2013 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 both undergraduate interns and a graduate student, and the incorporation of 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 microarray techniques into curricula, and educate students on the theory and application of microarrays.
The probe sequences designed for the UPVM were used as electronic probes of deep sequencing datasets derived from single symptomatic plants. Three 454 sequence datasets, RL-20, RL-24, and RL-26 were provided by USDA-APHIS. The only information provided on the samples was that each sample came from a different plant suspected of being infected with a virus. Each dataset was analyzed using a pipeline designed to detect known infectious agents in plant material (EDNA) with the UPVM e-probe set. The EDNA pipeline revealed 10 and 12 positive plant control e-probes for RL-20 and RL-24, respectively, but no positive virus e-probes. Eighteen positive plant control e-probes were found positive for RL-26, with 5 separate viral e-probes (3 “Tymovirales”, and 2 ”Alphaflexiviridae, Potexvirus”). The sequence of the type member of the virus family was used as a scaffold for mapping reads. Querying with the Potato virus X (PVX) genome recovered 359 reads, assembled into 15 contigs, each matching PVX with over 90% identity. Mapping reads from the dataset to PVX genome resulted in eight contigs consisting of 317 reads mapping to PVX, with 92.3% of the PVX genome covered. The detection of PVX was corroborated independently using a commercially available potexvirus antibody detection assay (USDA-APHIS; personal communication). This evidence strongly suggests that the UPVM probe sequences can, in addition to their use in microarray hybridization screening of plants for viruses, be used to probe deep sequencing datasets from plants to ascertain whether they contain viruses and which viruses are present. Six blind cucurbit samples were provided by a University of Tulsa scientist and assayed by UPVM hybridization. One of the six failed to produce any viral hits when the data were analyzed by UChip. On decoding, this sample was revealed to be a healthy control. Two other samples decoded as being infected with single viruses and three with multiple viruses, gave virus identifications by UChip. However, the identifications were not those that had been obtained by serological assay for 14 common cucurbit viruses, and require verification by other methods. 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.