Cloning and Sequencing of CsSEP Genes

Based on the annotated coding sequence (CDS) in the genome database (Yang et al., 2021 (link)), we cloned the CDS of CsSEP1, CsSEP2, CsSEP3, and CsSEP4 using specific primers designed by PrimerPremier 5.0. Total RNA was extracted from natural flowers of C. sinense ‘Baimo’ using the RNAprep Pure Plant Kit (TIANGEN, China) following the manufacturer’s instructions. RNA content was measured with a Nano-Drop 2000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE), and 1 μg of RNA was used to synthesize first-strand complementary DNA (cDNA) with the HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, China). The cDNA was then used as a template to clone the CDS of CsSEP genes with high-fidelity Taq DNA polymerase (Vazyme, China), and the gene-specific primers used are listed in Supplementary Table S3. The PCR products were purified using the FastPure^® Gel DNA Extraction Mini Kit (Vazyme, China) and cloned into the pCE2 TA/Blunt-Zero Vector (Vazyme, China) for transformation into DH5α (Tiangen, China). Positive clones (8-10) were selected for identification and sequenced by the Sangon Company in Shanghai. The plasmids were extracted with the FastPure^® Plasmid Mini Kit (Vazyme, China).