Model 475

The experiments were performed as described previously (42 (link)). Briefly, HDA1 and HDA2-GC primers were used to assess microbial diversity in each sample. PCR products were separated in 8% polyacrylamide gels with a range of 30–50% denaturing gradient (100% denaturant consisted of 7 M urea and 40% deionized formamide) cast with Bio-Rad’s Model 475 gradient delivery system (BioRad, Hercules, CA, USA). The electrophoresis was performed in TAE 0.5× buffer for 5 h at a constant electric current of 125 mA and a temperature of 60°C with the DCode Mutation Detection System (Bio-Rad, Hercules, CA, USA). Clustering analysis was performed using the UPGMA (unweighted pair group method with arithmetic mean clustering algorithm) to calculate the dendrograms.

DGGE was performed using a Dcode universal mutation detection system (Bio-Rad Inc., Hercules, CA, USA). Samples containing approximately equal amounts of PCR amplification products were loaded on a 10% poly-acrylamide gel in TAE buffer with a denaturing gradient ranging from 30% to 60% of the denaturant (denaturation of 100% corresponded to 7 mol·L⁻¹ urea and 40% (v/v) formamide). DGGE was performed at 60 °C with a constant voltage of 160 V for 6 h, by using a gradient delivery system (Model 475, Bio-Rad Inc., Hercules, CA, USA. The gel was then stained by AgNO₃ and the images of the gel were captured by a bio-image system (Bio-Rad).
DGGE profiles were analyzed using the software Quantity One 4.6.2 (Bio-Rad Inc., Hercules, CA, USA). Dendrograms relating band pattern similarities were automatically calculated using the unweighted pair group method with the arithmetic average (UPGMA) clustering algorithm. UPGMA employs a sequential clustering algorithm, in which local topological relationships are identified in order of similarity, and the phylogenetic tree is built in a stepwise manner. Relative band densities, which were necessary to determine the Shannon diversity index (SDI), were quantified, and the statistical data were exported for further SDI analyses.