Primer pairs, PCR conditions, and peptide nucleic acid clamps affect fungal diversity assessment from plant root tissues
2024
Viotti, Chloé | Chalot, Michel | Kennedy, Peter, G. | Maillard, François | Santoni, Sylvain | Blaudez, Damien | Bertheau, Coralie | Laboratoire Chrono-environnement (UMR 6249) (LCE) ; Centre National de la Recherche Scientifique (CNRS)-Université de Franche-Comté (UFC) ; Université Bourgogne Franche-Comté [COMUE] (UBFC)-Université Bourgogne Franche-Comté [COMUE] (UBFC) | University of Minnesota [Twin Cities] (UMN) ; University of Minnesota System (UMN) | Amélioration génétique et adaptation des plantes méditerranéennes et tropicales (UMR AGAP) ; Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Institut Agro Montpellier ; Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Université de Montpellier (UM) | Laboratoire Interdisciplinaire des Environnements Continentaux (LIEC) ; Institut national des sciences de l'Univers (INSU - CNRS)-Université de Lorraine (UL)-Centre National de la Recherche Scientifique (CNRS) | This work was supported by the Ph-D grant from the Ministère de l’Enseignement supérieur, de la Recherche et de l’Innovation (MESRI); European Union’s Horizon 2020 research and innovation program under Grant Agreement ERA-NET Cofund SusCrop (N°771134); French PIA project “Lorraine Université d’Excellence” Deepsurf, reference ANR-15-IDEX-04-LUE. | ANR-15-IDEX-0004,LUE,Isite LUE(2015)
Raw sequence data for this project have been submitted to NCBI’s SRA archive under accession no. SUB12918264.
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Show more [+] Less [-]English. High-throughput sequencing has become a prominent tool to assess plant-associated microbial diversity. Still, some technical challenges remain in characterising these communities, notably due to plant and fungal DNA co-amplification. Fungal-specific primers, Peptide Nucleic Acid (PNA) clamps, or adjusting PCR conditions are approaches to limit plant DNA contamination. However, a systematic comparison of these factors and their interactions, which could limit plant DNA contamination in the study of plant mycobiota, is still lacking. Here, three primers targeting the ITS2 region were evaluated alone or in combination with PNA clamps both on nettle (Urtica dioica) root DNA and a mock community. PNA clamps did not improve the richness or diversity of the fungal communities but increased the number of fungal reads. Among the tested factors, the most significant was the primer pair. Specifically, the 5.8S-Fun/ITS4-Fun pair exhibited a higher OTU richness but fewer fungal reads. Our study demonstrates that the choice of primers is critical for limiting plant and fungal DNA co-amplification. PNA clamps increase the number of fungal reads when ITS2 is targeted but do not result in higher fungal diversity recovery at high sequencing depth. At lower read depths, PNA clamps might enhance microbial diversity quantification for primer pairs lacking fungal specificity.
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