Genetic linkage mapping of zebrafish genes and ESTs

Abstract

Genetic screens in zebrafish (Danio rerio) have isolated mutations in hundreds of genes essential for vertebrate development, physiology, and behavior. We have constructed a genetic linkage map that will facilitate the identification of candidate genes for these mutations and allow comparisons among the genomes of zebrafish and other vertebrates. On this map, we have localized 771 zebrafish genes and expressed sequence tags (ESTs) by scoring single-stranded conformational polymorphisms (SSCPs) in a meiotic mapping panel. Of these sequences, 642 represent previously unmapped genes and ESTs. The mapping panel was comprised of 42 homozygous diploid individuals produced by heat shock treatment of haploid embryos at the one-cell stage (HS diploids). This "doubled haploid" strategy combines the advantages of mapping in haploid and standard diploid systems, because heat shock diploid individuals have only one allele at each locus and can survive to adulthood, enabling a relatively large quantity of genomic DNA to be prepared from each individual in the mapping panel. To integrate this map with others, we also scored 593 previously mapped simple-sequence length polymorphisms (SSLPs) in the mapping panel. This map will accelerate the molecular analysis of zebrafish mutations and facilitate comparative analysis of vertebrate genomes.

Figure 1

Production of the homozygous diploid mapping panel. C32 and SJD fish were crossed to produce a heterozygous F₁ generation. Eggs from two F₁ females were fertilized with UV-irradiated sperm, which makes no genetic contribution to the progeny. The resulting haploid embryos were subjected to heat shock treatment, which doubles the haploid chromosome set. DNA for the mapping panel was prepared from homozygous diploid F₂ adults.

Figure 2

Examples of polymorphisms scored in 23 individuals in the heat shock (HS) mapping panel and C32. An SSCP in the wnt8 gene and the SSLP Z10663 are shown.

Figure 3

(See pages 561–565.) Genetic linkage map of the zebrafish genome. Positions of 1364 polymorphic markers scored on the HS panel are shown. GenBank accession numbers, UniGene numbers, and primer sequences for genes and ESTs are shown in Table 1 (available online at http://www.genome.org). SSLP markers were described by Shimoda et al. (1999).

Figure 3

(See pages 561–565.) Genetic linkage map of the zebrafish genome. Positions of 1364 polymorphic markers scored on the HS panel are shown. GenBank accession numbers, UniGene numbers, and primer sequences for genes and ESTs are shown in Table 1 (available online at http://www.genome.org). SSLP markers were described by Shimoda et al. (1999).

Figure 3

(See pages 561–565.) Genetic linkage map of the zebrafish genome. Positions of 1364 polymorphic markers scored on the HS panel are shown. GenBank accession numbers, UniGene numbers, and primer sequences for genes and ESTs are shown in Table 1 (available online at http://www.genome.org). SSLP markers were described by Shimoda et al. (1999).

Figure 3

(See pages 561–565.) Genetic linkage map of the zebrafish genome. Positions of 1364 polymorphic markers scored on the HS panel are shown. GenBank accession numbers, UniGene numbers, and primer sequences for genes and ESTs are shown in Table 1 (available online at http://www.genome.org). SSLP markers were described by Shimoda et al. (1999).

Figure 3

(See pages 561–565.) Genetic linkage map of the zebrafish genome. Positions of 1364 polymorphic markers scored on the HS panel are shown. GenBank accession numbers, UniGene numbers, and primer sequences for genes and ESTs are shown in Table 1 (available online at http://www.genome.org). SSLP markers were described by Shimoda et al. (1999).