Rapid mapping of zebrafish mutations with SNPs and oligonucleotide microarrays

Abstract

Large-scale genetic screens in zebrafish have identified thousands of mutations in hundreds of essential genes. The genetic mapping of these mutations is necessary to link DNA sequences to the gene functions defined by mutant phenotypes. Here, we report two advances that will accelerate the mapping of zebrafish mutations: (1) The construction of a first generation single nucleotide polymorphism (SNP) map of the zebrafish genome comprising 2035 SNPs and 178 small insertions/deletions, and (2) the development of a method for mapping mutations in which hundreds of SNPs can be scored in parallel with an oligonucleotide microarray. We have demonstrated the utility of the microarray technique in crosses with haploid and diploid embryos by mapping two known mutations to their previously identified locations. We have also used this approach to localize four previously unmapped mutations. We expect that mapping with SNPs and oligonucleotide microarrays will accelerate the molecular analysis of zebrafish mutations.

Figure 1

Zebrafish SNP map. Vertical lines represent the 25 linkage groups, dots represent individual SNPs. Red dots correspond to SNPs represented on the oligonucleotide microarray. The positions of sequenced markers were derived from the meiotic map of Woods et al. (2000). Names, sequences, positions, and strain data for SNPs are shown in Web Supplement A.

Figure 2

Distribution of transitions and transversions among SNPs.

Figure 3

(A) Oligonucleotide probes specific for two SNPs. (B) Mapping scheme with haploid crosses. Haploid embryos are generated from F₁ females heterozygous for the mutation and for many SNPs (two of which are shown). SNPs are scored in pooled wild-type and mutant samples by hybridizing differentially labeled fragments to the same microarray. Alleles of linked SNPs are differentially labeled, whereas unlinked SNPs have both labels on each allele. Putative locations indicated by the microarray analysis are then tested by scoring markers in the region in individuals (not shown). (C) Microarray data mapping the floating head mutation to LG13. A two-color image from the hybridized microarray and a graph of relative hybridization intensity for the four probes of two SNPs are shown. ZSNP1100 on LG 13 exhibited differential labeling characteristic of linked markers. Polymorphic markers at other positions, including ZSNP653 on LG7, showed labeling characteristic of unlinked markers. In another trial of this experiment, we obtained similar results: Differential labeling indicative of linkage was detected for ZSNP1100, and there were no false positive or false negative markers (data not shown).

Figure 4

(A) Mapping scheme for diploid crosses. Boxes represent F₂ progeny from an intercross of F₁ fish (not shown) heterozygous for the mutation and for many SNPs, two of which are shown. Numbers adjacent to the genotypes refer to the ratios at which they are present among F₂ progeny. SNPs from wild-type and mutant pools are differentially labeled as in Fig. 3. For recessive mutations, the SNP allele linked in cis to the mutation is present in both pools due to the presence of heterozygotes in the wild-type pool. Therefore, this allele is labeled with both colors, whereas its wild-type counterpart is labeled only with one color (green in this example). (B) Microarray data mapping the st11 mutation to LG2. A two-color image from the hybridized microarray and a graph of relative hybridization intensity for the four probes of two SNPs are shown. ZSNP163 on LG2 exhibited differential labeling characteristic of linked markers. Polymorphic markers at most other positions, including ZSNP1120 on LG13, showed labeling characteristic of unlinked markers. (C) Map of LG2 showing the positions of st11 and SNPs that showed differential labeling characteristic of linked markers. Analysis of ZSNP158, ZSNP163, ZSNP173, and SSLP marker Z11023 in individual embryos confirmed that st11 resides in this region of LG2 (distances indicated). In another trial of this experiment (data not shown), we detected differential labeling indicative of linkage for markers ZSNP163, ZSNP165, ZSNP171, and ZSNP173 on LG2; no base calls were generated for ZSNP158, ZSNP159, and ZSNP167, or for ZSNP196, which was detected as a false positive in the other trial.