【作者】 杜运鹏；

【导师】 贾桂霞；

【作者基本信息】 北京林业大学 ， 园林植物与观赏园艺， 2014， 博士

【摘要】 百合是世界重要的观赏花卉,具有重要的经济价值。我国具有丰富的野生百合资源,但是目前破坏比较严重,濒危种不断增多,此外,开发利用程度也很低。关于百合资源的调查与评价大多集中于区域性,缺乏全面系统的了解,而资源的调查、评价和利用是种质创新的基础；抗病育种一直是百合育种的热点,百合灰霉病在百合生产中危害严重而广泛,但是目前有关百合抗灰霉病资源评价与筛选、抗病品种选育、抗病基因定位与克隆等方面十分薄弱。基于以上问题,本研究在资源系统调查与收集的基础上,从表型性状和AHP评价、亲缘关系和分子系统学、灰霉病抗性评价三个层面对百合野生资源进行了系统评价；进行了百合抗病基因同源序列的克隆与表达分析,为百合抗灰霉病基因的克隆奠定基础。通过上述研究,得到如下主要结果：1.本研究对我国百合属植物分布集中的东北地区、中部地区以及西南地区进行了系统的调查,收集资源34个种/变种,86份资源。调查发现我国百合属植物资源水平分布广泛,西南地区分布最为集中；在垂直分布上,从200-4000m都有分布,尤其是L. lancifolium、L. brownii和L. sargentiae的垂直分布范围较大；具有分布生境多样、群体内变异丰富、鳞茎增值方式和地下茎特点多样等特征；百合属植物更新进程较慢,易受环境的影响。对原生境土壤特性分析发现,不同种类野生百合原生境土壤的全氮、有机质含量、有效磷、速效钾、全盐及pH值都存在一定的差异；同种不同种源地原生境土壤特性也存在差异,其中L. davidii可作为培育耐盐碱百合的优良亲本；L.regale、L. lancifolium、L. henryi、L. rosthornii、L. leucanthum、L. sargentiae的适应性都较强,可作为抗逆育种亲本和应用于园林绿化。2.对表型性状分析发现,野生百合资源表现出显著的形态多样性。主成分分析表明,花器官因子是百合资源形态特征分化和在分类上的相对重要的指标。此外,基于观赏特性、开发潜力和生态适应性三个方面16个指标,对收集的27个种,3个变种进行了AHP评价筛选出L. dauricum、L. henryi、L. rosthornii、L. tsingtauense、L. concolor var. pulchellum、L. lancifolium及喇叭组百合等一批开发利用潜力较大的资源,可为我国野生百合资源的开发、利用和保护提供借鉴和参考。3.通过对我们收集的分布于中国所有的组的代表种25个种和5个变种,以及从目前已登录在NCBI的170份材料,共计98个种,5个变种的214份材料构建了目前百合属最大的ITS进化树。对目前百合属分类系统系统存在的关于subsect.5c Comber的划分sect.Leucolirion的修正以及L. rosthornii的系统位置进行了研究。通过分析,认为subsect.5c Comber应该划分为subsect.5c和sect. Lophophorum:而后者又被分为三个亚组subsect. Lophophorum Ⅰ,、,subsect. Lophophorum Ⅱ,及subsect. Lophophorum Ⅲ;通过ITS进化树和FISH分析,证明L. henryi和L. rosthornii亲缘关系较近,应该被划入subsect. Leucolirion6a;研究结果支持Comber基于鳞茎颜色对sect. Leucolirion的分组。中国分布的百合分为5个组：sect. Archelirion, sect. Leucolirion, sect. Sinomartagon,和sect. Lophophorum;建议将L. ducharteri和L.lankongense划分为sect. Sinomartagon的一个新的亚组。4.通过扫描电镜观察,发现百合属植物花粉大多为单粒花粉,首次在L.bakerianum花粉中发现了四合花粉；首次在L. formosanum花粉中发现一种新的外壁状纹饰,命名为Formosanum-type。通过ITS进化树与花粉外壁纹饰对应,表明百合花粉外壁纹饰对某些种或亚组有一定的分类学意义。基于花粉进化理论,补充和完善了百合属花粉外壁纹饰演化趋势：Martagon→Callose→Concolor→Formosanum。方差分析和相关性分析表明,年降水量与花粉性状有着显著的相关性；发现花粉大小与环境的选择压有关,小的花粉粒适应极端环境的能力更强。5.对SSR分子标记的遗传学参数分析,发现百合SSR位点多态性较高；聚类分析表明我国野生百合资源具有丰富的遗传多样性；主坐标分析表明野生百合聚类有明显的地域特征,从等位基因层面证实L. henryi和L. rosthornii与subsect. Leucolirion6a亲缘关系较近,L. brownii应该划入subsect. Leucolirion6b;同时也表明SSR标记可以有效的应用于百合种质资源的遗传多样性和亲缘关系分析。6.通过离体接种,筛选出8个高抗资源：L. leucanthum、L. regale、 L. sargentiae、 L. henryi、 L. rosthornii、 L. taliense、 L. dauricum及L. tsingtauense,可为百合抗灰霉病育种提供亲本材料,以及用于挖掘相关抗病基因。建立了百合RGA-PCR反应体系；通过抗性表型与抗病基因同源序列的多态性分析,发现百合扩增谱带较为丰富,而且抗性相似的趋于聚在一起。首次基于转录组数据,分离和克隆了百合NBS-LRR抗病基因同源序列；挑取1000个克隆进行序列分析,从系统发育树可看出百合RGA分为5个家族,氨基酸序列比对发现百合RGA均属于non-TIR-NBS类型,是逐步进化趋异,而不是快速进化过程；通过不同家族的百合NBS型RGAs在不同灰霉菌侵染时间的样品的表达分析可看出,都不同程度的参与了抗病反应；Southern杂交分析表明,百合RGA都以多拷贝形式存在。更多还原

【Abstract】 Lilium spp., as an important ornamental flowers in the world, has an important economic value. There are abundant wild resources in China. However, the resources were destroyed severely, and the endangered species was growing. In addition, the utilization of Lilium resources was not optimistic. Investigation, evaluation and utilization of germplasm resources is the basis of innovation, yet the investigation and evaluation of the Lilium mostly concentrated on some region, lacking of a comprehensive and systematic understanding of Lilium resources. Resistance breeding has always been a hot spot for lily breeding. The harm of gray mold is severe and extensive in cutting flower production of lily, however, the evaluation and screening of Lilium resources resistance to Botrytis elliptica, breeding for gray mold resistance, as well as the mapping and cloning of resistance gene is very weak. Therefore, evaluation and excavation of excellent resistance resources is very important! Due to the above problems, on the basis of investigation and collection of Lilium resources, this research evaluated resources at three levels:the phenotypic traits and the AHP, the genetic relationships and molecular systematics and the resistance to gray mold. Moreover, cloning and expression analysis of resistance gene homologous from Lilium was carried out, laying a foundation for cloning gray mold resistant genes. Through these studies, the main results obtained are as follows:1. A systematic investigation was carried out in the Northeast, Central and Southwest regions of concentrated distribution of Lilium, a total of34species (varieties) representing86samples of Lilium were found. The survey found that the level of Lilium resources are widely distributed, especially the southwest China. Besides, Lilium resources occupy a wide variety of habitats consists of forests, mountain, shrub and meadows. L. brownii, L. lancifolium and L. sargentiae have very wide ranges on the vertical distribution. Rich variation within populations was found. Features of Bulbs added and characteristics of underground stemsbulbs were various. Although reproductive capacity of Lilium is strong, the update process is slow, and therefore it is vulnerable to the environment. An analysis of soil characteristics of the original habitat found that different kinds of wild lily in situ soil nitrogen, organic matter content, phosphorus, potassium, full of salt and pH value were different, and soil characteristics of the same species from different provenances were also different. L. davidii can be used as an excellent parent for salt resistance breeding. Soils in situ of L. regale, L.lancifolium, L. henryi, L. rosthornii, L. sargentiae and L. leucanthum was relatively infertile, suggesting that they have good adaptability.2. An analysis of phenotypic trait found that wild lily resources showed significant morphological diversity. Principal component analysis showed that floral organ factors were the relative importance of indicators in morphological differentiation and the classification of resources. An analytical hierarchy process (AHP) utilizing16indicator characteristics was used to evaluate the collected Lilium resources based on their ornamental value, utilization potential, and ecological adaptability. L. dauricum, L. henryi, L. rosthornii, L. tsingtauense, L. concolor var. pulchellum, L. lancifolium, and species with trumpet-shaped flowers were selected which showed better potential for exploitation than other species. This study also provides references regarding the effective exploitation and use of wild Lilium resources.3. The largest currently ITS phylogenetic tree of Lilium for214samples representing98species and five varieties, including44species and five varieties native to China was constructed. The results suggest that the subsection (subsect.)5c Comber should be classified into the true subsect.5c and the section (sect.) Lophophorum. And the latter was divided into three subsections (subsect. Lophophorum Ⅰ, subsect. Lophophorum Ⅱ, and subsect. Lophophorum Ⅲ). Based on molecular phylogenetic analysis and fluorescence in situ hybridization, L. henryi and L. rosthornii are closely related, and we propose their classification into subsect. Leucolirion6a. The results support Comber’s subdivision of sect. Leucolirion, which was primarily based on bulb color. Chinese species were divided into five sections: sect. Martagon, sect. Archelirion, sect. Leucolirion, sect. Sinomartagon, and sect. Lophophorum. These findings contribute to our understanding of the phylogeny, origin, and classification of Lilium.4. Though the scanning electron microscopy (SEM), pollen tetrads was first found in L. bakerianum. Based on present and previous studies, our results suggest that pollen from L. formosanum should be classified as a new type, Formosanum. Combined with morphological and molecular evidence, pollen sculpture patterns appear to reflect phylogenetic relationships and are useful for species or subsection delimitation. Based on a comprehensive survey and correlation with potential functional implications, the following hypothesis was proposed:evolution of an exine sculpture shows pollen type trends from Martagon→Callose→Concolor→Formosanum. The evolutionary trend regarding pollen sculpture and size could be related to selective pressure to adapt to environmental conditions. Pollen size and shape showed a significantly positive correlation with annual precipitation, and smaller pollen grains appear to adapt better in habitats with extreme conditions.5. Based on the analysis of genetic parameters of SSR markers, the results show that the SSR loci were polymorphism. Cluster analysis indicate that the resources of wild lily has a rich genetic diversity, Principal coordinate analysis showed a significant clustering of wild lily with geographical features; L. henryi and L. rosthornii are closely related, and they showed close relationship with subsect. Leucolirion6a, moreover, L. brownii should be classisfied into subsect. Leucolirion6b. Besides, SSR markers in genetic analysis and phylogenetic analysis can be effectively used for lily evaluating, genetic diversity analysis as well as germplasm identification.6. Eight high resistance resources (L. regale, L. leucanthum, L. sargentiae L. dauricum, L. henryi. L. rosthornii, L. tsingtauense, L. taliense) were selected based on in vitro inoculation by mycelium block. These resources could provide for materials for resistance breeding and excavation of disease resistance genes. RGA-PCR amplification reaction system of lily was established. Resistance evaluation and cluster analysis by RGA fingerprinting showed similar resistance tending to gather together.7. Based on transcriptome sequencing, the NBS-LRR resistance gene analogs from Lilium was firstly isolated and cloned. Phylogenetic studies revealed close relationships of some Lilium RGAs to R genes and classified these sequences into five main subclasses. Pairwise comparisions indicated that the deduced amino acid sequences of RGAs contain the conserved domains such as P-loop, Kinase-2, Kinase-3a and GLPL, and the RGAs all belonged to nonTIR-NBS type resistance gene segments. Southern blot analyses showed that the RGAs were organized as multicopy loci in the Lilium genome. Quantitative real-time PCR analysis revealed dramatic variations in the transcript level of RGAs with the increase in the level of resistance of the hosts against gray mold, indicating its close relationship with the resistance against this pathogen. These RGAs are of great value for positional cloning of disease resistance genes. Our results provide a primary RGA pool for the further functional validation of resistance genes in Lilium.更多还原