Establishment of AFLP Reaction System for Salix integra and Primer Selection

Taking Salix integra F1 progeny as experimental material, the genomic DNA was extracted from the leaves by modified CTAB extraction method. Subsequently, the optimized AFLP experimental protocol was established through enzyme incision, conjunction, preamplification and selective amplification. 256 pairs of primer combinations were composed with 16 EcoRI and 16 MseI selective amplification primers respectively. The results showed that clear and repeatable gel profiles could be obtained under the reaction protocol as: 320 ng genomic DNA was digested at 37℃ for 6 hours by 5U of EcoRI and 5U MseI, then conjugated at 20℃ for 12 hours, the ligation products were 10 times diluted, and used as the template for preamplification. After preamplification, the products were diluted for 15 times, then used as templates for selective amplification. The selective amplification products were detected by ABI-3130 electrophoresis and the clear and repeatable gel profiles were obtained. 98 pairs of primer combinations that yielded high polymorphic bands were selected from the 256 pairs of AFLP primer combinations for AFLP analysis of S. integra.