Recombination experiments with inversion heterozygotes
Koury, Spencer (2023), Recombination experiments with inversion heterozygotes, Dryad, Dataset, https://doi.org/10.5061/dryad.x3ffbg7n3
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: Inbred lines carrying the standard arrangement and four different cosmopolitan paracentric inversions In(3R)C, In(3R)K, In(3R)Mo, and In(3R)P were drawn from the Drosophila melanogaster Genetic Reference Panel (DGRP). Inversions 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 dominant phenotypic markers (Gl1, Sb1, and Dr1) were selected due to their position relative to inversion breakpoints. These markers were introgressed onto both the standard and inverted arrangements 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. All three dominant markers (Gl1, Sb1, and Dr1) were also introgressed into the common tester stock Canton-S. 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 genotype Canton-S were crossed to three males homozygous for a given gene arrangement: Standard, In(3R)C, In(3R)Mo, In(3R)P, or In(3R)K, 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 viadominant markers and non-disjunction via white-eyed patroclinous exceptions. Non-disjunction rates among gene arrangements not differ with statistical significance (F4,97 = 0.537, p = 0.709) and were not considered further.
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 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 established by Bridges and Brehme. 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 were 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 (viawhite-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.
These are classical single interval (two marker) recombination experiments in Drosophila melanogaster. The experiment was conducted in the presence of heterozygous chromosomal inversions so some classes of recombinants are rare. Genotypes have number identifiers that correspond to the Drosophila melanogaster Reference Panel line number from whence the gene arrangement originated.
The experiments are balanced (i.e., full factorial design) except for two inversion-marker combinations. In these two instances, recombination can still be measured in half-balanced design (cis-trans design). In this case, the untested genotypes have "-----" entered to draw a distinction from the real observation of "0" recording the absence of recombination when tested.
Data are raw counts of F2 individuals in four possible phenotypic class (two non-recombinant classes and two complementary recombinant classes). The phenotypic classes can be classified as non-recombinant or recombinant based on F1 genotype (specifically cis versus trans orientation of visible mutations). All possible combinations of linkage arrays of markers and inversions were tested, and each combination has three replicates.
Further information about genomic location of markers and inversions can be obtained via Flybase.org.