Abstract
Arbuscular mycorrhizal fungi (AMF; Glomeromycota) are difficult to culture; therefore, establishing a robust amplicon-based approach to taxa identification is imperative to describe AMF diversity. Further, due to low and biased sampling of AMF taxa, molecular databases do not represent the breadth of AMF diversity, making database matching approaches suboptimal. Therefore, a full description of AMF diversity requires a tool to determine sequence-based placement in the Glomeromycota clade. Nonetheless, commonly used gene regions, including the SSU and ITS, do not enable reliable phylogenetic placement. Here, we present an improved database and pipeline for the phylogenetic determination of AMF using amplicons from the large subunit (LSU) rRNA gene. We improve our database and backbone tree by including additional outgroup sequences. We also improve an existing bioinformatics pipeline by aligning forward and reverse reads separately, using a universal alignment for all tree building, and implementing a BLAST screening prior to tree building to remove non-homologous sequences. Finally, we present a script to extract AMF belonging to 11 major families as well as an amplicon sequencing variant (ASV) version of our pipeline. We test the utility of the pipeline by testing the placement of known AMF, known non-AMF, and Acaulospora sp. spore sequences. This work represents the most comprehensive database and pipeline for phylogenetic placement of AMF LSU amplicon sequences within the Glomeromycota clade.
Highlights
It has been suggested that the internal transcribed spacer (ITS) region of the rRNA gene should be adopted as the universal fungal marker (Schoch et al 2012; Lindahl et al 2013), for the Arbuscular mycorrhizal fungi (AMF) in Glomeromycota this region is not optimal for two major reasons
We have increased the utility of this pipeline by providing amplicon sequencing variant (ASV) as well as operational taxonomic units (OTUs) adapted versions and by including code to extract clades at the family level
There are two major advantages to working with ASVs: increased resolution of data and possibility to compare across studies (Callahan et al 2017)
Summary
Arbuscular mycorrhizal fungi (AMF) are ubiquitous plant symbionts, associating with approximately 70% of plant species worldwide (Brundrett and Tedersoo 2018), yet difficulties associated with culturing and identifying these. A useful alternative to the ITS, SSU, or long reads is the large subunit (LSU) This region consistently shows utility for taxonomic resolution for AMF (Krüger et al 2012; Hart et al 2015; House et al 2016; Delavaux et al 2021), making it useful to access species diversity and community composition based on environmental AMF sequencing. Use of the LSU region for environmental sequencing of AMF is not common (Gollotte et al 2004; Lekberg et al 2013; House and Bever 2018; Vieira et al 2018; Schütte et al 2019), potentially due to bioinformatic challenges in implementation To address this gap, we previously outlined the use of a newly created database and pipeline for the LSU region for placement in the Glomeromycota phylum (Delavaux et al 2021). This work improves the utility of the database and pipeline, thereby making the LSU an attractive alternative for environmental sequencing of AMF
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