【作者】 郭翎；

【导师】 束怀瑞； 沈向；

【作者基本信息】 山东农业大学 ， 果树学， 2009， 博士

【摘要】 观赏苹果(海棠)是我国传统名花,在我国传统园林中占很重要位置。近百年来,由于各种原因我国园艺水平与世界先进国家的距离加大了。西方人利用从我国及东亚收集的苹果属植物种质资源培育出大量的观赏苹果新品种。自改革开放以来,国民经济有了翻天覆地的变化,园艺水平也在不断提高,整理传统观赏苹果品种,引进及培育新品种是十分必要的。引种是育种的手段之一,也是收集、保存观赏植物种质资源的途径之一。大量引进国外品种,丰富我国观赏苹果品种多样性,同时挖掘整理现有国内的观赏苹果品种资源是非常必要的。观赏苹果品种经济价值高,通过研究其繁殖及栽培特性,繁殖大量苗木,推广到我国北方各省,所产生的经济价值不可估量。因此及时总结、分析、对比现有已引进的观赏苹果品种,总结引种经验,确定推广品种及范围,也是园艺工作者紧迫而现实的任务。苹果属植物分类系统多样而混杂,亲缘关系复杂。经典分类学对于处理好苹果属植物种、变种、杂交种及品种有其局限性,通过DNA指纹技术分析该属的亲缘关系,可以从分子水平对其分类及其亲缘关系的认识进行辅助性研究,对有目的的选择亲本组合,提高育种效率,对创造自主知识产权品种具深刻意义。本论文重点研究现代观赏苹果品种的引种、驯化与推广的理论与实践。并用AFLP和TRAP DNA指纹技术分析苹果属植物的亲缘关系及品种的起源等方面进行了探讨。并得出如下结果:1.本论文介绍了从美国、荷兰、比利时等国引进观赏苹果品种78个种及品种的引种过程,阐述了北京与引进品种选择、试种、观察记载、繁殖、中间试验及推广等引种过程的科学性;通过比较栽培地北京与引种地间光、热、水、湿差异,说明了环境因子对观赏苹果品种引种的影响;总结了部分观赏苹果品种在推广到青海、新疆、上海、黑龙江等我国北方地区的生长情况。2.本论文总结了33种观赏苹果新品种的形态学性状,36个新品种的物候特征及抗性指标;调查了65种引种到北京5年到20年的苹果属植物种及品种的生长情况,以及12种观赏苹果新品种推广到哈尔滨等我给北方各城市的情况;建立了观赏苹果品种引种成功的评价体系及评价标准,利用该标准对37个引进北京7年以上的观赏苹果做了评价,得出在所引品种中‘塞山’得分最高,是在北京表现最好的观赏苹果品种。为在我国北方进一步引、育及推广观赏苹果品种,为在此基础上培育我国具有自主知识产权的新观赏苹果品种提供了翔实的资料及科学基础。3.用AFLP (Amplified Fragment Length Polymorphism)技术,对72个苹果属植物样本(34个种、变种、杂交种及38个品种)进行遗传多样性分析。在64对AFLP引物中选出6对引物进行扩增,得到有效谱带1,692条,其中多态谱带占1,547条,多态谱带比率为91.4%。对72个样品的试实验数据进行聚类分析,它们的相似系数为从0.54到～0.82。研究发现:相似系数为0.60处时参试材料被分为4大类,佛罗伦萨苹果(Malus florentina)为一类;绿苹果组(Sect.Choromeles)和多胜苹果组(Sect. Docyniopsis)分别各聚一类;花楸组(Sect. Sorbomalus)、脱萼组(Sect. Gymnomeles)和苹果组(Sect. Malus)试材聚为一类,所有苹果品种和观赏苹果品种全聚在原产亚洲的花楸组、脱萼组及苹果组中。亚洲原产的苹果属植物对于苹果及观赏苹果品种的发展有着重要作用。根据AFLP试实验结果,及文献资料,支持建议将佛罗伦萨苹果组(Sect. Florentinae Cheng M.H.)成立单种组;建议将原属花楸组植物除佛罗伦萨苹果外,全部并入苹果组;取消脱萼组,将其组内的植物全部并入苹果组。4. TRAP是一种新的基于PCR的植物基因型标记技术,该技术采用两个18核苷酸引物产生标记,一个为固定引物,依据EST序列设计;另一个为随机引物,针对外显子和内含子的特点,设计为分别富含GC或AT核心区的任意序列。本论文用TRAP (Target region amplification polymorphism)技术对苹果属42种、变种、杂交种及品种进行分析。两个固定引物是用三叶海棠(Malus sieboldii)的matK基因片段设计而来,固定引物为AF309181L (5’GATTTGCAGTCATTGTGG-3)和AF309181R( 5’AAATGTGGGAATTTGTCC-3 ) ,随机引物为Sa12-700 (TTCTAGGTAATCCAACAACA)和Ga5-800 (GGAACCAAACACATGAAGA),Trap PCR扩增共得到197个清晰谱带,其中182个具多态性(92.4%),扩增片段大小从75bp到700 bp。用Crosschecker程序对扩增产物进行分析。将所得2进制的数据传入NTSYSpc 2.11S得出相似性系数SC(similarity coefficient)矩阵,并用非加权算术平均法(UPGMA)进行聚类分析。TRAP在研究苹果属植物上是非常有用的标记新技术。与AFLP实验相同,TRAP实验中也证实佛罗伦萨苹果不适合放在花楸组或苹果组,而应单独成立佛罗伦萨苹果组。更多还原

【Abstract】 1. Seventy Eight species and cultivars in Malus genus were introduced from abroad. The morphological characteristics, cold of hardiness, drought resistance, waterlogging tolerance and the occurrence and control of pests and diseases were studied. A system for evaluation of ornamental apple cultivar was set up and used in selecting 37 species and cultivars. Propagation methods and cultivation techniques of ornamental apple were studied.2. Amplified Fragment Length Polymorphism (AFLP) analysis was used to reveal the relationships of species in Malus. A total of 72 samples representing 34 species, varieties, and hybrid species, and 38 cultivars and an outgroup were assayed. Six pairs of primers combinations selected from 64 combinations tested produced 1,692 legible loci, of which 1,547 (91.4%) were polymorphic. The genetic similarity coefficient among these 72 samples varied from 0.54 to 0.82. Four groups of taxa were really recognizable based on the results of cluster analysis. First one is Malus florentina. The second clade contains the taxa of Sect. Choromeles. And the third includes the taxa of Sect. Docyniopsis. The taxa in Sect. Sorbomalus, Sect. Gymnomeles and Ssect. Malus of Malus gather in clade fourth. All cultivars of apples and flowering crabapples distributed in Asia into the last two clades. The wild species in Asia played a very important role in the development of cultivars in Malus. A new section (Sect. Florentinae Cheng M. H.) is supportedproposed based on by AFLP result. The Ssect. Sorbomalus and Ssect. Gymnomeles are merged into Sect. Malus (except Malus florentina).3. TRAP (Target region amplification polymorphism) was applied to elucidate relationships in Malus genotype in this article. TRAP is a novel PCR-based molecular marker technique which uses gene-based information for primer design. 42 Malus samples of 27 species, varieties, 8 hybrid species and 7 cultivars were chosen for using TRAP maker system analysis. Two fixed primers, designed by using matK gene sequences of Malus sieboldii, paired with two arbitrary primers, were used to apply TRAP PCR. A dendrogram was generated using the UPGMA method of the SAHN function depends on a total of 197 unambiguous bands of which 182 were polymorphic with an average of 45 polymorphic bands per primer combination and the percentage of polymorphic bands was 92.4%. The bands ranged in size from 75 to 700 bp. On the basis of TRAP PCR cluster analysis revealed that the closer relationships in the dendrodram of Malus samples are in agreement with the origin of these genotypes. TRAP is a potentially useful marker technique for genetic diversity studies in Malus genus.更多还原