【作者】 江鑫；

【导师】 杨官品；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2009， 博士

【摘要】 分子生态学是分子生物学技术应用于生态学与种群生物学领域的产物，每一项分子生物学技术的出现都会带来分子生态学的进步。在众多现代分子生态学研究工具中，微卫星DNA标记系统是最成熟，高效且适合于长期海洋鱼类群体生态学研究的工具之一。虽然个别微卫星DNA标记位点背离理想标记性质的特性会影响其表现，但通过缜密的检测步骤后选择的一套合适微卫星标记能够对目标群体的各项指标做出准确测度。花鲈是栖息于中国沿岸水域的鱼类。大量中国沿岸野生鱼苗被捕获，贩卖并进行养殖。其遗传资源的可持续利用急需科学的管理。本研究为了客观的对花鲈资源状况进行评估而开发微卫星引物。我们在构建的花鲈的基因组微卫星DNA富集文库中，分离出22个位点并在35个中国沿岸花鲈个体上进行了检测。22个位点上等位基因数由3到23，期望杂合度由0．111到0．938。22个位点均不受选择并相互独立。但两个位点上的基因型分布偏离HWE。针对花鲈微卫星DNA位点开发中出现的阳性克隆率低的问题，我们在金鱼微卫星DNA标记筛选的碱洗脱后步骤后，增加一步更严谨的热洗脱步骤。经检验，洗脱产物中包含的阳性克隆率显著高于传统步骤（P=0．0003）。另外，我们尝试提高DSPT微卫星DNA标记开发的效率。（TC）₆（AC）₅和（AC）₁₂B分别搭配EcoR I引物对同一克隆进行PCR检测的产物信号强度差在内侧微卫星DNA重复数n=5和n＞5划分的两组间差异显著（P=0．0036）。二者信号强度差可作为控制内侧微卫星DNA重复数目的有效标准。在22个开发的微卫星DNA标记中，19个能够应用于花鲈与日本鲈的群体遗传分析。所有8个群体样本利用这19个标记对等位基因数和期望杂合度进行了测度。19个微卫星DNA标记对8个群体样品进行分配模式检验发现，大部分花鲈（93．8-98．8％）与日本鲈（90．9-94．6％）能够准确的分入相应的遗传群中。瓶颈检验发现花鲈经历过因过量捕捞或（和）冰期导致的瓶颈事件（P=0．00357）。花鲈群体样本间配对F_ST（0．01 9-0．029）和日本鲈群体样本间配对F_ST（0．003-0．021）显著小于花鲈与日本鲈群体样本间的配对F_ST（0．076-0．101），花鲈与日本鲈群体样本间Nei氏遗传距离（0．326-0．450）显著大于花鲈群体间（0．123-0．145）和日本鲈群体间（0．055-0．123）的Nei氏遗传距离。花鲈与日本鲈群体样本间平均配对F_ST为0．075，样本间变异占样本总变异的7．16％。花鲈与日本鲈有穿越其栖息地间地理间隔的迁移能力，出现的分化说明中日水域间存在其他基因交流屏障。对花鲈与日本鲈样品单独进行分配模式检验发现，舟山群体、有名海群体、东京湾和石川县样品中的个体被分入不同遗传群。花鲈与日本鲈可能在这些地点进行聚集。用23个微卫星DNA标记分析了2000年前后北海、威海和舟山（A组）及2006年烟台（B组）花鲈地理群体遗传结构。A组群体期望杂合度相似，有轻微遗传分化，属同一自由交配群；组内及威海和北海群体内存在瓶颈事件。B组群体Nei氏期望杂合度、平均等位基因数相似，无遗传分化；B组群体与威海群体属同一自由交配群，表明山东半岛沿海花鲈遗传结构从2000到2006年没有明显变化。虽然花鲈与日本鲈群体多样性与遗传结构研究结果明确，但二者在朝鲜半岛南部水域出现的同域分布原因仍不明确。花鲈与日本鲈栖息区域很多海洋生物分布模式复杂。分析其形成过程需要对混合的种群分化原因进行解析，并了解该区域海陆演化历史。江豚对海洋环境变化的直接响应，对小规模扰动的抵御能力和与沿岸内水生活特性使其能够准确记录栖息区域的生物地理学事件。中日沿岸与长江水域江豚群体中，单倍型C分布最广，出现最多，最为古老。中日沿岸与长江水域江豚群体的NCPA分析发现，带有C单倍型的古老种群分别在日本群岛太平洋沿岸区域，日本南部岛弧后边缘海沿岸区域和长江流域分化出包含新单倍型的独立群体。NCPA还发现，栖息于有名海-立花湾和黄海水域的群体与外界存在的基因交流障碍，解释了这两个区域较高的核苷酸多样性和单倍型多样性的成因。祖先种群与这两个区域的种群的分化原因在本研究中无法得到。虽然本研究仍然无法明晰花鲈与日本鲈群体复杂的分化模式成因。但本研究的结果与将来其他领域相关的研究结果整合后，对西北太平洋边缘海水域海陆演化历史背景的明确描绘。将为该区域复杂的海洋生物分化模式成因提供解释依据。更多还原

【Abstract】 Molecular ecology is a new branch of modern ecology, combines ecological and population biology theories with modern molecular biology techniques. In all technology used in molecular ecology researches, SSR markers are one of the most suitable and efficient tools for researches of marine fishes. Although recent papers report behaviors of SSR loci violated ideal marker properties, a set of SSR loci passed rigorous screening protocols for given samples will perform well in population parameter estimating.Spotted sea bass （Lateolabrax maculatus） , which inhabits East Asia coasts and cage-cultured as well in China currently, needs scientific management for sustainable exploration urgently. A set of SSR marker will certainly facilitate the management and exploitation of the genetic resource of L. maculatus. 22 microsatellite DNA markers are developed and used to type 35 individuals collected along the Chinese coast. The number of alleles per locus range from 3 to 23. The expected heterozygosities range from 0.111 to 0.938. The observed heterozygosities range from 0.114 to 0.914. All 22 loci are neutral and independent each other, of them, 2 deviate significantly from Hardy-Weinberg equilibrium. These microsatellite DNA markers are moderately informative. For the low ratio of positive clone in FIASCO used in spotted sea bass SSR isolation, a more stringent heating elution protocol is added after the alkaline elution. The fragments eluted produce more positive clones than original protocols significantly （P=0.0003） . In addition, a more convenient method named DSPT are used and modified. To selecting fragments with more than 5 inner repeats of combined SSR, EcoR I adapter primer combined with （TC）₆ （AC ）₅ （system A ） and （AC）₁₂B （system C ） are used in two different RCR screening systems for the same clone. The group of SSR loci with more than 5 inner repeats has significantly differences in signal deference A-C with Loci group with inner repeats less than 5 repeats （P=0.0036） . The different between A and C signals can be criterion for suitable combined SSR loci selection.Of 22 microsatellite DNA markers developed from spotted sea bass （L. maculatus） , 19 are independent from each other and at Hardy-Weinberg equilibrium across the 3 populations of spotted sea bass inhabiting Chinese coasts and the five populations of Japanese sea bass （L. japonicus） inhabiting Japanese coasts. These 19 markers are used to determine the number of alleles and the expected heterozygosity across the eight populations. The majority of individuals （93.8-98.8%） of the three populations of spotted sea bass are assigned to an inferred cluster, and 90.9-94.6% of the individuals of the five populations of Japanese sea bass are assigned to the other. The average number of alleles across the 19 loci is significantly lower in spotted sea bass than in Japanese sea bass （10.3 vs 15.4） , however, the average expected heterozygosity across 19 loci of spotted sea bass is similar to that of Japanese sea bass populations （0.743 vs 0.750）. An effective population size reduction （i.e. bottleneck effect） is detected in spotted sea bass （P = 0.00357）, which may have resulted from either over-catching or glaciations or both. The pair-wise F_ST among populations of spotted sea bass （0.019-0.029） and among populations of Japanese sea bass（0.003-0.021） are lower than those between the populations of spotted and Japanese sea bass （0.076-0.101） . The average pair-wise F_ST between populations of spotted and Japanese sea bass reaches 0.075 and the variation between spotted and Japanese sea bass accounts for 7.16% of the total. Nei’s original measures of genetic distances among populations of spotted and Japanese sea bass range from 0.123 to 0.145 and from 0.055 to 0.123, respectively, while that between the populations of spotted and Japanese sea bass group range from 0.326 to 0.450. Both species are able to disperse over a long distance; however, our observations demonstrate that they cannot migrate across a possible barrier existing between Chinese and Japanese coasts. Most individuals of Zhoushan population of spotted sea bass are assigned to two inferred clusters, indicating that Zhoushan is gathering ground of spotted sea bass. Most individuals of Ariake Sea, Tokyo Bay and Ishikawa populations of Japanese sea bass are assigned to three inferred clusters, indicating that these locations are gathering grounds of Japanese sea bass. The genetic structure of spotted sea bass （L. maculatus） inhabiting Chinese coast is determined with 23 microsatellite DNA markers. The Nei’s expected heterozygosities of Beihai, Weihai and Zhoushan geographic populations （Group A） are similar each other. These 3 populations differentiate slightly; however, the individuals collected around year 2000 belonged to a single panmictic group. The bottleneck event is detected in Group A as a whole and in Weihai and Beihai geographic populations. Two Yantai populations （Group B） are genetically identical; Both Nei’s expected heterozygosities and mean numbers of alleles of them are similar. The individuals of Group B and those of Weihai geographic population belong to a single panmictic group, indicating that the genetic structure of the spotted sea bass along the coast of Shandong Peninsula is unchanged from 2000 to 2006.The conducted population structure researches can not answer questions of sympatric distribution of spotted and Japanese sea bass in southern Korean peninsula. A lot of species inhabit Northwest Pacific marginal sea areas show profound distribution patterns that can not be clearly understood. To resolve these problems, the history of sea-land changes must be clarified and the special-temporal forming factors of population division must be separated. The direct response to marine environment changes, the resistance to small scale disturbance and inshore and inland water inhabiting behaviors guarantee finless porpoise a suitable model species for historical biogeographical event recording. In all population inhabit Yangtze River and Chinese and Japanese coasts, haplotype C is the most ancient haplotype belongs to ancestor population. NCPA of finless population inhabit Chinese and Japanese coasts reveals that, the ancient population with C haplotype expanded to pacific coasts of Japanese islands, Southern Japanese coasts of post-arc marginal sea and Yangtze River and divided in different populations with new haplotypes. Populations inhabiting Ariake Sound-Tachibana Bay and Yellow Sea isolated from others according to NCPA results, which explained the high nucleotide and haplotype diversities detect in these populations. But the deviation factors of these two from ancient population cannot be deduced in this study. This study alone cannot clarify the factors forming current distribution patterns of spotted and Japanese sea bass. But after being integrated with results of future studies, the clarified background of sea-land changes in Northwest Pacific marginal sea will greatly facilitate the interpretation of complicated distribution patterns of species inhabiting these areas.更多还原