Tetraodon nigroviridis , belongs to the order Tetraodontiformes, to the family Tetraodontidae, and is known as the "green spotted puffer fish". It is a small fish (less than 10cm long), which lives 5-8 years. Tetraodon nigroviridis is a carnivore, its diet consists mainly of snails, worms, small fish and crustacea. It is found in freshwater streams, rivers and in brackish waters (= water with more salinity than fresh waters, but not as much as sea water). It originally comes from Asia (Sri Lanka, Indonesia, North China), but in 2009 it was succesfully bred in captivity.

Tetraodon projectEdit

The first interest in the Tetraodon nigroviridis genome appeared in 1968, when R. Hinegardner discovered its small size. The Tetraodon nigroviridis genome sequencing project was launched by Genoscope and by the Broad Institute of MIT and Harvard in 1997. The purpose of this project was to discover similarities between the genomic sequence of the pufferfish and humans. This would help with a better annotation of human genes and estimation of their number. The sequencing was carried out as a collaboration between Genoscope and the Whitehead Institute Center for Genome Research and was finished in 2002.

Tetraodon genomeEdit

Tetraodon chromosomes The centromere is in black, and the chromosome coverage is shown in blue

The pufferfish Tetraodon nigroviridis has a genome of 350Mb, which is the smallest vertebrate genome discovered up to date. As a comparison, the zebrafish (Danio rerio ) has a genome four times larger than that of Tetraodon. The genome of Tetraodon is very compact, therefore heterochromatin regions seem to be very limited and the percentage of GC base pairs is higher than of the large mamalian genomes (this percentage correlates with gene density in vertebrates). The genome of Tetraodon nigroviridis contains a high diversity of transposable elements (75 types), but these elements occur with low abundance. Transposable elements are very rare in the Tetraodon genome. The transposable elements are mostly present in heterochromatin regions and some of them remain active. Another factor explaining, why the genome of the pufferfish is so compact, is is high resistance to insertions, which was observed in this lineage (this was suggested by a comparative study with diodons and a sunfish).


Genes represent 40% of the Tetraodon genome and are not homogenously distributed. It was predicted that all vertebrates posses more or less a comparable repertoire of genes. Therefore, in case of the pufferfish, the common genetic heritage of the vertebrates is gathered in a genome, which is eight times smaller than that of humans. Consequently, the small size of the Tetraodon genome is not generated by a reduction of the number of genes, but it is generated by a reduction in con-coding sequences - Tetraodon possesses only small intrones and intergenic regions.

Tetraodon nigroviridis x Human genome comparisonEdit

Although the length of exones is similar between humans and the pufferfish, the size of intrones and the size of the intergenic sequences is reduced in the pufferfish. While most of the non-coding sequences diverged completely after the separation of the lineages leading to humans and to tetraodon, the coding sequences were conserved.

EXOFISH (=EXOn Finding by Sequence Homology)Edit

Figure gene,387-.html Comparison of the human gene SPAG7, encoding a sperm protein, with the orthologous gene in Tetraodon nigroviridis. The gene is eight times smaller in the fish, but exhibits the same exon structure. The difference resides in the intron size, especially in the first intron which is 6867 pb in human and 628 pb in the fish. The decrease in intergenic distances also contributes to the compactness of the Tetraodon genome.

The above mentioned facts were discovered by using an Exofish method, a procedure developed at Genoscope, which is based on homology searches by the BLAST algorithm. This approach is based on the hypothesis, that genomic regions which are constrained by their function (coding sequences) show less mutations in comparison to other regions. This method therefore relies on the comparison between genomic sequences of the fish and the target human sequences to detect conserved sequences (Ecores =(Evolutionary COnserved REgions) with a very low background. Ecores are alignments of Tetraodon sequences with conserved regions in the human genome. Exofish can be used not only for comparing the human genome to the one of Tetraodon, but also for comparison of other mammals - rats, mice,...

Estimation of the number of human genesEdit

Exofis was first applied in 2000, in this time the whole human genome sequence, as well as the sequence of Tetraodon were still not known. It was revealed that the human genome contains only from 28,000 to 34,000 genes, not 50,000-90,000 as previously proposed. In 2003, Genoscope repeated the experiment, the data obtained corresponded to the previously gained results: human gene number is ranging from 22,500 to 29,500.


It was discovered by synteny experiments (= physical co-localization of genetic loci on the same chromosome within an individual or species) that a whole genome duplication occured in T. nigroviridis. The last common ancestor genome of all bony vertebrates was predicted by using these high resolution synteny maps. The 21 Tetraodon chromosomes are all derived by whole genome duplication and a small number of interchromosomal rearrangements from this ancestral karyotype, which contained 12 chromosomes. The distribution of duplicated genes in the T. nigroviridis genome shows striking similarity to human orthologues in a manner that supports the theory of whole genome duplication followed by a massive loss of duplicated genes. While the composition of the Tetraodon chromosomes is based on their duplication pattern, composition of human chromosomes is based on distribution of Tetraodon orthologues and interchromosomic rearrangements - large increase in repetitive sequences (for example human chromosome 16 is a combination of T. nigroviridis chromosomes 13 and 15) . Human genomes contains a great number (45%) of repetitive sequences, whereas as mentioned above, transposable elements in the pufferfish genome are very scarce (only 3,8%).


Jaillon et al., 2004 How to pass from the bony vertebrate’s last common ancestor genome (12 chromosomes) to the genomes of humans and Tetraodon. Model derived from the study of synteny groups between those two modern vertebrates

Human chromosomes x tetraodon

Jaillon et al., 2004 Synteny maps - human chromosomes

Tetraodon chromosomes

Jaillon et al., 2004 synteny maps - Tetraodon chromosomes

After WGD (Whole Genome Duplication) the resulting polyploid genome returns to a diploid state via extensive gene deletion. Only few genes are maintained in duplicated copies and serve as a source of innovation. WGD can be therefore determined by occurence of two signs: discovery of gene duplication on paralogous chromosomes a comparis of the genome with a related species that did not undergo WGD. The synteny maps for comparison of the human and Tetraodon genomes typically show association of two regions in Tetraodon and with one region in the human.


Mulley J. and Holland P., Comparative genomics: Small genome, big insights , Nature, 431: 916-917 (2004)

Jaillon et al., Genome duplication in the teleost fish Tetraodon nigroviridis reveals the early vertebrate proto-karyotype , Nature, 431: 946-957 (2004)

Genoscope project

Crollius H.R., Jaillon O., Dasilva, et al., Characterization and repeat analysis of the compact genome of fresh water pufferfish Tetraodon nigroviridis, Genome res., 10: 939-949 (2000)

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