P-270 - Accessing the Duckweed microbiome: Comparison of DNA extraction and 16S rRNA gene amplification methods to sequence the Duckweed-associated microbiome

The duckweed microbiome plays a key role in bioremediation, nitrogen fixation, and biomolecule production. Despite its ecological importance, knowledge of its composition and dynamics, particularly endophytic bacteria, remains limited. These bacteria are difficult to study due to their low abundance relative to plant cells and organelles. Standard methods, such as 16S rRNA gene amplification, often fail to accurately capture their diversity and abundance.This study aimed to refine methodologies for quantifying duckweed endophytes, improving accuracy in diversity and abundance estimates. Using Spirodela polyrhiza as a model, we compared three DNA extraction techniques: two commercial kits and phenol-chloroform extraction. To enhance precision, we assessed four 16S rRNA primer sets (V3-V4, V4, V5-V6) with and without PNA/LNA blocking agents to reduce chloroplast and mitochondrial interference.Our findings identified the most effective approach: a commercial DNA extraction kit paired with a V3-V4 primer set designed to exclude chloroplast 16S rRNA. This method provided the most comprehensive, reproducible bacterial profile, eliminating the need for blocking agents, thus simplifying and reducing costs.This work presents a cost-effective, reliable approach that could be used to unravel the endophytic microbiome of duckweed as well as other aquatic plant species, paving the way for future applications in environmental sustainability and biotechnology.