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

Abstract

Viroids are small circular single-stranded infectious RNAs that are characterized by a relatively high mutation level. Knowledge of their sequence heterogeneity remains largely elusive, and, as yet, no strategy attempting to address this question from a population dynamics point of view is in place. In order to address these important questions, a GF305 indicator peach tree was infected with a single variant of the Avsunviroidae family member Peach latent mosaic viroid (PLMVd). Six months post-inoculation, full-length circular conformers of PLMVd were isolated, deep-sequenced and the resulting sequences analyzed using an original bioinformatics scheme specifically designed and developed in order to evaluate the richness of a given the sequence's population. Two distinct libraries were analyzed, and yielded 1125 and 1061 different PLMVd variants respectively, making this study the most productive to date (by more than an order of magnitude) in terms of the reporting of novel viroid sequences. Sequence variants exhibiting up to ~20% of mutations relative to the inoculated viroid were retrieved, clearly illustrating the high divergence dynamic inside a unique population. Using a novel hierarchical clustering algorithm, the different variants obtained were grouped into either 7 or 8 clusters depending on the library being analyzed. Most of the sequences contained, on average, between 4.6 and 6.3 mutations relative to the variant used initially to inoculate the plant. Interestingly, it was possible to reconstitute the sequence evolution between these clusters. On top of providing a reliable pipeline for the treatment of viroid deep-sequencing, this study sheds new light on the importance of the sequence variation that may take place in a viroid population and which may result in the formation of a quasi-species.