【作者】 杨永超；

【导师】 李焕秀；

【作者基本信息】 四川农业大学 ， 蔬菜学， 2007， 硕士

【摘要】 本试验采用了22个番茄材料，调查了它们的生物学性状和室内品质，运用CTAB法提取了材料的DNA，并对RAPD反应条件，各反应试剂浓度和琼脂糖浓度进行了优化，其主要结果如下：1．利用改良CTAB法提取了材料的DNA，其OD₂₆₀／OD₂₈₀比值在1.8～2.0之间，OD₂₆₀／OD₂₃₀大于2，而且电泳图谱清晰可见，所提取的DNA含有较少的杂质，符合RAPD的要求。2．采用正交试验设计的方法，建立了RAPD优化反应体系。所得最优体系为：25ml中含有模板DNA40ng，引物0.25μmol／L，dNTP200μmol／L，Tapase1.25U，10×PCRbuffer2.5μL，25m mol／LMgCl₂，1.5μL，ddH₂O14μL。适宜的扩增条件为94℃预变性5 min，94℃变性30s，36℃复性30s，72℃延伸1min，40个循环后，在72℃保持7分钟。3．琼脂糖浓度在1.6％和2.0％时都能将不同的DNA分开，而且电泳图谱清晰可见，本试验选用1.6％的琼脂糖浓度。4．从40个随机引物中筛选出11个多态性好的随机引物，用这11个引物对22个番茄材料进行扩增，11个引物共扩增出了74条DNA带，平均每个引物6.72条，其中61条具有多态性，占总条带数的82.4％，平均每个引物5.1条多态性带。5．对供试22个材料进行了聚类分析，亲缘关系分析，发现F1有偏向于父本遗传的趋势。对遗传距离和单果重、株高、可溶性固性物含量进行了相关性分析，结果表明均呈不显著相关。用RAPD遗传距离对杂种优势的预测有待进一步研究。更多还原

【Abstract】 Twenty two tomato materials were adopted and their agronomic traits and quality were surveyed. CTAB was applied to extraction materials’ DNA, then, the reaction of RAPD, the concentration of agent that referred to the RAPD and concentration of agarose were optimized. The results are as follows:1. The improved CTAB was applied to extract DNA. The ratio of OD260/OD280 was from 1.8 to 2.0 and OD260/OD230 was more than 2. Furthermore, the results of electrophoresis was clear. All above indicated that the DNA was qualified for RAPD analysis.2. Orthogonal experiment design was used for setting up optimized RAPD reaction. Each sample was amplified in a reaction mixture （25μL in total volume） containing 40ng template DNA, 200μmol/L, deoxynucleoside triphosphate （SBS）, 1.25U of Taq polymerase, 5μL of 10×PCR buffer, 1.5μL of MgCl₂ and 14μL of ddH₂O. The reaction mixture was cycled through the following temperature profiles: 94℃for 5min, 94℃for 30s （predenaturation）, 36℃for 30s, and 72℃for 1min for 40 cycles. The PCR was terminated at 72℃for 7min.3. Both concentrations 1.6% and 2.0% of agarose can distinguish various DNA, but 1.6% was adopted for the sake of cost.4. 11 primers were screened out of 40 primers. The 11 primers amplified 74 DNA fragments and each primer can amplify 6.67 fragments. There were 61 polymorphic fragments among total 74 fragments, that accounted for 82.4% and each primer could amplify 5.1 polymorphic fragments average.5. The cluster analysis and relationship analysis were carried out and the results showed that the hybrid progenies F1 inclined to male inbred lines in heredity. The correlation analysis was performed between the genetic distance and plant height, mean fruit weight and total soluble solid, but the correlation coefficient was not significant, so the work of heterosis predicted by RAPD should be studied further.更多还原