Genome Walking for GhCYC Promoter Regions

We applied genome walking for identification of promoter sequences for the gerbera CYC clade genes (Tähtiharju et al., 2012). Genomic DNA was extracted by the cetyl-trimethyl-ammonium bromide method (Chang et al., 1993), and depending on the given gene sequence, digested with a restriction enzyme (either EcoRV, DraI, PuvII, StuI, HindII, SspI, NaeI, or Eco47III). The Genome Walker Adaptor was ligated according to the instructions of the GenomeWalker Universal kit (Clontech Laboratories). The first PCR was performed with the corresponding adaptor primer1 (AP1, GER1) and gene-specific primer1 (GSP1), and the second PCR with the AP2 (GER2) and gene-specific primer2 (Supplemental Table S6), using the recommended conditions and the Advantage 2 Polymerase Mix (Clontech Laboratories). The PCR products for each GhCYC promoter were cloned into pGEM-T Easy Vector (Promega).