Methodology for Single-Cell Genetic Analysis of Planktonic Foraminifera for Studies of Protist Diversity and Evolution
Methodology for Single-Cell Genetic Analysis of Planktonic Foraminifera for Studies of Protist Diversity and Evolution
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DOI：
10.3389/fmars.2016.00255
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发表时间：
2016-12
影响因子：
3.7
通讯作者：
Agnes K. M. Weiner;R. Morard;M. Weinkauf;K. Darling;Aurore André;F. Quillévéré;Y. Ujiié;C. Douady;C. de Vargas;M. Kučera
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文献类型：
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作者：
Agnes K. M. Weiner;R. Morard;M. Weinkauf;K. Darling;Aurore André;F. Quillévéré;Y. Ujiié;C. Douady;C. de Vargas;M. Kučera
单细胞遗传分析是研究海洋原生生物多样性和进化生态学的重要方法。在实验室条件下不能繁殖的原生生物群体中，这种方法提供了将分子序列与细胞形态直接关联的唯一方法。由此产生的 DNA 序列的明确分类学鉴定是环境宏基因组数据条形码和分析的先决条件。在过去的 20 年里，人们对浮游有孔虫进行了广泛的单细胞遗传学研究，以阐明它们的系统发育、神秘多样性、生物地理学以及遗传和形态变异之间的关系。在这些研究过程中，很明显，个体样本水平的遗传分析面临着无数的挑战，从单细胞中存在的 DNA 量可以忽略不计，到内共生体或食物颗粒引入的大量 DNA 污染。因此，多年来，人们开发并应用了一系列方法来进行浮游有孔虫的遗传分析，以提高 DNA 扩增的成功率。然而，文献中对这些方法的描述很少有同等程度的细节，并且从未对不同方法的效率和可重复性进行比较。在这里，为了方法的标准化，我们对浮游有孔虫单细胞遗传分析所采用的所有方法进行了全面的回顾。我们收集有关 DNA 扩增成功率的数据，并使用这些数据来评估与单细胞 DNA 样本收集、储存和提取方法相关的关键参数的影响。我们表明，所选择的方法会影响单细胞遗传学研究的成功率，但它们之间的差异不足以阻碍不同方法进行的研究之间的比较。因此，该综述不仅为未来有孔虫遗传学研究提供了全面的参考指南，而且还为使用现有单细胞数据集的研究建立了重要基准。这些方法广泛适用，审查可能有助于建立类似的标准原则，以便在其他原生生物群体中使用。
Single-cell genetic analysis is an essential method to investigate the biodiversity and evolutionary ecology of marine protists. In protist groups that do not reproduce under laboratory conditions, this approach provides the only means to directly associate molecular sequences with cell morphology. The resulting unambiguous taxonomic identification of the DNA sequences is a prerequisite for barcoding and analyses of environmental metagenomic data. Extensive single-cell genetic studies have been carried out on planktonic foraminifera over the past 20 years to elucidate their phylogeny, cryptic diversity, biogeography and the relationship between genetic and morphological variability. In the course of these investigations, it has become evident that genetic analysis at the individual specimen level is confronted by innumerable challenges ranging from the negligible amount of DNA present in the single cell to the substantial amount of DNA contamination introduced by endosymbionts or food particles. Consequently, a range of methods has been developed and applied throughout the years for the genetic analysis of planktonic foraminifera in order to enhance DNA amplification success rates. Yet, the description of these methods in the literature rarely occurred with equivalent levels of detail and the different approaches have never been compared in terms of their efficiency and reproducibility. Here, aiming at a standardization of methods, we provide a comprehensive review of all methods that have been employed for the single-cell genetic analysis of planktonic foraminifera. We compile data on success rates of DNA amplification and use these to evaluate the effects of key parameters associated with the methods of sample collection, storage and extraction of single-cell DNA. We show that the chosen methods influence the success rates of single-cell genetic studies, but the differences between them are not sufficient to hinder comparisons between studies carried out by different methods. The review thus not only provides a comprehensive reference with guidelines for future genetic studies on foraminifera, but it also establishes an important benchmark for investigations using existing single-cell datasets. The methods are widely applicable and the review may help to establish similar standard principles for their utilization in other protist groups.