Fungal Genomic DNA Extraction Methods for Rapid Genotyping and Genome Sequencing.

Isolation of fungal genomic DNA of high quality is required for a number of downstream biotechnology-derived applications such as genome sequencing, microarrays, and digital PCR technologies, to only name a few. In most cases, not only a high molecular weight DNA of superior grade is required but also large quantities. On the other hand, a number of laboratory experiments, such as polymerase chain reaction (PCR) for medical diagnostic or for genotyping, have to be conducted in a limited amount of time and can provide complete results with the use of lower quality DNA. We describe here two different fungal DNA extraction approaches, which are applicable to a wide range of fungal species.First, we adapted a DNA extraction method for PCR-based genotyping which allows analysis of single to hundreds of colonies simultaneously. Cells are disrupted in the presence of sodium dodecyl sulfate and Proteinase K which are then removed by precipitation and centrifugation. The cleared lysate is used for PCR reaction.Secondly, we describe a method to obtain genome sequencing quality grade DNA from fungal liquid cultures. Mycelia are harvested by either filtration or centrifugation. Cells are mechanically disrupted by liquid nitrogen grinding, followed by genomic DNA extraction using the QIAGEN's DNeasy® Plant Kit. The quality and quantity of genomic DNA is monitored by fluorometry.

Comparative genomic analysis of the thermophilic biomass-degrading fungi Myceliophthora thermophila and Thielavia terrestris.

Thermostable enzymes and thermophilic cell factories may afford economic advantages in the production of many chemicals and biomass-based fuels. Here we describe and compare the genomes of two thermophilic fungi, Myceliophthora thermophila and Thielavia terrestris. To our knowledge, these genomes are the first described for thermophilic eukaryotes and the first complete telomere-to-telomere genomes for filamentous fungi. Genome analyses and experimental data suggest that both thermophiles are capable of hydrolyzing all major polysaccharides found in biomass. Examination of transcriptome data and secreted proteins suggests that the two fungi use shared approaches in the hydrolysis of cellulose and xylan but distinct mechanisms in pectin degradation. Characterization of the biomass-hydrolyzing activity of recombinant enzymes suggests that these organisms are highly efficient in biomass decomposition at both moderate and high temperatures. Furthermore, we present evidence suggesting that aside from representing a potential reservoir of thermostable enzymes, thermophilic fungi are amenable to manipulation using classical and molecular genetics.

Generation, annotation, and analysis of an extensive Aspergillus niger EST collection.

BACKGROUND: Aspergillus niger, a saprophyte commonly found on decaying vegetation, is widely used and studied for industrial purposes. Despite its place as one of the most important organisms for commercial applications, the lack of available information about its genetic makeup limits research with this filamentous fungus. RESULTS: We present here the analysis of 12,820 expressed sequence tags (ESTs) generated from A. niger cultured under seven different growth conditions. These ESTs identify about 5,108 genes of which 44.5% code for proteins sharing similarity (E < or = 1e(-5)) with GenBank entries of known function, 38% code for proteins that only share similarity with GenBank entries of unknown function and 17.5% encode proteins that do not have a GenBank homolog. Using the Gene Ontology hierarchy, we present a first classification of the A. niger proteins encoded by these genes and compare its protein repertoire with other well-studied fungal species. We have established a searchable web-based database that includes the EST and derived contig sequences and their annotation. Details about this project and access to the annotated A. niger database are available. CONCLUSION: This EST collection and its annotation provide a significant resource for fundamental and applied research with A. niger. The gene set identified in this manuscript will be highly useful in the annotation of the genome sequence of A. niger, the genes described in the manuscript, especially those encoding hydrolytic enzymes will provide a valuable source for researchers interested in enzyme properties and applications.