Selective chemical labeling reveals the genome-wide distribution of 5-hydroxymethylcytosine in rice
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ABSTRACT: We generated a genome-wide map of 5hmC in rice panicle using a sensitive chemical labeling method to capture DNA fragments with 5hmC modification, followed by NGS sequencing. We showed that the 5hmC peaks distribution is non-random, about 52% 5hmC was located in gene bodies, 25% in intergenic regions, 11% in promoters, and 12% in immediate downstream. It was significantly enriched in heterochromatic regions of chromosomes 4 and 10, and especially located around TE genes. Combined with RNA-seq transcriptome data, we found that the gene-specific alterations of 5hmC can result in gene activation or repression, suggesting a potential role for 5hmC in gene regulation. From the compared distribution analysis among super-hybrid rice cultivars LYP9, and its parental lines 93-11 and PA64s, we investigated the cultivar-specificity of both the global levels and locus-specific distribution of 5hmC. Our data provided an overview of the genomic distribution of 5hmC in rice panicle. This will set the stage for further functional characterization of this novel DNA modification and its biosynthesis in rice. Examination of 5hmC modifications in rice panicle This submission represents ChIP-Seq component of study.
Project description:We generated a genome-wide map of 5hmC in rice panicle using a sensitive chemical labeling method to capture DNA fragments with 5hmC modification, followed by NGS sequencing. We showed that the 5hmC peaks distribution is non-random, about 52% 5hmC was located in gene bodies, 25% in intergenic regions, 11% in promoters, and 12% in immediate downstream. It was significantly enriched in heterochromatic regions of chromosomes 4 and 10, and especially located around TE genes. Combined with RNA-seq transcriptome data, we found that the gene-specific alterations of 5hmC can result in gene activation or repression, suggesting a potential role for 5hmC in gene regulation. From the compared distribution analysis among super-hybrid rice cultivars LYP9, and its parental lines 93-11 and PA64s, we investigated the cultivar-specificity of both the global levels and locus-specific distribution of 5hmC. Our data provided an overview of the genomic distribution of 5hmC in rice panicle. This will set the stage for further functional characterization of this novel DNA modification and its biosynthesis in rice.
Project description:To comprehend the profile of rice gene expression at reproductive stage under high temperature, Agilent 4x44k rice oligo microarray experiments were carried out using rice panicle of developmental stage 7-9 at 0min, 10min, 20min, 60min, and 2hr after the treatment of 40 degree centigrade, and the significantly expressed genes mainly involved in transcriptional regulation, transport, cellular homeostasis, and stress response were identified. Among them, the predominant transcription factor gene families were Hsf, NAC, AP2/ERF, WRKY, MYB, and C2H2. KMC analysis discovered the time-dependent gene expression pattern under heat. The results of motif co-occurrence on the promoters of genes from an early up-regulated cluster showed the important roles of GCC box, HSE, ABRE, and CE3, and unraveled the possible cross-talk mechanism during heating. The response model central to ROS combined with transcriptome data indicated the great importance to maintain ROS balance in heat response in rice panicle and the wide existing of cross-talks. Heat shock induced gene expression in rice panicle of late developmental stage was measured at 0min, 10min, 20min, 60min, and 2hr after the treatment of 40 degree centigrade in plant growth chamber. Two independent replicate experiments were performed at each time point.
Project description:To comprehend the profile of rice gene expression at reproductive stage under high temperature, Agilent 4M-CM-^W44k rice oligo microarray experiments were carried out using rice panicle of developmental stage 7-9 at 0min, 20min, 1hr, 2hr, 4hr, and 8hr after the treatment of 40 degree centigrade, and the significantly expressed genes mainly involved in transcriptional regulation, transport, cellular homeostasis, and stress response were identified. Among them, the predominant transcription factor gene families were Hsf, NAC, AP2/ERF, WRKY, MYB, and C2H2. KMC analysis discovered the time-dependent gene expression pattern under heat. The results of motif co-occurrence on the promoters of genes from an early up-regulated cluster showed the important roles of GCC box, HSE, ABRE, and CE3, and unraveled the possible cross-talk mechanism during heating. The response model central to ROS combined with transcriptome data indicated the great importance to maintain ROS balance in heat response in rice panicle and the wide existing of cross-talks. Heat shock induced gene expression in rice panicle of developmental stage 7-9 was measured at 0min, 20min, 1hr, 2hr, 4hr, and 8hr after the treatment of 40 degree centigrade in plant growth chamber. Two independent replicate experiments were performed at each time point.
Project description:Rice false smut is a common fungal disease caused by Ustilaginoidea virens. As it only scatter occured in panicle at florescence, little information is known about this crop disease. Here, we injected suspension spores into a susceptible indica rice cultivar 9311 booting panicle (infected water as mock) and divided the early disease symptom into 3 uninterrupted stages(S) at 6 day post inoculation (dpi): the infected pistil became expand (S1), the hyphae began to infect the bottom of anthers (S2) and the hyphae growth went on and surrounded the floral organ forming a floral-hyphae complex (S3). To gain insight into rice putatively differential responses to U. virens, all 3 infected and mock spikes with same spike length were collected and analyzed by Solexa/IlluminaM-CM-"M-BM-^@M-BM-^Ys digital gene expression (DGE) system, BGI. Our results contribute to the knowledge of understanding rice molecular mechanism in response to U. virens infection. Four samples including one control (CK) and three infected (S1, S2, S3) were analyzed
Project description:The profiling was conducted with the Rice 3'-Tiling 135k Microarray designed from 31,439 genes deposited at IRGSP, RAP2 database (http://rapdb.lab.nig.ac.jp). In this research, an array of 31,439 rice genes was used to elucidate gene expression in leaf and panicles of non-transgenic and HMB4 over-expression line. The analyses show that transgenic rice induces early flowering due to an enhancement of stress response. A total of 20 chips were used for microarray. Total RNAs were extracted from rice leaf and panicle. Experiments were duplicated.
Project description:In rice (Oryza sativa L.), chilling-induced male sterility increased when plants experienced low water temperature (Tw, 18 M-BM-0C for 14 days) before panicle initiation. The number of mature pollen grains after chilling at the booting stage (12 M-BM-0C for 5 days) was only approximately 45% of total pollen grains in low-Tw plants, whereas it was approximately 71% in normal-Tw plants (Tw not controlled; approximately 23 M-BM-0C under air temperature of 26 M-BM-0C/21 M-BM-0C, day/night). Microarray and quantitative PCR analyses showed that many stress-responsive genes (including OsFKBP65 and genes encoding a large heat shock protein OsHSP90.1, heat shock factors, and many small heat shock proteins) were strongly up-regulated by chilling in normal-Tw spikelets, but were not or rather down-regulated by chilling in low Tw spikelets. OsAPX2 and genes encoding some other antioxidative enzymes were also significantly down-regulated by low Tw in the chilled spikelets. In low-Tw plants, lipid peroxidation products (malondialdehyde equivalents) were significantly increased in the spikelets after chilling, and ascorbate peroxidase activity in the chilled spikelets was significantly lower than that in normal-Tw plants. Our data suggest that an OsFKBP65-related chilling response, which protects proteins from oxidative damage, is indispensable for chilling tolerance but is lost in low-Tw spikelets. Four conditions used: low Tw and chilled, low Tw and not chilled, normal Tw and chilled, normal Tw and not chilled, before panicle initiation and at the booting stage, respectively. Biological replicates: 4 for each treatment.
Project description:Rice genome contains three genes that encode for glutathione reductase (GR) viz., OsGR1, 2 and 3. GR is an important component of the anti-oxidative machinery of plant cells. GR2 down-regulated plants were produced by RNAi mediated down-regulation of GR2 (GR2-Ri). GR2-Ri plants were significantly smaller and have significantly lower grain yield (grain number per panicle) compared to WT(Wild type), under control conditions. RNA-Seq analysis of panicles (differentiation stage) identified genes known to affect grain size to be differentially regulated in GR2-Ri compared to WT, respectively, under control conditions.
Project description:To examine the rice genome methylation landscape and assess its functional significance, we generated the first single-base resolution genome methylation maps for Oryza sativa ssp. japonica, indica and their wild relatives, Oryza rufipogon and Oryza nivara. The methylation level of rice genomes is four times higher than that of Arabidopsis. Methylation in the promoter and gene body regions have similar patterns and effects on gene expression as those in Arabidopsis but different from a previous study on rice chromosomes 4 and 10. Most interestingly, we discovered for the first time that methylation in gene transcriptional termination regions can significantly repress gene expression, and the effect is even stronger than promoter methylation, which opens a new direction in the study of epigenetic regulation of gene expressions. Through integrated analysis of genetic, methylome and expression variation between cultivated and wild rice, we found that the genetic factor reflected by DNA variations may be the major determinant for methylation patterns at the whole-genome level and that methylation variation can only account for limited expression variation of genes between cultivated and wild rice. A single young panicle from each of the cultivated rice subspecies and the two wild rice species was ground in liquid nitrogen to fine powder using mortar and pestle. Total RNAs were isolated using the RNeasy Plant Mini Kit (Qiagen). DGE-tag libraries were constructed using the DGE-Tag Profiling NlaIII Sample Prep Kit (Illumina) according to the manufacturer's instructions. This submission represents the gene expression component of the study.
Project description:In the current study we did microarray of upland rice cultivar Nagina22 for drought stress at reproductive stage (panicle initiation) and analyzed drought stress responsive genes. We have taken flag leaf for our study as it is most essential organ for photosynthesis in rice. Normal watering Vs Drought Stress Flag leaf of Control (Three biological replicates) plant of Nagina22: C1, C2, C3 Flag leaf of drought stressed (Three biological replicates) plant of Nagina 22: S1, S2, S3
Project description:Studies on the epigenetic regulation of gene expression during germ cell development are still at the beginning stage. In the present study, we used our highly specific 5hmC-labeling and enrichment technique coupled with DNA deep-sequencing to profile the global 5hmC distribution in 8 serial stages of male germ cells during spermatogenesis, as well as in the the Sertoli cells (SE) which are the only somatic cell type inside seminiferous tubules. We analyzed the genomic distribution and dynamic changes of 5hmC during spermatogenesis. Moreover, to dissect the functional significance of 5hmC modifications for transcriptional regulation of gene expression, we also performed RNA-Seq transcriptome analysis in all of the 8 corresponding stages of male germ cells and found that 5hmC is positively correlated with gene expression. RNA-seq: Examination of the transcriptomes during mouse spermatogenesis. 5hmC-seq: Identification of 5hmC enriched genmoic regions in mouse germ cell.