2.4. Microbial diversity analysis
2.4.1. Microbial DNA isolation
For analyzing the microbial CAY10505 composition during the silage process, samples of B and A2 were collected on days 1, 3, 6, 10, 15, and 30, and stored at -20 °C before further analysis.
2.4.2. PCR amplification
Primer 515F (5′-GTGCCAGCMGCCGCGG-3′) and 907R (5′-CCGTCAATTCMTTTRAGTTT-3′) was used to amplify the V4-V5 regions of the bacteria 16S ribosomal RNA gene. PCR equipment (ABI GeneAmp® 9700) was used to perform polymerase chain reaction (PCR). The steps included hotstart at 95 °C for 2 min, followed by 28 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, elongation at 72 °C for 45 s and extension at 72 °C for 10 min. The reaction were performed in a 20 μL mixture containing 4 μL of 5× FastPfu Buffer, 2 μL of 2.5 mMdNTPs, 0.4 μL of each primer (5 μM), 0.4 μL of FastPfu Polymerase, and 10 ng of template DNA. For minimizing PCR bias, PCR reactions for each sample were conducted in triplicate, and the mixtures of three PCR products were used for measuring DNA concentration and sequencing.