Project description:We performed a transcriptome analysis of interior spruce (Picea glauca x engelmannii) bark response to weevil (Pissodes strobi) feeding using 21.8K spruce microarray (that contains 21.8 thousand unique transcripts). This microarray study revealed a large rearrangement of the interior spruce bark transcriptome in response to weevil feeding involving differential expression of close to 20% of the studied transcriptome.
Project description:Comparison of transcriptomes from bark, developing xylem and xylem of P. radiata saplings exposed to 0 or 1mg of Ethephon in lanolin for 1 or 8 weeks We developed an oligonucleotide microarray using sequences (mostly from Pinus taeda) from public sequence databases. These sequences were reconstituted into a non-redundant database by CAP3 assembly and used as templates for automated design of 60-mer oligonucleotide probes through eArray, Agilent’s online facility. The microarray slides, manufactured by Agilent, were used to monitor gene expression in an Ethephon-induction experiment. Ethephon was dispersed in lanolin paste and applied in a 3 cm band near the base of the stem of 2-year old Pinus radiata saplings. RNA was extracted from bark, cambial region, also known as “developing xylem”, and xylem tissues exposed for 1 or 8 weeks to Ethephon. The transcriptomes from these extracts were compared by hybridization onto the All-Pinus microarray slides. Statistically significant differentially expressed genes identified by limma (Linear Models for Microarray Data) were subsequently analysed by singular enrichment analysis through the Database for Annotation, Visualization and Integrated Discovery (DAVID) portal. Results revealed that bark, cambial region and xylem generate mostly mutually exclusive cohorts of genes and Gene Ontology (GO) classes. Ethephon induction led to the upregulation of xylem genes related to the metabolism of phenylpropanoids and flavonoids and to defence responses, specifically, fungal/insect attack and oxidative stress. Independent validation of the microarray data for five genes was obtained by quantitative RT-PCR. The results are also interpreted in reference to gross and microscopic morphological changes. These results confirm the utility of the All-Pinus microarray for transcriptomic research in P. radiata.
Project description:Comparison of transcriptomes from bark, developing xylem and xylem of P. radiata saplings exposed to 0 or 1mg of Ethephon in lanolin for 1 or 8 weeks We developed an oligonucleotide microarray using sequences (mostly from Pinus taeda) from public sequence databases. These sequences were reconstituted into a non-redundant database by CAP3 assembly and used as templates for automated design of 60-mer oligonucleotide probes through eArray, AgilentM-bM-^@M-^Ys online facility. The microarray slides, manufactured by Agilent, were used to monitor gene expression in an Ethephon-induction experiment. Ethephon was dispersed in lanolin paste and applied in a 3 cm band near the base of the stem of 2-year old Pinus radiata saplings. RNA was extracted from bark, cambial region, also known as M-bM-^@M-^\developing xylemM-bM-^@M-^], and xylem tissues exposed for 1 or 8 weeks to Ethephon. The transcriptomes from these extracts were compared by hybridization onto the All-Pinus microarray slides. Statistically significant differentially expressed genes identified by limma (Linear Models for Microarray Data) were subsequently analysed by singular enrichment analysis through the Database for Annotation, Visualization and Integrated Discovery (DAVID) portal. Results revealed that bark, cambial region and xylem generate mostly mutually exclusive cohorts of genes and Gene Ontology (GO) classes. Ethephon induction led to the upregulation of xylem genes related to the metabolism of phenylpropanoids and flavonoids and to defence responses, specifically, fungal/insect attack and oxidative stress. Independent validation of the microarray data for five genes was obtained by quantitative RT-PCR. The results are also interpreted in reference to gross and microscopic morphological changes. These results confirm the utility of the All-Pinus microarray for transcriptomic research in P. radiata. Series of 2-color, 2 condition experiments in 12 180k arrays. Main comparison is within tissues exposed to 0 [control] or 1 mg Ethephon. 2nd level of comparison is between tissues [bark, xylem scraping, xylem]. Third level of comparison is between time [1 or 8 week exposure]. One slide is hybridized with cRNA generated from control and treated tissues with the same duration of exposure to Ethephon. Two biological replicates [each biological rep is a 2 y old cutting propagated clone] for treated plants whilst control consists of RNA pooled, in equal proportions [estimated by UV absorbance], from 2 untreated biological replicates.]. Dyes used for each sample are indicated in sample description.
Project description:We performed a transcriptome analysis of interior spruce (Picea glauca x engelmannii) bark response to weevil (Pissodes strobi) feeding using 21.8K spruce microarray (that contains 21.8 thousand unique transcripts). This microarray study revealed a large rearrangement of the interior spruce bark transcriptome in response to weevil feeding involving differential expression of close to 20% of the studied transcriptome. RNA was isolated from the bark of interior spruce exposed to weevil feeding and from the bark of untreated trees at three time points (6 hours, 2 days and 2 weeks). Four independent biological replicates were included for treatment and control at each time point. Four hybridizations were performed for treatment and control comparison within each time point (6 hours, 2 days, 2 weeks) and one hybridization was performed for each comparison between time points for both treatment and control (total 18 hybridizations/slides).
Project description:Using 21K spruce microarray (that contains 21.8 thousand unique transcripts) we performed analysis of the transcriptome response of lodgepole pine (Pinus contorta) inoculated with the mountain pine beetle (Dendroctonus ponderosae) vectored fungal pathogen Grosmannia clavigera or treated with wounding. This microarray analysis revealed large transcriptome reorganization with close to 2000 transcripts (10% of the studied transcriptome) differentially expressed within two weeks of treatment, with the wounding response affecting close to 2% of the lodgepole pine transcriptome. RNA was isolated from the bark of lodgepole pine inoculated with Grosmannia clavigera, treated with wounding, or untreated control for three time points (6h, 2days and 2 weeks). Three independent biological replicates were included for each treatment and each time point. Three hybridizations were performed for each comparison of different treatments (fungal, wounding, control) within each time point (6 hours, 2 days, 2 weeks) and one hybridization was performed for the comparison of the same treatments between time points (total 36 hybridizations/slides).