DIFFERENTIATION OF CITRUS TRISTEZA CLOSTEROVIRUS (CTV) ISOLATES BY SINGLE-STRAND CONFORMATION POLYMORPHISM ANALYSIS OF THE COAT PROTEIN GENE

Citrus tristeza closterovirus (CTV) isolates of several geographical o rigins were compared for variations in their coat protein (CP) gene by analysis of single-strand conformation polymorphism (SSCP). The CP ge ne of 17 isolates was reverse transcribed, amplified by polymerase cha in reaction (PCR), and 22 clones were inserted into a plasmid vector. These clones were sequenced and found to have between 91.7% and 99.8% sequence homology. Clones were amplified and the PCR products denature d and compared by SSCP analysis in 8% polyacrylamide gels. Using two d ifferent electrophoretic conditions, the patterns were different for 1 6 or 17 clones. Four pairs of clones (T36/T66, P1/Q2, O3/8Q, and E1/E2 ) differing by 10, 2, 1 and 1 nucleotides, respectively, could not be distinguished using either condition. When these clones were compared by SSCP after digestion with Eco91I (BstEII) three of the pairs (T36/T 66, P1/Q2, and O3/8Q) could be differentiated, whereas the clones E1 a nd E2 (differing by 1 nucleotide) remained indistinguishable. Thus, SS CP analysis combining two electrophoretic conditions and restriction o f eight clones with Eco91I allowed discrimination between 21 of the 22 CP gene clones selected. SSCP analysis may provide a procedure to ide ntify and differentiate CTV isolates based on comparisons of several g enes or gene regions. It is rapid and cheap and may drastically reduce the amount of sequencing necessary for accurate comparisons.