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Drosophila melanogaster recombination experiments with inversion heterozygotes

Cite this dataset

Koury, Spencer (2023). Drosophila melanogaster recombination experiments with inversion heterozygotes [Dataset]. Dryad.


Recombination suppression in chromosomal inversion heterozygotes is a well-known but poorly understood phenomenon. Surprisingly, recombination suppression extends far outside of inverted regions where there are no intrinsic barriers to normal chromosome pairing, synapsis, double-strand break formation, or recovery of crossover products. The interference hypothesis of recombination suppression proposes heterozygous inversion breakpoints possess chiasma-like properties such that recombination suppression extends from these breakpoints in a process analogous to crossover interference. This hypothesis is qualitatively consistent with chromosome-wide patterns of recombination suppression extending to both inverted and uninverted regions of the chromosome. The present study generated quantitative predictions for this hypothesis using a probabilistic model of crossover interference with gamma-distributed inter-event distances. These predictions were then tested with experimental genetic data (>40,000 meioses) on crossing-over in intervals that are external and adjacent to four common inversions of Drosophila melanogaster. The crossover interference model accurately predicted the partially suppressed recombination rates in euchromatic intervals outside inverted regions. Furthermore, assuming interference does not extend across centromeres dramatically improved model fit and partially accounted for excess recombination observed in pericentromeric intervals. Finally, inversions with breakpoints closest to the centromere had the greatest excess of recombination in pericentromeric intervals, an observation that is consistent with negative crossover interference previously documented near Drosophila melanogaster centromeres. In conclusion, the experimental data support the interference hypothesis of recombination suppression, validate a mathematical framework for integrating distance-dependent effects of structural heterozygosity on crossover distribution, and highlight the need for improved modeling of crossover interference in pericentromeric regions.


Stock Construction: Five inbred lines each carrying a different cosmopolitan paracentric inversion In(3R)K,In(3R)P, In(3R)C, In(3R)Mo, or the Standard arrangement (as an inversion-free negative control) were drawn from the Drosophila melanogaster Genetic Reference Panel (DGRP). Inversions and the inversion-fee negative control were identified by polytene chromosome squashes of third instar larva salivary glands and confirmed with PCR amplification of inversion breakpoints. Focal third chromosomes were isolated by balancer chromosome-assisted extraction and placed on a common, standard arrangement genetic background for the X, Y, mitochondrial, and second chromosomes (from DGRP line 399).

Three fully penetrant dominant phenotypic markers (Gl1Sb1, and Dr1) were selected due to their position relative to inversion breakpoints. These markers were introgressed onto both the inverted arrangements and the standard arrangement (negative control) followed by repeated backcrossing for a minimum of ten generations. Two exceptions were made, Dr1 on In(3R)Mo and Sb1 on In(3R)P, because >10,000 meioses failed to produce desired marker-inversion recombinant. Excepting combinations involving Dr1 on In(3R)Mo and Sb1 on In(3R)P, a derived series of stocks was generated with all possible pairwise and three-way marker/inversion combinations.

Independently, all three dominant markers (Gl1Sb1, and Dr1) were also introgressed into a sixth inbred line for use as a common tester strain (Canton-S which also has the standard arrangement). Similarly, a derived series of stocks with all pairwise combinations of markers on the Canton-S genetic background were generated so the common tester strain could be used in factorial design described below. Finally, the isogenic stock w1118; 6326; 6326 was used for outcrossing the F1 experimental females. 

Crossing Design: To generate F1 experimental genotypes, three virgin females of standard arrangement tester strain Canton-S were crossed to three males homozygous for a given gene arrangement: Standard (inversion-free negative control), In(3R)KIn(3R)PIn(3R)C, or In(3R)Mo and hereafter collectively referred to as In(3R)x. Virgin female F1 experimental genotypes were selected and outcrossed to male w1118; 6326; 6326. The progeny of this cross (F2) were scored for recombination via dominant markers and non-disjunction via white-eyed patroclinous exceptions.

A balanced design was employed to estimate recombination fractions while simultaneously controlling for viability effects of dominant phenotypic markers, genetic backgrounds, and the inversions themselves. This balanced design included both “marker-switching” and “cis-trans” recombination experiments with all possible marker-inversion combinations on a common genetic background. Four different crosses were performed to generate F1 females with all markers and inversions in a full factorial design. In the Pgeneration, virgin Canton-S females with marker genotypes Sb+ Dr+, Sb1 Dr+, Sb+ Dr1, or Sb1 Dr1 were mated with males homokaryotypic for one of the five gene arrangements [StandardIn(3R)K, In(3R)P, In(3R)C, or In(3R)Mo] carrying either Sb1 Dr1, Sb+ Dr1, Sb1 Dr+, or Sb+ Dr+, respectively. Thus, selected F1 experimental females were always heterozygous for Dr1, Sb1, and In(3R)x in all possible linkage arrays on a common genetic background. For each gene arrangement, a second experiment was conducted independently following the same methods, but using Gl1, Sb1, and In(3R)x in all possible combinations.

Experimental Conditions: Experimental conditions followed the standard methods for mapping. Five virgins of the desired F1 genotype were collected over a three-day period, aged an additional three days, then outcrossed to five males from isogenic stock 6326, which had the standard arrangement on all chromosome arms and the X-linked mutation w1118.  Crosses were conducted using light CO2 anesthesia. After allowing 24 hours for recovery, the mated group of ten individuals was tap transferred into half-pint bottles with 30–40 ml of standard cornmeal-agar Drosophila food. Three replicate bottles were set for each cross. After five days of egg laying, the F1 adults were removed from bottles. A 2.5-inch x 2.5-inch blotting paper square was added to provide ample pupation sites with 0.05% v/v propionic acid added as needed to hydrate food. Emerging progeny (F2) were then scored daily for recombination (via dominant markers) and non-disjunction (via white-eyed patroclinous exceptions) for 15 days after the last eggs were laid. All vials and bottles were held at 25° C, greater than 50% relative humidity, under 24-hour light in a Percival Scientific incubator. Under these conditions, each bottle yields between 100–1000 F2 offspring for scoring. With a per locus gene conversion rate of approximately 2 in 44,230 total F2 offspring should produce false positives due to gene conversion at a visible marker.