2.2. DNA extraction, AFLP and marker scoring
The total DNA was extracted from the dried leaves by using a Plant Genomic DNA Kit (Bioteke Beijing Co. Ltd., Beijing, China). The DNA samples were diluted to 10 ng/μl and stored at -20°C until further analysis. AFLP marker was carried out according to the method of Vos et al. [30] with the following little modifications: the digestion–ligation reaction was performed in a 10 μl containing 1μl of 10 × T4 ligation buffer, 0.2 mM of ATP, 50 ng DNA, 1 U of T4 DNA ligase (Fermentas, Shenzhen, China), 1 U of Eco RI (Fermentas, Shenzhen, China), 1 U of Mse I (New England Biolabs, Beijing LTD), 1.0 μM ofMseI -Adapter and 0.1 μM of Eco RI-Adapter and double-distilled water. The reaction was incubated at 37 °C for 8 h, 16 °C for 4 h, inactivated at 65 °C for 10 min, and stored at 4 °C. Pre-amplification was performed in a 25μl containing 2.5 μl of 10× PCR buffer and 0.5 U of Taq polymerase(Transgen Biotech Beijing Co. Ltd., China ), 2.5μl of diluted digestion–ligation product, 0.2 μM of dNTPs, 0.3 μM each of primers with a single selective nucleotide. The pre–amplification conditions was as follows: pre-denatured at 94 °C for 5 min and 30 cycles of 94 °C for 30 s, 56 °C for 60 s, and 72 °C for 80 s, with a final extension for 10 min at 72 °C. The pre-amplification products were diluted 1 to 40 (v/v) with ddH2O.
The selective amplification was essentially the same as that for pre-amplification except that 2 μL diluted pre-amplification product was used as template, and 2 μM Eco RI and Mse I selective primer were used. 10 pairs of primers were selected for selective amplification (Table A ). The selective amplification reaction had two cycle sets: pre-denatured at 94 °C for 5 min, 13 cycles of 94 °C for 30 s, 65 °C (which was lowered 0.7 °C at each cycle) for 30 s and 72 °C for 60 s, followed by 18 cycles of 94 °C for 30 s, 56 °C for 30 s and 72 °C for 80 s. After selective amplification, the products were added 25 μl of formamide loading buffer (98% deionized formamide, 10 mM EDTA, 0.1% bromophenol blue, and 0.1% xylene cyanol). Then the products were denatured at 95 °C for 5 min and quickly cooled on ice, and separated on 6% denaturing polyacrylamide gel in 1× TBE buffer at 70 W for 4.5 h. Gels were stained according to the silver staining method(Bassam BJ, 1991).