Multiply inverted balancer chromosomes that suppress exchange with their homologs are an essential part of the
Drosophila melanogaster genetic toolkit. Despite their widespread use, the organization of balancer chromosomes has not been characterized at the molecular level, and the degree of sequence variation among copies of balancer chromosomes is unknown. To map inversion breakpoints and study potential diversity in descendants of a structurally identical balancer chromosome, we sequenced a panel of laboratory stocks containing the most widely used
X chromosome balancer,
First
Multiple 7 (
FM7). We mapped the locations of
FM7 breakpoints to precise euchromatic coordinates and identified the flanking sequence of breakpoints in heterochromatic regions. Analysis of SNP variation revealed megabase-scale blocks of sequence divergence among currently used
FM7 stocks. We present evidence that this divergence arose through rare double-crossover events that replaced a female-sterile allele of the
singed gene (
snX2) on
FM7c with a sequence from balanced chromosomes. We propose that although double-crossover events are rare in individual crosses, many
FM7c chromosomes in the Bloomington
Drosophila Stock Center have lost
snX2 by this mechanism on a historical timescale. Finally, we characterize the original allele of the
Bar gene (
B1) that is carried on
FM7, and validate the hypothesis that the origin and subsequent reversion of the
B1 duplication are mediated by unequal exchange. Our results reject a simple nonrecombining, clonal mode for the laboratory evolution of balancer chromosomes and have implications for how balancer chromosomes should be used in the design and interpretation of genetic experiments in
Drosophila.Balancer chromosomes are genetically engineered chromosomes that suppress crossing over with their homologs and are used for many purposes in genetics, including construction of complex genotypes, maintenance of stocks, and estimation of mutation rates. Balancers typically carry multiple inversions that suppress genetic exchange or result in the formation of abnormal meiotic products if crossing over does occur (); for example, single crossovers inside the inverted segment create acentric or dicentric chromosomes that will fail to segregate properly during meiosis or large deletions or duplications that will likely result in inviable gametes (
1,
2). Balancers also often carry recessive lethal or sterile mutations to prevent their propagation as homozygotes as well as dominant markers for easy identification. First developed for use in
Drosophila melanogaster, balancer chromosomes remain some of the most powerful tools for genetic analysis in this species (
3).
Open in a separate windowConsequences of a single or double crossover between a WT
X chromosome and an
X chromosome carrying a single inversion,
In(1)dl-49. Euchromatin is shown in blue, heterochromatin is shown in gray, and centromeres are depicted as circles. Thin white lines mark locations of inversion breakpoints, and yellow crosses/thin lines mark locations of crossover events. (
A) A single crossover event within the inverted segment results in the formation of chromosomes with deletions and zero (acentric) centromeres or duplications and two (dicentric) centromeres, neither of which will segregate properly during meiosis. (
B) A double crossover within an inverted segment results in intact chromosomes with one centromere that will segregate properly during meiosis.Despite their widespread use, very little is known about the organization of
Drosophila balancer chromosomes at the molecular level. Since their original syntheses decades ago, balancers have undergone many manipulations, including the addition or removal of genetic markers. Moreover, rare recombination events can cause spontaneous loss of deleterious alleles on chromosomes kept over balancers in stock, as well as loss of marker alleles on balancer chromosomes themselves (
3). Likewise, recent evidence has shown that sequence variants can be exchanged between balancer chromosomes and their wild type (WT) homologs via gene conversion during stock construction or maintenance (
4,
5). Thus, substantial variation may exist among structurally identical balancer chromosomes owing to various types of sequence exchange.To gain insight into the structure and evolution of balancer chromosomes, we have undertaken a genomic analysis of the most commonly used
X chromosome balancer in
D. melanogaster,
First Multiple 7 (
FM7). We have focused on
FM7 because this
X chromosome balancer series lacks lethal mutations and thus can be easily sequenced in a hemizygous or homozygous state. In addition, the
FM7 chromosome has been shown to pair normally along most of its axis with a standard
X chromosome, providing a structural basis for possible exchange events (
6). Moreover, although details of how early balancers in
D. melanogaster were created are not fully recorded, the synthesis and cytology of the
FM7 series is reasonably well documented (
3).The earliest chromosome in the
FM7 series,
FM7a, was constructed using two progenitor
X chromosome balancers,
FM1 and
FM6, to create a chromosome carrying three inversions—
In(1)sc8,
In(1)dl-49, and
In(1)FM6—relative to the WT configuration (
7,
8) (). Subsequently, a female-sterile allele of
singed (
snX2) was introduced onto
FM7a to create
FM7c, which prevents the loss of balanced chromosomes carrying recessive lethal or female-sterile mutations (
9). More recently, versions of
FM7a and
FM7c have been generated that carry transgene insertions that allow the determination of balancer genotypes in embryonic or pupal stages (
10–
14).
Open in a separate windowStructure of the
FM7 balancer chromosome. Euchromatin is shown in blue, and heterochromatin is shown in gray. (
A) Schematic view of the organization of WT and
FM7 X chromosomes.
FM7 contains three inversions—
In(1)sc8,
In(1)dl-49, and
In(1)FM6—relative to WT. The six breakpoint junctions for the three inversions are numbered 1–6 and are shown in detail in
B. (
B) Location and organization of inversion breakpoints in
FM7. Each inversion has two breakpoints that can be represented as A/B and C/D in the standard WT arrangement and as A/C and B/D in the inverted
FM7 arrangement, where A, B, C, and D represent the sequences on either side of the breakpoints. Locations of euchromatic breakpoints are on Release 5 genome coordinates, and the identity of the best BLAST match in FlyBase is shown for heterochromatic sequences. Primers used for PCR amplification are shown above each breakpoint; details are provided in
Methods and
Datasets S2 and
S3. Forward and reverse primers are named with respect to the orientation of the assembled breakpoint contigs, not the orientation of the WT or
FM7 X chromosome.To identify the inversion breakpoints in
FM7 balancers and to study patterns of sequence variation that may have arisen since the origin of the
FM7 series, we sequenced genomes of eight
D. melanogaster stocks carrying the
FM7 chromosome (four
FM7a and four
FM7c). We discovered several megabase-scale regions in which
FM7c chromosomes differ from one another, which presumably arose via double-crossover (DCO) events from balanced chromosomes (). These DCOs eliminate the female-sterile
snX2 allele in the centrally located
In(1)dl-49 inversion and are expected to confer a fitness advantage to
sn+ chromosomes, either by allowing propagation of
sn+
FM7 as homozygotes in females or by
sn+
FM7 males outcompeting
snX2
FM7 males in culture. We found that loss of the
snX2 allele is common in
FM7c chromosomes by screening other
FM7c-carrying stocks at the Bloomington
Drosophila Stock Center. We also identified the breakpoints of the
B1 duplication carried on
FM7, and found direct molecular evidence for the role of unequal exchange in the origin and reversion of the
B1 allele (
15–
19). Our results provide clear evidence that the common assumption that balancers are fully nonrecombining chromosomes is incorrect on a historical timescale, and that substantial sequence variation exists among balancer chromosomes in circulation today.
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