About 5 μl of the cell suspension was used to amplify the 16S rRNA gene of WYHC-5 cells by polymerase chain reaction with the universal bacterial primers 27F (5-′AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) (24 (link)), as described in detail by Li et al. (32 (link)). The amplification products were purified using a QIAquick Gel Extraction Kit (QIAGEN, Germany), ligated with the pMD19-T vector (TaKaRa, Japan), and then cloned in Escherichia coli (strain DH5α)–competent cells (Huada, Beijing, China) according to the manufacturer’s instructions. A total of 30 clones were picked randomly and were sequenced using the vector primers M13-47 (5′-CGCCAGGGTTTTCCCAGTCACGAC-3′) and RV-M (5′-GAGCGGATAACAATTTCACACAGG-3′) at the Beijing Huada Genomics Research Center (Beijing, China).
After discarding sequences of insufficient length (<1300 bp), remaining sequences were aligned with close relatives using the Gblocks 0.91b online algorithm with default parameters to eliminate poorly aligned positions (67 (link)). The maximum likelihood method phylogenetic tree was constructed using the MEGA Software package (version 7.0) (68 (link)). Bootstrap values were calculated with 1000 replicates.
After discarding sequences of insufficient length (<1300 bp), remaining sequences were aligned with close relatives using the Gblocks 0.91b online algorithm with default parameters to eliminate poorly aligned positions (67 (link)). The maximum likelihood method phylogenetic tree was constructed using the MEGA Software package (version 7.0) (68 (link)). Bootstrap values were calculated with 1000 replicates.