PubMed · 14190877
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M FEIWEL. 1964. GENERALIZED MORPHOEA.. https://pubmed.ncbi.nlm.nih.gov/14190877/
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High-quality genomic DNA extraction from basidiomycete fungi remains challenging due to polysaccharide co-purification and nuclease-mediated DNA degradation. We systematically compared 22 experimental conditions combining five DNA extraction methods (one Dellaporta-based method, three CTAB-based variants, and one commercial kit) with five mycelium preparation techniques (scalpel scraping, fungal suspension, silica bead homogenization, liquid fermentation, and liquid nitrogen treatment) for Phanerodontia australis (BRM62389). DNA quality was assessed by 1% agarose gel electrophoresis, NanoDrop spectrophotometry, and Qubit fluorimetry. The optimal protocol combined liquid-fermentation mycelium with an adapted CTAB method, yielding Qubit-quantified concentrations of 34.2 and 62.6 ng/µL (samples S21 and S22), 260/280 ratios of 2.06 and 2.05, 260/230 ratios of 1.88 and 1.85, and no detectable DNA degradation by agarose gel electrophoresis. This protocol enabled whole-genome sequencing, yielding a 37.62 Mb assembly with 98.5% completeness as assessed by BUSCO v5 (basidiomycota_odb10 lineage dataset, n = 1,764). Liquid fermentation is associated with the production of younger, actively growing hyphae with reduced cell wall thickness, and the adapted CTAB method effectively removed residual polysaccharides. This protocol provides a reliable, cost-effective approach for obtaining sequencing-grade DNA from P. australis.
Expressed sequence tag analysis (EST) is a robust approach for detecting novel and potentially important genes during fungal development. The technique was used in this study to identify and characterise genes induced during spore germination and hyphal morphogenesis under different culture conditions. For this study, a cDNA library was constructed from the germinated spores of S-type of Heterobasidion annosum. In total, 12,288 clones of the H. annosum germinated spores (HAGS) cDNA library were arrayed on a high-density nylon membrane filter. A total of 1248 trimmed ESTs were randomly selected and sequenced. These ESTs were composed of 343 contigs from a total of 883 EST sequences. The BLASTX analysis of these sequenced clones revealed several kinds of genes with diverse functions (e.g., MAP kinase and endoglucanase, etc.). Differential hybridization assay on the HAGS cDNA library using cDNA probes made from H. annosum pre-grown under different culture conditions, such as 1.0% glucose, 0.01% ferulic acid or 0.01% oxalic acid revealed 20, 37, 44 positive hybridizing clones, respectively. Among the positively hybridizing clones 15 gene clones were common to all conditions whereas 4, 9 and 15 clones were unique in glucose, ferulic acid and oxalic acid medium, respectively. BLASTX analysis of the differentially expressed clones led to the identification of genes with significant homologue to cytochrome P450 monooxygenase and transcript antisense to ribosomal RNA. Northern analyses confirmed that expression of both genes were elevated in ferulic acid and oxalic acid culture conditions than in glucose medium. The enhanced accumulation of the gene transcripts at the different culture conditions is discussed with respect to their potential role during fungal morphogenesis and in planta growth.
A plant's capability to develop ectomycorrhizal symbiosis is under the control of both genetic and environmental factors. In order to determine the roles played by these different factors, we have performed a quantitative genetic analysis of the ability of poplar trees to form ectomycorrhizas. Quantitative genetics were applied to an interspecific family of poplar for which the two parental genetic maps had already been described, and for which data analyses concerning fungal aggressors were obtained. Quantitative trait loci (QTL) related to ectomycorrhiza formation were identified and located in the genetic maps of the two parents. One QTL was located at a linkage group of the genetic map of Populus trichocarpa showing a high concentration of several QTL involved in the pathogenic interaction with the fungus Melampsora larici-populina, the causal agent of leaf rust.