Differentiation of citrus tristeza closterovirus (CTV) isolates by single‐strand conformation polymorphism analysis of the coat protein gene

Abstract

Summary.Citrus tristeza closterovirus (CTV) isolates of several geographical origins were compared for variations in their coat protein (CP) gene by analysis of single‐strand conformation polymorphism (SSCP). The CP gene of 17 isolates was reverse transcribed, amplified by polymerase chain 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 denatured and compared by SSCP analysis in 8% polyacrylamide gels. Using two different electrophoretic conditions, the patterns were different for 16 or 17 clones. Four pairs of clones (T36/T66, P1/Q2, 03/8Q, 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/T66, P1/Q2, and 03/8Q) could be differentiated, whereas the clones E1 and E2 (differing by 1 nucleotide) remained indistinguishable. Thus, SSCP analysis combining two electrophoretic conditions and restriction of eight clones with Eco91I allowed discrimination between 21 of the 22 CP gene clones selected.SSCP analysis may provide a procedure to identify and differentiate CTV isolates based on comparisons of several genes or gene regions. It is rapid and cheap and may drastically reduce the amount of sequencing necessary for accurate comparisons.

Stability of Citrus tristeza virus protective isolates in field conditions

“…The complex population of CTV RNA and its characteristic pathogenicity may suffer alterations due to host changes or aphid transmission, which suggests great genetic variation (Albiach-Martí et al, 2000). The genomic structure of CTV populations, and their alterations have been detected by the single strand conformational polymorphism (SSCP) technique (Rubio et al, 2000;D'urso et al, 2003;Davino et al, 2005). SSCP is also used in cross-protection studies to monitor the protective isolate in preimmunized plants (Souza et al, 2000a;Corazza-Nunes et al, 2001;Kim et al, 2006), and for virus identification and characterization (Vives et al, 2005;Iglesias et al, 2008).…”

Stability of Citrus tristeza virus protective isolate 'Pêra IAC' according to SSCP analysis of old and new lines of three sweet orange varieties

“…Diversity and stability of CTV isolates based on SSCP analysis have been evaluated worldwide in citrus growing areas (6,8,17,19). In the present study, SSCP analysis showed, in general, the occurrence of distinct electrophoretic patterns among the three sweet orange varieties (Figures 1).…”

Genotypic diversity of Citrus tristeza virus within red grapefruit, in a field trial site in South Africa

“…These include single-strand conformation polymorphism (Rubio et al 1996), restriction fragment length polymorphism (RFLP), bi-directional polymerase chain reaction (BD-PCR) (Jiang et al, 2008) and nucleotide sequence analysis (Iglesias et al 2008, Scott et al 2012, Wang et al 2013. While techniques such as SSCP and RFLP can indicate differences between CTV populations, the identity of CTV strains can be difficult to determine without actual nucleotide sequencing data.…”