Characterisation of the genetic diversity of citrus viroid VII using amplicon sequencing

Viroids occur in plants as swarms of sequence variants clustered around a dominant variant, leading to adoption of the term ‘quasispecies’ to describe the viroid population in an individual host. The composition of the quasispecies can potentially change according to the age of the infection, the position of the leaf or branch in the canopy, and the host species. The primary aim of this study was to investigate the quasispecies concept for citrus viroid VII (CVd-VII), a recently discovered member of the family Pospiviroidae. Three experiments were conducted to determine factors affecting viroid variability (i) within different tissues of a lemon plant, (ii) among different plants of the same species (citron), and (iii) among different species and hybrids of citrus. Using two primer sets to produce amplicons for high-throughput sequencing, viroid population profiles were generated for each sample. The number of variants that were identified with both primer sets ranged from 2 to 13 per sample, and each sample comprised 1 to 4 major (> 10% sample) variants. The composition of variants differed in samples from different plants and among tissue types of a single plant. Single-nucleotide polymorphisms (SNPs), mostly in the form of substitutions, were the primary source of variation; in this study, SNPs were observed in approximately 10% of the viroid genome. The results of the three experiments indicate that CVd-VII follows the quasispecies model as reported for other viroids and that variability occurs in viroid populations in different tissue types and host species.