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The effects of primer pairs, PCR conditions, and peptide nucleic acid clamps on plant root fungal diversity assessment

The effects of primer pairs, PCR conditions, and peptide nucleic acid clamps on plant root fungal diversity assessment
Abundance, richness, and diversity of fungi in Urtica dioica roots for the three primer pairs tested fITS7/ITS4, gITS7/ITS4, and 5.8S-Fun/ITS4-fun (Ta = 57 °C), the addition of PNA clamps (+PNA) and the increase of Ta (Ta = 68 °C or 63 °C). (a–c) Relative abundance of reads of Viridiplantae, fungi, and other phyla (i.e. Amoebozoa, Choanoflagellozoa, Heterolobosa, Ichthyosporia, Metazoa, Protista, Rhizaria, rhodoplantae, Stramenopila, and NA); (d–f) Richness and (g–i) Shannon's index. Boxes with the same letters did not differ significantly from each other using a Tukey-adjusted comparison and Kruskal-Wallis analysis followed by a post-hoc test using Fisher's least significant difference, respectively, P < 0.05. Credit: Dr. Coralie Bertheau, CNRS, Chrono-environnement, Université de Franche-Comté, Montbéliard, France

Fungi are frequently found both around and within plant tissues (especially in roots) and are involved in both plant nutrient acquisition and resistance to pathogens. Thus, characterizing the diversity and composition of plant-associated fungal communities has been a growing interest in recent years.

High-throughput sequencing (HTS), also called metabarcoding, has become a prominent tool to assess complex microbial communities from environmental samples. However, HTS applied to plant-associated is challenging due to plant and fungal DNA co-amplification. Fungal-specific primers, Peptide Nucleic Acid (PNA) clamps, or adjusting PCR conditions are described as efficient approaches to limit plant DNA contamination.

This study, led by Dr. Coralie Bertheau (Université de Franche-Comté), evaluated the combined effects of primer pairs, associated annealing temperature (Ta), and PNA clamps in determining the fungal community diversity and composition associated with .

Three primers (fITS7/ITS4, gITS7/ITS4, and 5.8S-Fun/ITS4-Fun) targeting the ribosomal internal transcribed spacer (ITS) 2 region were evaluated alone or in combination with PNA clamps both on nettle (Urtica dioica) root DNA and a mock fungal community.

The team found that 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.

"The results demonstrated 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," Dr. Coralie Bertheau said.

The work is published in the journal Mycology.

More information: Chloé Viotti et al, Primer pairs, PCR conditions, and peptide nucleic acid clamps affect fungal diversity assessment from plant root tissues, Mycology (2024). DOI: 10.1080/21501203.2023.2301003

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Citation: The effects of primer pairs, PCR conditions, and peptide nucleic acid clamps on plant root fungal diversity assessment (2024, February 26) retrieved 16 April 2024 from
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