An oligo-based microarray offers novel transcriptomic approaches for the analysis of pathogen resistance and fruit quality traits in melon (Cucumis melo L.)

Abstract

Background: Melon (Cucumis melo) is a horticultural specie of significant nutritional value, which belongs to the Cucurbitaceae family, whose economic importance is second only to the Solanaceae. Its small genome of approx. 450 Mb coupled to the high genetic diversity has prompted the development of genetic tools in the last decade. However, the unprecedented existence of a transcriptomic approaches in melon, highlight the importance of designing new tools for high-throughput analysis of gene expression.

Results: We report the construction of an oligo-based microarray using a total of 17,510 unigenes derived from 33,418 high-quality melon ESTs. This chip is particularly enriched with genes that are expressed in fruit and during interaction with pathogens. Hybridizations for three independent experiments allowed the characterization of global gene expression profiles during fruit ripening, as well as in response to viral and fungal infections in plant cotyledons and roots, respectively. Microarray construction, statistical analyses and validation together with functional-enrichment analysis are presented in this study.

Conclusion: The platform validation and enrichment analyses shown in our study indicate that this oligo-based microarray is amenable for future genetic and functional genomic studies of a wide range of experimental conditions in melon.

Figure 1

Quality analyses between biological and technical hybridization replicates. Box-plots of the samples before (A) and after (B) the normalization step. The baseline is set to a similar raw expression level, allowing the inter-chips comparison. (C) Hierarchical clustering (HC) of the samples performed using the whole expression data after normalization. Different conditions are separated and replicates cluster together. (D) Pearson correlation at gene-level for all the probes in the replicates of the microarray. All the replicates showed a correlation value greater than p > 0.95 thus showing a high level of similarity.

Figure 2

Analyses of differential transcription for the three different data set used in the microarray. (A) Principal Component Analysis (PCA) of the transcript profiles from all the melon samples in the microarray, showing a high separation for each experimental set up. The array data used for this analysis were normalized using the RMA algorithm implemented in the oligo R package for Nimblegen^® arrays. Pannels (B), (C) and (D) display support trees (ST) of the deregulated genes for all the conditions. (B) ST of the 937 deregulated genes with a q < 0.01 using the SAM in the 46d vs 15d melon fruit. (C) ST of the 198 deregulated genes with a q < 0.01 using the SAM in the M. cannonbalus infection. (D) ST of the 1182 deregulated genes with a q < 0.01 using the SAM in the CMV infection.

Figure 3

Venn Diagram addresses the high experiment-specificity of the microarray. Venn Diagram using all deregulated genes for all the experiments showed that little overlapping exists. Only four genes were shared between the three experiments, and overlapped genes in pairwise comparison did not exceed 10% of total deregulated genes.

Figure 4

Gene Ontology functional enrichment. (A) Fruit ripening experiment was analyzed for functional enrichment using Gene Ontology terms and Z-score statistics calculation (see Methods). Resulting p-values after FDR multiple testing adjustments are visualized in a colour code scale; colours toward red signify enrichment for a particular GO term. Gray means no statistically significant enrichment. (B) Enrichment analysis, using Z-score statistics, of the M. cannonbalus infection in the Agrestis accession roots. (C) Enrichment analysis of the CMV infection in photosynthetic cotyledons in C. melo var. tendral.