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

## TL;DR

This study improves molecular tools for identifying ascidians, a group of marine organisms that are hard to distinguish and often invasive.

## Contribution

The study introduces and validates a new primer pair, AscCOI2, specifically optimized for ascidian biodiversity assessment.

## Key findings

- The AscCOI2 primers showed an 82.42% amplification success rate at the species level, a significant improvement over previous methods.
- The redesigned primers maintained high specificity for ascidians while failing to amplify non-ascidian taxa.
- A clear barcoding gap of 0.015 was observed between intraspecific and interspecific genetic distances.

## Abstract

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 in silico and in vitro 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. In vitro 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.