Background

Nucleotide sequences are not the only form of genomic information: DNA methylation, histone proteins, enzymes that modify histones and nucleotide residues on DNA and even RNAs, influence gene activity and provide another layer of instructions to the cell. Epigenetic changes, also called epimutations, can be inherited and have important phenotypic consequences. In plants, methylation reactions modify cytosine residues into 5-methylcytosines. This epigenetic mechanism is essential to maintain the genomic integrity and contributes to regulate gene expression in developmental processes and in response to biotic and abiotic stresses. In addition, changes in DNA methylation are triggered by genomic shocks like hybridization and polyploidization, two vital phenomena in plant evolution (Cara et al., 2019).

There are different alternatives to study changes in DNA methylation. Global cytosine methylation can be assessed by using high-performance liquid chromatography (HPLC), analytical methods that allow to quantify cytosines and 5-methylcytosines and to calculate the percentage of methylated residues in the genome. For studying the DNA methylation at specific positions on the genome two alternatives can be mentioned. One is to use isoschizomers with different sensitivities to methylation at cytosines of the restriction site. For example, Methylation-Sensitive Amplification Polymorphism (MSAP) markers characterize the methylation pattern at anonymous 5′-CCGG sequences from random genomic DNA. This is an adaptation of the original AFLP protocol (Vos et al., 1995) substituting the frequent cutter enzyme MseI by HpaII and MspI. These enzymes recognize the same tetranucleotide restriction site (5′-CCGG), but HpaII is sensitive to full methylation (both strands methylated) and cleaves the hemi-methylated external cytosine, whereas MspI is sensitive only to methylation of the external cytosines of the restriction site. Another possibility to study site-specific methylation status is by using a bisulphite sequencing methodology. Bisulphite treatments on DNA convert cytosines into uracils while 5-methylcytosines remain unchanged. Then, by sequencing amplicons (i.e., a target gene or promoter) of bisulphited and control DNA, it is possible to distinguish between methylated and unmethylated cytosines. With the development of next generation sequencing technologies, whole-genome bisulphite sequencing, or WGBS, can be implemented to infer the position for all 5-methylcytosines in a genome. However, this approach requires a high-quality reference genome to perform epigenetic analyses. Although the increasing number of draft genomes and the reduction in sequencing costs offer possibilities to implement massive methylation analyses in non-model species, the use of MSAP markers continues to be a valuable tool in many laboratories.

Wild potatoes (Solanum, section Petota) are a group of species related to the cultivated potato Solanum tuberosum L. Internal breeding barriers can be incomplete, thus, interspecific hybridization occurs in areas of sympatry (Camadro et al., 2012). Epigenetic changes in response to interspecific hybridization have been documented in synthetic and natural hybrids of wild potato species (Cara et al., 2019). Solanum x rechei H. & H. is a hybrid species that grows in sympatry with its wild progenitors, Solanum kurtzianum B. & W. and Solanum microdontum B. Here, we present a protocol for analyzing DNA methylation patterns through MSAP markers in hybrids and their parental genotypes. Using an R script, fragments present in the synthetic hybrids are categorized as S. microdontum or S. kurtzianum species-specific if they are present on the parental genotypes, S. x rechei species-specific, if they are present in at least one of the S. x rechei evaluated genotypes or as novel if they are only observed in the synthetic hybrids.