【作者】 袁军辉；

【导师】 成仿云； 李嘉珏；

【作者基本信息】 北京林业大学 ， 园林植物与观赏园艺， 2010， 博士

【摘要】 牡丹(tree peonies)是原产中国的世界著名花卉之一,因其悠久的栽培历史以及特殊的历史文化价值,成为研究栽培植物(花卉)起源与演化的良好素材,具有重要学术研究与应用价值。牡丹野生种全部为中国特有,对其与栽培牡丹之间遗传关系的深入研究,是揭示栽培牡丹起源,科学保护与利用野生种质的有效途径。野生紫斑牡丹(Paeonia rockii)是栽培牡丹品种起源、形成的重要原种(野生祖先)之一,栽培紫斑牡丹是栽培牡丹品种中最重要的组成部分之一,延安牡丹(P.×yananensis)是与栽培牡丹品种形成具有重要关系的杂交种。本研究所用的600多份实验材料,涵盖了野生紫斑牡丹的整个分布区,栽培紫斑牡丹的主要栽培品种与所有栽培牡丹的近缘(祖先)种,以及延安牡丹及其16个变异类型。采用经典的形态学资源调查和数量分类学方法,结合现代分子生物学技术和手段,应用3个叶绿体基因片段(cpDNA)(petB-petD, rps16-trnQ和psbA-trnH)和共显性的14个微卫星(SSR)(核基因)分子标记,对野生紫斑牡丹遗传结构及生态成因、紫斑牡丹栽培品种和延安牡丹杂交起源等科学问题进行了研究,主要研究结果如下：1野生紫斑牡丹遗传结构及其成因紫斑牡丹遗传结构据cpDNA可以区分为两支,即东部分支(陕甘子午岭、河南伏牛山及湖北神农架林区)和西部分支(秦岭中部太白山及甘肃南部山区)而核基因(SSR)则可区分为与地理分布区相一致的3组,即北部组(秦岭中部太白山及陕甘子午岭)、西部组(甘肃南部山区)和东部组(河南伏牛山及湖北神农架林区)。物种进化历史研究表明北部组中,位于陕甘黄土高原南端的子午岭林区的现存野生群体可能是该种内最古老祖先类群的孑遗,其祖先类群可能在1.44(±0.54)百万年前,因受气候波动的影响,开始了向南迁移,继而沿着秦岭山脉向东部和西部迁移。空间遗传格局是长期的生境片段化、气候波动、物种低的自然结实率、差的种子发芽率与弱的居群间基因流及适应性进化等因素共同作用的结果。在物种水平上cpDNA (Hd=0.8875)和SSR (PIC=0.71)核基因均有较高的遗传多样性,在居群水平上cpDNA表明居群间显著的遗传分化(FST=0.971),居群内的遗传多样性极低(20个居群有16种单倍型),而SSR表明居群水平的遗传分化主要表现在居群内(Variation,67.45%),居群间(21.22%)及地区间(11.32%)的遗传分化较小,但差异仍然显著。2紫斑牡丹栽培品种起源基于3个cpDNA单倍型和14个SSR等位基因频率数据的多种系统发育和遗传结构分析表明,105个紫斑牡丹栽培品种的近缘(祖先)种主要是中原牡丹(P.cathayana)、杨山牡丹(P.ostii)和裂叶紫斑牡丹(P.rockii subsp. atava),前二者是主要的母系来源(占栽培品种约87.96%),后者是其核基因(父系)的主要来源(100%)和少量母系来源(7.4%)。全缘叶紫斑牡丹(P.rockii ssp.rockii)几乎没有参与现有紫斑牡丹栽培品种的形成,仅1个新品种和4个传统品种的母系亲本有可能与该亚种甘肃南部的居群有关；稷山牡丹(矮牡丹)(P.jishanensis)、卵叶牡丹(P.qiui)和四川牡丹(P.decomposita)可能没有直接参与品种形成。紫斑牡丹栽培品种为多系杂交起源,可能是在直接引种某些栽培品种的基础上,通过反复杂交和选择发展而来。3延安牡丹杂交起源通过22个形态性状、3个cpDNA和14个SSR分子标记的综合分析,表明延安牡丹是以延安万花山当地的稷山牡丹为母系亲本,以其临近区域分布的紫斑牡丹为父系亲本经杂交起源的,是一个分类学上的杂交好种。本研究也证明利用母系遗传的cpDNA结合SSR标记,辅以表型分析,是一种有效研究植物杂交机制的新方法。本研究还表明：紫斑牡丹是一个并系类群；四川牡丹的cpDNA表现出与稷山牡丹和卵叶牡丹关系更近,而SSR则表现出与紫斑牡丹的西部居群(甘肃南部)有更近的亲缘关系；中原牡丹和杨山牡丹亲缘关系更近,二者核基因具有复杂的杂交遗传背景,与其长期栽培的身份相吻合。此外,本研究还开发了一个叶绿体基因片段(petB-petD),并筛选、再克隆验证了14个SSR引物可以在芍药属内通用,可以用于遗传资源的种质分析和品种鉴定。更多还原

【Abstract】 Tree peonies are one of the most famous flowers in the world but little is known about the origin of cultivated tree peonies (CTP). With the historic, cultural and ornamental values, tree peonies are typical materials fto investigate the origin and evolution of cultivated plants (especially in floricultural crops) and, therefore, become of importantance in academic researches and ornamental application. The study on the relationship between species, all of them endemic to China, and cultivars is an effective way to probe into the origin of CTP and to conserve and utilize the wild germplasm. Paeonia rockii was one of important wild ancestral species involved in the origin of CTP, cultivated flare tree peonies (CFTP) are important composition of CTP and P.×yananensis is a plausible hybrid closely related to CTP, all of which constructured the main contents of this study. Finally, a total of more than six hundred samples, including collections from wild P. rockii throughout the distribution area, CBTP, varieties of P. yananensis and all possible ancestral species, such as P. cathayana, P. ostii, P. jishanensis, P. qiui and P. decomposita were involved in this study using integrated methods such as morphological investigation, chloroplast (cp) DNA gene fragments (petB-petD, rps16-trnQ and psbA-trnH) and fourteen microsatellites to assert the genetic structure of wild P. rockii, the origin of CFTP and hybrid origin of P.yananensis. The results are as follow:1.The genetic structure and diversity of wild P. rockiiThe population genetic structure of P. rockii were distinctly divided into two clades (eastern clade and western clade) as revealed by 3 cpDNA. But based on the microsatellite loci, the population genetic structure were mainly divided into three groups, northern group (NG), eastern group (EG) and western group (WG) in accord with the geographic distribution. The relict populations distributed Ziwuling forestry area in the south to Shan-Gan loess plateau indicated the oldest populations, which moved to south direction driven by cold climatic fluctuations in 1.44(+0.54) milion year ago, and continually moved to eastern and western directions along Qinling Moutation. This could be attributed to integrated factors, such as allopatric fragmentation, climatic fluctuations, lower germinating capacity and ripen rate of seeds, and lack of gene flow among populations, as well as the result of adapted evolution of P. rockii.The haplotypes diversity (Hd,0.8875) based on 3 cpDNA and polymorphism informative content (PIC=0.71) based on 14 microsatellites loci were higher at species level of P. rockii, while Hd based on 3 cpDNA were lower at population level. The genetic differentiation(FST=0.971) between pairwise populations were clearly shown higher as revealed by 3cpDNA. Sixteen haplotypes were found in 20 populations, of which the majority were single, suggesting that the germplasm almost in every population is an exclusive resource that should be more protected from disappearing by strict conservation policies. The analysis of the genetic differentiation based on microsatellites markers were significant, revealing that 67.45% of the total variance was partitioned within populations, whereas 11.32% and 21.22% of the total variance was explained by differences among groups and among populations within groups, respectively.2.The origin of cultivated flare tree peoniesIt were indicated that P. cathayana, P. ostii and P. rockii ssp. atava were three of the most important ancestral species for cultivated blotched tree peonies based on three cpDNA and fourteen SSR data. The former two species served mainly as maternal donation for almost 87.96% of 105 cultivars while the latter was mainly nuclear genome resource as paternal donation for all of CFTP and also as biparently donation for 7.4% of 105 cultivars. P. rockii ssp. rokii nearly was not involved into CFTP, except little relationship with one new cultivar and four tradiational cultivars. Moreover, P. jishanensis, P. qiui and P. decomposita were not directly involved into CFTP. Therefore, it was suggested that CFTP were originated from repeated hybridization and selection at the base of some introduced cultivars of CTP.3.Hybrid origin of P. yananensisOur results showed that P. yananensis as a good morphological species was a hybrid between P. jishanensis as maternal donation grown aroud Wuanhua Mountain in Yan’an City and P. rockii as paternal donation. The case also demonstrated that combined morphological data, cpDNA and SSR were powerful methods for studies on hybrid issues.The study also showed that P. rockii was a paraphyletic species, P. decomposita show close relationship with P. jisahanensis and P. qiui based cpDNA, and also close relationship with western population group of P. rockii ssp. rockii based on SSR. P. cathayana and P. ostii with more close relationship with each other show complicated hybrid background of nuclear genome which was consistent with their status being long cultivated. Moreover, a new chloroplast fragment, petB-petD, and 14 microsatellites were developed for genetic germplasm analysis and identification of cultivated tree peonies.更多还原