DNA Methylation Analysis in Barley and Other Species with Large Genomes
Detailed DNA methylation analyses in plant species with large and highly repetitive genomes can be challenging as well as costly. Here, we describe a complete protocol for a high-throughput DNA methylation changes analysis using Methylation-Sensitive Amplification Polymorphism Sequencing (MSAP-Seq; Chwialkowska et al., Front Plant Sci. 8: 2056 (2017)). This method allows detailed information about DNA methylation changes in large and complex genomes to be obtained at a relatively low cost. MSAP-Seq is based on conventional MSAP marker analysis and employs all its basic steps such as restriction cleavage with methylation-sensitive restriction enzyme, ligation of universal adapters, and PCR amplification. However, the traditional gel-based amplicon separation is replaced by direct, global sequencing with next-generation sequencing (NGS) methods. Consequently, MSAP-Seq allows for parallel analysis of hundreds of thousands of different CCGG sites and evaluation of their DNA methylation state. This technique especially targets to genic regions, so it is well suited for large genomes with low gene density, such as barley and other plants with large genomes.
Key wordsDNA methylation MSAP Next-generation sequencing Large genomes Barley
This work was supported by the European Union within The 7th Framework Programme under project no. 289300 “EURoot: Enhancing resource Uptake from Roots under stress in cereal crops,” the Ministry of Science and Higher Education grant 2486/7.PR/2012/2, and by the Polish National Science Centre (NCN) grant no. 2014/13/N/NZ2/01153. KC and UK were granted a scholarship co-funded by the European Union within the framework of the European Social Fund “DoktoRIS Scholarship Program for Innovative Silesia.” KC was a beneficiary of the ETIUDA scholarship funded by the Polish National Science Centre (NCN) under grant no. 2016/20/T/NZ2/00577.