【作者】 段娟；

【导师】 汤浩茹； 王小蓉；

【作者基本信息】 四川农业大学 ， 果树学， 2007， 硕士

【摘要】 我国西南地区是悬钩子属植物（Rubus L．）的多样性中心，拥有丰富的种质资源，其野生资源在各种抗性、易成活性、果实风味都优于从国外引进的栽培品种，具有重要的栽培价值和育种价值。本实验采用RAPD分子标记方法，对采自西南地区空心莓组（Sect．Idaeobatus）和木莓组（Sect．Malachobatus）10个亚组的33份野生悬钩子属植物材料进行遗传多样性分析，以期为合理利用西南地区悬钩子属野生种质资源和将我国特有的悬钩子属植物基因资源引入现有优良品种，创造出新的适合我国悬钩子属植物生产的优良品种提供参考。其试验结果如下：（1）通过比较CTAB法、SDS法和核DNA法等三种不同DNA提取方法表明，核DNA法是提取悬钩子属植物DNA的一种理想方法，能得到高纯度高质量的DNA。（2）从85条引物中筛选出扩增效果较好的25条引物用于扩增供试材料，所有引物能够对材料进行有效的分离。得到扩增条带589条，每个引物扩增的DNA带数在19～33条之间，平均每个引物扩增23.56条带，其中多态性带580条，多态性为98.47％，其中16条引物扩增的多态性条带百分率高达100％，表现出较高的多态性。（3）通过反复试验建立的适用悬钩子属植物的RAPD技术最佳反应体系为：25μL反应体系中含模板DNA20ng，10×buffer2.5μL，Mg²⁺2.0mmol／L，引物0.6μmol／L，dNTPs0.24mmol／L，TaqDNA聚合酶1.5U。反应程序为：94℃预变性4min；94℃变性1min，36℃退火50sec，72℃延伸2min，45个循环；完成最后一个循环后，在72℃继续延伸10min，然后在4℃保温。最后用1.5％的琼脂糖凝胶电泳检测其扩增产物。（4）PCR-RAPD结果分析表明，我国西南地区的野生悬钩子属植物遗传多样性非常丰富，从分子水平上支持西南地区是悬钩子属起源中心的观点，且能为扩大树莓育种范围提供多种选择。（5）遗传差异分析表明，组和亚组间的进化顺序与形态学标记、孢粉学标记和细胞学标记相吻合，这为研究悬钩子属的系统演化提供了分子生物学依据。（6）33份材料可分为9类，空心莓组（Sect．Idaeobatus）和木莓组（Sect．Malachobatus）的亚组间交叉聚类，表明了野生悬钩子属植物遗传关系的复杂性。供试材料间的遗传距离（GD）变异范围为0.1957～0.9088，平均为0.5397。来源于雅安老板山和张家山的红泡刺藤（R．niveus）遗传距离最近，GD值为0.1957，来源于雅安的山莓（R．corchorifolius）与来源于西充的陕西悬钩子（R．piluliferus）的遗传距离最远，GD值为0.9088，聚类结果与形态学标记结果基本相似。（7）以上结果表明了RAPD技术在悬钩子属植物资源的鉴定、分类及遗传多样性研究上应用的可行性。更多还原

【Abstract】 The southwest of China was the center of diversity of Rubus L. and its gerrnplasm resource was abundant. Their resistance to various enviroments, survival rate and fruit flavour were better than the introduced cultivars from abroad, which were a very important characteristic for Rubus breeding. The genetic diversity of 33 materials belonging to 10 subsetion of Sect. Idaeobatus and Sect. Malachobatus collected from southwest of China were studied by using RAPD analyses in order to utilizate the wild germplasm resource better and introduce their special gene to varieties and create new superior cultivars. The results were as follows:（1） Compared DNA extraction of CTAB method, SDS method and nuclear DNA method. The results were shown that nuclear DNA method was a better DNA extraction method of Rubus L., the purity and quality of DNA was high.（2） The 85 random primers screened, 25 primers exhibited sufficient polymorphic band patterns. All the primers can discriminate the materials. Total 589 bands were produced, in which 580 bands were polymorphic. The numbers of DNA bands amplified by each primer were between 19 and 33. The average numbers of DNA bands amplified by each primer were 23.56, the percentage of polymorphic bands was 98.47%, in which the percentage of polymorphic bands of 16 primers was 100%. The materials showed high polymorphism.（3） The optimum reaction system of RAPD-PCR and program for Rubus L. were established. DNA was amplified in 25μL using the reaction mixtures containing 1×PCR buffer, 2.0mmol/L Mg²⁺, 0.24mmol/L of dNTPs, 1.5U of Taq Polymerase, 0.6μmol/L of primer, 20ng of template DNA. The PCR reactions were performed in a thermal cycler （eppendorf） programmed for 1 cycle of 4 min at 94℃followed by 45 cycles of 1 min at 94℃, 50 sec at 36℃and 2 min at 72℃for denaturing, primer annealing and extension, respectively. The last cycle was followed by incubation for 10 rain at 72℃. Amplification products were conserved at 4℃. Amplification RAPD products were analysed by gel electrophoresis run in 1.5% agarose.（4） The result of PCR-RAPD showed the genetic diversity of Rubus L. was abundant in the southwest of China, and it supported the view that the southwest of China was the center of the origin of Rubus L. at molecule level. Abundant germplasm resource could provide more breeding materials and enlarge breeding extent.（5） The genetic diversity of materials was studied and obtained evolution seriatim of subsetion. The results of RAPD analyses were coincident with morphological markers, pollen morphological markers and cytological markers. It demonstrated molecule markers could supply the molecular biology evidence for studying phylogeny of Rubus L..（6） The 33 materials were clustered into 9 groups. The results showed that the genetic relationship and diversity of wild Rubus L. were very complex. The genetic distance among 33 materials were between 0.1957～0.9088. The genetic distance between R. niveus from ya’an laoban mountain and zhangjia mountain was the nearest （0.1957）, while R. corchorifolius from ya’an and R. piluliferus from xichong was the farthest （0.9088）. The RAPD results were roughly resemble with the materials’ morphology.（7） RAPD analyses were useful for identification, classification and genetic diversity analyses of germplasm resource of Rubus L..更多还原