【作者】 刘全兰；

【导师】 卢宝荣； 陈家宽； 钟扬；

【作者基本信息】 复旦大学 ， 植物学， 2005， 博士

【摘要】 小麦族(Triticeae Damort.)属于禾本科(Poaceae)的禾亚科(Pooideae)，包含着与人类的生活密切相关的三种重要粮食作物，如：小麦、大麦和黑麦，小麦族还包含了许多有重要经济价值的牧草种类。按照L(?)ve(1984)的系统分类法，披碱草属(Elymus L.)是小麦族中种类最多和分布最广的属，在全世界约有150多种，广泛分布于全球特别是北半球温带地区。披碱草属植物不仅包含了优异的牧草种类，而且拥有抗病、抗虫和抗环境胁迫的重要基因资源，为麦类作物和牧草品种的遗传改良提供重要的资源保障。许多披碱草属植物是草原的重要组成部分，在我国西北部草原的合理开发利用、草原生态系统平衡的维持、水土保持等方面有着不可低估的作用。除含有如此众多的重要的应用价值外，披碱草属还是研究多倍体物种形成的理想材料。披碱草属是异源多倍体属，由四倍体(2n=4x=28)和六倍体(2n=6x=42)物种组成，含5个含不同基因组，即St，H，P，W和Y，以不同的组合方式经天然杂交和染色体加倍而形成。H基因组来自大麦属(Hordeum L.)，St基因组来自拟鹅观草属(Pseudoroegneria(Nevski)(?)．L(?)ve)、P基因组来自冰草属(Agropyron(L.)Gaertner)，W基因组则来自澳大利亚冰草属(Australopyrum(Tsvelev)(?)．L(?)ve)但是含Y基因组的祖先种却一直没有发现。St基因组遍布于所有披碱草属物种中，是基础基因组(funda-mental genome)。据目前资料记载四倍体物种的披碱草属含有StH或StY基因组，六倍体披碱草属物种含有StHH、StStY、StHY、StPY和StWY等基因组。亚洲特别是中亚山区被认为是披碱草属植物的起源和多样性中心。这主要基于以下事实：(1)这个地区的物种数目占世界上披碱草属物种的一半；(2)披碱草属分类学上的重要形态性状都可以在这个地区物种中找到；(3)这个地区存有不同倍性的披碱草属物种，2n=4x=28，2n=6x=42，2n=8x=56；(4)披碱草属物种的大部分基因组组成都可以在亚洲找到；(5)存在于大多数亚洲物种的Y基因组，被认为起源于中亚或喜马拉雅地区。这表明亚洲披碱草属植物的系统学研究将有助于了解整个披碱草属植物的演化关系。StY基因组四倍体物种有30多个种，主要分布于亚洲，是披碱草属的重要组成部分。与StH基因组物种相比较，StY基因组四倍体物种的形态和细胞学特征说明它是研究多倍化的非常好的材料。形态上表现出明显的过渡型：体积较大和体积较小的物种群，还有处于二者中间表型的物种群。在细胞学方面，StY基因组四倍体物种表现出两个亮点：一是这些物种的亲缘关系与它们的发生地呈显著相关性，St和Y基因组都出现明显的地理分化。但这种关系还没有用分子系统学的方法验证，以及出现这种分化的机制也没有报道。二是Y基因组的来源，这是披碱草属研究中比较有挑战性的课题，是一个任重道远的课题，也是一个意义深远的课题。细胞学的种种数据表明，Y基因组与St基因组的系统学关系非常近。本文通过核基因ITS、Adh和Waxy基因以及叶绿体基因trnL-F和marK的序列分析了披碱草属相关二倍体种的系统发育关系和披碱草属植物的起源。本文侧重于研究StY基因组的四倍体物种的系统发育关系和Y基因组的来源。主要研究结果如下：1．利用叶绿体基因trnL-F、nrDNA内转录间隔区(ITS)和三个单拷贝核基因(Adh2、Adh3和Waxy基因)的同源序列，探讨了披碱草属植物的相关二倍体属的二倍体种的系统发育关系。这些多基因树都显示了一致的结果，即(1)P、W、St和H基因组都是单系的；(2)H基因组处于系统发育树的基部，显示出它与其它三个基因组较远的系统学关系；(3)P和W基因组的关系最近；(4)St基因组和P—W基因组的分支表现出比较近的系统发育关系。2．披碱草属植物的分子系统发育和进化研究中，选用了含St、StSt、H、P和StY、StH、StStY、StHY、StPY、StWY基因组的38个物种的54份样品，其中包括45份披碱草属的样品。这些披碱草属样品包括39份的具明显地理分布格局的StY基因组样品。这些样品的nrDNA内转录间隔区(ITS)和两个叶绿体片段(trnL-F、marK)被测序。基于简约法和邻接法的系统发育分析都说明：拟鹅观草属的二倍体祖先种参与了StY、StStY、StH、StHY、StPY和StWY等基因组的物种形成，而且提供了母本。此外，我们还揭示出StH和StHY基因组物种的另一个二倍体祖先种来自大麦属，StPY基因组物种的另一个二倍体祖先种来自冰草属，而澳大利亚冰草属的二倍体祖先种参与了StWY基因组的物种形成。这说明披碱草属植物是一个典型的属间杂种多倍体。同时，系统发育树还赋予了Y基因组的来源和StY基因组物种的起源的重要内容。在系统发育树的拓朴结构上，有明显的P、W、H和St的分支，但没有Y基因组的分支。对比StH、StHY、StPY和StWY基因组的ITS序列特征，我们推断Y和St基因组可能共起源。系统发育树还揭示出StY基因组物种经历了多次杂交，是多次起源的四倍体物种，而且在进化中经历了由中亚向东亚和西亚的快速辐射过程，是最近起源的物种，它们的系统发育关系与物种的地理分布位置呈明显的地理相关性，这进一步支持了细胞学的观点。3．鉴于rDNA内转录间隔区和两个叶绿体片段对Y基因组的起源和StY基因组披碱草属植物的进化的重要提示，我们决定用系统发育信息更多的单拷贝基因Adh2、Adh3和Waxy来进一步研究Y基因的来源和StY基因组披碱草属植物的系统发育关系。这三个核基因提供的系统发育信息要远远高于rDNA内转录间隔区和两个叶绿体片段的。三个基因构建的系统发育树都以高的支持率说明Y基因组来自St基因组。物种以中亚为中心向四周迁移，但迁移过程中，受到环境适应的选择，在西亚保留了的相对较少的基因型的中亚物种并进化发展成西亚物种，东亚几乎保留了所有单倍型的中亚物种并发展成东亚物种。树的拓朴结构说明杂交、迁移和分化是StY基因组披碱草属植物的进化主动力。通过目前的研究，我们得出四个主要结论：(1)披碱草属多倍体植物是典型的属间和种间杂交的结果。分子系统学的研究结果表明大麦属(Hordeum L.，H基因组)、拟鹅观草属(Pseudoroegneria(Nevski)(?).L(?)ve，St基因组)、冰草属(Agropyron(L.)Gaertner，P基因组)和澳大利亚冰草属(Australopyrum(Tsvelev)(?).L(?)ve，W基因组)的二倍体种是该属植物的祖先。(2)StY基因组之间没有明显的分化，因此推断Y和St可能有共同的起源，即StSt基因组的同源四倍体物种经历了基因组的一系列的分化，如基因突变的积累，染色体片段的倒位和易位和基因组间的变化等。随着这种变化的逐渐积累，以致于形成了现在的StY基因组。由此，我们提出多倍体进化的一种新机制：同源多倍体的基因组在同一物种内的不断分化以及这种分化的逐渐积累形成了今天的一些异源多倍体物种。(3)分子证据表明在StY基因组的物种之间有明显的地理分化，中亚可能是StY基因组披碱草属植物的分化中心，并在此基础上，StY基因组的披碱草属植物向东亚和西亚进行了快速的辐射，形成了现代StY基因组物种的分布格局；多次杂交、迁移和分化是StY基因组的披碱草属植物进化的主要动力。(4)拟鹅观草属物种可能是所有披碱草属植物的母本供体。更多还原

【Abstract】 The wheat tribe Triticeae Damort., in the subfamily Pooideae, Poaceae, includes three of the most important cereal crops, i.e., wheat, barley, and rye, as well as many economically valuable forage grasses. The genus Elymus L. is the largest genus in the Triticeae Damort., following the circumscription of Love and Dewey, encompassing approximately 150 taxa, and occurring from the Arcic and temperate to subtropical regions. As an exclusively allopolyploid genus, Elymus has its origin in other groups, and thus it has close relationships with other genera in Triticeae. Cytological studies suggest that five basic genomes, namely, the St, Y, H, P, and W in various combinations constitute Elymus species, i.e. the StH and StY genomes in tetraploids, and the StStY, StHY, StPY, and StWY in hexaploids. The St genome is a fundamental genome that exists in all Elymus species and was donated by the genus Pseudoroegneria (Nevski) A.Love. The H, P, and W genomes are derived from the genera Hordeum L., Agropyron (L.) Gaertner, and Australopyrum (Tsvelev) A. Love of Triticeae, respectively. However, the donor of the Y genome that is present in the majority of the Asiatic Elymus species has not yet been identified, although extensive investigations have been carried out.Asia, particularly the central Asiatic mountain region, is an important center for the origin and diversity of Elymus L., which is reflected by the following facts: (1) more than half of the world number of Elymus species occurs in this area; (2) many different morphological forms, which are used as key characters in the taxonomy, are found in this region; (3) the different polyploid levels have been reported from Asia; (4) most genomic combinations known in this genus are found in Asia; (5) the "Y"genome, which is present in the majority of Asiatic Elymus species, is thought to have its origin in Central Asia or the Himalayan region. The StY-genome Elymus species, being one of the most important genomic combinations, are restricted to Asia with one exception of E. panormitanus (Parl.) Tzvelev, which is also found in South East part of Europe.Data from extensive morphological and cytogenetic analyses have been used to illustrate systematic relationships of the genus and to clarify the ancestry of polyploid species. Compared with StH-genome species, StY-genome species show unique systematic relationships. There had three distinct morphological groups. The cytogenetic analyses showed the StY-genome Elymus species from the same geographic regions, e.g. within eastern or western Asia, have a relatively closegenomic relationship, whereas those from different regions have a more distant relationship, and the St and Y genomes have close affinities. In this work, we used the sequences of nuclear genes (ITS, Adh2, Adh3 and Waxy) and cpDNA (trnL-F and matK) to explore the origin of Elymus species and the Y genome, and to reveal the mechanism of the geographical differentiation of the StY-genome Elymus species. The main results are summarized as follows:1. Based on multiple-gene sequence data, including one chloroplst trnL-F gene, one ITS fragments of nuclear rDNA, and three single copy nuclear gene Adh2, Adh3, and Waxy, the phylogenetic relationships of the four related genera diploids of Elymus were inferred. The phylogenic trees indicated that: (i) each genome group is monophyletic; (ii) H genome is basal; (iii) P genome shows a closer affinity to W genome; (iv) St genome has a close affinity to P-W genome clade.2. To explore the origin of Elymus species and the mechanism of the geographical differentiation of StY-genome Elymus species, we selected 54 taxa with St, H, P, StSt, StY, StH, StStY, StHY, StPY and StWY genome constitutions, including 45 Elymus taxa. In the present study, the ITS, trnL-F and matK were sequenced. The ITS sequences revealed a distinct phylogenetic relationship of the polyploid Elymus and their diploid donor genera, although the St and Y genomes did not show any separation. The ITS tree indicated a certain degree of geographic differentiation of the StY-genome species. The trnL-F and matK sequences revealed an especially close relationship of Pseudoroegneria to all Elymus species included. All these trees indicated multiple origins and recurrent hybridization of Elymus species.3. In the ITS sequence analysis, the phylogenetic tree indicated the correlation between geographic distribution and species relationships exists, and the Y and St genomes may have the same origin. Considered the weak support in the ITS tree, we selected single copy genes (Adh2, Adh3, and Waxy) to give further research about these scientific questions. The three single copy genes had higher phylogenetic information sites and gave good support for these two questions. In phylogenetic trees, there had distinct genomic clades clustered the diploid genomic species and the polyploids with one of genomes similar to the corresponding diploid genome. In other words, the StH- and StHY-genome polyploid species were grouped with St- and H-genome diploids, respectively; the StPY-genome polyploid species clustered with St- and P-genome diploids, respectively; the StY-genomepolyploid species grouped with St- and StSt- genome species. Still, there had no Y genome clade. Based on the ample data about these three genes, combined with the ITS sequences, and cytological data and C values, we thought the Y genome may be the differentiated St genome after St genome polyploidization. So, the StY-genome allotetraploids may be the StSt-genome autotetraploids or segmental autotetraploids.The phylogenetic trees showed the species distributed in western Asia all grouped with the central Asiatic species and formed the basal clade in the Adh gene trees, and some central Asiatic species grouped with eastern Asiatic species. These indicated the genetic diversity is the highest in the central Asiatic species, and the Central Asia may be the original center.In summary, this study suggests that: (i) Elymus L. has its origin through a typical intergeneric/interspecific hybridization, and the diploids of Pseudoroegneria (Nevski) A.Love, Hordeum L., Agropyron (L.) Gaertner, and Australopyrum (Tsvelev) A. Love of Triticeae are its ancestors; (ii) Pseudoroegneria (Nevski) A.Love was the maternal donor of the polyploid Elymus; (iii) the "Y" genome existed in the StY-genome Elymus may be differentiated from the St genome, i.e., the autopolyploid StSt-genome species probably experienced genomic differentiation within a species after autopolyploidy, leading to the significant genomic change and resulting the StY-genome allotetraploids; (iv) the StY genome species distributed in the Central Asia with a high level of genetic diversity radiated to the Western and Eastern Asia, under the environmental selection and other factors, and only a few species spread to the western Asia and most species have remained in the Central and eastern Asia.更多还原