The use of SNP genotyping by HRMA is becoming increasingly accessible as equipment capable of performing HRMA becomes more common in research institutions around the world. A search of the PubMed database in October 2011 identified over 240 publications using high resolution melting in the past year alone, primarily relating to human diagnostics. For this purpose, the SNPs of interest are pre-defined due to their established linkage to or causation of human disease. While we have found that a large majority of SNPs can be genotyped in this way, not every such SNP is amenable to HRMA genotyping, depending upon the unique characteristics and complexity of surrounding DNA sequence. However, for the purposes of mapping inbred congenic mouse strains, the genomic location of a SNP rather than its potential biological function is the primary consideration, allowing flexibility for several different SNPs to be evaluated in a given area. Based on the Perlegen high-density resequencing project, SNPs differing between any two pairwise combinations of 12 classic inbred strains occur with an average frequency ranging from 1 in 440 bp to 1 in 21,000 bp in high-SNP or low-SNP regions of the mouse genome, respectively (13). Even in low frequency regions, this represents a potential resolution for congenic mapping on the order of 0.01 – 0.10 cM.
The Bbaa2 region of mouse chromosome 5 is complex. It exhibits a nonlinear relationship between basepair position and centiMorgan distance (Figure 1A), suggesting that meiotic recombination does not occur with uniform frequency across the interval. It also exhibits a highly variable SNP density, with some areas highly conserved between our parental B6 and C3H inbred strains of interest (Figure 1B). These characteristics impact the capacity to generate congenic animals and then to identify where recombination has occurred. Despite this complexity, diagnostic SNP genotyping assays were successfully designed at regular intervals. A total of 26 SNP genotyping assays across the Bbaa2 region are now used for routine genotyping of backcross progeny in our congenic colony. By adopting SNP genotyping methods, all previously used microsatellite markers have been replaced with approximately equivalent high-throughput SNP assays, and 15 additional landmarks have been added. As shown in Table 2, the average and median resolutions between genotyping landmarks across the Bbaa2 interval have improved from 2.20 Mbp and 1.56 Mbp to 0.88 Mbp and 0.75 Mbp, respectively. The ability to readily design new genotyping assays targeted to intervals containing novel break-points in individual mice contributes to a harmonious and efficient refinement process. In many cases, the prevalence of discernable SNPs will provide great enough resolution to exclude single genes, or even single SNPs within genes, from a congenic interval. This is especially important when attempting to precisely define the boundaries of similar or overlapping recombinant congenic lines. The use of HRMA based SNP genotyping may also be of special interest to anyone working with recombinant inbred (RI) lines such as those produced by the Collaborative Cross (14), or performing iterative backcrossing, such as during the transfer of a targeted gene knockout to a specific genetic background. A basic SNP genotyping panel for a broad interval surrounding the gene of interest may help speed up the backcrossing process by quickly identifying which individuals within litters inherited the narrowest surrounding interval, and would therefore best serve as breeders for successive backcrosses.
Our adoption of HRMA-based SNP genotyping has facilitated the generation, identification, and more precise discernment of 20 novel ISCLs carrying unique subintervals of the Bbaa2 locus, as shown in Table 3. Many of these newly defined advanced congenics coincide with regions previously predicted to encode loci with positive and negative effects on Lyme arthritis severity, thereby establishing tools for the formal analysis of regulatory regions on chromosome 5. Current congenic lines allow the pairwise interrogation of intervals as small as approximately 0.6 Mbp, which will greatly facilitate efforts to identify causal genes in the region, and may be of general interest for the investigation of any phenotypic differences assigned to this region of mouse chromosome 5.