Genomic structure of new Tunisian peach latent mosaic viroid variants

Peach latent mosaic viroid (PLMVd) is a single-stranded circular RNA that do not code for proteins and ranges in size from 335 to 351 nucleotides. It mainly infects peach. In this study, the sequence of 20 complete cDNA clones derived from seven PLMVd isolates detected in five Tunisian peach cultivars was analysed in 3 steps: primary structure, phylogeny and secondary structure. The analysis of the primary structure revealed that all the 20 cDNA clones sequences corresponded to different variants. They ranged in size from 336 to 341 nt. Sequence alignment of our variants with reference sequences revealed 81 polymorphic positions. Among them, 15 were never described in the literature so far. The variable positions are scattered all around the RNA molecules, but the majority of them were concentrated in the region corresponding to nucleotides 1 to 70 and 170 to 346 in the alignment. Sequence homologies between variants of the same isolate or variants of different isolates ranged from 96% to 100%. This confirms that a PLMVd isolate is composed by a complex mixture of closely related molecules. Moreover, some variants isolated from different cultivars were found to be similar, indicating that a sequence is not exclusive to a cultivar. Phylogenetic analysis of our sequences allowed their clustering into two groups: group I (16 variants) and group II (4 variants) that differed by 18 polymorphic positions. Further phylogenetic analysis and sequence alignment of our sequences and the reference sequences were done. It revealed that our sequences were similar to the reference sequence Hd8 in the regions delimited by nucleotides 1 to 69 (region 1) and 268 to 343 (region 5) and to the reference sequence Hd6 in the region between nucleotides 150 and 200 (region 3). The other regions corresponding to nucleotides 70 to 149 (region 2) and 201 to 267 (region 4) were similar for all the sequences. These observations revealed that our Tunisian PLMVd variants correspond to a new population never reported in the literature. Analysis of the secondary structure confirmed that all PLMVd Tunisian variants presented a branched secondary structure and revealed a new potential pseudoknot-like interaction between two loops.