Transcription profiling of 100 Arabidopsis Col/Ler recombinant inbred lines to generate a high-density genetic linkage map
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ABSTRACT: The aim of this study was to generate a high-density genetic linkage map of Arabidopsis. A custom-designed exon-specific whole-genome array was used to identify ~16,000 significant SFP markers between the parental accessions Col and Ler. Subsequently 100 Col/Ler recombinant inbred lines (RILs) were hybridized to the whole genome exon arrays and the SFP-markers genotyped in all lines.
Project description:Hair Follicle regeneration relies on both epithelial components (bulge and hair germ cells) and a mesenchymal one (dermal papilla cells). We used microarrays to detail the global programme of gene expression underlying organ regeneration at the transition between quiescent stages (early and middle telogen) and the initiation of a new growth (late telogen). Experiment Overall Design: These microarray at the 3 different stages were designed to identify signals released by the mesenchymal dermal papilla cells to activate epithelial growth, their target genes in the hair germ and bulge compartments, and to get at gene signature differences and similarities between hair germ and bulge cells.
Project description:We investigated gene expression levels in Heliconius erato butterflies with divergent wing patterns across a 656KB genomic interval linked to the red color pattern wing polymorphism. This included comparison of expression between two H. erato color pattern populations (H. e. petiverana and a H.e. etylus x H. himera hybrid) across three sections of the forewing that differed in pigmentation (the basal, mid, and distal wing sections) and five different stages of pupal development (Day 1, 3, 5 pupae and ommochrome and melanin pigmentation stages). These results allowed us to determine whether certain genes in this interval were differentially expressed between the wing pattern elements, and, therefore, potentially responsible for adaptive color pattern variation in these butterflies. Forewings from a total of 29 individuals, covering three biological replicates of five developmental time points for each of the two H. erato distinct phenotypes were dissected, with the only exception being that there were only two replicates of the day 1 hybrid phenotype. Individuals were reared at 25˚C and dissected at the following stages: a) day 1 = 12 hr after pupation; b) day 3 = 60 hr after pupation; c) day 5 = 108 hr after pupation; d) early ommochrome = ~ 156 hr after pupation when red scales in forewing partially mature, showing a pale orange color; and e) early melanin = ~ 180 hr after pupation melanic scales begin to turn black and are present primarily at the center of the wing. Using wing veins as landmarks, each forewing was cut into three sections corresponding to the color pattern boundaries: basal (F1), middle (F2), and apical (F3). A eight custom-designed Roche NimbleGen 12x135K format microarrays with probes spanning a 656,307bp genomic region (Roche NimbleGen Inc., Madison, Wisconsin, United States) were used to hybridize double stranded cDNA from 87 tissue samples. Repetitive sequence elements found more than five times across all currently available H. erato genomic sequences, including the probed region as well as additional genomic BAC sequences, were masked from the tiling region. The remaining unmasked non-repetitive genomic sequences were tiled using 60 bp probes staggered every 13 bp on average, with slight modifications to ensure probe quality, for a total of 48,547 probes. Microarry design and printing was performed by Roche NimbleGen. cDNA labeling, hybridization, and array scanning was performed by the City of Hope Microarray Facility (Duarte, California, United States). In addition to the probes from the red color pattern intervals, the arrays also include 40,763 probes across two other genomic intervals not addressed in this study, 45,046 probes representing 12450 transcripts from a recent transcriptome assembly at 1-6X coverage, and 3248 random probes. Results from the transcriptome and other color pattern intervals will be published separately, however, we analyzed all probes together for array normalization and quality control.
Project description:Accumulation of unfolded/misfolded proteins in endoplasmic reticulum (ER) elicits a well conserved response called the Unfolded Protein Response (UPR), which triggers the up-regulation of downstream genes involved in protein folding, vesicle trafficking, and ER-Associated Degradation (ERAD). Although the dynamic transcriptomic responses and underlying major transcriptional regulators in ER stress response in plants have been well established, the proteome changes induced by ER stress have not been reported in plants. In the current study, we found that the Arabidopsis Ler ecotype is more sensitive to ER stress than the Col ecotype. Quantitative mass spectrometry analysis with Tandem Mass Tag (TMT) isobaric labeling showed that totally 7439 and 7035 proteins were identified from Col and Ler seedlings, with 88 and 113 differentially regulated (FC>1.3 or <0.7, P<0.05) proteins by ER stress in Col and Ler, respectively. Among them, 40 proteins were commonly up-regulated in Col and Ler, of which 10 were not up-regulated in bzip28 bzip60 double mutant (Col background) plants. Of the 19 specifically up-regulated proteins in Col comparing to that in Ler, components in ERAD, N-glycosylation, vesicle trafficking and molecular chaperones were represented. Quantitative RT-PCR showed that genes encoding 7 out of 19 proteins were not up-regulated (FC>1.3 or <0.7, P<0.05) by ER stress in both ecotypes while genes encoding 12 out of 19 proteins were up-regulated by ER stress with no obvious differences in fold change between Col and Ler. Our results experimentally demonstrated the robust ER stress response at proteome level in plants and revealed differentially regulated proteins that may contribute to differed ER stress sensitivity between Col and Ler ecotypes in Arabidopsis.
Project description:HSC (Sca+ SP) were isolated from 8-12 week C57B6 mice at various time points after treatment with 5-Fluorouracil. RNA was isolated from 50,000-100,000 FACS sorted cells and subjected to two rounds of T7 based linear amplification using Ambion's Message Amp kit. Two replicates from each time point were analyzed. http://www.plosbiology.org/plosonline/?request=get-document&doi=10.1371%2Fjournal.pbio.0020301
Project description:The aim of this study was to analyze the impact of autotetraploidy on gene expression in Arabidopsis thaliana by comparing diploid versus tetraploid transcriptomes. In particular, this included the comparison of the transcriptome of different tetraploid A. thaliana ecotypes (Col-0 vs. Ler-0). The study was extended to address further aspects. One was the comparison of the transcriptomes in subsequent generations. This intended to obtain information on the genome wide stability of autotetraploid gene expression. Another line of work compared the transcriptomes of different diploid vs. tetraploid tissues. This aimed to investigate whether particular gene groups are specifically affected during the development of A. thaliana autotetraploids. Samples 1-8: Arabidopsis thaliana Col-0 tetraploid transcriptome. Transcriptional profiling and comparison of diploid vs. tetraploid Col-0 seedlings. The experiment was carried out with pedigree of independently generated and assessed tetraploid Col-0 lines. Samples 9-12: Arabidopsis thaliana Ler-0 tetraploid transcriptome. Transcriptional profiling and comparison of diploid vs. tetraploid Ler-0 seedlings. The experiment was carried out with pedigree of independently generated and assessed tetraploid Ler-0 lines. Samples 13-24: Arabidopsis thaliana Col-0 tetraploid transcriptome. Transcriptional profiling and comparison of diploid vs. tetraploid Col-0 leaves (6th - 8th). The experiment was carried out with pedigree of independently generated and assessed tetraploid Col-0 lines. Samples 25-32: Arabidopsis thaliana Ler-0 tetraploid transcriptome. Transcriptional profiling and comparison of diploid vs. tetraploid Ler-0 leaves (6th - 8th). The experiment was carried out with pedigree of independently generated and assessed tetraploid Ler-0 lines. Samples 33-36: Arabidopsis thaliana Ler-0 tetraploid transcriptome. Transcriptional profiling and comparison of tetraploid vs. tetraploid Ler-0 seedlings from the second (F2) and third (F3) generation after induction, respectively. The experiment was carried out with pedigree of independently generated and assessed tetraploid Ler-0 lines. Samples 37-40: Arabidopsis thaliana Col-0 tetraploid transcriptome. Transcriptional profiling and comparison of tetraploid vs. tetraploid Col-0 seedlings from the second (F2) and third (F3) generation after induction, respectively. The experiment was carried out with pedigree of independently generated and assessed tetraploid Col-0 lines. Samples 41-44: Arabidopsis thaliana Col-0/Ler-0 diploid transcriptome. Transcriptional profiling and comparison of diploid Col-0 vs. diploid Ler-0 seedlings. The experiment was carried out with pedigree of esrablished lines. Samples 45-48: Arabidopsis thaliana Col-0/Ler-0 tetraploid transcriptome. Transcriptional profiling and comparison of tetraploid Col-0 vs tetraploid Ler-0 seedlings. The experiment was carried out with pedigree of independently generated and assessed tetraploid Col-0 and Ler-0 lines.
Project description:While the close relationship between BRs and auxin has been widely reported, the molecular mechanism for combinatorial control of shared target genes has remained elusive. In this work, we demonstrate that BRs synergistically increase seedling sensitivity to auxin and show that combined treatment with both hormones can increase the magnitude and duration of gene expression. arf2 mutants are less sensitive to changes in endogenous BR levels, while a large number of genes affected in an arf2 background are returned to near wild-type levels by altering BR biosynthesis. Together, these data suggest a model where BIN2 increases expression of auxin-induced genes by directly inactivating repressor ARFs, leading to synergistic increases in transcription. Experiment Overall Design: Total RNA was extracted from 4-day-old, etiolated Arabidopsis seedlings grown on 0.5 µM BRZ or mock treatments and used to probe ATH1 microarrays (Affymetrix), according to manufacturerâ??s protocols. There are three independent biological replicates for each genotype and treatment, except for arf2 mutants with BRZ where only two arrays passed quality control. We performed standard Affymetrix quality-control procedures using the BioConductor packages simpleaffy. Expression was normalized and estimated using the gcRMA package of BioConductor.
Project description:Polyploidy is a widespread phenomenon in flowering plant species. Polyploid plants frequently exhibit considerable transcriptomic alterations after whole-genome duplication (WGD). It is known that the transcriptomic response to tetraploidization is ecotype-dependent in Arabidopsis. Nevertheless, the biological significance and the underlying mechanism are unknown. Here, we showed that 4x Col-0 and 4x Ler presented different flowering times, with a delayed flowering time in 4x Col-0 but not in 4x Ler. We found that the expression of FLOWERING LOCUS C (FLC), the major repressor of flowering, was significantly increased in 4x Col-0 but subtle change in 4x Ler. Moreover, the level of a repressive epigenetic mark, trimethylation of histone H3 at lysine 27 (H3K27me3), was significantly decreased in 4x Col-0 but not in 4x Ler, potentially leading to different transcription levels of FLC and flowering time in 4x Col-0 and 4x Ler. Apart from the FLC locus, hundreds of genes showed differentially H3K27me3 alterations in 4x Col-0 and 4x Ler. Comparably, LIKE HETEROCHROMATIN PROTEIN 1 (LHP1) and transcription factors required for H3K27me3 deposition presented differential transcriptional changes between 4x Col and Ler, potentially account for differential H3K27me3 alterations in 4x Col-0 and Ler. Last, we found that the natural 4x Arabidopsis ecotype Wa-1 presented early flowering time, associated with low expression and high H3K27me3 of FLC. Taken together, our results showed a role of H3K27me3 alterations in response to genome duplication in Arabidopsis autopolyploids and that flowering time variation potentially functions in autopolyploid speciation.
Project description:The global gene expression profiles of human umbilical cord blood and adult bone marrow CD34+CD33-CD38-Rho(lo)c-kit+ cells, enriched for hematopoietic stem/progenitor cells (HSC) with CD34+CD33-CD38-Rho(hi) cells, enriched in committed hematopoietic progenitor cells (HPC), were compared to identify candidate regulators of HSC self-renewal versus differentiation fate decisions.
Project description:Cutis laxa (CL) syndromes are a heterogenous group of connective tissue disorders that share a loose, redundant skin as a common clinical feature. The systemic features vary among the different subtypes. CL is caused by mutations in genes encoding for components of the extracellular matrix (FBLN4, FBLN5, LTBP4 and ELN), encoding for elastin-modifying enzymes (ATP7A) or encoding for components that influence cellular trafficking and metabolism (ATP6V1E1, ATP6V1A, ATP6V0A2, ALDH18A1, RIN2, GORAB, PYCR1 and SLC2A10). ATP6V1E1–related CL cause loose redundant skin folds, variable mental disability, typical facial characteristics, lipodystrophy, hypotonia, and cardiopulmonary involvement including pneumothorax, hypertrophic cardiomyopathy and aortic root dilatation. The intent of this study is to investigate which genes are up- or downregulated in atp6v1e1b-deficient zebrafish larvae compared to wild-type controls. Via transcriptome analysis, we want to study the pathogenic mechanism of ATP6V1E1-induced CL syndrome. We use a zebrafish line with viral insertion in the 5’UTR of atp6v1e1b, disrupting transcription (atp6v1e1bhi577aTg/+), from the Zebrafish International Research Center (ZIRC) and we use a line harboring a two base-pair insertion followed by a three base-pair deletion in exon 5 of atp6v1e1b, c.334insGG; c.337-340delCGG, predicted to result in p.R111WfsX2 (atp6v1e1bcmg78/+) which we created ourselves by CRISPR-Cas9 mutagenesis. Overview of the experimental work-flow: - Sample collection: pool of 10 zebrafish larvae of 3 dpf/genotype in RNA-later - RNA extraction: TRIzol® Reagent,RNeasy mini kit (Qiagen) according to manufacturer’s instructions - RNA integrity: 2100 Bioanalyzer (Agilent) - Sequencing library: TruSeq® Stranded mRNA Library Prep (Illumina, San Diego, California, United States) supplemented with TruSeq® RNA Single Indexes Set A (Illumina) - Sequencing: HiSeq 3000 sequencer (Illumina) - paired-end 150 bp - sequencing facility of the Center of Medical Genetics Ghent - alignement to zebrafish GRCz10 reference genome to generate bam files - RNA-seq pipeline was used that was published by the nf-core community. This pipeline was executed using the Nextflow engine for computational workflows and comprises several processing steps. QC analysis of the RNA-seq data was performed with FastQC and MultiQC. TrimGalore was used to remove adapter contamination and to trim low-quality regions. Duplicate reads were identified with MarkDuplicates. Subsequently, all cleaned and trimmed reads that passed QC were aligned to GRCz10 using STAR aligner. Gene counts were computed using the featureCounts package. Differential expression analysis subsequently was performed on these gene counts using DESeq2. Differentially expressed genes were identified using a fold change cut-off >1 and FDR=0.05. Finally, GO enrichment & pathway analysis were performed on differentially expressed gene sets using the Generally Applicable Gene-set Enrichment for Pathway Analysis (GAGE) algorithm.