A direct PCR approach with low-biomass insert opens new horizons for molecular sciences on cryptogam communities

Abstract

Cyanobacteria, green algae, lichens, and other cryptogams play crucial roles in complex microbial systems such as biological soil crusts of arid biomes or biofilms in caves. Molecular investigations on environmental samples or isolates of these microorganisms are often hampered by their dense aggregation, small size, or metabolism products which complicate DNA extraction and subsequent PCRs. Our work presents various examples of how a direct DNA extraction and PCR method relying on low biomass inserts can overcome these common problems and discusses additional applications of the workflow including adaptations.

The underestimated fraction: diversity, challenges and novel insights into unicellular cyanobionts of lichens

“…All sequences in bold are from isolates held by INCb. Sequences labeled with the strain identifier “INCb” were generated following the workflow described in [ 61 ] and the methodology therein was followed for the construction of the phylogenetic tree. Black squares indicate lichen symbionts (cyanobionts).…”

“…This, along with the small size of most cyanolichens, highlights the requirement for methods that allow an accurate differentiation between lichen photobionts and their free-living members that are often adhered to the substrate or epiphytically to the lichen. Such a method, using a direct PCR technique on a small scale has recently been introduced [ 61 ], which could, in the future, be complemented by a culture attempt based on a micromanipulator that allows picking of single cells directly from inner parts of lichens comparable with the technique described in [ 62 ]. The direct PCR method which requires low biomass inserts has been shown to be suitable for the target organisms because all sequences of strains labeled with “INCb” in Fig.…”

“…The direct PCR method which requires low biomass inserts has been shown to be suitable for the target organisms because all sequences of strains labeled with “INCb” in Fig. 2 were generated using the workflow as described in [ 61 ].…”