# Genetics - epigenetics

### Tight link between genetics and epigenetics

An important complicating factor, hindering advances in determining the importance of epigenetics in evolution, is posed by the tight link between the genome and the epigenome. In simple words, when a genetic polymorphism is controlling an epigenetic pattern, the latter will seem to be heritable and under selection, while in reality this is only true for the causal genetic polymorphism. This can be caused by trans-acting elements at the genome level, for example when a mutation affecting a methyltransferase is responsible for the induction of different DNA methylation patterns and different global DNA methylation (Dubin et al. 2015). Alternatively, this can also happen at the level of a specific locus, driven by cis-acting elements such as TE insertions causing DNA methylation of the neighbouring region. For example, in melon, a TE insertion next to the promoter of CmWIP1 causes silencing of this gene, resulting in a switch from male to female flowers (Martin et al. 2009).

Looking at this the other way around, epigenetic variation can also be in control of genetic variation, as for example TE activity is controlled by DNA methylation (Noshay et al. 2019). This link poses difficulties when studying the phenotypic effects of DNA methylation variation, as an induced change in the DNA methylation patterns (see chemical demethylation), often used to prove its contribution to phenotypic changes, may release TEs. It is therefore difficult to determine whether the phenotypic effect following a DNA methylation change is truly due to this change or due to an unseen TE insertion.

### Controlling or excluding genetic variation

To overcome these difficulties, different approaches have been used and studies have found that epigenetic variation can, at least in some cases, be independent from DNA sequence variation (Cubas, Vincent, and Coen 1999; Riddle and Richards 2002; Shindo et al. 2006; Vaughn et al. 2007). These approaches are either based on excluding genetic variation, by looking at epigenetic variation in populations with the same genetic background or accounting for genetic variation. Some common approaches are listed below:

1\.         **Excluding genetic variation:** these are the most common approaches because they are simpler and normally less expensive. The downside of these methods is that they do not allow to look at epigenetic variation in natural populations of genetically variable species.

1. Using asexually reproducing species as these have nearly identical genetic background (Vanden Broeck et al. 2018; Heer 2018; Shi et al. 2019).
2. Chemical demethylation: Demethylating agents such as Zebularine and 5-azacytidine can be used to extensively, but randomly, erase DNA methylation from genomes without modifying the genetic background, except for the above-mentioned possibility of TE activation (Latzel 2016; Münzbergova et al. 2018).
3. Methylation mutants: Introducing epigenetic variation through a mutation in the epigenetic machinery and then removing the mutation (Johannes et al. 2009, Cortijo et al. 2014, Kooke el al. 2015, Zhang et al. 2019).
4. Targeted DNA methylation: Using molecular biology techniques, such as transformation with inverted repeat (IR) sequences (Mette et al. 2000; Zicola et al. 2019) or Cas9-methyltransferase complexes, It is possible to methylate specific genomic regions. While Cas9 complexes can be used for any kind of sequence, IR insertions should only be used, when targeting non-transcribed regions or they will also degrade mRNAs through post transcriptional gene silencing.

2\.         **Accounting for genetic variation:** this strategy is used to study epigenetic variation in natural populations of species harbouring genetic variation. It is based on statistical methods allowing to determine whether the observed epigenetic variation is controlled by underlying genetic polymorphisms. This approach faces what we will call the large-scale vs high-accuracy problem, meaning it requires both large-scale collections and high-accuracy genomic tools. Large-scale surveys are fundamental to capture enough variation to provide a representative sample of the adaptive ability of a certain species. High-accuracy genomic tools are required at the DNA methylation level in order to capture the majority of the variation and to draw information about a variety of factors. Moreover, high-accuracy at the DNA sequence level, and therefore a reference genome, are also required due to the tight link between genetics and epigenetics. Some papers that adopted this approach in Arabidopsis are (Dubin et al. 2015, Sakani et al. 2019, and Kawakatsu et al. 2016).
