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Analysis of Gene Expression during Brassica Seed Germination Using a Cross-Species Microarray Platform

^a Torrey Mesa Research Institute, Syngenta Research and Technology, 3115 Merryfield Row, San Diego, CA 92121
^b Dep. of Crop Sciences, Univ. of Illinois, 334 NSRC, 1101 W. Peabody Blvd., Urbana, IL 61801
^c Syngenta Biotechnology, 3054 Cornwallis Rd., Research Triangle Park, NC 27709
^d Syngenta Seeds B.V., Westeinde 62, Enkhuizen, the Netherlands

View larger version (33K): [in this window] [in a new window]   Figure 1. Correlation of Brassica genomic and mRNA microarray hybridizations with Arabidopsis genomic DNA and with Brassica replicates. Scatterplots of expression signal values derived from Arabidopsis and Brassica genomic DNA hybridization to the Arabidopsis microarray are shown. Expression metrics for probesets were calculated from the 72nd percentile value of all perfect match probe tiles for each transcript. This was done either including all probes in each probeset (top left) or after excluding "bad" probes that show less than 75% of the hybridization signal to Brassica genomic DNA compared to Arabidopsis genomic DNA (top right). The bottom scatterplots show the reproducibility of replicate hybridizations to Brassica mRNA samples using the standard analysis (bottom left) and excluding "bad" probes (bottom right).

View larger version (14K): [in this window] [in a new window]   Figure 2. Seed germination after accelerated aging. The germination of Brassica oleracea seed from cultivars Lintop and Maverick was assayed after seeds were stored for 2 to 7 d, under conditions designed to accelerate deterioration in viability (46°C and 75% relative humidity). Unbroken line: untreated seeds; dotted line: partially hydrated (primed) seeds; broken line: primed seeds in which shelf life was induced using a slow drying and heating treatment.

View larger version (42K): [in this window] [in a new window]   Figure 3. Exploratory analysis of array data from pre-germination treatment. (A) Superimposed expression pattern of all transcripts detected using one or more probes with hybridization to Brassica genomic DNA. (B) Functional classification of the detected genes, using Arabidopsis annotation. The pie chart shows the breakdown by functional category (the highest-level MIPS category) of all the detected transcripts. The percentages in parentheses indicate the percentage of the categories in the whole genome annotation. (C) Clustering of normalized expression data. The graph at top left shows the median-centered expression profiles of all the detected transcripts at the five experimental points: (1) untreated seed, (2) primed seed, (3) primed and dried seed, (4) primed seed after shelf-life induction, and (5) primed seed after shelf-life induction followed by drying. The lower graphs show how these profiles are separated into three distinct patterns, which can be resolved using self-organizing map clustering. The charts to the right of each cluster graph show the relative percentage enrichment or depletion of genes in the functional categories shown in B, in each of the three clusters.

View larger version (27K): [in this window] [in a new window]   Figure 4. Expression patterns of probesets from key functional categories showing statistically significant changes. The expression signals for particular probesets corresponding to known Arabidopsis genes are plotted against the treatment of the seed before RNA extraction. The five points on each curve correspond to (1) untreated seed, (2) primed seed, (3) primed and dried seed, (4) primed seed after shelf-life induction, and (5) primed seed after shelf-life induction followed by drying. All probesets show statistically significant differences in expression. Further details and gene lists are available in the supplementary material. Error bars represent the deviation from the mean of two replicate hybridizations.

View larger version (22K): [in this window] [in a new window]   Figure 5. Relative expression levels of four validated Brassica orthologs of Arabidopsis genes in functional categories found to be strongly responsive to priming treatment, assayed by real-time quantitative reverse transcription polymerase chain reaction (RT–PCR). The data represent the mean of five replicates, with error bars representing the standard error of the mean. The five points on each curve correspond to (1) untreated seed, (2) primed seed, (3) primed and dried seed, (4) primed seed after shelf-life induction, and (5) primed seed after shelf-life induction followed by drying. The expression levels were normalized first to actin measured from the same RNA sample and then to the mean of all points for each genotype.