Conclusion: Successfully used the wheat microarray to detect expressed oat genes.

1. Oat 24hr C R2-AS Oat 96hr C R2-AS Oat 24hr V R1-AS Oat 0hr V R2-AS Oat 0hr C R2-AS Oat 96hr V R1-AS Oat 0hr V R1-AS Present Detection Oat 24hr C R1-AS Oat 24hr C R1-AS Oat 0hr V R1-AS Oat 96hr C R1-AS Oat 0hr C R1-AS Oat 0hr C R1-AS Oat 96hr C R1-AS Absent Detection Marginal Detection Fig 4:Pie chart showing the percent of genes whose level of expression can be detected after infestation. 1% 10x-fold change 30x-fold change 8% 2x-fold change 91% Probesets Detected 96hr after infestation Oat 24hr V R-AS Oat 24hr V R1-AS Oat 96hr V R2-AS Oat 96hr V R1-AS The susceptibility response to BYDV in Avena sativa: Using the wheat gene microarray as a tool for measuring gene expression in oat in response to BYDV infection Joseph M. Anderson1 and Elizabeth Buescher2 USDA-ARS1 & Department of Agronomy, Purdue University2, West Lafayette, IN Abstract: The development of wheat and barley microarrays for gene expression analyses have opened the ability to identify genes whose expression patterns change relative to some applied treatment. The development of these gene arrays has only been possible because of the large amount of EST sequencing. Because of very limited resources a relatively low level of sequencing has been done in oat. In this study, the wheat microarray was examined as a potential tool for examining global gene expression in oat. The wheat microarray was chosen because it has approximately 55,000 genes arrayed compared to the 23,000 gene barley array. Anecdotal evidence suggests that oat is inherently more susceptible to BYDV than other cereals such as wheat. My laboratory has preliminary data that suggests that Clintland 64, a variety that is highly susceptible to BYDV-PAV, accumulates more of this virus than comparable susceptible wheat lines and some defense response genes are down-regulated after infection. The aim of this project is to begin examining the susceptible response to infection with BYDV-PAV. RNA isolated from Clintland 64 plants that were untreated, infested with nonviruliferous aphids and infested with aphids viruliferous with BYDV-PAV at ten times points post-infestation were initially examined for virus accumulation. Samples before, during and virus accumulation were used to hybridize to Affymetrix wheat microarrays. The data from these experiments will determine which percentage of the wheat array will be useful in examining oat gene expression and how the plant is responding to virus infection. Figs 2: Scatter plots of probesets detected following hybridization to demonstrate reproducibility between biological replications: Red are the probesets (genes) showing detectable hybridization above background in both arrays. Blue is not reliably detected and yellow are probesets not detected. Materials and Methods: A Randomized Block Design was discussed for this study. The RBD is as follows: There are three blocks (one for each biological rep) and within each block there are three treatment groups (Control, Non-viruliferous, and Viruliferous). There are three biological reps with three treatment types over twelve time points. Each time point is considered a sample within a treatment (C, NV, V). Leaf tissue was ground in liquid nitrogen and total RNA isolated using Trizol for virus quantification and gene expression analysis. Affymetrix Wheat arrays were hybridized with labeled cRNA as specified by the manufacturer in the Purdue University Genomics facility. The microarrays were scanned using the Genescanner 4000 and the data analyzed using Microarray Suite 5.0 and GCOS. Figs 3: Scatter plots comparing expression levels of probesets between control (untreated) and virus infected Clintland 64 plants post infestation with BYDV-PAV viruliferous aphids. Red are the probesets (genes) showing detectable hybridization above background in both arrays. Blue are probesets not reliably detected and yellow are probesets not detected. • Conclusion: • Successfully used the wheat microarray to detect expressed oat genes. • The data shows high reproducibility between biological replications. • Approximately 10% of the genes contained on the array were detected. • Provides another tool to study gene expression in oat. Fig 1: Quantitative RT-PCR of BYDV-PAV over time after infestation with viruliferous aphids.