Evaluation of soil fungal communities using the ITS2 sublocus and 18S gene primers under different amplification methods

Primer selection and PCR methods can potentially lead to biased descriptions of microbial communities. Here, we investigated soil total fungi and arbuscular mycorrhizal fungal (AMF) community composition and diversity from diverse sites using several primers and amplification methods. The results showed that regardless of site, the ITS3tagmix1-5/ITS4ngs primer set generated a higher proportion of high-quality reads, targeted more fungal ASVs and revealed a higher total fungal alpha diversity compared to ITS3tagmix4/ITS4ngs. Among specific primers targeting the 18S rRNA gene, AMV4.5NF/AMDGR had differential specificity for Glomeraceae, whereas SSU515Fngs/Euk742R had differential specificity for Paraglomeraceae. Regardless of site, PCR approaches (nested vs non-nested) had higher influence on the AMF community structure than primer selection, though primer selection significantly influenced arbuscular mycorrhizal fungi richness. Overall, the findings suggest that the specificity of amplification in relation to primer selection and PCR stringency should guide the best interpretation of fungal community diversity data from high-throughput sequencing of environmental samples.