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以加工番茄为试验材料,采用PCR-SSCP分子标记技术,对DNA提取方法、PCR反应程序、聚合酶链式反应-单链构象多态性分子标记体系的电泳条件、变性剂等诸多因素进行了研究,筛选和建立了多态性检出率高、带型清晰、重复性好的PCR-SSCP反应体系和反应程序。结果表明:采用CTAB法提取番茄叶片DNA时,在CTAB缓冲液中加入5mol/L NaCl,第一次抽提离心速度和离心时间分别为8 000r/min和15min,可以获得高质量的DNA,PCR扩增程序为94℃5min,94℃1min,52℃30s,72℃1min,72℃10min,29个循环,退火温度52℃,引物大小100~300bp、98℃变性10min,非变性聚丙烯酰胺凝胶浓度8%,电泳1.5~2.5h,聚丙烯酰胺凝胶中不添加甘油为加工番茄PCR-SSCP分子标记体系最佳的条件组合。
The processing tomato was used as experimental material, and the factors such as DNA extraction method, PCR reaction program, electrophoresis conditions of PCR-SSCP polymorphic marker system and denaturing agent were carried out by using PCR-SSCP molecular marker technology The PCR-SSCP reaction system and reaction program with high detection rate, clear band pattern and good repeatability were established, screened and established. The results showed that when CTAB method was used to extract DNA from tomato leaves, 5 mol / L NaCl was added into CTAB buffer. The first centrifugation speed and centrifugation time were 8 000 r / min and 15 min, respectively. The amplification procedure was 94 ℃ for 5min, 94 ℃ for 1min, 52 ℃ for 30s, 72 ℃ for 1min, 72 ℃ for 10min, 29 cycles, annealing temperature 52 ℃, primer size 100 ~ 300bp, 98 ℃ denaturation 10min, non-denaturing polyacrylamide gel Gel concentration of 8%, electrophoresis 1.5 ~ 2.5h, polyacrylamide gel is not added for processing PCR PCR-SSCP molecular marker system the best combination of conditions.