Development and Application of Next Generation Sequencing to Facilitate the Release of New Grapevine Accessions in Quarantine a

This project involved the characterization of the technical tool “Next Generation Sequencing” (NGS) as a method of detection and identification of grapevine viral pathogens. The objective was to demonstrate that NGS is superior to the current biological indexing screen for the quarantine screening and certification of grapevine accessions for release to growers. The project has run side-by-side comparisons of the NGS analysis vs. biological indexing and PCR tests. The biological index test identifies virus infections, with the plants in the field expressing disease symptoms characteristic of particular viruses. PCR identifies individual virus species by the amplification of a DNA sequence specific to the genomes of each. NGS identifies viruses through the sequences of their genomes generated by total genomic deep sequencing analysis. That sequencing is directed by viral genomic RNAs extracted from individual infected vines. We have generated the biological index screening results, which took two years to produce from field trials begun during the course of this project. We have prepared PCR analyses of the viruses present in the samples. And we have prepared dsRNA or total RNA of samples from the 52 grapevine cultivars and accessions, and sequenced those using an Illumina platform to produce an NGS census of all the viral genome sequences from all 52 plants. The analysis of all the samples showed that more virus species were revealed by deep sequencing than by the other methods. (Bioassay is restricted by the range of symptoms that can be produced in the panel of indicator hosts; PCR is restricted by the selection of known viral genomic sequences, from which the primers can be designed). The side-by-side comparative analysis of the three assay procedures showed slight inconsistencies in each of them, as assessed by comparisons among them. All three of the procedures were generally very reliable. The conclusions to be drawn were that NGS analysis will speed the release of the material from quarantine and the certification process in two ways.

  1. For plants identified by NGS as infected, the tissue culture process to produce qualified and certifiable material can begin a few weeks after NGS analysis, a savings of the two years that the bioassay would take to identify infections.
  2. For plants identified by NGS as uninfected, a two year conditional certification and release from quarantine can be issued a few weeks after said analysis.

The certificate and quarantine release will be contingent on confirmation of the uninfected status of the vines by the bioassay process.