PCR-amplification and restriction fragment length polymorphism (RFLP) of the inter-transcribed spacer (ITS) of 16S–23S rRNA region

The 16S–23S rRNA inter-transcribed spacer (ITS) region of the genomic DNA was amplified using rhizobial specific respective primer pairs (Table S2). The ITS region was amplified by using a Thermal Cycler (Bio-RAD T100) in a 25 μl reaction mixture containing 40–50 ng DNA template, 5X MyTaq PCR buffer (1X), 10 pM of each of the primer, 0.5U Taq polymerase (Bioline, USA) and PCR grade water in 0.2 ml PCR tube. The PCR amplification was done using the temperature profile described in Table S2. The PCR-amplified products were digested with three endonucleases (TaqI, HindIII and MspI) separately (Thermo Scientific, Lithuania). The amplified polymorphic bands obtained from different nodule DNAs were considered as the ITS types of the bacterial population. The restriction enzyme-digested ITS fragments (ITS-RFLP patterns) were analysed after migration in 3% (w/v) agarose gel at 85 V for 2.5 h. The presence or absence of homologous bands was scored using a binary approach, and a UPGMA based dendrogram constructed with NTSYSpc 2.2 programme [46].