Definition of the zebrafish genome using flow cytometry and cytogenetic mapping
- Jennifer L Freeman1, 2,
- Adeola Adeniyi1,
- Ruby Banerjee3,
- Stephanie Dallaire1,
- Sean F Maguire3,
- Jianxiang Chi3,
- Bee Ling Ng3,
- Cinthya Zepeda4,
- Carol E Scott3,
- Sean Humphray3,
- Jane Rogers3,
- Yi Zhou5,
- Leonard I Zon5,
- Nigel P Carter3,
- Fengtang Yang†3 and
- Charles Lee†1, 2Email author
© Freeman et al; licensee BioMed Central Ltd. 2007
Received: 07 February 2007
Accepted: 27 June 2007
Published: 27 June 2007
The zebrafish (Danio rerio) is an important vertebrate model organism system for biomedical research. The syntenic conservation between the zebrafish and human genome allows one to investigate the function of human genes using the zebrafish model. To facilitate analysis of the zebrafish genome, genetic maps have been constructed and sequence annotation of a reference zebrafish genome is ongoing. However, the duplicative nature of teleost genomes, including the zebrafish, complicates accurate assembly and annotation of a representative genome sequence. Cytogenetic approaches provide "anchors" that can be integrated with accumulating genomic data.
Here, we cytogenetically define the zebrafish genome by first estimating the size of each linkage group (LG) chromosome using flow cytometry, followed by the cytogenetic mapping of 575 bacterial artificial chromosome (BAC) clones onto metaphase chromosomes. Of the 575 BAC clones, 544 clones localized to apparently unique chromosomal locations. 93.8% of these clones were assigned to a specific LG chromosome location using fluorescence in situ hybridization (FISH) and compared to the LG chromosome assignment reported in the zebrafish genome databases. Thirty-one BAC clones localized to multiple chromosomal locations in several different hybridization patterns. From these data, a refined second generation probe panel for each LG chromosome was also constructed.
The chromosomal mapping of the 575 large-insert DNA clones allows for these clones to be integrated into existing zebrafish mapping data. An accurately annotated zebrafish reference genome serves as a valuable resource for investigating the molecular basis of human diseases using zebrafish mutant models.
The zebrafish (Danio rerio) has long been appreciated as a unique model system for vertebrate genetics and developmental biology. The zebrafish genome is about half the size of most mammalian genomes  containing some 4.6 pg of DNA  distributed across 25 pairs of chromosomes (2n = 50) . Initial comparisons of zebrafish and mammalian gene maps have revealed extensive conservation of syntenic chromosome regions among vertebrates [4–10]. Hence, through the use of zebrafish mutants and a well annotated zebrafish reference genome, genetic mechanisms that are conserved among all vertebrates, can be investigated.
Since the launch of the Trans-NIH Genome Initiative in 1997 , excellent genetic and genome resources have been established to facilitate the genetic analysis of zebrafish. The first genetic linkage map was constructed using haploid genetics with random amplified polymorphic DNA (RAPD) markers [12, 13]. Several meiotic panels (e.g., the Boston MGH Cross, GAT, Heat Shock, and Mother of Pearl) and radiation hybrid (RH) panels (e.g., T51 and LN54) have been generated [9, 14–25]. Most recently, the Wellcome Trust Sanger Institute (Hinxton, United Kingdom) released the sixth zebrafish genome assembly (Zv6), comprising of 1.6 Gb . This assembly was generated from 7,615 BAC clones, which were fingerprinted and then arranged into contigs by overlap analysis to generate a physical map. DNA sequence data from whole genome shotgun contigs were then used to fill gaps. While more high-throughput methods for DNA sequencing are now emerging (e.g., the 454 sequencing platform – which can sequence 25 million bases in about four hours ), a major limitation of many of these technologies is the provision of relatively short DNA sequences (about 100–200 bases per read), making such sequencing technologies problematic with the assembly of repeated DNA elements .
Cytogenetic methodologies provide a complementary mapping approach for assembly of reference genomes. In this study, we have employed two cytogenetic approaches to define the zebrafish genome: flow karyotype analysis and chromosomal mapping by fluorescence in situ hybridization (FISH). In flow karyotyping, each chromosome is labeled directly with two DNA-binding fluorochromes (i.e., Hoechst 33258 and Chromomycin A3) and then analyzed on a flow cytometer. The fluorescence from the two DNA-binding dyes enables different chromosome types to be identified by both DNA content and base-pair ratio, providing a means to estimate the relative size of each zebrafish chromosome. In FISH, cloned DNA sequences are hybridized to their complementary sequences and visually mapped onto metaphase chromosomes. In this study, we have unequivocally mapped 575 large-insert DNA clones onto zebrafish metaphase chromosomes using multi-color FISH methods. These clones can be used as anchors for annotation of the zebrafish reference genome and provide independent validation of the sequence map, in a fashion similar to how cytogenetic data from the human genome was integrated into the human DNA sequence map . These data reinforce the use of cytogenetic methodologies for the assembly of complex genomes.
Estimation of chromosome sizes based on flow cytometry
BAC clones cytogenetically mapped by FISH to a unique chromosomal location
Chromosome size determined by flow cytometry (Mb)
Number of BAC clones uniquely mapped by FISH
Percent coverage within a chromosome*
6.1% of genome
Chromosomal mapping of 575 BAC clones by FISH
510 uniquely mapped BAC clones compared to LG chromosome assignment in four zebrafish genome databases
No. of clones*
Percent of clones
UCSC genome browser
Not in database
Present in database
Mapped to "U" †
Ensembl (Zv6-Release 40) ‡
Not in database
Present in database
Mapped to "U"
Sanger WebFPC (August 2006)
Not in database
Present in database
Mapped to "U"
Vega (version 20)
Not in database
Present in database
Mapped to "U"
Thirty-one clones (4.6%) mapped cytogenetically to multiple chromosomal locations (Additional file 3). One of the clones had two signals on the same chromosome (Figure 2B), 17 clones had hybridization signals on two different, non-homologous chromosomes (e.g., Figure 2C), 1 clone had signals on three different, non-homologous chromosomes (data not shown), and 2 clones had signals on four different, non-homologous chromosomes (data not shown). Five BAC clones were pan-centromeric (i.e., hybridized near the centromere of all chromosomes; e.g., Figure 2D) and five BAC clones were peri-centromeric (i.e., hybridized near the centromere of multiple chromosomes; e.g., Figure 2E).
A total of 95 BAC clones (14.2%) failed to produce distinct and reproducible FISH signals, even after multiple hybridization attempts. These clones may be shorter than the average BAC clone or could contain substantial amounts of highly repetitive elements that are largely out competed by unlabeled Cot1 DNA during the FISH procedure.
Development of a second generation probe panel
Second generation near-centromere and near-telomere zebrafish BAC clone probe panel*
Near-telomere short arm (p)
Near-telomere long arm (q)
Following analysis of the initial zebrafish gene maps, it has been suggested that during the course of evolution, the zebrafish and human lineages shared two rounds of whole genome duplication with a third whole genome duplication event occurring before the last teleost radiation [10, 35]. The duplicated genome of the zebrafish has contributed to the increased difficulty in constructing an accurate genetic map for this organism, in comparison to the mouse and human. In addition, similarity in the size, morphology, and banding pattern of the zebrafish chromosomes has resulted in at least 12 different published zebrafish karyotypes [reviewed in ]. Phillips et al.  used a combination of relative size and chromosome arm ratios to estimate the sizes of each zebrafish chromosome. When comparing the chromosome size estimates from our flow cytometry data to the size estimates reported by Phillips et al. , the four largest LG chromosomes (i.e., LG chromosomes 5, 7, 4, and 3) and the three smallest LG chromosomes (i.e., LG chromosomes 22, 24, and 25) are in agreement. However, the ranking of sizes of most other LG chromosomes are discrepant and should be investigated further by other independent studies.
Cytogenetic mapping is a complementary approach to other mapping strategies (e.g., genetic and radiation hybrid mapping) and can be used to confirm existing framework maps. Using fluorescence in situ hybridization (FISH), 510 BAC clones were assigned to unique LG chromosome locations in this study. This suggests that if there is widespread duplication in the zebrafish genome, a large proportion of these appear to be syntenic and localized (e.g., tandem duplications). FISH mapping data was over 85% concordant for three of the four zebrafish genome databases (i.e., Ensembl, Vega, and WebFPC) with 75.9% concordance for the UCSC Genome Browser, when evaluating the BAC clones present in these databases (Table 2). Discrepancies between the FISH mapping data and the LG chromosome assignment by the zebrafish genome databases may be attributed to cross-contamination of the clones, clone tracking inconsistencies, incorrect assignment by previous mapping efforts, or inconsistencies among the four zebrafish genome databases. From the comparisons, it became evident that at the time of analysis, there is some degree of discrepancy among all four of the zebrafish genome databases. In addition, the Ensembl Zv6 (version6-release 40, August 2006) used in this analysis did not contain any of the BAC clones of the zK library. Of the 510 BAC clones that were assigned to specific LG chromosomes by FISH, only 3 clones did not have mapping information in any of the databases. In addition for 53 of the 507 clones with database mapping information, none of the databases had the same chromosomal assignment as was assigned by FISH (Additional file 1).
A small percentage of the clones were observed to hybridize to multiple locations within the zebrafish genome (Additional file 3). One clone contained duplicated regions within the same chromosome, while the remaining thirty clones hybridized to more than one chromosome. Many of the BAC clones that hybridized to multiple chromosomal locations localized near the centromeres, providing further evidence for the lack of chromosome-specific repetitive DNA sequences near these regions. These clones provide clues to duplications and exchanges that have occurred within and between chromosomes. Further investigation is needed to determine the functional significance of these duplications.
Some BAC clones yielded weak or no fluorescence signals. The lack of signals for these clones may be due to poor quality of probe DNA, contamination with non-zebrafish DNA, loss of insert DNA or poor hybridization due to factors such as increased repetitive DNA elements within a clone.
One particularly fascinating observation was a peri-centromeric inversion on LG chromosome 3 (Figure 3). Interestingly, this heteromorphism was seen only in the AB strain and not in Tu embryos. This finding suggests that there are strain-specific chromosomal differences in the zebrafish. Large chromosomal rearrangements (e.g., inversions) are important for evolution and are suspected to play a role in speciation. Studies have suggested that inversions can influence patterns of nucleotide diversity between diverging taxa  and encourage speciation by suppressing recombination in heterozygotes . Chromosomal inversions can also play a role in fertility. For example, peri-centromeric inversion carriers may have a reduction in fertility due to the production of chromosomally unbalanced gametes. In a recent study, it has also been suggested that chromosomal inversions may be beneficial. Carriers for an inversion on human chromosome 17 were reported to have increased fertility rates in comparison to non-carriers .
Chromosomal mapping of zebrafish BAC clones by FISH in this study resulted in a second generation near-centromere and near-telomere BAC clone probe panel, which includes 41.3% new clones. These new BAC clones are visually mapped closer to the centromere or telomeres of each LG chromosome than those in the original probe panel. Furthermore, a near-telomeric BAC clone was identified at the long arm terminus of LG chromosome 4 (Figure 4). The long arm of LG chromosome 4 (i.e., chromosome 3 as denoted by the karyotype of Gornung et al.  and Phillips et al. ) has been previously shown to contain a substantial amount of constitutive heterochromatin . Regions of constitutive heterochromatin often consist of highly repetitive DNA and are largely transcriptionally silent. The heterochromatic region of LG chromosome 4 is late replicating , stains darker than the short arm by C-banding , and is reported to also harbor 5S rDNA [43, 44]. In the current study, 25 BAC clones mapped uniquely to the heterochromatic region of LG chromsome 4 (e.g., Figure 4). Moreover, five BAC clones that mapped to multiple chromosome locations included this heterochromatic region on LG chromosome 4 (Additional file 3; Figure 2C). This would suggest a complex genome structure of multiple repetitive DNA families in this region on LG chromosome 4, some of which are shared sequences at other chromosomal regions.
We have used flow cytometry and chromosomal mapping to develop a cytogenetic map of the zebrafish genome. These data can be integrated with genetic and radiation hybrid mapping data to produce a refined sequence map and to confirm the existing genomic framework of the zebrafish. A refined zebrafish genome serves as a valuable resource for genetic research that utilizes this model organism for understanding of the molecular basis of human diseases. For example, the mapped clones can be used to narrow the search for candidate genes, which may be altered in zebrafish mutant models using FISH assays. This strategy was recently used to identify a loss of one allele of the separase gene, when a probe containing the separase gene locus was hybridized upon epithelial tumor specimen, indicating that a loss of one copy of separase is sufficient to promote tumorigenesis in the zebrafish . The cytogenetically mapped clones can also be used in the development of a zebrafish array comparative genomic hybridization platform for use in identifying genomic imbalances in developmental and disease zebrafish models. Integrating these BAC clones with the zebrafish reference sequence will allow investigators to directly determine the genomic regions that are gained or lost. Overall these data contribute towards the ultimate defintion of a reference zebrafish genome and provide insight into the evolution of vertebrate chromosomes.
AB fibroblast cell line
The primary fibroblast cell line was established from approximately 100 embryos of the AB strain fishes, and grown in Advanced DMEM (Gibco) medium, supplemented with 15% fetal bovine serum (FBS, Gibco) and antibiotics (Penicillin and Streptomycin, Sigma). At about 70% confluency, cells were treated with 0.1 μg/ml colcemid, then placed in a hypotonic solution (0.067 M potassium chloride) at room temperature for 15 min., and then fixed in ice-cold Carnoy's methanol:glacial acetic acid (3:1) fixative. Chromosomes were dropped onto slides and allowed to dry overnight at 37°C.
AB and Tuebingen embryos
Methods are adapted from Lee and Smith . At 23 hours post-fertilization, 100 embryos were treated with colchicine (4 mg/ml) for 6 hours. Embryos were then dechorionated by pronase treatment  and lightly homogenized. Ice-cold 0.9× phosphate buffered saline (PBS; Sigma, St. Louis, MO), 10% fetal bovine serum (BioWhittaker, Walkersville, MD) was added and embryos filtered subsequently through a 100 μm and 50 μm mesh. Embryos were then centrifuged at 250 rcf for 10 min. at 4°C. Supernatant was decanted and embryos were treated for 25 min. at room temperature in a hypotonic solution (1.1% sodium citrate, 4 mg/ml colchicine). Cells were centrifuged at 450 rcf for 10 min. at 4°C and ice-cold Carnoy's methanol: glacial acetic acid fixative (3:1) was added. Centrifugation was repeated and fresh fixative added. Chromosomes were then dropped onto slides and allowed to dry overnight at 37°C.
Characterization of zebrafish chromosome sizes by flow cytometry
Chromosomes were prepared from human lymphoblastoid HRC159 cells and from the zebrafish AB strain fibroblast cell line. Twenty-four hours after subculturing, the cell cultures were treated with colcemid (0.1 μg/ml) for 6 hours. Chromosomes were prepared by using modifications of a polyamine isolation method . For the HRC159 cell line, 50 ml of blocked cell culture was centrifuged at 289 rcf for 5 min. and the cell pellet was resuspended in 5 ml of hypotonic solution (75 mM KCl, 10 mM MgSO4, 0.2 mM spermine, 0.5 mM spermidine, pH 8.0) and incubated at room temperature for 10 min. For the zebrafish cell lines, the supernatant from six 150 cm2 flasks after mitotic shake-off was collected and centrifuged at 289 rcf for 5 min. The cell suspensions were pooled together into one tube after resuspending the cell pellets in hypotonic solution (5 ml). The suspension of swollen cells was centrifuged at 289 rcf for 5 min. The cell pellets of HRC159 cells and zebrafish were each resuspended in 3 ml and 1 ml of ice cold polyamine isolation buffer (PAB, containing 15 mM Tris, 2 mM EDTA, 0.5 mM EGTA, 80 mM KCl, 3 mM Dithiothreitol, 0.25% Triton X-100, 0.2 mM Spermine, 0.5 mM spermidine, pH 7.5), respectively, and vortexed for 20 sec. For the zebrafish cells, this was followed by forcing the suspensions twice through a 21 gauge needle (Becton Dickinson, Microlance™ 3) attached to a 2 ml syringe (Becton Dickinson, Plastipak™).
All chromosomes suspensions were then briefly centrifuged (201 g, 2 min.) and the supernatant was filtered through a 20 μm mesh filter (Celltrics, Partec). An aliquot of zebrafish chromosome suspension was mixed with HRC159 and stained for at least 2 hours with 5 μg/ml of Hoechst (Sigma) and 40 μg/ml of Chromomycin A3 (Sigma). 10 mM MgSO4, 10 mM sodium citrate and 25 mM sodium sulphite was added to the stained preparation one hour before flow analysis. The stained chromosome suspension was analyzed on a flow cytometer (MoFlo®, DAKO) as described elsewhere . A total of 100,000 events were acquired for each cell line at a rate of 1000 events per second. The flow karyotype of the stained chromosomes was displayed as a bivariate flow karyogram of Hoechst versus Chromomycin fluorescence. Data collected from the experiments were analysed using Summit V3.1 (analysis software from DAKO) and the size of zebrafish chromosomes in mega-basepairs of DNA was estimated based on a method described previously [48–51] using the distribution of human chromosomes as the reference. Briefly, this method uses the projection of each flow peak in the flow karyotype onto the 'DNA line' which passes chromosome 4 and the origin. The distance from the origin to the point of projection on the 'DNA line' is proportional to the DNA content of the chromosome. Since the largest LG chromosomes and the smallest LG chromosomes of zebrafish are close to the human chromosomes 18 and 21, respectively, the sizes of human chromosomes 18 (76.12 Mb) and 21 (46.94 Mb) were used as the references.
Bacterial Artificial Chromosome (BAC) clone DNA preparation
Clones from the CHORI-211 (zC), Danio Key (zK), Danio Key Pilot (zKp), RPCI-71 (bZ), and CHORI-73 (zH) zebrafish BAC DNA libraries were streaked on LB-Agar plates containing the appropriate antibiotic and grown overnight at 37°C. A single colony was then inoculated in TB media containing the appropriate antibiotic and placed in a shaking incubator for 16 hours at 37°C. DNA was isolated by the Qiagen Plasmid Midi Kit protocol (Valencia, CA), amplified using the Repli-G Midi protocol (Qiagen, Valencia, CA) and submitted for sequencing (Additional file 4). Alternatively, the DNA was prepared using the BAC Phase-Prep kit (Sigma).
Fluorescence in situ Hybridization (FISH)
BAC DNA probe was labeled by adding 1 μg of DNA, 5 μl of 10× translation buffer (500 mM Tris-HCl, 100 mM MgSO4, 1 mM dithiothreitol), 10 μl dNTP mix (0.1 mM dATP, 0.1 mM dCTP, 0.1 mM dGTP), 5 μl of 0.1 mM dTTP, 10 μl of nick translation enzyme (5 units DNA polymerase I, 0.1 units DNAse I, 50 mM Tris-HCl, 10 mM MgSO4, 0.1 mM dithiothreitol, 0.5 mg/ml bovine serum albumin), 2.5 μl of 0.2 mM Spectrum Orange dUTP (Abbott Vysis, Des Plaines, IL) or Spectrum Green dUTP (Abbott Vysis, Des Plaines, IL), and water to a volume of 50 μl. The mixture was incubated at 15°C for 11 hours and the DNA cleaned using a Sephadex-G50 column (Zymo Research, Orange, CA). Alternatively, the BAC DNA was labeled with biotin- and digoxigenin-dUTP by nick translation using the Roche kits following the protocols supplied by the manufacturer. Twenty-five micrograms of Cot1 DNA was added and DNA lyophilized. DNA pellets were resuspended in hybridization buffer (50% formamide, 2× SSC, 10% Dextran Sulfate).
For hybridization, slides were pretreated for 15 min. in ice-cold Carnoy's methanol:glacial acetic acid (3:1) fixative, washed briefly in PBS, digested at 37°C in Protease II for 5 min., and then dehydrated in an ethanol series (70%, 90%, 100%). Following slide pretreatment, 5 μl (0.25 μg) of the DNA probe was added onto the slides under a coverslip and sealed with rubber cement. The DNA probe was denatured for 3 min. at 70°C and then incubated in a humidified chamber in the dark at 37°C for at least 24 hours.
Post-hybridization washes comprised of 50% formamide, 2× SSC at 45°C, 2× SSC at 45°C, and then 4× SSC, 0.05% Tween 20 at 37°C. Slides were counterstained in DAPI (Vector Laboratories, Burlingame, CA), and then analyzed by fluorescence microscopy on an Olympus BX-51 fluorescence microscope equipped with narrow band pass filters for Spectrum Orange and Spectrum Green dyes. Images were captured using a Photometrics KAF1400 CCD camera and Applied Imaging Genus Software system. At least 10 metaphase spreads were analyzed per BAC clone.
Bacterial artificial chromosome
fluorescence in situ hybridization
- p arm:
short chromosome arm
- q arm:
long chromosome arm
CHORI 211 zebrafish BAC library
Danio Key zebrafish BAC library
We thank Amanda Smith for her technical assistance; Cassandra Belair, Howard Stern, and Barry Paw for many helpful discussions during the early stages of these experiments; and David Elliott, Clare Riddle, Glen Threadgold, and Carol Carder for providing part of the BAC clones used for FISH mapping. We thank Shona Hislop for her assistance with the figures. This work was supported in part by National Institutes of Health grant R01-CA111560 (CL) and by a Ruth L. Kirschstein National Research Service Award (JLF). BLN, CES, FY, JC, JR, NPC, RB, SFM, and SH are funded by the Wellcome Trust.
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