Abstract
Aims: To develop a simple and rapid genomic DNA extraction technique for dried (≈ 1 year old) mushroom fruiting bodies that yields high-quality DNA, suitable for use by post-graduate institutions. Method: Small amounts (0.04 g) of pulverized dried mushroom sample were incubated in a Tris /EDTA/SDS lysis buffer (100mM:10mM: 2%) at 65°C to lyse the chitinous fungal cell walls. Genomic DNA purification was performed using chloroform isoamyl alcohol (24:1), and DNA was precipitated using 100% ethanol. Results: Genomic DNA was successfully extracted under 70 minutes from 16 samples morphologically identified as Panaeolus, Copelandia, Gymnopilus, Pluteus and Favolus species. DNA concentrations were on average of 696.9ng/µL. PCR successfully amplified the ITS-5.8S region. The protocol has been successfully used by numerous post-graduate students in our research programme. Conclusion: The rapid and easy protocol produced high-quality genomic DNA void of any inhibitors that is suitable for downstream molecular implications across multiple mushroom genera. Noticeably, this method requires only minute quantities (0.04g) of starting material and is ideal for student training in higher academic institutions.
Highlights
Fungi play essential roles in all components of ecosystems as nutritional sources, decomposers, mutualists and pathogens, as well as in economic sectors where they are sources of food, medicine, biological agents, and bioactive compounds (Cheng, 2021; de Mattos-Shipley et al, 2016; Enow Andrew et al, 2013; Gryzenhout et al, 2012)
The samples were airdried at room temperature stored in the Herbarium (Fungarium) of Marieka Gryzenhout (HMG), Genetics Department, University of the Free State
1.5mL Eppendorf tubes were used and the lysis buffer was prepared in a mass master mixture from which was allocated to each sample
Summary
Fungi play essential roles in all components of ecosystems as nutritional sources, decomposers, mutualists and pathogens, as well as in economic sectors where they are sources of food, medicine, biological agents, and bioactive compounds (Cheng, 2021; de Mattos-Shipley et al, 2016; Enow Andrew et al, 2013; Gryzenhout et al, 2012). DNA extraction is aiding research institutions, universities and biology students to identify fungi during biodiversity, ecological or applied studies, complimenting morphological methods (Badotti et al, 2017; Mullineux and Hausner, 2009; Zhang et al, 2016). Higher institutions have been implementing more laboratory orientated research by students, including performing DNA extraction from fungi for various types of studies. Fungal material is collected during fieldwork or retrieved from civilian scientists and is usually stored at room temperature for extended periods or stored erratically, especially for macrofungal samples. Students perform DNA extraction on fungal material that is no longer living. Culturing is a skill set not frequently found among biology students and non-taxonomy orientated researchers, while these cultures need to be maintained following specialist protocols
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