Bacterial 16S rDNA Profiling by RFLP

DNA was extracted from purified endophytic bacterial strains, following the procedure by Chen et al. (2018 (link)). Then, full length 16S rDNA was PCR amplified by the primer pairs 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGCTACCTTGTTACGAC TT-3′) in a 30 μL PCR reaction mixture by the following procedure: one cycle at 95°C for 5 min, followed by 30 cycles of 94°C for 30 s, 50°C for 30 s, 72°C for 2 min, and a final extension at 72°C for 10 min. The PCR products (~1,500 bp) were checked in 1% agarose gel and purified for restriction fragment length polymorphism (RFLP) analysis. Three restriction enzymes (HaeIII, Hinf I, and TaqI) were used for the digestion of 16S rDNA, following the manufacturer's instructions (Fermentas, EU). Fragments from the digestion were separated by gel electrophoresis in 2% agarose at 80 V for 3 h and photographed. The 16S rDNA-RFLP analysis was carried out by combining the results of three restriction enzymes. Then, the cluster analysis of 16S rDNA-RFLP was conducted by the UPGM clustering algorithm in a NTSYS program (Chen et al., 2018 (link)).