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).