甘薯AFLP分子标记体系建立

The Establishment of Technical System of AFLP Markers for Sweet Potato

AFLP是目前最常用的几种标记之一。本文以甘薯为材料,通过DNA提取、酶切、PCR扩增、凝胶电泳等系列程序摸索和优化,建立了甘薯的AFLP分子标记体系。优化的甘薯AFLP分子标记体系的程序如下:将4μl100ng/μl的甘薯DNA用EcoRⅠ酶、MseⅠ酶各5U进行双酶切,在37℃下酶切3h后再在65℃下酶切3h;然后加入10μl连接混合液在22℃下连接3h后在16℃下连接10h;取连接产物5μl,10μmol/LEcoRⅠ预扩引物和10μmol/LMseⅠ预扩引物各1.5μl,PCR缓冲液25μl,ddH2O17μl进行预扩增;取5μl稀释20倍后的预扩增产物,50ng/μlEcoRⅠ选扩引物和50ng/μlMseⅠ选扩引物各1μl,PCR缓冲液10μl,ddH2O3μl,进行选择性扩增。本研究为甘薯黑斑病和其他真菌性病害的分子标记克隆及抗病育种的辅助选择提供了有力工具。

Nowadays amplified fragment length polymorphism (AFLP) is one of the most regularly used molecular markers. In this paper,we used sweet potato as materials. Through the extraction of DNA、the digestion of DNA、PCR amplification and so on, we established the optimization technical system of AFLP markers for sweet potato:400ng sweet potato DNA were digested with 5U EcoRⅠand 5U MseⅠat 37℃ for 3 hours and then 65℃ for 3 hours, then added ligation mixture at 22℃ for 3 hours and 16℃ for 10 hours;pre-selective amplification was set up with 5μl restriction-ligation samples DNA, 10μmol/L pre-selective amplification primers 1.5μl respectively, PCR mixture 25μl, and ddH2O 17μl; after diluting the pre-selective amplification samples, selective amplification was set up with DNA of pre-selective amplification 5μl, selective amplification primers 1μl respectively, PCR mixture 10μl, ddH2O 3μl. Our results provided an useful tool to study the molecular cloning of sweet potato black rot and other fungus disease and were helpful for molecular assisted selection of sweet potato resistant breeding.