Retention of induced mutations in a Drosophila reverse-genetic resource

Abstract

Targeting induced local lesions in genomes (TILLING) is a reverse-genetic method for identifying point mutations in chemically mutagenized populations. For functional genomics, it is ideal to have a stable collection of heavily mutagenized lines that can be screened over an extended period of time. However, long-term storage is impractical for Drosophila, so mutant strains must be maintained by continual propagation of live cultures. Here we evaluate a strategy in which ethylmethane sulfonate (EMS) mutagenized chromosomes were maintained as heterozygotes with balancer chromosomes for >100 generations before screening. The strategy yielded a spectrum of point mutations similar to those found in previous studies of EMS-induced mutations, as well as 2.4% indels (insertions and deletions). Our analysis of 1887 point mutations in 148 targets showed evidence for selection against deleterious lesions and differential retention of lesions among targets on the basis of their position relative to balancer breakpoints, leading to a broad distribution of mutational densities. Despite selection and differential retention, the success of a user-funded service based on screening a large collection several years after mutagenesis indicates sufficient stability for use as a long-term reverse-genetic resource. Our study has implications for the use of balancer chromosomes to maintain mutant lines and provides the first large-scale quantitative assessment of the limitations of using breeding populations for repositories of genetic variability.

Figure 1.—

Distribution of the number of mutations discovered per TILLed Z3 locus. The top histogram displays the distribution of the number of mutations for the 164 chromosome 3 screens performed (98 Z3 amplicons were TILLed on half of the Z3 lines and 66 amplicons were additionally TILLed on the remainder). For comparison, the bottom histogram displays the number of mutations discovered from screens of ∼3000 Arabidopsis lines using 164 different Arabidopsis amplicons, each paired with a Drosophila amplicon of the same length (±5 bp).

Figure 2.—

Chromosomal positions of TILLed loci. Diamonds indicate the positions of 44 loci TILLed on the Z2 lines (top pair) and 104 loci TILLed on the Z3 lines (bottom pair). The loci are positioned on the physical (gray) and genetic (black) maps of Drosophila chromosomes 2 and 3. The cytogenetic positions of the breakpoints for the balancer chromosomes CyO and TM6B are indicated as landmarks. The dashed lines connect positions of these breakpoints on the physical map (in megabases, Mb) and genetic map (in centimorgans, cM).