Cannabis Virome Reconstruction and Antiviral RNAi Characterization through Small RNA Sequencing

Viral infections pose an emerging threat to hemp ( ) cultivation. We used Illumina small (s)RNA sequencing for virome reconstruction and characterization of antiviral RNA interference (RNAi) in monoecious and dioecious hemp varieties, which exhibited different virus-like symptoms. Through de novo and reference-based sRNA assembly, we identified and reconstructed Cannabis cryptic virus (family ), mitovirus 1 ( ) and Grapevine line pattern virus ( ) as well as a novel virus tentatively classified into . Members of both and were targeted by antiviral RNAi, generating 21 nt and, less abundant, 22 nt sRNAs from both strands of the entire virus genome, suggesting the involvement of Dicer-like (DCL) 4 and DCL2 in viral sRNA biogenesis, respectively. Mitovirus sRNAs represented predominantly the positive-sense strand and had a wider size range, with the 21 nt class being most abundant on both strands. For all viruses, 21 and 22 nt sRNAs had predominantly 5'-terminal uridine or cytosine, suggesting their binding to antiviral Argonaute (AGO) 1 and AGO5, respectively. As no clear association of any virus with symptoms was observed, further studies should clarify if these viruses individually or in combination can cause hemp diseases.