Cloning, sequencing and molecular characterization of 5S ribosomal RNA genes

For analysis of S. polyrhiza 5S rRNA genes, the specific DNA fragments were amplified by PCR from the same samples of genomic DNA used for the barcoding (Chen et al., 2022). In the standard PCR, we used the primers DW-5S-F and DW-5S-R, which are specific for 5S rDNA ( Supplementary Table 1 ), and followed the protocol of Borisjuk et al. (2018). The optimized protocol included the specific Taq polymerase buffer GCI (Takara, Dalian, China), which is designed to amplify GC-rich regions, and an increase in the number of amplification cycles to 40, as applied in (Stepanenko et al., 2022). After separately cutting PCR products of ~0.5 and 1–1.2 kb out of the gel and DNA purification using AxyPrep DNA Gel Extraction Kit (Axygen, United States), the generated fragments were cloned into the vector pMD19 (Takara, Dalian, China) and sequenced using a custom service provided by Sangon Biotech (Shanghai, China). The obtained forward and reversed sequences were assembled and analyzed using the CLC Main Workbench (Version 6.9.2, Qiagen) software. The sequencing data for the S.polyrhiza 5S rDNA clones are available at the NCBI Database (accession numbers {"type":"entrez-nucleotide","attrs":{"text":"OR841168","term_id":"2624773547"}}OR841168 through {"type":"entrez-nucleotide","attrs":{"text":"OR841270","term_id":"2624773649"}}OR841270).

The secondary structure of the 5S rRNA was analyzed using CLC Main Workbench (Version 6.9.2, Qiagen) software, based on a modified version the dynamic programming algorithm for free energy minimization (Zuker, 1989).