Bulk segregation mapping of mutations in closely related strains of mice

Abstract

Phenovariance may be obscured when genetic mapping is performed using highly divergent strains, and closely similar strains are preferred if adequate marker density can be established. We sequenced the C57BL/10J mouse genome using the Applied Biosystems SOLiD platform and here describe a genome-wide panel of informative markers that permits the mapping of mutations induced on the closely related C57BL/6J background by outcrossing to C57BL/10J, and backcrossing or intercrossing. The panel consists of 127 single nucleotide polymorphisms validated by capillary sequencing: 124 spaced at ∼20-Mb intervals across the 19 autosomes, and three markers on the X chromosome. We determined the genetic relationship between four C57BL-derived substrains and used the panel to map two N-ethyl-N-nitrosourea (ENU)-induced mutations responsible for visible phenotypes in C57BL/6J mice through bulk segregation analysis. Capillary sequencing, with computation of relative chromatogram peak heights, was used to determine the proportion of alleles from each strain at each marker.

Figure 1.—

Sample DNA sequence trace chromatograms. The region containing the SNP with strongest linkage to the june gloom phenotype from pooled DNA of 12 mice with normal phenotype (black coat) and 13 mice with the mutant phenotype (gray coat). Note that because this marker is linked to the phenotype, the sample from mice with gray coats is expected to contain the C57BL/6J allele exclusively, whereas the sample from mice with black coats is expected to contain C57BL/6J and C57BL/10J alleles at a ratio of 33.3:66.7.

Figure 2.—

Estimated C57BL/6J and C57BL/10J allele frequencies in june gloom and mayday circler mutant and normal phenotype pools at 124 SNP sites. (A) Normalized peak heights, calculated as described in materials and methods, for each of 124 markers in samples with known ratios of C57BL/6J:C57BL/10 genomic DNA. For each individual marker, a standard curve was generated and fitted to a line by linear regression. Values, mean ± SD; lines represent mean. (B and C) C57BL/6J and C57BL/10J allele frequencies at each of 124 markers in the mutant and normal phenotype DNA pools, respectively. Shown in red are data points representing the marker with the highest linkage score and those flanking it. Values, mean ± SD; lines represent mean. june gloom samples (B) and mayday circler samples (C) are shown.

Figure 3.—

Linkage scores for june gloom (intercross genetic mapping). Scores for all mice combined (A), for the mutant phenotype pool (B), and for the normal phenotype pool (C) are shown.

Figure 4.—

Linkage scores for mayday circler (backcross genetic mapping). Scores for all mice combined (A), for the mutant phenotype pool (B), and for the normal phenotype pool (C) are shown.