Hamilton Paul B, Lefebvre Keely E, Bull Roger D
Research and Collections, Canadian Museum of Nature Ottawa, ON, Canada.
Research and Collections, Canadian Museum of Nature Ottawa, ON, Canada ; Biology and Environmental Science, Center for Advanced Research in Environmental Genomics, University of Ottawa Ottawa, ON, Canada.
Front Microbiol. 2015 Oct 14;6:1084. doi: 10.3389/fmicb.2015.01084. eCollection 2015.
Single cell Chelex® DNA extraction and nested PCR amplification were used to examine partial gene sequences from natural diatom populations for taxonomic and phylogenetic studies at and above the level of species. DNA was extracted from cells that were either fresh collected or stored in RNAlater. Extractions from Lugol's fixation were also attempted with limited success. Three partial gene sequences (rbcL, 18S, and psbA) were recovered using existing and new primers with a nested or double nested PCR approach with amplification and success rates between 70 and 96%. An rbcL consensus tree grouped morphologically similar specimens and was consistent across the two primary sample treatments: fresh and RNAlater. This tool will greatly enhance the number of microscopic diatom taxa (and potentially other microbes) available for barcoding and phylogenetic studies. The near-term increase in sequence data for diatoms generated via routine single cell extractions and PCR will act as a multiproxy validation of longer-term next generation genomics.
采用单细胞Chelex® DNA提取和巢式PCR扩增技术，对自然硅藻种群的部分基因序列进行检测，用于物种及以上水平的分类学和系统发育研究。DNA从新鲜采集或保存在RNAlater中的细胞中提取。也尝试从卢戈尔固定液中提取DNA，但成功率有限。使用现有引物和新引物，通过巢式或双重巢式PCR方法，成功获得了三个部分基因序列（rbcL、18S和psbA），扩增成功率在70%至96%之间。一个rbcL一致性树将形态相似的标本归为一类，并且在两种主要样本处理方式（新鲜样本和保存在RNAlater中的样本）中保持一致。该工具将极大地增加可用于条形码和系统发育研究的微观硅藻分类单元（以及可能的其他微生物）数量。通过常规单细胞提取和PCR生成的硅藻序列数据在短期内的增加，将作为对长期下一代基因组学的多指标验证。
