Streamlined and Cost-Effective Genomic DNA Extraction Method for Lichens, Mushrooms, and Endolichenic Fungi: Enabling DNA Barcoding and Molecular Research

Abstract

Extraction of nucleic acids in pure form from organisms is of paramount importance for DNA based identification and other molecular studies. Over the past few decades, DNA-barcoding has emerged as a powerful technique, facilitating species identification across various ‘difficult to identify’ life-forms. Fungi, being an immensely diverse group of microorganisms, contribute significantly to global biodiversity, with estimates ranging from 2.2 to 3.8 million species. However, a vast majority of this diversity remains unidentified, and many fungal species are considered cryptic. Therefore, numerous large- and small-scale DNA-barcoding projects are being conducted worldwide to unravel this rich biodiversity. However, the rigidity and high complex polysaccharides content of fungal cell-wall presents a significant obstacle, making the extraction of high-quality genomic DNA a challenging task across varied fungal organisms. In this study, we employed a modified CTAB based method to isolate and purify high-quality PCR-amplifiable genomic DNA primarily from lichens and tested it on other fungal life forms as well, including, mushrooms, endolichenic fungi, and parasitic fungi. Remarkably, the isolated DNA proved successful as a template in PCR reactions, serving the purposes of DNA barcoding, RAPD as well as for metagenomic analysis effectively. This versatile protocol demonstrated its utility across all the fungal life forms investigated in this study, offering a universal, cost-effective, and efficient approach for fungal DNA isolation.