A comprehensive fungi-specific 18S rRNA gene sequence primer toolkit suited for diverse research issues and sequencing platforms

Abstract

Background: Several 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.

Results: We 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.

Conclusions: The 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.

Keywords: 18S rRNA gene sequence (SSU) primer; Annealing blocking oligonucleotides; Co-amplification; Community survey; FF390; FR-1; Fungal biodiversity; Fungi; Real-time Q-PCR; Taxonomic classification.

Fig. 1

Primer pairs covering the different variable regions of the fungal 18S rRNA gene sequence. The fungal 18S rRNA gene sequence possess eight different variable regions, V1-V9 (V6 does not exist), colored differently. The barchart indicates the number of tested primer pairs covering a variable region with their amplicon. Amplicons produced by the seven top primer pairs as arrow lines. Primer names beside the arrow lines

Fig. 2

Taxonomic composition of three environmental samples split into fungal and co-amplified sequences. Barchart indicates relative sequence abundance of the different a eukaryotic groups, and b fungal classes amplified by the primer pair nu-SSU-1333-5′/nu-SSU-1647-3′ (FF390/FR-1) after rarefying sequences. Two libraries were prepared on the same template DNA: (i) solely with the primer pair, and (ii) primer pair and four different blocking oligonucleotides designed for Stramenopiles, Alveolata, Rhizaria and Telonema (BO). Others in a: Centrohelida, Choanoflagellida, Corallochytrea, Discicristoidea, Excavata, Freshwater Opisthokonta, Filasterea, Picozoa, Telonema. Others in b: Pezizomycotina incertae sedis, Lecanoromycetes, Lichinomycetes, Pezizomycetes, Agaricomycetes, Cystobasidiomycetes, Agaricostilbomycetes, Microbotryomycetes, Pucciniomycetes, Wallemiomycetes, Pucciniomycotina incertae sedis, Ustilaginomycetes, LKM15

Fig. 3

Comparison of three detection methods. Subsampled fungal communities were described by a eukaryote-specific primer (TAReuk454FWD1/TAReukREV3_modified), a fungi-specific primer (nu-SSU-1333-5′/nu-SSU-1647-3′ (FF390/FR-1)), and a metagenomics approach. Taxonomic composition as relative sequence abundance of sample a OSD36 and b OSD28. Colored shadows between bars show which proportion (approximated) the fungal sequences detected by the eukaryote-specific and metagenomics approach represent in the non-subsampled community of the fungi-specific approach. Others in a: Dothideomycetes, Sordariomycetes. Others in b: Eukaryote-specific approach: Agaricomycetes. Fungal approach: Arthoniomycetes, Lecanoromycetes, Pezizomycetes, Agaricomycetes