The generation of chromosomal deletions to provide extensive coverage and subdivision of the Drosophila melanogaster genome

Abstract

Background: Chromosomal deletions are used extensively in Drosophila melanogaster genetics research. Deletion mapping is the primary method used for fine-scale gene localization. Effective and efficient deletion mapping requires both extensive genomic coverage and a high density of molecularly defined breakpoints across the genome.

Results: A large-scale resource development project at the Bloomington Drosophila Stock Center has improved the choice of deletions beyond that provided by previous projects. FLP-mediated recombination between FRT-bearing transposon insertions was used to generate deletions, because it is efficient and provides single-nucleotide resolution in planning deletion screens. The 793 deletions generated pushed coverage of the euchromatic genome to 98.4%. Gaps in coverage contain haplolethal and haplosterile genes, but the sizes of these gaps were minimized by flanking these genes as closely as possible with deletions. In improving coverage, a complete inventory of haplolethal and haplosterile genes was generated and extensive information on other haploinsufficient genes was compiled. To aid mapping experiments, a subset of deletions was organized into a Deficiency Kit to provide maximal coverage efficiently. To improve the resolution of deletion mapping, screens were planned to distribute deletion breakpoints evenly across the genome. The median chromosomal interval between breakpoints now contains only nine genes and 377 intervals contain only single genes.

Conclusions: Drosophila melanogaster now has the most extensive genomic deletion coverage and breakpoint subdivision as well as the most comprehensive inventory of haploinsufficient genes of any multicellular organism. The improved selection of chromosomal deletion strains will be useful to nearly all Drosophila researchers.

Figure 1

Generating molecularly defined deletions using Exelixis FRT-bearing transposon insertions. (a) The structure of the P{XP}, PBac{RB}and PBac{WH} constructs. The miniwhite-marked P element or piggyBac (PBac) constructs carry one or two FRT sequences with the indicated orientations. (miniwhite is a version of the white gene engineered for compact size.) P{XP} and PBac{WH} constructs also carry UAS sequences oriented to allow GAL4-induced expression of genes near the genomic insertion sites of the constructs. One UAS sequence in P{XP} can be removed by FLP-mediated recombination and it is likely that this cassette is absent from most deletion chromosomes, though we did not assay for it in our deletion chromosomes. (b-d) Simplified diagrams of FLP-mediated recombination events generating deletion chromosomes with different miniwhite copy numbers. (b) Our most frequently used screening strategy, where deletions are identified based on loss of miniwhite and the resulting white eye color. (c) The alternative strategy of identifying deletions based on increased miniwhite copy number relative to progenitor chromosomes and the resulting darker eye color. (d) Deletions can be recovered without a decrease or increase in miniwhite copy number, though we did not undertake such screens.

Figure 2

Frequency distribution of the number of genes between molecularly defined deletion breakpoints. The number of genes in a chromosomal interval between adjacent deletion breakpoints is shown on the x-axis and the number of intervals in the Drosophila genome with those sizes is shown on the y-axis. Because BSC, Exelixis and DrosDel deletions overlap extensively, the intervals were usually defined by breakpoints of different deletions. The median interval size is only nine genes. For simplicity, the Stellate gene cluster in chromosomal region 12E, the histone gene cluster in 39D and the 5S rRNA gene cluster in 56E were counted as single genes.