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 perform analysis of viral sequences to identify suitable sequences for the development of oligonucleotides, provide the facilities for production of the microarrays based on the selected oligonucleotides,and participate in 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 USDA-ARS scientists at Beltsville, MD, and scientists at the Donald Danforth Plant Science Center, Washington University, the University of Utah, Oklahoma State University, and Cornell University. The overall goals are to be able to detect any plant virus in extracts of infected plants, and to identify previously characterized viruses to the species level, or previously uncharacterized viruses to at least the viral family or genus level.
Progress was made towards the evaluation in silico of the characteristics of the Universal Plant Virus Microarray probe set. The ability to control plant viral and viroid diseases requires detection and correct identification of the virus or viroid to 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. A collaborating scientist at the University of Utah selected 9,392 oligonucleotides as the initial UPVM core probe set. Each of these probe sequences was evaluated against all available plant virus isolates of the major virus families. Satellite DNAs of the family Geminiviridae were also analyzed. The analysis showed that some geminivirus sequences appear to be mis-classified (incorrectly named), and other sequences appear to represent recombinants between recognized species. Discrimination between species and isolates of species were also observed within the family Potyviridae. 23 isolates representing 12 species (out of 599 potyviral sequences examined) may represent either mis-classified or recombinant isolates. Based on these tests, additional probes were tested and found to have broad recognition of multiple species (i.e. genus- or family-level probes) so were added to the initially-selected UPVM set. A total of 9600 probes, including approximately 50 host-specific control probes, will produce the first generation UPVM for validation. This information will be of most immediate application to the UPVM collaborators, but will also be of value to regulatory agencies, plant 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.