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DNA barcoding and eDNA barcoding in diatoms

机译:硅藻中的DNA条形码和eDNA条形码

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摘要

The microscopical analysis of diatom diversity is a well established approach in the assessment of the ecological status of water bodies and implemented e.g. in the European Water Framework Directive. This dissertation presents environmental DNA (eDNA) barcoding of diatom communities as an alternative methodology to the routine use of light microscopy.The V4 locus of the 18S rRNA gene is introduced in this work as an adequate DNA barcoding marker for diatoms: the locus is flanked by regions that are conserved throughout the diatoms, thus serving as universal primer binding sites. The 18S V4 region also contains sufficient sequence variation for diatom discrimination on a stretch of only approximately 390 bp; therefore it is also suitable for next generation sequencing. A standard laboratory protocol for the amplification of this marker from diatoms in culture as well as from environmental samples is specified in the present dissertation. Furthermore, this work includes the agreement of the CBOL Protist Working Group (ProWG) on the 18S V4 locus as pre]barcode for the assessment of general protist diversity. In addition to that each protist group could apply a second marker region to further examine taxon delimitation and diversity, if necessary.eDNA barcoding relates unidentified sequences from an environmental sample to identified sequences in a reference database to obtain an assignment of the environmental sequences to a respective taxon. The present study investigates the taxonomic discriminatory power of eDNA barcoding in comparison to light microscopy. It is demonstrated that the DNA based approach provides a more refined taxon detection than the microscopical approach. As the simple BLAST algorithm proved to be insufficient for the assignment of sequences to taxonomic entities, the phylogenetic]based coalescent model approach (PCMA) is introduced. The PCMA combines the general mixed Yule]coalescent (GMYC) model . a tree based approach ] with the statistic evaluation of genetic clusters via bootstrap support.It is also shown that the quality of sequence assignment to taxonomic entities is directly related to the quality of the taxonomic treatment of the reference sequences; and the information in the most commonly used reference database INSDC (International Nucleotide Sequence Database Collaboration; incl. Genbank, EMBL/ENA and DDBJ) is of questionable quality.This work therefore includes best practice guidelines for the deposition of sequence information in taxonomic reference libraries. In diatoms reference sequences are almost always obtained from clonal cultures permitting good documentation possibilities. Along with the reference sequences, physical vouchers in form of herbarium specimen and the DNA extract need to be deposited in scientifically curated collections. Also minimum requirements for metadata are suggested: these include e.g. collection data, cultivation data, primer and amplification details, pherograms, as well as photographic documentation of microstructures important for identification. Even though these specifications are proposed for diatoms, they are transferable to other protist groups and also beyond that. As parts of the specified metadata cannot be deposited in the INSDC along with the sequence data, AlgaTerra and the database of the DNA Bank Network are discussed as complements.The publications assembled in the present dissertation contribute towards the establishment of the standard application of eDNA barcoding in water quality assessments via diatom community analysis by introducing (a) a methodological approach and (b) best practice guidelines for the deposition of reference sequences.
机译:硅藻多样性的微观分析是评估水体生态状况的公认方法,并已实施(例如)在欧洲水框架指令中。本论文提出了硅藻群落的环境DNA条形码技术,作为常规光学显微镜的替代方法。本研究介绍了18S rRNA基因的V4基因座,作为硅藻的适当DNA条形码标记:基因座位于两侧通过在整个硅藻中保守的区域,可以用作通用引物结合位点。 18S V4区在大约390 bp的片段上也包含足够的序列变化,可用于区分硅藻。因此,它也适用于下一代测序。本论文规定了从培养物中的硅藻以及环境样品中扩增该标记物的标准实验室方案。此外,这项工作还包括CBOL Protist工作组(ProWG)关于18S V4基因座的协议,作为评估一般Protist多样性的条形码。此外,如果需要,每个保护者组可以应用第二个标记区域来进一步检查分类单元的界定和多样性。eDNA条形码将来自环境样本的未识别序列与参考数据库中的已识别序列相关联,以获得将环境序列分配给各自的分类单元。本研究调查了eDNA条形码与光学显微镜相比的分类学鉴别能力。证明基于DNA的方法比微观方法提供了更精细的分类单元检测。由于简单的BLAST算法不足以将序列分配给生物分类实体,因此引入了基于系统发育的聚结模型方法(PCMA)。 PCMA结合了一般的混合Yule] Colescent(GMYC)模型。基于树的方法,通过引导支持对遗传簇进行统计评估。还表明,分类实体的序列分配质量与参考序列的分类处理质量直接相关;并且最常用的参考数据库INSDC(国际核苷酸序列数据库协作;包括Genbank,EMBL / ENA和DDBJ)中的信息质量令人怀疑。因此,该工作包括在分类参考数据库中存储序列信息的最佳实践指南。 。在硅藻中,参考序列几乎总是从克隆培养物中获得,从而有很好的文献记载的可能性。除参考序列外,植物标本室标本和DNA提取物形式的物理凭证需要存放在科学策划的馆藏中。还建议了对元数据的最低要求:这些要求包括收集数据,培养数据,引物和扩增细节,电泳图以及对识别重要的微结构的照相记录。即使为硅藻提出了这些规范,但它们仍可以转移到其他有机主义者群体中,甚至超出这些范围。由于指定的元数据的一部分无法与序列数据一起存储在INSDC中,因此我们对AlgaTerra和DNA Bank Network数据库进行了补充讨论。本论文中的出版物对建立eDNA条形码的标准应用程序做出了贡献通过引入(a)方法学方法和(b)沉积参考序列的最佳实践指南,通过硅藻群落分析对水质进行评估。

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    Zimmermann Jonas;

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  • 年度 2014
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  • 正文语种 eng
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