Reagieren langlebige Koniferen auf Umweltstressoren mit somatisch epigenetischem Priming? Genomweite basengenaue Analyse von Methylierung mit Hilfe von Exome Capture und Bisulfitsequenzierung bei Fichte.
Genetik und Genomik der Pflanzen
Zusammenfassung der Projektergebnisse
In recent years, evidence accumulated that epigenetic mechanisms might play an important role for the response of plants to environmental conditions. This might be particularly relevant in trees which are confronted with changing environmental conditions over their long life span without the opportunity to migrate. However, most of our current knowledge on epigenetic mechanisms in plants is limited to a few model plant species. In recent years, genomic resources became available for a number of tree species, which now permits to transfer the approaches and methods from model species, for example to study methylation patterns at single-base resolution. However, large genome sizes, for example of conifer species, and the large number of individuals that need to be targeted in population epigenetic studies render whole genome approached too expensive. Therefore, in a proof-of-principle study, we tested the applicability of a combined exome capture and bisulfite sequencing approach (targeted bisulfite sequencing, TBS) to analyze variation of methylation patterns in Norway spruce (Picea abies). Furthermore, we tested whether methylation patterns change in a predictable manner in response to environmental cues among pairs of clones from contrasting environments. For TBS, we used a commercially available kit (SeqCap Epi Developer, NimbleGen) with customdesigned probes that targeted ~ 26.000 genes that were annotated with high confidence in the Norway spruce genome. In total, we included samples of eight clones that were grown at two and three sites respectively, and included replicates from the same sites and individuals which summed up to a total of 29 sequenced individuals. At the time of report writing, we had focused our analysis on four clones whose source trees (ortets) grow in the Bavarian Forest National Park, and its cuttings (ramets) in a seed orchard in Übersee, Germany. We obtained data on the methylation status for more than 6 million cytosines in ~26.000 genes. Similar to other plants, Cs in gene bodies had a higher methylation percentage in the CG than in the CHG and CHH context. When we compared methylation profiles between individuals, we found that methylation in CG was highly conserved between ortets and ramets, while there were slightly more differences in CHG methylation and substantial variation in CHH methylation. When we tested for methylation differences between sites, we found 334 positions which differed coherently in their methylation percentage, most of them in the CG and CHG context thus showing that single base resolution methods can detect somatic mutations which might pass undetected by global methylation approaches. Overall, we found that TBS permits to generate reproducible data on methylation status for a large fraction of the targeted regions from the Norway spruce genes. We are therefore convinced that TBS can be an important bridging technology for non-model species in the coming years. Further, our data indicated, that methylation patterns were highly similar between ortets and ramets. Yet, despite very stringent thresholds, we found 334 differentially methylated positions between sites which supported our hypothesis that environmental differences induce changes in methylation patterns, although this seems to be limited to a small fraction of the genic regions.
