Rapid gynogenetic mapping of Xenopus tropicalis mutations to chromosomes

Abstract

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.

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 UV-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 non-centromeric 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).

Figure 2. Cytological Localization of Centromeric Markers

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

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.

Figure 4. Interference in X. tropicalis

A. Individual gynogenotes (48 lanes total) scored with 4 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 ‘2-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 ‘4-strand double crossover’ (right), mutant gynogenote shows dark non-parental ditype (NPD) centromeric alleles. C. Multiple chiasmata (white arrowheads) in a DAPI-stained lampbrush preparation of Chromosome 1.

Figure 5. Sex-specific Recombination Rates

Crossover frequencies in male and female germlines were compared using a set of homozygous mutant cyd embryos and a ‘3-allele’ polymorphism (SSLP 011A08) ~10cM away, schematized in A. 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 grey box; paternal allele, hollow grey 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 grey 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.