【作者】 陈仕勇；

【导师】 张新全；

【作者基本信息】 四川农业大学 ， 草业科学， 2013， 博士

【摘要】 小麦族(Triticeae Dumort.)是禾本科(Poaceae)植物中十分重要的一个类群,它不仅包括有重要的粮食作物,还包括许多具有重要经济和生态价值的牧草资源。这其中披碱草属(Elymus L.)和鹅观草属(Roegneria C. Koch)是小麦族中两个重要的多年生属。披碱草属和鹅观草属的种属界限和系统关系一直存在争议,其生物系统学也是禾草学家长期研究的热点。目前的相关研究主要集中在披碱草属和鹅观草属四倍体物种中,但对于形态变异较大、分类争议较多的六倍体物种间的系统关系研究却少有报道。本论文广泛收集了来自世界范围的26个披碱草属、鹅观草属六倍体物种及相关基因组二倍体和四倍体材料展开了六倍体物种的分子系统学研究。论文分别采用分子标记(SSR、AFLP标记)、核糖体DNA序列(ITS)、叶绿体序列(trnL-F)及核单拷贝基因序列(DMC1)研究该类群六倍体物种的分子系统学关系,目的在于揭示披碱草属、鹅观草属六倍体物种的遗传变异和种间亲缘关系,探讨六倍体物种的系统进化关系及St、H、Y基因组的遗传分化。主要研究结果如下：1.选用了来自小麦族5个属的230个SSR和EST-SSR标记对小麦族St、H、Y基因组的23个物种进行了通用性分析。结果表明：(1)在筛选的230个标记中,163个(70.87%)SSR和EST-SSR标记能在所有的供试物种中扩增出清晰的条带,在St、 H、Y基因组物种中表现出了较高的通用性；(2)在扩增出的163个标记中,EST-SSR(87.6%)比基因组SSR(49.5%)标记在种属间表现出了较高的通用性,而基因组SSR标记(86%)则比EST-SSR (79.8%)标记表现出更高的多态性；(3)基于Dice遗传相似系数的UPGMA聚类结果与供试物种的基因组之间表现出了较高的相关性,相同或相似基因组物种间表现出较近的亲缘关系。2.采用27个高通用性的SSR标记对80份披碱草属、鹅观草属及其近缘属材料其中包括47个披碱草属、鹅观草属六倍体物种进行种间系统关系研究。结果表明：(1)27个SSR标记在供试材料中共扩增出234条带,基于Dice遗传相似系数,供试材料的遗传相似系数变化为0.286-0.981,平均值为0.599,其中披碱草属、鹅观草属六倍体物种之间的变化为0.479-0.981,平均值为0.670,供试材料间表现出了丰富的遗传变异；(3)基于Dice遗传相似系数的UPGMA聚类分析结果表明相同或者相似基因组物种能聚在一起,其中H基因组二倍体物种与供试材料间的亲缘关系较远；(4)具有StHY基因组的物种表现出了明显的分化,聚类分析将其明显的分为3类,即E. dahuricus类、E. nutans类及R. kamoji类,这种分化与其形态学特征间表现了高度的一致。3.采用AFLP标记对80份披碱草属、鹅观草属及近缘属材料进行种间系统关系研究。结果表明：(1)4对高多态的引物组合共扩增出214条带,其中多态性条带比率为99.1%,在披碱草属、鹅观草属六倍体物种共扩增出199条带,其中多态性条带比率为94.5%；(2)基于Dice遗传相似系数,供试物种间遗传相似系数变化为0.215-0.964,平均值为0.605,披碱草属、鹅观草属六倍体物种中变化为0.391-0.982,平均值为0.626；(3)基于Dice系数的UPGMA聚类分析同样与其物种的基因组间表现出一定的相关性,同时也揭示了StHY基因组物种间的明显分化；(4) AFLP标记与SSR标记在本研究中均得到了相似的研究结果,表现出了较高的相关性(r=0.734, p=0.024), AFLP标记在本研究中表现出比SSR标记更高的标记效率。4.采用ITS序列对43个披碱草属、鹅观草属六倍体及相关St、H基因组二倍体和四倍体物种进行了种间关系及系统进化关系分析。结果表明：(1)26个六倍体物种分离得到不同类型的ITS序列40条,披碱草属、鹅观草属六倍体物种不同类型的ITS序列之间协同进化不完全,并呈现出偏向St类型的进化趋势；(2)系统发育分析表明St基因组与H基因组之间的关系较远：(3)StHY基因组物种间存在分化,可能与其不同的系统演化途径及不同的亲本来源有关。5.采用叶绿体trnL-F序列对50个披碱草属、鹅观草属六倍体及相关二倍体和四倍体物种进行系统进化分析。结果表明：(1)供试的披碱草属、鹅观草属六倍体物种的母本供体基因组均来自于拟鹅观草属的St基因组；(2)拟鹅观草属二倍体物种间存在分化,不同的St基因组物种参与了披碱草属、鹅观草属六倍体物种的形成。6.采用核单拷贝基因DMC1序列对40个披碱草属、鹅观草属六倍体及相关二倍体物种进行系统发育分析。结果表明：(1)26个六倍体物种分离得到不同拷贝的61条DMC1序列,包括25条St拷贝类型、20条Y拷贝类型以及16条H拷贝类型；(2)六倍体物种的St、H、Y基因组之间存在明显的分化,DMC1基因序列的H拷贝类型的变异最大；(3)六倍体物种中St、H拷贝类型序列的多态性均明显小于相关二倍体物种,这可能表明少数几个St及H基因组二倍体物种参与了六倍体物种的形成；(4)进一步揭示了StHY等基因组物种间的分化。更多还原

【Abstract】 The Triticeae is a very important goup in Poaceae, which not only includes some important cereal crops, but also cmparises many forage grasses with high values in economy and ecology. The Elymus L. and Roegneria C. Koch are two important perennial genera in Triticeae, which also include many excellent cultivated forage grasses and the special gene pool for genetic breeding and improvement of crops and forages. However, there is a controversy in circumscription of related species and genera and phylogenetic raltionships between these two genera, which is also a hotpot for grass taxomomists. Although, there had been reported many studies about phylogenetic relationships and species circumscription in tetraploid species with StH and StY genome constitution, there is few studies inferred to hexaploid species with the large variation in morphology and taxonomy controversy.A total of26hexaploid species from Elymus and Roegneria, as well as the related tetraploid and diploid species were widely collected from worldwide. The molecular marker including SSR and AFLP, sequence of ITS, trnL-F and low copy gene DMC1were applied for their molecular phylogeny, which could reveal the relationships among the hexaploid species, disscuss the relationships among the St, H, Y genome and detect the related differentiation. The main results were as follows:1. A total of230SSR and EST-SSR markers from5different genera were used to study the transferability to species with St, H, Y genome. Among the230SSR markers,163(70.87%) marker could generate the clear bands, which showed a high transferability for the selective markers. The EST-SSR marker (87.6%) showed a higher transferability than genomic SSR (49.5%), the genomic SSR (86%) also showed a higher polymorphism than EST-SSR (79.8%). The results of cluster showed a related relationship with the genome consititution of the species, the species with same or similar genome could be groups together.2. A total of80materials including47hexaploid species from Elymus and Roegneria were selected to study the phylogenetic relationships using the27high transferability SSR markers. The Dice genetic similarities ranged from0.286-0.981with mean of0.599. Among the hexaploid species, the GS coefficients ranged from0.479-0.981. A high level of genetic diversity had been revealed among the materials. The results of cluster showed that the species with same or similar genome could be grouped together. Then, three different variation types had also been revealed among the species with StHY genome, which also was consistent to the morphology.3. A total of80materials including47hexaploid species from Elymus and Roegneria were also selected to study the phylogenetic relationship using AFLP markers. The Dice genetic similarities ranged from0.215-0.964with mean of0.605. Among the hexaploid species, a total of199bands with94.5%polymorphism were also generated, the GS coefficients ranged from0.391-0.982with an average of0.626. A high level of genetic diversityand genetic divergence had also been revealed among the hexaploid species. The results of AFLP waere agree with the SSR. The AFLP showed a higher analysis efficiency than SSR, which also showed a high related with SSR markers (r=0.734, p=0.024).4. The ITS sequence from43species including26hexploid species from Elymus and Roegneria were used to study the phylogenetic relationships among these materials. A total of40ITS sequences were selected for26hexaploid species. It showed an incomplete converted evolution and St bias pattern of ITS sequence among the hexaploid species. The related far relationship was revealed between St and H genome. The genome differentiation of StHY species was detected and maybe related with the different complex evolution and speciation pattern.5. The chloroplast trnL-F sequence from50species with St. H. Y genome were used to study the phylogenetic relationships among the materials. The results showed that the St genome from Pseudoroegneria was the female parent for all the hexaploid species of Elymus and Roegneria. Then, a significant genome differentiation had alao been revealed, which also indicated that different species with St genome involved inthe speciation of hexaploid species.6. The sequences of low-copy gene DMC1from40speices with St, H, Y genome were applied for elaborating the phylogenetic relationships among the materials. The results showed that a total of61sequences were gained for26hexaploid species, which included25St-copy,20Y-copy,16H-copy. A significant differentiation of St, H, Y genome were also detected, of which H-copy sequences showed the largest variation. Then, the sequences of St and H-copy from hexaploid speices showed a lower variation than relative sequences in diploid speices, which showed that a few donor species from Pseudoroegneria and Hordeum might be involved the formation of hexaploid species. The low-copy nuclear gene also demonstrated the divergence among the species with different genome constitution, such as the different types of hexaploid species with StHY.更多还原