ITS3/ITS4 outperforms other ITS region and 18S rRNA gene primer sets for amplicon sequencing of soil fungi

Abstract The primer sets of 18S rRNA genes and Internal Transcribed Spacer (ITS) regions are universal primers for amplicon sequencing in soil fungal diversity studies. The selection of proper primer sets has been identified as one of the main drivers affecting effective cross‐study comparisons in various types of soils. However, few studies have provided guidance for the selection of fungal primers in soil fungal biodiversity studies. The aim of this study was to evaluate and optimise fungal sequencing methods with three primer sets in desert, forest, grassland and farmland soils. Melting curve analyses were used to evaluate the specificity of each primer set in different soil ecosystems. The quality of fungal sequencing with three primer sets was assessed with the same conditions on the Illumina platform. The melting curve analysis showed that both the FR1/FF390 and the ITS3/ITS4 primer sets have better specificity than the ITS1/ITS primer set. 18S rRNA amplicon sequencing produced less fungal phylotypes compared to the amplicon sequencing data of the ITS1/ITS2 and ITS3/ITS4 primer sets. Our study suggests that the ITS3/ITS4 primer set was more suitable for the analysis of soil fungal biodiversity in desert, forest, grassland and farmland soils on the Illumina sequencing platform. Highlights Comparison of three existing primer sets for soil fungal amplicon sequencing. ITS region has better performance than 18S rRNA gene in amplicon sequencing. ITS3/ITS4 primer set is more suitable for the analysis of soil fungal biodiversity.