Evolutionary studies are often limited by missing data that are critical to understanding the history of selection. Selection experiments, which reproduce rapid evolution under controlled conditions, are excellent tools to study how genomes evolve under selection. Here we present a genomic dissection of the Longshanks selection experiment, in which mice were selectively bred over 20 generations for longer tibiae relative to body mass, resulting in 13% longer tibiae in two replicates. We synthesized evolutionary theory, genome sequences and molecular genetics to understand the selection response and found that it involved both polygenic adaptation and discrete loci of major effect, with the strongest loci tending to be selected in parallel between replicates. We show that selection may favor de-repression of bone growth through inactivating two limb enhancers of an inhibitor, Nkx3-2. Our integrative genomic analyses thus show that it is possible to connect individual base-pair changes to the overall selection response.

F 1 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 F 1 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 DMC1defined 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.

F1 hybrids between mouse inbred strains PWD and C57BL/6 represent the 37 most thoroughly genetically defined model of hybrid sterility in vertebrates. Hybrid male 38 sterility can be fully reconstituted from three components of this model, namely the Prdm9 39 hybrid sterility gene, intersubspecific homeology of Mus musculus musculus and Mus 40 musculus domesticus autosomes, and the X-linked Hstx2 locus. Hstx2 modulates the extent of 41Prdm9-dependent meiotic arrest and harbors two additional genetic factors responsible for 42 intersubspecific introgression-induced oligospermia (Hstx1) and reduced global meiotic 43 recombination rate (Meir1). To facilitate positional cloning and to overcome the 44 recombination suppression within the 4.3 Mb genomicDob interval encompassing the Hstx2 45 locus we designed Hstx2-CRISPR and SPO11/Cas9 transgenes aimed to induce DNA double-46 strand breaks specifically within the Hstx2 locus. The resulting recombinant reduced the 47Hstx2 locus to 2.70 Mb (Chr X:66.51-69.21 Mb). The newly defined Hstx2 still operates as 48 the major X-linked factor of the F1 hybrid sterility, controls meiotic chromosome synapsis, 49 and modifies meiotic recombination rate. Despite extensive further crosses, the 2.70 Mb 50Hstx2 interval behaved as a recombination cold spot with reduced PRDM9-mediated H3K4 51 hotspots and absence of DMC1-defined DNA DSB hotspots. To search for structural 52 anomalies as a possible cause of recombination suppression we used optical mapping of the 53 Hstx2 interval and observed high incidence of subspecies-specific structural variants along the 54 X chromosome, with a striking copy number polymorphism of the microRNA Mir465 cluster. 55Finally, we analyzed the role of one of the Hstx2 candidate genes, the Fmr1 neighbor 56 (Fmr1nb) gene in male fertility. 57 58 Article summary 59 Early meiotic arrest of mouse intersubspecific hybrids depends on the interaction 60 between the Prdm9 gene and Hybrid sterility X2 (Hstx2) locus on chromosome X. 61 4 Lustyk et al. conducted high-resolution genetic and physical mapping of the Hstx2 62 locus, reduced it to 2.7 Mb interval within a constitutive recombination cold spot and 63 found that the newly defined Hstx2 still operates as the X-linked hybrid sterility factor, 64 controls meiotic chromosome synapsis, and modifies recombination rate. Optical 65 mapping of the Hstx2 genomic region excluded inversion as a cause of recombination 66 suppression and revealed a striking copy number polymorphism of the microRNA 67 Mir465 cluster. 68 69 70 REPRODUCTIVE isolation is a basic prerequisite of speciation implemented by a range of 71 prezygotic and postzygotic mechanisms under complex genetic control (DOBZHANSKY 1951; 72 DION-COTE AND BARBASH 2017). Hybrid sterility, one of the reproductive isolation 73 mechanisms between closely related taxa appears in the early stages of speciation. Hybrid 74 sterility is a universal phenomenon, since infertility of many animal and plant species hybrids 75 share common features, such as preferential involvemen...