Abstract
BackgroundThe 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.ResultsHere, 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.ConclusionThe 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.
Highlights
The zebrafish (Danio rerio) is an important vertebrate model organism system for biomedical research
Estimation of chromosome sizes based on flow cytometry Zebrafish chromosomes were analyzed by flow cytometry and the bivariate flow karyotypes obtained for the mixed human-zebrafish and zebrafish chromosome preparations are shown in Figures 1A and 1B, respectively
It should be noted that since there is an absence of a consensus karyotype, the zebrafish community has chosen to name each chromosome based upon syntenic markers to a given linkage group (LG)
Summary
The zebrafish (Danio rerio) is an important vertebrate model organism system for biomedical research. To facilitate analysis of the zebrafish genome, genetic maps have been constructed and sequence annotation of a reference zebrafish genome is ongoing. Through the use of zebrafish mutants and a well annotated zebrafish reference genome, genetic mechanisms that are conserved among all vertebrates, can be investigated. The Wellcome Trust Sanger Institute (Hinxton, United Kingdom) released the sixth zebrafish genome assembly (Zv6), comprising of 1.6 Gb [26]. This assembly was generated from 7,615 BAC clones, which were fingerprinted and arranged into contigs by overlap analysis to generate a physical map. While more high-throughput methods for DNA sequencing are emerging (e.g., the 454 sequencing platform – which can sequence 25 million bases in about four hours [27]), 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 [28]
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