Marine Biotech Fellowship: Genetic Population Structure and Gene Flow in Deep-Sea Invertebrates

[212994]法国深海是地球上最大的环境，以其无脊椎动物的高度多样化而闻名。尽管在测量物种多样性的模式方面已经付出了相当大的努力，但对产生深海动物群的进化力量的研究仍然很少。为了估计基因流动和种群分化和物种形成的潜力，需要对地理变异的遗传模式进行研究。然而，在深海中进行的此类研究屈指可数。对深海物种遗传种群结构进行量化的主要障碍之一是难以获得足够数量的适当保存的遗传研究组织。大多数深海底栖生物是由小型、稀疏分布的生物组成的，在典型的采样中，它们被发现是死亡或垂死的；这些标本对于同酶电泳的标准技术来说是不够的。然而，随着新的分子生物学技术的出现，从古代、干燥和酒精保存的组织中鉴定DNA序列成为可能，对深海物种的种群遗传学研究现在是可行的。特别是，从数十个深海取样工作中收集的大量标本提供了丰富的潜在信息，可供分析。本项目将应用聚合酶链反应（PCR）技术对酒精保存的深海片脚类标本的遗传变异进行表征，目的是估计海洋内和海洋间地理尺度上的遗传种群结构和基因流动。为了实现这一目标，本项目将：1.利用PCR技术，利用从果蝇mtDNA序列构建的通用引物，扩增每个种群中15-25个个体的线粒体DNA （mtDNA）的快速进化部分；2)检查扩增片段的高变区；3)确定15-20个碱基对区域，其中大多数变异仅限于1或2个多态性核苷酸，并为每个等位基因设计等位基因特异性寡核苷酸（ASO）探针；4. 将ASO探针杂交到每个群体100-200个扩增的高变区；5)计算各种群等位基因和基因型频率，评价种群结构。该项目的结果应提供跨深海盆地和世界海洋之间的遗传种群结构的第一次详细检查。此外，该项目有可能证明分子生物学技术对深海生态问题的适用性，并促进进一步利用现有资源来解决海洋中的重要进化问题。＊＊＊

9212994 France The deep sea is the largest environment of Earth and is known to be highly diverse in its invertebrate fauna. Although considerable effort has been devoted to measuring the patterns of species diversity, the evolutionary forces which have generated the deep- sea fauna remain poorly studied. In order to estimate gene flow and the potential for population differentiation and speciation, studies of the genetic patterns of geographic variation are required. Yet only a handful of such studies have been done in the deep sea. One of the major obstacles to quantifying genetic population structure in deep-sea species has been the difficulty of obtaining sufficient quantities of appropriately preserved tissue for genetic studies. Most of the deep-sea benthos is comprised of small, sparsely distributed organisms which are recovered dead or moribund during typical sampling; these specimens have been inadequate for the standard techniques of allozyme electrophoresis. However, with the advent of new molecular biological techniques which make possible the characterization of DNA sequences from ancient, dried, and alcohol-preserved tissues, studies of the population genetics of deep-sea species are now feasible. In particular, the extensive collections of specimens from dozens of deep-sea sampling efforts provide a wealth of potential information ready to be analyzed. This project will apply the techniques of the polymerase chain reaction (PCR) to characterize genetic variation from alcohol-preserved specimens of deep-sea amphipods, with the goal of estimating genetic population structure and gene flow at intra-ocean and inter-ocean geographic scales. To accomplish this goal, this project will, 1.) with PCR, amplify rapidly evolving portions of the mitochondrial DNA (mtDNA) from 15-25 individuals in each population with universal primers constructed from mtDNA sequences of Drosophila; 2.) inspect amplified segments for hypervariable regions; 3.) identify 15-20 base pair regions where most variation is confined to 1 or 2 polymorphic nucleotides and design allele-specific oligonucleotide (ASO) probes for each allele; 4. hybridize ASO probes to amplified hypervariable regions from 100-200 individuals per population; 5) calculate allele and genotype frequencies in each population and evaluate population structure. The results of this project should provide the first detailed examination of genetic population structure across deep ocean basins and between world oceans. In addition, this project has the potential to demonstrate the applicability of molecular biological techniques to problems in deep-sea ecology and to promote further use of available resources in addressing important evolutionary issues in the oceans. ***