An algorithm for analyzing linkages affected by heterozygous translocations: QuadMap.

Heterozygous chromosome rearrangements such as reciprocal translocations are most accurately displayed as two-dimensional linkage maps. Standard linkage mapping software packages, such as MapMaker, generate only one-dimensional maps and so reciprocal translocations appear as clusters of markers, even though they originate from two nonhomologous chromosomes. To more accurately map these regions, researchers have developed statistical methods that use the variance in map distance to distinguish among the four segments (two translocation, two interstitial) of the translocation. In this study, we describe modifications to one of these protocols, that proposed by Livingstone et al. (2000). We also introduce QuadMap, a new software application for dissecting heterozygous translocation-affected linkage maps.

Candidate gene analysis of organ pigmentation loci in the Solanaceae.

Ten structural genes from the Capsicum (pepper) carotenoid biosynthetic pathway have been localized on a (Capsicum annuum x Capsicum chinense)F(2) genetic map anchored in Lycopersicon (tomato). The positions of these genes were compared with positions of the same genes in tomato when known, and with loci from pepper, potato, and tomato that affect carotenoid levels in different tissues. C2, one of three phenotypically defined loci determining pepper fruit color, cosegregated with phytoene synthase. The capsanthin-capsorubin synthase (Ccs) locus, shown previously to cosegregate with Y, another pepper fruit color locus, mapped to pepper chromosome 6. Other structural genes in pepper corresponded to loci affecting carotenoid production as follows: Ccs in pepper and the B locus for hyperaccumulation of beta-carotene in tomato fruit mapped to homeologous regions; the position of the lycopene beta-cyclase gene in pepper may correspond to the lutescent-2 mutation in tomato; and the lycopene epsilon-cyclase locus in pepper corresponded to the lycopene epsilon-cyclase locus/Del mutation for hyperaccumulation of delta-carotene in tomato fruit. Additional associations were seen between the structural genes and previously mapped loci controlling quantitative variation in pepper and tomato fruit color. These results demonstrate that comparative analyses using candidate genes may be used to link specific metabolic phenotypes and loci that affect these phenotypes in related species.

Genetic mapping of the Tsw locus for resistance to the Tospovirus Tomato spotted wilt virus in Capsicum spp. and its relationship to the Sw-5 gene for resistance to the same pathogen in tomato.

The Tsw gene conferring dominant resistance to the Tospovirus Tomato spotted wilt virus (TSWV) in Capsicum spp. has been tagged with a random amplified polymorphic DNA marker and mapped to the distal portion of chromosome 10. No mapped homologues of Sw-5, a phenotypically similar dominant TSWV resistance gene in tomato, map to this region in C. annuum, although a number of Sw-5 homologues are found at corresponding positions in pepper and tomato. The relationship between Tsw and Sw-5 was also examined through genetic studies of TSWV. The capacity of TSWV-A to overcome the Tsw gene in pepper and the Sw-5 gene in tomato maps to different TSWV genome segments. Therefore, despite phenotypic and genetic similarities of resistance in tomato and pepper, we infer that distinct viral gene products control the outcome of infection in plants carrying Sw-5 and Tsw, and that these loci do not appear to share a recent common evolutionary ancestor.

Linkage mapping in populations with karyotypic rearrangements.

A simulation study was used to examine the consequences of karyotypic rearrangements on molecular genetic map construction. Two groups of 50 datasets were created for F2 populations segregating for a reciprocal translocation of chromosomal segments or a reciprocal translocation and inversion. Multiple attempts were made to construct maps for each dataset using MapMaker/EXP. As expected, the markers from segments involved in the translocation formed one linkage group. Maps that corresponded to the known marker order within a segment could be constructed by the following method. The separation of markers distal to the translocation breakpoints into their respective segments could be made by constructing multiple maps, using distinct orders of marker entry, and observing the variances in intermarker distances: variances between pairs of markers from the same segment were an order of magnitude less compared to pairs where markers were from different segments. The order of markers within a segment could be determined from combining the pairwise linkage results from multiple maps, or from maps including all markers from a segment. No bias in map distances was observed. These results indicate that, under conditions similar to those tested, genetic maps corresponding to the segments conserved in translocations can be constructed.

Genome mapping in capsicum and the evolution of genome structure in the solanaceae.

We have created a genetic map of Capsicum (pepper) from an interspecific F2 population consisting of 11 large (76.2-192.3 cM) and 2 small (19.1 and 12.5 cM) linkage groups that cover a total of 1245.7 cM. Many of the markers are tomato probes that were chosen to cover the tomato genome, allowing comparison of this pepper map to the genetic map of tomato. Hybridization of all tomato-derived probes included in this study to positions throughout the pepper map suggests that no major losses have occurred during the divergence of these genomes. Comparison of the pepper and tomato genetic maps showed that 18 homeologous linkage blocks cover 98.1% of the tomato genome and 95.0% of the pepper genome. Through these maps and the potato map, we determined the number and types of rearrangements that differentiate these species and reconstructed a hypothetical progenitor genome. We conclude there have been 30 breaks as part of 5 translocations, 10 paracentric inversions, 2 pericentric inversions, and 4 disassociations or associations of genomic regions that differentiate tomato, potato, and pepper, as well as an additional reciprocal translocation, nonreciprocal translocation, and a duplication or deletion that differentiate the two pepper mapping parents.

Evidence that Tmevd2 and eae3 may represent either a common locus or members of a gene complex controlling susceptibility to immunologically mediated demyelination in mice.

Theiler's murine encephalomyelitis virus (TMEV)-induced demyelination and experimental allergic encephalomyelitis are the principal immunologically mediated, genetically controlled models of multiple sclerosis. Previous studies using different mapping techniques identified susceptibility loci for both diseases on chromosomes 3, 6, and 17. To more precisely map these TMEV and experimental allergic encephalomyelitis loci relative to each other, linkage analysis using microsatellite markers and a (BALB/cByJ x DBA/2J) x BALB/cByJ backcross population segregating for TMEV-induced disease was conducted. Comparisonwise and chromosomewise critical values based on permutation theory were estimated for each chromosome. Evidence for linkage to markers on chromosome 17 was not seen. Chromosomewise linkage (p = 0.13) was detected with D6 Mit36 and D6 Mit149 (marker-specific chromosomewise p values = 0.02) at 40.4 cM on chromosome 6. Chromosomewise linkage (p < 0.01) (marker-specific chromosomewise p value = 0.0) and comparisonwise linkage (p < < 0.0001) to D3 Mit156 at 33.9 cM on chromosome 3 were observed along with chromosomewise linkage (p < 0.05) and comparisonwise linkage (p < < 0.0001) to D3 Mit29, D3 Mit311, D3 Mit28, and D3 Mit11 from 33.9 to 37.2 cM, respectively. Significant linkage to D3 Mit156 places Tmevd2 1.1 cM proximal of D3 Mit101 (35 cM), the maximally linked marker to the eae3 susceptibility gene. Maximum likelihood estimates conducted by multilocus linkage analysis localized Tmevd2 within a 95% confidence interval bordered by D3 Mit29 and D3 Mit10, at 33.9 and 37.2 cM, respectively. Taken together these results suggest that Tmevd2 and eae3 may represent either a single, common susceptibility gene or members of a gene complex involved in central nervous system immunopathology.

Susceptibility to actively-induced murine experimental allergic encephalomyelitis is not linked to genes of the T cell receptor or CD3 complexes.

The role of the T cell receptor (TCR) in the genetic control of susceptibility to autoimmune demyelinating diseases remains shrouded in controversy. We have used the CXD2 series of recombinant inbred lines (RIL) and a (B10.S/DvTe x SJL/J) x B10.S/DvTe backcross (BC1) population to test for linkage between susceptibility to actively-induced EAE and the different TCR and CD3 loci. The two populations were inoculated for induction of EAE, phenotyped for both clinical and histological parameters of disease, and genotyped using markers flanking the loci of interest in the CXD2 RIL and an SJL/J allele-specific TCR V beta assay in the BC1 mice. Comparisons between the CXD2 strain distribution pattern (SDP) for disease and the SDPs for the chromosomal regions containing the TCR alpha, beta, gamma, delta, and CD3 delta, epsilon, gamma and zeta loci showed no linkage to these loci. Additional tests between EAE susceptibility and several other immunologically important loci for which the SDPs were known also showed no linkage to the minor lymphocyte-stimulating antigen gene Mlsl, Hc, the gene encoding complement component C5, Cd8a, or Cd5. Furthermore, our data from the BC1 mice demonstrate that the Tcrb locus segregates independent of disease and does not modulate disease severity. We conclude that while autoreactive TCRs are undoubtedly necessary for disease pathogenesis, the principle non-MHC-linked loci controlling susceptibility to murine EAE in BALB/c mice are not linked to any of the individual TCR-CD3 complex genes. Similarly, the major disease genes in the SJL/J mouse are not linked to TCR V beta. Our data cannot, however, preclude the possibility that TCR/CD3 alleles are involved in epigenetic phenomena or susceptibility in other mouse strains or animal systems.

Experimental allergic orchitis in mice. VII. Preliminary characterization of the aspermatogenic autoantigens responsible for eliciting actively and passively induced disease.

Experimental allergic orchitis (EAO) can be induced actively and passively in mice by either immunization with mouse testicular homogenate (MTH) in conjunction with the appropriate adjuvants or by transferring CD4+ T cells isolated from sensitized donors into non-immunized, naive recipients. The distribution of inflammatory lesions seen in active and passive EAO are markedly different. In active EAO maximal disease is observed in the seminiferous tubules, whereas in passive EAO lesions occur primarily in the straight tubules, rete testis, and ductus efferentes. These observations suggest that different immunopathogenic mechanisms and/or aspermatogenic autoantigens may be responsible for the distinct histopathologic profiles. Two murine testis-specific aspermatogenic autoantigens (mAP1 and mAP2) were partially purified from MT acetone powder by extraction in 7-M urea under reducing conditions, gel filtration, ion-exchange chromatography, and preparative isoelectric focusing from pH 3 to 10. In gel filtration on Sephacryl S-400 in 7-M urea, mAP1 is confined to the V0 peak, while mAP2 is in the major included peak. mAP1 has an isoelectric point of 4.4-4.9, is sensitive to both pronase and DNase but not RNase, and is active at a minimal dose of 250-500 micrograms (dry wt). Dose-response bioassays for active and passive EAO revealed that mAP1 preferentially elicits active disease, whereas mAP2 is most effective at eliciting passive disease. These results support the concept that the different histopathologic profiles seen in active and passive EAO are, in part, the result of different immunopathologic responses elicited by separate aspermatogenic autoantigens.