Contribution of Asian mouse subspecies Mus musculus molossinus to genomic constitution of strain C57BL/6J, as defined by BAC-end sequence-SNP analysis

Abstract

MSM/Ms is an inbred strain derived from the Japanese wild mouse, Mus musculus molossinus. It is believed that subspecies 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 the molossinus genome is thus essential not only for genetic studies involving molossinus but also for characterization of common laboratory strains. Here, we report the construction of an arrayed bacterial artificial chromosome (BAC) library from male MSM/Ms genomic DNA, covering approximately 1x 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, we established 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: The majority 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.

Figure 1.

Estimation and distribution of insert sizes in MSM BAC clones. (A) Randomly chosen BACs were digested with NotI that flank the cloning site (EcoRI) in the pBACe3.6 vector. The digested DNAs were analyzed by pulsed-field gel electrophoresis on a 1% agarose gel in 0.5× TBE at 6V/cm, with 0.1 sec to 40 sec pulse time for 16 h at 14°C. The DNA markers are λ concatemers plus HindIII-digested λDNA. Horizontal black bars indicate positions of the concatemers. The arrow indicates the position of the vector. Distribution of insert size is shown in (B).

Figure 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 corresponding genomic region can be viewed (A). The red triangles represent the `unique paired-end' BAC clones; the pink triangles represent `ambiguous' BESs. Locations of `one-ended' BAC clones are shown by the orange triangles (B). Clone MSMg01-114N13 is an example of such `one-ended' clones; its size is ∼150 kb long, extending toward the centromere, and the clone was found to contain the Rps28, Angptl4, Rab11b, and 9530046H09Rik genes.

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 this enlarged 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.

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).