Designing more ambitious studies

In a natural setting, plants are exposed to a complex environment, often with multiple stress conditions, and thus, to simulate ecological realism, we should have more than one stress factor in experimental design (see Lampei 2019). To study plant stress response, it is important to establish the causal links between epigenetic variation and phenotypes, as well the interference of genetic and epigenetic variation. Thus, one challenge lies in identifying the relevance of epigenetic factors for modulating the phenotypic response to specific stress factors. To this aim, it may be beneficial to include plant species with different life histories and genomic features. A few approaches have been reported by which epigenetic variation can be potentially measured for larger populations in an ecological setting.

Study system

Where possible, experiments should include several genetically divergent lines or diverse natural populations from one species. Single line, or genotype, experiments may provide very detailed information but lack generality of conclusion. Often, repeating the experiment with another genotype of the same species yields different results. This is not only true for epigenetic experiments but even more important in these because of frequent interactions with the genomic background. However, the enhanced generality comes with an increased complexity of the study. Therefore, epigenetic studies should preferably be conducted in ecological settings that control genotypic variability. For example, non-model species or natural populations that contain a low level of genetic diversity and reproduce asexually like, for instance, clonal plants, provide these properties (Richards, Bossdorf, et al. 2010).

‌Another approach is to study outcrossed species by evaluating both the DNA sequence and DNA methylation profiles of individuals using statistical approaches to understand the relation of genetic and epigenetic variation (Herrera and Bazaga 2011; Schulz et al. 2019). As the field is still developing for non-model species, we must be aware of the trade-off between the depth/resolution of functional information and cost-effectiveness. Following experimental methods to study epigenetic inheritance could help to fill the gaps in the field (Bossdorf et al. 2008).

Experimental method

An interesting option is the use of inhibitors for epigenetic factors such as the DNA methyltransferases (e.g., 5-azacytidine, zebularine) or histone deacetylases (e.g., Trichostatin). Together with knockout mutants, experimental demethylation can be used to establish the link between epigenetic factors and phenotypic response. However, this method has disadvantages in the first place because it is not targeted and reduces DNA methylation scattered across the genome so that connections between phenotype and methylation can be made, but it is often difficult to identify the specific methylation changes that were involved in the response. ‌

The study should choose the molecular mechanisms that are confirmatory and cost-effective to study epigenetics in natural populations. For example, for evaluating the methylation status, Reduced Representation Bisulfite Sequencing methods (RRBS) can also work without the availability of a high-quality genome (Niederhuth and Schmitz 2017), and it is cost-effective. This is a very nice option for studying methylation in non-model species. However, we need more methods with such criteria for studying other epigenetic factors. Quantitative genetics mapping approaches such as Epigenome-wide association studies (EWAS) can be another method to study the approximate genetic and epigenetic associations with the phenotype (Kreutz et al. 2020).