Abstract
Many studies describing communities of arbuscular mycorrhizal fungi (AMF, Glomeromycota) based on high-throughput sequencing target the V4 variable region of the 18S ribosomal gene. However, an accurate taxonomic assignment of these short 18S sequences is challenging. Here we describe a simple approach based on a phylogenetic analysis using a backbone of reference sequences with taxonomic names updated in MycoBank to improve the taxonomic assignment of amplicon sequence variants (ASVs). As a case study, paired-end sequencing (2 × 250 bp) was carried out to describe the community of AMF associated with Tamarix aphylla from Algerian steppe ecosystems. AMF from root and soil samples were targeted with a nested PCR, using the AMF-discriminating primers pair AML1/AML2 for the first amplification and a new primer pair for the second. The proportion of the sequences assigned to Glomeromycota was 85.9%, representing a total of 87 ASVs. Seven well-defined genera (Claroideoglomus, Dominikia, Funneliformis, Innospora, Microkamienskia, Rhizophagus, Septoglomus) and seven phylogenetic divergent clades of Glomus (27% of the ASVs) were identified with the proposed approach. This taxonomic assignment was in sharp contrast with querying the MaarjAM or GenBank databases. Out-of-date taxonomy led the MaarjAM database to attribute 85% of the ASVs to the genus Glomus and the GenBank database to assign 18% of the ASVs to unclassified taxa. We recommend using the simple workflow presented in this study so that up-to-date taxonomic information is accurately assigned to AMF communities analyzed by high-throughput sequencing.
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Acknowledgments
We are grateful to Marie-Josée Bergeron and James Tambong for insightful comments on an early draft of the manuscript; Susan Young for her assistance in literature review; Kitty Cheung and Julie Chapados, from the Molecular Technology Laboratory (Ottawa-RDC, AAFC), for their assistance with MiSeq library preparation and Illumina sequencing. The authors thank the two anonymous reviewers for their useful comments.
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This work was supported by funding from the Agriculture and Agri-Food Canada through the projects J-002272 and J-002295.
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Franck Stefani is the Curator of the Canadian Collection of Arbuscular Mycorrhizal Fungi.
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Stefani, F., Bencherif, K., Sabourin, S. et al. Taxonomic assignment of arbuscular mycorrhizal fungi in an 18S metagenomic dataset: a case study with saltcedar (Tamarix aphylla). Mycorrhiza 30, 243–255 (2020). https://doi.org/10.1007/s00572-020-00946-y
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DOI: https://doi.org/10.1007/s00572-020-00946-y