Aatii

To confirm the homologous integration of the deletion construct into the genome, gel-purified gDNA from potential Δpks1 and Δku80_Δpks1 transformants and their parental strains were digested overnight by PstI (Thermo). gDNA of Δku80 strains was digested overnight with AatII (Thermo). Digested gDNA was loaded on a 0.8% agarose gel, separated by electrophoresis, and transferred to a nylon membrane Hybond N⁺ (GE Healthcare). The probes for the upstream regions of pks1 (oligonucleotides 9 and 10) and ku80 (oligonucleotides 24 and 25) deletion constructs were synthetized using PCR DIG DNA Labelling Mix (Roche) and Taq DNA Polymerase (NEB). Probes were hybridized overnight in DIG Easy Hyb (Roche) at 42°C and detection was performed by chemiluminescence using anti-Digoxigenin-AP Fab fragments (Roche) and CDP-Star chemiluminescent substrate (Roche) in a ChemiDoc MP (Bio-Rad).

To find out the association between SiCYC1A and SiCYC1B alleles and floral symmetry phenotypes among F1 individuals, PCR-based restriction fragment length polymorphism (RFLP) was conducted. This PCR-RFLP procedure was divided into 4 parts: (1) isolation of genomic DNA, (2) performing a standard PCR, (3) digestion with appropriate enzymes, and (4) resolution digest of PCR products. The enzymes used for digesting SiCYC1A and SiCYC1B PCR product were AatII (#ER0991, Thermo) and PstI(#ER0611, Thermo), respectively. The two chosen enzymes were determined by the allelic variation of DA SiCYC1A and SiCYC1B. The protocol for digestion of PCR products after amplification followed the manufacturer's instructions.

To confirm the homologous integration of the deletion construct into the genome, gel-purified gDNA from potential Δpks1 and Δku80_Δpks1 transformants and their parental strains were digested overnight by PstI (Thermo). gDNA of Δku80 strains was digested overnight with AatII (Thermo). Digested gDNA was loaded on a 0.8% agarose gel, separated by electrophoresis, and transferred to a nylon membrane Hybond N⁺ (GE Healthcare). The probes for the upstream regions of pks1 (oligonucleotides 9 and 10) and ku80 (oligonucleotides 24 and 25) deletion constructs were synthetized using PCR DIG DNA Labelling Mix (Roche) and Taq DNA Polymerase (NEB). Probes were hybridized overnight in DIG Easy Hyb (Roche) at 42°C and detection was performed by chemiluminescence using anti-Digoxigenin-AP Fab fragments (Roche) and CDP-Star chemiluminescent substrate (Roche) in a ChemiDoc MP (Bio-Rad).