Development of HG.CAST speed congenic strains for MMU1, 5, 8, 9, 11 and 17
All black N2 males from the first two crosses described above were genotyped for 12 markers (D1Mit432, -480, D5Mit353, -311, D9Mit60, -262, D11Mit5, -67, D8Mit234, -211 and D17Mit28 and -142), two spanning each of the six QTL harboring regions (MMU1, 5, 8, 9, 11 and 17; Table 1 and Additional File 1). Markers were selected to capture, at a minimum, the 2-LOD support interval. Two N2 males were selected to propagate the N3 generation; one heterozygous for QTL on MMU1 and 9 and the other heterozygous for QTL on MMU5, 8, 11 and 17 (Figure 1). Both males were homozygous for HG alleles at all other known QTL. These males were backcrossed to HG females and two of the resulting N3 males inheriting the same sets of QTL as their sire were selected for breeding. These males were subsequently backcrossed to HG females and three N4 males were identified heterozygous for the following regions: 1) MMU1 and 9; 2) MMU5 and 11; 3) MMU8, 11 and 17 (Figure 1). Starting at N4 and continuing through N6, the "best" male with the lowest percent of unwanted donor alleles was selected after performing a genome scan using the remaining 67 genome-wide markers (79 total markers minus the 12 markers genotyped in the first two backcrosses spanning the know QTL intervals). At N5 a distinct strain was created for each of the six individual donor regions and heterozygous mice were intermated (Figure 1). Homozygous HG.CAST speed congenic strains were maintained through brother-sister mating. Once each congenic was stabilized, 19 additional microsatellite markers were used to refine the position of each congenic recombinant end point (Additional File 2).