DOI: 10.5061/dryad.x3ffbg7n3
title: 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: 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 &gt;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.
description: 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.