Characterisation of the Feminine (45S-M) Ribosomal DNA Array and Creation of a Synthetic Sex Determination System in Zebrafish

Danio rerio (zebrafish) have become a cornerstone model in developmental biology, yet, sex determination in this species remains poorly understood. Although common laboratory strains (AB and TU) have lost chromosomal sex determination, wild strains possess a ZZ/ZW sex- determination system, with a sex-linked genetic variant found in the sub-telomeric region of chromosome 4. Interestingly, overlapping this region is the 45S-M ribosomal DNA locus. Ribosomal DNA has previously been overlooked as a candidate for sex determination as ribosomes are almost always viewed as universal and non-specialised. Nevertheless, recent experiments from the Hore laboratory have shown that the 45S-M ribosomal DNA loci is not essential for life, as when mutated, male zebrafish can develop normally; however, female development is almost completely suppressed. This implies that 45S-M is both a truly specialist ribosome locus and also heavily implicated in sex determination. The poor resolution of rDNA in standard genome assemblies has proven a major barrier for defining rDNA function, especially at specialised ribosomal DNA loci. Here, I used long-read sequencing to resolve the previously intractable 45S-M rDNA locus, revealing its organisation as a large subtelomeric tandem-repeat array. I further examined the intersection between sex determination and ribosome heterogeneity by (i) characterising the stability and inheritance of 45S-M modification classes across generations, (ii) quantifying relationships between sex, modification, and rDNA copy number, and (iii) integrating long read sequencing to interpret 45S-M rDNA variation in its chromosomal context. Amplicon sequencing revealed that CRISPR–Cas9 editing of the 45S-M rDNA locus generates discrete modification classes that are stable and inherited across generations. Across the F2 population, adults rarely hold mixed modification classes, indicating strong suppression of heterozygosity at 45S-M. Population-scale qPCR reveals copy number reduction of the 45S-M rDNA array to a single copy in modified fish, against a background of natural copy number variation in wild-type fish. Targeted telomere-sequencing long reads spanning the subtelomeric locus directly resolve the copy number reduction, and confirm that suppression of heterozygosity is consistent in whole fish. Importantly, 45S-M rDNA reduced to a single mutated repeat unit remains strongly sex-biased, being tolerated exclusively in males. Outcrossing experiments revealed the creation of an unexpected synthetic sex determination system, which due to some undefined gene conversion mechanism, features a peculiar form of non-Mendelian inheritance. Overall, these findings establish the 45S-M knockout line as a robust and genetically tractable system in which rDNA array modifications can be resolved, quantified, and tracked across generations in relation to sex. This positions zebrafish and the 45S-M mutant lines as a powerful vertebrate model for investigating rDNA heterogeneity and inheritance using a clear, measurable developmental phenotype and tailored sequencing assays.