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Environmental DNA metabarcoding primers for freshwater fish detection and quantification: In silico and in tanks

机译：用于淡水鱼检测和量化的环境DNA地区促进引物：在硅和罐中

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摘要

Environmental DNA (eDNA) techniques refer to utilizing the organisms’ DNA extracted from environment samples to genetically identify target species without capturing actual organisms. eDNA metabarcoding via high‐throughput sequencing can simultaneously detect multiple fish species from a single water sample, which is a powerful tool for the qualitative detection and quantitative estimates of multiple fish species. However, sequence counts obtained from eDNA metabarcoding may be influenced by many factors, of which primer bias is one of the foremost causes of methodological error. The performance of 18 primer pairs for COI, cytb, 12S rRNA, and 16S rRNA mitochondrial genes, which are all frequently used in fish eDNA metabarcoding, were evaluated in the current study. The ribosomal gene markers performed better than the protein‐coding gene markers during in silico screening, resulting in higher taxonomic coverage and appropriate barcode lengths. Four primer pairs—AcMDB07, MiFish‐U, Ve16S1, and Ve16S3—designed for various regions of the 12S and 16S rRNA genes were screened for tank metabarcoding in a case study targeting six freshwater fish species. The four primer pairs were able to accurately detect all six species in different tanks, while only MiFish‐U, Ve16S1, and Ve16S3 revealed a significant positive relationship between species biomass and read count for the pooled tank data. The positive relationship could not be found in all species within the tanks. Additionally, primer efficiency differed depending on the species while primer preferential species varied in different fish assemblages. This case study supports the potential for eDNA metabarcoding to assess species diversity in natural ecosystems and provides an alternative strategy to evaluate the performance of candidate primers before application of eDNA metabarcoding in natural ecosystems.
机译：环境DNA（EDNA）技术是指利用从环境样品中提取的生物体DNA，在不捕获实际生物的情况下从环境样本中提取到遗传识别靶物种。通过高通量测序的EDNA地区可以同时检测来自单个水样的多种鱼类，这是一种强大的多种鱼类定性检测和定量估计的强大工具。然而，从EDNA地区获得的序列计数可能受到许多因素的影响，其中引物偏压是方法论误差的最重要原因之一。在目前的研究中评估了COI，CYTB，12S rRNA和16S rRNA线粒体基因的18个引物对的性能，这些成对常常用于鱼EDNA地区的鱼EDNA地区。在硅筛选期间，核糖体基因标记物比蛋白质编码基因标志物更好，导致分类覆盖率更高，条形码长度。在靶向六种淡水鱼类的情况下，筛选用于12S和16S rRNA基因的各个区域的四个引物对 - ACMDB07，MIFISH-U，VE16S1和VE16S3设计。四个引物对能够精确地检测不同罐中的所有六种物种，而只有MIFISH-U，VE16S1和VE16S3揭示了物种生物质和汇集坦克数据的读数之间的显着阳性关系。在坦克内的所有物种中找不到正面关系。另外，引物效率根据物种不同，而引物优先种类在不同的鱼组合中变化。本案例研究支持EDNA地区的潜力评估自然生态系统中的物种多样性，并提供替代策略，以评估候选引物在自然生态系统中的edna地区施用之前的候选底漆的性能。

著录项

  • 期刊名称 Ecology and Evolution
  • 作者

    Lu Shu; Arne Ludwig; Zuogang Peng;

  • 作者单位
  • 年(卷),期 2021(11),12
  • 年度 2021
  • 页码 8281–8294
  • 总页数 14
  • 原文格式 PDF
  • 正文语种
  • 中图分类
  • 关键词

    机译：环境DNA;在Silico;Metabarcoding;底漆选择;坦克;

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