【作者】 王晓丽；

【导师】 周永红；

【作者基本信息】 四川农业大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 小麦族(Triticeae Dumortier)是禾本科(Poaceae)植物中一个非常重要的类群。该族约有350-450多个种,广泛分布于世界各地,特别是北半球温带地区。小麦族多年生植物含有Ns、H、P、St, Y、W、E等20多个基本基因组,这些基因组的二倍体供体通过不同天然杂交组合形成了大量的含有不同基因组组成,如StP、StY、StH、StYW、StYP等多倍体植物,进而形成了大约70%-75%的小麦族多倍体物种。其中,具有StH基因组多倍体物种由二倍体的拟鹅观草属植物(St基因组)和二倍体的大麦属植物(H基因组)经异源多倍化形成。这些物种多为草原和草甸的优良牧草,具有较高的经济和生态效益,同时还具有抗寒、抗旱、耐盐碱等特性,以及具有麦类作物缺乏的优良抗病虫害和抗逆基因,是牧草遗传育种和麦类作物遗传改良重要的基因资源。具有StH基因组多倍体物种全世界约有20种,分布于全球温带和暖温带地区,生长在草原、山谷、林下、草地边缘等环境中。形态上,含StH基因组的小麦族植物存在每穗节小穗从单小穗(如Elymus caninus L.)到着生2枚小穗(如Elymus sibiricusL.)到着生3枚小穗(如Elymus canadensis L.);颖从狭长(如Elymus elymoides (Raf.) Swezey)到缺失(如Elymus hystrix L.)等一系列形态特征变异。含StH基因组物种拥有两个地理分布区：欧亚大陆和北美。尽管它们含有相同的基因组组成,却表现出不同的形态特征、不同的地理分布和生境。此外,不同学者对StH基因组植物具有不同的分类处理,在小麦族分类历史上,具StH基因组物种的分类处理存在很大的分歧,它们曾被归在披碱草属(Elymus)、猬草属(Hystrix)、裂颖草属(Sitanion)及偃麦草属(Elytrigia)等不同的属中,且许多物种还作为属的模式种处理。因此,不同学者不同的分类处理,使得它们间的关系显得非常复杂,分类处理困难。本研究基于多拷贝的细胞核ITS序列、单拷贝的细胞核Accl(plastid acetyl-CoA carboxylase)和DMC1(disrupted meiotic cDNA)基因序列、叶绿体trnL-F以及线粒体CoxII序列的分子系统发育分析,结合胚乳细胞学特征和杂种染色体配对资料,探讨含StH基因组物种的种间系统关系、不同地理分布下植物类群间的系统关系、基因组供体来源、物种形成的母本供体、复制基因的进化动力学、植物类群发生地理分化的生物学理论依据。主要结果如下：1.运用nrDNA ITS序列对16个具StH基因组组成的物种及5个拟鹅观草属(St)和3个大麦属(H)的二倍体物种进行了系统发育分析。结果表明：(1)过去归属于裂颖草属的Sitanion jubatum、Sitanion hystrix和猬草属的Hystrix patula与披碱草属物种关系较近,应当划入披碱草属中；(2)地理分布相同或相近的StH基因组物种聚类在一起,其亲缘关系较近；(3)北美StH基因组物种与欧亚大陆StH基因组物种在ITS序列上存在地理分布的异质性；(4)St基因组和H基因组的二倍体物种分别存在较大的分化；(5)探讨了造成小麦族StH基因组物种变异和分化的可能原因。2.获得了16个StH基因组物种的叶绿体trnL-F和线粒体CoxII内含子序列,并与小麦族二倍体供体物种的trnL-F和CoxII序列构建ML系统发育树、BI系统发育树和MJ网状支系结构。结果显示：(1)所有具StH基因组组成的物种与拟鹅观草属物种聚在一起,表明了StH基因组物种母本为St基因组供体；(2)StH基因组物种的母本来源存在多源化,其中Pseudoroegneria spicata或其祖先种可能充当了部分StH基因组物种的母本供体；(3)具St基因组的二倍体物种和StH基因组物种发生了一定程度的分化；(4)Hystrix patula、Sitanion hystrix和Sitanion jubatum与披碱草属物种聚在一起,将它们划入到披碱草属中是合适的;(5)Elytrigia repens与披碱草属物种存在差异,其系统地位还有待进一步研究。3.从15个含StH基因组物种中分离到29个Accl基因单倍型序列,单倍型序列覆盖了所有供试物种的Accl基因在不同基因组上的直系同源序列。将这29个单倍型序列与5个拟鹅观草属物种和3个大麦属植物类群的Accl基因序列进行了序列、系统发育及基因多态性分析,结果显示：(1)Elymus virginicus (H基因组拷贝类型)和Elvmus hystrix(H基因组拷贝类型)中拥有173 bp的反转录转座插入序列；(2)北美StH基因组物种和欧亚的StH基因组物种的Accl基因在长期的进化过程中己发生了遗传分化；(3)不同的拟鹅观草属植物可能作为St基因组供体参与了StH基因组物种的形成；(4)地理分布上相近的大麦属二倍体供体与StH基因组物种具有较近的亲缘关系；(5)Accl基因在StH基因组物种中多态性水平高于二倍体的基因多态性水平。4.从16个含StH基因组物种中分离到31个DMCl基因单倍型序列。将这31个单倍性序列与4个拟鹅观草属物种和3个大麦属植物类群的DMCl基因序列进行了序列和系统发育分析,结果显示：(1)北美StH基因组物种和欧亚的StH基因组物种在长期的进化过程中己发生了遗传分化；(2)不同的拟鹅观草属植物可能作为St基因组供体参与了StH基因组物种的形成；(3)Elymus sibiricus的H基因组可能存在多重起源；(4)DMC1基因比Accl基因在StH基因组物种中具有更多的简约信息位点比率。5.对小麦族含StH、St、H基因组的16个物种的胚乳细胞特征进行观察测量,结果表明StH基因组植物种子胚乳细胞存在丰富的多样性,不同物种的胚乳细胞在大小、形状和数量上均表现出明显的差异,但不能很好的反映属以及基因组间的差异。6.采用属间和种间杂交的方法,对来自6个不同地区的拟鹅观草属二倍体物种Pseudoroegneria libanotica、Pse.cognata、Pse. tauri、Pse. spicata、Pse. strigosa和Pse. stipifolia,和4个不同地区的大麦属二倍体物种Hordeum bogdanii、H. brevisubulatum、H. chilense、H. stenostachys与16个StH基因组物种进行相互杂交,目前仅得到了E.wawawaiensis和E. Hystrix与亚洲分布的拟鹅观草属物种间的4组杂交组合。对其杂种F1花粉母细胞减数分裂中期I染色体配对行为及繁育特性进行分析,结果表明：(1)分布于中东和中亚的拟鹅观草属二倍体物种间St基因组同源性较高,而与分布于北美的Pseudoroegneria spicata间同源性稍低,表明二倍体St基因组间存在分化;(2)来自于北美的Elymus hystrix和E. wawawaiensis基因组同源程度较高,而北美的Elymus hystrix和亚洲的E. sibiricus的同源程度相对较低,推测来自于北美和亚洲的StH基因组物种发生了一定程度的分化。更多还原

【Abstract】 The tribe Triticeae, including about 350-450 species distributed in the temperate and subtropical and tropic regions, is of enormous important group in Poaceae. Triticeae includes about 20 basic genomes (Ns, H, P, St, Y, W, E).These basic genomes in various combinations constitute numerous polyploid speceis, such as StP, StY, StH, NsXm, StYW, StYP species, leading to 70%～75% of Triticeae species are of polyploidy origin. One important group of polyploids in Triticeae is the species with StH genomes, which orignated from hybridization between the species from Pseudoroegneria and Hordeum. They are distributed from the Arctic, to temperate and subtropical regions, and occurred on grassland, semi-desert, mountain slopes, valleys, and forest regions. Most species with StH genomes are fine herbages, and some species have disease resistance, insect resistance and stress tolerance, which are precious germplasm resources in crop improvement and forage breeding.The species with StH genomes, including about 20 allotetraploids (2n=4x=28)and allohexaploids (2n=6x=42),was constituted by the St and H genomes in various combinations in tribe Triticeae. Morphologically, the species with StH genomes exhibit different variations with one (Elymus caninus L.)to two (Elymus sibiricus L.)to three (Elymus canadensis L.)spikelets per node, and narrow and long (Elymus elymoides (Raf.) Swezey) to reduced (Elymus hystrix L.)glumes.All the species with StH genomes were in the inter-continental disjunction between Eurasian and North American. Despite the similarity of genomic constitution, different species with StH genomes have different morphological characters, distribution and habits. In addition, the taxonomic treatment and phylogenetic relationships of the species with StH genomes are still in dispute. The StH genome species were once included in Elymus, Hystrix, Sitanion and Elytrigia, and many species were treated as the type species. The sectional delimitation of the species with StH genomes has been proposed by different scholars, which increase the uncertainties regarding the phylogenetic relationships among these species.In this study, we carried out the phylogenetic analysis of the species with StH genomes based on multiple-copy nuclear ITS,single-copy nuclear Accl and DMC1, chloplastid trnL-F and mitochondrial CoxII sequences.Data from these molecular phylogenetic evidences were analysed together with the endosperm cytological structures and meiotic pairing in hybrids.The objectives were:(1)to elucidate the phylogenetic relationships of the species with StH genomes;(2)to investigate whether the phylogenetic relationships of the StH-genome species are linked with the inter-continental disjunction between Eurasian and North American; (3)to reveal the origin of the genome in the species with StH genomes;(4) to identify the maternal genome donor to the species with StH genomes; (5) to examine the level of nucleotide polymorphism in St and H genome from the species with StH genomes;(6) to document geographic diversification patterns of the species with StH genomes.The main results are as follows:1.Based on nrITS sequences, phylogenetic analysis was performed on sixteen species with StH genome, five Pseudoroegneria (St) species and three Hordeum (H) species in the tribe Triticeae. Phylogenetic analysis suggests that:(1)Sitanion jubatum,Sitanion hystrix and Hystrix patula were closely related to the species of Elymus, and should be included in the genus Elymus; (2) the StH-genome species from the same areas or neighboring geographic regions are closely related to each other;(3)the ITS sequence of the StH-genome species distributed in North and Eurasian is heterogeneous geographically; (4) the ITS sequence of the St and H genome species were evolutionarily distinct; (5)why the variation and differentiation occurred in the species with StH genome were also discussed.2.The chloroplast trnL-F sequences and mitochondria CoxⅡintron and the combined data set of 16 StH-genome species and the related diploid species were analyzed using Maximum Parsimony (ML),Bayesian Inference (BI) and Median-joining (MJ)network approaches.Phylogenetic analysis and MJ network analysis suggest that:(1)the StH-genome species were clustered with diploid Pseudoroegneria species, indicating the St genome served as the maternal parent to the species with StH genome; (2)the StH-genome species have different maternal lineages and Pseudoroegneria spicata or its ancestry was the most possible maternal donor of some species; (3)the degree of differentiation occurred the diploid species with St genome and polyploid species with StH genome; (4)Hystrix patula and Sitanion hystrix and Sitanion jubatum were clustered with Elymus species, which is reasonable to transfer them to Elymus;(5)Elytrigia repens is different from Elymus species and its taxonomic rank is still ambiguous.3.Twenty-nine haplotyps of the Accl gene were isolated from fifteen StH-genome species.These haplotypes were analysis together with the haplotypes from five Pseudoroegneria and three Hordeum taxa. The results suggested that:(1)the Accl sequences from Elymus virginicus (H-copy) and E. hystrix (H-copy) had a 173 bp insertion; (2) the Accl sequence of the StH-genome species from Eurasian were evolutionarily distinct from those from North America;(3)different Pseudoroegneria species might sever as the donor during the speciation of StH-genome species; (4) the StH-genome species are closely related to the Hordeum species from the same areas or neighboring geographic regions; (5) nucleotide sequence diversity in the StH-genome species was higher than that in the St genome of diploid Pseudoroegneria and the H genome of diploid Hordeum.4.Thirty-one haplotyps of the DMC1 gene were isolated from 16 StH-genome species. These haplotypes were analysed together with the haplotypes from four Pseudoroegneria and three Hordeum taxa. The results suggested that:(1)the DMC1 sequences of the StH-genome species from Eurasian were evolutionarily distinct from those from North America; (2) different Pseudoroegneria species might serve as donor during the speciation of StH-genome species;(3)the StH-genome species might experience recurrent hybridization with different donor of Hordeum species; (4) the ratio of parsimony-informative sites were higher in the DMC1 sequences than that in the Accl sequences.5.The endosperm cytological structures of 16 Triticeae species with StH, St or H genomes were observed. The results showed that significant variations existed in the size, forms and quantity of endosperm cells in those species. The differences in genera are smaller than those in species, indicating that the characters of endosperm cells have less value in the systematic studies of those genera than in the studies of those species in Triticeae.6.To evaluate the genomic differentiation among the StH-genome species and to study the relationships between the StH-genome species and the species of the genus Pseudoroegneria and Hordeum,interspecific and intergeneric hybridizations were carried out among the 16 StH-genome species and between the StH-genome species and the diploid donor of Pseudoroegneria (Pseudoroegneria libanotica, Pse. cognata、Pse. tauri, Pse. spicata, Pse. strigosa and Pse. stipifolia) and Hordeum(Hordeum bogdanii, H. brevisubulatum, H. chilense and H. stenostachys).In the previous study, four intergeneric hybridizations of E. wawawaiensis and E. Hystrix were successfully carried out with Pseudoroegneria species in Asia. The fertility and chromosome pairing behavior in meiosis of the parents and their hybrids F1 were investigated. The results showed that:(1)a high homology existed in the St genome donored by Pseudoroegneria species from the middle East and central Asia, and a low homology existed in the St genome donored by Pseudoroegneria species from the middle East and North America, indicating the differentiation among the St genome; (2) a high homology existed between the North America Elymus hystrix and Elymus wawawaiensis, and a low homology existed between the North America Elymus hystrix and Asia Elymus sibiricus, which is indicative of the differentiation of StH-genome species distributed between North America and Asia.更多还原