Peptide nucleic acid (PNA) clamps enhance root microbiome profiling in wheat and maize.

BACKGROUND: Peptide Nucleic Acid (PNA) clamps represent a crucial molecular tool for reducing host DNA contamination during plant tissue microbiome profiling. This is particularly important when there is sequence similarity between the host organellar DNA (e.g., mitochondrial) and the targeted PCR sequences. However, the effectiveness and optimal concentration of universal PNA clamps can vary between plant species, necessitating a case-by-case evaluation. Here, we assessed the effectiveness of five concentrations (0.0, 0.25, 1.0, 2.0 and 4.0 µM) of mitochondrial and chloroplast PNA blockers (mPNA and pPNA) in reducing the amplification of organellar DNA and enhancing the profiling of prokaryotic communities across root tissues from 34 maize and 27 wheat samples cultivated under various soil and climatic conditions. RESULTS: We observed that host plant contamination in root samples was consistently high, with an average rate exceeding 95% across all samples. The application of PNA clamps significantly reduced plant host contamination by 2.4-27.2 times in a concentration-dependent manner. This reduction was more pronounced in maize samples than in wheat samples, particularly at lower doses (PNA ≤ 1.0 µM). PNA clamps also increased the read abundance of more than half of the observed microbiome phyla in the root tissues. The most substantial increase in prokaryotic read abundance was observed at a PNA concentration of 1.0 µM, without introducing significant bias to the prokaryotic community. CONCLUSIONS: In conclusion, the introduction of universal PNA clamps during PCR assays significantly reduced amplification of host contamination and enhanced the detection of low-abundance microbiome and the depth of microbial profiling in both maize and wheat root tissues, with effects being concentration- and crop-specific.