Genomic Profiling of Empurau (Tor tambroides) for the Discovery of Single Nucleotide Polymorphisms (SNPs) Associated with Resistance to Pseudomonas koreensis

Melinda Mei Lin, Lau (2024) Genomic Profiling of Empurau (Tor tambroides) for the Discovery of Single Nucleotide Polymorphisms (SNPs) Associated with Resistance to Pseudomonas koreensis. PhD thesis, Universiti Malaysia Sarawak.

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Abstract

Empurau (Tor tambroides) is highly sought for its high nutritional value and unique flesh taste, allowing it to earn a top place among the most expensive freshwater fishes among aquaculture industry in Malaysia. Uncovering and characterising its genomic data is crucial for unmasking ambiguous taxonomy within Tor genus, while resolving its species identification for conservation purposes. A combination of Nanopore long reads and Illumina short reads had reported on an estimated size of 1.23 Gb with 87.6 % completeness of T. tambroides genome. The genomic data had been mapped to various pathways and showed significant similarity to published immune- and growth-related genes. The causative agent of the diseased empurau at the local aquaculture farm was identified as Pseudomonas koreensis with its virulence, antibiotic sensitivity and growth characteristics being characterised. It displays notable virulence characteristics that led to the acute infection of the fish. The isolated strain, CM-01, had been characterised on its virulence genes, antibiotics susceptibility testing and growth. Experimental infection had revealed the lethal dosage of CM-01 strain as 5.01 x 107 CFU/mL along with significant histological lesions observed. Its pathogenicity was found to be correlated with twelve virulence genes identified which encodes for iron uptake, protease, acylhomoserine lactone synthase gacS/gacA component regulation system, type IV secretion system, hydrogen cyanide production, exolysin, alginate biosynthesis, flagella and pili. P. koreensis was tested susceptible to beta-lactam combination agents which further suggested as its drug of choice. Its growing features had shown its optimal growth rate at pH 7 and 30 °C. These findings could establish a scientific foundation for the recognition of the pathogen and fish management. Given antibiotics administration as the common solution to disease outbreaks, contributing to the worsening of antimicrobial resistance (AMR), the development of disease-resistant broodstock through the identification of SNP markers appeared as one of a promising strategy. The SNP markers can be identified via whole genome pooled sequencing on samples treated with LD50, forming resistant and susceptible group. Multiple tests had been 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 had been performed to validate the significance of the candidate marker. With the successful validation of the 19564 G/C SNP marker, it is hoped to contribute to further studies considering disease-resistant association SNPs and can be used into breeding programs to integrate genomic information into artificial selection decisions in empurau breeding.

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