2.4. SSR and SRAP genotyping
Twenty-four primers (Supplementary Table 2) selected from recently published 53 SSR primers were used in this study (Narina et al., 2011; Nemorin et al., 2013; Loko et al., 2016). The primers were synthesized by Shanghai Sangon Biotech, Co., Ltd. PCR amplification reactions were performed using a master mix solution of 10 μL containing 5 μL of 2 × Master Mix Blue (TSINGKE, China), 0.25 μL of each prime (10 mM), and 0.75 μL of DNA template (20 ng/μL), and the remaining volume was supplemented with ddH2O. The following cycling parameters were used in the amplification reaction: first pre-denaturation at 94 °C for 5 min, followed by 40 cycles of 30 s at 94 °C, annealing for 30 s at 54 °C, 30 s at 72 °C, and a final extension of 10 mins at 72 °C. The amplified PCR products were detected on an 8% non-denaturing polyacrylamide gel. Silver nitrate staining was employed, and the image was captured for analysis.
Forty-nine different SRAP primer combinations (Supplementary Table 3), seven forward primers (Li and Quiros, 2001), and seven reverse primers were employed. Twenty-nine primer combinations with good repeatability and high polymorphism were also selected. Each 14 μL of the PCR reaction mixture consisted of 7 μL of 2 × Master Mix Blue, 0.35 μL of each primer (10 mM), and 1.4 μL of DNA template (20 ng/μL), and the remaining volume was supplemented with ddH2O. PCR amplification was performed under the following conditions: denaturation at 94 °C for 5 min, five cycles of three steps: denaturation at 94 °C for 1 min, annealing at 35 °C for 1 min, and elongation at 72 °C for 1 min. In the following 30 cycles, the annealing temperature was increased to 56 °C, with the final extension step of 10 min at 72 °C. The amplified products were analyzed through 3% agarose gel electrophoresis prepared in 1 × TBE buffer. The gels were then visualized in a UV transilluminator (Bio-Rad GeL Doc XR+, USA) and photo-documented.