In silico identification, characterization and expression analysis of miRNAs in Cannabis sativa L.

• Homology based computational approach of miRNA identification was conducted in Transcript Sequence Assemblies (TSA) and ESTs of Cannabis sativa L.
• Eighteen miRNAs belonging to 9 independent miRNA families were identified in C. sativa L. for the first time.
• Expression of 6 predicted miRNAs was confirmed in young and mature leaf tissues of C. sativa seedlings using Real Time-PCR.
• A total of 80 potential target genes were identified for the predicted miRNAs in Arabidopsis genome.

Cannabis sativa L. is an annual herb and economically important as a source of fiber, oil, food and for its medicinal and intoxicating properties. MicroRNAs are a class of short (~ 21 nt), non-coding regulatory RNAs that play a major role in post-transcriptional gene silencing. By in silico analysis of the publically available Transcript Sequence Assemblies (TSA) and Expressed Sequence Tags (ESTs) of C. sativa, a total of 18 conserved miRNAs belonging to 9 independent families were identified. To validate the predicted miRNAs, SYBR green based assay of qPCR was applied to detect the tissue-specific (young and mature leaf) expression of 6 putative miRNAs (csa-miR156, csa-miR159a, csa-miR171b, csa-miR172a, csa-miR5021a, csa-miR6034) in C. sativa. A total of 80 target genes were also recognized for the newly identified miRNAs, and subsequently assigned to three broad functional categories: biological processes, cellular components and molecular functions as defined for the Arabidopsis proteome. The potential target genes consist of transcription factors (33.75%), transporters (5%), kinase and other enzymes (20%) as well as signaling and other functional proteins (32.50%). The findings in this study on C. sativa miRNA precursors, mature miRNAs, and miRNA targets will be helpful for future research on miRNA-mediated gene regulation in this important plant species.