Comparison of 18S rDNA primers for estimating fungal diversity in agricultural soils using polymerase chain reaction-denaturing gradient gel electrophoresis

Abstract

Direct DNA extraction followed by 18S rDNA polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) is widely used for the analysis of fungal diversity in agricultural soil ecosystems. Various PCR primer sets have been used for fungi, but few reports have compared the properties of the primers. We investigated the properties of four widely used primer sets for fungal 18S rDNA DGGE: (1) NS1/GCFung, (2) FF390/FR1(N)-GC, (3) NS1/FR1(N)-GC (for single PCR) and (4) NS1/EF3 for the first PCR and NS1/FR1(N)-GC for the second PCR (for nested PCR). Using six soil samples from upland and paddy fields in Japan, the primers were compared in terms of PCR amplification efficacy, detection and reproducibility of the DGGE banding profiles, the obtained diversity indices, and the discrimination ability of the fungal communities using DGGE. The efficacy of PCR amplification using primer set 1 and the first PCR of primer set 4 was better than that using primer sets 2 and 3. In DGGE analysis, the PCR products of primer sets 3 and 4 showed the highest diversity indices. However, these primer sets had drawbacks, namely, the presence of non-specific aggregates and poor reproducibility of the DGGE profiles. Although primer sets 1 and 2 yielded shorter sequences of similar length, the PCR products with primer set 1 showed higher diversity indices than those with primer set 2. Multidimensional scaling analysis of the DGGE profiles indicated that primer set 1 could most clearly discriminate each fungal community in the soil samples. Although each primer set had advantages and disadvantages, together our analyses indicated that primer set 1 is the most suitable for detecting fungal diversities in soil using DGGE analysis. Our results are useful for selecting primers according to the aim of a particular study.