Identification of miRNAs involved in fruit ripening by deep sequencing of Olea europaea L. transcriptome

The ripening process of olive fruits is associated with chemical and/or enzymatic specific transformations, making them particularly attractive to animals and humans. In olive drupes, including 'Cassanese' ones, ripening is usually accompanied by progressive chromatic change, resulting in a final red-brown colourization of both epidermis and mesocarp. This event has an exception in the 'Leucocarpa', in which we observed the destabilization in the equilibrium between the chlorophyll metabolism and that of the other pigments, particularly the anthocyanins, whose switch-off during maturation promotes the white colouration of the fruits. Recently, transcription profiling of 'Leucocarpa' and 'Cassanese' olives along ripening, performed through an Illumina RNA-seq approach, has provided useful insights on genes functions involved in fruit maturation such as those related to the biosynthesis of flavonoids and anthocyanins.

To assess expression alterations of genes involved in flavonoids and anthocyanins biosynthetic pathways during ripening, possibly caused by small nuclear RNA (snRNA) in olive drupes, snRNA libraries from 'Leucocarpa' and 'Cassanese' were constructed with RNAs extracted at 100 and 130 Days After Flowering (DAF) and sequenced by an Illumina approach. 130 conserved microRNAs (miRNA) in the Viridiplantae belonging to 14 miRNA families were identified. Regarding the 130 conserved miRNAs, approximately the 48% were identified in all libraries, 5 and 18 miRNAs were shared between the "Cassanese" (C100, C130) and "Leucocarpa" (L100, L130) libraries, respectively.

For the remaining reads not-matching with known miRNAs in the Viridiplantae, we combined secondary structure and minimum free energy to discover novel olive miRNAs. Based on these analyses, 492 sequences were considered as putative novel miRNAs. The putative target genes of identified miRNA were computationally predicted by alignment with the olive drupe transcripts obtained from the same samples. A total of 218 transcripts were predicted as targets of 130 known and 492 putative novel miRNAs. Interestingly, some identified target genes are involved in negative regulation of anthocyanin metabolic process. Quantification of the expression pattern of three miRNA and their target transcripts by qRT-PCR assay confirmed the results of Illumina sequencing.