The Molecular and Genetic Characterization of Second Chromosome Balancers in Drosophila melanogaster

Abstract

Balancer chromosomes are multiply inverted and rearranged chromosomes used in Drosophila melanogaster for many tasks, such as maintaining mutant alleles in stock and complex stock construction. Balancers were created before molecular characterization of their breakpoints was possible, so the precise locations of many of these breakpoints are unknown. Here, we report or confirm the positions of the 14 euchromatic breakpoints on the 2nd chromosome balancers SM1, SM5, CyO, and SM6a. This total includes three breakpoints involved in a complex rearrangement on SM5 that is associated with the duplication of two genomic regions. Unbiased sequencing of several balancers allowed us to identify stocks with incorrectly identified balancers as well as single and double crossover events that had occurred between 2nd chromosome balancers and their homologs. The confirmed crossover events that we recovered were at least 2 Mb from the closest inversion breakpoint, consistent with observations from other balancer chromosomes. Balancer chromosomes differ from one another both by large tracts of sequence diversity generated by recombination and by small differences, such as single nucleotide polymorphisms (SNPs). Therefore, we also report loss-of-function mutations carried by these chromosomes and unique SNP and InDel polymorphisms present on only single balancers. These findings provide valuable information about the structure of commonly used 2nd chromosome balancers and extend recent work examining the structure of X and 3rd chromosome balancers. Finally, these observations provide new insights into how the sequences of individual balancers have diverged over time.

speck, first identified in Drosophila melanogaster in 1910, is encoded by the Arylalkalamine N-acetyltransferase (AANAT1) gene

“…Assembly of the AANAT1 loci from both and comparison to the s peck 1 reference strain did not yield any candidate changes (data not shown). In addition, examination of SNPs between balancers carrying speck 2 and the reference strain carrying speck 1 showed no SNPs within the AANAT1 locus (Miller et al, 2018). Because there were no changes in the genomic interval that short-read, whole genome sequencing could identify, we looked to see if 412{881} had a second retrotransposon insertion, similar to AANAT1 lo (Brodbeck et al, 1998) .…”

Identification and Characterization of Breakpoints and Mutations onDrosophila melanogasterBalancer Chromosomes

“…Despite the widespread use of balancers, the genomic positions of many of the breakpoints of the most commonly used balancers were identified only recently, with many of the breakpoints found to lie within protein-coding genes (Miller et al 2016b(Miller et al , 2016a(Miller et al , 2018aGhavi-Helm et al 2019). Miller et al (2018a) assessed the phenotypic consequences of the breakpoints on second chromosome balancers by complementation testing the balancers against chromosomal deletions for breakpoint regions and showed that most breakpoints were not associated with severely deleterious phenotypes, but that the disruption of some genes by breakpoints caused recessive lethality or sterility. For example, the 22E breakpoint on the second chromosome balancer SM5 disrupts dachsous, resulting in lethality with escapers having shortened appendages, and the 22A breakpoint on SM1, SM5 and SM6a disrupts no individualized sperm resulting in male sterility.…”

“…In addition to identifying inversion breakpoints, sequencing the second chromosome balancers CyO, SM5 and SM6a identified potentially damaging missense, splice site and nonsense polymorphisms (Miller et al 2018a). Some polymorphisms were shared by some, but not all of the balancers sequenced.…”

“…It can also alert researchers to potential dosedependent effects of heterozygous balancer-borne mutations and potential dominant interactions between mutations on balancers and other chromosomes. Moreover, breakpoint information has revealed previously unknown duplicated chromosomal segments such as the region containing 117 protein-coding genes present in two copies on all SM5 balancers (Miller et al 2018a).…”

GRIBCG: A software for selection of sgRNAs in the design of balancer chromosomes

“…They also contain dominant mutations that allow their unambiguous tracking during crosses, and recessive lethal mutations that prevent the recovery of homozygous progeny. These features make balancer chromosomes particularly useful in preventing the loss of recessive lethal or sterile mutations from a population (without manual selection) and during saturation mutagenesis screens [1][2][3]. In plant breeding, balancer chromosomes could help preserve the advantages of heterosis without full apomixis [4].…”