*2.7. BrHSP18.2 Promoter Analysis*

The differential expression levels of the three *BrHSP18.2*s upon warming treatment prompted us to analyze cis-elements in their promoters. We designed a common forward primer based on a comparison of known *B. oleracea* genes, *B. napus* genes, three *B. rapa* genes (*BrHSP18.2A*: *Bra002539*, *BrHSP18.2B*: *Bra020295*, *BrHSP18.2C*: *Bra006697*), and AT5G59720, as well as specific reverse primers for each gene (Table S1). We obtained promoter sequences of different sizes (from the ATG start codon): 1257 bp for *BrHSP18.2A*, 969 bp for *BrHSP18.2B*, and 1199 and 1236 bp for *BrHSP18.2C*\_Chiifu and Kenshin, respectively (NCBI accession MH310903-8). The promoter sequences of *BrHSP18.2A* and *B* were identical between Chiifu and Kenshin, but the promoter sequences of *BrHSP18.2C* were not similar between the two inbred lines. However, the promoters of the three genes were different from each other but with partially conserved regions.

Despite sequence difference among the *BrHSP18.2A*, *B*, and *C* promoters, four HSE-binding modules [25] were present between −53 and −194 upstream of the ATG start codon: two head-to-head (nGAAnnTTCn) and two tail-to-tail (nTTCnnGAAn) modules (Figure S4). Two modules at −194 to −180 were overlapping. In *Arabidopsis*, eight HSEs (a(g,t,c)GAAn, a(g,t,c)GnAn, or a(g,t,c)Gann) have been detected between −97 and −53 bp in *HSP18.2*, and six HSE deletions were detected, leading to a loss of promoter activity [26]. In *BrHSP18.2*s, seven HSEs were present. The finding that *BrHSP18.2A*, *B*, and *C* possess sufficient numbers of HSEs for HSF binding, as well as possessing identical HSEs, points to the importance of having sufficient numbers of HSFs (or other elements) to control *HSP* expression levels.
