Molecular phylogeography of endangered sharp-snouted pitviper (Deinagkistrodon acutus; Reptilia, Viperidae) in Mainland China

Using phylogenetic and population genetic approaches, the present study reports the phylogeographic structure of the sharp-snouted pitviper (Deinagkistrodon acutus), a threatened snake species with commercial and medicinal importance in China. The entire mitochondrial ND2 gene (NADH dehydrogenase subunit 2) sequences of 86 individuals of D. acutus from 14 localities across its range in China were determined. Based on the results of phylogenetic analyses, distribution of diagnostic sites, haplotype network, and AMOVA hierarchical analysis, an cast-west division of the whole D. acutus population could be observed. Geographically, a line formed by a lake, river, and mountain chain (the Poyang Lake, Gan River to the southern end of the Wuyi Mountains), results in vicariance and approximately vertically splits the range into two and the whole population into two main lineages (western and eastern). The bifurcating tree suggested generally west to east dispersal trend. The data fit the isolation by distance (IBD) model well. Star-like clusters in haplotype network, significantly negative values of Fs statistics, and unimodal mismatch distributions all suggest recent demographic expansions in four areas. The results show that isolation, dispersal, bottleneck, and expansion jointly constitute the history of D. acutus. In a haplotype network, the excessive predominance of central haplotypes, few medium-frequency haplotypes, predominance (73.1 %) of the singletons among the derived haplotypes, most of which are connected to the central haplotype by only one mutational step, unsymmetrical campanulate unimodal curve of mismatch distributions and leftwards shift of the peaks, all suggest that the whole D. acutus population is a young population with low genetic diversity. Based on the data, the first priority for conservation action should be given to the Huangshan unit. (c) 2007 Elsevier Inc. All rights reserved.

Characterization of novel microsatellite loci in rare minnow (Gobiocypris rarus) and amplification in closely related species in Gobioninae

Rare minnow (Gobiocypris rarus) is an endangered small fish endemic to upper reach of the Yangtze River. From a (GT)n enriched genomic library, 32 microsatellites were isolated and characterized. Nineteen of these loci were polymorphic in a test population with alleles ranging from 2-7, and observed and expected heterozygosities from zero to 0.8438, and 0.2679 to 0.8264, respectively. In the cross-species amplifications, 13 out of 19 polymorphic loci were found to be also polymorphic in at least one of the 7 closely related species of the subfamily Gobioninae. These polymorphic microsatellite loci should provide sufficient level of genetic diversity to evaluate the fine-scale population structure in rare minnow and its closely related species for the conservation purpose.

Isolation and characterization of polymorphic microsatellites in a Yangtze River fish, brass gudgeon (Coreius heterodon Bleeker)

The brass gudgeon (Coreius heterodon) is a fish of economic importance in the Yangtze River. From a (GATA)(n)-enriched genomic library, 25 microsatellites were developed by employing the fast isolation by AFLP of sequences containing repeats (FIASCO) protocol. Nine loci exhibited polymorphism with two to 12 alleles (mean 3.9 alleles/locus) in a test population, and observed heterozygosity ranged from 0.1111 to 0.9630 (mean 0.4426). Three of the nine loci showed polymorphism in a congeneric species, the largemouth bronze gudgeon Coreius guichenoti. These loci should provide sufficient level of genetic diversity to evaluate the fine-scale population structure of C. heterodon.

Isolation and characterization of microsatellites in Chinese sturgeon, Acipenser sinensis

From (GATA)(n) and (AAAG)(n) enriched genomic libraries for the Chinese sturgeon (Acipenser sinensis), 50 primer pairs were developed using the fast isolation by AFLP of sequences containing repeats (FIASCO) protocol. Forty-six primer pairs exhibited highly polymorphic with two to 11 alleles per locus, while the rest four displayed monomorphic. These markers yielded 246 alleles in a survey of eight specimens of wild A. sinensis. Average observed heterozygosity ranged from 0.13 to 1.00. These loci should provide sufficient levels of genetic diversity to allow parentage analysis for artificial stocking management and delineation of fine-scale population structure.

Identification, cloning, and characterization of microsatellite DNA in Euglena gracilis

Microsatellite DNA has been developed into one of the most popular genetic markers. We have identified and cloned microsatellite loci in the genome of a free-living protozoan Euglena gracilis FACHB-848, using the random amplified microsatellites method (RAMS). The digoxigenin-labelled oligonucleotides(CT)(10) and (GT)(10) served as probes to detect complementary sequences in the randomly amplified polymorphic DNA (RAPD) fingerprints produced by means of Southern blotting. Subsequently, positive RAPD fragments were cloned. From a total of 31 RAPD primer profiles, eight microsatellite loci of E. gracilis were detected and characterized. Further, six sites (i.e. EGMS1, EGMS3, EGMS4, EGMS5, EGMS6, and EGMS7) showed polymorphisms. We found a GT or CT microsatellite every 10.5 kb in the genome of E. gracilis, and similar to animal genomes, the (GT)(n) motif was much more abundant than the (CT)(n) motif. These polymorphic microsatellite DNA will serve as advantageous molecular markers for studying the genetic diversity and molecular ecology of Euglena.

Geographic distribution of Pelteobagrus fulvidraco and Pelteobagrus vachelli in the Yangtze River based on mitochondrial DNA markers

Three populations of Pelteobagrus vachelli and Pelteobagrus fulvidraco of the Yangtze River were examined by PCR-RFLP analysis of mitochondrial DNA fragments. ND5/6 and D-loop fragments were digested by 10 restriction endonucleases. Significant geographic variations between upstream and mid-downstream populations in the haplotype frequencies and restriction patterns were revealed. This suggested that the diversity of P. vachelli was high; 11 haplotypes were obtained from all the samples. The upstream population shared seven haplotypes and the middle and downstream populations shared another four haplotypes. Among all of the haplotypes, one haplotype was shared in 30 samples of the populations from middle and downstream, but it was not found in the upstream population. Any haplotype found in the upstream population was not detected in the middle and downstream populations. Genetic diversity of P. fulvidraco was low and only five haplotyes were detected from all 60 samples. Phylogenic relationships also indicated that the fishes from upstream and mid-downstream were apparently divided into two populations.

Rapid establishment of pure lines of silver carp (Hypophthalmichthys molirix) and bighead carp (Aristichthys nobilis)

The diversity of gynogenetic, artificial sex reversal and natural silver carp and bighead carp is examined using randomly amplified polymorphic DNA (RAPD) method. All of the 187 bands are obtained and 19 (10.16%) of them are polymorphic in gynogenetic silver carp. Meanwhile 32 (15.61%) out of 205 bands are polymorphic in control group. In gynogenetic bighead carp a total of 232 bands are identified and 11 (4.74%) out of them are polymorphic, while 25 (10.37%) out of 241 bands are polymorphic in control group. The genetic distance of four populations is calculated and it is 0.102 and 0.023 for gynogenetic silver carp and gynogenetic bighead carp respectively. The values of natural silver carp and bighead carp are 0.161 and 0.104. From the UPGMA trees constructed based on genetic distance, the sex reversal individuals that match with the gynogenetic female individuals are picked out. A new breeding process of establishing a pure line is developed.

猕猴属的分子系统学与遗传多样性研究

用六个线粒体基因片段序列数据，对称猴属的分子系统发育关系进行了分析。8种称猴：台湾猴，北平顶猴，藏酋猴，熊猴，红面猴，J厦河猴，狮尾猴和食蟹猴共40个样本用于本研究。结合从GenBank里下载的雯猴的相应序列，用拂拂作为外群构建了系统发育树。由于六个目的片段均为线粒体基因片段，缺乏重组且作为单一位点遗传，因此我们将六个数据集合并为一个数据集进行分析。我们获得了比前人置信度更高的线粒体系统树。除了红面猴，我们的结果支持Delson（1980）的分组。与前人研究结果一致，红面猴与食蟹猴组成员关系较近（尤其是食蟹猴）。恒河猴（相对于台湾猴），熊猴（相对于藏酋猴）和北平顶猴（相对于狮尾猴）都形成了并系。通过线粒体控制区序列和20个微卫星位点对14个恒河猴地理群体的群体结构进行了研究。微卫星数据和线粒体控制区数据均显示恒河猴有较高的遗传多样度。线粒体数据表明各地区的恒河猴群体间的差异显著大于微卫星数据显示的结果。也就是说，基于微卫星数据所得到的群体间基因流远大于基于线粒体数据所得到的群体间基因流。两种分子标记所得到的结果差异应归因为雌性恒河猴的归家冲动和雄性恒河猴的扩散。线粒体数据显示所有恒河猴群体近期没有发生过快速扩张。微卫星数据显示大部分恒河猴群体近期没有经历过瓶颈效应。恒河猴是分布较广的非人灵长类动物，但它的地理差异研究一直颇有争论且不足以划分其亚种。我们对采自包括印度、越南、缅甸和18个中国地区在内的21个地区共35个恒河猴样品的四个线粒体基因片段（COI，Cofl，COlll和12srRNA）进行了研究。食蟹猴、红面猴、台湾猴和史猴的相应序列作为外群。大约长为100bp的序列用于构建最大似然树和贝叶斯树。与前人结果一致（彭燕章等，1993；蒋学龙等，1995；Groves，2001），西部恒河猴（印度）．相当于M.m.mulatta。根据王应祥（2003）的分类，我们的东部恒河猴可分为四组：M．最早分离出来且一个来自缅甸的恒河猴位于该组基部；M.m.brachyurus和M.m．littoralis组成一个大枝，这个大枝与M.m，lasiotus构成姐妹群。

东亚人群 LMBR1 基因 Intron 5的遗传多样性研究

人LMBR1(Limb region 1 homolog (mouse)) 基因位于染色体7q36区域，全长约210.2 kb，含17个外显子，编码一个由490个氨基酸构成的跨膜蛋白。研究表明，LMBR1 基因的表达活性与脊椎动物四肢的手指或脚趾数目变化有关；另外，发生在其重要元件——intron 5 内的许多变异与多种表型的轴前多指症((PPD, Preaxial polydactyly)存在相关性，这主要是因为LMBR1 intron 5 内含有一个与骨骼系统发育有关的基因（SHH(Sonic hedgehog)基因）的远程顺式调控元件。本研究旨在探究LMBR1基因 intron 5 内的遗传多样性，进而评估HapMap计划的样本选择策略，并检测该区域是否受自然选择的作用。 国际人类基因组单体型图计划(HapMap Project，The International Haplotype Map Project) 于2002年10月正式启动，该计划旨在构建人类基因组中常见变异的遗传图式。自其数据发布以来，广泛应用于生物医学、群体遗传学等领域，在复杂疾病的遗传机理研究、自然选择的检测等方面做出了前所未有的贡献；但是HapMap计划中样本的代表性有待评估。 本研究中，我们综合考虑地理来源信息及线粒体单倍型类群 (Haplogroup)信息选择了41个东亚人作为样本（以保证样本的代表性），测定位于LMBR1 基因intron 5 内的目的片段中存在的单核苷酸多态性(SNP, Singe nucleotide polymorphism)位点，通过所得数据与HapMap数据的比较，发现二者之间差异显著且HapMap数据不能覆盖所有我们得到的常见变异，因而我们认为：HapMap计划中国部分的样本选择策略有待进一步完善。 关于自然选择的研究不仅可以使我们了解生物的进化机制，同时还对复杂疾病的遗传机理研究具有重要的提示作用，因而，对于自然选择的检测，一直以来都是生物学研究的重点。平衡选择是一种维持遗传多态性的自然选择方式，现已发现很多与特定疾病或性状相关的基因或调控序列受平衡选择的作用，如 G6PD 基因、PTC 基因、FMO3 基因、FSHB 基因及 CCR5 基因5’端顺式调控区等我们对41个东亚样本中LMBR1 intron 5 内一段长为9256 bp (Chr7: 156280954-156271699 (Build36))的序列进行以 Tajima’s D 检验为主的群体遗传学分析，发现该区域在进化历程中受到平衡选择的作用。LMBR1 intron 5 内的多态位点与多种表型的多指症存在相关性，受其调控的 SHH 基因在骨骼系统发育中具有重要作用，人类骨骼系统的适应性进化等三方面的因素为该区域受平衡选择的作用提供了进一步的佐证。 总之，本研究对HapMap计划的样本选择策略和数据应用提供了一定的参考；同时还发现一个与骨骼系统发育有关的基因调控元件受平衡选择的作用。

rep-PCR对赤杨根瘤内局Frankia菌基因多样性研究

本文应用重复基因外回纹序列rep-PCR方法直接扩增长日山三种不同赤杨和云南不同地区早冬瓜根瘤内的Frankia菌基因，所获得的REP指纹图谱揭示Frankia菌具有丰富的基因多样性。研究结果表明Frankia菌多样性随地域，海拔，坡问不同而变：多样性指数与自然选抒）玉力大小成正相关：自然选择压力是产生和保持Frankia菌基因多样性的重要因子。同时发现Frankia菌基因型与地域有紧密关系：所观察的云南各地区中，感染高黎贡山旱冬瓜的Frankia菌基因多样性最为丰富。推测与高黎贡山旱冬瓜共生的Frankia可能作为种储备库，为其它地区的寄主植物提供了祖先菌株。在对长白山三种赤杨（东北赤杨，西伯利亚赤杨和色赤杨）根瘤内几百游殆菌基因多样性考察过程中发现，Frankia菌基因类型与寄主赤杨种型关系密切。同时海拔对感染东北赤杨的Frankia菌基因多样性也有一定影响。感染西伯利亚赤杨和色赤杨的Frankia菌享有相同的基因型，可能有较近的亲缘关系。

云南横断山与吉林长白山赤杨根瘤内Frankia菌遗传多样性

本文应用nifD-nifK IGS及16S-23S rDNAIGSPCR-RFLP方法，系统研究了分布于云南横断山的旱冬瓜以及吉林长白山的西伯利亚赤杨、色赤杨和东北赤杨根瘤内Frankia菌的遗传多样性。该研究可加深对Frankia菌起源与进化的认识，并为保护和利用这一固氮共生资源提供科学依据。从113株赤杨的根瘤样品中共检测到48种基因型的Frankia菌株。首次确切报道了自然条件下不同基因型Frankia菌可与同一宿主植株同时共生，并且这种混乱性在横断山区普遍存在。通过对Frankia菌群体遗传结构的分析，查明云南省横断山旱冬瓜Frankia菌群体内遗传变异水平由高到低的顺序是苍山群体、高黎贡山群体、鸡足山群体、来凤山群体和无量山群体。酶切带型9为云南横断山旱冬瓜Frankia菌群体中最古老的类型；大部分（75.63％）遗传变异发生在Frankia菌的各群体内，同一气候区的Frankia菌群体有较近的亲缘关系。旱冬瓜Fran辰a菌遗传多样性特点与横断山气候特征及冰川运动历史密切相关。宿主赤杨的进化地位不同，其共生Frankia菌的群体遗传多样性水平也不同。古老的旱冬瓜共生Fran舫“菌群体内遗传分化最大，分化最晚的东北赤杨共生Frankia菌群体内遗传变异最小；属同一生活型的西伯利亚赤杨和色赤杨共生Fronkia菌间存在着较近亲缘关系；早冬瓜Fran舫。菌群体可能为其它赤杨共生Fran舫“菌提供祖先，因此认为赤杨与其共生Frankia菌间存在着协同进化。

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.