环境DNA(eDNA)宏条形码(Metabarcoding)技术越来越多地被应用于环境中物种定性识别,但如何定量监测物种在环境中的丰度尚未得到解决。本研究以太湖流域常见的5种浮游动物拟同形溞、大型溞、蚤状溞、多刺裸腹溞、老年低额溞为研究对象,...环境DNA(eDNA)宏条形码(Metabarcoding)技术越来越多地被应用于环境中物种定性识别,但如何定量监测物种在环境中的丰度尚未得到解决。本研究以太湖流域常见的5种浮游动物拟同形溞、大型溞、蚤状溞、多刺裸腹溞、老年低额溞为研究对象,建立了一种基于eDNA宏条形码技术的物种定量方法,并与实时荧光定量PCR(qPCR)相比较,研究了eDNA宏条形码技术多物种定量的准确性。结果表明,PCR引物对eDNA宏条形码的物种检测和定量影响显著。313 bp COI313引物对浮游动物物种覆盖度高,但是物种间DNA扩增的偏好性大,不适用于eDNA宏条形码定量检测。基于COI序列重新设计的短COI116引物能够同时检测出所有5个物种。荧光定量PCR(qPCR)物种拷贝数与物种相对占比呈正相关。eDNA宏条形码所检出每个物种的序列数与qPCR定量拷贝数高度一致。综上,eDNA宏条形码技术可实现对浮游动物物种的半定量检测,在生物多样性监测和生物完整性评价有显著的应用价值。展开更多
Environmental DNA(eDNA)integrated with metabarcoding is a promising and powerful tool for species composition and biodiversity assessment in aquatic ecosystems and is increasingly applied to evaluate fish diversity.To...Environmental DNA(eDNA)integrated with metabarcoding is a promising and powerful tool for species composition and biodiversity assessment in aquatic ecosystems and is increasingly applied to evaluate fish diversity.To date,however,no standardized eDNA-based protocol has been established to monitor fish diversity.In this study,we investigated and compared two filtration methods and three DNA extraction methods using three filtration water volumes to determine a suitable approach for eDNA-based fish diversity monitoring in the Pearl River Estuary(PRE),a highly anthropogenically disturbed estuarine ecosystem.Compared to filtration-based precipitation,direct filtration was a more suitable method for eDNA metabarcoding in the PRE.The combined use of DNeasy Blood and Tissue Kit(BT)and traditional phenol/chloroform(PC)extraction produced higher DNA yields,amplicon sequence variants(ASVs),and Shannon diversity indices,and generated more homogeneous and consistent community composition among replicates.Compared to the other combined protocols,the PC and BT methods obtained better species detection,higher fish diversity,and greater consistency for the filtration water volumes of 1000 and 2000 mL,respectively.All eDNA metabarcoding protocols were more sensitive than bottom trawling in the PRE fish surveys and combining two techniques yielded greater taxonomic diversity.Furthermore,combining traditional methods with eDNA analysis enhanced accuracy.These results indicate that methodological decisions related to eDNA metabarcoding should be made with caution for fish community monitoring in estuarine ecosystems.展开更多
文摘环境DNA(eDNA)宏条形码(Metabarcoding)技术越来越多地被应用于环境中物种定性识别,但如何定量监测物种在环境中的丰度尚未得到解决。本研究以太湖流域常见的5种浮游动物拟同形溞、大型溞、蚤状溞、多刺裸腹溞、老年低额溞为研究对象,建立了一种基于eDNA宏条形码技术的物种定量方法,并与实时荧光定量PCR(qPCR)相比较,研究了eDNA宏条形码技术多物种定量的准确性。结果表明,PCR引物对eDNA宏条形码的物种检测和定量影响显著。313 bp COI313引物对浮游动物物种覆盖度高,但是物种间DNA扩增的偏好性大,不适用于eDNA宏条形码定量检测。基于COI序列重新设计的短COI116引物能够同时检测出所有5个物种。荧光定量PCR(qPCR)物种拷贝数与物种相对占比呈正相关。eDNA宏条形码所检出每个物种的序列数与qPCR定量拷贝数高度一致。综上,eDNA宏条形码技术可实现对浮游动物物种的半定量检测,在生物多样性监测和生物完整性评价有显著的应用价值。
基金supported by the National Natural Science Foundation of China(32102793)National Key R&D Program of China(2018YFD0900802)+4 种基金Central Public-Interest Scientific Institution Basal Research FundSouth China Sea Fisheries Research Institute,CAFS(2019TS13,2021SD18)Key Special Project for Introduced Talents Team of Southern Marine Science and Engineering Guangdong Laboratory(Guangzhou)(GML2019ZD0605)Open Fund Project of Key Laboratory of Offshore Fishery Development of Ministry of Agriculture and Rural Affairs(LOF 2020-02)China-ASEAN Maritime Cooperation Fund(CAMC-2018F)。
文摘Environmental DNA(eDNA)integrated with metabarcoding is a promising and powerful tool for species composition and biodiversity assessment in aquatic ecosystems and is increasingly applied to evaluate fish diversity.To date,however,no standardized eDNA-based protocol has been established to monitor fish diversity.In this study,we investigated and compared two filtration methods and three DNA extraction methods using three filtration water volumes to determine a suitable approach for eDNA-based fish diversity monitoring in the Pearl River Estuary(PRE),a highly anthropogenically disturbed estuarine ecosystem.Compared to filtration-based precipitation,direct filtration was a more suitable method for eDNA metabarcoding in the PRE.The combined use of DNeasy Blood and Tissue Kit(BT)and traditional phenol/chloroform(PC)extraction produced higher DNA yields,amplicon sequence variants(ASVs),and Shannon diversity indices,and generated more homogeneous and consistent community composition among replicates.Compared to the other combined protocols,the PC and BT methods obtained better species detection,higher fish diversity,and greater consistency for the filtration water volumes of 1000 and 2000 mL,respectively.All eDNA metabarcoding protocols were more sensitive than bottom trawling in the PRE fish surveys and combining two techniques yielded greater taxonomic diversity.Furthermore,combining traditional methods with eDNA analysis enhanced accuracy.These results indicate that methodological decisions related to eDNA metabarcoding should be made with caution for fish community monitoring in estuarine ecosystems.
