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Improved Extraction Methods to Isolate High Molecular Weight DNA From Magnaporthaceae and Other Grass Root Fungi for Long-Read Whole Genome Sequencing

Summary

Scientists have developed improved methods to extract high-quality DNA from pathogenic fungi, which is essential for sequencing and understanding how these fungi cause plant diseases. Two different extraction techniques were refined and tested on Magnaporthaceae fungi: one that works with small amounts of fungal material, and another that produces longer DNA strands when more material is available. The key to success is harvesting the fungi before they accumulate dark pigments that interfere with DNA extraction. These protocols will help researchers create better genome maps of disease-causing fungi, leading to improved strategies for crop protection.

Background

High-quality fungal genome assemblies are crucial for understanding fungal evolution and pathogenicity. Short-read sequencing data produces fragmented assemblies unable to span structural variants and repetitive regions. Long-read sequencing requires high molecular weight (HMW) DNA, which is challenging to extract from fungi due to their cell walls, secondary metabolite production, and limited starting material.

Objective

To develop and optimize modified DNA extraction protocols using two commercial kits (Cytiva Nucleon PhytoPure and Macherey-Nagel NucleoBond HMW DNA) specifically for isolating high-quality HMW DNA from Magnaporthaceae fungi and other grass root fungi suitable for long-read whole genome sequencing.

Results

The modified Macherey-Nagel protocol yielded superior results with >50% of DNA strands exceeding 40 kbp and 90% of samples meeting optimal purity ratios (260/280). The Cytiva Nucleon PhytoPure method successfully recovered HMW DNA for 50% of fungal species tested with lower starting material requirements, while the Macherey-Nagel method achieved 70% success rate. Both methods benefited from harvesting mycelium before melanin accumulation and careful DNA handling to preserve strand integrity.

Conclusion

Two optimized HMW DNA extraction protocols have been successfully developed and validated for Magnaporthaceae and related fungal species. The choice between methods depends on available starting material and time constraints: the Cytiva Nucleon PhytoPure method for limited biomass and shorter cultivation periods, and the Macherey-Nagel NucleoBond HMW DNA method for high-quality long-stranded DNA when sufficient material is available. These protocols are readily adaptable for other ascomycete fungi requiring long-read genome sequencing.
  • Published in:Bio Protoc (BioProtocol),
  • Study Type:Protocol Development Study,
  • Source: PMID: 40395841, DOI: 10.21769/BioProtoc.5245
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