obtained CENH3 immunoprecipitation data we identified two long (1.2 Kb) and high-copy repeats, AfCen1K and AcCen1K. FISH experiments showed that AfCen1K is located in all centromeres of A. fistulosum chromosomes while no AcCen1K FISH signals were identified on A. cepa chromosomes. Our molecular cytogenetic and bioinformatics survey demonstrated that these repeats are partially similar but differ in chromosomal location, sequence structure and genomic organization. In addition, we could conclude that the repeats are transcribed and their RNAs are not polyadenylated. We also observed that these repeats are associated with insertions of retrotransposons and plastidic DNA and the landscape of A. cepa and A. fistulosum centromeric regions possess insertions of plastidic DNA. Finally, we carried out detailed comparative satellitome analysis of A. cepa and A. fistulosum genomes and identified a new chromosome- and A. cepa-specific tandem repeat, TR2CL137, located in the centromeric region. Our results shed light on the Allium centromere organization and provide unique data for future application in Allium genome annotation.

LTR retrotransposons (RTEs) play a crucial role in plant genome evolution and adaptation. Although RTEs are generally silenced in somatic plant tissues under non-stressed conditions, some expressed RTEs (exRTEs) escape genome defense mechanisms. As our understanding of exRTE organization in plants is rudimentary, we systematically surveyed the genomic and transcriptomic organization and mobilome (transposition) activity of sunflower (Helianthus annuus L.) exRTEs. We identified 44 transcribed RTEs in the sunflower genome and demonstrated their distinct genomic features: more recent insertion time, longer open reading frame (ORF) length, and smaller distance to neighboring genes. We showed that GAG-encoding ORFs are present at significantly higher frequencies in exRTEs, compared with non-expressed RTEs. Most exRTEs exhibit variation in copy number among sunflower cultivars and one exRTE Gagarin produces extrachromosomal circular DNA in seedling, demonstrating recent and ongoing transposition activity. Nanopore direct RNA sequencing of full-length RTE RNA revealed complex patterns of alternative splicing in RTE RNAs, resulting in isoforms that carry ORFs for distinct RTE proteins. Together, our study demonstrates that tens of expressed sunflower RTEs with specific genomic organization shape the hidden layer of the transcriptome, pointing to the evolution of specific strategies that circumvent existing genome defense mechanisms.