大白菜基因组DNA的提取及AFLP反应体系的建立

1材料与方法1．1材料供试大白菜种质材料32份，分别由北京市农林科学院蔬菜研究中心品种资源库及蔬菜研究中心大白菜育种课题组和中国农业科学院蔬菜花卉研究所国家种质资源中期库提供。材料编号、品种名称、来源地见表1。

Studies on Genomic DNA Extraction and Establishment of AFLP Reaction System in Chinese Cabbage

Objective] The obtained clear AFLP fingerprint of Chinese cabbage provided basis for studies on the molecular markers of Chinese cabbage cultivars and the phylogenetic relationship among Chinese cabbage cultivars. Method] With the test materials of leaves of Chinese cabbages, the high-quality total DNA from leaves of Chinese cabbages was extracted by the modified CTAB method. DNA restriction-ligase reaction, pre-amplification and selective amplification were optimized, and the AFLP silver-staining reaction system for Chinese cabbage was established. Result] The quality of DNA template influenced restriction enzyme digestion and the subsequent ligase amplification reaction, while the modified CTAB extraction method could be used in AFLP analysis of Chinese cabbage to obtain a clear AFLP fingerprint. The optimum conditions for restriction enzyme digestion of genomic DNA from Chinese cabbage were as follows： 150 g DNA template, 12.5 μl reaction volume, 1.25 U Eco R Ⅰ, 1.25 U Mse Ⅰ and 5×Reaction Buffer with 4 h at 37 ℃. The ligation reaction with 2.5 h at 20 ℃ was the optimum condition. Six pairs of primers including E-AAC/M-CAG, E-AAG/M-CAC, E-ACA/M-CTG, E-ACT/M-CAC, E-ACT/M-CTT and E-ACT/M-CTC all had its own stable and clear patterns. Conclusion] With abundant bands and high polymorphism, AFLP selective amplification is an efficient molecular marker for genomic polymorphism of Chinese cabbage.