4.5.3. Strains Identified from Daqu

For the extraction of ribosomal DNA fragments, spores on the activated PDA plate were eluted with 1.5 mL methanol and collected in a centrifugal tube to centrifugate for 2 min at 12,000 rpm. The formed supernatant was discarded after centrifugation. The precipitate was washed by adding 1 mL Milli-Q water and centrifuged for 2 min at 12,000 rpm. The bacterial precipitate was obtained after centrifugation, followed by the addition of 0.2 mL Milli-Q water, 0.3-g glass beads (diameter 0.4 mm) and 0.3 mL of a phenol(Sigma-Aldrich, St. Louis, MO, USA):chloroform(Sigma-Aldrich, St. Louis, MO, USA) (1:1) mixed solution and then broken for 30 s by a cell analyzer. After that, 0.2 mL ultrapure water was added, mixed equally, and centrifuge for 2 min at 12,000 rpm. The supernatant was obtained and twice the volume of ice ethanol was added and left to stand for 2 h at 4 °C before being centrifuged for 15 min at 12,000 rpm. The supernatant formed was discarded again after centrifugation. The precipitate was dried under vacuum and dissolved using 30~50 μL Milli-Q water.

PCR amplification was performed with an ITS sequence experiment, amplified using ITS1 and ITS4 primer. The reaction system used was as follows: ITS: 5 min at 94 °C; 94 °C 30 s, 50 °C 30 s, 72 °C 60 s, for 30 cycles; the composition of the 25 μL of reaction system used was 10.5 μL sterile water, 12.5 μL mix enzyme, 0.5 μL ITS1, 0.5 μL ITS4 and 1 μL template.

The recovery and sequencing of the PCR amplification products consisted of electrophoresis onto 1% agarose gel. Then, the target product was sequenced at the Beijing Genomics Institute.