【作者】 张辕；

【导师】 戴思兰；

【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】 观赏植物种质资源的鉴定和分类是育种研究的必要前提,品种鉴定是品种分类的重要基础。将观赏植物种质资源的鉴定与品种分类相结合进行研究,将会最大限度地发挥种质的利用潜力,并为观赏植物的遗传育种研究奠定基础。菊花(Chrysanthemum X morifolium Ramat.)是菊科菊属多年生宿根草本植物。中国传统菊花品种数量繁多、变异丰富,是极为宝贵的栽培植物种质资源,对菊花品种进行鉴定和分类研究对于开发其优异的遗传潜力并应用于产业化生产具有重要的实际意义。前人在菊花品种鉴定和分类的研究中,缺乏对鉴定指标和分类标准的准确定义,遗漏了很多明显具有鉴定和分类价值的指标,对各指标的有效性和稳定性缺乏客观的评价；另外,品种分类的研究方法也较为单一,综合性不强,已建立的品种分类体系均强调实用性,缺乏科学依据。基于这些问题,本研究使用形态学标记、分子标记和细胞学标记3种研究方法对中国传统菊花品种进行了综合鉴定和分类,最终通过对各项研究结果的拟合构建了菊花品种分类新体系,以期为菊花的品种培育、品种保护、品种登录和国际交流奠定基础。本研究获得的主要成果如下。1.以735个中国传统菊花品种为试验材料,采用多元统计分析方法对20个花部形态性状和18个叶部形态性状连续5年的形态学测试数据进行了计算分析,筛选出了品种内一致性强、品种间差异显著并具有较大权重值的10个花部形态性状和10个叶部形态性状作为最具品种鉴定价值的形态性状,并将花部形态性状、花色和叶部形态性状分别作为菊花品种鉴定的一级、二级和三级标准,对所选735个中国传统菊花品种进行了逐级鉴定,综合分辨率达到79.18%,这一结果表明分级标准的划分是合理的；通过对花部形态性状的线性回归分析发现,菊花的瓣型和花型是品种分类的重要要素,通过聚类分析和判别分析将所选品种分为4类瓣型和18类花型。2.通过微卫星磁珠富集法成功构建了一个富含AC和GT基序的微卫星文库,在此基础上成功开发了35个菊花特异性SSR标记,经检测其中26个位点具有多态性。使用其中20个特异性SSR位点对形态学标记已鉴定的327个品种的基因组DNA进行了扩增,利用理想解法筛选出了5个具有较高多态性和鉴别力的位点作为核心位点,在此基础上建立了所选品种的SSR指纹图谱和19位的十进制分子身份证,并在形态学标记无法鉴别的153个品种中进行了验证,鉴别率达到100%。3.在对易混淆品种进行形态学鉴定的基础上,利用204个多态性位点对480个菊花品种进行了Q聚类分析,将所选品种划分为10个类群,其中区分明显的品种群分别为：具有管瓣、桂瓣和畸瓣等3类瓣型的品种,具有飞舞型、龙爪型和毛刺型等3类花型的品种以及具有红色、橙色、粉色、棕色和墨色等5类花色的品种,但这些品种群的基因多样性均较低；之后将聚类结果与群体遗传结构和主成分分析的研究结果进行比较,分类结果基本吻合；形态学标记与分子标记的关联分析结果表明,共有37个多态性位点与16个表型性状相关联,并且发现一些位点同时与多个性状相关联。4.将形态学标记和分子标记的分类结果进行综合比较分析发现,瓣型和花型分类结果的整体拟合率分别为74.84%和66.58%;综合形态学和分子标记研究的分类结果,将瓣型和花型分别作为菊花品种分类的一级和二级标准,进一步验证了4类18型的菊花品种分类体系。5.将常规压片法和去壁低渗火焰干燥法的关键步骤相结合,获得了较为清晰、分散的根尖细胞染色体中期分裂相。在此基础上,使用多种分析软件和统计学方法对70个品种的核型多样性进行了分析,筛选出了6个最具分类价值的核型参数。核型参数的聚类结果能够将平匙瓣类、管瓣类和桂瓣类品种明显区分开,在很大程度上证实了4类18型的菊花品种分类体系的准确性。本研究通过拟合形态学标记、SSR分子标记和细胞学标记的研究结果,建立了菊花品种的分级鉴定体系和“4类18型”的菊花品种分类体系,为菊花品种的合理利用和国际交流奠定了基础。更多还原

【Abstract】 The germplasm identification and classification of ornamental plants is an essential precondition for their breeding study, of which the former is an important basis for the latter. The combination of these two plays a decisive role in the exploration of utility potential of germplasm to the utmost extent, and also lays foundations for the genetic breeding of ornamental plants. Chrysanthemum×morifolium Ramat.(Asteraceae, Chrysanthemum L.) is a perennial herbaceous crop. The Chinese traditional chrysanthemum cultivars had been considered to be precious germplasm for their large quantities and rich variations. The identification and classification studies on this marvelous crop are of important practical significances for the exploitation of their excellent genetic potential and the application on industrial production. In the previous identification and classification studies on chrysanthemum cultivars, the accurate definitions for identification indicators and classification criteria were lack; many indicators which are of great values for cultivar identification and classification were overlooked; the objective evaluation for the effectiveness and stability of each indicator was also deficient. Moreover, the study methods for cultivar classification were monotonous; the present Chinese classification systems of chrysanthemum paid much attention to the practicability, while lack scientificalness. Based on these problems, in the present study, we used three types of genetic markers, the morphological markers, molecular markers and cytological markers, to identify and classify the Chinese traditional chrysanthemum cultivars comprehensively, and then constructed a new classification system of chrysanthemum after fitting the results together. This work aims at laying foundations for the breeding, protection, registration and international communication of chrysanthemums. The main results we obtained are the followings.1. As many as735Chinese traditional chrysanthemum cultivars were selected as materials, whose five years of morphological data were analyzed using multivariate statistical analyses, including20floral and18foliar morphological characters. After analyzing, a total of10floral and10foliar valuable morphological characters were screened out, which were of strong uniformity within cultivars, significant differences among cultivars and high weight values. Then, using the floral morphological characters, flower color phenotypes and foliar morphological characters as the first, second, and third identification criterion respectively, the selected735cultivars were identified hierarchically, and a comprehensive resolution of79.18%was obtained, indicating a good rationality of the hierarchical identification criteria. Moreover, the composing elements of petal type and flower head type as well as their significances were revealed after analyzing the floral morphological characters. Finally, the selected cultivars were classified into four petal types and18flower head types using the clustering and the discriminant analyses.2. An enriched genomic library with AC and GT microsatellite motifs was constructed using a magnetic beads enrichment method, and35chrysanthemum-specific SSR markers were exploited accordingly. Among these markers,26ones were proved to be polymorphic. Using20chrysanthemum-specific SSR loci, we amplified the genomic DNA of327cultivars which had been identified by the morphological markers previously. Then, five core loci were screened out using the TOPSIS method, which possessed higher polymorphism and discriminability simultaneously, compared with others. On this basis, the SSR fingerprints and molecular identities with19digits of the selected cultivars were established. Finally, we tested and verified the practicability of the molecular identities among the153cultivars which could not be identified by the morphological markers, and obtained an identification rate of100%.3. On the basis of morphological identification for the confused cultivars, a cluster analysis of480cultivars was performed according to the204polymorphic loci. The result showed that the selected cultivars were classified into10groups clearly, in which the cultivars with the tubular, anemone and peculiar of petal types, fluttered form, dragon-claw-like form and aristate form of flower head types, and red, orange, pink, brown and dark-red of flower colors could be distinguished clearly. However, the gene diversities of these groups remained low. After comparing the results of the cluster, population structure and principal component analyses, similar classification results were obtained. The trait-marker association analysis showed that as many as37polymorphic loci were associated with16traits. Moreover, some loci were associated with more than one trait.4. We comprehensively compared the classification results between the morphological and molecular marker studies, and obtained an integral fitting rate of74.84%for petal type and66.58%for flower head type. Based on the above results, the ’four types and eighteen forms’classification system was further approved using the petal type and flower head type as the first and second classification criterion, respectively.5. After integrating the key processes of the normal flaking method and the wall degradation hypotonic method together, we obtained many more clear and dispersed split phases. On this basis, the karyotype diversity of70typical cultivars was analyzed using many software and statistical methods. A total of six valuable karyotype parameters were screened out, in which the chromosome number and the long-to short-arm ratio were the key indicators for the cytotaxonomy of chrysanthemums. Cultivars with the flat-spoon, tubular and anemone of petal types could be distinguished clearly in the clustering depiction, proving the accuracy of the’four types and eighteen forms’classification system to a large extent.In the present study, after the fitting of the results obtained from the morphological marker, SSR molecular marker and cytological marker, we successfully constructed a hierarchical identification system and a’four types and eighteen forms’classification system for chrysanthemum cultivars, laying foundations for the rational utilization and international communication of chrysanthemums.更多还原