Characterization and Classification of Citrus Tristeza Virus Isolates by Amplification of Multiple Molecular Markers

Abstract

Nucleotide sequence and hybridization data using cDNA clones from the non-conserved regions of the genomes of the Israeli CTV isolate VT and the Florida isolates T3, T30, and T36 were used to develop sequence specific primers for PCR-based differentiation of CTV isolates of unknown genetic relatedness. Virus isolates from outside Florida were obtained from the exotic CTV collection at Beltsville, MD. Virions were captured from tissue extracts with antibodies, and reverse transcription and PCR were performed on captured virions with random and selective primers, respectively. Isolates were evaluated for the amplification of one common and 11 sequence-specific PCR products (markers) with primers derived from analogous sites within the genomes of the T3, T30, T36, and VT isolates. This set of amplified markers created a distinct marker profile for each isolate and this was termed the isolate "genotype". Analyses of 44 isolates yielded 14 different genotypes, but some clearly were minor variants of the T3, T30, T36, and VT genotypes. While the majority of isolates tested produced a product with at least one selective primer pair, at least three isolates did not produce products with any selective primers, suggesting that there are CTV sequences distinct from those of the isolates already sequenced. Isolates obtained from the same region or country often had the same pattern of markers, suggesting a common origin and possible extensive spread of a common CTV genotype in that location. Similar patterns of markers from isolates from different regions suggested a possible connection, such as movement of plant material between these regions. Isolates from other regions showed more diverse patterns of markers, suggesting greater diversification of CTV in that region or possible multiple introductions of different CTV isolates. We suggest that the use of selective primers in PCR-based assays provides a reliable and rapid method of assessing the degree of genetic variability present in global CTV populations as well as the relatedness of individual isolates from different citrus-growing regions.