Unveiling soil protist diversity: rational primer selection and peptide nucleic acid probe application

During the past decades, studies on the diversity of the polyphyletic unicellular soil-inhabiting eukaryotes (protists) strongly relied on analyzing PCR-DNA sequences amplified with primers binding to phylogenetically conserved regions of the 18S rRNA gene. In soils, however, such analyses can be confounded and distorted because fungal 18S rRNA gene sequences usually predominate. To overcome this challenge, we designed in this study a peptide nucleic acid (PNA) probe for inhibiting the PCR amplification of fungal sequences from soil DNA. PNA probes bind covalently to complementary single-strand DNA and thereby inhibit their amplification during thermocyling. In silico analyses indicated that our PNA probe was complementary to 63.5% of all fungal sequences, especially binding to 81.3% of Ascomycota and 65.4% of Basidiomycota, respectively. To test its efficacy, this PNA was then applied to an agricultural soil DNA samples subjected to a conservation or conventional tillage, and to soils from a paired field site representing a cropland and a forest land-use We utilized two 18S rRNA universal primer sets (TAReuk454FWD1/TAReukREV3 and EK-565F/EUK-1134R) for comparison. Our findings indicated that the PNA application did not significantly alter soil eukaryotic communities, and community profiles were primarily shaped by the choice of primers. Notably, the Conosa and Lobosa groups (Amoebozoa) exhibited the greatest bias, and the relative abundance of Cercozoa in clay soil was markedly lower compared to loam soil when using these universal primer sets. We assert that a judicious primer selection holds the potential to capture previously underrepresented soil protist groups effectively when using 18S rRNA universal primer.