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The optimized AFLP analysis system was established for Alnus.A.glutinosa was used to comparatively study and analyze key factors influencing the results of AFLP,including quality and concentration of extracted DNA,enzyme digestion time and amplification system that influenced the results of the AFLP.The results suggested an amount of DNA template of 300 ng;reaction time for enzyme digestion of 5 h;ligation time of 16 h;a 10 times dilution of ligation products for pre-amplification;and a 20 times dilution of pre-amplification products for selective amplification.The AFLP-PCR reaction system(20 μ1) for Alnus was determined as follows:DNA template 300 ng,10×Taq DNA polymerase Buffer(containing Mg~(2+)) 2.0 μl,dNTPs 0.3 μl,primers 1.0 μl,Taq DNA polymerase 0.6U,and ddH_2O in balance.Seven pairs of primer combinations were selected with many clear bands and high polymorphism.By using the AFLP reaction system,primers suitable for Alnus were obtained.Those results provided certain basis for further molecular studies on Alnus using AFLP markers.
The optimized AFLP analysis system was established for Alnus. A. glutinosa was used to comparatively study and analyze key factors influencing the results of AFLP, including quality and concentration of extracted DNA, enzyme digestion time and amplification system that influenced the results of the AFLP. The results suggested an amount of DNA template of 300 ng; reaction time for enzyme digestion of 5 h; ligation time of 16 h; a 10 times dilution of ligation products for pre-amplification; and a 20 times dilution of pre-amplification products for selective amplification.The AFLP-PCR reaction system (20 μl) for Alnus was determined as follows: DNA template 300 ng, 2.0 μl of 10 × Taq DNA polymerase Buffer (containing Mg 2+), 0.3 μl of dNTPs, primers 1.0 μl , Taq DNA polymerase 0.6U, and ddH_2O in balance. Seventh pairs of primer combinations were selected with many clear bands and high polymorphism. By using the AFLP reaction system, the primers suitable for Alnus were obtained.Those results provided certain basis for further molecular studies on Alnus using AFLP markers.