Stable Epigenetic Variants Selected from an Induced Hypomethylated Fragaria vesca Population

Abstract

Epigenetic inheritance was transmitted through selection over five generations of extreme early, but not late flowering time phenotypic lines in Fragaria vesca. Epigenetic variation was initially artificially induced using the DNA demethylation reagent 5-azacytidine (5-azaC). It is the first report to explore epigenetic variant selection and phenotypic trait inheritance in strawberry. Transmission frequency of these traits was determined across generations. The early flowering (EF4) and late stolon (LS) phenotypic traits were successfully transmitted across five and three generations through meiosis, respectively. Stable mitotic transmission of the early flowering phenotype was also demonstrated using clonal daughters derived from the 4th Generation (S4) mother plant. In order to further explore the DNA methylation patterns underlying the early flowering trait, the standard MSAP method using isoschizomers Hpa II/Msp I, and newly modified MSAP method using isoschizomers Tfi I/Pfe I which detected DNA methylation at CG, CHG, CHH sites were used in two early flowering lines, EF lines 1 (P2) and EF lines 2 (P3), and control lines (P1). A significant reduction in the number of fully-methylated bands was detected in P2 and P3 when compared to P1 using the novel MSAP method. In the standard MSAP, the symmetric CG and CHG methylation was maintained over generations in the early flowering lines based on the clustering in P2 and P3, the novel MSAP approach revealed the asymmetric CHH methylation pattern was not maintained over generations. This study provides evidence of stable selection of phenotypic traits, particularly early flowering through both meiosis and mitosis, which is meaningful to both breeding programs and commercial horticulture. The maintenance in CG and CHG methylation over generations suggests the early flowering phenotype might be related to DNA methylation alterations at the CG or CHG sites. Finally, this work provides a new approach for studying the role of epigenetics on complex quantitative trait improvement in strawberry, as well as providing a tool to expand phenotypic diversity and expedite potential new horticulture cultivar releases through either seed or vegetative propagation.

Tissue-Specific Response to Experimental Demethylation at Seed Germination in the Non-Model Herb Erodium cicutarium

“…In particular, experimental demethylation with DNMTi has contributed to reveal the broad range of plant traits and developmental processes that are regulated through DNA cytosine methylation, because such experiments have been able to, among others, alter sex expression [15], modify growth, leaf traits, flowering time or fruit ripening (e.g., [17,18,21,[41][42][43][44]), and induce dwarfism at maturity [14,17]. The phenotypic consequences of the cell reprogramming associated to genome-wide demethylation, however, are not necessarily the same across species, genetic backgrounds, and cell lines [13].…”

“…In plants, DNMT inhibitors (DNMTi hereafter), mainly 5-azacytidine, 5-aza-2 -deoxycytidine, and zebularine, have proven useful to obtain epimutants with characteristic phenotypes that, at least in some cases, remained stable across multiple generations (e.g., [14][15][16][17][18]). Zebularine is chemically more stable, forms a reversible complex with DNMTs, and exhibits a weaker inhibition activity and cytotoxicity than azacytosine analogs [13].…”

Variability in DNA Methylation and Generational Plasticity in the Lombardy Poplar, a Single Genotype Worldwide Distributed Since the Eighteenth Century

“…Moreover, methyl polymorphisms at CpG sites (cytosine-phosphate-guanine sites where a cytosine is directly followed by a guanine in the DNA sequence) have recently been proposed to a play a crucial role in rapid environmental adaptation (Huang et al, 2017 ) and may provide asexual organisms with additional sources of variation to cope with contrasting or shifting environmental conditions (e.g., Castonguay and Angers, 2012 ; Richards et al, 2012 ; Verhoeven and Preite, 2014 ). Recently, some studies have indeed shown that epigenetic effects can result in novel phenotypes without any variation in the DNA sequence (Cubas et al, 1999 ), and that epigenetic states may persist after the initiating factor causing the epigenetic effects disappeared (e.g., Verhoeven et al, 2010 ; Xie et al, 2015 ; Xu et al, 2016 ).…”

Epigenetic variation for agronomic improvement: an opportunity for vegetatively propagated crops

“…Epigenetic variants with potential agronomic interest that have been identified in several crop species include, for example, dwarf phenotypes in rice (Chen and Zhou, ), anthocyanin production in apple (Telias et al., ), an increased seed protein content and decreased oil content in oilseed rape (Long et al., ), sex determination in melon (Martin et al., ), and fruit ripening in tomato (Liu et al., ). Epigenetic variants of agronomic interest can also be obtained by using chemical treatments such as the exposure to the methylation inhibitor 5‐azacytidine, e.g., early flowering in strawberry (Xu et al., ). Locus‐specific epigenome editing techniques such as transcription activator‐like effectors nucleases (TALENs), zinc fingers nucleases (ZFNs) or CRISPR‐Cas9 (Hung et al., ) might also be used in the near future.…”

“…As a result, the transmission of epigenetic variants to the next generation in clonally multiplied plants (e.g., cuttings, in vitro propagation) is stable. There is evidence of this stability lasting up to five rounds of clonal propagation, such as genomewide DNA methylation modifications associated with biomass changes induced by maternal stress (drought, soil contamination, and shading) in the clonal plant Trifolium repens L. (Rendina González et al., ) and global demethylation associated with early flowering in the clonally propagated plant Fragaria vesca (Xu et al., ).…”