Plant-RRBS, a bisulfite and next-generation sequencing-based methylome profiling method enriching for coverage of cytosine positions

BMC Plant Biology

Methodology Article

Cindy Martens¹  Marc De Block¹  Magdalena Woloszynska²  Bram Slabbinck²  Mieke Van Lijsebettens²  Michiel Van Bel²  Frederik Coppens²  Martin Schmidt² 

[1] Bayer CropScience N.V., Innovation Center, Technologiepark 38, 9052, Ghent, Belgium;Department of Plant Biotechnology and Bioinformatics, Ghent University, Technologiepark 927, 9052, Ghent, Belgium;VIB Center for Plant Systems Biology, Technologiepark 927, 9052, Ghent, Belgium;

关键词: DNA methylation;    Reduced representation bisulfite sequencing;    RRBS;    Oryza sativa;    Epiline;    Cytosine methylation;    Rice;    Plant;   

DOI  :  10.1186/s12870-017-1070-y

 received in 2017-04-28, accepted in 2017-06-26,  发布年份 2017

来源: Springer

【 摘 要 】

BackgroundCytosine methylation in plant genomes is important for the regulation of gene transcription and transposon activity. Genome-wide methylomes are studied upon mutation of the DNA methyltransferases, adaptation to environmental stresses or during development. However, from basic biology to breeding programs, there is a need to monitor multiple samples to determine transgenerational methylation inheritance or differential cytosine methylation. Methylome data obtained by sodium hydrogen sulfite (bisulfite)-conversion and next-generation sequencing (NGS) provide genome-wide information on cytosine methylation. However, a profiling method that detects cytosine methylation state dispersed over the genome would allow high-throughput analysis of multiple plant samples with distinct epigenetic signatures. We use specific restriction endonucleases to enrich for cytosine coverage in a bisulfite and NGS-based profiling method, which was compared to whole-genome bisulfite sequencing of the same plant material.MethodsWe established an effective methylome profiling method in plants, termed plant-reduced representation bisulfite sequencing (plant-RRBS), using optimized double restriction endonuclease digestion, fragment end repair, adapter ligation, followed by bisulfite conversion, PCR amplification and NGS. We report a performant laboratory protocol and a straightforward bioinformatics data analysis pipeline for plant-RRBS, applicable for any reference-sequenced plant species.ResultsAs a proof of concept, methylome profiling was performed using an Oryza sativa ssp. indica pure breeding line and a derived epigenetically altered line (epiline). Plant-RRBS detects methylation levels at tens of millions of cytosine positions deduced from bisulfite conversion in multiple samples. To evaluate the method, the coverage of cytosine positions, the intra-line similarity and the differential cytosine methylation levels between the pure breeding line and the epiline were determined. Plant-RRBS reproducibly covers commonly up to one fourth of the cytosine positions in the rice genome when using MspI-DpnII within a group of five biological replicates of a line. The method predominantly detects cytosine methylation in putative promoter regions and not-annotated regions in rice.ConclusionsPlant-RRBS offers high-throughput and broad, genome-dispersed methylation detection by effective read number generation obtained from reproducibly covered genome fractions using optimized endonuclease combinations, facilitating comparative analyses of multi-sample studies for cytosine methylation and transgenerational stability in experimental material and plant breeding populations.

【 授权许可】

CC BY   
© The Author(s). 2017