To Clamp or Not to Clamp: Enhancing Seed Endophyte Metabarcoding Success

Seed microbes play crucial roles in plant health, but studying their diversity is challenging due to host DNA contamination. This study aimed to optimise methodologies for investigating seed microbiomes across diverse plant species, focusing on the efficacy of peptide nucleic acid (PNA) clamps to reduce host DNA amplification. We tested PNA clamps on three plant species: Melaleuca quinquenervia (tree), Microlaena stipoides, and Themeda triandra (grasses). The effectiveness of PNA clamps was assessed through in silico analysis, axenic tissue culture, and metabarcoding techniques. In silico analysis confirmed the specificity of PNA clamps to the 16S rRNA gene V4 region of chloroplasts in the grass species. Axenic tissue culture experiments showed that applying PNA clamps at both 1 µM and 0.25 µM concentrations significantly reduced plant DNA amplification. Metabarcoding analyses further confirmed that PNA clamps effectively suppressed host DNA, enhancing microbial diversity estimates across all three species while preserving core microbial taxa. The efficacy of the clamps varied among host species, with T. triandra exhibiting the highest blocking efficacy, and chloroplast clamps outperforming mitochondrial ones. This study demonstrates that PNA clamps are a useful for improving seed endophyte metabarcoding datasets, although they require optimisation for some plant species. This knowledge will contribute to enhancing our understanding of seed microbiome diversity and its ecological implications.