2.6 Cloning, sequencing, and phylogenetic analyses
In order to further identify the main T-RFs appearing in the profiles, clone libraries were established both the cbbL and nifHgenes.The DNA extracted from the three replicates of each IM treatment were mixed and served as the template for PCR. The PCR amplification was performed with the same primers as before but without the fluorescent labels. Subsequently, the PCR products were ligated into the plasmid vector pGEM-T, and transformed into Escherichia coliJM109 (Takara Bio Inc, Shiga, Japan) by the manufacturer’s instructions. Randomly selected clones (approximately 200 clones for each clone library) were screened through the reamplification with the vector-specific primers M13F and M13R and sequenced by the Sangon Biotech (Shanghai) Co., Ltd. Chimeras were detected using Decipher (Wright, Yilmaz, & Noguera, 2012) , and the BLASTn program was used to identify the most similar sequences from GenBank (NCBI). Based on 97% sequence similarity, 45 and 49 operational taxonomic unit (OTU) were obtained from 95 to 246 clones in the cbbL and nifH clone library, respectively. Only those OTUs containing at least two sequences were selected for phylogenetic analysis and T-RF assignment, and one representative sequence of these OTUs were defined for T-RFLP analysis as described above. Phylogenetic trees were constructed by the neighbor-joining method using MEGA 5.0 (Tamura et al., 2012). Bootstrapping (1000 replicate reconstructions) was used to test the reliability of phylogenetic reconstructions. All of the sequences generated in this study have been deposited in the GenBank database under accession numbers MF430858 to MF430936 for cbbL and MF6633454 to MF6633523 for nifH .