Novel genes in Sitka spruce associated with wood density identified from global transcriptomic analysis
ABSTRACT: The current study uses a transcriptomic approach to identify genes associated with differences in wood density, that are likely to be of value as candidate genes in Sitka breeding programmes for improved wood density. Following extensive wood density analysis from a Sitka spruce (Picea sitchensis (Bong) Carr.) field grown clonal trial, three detailed microarray studies were conducted to compare the transcriptome of cambial tissue from contrasting clonal lines with high and low wood density. Twenty five genes exhibited differential expression, reaching as high as 50 fold, in at least two of the three microarray experiments and this was verified using real-time PCR. Identified genes functioned in cell wall synthesis, transcriptional regulation and plant pathogen defence, amongst others. These results confirm the importance of previously-identified density-related genes, and highlight a number of novel genes with a putative role in wood quality. A wide range of processes influence wood density, but this study has allowed the identification of potential regulators in these pathways. Future studies may now use this information to understand the control of natural variation in wood density, and manipulate the expression of these genes to improve timber quality. The Sitka spruce (Picea sitchensis (Bong) Carr.) ‘Experiment 35’ clonal trial was grown at Newcastleton, Scotland (OS grid reference: NY506881, latitude: 55.1847, longitude: -2.77892) and set up by Forest Research, an agency of the Forestry Commission, which has been described previously (Mboyi and Lee 1999). A total of 750 trees were established from cuttings taken in 1989 of genotypes belonging to 6 unrelated full-sib families with 8 genotypes per family and 15 replicate ramets per genotype. Cambial cell scrapes were taken in summer 2004 when trees were 15 years old. A 5x2cm section of bark was cut away at a height of 1.3m from the ground for each tree selected for microarray analysis. The exposed xylem was immediately excised using a clean sharp razor blade and snap frozen in liquid Nitrogen. Samples were transported to the laboratory and ground in liquid nitrogen using a chilled mortar and pestle and stored at -80oC prior to RNA extraction. RNA was extracted and microarray hybridisation performed as described within.
Project description:The goal of this experiment is to assess tissue preferential transcript accumulation and fold difference between two tissues that support secondary vascular growth in three spruces: Picea glauca, Picea sitchensis and Picea mariana. Tissues compared are secondary xylem (wood forming tissue located on the internal side of the cambial meristem) and phelloderm (composite sample of the phloem and phelloderm tissues located on the outer side of the cambial meristem). One-color comparison of 3 spruce species in 2 tissue types: xylem and phelloderm. 20 biological repetitions per tissue for Picea glauca and 15 for Picea sitchensis and Picea mariana, for a total of 100 slides.
Project description:The current study uses a transcriptomic approach to identify genes associated with differences in wood density, that are likely to be of value as candidate genes in Sitka breeding programmes for improved wood density. Following extensive wood density analysis from a Sitka spruce (Picea sitchensis (Bong) Carr.) field grown clonal trial, three detailed microarray studies were conducted to compare the transcriptome of cambial tissue from contrasting clonal lines with high and low wood density. Twenty five genes exhibited differential expression, reaching as high as 50 fold, in at least two of the three microarray experiments and this was verified using real-time PCR. Identified genes functioned in cell wall synthesis, transcriptional regulation and plant pathogen defence, amongst others. These results confirm the importance of previously-identified density-related genes, and highlight a number of novel genes with a putative role in wood quality. A wide range of processes influence wood density, but this study has allowed the identification of potential regulators in these pathways. Future studies may now use this information to understand the control of natural variation in wood density, and manipulate the expression of these genes to improve timber quality. Overall design: The Sitka spruce (Picea sitchensis (Bong) Carr.) ‘Experiment 35’ clonal trial was grown at Newcastleton, Scotland (OS grid reference: NY506881, latitude: 55.1847, longitude: -2.77892) and set up by Forest Research, an agency of the Forestry Commission, which has been described previously (Mboyi and Lee 1999). A total of 750 trees were established from cuttings taken in 1989 of genotypes belonging to 6 unrelated full-sib families with 8 genotypes per family and 15 replicate ramets per genotype. Cambial cell scrapes were taken in summer 2004 when trees were 15 years old. A 5x2cm section of bark was cut away at a height of 1.3m from the ground for each tree selected for microarray analysis. The exposed xylem was immediately excised using a clean sharp razor blade and snap frozen in liquid Nitrogen. Samples were transported to the laboratory and ground in liquid nitrogen using a chilled mortar and pestle and stored at -80oC prior to RNA extraction. RNA was extracted and microarray hybridisation performed as described within.
Project description:Wood density is a foundamental quality trait for structural timber, bioenergy and pulp industries. We investigated genes differentially transcribed in radiate pine juvneile trees with distinct wood density using cDNA microarrays. Radiata pine trees were selected from a progeny trial planted at Flynn, Australia. Based on the gravitical measurement of wood cores, 12 families with highest and lowest density each were selected, representing two groups of trees with contrasting wood density. One individual with higher or lower density were further sampled in each selected family. Developing xylem tissues of selected trees were sampled in autumn (April) when latewood (LW) was formed. The xylem tissues were scraped at breast height with a sharp chisel after the bark was removed. Wood cores of the sampled trees were further measured using SilviScan 2. Total RNA extracted from ten developing xylem tissues with confirmed distinct density in each tree group were pooled into two bulks (five trees each), and the two bulks of HD were compared with two LD bulks in the microarray experiment (named the bulk experiment). Six developing xylem tissues with the most distinct density from each tree group were further chosen. Six xylem tissues with HD were individually compared with bulked six xylem tissues with LD in the second microarray experiment (named individual experiment). These two different pooling strategies can partly minimize the genetic variation among different genotypes. Dye swaps were applied in each biological replicate.
Project description:Secondary vascular system (SVS) development resulted from cambial growth is a currently not well understood process. Therefore, more studies are needed to shed more lights on the molecular mechanisms underpinning the cambial activity. The regeneration of SVS from debarked trunk that can mimic the vascular cambium-driven wood formation has developed and could be used to revealed a larger number of differentially expressed genes during the stages of cambium formation and xylem differentiation in Populus tomentosa. We used microarrays to detail the global programme of gene expression in 6 time points during the regeneration of SVS. Overall design: A 1-m strip of bark was peeled from 65 healthy P. tomentosa plants in a 4-year-old single clonal plantation (located in Tangshan, Hebei Province, China), starting 0.5 m from the tree base. Exposed tissue was wrapped with transparent plastic sheets to prevent desiccation. Samples were subsequently collected by scraping regenerating tissues from the whole surfaces of the trunks at the same daytime (between 10:00-11:00 am) at 7 days (D7), 10 days (D10), 12 days (D12), 16 days (D16), 18 days (D18), and 21 days (D21) after girdling (AG), and immediately frozen and stored in liquid nitrogen. Sampling was performed on three clonal trees at one time.
Project description:(paper abstract) Wood is a renewable resource that serves as a sustainable raw material for energy, fuels and materials. A basic understanding of the cambial meristem and the origin of wood cells is critical for forest tree molecular breeding to increase the availability of woody raw materials. Gene expression per cell type in the cambium of poplar (Populus trichocarpa x P. deltoïdes cultivar Boelare) was investigated using poplar microarray. Cells were hand microdissected from lyophilized sections of vascular cambial zone sampled in growing poplar. Two cell types were isolated, fusiform cambial cells (FCCs) and ray cambial cells (RCCs). Results showed about one thousand genes differentially expressed per cell type. Microarray expression results were validated by a second cell sampling used for reverse northern dot blot and RT-PCR analysis. RCCs revealed specific photosynthetic competences involving at least 35 genes of the photosystems. RCCs revealed also a marked difference for intercellular transport involving notably aquaporin genes. Distinct classes of cell-wall related genes were detected in RCCs and FCCs. A candidate xyloglucan endotransglycosylase showed to be upregulated and highly expressed in FCCs and an extensin family gene was also very highly expressed in these cells. A candidate polygalacturonase was strikingly expressed specifically in RCCs. A marked expression of cytoskeleton genes was also reported for FCCs where profilin family genes were very highly expressed. Related to cell signaling and regulation, one of the most intriguing result was the observation of LAX1 gene specificity in FCCs whereas PIN genes were expressed in both cell types.
Project description:Wood in conifers is mainly composed of tracheids. Some taxa, such as Pinus, present tracheids also in the rays, but are axial tracheids which constitute the vast majority of secondary xylem. Nevertheless, radial and axial parenchyma surrounding constitutive and traumatic resin ducts is known to serve as crucial reserve storage. These reserves are mobilized in response to traumatism, insect and pathogen attacks and defoliation, allowing the synthesis of resin, healing and, in few taxa, even resprouting. However, due to the low proportion of parenchymatic cells in secondary xylem relevant genes involved in their differentiation may have been missed in studies of transcriptomics of conifer wood formation. In this study we have used Pinus canariensis as a model species, given its comparatively high proportion of axial parenchyma. We have prepared two normalized libraries from its cambial zone, covering early- and late-wood differentiation. We have de novo assembled a transcriptome, and have analyzed the transcriptional profiles during the growing season, getting a more complete picture of wood formation in conifers. SUBMITTER_CITATION: Chano, V., López de Heredia, U., Collada, C., et al. (2017). Transcriptomic analysis of juvenile wood formation during the growing season in Pinus canariensis. Holzforschung, 0(0), pp. -. Retrieved 8 Aug. 2017, from doi:10.1515/hf-2017-0014 Overall design: mRNA samples were collected at 6 dates during the growing season in Pinus canariensis, supported by microscopical observation of cambial activity: March, April, May, June, July and September. Three biological replicates were collected for each time sampled, and therefore 18 mRNA samples were hybridazed in a one-color microarray experiment.
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:Cold acclimation in conifers is a complex process, the timing and extent of which reflects local adaptation and varies widely along latitudinal gradients for many temperate and boreal tree species. In spite of their ecological and economic importance, little is known about the global changes in gene expression that accompany autumn cold acclimation in conifers. Using three populations of Sitka spruce (Picea sitchensis) spanning the species range, and a Picea cDNA microarray with 21,840 unique elements, we monitored within and among-population gene expression during the fall. Microarray data were validated for selected genes using real-time PCR. Similar numbers of genes were significantly two-fold upregulated (1,257) and downregulated (967) between late summer and early winter. Among those upregulated were dehydrins, pathogenesis-related/antifreeze genes, carbohydrate and lipid metabolism genes, and genes involved in signal transduction and transcriptional regulation. Among-population microarray hybridizations at early and late autumn time points revealed substantial variation in the autumn transcriptome, some of which may reflect local adaptation. Our results demonstrate the complexity of cold acclimation in conifers, highlight similarities and differences to cold tolerance in annual plants, and provide a solid foundation for functional and genetic studies of this important adaptive process in conifers. Keywords: Time course Foliage for RNA extraction was obtained from four-year-old Sitka spruce seedlings, which were grown from seed collected across the species range in a raised-bed outdoor common garden in Vancouver, BC, Canada (49º N) (Mimura and Aitken, 2007, Heredity). Needle tissues from current year upper lateral shoots, separated from stem and bark tissue, were collected at five time points between late summer and early winter, 2004. Three populations were chosen for sampling: Valdez, Alaska, USA (61º N) (AK), Prince Rupert, British Columbia, Canada (54º N) (BC) and Redwood, California, USA (41º N) (CA) (Figure 1a). Needle samples (~1g) were taken from eight individuals in the BC population on each of the five dates (Aug. 30, Oct. 18, Nov. 24, Dec. 1, and Dec. 13). Eight individuals from the AK and CA populations were also sampled on the second and fourth dates, as well as eight additional individuals from the BC population. Tissues were flash frozen in an N2 vapor tank immediately upon collection, and subsequently stored at -80°C until processing. To decrease the effects of biological variance among individual seedlings within populations, equal amounts of foliar tissue were pooled from four individuals prior to RNA extraction. Two pools were collected at each time point for each population, and total RNA was extracted following a previously published protocol (Kolosova et al., 2004). Slides were scanned and spot intensity quantified using ImaGene software (BioDiscovery, Inc., El Segundo, CA). To correct for background intensity, the lowest 10% of median foreground intensities was subtracted from the median foreground intensities. Data were then normalized by variance stabilizing normalization (VSN) to compensate for nonlinearity of intensity distributions (Huber et al., 2002). To identify significant changes in gene expression, a linear mixed-effects model was fit to the normalized intensities in the Cy3 and Cy5 channels of the 32 microarray slides. The model contained an adjustment for dye bias, an array effect indicating which Cy5/Cy3 pair was on each array, a treatment effect indicating sample population and time point, and a random effect to adjust for repeated measures on the same biological sample (Kerr et al., 2000). P-values were computed for each gene-by-treatment effect and Q-values were calculated to adjust for the false discovery rate (FDR) (Storey & Tibshirani, 2003).
Project description:The brown rot wood decay fungus, Fomitopsis pinicola strain FP-58527, was cultivated for five dayes in media containing ground Populus tremuloides, Pinus taeda or Picea glauca wood as sole carbon source. Extracellular proteomic component was extracted and analyzed by LC-MS/MS.
Project description:Inappropriate response to normal intestinal bacteria is involved in the development of Inflammatory Bowel Diseases (IBD, e.g. Crohn’s Disease (CD), ulcerative colitis (UC)) and variations in the host genome may mediate this process. IL-10-/- mice develop CD-like colitis mainly in the colon, in part due to inappropriate responses to normal intestinal bacteria including Enterococcus strains. Comprehensive characterization of changes in gene expression associated with the observed inflammation in the IL-10-/- mouse model has yet to be reported. Our aim was to characterize changes in colonic gene expression in IL-10-/- and C57BL/6J (C57; control) mice resulting from oral bacterial inoculation with 12 Enterococcus faecalis and faecium (EF) strains isolated from calves or poultry, complex intestinal flora (CIF) collected from healthy control mice, or a mixture of the two (EF•CIF). At 12 weeks of age, total RNA extracted from intact colon was hybridized to Agilent 44k mouse arrays. Lists of differentially expressed genes were generated using linear models for microarray analysis (Bioconductor), and genes clustered using GeneSpring GX and Ingenuity Pathways Analysis software. Intestinal inflammation was increased in IL-10-/- mice as a result of inoculation, with the strongest effect being in the EF and EF.CIF groups; expression of >4,000 gene probes was altered in colon tissue compared with similarly inoculated C57 mice. Genes differentially expressed in IL-10-/- mice as a result of EF or EF.CIF inoculation were associated with the following pathways: inflammatory disease (111 genes differentially expressed), immune response (209 genes), antigen presentation (11 genes, particularly major histocompatability complex Class II), fatty acid metabolism (30 genes) and detoxification (31 genes). Our results suggest that the colonic inflammation in EF and EF.CIF-inoculated IL-10-/- mice is mediated by mechanisms similar to those of human IBD, in particular CD. Example logFC calculation; This example describes the calculation of the fold-change value (logFC) for a single spot (in this case, spot 27222) for a single comparison. This calculation method was applied to generate all of the comparison data shown in the supplementary data files. The comparison in question is for:; colon tissue from C57BL/6J mouse with EF.CIF treatment (represented by duplicate samples GSM307282 (Title: C57BL/6J_Colon_EF.CIF_Rep1_(Pool17)) and GSM307283 (Title: C57BL/6J_Colon_EF.CIF_Rep2_(Pool18)); vs. colon tissue from C57BL/6J mouse under conventional conditions (represented by duplicate samples GSM307270 (Title: C57BL/6J_Colon_Conventional_Rep1_(Pool5)) and GSM307271 (Title: C57BL/6J_Colon_Conventional_Rep2_(Pool6)); Important Note: Limma analysis maps the treatments on each slide to the M values which are by convention always log2(R) - log2(G). Because this experiment is a reference design, with the reference RNA always Cy5 (i.e. red, channel 2) and the treatment sample Cy3 (i.e. green, channel 1), and we are interested in the values of the treatment samples compared with the reference, the M values in the data files are displayed as log2(G) - log2(R); The average expression is what is important here, which is calculated from the normalized M values for the appropriate comparison of interest as follows:;  Mean value for Spot 27222, for each treatment:; C57BL/6J mouse, EF.CIF: = mean (0.26363587 (GSM307282), 0.05084283 (GSM307283)) = 0.15723935 C57BL/6J mouse, conventional = mean (1.42951674 (GSM307270),1.15444106 (GSM307271)) = 1.2919789;  Calculation of LogFC for the comparison of interest:; The contrast of interest is C57BL/6J: EF.CIF vs Conventional, so by performing a subtraction of these values for the comparison of interest (i.e. (mean for EF.CIF) - (mean for Conventional), the common reference is cancelled out, thus:; C57BL/6J: EF.CIF vs Conventional = 0.15723935 - 1.2919789 = -1.13473955; So, the fold-change value (logFC) for spot 27222 for the comparison in question (C57BL/6J: EF.CIF vs. Conventional) is: -1.13473955 Experiment Overall Design: Animals. Twenty five male IL-10-/- (C57BL/6J background, formal designation B6.129P2-IL-10<tm1Cgn>/J) mice and twenty five male C57BL/6J (C57) control mice were received from The Jackson Laboratory (Bar Harbor, Maine, USA) at approximately five weeks of age. Mice were housed either in pairs or groups of three (5 mice per treatment) in shoebox-style cages containing untreated wood shavings and a plastic tube for environmental enrichment. The animal room was maintained at a temperature of ~22°C and humidity of ~50% with a 12 hour light/dark cycle. All mice had ad libitum access to water, which was refreshed twice a week. An AIN-76A powdered diet was supplied twice a week, with sufficient provided to meet the daily intake of IL-10-/- mice, as determined in a previous feeding trial (data not shown). The diet for all groups was sterilized by gamma irradiation (Schering-Plough, Wellington, New Zealand) to a level required for SPF conditions, to minimize the possibility of bacterial introduction to the SPF group of animals. All mice were weighed three times weekly and carefully monitored for disease symptoms (weight loss, soft faeces and inactivity). Experiment Overall Design: Experimental design. Both IL-10-/- and C57 mice were randomly divided into five treatment groups with five animals per group. Experiment Overall Design: SPF. Housed in Specific Pathogen Free (SPF) conditions (isolator cages supplied with HEPA filtered air (Tecniplast SpA, Buguggiate, Italy)). Experiment Overall Design: C. Maintained under conventional conditions. Experiment Overall Design: EF. Maintained under conventional conditions and orally inoculated (200 microlitres) with a solutions containing 12 pure strains of Enterococcus (1.2 x 10^8 colony forming units (CFU)). Experiment Overall Design: CIF. Maintained under conventional conditions and orally inoculated with conventional intestinal flora (CIF) derived from healthy age-matched C57BL/6 mice which had been raised under conventional conditions. The CIF inoculation protocol was included to better mimic the complete microbiota associated with the mouse gastrointestinal tract. Experiment Overall Design: EF•CIF. Maintained under conventional conditions and orally inoculated with a combination of the EF and CIF inoculation solutions (6.0 x 10^7 CFU from the EF inoculum). Experiment Overall Design: All mice were euthanised via CO2 asphyxiation and cervical dislocation at 12 weeks of age. Blood was collected from euthanised mice by cardiac puncture. Plasma was separated from red blood cells by centrifugation (2000 g, 10 min, 4 ºC), frozen in liquid nitrogen and stored at - 80 ºC. The intestine was removed, cut open lengthwise and flushed with 0•9 % NaCl to remove digesta residues. The proximal half of the colon was cut in two pieces, one for histological evaluation and other for gene expression studies. Samples of the colon tissue for gene expression analysis were frozen in liquid nitrogen and stored at - 80 ºC. Samples for histological analysis were stored in formalin solution (10 % neutral buffered) at room temperature. Experiment Overall Design: Total RNA was extracted from the colon tissues using Trizol (Invitrogen, Carlsbad, CA, USA) as described by the manufacturer, with a subsequent purification step using RNeasy columns (Qiagen, San Diego, CA, USA). RNA was quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and RNA quality was examined using a RNA 6000 LabChip Kit and a 2100 Bioanalyser (Agilent Technologies, Palo Alto, CA, USA). Experiment Overall Design: The Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies Inc., Palo Alto, California, USA) was used to synthesize cDNA and fluorescent cRNA. Labelled cRNA was made on the same day for all sample pools, including the reference sample. cDNA was synthesized from 500 ng of purified total RNA from each pool using T7 promotor primer and Moloney Murine Leukemia Virus Reverse Transcriptase according to the manufacturer’s protocol. Cy3 (PerkinElmer, Waltham, Massachusetts 02451, USA) was used to label sample groups, while the reference RNA was labeled with Cy5 (PerkinElmer, Waltham, Massachusetts 02451, USA). Experiment Overall Design: HYBRIDIZATION Experiment Overall Design: Microarray hybridization was performed according to a reference design without dye swap. The in situ hybridization kit-plus (Agilent Technologies Inc., Palo Alto, California, USA) was used to hybridize cRNA samples to Agilent Technologies Mouse G4121A - 44k 60mer oligonucleotide arrays. Cy3-labelled cRNA (sample, 0.75 µg) and Cy5-labelled cRNA (reference, 0.75 µg) were hybridised onto the microarray according to the manufacturer’s protocol. Following hybridisation, slides were washed in solutions I, II and III (Agilent Technologies, Santa Clara, CA, USA) and air-dried. Experiment Overall Design: SCAN PROTOCOL Experiment Overall Design: Slides were scanned using a GenePix 4200A scanner (Molecular Devices Corporation, Sunnyvale, CA, USA) at a photomultiplier tube (PMT) setting of 450 V. Spot identification and quantification were performed using GenePix 6.0 software (Molecular Devices Corporation, Sunnyvale, CA, USA). Spot identification and quantification was performed using GenePix 6.0 software (Molecular Devices). All slides were individually checked and manually flagged for abnormalities. Experiment Overall Design: DATA PROCESSING Experiment Overall Design: Statistical analysis was performed using linear models for microarray analysis (limma) within the Bioconductor framework. Before analysis, poor quality spots were manually flagged and filtered out. Quality of the microarray data was assessed on diagnostic plots (boxplots and density plots) and spatial images generated from the raw (non-processed) data. All twenty arrays passed these strict criteria and were included in the analyses. Intensity ratio values for all microarray spots were normalized using a within-slide global Locally Weighted Scatterplot Smoothing (Loess) procedure to remove the effect of systematic variation in the microarrays; no background correction was necessary due to homogeneous hybridization. The normalized data from the arrays of each treatment group were averaged. For each comparison, differentially expressed genes were identified using false discovery rate (FDR) control with a threshold of q < 0.05. Data were log transformed before analysis and the mean difference between treatments calculated on this scale, resulting in a log ratio for each probe. The normalized values in the database consist of these log ratios. MA plots of the microarray data were drawn in order to check that there was no dependence of the log ratio on the intensity for any slide. The significance of the log ratio for each probe was determined by calculating one modified t-statistic per probe using an empirical Bayes approach. Experiment Overall Design: The probability values were then corrected for multiple testing using the Benjamini and Hochberg correction, and a false discovery rate (FDR) calculated. Probes that had an FDR of less than 5 % (q<0*05) were considered to be differentially expressed between treatment.