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中国芍药品种遗传多样性SRAP分析和核心种质的初步构建

Analysis of Genetic Diversity and Construction of the Core Collection for Chinese Herbaceous Peony Cultivars Using SRAP Marker

作者: 专业:园林植物与观赏园艺 导师:郭先锋 年度:2010 学位:硕士  院校: 山东农业大学

Keywords

Herbaceous peony, SRAP marker, genetic diversity, core collection

        芍药(Paeonia lactiflora Pall.)是我国的一种重要草本花卉,我国芍药种质资源十分丰富。在长达数千年的栽培历史中,受地域限制及多变气候的影响,在自然选择和人工选育的基础上,其群体已经产生了大量遗传变异,形成了丰富的遗传多样性,有大量的传统品种和珍贵品种亟待开发和利用。但随着现代农业的发展和社会经济的导向作用,芍药的种质资源不断遭到破坏或丧失,因此重视和开展芍药种质资源的收集和保存工作,并同时进行芍药品种遗传多样性的研究,有利于我国芍药种质资源的高效利用。本研究以中国芍药种质的150个品种为研究对象,以SRAP分子标记为主要研究方法,对其资源的遗传多样性进行研究,以期揭示不同品种间的亲缘关系及其分类地位,为今后的资源研究、品种鉴定、育种亲本选择及优异基因资源发掘利用提供依据;并在此基础上初步构建芍药的核心种质,以便资源的高效管理及遗传多样性的准确利用。主要研究结果如下:1.确定了芍药稳定的SRAP-PCR扩增试验。所得反应体系为:总体系体积25uL,其中10×PCR Buffer(100mmol·L-1 Tris-HCl pH8.3,500 mmol·L-1 KCl)2.5uL,MgCl2 2.5 mmol·L-1,dNTP各0.25 mmol·L-1,引物各0.3μmol·L-1,TaqDNA聚合酶1U,DNA模板120ng。反应程序为:95℃变性5分钟,反应前5个循环在94℃1min,35℃1min,72℃1min条件下运行;之后40个循环复性温度为50℃;最后72℃延伸10min,4℃保存。2.利用SRAP技术对我国芍药150份种质资源进行了遗传多样性研究,从120个引物对中经过两次筛选,得到8个引物组合用于正式扩增,共产生303条带,其中286条为多态性条带,多态性比率为94%,平均每个引物组合产生35.8个多态性条带。每个组合的多态性条带数24~44不等,单个引物组合的多态性比率在88.9%~100%之间。3.通过UPGMA聚类分析,芍药种质的遗传相似性系数在0.63~0.88之间。从聚类结果中可以看出,在相似性系数0.66处,所有品种被划分为5大类群。该聚类结果非常复杂,但仔细深入分析,可以发现各分类群与地域来源和花色分类方案呈现一定的关联,具有在地域性基础上按花色进行聚类的一般规律和特点,显示中国芍药品种地理资源的遗传多样性。4.尝试对芍药种质资源进行核心种质构建,在地域性分组的基础上以分子标记数据为主、表型性状等传统数据为辅的方法进行聚类分层取样,按照12%、16%、21%、26%和31%的抽样比例获得5组核心种质,并对核心种质的多样性和实用性进行了检验。根据检验数据,确定最优核心种质为:26%抽样比例,筛选出39个品种作为芍药核心种质。从而为芍药种质资源的保护和利用奠定了分子标记研究的理论基础,同时,为进一步分子标记辅助育种、重要基因的克隆等奠定了基础。
    Herbaceous peony (Paeonia lactiflora Pall.) is an important herbal flower in China and its germplasm resources are very rich. In the long history of thousands of years of cultivation, subjecting to geographical constraints and changing climate, the group of herbaceous peony have produced a great deal of genetic variation and formed a rich genetic diversity on the basement of natural selection and artificial breeding. A large number of traditional cultivars and rare species were needed to be exploited and applied urgently. But with the development of modern agriculture and guidance of socio-economic, the germplasm resources of herbaceous peony are constantly being damaged or lost. Therefore, it is helpful to use the germplasm resources effectively by attaching importance to and carrying out collection and preservation of germplasm resources of herbaceous peony, and carrying on the research of genetic diversity of herbaceous peony simultaneously.In this research, with the objects of 150 cultivars of herbaceous peony germplasm and the main method of SRAP molecular marker, the genetic diversity of the resources was studied to reveal the genetic relationship between different species and their taxonomic status, and it could provide the basis for the future resources research、species identification、selection of breeding parents and exploration and utilize of excellent genetic resources; meanwhile, the core of herbaceous peony germplasm was initially built on this basis to manage resources effectively and make use of genetic diversity precisely. The main results were as follows:1. SRAP-PCR amplification experiment of herbaceous peony was established steadily. The reaction system was: 25μl of total reaction volume, including 2.5μl of 10×PCR buffer (100mmol·L-1 Tris-HCl pH8.3, 500 mmol·L-1 KCl), 2.5 mmol·L-1 of MgCl2, 0.25 mmol·L-1 of each dNTP, 0.3μmol·L-1 of each primer, 1U of rTaq, 120ng of DNA template. The reaction procedure was: denatured for 5 minutes at 95℃, the primer 5 reaction cycles were operated under the conditions of 94℃1min, 35℃1min, 72℃1min ;then, 40 cycles of annealing temperature is 50℃; at last, extended for 10min at 72℃, and preserved at 4℃.2. The genetic diversity of 150 herbaceous peony germplasm resources was studied with SRAP technology. After a double-selection in 120 primer pairs, 8 primer combinations were chosen to be used in the formal expansion, and 303 bands were produced, among which 286 were polymorphic bands, the polymorphism ratio was 94%, each primer combination produced 35.8 polymorphic bands. The number of polymorphic bands produced of every combination ranged from 24 to 44, and the polymorphism ratio of single primer combination was between 88.9% and 100%.3. By UPGMA cluster analysis, the genetic similarity coefficient of herbaceous peony ranged from 0.63 to 0.88. The result showed that all cultivars were divided into 5 major groups in the similarity coefficient of 0.66, The clustering result was very complex, but in-depth analysis carefully, it was found that each taxonomic group was be connected with geographic origin and color classification scheme,and it had general regulation and characteristic of color clustering on base of locality. It showed genetic diversity of geographical resources on Chinese herbaceous peony cultivars.4. The core germplasm resources of herbaceous peony was tried to be constructed. On the basis of regional groups, stratified cluster sampling was carried out by dominating molecular data and supplementing the traditional data such as phenotypic character. 5 groups of core germplasm was gained according to the sampling ratio 12%、16%、21 %、26% and 31%, and the diversity and practicality of the core ration was verified. According to laboratory data, the optimal core optimal was determined: 26% of the sampling ratio and 39 cultivars were selected as the core germplasm of herbaceous peony. Thus the theoretical foundation on molecular markers was established to protect and use herbaceous peony germplasm resources, at the same time, it was established the basis of further molecular marker-assisted breeding and important gene cloning.
        

中国芍药品种遗传多样性SRAP分析和核心种质的初步构建

中文摘要7-9
Abstract9-11
1 引言12-26
    1.1 芍药概况12-13
        1.1.1 芍药的栽培简史12
        1.1.2 芍药种质资源概况12-13
    1.2 遗传多样性的研究进展13-21
        1.2.1 遗传多样性的概念13
        1.2.2 遗传多样性的意义13
        1.2.3 遗传多样性的研究方法13-16
            1.2.3.1 形态学水平14
            1.2.3.2 细胞学(染色体)水平14
            1.2.3.3 蛋白质(等位酶)水平14-15
            1.2.3.4 DNA 水平15-16
        1.2.4 遗传多样性的 SRAP 分析研究进展16-21
    1.3 核心种质的研究进展21-25
        1.3.1 核心种质的概念21-22
        1.3.2 核心种质的研究进展22-24
        1.3.3 核心种质的构建24-25
            1.3.3.1 数据收集整理24
            1.3.3.2 收集数据分组24
            1.3.3.3 样品选择24-25
            1.3.3.4 核心种质的管理25
            1.3.3.5 核心种质的遗传多样性检验与评价25
        1.3.4 核心种质研究意义25
    1.4 研究的目的和意义25-26
2 材料与方法26-43
    2.1 试验材料26
    2.2 主要试剂和器材26-37
        2.2.1 主要试剂26-28
            2.2.1.1 提取试剂26-27
            2.2.1.2 扩增试剂27
            2.2.1.3 电泳试剂27-28
        2.2.2 主要器材28-37
            2.2.1.1 提取器材28
            2.2.1.2 扩增器材28
            2.2.1.3 电泳器材28-37
    2.3 试验方法37-43
        2.3.1 芍药DNA 的提取与质量检测37-38
            2.3.1.1 DNA 的提取37-38
            2.3.1.2 DNA 的质量和浓度检测38
        2.3.2 芍药SRAP-PCR38-41
            2.3.2.1 SRAP-PCR 反应体系38
            2.3.2.2 SRAP-PCR 反应程序38-39
            2.3.2.3 SRAP-PCR 引物筛选39-40
            2.3.2.4 SRAP-PCR 扩增电泳分析40-41
        2.3.3 数据统计分析41
        2.3.4 核心种质构建方法41-43
            2.3.4.1 数据的收集整理41-42
            2.3.4.2 种质资源的分组42
            2.3.4.3 核心样品的选择42
            2.3.4.4 核心种质的检验与评价42-43
3 结果与分析43-55
    3.1 DNA 质量检测结果43
    3.2 SRAP-PCR 引物筛选结果43-44
    3.3 基于SRAP 分子标记的遗传多样性分析44-47
    3.4 基于SRAP 分子标记的聚类结果分析47-49
        3.4.1 产地分析47-48
        3.4.2 花色分析48-49
    3.5 芍药核心种质的初步构建49-55
        3.5.1 核心种质的初步构建49-54
        3.5.2 芍药核心种质的有效性检验54-55
            3.5.2.1 对芍药核心种质的遗传多样性检验54-55
            3.5.2.2 对芍药核心种质的实用性检验55
        3.5.3 芍药核心种质的最优确定55
4 讨论55-58
    4.1 遗传多样性分析55-56
    4.2 聚类结果分析56-57
    4.3 核心种质初步构建分析57-58
5 结论58-59
    5.1 遗传多样性58
    5.2 聚类结果58
    5.3 核心种质的初步构建58-59
参考文献59-65
致谢65-66
攻读硕士学位期间发表的学术论文目录66
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