Top10 competent cells

Candidate gene prediction was based on the Brassica oleracea genome database (ftp://ftp.ensemblgenomes.org/pub/release-38/plants/genbank/brassica_oleracea, accessed on 1 October 2022). The functions of the genes in the interval were analyzed using the BLASTP tool from TAIR. The gene and promoter sequence of the genes were amplified from two parents genomic DNA with PrimeSTAR^®vMax DNA Polymerase (TAKARA, Dalian, China). All primers used for sequencing are listed in Supplementary Materials Table S3. The PCR products were purified using the Gel Extraction Kit (CWBIO, Beijing, China), introduced into the PMD 18-T Vector (Takara, Dalian, China), and transformed into TOP10 competent cells (CWBIO). The recombinant plasmids were sequenced by Genewiz (Tianjin, China) and sequences were aligned using DNAMAN 6 (https://www.lynnon.com/, accessed on 1 October 2022).

Gene annotation information of the target region was obtained from the Brassica database (http://brassicadb.org/brad/index.php) and the Arabidopsis database (https://www.arabidopsis.org/). The candidate genes’ full-length sequence and a 2000 bp long promoter sequence were amplified with the specific primers using PCR, and the amplicons were purified using a Gel Extraction Kit (CWBIO, Beijing, China). The purified products were introduced into the pGEM^®-T Easy Vector (Promega, USA) and transformed into Top10 competent cells (CWBIO, Beijing, China). The colonies were sequenced at Sangon Biotech (Shanghai, China), and the sequences were aligned using DNAMAN 6.0 (Lynnon Biosoft, Canada). Meanwhile, PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) was used to predict the cis-regulatory elements in the promoter regions of the candidate genes.

The full length of candidate genes were amplified by full-lengths PCR primers in ‘FT’, wdm1, and wdm7. After the purification of PCR products with a Gel Extraction Kit (CWBIO, Beijing, China), we introduced the purified PCR products into a pGEM-T Easy Vector (Promega, USA). Then, the recombinant vector was transformed into the Top 10 competent cells (CWBIO, Beijing, China). The positive clones were sequenced by GENEWIZ (Tianjin, China).