This repository contains data files and scripts to reproduce the analysis for the manuscript titled: "refining the peak time point for recombination rate plasticity in Drosophila pseudoobscura"
Meiotic recombination rates vary in response to intrinsic and extrinsic factors. Recently, heat stress has been shown to reveal plasticity in recombination rates in Drosophila pseudoobscura. Here, a combination of molecular genotyping and X-linked recessive phenotypic markers were used to investigate differences in recombination rates due to either heat stress or advanced maternal age. However, haplotype frequencies deviated from equal proportions for crosses using phenotypic markers, which was compounded by stress, indicating fitness costsviability selection. Interestingly, skews in haplotype frequency were condition-dependent, consistent with the fixation of alleles in the wild type stocks used that are unfit at high temperature. Evidence of viability selection due to heat stress in the wild type haplotypes was most apparent on days 7-9 when more mutant non-crossover haplotypes were recovered in comparison to wild type (p=2.2e-4). Despite the condition-dependent mutational load in both wild type and mutant stocks, an analysis of recombination rate plasticity Using mixed model analysis, the odds of crossover formation was 1.55X higher during daysrevealed days 7-9 (p=0.0017) and and 1.41X higher on day 9 (p=0.037) post-mating in flies reared atto be significantly higher due to heat stress and days 1-3 as significantly higher due to maternal age (p=0.025) temperature as compared to control crosses, suggesting this time period as the timing of peak plasticity. Still, to confirm these findings, SNP genotyping markers were used to further investigate recombination rate. This analysis supported days 9-10 as significantly different due to heat stress in two pairs of consecutive SNP markers (p=0.018; p=0.015), suggesting this time period as when recombination rate is most sensitive to heat stress. This peak timing for recombination plasticity is consistent with D. melanogaster based on comparison of similarly timed key meiotic events, enabling future mechanistic work of temperature stress on recombination rate. For maternal age, previous negative results and a median survivorship of 42 days guided the choice of treatment age. Recombination rates were compared between females aged to 7 days (control) and 35 days prior to mating (N=23,559). Results revealed a 3.39% increase in recombination rate due to maternal age (p=0.025) for the first 72 hour time point in one of two marker intervals. However, haplotype frequencies deviated from equal proportions for crosses using phenotypic markers, which was compounded by stress, indicating fitness costs. Still, the timing difference for these two treatments suggest that the mechanisms responsible for recombination rate plasticity may differ between maternal age and heat stress in D. pseudoobscura.
The R code used for all statistical analysis and to produce all data figures and model tables is included in the folder "Scripts" as an R script divided into each section named after sequential experiments.
The folder "raw_data_files" includes the backcrosses, raw phenotyping results, fecundity, and female count data from the mutant screens. A separate csv file with treatment information includes dates and other metadata that would be needed to validate the analysis and conclusions herein. Additionally, a processed and cleaned up data file that includes sums of males and females, as well as crossover counts across each interval per time point, per replicate vial is also included. As an example a tutorial from experiment 3 in the manuscript can be found in the "Experiment3_Tutorial" folder with all of the results and figures for Experiment 3.