One of the reproductive barriers between diverging populations during formation of a new species is the sterility of their hybrids. The Prdm9-driven hybrid male sterility of Mus musculus musculus × Mus musculus domesticus hybrids depends on the interaction between PRDM9, a histone methyltransferase that determines the positions of meiotic recombination hotspots, and an as yet unknown X-linked genetic factor within the Hybrid sterility X2 (Hstx2) locus. Here, we report that the Mir465 microRNA (miRNA) gene cluster is the predicted Hstx2 hybrid sterility factor. We show that removal of the Mir465 genes restores the fertility of sterile hybrids and improves meiotic synapsis of homologous chromosomes. Mir465 knockout also restores spermatogenesis in sterile chromosomal translocation carriers, demonstrating that Mir465 acts as a meiotic checkpoint that can be activated independently of Prdm9 intersubspecific incompatibility. Furthermore, the Mir465 knockout increases the global recombination rate in hybrids and in parental Mus m. domesticus mice. This demonstrates that Mir465 is responsible for the phenotypes of the two overlapping genetic loci, the Hstx2 engaged in fertility of hybrids and the Meiotic recombination 1 (Meir1) controlling the recombination rate. The finding of enlarged Mir465 clusters in all European Mus m. musculus samples tested and the identification of differentially expressed targets suggest that the reproductive barrier between the two subspecies is sensitive to copy number variation of Mir465 genes. Together, the underdominant interaction between Prdm9 and Mir465 provides a rare example of Dobzhansky-Muller incompatibility in hybrids of closely related species, making it accessible for further analysis at the molecular level.

Doctoral Program in Medical Sciences Graduate School of Comprehensive Human Sciences University of Tsukuba Tsukuba 305 8575 Japan

Genomics Core Facility EMBL Heidelberg 69117 Germany

Laboratory Animal Resource Center in Transborder Medical Research Center Institute of Medicine University of Tsukuba Tsukuba 305 0006 Japan

Laboratory of Epigenetic Regulations Institute of Molecular Genetics Czech Academy of Sciences Praha 142 20 Czech Republic

Laboratory of Genomics and Bioinformatics Institute of Molecular Genetics Czech Academy of Sciences Praha 142 20 Czech Republic

Laboratory of Mouse Molecular Genetics Institute of Molecular Genetics Czech Academy of Sciences Praha 142 20 Czech Republic

Liston Dooley Laboratory Department of Pathology University of Cambridge Cambridge CB2 1QP United Kingdom

Max Planck Institute for Evolutionary Biology Ploen 24306 Germany

Studenec Research Facility Institute of Vertebrate Biology Czech Academy of Sciences Brno 675 02 Czech Republic

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Proc Natl Acad Sci U S A. 2025 Nov 25;122(47):e2526459122. doi: 10.1073/pnas.2526459122. PubMed

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