Dynamic Changes of DNA Methylation During Wild Strawberry (Fragaria nilgerrensis) Tissue Culture

Abstract

Tissue culture is an important tool for asexual propagation and genetic transformation of strawberry plants. In plant tissue culture, variation of DNA methylation is a potential source of phenotypic variation in regenerated plants. However, the genome wide dynamic methylation patterns of strawberry tissue culture remain unclear. In this study, we used whole-genome bisulfite sequencing (WGBS) to study genomic DNA methylation changes of a wild strawberry Fragaria nilgerrensis at six stages: from explants of shoot tips to outplanting and acclimation. Global methylation levels showed that CG sites exhibited the highest methylation level in all stages with an average of 49.5%, followed by CHG (33.2%) and CHH (12.4%). Although CHH accounted for the lowest proportion of total cytosine methylation, it showed the most obvious methylation change and the most of these changes occurred in the transposable element regions. The overall methylation levels alternately decreased and increased during the entire tissue culture process and the distribution of DNA methylation was non-uniform among different genetic regions. Furthermore, much more differentially methylated regions (DMRs) were detected in dedifferentiation and redifferentiation stages and most of them were transposable elements, suggesting these processes involved activating or silencing of amounts of transposons. The functional enrichment of the DMR-related genes indicated that genes involved in hormone metabolic processes, plant development and the stress response changed methylation throughout the tissue culture process. Finally, the quantitative real-time PCR (qRT-PCR) was conducted to examine the association of methylation and gene expression of a set of different methylated genes. Our findings give deeper insight into the epigenetic regulation of gene expression during the plant tissue cultures process, which will be useful in the efficient control of somaclonal variations and in crop improvement.

Phenotypic variation and epigenetic insight into tissue culture berry crops

“…Additionally, during the dedifferentiation and redifferentiation stages of the tissue culture process, differentially methylated regions were detected, which were mostly transposable elements. This finding suggests that dedifferentiation and redifferentiation processes were involved in activation or silencing of transposable elements (Cao et al, 2021). Sikdar et al (2022) demonstrated the interaction between cytosine methylation and secondary metabolites in tissue-culture shoots, lingonberry plants, and cutting propagated plants under in vitro and ex vitro conditions.…”

“…Among these two tissue culture systems, explants from the bioreaction system showed increased levels of methylation variation and polymorphism than explants derived from the semisolid media (Ghosh et al, 2021a). Cao et al (2021) studied genomic DNA methylation using whole genome bisulfite sequencing (WGBS) in wild strawberry (F. nilgerrensis) at various tissue culture stages, starting from use of the shoot tip explants to out planting and acclimation. The authors found that the most obvious methylation changes occurred in the transposable element region of the genome, and the global methylation levels alternately increased and decreased during the tissue culture process.…”

“…Paramutation in maize and tomato is another epigenetic phenomenon, which displays non-Mendelian epigenetic inheritance ( Chandler et al, 2000 ). Later, paramutation has also been detected in mammals, flies, and plants ( Cao et al., 2021 ). Understanding the paramutation phenomenon led several scientists who had been using plants as a model system to study epigenetic regulations.…”

“…However, propagation strategies have been developed to minimize variations in micropropagated berry plants ( Debnath and Goyali, 2020 ). Although a number of DNA-based molecular markers have been used to monitor the clonal fidelity in berry crops including raspberry ( Debnath, 2014 ), lingonberry ( Arigundam et al., 2020 ), blueberry ( Goyali et al., 2015 ; Debnath, 2017a ), and strawberry ( Debnath, 2009a ), tissue culture-induced epigenetic variation such as DNA methylation has been studied so far only in blueberry ( Ghosh et al., 2017 ; Goyali et al., 2018 ; Ghosh et al., 2021a ), strawberry ( Cao et al., 2021 ), and lingonberry ( Sikdar et al., 2022 ). The present review deals with in-depth variations during in vitro culture of berry crops along with their analysis at morphological, biochemical, and molecular levels.…”

Integrated transcriptome and methylome analyses reveal the molecular regulation of drought stress in wild strawberry (Fragaria nilgerrensis)

“…High quality RNA was extracted from F. nilgerrensis leaves and reversely transcribed into cDNA and used as template for qRT-PCR. Consistent with the qRT-PCR method described in our previous study [90], FnACTIN was selected as an internal reference to normalize the relative expression of each gene. The primer sequences used for qRT-PCR validation are shown in Supplementary Table S7.…”