Project description:In order to pinpoint the most differentially expressed genes between Eucalyptus grandis leaf blades and vascular (xylem) tissues as well as between E. grandis and Eucalyptus globulus xylem tissues, a total number of nine 50mer-oligoprobes covering the length of each one of 21,432 unique sequences derived from the Genolyptus EST dataset were synthesized “on-chip” in duplicate, randomly distributed in two blocks of each slide. Probes were also synthesized from ten cDNA sequences encoding known human proteins as negative controls, totaling 21,442 sequences. Leaves and xylem samples were taken from two E. grandis clonal trees, i.e., both derived from the same matrix tree and harboring the same genotype. Two additional xylem samples were collected from two other E. grandis clonal trees of a different genotype, as well as from two E. globulus clonal trees. Therefore, ten cDNA samples and ten identical chips were produced at Roche NimbleGen for the microarray assays, with a total number of 385,956 features per slide. Besides the discovery of differentially expressed genes between leaf and xylem, we wanted to test the validity of the assumed “technical” and “biological duplicates” since all trees were field-grown and four years-old in age. A ten chip study using total RNA recovered from mature leaf and vascular (xylem) tissues of Eucalyptus grandis and xylem from Eucalyptus globulus trees. Two clonal trees of E. grandis (E.grandis_Clone A_Ramet 1 and E.grandis_Clone A_Ramet 2), derived from a single matrix tree and therefore genomically identical, were the source of two samples of leaf RNA and two samples of xylem RNA, individually hybridized to four chips after cDNA synthesis/Cy3 labeling. Two other clonal trees of E. grandis (E.grandis_Clone B_Ramet 1 and E.grandis_Clone B_Ramet 2), derived from a different matrix tree, were the source of two additional samples of xylem RNA individually hybridized to four chips after cDNA synthesis/Cy3 labeling. Likewise, two pairs of clonal trees of E. globulus (E.globulus_Clone A_Ramet 1 and E.globulus_Clone A_Ramet 2/ E.globulus_Clone B_Ramet 1 and E.globulus_Clone B_Ramet 2), derived from two distinct matrix trees, were the source of four additional samples of xylem RNA, individually hybridized to four chips after cDNA synthesis/Cy3 labeling. Each chip measures the expression level of 21,432 genes from Eucalyptus sp. and ten human genes (negative controls) with nine 50-mer probe pairs (PM/MM) per gene in two separate blocks per chip (technical duplicate), totalizing 18 hybridization signal values per gene per chip.

Project description:Fast-growing Eucalyptus grandis trees are one of the most efficient producers of wood in South Africa. It is essential to maximize the effectiveness of these plantations by increasing their productivity, the quality and value of their products. We used microarray-based DNA-amplified fragment length polymorphism (AFLP) analysis in combination with expression profiling to develop fingerprints and profile gene expression of wood-forming tissue of seven individual E. grandis trees. A 1532-probe cDNA microarray was constructed by arraying 768 cDNA-AFLP fragments and 810 cDNA library clones from seven individual E. grandis trees onto silanised slides. The results revealed that 32% of the spotted fragments showed distinct expression patterns (with a fold change of at least 1.4 or -1.4 and a p value of 0.01) and could be grouped into clusters representing co-expressed genes. Evaluation of the binary distribution of cDNA-AFLP fragments on the array showed that the individual genotypes could be discriminated. A simple, yet general method was developed for genotyping and expression profiling of wood-forming tissue of E. grandis trees differing in their splitting characteristics and in their lignin contents. Evaluation of gene expression profiles and the binary distribution of cDNA-AFLP fragments on the chip suggest that the prototype chip developed could be useful for transcript profiling and for the identification of Eucalyptus trees with preferred wood quality traits in commercial breeding programmes. Transcriptional profiling and genotyping of seven E. grandis trees , namely (741-H (high lignin), 108-L (low lignin), 243-L (low lignin), 1/23/4-HS (high splitting), 1/71/6-HS (high splitting), 1/91/7-LS (low splitting) and 1/92/7-LS (low splitting), were performed with an in-house spotted cDNA microarray. All trees were characterized for their splitting qualities and lignin content and only the trees best corresponding to the selected traits were used for further analysis (Verryn and Turner 2000). RNA was isolated and cDNA synthesis was performed. A reference design microarray experiment was conducted and tree 1/23/4-HS served as a reference. Samples were fluorescently labelled with Cy5 or Cy3 dye and co-hybridized overnight. Two biological and one technical replicate (using independent labelling reactions) was performed, each replication consisting of a reverse labelling experiment.

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Eucalyptus grandis

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