中国西南地区青杨(Populus cathayana Rehd)遗传多样性的ISSR研究

青杨作为一个本土树种，能较好的适应潮湿和寒冷的环境，对中国西部的人工造林有着重要的参考价值。在本实验中，选取7个中国西南地区分布的自然群体，用ISSR(inter-simple sequence repeats)作为分子标记研究其遗传多样性水平和遗传结构。通过筛选的8个ISSR引物，获得了158条清晰可重复的DNA条带，其中有156条具有多样性(占98.7%)。平均的Nei’s遗传多样性(h)为0.331；遗传分化系数(GST)为0.477，这表明有47.7％的遗传多样性发生在群体间。这种高水平的分化可能是由于当地复杂多变的地形和气候特点阻碍了基因流而引起的。此外在这7个青杨群体中，遗传距离和地理距离并未体现出有显著相关性（r=0.3122, P>0.05)。联合遗传距离和地理距离分析，鉴定出两处低水平基因交流的地区, 探讨其遗传障碍形成原因。 As a native species to China, Populus cathayana Rehd is well-adapted to the wet and cold environments where it occurs. It is considered to be an important reforestation species in western China. In the present study, we surveyed the level of genetic variation and the pattern of genetic structure in seven natural populations of P. cathayana, originating from the southeastern Qinghai-Tibetan Plateau of China, by using ISSR (inter-simple sequence repeats) markers. Based on eight primers, 158 clear and reproducible DNA fragments were generated, of which 156 (98.7%) were polymorphic. The average value of Nei's gene diversity (h) equaled 0.331. The coefficient of genetic differentiation (GST) equaled 0.477, which means that 47.7% of the total molecular variance existed among populations. Such a high level of divergence present among populations may be caused by the complex topography and variable climatic conditions present in the southeastern Qinghai-Tibetan Plateau which effectively restrict gene flow. Moreover, there is a lack of significant association between genetic and geographical distances (r=0.3122, P>0.05) in the populations of P. cathayana. The application of a novel method, which combines geographical coordinates and genetic differentiation to detect barriers for gene flow, allowed us to identify two zones of lowered gene flow.

青藏高原东缘青杨（Populus cathayana Rehd.）遗传多样性研究

青杨（Populus cathayana Rehd.）是青杨派杨树的主要树种之一，为我国特有乡土树种，其主要分布区之一是我国的青藏高原，集中分布地带在甘肃省中部及青海省东部，四川省西北部岷江上游和松潘等地区。本研究以青藏高原东缘青杨天然分布区的6个群体143个个体为材料，用AFLP、SSR和叶绿体SSR分子标记分析青杨天然群体的遗传多样性，分析其遗传结构和分化，比较6个群体间遗传多样性的高低和群体间的遗传关系。旨在为青杨基因资源评价、保护与保存、遗传改良策略制定等提供科学理论依据。通过以上研究，得出如下主要研究结果： 1 AFLP分子标记研究结果 采用4对选择性引物对6个青杨天然群体143个个体进行分析，扩增谱带分析共检测到175个位点，其中173个位点表现为多态，多态位点百分率高达98.9%。从整体上表现出较高的遗传多样性，Nei’s基因多样度(h)水平为0.306。从青杨天然群体位点分布来看，有高达20%的位点（32位点）为群体所特有，仅有9.14%的位点（16位点）在所有群体中存在。群体间的遗传分化极大，所有遗传变异中，有48.9%的遗传变异存在于群体间。在个体群丛（Individuals cluster）和主坐标（PCO analysis）分析中，青杨各群体未呈现任何地理模式，Mantel检测也显示各群体间遗传距离与地理距离无明显相关。研究认为，由于地理和空间上大尺度的隔离和地形地貌复杂使得群体间无法进行基因交流，导致群体间遗传分化极大，另外各群体在不同的选择压力下，经历各自独立的进化历程，这些都可能导致群体间遗传距离与地理距离的不相关。 2 SSR分子标记研究结果 在SSR分析中，7个位点在6个青杨天然群体143个个体中共检测到79个等位基因，每位点检测到的等位基因数在5-16之间，平均11.3个，总体上多态位点百分率达100%。平均观察杂合度和期望杂合度分别为0.792和0.802。Hardy-Weinberg平衡检验表明青杨大部分群体都处于非平衡状态，群体大部分位点都是偏离哈迪-温伯格平衡（76.3%），只有23.7%的测验满足哈迪-温伯格平衡。分析青杨天然群体内和群体间的遗传变异，基因分化系数（GST）为0.373，即有62.7%的遗传变异存在群体内，37.3%的遗传变异存在群体间。群体内的遗传变异高于群体间水平。根据各群体遗传距离UPGMA聚类分析，有来自相临分布区、近似气候类型的群体聚在一起的趋势，但Mantel检测反映遗传距离与地理距离间并无明显相关性。 3 cpSSR分子标记研究结果 分析来自青藏高原东缘6个青杨天然群体，所用cpSSR引物中有5对cpSSR引物（CCMP2、CCMP5、SCUO01、SCU03、SCU07）都表现较高的多态性，单个引物检测的片段数都在4以上。5对cpSSR引物共检测片段数26个，组成了12种叶绿体DNA单倍型。各群体的单倍型分布和频率有较大差异，群体单倍型多样性范围为0-0.4926，TS、JZ、PW和SHY群体单倍型多样性高于QHY和LED群体水平。本研究发现，分布在青藏高原东缘的青杨天然群体，群体间不存在共享的单倍型，各群体间存在极大的遗传分化（GST=0.9223）。从青藏高原东缘地区经历的地质历史事件来看，第四纪的冰期气候变迁可能是造成青杨现今遗传结构模式的主要因素之一。根据单倍型在各群体的分布情况，进行青杨群体聚类分析结果，各群体无明显的分组现象，青杨各群体也未呈现任何清晰地理模式。 由于不同分子标记在对群体遗传多样性检测能力与效率上存在差异，所以三种标记检测的青藏高原东缘青杨天然群体遗传多性水平也不尽一致，但在与用同种方法检测其它物种或同一物种不同种源群体比较，三种分子标记方法都揭示了青藏高原东缘青杨天然群体具有中等偏上的遗传多样性水平。结果分析表明，群体间遗传分化极大，这是由于青杨天然群体分布于青藏高原东缘，既有高原又有高山峡谷，由于地理和空间上大尺度的隔离和地形地貌复杂导致了基因流物理上的阻隔。三种分子标记研究结果经Mantel分析检测，遗传距离与地理距离之间都无明显相关性。较为一致的解释是，青杨分布区域地理和空间上大尺度的隔离和和地形地貌复杂导致群体之间不存在均匀扩散现象，另外各群体在不同的选择压力下，经历各自独立的进化历程，这些都可能导致群体间遗传距离与地理距离的不相关。 The wide geographical and climatic distribution of P. cathayana Rehd. indicates that there is a large amount of genetic diversity available, which can be exploited for conservation, breeding programs and afforestation schemes. The results are as follows: 1 Research results of AFLP genetic diversity In present study, genetic diversity was evaluated in the natural populations of P. cathayana originating from southern and eastern edge of the Qinghai-Tibetan Plateau of China by means of AFLP markers. For four primer combinations, a total of 175 bands were obtained, of which 173 (98.9%) were polymorphic. Six natural populations of P. cathayana possessed different levels of genetic diversity, high level of genetic differentiation existed among populations (GST=0.489) of P. cathayana. Individuals cluster and PCO analysis based on Jaccard’s similarity coefficient also showed evident population genetic structure with high level population genetic differentiation. The long evolutionary process coupled with genetic drift within populations, rather than contemporary gene flow, are the major forces shaping genetic structure of P. cathayana populations. Moreover, there is no correspondence between geographical and genetic distances in the populations of P. cathayana, seldom gene exchange among populations and different selection pressures may be the causes. Our finding of different levels of genetic diversity within population and high level of genetic differentiation among populations provided promising condition for further breeding or conservation programs. 2 Research results of SSR genetic diversity In this study, the genetic diversity of P. cathayana was investigated using microsatellite markers. In a total of 150 individuals collected from six natural populations in the southeastern part of the Qinghai-Tibetan Plateau in China, a high level of microsatellite polymorphism was detected. At the seven investigated microsatellite loci, the number of alleles per locus ranged from 5 to 16, with a mean of 11.3, the observed heterozygosities across populations ranged from 0.408 to 0.986, with a mean of 0.792, and the expected heterozygosities across populations ranged from 0.511 to 0.891, with a mean of 0.802. The proportion of genetic differentiation among populations accounted for 37.3% of the whole genetic diversity. The presence of such a high level of genetic diversity could be attributed to the features of the species and the habitats where the sampled populations occur: The southeastern part of the Qinghai-Tibetan Plateau is regarded as the natural distribution and variation center of the genus Populus in China. Variation in environmental conditions and selection pressures in different populations, and topographic dispersal barriers could be factors associated with the high level of genetic differentiation found among populations. The populations possessed significant heterozygosity excesses, which may be due to extensive population mixing at the local scale. The cluster analysis showed that the populations are not strictly grouped according to their geographic distances but the habitat characteristics also influence the divergence pattern. In addition, we suggest that population SHY should be regarded as an ecologically divergent species of P. cathayana. 3 Research results of cpSSR genetic diversity Genetic diversity of six natural populations of P. cathayana originating from the southeastern part of the Qinghai-Tibetan Plateau in China was studied by use of cpSSR markers. Based on 5 pairs of polymorphic primers screened from 12 pairs of primers, twenty-six different length fragments and twelve different kinds of haplotypes were reduced in 143 samples. There were significant variant haplotypes among the populations．There were no shared haplotypes found among populations, analysis of molecular variance indicated that a high proportion of the total genetic variance was attributable to variations among populations (92.23%). The pattern of genetic structure which is associated with spatial separation, variation in environmental conditions and selection pressures in different populations, is also the result of geological historical factor. A molecular phylogenetic tree based on the 12 haplotypes showed that the populations are not strictly grouped according to their geographic distances.

粗枝云杉( Picea asperata Mast.)天然群体的遗传变异

西南地区在我国的经济发展和生态环境建设中占重要地位，但也是我国生态环境最脆弱的地区之一，生态系统退化，生态功能减弱，严重制约着西南林业的可持续经营与发展。本项目采用DNA 分子标记SSR 研究不同生境条件下粗枝云杉群体的遗传变异及其时空分布格局，考察遗传变异与复杂的山地生态环境间的潜在联系，系统地揭示粗枝云杉天然群体与环境系统相互作用的生态适应与分子进化机制。粗枝云杉适应性强，生长迅速，在植树造林和工业用材方面占有重要地位，研究成果可为中国西南部亚高山天然林的可持续经营及退化生态系统的恢复与重建提供理论依据和科学指导。主要研究结果如下： 1. SSR 位点变异丰富，等位基因频率的分布格局多样。7 个SSR 标记全是多态位点，每位点的等位基因数变化范围为13~24，平均为19.9 个。SSR 位点的等位基因片段长度范围变化较大。73.1%的等位基因变异遵循逐步突变模型(SSM)而发生1 个重复基元的变化，22.3%和4.6%的变异分别按两阶段突变模型(TMP)发生1 个重复基元以上的变化和在SSR 位点侧翼区发生1 个碱基变化的插入-删除事件。 2. 粗枝云杉拥有中等偏高水平的遗传多样性和相对大的群体间遗传分化。通过分析代表10 个群体的250 个个体在7 个SSR位点的变化，调查了源自中国西南山区的粗枝云杉的微卫星变异。相当高的遗传多样性和强烈的群体分化发生在粗枝云杉中， 其群体平均Nei's 期望杂合度为0.707 ， 群体间遗传距离为0.121~0.224(FST)和0.100~0.537(RST)。然而，群体间遗传距离与地理距离之间无相关性，从而排除了简单的距离分离模式并暗示迁移不是影响粗枝云杉遗传变异格局的主要因素。事实上，使用私有等位基因估算的基因流数量非常低，仅等于0.753。等位基因置换检验(Allele permutation tests)揭示逐步突变及遗传漂变都对群体间分化有贡献。另外，在多数位点检测到显著的群体间遗传差异，这个结果说明自然选择，假设通过环境压力，是引起粗枝云杉微地理分化的主要因素之一。根据SSR基因型，250 个粗枝云杉个体的70%被正确地归类入其各自的来源群体，结果表明微卫星(SSR)对区分来自中国不同生态地理位点的粗枝云杉基因型是有效的。 3. 在SSR、RAPD 和AFLP 位点，显著的群体间遗传结构被发现的，但三种标记间遗传分化程度和群体遗传关系有差异。利用来自10 个群体的247 个个体，我们报告了关于样本粗枝云杉群体间遗传关系的总体看法。根据各自对评价遗传关系的信息能力和适用性，SSR、RAPD 和AFLP 标记被选用，三种技术非常有效地区别这些基因型。使用的SSR、RAPD 和AFLP 标记分别估计平均Dice 相似性系数。Mantel 检验产生显著但相对低的共表型适合度(RAPD = 0.63£AFLP = 0.60和SSR = 0.75)。比较三种标记系统，RAPD 和AFLP 共表型指数相对高地相关(r =0.59)，而RAPD 和SSR 及SSR 和AFLP 之间的相关系数分别是0.53 和0.35。所有系统树，包括不同标记资料结合获得的系统树，反映了多数群体依据它们的地理条件而成某种特定关系。结果暗示单个或结合标记系统能用来深入洞察粗枝云杉遗传研究，并且不同标记系统合并资料能提供更可靠的信息。 Southwestern region plays an important role in economic developmentand ecological construction in China. Yet, it is also one of the weak regionsof ecological environment in China with degraded ecosystem and imperfectfunction, which restricts the sustaining management and development ofsouthwestern forestry. The genetic variation and spatial distribution patternof P. asperata populations originating from different habitats wereinvestigated using SSR molecular markers in this study. The correlationsbetween genetic variation and ecological and environmental conditionswere detected, and the interaction between P. asperata populations andenvironmental system and the mechanism of ecological adaption -molecular evolution were revealed. Given the significant ecological andeconomic roles of the fast-growing and wide-adaptive species in reforestation and production of pulp wood and timber, the study couldprovide a strong theoretical evidence and scientific direction for thesustaining management of subalpine natural forest, and the afforestationand rehabilitation of degraded ecosystem. The results are as follows: 1. The genetic variation at SSR loci was abundant and the distributionof allelic frequencies was uneven. All seven loci were polymorphic, and thenumber of alleles per locus varied from 13 to 24 with a mean valueequaling 19.9. The allele sizes at SSR loci were found to vary widely.73.1% of allelic variation followed stepwise mutation model (SSM) whichresults increase or decrease by one repeat type, and 22.3% and 4.6% wereresulted from two-phase mutation model (TMP) with allele size varying bymore than one repeat type and from insertion-deletion events in theflanking regions at SSR loci with a single basepair changing, respectively. 2. P. asperata possessed a moderate to high level of genetic diversityand considerable genetic differentiation. Microsatellite variation of P.asperata. originating from the mountains of southwestern China wasinvestigated by analyzing variation at seven SSR loci in 250 individualsrepresenting ten populations. A fair degree of genetic diversity and strongpopulation subdivision occurred with the mean gene diversity (H) of 0.707,and genetic distances among populations varying between 0.121 and 0.224(FST) and between 0.100 and 0.537 (RST). However, inter-populationgenetic distances showed no correlation with geographic distances between the population sites. This ruled out a simple isolation by distance modeland suggested that migration does not have a great impact. In fact, theamount of gene flow, detected using private alleles, was very low, equalingonly 0.753. Allele permutation tests revealed that stepwise-like mutations,coupled with genetic drift, could contribute to population differentiation.Moreover, significant genetic differences between populations weredetected at most loci. The results indicate that natural selection, presumablythrough environmental stress, may be one of the main factors causingmicro-geographical differentiation in the genetic structure of P. asperata.Based on SSR genotypes, 70% of the 250 individuals were correctlyclassified into their sites of origin. This suggests that microsatellites (SSRs) are effective in distinguishing genotypes of P. asperata originating fromdiverse eco-geographical sites in China. 3. Using a set of 247 individuals from ten P. asperata populations wereport an overview on the genetic relationship among the sampled P.asperata populations. RAPD, AFLP and SSR were used in terms of theirinformativeness and applicability for evaluate relationship and all threetechniques discriminated the genotypes very effectively. Mean Dicesimilarities coefficient were estimated using RAPD, AFLP and SSR,respectively. The Mantel test resulted in a significant but relatively low fit(RAPD = 0.63, AFLP = 0.60 and SSR = 0.75) of cophenetic values.Comparing the three marker systems to each other, RAPD and AFLP cophenetic indices were highly correlated (r = 0.59), while correlationcoefficient between RAPD and SSR was r = 0.53 and between SSR andAFLP was r = 0.35. For all markers a relatively high similarity indendrogram topologies was obtained although some differences wereobserved. All the dendrograms, including that obtained by the combineduse of all the marker data, reflect some relationships for most of thepopulations according to their geographic conditions. The results indicatethat single or combined marker system could be used to insight into geneticstudy in P. asperata and the combined data of different marker systems canprovide more reliable information.

川西云杉(Picea balfouriana)天然群体的遗传变异

青藏高原东南缘由于特殊的生态地理条件，有着丰富的森林资源，这些资源是长江上游涵养水源、保持水土的生态屏障，是生物多样性的资源宝库。但随着过量的森林采伐，使该区曾经丰富的生物多样性资源遭受了前所未有的破坏，天然林的质量严重下降，生态系统退化，功能减弱。与此同时，许多物种的种群规模正在锐减，物种的遗传多样性也严重丧失。川西云杉是西部地区分布最广的云杉树种之一，在较高海拔的地区有着重要的生态学功能，是一种适应性很强的乡土树种。本项目采用简单序列重复标记(SSR)和特定序列位点(STS)研究不同生境条件下川西云杉群体的遗传变异及其时空分布格局，考察遗传变异与复杂的山地生态环境间的潜在联系，系统地揭示川西云杉天然群体与环境系统相互作用的生态适应与分子进化机制。研究成果能有效地为该树种遗传资源的科学保护与合理利用提供理论依据和科学指导，可为中国西南部亚高山天然林的可持续经营及退化生态系统的恢复与重建提供依据。主要研究结果如下： 1 STS和SSR两种分子标记的研究结果表明：川西云杉群体拥有中等水平的遗传多样性(基于SSR标记，平均He = 0.640；基于STS标记，平均He = 0.553)。造成这种中等水平的遗传多样性，可能是由于历史原因，川西云杉天然林被大量采伐，导致了遗传多样性的丧失。两种方法都检测出群体BT有着最高水平的遗传多样性。 2 两种标记的结果都一致说明：检测的10个川西云杉群体间遗传分化比较高，其存在群体间的遗传变异比例要明显地高于广泛分布的挪威云杉、横贯大陆的黑云杉以及兼有连续分布和不连续分布的西加云杉等，但要低于分布范围狭窄且呈不连续分布的粗枝云杉。青藏高原东南部的片段化生境可能是导致高水平遗传分化的主要原因。 3 基于两种标记的UPGMA 聚类和PCA分析结果，以及基于SSR标记的FCA分析结果都显示：群体BY遗传上明显区别于其它群体，其可能原因是青藏高原东南缘山脉阻隔而导致的生殖隔离的结果。 4 根据软件Bottleneck 1.2.02的检测以及不依赖哈迪一温伯格平衡的M比率检测结果：川西云杉种群很有可能经历了近期的遗传瓶颈效应。在本研究中遗传瓶颈效应并没有显著影响到物种的遗传多样性。然而，在这些片断化群体未来的子代群体中很可能出现遗传瓶颈导致的遗传多样性下降的效应。 Due to the extremely complex topography and climatic conditions, the southeast of the Qinghai-Tibet Plateau is region abundant in forest resource, which is benefit to the upper reaches of the Yangtze River water conservation, and protecting natural environment and biodiversity resources. However, by the reason of a plenty of trees were cut in these yeas, the biodiversity resource was destroy with degraded ecosystem and imperfect funcation. Picea balfouriana is one of the regionally distributed conifer species in the southeast of the Qinghai-Tibet Plateau and considered as a constructive species within its distribution area. It is an optimal species for the production of biomass. Moreover, it is well adapted to stressful environments at high altitude, especially to cold and drought conditions which are generally harsh for other trees. In our study, two types of molecular markers (SSR and STS) were used to estimate the genetic diversity and genetic structure of P. balfouriana populations originating from the southeast of the Qinghai-Tibet Plateau with varying climatic and geographical conditions. The results will not only provide a deep insight into its genetic diversity and population genetic structure but also valuable information for further management and breeding programs in P. balfouriana. In summary, the results obtained in this study revealed that: 1 A moderate degree of genetic variation is present in P. balfouriana in the southeast of the Qinghai-Tibet Plateau and it may caused by many trees were cut in the past. Population BT owns the highest level of genetic diversity by both two types of markers. 2 Considerable population differentiation exists among the ten P. balfouriana populations based on both SSR and STS markers, possibly caused by habitat fragmentation and heterogeneous environments. 3 The UPGMA clustering and PCA analyses based on SSR and STS marker, and FCA analyses based on SSR marker congruously separated population BY from other populations, which is likely due to the presence of mountain barriers. 4 The results of bottleneck analysis indicated that P. balfouriana populations that have undergone recent declines. In our research, the bottleneck effect don’t have a significant impact on the genetic diversity of species, however, the level of genetic diversity of P. balfouriana offspring populations growing in the fragmented habitats will decline in the future.

若尔盖自然保护区高原林蛙(Rana kukunoris)扩散模型与景观连接分析

揭示水体中繁殖的两栖动物在异质性景观中的空间扩散特点，探讨景观面积丧失和破碎化对于两栖动物的影响，为两栖动物的保护提供理论依据。本文以四川西北部若尔盖湿地自然保护区的高原林蛙(Rana kukunoris)为研究对象，通过运用地理信息系统及建立景观模型等方法，在分析若尔盖湿地自然保护区范围内现有景观格局的基础上，建立了高原林蛙的景观扩散模型，并模拟了“沼泽→草甸”的湿地逆演化过程下高原林蛙的空间分布与景观连接的变化特点。主要结果是： 1．若尔盖湿地自然保护区呈典型的沼泽—草甸式斑块—基质景观格局。草甸面积占整个景观面积的79.42%，景观蔓延度指数（CONTAG）为79.00远离最小值0而更趋向于最大值100，面积和景观蔓延度指数表明草甸是整个景观中面积占绝对优势且景观连接好的类型，构成了景观的基质，对景观的动态格局演变起主导作用。沼泽面积仅占整个景观面积的18.08%，但却是整个景观中斑块数目最多的单元，占所有斑块数的82.9%。因此沼泽斑块与草甸基质之间的动态结构对高原林蛙的扩散起着决定性的作用。 2．空间扩散模型表明，其它类型的景观不但扩展了高原林蛙的活动范围，而且也为高原林蛙在不同沼泽斑块间的连接提供了通道。高原林蛙的空间扩散区域使得彼此间成斑块化隔离状分布的沼泽形成了潜在景观功能连接，促进了不同斑块间物种的交流。小型沼泽作为垫脚石（stepping-stone），使得整个景观中的相隔距离较远的大型斑块联结为一个功能整体，促进了高原林蛙在整个景观中的相互动态联系。 3．模拟“沼泽→草甸”的湿地逆演化过程表明，大量小型沼泽湿地的消失将会 对在沼泽中繁殖并扩散到其它景观类型中去的高原林蛙造成潜在影响。逆演化过程不仅使沼泽斑块的分布范围，沼泽源斑块的面积和空间扩散面积减少，而且对景观连接也有很大影响。小型沼泽的消失，将使得景观斑块的功能连接变小，使得依靠小型沼泽作为跳板的动物在沼泽斑块之间的移动将变得更加困难。 本文是对生境丧失与破碎化影响下两栖动物的行为反应的一种尝试。影响模型的因素很多，包括动物对各种类型景观的偏好程度，地理数据的精度，及模型的可靠程度都是制约模型准确度的因素。 The spatial diffusion of water—breeding amphibian through heterogeneous landscape and the effects of landscape losing and fragmentation to amphibian were the core theory of the landscape ecology of amphibian. Geographical information system (GIS) and landscape model were used to model the diffused area of Rana kukunoris in Zoige Wetland Natural Reserve. Model was also used to analysis the spatial distribution variation of R. kukunoris and the change of landscape connectivity when simulated the retrogressive succession of landscape. The main results are below: 1. There was peatland—meadow pattern which was typical patch—matrix landscape pattern in Zoige Natural Reserve. The meadow area occupied 79.42% of the entire landscape area, contagion index (CONTAG) was 79.00 which was far away the minimum value (0) but tend to the maximum value (100). Both of these showed that meadow was the largest part and the most continue units. It was shown that meadow was matrix of the landscape, which evolved the leading role to the landscape dynamic pattern. Though their area only occupies 18.08% of entire landscape area, peatlands were according to 82.9% of the total patches. Dynamic of the pattern between peatlands and meadows decided the spatial diffusion of R. kukunoris. 2．The model indicated that the other types of landscape not only expanded diffusion of R. kukunoris, but also have provided the potential channels for frog's connections among different peatlands. The spatial diffusion zone of R. kukunoris forced isolated patch peatlands to be potential landscape functional connectivity. The small peatlands, as stepping-stone, made the large peatlands connect as a functional one and promoted the integrated and dynamic connectivity of R. kukunoris in the whole landscape. 3. The simulation of “peatlands→meadows”retrogressive succession process indicated that the decrease of small peatlands will have potential effect to R. kukunoris because they must bred in peatlands and diffuse to other type of the landscape. Retrogressive succession process not only made the decrease of distribution of peatlands, patches number of peatlands and diffused area of R. kukunoris, but also reduced the connectivity among source patches. As stepping-stone, the disappearance of small peatlands will made the migration of R. kukunoris among patches more difficult. The model was an experiment of the amphibian behavior reaction to habitat losing and fragmentation. There were many factors that could influence the accuracy of model, such as the preference of animals to each type of landscape, the geographical data precision, reliable degree of model.

青藏高原栽培青稞遗传多样性研究

青稞，是我国藏区居民对裸大麦的称谓，它不仅是藏民的主要食粮、燃料和牲畜饲料，而且也是啤酒、医药和保健品生产的原料；青稞不仅为藏区人民的健康和经济发展做出了很大的贡献，而且对人类健康和社会经济的可持续发展都有重要的意义。青藏高原是我国及世界上青稞分布和种植面积最大的地区，资源极其丰富。虽然从经典遗传直到分子标记对我国大麦遗传多样性都有研究，但研究手段、数量仍然不够深入，对我国大麦资源遗传多样性研究的信息非常有限，不能很好地满足大麦遗传研究和育种应用的需要，尤其是对西藏栽培大麦的遗传多样性的研究还只是刚刚开始，关于栽培青稞多态性的研究报道很少。本研究采用SSR标记和蛋白质电泳两类技术，从SSR标记位点、单体醇溶蛋白、B组醇溶蛋白和淀粉粒结合蛋白（SGP）等四个方面对我国青藏高原栽培青稞的遗传多样性进行了综合评价。 SSR标记具有基因组分布广泛、数量丰富、多态性高、容易检测、共显性、结果稳定可靠、实验重现性好、操作简单、经济、易于高通量分析等许多优点，被认为是用于遗传多样性、品种鉴定、物种的系统发育、亲缘关系及起源等研究的非常有效的分子标记。本研究采用SSR标记分析了64份青藏高原栽培青稞的遗传多样性，同时评估SSR标记在我国大麦育种和品种鉴定中的应用潜力。选择了30个已知作图位点SSR标记，其中25个标记与重要性状的控制位点连锁紧密。选择的30个SSR标记，5个未得到很好的扩增产物，3个无多态性。22个多态性SSR标记位点中，每位点检测出等位基因2~15个，共检测出等位基因132个，平均每位点6.0 个。各多态位点检测出基因型为2~11种，位点HVM33的基因型最多。各多态位点的多态信息指数为0.16~0.91, 平均为0.65。根据PIC值选择了13个SSR标记用于我国青藏高原栽培青稞基因型鉴定，这些标记的PIC值为0.6以上。结合PIC值和基因型差异，选择了8个多态信息含量高的SSR标记，构建了高效指纹图谱，此图谱能把64份材料完全区分。 贮藏蛋白电泳分析是研究相关编码蛋白基因多态性的非常有效的方法。大麦单体蛋白与小麦醇溶蛋白相对应，具有丰富的多态性，可用于大麦遗传多样性、品种鉴定和群体进化等研究。本研究通过A-PAGE电泳技术研究了84份青藏高原栽培青稞的单体醇溶蛋白多态性。大麦单体醇溶蛋白图谱与小麦醇溶蛋白电泳图谱类似，所分离的蛋白清晰地分为ω－，γ－，β－和α－四个部分。青藏高原栽培青稞单体醇溶蛋白具有丰富的多态性，84份青稞材料中存在43条不同的蛋白带，75种组合带谱；其中67种为单一材料所独有，另8种则分别包含了2-3份材料。每份材料中拥有醇溶蛋白带为6－16条，含有6-10条单体醇溶蛋白带材料较多。西藏和四川材料群体单体醇溶蛋白多态性不同，具有区域特异性。西藏材料中发现了40条不同蛋白带，3条特异带，46 种蛋白组合；四川材料中出现了40种不同蛋白带，26种条带组合, 3条特异带。基于单体蛋白多态性的聚类与材料的来源有一定的相关性。A-PAGE单体蛋白具有丰富的多态性，可作为遗传研究和品种鉴定的标记。 大麦醇溶蛋白（hordein）是大麦籽粒的主要贮藏蛋白，与大麦的营养品质和加工品质密切相关，而且具有丰富的多态性，广泛用于品种鉴定、种质筛选、遗传多样性和亲缘关系研究。B组醇溶蛋白是主要的醇溶蛋白组份，约占总醇溶蛋白的80%，而且具有丰富的多态性。本研究采用SDS-PAGE分析了72份青藏高原栽培青稞B组醇溶蛋白的遗传多样性。青藏高原栽培青稞B组醇溶蛋白具有丰富的多态性，72份青稞材料中存在15种蛋白带，30种组合带谱，其中15种为单一材料所独有，另15种则分别包含了2-10份材料。每份材料中B组醇溶蛋白条带数为4-8条，含5、6条的材料较常见。不同来源的群体材料间B组醇溶蛋白组成存在差异，西藏青稞含有26种蛋白组合带谱，其中有19种特异带谱；四川群体中共发现11种蛋白组合带型，其中有4种特有带谱。两群体中都存在稀有条带。聚类分析将材料分成三组，材料聚类与材料来源地没有明显的相关性。 淀粉粒蛋白（Starch granule proteins, SGPs）是一类与淀粉粒结合的微量蛋白，一些淀粉粒蛋白具有淀粉生化合成中主要的酶蛋白功能，其变异会影响淀粉含量和特性，从而影响淀粉的应用。关于我国大麦淀粉粒组成研究还未见报道。本实验首次开创了我国大麦淀粉粒结合蛋白的研究工作。采用SDS-PAGE电泳技术研究了青藏高原栽培青稞的SGP组成，并分析了不同SGP组合间淀粉含量的差异，初步探索了所分离的SGP蛋白与淀粉合成的关系。66份青稞材料中分离了10种主要的SGP，其表观分子量为40-100KD，低于60KD的SGP带有7条，共有16种组合带谱；各SGP蛋白和组合带谱出现的频率存在差异，青藏高原青稞的SGP组成存在多态性。西藏青稞和四川青稞的SGP组成有很大差异，SGP组成具有地域差异性，西藏青稞含有12种蛋白组合带谱，其中有9种特异带谱；四川群体中共发现7种蛋白组合带型，其中有4种特有带谱；两群体中仅有3种共同的蛋白组合带谱。SGP蛋白特性将66份青稞分为三组, 即Ⅰ、Ⅱ、Ⅲ，材料聚类与材料来源具有一定的相关性。不同组合带谱材料间淀粉含量差异显著性检验结果显示，不同带谱间材料的总淀粉含量、直链淀粉含量和支链淀粉含量有差异，带谱2（SGP1+3+7+9+10）和8（SGP1+2+4+6+8）的总淀粉含量及支链淀粉含量显著大于组合带谱3（SGP1+3+7+10）的总淀粉含量。组合带谱7（SGP1+2+6+8）的直链淀粉含量显著低于带谱11（SGP1+5+8）的直链淀粉。带谱SGP2、3、4、5、6、7、8、9、10可能参与淀粉合成，SGP9可能与高支链淀粉的合成相关。 SSR标记位点、单体醇溶蛋白、B组醇溶蛋白、淀粉结合蛋白等四个方面的研究结果表明青藏高原SSR标记多态性、单体醇溶蛋白多态性、B组醇溶蛋白多态性和SGP多态性都非常丰富，与青藏高原是栽培青稞的多样性分布中心的观点一致。 青藏高原栽培青稞的SSR标记、单体醇溶蛋白、B组醇溶蛋白和SGP多态性表现出很大差异。SSR标记覆盖了整个基因组，多态性非常高。单体蛋白、B组醇溶蛋白、SGP蛋白是育种中非常关注的性状，他们只是代表基因组中的某一区域或位点，多态性相对较低。但单体蛋白多态性很高，84份材料中检测出43条不同蛋白带，75种不同的组合带谱。SSR标记技术和单体蛋白技术都是遗传多样性研究的有力工具，但单体蛋白技术不仅多态性高，而且经济、操作简便，是种质鉴定的理想方法。 对不同标记的多态性材料数据进行聚类，聚类图能为我们提供各材料间的遗传相似信息，为材料选择提供参考。但材料聚类与材料来源的地理区域的相关性表现不一致。SSR聚类和B组醇溶蛋白聚类与材料的来源地无相关性，而单体醇溶蛋白和SGP聚类与材料来源地有一定相关性，即西藏群体和四川群体分别有集中类群，这可能是人为选择的附加效应。 不同来源的群体材料的遗传多样性不同，具有区域特异稀有基因，加强不同地区间资源的交换和配合使用，有利于增加群体遗传多样性和新品种培育。 青藏高原栽培青稞的麦芽浸提性状、淀粉性状、病虫及裸粒等重要农艺性状控制位点存在丰富的变异，遗传基础宽广，可能蕴藏着多种不同的等位基因，是研究重要性状遗传特性、基因资源挖掘和遗传育种的宝贵资源库。 Hulless barley, due to its favorable attributes such as high feed value, good human nutrition，rich dietary fiber and ease processing, attracts people,s attention . Hulless barley plays a very important role in Tibetan life, used as essential food crop, main animal feed and important fuel. In addition to tsampa (roasted barley flour), a main food for Tibetan, hulless barley is also made into cake, soup, porridge, recent naked barley liquor and cornmeal. Qinghai-Tibet Plateau is one of a few areas which plant naked barley widely in the world and also has a long growing history. Genetic diversity of the cultivated hulless barley in this region , however, has not been documented. The study of genetic diversity existing within this population is of particular interest in germplasm identification, preservation, and new cultivar development. This study analyzed the genetic diversity of the cultivated naked barley from Qinghai-Tibet plateau through the study of SSR marker loci and monomeric prolamins, B-horden and starch granule proteins. SSRs are present abundantly in genomes of higher organisms and have become a popular marker system in plant studies. SSRs offer a number of advantages, such as the high level of polymorphisms, locus specificity, co-dominance, reproducibility, ease of use through PCRand random distribution throughout the genome. In barley, several hundred SSRs have been developed and genetically mapped and can therefore be selected from specific genomic regions. The genetic diversity of 64 cultivated naked barley from Tibet and Sichuan was studied with 30 SSRs of known map location.Among the selected SSR markers, PCR products of 5 SSR markers were not obtained and 3 SSR marker loci were monomeric. A total of 132 alleles were identified at 22 polyomeric SSR loci. The number of alleles per locus ranged from 2 to 15, with an average of 6.0. The polymorphism information content values for the SSRs ranged from 0.08 to 0.94, with an average of 0.65. 13 SSR markers with the PIC value >0.6 have been selected for discrimination of Qinghai-Tibet naked barley genotypews. A finger Print map was developed through 7 SSR markers with the high PIC value. It could be used as an efficient tool for gene discovery and identification of gernplasm. Hordeins, the main storage proteins of the barley seed, are composed of momomeric and polymeric prolamins and divided into -A, B, C and D groups in order of decreasing electrophoretic mobility. Hordeins show high inter-genotypic variation and have been extensively used as markers for cultivar identification and analyzing the genetic diversity. This study analyzed the genetic diversity of B-hordein in 72 naked barley from Qinqhai-Tibet Plateau. Extensive diversity was observed. A total of 15 different bands and 30 distinct patterns were found. Jaccard's coefficient of similarity was calculated, and the accessions were divided into three　main groups by cluster analysis using UPGMA. Differentiation among the populations from different collecting regions based on the polymorphism of B-hordein was investigated. Monomeric prolamins show high inter-genotypic variation and have been used as molecular markers for cultivar identification, analyzing the genetic diversity in collections and investigating the evolution processes and structure of populations However, the cultivated hulless accessions from Qinghai-Tibet Pateau in China have never been examined with respect to monomeric prolamins. This study analyzed the genetic diversity of monomeric prolamins (protein fraction corresponding to wheat gliadins) using the Acid -PAGE technique in eighty-four cultivated hulless barley from Qinqhai-Tibet Plateau in China. Extensive diversity was observed. A total of 43 different bands were found, of which 21 different bands were in the region of ω group, 8 in the region of γ, 8 in the region of β, and 6 in the region of α group. Among the 86 accessions, 75 distinct patterns were identified. The number of bands ranged from 6 to 16, depending on the variety. Jaccard’s coefficient of similarity was calculated, and the lines were grouped by cluster analysis using UPGMA. A dendrogram was obtained from the analysis of the groups and five main clusters were identified. No relationship between the distribution in the dendrogram and growth habits and origins of the cultivars could be detected. Starch is the major constituent of the cereal endosperm, comprising approximately 65% of the dry weight of the mature wheat grain. The starch formed in all organs of plants is packaged into starch granules, which vary widely between species and cultivars in size and shape. Wheat endosperm starch granules contain about corresponding to the main biosynthase of starch. This report firstly dealed with intraspecific variation of the major SGPs in cultivated naked barley from Qinghai-Tibet plateau. A total of 10 major SGPs were observed in the range of 40KD-100KD and 16 types of patterns were found. Based on the variation of SGPs, accessions studied were classified into 3 groups. A geographical cline of electrophoregram was observed. In addition, significance test of the difference of starch content among groups and types of patterns were done, and the results indicated those SGPs could be related to the content of starch. Diagram obtained through cluster analysis exhibited a structuration of diversity and genetic relationship among cultivated hulless accessions. In breeding program, parents with genetically distant relationship for hybridization will increase genetic diversity of progenies. In conclusion, cultivated naked barley from Qinghai-Tibet Plateau in China presents a high variability with respect to monomeric prolamins，SSR markers , B- hordeins and SGPs. The result of this study supports Qinghai-Tibet Plateau is the center of cultivated hulless barley and the cultivated naked barley is considered to be a gene pool with large diversity and could be applied to breeding for cereal.