Identification of SNP Markers in the Glutamate Dehydrogenase (GDH) and Aspartate Aminotransferase 2 (AST2) Associated with Ammonia Nitrogen Tolerance in Penaeus monodon

Black tiger shrimp (<i>Penaeus monodon</i>) is the largest species of penaeid, being commercially cultured globally, ranking as the second most farmed species in the shrimp industry. However, with the transformation of shrimp aquaculture from semi-intensive to high-density farming, the concentration of ammonia nitrogen in the aquatic environment has increased, severely affecting the growth and survival of shrimps. To increase production efficiency, breeding new strains of shrimp with the trait of tolerance to high ammonia nitrogen is desired in the black tiger shrimp aquaculture. Previous studies have shown that glutamate dehydrogenase (GDH) and aspartate aminotransferase 2 (AST2) play important roles in the metabolism of ammonia nitrogen in crustaceans. In the present study, we conducted synteny analysis of <i>PmGDH</i> and <i>PmAST2</i> in different species. The interactions of <i>PmGDH</i> with ammonium (NH₄⁺) and <i>PmAST2</i> with aspartate were analyzed by docking. To develop molecule markers associated with ammonia nitrogen tolerance, SNPs of <i>PmGDH</i> and <i>PmAST2</i> were identified by direct sequencing, genotyped by the SNaPshot technique, and characterized through genotype-phenotype association analysis by PLINK software version 1.9 in the three geographical populations, two families with different ammonia tolerance, and 120 susceptible and resistant individuals of black tiger shrimp. The results indicate that the <i>GDH</i> and <i>AST2</i> genes are evolutionarily conserved in vertebrates, except for the black tiger shrimp, which suggests divergence in selective pressure between invertebrates and vertebrates. Notably, <i>PmGDH</i> may interact with NH₄⁺ via the residue Asp178 within loop 1 in the GdhA domain through a hydrogen bonding interaction, and <i>PmAST2</i> may interact with aspartate via helix 1, sheet 1, loop 1, and loop 2, through both hydrogen bonding interactions and a salt bridge interaction. A total of 12 SNPs were detected in the exons of <i>PmGDH</i> and <i>PmAST2</i>. Among these candidate SNPs, the distributions of <i>PmGDH</i>-1227 and <i>PmAST2</i>-132 showed both significant differences in the genotype and allele association analysis between susceptible and resistant groups. Haplotype association analysis showed that three haplotypes exhibited significantly different distributions between susceptible and resistant groups. In conclusion, <i>PmGDH</i>-1227 and <i>PmAST2</i>-132 were associated with ammonia nitrogen tolerance, and these SNP markers are expected to contribute to marker-assisted selection (MAS) breeding programs to obtain new strains of <i>Penaeus monodon</i>.