Development and Application of Next Generation Sequencing to Facilitate the Release of New Grapevine Accessions in Quarantine and Certification Programs
This project is involved with the characterization of the technical tool “NGS” as a method of detection of grapevine viral pathogens. The objective is to demonstrate that, by every measure, NGS is superior to the current biological indexing screen for the certification of grapevine accessions for release into the field. The project is proceeding with the side-by-side comparison of the two diagnostic procedures. Both procedures focus on the identification of viral pathogens. The bioassay identifies virus infections through the symptoms caused in indicator plants in the field. NGS identifies viruses through the sequences of their genomes, found in the laboratory by total genomic deep sequencing analysis of extracts of DNA and RNA from infected vines. We have not yet described the biological index screening results, since it will take two years to get those results from field trials begun during the course of this project. In the past year, dsRNA samples were prepared from54 grapevine cultivars and accessions and sequenced by an Illumina platform. Sequences from 15 of these accessions have been analyzed and work on remaining 39 samples is in progress. For a comparison and optimization of the NGS analysis, small interfering RNA and total RNA were also prepared from selected grapevine accessions in our list and sequenced and the analysis is pending. The initial NGS data analysis of the subset of the samples (15 samples) from this project already suggests that many more virus species will be revealed by deep sequencing than can be identified by the biological field assay on indicator plants. We will have to wait for the bioassay results to be scored before we can make quantitative deductions about the comparative sensitivity, accuracy, and comprehensiveness of the two methods.