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Protein coding genes have been extensively studied in both plant and animal genomes, while non-coding portions of the genomes were considered not relevant for a long time. This was due to the fact that non-coding led immediately to not functional, until the discovery of let-7, the first conserved miRNA, in Caenorhabditis elegans. From here on, several studies on small RNAs (sRNAs) were performed, while long non-coding RNAs (lncRNAs) have risen to attention in the last two decades, also because of their usage as diagnostic biomarkers in cancer. Studies to assign function to RNAs have progressed more slowly in plants compared to the animal kingdom and there is still a lot to explore even in the protein coding space, above all if we consider huge genomes like Norway spruce and Scots pine, so the non-coding part of the genome still represents an abyss to discover. In my PhD I mostly focused on a subclass of non-coding RNAs in Norway spruce and aspen. Long non-coding RNAs are considered arbitrarily longer than 200 nucleotides (nt) and can have one small open reading frame (sORF, length < 300 nt) coding for a short peptide (not a complete protein). lncRNAs tend to be expressed at lower levels than genes, but with precise spatio-temporal patterns. They are mostly expressed in particular tissues, stages of a biological process and/or particular conditions, that are often related to biotic or abiotic stresses. They have low levels of sequence homology conservation, even in close related species. In particular, I studied the class of lncRNAs located in the intergenic space, the long intergenic non-coding RNAs (lincRNAs). 

In the first part of this thesis, I developed a pipeline to identify lincRNAs. This pipeline allows to identify in silico bona fide lincRNAs starting from an RNA-Sequencing dataset. It is an ensemble method, considering different tools and the characteristics of lincRNAs. 

In the second part of this thesis, I focused on functionally annotating lincRNAs. To achieve this challenge, I decided to use the guilt-by-association strategy. This method relies on a co-expression network containing both lincRNAs and protein coding genes. Through a functional enrichment of the protein coding genes, it is possible to transfer the same annotation to a lincRNA co-expressed in the same module. I have also tried to relate lincRNAs to a possible function in the de novo methylation of DNA via the RdDM pathway in Norway spruce.

In the last part of this thesis, I identified lincRNAs expressed during leaf development in aspen and produced CRISPR-Cas9 mutants lacking the sequence of two lincRNAs in order to provide a functional validation. 

In general, RNA-Sequencing has enabled and advanced the identification of lincRNAs, and this thesis demonstrates an implemented strategy to identify and assign putative functional information to lincRNAs, deepening the knowledge in the non-coding abyss.