Population-level annotation of lncRNAs in Arabidopsis thaliana reveals extensive expression and epigenetic variability associated with TE-like silencing [RNA-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 RNA-seq to map lncRNAs and study their inter- and intra-accession expression variation in Arabidopsis thaliana. Plants from 27 different A.thaliana natural accessions 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). For the “9-leaf rosette” samples we collected the full rosette at the 9-leaf stage with one plant harvested per sample. Seedlings were collected at 7 days post sowing (~5 days post germination). The full seedling with the root was collected with approximately 10 individuals/seedlings harvested per sample. For the “flower” samples we collected flowers and flower buds from approximately 5 individuals per sample. Accessions 1741, 6024, 6244, 9075, 9543, 9638, 9728, 9764, 9888, 9905, 22003, 22004, 22005, 22006, 22007 were cold-treated by placing them into 10oC growth chambers (long-day conditions) for ~ 4 weeks to induce flowering. Pollen was collected using the method described in (Johnson-Brousseau and McCormick, 2004) that uses vacuum suction and a series of filters to harvest dry pollen from flowering plants. We used polyester mesh filters of 3 sizes - 150mm, 60mm and 10mm - for the collection. Collected pollen was snap-frozen in liquid nitrogen, stored at -70oC and ground for RNA-sequencing using ~0.5ml of 0.5mm glass beads (Scientific Industries, Inc.). Frozen tissue was ground with a Retsch machine using steel beads. RNA was isolated using an in-house RNA isolation kit (magnetic beads based) and DNAse treated with DNAse I (New England Biolabs). We did not manage to get accessions 6069 and 6124 to flower, so for these two accessions we only have rosette and seedling samples.