by Chen Ming-jie. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 81-95). / List of Abbreviations --- p.I / List of Tables --- p.II / List of Figures --- p.III / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Background of Volvariella volvacea and the purposes of this study --- p.1 / Chapter 1.1.1 --- Background of Volvariella volvacea --- p.1 / Chapter 1.1.2 --- Purposes of this molecular study on Volvariella volvacea --- p.5 / Chapter 1.2 --- Molecular studies in edible mushrooms --- p.5 / Chapter 1.2.1 --- Recombinant DNA technology --- p.5 / Chapter 1.2.2 --- Restriction fragment length polymorphisms (RFLPs) --- p.6 / Chapter 1.2.3 --- Polymerase chain reaction (PCR) --- p.7 / Chapter 1.2.3.1 --- Ribosomal RNA gene-PCR (rDNA-PCR) --- p.8 / Chapter 1.2.3.2 --- Random amplified DNAs by polymerase chain reaction --- p.10 / Chapter 1.2.3 --- Pulsed field gel electrophoresis --- p.12 / Chapter Chapter 2 --- Materials and Methods --- p.17 / Chapter 2.1 --- Organisms --- p.17 / Chapter 2.2 --- Cell cultivation and maintenance --- p.17 / Chapter 2.3 --- Solutions and chemicals --- p.17 / Chapter 2.3.1 --- Solutions for DNA extraction --- p.17 / Chapter 2.3.2 --- Solutions for agarose gel electrophoresis --- p.18 / Chapter 2.3.3 --- Solutions for DNA labeling and detection --- p.18 / Chapter 2.3.3.1 --- Colorimetry --- p.18 / Chapter 2.3.3.2 --- Chemiluminescence --- p.19 / Chapter 2.3.4 --- Hybridization solution --- p.19 / Chapter 2.3.5 --- PCR primers --- p.19 / Chapter 2.3.6 --- SOC medium --- p.20 / Chapter 2.4 --- Agarose gel electrophoresis --- p.20 / Chapter 2.5 --- DNA extraction and purification --- p.20 / Chapter 2.5.1 --- Genomic DNAs --- p.20 / Chapter 2.5.2 --- Plasmid DNA --- p.21 / Chapter 2.6 --- Formation of complementary ends --- p.23 / Chapter 2.6.1 --- Partial digestion of genomic DNA with the restriction enzyme Sau3A I --- p.23 / Chapter 2.6.2 --- Production of vector arms --- p.23 / Chapter 2.7 --- Ligation --- p.24 / Chapter 2.8 --- Transformation --- p.24 / Chapter 2.8.1 --- Chemical transformation method --- p.24 / Chapter 2.8.1.1 --- Preparation of competent E. coli cells --- p.24 / Chapter 2.8.1.2 --- Transformation --- p.25 / Chapter 2.8.2 --- Electroporation --- p.25 / Chapter 2.8.2.1 --- Preparation of electro-competent cells --- p.25 / Chapter 2.8.2.2 --- Electroporation --- p.26 / Chapter 2.9 --- Southern transfer and hybridization using non- radioactive method --- p.27 / Chapter 2.9.1 --- Random labeling the V.volvacea genomic DNA by digoxigenin-11-dUTP --- p.28 / Chapter 2.9.2 --- Conventional PCR to amplify and label cloned DNA inserts --- p.28 / Chapter 2.9.3 --- Southern blotting --- p.29 / Chapter 2.9.4 --- Prehybridization --- p.29 / Chapter 2.9.5 --- Hybridization --- p.30 / Chapter 2.9.6 --- High stringency washing --- p.30 / Chapter 2.9.7 --- Detection --- p.30 / Chapter 2.9.7.1 --- Color detection --- p.30 / Chapter 2.9.7.2 --- Chemiluminescent detection --- p.31 / Chapter 2.9.8 --- Reprobing --- p.31 / Chapter 2.9.9 --- Colony hybridization --- p.31 / Chapter 2.10 --- Polymerase chain reaction (PCR) --- p.32 / Chapter 2.10.1 --- Arbitrarily primed polymerase chain reaction (AP- PCR) --- p.32 / Chapter 2.10.2 --- Random amplification of polymorphic DNA (RAPD) --- p.32 / Chapter 2.10.3 --- Amplification of ribosomal RNA gene (rDNA- PCR) --- p.33 / Chapter 2.11 --- Pulsed field gel electrophoresis --- p.33 / Chapter 2.11.1 --- Preparation of protoplasts --- p.33 / Chapter 2.11.2 --- Embedding of chromosomal DNAs --- p.34 / Chapter 2.11.3 --- Electrophoresis --- p.34 / Chapter 2.11.4 --- Southern blotting and hybridization --- p.35 / Chapter Chapter 3 --- Results --- p.36 / Chapter 3.1 --- Construction of a partial genomic library for Volvariella volvacea --- p.36 / Chapter 3.1.1 --- Genomic DNA purification and restriction enzyme digestion --- p.36 / Chapter 3.1.2 --- Preparation of vector arms --- p.36 / Chapter 3.1.3 --- Ligation and transformation --- p.36 / Chapter 3.2 --- Characterization of clones in the genomic library --- p.42 / Chapter 3.3 --- Fishing out ribosomal RNA gene from the genomic library by homologous rDNA probe --- p.45 / Chapter 3.4 --- Strain typing --- p.50 / Chapter 3.4.1 --- Strain typing by RFLPs using moderately repetitive probes --- p.50 / Chapter 3.4.2 --- Strain typing by PCR-based protocols: AP-PCR and RAPD --- p.50 / Chapter 3.4.3 --- Strain typing by PCR- RFLPs --- p.56 / Chapter 3.5 --- Electrophoretic karyotype analysis by pulsed field gel electrophoresis --- p.56 / Chapter 3.5.1 --- Protoplast preparation --- p.56 / Chapter 3.5.2 --- The electrophoresis condition --- p.56 / Chapter 3.5.3 --- Southern hybridization --- p.65 / Chapter Chapter 4 --- Discussion --- p.68 / Chapter 4.1 --- Genomic library --- p.68 / Chapter 4.2 --- Generation of molecular markers --- p.70 / Chapter 4.2.1 --- RFLPs --- p.70 / Chapter 4.4.2 --- AP-PCR and RAPD methods --- p.71 / Chapter 4.2.3 --- PCR- RFLP of rRNA gene --- p.72 / Chapter 4.2.4 --- Comparison of the four types of molecular markers --- p.72 / Chapter 4.3 --- Electrophoretic karyotype by PFGE --- p.74 / Conclusion --- p.80 / References --- p.81