Studying DNA Methylation for Transgenerational Inheritance.

A subset of cytosine bases in plant nuclear genomes is methylated at the fifth position, forming 5-methylcytosine (5mC). DNA methylation is a conserved epigenetic mark important for genome stability and gene transcription. The genome methylation status is altered under conditions of stress, affecting gene expression patterns as a resistance response. Furthermore, changes in genome methylation status can be transmitted to germline cells in many plant species by overcoming the restraints on meiosis, resulting in the inheritance of acquired resistance traits in the next generation. Arabidopsis (Arabidopsis thaliana) is an attractive research model plant for studies of transgenerational inheritance due to its short life cycle (8-10 weeks). Isogenic lines of plants should be used throughout a transgenerational inheritance study to avoid genetic differences between individuals in the descendant generations. In addition, plants should be germinated and grown under optimal conditions to minimize environmental factors that may cause DNA methylome variations between the generations. 5mC in genomic DNA can be experimentally distinguished from unmethylated cytosine by treatment with sodium bisulfite, which converts unmethylated cytosine, but not 5mC, to uracil. Genomic regions with differential methylation between generations can be identified via whole-genome bisulfite sequencing and alignment of sequences between generations. Apart from whole-genome sequencing, particular chromatin regions of interest can be amplified by methylation-specific polymerase chain reaction (PCR). In this chapter, experimental steps for studying transgenerational inheritance of gene methylation are described using Arabidopsis as a model plant species.