How to Isolate a Plant’s Hypomethylome in One Shot

Abstract

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.

Epigenetic Variability Among Saffron Crocus (Crocus sativus L.) Accessions Characterized by Different Phenotypes

“…The obtained reference hypomethylome was then used for the annotation step and the subsequent analyses. As already described in similar studies, the coverage was low (close to the 0.2 X-value reported in Wischnitzki et al, 2015), thus we did not expect to have reconstructed the complete hypomethylome; however, we still found a depletion in transposable elements (less than 1% of sequences derived from repetitive elements) and an enrichment for gene-associated regions close to the percentage reported in the literature (Wischnitzki et al, 2015). Globally, 11,569 contigs, corresponding to 26.35% of the total, could be annotated as being at least partially protein coding by comparing our sequences with online resources such as the NCBI NR protein and NT nucleotide databases, the O. sativa genome, GO, KEGG -Kyoto Encyclopedia of Genes and Genomes and the available saffron sequences (Table 4).…”

“…To carry out the methyl filtration enrichment of gene-related sequences, genomic DNA was digested using the restriction enzyme Hpa II. This enzyme was selected because it is able to achieve very good gene space coverage and very good depletion of transposable elements, as also reported in Wischnitzki et al (2015) . In brief, the steps of the MRE-seq analysis are: (1) digestion of 300 ng of genomic DNA with the restriction enzyme Hpa II and contemporary ligation of double strand adaptors to the generated fragments; (2) PCR amplification of the adaptor-ligated DNA; (3) removal of the adaptor sequences by Pme I digestion (the sequence of the adaptor includes the rare cutting site of the enzyme GTTTAAAC).…”

“…Epigenetic marks are defined as a series of chemical modifications of DNA and histones, but in plants, most studies have mainly focused on DNA methylation, which presents inheritance mechanisms and is relatively simple to investigate genome-wide ( Seymour and Becker, 2017 ). The most frequent mark of DNA methylation is 5-methylcytosine (5-mC), which in plants can be methylated at different sites containing CG, CHG and CHH (H represents any base but G) sequences ( Wischnitzki et al, 2015 ). The extent of cytosine methylation varies among plant species and along the chromosomes, but the functional consequences for gene expression are strongly influenced by the location of 5-mCs relative to the gene.…”

“…Given that the gene body in plants is characterized by rather low methylation levels (hypomethylation) and that, in contrast, cytosine methylation is most frequent in repetitive elements (e.g., transposable elements), studies of the hypomethylome can provide interesting information about the genes and regulatory regions subjected to epigenetic regulation and variation. Several methods have been developed to study the plant hypomethylome, one of which is the methylation-sensitive restriction enzyme-sequencing (MRE-seq) approach, MRE-seq ( Wischnitzki et al, 2015 ). This method has been applied successfully to different plant species including saffron, making it possible, even in the absence of a reference genome and low coverage of the generated sequences, to isolate and sequence genes and regulatory elements, representing 26 and 47% of the total reads, respectively ( Wischnitzki et al, 2015 ).…”

“…Several methods have been developed to study the plant hypomethylome, one of which is the methylation-sensitive restriction enzyme-sequencing (MRE-seq) approach, MRE-seq ( Wischnitzki et al, 2015 ). This method has been applied successfully to different plant species including saffron, making it possible, even in the absence of a reference genome and low coverage of the generated sequences, to isolate and sequence genes and regulatory elements, representing 26 and 47% of the total reads, respectively ( Wischnitzki et al, 2015 ). To gain a deeper insight into the epigenetics of saffron, the current study compared the epigenetic profile of five phenotypically different, but genetically similar by AFLP analysis ( Busconi et al, 2015 ), accessions from the WSCC germplasm using MRE-seq focusing on differentially methylated regions.…”