Single nucleotide polymorphism markers for genetic mapping in Drosophila melanogaster

Abstract

For nearly a century, genetic analysis in Drosophila melanogaster has been a powerful tool for analyzing gene function, yet Drosophila lacks the molecular genetic mapping tools that recently have revolutionized human, mouse, and plant genetics. Here, we describe the systematic characterization of a dense set of molecular markers in Drosophila by using a sequence tagged site-based physical map of the genome. We identify 474 biallelic markers in standard laboratory strains of Drosophila that span the genome. Most of these markers are single nucleotide polymorphisms and sequences for these variants are provided in an accessible format. The average density of the new markers is one per 225 kb on the autosomes and one per megabase on the X chromosome. We include in this survey a set of P-element strains that provide additional use for high-resolution mapping. We show one application of the new markers in a simple set of crosses to map a mutation in the hedgehog gene to an interval of <1 Mb. This new map resource significantly increases the efficiency and resolution of recombination mapping and will be of immediate value to the Drosophila research community.

Figure 1

Distribution in the Drosophila genome of SNPs identified in this study. The euchromatic portion of the genome is represented by horizontal bars, with the extent of each cytological division representing the genomic extent as estimated by Sorsa (1988). The positions of SNP markers identified in this study are represented by vertical hatch marks: SNPs for which sequence has been determined in all strains are represented by red hatch marks; those that were identified in pairwise subsets of strains are indicated by blue or green. The distribution is relatively even throughout the genome, with the exception of cytological divisions 78–83 near the centromere of chromosome 3. Strain designations (e.g., Q1040) and a small downward arrow indicate the positions of P elements useful for fine-scale mapping.

Figure 2

Recombination mapping of a recessive lethal mutation by using SNP markers. Chromosomes (bars) and molecular markers (vertical hatch marks) are shown. The mapping process occurs in two stages. (A) The mutation (asterisk) induced in the w;iso2;iso3 background (black bar) that has been mapped previously to a chromosome and balanced is mapped relative to a polymorphic mapping strain (open bar). Single flies heterozygous for the mutation-carrying chromosome and the mapping chromosome are crossed to flies homozygous for the parental w;iso2;iso3 strain to generate 96 recombinant flies. The four recombinant classes are represented. Each recombinant fly strain is assayed for a low-density set of markers that span the chromosome. These markers can be of any type, including SNPs or P-element insertions. We have typically tested six markers spaced at ∼10-Mb intervals on these 96 recombinants, for a total of 576 assays. Each recombinant also is assayed for presence or absence of the mutation by outcrossing. From the outcross data and initial marker data on this set of 96 recombinants, the mutation can be assigned to an interval of 10–20 Mb. (B) A higher density set of SNPs then is assayed on recombinants from A that break in the appropriate interval. An example of mapping a mutation in the hedgehog gene is shown. SNP markers are indicated by the STSs from which they are derived. Two P elements (Q 1059 and Q 1058) that were used to localize these mutations also are indicated. The chromosomal compositions of three recombinant (#6, #25, and #42) and two control (ISO and HET) flies are represented. Asterisks indicate chromosomes that carry the mutation as defined by outcrossing. The SNP markers shown were scored using DHPLC. The recombinants shown delimit the position of these mutations to between Dm1601 and Dm1655, a region of ∼984 kb. Subsequent complementation testing showed that these mutations are alleles of the hedgehog gene, which lies between Dm1601 and Dm1655. (C) DHPLC scoring of SNPs. PCR products were amplified from recombinants and analyzed under partially denaturing conditions. Data are shown for Dm1655, a C/T dimorphism. Run time in minutes is shown on the X axis and ultraviolet absorbance on the Y axis. Dm1655 was analyzed from the following strains: w;iso2;iso3 (ISO), a w;iso2;iso3/Q1059 heterozygote (HET), and hedgehog recombinants 6 and 25 (#6 and #25). In this example, the sample from recombinant 25 shows a heteroduplex pattern and therefore is scored as a heterozygote. (DHPLC) denaturing high performance liquid chromatography.