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Letter

Genetic variation in the zebrafish

¹ Hubrecht Laboratory, Netherlands Institute for Developmental Biology, 3584CT, Utrecht, The Netherlands; ² Departmentof Genetics, Washington University Medical School, St. Louis, Missouri 63130, USA

	ABSTRACT

Top ABSTRACT Results and Discussion Methods Acknowledgments References

 
Although zebrafish was introduced as a laboratory model organism several decades ago and now serves as a primary model for developmental biology, there is only limited data on its genetic variation. An establishment of a dense polymorphism map becomes a requirement for effective linkage analysis and cloning approaches in zebrafish. By comparing ESTs to whole-genome shotgun data, we predicted >50,000 high-quality candidate SNPs covering the zebrafish genome with average resolution of 41 kbp. We experimentally validated 65% of a randomly sampled subset by genotyping 16 samples from seven commonly used zebrafish strains. The analysis reveals very high nucleotide diversity between zebrafish isolates. Even with the limited number of samples that we genotyped, zebrafish isolates revealed considerable interstrain variation, ranging from 7% (inbred) to 37% (wild-derived) of polymorphic sites being heterozygous. The increased proportion of polymorphic over monomorphic sites results in five times more frequent observation of a three allelic variant compared with human or mouse. Phylogenetic analysis shows that comparisons between even the least divergent strains used in our analysis may provide one informative marker approximately every 500 nucleotides. Furthermore, the number of haplotypes per locus is relatively large, reflecting independent establishment of the different lines from wild isolates. Finally, our results suggest the presence of prominent C-to-U and A-to-I RNA editing events in zebrafish. Overall, the levels and organization of genetic variation between and within commonly used zebrafish strains are markedly different from other laboratory model organisms, which may affect experimental design and interpretation.

There are several key advantages that distinguish another type of marker, single nucleotide polymorphism (SNP), as a marker of choice for many genetic studies. To mention a few, SNPs are the most common type of variation in genomes, allowing the generation of ultra-dense genetic maps, and there are efficient low- and high-throughput typing procedures for SNPs currently available (for review, see Vignal et al. 2002). Until now only a low-density SNP-based mapping panel with 2000 polymorphisms was available (Stickney et al. 2002); the SNP-map contains large gaps up to 58 cM and needs further refinement for routine applications.

In addition to simplifying genetic mapping experiments, studies on genetic variation in model organisms can clarify rate and composition as well as distribution and organization of polymorphic loci in the genome. In particular, it is not clear how much variation still persists in zebrafish laboratory inbred and outbred strains and how it compares to that present in wild isolates. The discovered variation at 9% of tested polymorphic loci in initially homozygous zebrafish C32 strain (Streisinger et al. 1981) raised a discussion of whether high mutation rate (Buth et al. 1995) or introgression (Nechiporuk et al. 1999) has introduced polymorphisms to this strain. However, the abundance of genetic variation in zebrafish inbred strains alone suggests that individuals within a strain have a diverse genetic background. From this perspective, zebrafish inbred strains differ from other commonly used vertebrate laboratory animals such as inbred mouse or rat strains.

Finally, the analysis of genotype data contributes to better understanding of strain history and the degree of interstrain variation. The variety of methods used to generate inbred lines, e.g., gynogenetic diploids and half-tetrad diploids, inbreeding (for review, see Beier 1998), different natural sources of animals, and breeding regimes, is likely to influence the allele fixation rates in different zebrafish isolates and strains. Knowledge of the phylogenetic relationships between laboratory strains greatly facilitates the choice of strains, which will be most informative for a genetic experiment.

	Results and Discussion

Top ABSTRACT Results and Discussion Methods Acknowledgments References

 
Candidate SNP discovery
We have developed a computational SNP discovery pipeline and candidate SNP database named CASCAD (Cascad Snp Candidate Database, http://cascad.niob.knaw.nl) (Guryev et al. 2004, 2005). Here, we report an application of this pipeline for construction and verification of a single-nucleotide variation database for zebrafish (D. rerio). Publicly available EST and mRNA sequences were compared to each other and to whole-genome shotgun (WGS) trace sequences for the occurrence of SNPs (Table 1). We did not predict variations within the WGS set. The polymorphisms that are observed in expressed sequences may be particularly useful for genetic mapping studies as well as for drawing inferences about functional differences of genes in different strains.

View this table: [in this window] [in a new window]   Table 1 Input and output statistics for the computational prediction of zebrafish candidate SNPs using the CASCAD pipeline

The average frequency of candidate SNP is 1 per 41 kbp, and the largest gap between two adjacent markers is 2 Mb on linkage group 14. About one-third of the candidates reside in genomic regions that are annotated as protein coding, including 9111 synonymous and 6375 nonsynonymous changes. The candidate SNPs cover 13,016 of 31,219 UniGene clusters and 7841 of 22,877 predicted Ensembl genes, or approximately one-third of zebrafish genes.

Over 66% of the candidate SNPs could be assigned to unique positions in the current zebrafish genome build (Zv5; http://www.sanger.ac.uk/Projects/D_rerio/ Zv5_assembly_information. shtml). We failed to place 4% of the candidate SNPs to any location on the assembly, and a further 19% of the candidate SNPs mapped to multiple locations. Presumably, the major part of the nonunique fraction was assigned to fragments that are present redundantly in Zv5 as an artifact of the assembly process in its intermediate stage, although a small fraction may result from sequence difference between otherwise highly similar paralogs. We should mention here that Zv5 is a draft assembly and in addition to false duplications also contains other misassemblies and dropouts, meaning that all interpretations based on it should be treated with caution. Our analysis indicates that 73% candidate SNPs map to the same linkage group in Zv5 as they would be placed on gene-based meiotic map of Woods and coworkers (2005), considerably better than 66% overlap between this meiotic map and our candidates mapped on previous Zv4 assembly.

Validation of SNPs
To validate the computationally predicted SNPs, we assayed 398 candidate SNP-containing amplicons evenly distributed over the 25 zebrafish linkage groups (Fig. 1). By resequencing these regions in a panel of 16 individuals representing seven widely used laboratory strains, we were able to confirm 65% (256) of them. This relatively low confirmation rate may at least partially be explained by the presence of false negatives due to high intrastrain variation (see below) in combination with the small sampling size per strain (typically two individuals). In addition, the origins of some of the strains used in EST library construction are unknown, or samples from the same population were not available to us. Although segmental genomic duplications could potentially result in false positives, we did not observe evident deviations from Hardy-Weinberg equilibrium of allele frequencies, such as excess of heterozygotes that distinguishes paralogous sequence variants from true SNPs, in our verification experiments.

View larger version (60K): [in this window] [in a new window]   Figure 1. Distribution of candidate and verified SNPs on zebrafish physical map (working draft genome assembly Zv5). Vertical bars represent zebrafish linkage groups; horizontal bars on left side of each linkage group show candidate SNP density given in red for coding and in blue for noncoding candidates (according to Ensembl genome annotation 35.5b, window size 280 kbp). The filled and open boxes to the right of the linkage group correspond to amplicons with confirmed and nonconfirmed candidate SNPs, respectively. The number in each box indicates the total number of confirmed polymorphisms in each amplicon. Genotype information and oligonucleotide primers are available from http://cascad.niob.knaw.nl/snpview.

More than 96% of all polymorphic loci were diallelic (2118/2198), and the remainder consisted predominantly of short SSLPs. One-tenth of the variants observed (228) were due to small insertions or deletions (indels), displaying an intermediate indel frequency if compared to human and chicken (6.6% and 13.9%, respectively; source, dbSNP build 124). Only a small fraction of polymorphisms identified in this study was observed within coding sequence as annotated in the Ensembl database, with 178 of them being silent, 85 missense, and two frameshift mutations.

We have designed a Web interface (http://cascad.niob. knaw.nl/snpview) that facilitates the selection and use of the validated SNPs in genetic experiments. This tool allows the interactive retrieval and visual representation of validated SNPs for arbitrary combinations of strains.

Candidate SNP characteristics and validation
A comparison of SNP prediction and its verification results for different organisms can shed light on species-specific characteristics of variation. Our CASCAD SNP discovery pipeline (Guryev et al. 2005) used a comparable amount of input data but resulted in many more candidate polymorphisms for zebrafish than for rat, providing indirect evidence for higher nucleotide diversity in zebrafish. A comparison of the verification experiment results in rat and zebrafish can reveal classes of candidate polymorphisms with increased or reduced confirmation success rates. We calculated the correlation between various SNP characteristics and the confirmation status (Table 2), potentially revealing driving forces that shape polymorphism composition in these two organisms. In addition, these correlations were used to define a confirmation likelihood score (categories 0–9), allowing database users to restrict their search to a subset of SNPs with higher expected validation rates.

View this table: [in this window] [in a new window]   Table 2 Correlation between validation of candidate SNPs and their characteristics

Surprisingly, transitive substitutions were less frequently confirmed in zebrafish in contrast to rat, for which they had a higher verification level. As the ratio between transitions and transversions is similar for both organisms, an organism-specific mechanism is suspected. Interestingly, we found two classes of frequently nonconfirmed transitive variants in our verification set, and these correspond to the most frequent type of vertebrate RNA editing events: A_DNA to G_cDNA (P < 0.1) and C_DNA to T_cDNA (P < 0.01) due to A-to-I editing and C-to-U editing, respectively. As editing events usually affect multiple consecutively located sites, many of these events may easily be filtered out by our stringent filtering for candidates. Therefore, we performed a computational whole-genome screen for individual mismatches between EST sequences and the zebrafish genome assembly. The search was restricted to the sense strand as annotated in Ensembl build 31 and showed 8% overrepresentation of A-to-G over G-to-A substitutions and 11% excess of C-to-T versus T-to-C substitutions. From the analysis of this limited set of ESTs, we estimate that there are at least 2600 editing sites (C-to-U and A-to-I). Similarly to primates, RNA editing may be very abundant in zebrafish, with a frequency of A-to-I editing of one order of magnitude larger compared with that of mouse, rat, chicken, or fly (Eisenberg et al. 2005). However in contrast to primates, C-to-U editing events seem to be more common than are A-to-I editing in zebrafish.

When we now eliminate all nonconfirmed polymorphisms from our confirmation experiment that may have been due to RNA-editing events, we observe a positive, although not significant (possibly due to lower sample size, n = 339) verification correlation with both CpG sites and transitive mutations, similar as for the rat. These results strongly suggest that high rates RNA editing events in zebrafish account for the observed relatively low confirmation rate of transitive candidate SNPs. We need to note that in absence of solid experimental data, one cannot exclude an alternative explanation for this apparent bias between ESTs and genomic sequence, namely, the occurrence of cytosine deamination during sample preparation and library construction, but it seems unlikely as it is observed for two independent EST data sets (Washington University EST project, http://genome.wustl.edu/est) (Lo et al. 2003).

Nucleotide diversity
Our validation assay employed 400 amplicons evenly distributed throughout all zebrafish linkage groups and covering coding, intronic, untranslated, and intergenic parts of its genome with the great prevalence of newly discovered variants over computationally predicted SNPs. This justifies the use of our genotyping results for establishing an estimate of nucleotide diversity in zebrafish. To this end, we used only regions for which high-quality sequence data were obtained for at least half of the samples that were tested (total, 105 kbp). Gene annotation from the Ensembl zebrafish genome database build 33 enabled us to calculate the nucleotide diversity for functionally different fractions of the genome (Table 3). Polymorphisms are distributed in a nonrandom fashion between coding and noncoding parts of the genome (P < 0.001) at a rate of one per 82 and 47 bp, respectively. Within coding sequences, there is a strong bias toward synonymous substitutions: The ratio between nonsynonymous and synonymous substitutions per available site (K_a/K_s) is 0.142. This value is similar in other vertebrate organisms and illustrates a pronounced negative selection on coding regions. Extrapolation of the variation data over the complete genome suggests the presence of 425,000 coding SNPs, including 146,000 missense variants that may result in phenotypic effects. It should be mentioned that these numbers are likely to be an underestimation because some polymorphisms that were missed in our study assayed only two samples per strain, but also because of incomplete genome assembly and annotation. The incompleteness is exemplified by the fact that although all candidate regions are associated with an EST read, only 206, roughly half of the tested amplicons, correspond to an annotated Ensembl transcript (based on zebrafish mRNAs and ESTs), while 57 of them overlap with predicted transcripts (based on ESTs and predicted open reading frames) and the remaining 135 were not associated with any known or predicted transcript.

View this table: [in this window] [in a new window]   Table 3 Estimates of nucleotide diversity for different functional fractions of the zebrafish genome

The high nucleotide diversity in zebrafish also results in more frequent occurrence of three alleles at a single locus. About 1% of single nucleotide variants had three alleles, which is significantly higher than observed in mouse, human, or chicken (0.19%, 0.22%, and 0.28%, respectively, as calculated from NCBI dbSNP build 124). The observed number of triallelic SNPs in zebrafish is close to an estimate based on diallelic SNPs frequency (18–21), suggesting that most triallelic SNPs result from independent, unselected mutations, rather than the identification of sites of strong positive selection. Although such triallelic SNPs are mostly neglected in genetic studies in other vertebrates where they are rare, in zebrafish they may prove advantageous in designing mapping probes sets useful for a greater fraction of loci tested across a wider variety of genetic backgrounds used.

Phylogenetic relationships
To assess the degree of similarity between different strains, we used 2120 confirmed SNPs that were genotyped in 16 zebrafish samples representing seven widely used laboratory isolates for construction of a phylogenetic tree (Fig. 2). The tree is mostly consistent with previously described CA-repeat and SSCP-based results (Knapik et al. 1998; Nechiporuk et al. 1999). The relatively large distance between most of the lines can be explained by the high nucleotide diversity in combination with the recent independent establishment of different laboratory lines from wild populations from around the world (Trevarrow and Robison 2004). This is fundamentally different from the history of laboratory mice and rats, which are thought to have originated from a limited source of domesticated animals (Beck et al. 2000; Hedrich 2000).

View larger version (17K): [in this window] [in a new window]   Figure 2. Neighbor-joining tree for 16 zebrafish samples representing seven different strains, based on genotyping of 2120 SNPs. Coefficients represent bootstrap test support values for tree nodes.

Although this phylogenetic tree can be used to choose optimal pairs of strains for setting up genetic mapping experiments, the diversity between any of the lines will in most cases already be ample for the selection of sufficient SNP markers. For example, the rate of polymorphisms homozygous in both closely related AB and Tu strains is estimated to be about one per 500 bp.

Intrastrain variation
Most zebrafish lines originate and are maintained as outbred stocks. Only C32 and SJD have been bred to obtain inbred lines. Although most strains are kept as independent stocks at many laboratories worldwide, only very limited data are available on the degree of genetic variation within a line and the potential genetic differences between various (sub-)stocks. As expected, the Singapore local wild-type isolate (n = 4) was found to be the most heterozygous "strain," with 37% of the SNPs being polymorphic; 14.1%, 14.6%, 17.6%, and 24.8% of the SNPs are heterozygous in WIK, Tu, TL, and AB, respectively (n = 2 per strain). For the inbred strains, we found that 7% and 11% of the loci are polymorphic in SJD and C32, respectively, which is in line with previous observations showing that inbred zebrafish strains are not genetically uniform (Buth et al. 1995; Nechiporuk et al. 1999). Interestingly, most of the heterozygous loci (172/184) in the C32 strain are also polymorphic in the other samples, supporting the hypothesis that these polymorphisms originate from a common origin, were inherited, and did not appear in this strain due to mutation process as was proposed earlier (Buth et al. 1995).

Structure of genetic variation
An important question for any model organism is organization of its genetic variation. A limited number of founder animals and continuous inbreeding result in genome blocks with limited haplotype diversity that can greatly simplify genetic and QTL mapping in laboratory strains. Data available for eight mouse laboratory strains (Yalcin et al. 2004), show that most multi-SNP regions resequenced (136/226; average, 5.32 SNPs/region) contain only two haplotypes. On the opposite, only a small fraction of zebrafish amplicons containing multiple SNPs (19/334; average, 5.74 SNPs/amplicon; Singapore wild-types were excluded from the analysis) is compatible with the presence of only two haplotypes, suggesting that zebrafish strains used in a laboratory will not reveal a pronounced high-level structure of genomic variation, providing little reason for building a detailed haplotype map for this organism.

Conclusions
The degree and organization of genetic variation between and within zebrafish strains was not found to be comparable to other commonly used vertebrate model organisms. Therefore, one should take into account possible effects of genetic variation in experimental design and interpretation and should be careful when comparing results from different laboratories using different (sub-)strains. The development and use of well-characterized inbred zebrafish lines, preferentially marked with a unique recessive phenotype, could significantly reduce confounding effects resulting from genetic heterogeneity.

	Methods

Top ABSTRACT Results and Discussion Methods Acknowledgments References

 
SNP discovery
The mRNA and EST sequence data used in this study were downloaded from NCBI GenBank (http://www.ncbi.nlm.nih.gov/Genbank) and Ensembl trace repository (http://trace.ensembl.org). EST sequences and quality data from Singapore isolate were provided by Dr. Jinrong Peng (Institute of Molecular and Cell Biology, Singapore). We used Ensembl trace archive (http://trace. ensembl.org) as a source of genomic traces. EST and mRNA sequences were masked for zebrafish-specific repeats, low-complexity regions, and zebrafish mitochondrial DNA by using RepeatMasker. Local SSAHA searches were performed to collect hits with nearly exact homology containing a single mismatch in mRNA/EST subset and remote searches (using Ensembl SSAHA search server) in case of mRNA/EST versus WGS comparison. Only hits with a high-quality mismatch (phred score >20 for both reads) within a sequence stretch of >80-bp identity were retained. The mRNA subset that is not annotated for base-calling quality data was treated as having a reliable overall quality. Hits were clustered to represent unique variations and stored in a MySQL database. Candidate SNPs were annotated and placed on the Zv5 genome assembly by using methods reported previously (Guryev et al. 2004).

Predicted and discovered SNPs as well as genotype data obtained in this study were submitted to dbSNP under the following accession numbers: ss49785942–ss49839678. The CASCAD database of candidate SNPs and underlying supporting information is publicly available at http://cascad.niob.knaw.nl. All scripts are freely available upon request.

SNP validation
For the verification experiment, we used 16 samples from seven different zebrafish isolates: AB (two individuals), C32 (two), SJD (two), TL (two), Tu (two), WIK (two), and Singapore wild type (four). AB, TL, Tu, and WIK samples were taken from the colony kept at the Hubrecht Laboratory, C32 and SJD originated from Washington University, and the Singapore wild types were kindly provided by Dr. Jinrong Peng. DNA isolation was done by using the protocol described in Westerfield (2000).

We have semirandomly sampled candidate SNPs to generate a set of markers with even distribution throughout the zebrafish linkage groups. For this purpose we have divided the assembled zebrafish genome into equally sized bins and randomly selected a candidate from every bin. Primers for PCR amplification and sequencing of the genomic region were designed by using a customized Web interface (http://primers.niob.knaw.nl) to the Primer3 program (http://www-genome.wi.mit.edu/genome_ software/other/primer3.html). Primer sequences can be obtained upon request or retrieved interactively from the Web interface (http://cascad.niob.knaw.nl/snpview) that allows the retrieval and visual representation of validated SNPs between arbitrary combinations of strains.

PCRs were carried out by using a touchdown thermocycling program (60 sec at 92°C; 30 cycles for 20 sec at 92°C, 20 sec at 65°C with a decrement of 0.4°C per cycle, and 30 sec at 72°C; followed by 10 cycles of 20 sec at 92°C, 20 sec at 58°C, and 30 sec at 72°C; and 18 sec at 72°C; GeneAmp9700, Applied Biosystems) and contained 30–50 ng genomic DNA, 0.2 µM of each forward primer and 0.2 µM of each reverse primer, 400 µM of each dNTP, 25 mM Tricine, 7.0% glycerol (w/v), 1.6% DMSO (w/v), 2 mM MgCl₂, 85 mM ammonium acetate (pH 8.7), and 0.2 U Taq polymerase in a total volume of 10 µL. After thermocycling, the PCR reactions were diluted with 25 µL water and mixed by pipetting, and 1 µl was used as template for dideoxy cycle sequencing, as recommended by the manufacturer (BigDye v3.1, Applied Biosystems) using one of the primers used for the PCR amplification. Sequencing reactions were analyzed on an ABI3730XL capillary sequencer (Applied Biosystems), and the obtained sequences were scored for polymorphic positions by using the PolyPhred program (Nickerson et al. 1997) followed by manual inspection.

Phylogenetic reconstruction
Sequence alignments of 2120 confirmed variable positions were used as an input for the MEGA3 program (Kumar et al. 2004). Phylogenetic tree was built with a Neighbor-joining algorithm using p-distances with a pair-wise deletion option. Support for each node was determined by a bootstrap test.

Whole-genome mutation-type screen
EST sequences were mapped to zebrafish assembly Zv4 by using the GMAP program (Wu and Watanabe 2005). We scored only candidate SNPs occurring in exons having at least 90% identity between EST and genome sequences. Genome annotation from Ensembl build 31.4d was used to deduce alleles observed in genomic and cDNA-based reads.

	Acknowledgments

Top ABSTRACT Results and Discussion Methods Acknowledgments References

 
We thank Dr. Jinrong Peng (Institute of Molecular and Cell Biology, Singapore), Washington University St. Louis, and Agencourt Bioscience Corporation for providing zebrafish EST sequence and/or quality data, and the Zebrafish Sequencing Group at the Wellcome Trust Sanger Institute for making the WGS trace data and zebrafish genome assemblies publically available before publication. This work was supported by NWO genomics grant 050-10-024.

	Footnotes

 
³ Corresponding author.

E-mail ecuppen{at}niob.knaw.nl; fax 31-30-251-6554.

[The polymorphism and genotype data from this study have been submitted to dbSNP under accession nos. ss49785942–ss49839678.]

Article published online ahead of print. Article and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.4791006

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Received October 10, 2005; accepted in revised format January 18, 2006.

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