Abstract
Oligonucleotide-based microarrays with accurate gene coverage represent a key strategy 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 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 (www.blast2go.de). 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.
Highlights
Sunflower (Helianthus annuus L.) is an important source of edible oil and its uses have expanded to include new markets like biofuels, biolubricants and biopharmaceuticals [1]
We present the development of a comprehensive Sunflower Unigene Resource, its functional annotation and the design and validation of a custom sunflower oligonucleotide-based microarray for identification of key regulatory genes for molecular breeding and examination of concerted transcriptional responses such as those associated with leaf senescence
EST datasets were used to create a comprehensive unigene collection. These datasets include ESTs from cDNA libraries described at NCBI
Summary
Sunflower (Helianthus annuus L.) is an important source of edible oil and its uses have expanded to include new markets like biofuels, biolubricants and biopharmaceuticals [1]. Arrays based on cDNAs were developed to carry out studies in wild Helianthus [11,12] including hybrid species [13]. This approach useful is confined to the analysis of a limited set of genes. The shortage of candidate genes underlying agronomically important traits represents one of the main drawbacks in sunflower molecular breeding In this context, functional tools such as a high-density oligonucleotide microarray would enable the discovery and characterization of important novel genes affecting key agronomic traits. Condensed gene indexes based on small and large scale assemblies were successfully used in model plants for the development of microarray analysis, e.g., Arabidopsis, tobacco, melon and rice; [17,18,19,20] and non-model economically relevant plants such as maize, tomato, cotton, citrus, cucumber, Brassica, wheat, flax and coffee [21,22,23,24,25,26,27,28,29]
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