Abstract
Citrus tatter leaf virus (CTLV) threatens citrus production worldwide because it induces bud-union crease on the commercially important Citrange (Poncirus trifoliata × Citrus sinensis) rootstocks. However, little is known about its genomic diversity and how such diversity may influence virus detection. In this study, full-length genome sequences of 12 CTLV isolates from different geographical areas, intercepted and maintained for the past 60 years at the Citrus Clonal Protection Program (CCPP), University of California, Riverside, were characterized using next generation sequencing. Genome structure and sequence for all CTLV isolates were similar to Apple stem grooving virus (ASGV), the type species of Capillovirus genus of the Betaflexiviridae family. Phylogenetic analysis highlighted CTLV's point of origin in Asia, the virus spillover to different plant species and the bottleneck event of its introduction in the United States of America (USA). A reverse transcription quantitative polymerase chain reaction assay was designed at the most conserved genome area between the coat protein and the 3'-untranslated region (UTR), as identified by the full genome analysis. The assay was validated with different parameters (e.g. specificity, sensitivity, transferability and robustness) using multiple CTLV isolates from various citrus growing regions and it was compared with other published assays. This study proposes that in the era of powerful affordable sequencing platforms the presented approach of systematic full-genome sequence analysis of multiple virus isolates, and not only a small genome area of a small number of isolates, becomes a guideline for the design and validation of molecular virus detection assays, especially for use in high value germplasm programs.
Highlights
Citrus tatter leaf virus (CTLV), a Capillovirus belonging to the family Betaflexiviridae, is considered to be a strain of Apple stem grooving virus (ASGV) [1, 2]
Full-length viral genome sequences of 12 CTLV isolates were obtained by RNA-Seq and the average total reads generated was 27,158,037 which covered 74% to 100% of the viral genome
Sequence analysis showed the CTLV genome was similar to other capilloviruses, including ASGV and pear black necrotic leaf spot virus (PBNLSV), with two overlapping open reading frames (ORFs) (Fig 1)
Summary
The purpose of this study was to characterize and further develop a robust CTLV RTqPCR detection assay based on the systematic analysis of newly generated full-length genome data from multiple virus isolates maintained for the past 60 years at the CCPP. Since the genetic variation within the targeted virus population can lead to false negative RT-qPCR results, for the design of the CTLV detection assay we aimed to locate the most conserved region on the virus genome beyond the traditional approaches that focus on individual genes presumed conserved due to their function [64]
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