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Natural epigenetic variation: a driver for adaptation of wild plant populations?
Sammarco, Iris ; Latzel, Vít (advisor) ; Johannes, Frank (referee) ; Pečinka, Aleš (referee)
A growing body of literature suggests that epigenetic variation might contribute to local adaptation of natural plant populations. Epigenetic mechanisms, such as DNA methylation, can in fact quickly alter phenotypes in response to environmental changes. Furthermore, these changes can be inherited across several generations (especially clonal), suggesting that DNA methylation may enable heritable phenotypic variation and eventually contribute to adaptation. However, it is still unclear whether epigenetic mechanisms can have an adaptive potential in plants, or in other words, whether they present variation among natural populations, are inherited across generations and have fitness effects. During my PhD project, together with my co-authors, I tested these conditions on several natural populations of two non-model clonal plant species, the wild strawberry (Fragaria vesca) and the black poplar (Populus nigra). To do so, I used the current gold standard for DNA methylation analysis (Whole Genome Bisulfite Sequencing), which allowed me to characterize the genome-wide DNA methylation level of individual plants at the single base-resolution. For both species, I found extensive DNA methylation diversity among populations, which was partially affected by the natural climatic conditions of the populations...
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Dynamics of de novo DNA methylation and its impact on transgene expression and CRISPR/Cas9 mutagenesis
Přibylová, Adéla ; Fischer, Lukáš (advisor) ; Pečinka, Aleš (referee) ; Fajkus, Jiří (referee)
Genetic information must be protected, maintained and copied from cell to daughter cells, from generation to generation. In plants, most of the cells contain complete genetic information, and many of these cells can regenerate to a whole new plant. Such a feature leads to the need for precise control of which genes will be active and which not because in growth and differentiation, only the activity of specific genes for the individual cells, tissues, organs are required. One of the mechanisms controlling the gene activity is RNA interference (RNAi), which down- regulates or blocks the expression of specific genes at the transcriptional or post-transcriptional level. The crucial part of the RNAi is guiding the RNAi machinery to the target. It is mediated via sequence complementarity of the target with a small RNA (sRNA), which is diced from a double- stranded RNA (dsRNA) precursor. The molecular mechanism of dsRNA and sRNA formation and also the target origin predestinates the subsequent silencing pathway. In transcriptional gene silencing (TGS), the gene expression is regulated through chromatin epigenetic modifications. One of the epigenetic marks is cytosine methylation, which is established mainly by RNA-directed DNA-methylation (RdDM) pathway. Although the protein machinery was relatively...
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Dynamics and variability of induced transgene silencing in tobacco cell line BY-2
Čermák, Vojtěch ; Fischer, Lukáš (advisor) ; Pečinka, Aleš (referee) ; Lafon Placette, Clément (referee)
RNA interference (RNAi) is an important mechanism regulating gene expression. In plants, RNAi is triggered by double-stranded RNA (dsRNA) which is processed into small RNAs (sRNAs), usually 21-24 nt long. The sRNAs are loaded into Argonaut (AGO) protein and recognize the target based on sequence complementarity. When the target is mRNA, they can slice it or block translation leading to posttranscriptional gene silencing (PTGS). When the target is DNA, they can induce DNA methylation and chromatin changes, which when present in the promoter can lead to transcriptional gene silencing (TGS). The individual components of RNAi are well described, but less is known about the impact of different types of dsRNA precursors on the dynamics of RNAi. To study these aspects of RNAi, we used tobacco BY-2 cell line expressing GFP reporter and inducible silencers. The silencers used different ways of triggering the dsRNA formation by transcripts from antisense (AS), unterminated sense (UT) and inverted repeat (IR) GFP sequence to initiate PTGS. Additionally, one IR silencer based on the CaMV 35S promoter initiated TGS. This allowed us to study RNAi from the beginning throughout the steady state level and till the recovery phase, all in the highly homogeneous system. Using this system, we described several features...
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