Design and validation of a Peptide Nucleic Acid clamp of barley and wheat ITS2 for fungal microbiome surveys

Authors: Whitaker, Briana

Submitted to: PhytoFrontiers
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/7/2025
Publication Date: 9/10/2025
Citation: Whitaker, B.K. 2025. Design and validation of a Peptide Nucleic Acid clamp of barley and wheat ITS2 for fungal microbiome surveys. PhytoFrontiers. https://doi.org/10.1094/PHYTOFR-07-25-0066-SC.
DOI: https://doi.org/10.1094/PHYTOFR-07-25-0066-SC

Interpretive Summary: Advances in next generation sequencing technologies over the last 20 years have greatly improved our ability to study crop microbiomes. This research has shown that microbial communities greatly contribute to overall plant health and productivity. However, crop microbiome surveys often lose many reads due to unwanted amplification of host DNA, especially when studying the fungi living on crops. To fix this problem, scientists can use peptide nucleic acid (PNA) clamps during PCR amplification. These clamps bind to host DNA and block its amplification. Therefore, ARS researchers in Peoria, Illinois, designed and tested a new PNA clamp that targets the ITS2 gene region in barley and wheat. The PNA reduced unwanted plant DNA amplification by up to 25% and did not affect scientists’ ability to measure fungal species variation. The design process for the PNA is simple to follow and uses free software, so it can be adapted for other host systems or gene regions. Ultimately, the development of the barley-wheat PNA for fungal microbiome surveys will allow American researchers and researchers worldwide to improve the cost efficiency of fungal surveys in barley and wheat, which are two of the world’s most important small grain crops.

Technical Abstract: Next generation sequencing surveys of plant microbiomes often experience considerable loss of reads due to non-target amplification of host DNA. To alleviate this, peptide nucleic acid (PNA) clamps can be included during PCR amplification to specifically bind and block amplification of host DNA. However, fewer PNA clamps of the plant ITS genomic regions exist for fungal microbiome surveys or are available for multi-host comparisons. Here, we designed and validated a novel PNA targeting the ITS2 gene region in barley (Hordeum vulgare) and wheat (Triticum aestivum). The PNA reduced non-target plant amplification by up to 25% in high fungal load samples and did not affect common metrics of microbial diversity (Shannon diversity and community structure). The PNA design pipeline itself is straightforward to implement and requires only freely available software (R, BLASTn, MAFFT); thus, it could be applied to other host systems or gene regions. Development of this PNA will substantially improve the cost efficiency of fungal surveys in two globally important small grain crops, barley and wheat.