High-throughput gene mapping in Caenorhabditis elegans - PubMed (original) (raw)

High-throughput gene mapping in Caenorhabditis elegans

Kathryn A Swan et al. Genome Res. 2002 Jul.

Abstract

Positional cloning of mutations in model genetic systems is a powerful method for the identification of targets of medical and agricultural importance. To facilitate the high-throughput mapping of mutations in Caenorhabditis elegans, we have identified a further 9602 putative new single nucleotide polymorphisms (SNPs) between two C. elegans strains, Bristol N2 and the Hawaiian mapping strain CB4856, by sequencing inserts from a CB4856 genomic DNA library and using an informatics pipeline to compare sequences with the canonical N2 genomic sequence. When combined with data from other laboratories, our marker set of 17,189 SNPs provides even coverage of the complete worm genome. To date, we have confirmed >1099 evenly spaced SNPs (one every 91 +/- 56 kb) across the six chromosomes and validated the utility of our SNP marker set and new fluorescence polarization-based genotyping methods for systematic and high-throughput identification of genes in C. elegans by cloning several proprietary genes. We illustrate our approach by recombination mapping and confirmation of the mutation in the cloned gene, dpy-18.

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Figures

Figure 1

Figure 1

Distribution of single nuclear polymorphism (SNP) markers across the Caenorhabditis elegans genome. (A) Marker distribution along the physical map of chromosome. The location of the Tier 1 SNP markers are shown with open squares, and the location of Tier 2 markers are shown by open circles. The location of each of the validated 1099 markers is shown along the chromosomal axis. The largest gap is 449 kb (chromosome I), and the average validated marker spacing is 91 kb ± 56 kb. (B) Recombination rates across the six chromosomes. The physical (Mb) location of all predicted polymorphisms (y-axis) is plotted versus the extrapolated genetic (cM) location (x-axis) of the associated cosmid across the genome (Genetic Map Position from Wormbase; Stein et al. 2001). This illustrates the rate of change in the recombination rate across the chromosome and shows the “gene cluster” effect in the center of the autosomes (Barnes et al. 1995). This information is used during mapping, because the local recombination rate affects the number of putative recombinants that must be genotyped to obtain a 100-kb interval.

Figure 1

Figure 1

Distribution of single nuclear polymorphism (SNP) markers across the Caenorhabditis elegans genome. (A) Marker distribution along the physical map of chromosome. The location of the Tier 1 SNP markers are shown with open squares, and the location of Tier 2 markers are shown by open circles. The location of each of the validated 1099 markers is shown along the chromosomal axis. The largest gap is 449 kb (chromosome I), and the average validated marker spacing is 91 kb ± 56 kb. (B) Recombination rates across the six chromosomes. The physical (Mb) location of all predicted polymorphisms (y-axis) is plotted versus the extrapolated genetic (cM) location (x-axis) of the associated cosmid across the genome (Genetic Map Position from Wormbase; Stein et al. 2001). This illustrates the rate of change in the recombination rate across the chromosome and shows the “gene cluster” effect in the center of the autosomes (Barnes et al. 1995). This information is used during mapping, because the local recombination rate affects the number of putative recombinants that must be genotyped to obtain a 100-kb interval.

Figure 2

Figure 2

Tiered mapping strategy. Shown is a schematized mapping workflow. (A) Tier 1 mapping localizes the gene of interest to a chromosome by assessing linkage to one centrally located SNP. (B) Tier 2 mapping localizes the gene to a subregion of the chromosome. This region can vary in size between 1 and 6.7 Mb. In the dpy-18 example shown, the gene falls between two SNP markers that are 2 Mb apart on chromosome III. This resolution is routinely achievable by genotyping 30–60 recombinants. (C) Tier 3 mapping begins by identification of informative animals with recombination breakpoints within the region defined by Tier 2 and then fine mapping with Tier 3 markers to narrow the region of interest. For dpy-18, Tier 3 mapping localizes the gene to a region as small as 97 kb and thus narrowed the candidate region to ∼0.1% of the worm genome. The number of recombinants required to achieve this mapping resolution will depend on the local recombination rate (Fig. 1B; Genetic Map Position from Wormbase; Stein et al. 2001) along the chromosome in the vicinity of the mutated gene. (D and E) Tier 4 mapping and/or mutation detection. Further refinement of the candidate interval occurs by validation of additional SNP markers and genotyping of informative recombinants. For dpy-18, the location of the predicted substitution SNPs located within the 97-kb region of chromosome III are shown. During this fine-mapping process, candidate gene approaches such as cosmid rescue or RNA interference (RNAi) can also be used to help identify the mutation.

Figure 3

Figure 3

Sample fluorescence polarization-template directed incorporation (FP-TDI) mapping data. (A) High quality discrimination data from the FP-TDI assay of 48 random SNP markers on control genomic DNA from N2 and CB4856 strains. SNPs are detected through analysis of a single base pair extension from a sequencing primer that hybridizes just adjacent to the polymorphic nucleotide (Chen et al. 1999). Clusters are readily identified and unambiguous. (B) FP-TDI data from eight recombinant samples (crude worm lysates), each tested with six SNP markers. In this case, the FP-TDI kit used distinguishes A from T, the most prevalent SNP change in C. elegans. (C) FP-TDI analysis with the four Tier 2 SNP markers on chromosome III using 35 samples from a mapping cross to localize dpy-18. We localized the recessive gene to a 2.0-Mb subregion of chromosome III. SNP data are converted to table format for interpretation, and the informative recombinants that defined the interval are labeled Left 1, Left 2, and Right 1. (D) The 97-kb interval defined by the Tier 3 markers CE3–194 and CE3–195 is shown. There are 16 predicted genes in this region (AceDB version WS-48); for Tier 4 mapping there are nine predicted substitution SNPs in this interval. RNAi of the predicted gene sequence Y47D3B.10 is known to give a Dumpy (Dpy) visible phenotype (Hill et al. 2000), indicating that this gene is a strong candidate for dpy-18. We confirmed by directed sequencing that dpy-18(e364) did contain a mutation G to A in exon 3 altering the TGG(Trp) codon to TAG(Stop).

References

    1. Barnes TM, Kohara Y, Coulson A, Hekimi S. Mitotic recombination, noncoding DNA and genomic organization in Caenorhabditis elegans. Genetics. 1995;141:159–179. - PMC - PubMed
    1. C. elegans Sequencing Consortium. Genome sequence for the nematode C. elegans: A platform for investigating biology. Science. 1998;282:2012–2018. - PubMed
    1. Chen X, Levine L, Kwok PY. Fluorescence polarization in homogeneous nucleic acid analysis. Genome Res. 1999;9:492–498. - PMC - PubMed
    1. Collins FS. Positional cloning moves from perditional to traditional. Nat Genet. 1995;9:347–350. - PubMed
    1. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998;391:806–811. - PubMed

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