  Multiply inverted balancer chromosomes that suppress exchange with their
homologs are an essential part of the genetic toolkit in Drosophila
melanogaster. 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 any given balancer chromosome is unknown.
To map inversion breakpoints and study potential sequence diversity in the
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 by rare double crossover events
that replaced a female-sterile allele of the singed gene (sn[X2]) on FM7c with
wild type sequence from balanced chromosomes, and propose that many FM7c
chromosomes in the Bloomington Drosophila Stock Center have lost sn[X2] by this
mechanism. Finally, we characterize the original allele of the Bar gene (B[1])
that is carried on FM7 and validate the hypothesis that the origin and
subsequent reversion of the B1 duplication is mediated by unequal exchange. Our
results reject a simple non-recombining, 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.
