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

## TL;DR

This study identifies genetic markers in black tiger shrimp linked to ammonia tolerance, which can help improve breeding for better survival in high-density aquaculture.

## Contribution

The study discovers specific SNPs in GDH and AST2 genes associated with ammonia nitrogen tolerance in black tiger shrimp.

## Key findings

- Two SNPs, PmGDH-1227 and PmAST2-132, show significant differences between ammonia-resistant and susceptible shrimp.
- PmGDH and PmAST2 are evolutionarily conserved but show divergence in selective pressure in invertebrates.
- Three haplotypes were found to have significantly different distributions between resistant and susceptible shrimp.

## Abstract

Black tiger shrimp is a major farmed shrimp species in the world, which is negatively impacted by high ammonia nitrogen levels in high-density aquaculture. This study investigated the genetic basis of ammonia tolerance, focusing on two conserved genes (glutamate dehydrogenase and aspartate aminotransferase 2) involved in ammonia metabolism. Single nucleotide polymorphisms (SNPs) in these two genes were identified, genotyped and analyzed in different shrimp populations. Two specific SNPs (PmGDH-1227 and PmAST2-132) showed a significant distribution difference between ammonia-susceptible and ammonia-resistant shrimp. This study intuitively represented the interaction of PmGDH and PmAST2 to ammonia nitrogen and developed SNP markers linked to ammonia tolerance in these two genes from black tiger shrimp, providing valuable tools for molecular marker-assisted breeding programs to develop new strains with ammonia tolerance, thereby improving the survival rate and yield of black tiger shrimp under high-density farming conditions.

Black tiger shrimp (Penaeus monodon) 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 PmGDH and PmAST2 in different species. The interactions of PmGDH with ammonium (NH4+) and PmAST2 with aspartate were analyzed by docking. To develop molecule markers associated with ammonia nitrogen tolerance, SNPs of PmGDH and PmAST2 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 GDH and AST2 genes are evolutionarily conserved in vertebrates, except for the black tiger shrimp, which suggests divergence in selective pressure between invertebrates and vertebrates. Notably, PmGDH may interact with NH4+ via the residue Asp178 within loop 1 in the GdhA domain through a hydrogen bonding interaction, and PmAST2 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 PmGDH and PmAST2. Among these candidate SNPs, the distributions of PmGDH-1227 and PmAST2-132 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, PmGDH-1227 and PmAST2-132 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 Penaeus monodon.