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Methodology for Extracting High-Molecular-Weight DNA from Field Collections of Macrofungi

Summary

Scientists developed a practical method to extract high-quality DNA from mushrooms collected in the wild, even in remote locations without refrigeration. The technique involves preserving fresh samples in alcohol and then carefully purifying the DNA through multiple steps. This method successfully produced DNA suitable for reading the complete genetic code of 23 different fungal species, particularly those that cannot be grown in laboratories.

Background

Many macrofungi, particularly ectomycorrhizal species, cannot be cultured in the laboratory, requiring DNA extraction from field-collected samples. Field collections present unique challenges including environmental damage, metabolite contamination, and the practical absence of refrigeration in remote locations.

Objective

To develop and validate a practical methodology for extracting high-molecular-weight DNA from field-collected macrofungi that is suitable for long-read sequencing and high-quality genome assembly.

Results

64 DNA preparations were made from 48 field collections of 33 macrofungi species, yielding 23 high-quality genomes. DNA yields ranged from 25-80 µg per 200 mg dry fungus with modal molecular lengths up to 38,000 bp. Genome quality scores ranged from 97.8-99.6% for CEGMA and 95.5-98.9% for BUSCO metrics.

Conclusion

The described protocol successfully produces high-molecular-weight DNA suitable for long-read sequencing from field-collected macrofungi. Success depends primarily on sample quality and fungal species characteristics, with cultured mycelium yielding more consistent results than field-collected material.
  • Published in:Journal of Fungi,
  • Study Type:Methodology Paper,
  • Source: 10.3390/jof11070490, PMID: 40985414
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