Mammalian meiotic recombination, which preferentially occurs at specialized sites called hotspots, assures the orderly segregation of meiotic chromosomes and creates genetic variation among offspring. A locus on mouse Chr 17, that controls activation of recombination at multiple distant hotspots, has been mapped within a 181 Kb interval, three of whose genes can be eliminated as candidates. The remaining gene, Prdm9, codes for a zinc finger containing histone H3K4 trimethylase that is uniquely expressed in early meiosis and whose deficiency results resulting in sterility in both sexes. Mus musculus exhibits five alleles of Prdm9; human populations exhibit two predominant alleles and several minor alleles. The discovery of Prdm9 as the first protein regulating mammalian recombination hotspots initiates molecular studies of this important biological control system. TextGenetic recombination is an essential biological process among eukaryotes. Mammalian meiotic recombination, which preferentially occurs at specialized sites, 1-2 Kb long, known as hotspots, assures the orderly segregation of meiotic chromosomes and creates genetic variation among offspring. However, despite their importance in determining the recombination landscape of the organism, we have little understanding of the elements determining the location and relative activity of hotspots. That the location of a hotspot is not determined simply by its internal DNA sequence is supported by both yeast 1 and mammalian studies 2,3 . Two recent reports 4,5 have described the existence of trans-acting loci that control the activation of specific hotspots elsewhere. The loci, Dsbc1 and Rcr1, are located in overlapping 5.4 Mb and 6.3 Mb regions on mouse chromosome 17.We have now extended the mapping of both Rcr1 and Dsbc1 using 1580 progeny of a cross between the C57BL/6J (B6) and CAST/EiJ (CAST) mouse strains differing in activity of the Rcr1/ Dsbc1 controlled hotspots Hlx1, Esrrg-1 and Psmb9 (Fig. S1). We located both loci to a common 181 Kb region containing four protein coding genes ( Fig. 1): Psmb1, Tbp, Pdcd2, and Prdm9. We found no differences in expression levels of the transcripts of any of these proteins in testes when assayed by RT-PCR (Table S1).Psmb1, proteasome beta type 1 subunit, is a ubiquitously expressed protein that functions as a structural component of an organelle responsible for generalized protein degradation. Tbp, TATA binding protein, is an essential component of the TFIID transcription initiation complex which has considerable DNA binding specificity. Pdcd2, programmed cell death protein 2, is widely expressed, repressed during B cell lymphomagenesis. Based on DNA sequencing of the entire coding regions of these three proteins, we found only five coding SNPs, all synonymous, resulting in no amino acid differences between the B6 and CAST strains (Table

A set of 1638 informative SNP markers easily assayed by the Amplifluor genotyping system were tested in 102 mouse strains, including the majority of the common and wild-derived inbred strains available from The Jackson Laboratory. Selected from publicly available databases, the markers are on average ∼1.5 Mb apart and, whenever possible, represent the rare allele in at least two strains. Amplifluor assays were developed for each marker and performed on two independent DNA samples from each strain. The mean number of polymorphisms between strains was 608±136 SD. Several tests indicate that the markers provide an effective system for performing genome scans and quantitative trait loci analyses in all but the most closely related strains. Additionally, the markers revealed several subtle differences between closely related mouse strains, including the groups of several 129, BALB, C3H, C57, and DBA strains, and a group of wild-derived inbred strains representing several Mus musculus subspecies. Applying a neighbor-joining method to the data, we constructed a mouse strain family tree, which in most cases confirmed existing genealogies.

In mammals, genetic recombination during meiosis is limited to a set of 1-to 2-kb regions termed hotspots. Their locations are predominantly determined by the zinc finger protein PRDM9, which binds to DNA in hotspots and subsequently uses its SET domain to locally trimethylate histone H3 at lysine 4 (H3K4me3). This sets the stage for double-strand break (DSB) formation and reciprocal exchange of DNA between chromatids, forming Holliday junctions. Here we report genomewide analyses of PRDM9-dependent histone modifications using two inbred mouse strains differing only in their PRDM9 zinc finger domain. We show that PRDM9 binding actively reorganizes nucleosomes into a symmetrical pattern, creating an extended nucleosome-depleted region. These regions are centered by a consensus PRDM9 binding motif, whose location and identity was confirmed in vitro. We also show that DSBs are centered over the PRDM9 binding motif within the nucleosome-depleted region. Combining these results with data from genetic crosses, we find that crossing-over is restricted to the region marked by H3K4me3. We suggest that PRDM9-modified nucleosomes create a permissible environment that first directs the location of DSBs and then defines the boundaries of Holliday junction branch migration.[Supplemental material is available for this article.]

Among mammals, genetic recombination occurs at highly delimited sites known as recombination hotspots. They are typically 1–2 kb long and vary as much as a 1,000-fold or more in recombination activity. Although much is known about the molecular details of the recombination process itself, the factors determining the location and relative activity of hotspots are poorly understood. To further our understanding, we have collected and mapped the locations of 5,472 crossover events along mouse Chromosome 1 arising in 6,028 meioses of male and female reciprocal F1 hybrids of C57BL/6J and CAST/EiJ mice. Crossovers were mapped to a minimum resolution of 225 kb, and those in the telomere-proximal 24.7 Mb were further mapped to resolve individual hotspots. Recombination rates were evolutionarily conserved on a regional scale, but not at the local level. There was a clear negative-exponential relationship between the relative activity and abundance of hotspot activity classes, such that a small number of the most active hotspots account for the majority of recombination. Females had 1.2× higher overall recombination than males did, although the sex ratio showed considerable regional variation. Locally, entirely sex-specific hotspots were rare. The initiation of recombination at the most active hotspot was regulated independently on the two parental chromatids, and analysis of reciprocal crosses indicated that parental imprinting has subtle effects on recombination rates. It appears that the regulation of mammalian recombination is a complex, dynamic process involving multiple factors reflecting species, sex, individual variation within species, and the properties of individual hotspots.

Genotyping microarrays are an important resource for genetic mapping, population genetics, and monitoring of the genetic integrity of laboratory stocks. We have developed the third generation of the Mouse Universal Genotyping Array (MUGA) series, GigaMUGA, a 143,259-probe Illumina Infinium II array for the house mouse (Mus musculus). The bulk of the content of GigaMUGA is optimized for genetic mapping in the Collaborative Cross and Diversity Outbred populations, and for substrain-level identification of laboratory mice. In addition to 141,090 single nucleotide polymorphism probes, GigaMUGA contains 2006 probes for copy number concentrated in structurally polymorphic regions of the mouse genome. The performance of the array is characterized in a set of 500 high-quality reference samples spanning laboratory inbred strains, recombinant inbred lines, outbred stocks, and wild-caught mice. GigaMUGA is highly informative across a wide range of genetically diverse samples, from laboratory substrains to other Mus species. In addition to describing the content and performance of the array, we provide detailed probe-level annotation and recommendations for quality control.