• RNA-directed DNA methylation
• Norway spruce
• small RNAs
• transposable elements

The recently sequenced Norway spruce genome showed some puzzling features:
- a quite large and complex genome mostly populated by repetitive sequences,
- abundant and heterogeneous population of LTR-RTs that mostly inserted in distant evolutionary times and appears to be inactive,
- contrary to previous reports (Dolgosheina et al. 2008) new insights demonstrated that the small RNA population in conifers includes size categories other than the 21 nucleotide one, once thought to be the only significantly represented in these genomes.
Small RNAs have a major role in the regulation of the transposable elements activity through the transcripts degradation or through the methylation of the chromatin region where the transposable element is located. Small RNAs profiling and analyses have been extensively carried out in angiosperms but there is a paucity of similar efforts for gymnosperms. In order to bridge this gap, we deeply characterized the small RNA fraction of two RNA libraries: Spruce Project library and Somatic Embryogenesis library. Spruce Project library, was produced in the frame of the Norway spruce genome sequencing project and includes 22 different spruce tissues. The Somatic Embryogenesis library includes 11 different spruce tissues sampled during the somatic embryogenesis process. For each of the two libraries the size classes in the 19 to 25 nucleotide length range have been quantified and subsequently aligned to the Norway spruce LTR-RTs Gypsy and Copia elements and to other repetitive sequences. The amount of mapped small RNAs, for each of the size classes have been quantified. Furthermore, given the considerable amount of samples tested in both libraries and obtained from different tissues and conditions, several comparisons were carried out to obtain insights about the behaviour of the different small RNA size classes. The results obtained allowed to identify 21-nt as the most abundant small RNA size class. However, other size classes than this one have shown to be considerably represented revealing novel features of the Norway spruce small RNA complement. The comparisons of samples collected from different tissues demonstrated that, in some cases, the length distribution profile of small RNA targeting repetitive sequences is tissue specific and vary across the time. Furthermore, the quantification of the 19 to 25 nucleotide long small RNAs during different stages of the somatic embryogenesis process showed how the regulation of the transposable elements is quite dynamics. The overall results suggest the important and extensive role of RNA-directed DNA methylation in gene and transposable elements transcriptional silencing in the Norway spruce genome.