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Updated: Jan 17, 2026
Isolation, Characterization, and Total DNA Extraction to Identify Endophytic Fungi in Mycoheterotrophic Plants
Published on: May 5, 2023
Methodology for Extracting High-Molecular-Weight DNA from Field Collections of Macrofungi
Leigh A Burgoyne1, Andy R Nilsen2, Teresa Lebel1,3
1Biological Sciences, College of Science and Engineering, Flinders University, Adelaide 5001, Australia.
Abstract:
Many macrofungi are impractical or impossible to culture. Consequently, DNA for long-read sequencing required for the assembly of high-quality genomes must be isolated from samples taken from the environment. Collection is often in remote locations, limiting the options for stabilising samples to methods that do not require refrigeration. Fungi contain species-specific arrays of metabolites that may complicate purification techniques and call for judgement to be made to apply appropriate modifications to the DNA extraction protocol in specific cases. The protocols and commentary we describe are informed by the preparation of DNA from a range of Australasian ectomycorrhizal and saprotrophic macrofungi. We collect samples into isopropanol at ambient temperature and employ a strategy of chromatin isolation followed by the sequential removal of unwanted molecular components to purify DNA.
Insights
Isolating fungal DNA for genome sequencing from environmental samples is challenging. This study presents a robust DNA extraction protocol for macrofungi, enabling high-quality genome assembly from non-refrigerated samples.
Area of Science:
- Mycology
- Genomics
- Molecular Biology
Background:
- Many macrofungi cannot be cultured, necessitating environmental DNA (eDNA) isolation for genomic studies.
- Collecting samples in remote locations often precludes refrigeration, requiring alternative preservation methods.
- Fungal secondary metabolites can interfere with DNA purification, demanding optimized extraction protocols.
Purpose of the Study:
- To develop and describe a reliable DNA extraction protocol for environmental macrofungi samples.
- To enable high-quality genome assembly from non-refrigerated fungal specimens.
- To provide a practical method for researchers working with challenging fungal samples.
Main Methods:
- Samples collected in isopropanol at ambient temperature.
- Chromatin isolation strategy employed.
- Sequential removal of unwanted molecular components for DNA purification.
Main Results:
- Successful DNA isolation from diverse Australasian ectomycorrhizal and saprotrophic macrofungi.
- Protocol adaptable to species-specific metabolite profiles.
- Yielded DNA suitable for long-read sequencing and genome assembly.
Conclusions:
- The described protocol is effective for extracting high-quality DNA from environmental macrofungi.
- This method overcomes limitations of culturing and sample preservation.
- Facilitates advancements in fungal genomics and biodiversity research.
