Taxonomic identification of the candidate endophyte based on 16S rRNA

Though the taxonomy of the candidate P-solubilizing endophyte was previously reported²⁵, the previous 16S rRNA sequence was short. To obtain longer sequence reads and to verify the identity, DNA was extracted using a Bacterial Genomic Miniprep Kit (NA2110, Sigma). DNA was quantified using a NanoDrop ND-1000 machine (Thermo Scientific, USA) then 100 ng of DNA were used in a PCR reaction with universal 16S rRNA primers^70,71 in a total volume of 40 µl. The reaction mixture contained: 20 µl of GoTaq® Green Master Mix (M712C, Promega), 1 μl of 10 μM 27f primer with sequence AGAGTTTGATCMTGGCTCAG, 1 μl of 10 μM 1492r primer with sequence GGTTACCTTGTTACGACTT, and double distilled water up to 40 μl. A PTC200 DNA Thermal Cycler (MJ Scientific, USA) was used with the following amplification conditions: 94 °C for 5 min, 35 amplification cycles (94 °C for 45 sec, 50 °C for 1 min, 72 °C for 2 min), with a final extension at 72 °C for 7 min. PCR products were gel purified (Illustra GFX, GE Healthcare, USA), submitted for sequencing at the Genomics Facility at the Advanced Analysis Centre (AAC) at the University of Guelph and identified using BLAST searches. Following sequencing, related 16S sequences were obtained from GenBank. These sequences were then used to construct a phylogenetic tree using Phylogeny.lirmm.fr using default parameters^72–74.