Development of High Throughput Analysis of Plankton Communities

在过去十年中，采用分子方法和其他新技术进行的研究提高了对浮游生物多样性的认识，并导致在全世界海洋中发现了重要的新分类群。在理解这种新发现的多样性的重要性的进展一直受到阻碍，缺乏高通量的技术，同时识别和量化的许多类群，包括这样的社区。微珠阵列技术为高通量分析多个分类群提供了一种很有前途的方法。本项目的目标是测试微珠阵列技术对不同海洋微生物群落的定量分析的适用性。如本文所应用的，珠阵列由一组附着于5微米珠的分类群特异性寡核苷酸组成。多达100种不同的寡核苷酸探针，每个连接到不同颜色的珠子，用于定量探测从散装海水过滤的浮游生物提取的DNA。具有不同分辨率水平的探针可以在单个多重测定中混合。将采用两个测试系统来开发这项技术。 将开发一个阵列来分析沿海环境中微型浮游植物多样性的季节性变化。第二个阵列将被开发出来，以区分硅藻属的25种伪菱形藻，其中10种已知会产生神经毒素软骨藻酸。多重微珠阵列技术将为海洋微生物群落分析提供一种新的方法学工具。计划中的调查还将为寻求将新分子技术应用于海洋生态学重要问题的本科生、研究生和博士后研究人员提供极好的培训机会。

Over the past decade, research employing molecular approaches and other new technologies has permitted increased resolution of plankton diversity and led to the discovery of important new taxa throughout the world's oceans. Progress in understanding the significance of this newfound diversity has been hampered by a lack of high throughput technologies for the simultaneous identification and quantification of the many taxa comprising such communities. Bead arrays offer a promising approach to the problem of high throughput analysis of multiple taxa. The goal of this project is to test the applicability of bead array technology to the quantitative analysis of diverse marine microbial communities. As applied here, a bead array consists of a set of taxon-specific oligonucleotides attached to 5 micron beads. Up to 100 different oligonucleotide probes, each attached to beads of a different color, are used to quantitatively probe DNA extracted from planktonic organisms filtered from bulk seawater. Probes with different levels of resolution can be mixed in a single multiplex assay. Two test systems for the development of this technology will be employed. An array will be developed to assay seasonal changes in picophytoplankton diversity in a coastal environment. A second array will be developed to distinguish among the 25 species of the diatom genus Pseudo-nitzschia, ten of which are known to produce the neurotoxin domoic acid. Multiplex bead arrays will provide a new methodological tool for the analysis of marine microbial communities. The planned investigations also will provide excellent training opportunities for undergraduate, graduate and postdoctoral researchers seeking to apply new molecular technologies to important questions in marine ecology.