Identification of circular RNAs of Cannabis sativa L. potentially involved in the biosynthesis of cannabinoids.

ABSTRACT

MAIN CONCLUSION: We identified circRNAs in the Cannabis sativa L. genome and examined their association with 28 cannabinoids in three tissues of C. sativa. Nine circRNAs are potentially involved in the biosynthesis of six cannabinoids. Cannabis sativa L. has been widely used in the production of medicine, textiles, and food for over 2500 years. The main bioactive compounds in C. sativa are cannabinoids, which have multiple important pharmacological actions. Circular RNAs (circRNAs) play essential roles in growth and development, stress resistance, and the biosynthesis of secondary metabolites. However, the circRNAs in C. sativa remain unknown. In this study, to explore the role of circRNAs in cannabinoid biosynthesis, we performed RNA-Seq and metabolomics analysis on the leaves, roots, and stems of C. sativa. We identified 741 overlapping circRNAs by three tools, of which 717, 16, and 8 circRNAs were derived from exonic, intronic, and intergenic, respectively. Functional enrichment analysis indicated that the parental genes (PGs) of circRNAs were enriched in many processes related to biological stress responses. We found that most of the circRNAs showed tissue-specific expression and 65 circRNAs were significantly correlated with their PGs (P < 0.05, |r|≥ 0.5). We also determined 28 cannabinoids by High-performance liquid chromatography-ESI-triple quadrupole-linear ion trap mass spectrometry. Ten circRNAs, including ciR0159, ciR0212, ciR0153, ciR0149, ciR0016, ciR0044, ciR0022, ciR0381, ciR0006, and ciR0025 were found to be associated with six cannabinoids by weighted gene co-expression network analysis. Twenty-nine of 53 candidate circRNAs, including 9 cannabinoids related were validated successfully using PCR amplification and Sanger sequencing. Taken together, all these results would help to enhance our acknowledge of the regulation of circRNAs, and lay the foundation for breeding new C. sativa cultivars with high cannabinoids through manipulating circRNAs.