House mouse Mus musculus dispersal in East Eurasia inferred from 98 newly determined complete mitochondrial genome sequences.

The Eurasian house mouse Mus musculus is useful for tracing prehistorical human movement related to the spread of farming. We determined whole mitochondrial DNA (mtDNA) sequences (ca. 16,000 bp) of 98 wild-derived individuals of two subspecies, M. m. musculus (MUS) and M. m. castaneus (CAS). We revealed directional dispersals reaching as far as the Japanese Archipelago from their homelands. Our phylogenetic analysis indicated that the eastward movement of MUS was characterised by five step-wise regional extension events: (1) broad spatial expansion into eastern Europe and the western part of western China, (2) dispersal to the eastern part of western China, (3) dispersal to northern China, (4) dispersal to the Korean Peninsula and (5) colonisation and expansion in the Japanese Archipelago. These events were estimated to have occurred during the last 2000-18,000 years. The dispersal of CAS was characterised by three events: initial divergences (ca. 7000-9000 years ago) of haplogroups in northernmost China and the eastern coast of India, followed by two population expansion events that likely originated from the Yangtze River basin to broad areas of South and Southeast Asia, including Sri Lanka, Bangladesh and Indonesia (ca. 4000-6000 years ago) and to Yunnan, southern China and the Japanese Archipelago (ca. 2000-3500). This study provides a solid framework for the spatiotemporal movement of the human-associated organisms in Holocene Eastern Eurasia using whole mtDNA sequences, reliable evolutionary rates and accurate branching patterns. The information obtained here contributes to the analysis of a variety of animals and plants associated with prehistoric human migration.

The ancestor of extant Japanese fancy mice contributed to the mosaic genomes of classical inbred strains.

Commonly used classical inbred mouse strains have mosaic genomes with sequences from different subspecific origins. Their genomes are derived predominantly from the Western European subspecies Mus musculus domesticus, with the remaining sequences derived mostly from the Japanese subspecies Mus musculus molossinus. However, it remains unknown how this intersubspecific genome introgression occurred during the establishment of classical inbred strains. In this study, we resequenced the genomes of two M. m. molossinus-derived inbred strains, MSM/Ms and JF1/Ms. MSM/Ms originated from Japanese wild mice, and the ancestry of JF1/Ms was originally found in Europe and then transferred to Japan. We compared the characteristics of these sequences to those of the C57BL/6J reference sequence and the recent data sets from the resequencing of 17 inbred strains in the Mouse Genome Project (MGP), and the results unequivocally show that genome introgression from M. m. molossinus into M. m. domesticus provided the primary framework for the mosaic genomes of classical inbred strains. Furthermore, the genomes of C57BL/6J and other classical inbred strains have long consecutive segments with extremely high similarity (>99.998%) to the JF1/Ms strain. In the early 20th century, Japanese waltzing mice with a morphological phenotype resembling that of JF1/Ms mice were often crossed with European fancy mice for early studies of "Mendelism," which suggests that the ancestor of the extant JF1/Ms strain provided the origin of the M. m. molossinus genome in classical inbred strains and largely contributed to its intersubspecific genome diversity.

A natural allele of Nxf1 suppresses retrovirus insertional mutations.

Endogenous retroviruses have shaped the evolution of mammalian genomes. Host genes that control the effects of retrovirus insertions are therefore of great interest. The modifier-of-vibrator-1 locus (Mvb1) controls levels of correctly processed mRNA from genes mutated by endogenous retrovirus insertions into introns, including the Pitpn(vb) tremor mutation and the Eya1(BOR) model of human branchiootorenal syndrome. Positional complementation cloning identifies Mvb1 as the nuclear export factor Nxf1, providing an unexpected link between the mRNA export receptor and pre-mRNA processing. Population structure of the suppressive allele in wild Mus musculus castaneus suggests selective advantage. A congenic Mvb1(CAST) allele is a useful tool for modifying gene expression from existing mutations and could be used to manipulate engineered mutations containing retroviral elements.

Clone identification in Japanese flowering cherry (Prunus subgenus Cerasus) cultivars using nuclear SSR markers.

Numerous cultivars of Japanese flowering cherry (Prunus subgenus Cerasus) are recognized, but in many cases they are difficult to distinguish morphologically. Therefore, we evaluated the clonal status of 215 designated cultivars using 17 SSR markers. More than half the cultivars were morphologically distinct and had unique genotypes. However, 22 cultivars were found to consist of multiple clones, which probably originate from the chance seedlings, suggesting that their unique characteristics have not been maintained through propagation by grafting alone. We also identified 23 groups consisting of two or more cultivars with identical genotypes. Most members of these groups were putatively synonymously related and morphologically identical. However, some of them were probably derived from bud sport mutants and had distinct morphologies. SSR marker analysis provided useful insights into the clonal status of the examined Japanese flowering cherry cultivars and proved to be a useful tool for cultivar characterization.

Detection of recombinant haplotypes in wild mice (Mus musculus) provides new insights into the origin of Japanese mice.

Japanese house mice (Mus musculus molossinus) are thought to be a hybrid lineage derived from two prehistoric immigrants, the subspecies M. m. musculus of northern Eurasia and M. m. castaneus of South Asia. Mice of the western European subspecies M. m. domesticus have been detected in Japanese ports and airports only. We examined haplotype structuring of a 200 kb stretch on chromosome 8 for 59 mice from throughout Eurasia, determining short segments (approximately 370-600 bp) of eight nuclear genes (Fanca, Spire2, Tcf25, Mc1r, Tubb3, Def8, Afg3l1 and Dbndd1) which are intermittently arranged in this order. Where possible we identified the subspecies origin for individual gene alleles and then designated haplotypes for concatenated alleles. We recovered 11 haplotypes among 19 Japanese mice examined, identified either as 'intact' haplotypes derived from the subspecies musculus (57.9%), domesticus (7.9%), and castaneus (2.6%), or as 'recombinant' haplotypes (31.6%). We also detected recombinant haplotypes unique to Sakhalin. The complex nature of the recombinant haplotypes suggests ancient introduction of all three subspecies components into the peripheral part of Eurasia or complicated genomic admixture before the movement from source areas. 'Intact'domesticus and castaneus haplotypes in other Japanese wild mice imply ongoing stowaway introductions. The method has general utility for assessing the history of genetic admixture and for disclosing ongoing genetic contamination.

Molecular cloning and characterization of the repetitive DNA sequences that comprise the constitutive heterochromatin of the A and B chromosomes of the Korean field mouse (Apodemus peninsulae, Muridae, Rodentia).

Three novel families of repetitive DNA sequences were molecularly cloned from the Korean field mouse (Apodemus peninsulae) and characterized by chromosome in-situ hybridization and filter hybridization. They were all localized to the centromeric regions of all autosomes and categorized into major satellite DNA, type I minor, and type II minor repetitive sequences. The type II minor repetitive sequence also hybridized interspersedly in the non-centromeric regions. The major satellite DNA sequence, which consisted of 30 bp elements, was organized in tandem arrays and constituted the majority of centromeric heterochromatin. Three families of repetitive sequences hybridized with B chromosomes in different patterns, suggesting that the B chromosomes of A. peninsulae were derived from A chromosomes and that the three repetitive sequences were amplified independently on each B chromosome. The minor repetitive sequences are present in the genomes of the other seven Apodemus species. In contrast, the major satellite DNA sequences that had a low sequence homology are present only in a few species. These results suggest that the major satellite DNA was amplified with base substitution in A. peninsulae after the divergence of the genus Apodemus from the common ancestor and that the B chromosomes of A. peninsulae might have a species-specific origin.

Disruption of genetic interaction between two autosomal regions and the X chromosome causes reproductive isolation between mouse strains derived from different subspecies.

Reproductive isolation that initiates speciation is likely caused by incompatibility among multiple loci in organisms belonging to genetically diverging populations. Laboratory C57BL/6J mice, which predominantly originated from Mus musculus domesticus, and a MSM/Ms strain derived from Japanese wild mice (M. m. molossinus, genetically close to M. m. musculus) are reproductively isolated. Their F1 hybrids are fertile, but successive intercrosses result in sterility. A consomic strain, C57BL/6J-ChrX(MSM), which carries the X chromosome of MSM/Ms in the C57BL/6J background, shows male sterility, suggesting a genetic incompatibility of the MSM/Ms X chromosome and other C57BL/6J chromosome(s). In this study, we conducted genomewide linkage analysis and subsequent QTL analysis using the sperm shape anomaly that is the major cause of the sterility of the C57BL/6J-ChrX(MSM) males. These analyses successfully detected significant QTL on chromosomes 1 and 11 that interact with the X chromosome. The introduction of MSM/Ms chromosomes 1 and 11 into the C57BL/6J-ChrX(MSM) background failed to restore the sperm-head shape, but did partially restore fertility. This result suggests that this genetic interaction may play a crucial role in the reproductive isolation between the two strains. A detailed analysis of the male sterility by intracytoplasmic sperm injection and zona-free in vitro fertilization demonstrated that the C57BL/6J-ChrX(MSM) spermatozoa have a defect in penetration through the zona pellucida of eggs.

Mosaic genealogy of the Mus musculus genome revealed by 21 nuclear genes from its three subspecies.

Patterns of genetic variation provide insight into the evolutionary history of a species. Mouse (Mus musculus) is a good model for this purpose. Here we present the analysis of genealogies of the 21 nuclear loci and one mitochondrial DNA region in M. musculus based on our nucleotide sequences of nine inbred strains from three M. musculus subspecies (musculus, domesticus, and castaneus) and one M. spicilegus strain as an outgroup. The mitochondrial DNA gene genealogy of those strains confirmed the introgression pattern of one musculus strain. When all the nuclear DNA data were concatenated to produce a phylogenetic tree of nine strains, musculus and domesticus strains formed monophyletic clusters with each other, while the two castaneus strains were paraphyletic. When each DNA region was treated independently, the phylogenetic networks revealed an unnegligibly high level of subspecies admixture and the mosaic nature of their genome. Estimation of ancestral and derived population sizes and migration rates suggests the effects of ancestral polymorphism and gene flow on the pattern of genetic variation of the current subspecies. Gene genealogies of Fut4 and Dfy loci also suggested existence of the gene flow between M. musculus and M. spicilegus or other distant species.

Origin and evolution of processed pseudogenes that stabilize functional Makorin1 mRNAs in mice, primates and other mammals.

We investigate the origin and evolution of a mouse processed pseudogene, Makorin1-p1, whose transcripts stabilize functional Makorin1 mRNAs. It is shown that Makorin1-p1 originated almost immediately before the musculus and cervicolor species groups diverged from each other some 4 million years ago and that the Makorin1-p1 orthologs in various Mus species are transcribed. However, Mus caroli in the cervicolor species group expresses not only Makorin1-p1, but also another older Makorin1-derived processed pseudogene, demonstrating the rapid generation and turnover in subgenus Mus. Under this circumstance, transcribed processed pseudogenes (TPPs) of Makorin1 evolved in a strictly neutral fashion even with an enhanced substitution rate at CpG dinucleotide sites. Next, we extend our analyses to rats and other mammals. It is shown that although these species also possess their own Makorin1-derived TPPs, they occur rather infrequently in simian primates. Under this circumstance, it is hypothesized that already existing TPPs must be prevented from accumulating detrimental mutations by negative selection. This hypothesis is substantiated by the presence of two rather old TPPs, MKRNP1 and MKRN4, in humans and New World monkeys. The evolutionary rate and pattern of Makorin1-derived processed pseudogenes depend heavily on how frequently they are disseminated in the genome.

Further evidence for recombination between mouse hemoglobin beta b1 and b2 genes based on the nucleotide sequences of intron, UTR, and intergenic spacer regions.

Nucleotide sequences of the intron regions and UTRs (Untranslated regions) of the hemoglobin beta adult genes, b1 and b2, and of the intergenic spacer region were determined for mouse strains representing the d, p, and w1 hemoglobin haplotypes defined by protein electrophoretic analyses. The hypothesis of recombination of the b1 and b2 genes between the d and w1 haplotypes previously reported in the cDNA nucleotide sequences was confirmed by neighbor-joining analyses of the intron regions and UTRs within the b1 and b2 genes, suggesting that all of the structures of hemoglobin beta adult genes support the hypothesis that the p haplotype was established by hybridization between d and w1 haplotype mice. The resultant recombinant of the p haplotype was found to have a d-like b1 gene and a w1-like b2 gene. In addition to the possible recombination, a break point was suggested around 2-3 kb downstream of the b1 gene within the intergenic spacer region, despite the absence of clear properties that could stimulate the recombination machinery. Some large insertions or deletions (indels) specific to the p or d haplotypes were located within the intergenic spacer region, in which the 1010-bp indel specific to the p haplotype was shared by all examined strains representing the p haplotype.

Mouse phenome research: implications of genetic background.

Now that sequencing of the mouse genome has been completed, the function of each gene remains to be elucidated through phenotypic analysis. The "genetic background" (in which each gene functions) is defined as the genotype of all other related genes that may interact with the gene of interest, and therefore potentially influences the specific phenotype. To understand the nature and importance of genetic background on phenotypic expression of specific genes, it is necessary to know the origin and evolutionary history of the laboratory mouse genome. Molecular analysis has indicated that the fancy mice of Japan and Europe contributed significantly to the origin of today's laboratory mice. The genetic background of present-day laboratory mice varies by mouse strain, but is mainly derived from the European domesticus subspecies group and to a lesser degree from Asian mice, probably Japanese fancy mice, which belong to the musculus subspecies group. Inbred laboratory mouse strains are genetically uniform due to extensive inbreeding, and they have greatly contributed to the genetic analysis of many Mendelian traits. Meanwhile, for a variety of practical reasons, many transgenic and targeted mutant mice have been created in mice of mixed genetic backgrounds to elucidate the function of the genes, although efforts have been made to create inbred transgenic mice and targeted mutant mice with coisogenic embryonic stem cell lines. Inbred mouse strains have provided uniform genetic background for accurate evaluation of specific genes phenotypes, thus eliminating the phenotypic variations caused by mixed genetic backgrounds. However, the process of inbreeding and selection of various inbred strain characteristics has resulted in inadvertent selection of other undesirable genetic characteristics and mutations that may influence the genotype and preclude effective phenotypic analysis. Because many of the common inbred mouse stains have been established from relatively small gene pools, common inbred strains have limitations in their genetic polymorphisms and phenotypic variations. Wild-derived mouse strains can complement deficiencies of common inbred mouse strains, providing novel allelic variants and phenotypes. Although wild-derived strains are not as tame as the common laboratory strains, their genetic characteristics are attractive for the future study of gene function.

Length variation of CAG/CAA triplet repeats in 50 genes among 16 inbred mouse strains.

CAG repeats coding for poly-glutamines have been studied by many groups as repeat length variations contributes to differences in protein function and disease outcome. In this study, we systematically searched public databases for genes carrying CAG repeats. For the genes obtained, we experimentally analyzed variations of length and the purity of the repeats in 62 loci among 16 inbred mouse strains, including wild-derived and laboratory strains. We found that length was conserved in 50% of the loci, especially among wild-derived strains. Of 496 polymorphic repeat alleles, 78% were uninterrupted and 22% were interrupted with non-CAG codons. Interruptions tended to occur in longer repeats and all repeats of greater length than 23 were interrupted. Although interruptions can act as suppressors for the expansion of CAG repeats, we found that the occurrence of the interruptions depended on the length of the CAG repeats. Furthermore, most poly-glutamines examined in this study existed in human orthologous genes, reflecting the functional significance of poly-glutamines in proteins.

Hybrid breakdown caused by substitution of the X chromosome between two mouse subspecies.

Hybrid breakdown is a type of reproductive failure that appears after the F2 generation of crosses between different species or subspecies. It is caused by incompatibility between interacting genes. Genetic analysis of hybrid breakdown, particularly in higher animals, has been hampered by its complex nature (i.e., it involves more than two genes, and the phenotype is recessive). We studied hybrid breakdown using a new consomic strain, C57BL/6J-X(MSM), in which the X chromosome of C57BL/6J (derived mostly from Mus musculus domesticus) is substituted by the X chromosome of the MSM/Ms strain (M. m. molossinus). Males of this consomic strain are sterile, whereas F1 hybrids between C57BL/6J and MSM/Ms are completely fertile. The C57BL/6J-X(MSM) males showed reduced testis weight with variable defects in spermatogenesis and abnormal sperm head morphology. We conducted quantitative trait locus (QTL) analysis for these traits to map the X-linked genetic factors responsible for the sterility. This analysis successfully detected at least three distinct loci for the sperm head morphology and one for the testis weight. This study revealed that incompatibility of interactions of X-linked gene(s) with autosomal and/or Y-linked gene(s) causes the hybrid breakdown between the genetically distant C57BL/6J and MSM/Ms strains.

Histological studies on male sterility of hybrids between laboratory and wild mouse strains: (hybrid sterility/Hst-1 locus/spermatogenesis/chimera/Sertoli cell).

In this study the cellular mechanisms of male sterility in F1 hybrids (BNF1) between BALB/c and wild-derived M.MUS-NJL (NJL) was investigated. Cell proliferation and differentiation in the sterile testis were examined by bromodeoxyuridine-labeling and use of germ cell stage-specific antibodies. In BNF1 testes, spermatogonia actively proliferated with a seminiferous epithelial cycle, and were retained in the basal layer of the tubules. However, preleptotene, leptotene and zygotene spermatocytes moved to the adluminal region. Immunohistological data with germ cell stage-specific antibodies indicated the presence of few, if any, pachytene spermatocytes in BNF1 testes. Thus, spermatogenesis seemed to be blocked at the zygotene stage. For examination of germ cell-Sertoli cell interactions, testes of aggregation chimeras between BNF1 and C3H/HeN were analyzed immunohistologically with C3H-specific antibody. Results showed that spermatogenesis of C3H-germ cells was normal, even when these cells in contact with BNF1-Sertoli cells. Differentiation of BNF1-germ cells progressed from zygotene to pachytene stage spermatocytes when these cells were surrounded by C3H-Sertoli cells, but never proceeded beyond the pachytene stage. These observations suggest that at least two different cellular factors may be involved in spermatogenesis, one acting in the germ cells and the other mediated by Sertoli cells. Furthermore, mating experiments revealed that the degree of spermatogenesis varied in different F1 hybrids, and that the major sterility factor was closely linked to the T-locus on chromosome 17.

The ter mutation first causes primordial germ cell deficiency in ter/ter mouse embryos at 8 days of gestation.

The ter (teratoma) mutation causes primordial germ cell (PGC) deficiency in ter/ter embryos at 9.5-12.5 days of post-coitum (dpc) in mouse strains 129/Sv-ter and LTXBJ-ter. To study the effects of the ter mutation on the PGC development more precisely, we examined the PGC number and distribution in 7.5-12.5 dpc embryo of ter congenic C57BL/6J-ter strain using their complete serial sections. The ter genotypes of embryos were identified by the polymerase chain reaction (PCR) polymorphisms of the microsatellite DNA of the Grl-1 locus mapped near the ter locus. Results showed that: (i) the PGC number in ter/ter embryos was similar to those of + /ter and + / + embryos at 7.5 dpc, and did not increase at 8.0-12.5 dpc, although those of normal littermates did usually; (ii) the PGC migration to genital ridges was never affected in all embryos; and (iii) the ter genotype difference in the PGC numbers was not recognized between + /ter and + / + embryos. We concluded that the ter mutation does not affect the PGC appearance around 7.5 dpc, but first causes PGC deficiency around 8.0 dpc at the beginning of their migration and proliferation, suggesting that the normal function of the ter gene may be essential for the proliferation or survival mechanisms of PGC.

The ter mutation responsible for germ cell deficiency but not testicular nor ovarian teratocarcinogenesis in ter / ter congenic mice.

The ter (teratoma) gene causes germ cell deficiency and a high incidence of congenital testicular teratomas derived from primordial germ cells in 129/Sv-ter strain mice. Ovarian teratomas in LTXBJ mice originate from ovarian parthenotes. In order to study the function of the ter gene in germ cell development and teratocarcinogenesis, we examined the influence of a foreign genetic background on the ter action by introducing the ter gene of 129/Sv-ter strain mice into C57BL/6J, LTXBJ and C3H/HeJ genetic backgrounds by the backcross method and by thus establishing B6-ter, LTXBJ-ter and C3H-ter ter congenic strains, respectively. Histological analysis showed that germ cell deficiency occurred in both sexes of the ter mutants, through the fetal stages to adulthood, but that congenital testicular teratocarcinogenesis did not occur after the fifth backcross generation. The ter/ter gonads were smaller than normal (+/+ or +/ter). Experimental testicular teratomas never developed from intratesticular grafts of B6-ter genital ridges. LTXBJ-ter/ter females had no ovarian teratomas. It is concluded that the ter gene is solely responsible for germ cell deficiency, but not testicular teratocarcinogenesis, in ter congenic strains having background genes other than 129/Sv-ter and that the ter gene is not involved in ovarian teratocarcinogenesis.

STRAIN-SPECIFIC APPEARANCE OF A POSSIBLE FETAL α-GLOBIN POLYPEPTIDE CHAIN IN MICE.

DDD mouse embryos at 12-16 days of gestation have a putative 'fetal'α-globin polypeptide chain, detected by its lower electrophoretic mobility than the authentic α-globin chain on acidic urea-Triton gel. When hepatic erythroid cells of DDD embryos were contaminated with yolk sac erythroid cells, two α-bands were visible, but only 'fetal'α seemed to be synthesized, because in embryos of strain ddY which share a common ancestry with DDD, only 'fetal'α was detectable by autoradiography when the hepatic erythroid cells of embryos of more than 15 days of gestation showed an authentic adult α-globin chain other than this 'fetal'α-globin chain. If this 'fetal'α-globin chain is structurally different from the yolk sac α and adult peripheral blood α-globin chains, then switching over of the transcription of α-globin polypeptide chain occurs in hepatic erythroid cells of mid-late DDD embryos. However, it is still possible that this 'fetal'α-globin chain is formed by post-translational modification of the authentic α-globin chain. The α-globin chains of hepatic erythroid cells of C57BL/6 and BALB/c embryos had similar mobilities to those of yolk sac and adult erythroid cells.

Genetic diversity underlying capsaicin intake in the Mishima battery of mouse strains.

Capsaicin is the active substance responsible for the pungent sensation produced by red pepper. In order to approach the underlying genetic mechanism for preference of red pepper, we conducted a 12-h, 1-bottle intake test of capsaicin solution using both male and female animals from the Mishima battery of mouse strains: 10 wild-derived inbred strains (PGN2, BFM/2, HMI, CAST/Ei, NJL, BLG2, CHD, SWN, KJR, MSM), 1 strain derived from the so-called fancy mouse (JF1), and 3 widely used laboratory strains (C57BL/6J, DBA/1J and BALB/cAnN). The concentration of capsaicin was increased from 0.5 to 15 microM successively. Gender differences were not observed in this test, but we found striking strain differences in capsaicin intake. Relative to baseline water intake, C57BL/6J and DBA/1J consumed 10%, whereas two wild strains, KJR and MSM, ingested approximately 60% of the 15-microM capsaicin solution. In a 2-bottle fluid preference test, both C57BL/6J and MSM strains reject capsaicin fluid even at the 0.5-microM concentration, which indicates that the receptors for capsaicin in these strains recognize capsaicin at a similar level. Thus, the strain differences at higher capsaicin concentrations in the 1-bottle test may reflect differences in central nervous system response to the capsaicin solution. The genetic difference in intake of capsaicin observed in these strains may provide a useful tool for identifying genes underlying response to red pepper in mice and other mammalian species.

Update of mouse microsatellite database of Japan (MMDBJ).

We updated a database of microsatellite marker polymorphisms found in inbred strains of the mouse, most of which were derived from the wild stocks of four Mus musculus subspecies, M. m. domesticus, M. m. musculus, M. m.castaneus and M. m. molossinus. The major aim of constructing this database was to establish the genetic status of these inbred strains as resources for linkage analysis and positional cloning. The inbred strains incorporated in our database are A/J, C57BL/6J, CBA/J, DBA/2J, SM/J, SWR/J, 129Sv/J, MSM/Ms, JF1/Ms, CAST/Ei, NC/Nga, BLG2/Ms, NJL/Ms, PGN2/Ms, SK/CamEi and SWN/Ms, which have not or have only been poorly incorporated in the Whitehead Institute/MIT (WI/MIT) microsatellite database. The number of polymorphic microsatellite loci incorporated in our database is over 1,000 in all strains, and the URL site for our database is located at http:// www.shigen.nig.ac.jp /mouse/mmdbj/mouse.html.

Prdm9 polymorphism unveils mouse evolutionary tracks.

PR/SET domain containing 9 (Prdm9) mediates histone modifications such as H3K4me3 and marks hotspots of meiotic recombination. In many mammalian species, the Prdm9 gene is highly polymorphic. Prdm9 polymorphism is assumed to play two critical roles in evolution: to diversify the spectrum of meiotic recombination hotspots and to cause male hybrid sterility, leading to reproductive isolation and speciation. Nevertheless, information about Prdm9 sequences in natural populations is very limited. In this study, we conducted a comprehensive population survey on Prdm9 polymorphism in the house mouse, Mus musculus. Overall M. musculus Prdm9 displays an extraordinarily high level of polymorphism, particularly in regions encoding zinc finger repeats, which recognize recombination hotspots. Prdm9 alleles specific to various M. musculus subspecies dominate in subspecies territories. Moreover, introgression into other subspecies territories was found for highly divergent Prdm9 alleles associated with t-haplotype. The results of our phylogeographical analysis suggest that the requirement for hotspot diversity depends on geographical range and time span in mouse evolution, and that Prdm9 polymorphism has not been maintained by a simple balanced selection in the population of each subspecies.