Single nucleotide polymorphisms based phylum specific PCR amplification technique (SPAT): its application to analyze polar microbial eukaryotic community

Abstract

Over the past several decades, many genes such as SSU rDNA were successfully used to resolve ambiguity of traditional morphology-based taxonomy. The next challenge in population genetics and molecular evolution is to investigate the diversity of eukaryotes at the community level using high-throughput sequencing technique. However, even though high-throughput sequencing technique has a powerful potential to study community diversity, it has not been successful to apply the technique to study eukaryotic community due to biased PCR enrichment of dominant species in the community. In this study, we found several taxon specific SNP sites from monophyletic phyla such as ciliates and diatoms which are most dominant in marine ecosystem. By using these taxon specific SNP sites, we developed SNP-based phylum specific PCR Amplification Technique (SPAT). We observed that our method has a higher specificity compared to traditional universal primer based PCR amplification method. We successfully characterize the diversity of diatoms and ciliates without isolation process of them from polar eukaryotic community. Combining phylum specific PCR amplification method with pyrosequencing technique, it will be feasible to monitor the change of the community structure of these important marine protists at the seasonal and annual level.