2010 Annual Report
1a.Objectives (from AD-416)
To demonstrate the ability of an oligonucleotide microarray to detect and differentiate plant viruses from random amplification of plant total nucleic acid extracts, and to provide education, through training, of undergraduate interns, and the incorporation of macroarray and microarray techniques into curricula.
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, and participate in analysis of microarray hybridization results to determine with a high degree of confidence which viruses were present in validation samples. The COOPERATOR will also integrate macroarray and microarray techniques into curricula, and educate students on the theory and application of macroarrays and microarrays.
This research is part of a larger collaboration including USDA-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 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 utilization of grapevine virus-specific probes from the Universal Plant Virus Microarray design in a macroarray format. 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 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 a previously uncharacterized species to the correct genus or family. A collaborating scientist at the University of Utah designed a set of oligonucleotides, each 60 nucleotides in length, and selected a subset of 9,392 oligonucleotides as the initial UPVM core probe set. From within this UPVM core set, those probes specific for viruses affecting grapevine were synthesized and included in a nylon membrane-based macroarray, and evaluated against amplified labeled extracts of various infected grapevines. The UPVM probes were observed to detect several grapevine viruses as expected based on predicted hybridization patterns. 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.