Arabidopsis thaliana
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PROVIDER: PRJNA352276 | ENA |
REPOSITORIES: ENA
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Project description:Arabidopsis thaliana EMS mutant mapping backcross method
| PRJNA215956 | ENA
Project description:We were interested in identifying targets of novel putative miRNAs we identified from small RNA sequencing libraries of Arabidopsis shoots. The small RNA (smRNA) sequencing libraries were made to identify changes in abundance of specific smRNAs in response to developmental transitions in Arabidopsis thaliana shoots, with special focus on vegetative phase change. We specifically wanted to separate the temporal changes in gene expression that result from vegetative phase change and those from flowering. Because of the close timing between the juvenile-to-adult and adult-to-reproductive developmental transitions in Arabidopsis grown under long day conditions, we used the late-flowering genotype FRI;FLC developed by the lab of Richard Amasino by introgressing the FRI allele from Sf-2 into the Col-0 genetic background, which is fri;FLC. For the early flowering genotype, we used FRI;flc-3, also developed by the Amasino lab by EMS-mutagenizing FRI;FLC, identifying early flowering mutants, and backcrossing multiple times to eliminate other EMS-induced mutations. The onset of vegetative phase change in FRI;FLC and FRI;flc-3 under our growth conditions was identical, but the progression was slower in FRI;FLC. By sequencing small RNAs from shoot apices at different time points and fully-expanded leaves at different positions on the shoot and comparing the results between the two genotypes, we were able to obtain a clear picture of changes in small RNA abundance in response to vegetative phase change and flowering in Arabidopsis. We then used the remaining RNA to make genome-wide mapping of uncapped and cleaved transcripts (GMUCT) 2.0 libraries of a subset of our samples. GMUCT 2.0 allows you to identify RNAs that are 1) uncapped and in the process of 5’->3’ exonuclease degradation and 2) miRNA and siRNA-mediated cleavage products. We wanted to use these GMUCT 2.0 libraries to identify targets of novel putative miRNAs discovered by our smRNA sequencing, thereby supporting the idea that these novel putative miRNAs are in fact functional.
2016-02-01 | GSE72505 | GEO
Project description:Eukaryotic genomes are heavily regulated by epigenetic marks that often act to modulate the transcriptional control of genetic elements. In Arabidopsis thaliana the ATXR5 and ATXR6 histone methyltransferases, and their cognate H3K27 monomethylation mark, act in transcriptional silencing while also maintaining genome stability by preventing generation of excess DNA corresponding to pericentromeric heterochromatin. In this study we characterize the atxr5 atxr6 transcriptome and its relationship to the DNA damage response which suggests that the atxr5 atxr6 transcriptional defects may be epistatic to the genome instability defects in the mutants. In addition we isolate several factors that modulate both the transcriptional and genomic instability phenotypes of atxr5 atxr6 mutants, which suggest a mechanism for atxr5 atxr6-induced extra DNA involving conflicts between the replicative and transcriptional processes in the cell. PolyA RNA sequencing (RNA-seq), whole-genome resequencing (DNA-seq), and whole-genome bisulfite sequencing (methyl-seq) was performed on Arabidospsis thaliana mutant and wildtype plants. DNA-seq was used to characterize DNA copy number and map EMS-induced mutations, RNA-seq was used to quantify transcript abundance and map EMS-induced mutations, and methyl-seq was used to assess DNA methylation. Details of the relationship between samples in this series and figures in the associated manuscript can be found in Supplemental Table 4 of the associated manuscript. Unless otherwise noted in the description all lines are ecotype Columbia, and all genotypes should be assumed homozygous unless otherwise indicated with a '/'.
2016-06-01 | E-GEOD-77735 | biostudies-arrayexpress
Project description:We were interested in changes in small RNA abundance changes in response to developmental transitions in Arabidopsis thaliana shoots, with special focus on vegetative phase change. We specifically wanted to separate the temporal changes in gene expression that result from vegetative phase change and those from flowering. Because of the close timing between the juvenile-to-adult and adult-to-reproductive developmental transitions in Arabidopsis grown under long day conditions, we used the late-flowering genotype FRI;FLC developed by the lab of Richard Amasino by introgressing the FRI allele from Sf-2 into the Col-0 genetic background, which is fri;FLC. For the early flowering genotype, we used FRI;flc-3, also developed by the Amasino lab by EMS-mutagenizing FRI;FLC, identifying early flowering mutants, and backcrossing multiple times to eliminate other EMS-induced mutations. The onset of vegetative phase change in FRI;FLC and FRI;flc-3 under our growth conditions was identical, but the progression was slower in FRI;FLC. By sequencing small RNAs from shoot apices at different time points and fully-expanded leaves at different positions on the shoot and comparing the results between the two genotypes, we were able to obtain a clear picture of changes in small RNA abundance in response to vegetative phase change and flowering in Arabidopsis. For the small RNA samples, we performed two replicates using two different indices in the 5'-adapter and ran each replicate pair on the same sequencing lane. For the cotyledon and leaf samples we only performed one replicate using the same index for all samples because we obtained significantly different results with the two adapters used for the shoot apices, preventing us from using them as true replicates.
2016-02-01 | GSE72303 | GEO
Project description:Arabidopsis mutants in the PMP/PNP oxidase PDX3 show abberant growth and development.RNA sequencing reveals strong induction of stress-related genes in pdx3, particularly those associated with biotic stress.
2016-02-02 | GSE77428 | GEO
Project description:Phenotype-driven forward genetic experiments are among the most powerful approaches for linking biology and disease to genomic elements. Although widely used in a range of model organisms, positional cloning of causal variants is still a very laborious process. Here, we describe a novel universal approach, named fast forward genetics that combines traditional bulk segregant techniques with next-generation sequencing technology and targeted genomic enrichment, to dramatically improve the process of mapping and cloning multiple mutants in a single experiment. In a two-step procedure the mutation is first roughly mapped by ‘light’ sequencing of the bulk segregant pool, followed by genomic enrichment and deep-sequencing of the mutant pool for the linked genomic region. The latter step allows for simultaneous fine-mapping and mutation discovery. We successfully applied this approach to three Arabidopsis mutants, but the method can in principle be applied to any model organism of interest and is largely independent of the genome size. Moreover, we show that both steps can be performed in multiplex using barcoded samples, thereby increasing efficiency enormously. Inducible overexpression of the RETINOBLASTOMA-RELATED (RBR-OE) gene in Arabidopsis roots causes the complete differentiation of stem cells and premature differentiation of daughter cells, leading to a full exhaustion of the primary root meristem. In order to identify regulators of RBR function in cell differentiation, RBR-OE plants in the Columbia background (Col0) were treated with EMS mutagenesis and a set of genetic suppressors of RBR-OE, which restores root growth capacity, were isolated. In this study, we used one the identified suppressor lines, which segregated as a recessive mutation. Mapping populations were generated by outcrossing to Ler ecotype. Seedlings from the F2 population were grown for 15 days post germination (dpg). A pool of 60 seedlings each with a clear suppressor phenotype (homozygous for suppressor mutation) and of 60 seedlings showing RBOE phenotype (Heterozygous for the suppressor mutation) were prepared and genomic DNA was isolated with the RNeasy Plant Mini Kit from QIAGEN according to manufacturer's protocol. The other two, mutants 136 and 193 were obtained in fluorescence based mutant screen and a QCmarker based mutagenesis, respectively. Mutants were generated by chemical mutagenesis (EMS) in Colombia (Col) genetic background. Mutants were subsequently crossed to the Landsberg (Ler) ecotype to create the mapping populations. Bulk-segregant pools of about 200 mutant as well as wild-type plants were generated for every mutant line.
2010-12-31 | E-GEOD-24511 | biostudies-arrayexpress
Project description:mRNA sequencing of Arabidopsis thaliana pdx3 mutants
| PRJNA310312 | ENA
Project description:Bisulfite sequencing of Arabidopsis T-DNA mutants
| PRJDB5210 | ENA