High-Throughput Sequencing Analysis of Small RNAs Derived from Coleus Blumei Viroids

Characterization of viroid-derived small RNAs (vd-sRNAs) is important to understand viroid–host interactions; however, vd-sRNAs belonging to the genus Coleviroid are yet to be identified and characterized. Herein, we used coleus plants singly infected with coleus blumei viroid (CbVd)-1, -5, or -6 and doubly infected with CbVd-1 and -5 to identify and analyze their vd-sRNAs. We found sense and antisense vd-sRNAs for CbVd-1, -5 and -6, and 22-nt vd-sRNAs were the most abundant; moreover, the 5′-terminal nucleotides (nts) of CbVd-1, -5, and -6 were biased toward U and C, and sRNAs derived from these three viroids were unevenly distributed along their genomes. We also noted that CbVd-5 and -6 share a fragment that forms the right half of the rod-like secondary structure of these viroids, which implied that they generated almost the same type of vd-sRNAs. This finding indicated that vd-sRNA biogenesis is mainly determined by the primary sequence of their substrates. More importantly, we found two complementary vd-sRNAs (22 nt) that were generated from the central conserved region (CCR) of these three viroids, suggesting an important role of CCR in vd-sRNA biogenesis. In conclusion, our results provide novel insight into the biogenesis of vd-sRNAs and the biological roles of CCR.