The production of exclusively female populations of rainbow trout (Oncorhynchus mykiss) is a key strategy in modern aquaculture due to the late maturation and larger size of females. However, the spontaneous appearance of males or intersex individuals in genetically XX batches represents a challenge.
A recent study published in PLOS One, by researchers from Université Paris-Saclay, INRAE, AgroParisTech, The University of Edinburgh, and Université Clermont-Auvergne, has delved into the genome of this species to refine the location of genetic regions (QTLs) associated with this phenomenon, identifying candidate genes that could be crucial to understand and, potentially, control spontaneous masculinization.
Sex Control in Rainbow Trout
Sex determination in fish is a remarkably plastic process, influenced by genetic and environmental factors. In rainbow trout, an XX/XY sex determination system predominates, where the genetic sex is male heterogametic. Despite this, in cultured populations theoretically composed only of XX females, aquaculture producers regularly observe a small percentage of individuals that develop male or intersex characteristics. This spontaneous masculinization is a highly heritable trait, controlled by minor modifier genes yet to be fully identified, although previous studies had already detected several implicated QTLs.
Sex control and the development of monosex populations are of great interest for research on reproductive development in vertebrates and for selective breeding in fish. Particularly in rainbow trout, the production of large specimens (over 1 kg) for fresh and smoked fillets is a growing market. A significant obstacle is the early maturation of males (1-2 years) compared to females (2-3 years), which stops growth, increases susceptibility to pathogens, and reduces meat quality. Therefore, all-female populations are preferred.
Currently, these populations are obtained by crossing hormonally reversed XX females (XX neomales) with XX females. However, concern about the potential risks to human and environmental health from the use of hormones such as 17-alpha methyltestosterone makes this method less sustainable. Finding safe and consumer- and environmentally-friendly alternatives to produce all-female populations is a fundamental challenge.
Delving into the Genome
The research team used genome-based approaches and various statistical methods to further investigate the QTLs previously identified in a French commercial population on chromosomes Omy1, Omy12, and Omy20.
The study had two main objectives:
- Validate the existence of four QTLs (two on Omy1, one on Omy12, and one on Omy20) in six different French rainbow trout populations.
- Refine the location of these QTLs in the original discovery population, using a new reference genome assembly (derived from the Arlee line, phylogenetically closer to French populations) and combining machine learning techniques (Random Forests) with discriminant analysis of principal components (DAPC).
For validation, they genotyped between 30 and 77 XX fish for each of the six populations, including neomales, using SNP (Single Nucleotide Polymorphism) panels. The scientists applied Fisher’s exact test to determine the association between genotypes at each SNP and the phenotypic sex of the fish.
For refinement, the scientists re-analyzed the genome sequences of 23 broodstock females from the discovery population with extreme proportions of sexually reversed offspring (very high or very low). They performed Fisher’s exact tests on all SNPs in extended genomic regions to define new boundaries for the QTLs.
Subsequently, they applied Random Forests analyses on haplotypes (combinations of SNPs) within these new QTL regions to identify the most relevant haplotypes and positional candidate genes. Finally, a Discriminant Analysis of Principal Components (DAPC) was used on the SNPs of the candidate genes to find the variants that best discriminated between broodstock with high or low masculinized progeny.
Genes within the QTL regions were annotated using the Arlee reference genome database, and functional information was extracted from databases such as GeneCards and UniProtKB/Swiss-Prot.
Towards the Identification of Sex-Modifying Genes
The research successfully validated the involvement of DNA markers, previously identified on chromosomes Omy1, Omy12, and Omy20, in spontaneous masculinization in six different farmed trout populations.
Validation of QTLs in Diverse Populations:
- Of the 173 SNPs tested, 117 showed a significant effect in at least one population.
- In population A, genetically close to the discovery population, 50 SNPs on Omy1 had an effect, and many of these also showed effects in populations B, C, and E.
- Although no SNP on Omy12 or Omy20 had an effect in population A (possibly due to sampling issues for rare variants), 7 SNPs on Omy12 and 13 SNPs on Omy20 did show effects in other populations (C, D, E, F).
- The study identified 90 SNPs with at least a putative associated effect in two validation populations, and 63 SNPs with a putative effect in four populations or a clear significant effect in two or more.
Refinement of QTL Regions and Candidate Genes
The study defined new extended boundaries for the QTLs, especially on chromosome Omy20, where three distinct regions are now suggested (Omy20_a, Omy20_b, Omy20_c).
According to the study’s results, there are several functional candidate genes that could be involved in spontaneous masculinization by reducing germ cell proliferation and repressing oogenesis in XX trout in the absence of the master sex-determining gene (sdY).
The authors suggest that the findings are consistent with a model where spontaneous female-to-male sex reversal in rainbow trout is associated with genetic factors capable of reducing germ cell proliferation and arresting oogenesis.
Implications for Trout Farming
The results of this study have significant implications for the rainbow trout industry. The identification of these candidate genes, particularly syndig1, tlx1, and hells on Omy1, as well as khdrbs2 and csmd1 on Omy20, deserves further investigation to validate their roles as sex modifiers and their interaction with environmental factors such as rearing temperature.
From the aquaculture producer’s perspective, greater knowledge about the combined effects of the genetic basis and environmental factors that determine spontaneous reversal is crucial. This could allow for:
- Eradicating spontaneous sex reversal in all-female production stocks.
- Developing methods to produce hormone-free neomales for breeding.
It is important to consider that the high heritability of spontaneous masculinization in XX individuals suggests that using these spontaneously masculinized individuals as progenitors would increase the frequency of undesired masculinized offspring in all-female stocks. Therefore, a balance is needed between genetic and environmental control depending on the destination of the fish (broodstock vs. all-female production stock).
Furthermore, in a context of global warming, understanding the interaction between genetics and the environment that controls spontaneous masculinization is essential for both cultured and wild populations. The impact of spontaneous masculinization on population dynamics and the fitness of wild rainbow trout remains unknown and deserves more attention, as this phenomenon, driven by environmental factors, may become more frequent with rising water temperatures.
Conclusion
This study represents a significant advance in understanding the genetic basis of spontaneous sex reversal in XX rainbow trout. By validating QTLs in multiple populations and refining their localization to pinpoint candidate genes with plausible biological functions, new avenues for research and practical application in aquaculture are opened.
The confirmation of the importance of genomic regions on chromosomes Omy1, Omy12, and Omy20 as minor sex-determining regions in rainbow trout, and the proposal of genes such as syndig1, tlx1, hells, gbf1, arfgef3, khdrbs2, dst, hmgcll1, csmd1, lrrc59, and caskin2, lay the groundwork for future studies focused on validating the precise causal variants and functional roles of these genes. This research not only benefits the trout industry but also contributes to the broader knowledge of the complex mechanisms of sex determination and differentiation in fish, an area where much remains to be discovered.
Contact
Florence Phocas
Université Paris-Saclay, INRAE, AgroParisTech
GABI, 78350, Jouy-en-Josas, France
Email: florence.phocas@inrae.fr
Reference (open access)
Dehaullon A, Fraslin C, Bestin A, Poncet C, Guiguen Y, Quillet E, et al. (2025) In-depth investigation of genome to refine QTL positions for spontaneous sex-reversal in XX rainbow trout. PLoS One 20(5): e0313464. https://doi.org/10.1371/journal.pone.0313464
Editor at the digital magazine AquaHoy. He holds a degree in Aquaculture Biology from the National University of Santa (UNS) and a Master’s degree in Science and Innovation Management from the Polytechnic University of Valencia, with postgraduate diplomas in Business Innovation and Innovation Management. He possesses extensive experience in the aquaculture and fisheries sector, having led the Fisheries Innovation Unit of the National Program for Innovation in Fisheries and Aquaculture (PNIPA). He has served as a senior consultant in technology watch, an innovation project formulator and advisor, and a lecturer at UNS. He is a member of the Peruvian College of Biologists and was recognized by the World Aquaculture Society (WAS) in 2016 for his contribution to aquaculture.
