Microbial Community Profiling by PCR Cloning

The PCR products of the expected size (~800 bp) were purified using the TaKaRa MiniBEST Agarose Gel DNA Extraction Kit (TaKaRa, Japan) and were cloned into pMD19-T Vector (TaKaRa, Japan). The vector was transformed into E. coli strain DH5α, which were spread on agar plates and incubated overnight at 37°C. Inserts from 80 randomly selected clones in each resulting SSU rRNA gene library (4 blocks × 4 growth stages × 2 years × 2 cultivars = 64 libraries) were sequenced using M13-f47 and M13-r48 primers on ABI 3730 genetic analyzer. The obtained sequences were trimmed to remove the vector sequence, and sequence similarities were determined using the Basic Local Alignment Search Tool for nucleotides (BLASTn) sequence similarity search tool provided by GenBank [24 (link)]. Sequences were clustered at 97% sequence similarity with QIIME [25 (link)] and a representative sequence from each OTU was picked for downstream analysis. Taxonomic assignments of OTUs were performed by using QIIME in accordance with SILVA 115 [26 (link)]. Representative sequences of the clones generated in this study were deposited at the National Centre for Biotechnology Information (NCBI) GenBank database under the accession numbers KJ740816 to KJ740970.