Trans-centromere effects on meiotic recombination in the zebrafish

Abstract

We report that lack of crossover along one chromosome arm is associated with high-frequency occurrence of recombination close to the opposing arm's centromere during zebrafish meiotic recombination. Our data indicate that recombination behavior on the two arms of a chromosome is linked. These results inform mapping strategies for telomeric mutants.

Figure 1.—

(A) Pedigree of early pressure (EP) gynogenetic half-tetrad progeny that were analyzed. Fish lines: asm was induced with ethylnitrosourea and mutants were distinguished from wild-type by morphology (small, protruding eyes and small head phenotypes) or by in situ hybridization (using an antisense RNA probe for the T cell-specific kinase lck) at 5 day postfertilization (dpf) (Trede et al. 2008). Correspondence between lack of T cells and head morphology is 100% (data not shown). The heterozygous asm mutation was induced in the WIK background and a mapping hybrid cross was generated for the present study by crossing a single asm^+/− heterozygote with a single wild-type Tu individual (Figure 1A, Figure S2). asm^+/− F₁ individuals were identified by progeny test crosses. OG076 was identified in the Tübingen 2005 screen by its lack of macrophages and neutrophils (P. Herbomel and M. Redd, personal communication). It was generated on the Tu background and crossed to WIK for mapping purposes. EP parthenogenesis: Sperm collection, egg extrusion, in vitro fertilization, and timing of pressure treatment were performed as described previously (Streisinger et al. 1981). Sperm was UV-treated in a Stratagene stratalinker set to deliver 7 × 10⁴ μJ. Pressures and hydraulic press equipment used were described previously (Gestl et al. 1997). Generation of mutant offspring: EP parthenogenotes were generated from heterozygous F₁ females and phenotypically classified as either mutant (asm) or wild type (WT). For analysis of recombination affecting the asm chromosome, EP was performed on multiple occasions on eggs derived from a total of 20 asm⁺ F₁ females (Figure 1A, Figure S2). Resulting gynogenetic diploid half-tetrad larvae were raised to 5 dpf when asm mutants are viable: among >4000 offspring produced from matings between heterozygotes, ∼25% were mutant at 5 dpf (data not shown). For analysis of the OG076 chromosome, eggs from four heterozygous females were subjected to EP, larvae were fixed at 4 dpf and assayed for presence of neutrophils by staining with Sudan Black (Le Guyader et al. 2008). (B) Nonindependence of recombination on the two arms of chromosome 18. Primers and PCR: Centromere-linked microsatellite marker loci were obtained from published studies (Shimoda et al. 1999; Mohideen et al. 2000). https://wiki.zfin.org/display/prot/MGH-CVRC+Mapping+Resources. PCR products were resolved by gel electrophoresis using 3% metaphor agarose. Recombination events in 34 mutant and 23 wild-type sibling EP offspring from asm⁺ heterozygous females were analyzed using the chromosome 18 SSR markers depicted in the bar graph on the left. Half-tetrad EP offspring were divided into two groups: mutant offspring whose chromosomes 18 were nonrecombinant along the right arm (no exchange on right arm), and wild-type offspring that harbored a single chromosome 18 that was recombinant along the right arm (exchange on right arm). Because recombination affecting either of the two left arms of the sister chromatid pair could produce a heterozygous half-tetrad, we would expect that 7.4% of the EP progeny would be heterozygous for marker Z9194. Chromosomes with a right arm exchange exhibited a frequency of exchange in the left pericentric interval very close to expectations on the basis of the published map distance. In contrast, the frequency of exchange in the left pericentric interval appeared aberrantly high among larvae lacking an exchange on the right arm of chromosome 18. The two groups differed significantly with respect to the occurrence of recombination in the left pericentric interval. *These larvae constituted all asm mutant EP progeny lacking exchanges on the right arm of chromosome 18 (Figure S3B). ^†These larvae were selected at random from the phenotypically wild-type EP progeny and were genotyped to verify that a right arm exchange had occurred. (C) Nonindependence of recombination on the two arms of chromosome 14. Recombination events in 14 mutant and 47 wild-type EP offspring of OG076⁺ heterozygous females were analyzed using the markers depicted in the bar graph on the left. Half-tetrad EP offspring were divided into two groups: mutant offspring whose chromosomes 14 were nonrecombinant along the left arm and wild-type offspring that harbored a single chromosome 14 that was recombinant along the left arm. Because recombination affecting either of the two right arms of the sister chromatid pair would produce a heterozygous half-tetrad, we would expect that 8.8% of the EP progeny would be heterozygous for marker Z22094. Chromosomes with a left arm exchange exhibited a frequency of exchange in the right pericentric interval very close to expectations. In contrast, the frequency of exchange in the right pericentric interval appeared aberrantly high among larvae lacking an exchange on the left arm of chromosome 14. The two groups differed significantly with respect to the occurrence of recombination in the right pericentric interval. *These larvae were homozygous mutant EP progeny and presumably most lacked exchanges on the left arm of chromosome 14. ^†These larvae were selected at random from the phenotypically wild-type EP progeny and genotyped to verify that a left arm exchange was present.