Primer pairs, PCR conditions, and peptide nucleic acid clamps affect fungal diversity assessment from plant root tissues

Chloé Viotti (1) , Michel Chalot (1) , Peter Kennedy (2) , François Maillard (2) , Sylvain Santoni (3) , Damien Blaudez (4) , Coralie Bertheau (1)

1 LCE - Laboratoire Chrono-environnement (UMR 6249)
2 UMN - University of Minnesota [Twin Cities]
3 UMR AGAP - Amélioration génétique et adaptation des plantes méditerranéennes et tropicales
4 LIEC - Laboratoire Interdisciplinaire des Environnements Continentaux

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.

Mots clés: ITS2; fungi; annealing temperature; metabarcoding; high-throughput sequencing; blocking primers; Urtica dioica

Domaines: Sciences de l'environnement; Sciences du Vivant [q-bio]

Chloé Viotti, Michel Chalot, Peter Kennedy, François Maillard, Sylvain Santoni, et al.. Primer pairs, PCR conditions, and peptide nucleic acid clamps affect fungal diversity assessment from plant root tissues. Experimental Mycology, In press, pp.1-17. ⟨10.1080/21501203.2023.2301003⟩. ⟨hal-04438059⟩