DOI: 10.5061/dryad.cc2fqz69t
title: Drosophila melanogaster recombination experiments with inversion heterozygotes

description: 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 (&gt;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.
description: <strong>Stock Construction:</strong> Five inbred
 lines each carrying a different cosmopolitan paracentric
 inversion <em>In(3R)K,In(3R)P, In(3R)C, In(3R)Mo</em>, or
 the <em>Standard</em> arrangement (as an inversion-free
 negative control) were drawn from the <em>Drosophila</em>
 <em>melanogaster</em> 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
 (<em>Gl<sup>1</sup></em>, <em>Sb<sup>1</sup></em>, and <em>Dr<sup>1</sup></em>) 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, <em>Dr<sup>1</sup></em> on <em>In(3R)Mo</em> and <em>Sb1</em> on <em>In(3R)P</em>, because &gt;10,000 meioses failed to produce desired marker-inversion recombinant. Excepting combinations involving <em>Dr<sup>1</sup></em> on <em>In(3R)Mo</em> and <em>Sb<sup>1</sup></em> on <em>In(3R)P</em>, a derived series of stocks was generated with all possible pairwise and three-way marker/inversion combinations. Independently, all three dominant markers (<em>Gl<sup>1</sup></em>, <em>Sb<sup>1</sup></em>, and <em>Dr<sup>1</sup></em>) were also introgressed into a sixth inbred line for use as a common tester strain (<em>Canton-S</em> which also has the standard arrangement). Similarly, a derived series of stocks with all pairwise combinations of markers on the <em>Canton-S</em> genetic background were generated so the common tester strain could be used in factorial design described below. Finally, the isogenic stock <em>w<sup>1118</sup></em>; <em>6326</em>; <em>6326</em> was used for outcrossing the <em>F<sub>1</sub></em> experimental females.  <strong>Crossing Design:</strong> To generate <em>F<sub>1</sub></em> experimental genotypes, three virgin females of standard arrangement tester strain <em>Canton-S</em> were crossed to three males homozygous for a given gene arrangement: Standard (inversion-free negative control), <em>In(3R)K</em>, <em>In(3R)P</em>, <em>In(3R)C</em>, or <em>In(3R)Mo</em> and hereafter collectively referred to as <em>In(3R)x</em>. Virgin female <em>F<sub>1</sub></em> experimental genotypes were selected and outcrossed to male <em>w<sup>1118</sup></em>; <em>6326</em>; <em>6326</em>. The progeny of this cross (<em>F<sub>2</sub></em>) 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 “<em>cis</em>-<em>trans</em>” 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 <em>P</em>generation, virgin Canton-S females with marker genotypes <em>Sb<sup>+</sup> Dr<sup>+</sup>, Sb<sup>1</sup> Dr<sup>+</sup>, Sb<sup>+</sup> Dr<sup>1</sup>,</em> or <em>Sb<sup>1</sup> Dr<sup>1</sup></em> were mated with males homokaryotypic for one of the five gene arrangements [<em>Standard</em>, <em>In(3R)K, In(3R)P, In(3R)C</em>, or <em>In(3R)Mo]</em> carrying either <em>Sb<sup>1</sup></em><em> </em><em>Dr<sup>1</sup></em><em>, </em><em>Sb<sup>+</sup></em><em> </em><em>Dr<sup>1</sup></em><em>, </em><em>Sb<sup>1</sup></em><em> </em><em>Dr<sup>+</sup></em>, or <em>Sb<sup>+</sup></em><em> </em><em>Dr<sup>+</sup></em>, respectively. Thus, selected <em>F<sub>1</sub></em> experimental females were always heterozygous for <em>Dr<sup>1</sup></em>, <em>Sb<sup>1</sup></em>, and <em>In(3R)x</em> 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 <em>Gl<sup>1</sup></em>, <em>Sb<sup>1</sup></em>, and <em>In(3R)x</em> in all possible combinations. <strong>Experimental Conditions: </strong>Experimental conditions followed the standard methods for mapping. Five virgins of the desired <em>F<sub>1</sub></em> 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 <em>w<sup>1118</sup></em>.  Crosses were conducted using light CO<sub>2 </sub>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 <em>Drosophila</em> food. Three replicate bottles were set for each cross. After five days of egg laying, the <em>F<sub>1</sub></em> 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 (<em>F<sub>2</sub></em>) 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 <em>F<sub>2</sub></em> offspring for scoring. With a per locus gene conversion rate of approximately 2 in 44,230 total <em>F<sub>2</sub></em> offspring should produce false positives due to gene conversion at a visible marker.