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Viroids are small circular single-stranded infectious RNAs that arecharacterized by a relatively high mutation level. Knowledge of their sequenceheterogeneity remains largely elusive, and, as yet, no strategy attempting toaddress this question from a population dynamics point of view is in place. Inorder to address these important questions, a GF305 indicator peach tree wasinfected with a single variant of the Avsunviroidae family member Peach latentmosaic viroid (PLMVd). Six months post-inoculation, full-length circularconformers of PLMVd were isolated, deep-sequenced and the resulting sequencesanalyzed using an original bioinformatics scheme specifically designed anddeveloped in order to evaluate the richness of a given the sequence'spopulation. Two distinct libraries were analyzed, and yielded 1125 and 1061different PLMVd variants respectively, making this study the most productive todate (by more than an order of magnitude) in terms of the reporting of novelviroid sequences. Sequence variants exhibiting up to ~20% of mutations relativeto the inoculated viroid were retrieved, clearly illustrating the highdivergence dynamic inside a unique population. Using a novel hierarchicalclustering algorithm, the different variants obtained were grouped into either7 or 8 clusters depending on the library being analyzed. Most of the sequencescontained, on average, between 4.6 and 6.3 mutations relative to the variantused initially to inoculate the plant. Interestingly, it was possible toreconstitute the sequence evolution between these clusters. On top of providinga reliable pipeline for the treatment of viroid deep-sequencing, this studysheds new light on the importance of the sequence variation that may take placein a viroid population and which may result in the formation of aquasi-species.

Deep-sequencing of the Peach Latent Mosaic Viroid Reveals New Aspects of Population Heterogeneity