The cytosine methylation landscape of spring barley revealed by a new reduced representation bisulfite sequencing pipeline, WellMeth

Abstract

Patterns and level of cytosine methylation vary widely among plant species and are associated with genome size as well as the proportion of transposons and other repetitive elements in the genome. We explored epigenetic patterns and diversity in a representative proportion of the spring barley (Hordeum vulgare L.) genome across several commercial and historical cultivars. This study adapted a genotyping‐by‐sequencing (GBS) approach for the detection of methylated cytosines in genomic DNA. To analyze the data, we developed WellMeth, a complete pipeline for analysis of reduced representation bisulfite sequencing. WellMeth enabled quantification of context‐specific DNA methylation at the single‐base resolution as well as identification of differentially methylated sites (DMCs) and regions (DMRs). On average, DNA methylation levels were significantly higher than what is commonly observed in many plants species, reaching over 10‐fold higher levels than those in Arabidopsis thaliana (L.) Heynh. in the CHH methylation. Preferential methylation was observed within and at the edges of long‐terminal repeats (LTR) retrotransposons Gypsy and Copia. From a pairwise comparison of cultivars, numerous DMRs could be identified of which more than 5,000 were conserved within the analyzed set of barley cultivars. The subset of regions overlapping with genes showed enrichment in gene ontology (GO) categories associated with chromatin and cellular structure and organization. A significant correlation between genetic and epigenetic distances suggests that a considerable portion of methylated regions is under strict genetic control in barley. The data presented herein represents the first step in efforts toward a better understanding of genome‐level structural and functional aspects of methylation in barley.

Methylome changes in Lolium perenne associated with long-term colonisation by the endophytic fungus Epichloë sp. LpTG-3 strain AR37

“…For the analysis of methylated cytosines, pair-end reads of 150 bp were processed using the WellMeth pipeline ( Malinowska et al., 2020 ) and mapped to the reference genome of L. perenne ( Nagy et al., 2022 ), which contains 7 pseudo-chromosomes and 9400 unanchored scaffolds. The identification of methylated sites was followed by PCR duplicate removal.…”

“…Transposable elements and repeats were identified using the RepeatMasker software tool (version open-4.0.6, Smit et al., 2013-2015) using the Liliopsida species model and RepBase Update 20160829. A custom pipeline was used to annotate the RepeatMasker output, as described in Malinowska et al. (2020) .…”

Exploring the crop epigenome: a comparison of DNA methylation profiling techniques

“…RRBS with optimized endonuclease combinations is used to explore the impact of DNA methylation on rice responses to salt stress ( Schmidt et al., 2017 ), and on the regulation of TEs in maize ( Hsu et al., 2018 ). RRBS has been also applied for identifying markers that aid in the breeding and improvement of crops ( Malinowska et al., 2020 ; Li et al., 2023 ). By detecting methylation patterns associated with specific agricultural traits, such as disease resistance or yield potential, valuable information for the development of new crop varieties that are more resilient to pests and diseases has been provided ( Turcotte et al., 2022 ).…”

“…This approach offers advantages over using a single restriction endonuclease that only covers a limited number of cytosine positions, hampering subsequent comparative analyses, as seen in the study of Quercus lobata Neé (Gugger et al, 2016). Furthermore, Malinowska et al (2020) introduced WellMeth, a RRBS pipeline specifically designed for methylation analysis of plants with available high-quality reference genomes. WellMeth enables quantification of methylation at singlebase resolution, identification of DMRs and cites, and has been successfully applied to spring barley.…”

Relative importance of genotype, gene expression, and DNA methylation on complex traits in perennial ryegrass

“…To obtain methylomic data, epiGBS libraries were prepared as previously described (Malinowska et al., 2020) and sequenced on the Illumina HiSeq4000 platform (2 × 150 bp) by Beijing Genomics Institute (Shenzhen, China). The sequencing data were processed using the WellMeth pipeline (Malinowska et al., 2020). Demultiplexed and trimmed reads were mapped to the perennial ryegrass pseudo‐chromosome assembly (Nagy et al., 2022).…”