Abstract
BackgroundSeveral fungi-specific primers target the 18S rRNA gene sequence, one of the prominent markers for fungal classification. The design of most primers goes back to the last decades. Since then, the number of sequences in public databases increased leading to the discovery of new fungal groups and changes in fungal taxonomy. However, no reevaluation of primers was carried out and relevant information on most primers is missing. With this study, we aimed to develop an 18S rRNA gene sequence primer toolkit allowing an easy selection of the best primer pair appropriate for different sequencing platforms, research aims (biodiversity assessment versus isolate classification) and target groups.ResultsWe performed an intensive literature research, reshuffled existing primers into new pairs, designed new Illumina-primers, and annealing blocking oligonucleotides. A final number of 439 primer pairs were subjected to in silico PCRs. Best primer pairs were selected and experimentally tested. The most promising primer pair with a small amplicon size, nu-SSU-1333-5′/nu-SSU-1647-3′ (FF390/FR-1), was successful in describing fungal communities by Illumina sequencing. Results were confirmed by a simultaneous metagenomics and eukaryote-specific primer approach. Co-amplification occurred in all sample types but was effectively reduced by blocking oligonucleotides.ConclusionsThe compiled data revealed the presence of an enormous diversity of fungal 18S rRNA gene primer pairs in terms of fungal coverage, phylum spectrum and co-amplification. Therefore, the primer pair has to be carefully selected to fulfill the requirements of the individual research projects. The presented primer toolkit offers comprehensive lists of 164 primers, 439 primer combinations, 4 blocking oligonucleotides, and top primer pairs holding all relevant information including primer’s characteristics and performance to facilitate primer pair selection.
Highlights
Several fungi-specific primers target the 18S Ribosomal ribonucleic acid (rRNA) gene sequence, one of the prominent markers for fungal classification
The non-fungal eukaryotic co-amplification of the three primer pairs was not acceptable and co-amplification targeted many more eukaryotic groups compared to the top primer pairs (Additional files 3 and 6). These results indicate the limitations of primer design for a relative conserved marker like the 18S rRNA gene aiming to target the great majority of sequences of a taxon-rich group as Fungi
Primer pairs are chosen based on comparable research studies, they may not be the best choice in terms of efficiency and target specificity
Summary
Several fungi-specific primers target the 18S rRNA gene sequence, one of the prominent markers for fungal classification. Fungi belong to a highly diverse kingdom providing key ecosystem functions Their biosynthesis of natural products relevant for biotechnological application renders them of great interest to the research community. They are a highly understudied group with an estimated species number of up to 3.8 million but Molecular taxon identification is mainly based on marker gene sequencing whose sensitivity, resolution and throughput are controlled by the choice of the marker gene and sequencing platform. Sequences originating from unknown fungal taxa can often be assigned to a lower taxonomic level Such a double-marker gene approach has been shown to be effective in surveys targeting communities mainly composed by undescribed fungal taxa [6, 7]. In the case that the aim of a research project is the analysis of the structure and dynamic of fungal communities rather than the monitoring of known fungal taxon groups, phylogenetic marker sequencing is a promising approach
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