Population-level annotation of lncRNAs in Arabidopsis thaliana reveals extensive expression and epigenetic variability associated with TE-like silencing [Bisulfite-seq]
Background Long non-coding RNAs (lncRNAs) are under-studied and under-annotated in plants. In mammals, lncRNA expression has been shown to be reaching the extent of protein-coding expression and be highly variable between individuals of the same species. Using A. thaliana as a model plant organism, we aimed to understand the true scope of lncRNA transcription across plants from different regions, characterize lncRNA natural expression variability, and study the causes of this variability. Results Using RNA-seq data spanning 499 natural lines and 4 different developmental stages to create a more comprehensive annotation of lncRNAs in A. thaliana, we found over 10,000 novel loci — three times as many as in the current public annotation. We showed that, while lncRNA loci are ubiquitous in the genome, most appear to be actively silenced and their expression and repressive chromatin levels are extremely variable between natural lines. It was particularly prominent in intergenic lncRNAs, where TE-like sequences present in 50% of the loci are associated with increased silencing and variation and such lincRNAs tend to be targeted by TE silencing machinery. Conclusion lncRNAs are ubiquitous in the A. thaliana genome, but their expression is highly variable between different lines and tissues. This high expression variability is largely caused by high structural and epigenetic variability of non-coding loci, especially those containing pieces of transposable elements. We create the most comprehensive A. thaliana lncRNA annotation to date and improve our understanding of plant lncRNA biology. Overall design: We performed this bisulfite-seq to analyze epignetic patterns and silencing of lncRNAs in Arabidopsis thaliana. plants from 32 different A.thaliana natural accessions (https://1001genomes.org/accessions.html) were grown at the growth chambers of GMI, Vienna, Austria. The growing conditions were 21 degrees Celcius, long-day (16-hour light, 8-hour dark). Mature leaves (not the oldest and not the youngest) from 2-3 individual plants were collected for each accession and snap frozen in liquid nitrogen. Leaves were collected when the plants reached 14-16-leaf stage prior to the start of bolting. Frozen tissue was ground with a Retsch machine using steel beads. DNA was isolated using Nuclear Mag Plant kit (Machery-Nagel) and the bisulfate libraries were prepared using a tagmentation method described in (Wang et al Nature Protocols 2013) using an in-house Tn5 transposase (IMBA-IMP-GMI Molecular Biology Services) and EZ-96 DNA Methylation-Gold Mag Prep kit (Zymo Research) for bisulfite conversion. The sowing, growing and sample collection was performed 2 to 4 times and this corresponds to 2 to 4 replicates per accession.