【作者】 黄子锋；

【导师】 周厚高； 刘丕庆；

【作者基本信息】 广西大学 ， 作物遗传育种， 2003， 硕士

【摘要】 新铁炮百合（Lilium formolongi）是台湾百合（L.formosanum）和麝香百合（L.Longiflorum）种间杂种，是以种子繁殖生产切花为特色。其株系间亲和性较大，又可保留种球，适合研究世代的遗传分化。本实验通过对自交株系的RAPD检测，探讨了其遗传多样性变异趋势和株系间的遗传分化。主要研究结果如下： （1）通过优化设计，确定了新铁炮百合的RAPD反应体系和反应程序参数。 单引物反应：dNTPs 150μmol／L，Bg²⁺1.5mmol／L，Taq聚合酶1U，引物primer 0.3μmol／L，模板DNA50ng，反应总体积为20μl。 双引物反应：dNTPs 200μmol／L，Mg²⁺2mmol／L，Taq聚合酶1.5U，引物primer 0.4μmol／L，模板DNA50ng，反应总体积为20μl。双引物的最佳反应成分浓度普遍比单引物的要高。 反应程序参数：（1）94℃预变性5min 1个循环；（2）94℃变性30sec，37℃退火50sec，72℃延伸1min，35个循环；（3）72℃延伸10min 1个循环；（4）4℃保存，待用。 （2）采用正交设计的极差法得出影响RAPD实验因素的大小顺序：①反应条件因素显著性程度依次为：Mg²⁺＞primer＞Taq＞dNTPs＞DNA；②反应参数因素显著性程度依次为：循环次数＞延伸时间＞复性温度＞复性时间。 门门 条单引物和 8个双引物用于新铁炮百合的 RAPD扩增，产生扩增总谱带达 198条，163条谱带是多态的，多态百分比达 引．8兄 双引物谱带的产生和单引物的谱带无必然关系。双引物可以扩增出单引物所没有的DNA片段，但也可能会连单引物的谱带也扩增不出来。 （4）用多态性百分率、相对多态性频度和 Shannon 遗传多样性指数评价世代间的遗传多样性变化。其中引物 SIg、5303＋S 366＼519＋5210、5469＋519、555＋530白 多态百分比 均为 100儿 羡现极高的多态性。F。世代、F。世代、F。世代多态性百分比分别为 5 9．1趴 5 6．6兄 4 8．0队 多态性百分比呈现出下＊ 白 趋势。 三个世代的 遗传多样性主 数分别为：1．749、1．530、1．292，也是呈现下降的趋势。在总变异中，群体内和群体间遗传多样性所占的比率分别为 76．2％和 23．8儿羡现出群体内大于群体间的趋势，这与新铁炮百合的繁育系统相符合的。 门)通过非加权算术平均数对群聚类法 uPGMA)构建品系间的系统聚类树。从三个数据集获得的聚类结果可以看出，其聚类树状图基本反应系谱关系：双引物扩增结果能较精确反应其系谱关系。在亲缘关系相近的品系，用RAPD方法能比较准确地推测到世代间的遗传多样性、遗传分化的细小变化。更多还原

【Abstract】 Lilium formolongi was an interspecific hybridization between Lilium formosanum and Lilium Longiflorum with the characteristics of producing cut-flower by seedlings. For strong compatibility between lines and preservable bulbs, Lilium formolongi was a good material for genetic diversity and genetic differentiation analysis. The trend of genetic diversity and genetic differentiation of selfed lines was studied based on RAPD markers. The results could be summarized as follows.1. By optimal experimental design, the conditions of RAPD were confirmed as follows.Single primer reaction: dNTPsl50 mol/L, Mg2+1.5mmol/L,Taq polymerase 1U, primer0.3 mol/L,DNA template 50ng,total volume 20 u 1 .Double prime reaction: dNTPs200 u mol/L, Mg2+2mmol/L, Taq polymerasel.5U, primer0.4 mol/L, DNA template 50ng, total volume 20 1 Concentrations of double primer were higher than those of single primer, except that of DNA template, for best effect.Reaction procedure parameters: (1)pre-denatured DNA at 94 C for 5 min,1 cycle; (2) denatured DNA 94 C for 30sec,annealed primer at 37C for 50sec,extended primers at 72 C for 1min ,35 cycles; (3)re-extended reaction at 72 C for 10min,l cycle; (4) Stored product at 4 C.2. A total of 163 polymorphic bands out of 198 reproducibleproducts were amplified from the selected 11 single primers and 8 double primers, corresponding to 81.8% polymorphism of the amplification bands. The bands generated by double primer had no obvious relationship with single primer. The bands generated by single primer had no relation with double primer either.3. To evaluate genetic diversity of generations, percentage of polymorphic bands, Shannon genetic diversity index and relative polymorphism frequency were studied. The percentage of polymorphic bands of primer S19, S303+S1366, S19+S210, S469+S19, S55+S30 were 100%. The percentage of polymorphic bands of F2, F3, F4 generations were 59.1%, 56.6%, 48.0% respectively, which showed reduction trend.4. The genetic diversities of three generations were 1.749, 1.530, 1.292, which also showed reduction trend. Among total variation, genetic diversity intrapopulation accounted for 76.2%, interpopulation for 23.8%. This result showed that genetic diversity of intrapopulations was higher than that of interpopulations.5. UPGMA cluster analysis could reveal original strain relationships. The data set from double primers revealed more accurately. RAPD markers could accurately reveal small change of genetic diversity and genetic differentiation among near genetic relationship.更多还原