Variability of Citrus Exocortis Viroid (CEVd)

Abstract

Single-strand conformation polymorphism (SSCP) has been adopted as a tool for viroid characterization. Nucleic acid extracts from a viroid field isolate containing CEVd was sub- jected to retrotranscription and PCR amplification using a set of contiguous CEVd-specific prim- ers. The analysis of cloned DNAs recovered from this isolate illustrates the extent of variability within a single CEVd isolate. The relationship between the SSCP profiles observed among clones and the variation in their nucleotide sequences was confirmed by sequence analysis of selected clones. The results indicate that field isolates are complex populations or quasispecies. Two highly conserved sequences (master sequences) as well as several less abundant variants were consis- tently identified. The sequence variants presented a base pairing arrangement in the P domain characteristic of severe strains of CEVd. A branched secondary structure rather than the rod-like structure was predicted to occur at 37 ° C. Index words. Variants, SSCP, quasispecies. Citrus exocortis viroid (CEVd), the causal agent of the exocortis dis- ease, was discovered in 1972 (12). Determination of its primary struc- ture (4, 14), showed that the CEVd molecule is a covalently closed circu- lar RNA with a highly base paired rod-like structure which conforms the model of five structural domains (T L , P, C, V, T R ) (7). Viroid replication which occurs in the absence of proof-reading mecha- nisms accounts for a high rate of variability generation. Although there are numerous structural con- straints, which impair infectivity