Validation of improved cytochrome c oxidase I (COI) primers for comprehensive biodiversity assessment of ascidians

Reliable molecular tools are needed for effective biodiversity assessment of marine organisms. These tools can be used in ascidians species, which are one of the most invasive taxa worldwide and morphologically difficult to identify. This study aimed to redesign and improve ascidian-specific primers and validate the new primer pair (AscCOI2) for comprehensive biodiversity assessment. To design an optimized primer, 3,948 COI sequences from 273 ascidian species were used as a dataset. The AscCOI2 pair was developed through strategic modifications to the binding site and validated using <i>in silico</i> and <i>in vitro</i> approaches. Analysis of penalty scores showed the improved efficiency of the redesigned AscCOI2 pair, with ascidians scoring less than 150 points for both forward and reverse primers and the non-target groups maintaining a score above 480. Primer-binding analysis results showed a significant improvement in amplification success rate from 47.99% to 82.42% at the species level. <i>In vitro</i> validation using conventional polymerase chain reaction (PCR) confirmed the successful amplification of six ascidian species and failure for a non- ascidian taxon. Barcoding gap analysis showed a clear gap of 0.015 between intraspecific and interspecific genetic distances. The species detection capability of the redesigned AscCOI2 pair greatly improved, and the high taxonomic specificity of ascidians is maintained. Overall, this study demonstrates that the AscCOI2 pair is an effective tool for both metabarcoding and mini-barcode applications in biodiversity assessment and molecular systematics research of ascidians.