Barcoded NS31/AML2 primers for sequencing of arbuscular mycorrhizal communities in environmental samples

Abstract

Premise of the study:Arbuscular mycorrhizal fungi (AMF) are globally important root symbioses that enhance plant growth and nutrition and influence ecosystem structure and function. To better characterize levels of AMF diversity relevant to ecosystem function, deeper sequencing depth in environmental samples is needed. In this study, Illumina barcoded primers and a bioinformatics pipeline were developed and applied to study AMF diversity and community structure in environmental samples.Methods:Libraries of small subunit ribosomal RNA fragment amplicons were amplified from environmental DNA using a single-step PCR reaction with barcoded NS31/AML2 primers. Amplicons were sequenced on an Illumina MiSeq sequencer using version 2, 2 × 250-bp paired-end chemistry, and analyzed using QIIME and RDP Classifier.Results:Sequencing captured 196 to 6416 operational taxonomic units (OTUs; depending on clustering parameters) representing nine AMF genera. Regardless of clustering parameters, ∼20 OTUs dominated AMF communities (78–87% reads) with the remaining reads distributed among other OTUs. Analyses also showed significant biogeographic differences in AMF communities and that community composition could be linked to specific edaphic factors.Discussion:Barcoded NS31/AML2 primers and Illumina MiSeq sequencing provide a powerful approach to address AMF diversity and variations in fungal assemblages across host plants, ecosystems, and responses to environmental drivers including global change.

Arbuscular mycorrhizal fungi as mediators of ecosystem responses to nitrogen deposition: A trait‐based predictive framework

“…() recorded positive relationships between mycelium extent and P uptake among Glomus species. As high‐throughput sequencing more common, so should information regarding the responses of AMF species/genotypes to N in the environment (Morgan & Egerton‐Warburton, ). In addition, we synthesized trait data from disparate studies, and study‐specific differences in experimental design and growth conditions could have influenced the patterns we observed.…”

User‐friendly bioinformatics pipeline gDAT (graphical downstream analysis tool) for analysing rDNA sequences

“…gDAT provides several trimming approaches: trim by length, trim by lowest quality score and trim by average quality window. Trimming can be useful when the amplicon is short and read length can be longer (e.g., in the ITS region), but is not suitable where it removes the overlap of paired‐end reads (e.g., for SSU on Illumina MiSeq supporting 2 × 300 bp), thus removing the possibility to combine the read pairs and resulting in orphan reads (Morgan, & Egerton‐Warburton, 2017).…”

“…The pipeline also incorporates an option to use the newer vsearch algorithm to perform combination of reads (Rognes et al 2016). Depending on the amplicon, paired‐end reads may exhibit insufficient overlap, meaning that a large fraction of sequences are rejected (Morgan, & Egerton‐Warburton, 2017). Among paired‐end reads, forward reads tend to have higher quality (Schirmer et al, 2015; Vasar et al, 2017) and for the AM fungal SSU rDNA amplicon forward Illumina reads (using the NS31 or WANDA primer, Dumbrell et al, 2011; Lee et al, 2008) closely match the variable region, making it possible to discard reverse reads and use only forward reads for analysis.…”

Trait‐based aerial dispersal of arbuscular mycorrhizal fungi

“…To extract genomic DNA, membrane filters containing dust samples were cut into 4 mm strips using sterile scissors and placed into sample collection tubes for DNA extraction using the DNeasy PowerSoil Kit (Qiagen) according to standard protocol. Sequencing libraries were then prepared by the Environmental Sample Preparation and Sequencing Facility at Argonne National Laboratory (https://www.anl.gov/bio/environmental-sample-preparation-and-sequencing-facility), using the protocol as laid out by Morgan & Egerton‐Warburton (2017). In brief, a segment of Glomeromycotinan small subunit ribosomal RNA (SSU rRNA) was amplified using the NS31 and AML2 primers (Simon et al ., 1992; Lee et al ., 2008) in a single‐step PCR reaction.…”