XB-ART-39681Dev Dyn. June 1, 2009; 238 (6): 1398-46.
Rapid gynogenetic mapping of Xenopus tropicalis mutations to chromosomes.
Pilot forward genetic screens in Xenopus tropicalis have isolated over 60 recessive mutations. Here we present a simple method for mapping mutations to chromosomes using gynogenesis and centromeric markers. When coupled with available genomic resources, gross mapping facilitates evaluation of candidate genes as well as higher resolution linkage studies. Using gynogenesis, we have mapped the genetic locations of the 10 X. tropicalis centromeres, and performed fluorescence in situ hybridization to validate these locations cytologically. We demonstrate the use of this very small set of centromeric markers to map mutations efficiently to specific chromosomes. Developmental Dynamics 238:1398-1406, 2009. (c) 2009 Wiley-Liss, Inc.
PubMed ID: 19441086
PMC ID: PMC2962985
Article link: Dev Dyn.
Grant support: 1R01DE018824 NIDCR NIH HHS, 7R01HD042276 NICHD NIH HHS , GM42341 NIGMS NIH HHS , Medical Research Council , R01 DE018824-04 NIDCR NIH HHS, R01 GM042341-24 NIGMS NIH HHS , R01 HD042276-04 NICHD NIH HHS , R01 DE018824 NIDCR NIH HHS, MC_U117560482 Medical Research Council , R01 GM042341 NIGMS NIH HHS , R01 HD042276 NICHD NIH HHS , T32 GM007205 NIGMS NIH HHS , R01 DE018824 NIDCR NIH HHS, GM42341 NIGMS NIH HHS , Medical Research Council , R01 GM042341 NIGMS NIH HHS , 1R01DE018824 NIDCR NIH HHS, R01 GM042341-24 NIGMS NIH HHS , R01 DE018824-04 NIDCR NIH HHS, T32 GM007205 NIGMS NIH HHS , 7R01HD042276 NICHD NIH HHS , R01 HD042276 NICHD NIH HHS , R01 HD042276-04 NICHD NIH HHS , MRC_MC_U117560482 Medical Research Council , R01 DE018824 NIDCR NIH HHS, GM42341 NIGMS NIH HHS , Medical Research Council , R01 GM042341 NIGMS NIH HHS , 1R01DE018824 NIDCR NIH HHS, R01 GM042341-24 NIGMS NIH HHS , MC_U117560482 Medical Research Council , R01 DE018824-04 NIDCR NIH HHS, T32 GM007205 NIGMS NIH HHS , 7R01HD042276 NICHD NIH HHS , R01 HD042276 NICHD NIH HHS , R01 HD042276-04 NICHD NIH HHS
Genes referenced: epb41 ezh1 ezh2 fbxl7 gemin5 mast3 mat1a naif1 olig3 stat4 znf423
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|Figure 1. Gynogenesis and centromere mapping. A:X. tropicalis N and IC strains differ at many sequence polymorphisms, schematized in red and blue. Hybrids contain both N and IC parental chromosomes, which recombine during meiosis I. Shortly after fertilization, one set of sister chromatids is extruded as a polar body, resulting in a haploid embryo if eggs are fertilized with ultraviolet-irradiated sperm. Cold shock suppresses polar body formation, resulting in a gynogenote containing the sister chromatids of meiosis II. Loci very close to centromeres are homozygous in gynogenotes, but meiotic recombination can produce heterozygous noncentromeric loci. B: Centromeres were mapped by assaying frequency of homozygosity at polymorphic loci in gynogenotes. Three markers on Chr 1/LG1 were tested on the same set of gynogenotes. Proximity to centromere is reflected by relative frequency of heterozygosity, with marker 013H11 (2/19) closer than 013D04 (9/19) and 003D01 (17/20).Download figure to PowerPoint|
|Figure 2. Cytological localization of centromeric markers. Fluorescence in situ hybridization (FISH) probes were generated from cDNAs of genes on centromere-linked scaffolds identified by gynogenesis. Probe name, location: Chr1/LG1: mast3, q0.13; Chr2/LG6: epb41, q0.22; Chr3/LG8: gemin5, p0.00; Chr4/LG7: znf423, p0.00; Chr5/LG9: olig3, p0.00; Chr6/LG2: fbxl7, q0.13; Chr7/LG4: mat1a, p0.12; Chr8/LG5: naif1, p0.00; Chr9/LG3: stat4, q0.09; Chr10/LG10: ezh1, q0.03.Download figure to PowerPoint|
|Figure 3. Mapping mutations with centromere markers and gynogenesis. Bulk segregant analysis of pools of 20 mrs lot mutant (mlo) and sibling wild-type (WT) gynogenotes scored with centromeric markers (LG1 013H11, LG2 025H10, LG3 025G03, LG4 010E04, LG5 049C11, LG6 016H09, LG7 047F06, LG8 051H02, LG9 026H07, LG10 028C03) from the 10 X. tropicalis linkage groups. mrs lot shows clear linkage to LG3.Download figure to PowerPoint|
|Figure 4. Interference in X. tropicalis. A: Individual gynogenotes (48 lanes total) scored with four markers from the q arm of Chr2/LG6 with centromere-proximal marker (top) to distal (bottom) (016H09, LG6 map position 0.00 cM, 043H07/2.72 cM, 020E05/27.46 cM, and 025E03/44.7cM). Crossovers can be detected by changes between homo- and heterozygosity within a lane; embryos 3, 8, 11, 15, 16, 19, and 27 show more than one crossover between the centromere and the most distal marker. Heterozygosity in the most proximal marker in lanes 8 and 19 reflects a crossover between the centromere and that marker. Lanes 5 and 23 do not display crossovers. B: Some species of double crossover uncouple linkage of distal loci to centromere markers. In the “two-strand double crossover” (left), a distal recessive mutant locus (asterisks) remains linked to the white parental centromere allele after two intervening crossovers (parental ditype, PD). In the “four-strand double crossover” (right), mutant gynogenote shows dark nonparental ditype (NPD) centromeric alleles. C: Multiple chiasmata (white arrowheads) in a 4′,6-diamidine-2-phenylidole-dihydrochloride (DAPI) -stained lampbrush preparation of Chromosome 1.Download figure to PowerPoint|
|Figure 5. A: Sex-specific recombination rates crossover frequencies in male and female germlines were compared using a set of homozygous mutant cyd embryos and a “three-allele” polymorphism (simple sequence length polymorphism [SSLP] 011A08) ∼10 cM away. Parental female and male chromosomes (left) bearing the mutant cyd allele share one 011A08 allele (thin black bar) but have distinct 011A08 alleles in repulsion to cyd (maternal allele, solid gray box; paternal allele, hollow gray box). cyd mutant embryos homozygous for the coupled 011A08 allele indicate no recombination (top right, 170/217 cyd embryos assayed); 47 embryos were heterozygous at 011A08, indicating an intervening crossover. Recombination in maternal germline denoted by the intermediate MW solid gray maternal 011A08 allele (middle right) was detected in 39/47 (83%) recombinants, and in the paternal germline (high MW allele) in 8/47 (17%) (bottom right). No embryos showed recombination in both parents. B: Representative genotyping results at marker 011A08: left lanes, female and male parents showing distinct intermediate and high MW alleles; progeny lanes: homozygous low MW band = no recombination; black asterisks, intermediate MW allele = maternal recombination; white asterisk, high MW allele = paternal recombination.Download figure to PowerPoint|