Development, Characterization and Experimental Validation of a Cultivated Sunflower (<em>Helianthus annuus</em> L.) Gene Expression Oligonucleotide Microarray

<div><p>Oligonucleotide-based microarrays with accurate gene coverage represent a key strategy for transcriptional studies in orphan species such as sunflower, <em>H. annuus L.</em>, which lacks full genome sequences. The goal of this study was the development and functional annotation of a comprehensive sunflower unigene collection and the design and validation of a custom sunflower oligonucleotide-based microarray. A large scale EST (>130,000 ESTs) curation, assembly and sequence annotation was performed using Blast2GO (<a href="http://www.blast2go.de">www.blast2go.de</a>). The EST assembly comprises 41,013 putative transcripts (12,924 contigs and 28,089 singletons). The resulting Sunflower Unigen Resource (SUR version 1.0) was used to design an oligonucleotide-based Agilent microarray for cultivated sunflower. This microarray includes a total of 42,326 features: 1,417 Agilent controls, 74 control probes for sunflower replicated 10 times (740 controls) and 40,169 different non-control probes. Microarray performance was validated using a model experiment examining the induction of senescence by water deficit. Pre-processing and differential expression analysis of Agilent microarrays was performed using the Bioconductor limma package. The analyses based on p-values calculated by eBayes (p<0.01) allowed the detection of 558 differentially expressed genes between water stress and control conditions; from these, ten genes were further validated by qPCR. Over-represented ontologies were identified using FatiScan in the Babelomics suite. This work generated a curated and trustable sunflower unigene collection, and a custom, validated sunflower oligonucleotide-based microarray using Agilent technology. Both the curated unigene collection and the validated oligonucleotide microarray provide key resources for sunflower genome analysis, transcriptional studies, and molecular breeding for crop improvement.</p> </div

qPCR results for ten selected differentially expressed genes.

<p>qPCR results for ten selected differentially expressed genes.</p

Box-plot of the normalized expression in each replicate of both treatments.

<p>Base line at 4.7 describes a sensitivity limit for detection of changes in RNA concentration.</p

CV estimation for technical and biological replicates.

<p>RC: relative concentration.</p><p>CT: control.</p><p>WD: water deficit.</p><p>Technical variability within biological replicates (columns: CT1, CT3, WD1, WD2); technical variability plus biological variability within each treatment (Columns: CT and WD).</p

Pie charts showing predicted gene products using Gene Ontology terms.

<p>Data was obtained from SUR v 1.0.</p