【作者】 白锦荣；

【导师】 张启翔；

【作者基本信息】 北京林业大学 ， 园林植物与观赏园艺， 2009， 博士

【摘要】 月季（Rosa spp.）是世界性的重要观赏植物之一,在切花生产、盆花栽培、城市园林绿化和香料生产中起到了非常重要的作用。中国蔷薇有2000多年的栽培历史,我国共有蔷薇属植物95种,其中64种为特有种,但野生蔷薇资源破坏严重,蔷薇资源现状不清。中国传统月季品种流传至今,适应性广,抗病性强,具备四季开花、花香芳郁宜人等优良的遗传品质。中国月季品种‘月月红’（R.chinensis’Slater’s CrimsonChina’）和‘月月粉’（R.chinensis’Parsons’Pink China’）,香水月季品种‘彩晕’香水月季（R.odorata’Hume’s Blush Tea-scented China’）和‘淡黄’香水月季（R.odorata’Parks’Yellow Tea-scented China’）在现代杂种香水月季的形成过程中曾起到里程碑的作用。利用中国特有蔷薇野生种或传统月季品种的优良种质进行育种,对创造具有我国自主知识产权的月季新品种具有重要意义。因此,为避免或减少蔷薇种质资源的进一步流失与破坏,保护其资源多样性,本研究连续3年对蔷薇野生种和传统月季品种资源进行了调查、整理、分析和评价,建立种质资源圃,并采用表型、孢粉学、SSR分子标记和nrITS序列对部分蔷薇属植物的亲缘关系进行研究。本文调查范围选择蔷薇属分布最为集中的云南、四川横断山脉地区,种质特点明显的新疆伊犁、阿尔泰,长白山和北京周边地区。结合资源评价,筛选出观赏性状优良、抗白粉病的优异亲本材料,与现代杂种香水月季进行杂交育种,创造抗白粉病的月季新种质,为月季育种提供物质基础。主要结果如下:1.此次调查共计搜集43种蔷薇属植物和7个变种,隶属7组7系,占我国蔷薇属种质资源的44.8%。蔷薇属垂直分布海拔变化幅度大,介于海拔700-4300m之间。从资源现状看,峨眉蔷薇（R.omeiensis）、川滇蔷薇（R.soulieana）、长尖叶蔷薇（R.longicuspis）、山刺玫（R.davurica）、钝叶蔷薇（R.sertata）等种水平分布广泛,适应性强,变异丰富;中甸刺玫（R.prelucens）、大花香水月季（R.odorata var.gigantea）、玫瑰（R.rugosa）、深山蔷薇（R.marretii）等种群数量少,资源破坏严重,急需加强保护。采用层次分析法对蔷薇野生资源进行综合评价,筛选出华西蔷薇（R.moyesii）、刺蔷薇（R.acicularis）、黄刺玫（R.xanthina）等10个种可直接应用于园林绿化,并可作为育种亲本加以利用;川滇蔷薇、疏花蔷薇（R.laxa）、复伞房蔷薇（R.brunonii）等15种观赏性状较好,可直接或改良后选择其优良性状进行杂交育种,其后代可作为育种的中间材料进一步杂交或回交。2.调查和整理出22个中国传统月季品种,这些品种适应性广,抗性强,花型独特,芳香浓郁。将Q型聚类应用于蔷薇22个品种和2个香水月季及其变种的分类,对27个性状进行了R型聚类分析和主成分分析,选出了影响力比较大的19个性状,并根据植株性状和花部特征将传统月季品种分为4类。3.利用扫描电镜对11个蔷薇野生种和14个中国传统月季品种的花粉形态进行了系统观察和比较分析。孢粉学研究结果表明:花粉均为单粒、等极、辐射对称。花粉长球形或超长球形,极面观三裂圆形,具三孔沟,沟宽因种和品种而异,赤道面观椭圆形或长矩形,属于N₃P₄C₅型花粉。外壁纹饰以条纹状纹饰为主,种间穿孔的有无、密度和大小有差异,传统品种间差异相对较小。根据条纹走向、粗细、间距和密度,嵴洼深浅和穿孔孔频,可将供试花粉分为5种类型。聚类分析结果表明,花粉大小和外壁纹饰特征在属内不同组间花粉形态差别较大,月季组（Sect.Chinensis）与合柱组（Sect.Synstylae）亲缘关系较近;传统月季品种中花型相似的聚为一类,亲缘关系较近,这与形态学分类基本一致。根据花粉形态演变规律可推断野生种中芹叶组（Sect.Pimpinellifoliae）为较原始的类型,桂味组（Sect.Cinnamomeae）次之,合柱组和月季组较进化。传统月季品种中‘四面镜’（R.’Si Mian Jing’）可能为最原始的品种类型,‘月月粉’、‘月月红’及‘匍匐红’（R.’Pufu Hong’）等品种较为进化。4.建立了适合于蔷薇属亲缘关系分析的SSR反应体系,筛选出蔷薇属种质资源SSR分析的引物,并利用荧光定量PCR技术,对64份蔷薇属种质资源的亲缘关系进行了研究。12对引物对供试材料具有多态性,扩增片段大小在100-400bp之间,共检测出116个等位基因变异,每个微卫星标记包含有4-15个等位基因,每对引物检测出的等位基因平均位点9.7个。聚类分析结果表明,传统月季品种分别与合柱组、月季组聚在一起,亲缘关系较近,而与木香组、小叶组（sect.Microphyllae）、芹叶组和金樱子组（sect.Laevigatae）亲缘关系较远:野蔷薇（R.multiflora）和淡黄香水月季（R.odorata var.erubescens）与现代杂种香水月季聚为一组;云南特有种中甸刺玫（小叶组）与桂味组的玫瑰和山刺玫聚为一类,而与缫丝花（R.roxburghii）（小叶组）遗传距离较远。其余聚类结果与植物学分类基本一致。5.为了进一步分析蔷薇属植物亲缘关系,本研究利用核糖体内转录间隔区分析了部分蔷薇属的nrITS序列。GeneBank登录36条nrITS序列,登录号为FJ527684-FJ527719,蔷薇属ITS序列长度范围为622—628bp,其中有85个变异位点,42个为信息位点,分别占13.5%和6.7%,G+C含量为55.9-58.4%。蔷薇属种间或品种间ITS序列变异主要存在于ITS1间隔区内。按照聚类结果,传统月季品种分别与合柱组和月季组聚在一起,与SSR分析结果基本一致,可以推导在传统月季品种的形成过程中,月季组和合柱组蔷薇具有重要作用。小叶组的中甸刺玫和缫丝花分别与不同的桂味组野生种聚在一起,这与传统分类不一致。木香组形成一个单支,与其它各组并列,显示出较远的遗传距离。6.在蔷薇属植物亲缘关系分析的基础上,统计田间自然发病率,在31个供试材料中筛选出5个高抗白粉病材料,18个抗病材料;结合观赏性状,选择11个抗病传统月季品种,与6个观赏性状优良、白粉病抗性较差的现代月季杂交,共计44个杂交组合,授粉563朵,其中29个组合获得杂交果实158个,杂交结实率为28%。并用SSR分子标记对10株杂种苗进行鉴定,其中6株具有父母本特异片段,初步推断是真杂种,有待于进一步形态学鉴定,其余4株只有母本片段。本研究为蔷薇属种质资源的保护、开发和利用、亲缘关系的研究提供了理论依据,为培育具有我国自主知识产权的月季新品种奠定了基础。更多还原

【Abstract】 The rose is one of the most prominent ornamental plants in the world.Roses are widely used as cut flowers and pot flowers,as well as garden plants,and whose extracts are used in a variety of industries. With over 2000 years of cultivation history,China has been a main distribution centre of the genus Rosa L.and holds 95 shrubs species,which includes 65 endemic in total.However,due to serious damages to natural habitats,ambiguous nomenclature,a complicated genetic background,and inadequate investigation into germplasm resources,the progress of researchers in breeding and exploitation were severely restricted.Chinese traditional roses,with favorable features such as strong disease resistance, adaptability and fragrance,have preserved up to now.R.chinensis ’Slater’s Crimson China’,R. chinensis ’Parsons’ Pink China’,R.odorata ’Hume’s Blush Tea-scented China’ and R.odorata ’Parks’ Yellow Tea-scented China’ have played a foremost role in the breeding development of modern roses. Therefore,in order to protect Rosa resources,conserve genetic diversity and classify different rose species,this research continued for three years and focused on the investigation,evaluation on the wild and cultivar Rosa resources,as well as their genetic relationships by using phenotypic,palynology,SSR molecular markers and nrITS sequence processes.The wild resource investigations were conducted in Northwestern Yunnan and Southwestern Sichuan areas where Rosa is concentrate distributed,Ili and Altay in Xinjiang,Changbai Mountain and Beijing suburban areas that all have distinctive rose resources.Furthermore,powdery mildew resistant plants were selected among the wild species and traditional roses as parents based on the resource evaluation,and new genotypes were then created by crossing with modern roses.The results are shown as follows:1.After 3 years of field survey in the areas mentioned above,43 Rosa species and 7 varieties belonging to 7 sections and 7 series were located and collected,these account for 44.8%of the total species number in China.The geographic distribution of Rosa featured a broad altitude range （700-4300m）.Species such as R.orneiensis,R.soulieana,R.longicuspis,R.davurica and R.sertata, with strong adaptability and abundant variation,are widly distributed.The natural habitats of Rosa, especially in R.prelucens,R.odorata var.gigantean,R.rugosa and R.marretii have been devastated severely and should be protected urgently.A multifactorial appraisal system based on the Analytic Hierarchy Process was applied against Rosa species.10 species including R.moyesii,R.acicularis,R. xanthina et al.were chosen for direct landscape application or crossing parents,while 15 species consist of R.soulieana,R.laxa,R.brunonii could be able to improve their hybrids by some potentially useful characteristics.2.An investigation and arrangement of existing Chinese traditional roses was carried out and 22 traditional Chinese roses were selected.22 rose cultivars and 2 R.odorata were classified by the Q cluster analysis.19 dominate characteristics among 27 were selected under R duster analysis and the principal component analysis.The traditional roses were classified into 4 categories by the differences of plant types and flower traits.3.Pollen morphology of 11 Rosa species and 14 Chinese traditional roses were examined under scanning electron microscope（SEM） for the purpose of systematic observation and comparative analysis.The features of pollen grains were goniotreme,prolate and perprolate.The equatorial view was ellipse,while the polar view was 3-1abed rounded with 3 narrow opereulums annularly distributed in equal space.Exine sculpture was striate with apertures.The pollen type was N₃P₄C₅ model.Variability among species and cultivars were showed by the features of the exine sculpture.The exine sculpture was mainly stripy,and the tested pollens could be divided into five categories according to the orientation,thickness,distance and density of the stripes.The results of cluster analysis indicated that the size and exine sculpture of pollen could reflect the differences between sections to a certain degree. According to the evolution of rose pollen,it can be inferred that sect.Pimpinellifoliae is more primitive than sect.Cinnamomeae,and sect.Synstylae,sister to sect.Chinensis,are more evolutive.R.chinensis ’Parsons’ Pink China’,R.chinensis ’Slater’s Crimson China’ and R.chinensis ’Pu Fu Hong’ were more evolutive than other cultivars,while R.chinensis ’Si Mian Jing’ could be regarded as an original cultivar.4.SSR reaction system for Rosa was established and the suitable SSR primers were screened.64 resources were amplified by fluorescence quantitative PCR（FQ-PCR） with 12 pairs of primers.All mierosatellite markers showed a high level of polymorphisrn（4-15 alleles per microsatellite marker, with an average of 9.7 per locus）.The fragment sizes ranged between 100-400bp.Cluster analysis revealed that Chinese traditional roses showed close genetic relationship with sect.Synstylae and sect. Chinensis,while showed dissimilarity with sect.Banksianae,sect.Cinnamomeae,sect.Microphyllae, sect.Pimpinellifoliae,and sect.Laevigatae.Meanwhile,R.multiflora,R.odorata var.erubescens formed a branch with modern roses.R.prelucens in sect.（endemic species in Yunnan） were in the same cluster with R.rugosa and R.davurica（both in sect.Cinnamomeae）,while showed dissimilarity with R. roxburghii（sect.Microphyllae）.Moreover,the rest of the cluster results coincided with traditional botanical classification.5.A further research of genetic relationships among Rosa L.was carried out by nrITS sequences. 36 ITS sequences were registered in GenBank and the accession number was FJ527684-FJ527719.In all species and cultivars,the length of ITS was 622-628bp,including 85 parsimony-variable sites and 42 parsimony-informative site,which were account for 13.5%and 6.7%respectively.The G+C content varied from 55.9%to 58.4%.The main variation of ITS sequences in Rosa was ITS1.In the ITS analysis,Chinese traditional roses,sect.Synstylae and sect.Chinensis comprise a single group,which agreed with the results of SSR molecular marker analysis and showed strong effects on the development of traditional roses.While R.prelucens and R.roxburghii（both in sect.Microphyllae,） were clustered together with wild species from sect.Cinnamomeae,which indicated that there was close genetic relationship between them.This result did not fully support the classification of the genus Rosa. Meanwhile,R.banksiae formed a single group by itself showed larger genetic distance with other groups.6.Resistance of 31 Rosa species and traditional roses to powdery mildew was studied by measuring the natural infection rate in the field.The results indicated that the resistance abilities varied among different materials.5 wild species were found to be highly resistant,while another 18 showed above-average resistance.11 traditional Chinese roses were selected for crossing with 6 modern roses with good ornamental characteristics but low resistant.From the above 44 cross combinations,563 flowers were pollinated,and 158 fruits were obtained from 29 hybrid combinations.The seed setting rate was 28%.The 10 obtained F1 hybrids were validated using the SSR molecular marker process. After amplification of 6 specific fragments,the results showed strong similarity with both parent fragments,thus demonstrating strong evidence of true hybrids.Further research is needed for the morphology identification with the other 4 hybrids that had only maternal fragments.The study provided the theory basis for the resource investigation,protection and utilization, genetic relationships analysis of genus Rosa L..Moreover,the research eatablished a material basis for new rose cultivars culitivation with our own independent intellectual property.更多还原