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Genome-wide high-density SNP map of Drosophila melanogaster

In Drosophila melanogaster, single nucleotide polymorphisms (SNPs) and insertions/deletions (InDels) can be found in high frequency, in average once every 200-300bp, when comparing two or more strains. Since the majority of these polymorphisms are also phenotypically neutral, and existing in two codominant variants, they are highly suitable as marker for positional cloning of mutations. We have created a genome-wide, high-density SNP map of Drosophila melanogaster consisting of 2300 SNP markers (1kb amplicons containing at least one SNP), evenly spaced across all major chromosome arms (X, 2L, 2R, 3L, and 3R), with an average distance of 49.7kb. For SNP detection, 1kb regions from three to seven standard laboratory strains per site were selectively amplified, sequenced and compared to the Release 4 genomic sequence from FlyBase. Amplification primers were designed so that they preferentially lie in non-repetitive and non-coding regions of the genome. After automatic SNP detection by the PolyBayes software package, quality criteria were applied to filter the most probable SNPs, followed by visual inspection of the alignments to increase reliablity of the data. With this procedure, 200.000 allele calls were reduced by ca. 50% to give a high-quality, reliable SNP map. When two strains are compared, one can find at least one polymorphism in 66%-86% of all 1kb segments. The polymorphisms are evenly distributed except for the regions lying close to the tip and the centromer of each chromosome arm.

Construction of the SNP map proceeded in three phases: an initial low-density map, an intermediate medium-density SNP map and a final high-density SNP map. The low-density map consisted of ca. 120 SNP markers, the medium-density map of >600 SNP markers, and the high-density map of 2300 SNP markers. The average distances between any two adjacent markers was 1 Mb, 200 kb, and 50 kb, respectively. Three different wild type, one balancer, one recessive marker, one FRT, and one dEP stock per chromosome arm were genotyped for the SNP map. For the low resolution map all listed stocks were analysed, for the medium resolution map all wild type and one marker, one FRT, and one dEP stock were used. For the high-resolution map, only the two most divergent stocks and a third stock which is useful for mapping purposes were analysed. All lines needed for the SNP map were isogenized and tested for the lack of any obvious developmental phenotype.

Dissemeniation of the SNP map occurs via the FLYSNP Database (http://flysnp.imp.univie.ac.at/snpdb.php) at the FLYSNP Website. SNPs from a specified region can be selected, and the corresponding data (allelic variants, coordinates, flanking regions, amplification primer sequences, genotyping assays etc.) downloaded. A direct link to FlyBase-GBrowse enables the user to open the graphical representation of the specified chromosomal segment in a separate window. Attempts to link the FLYSNP Database directly to FlyBase, and to enter the data to NCBI-dbSNP, are ongoing.

The website also includes an introduction to the FLYSNP project, a user guide, protocols, contact addresses, workshop information, and links to important relevant sites. Also, links to the FLYSNP Website were added to several other webpages, and the project was mentioned in listings of resources (e.g. FlyBase website, section External Resources, see http://www.flybase.net/allied-data/resources.html; publication "Research Resources for Drosophila: The Expanding Universe", Matthews K.A., Kaufman T.C., Gelbart W.M., Nature Reviews Genetics, 2005, Vol. 6, pp179-193.).

Mapping mutations from a mutagenesis screen used to be a very time-consuming task, which usually lasted for several months or even years. With the SNP mapping technique, which is now the state-of-the-art mapping method, one can reduce the process to a few months. Once genes involved in a certain process are identified, researchers can focus on further analysis of these genes, giving them much more time for downstream analyses and conclusions from their screens. Therefore, the genome-wide high-density SNP map created by FLYSNP provides a valuable and useful resource for researchers who need to locate mutations.

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IMBA, Austrian Academy of Sciences
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