The zebrafish reference genome sequence and its relationship to the human genome

Abstract

Zebrafish have become a popular organism for the study of vertebrate gene function. The virtually transparent embryos of this species, and the ability to accelerate genetic studies by gene knockdown or overexpression, have led to the widespread use of zebrafish in the detailed investigation of vertebrate gene function and increasingly, the study of human genetic disease. However, for effective modelling of human genetic disease it is important to understand the extent to which zebrafish genes and gene structures are related to orthologous human genes. To examine this, we generated a high-quality sequence assembly of the zebrafish genome, made up of an overlapping set of completely sequenced large-insert clones that were ordered and oriented using a high-resolution high-density meiotic map. Detailed automatic and manual annotation provides evidence of more than 26,000 protein-coding genes, the largest gene set of any vertebrate so far sequenced. Comparison to the human reference genome shows that approximately 70% of human genes have at least one obvious zebrafish orthologue. In addition, the high quality of this genome assembly provides a clearer understanding of key genomic features such as a unique repeat content, a scarcity of pseudogenes, an enrichment of zebrafish-specific genes on chromosome 4 and chromosomal regions that influence sex determination.

Figure 1. Landscape of chromosome 4

a, Exon coverage (blue), stacked with coverage by snRNA exons (black). b, Stacked repeat coverage, divided into type I transposable elements (red), type II transposable elements (grey) and other repeat types (blue), including dust, tandem and satellite repeats. c, Sequence composition (grey bars, clones; blue bars, WGS contigs). d, Genetic marker placements (red, SATmap markers; blue, heat shock meiotic map markers; black, Massachusetts General Hospital meiotic map markers). Marker placements have been normalized so that the maps can be compared. Near-centromeric clones are positioned at 20Mb (BX537156), 20.2Mb (Z10280) and 24.4Mb (Z20450). The x axis shows the chromosomal position in Mb. a and b were calculated as percentage coverage over 1-Mb overlapping windows (y axis), with a 100-kb shift between each window. c and d were calculated over 100-kb windows. The y axis for d shows the normalization of marker positions relative to the span of the individual map. Similar graphs for the other chromosome are provided in the Supplementary Information.

Figure 2. Sex determination signal on chromosome 16

a, Breeding scheme for SATmap. Double haploid generation zero (G₀) founders were sequenced to approximately 40× depth using Illumina GAII technology. We found approximately 7 million SNPs between the two SATmap founders. This number of SNPs between just two homozygous zebrafish individuals is far in excess of that seen between any two humans and is nearly one-fifth of all SNPs measured among 1,092 human diploid genomes. Genetically identical, heterozygous F₁ fish of both sexes resulted from crossing the founders. The F₁ individuals were crossed to generate a panel of F₂ individuals, each with its own unique set of meiotic recombinations between AB and Tübingen (Tü) chromosomes, which were uncovered by dense genotyping with a set of 140,306 SNPs covering most of the genome. b, Genome-wide P values for tests of genotype difference between sexes, arranged by chromosome. The dotted line corresponds to differences that are expected once in 100 random genome scans, and the dashed line corresponds to differences expected once in 1,000 random genome scans. The only locus that is statistically significant at these levels is on chromosome 16. c, Genotype frequencies for males and females on chromosome 16. The grey line at 0.5 corresponds to expectation for heterozygotes (solid lines) and the grey line at 0.25 corresponds to expectation for homozygotes (dashed and dotted lines). The light grey shaded box corresponds to the region in which empirical P < 0.01, the dark grey shaded box corresponds to the region in which P < 0.001.

Figure 3. Evolutionary aspects of the zebrafish genome

a, Orthologue genes shared between the zebrafish, human, mouse and chicken genomes, using orthology relationships from Ensembl Compara 63. Genes shared across species are considered in terms of copies at the time of the split. For example, a gene that exists in one copy in zebrafish but has been duplicated in the human lineage will be counted as only one shared gene in the overlap. b, The ohnology relationships between zebrafish chromosomes. Chromosomes are represented as coloured blocks. The position of ohnologous genes between chromosomes are linked in grey (for clarity, links between chromosomes that share less than 20 ohnologues have been omitted). The image was produced using Circos.