Mitotic exchange in female germline stem cells is the major source of Sex Ratio chromosome recombination in Drosophila pseudoobscura

Abstract

Sex Ratio chromosomes in Drosophila pseudoobscura are selfish X chromosome variants associated with 3 nonoverlapping inversions. In the male germline, Sex Ratio chromosomes distort the segregation of X and Y chromosomes (99:1), thereby skewing progeny sex ratio. In the female germline, segregation of Sex Ratio chromosomes is mendelian (50:50), but nonoverlapping inversions strongly suppress recombination establishing a 26-Mb haplotype (constituting ∼20% of the haploid genome). Rare crossover events located between nonoverlapping inversions can disrupt this haplotype, and recombinants have sometimes been found in natural populations. We recently reported on the first lab-generated Sex Ratio recombinants occurring at a rate of 0.0012 crossovers per female meiosis. An improved experimental design presented here reveals that these recombination events were at least 4 times more frequent than previously estimated. Furthermore, recombination events were strongly clustered, indicating that the majority arose from mitotic exchange in female germline stem cells and not from meiotic crossing-over in primary oocytes. Finally, asymmetric recovery of complementary recombinants was consistent with unequal exchange causing the recombination-induced viability defects. Incorporating these experimental results into population models for Sex Ratio chromosome evolution provided a substantially better fit to natural population frequencies and allowed maintenance of the highly differentiated 26-Mb Sex Ratio haplotype without invoking strong epistatic selection. This study provides the first estimate of spontaneous mitotic exchange for naturally occurring chromosomes in Drosophila female germline stem cells, reveals a much higher Sex Ratio chromosome recombination rate, and develops a mathematical model that accurately predicts the rarity of recombinant Sex Ratio chromosomes in natural populations.

Keywords: Drosophila pseudoobscura; Sex Ratio; germline stem cells; mitotic exchange.

Fig. 1.

Schematic representation for recombination experiments with metacentric X chromosomes of D. pseudoobscura. a) Centromeres are depicted as centrally placed circles and the 3 nonoverlapping inverted regions of the Sex Ratio chromosomes are shown in dark gray on the X chromosome right arm. Physical dimensions of Sex Ratio haplotype and colinear region are listed above the chromosome, below chromosomes are the genetic map position for the visible markers. b) Summary of Fuller et al. (2020) recombination experiment with raw counts provided in parentheses, the asymmetry of this result was very unexpected under a reciprocal-exchange model of meiotic crossing-over $\left({\chi }_{\left[1\right]}^{ 2}=12, P=2.85\times {10}^{-4}\right)$.

Fig. 2.

Cartoon sequence of germarium and single egg chamber development in anterior region of Drosophila ovariole. Mitotic exchange in GSCs (red on the far left) with their asymmetric division and self-renewal (reflexive arrow) can cause clustering in recombination datasets. Mitotic exchange in developing cystoblast (yellow in the center) cannot cause clustering as only 1 of 16 nuclei will develop into the oocyte (blue on the far right), with the other 15 becoming nurse cells (gray on the far right). Meiotic crossing-over in oocyte (blue) only occurs after development of the 16-cell cyst (yellow) and are causally independent among oocytes. Therefore, clustering of recombination events must originate from mitotic exchange in GSC or earlier in development. Cartoon does not represent a proportional scale but is meant to illustrate the sequential events allowing for interpretation of clustering in recombination data.

Fig. 3.

Factorial design matrix and crossing scheme for recombination experiments. The $3\times 3\times 3-9$ design produced 18 unique F₁ X_SR/X_ST genotypes with 9 inbred genotypes excluded. Color-coded crossing scheme shows Cross 1, see Supplementary File 3 for all 18, depicting full D. pseudoobscura karyotype consisting of a metacentric X chromosome, J-shaped Y chromosome, 3 acrocentric autosomes, and 1 pair of dot chromosomes. F₂ recombinants are only illustrated as female, but both F₂ sexes were scored and backcrossed to confirm recombinants.

Fig. 4.

Observed versus expected distribution of recombination events. a) Observed recombination events (red) were clustered among single-female crosses when compared with Poisson expectations (blue) based on experiment-wide recombination rate $\left({\chi }_{\left[4\right]}^{ 2}=21.03, P=1.43\times {10}^{-4}\right)$ (see Table 3). b) Proportion of recombinants $\left(q\right)$ carrying X_TO were clustered within single-female crosses when compared with expected proportions from the binomial distribution. Multiple recombinants from single-female crosses (red) were more likely to all be X_TO than expected by chance (blue) $\left({\chi }_{\left[4\right]}^{ 2}=64.30, P=1.75\times {10}^{-13}\right)$ (see Table 4). c) Recovery rates of complementary recombinants were neither equivalent between nor independent of F₂ progeny sex. The observed data (red) differed from uniform discrete expectations (blue) with statistical significance $\left({\chi }_{\left[3\right]}^{ 2}=55.9, P=2.15\times {10}^{-12}\right)$ (see Table 5). A deficit of both complementary recombinants (X_TO and X_BM) was detected in the hemizygous (male) state and was particularly severe for X_BM.

Fig. 5.

Modeled evolutionary trajectories for X chromosome variants in D. pseudoobscura. Change in population frequency of X_ST (black), X_SR (yellow), X_BM (red), and X_TO (blue) chromosomes with initial frequencies ranging from 0.01 to 0.99. For comparison, Beckenbach’s (1996) observations from AZ, USA are given as color-coded points on the far right with exact binomial 95% confidence intervals. a) Extension of Edwards (1961) population genetic model assuming recombination is reciprocal-exchange “meiotic crossing-over.” b) The same model incorporating experimentally determined asymmetries in recombination due to “unequal mitotic exchange.” The Edwards (1961) model incorporating unequal mitotic exchange is a much better fit to the frequencies observed in the wild (see Table 7).