2010 Annual Report
1a.Objectives (from AD-416)
To develop and validate a universal plant virus microarray for detection and differentiation of plant viruses. To demonstrate the ability of an oligonucleotide microarray to detect and differentiate plant viruses from random amplification of plant total nucleic acid extracts.
1b.Approach (from AD-416)
ARS will acquire the lists of viral taxa to be represented on the viral detection microarray, and virus-infected samples from which to amplify nucleic acids to validate the microarray. This information and material will be utilized by both ARS and the Cooperator to jointly develop and validate the microarray for detection of target viruses, and to make validation results available to collaborators via a web server. The COOPERATOR will further develop bioinformatic software (based on E-Predict and vTaxI) to perform analysis of viral sequences to identify suitable sequences for development of oligonucleotides, and for analysis of microarray hybridization results to determine with a high degree of confidence which viruses were present in validation samples.
This research is part of a larger collaboration including ARS scientists at Beltsville, MD; the Donald Danforth Plant Science Center, St. Louis, MO; Washington University, St. Louis, MO; the University of Utah, Salt Lake City, UT; Oklahoma State University, Stillwater, OK; and Cornell University, Ithaca, NY. The overall goals are to able to detect any plant virus in extracts of infected plants, and to identify previously characterized viruses to the species level, or previously uncharacterized virus to at least the viral family or genus level.
Progress was made towards the design and initial evaluation of oligonucleotide probes for a Universal Plant Virus Microarray. The ability to control plant viral and viroid diseases requires the ability to correctly detect and identify the virus or viroid, and thus obtain knowledge about the likely vectors or other means of transmission. The ability to assign a previously uncharacterized virus to a known taxonomic group allows inference of many important characters, and facilitates the selection of appropriate methods to obtain further specific sequence information to develop specific detection and identification reagents. The first step in developing a Universal Plant Virus Microarray (UPVM) is to design and select oligonucleotide probes that will collectively provide information allowing identification of isolates of a previously characterized virus to the correct species, and of previously uncharacterized species to the correct genus or family. Previously developed software was further customized for design of the probe sequences. All plant viral sequences in the core nucleotide set of GenBank as of December 2009, plus additional sequences obtained through UPVM project collaborators (over 34,000 sequences in total) were screened and reduced to a non-redundant set of 6,100 sequences. These genomic sequences yielded 9,600 probes, which include approximately 50 control probes for conserved healthy plant sequences, plus some special interest sequences, and the broadest specificity ViroTax probes. The broadest specificity probes were included to maximize the probability of detecting previously uncharacterized species within known genera. Each viral species and isolate is predicted to have a characteristic and distinct hybridization signature against the matrix of UPVM oligonucleotide probes, and during validation of the UPVM, observed hybridization profile results will be compared to predicted profiles using E-predict software, yielding a probability-based identification of the viral target from the energy profile calculations. This information will be of most immediate application to the UPVM collaborators, but will also be of value to regulatory agencies, diagnostic clinics, germplasm repositories, and producers operating plant certification schemes.
Communications to monitor progress were carried out by e-mail and conference calls between the various partners, by a group meeting during the Annual meeting of the American Phytopathological Society, and by written and oral reports to the NRI Plant Biosecurity Program.