High-Throughput Genotyping of Advanced Congenic Lines by High Resolution Melting Analysis for Identification of Bbaa2 , a QTL Controlling Lyme Arthritis

Abstract

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.

Genetic Control of Lyme Arthritis by Borrelia burgdorferi Arthritis–Associated Locus 1 Is Dependent on Localized Differential Production of IFN-β and Requires Upregulation of Myostatin

“…Interval specific recombinant congenic lines (ISRCL1-4) with Chr4 Bbaa1 intervals 11.6–77.8 Mbp, 76.48–93.46 Mbp, 83.7–93.46 Mbp, and 88.3–93.46 Mbp were independently generated through repeated backcrosses of B6.C3- Bbaa1 to the parental B6 line. Filial offspring were selected based on SNP identification by high-resolution melting analysis as described (28), and homozygous lines were fixed by mating littermates. All mice used in this study were housed in the University of Utah Animal Research Center (Salt Lake City, UT) and handled with protocols approved by the Institutional Animal Care and Use Committee at the University of Utah in accordance with the NIH guidelines for the care and use of animals.…”

Fine mapping of the Bmgr5 quantitative trait locus for allogeneic bone marrow engraftment in mice

“…SNP genotyping was performed by PCR of tail tip DNA for high-resolution melt curve analysis on a LightCycler 480 Instrument (Roche Applied Science, Indianapolis, IN) as previously described (Cao et al 2009b). In 23 of the 43 chromosome 16 SNP markers used in this study, SNP genotyping was also performed by high-resolution melt curve analysis using unlabeled 3′-phosphorylated oligonucleotide probes as described elsewhere (Bramwell et al 2012; Montgomery et al 2007). A list of chromosome 16 SNP markers, primers and probes used for genotyping is provided as Supplemental Table 1.…”

The Cdkn2a gene product p19 alternative reading frame (p19ARF) is a critical regulator of IFNβ-mediated Lyme arthritis

“…Continued backcrossing of B6.C3-Bbaa1 mice with B6 mice allowed development of interval-specific recombinant congenic lines (ISRCL1-4) with the indicated Chr4 Bbaa1 intervals: ISRCL 1 ( [20]. ISRCL5 (88.3-90.54 Mbp) was generated recently and the interval was fixed by filial mating as described previously [71]. C57BL/6 Arf -/-(B6 Arf -/-) mice (B6.129X1-Cdkn2a tm1Cjs /KaiJ) were from Jackson laboratory, and were originally derived by Kamijo et al by disruption of Exon 1β [72].…”