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

Background: Cytosine 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. Methods: We 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. Results: As 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. Conclusions: Plant-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

Genetic diversity and adaptive traits of European versus American Douglas-fir seedlings

Due to its productivity and potential to adapt to the expected climate change, the Douglas-fir is one of the most important commercial non-native forest tree species in Europe. Currently, seeds from both non-native European and native American seed stands are used for plantations. In this study, we investigate European seed lots for their native origin (variety and potential geographic origin in America) and assess the adaptability, growth and survival potential of European versus American Douglas-fir seed lots. We compare the genetic diversity, morphological characteristics such as height (h), root collar diameter (rcd) and the ratio of h/rcd, and the timing of bud burst. We investigate 852 1-year-old seedlings from 10 different US and European seed lots representing 5 provenance regions which are sold in Germany and Austria. Seedlings are genotyped for 13 nuclear SSRs and analysed together with reference data set and standard genetic structuring and assignment methods. Adaptive traits of morphological characteristics and timing of bud burst of the seedlings are recorded and statistically analysed. The results show that the investigated European seedlings originate from recommended American native seed sources and represent both varieties and inter-varietal admixed individuals. European seedlings have a lower genetic diversity versus the American seedlings and native populations. They show significant differences in the adaptive traits such as morphological characteristics and timing of bud burst. According to the genetic diversity indices, certified North American Douglas-fir seed sources should be preferred for planting in Central Europe