Genomic characterization of two introgression strains (B6.Cb4i5) for the analysis of QTLs

The effect of an individual locus that affects complex traits is difficult to detect because it is responsible for only a fraction of the total environmental, genetic, and interaction variation, and its contribution is therefore usually masked. In 1983, to diminish the above-mentioned difficulties, we began the development of a novel approach to QTL mapping, by providing the same standardized genetic background for each QTL that affects a particular trait, and by distributing the QTLs individually in recombinant quasi-congenic lines (Vadasz 1990; Vadasz et al. 1987, 1994a, 1994b). We assumed that the relatively low genetic signal of the distributed QTLs could be amplified in these lines by measuring a number of isogenic animals. To test this hypothesis, we constructed a series of inbred quasicongenic recombinant QTL introgression strains by repeated backcross-intercross cycles with concomitant selection for the extreme expressions of a quantitative trait, and fixation of the genes by strict brother-sister mating (Vadasz et al., 1994a). In the present pilot study, in order to assess the amount and distribution of the introduced donor genome, we tested the B6 background strain, the BALB/cJ (C) donor strain, and two strains of the B6.Cb4i5 series, which had been developed by four backcross-intercross cycles with concomitant selection for high midbrain tyrosine hydroxylase (TH) activity, followed by at least 19 generations of brother-sister matings. Currently, not including sublines, we have 14 strains of the B6.Cb4i5 series (Fn>20). We present evidence that the strains can be considered quasicongenic, inbred, and recombinant, and that the method has the potential for QTL mapping of both the differential trait and other quantitative traits affected by passenger genes. Also, we present phenotypic data with respect to the trait being selected for, and demonstrate that the two tested introgression strains have higher TH/MES than the background strain. Microsatellite markers were selected from the Whitehead Institute/MIT database (Dietrich et al. 1992; Whitehead Institute/ MIT 1994) on the basis of previously detected allelic differences between the B6 background and the C donor strain. A 10-cM (average) resolution map was created for two representative QTL introgression strains by testing 169 polymorphic markers in the B6.Cb4i5a-12A (a-12A) and B6.Cb4i5b-13 (b-13) strains and in their B6 background partner strain. PCR products were analyzed on 6% Nusieve agarose gels (Love et al. 1990). Because in the b-13 strain no PCR product was obtained for the most distal marker D19Mit6, we could not estimate the length of a segment on Chromosome (Chr) 19 marked by three consecutively positioned C-type alleles (D19Mit37, D19Mit36, D19Mit34). No heterozygous loci were found in the QTL introgression strains. The mapping results indicated that the maximum length of an introgressed donor segment, estimated by the distances of flanking background type markers from the centromere, varied from 3.3 cM to 22.5 cM on the Whitehead Institute/MIT map. The segments are shorter than the estimated average length of an introgressed chromosome segment that carries the differential locus after four backcrosses with concomitant selection for one donor-strain gene (39 cM; Ln 4 200 × (1–2)/n; n1 4 F1; Flaherty 1981), presumably because (i) during the development of the b4i5 series each backcross was followed by intercrosses (Vadasz et al. 1994a), and (ii) there were additional chances for recombination during fixation (Taylor 1978). Assuming one differential locus, the theoretically expected proportion of the fixed, nonselected, nonlinked donor genes was estimated as 3.0%, while the proportion of the linked donor genes is about 1.2% in inbred QTL introgression strains of the b4i5 series. Our results suggest that the total of the introduced donor genome carried by seven segments in each strain was about 73.7 cM (4.6%) in the a-12A strain, and (assuming that the problematic D19Mit6 marker was of B6-type) was about 117.3 cM (7.3%) in the b-13 strain. The a-12A strain carried C-type chromosome segments on Chrs 2, 8, 9 (2 segments), 13 (2 segments), and 18. The beta-13 strain carried C-type chromosome segments on Chrs 1, 2, 7 (2 segments), 15, 18, and 19. If the length of an introgressed chromosome segment after four backcrosses is estimated as 39 cM, and if during inbreeding four crossovers per 100 cM occurred (Taylor 1978), then the introgressed segment had a 1.5 chance on the average for meiotic recombination before fixation. Therefore, if the length of the differential segment is approximately 26 cM after fixation, and there are two unlinked QTLs with major effects (Vadasz et al. 1994a), the total length of the donor material (including the nonselected, nonlinked donor genes estimated as 3% of 1600 cM) would be about 100 cM. Assuming that the 169 microsatellite markers are randomly distributed in the genome of 1600 cM, the proportion (P) of the genome lying within ±10 cM from the markers is 88% (P 4 1 − e; Jacob et al. 1991). The above estimates indicate that about 12% of the donor segments have not been detected, and that about 240 markers will be needed to ensure that 95% of the genome will lie within ±10 cM of a marker. A detailed summary of the genotyping results of the informative polymorphic markers is provided in Fig. 1 and Fig. 2. Two loci were of identical C-type in the two strains: D2Nds1 (50 cM from the centromere, on a maximum 3.3 cM long segment) and D18Mit107 (26.2 cM from the centromere, on a maximum 9 cM [a-12A] and on a maximum 12.4 cM [b-13] long segment). If the probability of detecting a fixed, nonselected, nonlinked donor gene in one of the b4i5 strains is about 3.05% (p 4 0.0305), then their joint probability of occurrence is p 4 0.00093, while the probability of retaining in two strains the same passenger gene residing 10 cM from the differential gene is p > 0.3 (Flaherty 1981). Thus, it is possible that a maximum 3.3-cM region about 50 cM from the Correspondence to: C. Vadasz Mammalian Genome 7, 545–548 (1996).