Evaluation of primers for the detection of deadwood-inhabiting archaea via amplicon sequencing

Abstract

Archaea have been reported from deadwood of a few different tree species in temperate and boreal forest ecosystems in the past. However, while one of their functions is well linked to methane production any additional contribution to wood decomposition is not understood and underexplored which may be also attributed to lacking investigations on their diversity in this substrate. With this current work, we aim at encouraging further investigations by providing aid in primer choice for DNA metabarcoding using Illumina amplicon sequencing. We tested 16S primer pairs on genomic DNA extracted from woody tissue of four temperate deciduous tree species. Three primer pairs were specific to archaea and one prokaryotic primer pair theoretically amplifies both, bacterial and archaeal DNA. Methanobacteriales and Methanomassiliicoccales have been consistently identified as dominant orders across all datasets but significant variability in ASV richness was observed using different primer combinations. Nitrososphaerales have only been identified when using archaea-specific primer sets. In addition, the most commonly applied primer combination targeting prokaryotes in general yielded the lowest relative proportion of archaeal sequences per sample, which underlines the fact, that using target specific primers unraveled a yet unknown diversity of archaea in deadwood. Hence, archaea seem to be an important group of the deadwood-inhabiting community and further research is needed to explore their role during the decomposition process.