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Experimental alteration of DNA methylation is a suitable tool to infer the relationship between phenotypic and epigenetic variation in plants. A detailed analysis of the genome-wide effect of demethylating agents, such as 5-azacytidine (5azaC), and zebularine is only available for the model species Arabidopsis thaliana, which suggests that 5azaC may have a slightly larger effect. In this study, global methylation estimates obtained by high-performance liquid chromatography (HPLC) analyses were conducted to investigate the impact of 5azaC treatment on leaf and root tissue in Erodium cicutarium (Geraniaceae), which is an annual herb native to Mediterranean Europe that is currently naturalized in all continents, sometimes becoming invasive. We used seeds collected from two natural populations in SE Spain. Root tissue of the second generation (F2) greenhouse-grown seedlings had a significantly lower global cytosine methylation content than leaf tissue (13.0 vs. 17.7% of all cytosines). Leaf tissue consistently decreased methylation after treatment, but the response of root tissue varied according to seed provenance, suggesting that genetic background can mediate the response to experimental demethylation. We also found that both leaf number and leaf length were reduced in treated seedlings supporting a consistent phenotypic effect of the treatment regardless of seedling provenance. These findings suggest that, although the consequences of experimental demethylation may be tissue- and background-specific, this method is effective in altering early seedling development, and can thus be useful in ecological epigenetic studies that are aiming to investigate the links between epigenetic and phenotypic variation in non-model plant species.Peer reviewe

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