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Contribution of Asian mouse subspecies Mus musculus
molossinus to genomic constitution of strain C57BL/6J, as definedby BAC-end sequence–SNP analysis
Kuniya Abe, Hideki Noguchi, Keiko Tagawa, Misako Yuzuriha, Atsushi Toyoda, Toshio Kojima, Kiyoshi
Ezawa, Naruya Saitou, Masahira Hattori, Yoshiyuki Sakaki, Kazuo Moriwaki and Toshihiko Shiroishi
Genome Res. 2004 14: 2439-2447
Access the most recent version at doi:10.1101/gr.2899304

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LetterContribution of Asian mouse subspecies Mus
musculus molossinus to genomic constitutionof strain C57BL/6J, as defined by BAC-end
sequence–SNP analysis
Kuniya Abe,1,2,10 Hideki Noguchi,3,10 Keiko Tagawa,4 Misako Yuzuriha,1Atsushi Toyoda,3 Toshio Kojima,5 Kiyoshi Ezawa,6 Naruya Saitou,6 Masahira Hattori,5,7
Yoshiyuki Sakaki,3 Kazuo Moriwaki,1 and Toshihiko Shiroishi8,9,10
1

Technology and Development Team for Mammalian Cellular Dynamics, BioResource Center, RIKEN Tsukuba Institute, Tsukuba,
Ibaraki 305-0074, Japan; 2Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki
305-0006, Japan; 3Sequence Technology Team, Genome Core Technology Facility, RIKEN Genomic Sciences Center, Tsurumi-ku,
Yokohama, Kanagawa 230-0045, Japan; 4Department of Developmental Genetics, Institute of Molecular Embryology andGenetics, Kumamoto University, Kumamoto 862-0976, Japan; 5Human Genome Research Group, Genomic Sciences Center,
RIKEN Yokohama Institute, Tsurumi-ku, Yokohama 230-0045, Japan; 6Division of Population Genetics, National Institute of
Genetics, Mishima, Shizuoka 411-8540, Japan; 7Kitasato Institute for Life Science, Kitasato University, Sagamihara, Kanagawa
228-8555 Japan; 8Mammalian Genetics Laboratory, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan;
9
Mouse Functional Genomics Research Groups, Genomic Sciences Center, RIKEN Yokohama Institute,
Yokohama, Kanagawa 244-0804, Japan
MSM/Ms is an inbred strain derived from the Japanese wild mouse, Mus musculus molossinus. It is believed thatsubspecies molossinus has contributed substantially to the genome constitution of common laboratory strains of mice,although the majority of their genome is derived from the west European M. m. domesticus. Information on themolossinus genome is thus essential not only for genetic studies involving molossinus but also for characterization ofcommon laboratory strains. Here, we report the construction of an arrayed bacterial artificial chromosome (BAC)
library from male MSM/Ms genomic DNA, covering ∼11× genome equivalent. Both ends of 176,256 BAC clone
inserts were sequenced, and 62,988 BAC-end sequence (BES) pairs were mapped onto the C57BL/6J genome (NCBI
mouse Build 30), covering 2,228,164 kbp or 89% of the total genome. Taking advantage of the BES map data, weestablished a computer-based clone screening system. Comparison of the MSM/Ms and C57BL/6J sequences revealed
489,200 candidate single nucleotide polymorphisms (SNPs) in 51,137,941 bp sequenced. The overall nucleotide
substitution rate was as high as 0.0096. The distribution of SNPs along the C57BL/6J genome was not uniform: Themajority of the genome showed a high SNP rate, and only 5.2% of the genome showed an extremely low SNP rate
(percentage identity = 0.9997); these sequences are likely derived from the molossinus genome.
[Supplemental material is available online at www.genome.org, and the MSM BAC database is available at
http://stt.gsc.riken.jp/msm/ or http://analysis1.lab.nig.ac.jp/Mus_musculus/.]
A number of inbred mouse strains characterized by different heritable traits have been established, and these strains are extensively used in biomedical research (Atchley and Fitch 1991; Becket al. 2000). The distinct phenotypes manifested by these laboratory strains are attributable to differences in the genomic constitution carried by each strain. However, the gene pools of thecommonly used laboratory mouse strains are probably small, as
they were derived from a mixed, small number of fancy micebred at the beginning of the 20th century (Morse III 1981; Silver
1995).
10
Corresponding authors.
E-mail: abe@rtc.riken.jp; fax 81 29-836-9199.
E-mail: hide@gsc.riken.go.jp; fax 81 45-503-9170.
E-mail: tshirois@lab.nig.ac.jp; fax 81 55-981-6817.
Article and publication are at http://www.genome.org/cgi/doi/10.1101/
gr.2899304.

The house mouse, Mus musculus, is a complex species comprised of several subspecies. Results of our past studies (Yonekawa
et al. 1980, 1982; Moriwaki 1994) and those of others (Ferris et al.
1982; Bonhomme et al. 1987) suggest that commonly used laboratory strains were derived predominantly from the Mus musculus
domesticus subspecies, which is indigenous to Western Europe
and the Mediterranean Basin, and that there were some small
contributions from the Japanese fancy mouse, which originatedfrom the Mus musculus molossinus subspecies (Morse III 1981;
Silver 1995). Thus, the laboratory strains can be considered anatural set of recombinant inbred strains derived from a small
number of mice belonging to either M. m. domesticus or Asiansubspecies (Wade et al. 2002). Different combinations of genomic segments contributed by the two major ancestral sources
may have created the diverse phenotypic traits characteristic ofeach strain. Therefore, information as to how these inbred strains

14:2439–2447 ©2004 by Cold Spring Harbor Laboratory Press; ISSN 1088-9051/04; www.genome.org

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Abe et al.
in the Mouse Phenome Project, and therefore accumulating data
originated historically and how they are related to each ancestralof phenotypes will be available (Paigen and Eppig 2000).
subspecies is crucial in any genetic analyses of the phenotypes ofHere we describe the general characteristics of the MSM/Ms
interest.
BAC library, which is the first, representative BAC library from M.
Recent analyses using single nucleotide polymorphism
m. molossinus, and the results of BAC-end sequence (BES) analysis(SNP) markers across the whole mouse genome have revealed
uneven distributions of SNPs along the genomes of the laboraof about 180,000 clones. We mapped the MSM/Ms BESs onto thetory strains (Wade et al. 2002; Wiltshire et al. 2003). From pairC57BL/6J (B6) genome assembly (NCBI Build 30). Comparison ofwise comparisons of inbred strains, Wade et al. (2002) found that
the MSM/Ms sequences with the B6 data provided a vast number
the genomes of the laboratory strains have mosaic structures conof SNPs with defined genomic locations. Using this SNP inforsisting of long segments of either low or high polymorphism
mation, we present the fine details of the genetic variation berates, and they proposed that the high-SNP-rate regions are retween M. m. molossinus and C57BL/6J, one of the most widely
flections of intersubspecific polymorphisms between M. m. doused laboratory strains.
mesticus and Asian subspecies, M. m. molossinus or M. m. musculus. However, this notion is based on rather indirect evidence
Results
obtained mainly from the genomic data on common laboratory
strains, and the precise structures of such mosaic patterns in eachConstruction of MSM BAC librarystrain have not been presented. Currently, sizable data on genomic sequences are available only for the common laboratory
A BAC library was constructed from genomic DNA of male MSM/
strains, and our knowledge of the genomic constitutions of otherMs (henceforth MSM), an inbred strain derived from mouse submouse subspecies is very limited. Such scarcity of genomic inforspecies groups of M. m. molossinus (Moriwaki 1994). Highmation hampers our better understanding of the genomic evomolecular-weight DNA isolated from MSM male mice was parlution of M. musculus subspecies and the origin of the common
tially digested with a combination of EcoRI and EcoRI methylase
laboratory strains, as well as of the mosaic structures of variations
and cloned into pBACe3.6 vector (Osoegawa et al. 1998). The
among those strains.
library was composed of 210,432 BAC clones arrayed in 384-well
We therefore attempted to gain genomic information frommicrotiter plates. The average insert size of the library was 135.1
Asian subspecies. For this purpose, we chose MSM/Ms as a target
kb, as determined by pulsed-field gel electrophoresis analysis ofstrain, because there are several lines of evidence that M. m. mo162 randomly selected clones (Fig. 1). Thus this library has ∼11.9
lossinus predominantly contributed to the genomes of the com⳯ coverage of the haploid mouse genome. This library can be
mon laboratory strains (Nagamine et al. 1992; Floyd et al. 2003).
screened by hybridizing probes with high-density colony blot or
We constructed a bacterial artificial chromosome (BAC) genomic library, a fundamental resource for a variety of genomic research (Zhao et al. 2001) and subsequently
sequenced the BAC-ends of ∼180,000
clones. The MSM/Ms strain was establishedfrom Japanese wild mice, M. m. molossinus,
collected in 1978 in Mishima, Japan (Moriwaki 1994). The inbreeding generations of
this strain had reached N71 at the end of
2003, and it is now established as a pureinbred strain. Genetic analysis using the
MSM/Ms strain has several advantages: (1)
MSM/Ms has unique characteristics not observed in the other laboratory strains, forexample, extremely low incidence of tumor
development (Miyashita and Moriwaki
1987) and characteristic behavioral phenotypes (Koide et al. 2000); (2) it shows highbreeding performance (100–125 N2 progeny/pair/year); (3) it has a number of polymorphic DNA markers (more than 2000
SSLP markers are now available for the crossof MSM/Ms with the common laboratory
strains; Kikkawa et al. 2001 and see Mouse
Microsatellite Database of Japan [MMDBJ],
http://www.shigen.nig.ac.jp/mouse/
mmdbj/top.jsp); (4) a full series of consoFigure 1. Estimation and distribution of insert sizes in MSM BAC clones. (A) Randomly chosen
mic strains, in which each chromosome of
BACs were digested with NotI that flank the cloning site (EcoRI) in the pBACe3.6 vector. The
MSM/Ms is introduced into the B6 backdigested DNAs were analyzed by pulsed-field gel electrophoresis on a 1% agarose gel in 0.5⳯ TBE
ground by repeated backcross, is estabat 6V/cm, with 0.1 sec to 40 sec pulse time for 16 h at 14°C. The DNA markers are ␭ concatemers
lished (T. Shiroishi, unpubl.); and (5) MSM/ plus HindIII-digested ␭DNA. Horizontal black bars indicate positions of the concatemers. The arrow
Ms has been selected as a target strain
indicates the position of the vector. Distribution of insert size is shown in (B).

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Genomic constitution defined by BAC–SNP analysis
by performing PCR with DNA pools consisting of mixtures of the
BAC DNA isolated from each of the library plates (data notshown).

Derivation of BAC-end sequences and their mapping onto themouse genome sequence
To exploit this genomic resource fully, we sequenced both endsof the BAC clones and mapped the BESs onto the B6 genome
assembly (NCBI mouse Build 30). The precise assignment of each
BAC clone position along the chromosomes by the BES mapping
enabled computer-based screening of the library. BES mapping
can also provide information on chromosomal rearrangements
between the MSM and the B6 (Volik et al. 2003). More importantly, comparison of the MSM sequences with the B6 data will
generate a vast amount of SNPs with defined genomic locations.
The distribution patterns of the SNPs will reveal the fine details of
the genetic variation between the two M. musculus subspecies(Wade et al. 2002).
Both ends of 176,256 BAC clone inserts were sequenced,
corresponding roughly to 6.4% of the haploid mouse genome.
After we had trimmed the vector sequences and masked any
highly repetitive sequences, we performed a BLAST search of the
BESs against the B6 genome to determine the genomic locationsof these BAC clones. For this mapping, we considered the size ofhomologous sequences, percentage identity, sequence orientation, and the average size of the BAC inserts (<300 kb). If the BES
hit multiple locations, the position of the region showing thehighest BLAST score was assigned. The ‘paired-end’ sequenceswere selected from 62,988 BACs. From the ‘paired-end’ group,
38,611 clones with ‘unique paired-end’ sequences were further
selected; these were mapped unambiguously to one location in
the genome. Coverage of the mouse genome by the MSM BAC
clones was estimated on the basis of the results of the BES mapping. As shown in Table 1, 89% of the B6 genome sequence (i.e.,
2,228,164 kbp) was covered by the ‘paired-end’ clones. In otherwords, 62,988 ⳯ 2 = 125,976 BESs were interspersed across the2,228,164 kbp genomic sequence (i.e., one BES was found in

Table 1. Coverage of the genome by the MSM BAC clones

Chromosome

B6 genome
sequenced(kb)

MSM
covered(kb)

Coverage

No. ofclones

chr1
chr2
chr3
chr4
chr5
chr6
chr7
chr8
chr9
chr10
chr11
chr12
chr13
chr14
chr15
chr16
chr17
chr18
chr19
chrX
Total

191,720
177,924
156,874
149,422
145,320
145,901
128,702125,273
120,867
127,088
119,563
110,213112,393
112,144
100,91295,83789,43087,841
57,643
145,596
2,500,661 (kb)172,683
162,218
141,439
133,339
130,549
128,731
112,502113,156
109,326
114,979
111,820
100,598
104,410
102,497
94,315
88,132
79,65880,94953,08893,7752,228,164 (kb)

0.90
0.91
0.90
0.89
0.90
0.88
0.87
0.90
0.90
0.90
0.94
0.91
0.93
0.91
0.93
0.92
0.89
0.92
0.92
0.64
0.89

4,597
4,839
4,056
3,674
3,706
3,718
3,056
3,336
3,057
3,212
3,402
3,040
2,948
3,052
2,659
2,556
2,226
2,389
1,653
1,452
62,988every 17.68 kb). The sum of the ‘paired-end’ BES size was51,137,941 bp (average, 406 bp per BES), corresponding to 2.29%of the total genome size. Coverage for each chromosome wassimilar, with the exception of the X chromosome (Table 1),
which we would expect to be underrepresented in this librarybecause male genomic DNA was used as a source material. BESs
often carry highly repetitive sequences that prevent mapping
onto the genome (Zhao et al. 2001). Clones with unique hit
sequences at one end but unmapped sequences at the other andthose with unmapped sequences at both ends were excluded
from the analysis described above.
To show the effective coverage of the genome by paired-end
clones in detail and to establish a convenient means of identifying BAC clones, we made a Web browser-based BAC library
screening system (http://stt.gsc.riken.jp/msm/). In this system, it
is possible to view genomic regions of interest covered by MSM
BAC clones by either clicking on a region of interest on the chromosome map in the top menu or typing a gene symbol or asequence ID in the search window (Fig. 2A,B). In this example,
the genomic region covering H-2K gene of mouse major histocompatibility complex is shown (Fig. 2B). The BAC clones with a
red triangle at both ends represent the ‘unique paired-end’ BAC
clones, whereas the pink triangle represents ‘ambiguous’ BESs.
Orange triangles indicate the positions of ‘one-ended’ clones, ofwhich only one BAC-end was unambiguously mapped. Including
such clones, the coverage of total genomic regions by the MSM
BAC clones was practically extended (data not shown).

Collection of SNPs from the BAC-end sequencesand distribution of SNPs across the genome
The nucleotide substitutional difference between B6 and MSM
was calculated by comparing the MSM BESs with the B6 genome
sequences. Insertion or deletion of DNA sequences (so-called ‘indels’) was excluded from this analysis (see Discussion). From the‘paired-end’ BESs, 489,200 SNPs were detected in 51,137,941 bp
sequenced. For this SNP survey, we used BESs with PHRED (Ewing
et al. 1998) values equal to or higher than 30 (PHRED ⱖ30). The
calculation yielded a base substitution rate 0.0096. Of these
nucleotide substitutions, there were 323,416 transition changes
and 165,784 transversion changes. The ratio of transitionchanges to transversion changes was thus calculated to be 1.95.
Similar figures for substitutional difference (0.0093) and transition versus transversion ratio (1.97) were obtained from ‘uniquepaired-end’ data sets.
We next plotted the percentage identity of each of the MSM
BESs in comparison with the B6 sequences along the chromosomes (Fig. 3). Although most of the genomic regions contained
a high density of SNPs, as expected from the calculated substitutional differences, there were regions showing percentage
identities close to 1.00 (average percentage identity = 0.9997),
indicating that the distributions of the SNPs were not uniform. There were 66 such extremely low SNP regions acrossthe whole genome. The size of the low-SNP-rate regions varied
from ∼140 kb to 12 Mb, with an average size of 2 Mb (median = 1.37 Mb). The precise locations and sizes of the low-SNPrate regions are summarized in Supplemental Table S1. The proportion of low-SNP segments versus high-SNP regions also varied
for each chromosome. For example, 20% of chromosome 8
corresponded to low-SNP regions (Table 2), and the largest
consecutive low-SNP segment (12 Mb) was also found on chromosome 8 (Fig. 3, inset). In contrast, there were no low-SNP

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Abe et al.
then abruptly decreased, reaching ∼0.985
within the region between bases ∼800 and∼900. Although the percentage identity varied markedly in the next ∼80 kb, the average base substitutions remained high. An
essentially similar result was also obtained
from analysis of the clone MSMg01275M02 (chr1_176213505_176378730)(data not shown). These results confirmedthat the mosaic structures of low- and highSNP regions revealed by the BES SNP distributions were true reflections of the nucleotide sequence variations between the B6
and MSM genomes, and that nucleotide
substitutional differences between the twostrains were in the range of ∼1% in thehigh-SNP-rate genomic segments.
Sixty-seven BESs were mapped onto
the Y-chromosome sequence retrieved fromthe NCBI Build 32 data. The substitutional
differences of the Y-derived BESs (0.0025)
was much lower than those of the autosome and X chromosome sequences (Table2). The ratio of transition versus transversion changes for the Y-SNPs was 0.7, theinverse of those obtained from other chromosomes.
As described above, an unexpectedly
high incidence of nucleotide substitutions(i.e., 0.96%) was found between B6 andMSM. This nucleotide difference value wascomparable with that of the genomic difference (i.e., 1.23%) between humans andchimpanzees, which were estimated to diverge more than five million years ago(Mya) (Fujiyama et al. 2002). Because thetwo mouse subspecies groups were considered to diverge ∼1 Mya (Moriwaki 1994;
Silver 1995), mice (or rodents in general)
should have a higher evolutionary rate
than do hominoids. Alternatively (alFigure 2. BAC contigs retrieved by the web-based clone screening system. By clicking a genomic
region of interest on the chromosome map in the top menu, BAC contigs covering the correspondthough unlikely), some biological coning genomic region can be viewed (A). The red triangles represent the ‘unique paired-end’ BAC
straints could exist on the BAC-end seclones; the pink triangles represent ‘ambiguous’ BESs. Locations of ‘one-ended’ BAC clones arequences, resulting in deviations from theshown by the orange triangles (B). Clone MSMg01-114N13 is an example of such ‘one-ended’
normal evolutionary rate. To examine theseclones; its size is ∼150 kb long, extending toward the centromere, and the clone was found tocontain the Rps28, Angptl4, Rab11b, and 9530046H09Rik genes.
possibilities, we performed comparative
analyses of mouse and rat genomic sequences using two kinds of data set: one
was the MSM BESs presented in this study and their orthologous
regions on chromosomes 15 and 18 or on the X chromosome
sequences in the rat genome, and the other was the sequences of(Table 2). The overall occurrence of nucleotide substitutions did
the protein coding regions of the mouse and their orthologous
not vary significantly among the chromosomes. The X chromocounterparts in the rat. Only fourfold degenerate (4D) synonysome seemed to have a slightly lower substitutions than did themous sites were compared in the latter case (see Supplemental
autosomes. Low-SNP segments occupied a total of 5.2% of the B6material for detailed methods). Nucleotide differences calculated
genome (Table 2).
from either the BES data or 4D synonymous sites of proteinThe B6 genome appeared to be composed of genomic segcoding region sequences were very similar and were estimated toments with different SNP densities, according to the BES mapbe 13.66%∼14.61% (Supplemental Tables S2, S3).
ping data. To confirm this finding, we sequenced BAC clones
This amount of divergence between mice and rat is compatthat were on the borders of low-to-high SNP transitions andible with that between MSM and B6 strains if we consider a reafound that there were sharp transitions from low to high SNP fresonable range of divergence times (see Supplemental material
quency within those BAC finished sequences (Fig. 4). For example,
and Discussion for details). This fact, in turn, suggests that thein the clone MSMg01-122K03 (chr1_156195129_156370415),
BES data from the MSM library are likely to comprehensively
the percentage identity was close to 1.00 from bases 0 to 800,

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Genomic constitution defined by BAC–SNP analysis

Figure 3. Distribution of SNPs across the chromosomes; identification of low- and high-SNP regions. The percentage identity of each of the MSM BESs
in comparison with the B6 sequences was plotted along the chromosomes. Red dots represent the MSM BESs, and positions of the dots along the y-axis
indicate % identity of each BES. Examples of the low-SNP regions (chr8; 30440248–42461244) are enlarged and shown in the inset. Position of thisenlarged region on chromosome 8 is indicated by a dashed rectangle. Another dashed rectangle on chromosome 1 indicates position of a BAC clone,
MSMg01-122K03, which was completely sequenced (see Fig. 4). Horizontal bars represent chromosomes, and chromosome number is indicated at left
side. Blue bars represent genomic regions covered by the MSM BAC clones. Light blue bars indicate the low-SNP regions.

represent the neutral nucleotide sequences in the MSM genome,
and implies that rodents have a higher evolutionary rate than dohominoids (see Discussion for details).

Discussion
We constructed a representative BAC genomic library from
MSM/Ms. This is the first BAC library made from the M. m. molossinus subspecies. We sequenced both ends of 176,256 clone
inserts and mapped the end sequences onto the C57BL/6J mouse
genome assembly. Information on the genomic positions of the
BAC clones allowed us to establish a browser-based clone screening system. Such computer-based screening has been possible

only for the B6 BAC library until now. BES information is useful
not only for clone mapping but also for identification of novel
STSs and their corresponding EST markers (Zhao et al. 2001) or
genomic rearrangements (Volik et al. 2003). Furthermore, to extend the utility of BESs, we compared MSM BESs with the B6
whole mouse genome sequences, generating a vast number of
SNPs between the two strains. Thus the BAC library itself and thehigh-quality BES information obtained from this study will bevaluable resources to the mouse research community.
Because we used BESs with PHRED values of ⱖ30 for thisSNP survey, the incidence of “false-positives” in the 489,200 candidate SNPs was expected to be very small, if not negligible. This
number of SNPs—nearly half a million—is much larger than

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Abe et al.
Table 2. Size of low-SNP-rate regions relative toeach chromosome

chr1
chr2
chr3
chr4
chr5
chr6
chr7
chr8
chr9
chr10

% of
low SNPregions

Substitution
rate

7.30%
4.90%
3.70%
6.60%
1.00%
5.60%
1.10%
20.00%
5.30%
2.00%

0.00960.00950.01040.00970.011
0.00980.011
0.00880.01
0.0108

chr11
chr12
chr13
chr14
chr15
chr16
chr17
chr18
chr19
chrX
chrYa

% of
low SNPregions

Substitution
rate

3.20%
11.30%
5.40%
15.10%
0%
3.30%
1.60%
0%
3.50%
0%
ND

0.00960.00930.01020.00920.01080.01050.01070.011
0.011
0.00720.0025

Total: 5.2%b (Sum of the Low SNP regions relative to the whole B6
genome)aBased on NCBI build 32.
801 genes out of 12,389 known genes (loci) are present in this 5.2% of
the genome.

bthose previously reported (Lindblad-Toh et al. 2000; Wade et al.
2002; Wiltshire et al. 2003). The SNP markers found every 17.68
kb across the genome should facilitate any type of genetic analysis involving B6 and MSM. Moreover, as it is highly likely thatmost of the MSM-associated SNPs are not MSM strain-specific but
rather are shared by M. m. molossinus and M. m. musculus, they
will be useful in crosses of these subspecies-derived strains.
It is generally believed that Asian mice have made substantial genetic contributions to the genomes of laboratory strains,
although the majority of these genomes was derived from M. m.
domesticus of Western European origin (Yonekawa et al. 1980,
1982; Moriwaki 1994). Wade et al. (2002), through SNP analyses,
confirmed the notion that the genomes of laboratory strains area mosaic of genetic segments derived from either domesticus or
musculus (molossinus) ancestral sources, and suggested that the
SNPs found among the laboratory strains are reflections of ancestral intersubspecific differences, not mutations specific to aparticular strain. If this is the case, then most of the SNPs presented in this study can be used to type domesticus-molossinus
differences. The unprecedented number of these SNPs, in conjunction with a high-throughput SNP typing method (e.g., Lindblad-Toh et al. 2000; Matsuzaki et al. 2004) should greatly facilitate genotyping, QTL mapping, modifier mapping, and the defining of SNP haplotype patterns in commonly used laboratory
strains. We did not present details of indels, because (1) B6 genome assembly is not yet a ‘finished’ sequence, and the BAC-end
sequences used in this analysis are results of one-pass sequencing,and (2) long insertions may not be accurately assigned in the
short BAC-end sequences, which are only ∼400 bp-long on average. Our analysis revealed ∼164,000 indels between B6 and MSM(H. Noguchi, unpubl.). Distribution of size of indels is depicted in
Supplemental Figure S1. The majority of the indels were short,
but a small peak at around 200 bp was detected. This peak likely
was produced by the integrations of SINE, mainly B2 (Supplemental Fig. S1). A similar phenomenon due to Alu insertion was
revealed by human–chimpanzee comparison (Watanabe et al.
2004). Information on such indels is potentially important for
tracing evolutionary events after separation of two subspecies.
Wade et al. (2002) and Wiltshire et al. (2003) reported thatthe genomes of laboratory strains comprised blocks of low and

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high SNP occurrence, and that each strain appears to have acharacteristic pattern of SNP haplotype. Estimates of the sizes of
low and high regions, as well as of SNP densities, however, appeared to vary greatly in these studies and the present study. For
example, about one-third (Wade et al. 2002) or about 40%–70%(Wiltshire et al. 2003) of the genome has high SNP densities,
representing segments with intersubspecific difference, whereas
in this study the low-SNP regions occupy only 5% of the genome
in the B6-MSM comparison, representing molossinus-derived segments. These differences seem to be related to the extensivenessof the studies. For example, the sample sequence numbers andthe sizes of the regions analyzed were different in the above studies. Moreover, differences in the methods used to make comparisons may explain the variations in data. In the previous studies,
the B6 genome was compared with sequences from another common laboratory strain, whereas in our study the B6 genome was
directly compared with BAC-end sequences of a molossinusderived MSM strain. In our analysis, the low-SNP region (∼5% of
the B6 genome) simply corresponded to the genomic segments
inherited from M. m. molossinus. In the previous ‘indirect’ analyses, however, the situation is more complicated. As shown in
Supplemental Figure S2, the low- and high-SNP regions revealedby comparison of two laboratory strains rather reflect the composite patterns of SNP haplotypes from the two single strains. In
the low-SNP regions, the two strains share a common subspecies
origin (i.e., both domesticus or both molossinus). Conversely, thehigh-SNP regions are composites of segments from domesticus
and molossinus, although we cannot distinguish which laboratory strain carries which ancestral source. Previous reports (Wadeet al. 2002; Wiltshire et al. 2003) indicate that the distribution
patterns of the low- and high-SNP regions vary in the comparisonof different strain-pairs. Therefore, the sizes of the high-SNP regions revealed by comparison of any two laboratory strains will
be usually larger than those revealed by comparison of one of thelaboratory strains and an Asian wild mouse-derived strain (seeSupplemental Figure S2). This would reconcile discrepancies inthe extent of the contributions from the Asian mouse to the
genomes of laboratory strains. In theory, our ‘direct’ comparison

Figure 4. SNP distribution within the BAC clone MSMg01-122K03, on
the border of low-to-high SNP regions. MSMg01-122K03 is mapped at
chr1_156195129–156370415, which is on the border of low-to-high
SNP transition. Complete sequence of this clone was determined, compared with the B6 sequences, and % identity (y-axis) was plotted along
the entire sequence (x-axis).

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Genomic constitution defined by BAC–SNP analysis
of MSM-BES with the B6 genome should yield more straightforward interpretations, and our genome-wide, high-resolution data
are effective in refining the structures of genetic variations incommon laboratory strains. For example, our analysis identified
relatively small (<1000-kb) regions of co-ancestry, which would
be overlooked in other studies.
We estimated the base substitutional differences between
MSM and B6 to be 0.0096, which is likely to represent the divergence rate between domesticus and molossinus. In a previous study(Wade et al. 2002), SNP rates were calculated on a different basiswith different-sized data sets and were estimated to be lower thanthe value obtained in our study. Whether this difference is due totechnical reasons or to genetic contributions from ancestralsources other than molossinus remains to be elucidated.
Knowledge of the fine structure of shared haplotype blocks
will have a great impact on the design and interpretation of various genetical analyses (Wade et al. 2002; Wiltshire et al. 2003). In
this context, one of the most important findings of our study isthat we could define, for the first time, the precise structures of
SNP haplotypes in the C57BL/6J genome, because B6 is one of themost widely used laboratory strains. It will be perhaps equallyimportant to define two genomic segments with distant origins,which cannot coexist in the same genome. We found no molossinus-derived segments in the X chromosome of B6, consistent
with the observation of Wiltshire et al. (2003) that the rate ofpolymorphism in the X chromosome is exceptionally low in thecomparison of any strain combinations. This implies that interactions of X-linked genes of different evolutionary origins with
autosomal or Y-linked genes would disturb the survival or fertility of individuals. In fact, such genetic interactions have beenreported (Takagi et al. 1994; Oka et al. 2004).
In most of the present analyses, data from the Y chromosome were excluded, as the available data from this chromosome
were still very limited. However, even the limited data on the
Y-derived SNPs indicated that the Y chromosome of the B6 strain
was much more similar to that of the MSM/Ms than was the casewith the autosomes. This result is consistent with the previous
findings that the Y chromosomes of the majority of laboratory
strains, including B6, were derived from M. m. musculus (Bishop
et al. 1985). More specifically, they are derived from the molossinus population, because most common laboratory strains havemolossinus-specific polymorphisms of Y-linked genes such as SRY
and Zfy (Nagamine et al. 1992, 1994). This can be further validated by the Y-SNPs from MSM/Ms. It is also notable that theratio of transition versus transversion changes is quite different
from the ones obtained in the autosomes and the X chromosome, implying that the mechanisms by which nucleotide substitutions are generated may be different in the Y chromosome(Skaletsky et al. 2003).
Data from comparative genomic analyses on the MSM BESs
and rat sequences can be used for estimations of evolutionary
rates of rodents. Evolutionary distance between mouse and rat
was calculated to be 0.155 (Supplemental Table S2). This estimate
is compatible with that obtained for the rat–mouse genomic
comparison (Rat Genome Sequencing Project Consortium 2004).
If we assume that mouse subspecies groups diverged from theirancestors ∼1 Mya, then mouse–rat speciation can be estimated tohave occurred ∼16 (=0.155/0.0096 ⳯ ∼1) Mya, assuming that theevolutionary rate for rodents is constant during this period. Thisestimation of timescale for mouse–rat divergence appears to beconsistent with a previous assumption that mice and rats diverged from a common ancestor 10–15 Mya (Jaeger et al. 1986).

If we use a formula, ␭ = d/2T (␭, evolutionary rate; d, evolutionary distance; T, divergence time) (Nei 1987), the evolutionary rate of nucleotide substitution in mouse was assumed to be
4.8 ⳯ 10ⳮ9 in the above consideration, where d = 0.0096 andT = 1 million years (Myr). Alternatively, we also estimated theevolutionary rate for rodents (between mouse and rat), ␭, to be
2 ⳯ 10 ⳮ9 ∼8 ⳯ 10 ⳮ9 , based on the evolutionary distanced = 0.155, and mouse–rat divergence time T = 10 or a maximum
estimate of 40 Myr (Kumar and Hedges 1998). We therefore suggest that the estimated evolutionary rate in subfamily murinae is
two to eight times larger than the maximum evolutionary rate
(0.99 ⳯ 10ⳮ9) for humans and chimpanzees (Yi et al. 2002).

Methods
Construction of BAC library from MSM/Ms
The MSM/Ms BAC library was constructed in accordance withthe methods described by Osoegawa et al. (1998). Highmolecular-weight genomic DNA prepared from spleens and kidneys of MSM/Ms male mice was partially digested with a combination of EcoRI restriction endonuclease/EcoRI methylase(New England Biolabs) and size-fractionated by pulsed-field gelelectrophoresis. The partially digested DNA was then ligated to
EcoRI-digested and dephosphorylated pBACe3.6 vector and introduced into ElectroMAX DH10B competent cells (Invitrogen)by electroporation performed on a Gene Pulser (BioRad). Recombinant clones grown on agar plates containing 5% sucrose,and 20 µg/mL chloramphenicol were picked up with a Flexys
colony picker (Genomic Solutions), and individual clones werearrayed into 384-well plates and stored at ⳮ80°C. A browserbased clone screening system will be available at these Web sites:
http://stt.gsc.riken.jp/msm/ and http://shigen.lab.nig.ac.jp/
mouse/polymorphism/ and http://analysis1.lab.nig.ac.jp/
Mus_musculus/).
MSM BAC clones will be available from the RIKEN BioResource Center DNA bank (http://www.brc.riken.jp/lab/dna/).

BAC-end sequencing
The BAC library was rearrayed to 96-well plates with a Flexys
system equipped with a gridding tool (GeneMachines). BAC
clones were inoculated into a 96-deep-well plate and cultured
overnight at 37°C in 1.5 mL 2⳯ LB containing 12.5 µg/mL chloramphenicol. BAC DNA was isolated by using a Montage BAC96
Miniprep kit (Millipore) and dissolved in 35 µL of 10 mM TrisHCl buffer (pH 8.0). Cycle sequencing reaction was carried out ina 15-µL reaction volume containing 10 µL BAC DNA (∼300 ng),
1.5 µL Big Dye terminator Ready Reaction mix (Applied Biosystems), and 1.5 µL sequence primer (3.2 pmol/µL). Customdesigned sequence primers at the T7 and SP6 ends were TGACATTGTAGGACTATATTGC and ATCTGCCGTTTCGATCCTCC, respectively. PCR conditions were 95°C for 5 min, then 75 cycles of
95°C for 30 sec, 50°C for 10 sec, and 60°C for 4 min. The reaction
mixture was cleaned up by isopropanol precipitation followed by
70% ethanol wash. The reaction products were loaded on ABI
3700 automated capillary DNA sequencers (Applied Biosystems).

Sequence processing and bioinformatics
Raw sequence data were base-called using the Phred program(Ewing and Green 1998; Ewing et al. 1998), and the vector sequences were filtered out. All sequence reads were submitted to
the DNA Databank of Japan (DDBJ) under accession numbers
AG275743–AG613213. They can also be viewed at our Web site(http://stt.gsc.riken.jp/msm/).

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Abe et al.
For BES analyses, repetitive sequences in the BESs weremasked with RepeatMasker (ftp://ftp.genome.washington.edu),
and then a similarity search was performed against NCBI Build 30of the B6 genome assembly, using BLASTN (Altschul et al. 1997).
Some BLAST hits were split because of young repetitive elements
or other short indels. In such cases, those hits were merged into
one hit, and sum of their bit-scores was assigned as a score of
the merged hit. When a BAC-end was mapped to multiple locations in the genome, the hit with the highest score was selected
as the position of the end, and the BES was labeled an ‘ambiguous
hit’. BESs whose alignments were shorter than 50 bp were also
treated as ambiguous hits. Once locations of BESs had been determined, detailed alignments were calculated without mask ofrepeats using dynamic programming. When both ends of a BAC
clone were mapped with correct orientations and positions considering the possible insert size of BAC (<300 kb), the clone wasregarded as the ‘paired-end’ clone. For mapping of ‘one-ended’
clones, we regarded end sequences as singletons if they wereuniquely mapped onto B6 genome and had alignments longer
than 100 bp.
The nucleotide substitutional differences between B6 andMSM was calculated from the alignments of unique paired-end
sequences. For this calculation, we used nucleotide sequenceswith PHRED quality values of q ⱖ 30.

Sequencing and data assembly
MSM BAC clones were sequenced by the conventional shotgun
sequencing method. Briefly, shotgun sequencing was performed
to provide 8⳯ coverage of draft sequences. In addition, we constructed plasmid clone libraries from appropriate restriction fragments, and sequenced both ends of these clones to provide 2⳯
additional coverage. After assembly of all the sequence data using
Phred/Phrap/Consed (Ewing and Green 1998; Ewing et al. 1998;
Gordon et al. 1998), gap-filling and resequencing of low-quality
regions in the assembled data were performed by a nested deletion method (Hattori et al. 1997), primer-walking, and direct
sequencing of the BAC clone. Accession numbers for the clones
MSMg01-122K03 and MSMg01-275M02 are AP007207 andAP007208, respectively.

Rat–mouse comparative sequence analysis
Methods for the comparative analysis are described in the
Supplemental material.

Acknowledgments
We thank C. Kawagoe, X. Son, and all technical staff of the Genome Sequencing Team (Human Genome Research Group,
RIKEN Genomic Sciences Center, Japan) for their excellent sequencing work and support through computational data management; Dr. Hiromichi Yonekawa of The Tokyo Metropolitan
Inst. of Medical Science for valuable discussion about mouse subspeciation; Dr. Satoshi Oota of Bioresource Information Division
at RIKEN BRC for comments and discussion on mouse–rat speciation, and Drs. Kazutoyo Osoegawa and Pieter De Jong of Children’s Hospital Oakland Research Inst. for advice on BAC library
construction. This work was supported in part by Special Coordination Funds for Promoting Science and Technology from the
Ministry of Education, Culture, Sports, Science and Technologyof Japan to K.A. and T.S. and by the National Bio-Resources
Project of the Ministry of Education, Culture, Sports, Science andTechnology of Japan.

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Web site references
http://www.shigen.nig.ac.jp/mouse/mmdbj/top.jsp; Mouse Microsatellite
Database of Japan.
http://stt.gsc.riken.jp/msm/; Web browser-based BAC library screening
system.
http://shigen.lab.nig.ac.jp/mouse/polymorphism/; a browser-based clone
screening system site.
http://analysis1.lab.nig.ac.jp/Mus_musculus/; the MSM BAC database,and a browser-based clone screening system site.
http://www.brc.riken.jp/lab/dna/; the RIKEN BioResource Center DNA
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ftp://ftp.genome.washington.edu; RepeatMasker.

Received June 17, 2004; accepted in revised form September 27, 2004.

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