DNA Extraction and SSR Marker Genotyping

The total genomic DNA of 160 accessions was extracted from fresh young leaves using a DNAsecure plant kit (Tiangen Biotech, Beijing, China), following the manufacturer’s instructions. The quality of the extracted DNA was determined by electrophoresis on 2% agarose gels and visualization using a UnicoUV-visible Spectrophotometer (Agilent, Palo Alto, CA, United States). Then, the DNA were diluted with deionized water to 20–30 ng/μl and stored in a freezer at –20°C.

The polymorphism of 140 previously developed SSR markers (Homolka et al., 2010; Hou et al., 2011; Gai et al., 2012; Zhang et al., 2012; Yu et al., 2013; Wu et al., 2014; Liu and Cheng, 2020a) was evaluated using a random sample of 12 accessions. After screening, a total of 81 EST-SSRs (Supplementary Table S3) were used to reveal the polymorphism of these 160 accessions. The SSR-PCR amplification reaction was conducted in a 10-μl solution, including 5 μl of 2×Power Taq PCR Master MIX (Aidlab Biotechnologies, Beijing, China), 3 μl of ddH₂O, 1 μl of 20–25 ng/μl genomic DNA, and 0.5 μl of 10 μmol/L each of forward and reverse primers, and the procedure was performed as described by Wu et al. (2014). The PCR products were differentiated by capillary electrophoresis using an ABI3730xl DNA Analyzer (Applied Biosystems, Carlsbad, CA, United States). Micro-Checker 2.2.3 (Van Oosterhout et al., 2004) was applied to identify and correct genotyping errors.