*2.4. Genomic DNA Extraction, PCR Amplification, and Electrophoresis Detection*

Genomic DNA of all the materials was extracted using 1 g of young leaf sample with the cetyl trimethyl ammonium bromide (CTAB) method [30]. The extracted DNA was dissolved in 1× Tris-EDTA (TE) buffer (Solarbio, Cat: T1121). The concentration and purity were detected by the Nanodrop-2000 nucleic acid analyzer. The extracted DNA was diluted to 30 ng/μL as working solution and kept at 4 ◦C.

Each PCR reaction contained 1 μL of template DNA, 0.5 μM each of forward and reverse primers, 5 μL mastermix (GenStar, Cat: A012-105), and 3 μL ddH2O. The amplification was carried out as follows: An initial denaturing step at 95 ◦C for 5 min, 94 ◦C for 30 s, followed by 6 cycles of 68–58 ◦C for 45 s. Each cycle was reduced by 2 ◦C, each annealing time was 1 min, and 72 ◦C for 1 min; 30 cycles of 94 ◦C for 30 s, 50 ◦C for 30 s, and 72 ◦C for 1 min. In the last cycle, primer extension was performed at 72 ◦C for 10 min.

The PCR products were analyzed by 9% polyacrylamide gel electrophoresis, and a 100 bp DNA ladder was used as the reference marker. After electrophoresis, silver staining was performed to display the PCR products, and photos were taken for preservation.
