【作者】 刘维侠；

【导师】 谢宝贵；

【作者基本信息】 福建农林大学 ， 蔬菜学， 2004， 硕士

【摘要】 色泽是金针菇的重要性状之一，受一对等位基因的控制。本研究以FL19（黄色）、8801（白色）及其后代（F₁代、F₂代）菌株和相应单核菌株为材料，通过对金针菇DNA提取方法和RAPD扩增条件的优化，建立了一套适于金针菇RAPD反应的反应体系；在此基础上，通过RAPD分析和BSA法相结合，成功的筛选到一个与金针菇白色基因紧密连锁的RAPD标记S375₁₈₀₀，并根据序列分析结果将RAPD分子标记转化为重复性和特异性更好的SCAR分子标记。主要结果如下： 1．DNA提取条件优化 通过正交实验设计，在4个因子（菌丝培养方式、菌丝处理方式、氯化苄浓度、SDS浓度）的3个水平共9个处理方面对氯化苄法提取DNA进行了探讨，所提取的DNA在OD₂₆₀处的数据结果和电泳结果均显示，处理3（菌丝静止培养，液氮研磨，氯化苄浓度为5ml／g菌丝，SDS浓度为20％）效果明显优于其它8个处理。 2．RAPD扩增条件优化 运用单因素筛选方法，分别对不同的Mg²⁺浓度、模板DNA浓度、引物浓度、dNTP浓度、Taq酶浓度以及不同的退火温度进行了筛选，最后得到金针菇PCR扩增的最佳反应体系为：2.5μl Buffer，2mM MgCl₂，50ng DNA，0.2μM Primer，100μM dNTP，1.25U Taq酶，最后用无菌超纯水补至25μl。 3．色泽基因的RAPD标记筛选 根据BSA法的原理，分别从黄、白色菌株中选取5株（单核4株，双核1株），提取菌丝DNA混合组成两个基因池，然后用200个10碱基对的随机引物进行RAPD多态性分析，筛选到一个稳定的多态性标记S375₁₈₀₀，通过对FL19、8801及其后代共75株单核菌株和13株双核菌株的验证，证明该标记与白色基因是紧密连锁的。 4．RAPD标记转化为SCAR标记 对上述RAPD标记S375₁₈₀₀片段进行了克隆和两端测序，并根据序列分析结果将上述RAPD分子标记转化为重复性和特异性更好的SCAR分子标记。利用该标记对F₁代单核菌株，以及不同来源的32个双核菌株和其中6株的90个单核菌株分别进行了验证，证明了SCAR标记的可靠性，同时说明用SCAR标记可以快速、准确的鉴别出金针菇颜色的基因型，从而可以有效地指导白色金针菇品种选育，加速育种进程。更多还原

【Abstract】 Color is one of the most important traits of Flammulina velutipes, and it is controlled by a pair of alleles. In our studies, the strain FL19(yellow),8801(white), their monokaryotics and their offspring (F1, F2 generations),as well as the F1 generations’ monokaryotics were used. A reaction system which accommodated RAPD reaction was established by optimizing DNA extracting and RAPD conditions. Through the combination of RAPD and BSA, a molecular marker S3751800 linked tightly to the white gene was found, and was converted into SCAR marker, which performed better than RAPD marker at repetition and particularity. Major results are as follows:1. optimization of DNA extraction condition Experiment was designed by using table of orthogonal arrays for the extraction DNA with Benzyl chloride, at three levels , by four factors (mycelium cultivation, mycelium treating, concentration of Benzyl chloride, concentration of SDS) , nine treatments in total. The OD260 and electrophoresis results on the DNA extracted showed that treatment No.3 (mycelium was cultivated statically and abraded with liquid nitrogen, concentration of Benzyl chloride was 5ml/g mycelium, concentration of SDS was 20%) has better results than others.2. Optimization of RAPD amplifying conditions different concentration of Mg2+, dNTP, primer, template DNA, Taq DNA polymerase and different annealing temprature were tested respectively by momofactorial screening. Eventually we obtained the optimal reaction system as follows: 2.5 ul Buffer, 2mM MgCl2, 50ng DNA, 0.211M Primer, 100 u M dNTP, 1. 25U Taq DNA polymerase, and add ddH2O1 to 25 u1 as terminal volume.3. screening of RAPD markers linked to color gene By the principle of Bulked Segregant Analysis,five white color DNA samples and five yellow DNA samples were prepared and mixed into two DNA pools: one white, and the other yellow, 200 RAPD primers were screened to identify the markers linked to thecolor gene of Flammulina velutipes. A RAPD marker S3751800 which was tightly linked to the color gene was obtained, which was confirmed by testing 75 monokaryotics and 13 homokaryotics.4. Conversion of the RAPD marker into SCAR marker In order to convert the RAPD marker into SCAR marker, a 20-mer specific primer was constructed and used for PCR amplifying. The white color-linked SCAR marker was obtained. The test on the offspring of F1 generation, as well as 32 homokaryotics from different sources and 90 monokaryotics about 6 homokaryotics showed that monokaryotics and homokaryotics with different color could be identified with the SCAR marker. The molecular marker offered a powerful tool for selection of white color strains and could speed up the process of Flammulina velutipes Breeding programmes.更多还原