【作者】 邓立宝；

【导师】 何新华；

【作者基本信息】 广西大学 ， 作物遗传育种， 2013， 博士

【摘要】 广西是中国最大的柿产区之一,其悠久的柿栽培历史和独特的地理气候造就了丰富的柿种质资源。由于广西柿资源研究基础薄弱,种质信息缺乏,在一定程度上影响着广西柿种质资源的开发、利用和保护。为了揭示广西柿种质资源状况、遗传背景和多样性信息,为广西柿种质资源分类鉴定、开发利用与保护、引种驯化、抗病性早期鉴定、品种选育等提供依据,本文调查研究了广西柿种质资源和开发利用状况,收集不同生态气候区的柿种质资源并建立柿种质资源圃,运用表型系统聚类分析和模糊综合评价法对柿种质资源进行多样性研究和综合评价,利用目标起始密码子多态性分子标记(SCoT)和简单重复序列分子标记(SSR)对柿种质资源进行遗传多样性分析；同时在开展柿角斑病自然发病调查和病菌接种鉴定的基础上研究了柿种质叶片SOD、POD、CAT、PAL酶活性和MDA、GSH、叶绿素含量的变化与抗病性的关系,并首次运用改良的cDNA-SCoT技术对柿种质角斑病抗病性进行基因差异表达研究。主要研究结论如下：1、广西柿种质资源调查及开发利用现状调查、考察和收集广西不同生态气候区的柿种质资源并进行分类研究,收集到柿属植物中柿、君迁子、油柿、乌柿、毛柿和野柿5个种1个变种的104份种质,在广西大学建立了柿种质资源圃,为广西柿种质资源开发利用、引种驯化、种质保存和品种选育提供了材料和依据。调查发现近年广西柿生产取得迅速发展,在柿种植面积和投产面积变化不大的情况下,平均单位面积产量、总产量和产值均逐年增加,但广西柿产业在发展过程中仍存在柿种质资源开发和利用不充分、品种单一和结构不合理、标准化生产程度低、贮藏保鲜和加工产业链不深化及加工产品单一等问题,据此提出了促进广西柿产业发展的对策。2、广西柿种质资源表型多样性分析与综合评价广西柿种质资源在表型上具有丰富的遗传多样性,所选用的44个表型性状间的欧氏距离较远,各性状间相对独立,在柿种质分类中具有较好的辨别能力。表型系统聚类分析能将一些具有相似特征的柿种质聚在一起,可作为柿品种分类的重要依据,但在进一步明确亲缘关系时,还有赖于分子标记等其它分类和鉴定途径的有机结合。对不同柿种质的模糊综合评价结果表明,Yueshi、Niuxinshi、Dafangshi、Jingshi、Jixinshi、Xiaofangshi和Huoshi等柿品种及一些实生单株的模糊综合评价指数较高,它们的果实综合性状表现较优,建议作为优良品种和单株加以开发和利用；部分单个性状比较优良的实生单株虽不适合于直接推广种植,但可作为柿品种改良的资源加以利用。3、广西柿种质资源分子标记遗传多样性分析本研究首次利用SCoT分子标记技术对广西不同生态气候区的柿种质资源进行遗传多样性分析,并与SSR分子标记分析结果进行比较研究,结果表明：两种标记结果虽有一定的差异,但从总体上来看结果大致相同；两种分子标记都可以将供试材料分开,可以作为柿种质资源分类的有力工具。UPGMA聚类分析表明,广西柿种质资源具有丰富的遗传多样性,柿、油柿和君迁子分别聚到不同的组,野柿与柿种质间有交叉现象出现。广西各野生柿居群遗传背景宽广,多样性较为丰富,由于多山的地形和较高的海拔等原因使居群间的基因交流比较少,各居群间产生较大程度的遗传分化。4、广西柿种质资源角斑病抗病性研究不同柿种质间的抗病性差异比较大,总体上柿栽培品种和野柿种质对角斑病的抗性或耐性比较强,油柿和君迁子的抗病性比较弱。柿叶片接种角斑病菌后各种质SOD. POD、PAL酶活性和MDA含量均升高并呈一定规律的变化,这些指标的变化与柿种质间的抗病性存在相关性；CAT活性和GSH含量也呈现一定规律的变化,但这两个指标的变化与寄主的抗病性关系不密切。采用cDNA-SCoT技术对柿角斑病抗病相关基因进行差异显示分析,结果表明SCoT分子标记技术能够与柿叶片角斑病侵染应答产生关联,共获得13个柿叶片角斑病侵染相关基因片段,其中7个基因片段与已知的基因具有较高的同源性,包括一些在植物逆境反应中起重要作用的基因,其它6个基因差异片段在数据库中未找到已知功能的同源序列。更多还原

【Abstract】 Guangxi is one of the biggest persimmon production areas in China, it possesses ample persimmon germplasm resources due to its long histry of persimmon production and special geographical climate. The basis of research on persimmon resources in Guangxi is weak and the germplasm information is lack, which affects the exploitation, utilization and protection of persimmon germplasm resources in Guangxi. In order to reveal the situation, genetic background and diversity information of germplasm resources and provide the scientific basis of classification, preservation, utilization, introduction, early identification of disease resistance and breeding of persimmon germplasms in Guangxi, the study was conducted to investigate persimmon resources in Guangxi and their utilization condition. The persimmon germplasm resources from different eco-climatic zones were collected and preserved at the persimmon germplasm repository. Morphologic system cluster and fuzzy comprehensive evaluation analysis methods were used to analyze the diversity and comprehensive characters of the gernplasms. SCoT and SSR molecular markers technologies were used to study the diversity of the persimmon germplasm resources as well. Mean while, the effect of SOD, POD, CAT, PAL, MDA and GSH of persimmon leaves on the resistance of angular leaf spot disease were studied on the basis of survey of natural occurrence and artificial inoculation. cDNA-SCoT method was firstly used in persimmon germplasm research and studied the different expression of genes between the susceptive and resistant germplasms; the purpose of the study is to provide scientific basis of classification, preservation, utilization, introduction, early identification of resistance and breeding of persimmon germplasms. The Main conclusions are as follows:1. The Investigation of persimmon germplasm resources in Guangxi and its development and utilization stateIn order to provide materials and basis for persimmon resources utilization, introduction and domestication, conservation and breeding, the persimmon germplasm resources from different eco-climatic zones in Guangxi were surveied and investigated,104accessions of germplasms which belong to Diospyros. kaki Thunb., Diospyros.lotus Linn, Diospyros. oleifera Cheng., Diospyros. glaucifolia Mete, Diospyros. Virniana Liun. and Diospyros. rhombifolia Hems were collected, they were preserved in the persimmon germplasm repository in Guangxi university. The investigation indicated that the persimmon industry in Guangxi have got a rapid development in recent years, the change of persimmon planting area and production area was small, but the total yield and average yield per unit were increasing yearly. Meanwhile, there were some problems in the persimmon industry in Guangxi, such as the exploitation and utilization of persimmon germplasm resources was insufficient, and with single variety, unreasonably growing structure, low degree of cultivation standardization, the storage and processing was not deep and the processing products were few; for thess reasons, countermeasures to promote the development of persimmon industry in Guangxi were put forward.2. Analysis and comprehensive evaluation of the genetic diversity of persimmon Germplasm resources in GuangxiThe persimmon germplasm resources in Guangxi possessed ample diversity in phenotype, the Euclidean distances between44phenotypic traits used in the study were far, which indicated that these traits were independent and could distinguish the persimmon germplasms well. Phenotypic cluster analysis could cluster some germplasms which possessed some similar characteristics together, it could be important basis for classification of persimmon cultivars. Owing to the shortcoming of the phenotypic cluster analysis, it had better combined with some other identification methods to further distinguish the relationship, such as molecular markers. The result of fuzzy comprehensive evaluation analysis showed that the fuzzy comprehensive evaluation index of persimmon cultivars Yueshi, Niuxinshi, Dafangshi, Jingshi, Jixinshi, Xiaofangshi Huoshi and some seedlings were high, their fruit comprehensive characters were excellent, it’s suggested that these excellent varieties and seedling should be used in the persimmon exploitation and utilization. Some excellent seedling which possessed excellent single trait was not suitable for direct planting, but they could be used as materials in persimmon cultivar improvement.3. The genetic diversity of persimmon germplasm resources in Guangxi revealed by molecular markersThe persimmon germplasm resources collected from different eco-climatic zones in Guangxi were analysized and identified by start condone targeted (SCoT) polymorphism molecular marker; they were compared with SSR molecular markers. The result showed there were some differences between SCoT and SSR molecular markers, but the results of the two methods are consistent in whole; two molecular markers methods could distinguish the germplasms from each other, they could be used as powerful tools in the classification of persimmon germplasm resources. The cluster analysis conducted by UPGMA showed that the genetic diversity of persimmon germplasm resources in Guangxi was ample, the germplasms belong to Diospyros. kaki Thunb., Diospyros. oleifera Cheng, and Diospyros.lotus Linn were clustered into different groups, there was a crossover among Diospyros. kaki Thunb. and Diospyros. Virniana Liun. Guangxi natural persimmon germplasm resources possessed broad background and the genetic diversity was abundant; a great degree of genetic differentiation happened among the populations, it might due to the destruction of inhabit environment and the gene flow being blocked.4. Research on the resistance to persimmon angular leaf spot diease of persimmon germplasm resouces in GuangxiThere was ample diversity of resistance to persimmon angular leaf spot disease among different persimmon germplasms. Germplasms of Diospyros. kaki Thunb and some Diospyros. Virniana Liun seedlings had good resistance to angular leaf spot disease. The content or activity of SOD, POD, PAL, MDA in leaves of each persimmon germplasms increased and showed certain patterns, which indicated that the changes of activity or content of these enzyme was related to the resistance to angular leaf spot disease. The changes of CAT activity and GSH content also had some certain regularity, but they weren’t related to angular leaf spot disease resistance of susceptibility and resistance persimmon germplasms. Gene differential expression of susceptive and resistant persimmon germplasms was studied by cDNA-SCoT technology, the result showed that SCoT molecular marker technology could be associated with persimmon leaf spot disease stress response,13gene fragments related with persimmon angular leaf spot disease stress were obtained,7of them had a high homology with the known genes including some genes which played important roles in plant stress defence. The other6differential expression fragments had no homologous sequences with known function in the database.更多还原