Bioscience Methods 2025, Vol.16, No.3, 137-153 http://bioscipublisher.com/index.php/bm 140 3 Genome Variation and Evolutionary Divergence 3.1 Distribution of SNPs, InDels, and base substitutions The mitochondrial genome of abalone has accumulated a certain level of single nucleotide polymorphism (SNP) and insertion/deletion (InDel) variation in different species and populations. These variations are not randomly distributed throughout the genome, but have certain regularities. In general, the variation in the protein-coding gene region is mainly base substitution (SNP), and most of them are synonymous mutations, while InDel variation often occurs in the non-coding region. Taking the small abalone (H. diversicolor) as an example, a study determined the mitochondrial coding region sequence of the variegated abalone from southern China and Vietnam, and found that there were 111 SNPs in the whole genome, of which 94 occurred in the protein coding region and most of them were synonymous mutations. Only 7 variations were found in the non-coding control region, and the remaining 10 were located in the rRNA region (Xin et al., 2011). This shows that most of the variation in the abalone mitochondrial genome is concentrated in the coding region, but due to the redundant nature of the codons, the amino acid sequence is not significantly affected. Correspondingly, only a very small amount of amino acid differences were detected in the above-mentioned different geographical populations of Haliotis diversicolor, mainly distributed in some regions such as the ND series genes. InDel variation is relatively rare in the abalone mitochondrial genome and mostly occurs in non-coding spacer regions or control regions, which can be used as additional molecular markers. For example, in the mitochondrial control region of Haliotis discus, insertions/deletions of 1-2 bp in length were observed between different individuals, resulting in different haplotype lengths. The base substitution differences between different abalone species are more obvious. In general, the differences in mitochondrial sequences between species are mainly reflected in synonymous substitutions at the third codon position and variations in the control region, which together determine the genetic distance of the sequence. For example, the similarity of the mitochondrial genome sequences of the Chinese population and the Korean population of Haliotis discus is as high as more than 98.5%; while the mitochondrial sequences of two morphologically similar species (such as Haliotis discus and Haliotis diversicolor) differ by about 5%-8%, and dozens of fixed difference sites can often be detected on the DNA barcode (COI) fragment, which can clearly distinguish the two species. Interestingly, in artificial hybridization studies, the mitochondrial sequences of the first generation of abalone hybrids were completely derived from the mother, and no new mutations were detected, which is consistent with the law of strict maternal inheritance of mitochondria. Guo et al. (2019b) sequenced the mitochondrial genome of the hybrid offspring of Haliotis discus (mother) × Haliotis nigromaculata (father), and the results showed that the sequence consistency between the hybrid F1 and the mother Haliotis discus was as high as 99.40%, with only very small base differences. This further confirms that the variation of the abalone mitochondrial genome mainly comes from lineage accumulation rather than hybridization. 3.2 Non-synonymous mutations and potential adaptive implications Although most mitochondrial gene mutations are functionally neutral or nearly neutral, a small number of non-synonymous mutations may play a role in adaptive evolution. Abalone is distributed in vast seas from tropical to temperate zones. Different species have different ecological environments such as water temperature and food. Some changes in the mitochondrial genome may be related to environmental adaptation. For example, a study compared the mitochondrial gene sequences of various temperate and tropical marine mollusks and found that the mitochondrial genome GC content of species living in higher water temperature environments was slightly increased, which is believed to help improve the stability of mRNA secondary structure, thereby improving the efficiency of protein synthesis under high temperature conditions. A similar trend was also observed in abalone: the average GC content of mitochondrial gene sequences of tropical species (such as ear abalone H. asinina) was higher than that of temperate species (such as European abalone), which is speculated to be a molecular adaptation to water temperature differences (Zhang et al., 2025). Amino acid substitutions in specific mitochondrial protein genes may affect the performance of enzyme complexes. For example, the amino acid variation of NADH dehydrogenase subunit genes among different abalone species is mainly concentrated in certain transmembrane regions, and variations in these regions may change the function of proton pumps to adapt to different metabolic needs. The selective pressure analysis of 13 mitochondrial protein genes in four species of the genus Haliotis
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