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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 found in nature, which is essential for understanding fungal genomes. The technique works without needing freezers or ice by preserving tissue in alcohol at room temperature. The method successfully extracted usable DNA from 33 different mushroom species, including rare species that cannot be grown in laboratories, enabling researchers to sequence and study their complete genomes.

Background

Many macrofungi, particularly ectomycorrhizal species, are impractical or impossible to culture. DNA for long-read sequencing must be isolated from field-collected samples, often in remote locations where refrigeration and cryostorage are impractical. Fungal sporing bodies contain diverse metabolites and are subject to environmental damage that complicates DNA purification.

Objective

To develop and describe a practical methodology for extracting high-molecular-weight DNA suitable for long-read sequencing from field-collected macrofungi samples. The protocol addresses the challenges of ambient temperature sample preservation and removal of fungal metabolites without refrigeration infrastructure.

Results

64 DNA preparations from 48 field collections of 33 macrofungal species yielded DNA suitable for long-read sequencing of 23 genomes. Modal molecular lengths reached 38,000 bp. CEGMA and BUSCO quality scores ranged from 97.8-99.6% and 95.5-98.9% respectively. Cultured mycelium showed 100% success rate while field-collected material succeeded in 16 of 41 preparations (39%).

Conclusion

The chromatin-based isolation and sequential purification methodology successfully yielded high-quality genomic DNA from both cultured and field-collected macrofungi. Success depends on source material quality and species-specific metabolite composition, with resampling effective for improving outcomes when initial collections are compromised.
  • Published in:Journal of Fungi (Basel),
  • Study Type:Methodological Study,
  • Source: 10.3390/jof11070490, PMID: 40985414
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