A genome-wide 20 K citrus microarray for gene expression analysis

M Angeles Martinez-Godoy 1 Nuria Mauri 1 Jose Juarez 0 M Carmen Marques 1 Julia Santiago 1 Javier Forment 1 Jose Gadea 1 0 Instituto Valenciano de Investigaciones Agrarias (IVIA) , Carretera Moncada-Naquera, Km.4.5, E46113 Moncada, Valencia , Spain 1 Instituto de Biologia Molecular y Celular de Plantas (IBMCP), Laboratorio de Genomica (Universidad Politecnica de Valencia - Consejo Superior de Investigaciones Cientificas) , Avenida de los Naranjos s/n, E46022 Valencia , Spain Background: Understanding of genetic elements that contribute to key aspects of citrus biology will impact future improvements in this economically important crop. Global gene expression analysis demands microarray platforms with a high genome coverage. In the last years, genomewide EST collections have been generated in citrus, opening the possibility to create new tools for functional genomics in this crop plant. Results: We have designed and constructed a publicly available genome-wide cDNA microarray that include 21,081 putative unigenes of citrus. As a functional companion to the microarray, a webbrowsable database [1] was created and populated with information about the unigenes represented in the microarray, including cDNA libraries, isolated clones, raw and processed nucleotide and protein sequences, and results of all the structural and functional annotation of the unigenes, like general description, BLAST hits, putative Arabidopsis orthologs, microsatellites, putative SNPs, GO classification and PFAM domains. We have performed a Gene Ontology comparison with the full set of Arabidopsis proteins to estimate the genome coverage of the microarray. We have also performed microarray hybridizations to check its usability. Conclusion: This new cDNA microarray replaces the first 7K microarray generated two years ago and allows gene expression analysis at a more global scale. We have followed a rational design to minimize cross-hybridization while maintaining its utility for different citrus species. Furthermore, we also provide access to a website with full structural and functional annotation of the unigenes represented in the microarray, along with the ability to use this site to directly perform gene expression analysis using standard tools at different publicly available servers. Furthermore, we show how this microarray offers a good representation of the citrus genome and present the usefulness of this genomic tool for global studies in citrus by using it to catalogue genes expressed in citrus globular embryos. - Background In the last years, microarray technology has demonstrated the power of the high-throughput study of gene expression in the unravelling of key processes of plant biology [2-4]. Microarrays have become especially relevant for crop species where little genome information is available, and where intensive laboratory work is necessary to get insight into a particular biological process, as well as to identify candidate target genes for future breeding [5]. Citrus is the most economically important fruit crop in the world, with a total production of 105 million metric tons. There is a plethora of important commercial species and varieties, including sweet oranges, mandarins, lemons and grapefruits. Variety improvement efforts have been hampered by general characteristics of citrus biology, such as apomixis, sexual incompatibility or prolonged juvenility, that limit classical molecular biology approaches. Functional genomics is then viewed as a relatively easy way to move forward into the identification of candidate genes of agronomical relevance, and to the understanding of biological processes important for citriculture. Two years ago, aiming to develop genomic tools to assist future citrus research, we generated an EST collection covering a wide range of tissues and developmental stages, as well as biotic and abiotic stress situations, and constructed a first-generation cDNA microarray containing 6875 putative unigenes to initiate the characterization of citrus transcriptome [6]. This first microarray has been used so far to monitor the transcriptional response of citrus in ovaries and young fruit during development and ripening of citrus flesh [7], during CTV virus infection [8], or under water stress conditions [9], as well as to predict citrus varieties using expression profiles [10]. However, to perform expression analysis in citrus at a more global scale, new microarray platforms with increased genome representation are mandatory. cDNA microarrays are still a valuable tool for transcriptomic analysis in many species [11-14]. In plants, a cDNA array containing more than 10.000 unigenes has been recently generated for canola [15]. Although cDNA microarrays are being gradually substituted by oligo arrays due to reduction of manipulation steps during fabrication, and to their ability to detect similar members of some gene families, the validity of both platforms to perform reproducible and biologically consistent results has been clearly demonstrated, and the lack of concordance between microarray platforms has proven to be a failure of the metrics used to evaluate such concordance [16]. Moreover, cDNA microarrays seems to be the best option for comparative, evolutionary and ecological studies of closely related species [17], taking profit that cross-hybridization is expected to occur in cDNA arrays when sequence homology between targets and probes is higher than 70% [18]. This is especially relevant for citrus, a tree grown as a combination of the fruit-producing scion variety bud-grafted onto a rootstock variety adapted to the soil and environment, as many studies combine both parts of the tree. Here we describe the design and creation of a publicly available cDNA microarray that include 21,081 putative unigenes of citrus. Our microarray complements the recently released Citrus Affymetrix GeneChip [19] and provides an alternative tool to perform global transcriptomic assays in these species. Although the majority of gene fragments spotted on the array were isolated from Citrus clementina, the cDNA nature of our microarray extends its use to any citrus species [8,10], allowing also comparison of scion/ rootstock expression [9]. To illustrate their utility, we use this microarray to catalogue genes expressed in citrus globular embryos, and show how embryogenesis in citrus proceeds expressing a similar set of genes as it does in Arabidopsis. Results and Discussion Microarray design The starting material for the selection of probes to be printed in the microarray were the cDNA clone collection generated by the Citrus Functional Genomics Project (CFGP) [6], and a number of external clones integrated in this collection [20,21], as well as the 92,011 trace files generated from all of them. Details about the source cDNA libraries can be found at CFGP homepage [1]. Figure 1 shows the steps in the selection of citrus unigenes to be represented in the microarray. After vec (...truncated)