*4.4. Gene Cloning and Sequence Analysis*

To analyze the intron sequence of *BrHSFA2* and promoter sequences of *B. rapa* small *HSP18.2* genes (*BrHSP18.2*s), genomic DNA was cloned and analyzed. Genomic DNA was isolated from Chiifu and Kenshin leaves using a DNeasy Plant Mini kit (Qiagen GmbH, Hilden, Germany). Primers were designed based on sequences listed in the BRAD website (Table S1). Genomic PCR was performed under the following conditions: denaturation (5 min at 94 ◦C), 30 cycles of amplification (30 s at 94 ◦C, 30 s at 52 ◦C, and 3 min at 72 ◦C), and a final extension (7 min at 72 ◦C). The PCR products were purified using a MEGA-Spin Gel Extraction kit (Intron Biotech. Inc., Sungnam, Korea) and cloned into the TA-vector using a T&A Cloning kit (RBC Bioscience Corp., New Taipei City, Taiwan). *Escherichia coli* (DH5α) cells were transformed with plasmid DNA carrying the desired insert. Plasmid DNA was purified using DNA-Spin (Intron Biotech. Inc., Sungnam, Korea) prior to sequencing (Macrogen, Seoul, Korea). To eliminate PCR and sequencing errors, at least 10 clones per gene were sequenced and analyzed. Any possible PCR and/or sequencing errors were eliminated by aligning independent sequences (http://www.genome.jp/tools-bin/clustalw).
