Bioscience Methods 2025, Vol.16, No.3, 137-153 http://bioscipublisher.com/index.php/bm 150 7 Concluding Remarks The mitochondrial genome of the abalone genus is highly conserved and highly variable, making it an ideal molecular tool for species identification. It is precisely because the abalone mitochondrial genome retains a stable genetic composition and arrangement among species that we can use common molecular methods to obtain the corresponding sequences of different species and conduct reliable comparative analysis. At the same time, the sequence differences accumulated by different species (especially the differences in gene fragments such as COI and 16S) are sufficient to be used as species "barcodes" for identification. In the past decade, a large number of studies have demonstrated the accuracy and practicality of abalone species identification methods based on mitochondrial genes. For example, for young abalone or processed abalone products with extremely similar morphology, traditional methods cannot determine their species, while mitochondrial DNA sequence comparison can give a clear species conclusion. The evolutionary characteristics of the abalone mitochondrial genome, such as the combination of rapidly evolving control regions and highly conserved coding genes, provide the possibility of multi-level identification: sensitive distinction between species/populations can be achieved through highly variable regions, and the stability of comparisons and the consistency of traceability can be ensured by using conservative regions.The maternal monophyletic nature of abalone mitochondrial inheritance simplifies genetic interpretation and avoids the complexity brought about by nuclear gene recombination. This means that in practical applications, the use of mitochondrial markers can often directly and clearly reveal the sample's ownership. For example, if the mitochondrial haplotype of Haliotis discus is detected, it can be determined that its maternal species is Haliotis discus. The evolutionary pattern of the abalone mitochondrial genome provides valuable value for species identification: its degree of difference is moderate, and it can be stably distinguished at the species level; its molecular characteristics are clear, which facilitates the development of diverse detection methods. It can be foreseen that as more abalone species' mitochondrial genomes are sequenced and reference databases are improved, molecular identification will play an increasingly important role in abalone classification and resource management. Although the mitochondrial genome has outstanding advantages in the systematic classification and identification of abalone, various molecular methods also have their own limitations, which need to be fully understood. The main advantages of mitochondrial DNA markers are: high mutation rate, strong resolution, simple experimental operation and low cost, which are suitable for large-scale sample screening. In particular, the popularity of COI barcodes has enabled non-professional laboratories to easily carry out species identification. In addition, as a maternal genetic marker, the mitochondrial genome plays a unique role in tracing the maternal origin of hybrid offspring. For example, we can confirm the maternal component of abalone hybrid populations through mitochondrial haplotypes. In contrast, nuclear DNA markers are more complicated and need to consider parental inheritance and allele separation. They are often not as intuitive as mitochondria for interspecies identification. However, the mitochondrial method also has inherent limitations. First, mitochondrial DNA represents only the maternal genetic history and cannot reflect hybridization or paternal gene introgression. Once interspecies hybridization occurs, the offspring will carry the maternal mitochondria, so that the identification results may not match the morphological or nuclear gene information. Second, the mitochondrial genome sometimes has the problem of incomplete pedigree sorting between closely related species, which may lead to inconsistency between the single gene tree and the actual species tree (i.e., "DNA pedigree is inconsistent with species pedigree"). Although this situation is rare in abalone, the molecular tree results still need to be interpreted with caution. Third, there is the possibility of mitochondrial pseudogenes inserted into the nuclear genome (NUMTs), which will interfere with PCR and sequencing results and need to be avoided through primer design and analysis. Finally, different molecular methods have their own preferences: sequencing is accurate but time-consuming, suitable for laboratory identification; rapid PCR is convenient but requires pre-designed specific primers and probes, and is usually targeted at limited species. New technologies such as SNP chips and high-throughput sequencing can provide massive data, but the cost is high and the data analysis is complex, and they are generally used for research rather than daily identification.
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