论文部分内容阅读
利用不同的引物、引物浓度、复性温度、ｄＮＴＰｓ浓度、Ｔａｑ酶含量等对棉花品种鄂荆１号、ＰｉｍａＳ－３进行ＲＡＰＤ分析。结果表明，高温复性有利于提高扩增带型的特异性。低Ｍｇ２＋浓度易导致小片断扩增，高Ｍｇ３＋浓度易产生非特异带型。ｄＮＴＰｓ浓度增加导致扩增带型的数目减少。双引物扩增带型与卓引物相比可以检测到更多的品种间遗传差异。根据上述分析，棉花ＲＡＰＤ分析反应物组成为：１Ｘ反应ｂｕｆｆｃｒ、１．８ｍｍｏｌ．Ｌ－１Ｍｇ２＋、０．１６ｍｍｏｌ·Ｌ－１ｄＮＴＰｓ、４０ｎｇＤＮＡ、０．６μｍｏｌ．Ｌ－１随机引物、Ｔａｑ酶１．５单位，加双蒸水至２５μｌ反应体系。
The cotton cultivars E Jing 1 and PimaS-3 were analyzed by RAPD using different primers, primer concentration, refolding temperature, dNTPs concentration and Taq enzyme content. The results showed that refolding at high temperature was beneficial to improve the specificity of the amplified bands. Low Mg2 + concentration easily lead to small fragment amplification, high concentration of Mg3 + easy to produce non-specific banding. The increase in dNTPs concentration leads to a decrease in the number of amplified bands. Double-primer amplification bands can detect more genetic differences among cultivars than the same primers. Based on the above analysis, the cotton RAPD assay reagent composition: 1X reaction buffcr, 1.8mmol. L-1Mg2 +, 0.16 mmol·L-1 dNTPs, 40 ng DNA, 0.6 μmol. L-1 random primer, Taq enzyme 1.5 units, plus double distilled water to 25μl reaction system.