Dec 11, 2025

Public workspacePreparation of long rDNA amplicons of arbuscular mycorrhizal fungi (Glomeromycota) from soil and root DNA for high-fidelity long-read sequencing

DOI
https://dx.doi.org/10.17504/protocols.io.e6nvw4xjzlmk/v1
  • Nobuhiko Shigyo1
  • 1Department of Forest Soils, Forestry and Forest Products Research Institute
Nobuhiko Shigyo
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DOI: https://dx.doi.org/10.17504/protocols.io.e6nvw4xjzlmk/v1
Protocol Citation: Nobuhiko Shigyo 2025. Preparation of long rDNA amplicons of arbuscular mycorrhizal fungi (Glomeromycota) from soil and root DNA for high-fidelity long-read sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw4xjzlmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 09, 2025
Last Modified: December 11, 2025
Protocol Integer ID: 234562
Keywords: Forest soil, Plant root, Long-read sequencing, PacBio Revio, Arbuscular mycorrhizal fungi, arbuscular mycorrhizal fungi, arbuscular mycorrhizal, rrna gene, preparation of long rdna amplicon, phylum glomeromycota, long rdna amplicon, fungi, amf communities in both soil, glomeromycota, environmental dna, new phytologist, read sequencing, dna extracts in test run, gene, robust amplification from diverse soil, diverse soil, using dna, amf lineage, lsu for amf lineage, dna extract, specific second pcr, dna, nested pcr strategy, lsumar24 lsu primer design, lsumar24 lsu primer design of tedersoo et al, soil
Funders Acknowledgements:
JSPS Grants-in-Aid for Early-Career Scientists
Grant ID: 23K17070
Abstract
This protocol describes the preparation of long rDNA amplicons (~2.7 kb) of arbuscular mycorrhizal (AM) fungi (phylum Glomeromycota) from environmental DNA for high-fidelity long-read sequencing. The workflow targets AMF communities in both soil and fine roots, using DNA extracted with common commercial kits. The method employs a nested PCR strategy adapted from Kolaříková et al. (2021, New Phytologist), which amplifies a contiguous fragment spanning most of the SSU rRNA gene, the complete ITS region, and the D1–D2 domains of the LSU rRNA gene. In the first round, broad AMF primer combinations NS31/LSUmAr13+24 and AML1/LSUmAr13+24 are used, following the LSUmAr13 + LSUmAr24 LSU primer design of Tedersoo et al. (2018, New Phytologist) rather than the original LSUmAr1–4 mix. This primer scheme provided robust amplification from diverse soil and root DNA extracts in test runs. The pooled first-round products are then subjected to a Glomeromycota-specific second PCR with NS31_Glo3/wLSUmBr, yielding ~2.7 kb amplicons that cover SSU–ITS–LSU for AMF lineages. The resulting libraries are suitable for downstream long-read sequencing on PacBio Revio/Sequel IIe or Oxford Nanopore platforms. Because the primer scheme is AMF-focused but not site-specific, the protocol can be applied to a wide range of soil- or root-associated AMF communities and integrated with existing SSU, ITS, and LSU datasets for phylogenetic and ecological analyses.
Guidelines
This protocol describes a nested PCR workflow for amplifying near-full-length SSU–ITS–LSU rDNA of arbuscular mycorrhizal (AM) fungi (Glomeromycota) from soil and root DNA. It is optimized for environmental samples with low DNA yield or humic contamination and can be applied prior to long-read sequencing (e.g., PacBio or Nanopore). All steps should be carried out using sterile, DNA-free consumables to minimize contamination.
Materials
Materials and Reagents

  • 0.2 mL PCR tubes
  • Nuclease-free water
  • DNA extraction kit for soil: NucleoSpin Soil DNA Kit (Macherey-Nagel, Germany) or equivalent
  • PCR enzyme: KOD One PCR Master Mix (TOYOBO, Japan)
  • Magnetic bead purification: AMPure XP (Beckman Coulter, USA)
  • Primers (PAGE/HPLC purified):
1. NS31 (5′-TTGGAGGGCAAGTCTGGTGCC-3′; Simon et al. 1992)
2. AML1 (5′-ATCAACTTTCGATGGTAGGATAGA-3′; Lee et al. 2008)
3. LSUmAr13 (5′-GCTCDYACTCAAATCTATCAAA-3′; Krüger et al. 2009)
4. LSUmAr24 (5′-GCTCTDACTCAAAYCTATCGAT-3′; Tedersoo et al. 2017)
5. NS31_Glo3 (5′-AminoC6-TTGYTGCRGTTAAAAAGCTCG-3′; Kolaříková et al. 2021)
6. wLSUmBr (5′-AminoC6-AACACTCGCAYAYATGYTAGA-3′; Schlaeppi et al. 2016)
  • 70% ethanol (molecular grade)
  • Low-TE buffer for elution
  • Qubit dsDNA HS assay kit (Thermo Fisher Scientific, Waltham, MA, United States)
  • Qubit Assay Tubes (Thermo Fisher Scientific)
  • DNA ladder (1 kb–3 kb range)

Equipment

  • Thermal cycler (e.g., TaKaRa PCR Thermal Cycler Dice Touch, Takara Bio Inc.)
  • Micropipette (P1000, P200, P100, P20, P10, and P2)
  • Vortex mixer
  • Magnetic stand (e.g., NGS MagnaStand (YS-Model) 8Ch × 0.2 mL PCR tube, Nippon Genetics Co., Ltd., Tokyo, Japan)
  • Microcentrifuge
  • Qubit fluorometer (Qubit 4 fluorometer, Thermo Fisher Scientific)
  • Gel electrophoresis system
  • PacBio Revio sequencer (outsourced service)
Troubleshooting
Safety warnings
  • Avoid cross-contamination between PCR rounds; use separate work areas for template preparation and post-PCR handling.
  • Over-cycling (>35 cycles) may produce chimeric amplicons.
  • AMPure beads are sensitive to overdrying—dry only until the surface just turns matte.
  • The long rDNA amplicons (~2.7 kb) are prone to shearing; minimize pipetting and vortexing.
  • For root DNA, ensure that only fine roots (not woody tissue) are used; otherwise PCR inhibition may occur.
Ethics statement
No animal or human subjects were involved in this study. Soil and root samples were collected from managed research sites with permission from the responsible authorities.
Before start
  • Prepare all reagents, primers, and consumables in a clean workspace dedicated to pre-PCR operations.
  • Prepare 70% ethanol and magnetic beads (e.g., AMPure XP) for purification steps in advance.
  • Ensure that the thermocycler and magnetic stand are clean and functional before starting.
DNA extraction
4h
For soil samples:
Extract total genomic DNA from approximately Amount500 mg of soil using the NucleoSpin Soil DNA Kit (Macherey-Nagel, Germany) according to the manufacturer’s instructions. Perform all steps at room temperature unless otherwise noted, and elute DNA with Amount50 µL of SE buffer.

2h
For root samples:
Extract total gDNA from approximately Amount20 mg of fine roots using the ISOPLANT II Kit (Nippon Gene Corporation, Tokyo, Japan) following the manufacturer’s protocol. Elute DNA with Amount50 µL of the supplied buffer.

2h
Measure the extracted gDNA concentration using a Qubit fluorometer with the Qubit dsDNA HS assay kit (Thermo Fisher Scientific, Waltham, MA, USA).
1st PCR: NS31 / LSUmAr13 + LSUmAr24
45m
Prepare PCR-master mix in a 0.2 mL PCR tube with the following:
ComponentVolume (total 25 µl)
Nuclease-free waterX µl
Forward primer (NS31, 10 µM)1.25 µl
Reverse primer (LSUmAr13, 10 µM) 0.675 µl
Reverse primer (LSUmAr24, 10 µM)0.675 µl
2× KOD One mix12.5 µl
Template DNAY µl (20 ng)


5m
Run the following PCR program:
[Temperature98 °C Duration00:00:10 → Temperature66 °C Duration00:00:05 → Temperature68 °C Duration00:00:20 ] × 30 → Temperature8 °C ∞

40m
1st PCR: AML1 / LSUmAr13 + LSUmAr24
45m
Prepare PCR-master mix in a 0.2 mL PCR tube with the following:

ComponentVolume (total 25 µl)
Nuclease-free waterX µl
Forward primer (AML1, 10 µl)1.25 µl
Reverse primer (LSUmAR13 10µl)0.675 µl
Reverse primer (LSUmAr24, 10µl)0.675 µl
2 × KOD One mix12.5 µl
Template DNAY µl (20 ng)

5m
Run the following PCR program:
[Temperature98 °C Duration00:00:10 → Temperature53 °C Duration00:00:05 → Temperature68 °C Duration00:00:20 ] × 30 → 8℃ ∞

40m
Clean-up of 1st PCR products
30m
Clean up the 1st PCR products using AMPure XP (Beckman Coulter, USA).
Equilibrate the beads solution at room temperature and allow the beads to resuspend completely.
Add Amount12.5 µL beads solution to the Amount25 µL PCR products (0.5× bead solution/sample ratio) and mix by pipetting up and down multiple (10 to 20) times.

Incubate the tube at room temperature for 5 min.
Place the tube on a magnetic rack (NGS MagnaStand (YS-Model) 8Ch × 0.2 mL PCR tube, Nippon Genetics Co., Ltd., Tokyo, Japan) to capture the beads. Incubate until the liquid is clear (~Duration00:03:00 min).

3m
Discard the supernatant.
Wash beads by adding Amount200 µL 70% ethanol.

Incubate the tube on the magnetic rack at room temperature for Duration00:00:30 .

30s
Discard the supernatant.
Repeat the previous washing steps (8.6-8.8).
Remove the tube from the magnetic rack.
Elute the purified DNA by addingAmount25 µL of TE Buffer and mix by pipetting.

Incubate the tube at room temperature for Duration00:02:00 .

2m
Place the tube on the magnetic rack to capture the beads. Incubate until the liquid is clear (Duration00:01:00 ).

1m
Transfer the supernatant to a new PCR tube.
2nd PCR: NS31_Glo3 / wLSUmBr
50m
Prepare PCR-master mix in a 0.2 mL PCR tube with the following:

ComponentVolume (total 25 µl)
Forward primer (NS31_Glo3)1.25 µl
Reverse primer (wLSUmBr)1.25 µl
2 × KOD One mix12.5 µl
Template DNA (Cleaned 1st PCR products) NS31 / LSUmAr13 + LSUmAr245 µl
Template DNA (Cleaned 1st PCR products) AML1 / LSUmAr13 + LSUmAr245 µl

10m
Run the following PCR program:
[Temperature98 °C Duration00:00:10 → Temperature62 °C Duration00:00:05 → Temperature68 °C Duration00:00:20 ]×30 → Temperature8 °C ∞
40m
Clean-up of 2nd PCR products
20m
Clean up the 2nd PCR products using AMPure XP (0.5× bead solution/sample ratio) following the same procedure as in Section 8.
20m
Quality control
10m
Measure the DNA concentration of the purified 2nd PCR products using a Qubit fluorometer with the Qubit dsDNA HS assay kit.
10m
OPTIONAL:
Check the amplified fragments by gel electrophoresis.
Optional
Protocol references
Kolaříková Z, Slavíková R, Krüger C, Krüger M, Kohout, P (2021) PacBio sequencing of Glomeromycota rDNA: a novel amplicon covering all widely used ribosomal barcoding regions and its applicability in taxonomy and ecology of arbuscular mycorrhizal fungi. New Phytol 231:490–499. https://doi.org/10.1111/nph.17372

Krüger M, Stockinger H, Krüger C, Schüßler A (2009) DNA‐based species level detection of Glomeromycota : one PCR primer set for all arbuscular mycorrhizal fungi. New Phytol 183:212–223. https://doi.org/10.1111/j.1469-8137.2009.02835.x

Lee J, Lee S, Young JPW (2008) Improved PCR primers for the detection and identification of arbuscular mycorrhizal fungi: PCR primers for arbuscular mycorrhizal fungi. FEMS Microbiol Ecol 65:339–349. https://doi.org/10.1111/j.1574-6941.2008.00531.x

Schlaeppi K, Bender SF, Mascher F, Russo G, Patrignani A, Camenzind T, Hempel S, Rillig MC, van der Heijden MGA (2016) High-resolution community profiling of arbuscular mycorrhizal fungi. New Phytol 212:780–791. https://doi.org/10.1111/nph.14070

Simon L, Lalonde M, Bruns TD (1992) Specific amplification of 18S fungal ribosomal genes from vesicular-arbuscular endomycorrhizal fungi colonizing roots. Appl Environ Microbiol 58:291–295. https://doi.org/10.1128/aem.58.1.291-295.1992

Tedersoo L, Tooming-Klunderud A, Anslan S (2018) PacBio metabarcoding of Fungi and other eukaryotes: errors, biases and perspectives. New Phytol 217:1370–1385. https://doi.org/10.1111/nph.14776
Acknowledgements
This protocol was developed as part of a study supported by JSPS Grants-in-Aid for Early-Career Scientists (No. 23K17070).