Development of a new primer cocktail for improved DNA barcoding of brachyuran crabs (Decapoda: Brachyura)

Abstract DNA barcoding has revolutionized species identification and molecular phylogeny, offering immense potential for elucidating the diversity of brachyuran crabs. We explored the utility of a new primer cocktail-based approach for amplifying DNA barcode regions of mitochondrial cytochrome c oxidase I (CO1) from 28 specimens representing brachyuran species across 21 major families collected along the Korean coast. The primer cocktail, comprising three forward and two reverse degenerate primers with M13 adapter tails, exhibited superior PCR amplification efficiency, being 100% successful. Sequencing of the PCR products yielded high-quality, 726 bp, DNA barcode sequences, facilitating accurate species identification with 23 out of 28 specimens matching precisely with their morphological taxonomy. Barcoding gap analysis revealed a clear gap between mean intra- and interspecific K2P distances, with a 148-fold difference, confirming the reliability of CO1 barcodes for achieving 100% success in species identification within the dataset. The study highlights the effectiveness and reliability of the new primer cocktail as a robust molecular marker for DNA barcoding of brachyuran crabs, essential for ecological investigations and conservation endeavors, and elucidating the taxonomic complexities among them.