[Objective] This study aimed to establish an AFLP reaction system for spinach. [Method] Modified CTAB method was used for DNA extraction from six varieties of spinach. Experimental conditions in enzyme digestion and l...[Objective] This study aimed to establish an AFLP reaction system for spinach. [Method] Modified CTAB method was used for DNA extraction from six varieties of spinach. Experimental conditions in enzyme digestion and ligation, pre-amplification and selective amplification in the AFLP reaction system were analyzed and optimized. [Result] The results showed that： （1） enzyme digestion had better effects in the PCR instrument than in a water bath and it was not sensitive to the digestion time and DNA concentrations; （2） a total volume of 20 μl, containing 1.2 μl of Mg2＋ （25 mmol/L）, 1.6 μl of dNTPs （2.5 mmol/L） and 0.2 μl of Taq-polymerase （5 U/μl） was the optimal condition for pre-amplification and selective amplification reactions. [Conclusion] The AFLP system established in this study was stable and efficient, which provided a theoretical basis for AFLP molecular marker analysis of genetic relationship between spinach varieties and genetic breeding.展开更多
基金Supported by Beijing New Star Science and Technology Program(2008B37)Scientific and Technological Innovation Capacity Building Project of Beijing Academy of Agriculture and Forestry Sciences(KJCX201101010)~~
文摘[Objective] This study aimed to establish an AFLP reaction system for spinach. [Method] Modified CTAB method was used for DNA extraction from six varieties of spinach. Experimental conditions in enzyme digestion and ligation, pre-amplification and selective amplification in the AFLP reaction system were analyzed and optimized. [Result] The results showed that： （1） enzyme digestion had better effects in the PCR instrument than in a water bath and it was not sensitive to the digestion time and DNA concentrations; （2） a total volume of 20 μl, containing 1.2 μl of Mg2＋ （25 mmol/L）, 1.6 μl of dNTPs （2.5 mmol/L） and 0.2 μl of Taq-polymerase （5 U/μl） was the optimal condition for pre-amplification and selective amplification reactions. [Conclusion] The AFLP system established in this study was stable and efficient, which provided a theoretical basis for AFLP molecular marker analysis of genetic relationship between spinach varieties and genetic breeding.
