Large-scale mapping of mutations affecting zebrafish development

Selection of markers for genome scanning

The average distance between markers of the G4 set is 11.6 cM, and all distances are smaller than 36 cM, except for a 71.1 cM interval on LG21 (between Z4425 and Z1497). Within this particular interval few MGH markers are available, and no suitably polymorphic marker could be identified in our reference crosses. For the H2 set the average distance is 11.5 cM, and all distances are smaller than 53.8 cM, except for a 83.3 cM interval on LG21. The more uneven chromosomal distribution of markers in the H2 set reflects the fact that frequently the best markers available were already used in the G4 set.

Our mapping methodology as described below can theoretically detect significant linkage over a distance of approximately 36 cM (assuming the genotyping of 48 mutant individuals). However, since the LOD score is proportional to the number of individuals scored, this range can be easily increased by adding more mutant individuals if a linkage is questionable. Our marker sets therefore cover the genome adequately to detect significant linkage with the great majority of mutant loci. All the mutant loci mapped in this work have confirmed linkage to at least one G4 or H2 marker (not shown if the closest flanking markers were selected from outside the sets).

Mapping of mutant loci

We report here on the mapping of 319 mutant loci identified in the ENU-based Tübingen I mutagenesis screen [1, 2] and subsequent screening among the mutant collection (Additional file 1). For 42 of the loci the corresponding genes have already been identified by other researchers, as listed by the ZFIN database [10]; they are included as controls for our mapping procedure (see below). Not included are 70 successfully mapped loci for which the corresponding genes were already published by ourselves or such a publication is in preparation, or the carriers of which were lost after mapping.

For each mutation we crossed mutant carriers against the polymorphic reference line WIK which was established in our lab for this purpose [8]. Brother-sister matings were performed in the F1 and the F2 progeny was sorted by phenotype. DNA was prepared on 96-well plates, and aliquots of 36 – 48 mutant F2 individuals and their wildtype siblings were pooled. Genome scanning was performed by PCR of the mutant and sibling pools with the markers of the G4 marker set, and the band intensities on agarose gels were quantified semi-automatically using NIH Image software as well as visually assessed to identify potential linkages. Mutant and sibling pools representing up to 24 different mutations were tested in parallel. Verification of the best potential linkages (up to six) for each mutation was then attempted by performing PCR of the respective marker with the individual mutants and siblings that had been used for pooling, and counting the recombinant genotypes (for the genotype data see Additional file 2). Siblings were always included on the same gel as a control to confirm that the marker is polymorphic and the two polymorphic bands appear at the proper frequency. If no potential linkage could be verified for a mutation and sufficient material was available, the procedure was repeated once with the G4 marker set, and another two times with the H2 marker set. If possible, DNA was prepared from a different F1 pair for each genome scan, since the Tübingen and WIK lines used are not isogenic and markers that show no usable polymorphism in progeny of one F2 pair are therefore sometimes usable in progeny of another one.

A potential linkage was considered confirmed if it had a two-point LOD score equal or greater than 3. The individuals were then genotyped for all polymorphic markers from the same marker set and chromosomal region in order to identify, if possible, a pair of markers flanking the mutation, and if that was not possible, the two closest markers on one side of the mutation. Occasionally additional markers not in the chosen marker set were also included in the genotyping. Decisions on whether or not a mutation was flanked by two markers were based on whether recombinations with the markers occurred independently. For details of the mapping procedure and the calculation of map positions see the Methods section and [9].

In total, mapping was attempted for 486 mutations from the Tübingen I screen and subsequent screens of the mutant collection and successful for 389, giving a success rate of 80 %. 12 of these could be mapped only with the H2 set. Unsuccessful mapping experiments were due to difficulties in obtaining sufficient F2 individuals and to PCR problems as well as to a lack of polymorphic markers in our marker set. Among the mutations to be mapped, a group of 63 was prioritized based on interest in their phenotypes. For each of these several additional mapcrosses were set up (data not shown). 56 mutations of this group, or 89 % were successfully mapped, providing a lower limit for the percentage of mutations that our marker sets and methodology is capable of mapping if sufficient F2 individuals are available. The biggest distance to markers on either side at which we could confirm linkage was 31.9 cM (for the mutation spt), approaching the theoretical cutoff of 36 cM.

Chromosomal distribution of mutant loci

Between 1,400 and 2,400 zebrafish genes have been estimated to have visible mutant phenotypes in embryonic and early larval development [1, 11]. Therefore the loci reported in this work represent at least one eighth and possibly as much as quarter of all the loci that can be mutated to give a visible phenotype.

The number of mapped loci assigned to each chromosome is between 6 and 32 (on average 12.8 ± 5.8) (Figure 1). These numbers are not significantly correlated with the number of mutant loci per chromosome identified by insertional mutagenesis in the laboratory of N. Hopkins ([11, 12] and unpublished data, available from ZFIN [10]) (R² = 0.02 assuming a linear regression relationship) or with the number of Ensembl genes per chromosome in the Ensembl Zv6 assembly [13] (R² = 0.19); by comparison, the values of Amsterdam et al. have a slightly stronger correlation to the number of Ensembl transcripts (R² = 0.28). Because mapping with our methodology was successful for 80% of all mutations for which it was attempted, possible deficiencies of the mapping method cannot fully account for this low correlation. Rather, it probably reflects an uneven distribution of genes with specific, visible phenotypes in embryos or early larvae as identified in ENU mutagenesis screening, and the absence of such selectivity in the insertional mutagenesis experiment, demonstrating that both types of mutagenesis experiments complement each other in their coverage of their genome. Moreover, we cannot rule out region-specific differences in ENU mutagenesis efficiency.

Assessment of mapping quality

A comparison of the linkage group assignments shows that two of the 21 genes (frs/slc25a and ovl/ift88) are assigned to a different linkage group by ZMAP, in both cases based on results from the Heat Shock (HS) panel [14–16]. However, several published linkages to genetic markers support our linkage group assignment of frs/slc25a [17] while our assignment of ovl/ift88 is supported by the T51 panel (as shown on the ZFIN website) and by the latest version of the HS map [18]. In conclusion, none of our linkage group assignments is conclusively contradicted by gene mapping.

Next we compared the map positions of the mutations with those of the genes on ZMAP (using the median of the ZMAP positions if a gene was placed on more than one mapping panel). If we assume the gene positions to be correct, we obtain a standard error of our mutant map positions of 6.1 cM. Further assuming a normal distribution of errors, we can predict that approximately 95 % of the genes should be within 12.2 cM (two standard errors) of the rough mapping position of the mutation. Indeed, 17 out of the 19 genes mapped on the same chromosome (90 %) are within two standard errors of the mutation, and 16 out of 19 (84 %) within one standard error. Actually both mutation and gene mapping contribute to the observed errors to an unknown degree, so that 6.1 cM merely represents an upper limit for the standard error of our mapping procedure.

Fish breeding

Mapcrosses were set up between mutant carriers and the laboratory reference line WIK [8] and brother-sister matings were performed between F1 individuals following standard laboratory procedures [25].

DNA preparation

F2 embryos were sorted by phenotype and stored in Eppendorf tubes with 100 % MeOH at -70°C until use. Single embryos were arrayed on a 96-well microtiter plate with a glass Pasteur pipette. The MeOH was evaporated on a PCR block at 70°C and 25 μl of 1.7 mg/ml Proteinase K in 1 × TE was added to each well. The plate was covered with sealing film and heated to either 55°C or 70°C for 240 min and to 94°C for 10 min in a thermocycler. 10 μl of each of the sibling and mutant lysates was pooled and 45 μl sterile ddH₂O was added to the remainder. Plates were stored at -20°C.

Genotyping

PCR was initially performed on mutant and sibling pools for genome scanning, and subsequently on the individuals that had been used for the pooling in order to confirm potential linkages to specific markers. 20 μl PCR reactions were set up from 14.28 μl of reaction mix (2 μl of 10 × PCR buffer, 0.04 μl each of 100 mM dATP, dCTP, dGTP and dTTP, 12.12 μl water), 0.16 μl each of 20 mM forward and reverse primer, 0.4 μl of 5U/μl Taq polymerase, and 5 μl of template DNA. 10 × PCR buffer contained 100 mM Tris-HCl (pH 8.3), 500 mM KCl, 15 mM MgCl₂ and 0.1 % (w/v) gelatin. All pipetting was done with a Biomek 2000 robot. Cycling was carried out by initially denaturating at 94°C for 2 min, 35 cycles of denaturation at 94°C for 30 sec, annealing at 60°C for 30 sec and extension at 73°C for 1 min, and a final extension at 73°C for 5 min. 5 μl of 6 × loading buffer were added to each sample, and electrophoresis was carried out at 200 V for 45 min in 1 × TBE buffer, on 2 % agarose gels. Gels were imaged and scored semi-automatically with NIH Image and a set of custom-designed macros.

Calculation of map positions

Distances between mutations and markers were calculated by determining the recombination fraction in the mutant F2 individuals and applying the Kosambi mapping function. Linkages with a two-point LOD score equal or greater 3 were regarded as significant.

In order to place a mutation in the genetic interval between the closest marker and another linked marker we determined whether recombinations for both of them were correlated. For this purpose we considered only single recombinants for the closest marker, i.e. heterozygotes. If the majority of these were heterozygous for the second marker we regarded the recombinations as uncorrelated and placed the mutation in the interval between the markers. Otherwise we placed the mutation outside the interval in the direction opposite from the second marker.

Assuming complete meiotic interference, i.e. only a single recombination event per chromosome, all recombinants for the first marker should be either non-recombinant for the second marker if the markers flank the mutation, or heterozygous if both markers are on the same side of the mutation. In our data approximately half of the mutations gave results in between these extremes. This may be due to occasional contaminations of the PCR assays but also to less than complete meiotic interference, which would allow a second recombination in the same individual. We therefore did not eliminate any contradictory individuals from the calculation of genetic distances as they may represent a genuine second recombination.

If a mutation could be placed in an interval between two markers, a map position was calculated by scaling the observed distances between the mutation and the markers so as to fit into the published distance between the markers. In the remaining cases only the distance to the closest marker was used to calculate the map position. A FileMaker Pro 5 database was used to store the scoring data and perform the calculations [9]. The latest version of the MGH map, available through ZFIN [10], was used as a reference for calculating map positions.