تنوع ژنتیکی جدایه های Candidatus Phytoplasma aurantifolia همراه با بیماری جاروک لیموترش با استفاده از آنالیز چندشکلی ساختار تک رشته ای

Single-strand conformation polymorphism (SSCP) analysis is a sensitive and rapid technique for detecting DNA polymorphisms and mutations in PCR-amplified fragments. The aim of this work was to analyze the phytoplasma retroelements (bacterial group II introns) by SSCP for investigating the genetic variation of lime witches'-broom phytoplasma. Twenty samples collected from citrus plants exhibiting symptoms of lime witches' broom from Hormozgan and Kerman Provinces. DNA was extracted by CTAB method. A 900 bp fragment amplified by PCR using IntF1/IntR1 primer pair, was selected for genetic diversity analysis of the phytoplasma isolates by SSCP method. One to four μL (depending on the intensity of the band) of PCR product were mixed with 7 μL of denaturing buffer (90% formamide, 25 mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanole) and denatured at 99 °C for 10 min, cooled on ice and loaded on a polyacrylamide gel (8% acrylamide). Electrophoreses were performed at 4 °C at a constant voltage of 200 V for 3-4 hours. The SSCP profiles of the 900 bp retroelement fragments revealed a genetic diversity among Candidatus Phytoplasma aurantifolia isolates. SSCP analysis of the 20 lime phytoplasma isolates showed four different profiles. PCR products of ten samples were sequenced and phylogenetic tree were constructed. In general, identical SSCP profiles were indicative of identical sequences and positions in the phylogenetic tree. However, in some cases, relationship between the similarity of profiles and the proximity in the phylogram were in contrast with each other. The SSCP patterns of DE5 and NO1 isolates were different but clustered on the same branch of the phylogenetic tree.