Analysis of meiotic recombination in Drosophila simulans shows no evidence of an interchromosomal effect

Abstract

Chromosome inversions are of unique importance in the evolution of genomes and species because when heterozygous with a standard arrangement chromosome, they suppress meiotic crossovers within the inversion. In Drosophila species, heterozygous inversions also cause the interchromosomal effect, whereby the presence of a heterozygous inversion induces a dramatic increase in crossover frequencies in the remainder of the genome within a single meiosis. To date, the interchromosomal effect has been studied exclusively in species that also have high frequencies of inversions in wild populations. We took advantage of a recently developed approach for generating inversions in Drosophila simulans, a species that does not have inversions in wild populations, to ask if there is an interchromosomal effect. We used the existing chromosome 3R balancer and generated a new chromosome 2L balancer to assay for the interchromosomal effect genetically and cytologically. We found no evidence of an interchromosomal effect in D. simulans. To gain insights into the underlying mechanistic reasons, we qualitatively analyzed the relationship between meiotic double-stranded break (DSB) formation and synaptonemal complex (SC) assembly. We found that the SC is assembled prior to DSB formation as in D. melanogaster; however, we show that the SC is assembled prior to localization of the oocyte determination factor Orb, whereas in D. melanogaster, SC formation does not begin until the Orb is localized. Together, our data show no evidence that heterozygous inversions in D. simulans induce an interchromosomal effect and that there are differences in the developmental programming of the early stages of meiosis.

Keywords: Drosophila simulans; inversion; meiosis.

Fig. 1.

Results of testing for the ability of j2LM1 to prevent inheritance of recombination events and serve as a balancer chromosome. a) Flies carrying j2LM1 (3XP3-EYFP) were crossed to D. mauritiana males and then hybrid flies were backcrossed to D. mauritiana males for 10 generations. A total of 12 independent crosses were performed. b) Single flies carrying j2LM1 (3XP3-EYFP) were sampled from each backcross after 10 generations of backcrossing and subjected to Multiplex Shotgun Genotyping (Andolfatto et al. 2011). Ancestry plots for each of the 10 individuals are shown. Par1 is probability of homozygous D. simulans ancestry, which would be shown in red if any regions displayed homozygous homozygous D. simulans ancestry. Par2 is probability of homozygous D. mauritiana ancestry, shown in blue, revealing that many genomic regions have become homozygous for D. mauritiana DNA after 10 generations. In all flies, the left side of Chromosome 2 shows heterozygous ancestry (0 probability of homozygous ancestry for either parent), indicating that j2LM1 prevents inheritance of recombination events in this chromosomal region. In all lines, the region of heterozygosity extends for several Mb proximal to the most proximal breakpoint of the j2LM1 balancer, suggesting that recombination is suppressed also proximal to this breakpoint.

Fig. 2.

Chromosome sizes, positions of the recessive markers, and location of inversion breakpoints used in this study. Recessive markers and their physical position are shown below the chromosomes. Inversion breakpoints are depicted as brackets and their physical position is listed above the chromosome. The end of each chromosome assembly is shown below the chromosome. All physical positions were obtained from the D. simulans genome NCBI:GCF_016746395.2 Black circles represent the location of centromere. Hashed lines represent unassembled pericentric heterochromatin of unknown size.

Fig. 3.

Analysis of Orb and Corolla localization in full sibling wildtype and j2LM1 heterozygotes shows no delay in oocyte specification. a) Immunofluorescence in D. simulans germaria and Stage 2 egg chambers with antibodies against Orb and Corolla in full sibling wildtype (top) and +/j2LM1 heterozygotes (bottom). Orb accumulates in identical patterns to D. melanogaster. It first appears in the cytoplasm of all cells of a 16-cell cyst in region 2a. By region 2b, it has started to concentrate in the pro-oocytes, and by region 3 is concentrated in the designated oocyte. Different from D. melanogaster, Corolla is loaded onto full-length SC at the end of region 1, before Orb signal is detected. Note that because a single z-slice is shown, some signal is out of the focal plane and not all Corolla-positive nuclei are shown. b) and c) Quantification of the 2-oocyte phenotype using b) Orb localization and c) Corolla localization. There are no statistically significant differences in the frequency of the 2 oocytes in region 3 (Fisher's exact test, P = 0.15).

Fig. 4.

Qualitative analysis of DSB and synaptonemal complex formation suggests the SC may form prior to DSB formation in D. simulans. a) to d) A single z-slice of a germarium from a wildtype full sibling showing full-length Corolla and phosphorylated H2AV in region 1 before Orb signal appears. Asterisks: Future nurse cell showing phosphorylated H2AV but no Corolla signal. Dashed circle: One of the nuclei in Region 1 has a phospho-H2AV signal, while the other nucleus shows none. e) to h) Higher magnification of nuclei in dashed circle in D. i) to l) Projection of 5 z-slices of a germarium from a +/j2LM1 heterozygote showing full-length Corolla and phosphorylated H2AV in region 1 before Orb signal appears. Dashed circle: One of the nuclei in region 1 has a strong phospho-H2AV signal, while the other nucleus has a qualitatively weaker signal. m) to p) Higher magnification of nuclei in dashed circle in L.