Development and application of a DNA metabarcoding method for comprehensive analysis of soil nematode communities

Nematodes mediate soil ecosystem functions together with other soil biota and are employed as effective ecological indicators. Among nematodes, plant-parasitic nematodes are economically important due to the damage they cause to many crops. While morphological identification of nematodes after elutriation from soil has been widely used to assess their diversity and for diagnostic analyses, high-throughput sequencing techniques are becoming more widely available. In this study, highly sensitive primer sets using the 18S rDNA region as metabarcoding markers were developed specifically for soil nematode fauna using next generation sequencing (NGS). In silico analysis using Primer BLAST identified a primer set (F548_A/R1912) specific to nematodes and is effective in application to DNA directly extracted from soil as well as from elutriated nematode populations. In silico analysis also indicated that three primer sets (F548_A/R915, F548_A/SSU26R and F548_A/R1912) developed in this study were expected to properly amplify the target sequences of 97-98% of 88 selected free-living and 83-92% of 60 selected plant-parasitic nematode species. All the results of NGS using three primer sets applied to DNA from a nematode suspension and the nematode specific primer set applied to soil DNA showed significant (p < 0.01, linear mixed-effects model) correlations with the results of nematode enumeration using a morphological approach. Furthermore, all the NGS assays indicated population structures were 3-9% more diverse than the microscopic approach. The current NGS protocol detects a proper level of sequence reads to represent nematode fauna for diversity and diagnostic analyses in 5 soil sampling sites in Japan, based on the rarefaction curves. As a result, this study revealed that the primer set F548_A/R1912 is highly specific to nematode species and applicable to soil DNA, and further F548_A/R915 and F548_A/SSU26R may extend the coverage to detect more nematode species than that using only F548_A/R1912 with soil DNA. The three primer sets and the metabarcoding protocol examined appear suitable for comprehensive studies of nematode community structure.