Bioscience Methods 2025, Vol.16, No.3, 137-153 http://bioscipublisher.com/index.php/bm 145 within the species is significantly smaller than the distance between species. Ran et al. (2020) also confirmed that the COI barcode can correctly classify about 98% of the samples into species in a study of 11 species of shellfish (including abalone) along the coast of China. Therefore, COI has been widely regarded as the preferred gene for abalone species identification. However, in some cases a single COI fragment may not be sufficient to solve all problems. For example, for closely related species pairs (such as Taiwan's nine-hole abalone and mainland abalone), the COI sequence difference is very small (about 1%), and identification ambiguity may occur. For this reason, it is possible to consider introducing other mitochondrial gene fragments as an auxiliary means. The 16S rRNA gene is a common choice. Its evolutionary rate is slightly slower, but the secondary structure region is obviously different, and it has good discrimination for some closely related species. In addition, the cytochrome b (cyt b) gene is often used in fish classification and also shows high interspecific variation in abalone, making it suitable as a supplementary marker. In order to screen the best species identification gene combination, researchers usually evaluate the interspecific genetic distance and intraspecific variation level of different genes, as well as the convenience of amplification and sequencing success rate. The results often show that the combination of COI plus 16S can significantly improve the reliability of identification, and the credibility is extremely high when the identification conclusions of the two are consistent (Kannan et al., 2020). When the two are inconsistent or in doubt, a third marker (such as ND1 or cyt b) can be added for verification. In addition to traditional sequencing methods, some studies have developed species-specific PCR identification techniques based on core mitochondrial genes. For example, specific primers for Haliotis rubripes, Haliotis diversicolor, Haliotis ocellaris, etc. are designed, and the sizes of amplified products of different species are distinguished by multiplex PCR, so as to quickly identify the species components in mixed samples. There are also studies using high-fidelity probe qPCR methods to detect specific abalone DNA in processed aquatic products and distinguish Haliotis rubripes from other abalone raw materials (patent CN109680077B). The selection of core mitochondrial genes needs to take into account both discrimination and practicality. At present, COI is undoubtedly the first choice, and combining other markers can improve the accuracy in tricky situations. With the popularization of abalone mitochondrial genome sequencing, more highly variable markers can be discovered based on whole genome information in the future, such as specific fragments of the control region or species-specific SNPs in the nuclear genome, but these need to be verified by a large number of samples in practical applications. At this stage, the species identification strategy developed around mitochondrial core genes can meet most scientific research and law enforcement needs. 5.2 DNA barcoding applications in abalone taxonomy DNA barcoding technology refers to a method of identifying species using standard short gene sequences. Since Hebert et al. proposed using mitochondrial COI fragments as universal animal barcodes, this technology has been widely used in species diversity research and monitoring of fish, mollusks, etc. In abalone, DNA barcoding technology has shown great application potential. First, it provides an objective and quantifiable standard for species identification. By establishing an abalone barcode sequence library, any sample of unknown origin (whether it is larvae, fragments or even processed products) can be sequenced to determine the species identity. For example, for imported dried abalone and other aquatic trade products, barcode sequence comparison can be used to identify their species and reveal whether there is a phenomenon of cheap abalone impersonating precious abalone, thereby safeguarding consumer rights (Senathipathi et al., 2024). Secondly, DNA barcoding technology is also used in the discovery of cryptic abalone species. Some abalone that were previously believed to belong to the same species based on morphology may show obvious differentiation after barcode analysis, suggesting the existence of new species or evolutionary lineages that have not been recognized. Taiwan's nine-hole abalone and some Indonesian abalone are identified as different evolutionary groups through COI barcode differences (>10%) (Hsu and Gwo, 2017). Thirdly, barcode technology can assist in the management of abalone seedlings and cultured strains. Since the morphology of different species of abalone seedlings is extremely similar in the larval stage, DNA barcodes can be used to quickly screen the composition of larvae in the nursery pond to ensure the purity of the introduced species or seedlings. In genetic breeding, barcodes can also be used to verify the maternal species of hybrid offspring to track breeding pedigrees. It is worth mentioning that with the development of high-throughput sequencing, DNA barcoding technology has derived a variety of new forms, such as
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