【作者】 陈国娟；

【导师】 李春阳；

【作者基本信息】 中国科学院研究生院（成都生物研究所） ， 植物学， 2007， 博士

【摘要】 中国沙棘是一种雌雄异株、风媒传粉的灌木或乔木，在中国西南的卧龙自然保护区有广泛的分布。本研究以采集于四川卧龙自然保护区5个海拔(1800m、2200m、2600m、3000m、3400m)梯度的中国沙棘天然群体为材料，以ISSR和AFLP标记技术研究其遗传多样性水平及其遗传结构，旨在了解卧龙地区中国沙棘天然群体的遗传多样性水平以及遗传多样性在群体间、群体内以及雌雄亚群体间的分布和特征，为中国沙棘树种的遗传改良及种质资源保存提供遗传研究背景与实验依据。同时探讨ISSR、AFLP和RAPD三种标记对中国沙棘天然群体的遗传变异水平和群体间遗传结构的评估能力和各自的优缺点。研究得出以下主要结论：1．ISSR和AFLP分析都表明卧龙自然保护区的中国沙棘群体拥有较高的遗传变异水平(h=0．249，H_T=0．305)。出现这种结果的主要原因可能与卧龙自然保护区多变的气候条件和生境的异质度大有关。2．ISSR和AFLP都揭示出卧龙自然保护区中国沙棘群体的遗传多样性随着海拔的增加发生显著的变化，表现为中海拔群体(2200m和2600m)比高海拔群体(3000m和3400m)和低海拔群体(1800m)有更高的遗传多样性的趋势。出现这种趋势的可能解释是低海拔群体处在相对高温和相对干旱的环境，高海拔群体受到低温和紫外线胁迫，而中海拔群体存在中国沙棘生长的适宜环境。3．ISSR和AFLP分析都表明：卧龙自然保护区中国沙棘的遗传结构遵循分布范围广、交配系统以异交为主的木本植物的通常模式，即大多数的遗传变异存在于群体内，只有少部分的遗传变异存在于群体间。4．经Mantel检测表明，卧龙自然保护区中国沙棘群体间的海拔距离和对应遗传距离之间存在显著的正相关关系，即随着垂直海拔距离的增加，群体间的遗传距离也随之增加。Mantel检测结果以及聚类分析将卧龙自然保护区5个不同海拔的中国沙棘群体分为低、中、高海拔群体三组的研究结果都表明，海拔很可能是限制群体间基因交流的主要因素。5．ISSR分析发现同一海拔的雌雄亚群体首先聚类的研究结果表明，同一海拔的雌雄亚群体在遗传上最相似。方差分析结果表明只有3．8％的总遗传变异存在于雌雄亚群体间，这可能与雌雄植株间的交配和遗传物质的混合有关。6．ISSR、AFLP和RAPD分析都表明卧龙自然保护区不同海拔的中国沙棘天然群体的遗传多样性水平较高。它们的分析结果估算得到的Nei’s平均基因多样度(h)分别为0．249、0．214和0．170。从该结果可以看出ISSR和AFLP比RAPD检测到更多的遗传多态性，这很可能是不同标记检测的基因组的位点不同所致。7．依据对不同标记系统的比较分析，认为ISSR、AFLP和RAPD三种分子标记系统都能成功地用于调查卧龙自然保护区不同海拔的中国沙棘群体的遗传变异水平及遗传变异结构，提供关于中国沙棘天然群体多态性水平和遗传变异分布的有用信息。在三者中，AFLP具有最高效能指数和标记指数，在确定种间分类关系或鉴别个体方面是一种比较理想的标记。更多还原

【Abstract】 Hippophae rhamnoides subsp. sinensis, a dioecious and deciduous shrub species, occupies a wide range of habitats in the Wolong Nature Reserve, Southwest China. Our present study investigated the pattern of genetic variation and differentiation among five natural populations of H. rhamnoides subsp. sinensis, occurring along an altitudinal gradient that varied from 1,800 to 3,400 m above sea level in the Wolong Natural Reserve, by using ISSR and AFLP markers to guide its genetic improvement and germplasm conservation. And, comparative study of ISSR, AFLP and RAPD was performed to detect their capacity to estimating the level and pattern of genetic variation occurring among the five elevation populations of H. rhamnoides subsp. sinensis, and to discuss their application to the study on plant genetics. The results were list following:1. The ISSR and AFLP analysis conducted for the H. rhamnoides subsp. sinensis populations located in the Wolong Natural Reserve of China revealed the presence of high levels of genetic variation (h=0.249, H_T=0.305). Besides such features as relatively wide distribution, dominantly outcrossing mating system, and effective seed dispersal by small animals and birds, it is sometimes argued that hard climatic conditions and heterogeneous habitats may also contribute to high levels of diversity.2. Genetic diversity of H. rhamnoides subsp. sinensis populations was found to vary significantly with changing elevation, showing a trend that mid-elevation populations (2,200 m and 2,600 m) were genetically more diverse than both low-elevation (1,800 m) and high-elevation populations (3,000 m and 3,400 m). H. rhamnoides subsp. sinensis is thought to be stressed by drought and high temperature at low elevations, and by low temperature at high elevations. The high genetic variability present in the mid-elevation populations of H. rhamnoides subsp. sinensis is assumed to be related to a greater plant density in the middle altitudinal zone, where favorable ecological conditions permit its continuous distribution covering the zone from 2,200 m to 2,600 m above sea level.3. The genetic structure of H. rhamnoides subsp. sinensis revealed by ISSRs and AFLPs followed the general pattern detected in woody species with widespread distributions and outcrossing mating systems. Such plants possess more genetic diversity within populations and less variation among populations than species with other combinations of traits.4. In the present study, Mantel tests showed positive correlations between altitudinal distances and genetic distances among populations or subpopulations. The observed relationship between altitude and genetic distances, and the result of the cluster analysis including populations or male subpopulations and classifying the groups into three altitude clusters suggest that altitude is a major factor that restricts gene flow between populations and subpopulations.5. The analysis of molecular variance showed that only 3.8% of the variability resided between female and male subpopulations. Such a very restricted proportion of the total molecular variance between female and male subpopulations is due to common sexuality and mixing of genetic material between females and males. 6. The analysis based on ISSRs, AFLPs and RAPDs all revealed relatively high levels of genetic variation among different altitudinal populations of H. rhamnoides subsp. sinensis in Wolong Natural Reserve of China. Their estimates of mean Nei’s gene diversity is equal to 0.249, 0.214 and 0.170 respectively, suggesting the higher capacity of detecting genetic variation of ISSR and AFLP than RAPD. It might be ascribed to their distinct sensitivity to different type of genetic variation.7. Based on the coparative study on ISSR, AFLP and RAPD, we drew a conclusion that they all successfully reveal some useful information concerning the level and pattern of genetic vatiation occurring among different elevation populations of H. rhamnoides subsp. sinensis. AFLP is a ideal tool to taxonomic study and individual identification for their highest efficiency index and marker index among the three marker systems.更多还原