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Genome assembly remains a challenge for large and/or complex plant genomes due to their abundant repetitive regions resulting in studies focusing on gene space instead of the whole genome. Thus, DNA enrichment strategies facilitate the assembly by increasing the coverage and simultaneously reducing the complexity of the whole genome. In this paper we provide an easy, fast, and cost-effective variant of MRE-seq to obtain a plant's hypomethylome by an optimized methyl filtration protocol followed by next generation sequencing. The method is demonstrated on three plant species with knowingly large and/or complex (polyploid) genomes: Oryza sativa, Picea abies, and Crocus sativus. The identified hypomethylomes show clear enrichment for genes and their flanking regions and clear reduction of transposable elements. Additionally, genomic sequences around genes are captured including regulatory elements in introns and up- and downstream flanks. High similarity of the results obtained by a de novo assembly approach with a reference based mapping in rice supports the applicability for studying and understanding the genomes of nonmodel organisms. Hence we show the high potential of MRE-seq in a wide range of scenarios for the direct analysis of methylation differences, for example, between ecotypes, individuals, within or across species harbouring large, and complex genomes.

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Abstract: Long-term drought during one and two growing seasons, respectively strongly affected the timing and number of flushes of oak plants (Quercus robur L.), and negatively influenced subsequent growth. In contrast, strong compensation growth was observed upon rewatering. The defence response of experimental plants was assessed by analyzing stress metabolites and transcriptome changes. Our data revealed that while during mild and/or short drought stress only readily metabolizable carbohydrates e.g. glucose, fructose, and galactose are accumulated, prolonged drought stress results in accumulation of more stable and protective osmolytes such as quercitol and mannitol. At transcriptome level more than 3% of the expressed sequences tags (ESTs) represented on the microarray were differentially expressed reflecting the activation of defence responses in oak which appear to be stronger in the second year of persisting stress. The transcriptome changes are in good agreement with both the metabolic data as well as morphological observations. We conclude that mild long-term drought stress over two years triggers adaptation processes in oak plants that ensure survival, while irreversible damage is kept low facilitating full recovery upon rewatering. [Copyright &y& Elsevier]