【作者】 马啸；

【导师】 周永红； 白史且；

【作者基本信息】 四川农业大学 ， 草业科学， 2008， 博士

【摘要】 研究和了解植物种质间的遗传变异对于其在育种上的有效利用非常有益。自然种群的遗传多样性研究可以为物种保护计划的制定和实施提供参考和借鉴。老芒麦（Esibiricus L.）又名西伯利亚野麦草,是披碱草属的模式种,原生于欧亚大陆北部,具有多年生和自花授粉的习性,为具有StStHH染色体组构成的异源四倍体。其自然分布横跨欧洲的瑞典到东亚的日本,甚至到达北美的阿拉斯加和加拿大,向南可以延伸分布至中国青藏高原的海拔4000米以下的地区。老芒麦一般生长于湿润的草地、河滩、灌木丛中或森林边缘地带。国内对老芒麦的研究多集中在资源评价、栽培技术、种子生产、抗性生理生化等方面;国外的研究更多的是集中于其抗寒性的生化与分子生物学机理及包括老芒麦在内的披碱草属物种的系统分类与进化方面。目前对老芒麦种质的遗传多样性几乎没有相关研究。本研究利用形态-农艺学性状、种子醇溶蛋白和SRAP分子标记评价了来源广泛的老芒麦种质的遗传多样性;同时利用ISSR和RAPD分子标记对青藏高原东南部的8个老芒麦居群进行了群体结构和遗传变异分析。主要结果如下:1.以栽培品种川草2号为对照,观测34份老芒麦野生种质的13个形态和农艺学性状的基本数据,基于欧氏距离进行UPGMA聚类分析,揭示老芒麦各野生种质间的生物多样性。研究结果表明,老芒麦野生种质间表型差异明显,生物多样性丰富。根据聚类结果,供试种质可以划分成3个具有明显形态和农艺性状差异的类群。类群Ⅲ中3份来自新疆的种质和1份来四川红原的种质具有突出的农艺性状表现,其牧草和种子生产性能远高于其它居群。主成分分析的结果与聚类分析基本一致。另外还对野生老芒麦种质的收集和保护提出了建议。2.利用基于APAGE的醇溶蛋白标记对来自亚洲和北美的86份老芒麦种质的遗传多样性和遗传关系进行了分析。电泳共检测到52条醇溶蛋白条带,其中47条为多态性条带,多态性比率达90.4%。种质间遗传相似系数的变幅为0.108～0.952,平均值为0.373,这表明种质间的存在较高水平的遗传多样性。利用Shannon多样性指数反映了类似的结果,即供试种质间的多样性指数达到0.460的较高水平。基于多样性指数计算了老芒麦种质地理类群遗传分化程度,地理类群间和地理群内的遗传变异分别占总变异的55.9%和44.2%。对种质和地理类群的聚类分析结果均显示来源于青藏高原的种质与来源于其它地理来源的种质具有较大的差异,可以分成明显的两支。这种聚类结果可能与种质的地理来源与生态适应性有关。同时还就遗传多样性的范围及种质间的遗传关系做了进一步探讨。3.利用SRAP标记对来自亚洲的84份老芒麦种质的遗传多样性和遗传关系进行了分析。23个引物组合共产生337条扩增带,其中203条为多态性带,多态性比率为60.24%。各种质问遗传相似系数的变幅为0.783到0.965,平均值为0.865。来自于青藏高原和蒙古的种质间的平均GS值最小（0.830）,而来自于俄罗斯和和蒙古的种质间的平均GS值最大（0.897）。对84份种质的聚类分析表明,供试种质可以划分成2大类,而且聚类结果与原始相似性矩阵间的具有很高的吻合度（r=0.88）。同时,主向量分析（PCoA）也得到了与聚类分析类似的结果。分子方差分析（AMOVA）表明在总的遗传变异中有79.62%发生在地理类群内,有20.38%发生在类群间(Φ_ST=0.204),类群间和类群内的变异均为极显著（P＜0.0001）。基于各地理类群间Φ_ST值进行的聚类分析也表明青藏高原类群明显区别于其它地理类群。这种聚类模式可能依赖于种质的地理来源赋予其的特殊生态地理适应性。4.本研究利用ISSR分子标记对来自青藏高原东南部的8个老芒麦的遗传多样性和群体遗传结构进行了分析和评价。在100个ISSR引物中筛选出13个能扩增出高度重复性条带的引物。这13个引物共扩增出193条可分辨的条带,其中149条（占77.2%）具有多态性,表明老芒麦居群在物种水平上存在较高水平的变异。相反,各居群的多态性位点比率（P_P）在44.04%到54.92%之间变化,表明群体水平的变异较低。群体水平的平均基因多样性（H_E）为0.181（变幅为0.164～0.200）,而物种水平的平均基因多样性达0.274。基于Nei.氏基因多样性的群体分化系数达到33.1%,而基于Shannon指数、贝斯叶方法和分子方差分析（AMOVA）的群体分化系数分别为34.5%、33.2%和42.5%。AMOVA分析表明采样地区之间的ISSR变异不存在显著的统计学差异（P=0.08）,然而群体间和群体内的变异分别为42.5%和57.5%,均显示为差异极显著（P＜0.001）。各居群间存在较高的Nei氏遗传一致度。这种遗传变异模式不同于已报导的大多数小麦族自交物种。另外,基于聚类分析和主向量分析的结果表明各居群间存在较为明显的地理分化,即8个居群分化为采集地的南部和北部2个分支。总之,本研究结果表明来自青藏高原东南部的老芒麦居群内部存在较高水平的ISSR变异。5.本研究利用RAPD分子标记对来自青藏高原东南部的8个老芒麦的遗传多样性和群体遗传结构进行了分析和评价。在150个RAPD引物中筛选出25个能扩增出高度重复性条带的引物。这25个引物共扩增出370条可分辨的条带,其中291条（占78.65%）具有多态性,表明供试居群在物种水平上存在较高水平的变异。同时各居群的多态性位点比率（P_P）在46.49%到53.78%之间变化,表明群体水平的变异较低。居群水平的平均基因多样性（H_E）为0.176（变幅为0.159～0.190）,而物种水平的平均基因多样性达0.264。基于Nei氏基因多样性、Shannon指数和贝叶斯方法的群体分化系数分别为32.0%、33.7%和33.5%。AMOVA分析表明居群内遗传达到总变异的59.9%,而居群间变异仅有40.1%,但二者均达到极显著水平（P＜0.001）。居群间每世代迁入个体数（基因流）达到1.06个。各居群间存在较高的Nei氏遗传一致度。基于RAPD和ISSR的Nei基因多样性指数间存在显著相关。另外,基于聚类分析、主向量分析及AMOVA的结果均表明各居群间存在较为明显的地理分化,即8个居群分化为采集地的南部和北部2个分支,与RAPD结果存在相似性。对该地区老芒麦应尽量选择遗传多样性高的居群实施就地保护。更多还原

【Abstract】 The understanding and knowledge of genetic variation within germplasm collections are useful for the efficient utilization of genetic resources in breeding programs.A prerequisite for an efficient conservation of biodiversity is that there is knowledge on genetic variation of native populations.As the type species of the genus Elymus,E. sibiricus L.（Siberian wildrye）is a perennial,self-pollinating and allotetraploid grass indigenous to Northern Asia,possessing the St and H genome.Its natural geographic distribution extends from Sweden to Japan and even to parts of Alaska and Canada,and that extends southerly to Qinghai-Tibet Plateau,which is the highest plateau in the world. E.sibiricus usually grows on wet meadows,riverside sands,and among open forest or shrubs.In the subalpine meadows with less than 4000m altitude in Qinghai-Tibet Plateau, E.sibiricus usually serves as an important forage species.Resource evaluation,cultivation technique,and seed production has been studied for E.sibiricus in China,whereas overseas studies of this species focused on biochemical and molecular mechanism of cold resistance, and phylogegenetic and evolutionary relationships of Elymus species including E.sibiricus. At present,very little is known about the information of genetic diversity of E.sibiricus germplasm collections or populations.The genetic diversity of worldwide germplasm collections of E.sibiricus was evaluated based on morpho-agronomic traits,gliadin markers,and SRAP markers.In addition,population structure and genetic variation among E.sibiricus populations from the southeast of Qinghai-Tibet Plateau of China.The main results showed as follows:1.Based on investigation of thirteen morpho-agronomic traits of 35 native Elymus sibiricus accessions including cultivar Chuancao No.2 as a check,Euclidean distances were calculated to performing cluster analysis for.The results showed that there were significant phenotypic differences among the studied accessions,indicating rich within-species biodiversity.According to the cluster analysis,the 35 accessions were classified into three groups with great morpho-agronomic differences.In groupⅢ,three accessions from Xingjiang Province and one accession from Hongyuan,Sichuan Province, had the highest performance in terms of forage and seed yields.The principle component analysis revealed a similar result to cluster analysis.The collection and conservation of native germplasm resources of E.sibiricus was also discussed in this part.2.Gliadin markers based on A-PAGE were used to analyze genetic diversity and genetic relationship among eighty-six Elymus sibiricus accessions from Asia and North America. Total of 52 gliadin bands were scored,of which 47 were polymorphic（90.4%）.The generated similarity matrix showed that the genetic diversity within the accessions was fairly high（average similarity index=0.373）.Similarity values among the accessions ranged between 0.108 and 0.952.Similar results were obtained when genetic diversity was estimated using the Shannon-Weaver index of diversity.The total genetic diversity across all accessions was 0.460.The proportion of variation explained by within geographic group and between geographic groups diversity was 0.559 and 0.442,respectively.Cluster analysis showed a clear demarcation between accessions from Qinghai-Tibetan Plateau, China and the others as separate groups.The extent of genetic diversity and genetic relationship among accessions is discussed.The clustering pattern was probably dependent on geographic origin and ecological adaptability of the accessions.3.SRAP markers were used to analyze genetic diversity and genetic relationship among eighty-four Elymus sibiricus accessions from Asia.A set of 23 primer combinations yielded 337 bands,of which 203 were polymorphic（60.24%）.Genetic similarity values （GS）among the accessions ranged between 0.783 and 0.965 with a mean of 0.865.On the average,Mongolian and Russian accessions were the most similar while,Mongolian and Qingahi-Tibet Plateau accessions were the most distant ones.Cluster analysis grouped the 84 accessions into two clusters,which has quite a high fit（r=0.88）to the original similarity matrix.Results of cluster analysis which was supported strongly by the principal coordinate analysis.The molecular variance analysis（AMOVA）showed that the proportion of variation explained by within geographic group and between geographic groups diversity was 0.7962 and 0.2038,respectively.Similar results were obtained when genetic diversity was estimated using the Shannon’s index of diversity.Based on pairwiseΦ_STbetween geographic groups,cluster analysis showed a clear demarcation between accessions from Qinghai-Tibetan Plateau and the others as separate groups.The clustering pattern was probably dependent on geographic origin and ecological adaptability of the accessions.The results of present study can be useful in collecting germplasm and the establishment of core collections of E.sibiricus.4.Inter-simple sequence repeats（ISSR）markers were used to assess the genetic diversity and population structure in eight populations of Elymus sibiricus L.from the southeast of Qinghai-Tibet Plateau of China.Of the 100 primers screened,13 produced highly reproducible ISSR bands.Using these primers,193 discernible DNA fragments were generated with 149（77.2%）being polymorphic,indicating considerable genetic variation at the species level.In contrast,there were relatively low levels of polymorphism at the population level with the percentage of polymorphic loci（P_P）ranging from 44.04% to 54.92%.The mean gene diversity（H_E）was estimated to be 0.181 within populations （range 0.164 to 0.200）,and 0.274 at the species level.A high level of genetic differentiation among populations was detected based on Nei’s genetic diversity analysis （33.1%）,Shannon’s index analysis（34.5%）,Bayesian method（33.2%）and AMOVA analysis（42.5%）.No significant statistical differences（analysis of molecular variance [AMOVA],P=0.08）in ISSR variation was found between the sample collection regions. However,among populations（42.5%of the variance）and within populations（57.5%of the variance）,there were significant differences（P＜0.001）.Populations shared high levels of genetic identity.This pattern of genetic variation was different from that for most of inbreeding Triticeae species reported.In addition,a geographical pattern of population differentiation,where the populations from south and north of sampling sites were clearly separated from each other,was revealed by both the cluster and principal coordinates analyses.Generally,the result of this study indicates that E.sibiricus contains high molecular variation in its populations.5.Random amplified polymorphic DNA（RAPD）markers were also used to assess the genetic diversity and population structure in eight populations of Elymus sibiricus L.from the southeast of Qinghai-Tibet Plateau of China.Of the 150 primers screened,25 produced highly reproducible RAPD bands.Using these primers,370discernible DNA fragments were generated with 291（78.65%）being polymorphic,indicating considerable genetic variation at the species level.In contrast,there were relatively low levels of polymorphism at the population level with the percentage of polymorphic loci（P_P）ranging from 46.49% to 53.78%.The mean gene diversity（H_E）was estimated to be 0.176 within populations （range 0.159 to 0.190）,and 0.264 at the species level.A high level of genetic differentiation among populations was detected based on Nei’s genetic diversity analysis （32.0%）,Shannon’s index analysis（33.7%）,Bayesian method（33.5%）.The partitioning of molecular variance by AMOVA analysis indicated significant genetic differentiation within populations（59.9%）and among populations（40.1%;P＜0.001）.The average number of individuals exchanged between populations per generation（Nm）was 1.06.Populations shared high levels of genetic identity.The correlation between RAPD and ISSR gene diversity（H_E）showed a relative high correlation.In addition,a geographical pattern of population differentiation,where the populations from south and north of sampling sites were clearly separated from each other,was revealed by both the cluster,AMOVA,and principal coordinates analyses.Generally,the result of this study indicates that E.sibiricus contains high molecular variation in its populations.The implications of these results for the conservation of the species are also discussed.更多还原