环境DNA(eDNA)宏条形码技术对枝角类浮游动物物种鉴定及其生物量监测研究

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。

Identification and Biomass Monitoring of Zooplankton Cladocera Species with eDNA Metabarcoding Technology

Environmental DNA (eDNA) metabarcoding technology has been increasingly applied to qualitative identification of species. However, how to quantitatively monitor different species in the environment has not yet been solved. Here 5 common zooplankton species in the Lake Tai basin, including Daphnia magna, Daphnia similoides, Daphnia pulex, Moina macrocopa, Simocephalus vetulus, were used to develop a quantitative monitoring protocol. The accuracy of multi-species eDNA quantification of eDNA metabarcoding of two primer sets were validated by Real-time quantitative PCR (qPCR) methods. PCR primers significantly affected the species detection and quantification by eDNA metabarcoding. Although the COI³¹³ primer has high coverage of zooplankton species, it is not suitable for eDNA metabarcoding due to the bias of DNA amplification among species. The COI¹¹⁶ primer designed in this research was able to detect all 5 species simultaneously. The DNA copy numbers and count numbers of each species were positively correlated in the qPCR quantification. In eDNA metabarcoding, the reads number of each species increased with its relative proportion of morphological abundance, which was consistent with the results of qPCR. Therefore, eDNA metabarcoding provides a semi-quantitative detection of zooplankton with suitable primers, which is valuable in the monitoring of zooplankton biodiversity and biological assessment.