小麦基因组AFLP反应体系的建立

The optimization of AFLP system for wheat genome

利用小麦抗叶锈近等基因系研究了小麦基因组AFLP反应体系,建立了适于小麦基因组的AFLP反应体系:40μL酶切体系中采用7.5UPstⅠ和5UMseⅠ37℃3h,65℃3h双酶切0.1～0.2μgDNA,然后加入10μL接头、T4连接酶混合液,16℃连接过夜;吸取未稀释的酶切连接液4μL,加入75ng预扩增引物,10mmol/LdNTP,1.5UTaq酶,1×buffer,加PCR水至40μL进行预扩,将预扩产物稀释20倍,吸取5μL,加入40ng选择性引物,1.25UTaq酶,7.5mmol/LdNTP,2%去离子甲酰胺,1×buffer,加PCR水至25μL进行选择性扩增。本研究为小麦抗叶锈基因的分子标记克隆及抗病育种的辅助选择提供有力的工具。

Some researches on AFLP protocol for wheat genome were done in T-gradient Thermal cycler PCR with wheat leaf rust Near-Isogene Lines. A 0.1～0.2 μg quantity of genomic DNA was digested with 7.5 U of PstⅠand 5.0 U of MseⅠat 37℃ for 3 hours and then 65℃ for 3 hours, ligation was done with T4 DNA ligase at 16℃ for 3 hours or O/N. The pre-selective amplification were set up with 4 μL restriction-ligation samples DNA,75 ng pre-amplification primers each, 10 mmol/L dNTP,1.5 U of Taq DNA polymerase, 1×buffer and added PCR water to a total volume of 40 μL. Selective amplification was carried out using 5.0 μL of 20×diluted pre-amplification DNA, 40 ng selective amplification primers each,1.25 U of Taq DNA polymerase,7.5 mmol/L dNTP,2% deionized formamide,1×buffer and added PCR water to a 25 μL volume. It was a base work for further researches of AFLP for wheat and supplies a strong tool for molecular cloning and breeding of assisted selection.