Cas9-targeted Nanopore sequencing rapidly elucidates the transposition preferences and DNA methylation profiles of mobile elements in plants.

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 40× sequence coverage. Analysis of hemizygous T-DNA insertion sites and genetically inherited insertions of the EVD transposon in the ddm1 (decrease in DNA methylation 1) 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 ONSENs. Finally, we detected hypomethylation of novel somatic insertions for two ONSENs. 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.

Nanopore RNA Sequencing Revealed Long Non-Coding and LTR Retrotransposon-Related RNAs Expressed at Early Stages of Triticale SEED Development.

The intergenic space of plant genomes encodes many functionally important yet unexplored RNAs. The genomic loci encoding these RNAs are often considered "junk", DNA as they are frequently associated with repeat-rich regions of the genome. The latter makes the annotations of these loci and the assembly of the corresponding transcripts using short RNAseq reads particularly challenging. Here, using long-read Nanopore direct RNA sequencing, we aimed to identify these "junk" RNA molecules, including long non-coding RNAs (lncRNAs) and transposon-derived transcripts expressed during early stages (10 days post anthesis) of seed development of triticale (AABBRR, 2n = 6x = 42), an interspecific hybrid between wheat and rye. Altogether, we found 796 lncRNAs and 20 LTR retrotransposon-related transcripts (RTE-RNAs) expressed at this stage, with most of them being previously unannotated and located in the intergenic as well as intronic regions. Sequence analysis of the lncRNAs provide evidence for the frequent exonization of Class I (retrotransposons) and class II (DNA transposons) transposon sequences and suggest direct influence of "junk" DNA on the structure and origin of lncRNAs. We show that the expression patterns of lncRNAs and RTE-related transcripts have high stage specificity. In turn, almost half of the lncRNAs located in Genomes A and B have the highest expression levels at 10-30 days post anthesis in wheat. Detailed analysis of the protein-coding potential of the RTE-RNAs showed that 75% of them carry open reading frames (ORFs) for a diverse set of GAG proteins, the main component of virus-like particles of LTR retrotransposons. We further experimentally demonstrated that some RTE-RNAs originate from autonomous LTR retrotransposons with ongoing transposition activity during early stages of triticale seed development. Overall, our results provide a framework for further exploration of the newly discovered lncRNAs and RTE-RNAs in functional and genome-wide association studies in triticale and wheat. Our study also demonstrates that Nanopore direct RNA sequencing is an indispensable tool for the elucidation of lncRNA and retrotransposon transcripts.

Genomic and Transcriptomic Survey Provides New Insight into the Organization and Transposition Activity of Highly Expressed LTR Retrotransposons of Sunflower (Helianthus annuus L.).

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.

Functional Allium fistulosum Centromeres Comprise Arrays of a Long Satellite Repeat, Insertions of Retrotransposons and Chloroplast DNA.

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.