University of Colorado, Department of Ecology and Evolutionary Biology, Ramaley N122, Campus Box 334, Boulder, CO 80309-0334, USA.
BMC Ecol. 2009 Dec 11;9:25. doi: 10.1186/1472-6785-9-25.
The time it takes to isolate individuals from environmental samples and then extract DNA from each individual is one of the problems with generating molecular data from meiofauna such as eutardigrades and bdelloid rotifers. The lack of consistent morphological information and the extreme abundance of these classes makes morphological identification of rare, or even common cryptic taxa a large and unwieldy task. This limits the ability to perform large-scale surveys of the diversity of these organisms.Here we demonstrate a culture-independent molecular survey approach that enables the generation of large amounts of eutardigrade and bdelloid rotifer sequence data directly from soil. Our PCR primers, specific to the 18s small-subunit rRNA gene, were developed for both eutardigrades and bdelloid rotifers.
The developed primers successfully amplified DNA of their target organism from various soil DNA extracts. This was confirmed by both the BLAST similarity searches and phylogenetic analyses. Tardigrades showed much better phylogenetic resolution than bdelloids. Both groups of organisms exhibited varying levels of endemism.
The development of clade-specific primers for characterizing eutardigrades and bdelloid rotifers from environmental samples should greatly increase our ability to characterize the composition of these taxa in environmental samples. Environmental sequencing as shown here differs from other molecular survey methods in that there is no need to pre-isolate the organisms of interest from soil in order to amplify their DNA. The DNA sequences obtained from methods that do not require culturing can be identified post-hoc and placed phylogenetically as additional closely related sequences are obtained from morphologically identified conspecifics. Our non-cultured environmental sequence based approach will be able to provide a rapid and large-scale screening of the presence, absence and diversity of Bdelloidea and Eutardigrada in a variety of soils.
从环境样本中分离个体并从每个个体中提取 DNA 所需的时间是从缓步动物和蛭形轮虫等小型后生动物中生成分子数据的问题之一。这些类群缺乏一致的形态信息，而且数量极其庞大，使得对稀有甚至常见的隐生分类单元进行形态鉴定成为一项庞大而繁琐的任务。这限制了对这些生物多样性进行大规模调查的能力。在这里，我们展示了一种无需培养的分子调查方法，该方法可直接从土壤中生成大量的缓步动物和蛭形轮虫序列数据。我们的 PCR 引物针对 18S 小亚基 rRNA 基因，针对缓步动物和蛭形轮虫进行了特异性设计。
开发的引物成功地从各种土壤 DNA 提取物中扩增了目标生物的 DNA。这通过 BLAST 相似性搜索和系统发育分析得到了证实。缓步动物的系统发育分辨率明显优于蛭形轮虫。这两个类群的生物都表现出不同程度的特有性。
开发用于从环境样本中鉴定缓步动物和蛭形轮虫的类群特异性引物，将极大地提高我们在环境样本中鉴定这些分类单元组成的能力。这里展示的环境测序与其他分子调查方法不同，因为不需要预先从土壤中分离出感兴趣的生物来扩增它们的 DNA。从不需要培养的方法获得的 DNA 序列可以在获得更多形态鉴定的同种近缘序列后进行后置鉴定，并进行系统发育分析。我们的非培养环境序列为基础的方法将能够快速、大规模地筛选各种土壤中 Bdelloidea 和 Eutardigrada 的存在、缺失和多样性。
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