The novel hotspot at chr1 40.6 Mb, activated by PRDM9<sup>Cst</sup> on the B6 haplotype, shows transmission distortion to the CAST haplotype.

<p>(A) PRDM9<sup>Cst</sup> motif aligned to the DNA sequence for both B6 and CAST haplotypes at the peak of PRDM9 ChIP-seq signal (B) PRDM9 and H3K4me3 coverage profile at chr1 40.6 Mb (black lines – SNP positions between B6 and CAST, black box – PRDM9<sup>Cst</sup> motif). (C) Proportion of mapped reads to each haplotype (unassigned reads lack informative SNPs). (D) Cumulative frequency of recombination crossovers at hotspot chr1 40.6 Mb (n = 19 mice). (E) Transmission distortion at hotspot chr1 40.6 Mb favors the CAST haplotype at the PRDM9 binding site.</p>