Saitoh Seikoh, Aoyama Hiroaki, Fujii Saori, Sunagawa Haruki, Nagahama Hideki, Akutsu Masako, Shinzato Naoya, Kaneko Nobuhiro, Nakamori Taizo
a Tropical Biosphere Research Center, University of the Ryukyus, 1 Senbaru, Nishihara 903-0213, Japan.
b Department of Environment and Natural Sciences, Graduate School of Environment and Information Sciences, Yokohama National University, Yokohama 240-8501, Japan.
Genome. 2016 Sep;59(9):705-23. doi: 10.1139/gen-2015-0228.
We developed a novel protocol with superior quantitative analysis results for DNA metabarcoding of Collembola, a major soil microarthropod order. Degenerate PCR primers were designed for conserved regions in the mitochondrial cytochrome c oxidase subunit I (mtCOI) and 16S ribosomal RNA (mt16S) genes based on published collembolan mitogenomes. The best primer pair was selected based on its ability to amplify each gene, irrespective of the species. DNA was extracted from 10 natural communities sampled in a temperate forest (with typically 25-30 collembolan species per 10 soil samples) and 10 mock communities (with seven cultured collembolan species). The two gene regions were then amplified using the selected primers, ligated with adapters for 454 technology, and sequenced. Examination of the natural community samples showed that 32 and 36 operational taxonomic units (defined at a 90% sequence similarity threshold) were recovered from the mtCOI and mt16S data, respectively, which were comparable to the results of the microscopic identification of 25 morphospecies. Further, sequence abundances for each collembolan species from the mtCOI and mt16S data of the mock communities, after normalization by using a species as the internal control, showed good correlation with the number of individuals in the samples (R = 0.91-0.99), although relative species abundances within a mock community sample estimated from sequences were skewed from community composition in terms of the number of individuals or biomass of the species. Thus, this protocol enables the comparison of collembolan communities in a quantitative manner by metabarcoding.
我们开发了一种新方案，用于弹尾目（一种主要的土壤微型节肢动物目）的DNA宏条形码分析，该方案具有卓越的定量分析结果。基于已发表的弹尾目线粒体基因组，针对线粒体细胞色素c氧化酶亚基I（mtCOI）和16S核糖体RNA（mt16S）基因的保守区域设计了简并PCR引物。根据其扩增每个基因的能力（与物种无关）选择最佳引物对。从温带森林中采集的10个自然群落（每10个土壤样本中通常有25 - 30种弹尾目物种）和10个模拟群落（含有7种养殖弹尾目物种）中提取DNA。然后使用选定的引物扩增这两个基因区域，连接用于454技术的接头并进行测序。对自然群落样本的检测表明，从mtCOI和mt16S数据中分别恢复了32个和36个操作分类单元（定义在90%序列相似性阈值），这与25个形态物种的显微镜鉴定结果相当。此外，以一个物种作为内对照进行标准化后，模拟群落的mtCOI和mt16S数据中每个弹尾目物种的序列丰度与样本中的个体数量显示出良好的相关性（R = 0.91 - 0.99），尽管从序列估计的模拟群落样本中的相对物种丰度在物种个体数量或生物量方面与群落组成存在偏差。因此，该方案能够通过宏条形码分析以定量方式比较弹尾目群落。
