【作者】 武荣花；

【导师】 彭镇华； 王雁；

【作者基本信息】 中国林业科学研究院 ， 园林植物与观赏园艺， 2007， 博士

【摘要】 石斛属(Dendrobium)植物是兰科的附生植物，它是我国传统的名贵中药材，除了具有较高的药用价值外，它还具有较高的观赏价值。现在广泛栽培的石斛兰是以石斛属植物的原生种为亲本进行杂交选育出的优良观花品种。我国有丰富的石斛属植物资源，但观赏石斛的育种比较落后。针对这一问题，本研究进行了我国石斛属植物的种质资源及其亲缘关系的研究，以期为我国的观赏石斛兰育种奠定基础。本研究调查研究了我国石斛属植物资源现状及其保护措施，并从思茅、景洪和深圳等地收集引种石斛属植物31种1变种，并对其观赏特性进行综合评价。采用石斛叶绿体DNA trnL-trnF间隔区序列比较和采用ISSR分子标记对我国的部分石斛属植物资源进行亲缘关系分析，主要研究结果如下：调查收集并引种栽培了31个石斛原生种和1个变种，引种的石斛资源栽培于中国林业科学研究院科研温室。首次运用模糊数学的方法对所收集的石斛资源进行观赏特性综合评价。将32个石斛样本依据观赏特性划分为优、良、中、差4个等级。综合观赏特性优的有7种石斛；良的有14种石斛和1个变种；中等的有8种石斛；差的有2种石斛。对利用CTAB法从以下几种石斛(铁皮石斛、石斛、细茎石斛、齿瓣石斛、喉红石斛、黑毛石斛、翅梗石斛)的幼嫩叶片提取DNA的方法进行了改进，用改进后的方法提取的DNA数量多，质量好，纯度高。首次利用石斛属植物的叶绿体DNA trnL-trnF间隔区序列分析研究石斛属植物种间的亲缘关系。所测的30个石斛样本的叶绿体DNA trnL-trnF间隔区序列已登录GenBank。所测序列用CLUSTAL软件进行序列分析，分析了石斛种间的亲缘关系，总体上将30个石斛样本划分为4个相对独立的大组。石斛叶绿体DNA trnL-trnF间隔区序列分析确定出的石斛属植物部分种间的亲缘关系为：报春石斛和兜唇石斛亲缘关系较近；串珠石斛与大苞鞘石斛亲缘关系较近；黑毛石斛与长距石斛和喉红石斛亲缘关系较近；刀叶石斛与剑叶石斛亲缘关系较近；这与中国植物志石斛属形态分类相一致。而翅梗石斛与小黄花石斛亲缘关系较近；鼓槌石斛与短棒石斛亲缘关系较近；石斛与重唇石斛亲缘关系较近这与中国植物志的石斛属形态分类不一致。首次从60条ISSR引物中筛选出5条引物用于石斛属植物ISSR-PCR扩增。这5条引物扩增的条带清晰、多态性好，32个石斛样本共扩增出103个清晰、稳定的条带，其中有93个多态性条带，多态性条带百分率为90.2％，扩增的条带长度在200—2000bp之间。建立了稳定的、可重复的石斛属植物ISSR-PCR反应体系，为今后利用ISSR标记技术开展石斛种间亲缘关系的研究提供一个标准化程序。根据ISSR扩增结果，用Ntsys(2.0)软件计算出遗传相似系数和遗传距离，按UPGMA方法构建聚类树状图，32个石斛样本可分为4组。利用ISSR分子标记确定出的石斛属植物部分种间的亲缘关系为：细茎石斛与铁皮石斛和滇桂石斛亲缘关系较近；流苏石斛与叠鞘石斛亲缘关系较近；喉红石斛与黑毛石斛和长距石斛亲缘关系较近；刀叶石斛与剑叶石斛亲缘关系较近。这与中国植物志石斛属形态分类相一致。而翅梗石斛与小黄花石斛亲缘关系较近；束花石斛与罗河石斛亲缘关系较近；这与中国植物志石斛属形态分类不一致。两种分子标记方法都支持的石斛属植物亲缘关系为：黑毛石斛与长距石斛和喉红石斛亲缘关系较近；刀叶石斛与剑叶石斛亲缘关系较近；这与中国植物志石斛属形态分类相一致。翅梗石斛与小黄花石斛亲缘关系较近；鼓槌石斛与短棒石斛亲缘关系较近；这与中国植物志石斛属形态分类不一致。更多还原

【Abstract】 Dendrobium species are epiphytic plants of orchidaceae. Many wild species ofDendrobium are traditional precious Chinese medical herbals and precious ornamentalplants.Dendrobium orchid cultivers which are cultivated diffusely are artifical hybrids ofDendrobium species.There are rich Dendrobium resources in China, but Densrobium orchidbreeding in China drop behind other countries. Aim at this problem, this research studies on thegermplasm resources of Dendrobiun in China and their genetic relationships, in order toestablish the base of Dendrobium orchid breeding in China.The status quo of Dendrobium resources in China and their conservation measures wereinvestigated and studied. 31 Dendrobium species and 1 variety were collected from Simao andJinghong, Yunnan province and Shenzhen, Guangdong province. The ornamentalcharacteristics of the 32 spcimen were evaluated. Marking by the chloroplast DNA (cpDNA)trnL-trnF sequence and the inter-simple sequence repeat (ISSR) markers, their geneticrelationships were conducted. The main results were as follows.31 Dendrobium species and 1 variety were introduced and cultivated in the greenhouse forscience research in Chinese academy of forestry.It is the first time to evaluate the ornamental characteristics of the Dendrobium resourcesin China by fuzzy mathematics. 32 Dendrobium spp. samples were classified into 4 groups bytheir ornamental characteristics. GroupⅠ,7 species, which are best; GroupⅡ, 14 species and 1variety, which are better; GroupⅢ,8 species, which are medium; GroupⅣ,2 species, whichare disqualification.Ameliorated the DNA extraction method by CTAB from the young foliages of someDendrobium species, such as D. officinale, D. nobile, D. moniliforme, D. devonianum, D.christyanum, D. williamsonii, D. trigonopus.And it is also the first time to study on the genetic relationships inter the Dendrobiumspecies in China by chloroplast DNA (cpDNA) trnL-trnF sequence. 30 cpDNA trnL-trnFsequences of the Dendrobium resource samples had been accepted by GenBank of NCBI.The genetic relationships of 30 samples were analyzed by using software CLUSTALbased on the chloroplast DNA (cpDNA) trnL-trnF sequence.The results showed that 30 samples were classified into 4 groups. The geneticrelationships of 30 Dendrobium samples are as follow. D. primulinum is close with D. aphyllum; D..falconeri is close with D. wardianum; D. williamsonii is close with D. longicornuand D. christyanum; D. terminale is close with D. acinaciforme. These are according to withthe morphological taxonomy of Dendrobium of Chinese flora sinica. But D. trigonopus is closewith D. jenkinsi; D. chrysotoxum is close with D. capillipes; D. nobile is close with D.hercoglossum. The results are not support the Chinese flora sinica classification which isclassified the Dendrobium with the morphological taxonomy.The genetic relationships of Dendrobium resource in China were studied by ISSR marker.5 primers, which produced clear and rich polymorphism bands were chosen from 60ISSR-PCR primers. 103 bands were amplified from 32 samples, and 93 bands of them werepolymorphic (percentage of polymorphic bands was 90.2%). The length of the amplified bandswere between 200～2000bp. The stable and reproducible optimum reaction system ofISSR-PCR and PCR amplification parameter were established. The results provided astandardizing program for the analysis of inter species genetic relationships of Dendrobiumresources.According to the results of ISSR amplified, similarity coefficient and genetic distancewere calculated by using Ntsys(2.0) software.A dendrogram was constructed based onUPGMA cluster analysis. 32 Dendrobium spp. samples were classified into 4 major groups.The genetic relationships of Dendrobium species in China were as fallow: D. moniliforme isclose with D. officinale and D. guangxiense; D. fimbriatum is close with D. denneanum; D.christyanum is close with D. williamsonii and D. longicornu; D. terminale is close with D.acinaciforme. The result is coherency with the Chinese flora sinica. But D. trigonopus is closewith D. jenkinsii; D. chrysanthum is close with D. lohohense. It is incoherecy with theChinese flora sinica.Based on the results of two molecular markers, the genetic relationships of Dendrobiumspecies in China were as follow: D. williamsonii is close with D. longicornu and D.christyanum; D. terminale is close with D. acinaciforme. It is coherency with the Chinese florasinica. But D. trigonopus is close with D. jenkinsii; D. chrysotoxum is close with D. capillipes.It is incoherecy with the Chinese flora sinica.更多还原