【作者】 董晓莉；

【导师】 汤浩茹； 杨足君；

【作者基本信息】 四川农业大学 ， 果树学， 2009， 博士

【摘要】 悬钩子（Rubus L.）隶属于蔷薇科（Rosaceae）,全世界有悬钩子属植物750～1000种,中国有201种,98变种,分布遍及全国27个省区,尤以西南地区最为丰富。西南地区是中国悬钩子属植物的现代分布中心与多样性中心,不仅悬钩子属植物种类繁多,而且类型多样,兼有从草本到木本的一系列类群。但是,西南地区的悬钩子资源还未能得到很好的开发利用,其主要原因之一是对该地区悬钩子属植物的亲缘关系和系统发育还缺乏系统而深入的研究。本研究在综合分析已有悬钩子属植物系统学研究成果的基础上,依托国家自然科学基金项目“西南地区树莓植物遗传多样性研究与特异资源的筛选”建立的资源库,运用现代分子生物学技术,采用序列分析方法从核-质基因组入手开展西南地区悬钩子属植物的分子系统学研究,以期为该属植物的系统分类及资源鉴定积累一定的分子证据。同时采用分子标记方法对17个国外引进树莓品种与12个优良野生种质进行遗传差异分析,从而为种质资源的合理利用提供一定的参考。主要结果如下:1、对悬钩子属植物14个亚组的34个种（或变种）进行ITS序列测定结果表明:（1）34份材料的ITS（包括ITS1,5.8S和ITS2）序列长度范围为627～632bp,GC含量平均为55.7%。从ITS1和ITS2的长度变异来看,ITS1长度范围为254～260 bp,稍大于ITS2（208～211bp）。以路边青属Geum和蔷薇属Rosa为外类群,比对排序后ITS全长为639个位点,其中变异位点为120个,信息位点为83个,分别占18.8%和13.0%。（2）基于测定的34条ITS序列结合外类群构建的严格一致树表明木莓组与空心莓组的分化十分明显,属于木莓组的材料单独聚为一类,而空心莓组被划分为5类。就各组内部而言,各材料并没有完全按照亚组的划分聚类,而是相互交叉。其中柔毛叶亚组与绒毛叶亚组出现交叉聚类的现象较多,说明这两个组进化关系很近。但是,绒毛叶亚组的椭圆悬钩子并没有与其变种栽秧泡首先聚类,而是与分属于不同亚组的红毛悬钩子（柔毛叶亚组）以非常高的支持率（100%）聚在一起。（3）将测定的34份样品序列与从GenBank中下载的分属于5个组的10条序列进行比较分析,结果表明木莓组与刺毛莓组的关系较近,空心莓组与匍匐莓组的石生悬钩子关系较近,而单性莓组的兴安悬钩子与其余供试样品的关系均较远,且处于树的基部,推测该组植物具有不同的进化方式。对以上结果结合形态学、细胞学方面的资料进行了讨论。2、对悬钩子属植物13个亚组33份材料进行了trnL-trnF序列测定,结果表明:（1）33份材料trnL-trnF序列长度变化较大,从954bp（大红泡）到998 bp（栽秧泡）,相差44bp,GC含量平均为34%。以Geum和Rosa作为外类群,比对排序后trnL-trnF全长为1121个位点,其中变异位点为191个,信息位点为57个,分别占17.0%和5.1%。（2）基于测定的33条trnL-trnF序列结合外类群构建了严格一致树,结果表明以trnL-trnF序列为基础构建的严格一致树与以ITS序列为基础构建的严格一致树的拓扑结构很相似,仍然是属于木莓组的材料单独聚为一类,而空心莓组被划分为5类,只是各小组成员稍有变化。（3）将测定的33份样品的trnL-trnF序列与从GenBank中下载的分属于4个组的10条序列进行比较分析,结果表明木莓组与刺毛莓组的所有供试材料及矮生莓组的部分材料聚为一大类,空心莓组被划分为4类。另外,匍匐莓组的凉山悬钩子、石生悬钩子以及矮生莓组的匍匐悬钩子与木莓组的关系也较近,它们与木莓组聚在一起的支持率为66%。（4）将ITS序列与trnL-trnF序列均具备的所有样品的两种序列进行拼接,采用最大简约法构建了严格一致树。结果表明采用两种序列拼接后构建的严格一致树与单独用ITS序列或trnL-trnF序列构建的严格一致树相似度很高,均是木莓组单独聚类,空心莓组被划分为不同的组,且都以很高的支持率（100%）将椭圆悬钩子与红毛悬钩子聚在一起,说明悬钩子属植物核质进化的同步性。对以上结果结合形态学、细胞学方面的资料进行了讨论。3、采用ISSR分子标记技术对17个国外引进树莓栽培品种及12个悬钩子属植物优良野生种质进行了遗传差异分析。首先采用正交设计对ISSR反应条件进行了优化,优化后的体系为25μl反应体系中含有1×PCR Buffer,2mmol/L MgCl₂,1.5U Taq酶,0.2μmol/L primer,0.4 mmol/L dNTP和20ng DNA。从60条引物中筛选出10条扩增效果好的引物,共产生285条带,其中多态性带278条,占97.5%。各引物扩增的条带数在21～37条不等,平均每个引物扩增出的条带数为28.5条。聚类分析表明,空心莓组材料与树莓种群（Raspberry）品种的遗传关系较近,而木莓组材料与其它供试材料的遗传关系较远,ISSR标记对各大组和种群的划分与形态学分类基本一致。更多还原

【Abstract】 The genus Rubus belongs to the family Rosaceae and comprises 750～1000 species in the world,of which 201 species and 98 varieties have been found in China.Considered to be the major center of distribution and diversity of Rubus,Southwest China is exceptionally abundant in Rubus species,varieties and genotypes,from herbaceous plants to woody plants.However,the germplasm resources in this region were little-known and had not to be utilized,for the most important reason was that there was almost no deep study on them.Therefore,it is quite necessary to study the phylogeny and genetic diversity of Rubus distributed in this region,which will be beneficial to further utilization effectively. In this study,more than 30 species collected in Southwest China and 17 cultivars introduced from foreign countries were used as materials.The phylogenetic relationships of wild materials were revealed using sequencing techniques in nrDNA and cpDNA,and the genetic diversity between excellent wild germplasm and introduced cultivars were analyzed using molecular markers.The main results were as follow:1.The internal transcribed spacer（ITS） regions of nuclear ribosomal DNA（including ITS1,5.8S rRNA and ITS2） of 34 species or accessions from Sect.Malachobatus and Sect. ldaeobatus,representing 14 subsections of Rubus were sequenced.The results showed:（1） Length variation for the entire ITS region ranged from 627 to 632 bp.The ITS1 region （254-260bp） was longer than ITS2（207-211 bp）,and the GC content of ITS1（55.09%） was slightly lower than ITS2（57.29%）.（2） Together with the ITS sequences Rosa and Geum downloaded from GenBank as outgroups,the phylogenetic relationships within and between the 34 Rubus species were explored by employing maximum parsimony analysis using PAUP software.The length of complete ITS region was 639 sites after alignment, with 120 variable sites and 83 parsimony informative sites.The ITS-based tree inferred that Sect.Malachobatus was well-supported monophyletic group,and Sect.Idaeobatus was polyphyletic with five lineages.R.ellipticus and its variety species R.ellipticus var. obcordatus clustered separately.However,R.pinfaensis（Subsect.Pungentes） and R. ellipticus（Subsect.Stimulantes） formed into one group.（3） Ten sequences of other 5 sections of Rubus were downloaded from GenBank as comparison.The results showed that Sect.Malachobatus plus Sect.Dalibardastrum formed a strongly supported monophyletic group.Sect.Idaeobatus and Sect.Cylactis had close relationship.Sect.Chamaemorus had relatively far relationship with other sections,for it located at the basal clade of the ITS tree.The phylogenetic relationship of the materials were discussed according to the ITS data combining with their morphological characteristics and chromosome numbers.2.The trnL-trnF region of 33 species or accessions from Sect.Malachobatus and Sect. Idaeobatus,representing 13 subsections of Rubus were sequenced.The results showed:（1） Length variation for the entire trnL-trnF region ranged from 954 to 998 bp.The mean GC content was 34%.（2） Together with the trnL-trnF sequences Rosa and Geum downloaded from GenBank as outgroups,the phylogenetic relationships within and between the 33 Rubus species were explored by employing maximum parsimony analysis using PAUP software.The length oftrnL-trnF was 1121 sites after alignment,with 191 variable sites and 57 parsimony informative sites.The topological structure of trnL-trnF-based tree was very similar to ITS-based tree.It also supported that Sect.Malachobatus formed a monophyletic group and Sect.Idaeobatus was polyphyletic with five lineages,although some species had different position on the tree.（3） Ten sequences of 4 sections of Rubus were downloaded from GenBank as comparison.The results showed that Sect. Malachobatus plus Sect.Dalibardastrum and some species of Sect.Chamaebatus formed a strongly supported monophyletic group.Sect.Cylactis had parallel relationship with Sect. Malachobatus.（4） We combined ITS and trnL-trnF together to constructed a parsimony tree.It showed that the topological structure of ITS+trnL-trnF-based tree was very similar to the tree based on ITS data or trnL-trnF data separately.The phylogenetic relationships of the materials were discussed according to their morphological characteristics and chromosome numbers.3.Total 29 samples including 17 bramble cultivars introduced abroad and 12 wild excellent Rubus germplasm resources were analyzed by using inter-simple sequence repeat （ISSR）.The results showed:（1） By orthogonal design,the satisfactory ISSR reaction system was established,i.e.a total volume of 25μl system,it contained 1×PCR Buffer, 2mmol/L Mg²⁺,1.5U Taq DNA polymerase,0.2μmol/L primer,0.4 mmol/L dNTP and 20ng DNA template.（2） Of the 60 primers screened,10 primers exhibited sufficient polymorphic band patterns.A total of 285 ISSR bands were generated by 10 selected primers,among which 278 bands were polymorphic.The average number of band patterns observed per primer was 28.5 with a range from 21 to 37.The results showed that the genetic relationship between Sect.ldaeobatus and Raspberry cultivars was close,and Sect. Malachobatus had relatively far relationship with other materials.The cluster analysis results were consisted with the morphological classification.更多还原