title: Genome Sequencing of R. azygosporus CBS 357.93
design: Cultures of R. azygosporus strain CBS 357.93 and R. stolonifer strain LSU 92-RS-3 were grown on 1% potato dextrose agar (PDA, NEOGEN, Lansing, MI, USA). Three 0.5_0.5-cm pieces were cut from the edge of 5-day-old colonies and homogenized in a Waring Blender for further use as inoculum in liquid potato dextrose broth (0.5%). Cultures were grown in 250-mL Erlenmeyer flasks in 50 mL of medium on a shaker at room temperature for five days in three replicates. Before harvesting, the samples were examined microscopically to confirm the absence of bacterial or fungal contamination. Consolidated tissue was filtered through sterilized Miracloth and washed twice in sterile distilled water before DNA extraction. The mycelial biomass was then lyophilized for one to two days and ground in liquid nitrogen with a mortar, pestle, and sterilized sand. DNA was extracted with 2_ CTAB buffer following a modified DNA chloroform extraction technique. To prevent nucleic acid degradation, the samples were not incubated in a water bath prior to the addition of chloroform. The sample quality was verified by SYBR¨ Safe DNA and RNA staining on 0.8% agarose gels to detect nucleic acid contamination and traces of degradation. The total quantity of high molecular weight DNA was estimated using Quantity One¨ 1-D analysis software with a Gel Doc UV transilluminator (Bio-Rad, Hercules, CA, USA).