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

FERNANDEZ, P.; SORIA, M.; BLESA, D.; DI RIENZO, J.; MOSCHEN, S.; RIVAROLA, M.; CLAVIJO, B.; GONZALEZ, S.; PELUFFO, L.; PRINCIPI, D.; DOSIO, G.A.A.; AGUIRREZÁBAL, L.A.N.A.; GARCIA-GARCIA, F.; CONESA, A.; HOPP, H,E.; DOPAZO, J.; HEINZ, R.A.; PANIEGO N.

PLOS ONE

PUBLIC LIBRARY SCIENCE

Año: 2012 p. 1 - 11

1932-6203

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