IDENTIFYING QTLs FOR RESISTANCE TO RAINBOW TROUT FRY SYNDROME IN AN ORGANIC LINE OF RAINBOW TROUT Oncorhynchus mykiss

Introduction

Antibiotic use is restricted in organic aquaculture, increasing the need for sustainable strategies to enhance disease resistance. Selective breeding represents a promising approach to improve host disease resistance, and genomic tools such as quantitative trait locus (QTL) mapping have been widely used to identify genetic regions associated with disease resistance in aquaculture species. A globally prevalent and devastating disease in rainbow trout (Oncorhynchus mykiss) production is Rainbow Trout Fry Syndrome (RTFS), caused by Flavobacterium psychrophilum. This bacterial disease can result in high mortalities, particularly among fry and juvenile fish. Its impact on production can be substantial, leading to significant economic losses in commercial aquaculture. Currently, no commercial vaccine against RTFS is available. Control of the disease typically relies on antibiotic treatments, however their use raises environmental concerns and resistant strains of F. psychrophilum to various types of antibiotics have been reported (Barnes & Brown, 2011). The impact of RTFS is particularly concerning in organic aquaculture, where the number and type of treatments are limited, hence requiring alternative disease management strategies. Several QTLs for RTFS resistance were already identified in conventional strains of rainbow trout (Fraslin et al., 2019; Mathiessen et al., 2023; Palti, Vallejo, et al., 2015). However, it remains unknown whether these QTLs are present or effective in organic strains, which may differ in genetic architecture from conventional strains due to distinct selection pressures. Addressing this knowledge gap is critical for the development of targeted breeding programs in organic aquaculture. Therefore, this study aims to identify QTL associated with RTFS resistance in an organic rainbow trout line.

Materials and Methods

In this study, we sampled rainbow trout at an organic farm in Denmark during a natural outbreak of RTFS. The population consisted of 210 full- and half-sib families, produced in April 2025. For each family, 18 fish were randomly distributed across three tanks (six fish from each family per tank). The RTFS outbreak began in mid-September 2025 and ended in early November 2025, with a duration of 47 days in total. Throughout the outbreak, mortalities were sampled daily. Surviving fish were sampled after the disease outbreak. DNA was extracted from tissue samples and genotyped using the 57K SNP Axiom Trout Genotyping Array (Palti, Gao, et al., 2015) at a commercial laboratory (Eurofins, Galten, Denmark). Quality control on SNP level was performed using PLINK v1.9. SNPs were excluded based on the following criteria: (i) call rate <90%, (ii) deviation from Hardy–Weinberg equilibrium (P <1 ×10-5), and (iii) minor allele frequency (MAF) <0.05. We performed GWAS analysis for RTFS resistance defined as a binary trait (survival: 0/1). An association analysis was conducted using the mixed linear model association (MLMA) framework with a leave-one-chromosome-out (LOCO) approach, as implemented in GCTA . The model used was:

where y is the corrected phenotype, a is the mean term, b is the additive effect (fixed effect) of the candidate SNP to be tested for association, x is the SNP genotype indicator variable coded as 0,1 or 2, g- is the polygenic effect (random effect) of all SNPs except those on the chromosome where the tested SNP is located and e is the residual. A SNP was considered significant when exceeding a Bonferroni corrected threshold of P < 2.01 * 10^-6 (0.05/24,879).

Results and Discussion

In total, 295 survivors and 1,195 mortalities were sampled. After quality control, the final dataset comprised 1,490 samples and 24,879 SNPs. Significant SNPs associated with RTFS resistance were detected on chromosomes 13 and 25 (Fig. 1). The lead SNPs on chromosomes 13 and 25 explained 4% and 10% of the phenotypic variance, respectively. Significant SNPs on chromosome 25 have been reported previously (Mathiessen et al., 2023; Palti, Vallejo, et al., 2015), whereas chrosomome 13 has not previously been associated with RTFS resistance.

Figure 1. Results of GWAS for RTFS resistance as binary trait (survival: 0/1), with a Bonferroni-corrected significance threshold of p < 2.01 * 10^-6.

This study demonstrates that RTFS resistance is influenced by multiple loci explaining a relatively high proportion of the phenotypic variance. This indicates that these SNP markers can potentially be used to improve RTFS resistance in an organic rainbow trout breeding population. Because the sampling design reflected the natural infection route in an organic production system, the results can be directly applied to selective breeding programs targeting RTFS resistance in organically farmed rainbow trout.

Acknowledgment

This work was funded by GUDP/ICROFS as part of the Troutganic project (project nr. 34009-23-2174).

References

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Fraslin, C., Brard‐Fudulea, S., D'Ambrosio, J., Bestin, A., Charles, M., Haffray, P., Quillet, E., & Phocas, F. (2019). Rainbow trout resistance to bacterial cold water disease: two new quantitative trait loci identified after a natural disease outbreak on a French farm. Animal Genetics, 50(3), 293-297. https://doi.org/10.1111/age.12777

Mathiessen, H., Duan, Y., Marana, M. H., Zuo, S., Karami, A. M., Jafaar, R., von Gersdorff Jørgensen, L., Kania, P. W., Dalsgaard, I., & Madsen, L. (2023). Validation of a QTL for Flavobacterium psychrophilum resistance in rainbow trout Oncorhynchus mykiss. Aquaculture Reports, 30, 101573.

Palti, Y., Gao, G., Liu, S., Kent, M. P., Lien, S., Miller, M. R., Rexroad, C. E., & Moen, T. (2015). The development and characterization of a 57K single nucleotide polymorphism array for rainbow trout. Molecular Ecology Resources, 15(3), 662-672. https://doi.org/10.1111/1755-0998.12337

Palti, Y., Vallejo, R. L., Gao, G., Liu, S., Hernandez, A. G., Rexroad, C. E., & Wiens, G. D. (2015). Detection and Validation of QTL Affecting Bacterial Cold Water Disease Resistance in Rainbow Trout Using Restriction-Site Associated DNA Sequencing. PLOS ONE, 10(9), e0138435. https://doi.org/10.1371/journal.pone.0138435