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.
Hybridizations of virus-infected and control healthy extracts of grapevine, peanut, sweet potato, Brachypodium, Hibiscus and Citrus plants at the Donald Danforth Plant Science Center have been utilized to detect some non-specific reactions of a number of the 60-mer Universial Plant Virus Microarray (UPVM) probes; based on these data the per-probe noise models were refined which will aid interpretation of future UPVM results by enhancing the predictive power of the T-predict per-probe noise models. At least 162 hybridizations from 3 UPVM sites have been entered into Uchip so far in 2012. This sharing of the design methods and results led to rapid incorporation of the UPVM probe sequences into other plant virology projects headed by UPVM collaborators at Oklahoma State University and Cornell University. Based on feedback from the 2011 workshops, the Uchip software interface was improved, and one new computational feature, iterative T-Predict, was added. This feature allows the signature of a virus to be removed in silico before T-predict is rerun on an experiment. This procedure is supervised by the Uchip user and can be repeated, subsequently removing high titer viral signatures and revealing viruses that are present at relatively lower levels. Information on the design of the UPVM and analysis of UPVM hybridization results was presented at the UPVM Workshop and BARD Workshop ‘Microarrays and Next-Generation Sequencing for Detection and Identification of Plant Viruses’ held at Beltsville in November 2011. 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.