Fisheries Laboratory, Graduate School of Agricultural and Life Sciences, University of Tokyo, Hamamatsu, Japan.
Department of Applied Aquabiology, National Fisheries University, Shimonoseki, Japan.
Mol Ecol Resour. 2019 Sep;19(5):1153-1163. doi: 10.1111/1755-0998.13025. Epub 2019 May 29.
While various technologies for high-throughput genotyping have been developed for ecological studies, simple methods tolerant to low-quality DNA samples are still limited. In this study, we tested the availability of a random PCR-based genotyping-by-sequencing technology, genotyping by random amplicon sequencing, direct (GRAS-Di). We focused on population genetic analysis of estuarine mangrove fishes, including two resident species, the Amboina cardinalfish (Fibramia amboinensis, Bleeker, 1853) and the Duncker's river garfish (Zenarchopterus dunckeri, Mohr, 1926), and a marine migrant, the blacktail snapper (Lutjanus fulvus, Forster, 1801). Collections were from the Ryukyu Islands, southern Japan. PCR amplicons derived from ~130 individuals were pooled and sequenced in a single lane on a HiSeq2500 platform, and an average of three million reads was obtained per individual. Consensus contigs were assembled for each species and used for genotyping of single nucleotide polymorphisms by mapping trimmed reads onto the contigs. After quality filtering steps, 4,000-9,000 putative single nucleotide polymorphisms were detected for each species. Although DNA fragmentation can diminish genotyping performance when analysed on next-generation sequencing technology, the effect was small. Genetic differentiation and a clear pattern of isolation-by-distance was observed in F. amboinensis and Z. dunckeri by means of principal component analysis, F and the admixture analysis. By contrast, L. fulvus comprised a genetically homogeneous population with directional recent gene flow. These genetic differentiation patterns reflect patterns of estuary use through life history. These results showed the power of GRAS-Di for fine-grained genetic analysis using field samples, including mangrove fishes.
虽然已经开发出各种高通量基因分型技术用于生态研究，但仍缺乏简单的方法来耐受低质量的 DNA 样本。在本研究中，我们测试了一种基于随机 PCR 的基因分型测序技术，即随机扩增子测序直接法（GRAS-Di）的可用性。我们专注于河口红树林鱼类的种群遗传分析，包括两种定居物种，即 ambonina cardinalfish（Fibramia amboinensis，Bleeker，1853）和 Duncker's 河鲱（Zenarchopterus dunckeri，Mohr，1926），以及一种海洋洄游物种，黑尾笛鲷（Lutjanus fulvus，Forster，1801）。采集地点是日本琉球群岛。从约 130 个个体中提取 PCR 扩增子，在 HiSeq2500 平台的单个泳道中进行测序，每个个体平均获得 300 万个reads。为每个物种组装共识 contigs，并通过将修剪后的 reads 映射到 contigs 上来对单核苷酸多态性进行基因分型。经过质量过滤步骤，每个物种检测到 4000-9000 个可能的单核苷酸多态性。尽管 DNA 片段化会降低下一代测序技术分析时的基因分型性能，但影响较小。通过主成分分析、F 统计量和混合分析，在 F.amboinensis 和 Z.dunckeri 中观察到遗传分化和明显的距离隔离模式。相比之下，L.fulvus 由具有定向近期基因流的遗传同质种群组成。这些遗传分化模式反映了通过生活史对河口的利用模式。这些结果表明，GRAS-Di 具有使用野外样本进行细粒度遗传分析的能力，包括红树林鱼类。
