Bacterial 16S rRNA Gene Amplification and Sequencing

DNA was extracted from isolates using a Bacterial DNA Kit (Omega Bio-Tek) following the protocol of the manufacturer. PCR of 16S rRNA genes and sequencing were conducted by Majorbio Company (Shanghai, China). In brief, the almost complete bacterial 16S rRNA gene was amplified with Ex Taq^® (Takara Bio Inc.), using primers 27F (5’-AGAGTTYGATCCTGGCTCAG-3’) and 1492R (5’- GGTTACCTTGTTACGACTT-3’). Conditions comprised an initial denaturation at 95°C for 5 min, followed by 25 cycles of 95°C for 30 s, 56°C for 30 s, 72°C for 1.5 min, and a final extension step at 72°C for 10 min on the last cycle. The amplicons were purified and sequenced on a 3730XL DNA analyzer system (Applied Biosystems). Retrieved DNA sequences were manually edited using Seqman Pro (DNASTAR Lasergene package). The similarities of 16S rRNA genes between isolated endophytes and their phylogenetic neighbors were determined through the EzBioCloud Database (access time: January 2023) (Yoon et al., 2017 (link)), supplemented with the BLASTN (https://blast.ncbi.nlm.nih.gov/Blast.cgi) of the NCBI to search for the closest matching sequences.