Page 176 - 《水产学报》2025年第11期

杨锦毅，等 水产学报, 2025, 49(11): 119314

Species identification rates of commonly used Environmental DNA primers
for Yangtze River fishes

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YANG Jinyi 1,2,3 , YAN Mingying , GAO Lei , DUAN Xinbin , LIU Mingdian ,
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CHEN Daqing , WANG Dengqiang 1*
1. Fishery Resources and Environmental Science Experimental Station of the Upper-middle Reaches of Yangtze River,
Ministry of Agriculture and Rural Affairs, National Agricultural Science Observing and Experimental Station of Chongqing,
Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Science, Wuhan 430233, China;
2. National Experimental Teaching Demonstration Center of Aquatic Science,
Shanghai Ocean University, Shanghai 201306, China;
3. Xiaogan Academy of Agricultural Sciences, Xiaogan 432000, China

Abstract: Environmental DNA (eDNA) metabarcoding has emerged as a powerful approach for assessing aquatic biodiversity;
however, it remains limited by uncertainties in OTU-clustering thresholds and taxonomic assignment. The Yangtze River,
China’s longest river system, harbors over 4 300 aquatic species, with fish constituting a dominant group in freshwater ecosys-
tems. Due to their sensitivity to water quality, fish serve as reliable ecological indicators for monitoring environmental change.
To improve the reference value of eDNA in studying Yangtze River fish diversity, DNA sequences of 320 species were collec-
ted from Mitofish and NCBI databases. Five commonly used eDNA fragments (12S-AcDBM07, 12S-Mifish-U, 12S-Tele02,
16S-Ac16s, COI-PS1) were extracted from three genes (12S RNA, 16S RNA, COI) for genetic distance calculations under
thresholds of 0.03, 0.02, and 0.01. This aimed to evaluate identification efficiency and explore applications in fish diversity
studies. Results show that at thresholds of 0.03, 0.02, and 0.01, 137, 108, and 56 fish species could not be identified using the
five fragments. Among them, the protein-coding gene COI-PS1 exhibited the highest identification rate, reaching 53.44%,
60.63%, and 72.50% respectively. Among the five fragments, 12S-Tele02 with 12S-Mifish-U had the greatest overlap in identi-
fied species, with 98.54%, 100%, and 91.11% consistency across thresholds. When selecting fragment combinations, “12S-
Tele02” plus “COI-PS1” identified the most species under all thresholds (180, 206, and 257 species), differing only by 3, 6, and
7 species from the results of using all five fragments. In summary, this study highlights that the relationship between interspe-
cies difference thresholds and species identification must be carefully considered in applying eDNA for Yangtze River fish
diversity, to avoid false positives in OTU annotations. For species that are hard to distinguish but of high conservation value,
targeted primer design can enhance monitoring efficiency. Moreover, combining eDNA technology with traditional netting can
provide a more comprehensive assessment of local aquatic biodiversity.
Key words: fishes; environmental DNA; metabarcoding; interspecies difference threshold; Yangtze River
Corresponding author: WANG Dengqiang. E-mail: wdq@yfi.ac.cn
Funding projects: National Key R & D Program of China (2022YFC3202001); National Natural Science Foundation of China,
Ministry of Water Resources of the People's Republic of China and China Three Gorges Corporation-Yangtze River Water Sci-
ence Research Joint Fund Project (U2240214); Central Public-interest Scientific Institution Basal Research Fund, CAFS
(2023TD09)

https://www.china-fishery.cn 中国水产学会主办 sponsored by China Society of Fisheries
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