Optimizing primer selection for microbial diversity studies in freshwater lakes: a comprehensive evaluation of 16S rRNA, 18S rRNA, and ITS gene regions

Accurate assessment of microbial community structure via high-throughput sequencing is fundamentally dependent on primer selection; however, systematic primer optimization for freshwater lake ecosystems remains insufficiently explored. Using the representative Lake Qiandao as a model, this study provides an integrative comparison of multiple primer sets targeting the 16S rRNA gene (V1-V2, V3-V4, V4, and V5-V7), the 18S rRNA gene (V4-1 and V4-6), and the Internal Transcribed Spacer (ITS1 and ITS2) regions. Our results elucidate that the V3-V4 region yielded significantly higher bacterial diversity and a more biologically representative community structure. For eukaryotic microorganisms, the V4-1 primers exhibited superior performance, capturing a broader taxonomic spectrum of algae (e.g. Ochrophyta and Cryptophyta) and protozoan groups. Regarding fungi, the ITS2 region provided enhanced stability and more comprehensive taxonomic coverage compared to ITS1. This study establishes a standardized molecular framework for microbial diversity research in freshwater limnology and underscores the necessity of primer optimization to ensure the high-fidelity resolution of microbial community structures and their associated ecological function.