Vivien Régis, Ferrari Benoit J D, Pawlowski Jan
Swiss Centre for Applied Ecotoxicology (Ecotox Centre), Eawag/EPFL, Lausanne, Switzerland.
Department of Genetics and Evolution, University of Geneva, Geneva, Switzerland.
BMC Res Notes. 2016 Jul 13;9:342. doi: 10.1186/s13104-016-2140-1.
Oligochaetes are valuable bioindicators of the quality of watercourse and lake sediments. The morphological identification of aquatic oligochaetes is difficult, prompting the development of new molecular oligochaete indices based on DNA barcoding and Next-generation sequencing of sorted specimens. In general, the samples for DNA barcoding are fixed in absolute ethanol. However, in the case of aquatic oligochaetes, this medium is not appropriate as it can induce a modification of specimen abundances and of the composition of communities. Therefore, we investigated the possibility to amplify and sequence aquatic oligochaetes fixed in formalin for a short time. We performed guanidine extraction and polymerase chain reaction (PCR) amplification/sequencing of the cytochrome c oxydase I (COI) gene on tissue fragments fixed in formalin for different periods of time (from 1 h to 1 week) and in ethanol.
The large majority of aquatic oligochaete specimens fixed in formalin for up to 1 week could be successfully amplified and all obtained sequences were of high quality. The amplification and sequencing success rate of formalin-fixed samples and ethanol-fixed samples was similar. These results suggest that formalin fixation of aquatic oligochaete tissues for a short time does not cause serious damages to DNA and inhibit PCR amplification.
The possibility to fix aquatic oligochaetes with formalin before genetic analyses is very promising for diversity monitoring, for construction of a comprehensive DNA barcode library and for development of an index based on Next-generation sequencing analysis of samples composed of sorted specimens.
寡毛纲动物是河道和湖泊沉积物质量的重要生物指示物种。水生寡毛纲动物的形态鉴定较为困难，这促使人们基于DNA条形码技术和对分选标本的下一代测序技术开发新的分子寡毛纲动物指标。一般来说，用于DNA条形码分析的样本是用无水乙醇固定的。然而，对于水生寡毛纲动物而言，这种介质并不合适，因为它会导致标本丰度和群落组成发生改变。因此，我们研究了对短期保存在福尔马林中的水生寡毛纲动物进行扩增和测序的可能性。我们对保存在福尔马林不同时间（从1小时到1周）以及乙醇中的组织片段进行了胍提取和细胞色素c氧化酶I（COI）基因的聚合酶链反应（PCR）扩增/测序。
绝大多数保存在福尔马林中长达1周的水生寡毛纲动物标本能够成功扩增，并且所有获得的序列质量都很高。福尔马林固定样本和乙醇固定样本的扩增和测序成功率相似。这些结果表明，短期用福尔马林固定水生寡毛纲动物组织不会对DNA造成严重损伤并抑制PCR扩增。
在进行遗传分析之前用福尔马林固定水生寡毛纲动物的可能性对于多样性监测、构建全面的DNA条形码文库以及基于对分选标本组成的样本进行下一代测序分析来开发指标而言非常有前景。
