2.5 Terminal restriction fragment length polymorphism (T-RFLP) analysis
The microbial community structures ofcbbL-and nifH -carrying populations were estimated by terminal restriction fragment length polymorphism (T-RFLP) analysis. The PCR amplification was performed using primer pairs K2f / V2r for cbbLand PolF / PolR for nifH , labeled with 6-carboxy-fluorescein (6-FAM) at the 5’end. The labeled PCR amplicons were cleaned using a SanPrepPCR Purification Kit (Sangon Biotech, Shanghai, China), and then digested digested at 37°C for 4 h using the following restriction enzymes: Msp I for cbbL and Hae Ⅲ for nifH . The fluorescently labeled PCR products were cleaned as described above and then digested with Taq I (Takara Bio Inc, Shiga, Japan) by incubating at 65 °C for 6 h. Subsequently, DNA fragments were analyzed using capillary electrophoresis (3730 Genetic Analyzer; Applied Biosystems, CA), using a GeneScan ROX-labeled GS500 internal size standard. Peaks were manually edited, imported into the T-REX online software (Culman et al., 2009), filtered with one standard deviation height, and clustered at 1.0 bp. Only T-RFs with the relative abundance (RA) > 1 % in all three replicates were included for further analysis, and fragments with RA > 10 % were regarded as dominant T-RFs (Yuan et al., 2012).