2Department of Medicine, University of Vermont, Burlington, VT, USA
Congenic mapping is a powerful strategy to identify genomic loci regulating quantitative traits. Generating congenic lines is an iterative process of refinement that is both time and resource intensive. Here we report an alternative to traditional microsatellite marker analysis or costly high-density oligonucleotide single nucleotide polymorphism (SNP) arrays for congenic genotyping. The identification of inherited SNP variability in congenic lines using high resolution melting analysis (HRMA) represents a novel application of the method. The blocked probe HRMA approach offers a scalable, low cost, closed-tube system that benefits from rapid turnaround times, and unequivocal interpretation. The markedly higher prevalence of SNPs relative to microsatellites in the genome allows much greater flexibility for the identification of new genotyping landmarks as congenic intervals are refined. We have adopted this approach in our development of B6.C3-Bbaa2 congenic lines for the identification of loci regulating murine Lyme arthritis severity. As a result, we have been able to fully genotype individuals prior to weaning age, and expand our number of breeding cages without increasing our colony budget. Thus far, 26 SNP markers have been successfully mapped to the Bbaa2 locus. This has facilitated the identification of 20 novel B6.C3-Bbaa2 congenic lines spanning the original interval.
The central dogma of molecular biology is that heritable differences in DNA lead to changes in mRNA transcripts, proteins, and ultimately to observable variations in phenotypes such as disease susceptibility (1). Inbred mouse strains provide an invaluable resource of fixed genomic variability to scientifically investigate this genetic component of disease susceptibility. The ability to generate intercross populations between susceptible and resistant inbred mouse strains, combined with powerful statistical analysis, allows regulatory loci of quantitative traits to be mapped to regions of individual chromosomes. Once identified, the breeding of congenic mouse lines allows individual regulatory loci to be isolated in the context of a controlled genetic background, and then to be narrowed to finer resolution in an unbiased manner through additional backcrossing to parental lines. The genetic resolution is achieveable through such breeding strategies is limited by the number and position of experimentally discernable genetic landmarks across the region of interest. Microsatellite markers have long been a key resource for genetic mapping due to their prevalence and ease of analysis by PCR.
The Bbaa2 murine Lyme arthritis susceptibility locus was identified in multiple independent intercrosses between resistant and susceptible inbred strains (2,–3). Roper et al. used composite interval mapping (CIM) on reciprocal backcross populations between C3H/HeN (C3H) and either C57BL/6 (B6) or Balb/c to identify complex regulation by up to four putative QTL within Bbaa2Bbaa3, with a maximum LOD score of 10.2. Reciprocal interval specific congenic lines (ISCLs) for the Bbaa2Bbaa3 region on chromosome 5 developed on both the B6 and C3H backgrounds each retained a significant portion of the disease susceptibility phenotype conferred by the parental line (4). However, the full B6.C3-Bbaa2Bbaa3 congenic interval spanned 72.29 – 141.16 Mbp and included more than 870 genes and ESTs, making candidate gene selection impractical.
Efforts have been made to further refine regulatory loci within Bbaa2 to a resolution of 1 Mb or narrower through additional ISCL backcrossing. However, given the putative complexity of the Bbaa2 locus, the availability of microsatellite markers across the interval became a limiting factor, prompting the evaluation of alternative genotyping strategies. At present, the dbSNP database contains more than 8,000 single nucleotide polymorphisms (SNPs) which differ between B6 and C3H inbred lines across the Bbaa2 interval (5). Although the C3H/HeN mouse strain used to create our congenic lines is not one of the eighteen currently included in the Sanger SNP re-sequencing project (6), data for the closely related C3H/HeJ strain is available. The Sanger database contains over 19,000 high quality SNPs which differ between C3H/HeJ and C57BL/6NJ SNPs in the Bbaa2 interval. A SNP genotyping strategy thus offered the potential to dramatically improve genetic resolution by increasing the number of discernable landmarks available. Several previously described SNP genotyping methodologies were evaluated: amplification refractory mutation system (ARMS)-PCR (7), small amplicon high resolution melting analysis (HRMA) (8), and blocked probe HRMA (9). We found that both methods of HRMA were useful for the genotyping of inherited intervals in congenic animals, with blocked probe HRMA being especially practical. Recent studies have emphasized the application of HRMA analysis for clinical diagnostics, but its application toward precise characterization of congenic mice has not previously been described.Materials and methods
All mice used in this study were maintained in a pathogen free facility, and cared for in accordance with protocols approved by the University of Utah Institutional Animal Care and Use Committee (IACUC).
Genomic DNA was prepared from 2–3 mm tail clips from 14 to 17 day old mice by incubating in 600 µL of 50mM NaOH at 95C for one hour, following by neutralization with 50 µL of 1M Tris-HCl pH 8. Samples were then centrifuged at 6000 × g for 6 min in a tabletop centrifuge and transferred to a clean tube. Two µL of 1:10 diluted DNA was used in each PCR reaction.
ARMS-PCR is a tetra-primer SNP genotyping strategy that combines two inner SNP-specific and two outer primers in a single reaction volume (7). Reactions are designed to produce up to three amplicons of diagnostic sizes which are then evaluated by agarose gel electrophoresis, as shown in Supplemental Figure S-1A. ARMS-PCR primer sets for ten SNPs across the Bbaa2 region were developed using the recommended design program available online. (http://cedar.genetics.soton.ac.uk/public_html/primer1.html). ARMS-PCR primer sequences are available in Supplemental Table S-1. PCR was performed in 96-well plates on a PTC-200 Thermal Cycler using Platinum Taq (Invitrogen, Carlsbad, CA, USA) following the manufacturer's recommendations. PCR products were subjected to 1% agarose gel electrophoresis in 1X TBE buffer (89 mM Tris Base, 89 mM Boric Acid, 2 mM EDTA), and bands were visualized by ethidium bromide staining on a Gel Doc XR+ platform (Bio-Rad Laboratories, Hercules, CA, USA).