F1 hybrids between mouse inbred strains PWD and C57BL/6 represent the most thoroughly genetically defined model of hybrid sterility in vertebrates. Hybrid male sterility can be fully reconstituted from three components of this model, the Prdm9 gene, intersubspecific homeology of Mus musculus musculus and Mus musculus domesticus autosomes, and the X-linked Hstx2 locus. Hstx2 modulates the extent of Prdm9-dependent meiotic arrest and harbors two additional factors responsible for intersubspecific introgression-induced oligospermia (Hstx1) and meiotic recombination rate (Meir1). To facilitate positional cloning and to overcome the recombination suppression within the 4.3 Mb encompassing the Hstx2 locus, we designed Hstx2-CRISPR and SPO11/Cas9 transgenes aimed to induce DNA double-strand breaks specifically within the Hstx2 locus. The resulting recombinant reduced the Hstx2 locus to 2.70 Mb (chromosome X: 66.51-69.21 Mb). The newly defined Hstx2 locus still operates as the major X-linked factor of the F1 hybrid sterility, and controls meiotic chromosome synapsis and meiotic recombination rate. Despite extensive further crosses, the 2.70 Mb Hstx2 interval behaved as a recombination cold spot with reduced PRDM9-mediated H3K4me3 hotspots and absence of DMC1-defined DNA double-strand-break hotspots. To search for structural anomalies as a possible cause of recombination suppression, we used optical mapping and observed high incidence of subspecies-specific structural variants along the X chromosome, with a striking copy number polymorphism of the microRNA Mir465 cluster. This observation together with the absence of a strong sterility phenotype in Fmr1 neighbor (Fmr1nb) null mutants support the role of microRNA as a likely candidate for Hstx2.

Department Evolutionary Genetics Research Group Meiotic Recombination and Genome Instability Max Planck Institute for Evolutionary Biology Plön D 24306 Germany

Faculty of Science Charles University Prague CZ 12000 Czech Republic

Laboratory of Mouse Molecular Genetics Division BIOCEV Institute of Molecular Genetics Czech Academy of Sciences Vestec CZ 25250 Czech Republic

Molecular Basis and Evolution of Complex Traits Group Friedrich Miescher Laboratory of the Max Planck Society Tübingen 72076 Germany

The Czech Centre for Phenogenomics Division BIOCEV Institute of Molecular Genetics Czech Academy of Sciences Vestec CZ 25250 Czech Republic

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Anderson L. K., Reeves A., Webb L. M., and Ashley T., 1999.  Distribution of crossing over on mouse synaptonemal complexes using immunofluorescent localization of MLH1 protein. Genetics 151: 1569–1579. PubMed PMC

Baker C. L., Kajita S., Walker M., Saxl R. L., Raghupathy N. et al. , 2015.  PRDM9 drives evolutionary erosion of hotspots in Mus musculus through haplotype-specific initiation of meiotic recombination. PLoS Genet. 11: e1004916 10.1371/journal.pgen.1004916 PubMed DOI PMC

Balcova M., Faltusova B., Gergelits V., Bhattacharyya T., Mihola O. et al. , 2016.  Hybrid sterility locus on chromosome X controls meiotic recombination rate in mouse. PLoS Genet. 12: e1005906 10.1371/journal.pgen.1005906 PubMed DOI PMC

Ball R. L., Fujiwara Y., Sun F., Hu J., Hibbs M. A. et al. , 2016.  Regulatory complexity revealed by integrated cytological and RNA-seq analyses of meiotic substages in mouse spermatocytes. BMC Genomics 17: 628 10.1186/s12864-016-2865-1 PubMed DOI PMC

Baudat F., Buard J., Grey C., Fledel-Alon A., Ober C. et al. , 2010.  PRDM9 is a major determinant of meiotic recombination hotspots in humans and mice. Science 327: 836–840. 10.1126/science.1183439 PubMed DOI PMC

Bhattacharyya T., Gregorova S., Mihola O., Anger M., Sebestova J. et al. , 2013.  Mechanistic basis of infertility of mouse intersubspecific hybrids. Proc. Natl. Acad. Sci. USA 110: E468–E477. 10.1073/pnas.1219126110 PubMed DOI PMC

Bhattacharyya T., Reifova R., Gregorova S., Simecek P., Gergelits V. et al. , 2014.  X chromosome control of meiotic chromosome synapsis in mouse inter-subspecific hybrids. PLoS Genet. 10: e1004088 10.1371/journal.pgen.1004088 PubMed DOI PMC

Brick K., Thibault-Sennett S., Smagulova F., Lam K. G., Pu Y. et al. , 2018.  Extensive sex differences at the initiation of genetic recombination. Nature 561: 338–342. 10.1038/s41586-018-0492-5 PubMed DOI PMC

Campbell P., Good J. M., and Nachman M. W., 2013.  Meiotic sex chromosome inactivation is disrupted in sterile hybrid male house mice. Genetics 193: 819–828. 10.1534/genetics.112.148635 PubMed DOI PMC

Chan S., Lam E., Saghbini M., Bocklandt S., Hastie A. et al. , 2018.  Structural variation detection and analysis using Bionano optical mapping. Methods Mol. Biol. 1833: 193–203. 10.1007/978-1-4939-8666-8_16 PubMed DOI

Collaborative Cross Consortium , 2012.  The genome architecture of the Collaborative Cross mouse genetic reference population. Genetics 190: 389–401. 10.1534/genetics.111.132639 PubMed DOI PMC

Cong L., Ran F. A., Cox D., Lin S., Barretto R. et al. , 2013.  Multiplex genome engineering using CRISPR/Cas systems. Science 339: 819–823. 10.1126/science.1231143 PubMed DOI PMC

Coyne J. A., and Orr H. A., 2004.  Speciation, Sinauer Associates, Sunderland, Massachusetts.

Davies B., Hatton E., Altemose N., Hussin J. G., Pratto F. et al. , 2016.  Re-engineering the zinc fingers of PRDM9 reverses hybrid sterility in mice. Nature 530: 171–176. 10.1038/nature16931 PubMed DOI PMC

Dion-Côté A. M., and Barbash D. A., 2017.  Beyond speciation genes: an overview of genome stability in evolution and speciation. Curr. Opin. Genet. Dev. 47: 17–23. 10.1016/j.gde.2017.07.014 PubMed DOI PMC

Dobzhansky T., 1951.  Genetics and the origin of Species, Columbia University, New York.

Duvaux L., Belkhir K., Boulesteix M., and Boursot P., 2011.  Isolation and gene flow: inferring the speciation history of European house mice. Mol. Ecol. 20: 5248–5264. 10.1111/j.1365-294X.2011.05343.x PubMed DOI

Dzur-Gejdosova M., Simecek P., Gregorova S., Bhattacharyya T., and Forejt J., 2012.  Dissecting the genetic architecture of f(1) hybrid sterility in house mice. Evolution 66: 3321–3335. 10.1111/j.1558-5646.2012.01684.x PubMed DOI

Ernst C., Eling N., Martinez-Jimenez C. P., Marioni J. C., and Odom D. T., 2019.  Staged developmental mapping and X chromosome transcriptional dynamics during mouse spermatogenesis. Nat. Commun. 10: 1251 10.1038/s41467-019-09182-1 PubMed DOI PMC

Flachs P., Mihola O., Simecek P., Gregorova S., Schimenti J. et al. , 2012.  Interallelic and intergenic incompatibilities of the Prdm9 (Hst1) gene in mouse hybrid sterility. PLoS Genet. 8: e1003044 10.1371/journal.pgen.1003044 PubMed DOI PMC

Flachs P., Bhattacharyya T., Mihola O., Pialek J., Forejt J. et al. , 2014.  Prdm9 incompatibility controls oligospermia and delayed fertility but no selfish transmission in mouse intersubspecific hybrids. PLoS One 9: e95806 10.1371/journal.pone.0095806 PubMed DOI PMC

Flint J., Valdar W., Shifman S., and Mott R., 2005.  Strategies for mapping and cloning quantitative trait genes in rodents. Nat. Rev. Genet. 6: 271–286. 10.1038/nrg1576 PubMed DOI

Forejt J., 1996.  Hybrid sterility in the mouse. Trends Genet. 12: 412–417. 10.1016/0168-9525(96)10040-8 PubMed DOI

Forejt J., and Ivanyi P., 1974.  Genetic studies on male sterility of hybrids between laboratory and wild mice (Mus musculus L.). Genet. Res. 24: 189–206. 10.1017/S0016672300015214 PubMed DOI

Forejt J., Pialek J., and Trachtulec Z., 2012.  Hybrid male sterility genes in the mouse subspecific crosses, pp. 482–503 in Evolution of the House Mouse, edited by Macholan M., Baird S. J. E., Muclinger P., and Pialek J.. Cambridge University Press, Cambridge: 10.1017/CBO9781139044547.021 DOI

Fuller Z. L., Leonard C. J., Young R. E., Schaeffer S. W., and Phadnis N., 2018.  Ancestral polymorphisms explain the role of chromosomal inversions in speciation. PLoS Genet. 14: e1007526 10.1371/journal.pgen.1007526 PubMed DOI PMC

Garcia-Muse T., Galindo-Diaz U., Garcia-Rubio M., Martin J. S., Polanowska J. et al. , 2019.  A meiotic checkpoint Alters repair partner bias to permit inter-sister repair of persistent DSBs. Cell Rep. 26: 775–787.e5. 10.1016/j.celrep.2018.12.074 PubMed DOI PMC

Good J. M., Dean M. D., and Nachman M. W., 2008.  A complex genetic basis to X-linked hybrid male sterility between two species of house mice. Genetics 179: 2213–2228. 10.1534/genetics.107.085340 PubMed DOI PMC

Gregorova S., and Forejt J., 2000.  PWD/Ph and PWK/Ph inbred mouse strains of Mus m. musculus subspecies–a valuable resource of phenotypic variations and genomic polymorphisms. Folia Biol. (Praha) 46: 31–41. PubMed

Gregorová S., Mnuková-Fajdelová M., Trachtulec Z., Capková J., Loudová M. et al. , 1996.  Sub-milliMorgan map of the proximal part of mouse Chromosome 17 including the hybrid sterility 1 gene. Mamm. Genome 7: 107–113. 10.1007/s003359900029 PubMed DOI

Gregorova, S., V. Gergelits, I. Chvatalova, T. Bhattacharyya, B. Valiskova et al., 2018 Modulation of Prdm9-controlled meiotic chromosome asynapsis overrides hybrid sterility in mice. eLife 7: e34282. 10.7554/eLife.34282 PubMed DOI PMC

Haldane J., 1922.  Sex ration and unisexual sterility in hybrid animals. J. Genet. 12: 101–109. 10.1007/BF02983075 DOI

Hammer M. F., Mendez F. L., Cox M. P., Woerner A. E., and Wall J. D., 2008.  Sex-biased evolutionary forces shape genomic patterns of human diversity. PLoS Genet. 4: e1000202 10.1371/journal.pgen.1000202 PubMed DOI PMC

Harr B., Karakoc E., Neme R., Teschke M., Pfeifle C. et al. , 2016.  Genomic resources for wild populations of the house mouse, Mus musculus and its close relative Mus spretus. Sci. Data 3: 160075 10.1038/sdata.2016.75 PubMed DOI PMC

Hinch A. G., Zhang G., Becker P. W., Moralli D., Hinch R. et al. , 2019.  Factors influencing meiotic recombination revealed by whole-genome sequencing of single sperm. Science 363: eaau8861. 10.1126/science.aau8861 PubMed DOI PMC

Hooper D. M., Griffith S. C., and Price T. D., 2018.  Sex chromosome inversions enforce reproductive isolation across an avian hybrid zone. Mol. Ecol. 28: 1246–1262. PubMed

Janoušek V., Wang L., Luzynski K., Dufkova P., Vyskocilova M. M. et al. , 2012.  Genome-wide architecture of reproductive isolation in a naturally occurring hybrid zone between Mus musculus musculus and M. m. domesticus. Mol. Ecol. 21: 3032–3047. 10.1111/j.1365-294X.2012.05583.x PubMed DOI PMC

Jung M., Wells D., Rusch J., Ahmad S., Marchini J. et al. , 2019.  Unified single-cell analysis of testis gene regulation and pathology in five mouse strains. Elife. 25: 8 10.7554/eLife.43966 PubMed DOI PMC

Kirkpatrick M., 2010.  How and why chromosome inversions evolve. PLoS Biol. 8: e1000501. 10.1371/journal.pbio.1000501 PubMed DOI PMC

Lange J., Yamada S., Tischfield S. E., Pan J., Kim S. et al. , 2016.  The Landscape of Mouse Meiotic Double-Strand Break Formation, Processing, and Repair. Cell 167: 695–708.e16. 10.1016/j.cell.2016.09.035 PubMed DOI PMC

Larson E. L., Keeble S., Vanderpool D., Dean M. D., and Good J. M., 2016.  The composite regulatory basis of the large X-effect in mouse speciation. Mol. Biol. Evol. 34: 282–295. PubMed PMC

Mack K. L., and Nachman M. W., 2017.  Gene regulation and speciation. Trends Genet. 33: 68–80. 10.1016/j.tig.2016.11.003 PubMed DOI PMC

Macholán M., Baird S. J., Dufkova P., Munclinger P., Bimova B. V. et al. , 2011.  Assessing multilocus introgression patterns: a case study on the mouse X chromosome in central Europe. Evolution 65: 1428–1446. 10.1111/j.1558-5646.2011.01228.x PubMed DOI

Macholán M., Munclinger P., Sugerkova M., Dufkova P., Bimova B. et al. , 2007.  Genetic analysis of autosomal and X-linked markers across a mouse hybrid zone. Evolution 61: 746–771. 10.1111/j.1558-5646.2007.00065.x PubMed DOI

Maheshwari S., and Barbash D. A., 2011.  The genetics of hybrid incompatibilities. Annu. Rev. Genet. 45: 331–355. 10.1146/annurev-genet-110410-132514 PubMed DOI

Margolin G., Khil P. P., Kim J., Bellani M. A., and Camerini-Otero R. D., 2014.  Integrated transcriptome analysis of mouse spermatogenesis. BMC Genomics 15: 39 10.1186/1471-2164-15-39 PubMed DOI PMC

Mihola O., Trachtulec Z., Vlcek C., Schimenti J. C., and Forejt J., 2009.  A mouse speciation gene encodes a meiotic histone H3 methyltransferase. Science 323: 373–375. 10.1126/science.1163601 PubMed DOI

Miyata H., Castaneda J. M., Fujihara Y., Yu Z., Archambeault D. R. et al. , 2016.  Genome engineering uncovers 54 evolutionarily conserved and testis-enriched genes that are not required for male fertility in mice. Proc. Natl. Acad. Sci. USA 113: 7704–7710. 10.1073/pnas.1608458113 PubMed DOI PMC

Morgan A. P., Gatti D. M., Najarian M. L., Keane T. M., Galante R. J. et al. , 2017.  Structural variation shapes the landscape of recombination in mouse. Genetics 206: 603–619. 10.1534/genetics.116.197988 PubMed DOI PMC

Myers S., Bowden R., Tumian A., Bontrop R. E., Freeman C. et al. , 2010.  Drive against hotspot motifs in primates implicates the PRDM9 gene in meiotic recombination. Science 327: 876–879. 10.1126/science.1182363 PubMed DOI PMC

Nachman M. W., and Payseur B. A., 2012.  Recombination rate variation and speciation: theoretical predictions and empirical results from rabbits and mice. Philos. Trans. R. Soc. Lond. B Biol. Sci. 367: 409–421. 10.1098/rstb.2011.0249 PubMed DOI PMC

Orr H. A., 2005.  The genetic basis of reproductive isolation: insights from Drosophila. Proc. Natl. Acad. Sci. USA 102: 6522–6526. 10.1073/pnas.0501893102 PubMed DOI PMC

Ortiz-Barrientos D., Engelstadter J., and Rieseberg L. H., 2016.  Recombination rate evolution and the origin of species. Trends Ecol. Evol. 31: 226–236. 10.1016/j.tree.2015.12.016 PubMed DOI

Ota H., Ito-Matsuoka Y., and Matsui Y., 2019.  Identification of the X-linked germ cell specific miRNAs (XmiRs) and their functions. PLoS One 14: e0211739 10.1371/journal.pone.0211739 PubMed DOI PMC

Parvanov E. D., Petkov P. M., and Paigen K., 2010.  Prdm9 controls activation of mammalian recombination hotspots. Science 327: 835 10.1126/science.1181495 PubMed DOI PMC

Patten M. M., 2018.  Selfish X chromosomes and speciation. Mol. Ecol. 27: 3772–3782. 10.1111/mec.14471 PubMed DOI

Payseur B. A., Krenz J. G., and Nachman M. W., 2004.  Differential patterns of introgression across the X chromosome in a hybrid zone between two species of house mice. Evolution 58: 2064–2078. 10.1111/j.0014-3820.2004.tb00490.x PubMed DOI

Payseur B. A., Presgraves D. C., and Filatov D. A., 2018.  Introduction: sex chromosomes and speciation. Mol. Ecol. 27: 3745–3748. 10.1111/mec.14828 PubMed DOI PMC

Phifer-Rixey M., and Nachman M. W., 2015.  Insights into mammalian biology from the wild house mouse Mus musculus. eLife 4: e05959. 10.7554/eLife.05959 PubMed DOI PMC

Presgraves D. C., 2018.  Evaluating genomic signatures of “the large X-effect” during complex speciation. Mol. Ecol. 27: 3822–3830. 10.1111/mec.14777 PubMed DOI PMC

Royo H., Polikiewicz G., Mahadevaiah S. K., Prosser H., Mitchell M. et al. , 2010.  Evidence that meiotic sex chromosome inactivation is essential for male fertility. Curr. Biol. 20: 2117–2123. 10.1016/j.cub.2010.11.010 PubMed DOI

Royo H., Seitz H., ElInati E., Peters A. H., Stadler M. B. et al. , 2015.  Silencing of X–linked MicroRNAs by meiotic sex chromosome inactivation. PLoS Genet. 11: e1005461 10.1371/journal.pgen.1005461 PubMed DOI PMC

Sharan S. K., Thomason L. C., Kuznetsov S. G., and Court D. L., 2009.  Recombineering: a homologous recombination-based method of genetic engineering. Nat. Protoc. 4: 206–223. 10.1038/nprot.2008.227 PubMed DOI PMC

Smagulova F., Brick K., Pu Y. M., Camerini-Otero R. D., and Petukhova G. V., 2016.  The evolutionary turnover of recombination hot spots contributes to speciation in mice. Genes Dev. 30: 266–280. 10.1101/gad.270009.115 PubMed DOI PMC

Spies M., and Fishel R., 2015.  Mismatch repair during homologous and homeologous recombination. Cold Spring Harb. Perspect. Biol. 7: a022657 10.1101/cshperspect.a022657 PubMed DOI PMC

Srivastava A., Morgan A. P., Najarian M. L., Sarsani V. K., Sigmon J. S. et al. , 2017.  Genomes of the mouse collaborative cross. Genetics 206: 537–556. 10.1534/genetics.116.198838 PubMed DOI PMC

Storchová R., Gregorova S., Buckiova D., Kyselova V., Divina P. et al. , 2004.  Genetic analysis of X-linked hybrid sterility in the house mouse. Mamm. Genome 15: 515–524. 10.1007/s00335-004-2386-0 PubMed DOI

Trachtulec Z., Mnukova-Fajdelova M., Hamvas R. M., Gregorova S., Mayer W. E. et al. , 1997.  Isolation of candidate hybrid sterility 1 genes by cDNA selection in a 1.1 megabase pair region on mouse chromosome 17. Mamm. Genome 8: 312–316. 10.1007/s003359900430 PubMed DOI

Truett G. E., Heeger P., Mynatt R. L., Truett A. A., Walker J. A. et al. , 2000.  Preparation of PCR-quality mouse genomic DNA with hot sodium hydroxide and tris (HotSHOT). Biotechniques 29: 52, 54 10.2144/00291bm09 PubMed DOI

Tucker P., Sage R., Warner J., Wilson A., and Eicher E., 1992.  Abrupt cline for sex chromosomes in a hybrid zone between two species of mice. Evolution 46: 1146–1163. 10.1111/j.1558-5646.1992.tb00625.x PubMed DOI

Turner L. M., Schwahn D. J., and Harr B., 2012.  Reduced male fertility is common but highly variable in form and severity in a natural house mouse hybrid zone. Evolution 66: 443–458. 10.1111/j.1558-5646.2011.01445.x PubMed DOI

Wang L., Valiskova B., and Forejt J., 2018.  Cisplatin-induced DNA double-strand breaks promote meiotic chromosome synapsis in PRDM9-controlled mouse hybrid sterility. eLife 7: e42511. 10.7554/eLife.42511 PubMed DOI PMC

Wojtasz L., Cloutier J. M., Baumann M., Daniel K., Varga J. et al. , 2012.  Meiotic DNA double-strand breaks and chromosome asynapsis in mice are monitored by distinct HORMAD2-independent and -dependent mechanisms. Genes Dev. 26: 958–973. 10.1101/gad.187559.112 PubMed DOI PMC

Wu G., Wang W., Liu Y., Zhuang K., Cai T. et al. , 2018.  RETRACTED: NY-SAR-35 is involved in apoptosis, cell migration, invasion and epithelial to mesenchymal transition in glioma. Biomed. Pharmacother. 97: 1632–1638. 10.1016/j.biopha.2017.11.076 PubMed DOI

Zhang F., Zhang Y., Lv X., Xu B., Zhang H. et al. , 2019.  Evolution of an X–linked miRNA family predominantly expressed in mammalian male germ cells. Mol. Biol. Evol. 36: 663–678. 10.1093/molbev/msz001 PubMed DOI PMC

Zhang L., Sun T., Woldesellassie F., Xiao H., and Tao Y., 2015.  Sex ratio meiotic drive as a plausible evolutionary mechanism for hybrid male sterility. PLoS Genet. 11: e1005073 10.1371/journal.pgen.1005073 PubMed DOI PMC

A Minimal Hybrid Sterility Genome Assembled by Chromosome Swapping Between Mouse Subspecies (Mus musculus)

Natural variation in the zinc-finger-encoding exon of Prdm9 affects hybrid sterility phenotypes in mice

Meiotic Recognition of Evolutionarily Diverged Homologs: Chromosomal Hybrid Sterility Revisited

Genic and chromosomal components of Prdm9-driven hybrid male sterility in mice (Mus musculus)

Chromosome-wide characterization of meiotic noncrossovers (gene conversions) in mouse hybrids

Prdm9 Intersubspecific Interactions in Hybrid Male Sterility of House Mouse