| 摘要: |
| 基于环境DNA（eDNA）技术的鱼类多样性监测方法因具有灵敏度高和对目标生物无伤害且成本低等特点，近年来在国内外得到了广泛应用。eDNA方法的有效性往往取决于宏条形码引物的选择，尽管目前已经有了一些鱼类环境DNA宏条形码引物，但较少有研究综合评估这些引物的检出效果。本研究评估了来自COI、Cytb、12S rRNA和16S rRNA基因的29对鱼类eDNA宏条形码引物（包括本研究设计的2对和从国内外文献中引用的27对引物），首先基于计算机模拟PCR（in silico PCR）进行了初步的分析，随后通过高通量测序对其中效果较好的17对引物开展了更进一步的验证，结果显示：计算机模拟PCR结果良好的引物在进行高通量测序时并非全部表现良好，表明引物的筛选不能仅仅依靠计算机模拟PCR；具有更长扩增片段长度的宏条形码引物并没有获得理想中更好的扩增效果，表现效果好的引物大多是扩增片段长度处于200~300 bp之间的引物；相对于COI和Cytb而言，12S rRNA引物与16S rRNA引物均具有良好的扩增效果，适宜于作为鱼类环境DNA宏条形码而用于鱼类多样性研究；同时，鉴于当前的鱼类DNA条形码数据库尚不完备以及不同引物的特性不同，使用多对引物将大大增加物种的检出概率和eDNA研究的可信性。本研究展示了eDNA在评估生物多样性方面的潜力，有助于将来的鱼类eDNA研究，从而为鱼类多样性的保护提供参考。 |
| 关键词: eDNA DNA宏条形码 生物多样性 计算机模拟PCR 引物 |
| DOI： |
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| 基金项目:国家重点研发计划重点专项 |
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| Screening of primers for environmental DNA metabarcoding of freshwater fish and its application in Lake Qiandaohu |
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Zhou Yan1, Tong Lu1, Hu Wenjing1, Li Zhili1, Hao Lei1, Hu Zhongjun1, Liu Qigen2
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1.Shanghai Ocean University;2.上海海洋大学
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| Abstract: |
| Environmental DNA (eDNA)-based methods for monitoring fish diversity have been widely used in recent years at home and abroad because of its high sensitivity, harmlessness to target organisms and low cost. The effectiveness of eDNA methods often depends on the selection of macrobarcode primers, and although a number of fish environmental DNA macrobarcode primers are available, fewer studies have comprehensively evaluated the detection effectiveness of these primers. In this study, 29 pairs of fish eDNA metabarcoding primers from COI, Cytb, 12S rRNA and 16S rRNA genes (including 2 pairs designed in this study and 27 pairs cited from national and international literature) were evaluated. A preliminary analysis was performed in silico PCR, followed by further validation of the 17 primer pairs that worked well by high-throughput sequencing. The results showed that not all the primers with good results in silico PCR performed well in high-throughput sequencing, indicating that the selection of primers cannot rely on silico PCR alone; the metabarcoding primers with longer amplification fragment length did not obtain better amplification results, and most of the primers with good performance were in the 200~300 bp fragment length range. In contrast to COI and Cytb, 12S rRNA primers and 16S rRNA primers have good amplification effects and are suitable for use as environmental DNA metabarcoding for fish diversity studies; at the same time, the use of multiple primer pairs will greatly increase the detection probability of species and the credibility of eDNA studies, given the incompleteness of the current fish DNA barcode database and the different characteristics of different primers. This study demonstrates the potential of eDNA in assessing biodiversity, which will help future eDNA studies of fish and thus provide a reference for the conservation of fish diversity. |
| Key words: eDNA DNA metabarcoding biodiversity in silico PCR primers |
