Small RNAs of Nuclear Viroids Figure 3. Relative abundance of vd-sRNAs with different size and 59 termini. Histograms comparing the size distribution (20–24-nt) and nucleotide at the 59 termini of total (A and D), (+) (B and E) and (2) (C and F) HSVd-sRNAs reads (left panels) and GYSVd1-sRNAs reads (right panels) from tendril. doi:10.1371/journal.pone.0007686.g003 that the (+) sRNAs from HS1 and HS2 were not concurrently generated by DCLs targeting the structured circular (+) genomic RNA. The (2) HSVd-sRNAs hotspot (HS3) mapped to the lower strand of the viroid rod-like secondary structure, partially overlapping with HS2 (Fig. 5A). Also in this case, complementary vd-sRNAs with two 39-protruding nucleotides in each strand composed by (+) and (2) HSVd-sRNAs from the two overlapping hotspots were not identified, suggesting that these HSVd-sRNAs cannot be considered concurrent products of common DCL- mediated cleavages. HSVd-sRNAs of different sizes mapped to each hotspot (see below), suggesting that several DCLs accessed the same restricted RNA genomic regions. Taking advantage of the observation that the HS2 hotspot corresponds to a genomic sequence wherein a polymorphic position in the HSVd-infecting population was also mapped (Fig. S1), we could confirm that most vd-sRNAs (90%) from this region had a nucleotide composition corresponding to that of the identified HSVd master sequence (accession number: X06873), supporting the quantitative repro- ducibility of our data and their consistency with the genomic variability of the infecting HSVd population. Mapping GYSVd1-sRNAs from tendrils gave similar results: most (+) sRNAs derived from only two hotspots (GY1 and GY2), whereas those of the (2) polarity mostly concentrated at a single hotspot (GY3) (Fig. 5B). Similarly to HSVd, 79% and 75% of (+) PLoS ONE | www.plosone.org 5 November 2009 | Volume 4 | Issue 11 | e7686