Introduction

Fungal genomes are among the smallest genomes of eukaryotes. The sizes of fungal genomes range from less than 10 Mbp to hundreds of Mbp. The average genome size is approximately 37 Mbp in Ascomycota, 47 Mbp in Basidiomycota and 75 Mbp in Oomycota.

The sizes and gene numbers of the smallest genomes of free-living fungi such as those of Wallemia ichthyophaga, Wallemia mellicola or Malassezia restricta are comparable to bacterial genomes. Some fungi exist as stable haploid, diploid, or polyploid cells, others change ploidy in response to environmental conditions and aneuploidy is also observed in novel environments or during periods of stress

Principle

The method involves disruption of fungal cells by employing homogenizer followed by inactivation of proteins using CTAB/Proteinase K. Further purify by chloroform: iso amyl alcohol and precipitated with absolute alcohol. The DNA yields from fungal isolate varied from 310 to 1879 μgm / gm of dry mycelium. Absorbancy ratio of 260/280 ranged from 1.7 to 1.9 which indicates minimal presence of contaminating metabolites.

Requirements

1)    Growth of Aspergillus niger on PDA medium.

2)    Extraction buffer 

3)    Homogenizer

4)    Water bath maintained at 60°c

5)    phenol: chloroform: isoamyl alcohol (25:24:1)

6)    chloroform: isoamylealcohol (24:1) 

7)    100% Ethanol

8)    70% Ethanol

9)    TE buffer

10) Electrophoresis Unit

11) St. Microfuge tubes

12) St. Tip box

13) Autopipette

Procedure

1.     Take fungal mass (approx.200 mg) from cultured plate and place in microfuge tube containing 800 μl extraction buffer and homogenized for 10 minutes.

2.     Put the tubes in water bath, maintain at 60°c for 30 min.

3.     Centrifuge at 10,000 rpm for 10 min.

4.     Transfer supernatant in to fresh microfuge tube.

5.     Add equal volume of phenol: chloroform: isoamylealcohol (25:24:1) in supernatant mix well and centrifuge at 10,000 rpm for 10 min.

6.     Transfer supernatant in fresh tube.

7.     Add equal volume of chloroform: isoamylealcohol (24:1) in supernatant and mix well and do the centrifugation at 10,000 rpm for 10 min.

8.     Take upper aqueous layer in fresh microfuge tube and add equal volume of 100% Ethanol or Absolute Alcohol.

9.     Incubate the tube for precipitation at -20°c for 30 min

10.  Whole content centrifuge at 10,000 rpm for 10 min to pallet down DNA.

11.  DNA pallet is washed with 800 μl 70% Ethanol and centrifuge at 10,000 rpm for 5 min.

12.  Air dry DNA pallets and dissolve DNA pallets in 30 μl TE buffer.

13.  Analyze the fungal DNA by running Agarose gel (0.8%) electrophoresis and perform spectroscopy analysis by taking 260/280 ratio and quantify the fungal DNA.

Advantages of Fungi DNA

Fungi DNA, like DNA in all living organisms, serves as the genetic blueprint for these organisms. There are several advantages and unique characteristics associated with fungi DNA:

·       Biodiversity: Fungi represent a highly diverse group of organisms, and their DNA allows for the study of this diversity. Fungi play crucial roles in ecosystems as decomposers, symbionts, and pathogens, and understanding their genetic diversity helps researchers better comprehend their ecological and evolutionary significance.

·       Medicinal and Biotechnological Applications: Fungi produce a wide range of bioactive compounds, including antibiotics, immunosuppressants, and enzymes. Studying their DNA can lead to the discovery of new pharmaceuticals and biotechnological applications. For instance, penicillin, one of the most famous antibiotics, is derived from the fungus Penicillium.

·       Mycorrhizal Relationships: Many fungi form mutualistic relationships with plants, known as mycorrhizae, where they exchange nutrients with the host plant. Understanding the genetics of these interactions can help improve agriculture and forest management by enhancing nutrient uptake in plants.

·       Bioremediation: Some fungi can degrade complex organic compounds, making them useful in bioremediation efforts to clean up polluted environments. DNA analysis can help identify and manipulate these fungi for specific remediation purposes.

·       Food Production: Fungi are essential in various food processes, such as fermentation (e.g., brewing, baking, and cheese production) and the production of edible mushrooms. DNA studies can aid in selecting and breeding strains with desired characteristics, improving food production.

·       Disease Diagnosis and Management: Fungal pathogens can affect plants, animals, and humans. DNA-based techniques, such as PCR and DNA sequencing, are valuable tools for identifying and studying these pathogens, enabling better disease management strategies.

·       Evolutionary Studies: Fungi have a long evolutionary history, and their DNA provides insights into the evolution of life on Earth. By comparing fungal DNA with that of other organisms, researchers can unravel the evolutionary relationships among species.

·       Conservation: Understanding the genetic diversity of fungi is essential for conservation efforts, especially for endangered or rare species. DNA analysis can help assess population sizes, genetic variability, and potential threats to fungal species.

·       Drug Discovery: Fungi are a rich source of secondary metabolites with potential therapeutic properties. Investigating fungal DNA can lead to the discovery of novel compounds for drug development.

·       Environmental Monitoring: Fungi are sensitive to environmental changes, and their DNA can be used as a bioindicator to assess the health of ecosystems. Changes in fungal communities can provide early warnings of ecological disturbances.

In summary, studying fungi DNA offers a wide range of advantages, from advancing biotechnology and medicine to enhancing our understanding of ecology, evolution, and the environment. It has practical applications in various fields and contributes to our knowledge of these important and diverse organisms.

Limitations of Fungi DNA

Fungi DNA, like all DNA, has certain limitations that can affect research and applications related to fungi. Some of these limitations include:

·       Lack of complete genome sequences: While the number of fungal genome sequences has increased significantly over the years, there are still many fungi for which complete genome sequences are unavailable. This limits our understanding of their genetics and biology.

·       Heterozygosity: Many fungi have complex and highly heterozygous genomes. This can make it challenging to assemble their genomes accurately, which can affect our ability to study their genetic makeup and functions.

·       High diversity: Fungi represent a highly diverse group of organisms with a wide range of lifestyles and ecological roles. This diversity can make it difficult to generalize findings from one fungal species to another, as their genetics and biology can vary significantly.

·       Limited genetic tools: In comparison to model organisms like yeast and certain filamentous fungi, many fungi lack well-developed genetic tools and resources for manipulation. This can hinder research on less-studied fungi.

·       Fungal evolution: Fungi have undergone complex evolutionary processes, including horizontal gene transfer, hybridization, and gene duplication events. These processes can complicate the interpretation of fungal DNA and its evolution.

·       Non-coding regions: Fungal genomes, like other eukaryotes, contain a significant portion of non-coding DNA. Understanding the function and regulation of these non-coding regions can be challenging.

·       Epigenetic regulation: Fungi, like other organisms, have epigenetic mechanisms that play a crucial role in gene regulation. These mechanisms can involve DNA methylation, histone modifications, and other processes that add layers of complexity to the study of fungal DNA.

·       Environmental influences: Fungi are highly adaptable and can undergo significant changes in their gene expression and genetic makeup in response to environmental conditions. This plasticity can make it challenging to study their DNA under different contexts.

·       Lack of genetic diversity in culture collections: Many studies rely on fungal strains that have been isolated and maintained in culture collections for years. These strains may not represent the genetic diversity found in natural populations, which can limit the generalizability of research findings.

·       Ethical and practical challenges: In some cases, obtaining fungal DNA for research may involve ethical considerations, especially when dealing with rare or endangered species. Additionally, some fungi can be difficult to culture and work with in the laboratory.

Despite these limitations, advances in DNA sequencing technologies, bioinformatics, and fungal research techniques continue to improve our understanding of fungi and their genetics. Researchers are making strides in overcoming these challenges to unlock the potential benefits of fungi in various fields, including medicine, agriculture, and biotechnology.