Data from: High-throughput molecular identification of fish eggs using multiplex suspension bead arrays

The location and abundance of fish eggs provide information concerning the timing and location of spawning activities and can provide fishery-independent estimates of spawning biomass. However, the full value of egg and larval surveys is severely restricted because many species' eggs and larvae are morphologically similar, making species-level identification difficult. Recent efforts have shown that nearly all species of fish may be identified by mitochondrial DNA (mtDNA) sequences (e.g., via "DNA barcoding"). By taking advantage of a DNA barcode database, we have developed oligonucleotide probes for 23 marine fish species that produce pelagic eggs commonly found in California waters. Probes were coupled to fluorescent microspheres to create a suspension bead array. Biotin-labeled primers were used to amplify the mitochondrial cytochrome oxidase subunit I (COI) and 16S ribosomal rRNA genes from individual fish eggs. The amplicons were then hybridized to the bead array and after addition of a reporter fluorophore, samples were analyzed by flow cytometry with Luminex 100 instrumentation. Probes specifically targeted eggs that are abundant and/or from morphologically indistinguishable species pairs. Results showed the 33 different probes designed for this study accurately identified all samples when PCR was successful. Suspension bead arrays have a number of benefits over other methods of molecular identification; these arrays permit high multiplexing, simple addition of new probes, high throughput, and lower cost than DNA sequencing. The increasing availability of DNA barcode data for numerous fish faunas worldwide suggests bead arrays could be developed and widely used for fish egg, larval and tissue identifications.鱼卵的分布位置与丰度可提供鱼类产卵活动的时间与地点信息，还能开展独立于渔业捕捞的产卵生物量估算。然而，由于诸多鱼类的卵与幼体形态相似，导致物种级别的鉴定难度极大，因此鱼卵和仔鱼调查的全部潜力受到严重限制。近期研究表明，几乎所有鱼类均可通过线粒体DNA（mtDNA）序列进行物种鉴定，例如通过“DNA条形码（DNA barcoding）”技术。本研究借助DNA条形码数据库，为加州海域常见的23种产浮性卵的海洋鱼类设计了寡核苷酸探针。将探针与荧光微球偶联，构建悬浮微球阵列（suspension bead array）。采用生物素标记的引物，从单尾鱼卵中扩增线粒体细胞色素氧化酶亚基I（COI）以及16S核糖体RNA基因。随后将扩增子与悬浮微球阵列进行杂交，加入报告荧光基团后，使用Luminex 100仪器通过流式细胞术对样本进行分析。探针特意针对丰度较高的鱼卵，以及形态上难以区分的物种种对的鱼卵。实验结果显示，当聚合酶链式反应（PCR）扩增成功时，本研究设计的33种探针可准确鉴定所有样本。相较于其他分子鉴定方法，悬浮微球阵列具备诸多优势：可实现高多重检测能力、易于添加新探针、通量高且成本低于DNA测序。全球范围内众多鱼类区系的DNA条形码数据日益丰富，这意味着悬浮微球阵列可被开发并广泛应用于鱼卵、仔鱼以及组织样本的物种鉴定。