【作者】 李炎梅；

【导师】 郑郁善；

【作者基本信息】 福建农林大学 ， 园林植物与观赏园艺， 2012， 硕士

【摘要】 本研究以福建农林大学百竹园中竹亚科的30个属命名种为研究对象，从DNA多态性分析的ISSR分子标记方法着手，利用DNA分子标记技术系统的研究遗传背景十分复杂的竹子种质资源，对其遗传多样性、亲缘关系进行研究，开展种质资源的分子鉴定，并建立有效的DNA指纹图谱，同时研究竹子在园林上的应用。采用ISSR分子标记技术，对我国竹亚科30个属的命名种即：绿竹、短穗竹、业平竹、箬竹、寒竹、慈竹、牡竹、大节竹、瓜多竹、唐竹、酸竹、巨竹、矢竹、少穗竹、簕竹、刚竹、茶秆竹、梨竹、思劳竹、泰竹、泡竹、单枝竹、空竹、巴山木竹、镰序竹、倭竹、大明竹、箭竹、玉山竹、赤竹进行遗传多样性分析。实验研究竹亚科植物的DNA提取方法，研究竹类植物普遍适用的ISSR-PCR反应体系进行优化，筛选出18条条带清晰和稳定性较好的引物，使用Popgene32软件进行遗传多样性分析，获得遗传相似度和遗传距离的信息。用SPSS13.0软件，得出了30个竹种的ISSR树状聚类图。本实验得到结论如下：（1）采用单因素多水平梯度筛选法进行实验设计，对Mg2+、模板DNA、TaqDNA聚合酶、dNTPs、引物这5种因素进行体系优化，建立竹亚科30个属命名种的ISSR-PCR扩增体系，即20μl反应体系中模板DNA40ng，引物0.6μmol·L^-1，TaqDNA聚合酶1.0U，Mg2+2.5mmol/L，dNTPs0.25mmol·L^-1，1×buffer；并通过实验筛选得出相应引物的最佳退火温度。（2）从100条的ISSR随机引物中筛选出18条能够扩增出清晰的特异性条带的引物，对30个命名种进行遗传多样性的分析，一共扩增出253个清晰的位点，其中多态性位点有253个，占总位点的100%；每个引物平均扩增出14个位点，而多态性位点百分比都为100%。（3）30个竹种的多态性百分比变化范围在23.72％～44.27％之间。PPB最大的是箭竹，PPB最小的是绿竹，说明这两个群体间的适应能力差异性较大。其它竹种的多态性也都较高，30个竹种的遗传基础范围广，遗传多样性较丰富。（4）30个竹种的观测等位基因数（Na）为2.0000个，有效等位基因数（Ne）为1.5756个，Nei’s基因多样性指数（h）为0.3437，Shannon多样性指数（I）为0.5178，多态位点百分率（PPB，％）为100%，多态位点有253个，同时30个竹种间的遗传多样性Ht=0.3463。说明30个竹种的遗传多样性还是相当丰富，这为竹类植物多样性提供了分子基础。（5）利用软件Popgene32软件，分析30个命名种的遗传距离在之间0.104～0.6207，平均为0.4414，遗传距离最大0.6207，在9号瓜多竹和27号大明竹之间；遗传距离最小的是0.104，在28号箭竹和29玉山竹之间。（6）采用的DNA聚类分析结果和传统的分类很多地方比较吻合，在这次试验研究的竹种中，如传统分类中的刚竹亚族的竹种大节竹、唐竹、短穗竹、刚竹，具有较近亲缘关系，都聚集在第六组分类中；而且牡竹族的倭竹、业平竹、寒竹也都集中于第六组分类中；而遗传传距离最小的箭竹、玉山竹种也同时出现在第五组中；传统分类中的倭竹亚族：业平竹和寒竹也在实验结果出现于倭竹亚族中；梨竹族的竹种也集中在第二和第五组。该实验结果和传统分类结果基本吻合，由此可见，DNA分子标记用于分析竹种间的亲缘关系和遗传多样性，具有较高的精确性和可靠性，同时可以作为竹子分类的参考依据。（7）本实验使用的18个引物，即UBC810、UBC811、UBC825、UBC827、UBC835、UBC836、UBC841和UBC857引物都能独立构建基于ISSR-PCR30个属命名种的数字指纹识别码。（8）采用传统的方法分类，主要根据竹子外部显著的形态特征，采用二歧归类方法进行分类和命名，通俗易懂，直观，具有普遍适用性，产生时间较早。ISSR分子标记是一种完全不同于传统分类的方法，揭示30个竹种的内在联系，全面反映了竹种间遗传多样性。而且学术性强，针对性强，适用群体单一，分类的可靠性和准确性较高。ISSR分子标记分类法是一种新的识别技术手段，为竹子的研究开辟了新的道路。（9）本实验通过对竹子在园林造景方面的研究，从遗传多样性、美学特征、空间关系、运用形式、角色扮演、城市绿地等方面进行分析，将传统的造园“因地制宜，师法自然”的思想和现代的“生态、低碳”的设计理念巧妙的结合，注重因境置竹和生物多样性保护，扩宽竹子造景的视野，为竹子的园林运用开辟了新的途径。但是当前还需积极探索新的设计思想和造景手法，竹子造景要进一步发展，还是任重而道远的。更多还原

【Abstract】 This study’s research object is30named species of Bambusoideae from the Fujian Agriculture AndForestry University in bamboo garden as the，to bagin with the DNA polymorphism analysis of ISSRmolecular marker method，using DNA molecular marker to study bamboo’s germplasm resources whosegenetic background is very complicated，to study the genetic diversity，phylogenetic relationship，to carryout bamboo’s germplasm resources molecular identification，and establish effective DNA fingerprinting，at the same time Research on the application of bamboo in landscape.This study uses ISSR molecular marker technique to analyze China’s30named species ofBambusoideae，The species are as follows：Dendrocalamopsis oldhami，Brachystachyum densiflorum，Semiarundinaria fastuosa，Indocalamus tessellatus，C.marmorea，Neosinocalamus Keng f.，Dendrocalamusstrictu，Icrassiflora McClure，Guadua amplexifolia J.S.Presl，Sinobambusa tootsi，Acidosasa chinensis，Gigantochloa atter，Pseudosasa japonica，Oligostachyum sulcatum，Bambusa arundinacea，Phyllostachysviridis，Pseudosasa amabilis，Melocanna baccifera，Schizostachyum pseudolima，Thyrsostachys siamensis，Pseudostachyum Ploymorphum，Monocladus saxtilis，Cephalostachyum fuchsianum，Bashania fargesii，Drepanostachyum falcatum，Shibataea kumasasa，Pleioblastus. gramineus，Fargesia spathacea Franch.，Yushania niitakayamensis，Sasa longiligulata McClure.Analyzing their genetic diversity， Thisexperiment study the DNA extraction method of30named bamboo species，studying bamboo plantsgenerally applicable ISSR-PCR optimization reaction system，screening out of18bands of clear and goodprimers， using Popgene32software to analyze genetic diversity，Getting genetic similarity and geneticdistance informations. Using SPSS13.0software，Generating polygenetic tree of genetic relationship of30bamboo species. The experimental results are as follows：(1) With the one-single and multi-level gradient testing method，To optimize system in five factors ofMg2+，template DNA，TaqDNA polymerase，dNTPs，and primer. in a total volum of20μL system whichcontained40ng template DNA，0.6μmol/L primer1.0U TaqDNA polymerase，2.5mmol/L Mg2+，0.25mmol/L dNTPs，1×buffer. Through experiments，screening the corresponding primer’s annealingtemperature.(2) screened18primers from the100ISSR random primers which can amplify clear and specific ands，With the analyzation of30named species’ genetic diversity，there are253polymorphic bands in253bands，a polymorphic rate of100%. Each primer amplified14polymorphic bands on everage，the polymorphic rate are all of100%.(3)30bamboo species polymorphism percentage change in the range of23.72%～44.27%. PPBlargest is Fargesia spathacea Franch, PPB minimum is Dendrocalamopsis oldhami, the two groups ‘sadaptability is different. Other bamboo species polymorphism are much higher,30bamboo species havewide genetic basis, and rich genetic diversity.(4)30bamboo species observed number of alleles (Na) is2, the effective number of alleles (Ne) is1.5756, Nei ’ s gene diversity index (H) is0.3437, Shannon diversity index (I) is0.5178, the percentageof polymorphic loci (PPB,%) is100%polymorphic loci is253, and30bamboo species’ genetic diversityHt=0.3463.This description30species bamboo’s genetic diversity is quite rich, the bamboo diversityprovides a molecular basis.(5) With the software of Popgene32，analyze genetic distance of30named bamboo species between0.104~0.6207，the average is0.4414，the largest genetic distance is0.6207，that is between No.9Guaduaamplexifolia J.S.Presl and No.27P. gramineus，the smallest genetic distance is0.104，that is between28Fargesia spathacea Franch and29Yushania niitakayamensis.(6) the DNA cluster analysis results and the traditional classification in many places were compared,in this study of bamboo species, such as the traditional classification of Phyllostachys subfamily ofIcrassiflora McClure, Sinobambusa tootsik, Brachystachyum densiflorum, Phyllostachys viridis, havinga close phylogenetic relationship, gathered in the sixth group; and Dendrocalamus strictus subfamily’sShibataea kumasasa, Semiarundinaria fastuosa, Chimonolamluca marmoreal, gathered in the sixthgroup; and the smallest genetic distance is Fargesia spathacea Franch. And Yushania niitakayamensisalso appeared in the fifth group; the traditional classification Shibataea kumasasa subfamily: theexperimental results appear Semiarundinaria fastuosa and Chimonolamluca marmoreal be all inShibataea kumasasa subfamily. Melocanna baccifera subfamily are concentrated in the second and fifthgroups.The experimental results and the traditional classification results are basically consistent, therefore,DNA molecular markers for the analysis of bamboo species phylogenetic relationships and geneticdiversity, it has higher accuracy and reliability, and can be used as the reference basis of bambooclassification.(7) The experiment uses18primers，as follow，UBC810，UBC811，UBC825，UBC827，UBC835，UBC836，UBC841and UBC857primers all can independent constructe a digital fingerprint identificationcode of30named bamboo species based on ISSR-PCR.(8) Using the traditional method to classify, mainly according to the bamboo outside significantmorphological features, using two ambiguous classification method to classify and naming, easy, intuitive,and has general applicability, appear earlier. ISSR molecular marker is a method completely different fromthe traditional classification, revealing30bamboo’s immanent connection, fully reflects the bamboo genetic diversity. And the academic strong, pertinence strong, applicable groups is single, the classificationhas higher reliability and accuracy. ISSR molecular marker classification method is a new identificationtechnique means, opened up a new path to bamboo research.(9)The experiment based on bamboo in landscape studies， from genetic diversity， aestheticcharacteristics，space relations，application forms，cosplay，city green space and so on to analyse， thetraditional gardening thought about“suit the local conditions，obey the nature rule”and modern designconcepts about“ecological，low carbon”combined the two aspects together，pay attention to because ofHabitat home bamboo and biodiversity protection，broaden the horizons of bamboo landscape design，inorder to open a new way of bamboo garden using. But nowadays it need to explore the new design thoughtand design technique actively，to further development，it still has a long way to go.更多还原