Development of single nucleotide polymorphisms markers associated with disease resistance towards Pseudomonas koreensis in empurau (Tor tambroides) using T-Plex ARMS PCR assay

Empurau is a highly sought freshwater fish with high market value due to its unique flesh taste. However, the main challenge of the industry remains the infectious disease outbreak, with Pseudomonas species as one of the most threatening fish pathogens while residing in a wide range of environments. Antibiotic use is a common solution to disease outbreaks and leads to increased antimicrobial resistance. The development of disease-resistant broodstock through the identification of SNP markers has emerged as a promising strategy. In this study, a total of 1,048,576 SNP markers were identified via whole genome pooled sequencing on samples treated with LD50, forming resistant and susceptible groups. Multiple tests (pairwise FST test, CMH test, and FE test) and visualization were conducted to screen and select candidate SNP markers for further validation using T-plex ARMS real-time PCR assay. The genotyping results on the selected candidate markers were confirmed using Sanger sequencing. Statistical analysis was performed to validate the significance of the candidate markers. The successful validation of 19,564 G/C SNP markers is hoped to contribute to research focusing on disease-resistant association SNPs, enabling the application of genomic information to enhance artificial selection strategies in Tor tambroides (empurau) breeding.