【作者】 马艳芝；

【导师】 张玉星；

【作者基本信息】 河北农业大学 ， 果树学， 2003， 硕士

【摘要】 梨为蔷薇科（Rosaceae），梨亚科（Pomoideae）之梨属（Pyrus L．）植物。世界梨属植物约有60余种，分布于欧、亚、北美洲及南半球温带地区。我国是世界梨属植物的起源中心之一，蕴藏的梨属种质资源极为丰富。本研究借助RAPD技术对鸭梨及其9个变异系和其它44个梨属植物进行了DNA水平上的研究，旨在探讨RAPD技术在梨树芽变鉴定中的作用；通过对梨属部分种质进行鉴定和亲缘关系分析，解决例如苹果梨等在分类上存在争议的品种的归属问题，为梨属种质的科学利用和分子标记辅助育种提供参考。本研究取得的主要结果如下： 1．通过系统的比较，反复验证，建立起了梨属植物RAPD分析的优化反应体系，即在25μL的反应体系之中含有100 mM KCl、8 mM （NH₄）₂SO₄、10 mM Tris-HCl（pH9.0）、2.0 mM MgCl₂、200μM dNTP、0.4 μM引物、40ng模板DNA、1.5UTaq DNA聚合酶。经与前人结果进行比较，认为梨属植物RAPD分析的最佳反应条件在不同实验室之间有较强的一致性，只需对引物、Taq DNA聚合酶和Mg²⁺的用量进行小幅度调整。 2．从50个随机引物中筛选出18个多态性引物，并进行了44个供试梨属植物的RAPD扩增，每个引物扩增出的条带数在4—11之间，扩增出的DNA片段分子量大多在450bp—2000bp之间，共扩增出118个条带，其中样品间相同的条带数有11条，呈现多态性的条带107条，多态百分率达90.8％。 3．首次对9个鸭梨变异类型进行RAPD分析，并初步筛选出4个多态性引物即S28、S32、S176、S1450。研究发现： （1）芽变品种垂枝鸭梨（编号10）与其他类型相比较特异增加了1条条带，即S32-600。 （2）在芽变品种魏县巨鸭梨（编号2）、甜鸭梨（编号8）、垂枝鸭梨（编号10）的扩增产物中均少了2条特异性条带，即S28-400和S28-650。 （3）在芽变品种魏县巨鸭梨（编号2）和阎庄自花结实鸭梨（编号6）的扩增产物中均缺少了2条特异性条带，即S176-900和S176-1150。 （4）引物S1450的扩增产物在供试变异类型中虽未出现多态性，但是芽变品种晋县大鸭梨（编号4）的扩增产物在大约750bp处的条带丰度发生了变化，即晋县大鸭梨S1450-750是1条强带，而其它类型中此条带为1条弱带。 梨属（P）)ri’ L．埔物RAPD反应体系的建立及其应用研究 4．对供试44个梨品种资源的RAPD数据结果进行了聚类分析。结果表明，白梨系统与砂梨系统品种类型首先聚在一起，然后是秋子梨系统，最后与西样梨系统聚在一起。 5．通过聚类分析和遗传相似系数分析，重点探讨了苹果梨在DNA水平上的分类地位。认为将苹果梨归为白梨系统比较适宜。 6．通过聚类分析和遗传相似系数分析，探讨了库尔勒香梨在DNA水平上的分类地位。认为将库尔勒香梨归为白梨系统比较适宣。更多还原

【Abstract】 Pyrus plants belonged to Rosaceae, Pomaceae, Pyrus L. There were more than 60 species in the world, which were distributed over Europe, Asia, North America and the Temperate Zone area of the Southern Hemisphere. One of the origin centers of Pyrus was China, which was abundant in Pyrus germplasm resources deposits. Yali pear and it’s mutation types and other 44 Pyrus plants were studied using RAPD (Random Amplified Polymorphic DNA) technique. This study aimed at discussing the feasibility of RAPD applying on Pyrus germplasm resource, delving the function of RAPD in bud mutation identification of fruits. This study also aimed at delving and clearing the genetic relationships of partly Pyrus germplasm, which could find out the ownership of some cultivars, such as Pingguoli and so on. The main results were as follows:1. The optimized RAPD reaction system was established: in a total volume of 25 L, containing 100mM KC1, 8mM (NH4)2SO4, 10mM Tris-HCl(pH9.0), 2.0mM MgCl2, 200 M dNTP, 0.4 M primer, 40 ng template DNA, 1.5U Tag DNA polymerase. This condition was similar to those obtained in other labs, with little different in the concentrations of primer, Taq DNA polymerase and Mg2+.2. 18 primers selected from 50 ones were identified to be high polymorphic and were applied on RAPD amplification of 44 Pyrus plants. The amounts of bands were 4-11 and the molecular weight mainly was 450bp-2000bp. A total of 118 DNA bands were amplified, including 11 same bands and 107 polymorphic bands.3. Yali pear and it’s mutations were studied using RAPD technique at the first time. Four primers were selected from 50 ones. They were S28, S32, S176, and S1450. Theresults amplified based on the four primers as follows:(1) Compared to other mutations, the RAPD amplification of ChuizhiYali pear existed one specific band: S32-600.(2) Compared to other mutations, all of the RAPD amplifications of WeixianjuYali pear, TianYali pear and ChuizhiYali pear were short of two specific bands: S28-400 and S28-650.(3) Compared to other mutations, both of the RAPD amplifications of WeixianjuYali pear and Yanzhuangzihua Yali pear were short of two specific bands: SI76-900 and S176-1150.(4) There was no polymorphism among the mutation types, which were polymorphic by primer S1450, but the abundance of S1450-750 of JinxiandaYali pear was changed. The band S1450-750 of JinxiandaYali pear was one strong band, but that of others was one weak band.4. Basing on the RAPD data obtained, clustering analyses on the 44 materials of Pyrus were carried out, and the tree diagrams were established by Ward method. The relationships within different systems of Pyrus were discussed.5. Aiming at the existent demurral on systematic status of Pingguoli pear, we considered that Pingguoli should belong to White-pear system.6. Aiming at the existent demurral on systematic status of Ku’erlexiangli pear, we considered that Kuerlexiangli pear should belong to White-pear system.更多还原