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Construction of AFLP Silver-staining Analysis System in Tobacco

DU Chuan-yin1,3,4,WANG Yu-jun2,LI Si-shen3,LI Chang-bao3,LIU Hong-xiang4,TIAN Ji-chun3 (1.Weifang Tobacco Limited Corporation,Weifang 261031,China;2.Plant Protection College,Shandong Agricultural University,Taian 271018,China;3.Agronomy College,Shandong Agricultural University,Taian 271018,China;4.Qingzhou Tobacco Research Institute of CNTC,Qingdao 266061,China)  
AFLP(Amplified Fragment Length Polymorphism) silver-staining analysis system was established with 14 tobacco cultivars.The results of several reactions in the analysis system were as following: tobacco genomic DNA was extracted by CTAB method;the double enzymes restriction and the adapter-ligation were completed at the same time;E+A and M+C primer combinations were used in pre-amplification while E+3 and M+3 primer combinations in selective amplification;the PCR reaction products were analyzed on 6% denatured polyacrylamide gel and then stained by silver.With the optimized system,the AFLP fingerprinting of 3 tobacco cultivars amplified by 20 primer combinations had been obtained.
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