Development of a DNA mini-barcoding protocol targeting COI for the identification of elasmobranch species in shark cartilage pills

Abstract

Many elasmobranch (shark and ray) species are considered threatened and their identification in processed products is important for conservation and authentication purposes. However, identification of elasmobranch species in shark cartilage pills has proven difficult using existing methodologies. The objective of this study was to develop a DNA mini-barcoding protocol using a ~130 bp region of the cytochrome c oxidase subunit I (COI) gene for species identification in shark cartilage pills. A total of 22 shark cartilage products underwent DNA extraction in duplicate using the DNeasy Blood and Tissue Kit (Qiagen). The effectiveness of a clean-up step following DNA extraction was analyzed by comparing DNA purity values and polymerase chain reaction (PCR) amplification rates. Next, five different mini-barcode primer sets were compared based on amplification rates, and the three top-performing primer sets were used in DNA sequencing. The incorporation of a clean-up step following DNA extraction showed a slight advantage over DNA extraction alone, with a higher amplification rate (52.3% vs. 47.8%) and A260/A230 value (3.3 vs. 0.6). The three primer sets selected for DNA minibarcoding showed DNA sequencing rates of 54.5-65.9% among the 44 duplicate samples. When the results for all three primer sets were combined, 18 of the 22 shark cartilage products were identified to the species or genus level. On an individual basis, the best-performing primer set identified 16 of the 22 products to the species or genus level. Overall, the protocol developed in this study increased the identification rate for elasmobranches in cartilage products by more than 2-fold as compared to previous research.

Advancing Species Identification in Traditional Chinese Medicine: A Novel Mini-Barcode from Mitochondrial Genomes Combined with Meta-barcoding Technology for Leech Authentication

“…However, high-resolution mini-barcode is not easily found. Most researchers choose to develop mini-barcodes from the COI gene [42][43][44] , but considering some sequences were incorrectly taxonomically classi ed in the COI gene database 45 , we have chosen to develop mini-barcodes from the mitochondrial genome. Ultimately, we developed the mini-barcode from the 16S rRNA gene.…”

Efficiency of eDNA and iDNA in assessing vertebrate diversity and its abundance

“…Although COI presents the most extensive reference database for barcode studies (Hebert, Ratnasingham, & de Waard, 2003), there is a challenge in the development of primers that amplify a small fragment capable of identifying a high proportion of taxa (Deagle et al, 2014), avoiding COI primer mismatch (Kocher, de Thoisy, Catzeflis, Huguin, et al, 2017). Mini-COI sequences are usually developed to identify species within vertebrate groups, as recently developed to leporids (Rodrigues et al, 2020) and elasmobranchs (Zahn, Silva, & Hellberg, 2020). Nevertheless, a substantial amplification bias towards non-metazoan taxa has been identified with COI, which can hinder the recovery of all vertebrate taxa in an environmental sample (Harper et al, 2019).…”

Environmental DNA (eDNA) Metabarcoding in the Fish Market and Nearby Seafood Restaurants in Taiwan Reveals the Underestimation of Fish Species Diversity in Seafood

“…Amplified gene fragments were prepared for NovaSeq 6000 sequencing according to the Illumina Sequencing Library preparation guidelines (Illumina, Inc., San Diego, Califor-nia, USA). Three mitochondrial gene regions were amplified by 19 universal primers, including: 12S rRNA (MiFish-U/E), ND5 (MiFish-tuna-ND5), and COI (Fish-miniA/C/E; Fish1/2; Fish1F/2F with Shark COI-MINIR, Shark-MiniV1-R, and Shark-MiniV2-R) as described [ 25 , 26 , 27 , 28 ] with modification ( Table 1 ).…”