The volume per PCR was 20 μL. The reaction chemical components included 4 μL
10 ng/μL DNA, 2 μL 10X PCR buffer, 2 μL 25 mM Mg2+, 2.4 μL 2.5 mM dNTPs, 0.2 μL 5
U/μL Taq DNA polymerase, 1 μL 10 mM upstream primer, 1 μL 10 mM downstream primer,
and 7.6 μL of sterile water. One hundred and twenty primer pairs of 10 upstream primers and
12 downstream primers published by Li and Quiros (2001) were screened for effective amplification,
clear bands, high polymorphisms and reliability. The primers were synthesized by
Shanghai Biochemical Engineering Technology (Shanghai, China). The SRAP markers were
amplified using the following thermal cycle parameters: 5 min at 94°C; 5 cycles of 94°C for
1 min, 35°C for 1 min, and 72°C for 2 min; 35 cycles of 94°C for 1 min, 50°C for 1 min, and
72°C for 1 min; extension of 5 min at 72°C; and a final storage at 4°C. The PCR products
were separated and visualized using 2% agarose gel electrophoresis.