A universal method for automated gene mapping

Abstract

Small insertions or deletions (InDels) constitute a ubiquituous class of sequence polymorphisms found in eukaryotic genomes. Here, we present an automated high-throughput genotyping method that relies on the detection of fragment-length polymorphisms (FLPs) caused by InDels. The protocol utilizes standard sequencers and genotyping software. We have established genome-wide FLP maps for both Caenorhabditis elegans and Drosophila melanogaster that facilitate genetic mapping with a minimum of manual input and at comparatively low cost.

Figure 1

FLP detection of InDels of various sizes in homozygotes and heterozygotes. In each panel the top two graphs show the homozygotes and the bottom graph the heterozygote. Gray shaded areas mark the defined expected allele lengths and red lines indicate the borders of a predefined window of expected allele lengths. (a-c) Detection of InDels in C. elegans that show increasing levels of adenosine (A) addition. (a) 3-bp InDel ZH1-01 with no A addition; (b) 12-bp InDel ZH2-01 with A addition; (c) 2-bp InDel ZH3-05a with A addition. (d) 1-bp InDel ZH3-23 in C. elegans with A addition. An unambiguous allele-call can be made, irrespectively of the level of A addition: both homozygous samples consist of two peaks at different positions, whereas the heterozygous animal exhibits three peaks. (e) The 1-bp InDel 3R160 in Drosophila runs over a 12-13 nucleotide poly(T) stretch and exhibits stutter bands. Even in this case, a clear allele-call can be made (three peaks in homozygous and four peaks in heterozygous animals). (f) The 6-bp InDel ZHX-22 in C. elegans occurs in a poly(C) stretch and the FLP graph displays stutter bands. As expected, the longer fragment exhibits a higher degree of stuttering.

Figure 2

C. elegans and Drosophila FLP maps. (a) The C. elegans FLP map. Marker names comprise a ZH prefix followed by the chromosome number and a unique identifier number. Markers used in first-level assays (Tier 1) for determination of chromosomal linkage are in red, those used for second-level assays (Tier 2) for higher resolution mapping are in black. (b) The Drosophila FLP map of chromosomes 2 and 3. The FRT sites and EP elements are symbolized by blue and green triangles, respectively. The strains that were genotyped are shown below each chromosome. Green indicates the EP genotype, blue the FRT genotypes and new alleles are shown in other colors.

Figure 3

FLP mapping in C. elegans. (a) Crossing scheme used to map mutations generated in the N2 Bristol background. The different classes of recombinants recovered in the F₂ generation are shown. (b) Analysis of the zh41 mutation with Tier 1 assays establishes linkage to chromosome I. (c) Analysis with Tier 2 places zh41 between assays ZH1-01 and ZH1-15. ND, no data as a result of PCR reaction failure. (d) Ventral views of the vulva in wild-type and zh41 L4 larvae stained with the adherens junction antibody MH27 [44]. In the wild type, the vulval cells have fused to generate the torroids that appear as concentric rings. zh41 mutants exhibit the same fusion defects observed in other lin-11 alleles [33].

Figure 4

FLP mapping in Drosophila. (a) Crossing scheme used to map mutations generated in the FRT background and recombined with an EP line. The different classes of recombinants recovered in the F₂ generation are shown. (b) Big head phenotypes of the hippo null allele hpo^42-20 (1) and the VI.29 mutation (2). A wild-type control is shown in (3). (c) FLP mapping of the VI.29 mutation on chromosome 2R. Analysis of the different classes of recombinants places the mutation between markers 2R096 and 2R109 (dashed red line). Informative recombinants are boxed in red. ND, not determined or no data as a result of PCR reaction failure.