Molecular community analysis of arbuscular mycorrhizal fungi—Contributions of PCR primer and host plant selectivity to the detected community profiles

• PCR bias in molecular profiling of AMF communities remains poorly understood.
• Influence of primer choice was thus compared with selectivity due to plant species.
• The different primers demonstrated good agreement in detecting AMF dominants.
• AMF community profiles were more affected by primers than by host plants.
• Commonly used SSU primers missed 4 rare of the 11 AMF genera in the community.

Various primers targeting different regions of nuclear ribosomal DNA (rDNA) are commonly used in studies addressing diversity of soil- or root-associated fungi including the arbuscular mycorrhizal fungi (AMF) from the phylum Glomeromycota. Nevertheless, direct comparisons of the different primers remain rare. In this study, AMF community profiles were generated by 454 pyrosequencing of amplicons resulting from direct (i.e., single) PCR amplification with three commonly used primer pairs. Root DNA extracts from four different plant species growing in the same field soil were included into the study to address the following aspects: (1) specificity of the primers for Glomeromycota, (2) structure of the detected AMF communities, and (3) efficiency of the different primers for detecting specific AMF genera. The magnitude of the effect due to PCR selectivity was then compared with the well documented effect of host plant identity on the structure of AMF communities. The primers targeting the small ribosomal subunit (SSU) yielded almost exclusively glomeromycotan sequences. The primers targeting the large ribosomal subunit (LSU) were reasonably selective for Glomeromycota (75% of the operational taxonomic units [OTUs] detected), whereas only about 25% of the OTUs obtained by the primers targeting the internal transcribed region (ITS) of the rDNA belonged to the Glomeromycota. There was good agreement in detecting AMF community dominants between the different PCR primers. In comparison to the AMF selectivity due to host plant identity, the variation due to PCR primer choice was even larger. This was partly due to a large fraction of (mainly ITS) sequences that could not be assigned to validly described AMF taxa. The community profiles generated by the SSU and LSU primers also differed significantly from each other, mainly due to considerable SSU primer selectivity. The SSU primers in fact missed 4 rare AMF genera out of at least 11 genera present in the analyzed AMF community. Our study thus highlights the magnitude of possible bias in AMF community profiling that can occur using single-PCR amplification coupled with next-generation sequencing.