Abstract
Environmental DNA (eDNA) is a promising biomonitoring tool for marine ecosystems, but its effectiveness for North Pacific coastal fishes is limited by the inability of existing barcoding primers to differentiate among rockfishes in the genus Sebastes. Comprised of 110 commercially and ecologically important species, this recent radiation is exceptionally speciose, and exhibits high sequence similarity among species at standard barcoding loci. Here, we report new Sebastes-specific metabarcoding primers that target mitochondrial cytochrome B. Amongst the 110 Sebastes species, 85 unique barcodes (of which 62 are species-specific) were identified in our amplicon region based on available reference sequences. The majority of the remaining barcodes are shared by only two species. Importantly, MiSebastes yield unique barcodes for 28 of 44 commercially harvested species in California, a dramatic improvement compared to the widely employed MiFish-U 12 S primers which only recover one of 44. Tests of these primers in an aquarium mesocosm containing 16 rockfish species confirms the utility of these new primers for eDNA metabarcoding, providing an important biomonitoring tool for these key coastal marine fishes.
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Data Availability
Sanger sequence Cytochrome B barcode data generated for this study are publicly available on NCBI GenBank, bioproject number PRJNA733285. The MiFish-U and MiSebastes sequencing datasets are available on Dryad (https://doi.org/10.5068/D1J951).
Code availability
Code used for primer design and subsequent analyses are publicly available here: https://github.com/markusmin/MiSebastes.
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Acknowledgements
We thank Emily Curd and Meixi Lin for their assistance with the primer design process. We thank Andrew Thompson and Dovi Kacev for providing us with DNA extracted from tissue samples. We thank Adam Wall for his help processing our Sanger sequencing data. We thank Keira Monuki, McKenzie Koch, Beverly Shih, Cristopher Ruano, and Nikita Sridhar for their assistance with sample preparation in the lab.
Funding
Grants to MAM from the UCLA Undergraduate Research Scholars Program, to ZJG from the US-NSF Graduate Research Fellowship (DEG No. 1650604), and to PHB from the Howard Hughes Medical Institute Science Education Program supported this study.
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Contributions include: Conceptualization MAM, PHB, ZJG. Investigation MAM, ZJG. Funding Acquisition MAM, PHB, ZJG. Data Curation MAM, ZJG. Formal Analysis MAM, ZJG. Writing – Original Draft Preparation MAM, PHB, ZJG.
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12686_2021_1219_MOESM1_ESM.eps
Electronic supplementary material 1 Fig. 1 Detections of the 11 species identified by the MiSebastes primers by individual technical replicates from the kelp forest aquarium at the California Science Center. Samples are divided into “bottom” and “surface” samples. Biological replicates are indicated by the numbers in the sample names; technical replicates are indicated by the lettering following the biological replicate numbers (EPS 18 kb)
12686_2021_1219_MOESM2_ESM.xlsx
Electronic supplementary material 2 Table 1 DNA sequences for Sanger sequencing of tissue samples barcoded with the MiSebastes primer set, along with the following BLAST results: top three hits for each sequence, percent identity, query cover, and accession number of the matching sequences (XLSX 16 kb)
12686_2021_1219_MOESM3_ESM.xlsx
Electronic supplementary material 3 Supplementary Table 2 Amplicons for the region targeted by the MiSebastes primer set as predicted by in silico PCR results from ecoPCR. For each amplicon, the species that share the amplicon, the region where the species are found, and the number of mismatches in the priming regions are listed (XLSX 17 kb)
12686_2021_1219_MOESM4_ESM.xlsx
Electronic supplementary material 4 Supplementary Table 3 Inventory of the species in the kelp forest aquarium at the California Science Center, along with detections by MiFish-U (using both the Fish CARD and CRUX databases) and the MiSebastes primer sets (XLSX 17 kb)
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Electronic supplementary material 5 Supplementary Table 4 List of all samples in each of the two sequencing libraries in this study (one each for MiSebastes and MiFish-U), along with associated metadata (XLSX 15 kb)
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Min, M.A., Barber, P.H. & Gold, Z. MiSebastes: An eDNA metabarcoding primer set for rockfishes (genus Sebastes). Conservation Genet Resour 13, 447–456 (2021). https://doi.org/10.1007/s12686-021-01219-2
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DOI: https://doi.org/10.1007/s12686-021-01219-2