بررسی تغییرات نوکلئوتیدی ویروئید اگزوکورتیس مرکبات در نارنج با استفاده از چند شکلی فضایی تک رشته‌ای

Citrus exocortis viroid (CEVd) is the causal agent of exocortis disease and belongs to the genus Pospiviroid of the family Pospiviroidae. CEVd have a potential for high genetic diversity due to the absence of proofreading activity of the RNA polymerases, their large populations and rapid replication. The rapid evolutionary pattern of viroid fits the quasispecies model. In Iran, the majority of citrus species cultivated commercially are grafted onto sour orange (Citrus aurantium L.) rootstocks. In this study full-length in vitro transcript of single sequence CEVd-S1 originated form sweet orange tree on citrange rootstock of Mazandaran, was used to inoculate three sour orange seedlings. Seedlings were slash inoculated with RNA transcripts and sterile water (in negative control) using a razor blade. Following the RNA extraction from fresh bark tissues, cDNA synthesis and DNA amplification was performed using specific primers. The amplified DNA cloned into pJET1.2/ blunt vector. Generated variants in sour orange were detected in 60 cDNA clones by using single strand conformation polymorphism (SSCP). In SSCP, mutated sequence was detected as a change of mobility caused by its altered folded structure. Alongside the most frequent wild-type variant (68%), frequency of three generated variants was estimated as 20, 7 and 5 percent. Furthermore, analysis of thermodynamic secondary structures illustrated that nucleotide changes identified in this study do not induce major modifications in the viroid rod-like secondary structure because their structures play a critical role in their life cycles, or are compensatory mutations.
Key words: In vitro transcripts, Population structure, Thermodynamic secondary structures, Variant