2.3. SSR Primer Evaluation in Eight Cultivars

A total of 960 pairs of primers were selected for synthesis (Ruibiotech, Co., Ltd., Beijing, China). To improve the efficiency of primer fluorescence labeling, the thermocycler amplification protocol was conducted in two rounds. First, the primers synthesized for the DNA of the eight cultivars were used for amplification. The 10 μL PCR mixture consisted of 0.1 μL of 10 μmol/μL forward primer containing the M13(-21) tail at its 5′ end and reverse primer, 1 μL of 30 ng/μL DNA, 5 μL of 0.1 U/μL 2×Taq PCR MasterMix (containing 0.05 units/μL Taq DNA polymerase (recombinant), 4 mM MgCl₂ and 0.4 mM dNTPs, Aidlab Biotechnologies Co., Ltd., Beijing, China) and 3.8 μL of ddH₂O. After an initial denaturation step of 95 °C for 5 min, 20 cycles of 95 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s, as well as extension at 72 °C for 10 min, were performed. Second, to efficiently and economically analyze the length of PCR products, fluorescently labeled (i.e., FAM, HEX, TAMRA or ROX) M13(-21) universal primers were added to the PCR mix [30]. The 10 μL PCR mixture contained 0.15 μL of 10 μmol/μL M13(-21) universal primer and reverse primer, 2 μL of the PCR product from the first round, 5 μL of 0.1 U/μL 2×Taq PCR MasterMix and 2.7 μL of ddH₂O. In the thermocycler, amplification was performed at 95 °C for 5 min and followed by 35 cycles of 95 °C for 30 s, 52 °C for 30 s and 72 °C for 30 s, as well as extension at 72 °C for 10 min. After 3% agarose gel electrophoresis, the amplified loci of the final PCR product were detected by a 3730xl DNA Analyzer with 96 capillaries (Applied Biosystems, Foster City, CA, USA) and sized with GS500 LIZ. The amplified loci were analyzed by GeneMarker V2.2.0.