Bioscience Methods 2025, Vol.16, No.3, 137-153 http://bioscipublisher.com/index.php/bm 144 effects of isolation and gene flow on the genetic structure of abalone can be revealed. At the species level, abalones in different geographical regions show obvious genetic differentiation. For example, the mitochondrial sequences of the Halion discus off the coast of China and the red abalone in California are very different, corresponding to the long-term geographical isolation on both sides of the Pacific Ocean (Cook, 2025). Even within the region, geographical barriers can lead to genetic differentiation of populations. Taking the cultured population of Haliotis discus in China as an example, there is a significant difference in the mitochondrial haplotype frequency between the Dalian population in the north and the Fujian population in the south, and the FST value indicates that the two populations have a certain degree of genetic separation. This may be due to the long distance along the coast of China and the separation of cold and warm currents. For example, the Taiwan Strait was once a land bridge during the glacial period, and the abalone populations on different sides may have experienced isolation in history, thereby accumulating genetic differences. Mitochondrial DNA sequence analysis also revealed some interesting phenomena, such as genetic mixing between introduced populations and local populations. China introducedHaliotis discus seedlings from Japan for culture at the end of the 20th century. After many generations of reproduction, the mitochondrial genetic diversity of the current coastal cultured population is slightly lower than that of the original Japanese population, but it remains highly similar overall. This indicates that the initial maternal genetic contribution of the introduced population was dominant and did not hybridize with the local abalone. However, some studies have shown that natural hybridization between the introduced Haliotis discus and the local abalone discus was detected in some areas, and the mitochondria of their offspring were the same as those of the Haliotis discus mother, but the nuclear DNA showed recombinant characteristics (Yang et al., 2023). This suggests that we need to conduct a joint analysis of mitochondrial and nuclear genes to deeply evaluate the impact of introduction on the genetic pattern of local abalone. In general, the geographical pattern of mitochondrial variation supports isolation-induced differences: distant populations differ greatly, and adjacent populations are more similar. For example, the mitochondrial haplotype network of the abalone populations in East Asia is star-shaped, with the central type shared in all places, while the marginal type is regionally specific (Li et al., 2021). This shows that although there is a certain gene flow among populations in various places, local genetic differentiation has still occurred in the long run. Looking at the species level, the black abalone and the wrinkled abalone in Japan basically have no mitochondrial haplotype sharing in the cross-distribution area, suggesting that their reproductive isolation is effective (Hsu and Gwo, 2017). On the contrary, the Taiwan Jiukong and the mainland abalone share most haplotypes, indicating that gene exchange may have occurred in history through the cross-strait transport of planktonic larvae. The Mantel test found that the mitochondrial genetic distance between abalone populations is often positively correlated with the geographical distance, but the degree of influence varies from species to species. The longer the geographical isolation time and the greater the distance, the greater the difference in abalone mitochondria, which is consistent with the isolation-induced differentiation pattern in the marine environment. At the same time, ocean currents and human activities can also break the simple distance-gene relationship. For example, the artificial introduction of Japanese wrinkled abalone has made the East China population and the native Japanese population very different (Guo et al., 2019a). Therefore, when analyzing the geographical association of mitochondrial variation, it is necessary to comprehensively consider marine geographical barriers, ocean current paths, biological life history and human factors. 5 Development of Mitochondrial Markers for Species Identification 5.1 Screening and validation of core diagnostic genes Molecular identification of abalone species usually relies on differences in mitochondrial gene sequences, of which the most commonly used are mitochondrial encoded genes, such as the COI gene (i.e., animal DNA barcode standard fragments). To ensure the accuracy and efficiency of identification, it is necessary to screen out core marker genes that are sensitive to species differences and easy to amplify and sequence. A large number of studies have shown that COI gene fragments have a high success rate in abalone species identification, with species identification efficiency reaching more than 95%. For example, by determining the abalone COI sequence, researchers found that almost all abalone species have a "barcode gap" on this gene, that is, the genetic distance
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