DNA Extraction and SSR Genotyping of Camellia hainanica

The DNeasy Plant Mini Kit (Tiangen Biotech Co. Ltd., Beijing, China) was used to extract the DNA of the leaf sample, and the operation of this application was strictly conducted according to the manufacturer’s instructions. Likewise, a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA) and agarose gel assay were used to measure the quality of the DNA samples. Twelve highly polymorphic SSR markers were used to genotype 96 samples of C. hainanica (Table 1). The target DNA fragment was amplified using the fluorescently labelled TP-M13-SSR polymerase chain reaction (PCR) method (Schuelke, 2000 (link)). The universal M13 primers fluorescently labelled with 6-carboxy-x-rhodamine, 6-carboxy-fluorescein, tetramethyl-6-carboxyrhodamine, or 5-hexachloro-fluorescein and the forward primer with a universal M13 primer tail (5-TGTAAAACGACGGC CAGT-3) at the 5′ end were used for the previously described PCR method. The PCR procedure was as follows: denaturation for 2 min at 95 °C; 12 cycles of 30 s at 95 °C, 30 s at 64–59 °C (−0.5 °C per cycle), 1 min at 72 °C; then, 24 cycles of 30 s at 95 °C, 30 s at 65 °C, 1 min at 72 °C, and a extension of 2 min at 72 °C (Cui et al., 2021 (link)). PCR amplification was performed in a Thermal Cycler (Bio-Rad, Hercules, CA, USA), and an ABI 3730XL DNA Analyser (Applied Biosystems, USA) was used for the detection of PCR products. The fluorescence detection data from capillary electrophoresis were used for SSR analysis with the GeneScan™ 500 LIZ^® Size Standard as an internal standard, and allele analysis for each SSR locus was performed using GeneMapper version 3.7 (Applied Biosystems, Foster City, CA, USA).