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In 2008, there were several significant breakthroughs in production technology which we have utilized for expansion into new species. One of the most exciting breakthroughs has been in the spotted green puffer species Tetraodon nigroviridis. The green puffer is important to the ornamentals industry with an estimated quarter-million sold annually in the US alone.  More significantly however, is a new dimension to this species that has been discovered which now opens the door for expansion into new markets.  The spotted green puffer has the smallest genome of any vertebrate and has had its genome completely sequenced. The principal objective of the sequencing of the Tetraodon genome was to compare the genomic sequences of this fish with those of humans in order to facilitate the identification of human genes. Subsequent research has been expanded into ascertaining the mechanics of certain key genes as well.  However, research has been limited because until now, only wild caught specimens were available.  The consistency of supply for wild caught fish is very unreliable which has made it difficult for researchers to complete projects. However, because we are now able to spawn and rear them at our aquaculture facility using this new spawning technology, Tetraodon nigroviridis has become highly suitable for molecular analyses because the biological material is constantly accessible. In addition to providing a more reliable and biosecure product, we also now have the capability to supply gametes, embryos, and fish of specific and known parentage.
The pufferfish Tetraodon nigroviridis has a genome of 350 Mb, the smallest genome known to date in the vertebrates. This characteristic makes it very attractive for genomic studies, and inspired the launching of a sequencing project at Genoscope in 1997, the year that the center opened. The initial objective of Genoscope was to compare the genomic sequences of this fish to that of humans to help in the annotation of human genes and to estimate their number. This strategy is based on the common genetic heritage of the vertebrates: from one species of vertebrate to another, even for those as far apart as a fish and a mammal, the same genes are present for the most part. In the case of the “compact” genome of Tetraodon, this common complement of genes is contained in a genome eight times smaller than that of humans. Although the length of the exons is similar in these two species, the size of the introns and the intergenic sequences is greatly reduced in this fish. Furthermore, these regions, in contrast to the exons, have diverged completely since the separation of the lineages leading to humans and Tetraodon. The Exofish method, developed at Genoscope, exploits this contrast such that the conserved regions which can be identified by comparing genomic sequences of the two species, correspond only to coding regions. Using preliminary sequencing results of the genome of Tetraodon in the year 2000, Genoscope evaluated the number of human genes at about 30,000, whereas much higher estimations were current. The progress of the annotation of the human genome has since supported the Genoscope hypothesis, with values as low as 22,000 genes and a consensus of around 25,000 genes.
The sequencing of the Tetraodon genome at a depth of about 8X, carried out as a collaboration between Genoscope and the Whitehead Institute Center for Genome Research (now the Broad Institute), was finished in 2002, with the production of an assembly covering 90% of the euchromatic region of the genome of the fish. This has permitted the application of Exofish at a larger scale in comparisons with thegenome of humans, but also with those of the two other vertebrates sequenced at the time (Takifugu, a fish closely related to Tetraodon, and the mouse). The conserved regions detected in this way have been integrated into the annotation procedure, along with other resources (cDNA sequences from Tetraodon and ab initio predictions). Of the 28,000 genes annotated, some families were examined in detail: selenoproteins, and Type 1 cytokines and their receptors. The comparison of the proteome of Tetraodon with those of mammals has revealed some interesting differences, such as a major diversification of some hormone systems and of the collagen molecules in the fish.

A search for transposable elements in the genomic sequences of Tetraodon has also revealed a high diversity (75 types), which contrasts with their scarcity; the small size of the Tetraodon genome is due to the low abundance of these elements, of which some appear to still be active. Another factor in the compactness of the Tetraodon genome, which has been confirmed by annotation, is the reduction in intron size, which approaches a lower limit of 50-60 bp, and which preferentially affects certain genes.

The availability of the sequences from the genomes of humans and mice on one hand, and Takifugu and Tetraodon on the other, provide new opportunities for the study of vertebrate evolution. We have shown that the level of neutral evolution is higher in fish than in mammals. The protein sequences of fish also diverge more quickly than those of mammals. A key mechanism in evolution is gene duplication, which we have studied by taking advantage of the anchoring of the majority of the sequences from the assembly on the chromosomes. The result of this study speaks strongly in favor of a whole genome duplication event, very early in the line of ray-finned fish (Actinopterygians). An even stronger evidence came from synteny studies between the genomes of humans and Tetraodon. Using a high-resolution synteny map, we have reconstituted the genome of the vertebrate which predates this duplication - that is, the last common ancestor to all bony vertebrates (most of the vertebrates apart from cartilaginous fish and agnaths like lamprey). This ancestral karyotype contains 12 chromosomes, and the 21 Tetraodon chromosomes derive from it by the whole genome duplication and a surprisingly small number of interchromosomal rearrangements. On the contrary, exchanges between chromosomes have been much more frequent in the lineage that leads to humans. All these results are presented in an article published in the 21th October 2004 issue of Nature.

Teams
The sequencing of the genome of Tetraodon nigroviridis is one of Genoscope’s internal projects. In 2001, the Broad Institute of MIT and Harvard (formerly the Whitehead Institute Centre for Genome Research) has joined the project and has produced a substantial part of the reads. The assembly step has also been performed in collaboration with the Broad Institute. Several groups have contributed to the analysis and the annotation of the genomic sequence of Tetraodon nigroviridis:

•Laboratoire de Biologie Moléculaire de la Cellule, CNRS UMR 5161, INRA UMR 1237, Ecole Normale Supérieure de Lyon ;
•Service de Systématique Moléculaire, CNRS IFR 101, dans le Département Systématique et Evolution du Muséum National d’Histoire Naturelle, Paris ;
•Défenses Antivirales et Antitumorales, CNRS UMR 5124, Montpellier ;
•Grup de Recerca d’Informàtica Biomèdica, IMIM-UPF et Programa de Bioinformàtica i Genòmica à Barcelone ;
•Biométrie et Biologie Evolutive, CNRS UMR 5558, University Lyon 1 ;
•Laboratoire des Interactions Plantes-Microorganismes, INRA UMR 441, CNRS UMR 2594, Toulouse ;
•Agencourt, Massachusetts, Etats-Unis ;
•Groupe “Evolutionary Fish Genomics”, Biozentrum, Université de Würzburg, Allemagne.

Bibliography
•R. Hinegardner, Am. Nat. (1968) 102, 517-563.
•S. Brenner et al., Nature (1993) 366, 265-268.
•T. Crnogorac-Jurcevic et al., Genomics (1997) 41, 177-184.
•B. Ewing & P. Green, Nature Genet. (2000) 25, 232-234.
•S. Aparicio et al., Science (2002) 297, 1301-1310.
•K.L. Howe et al., Genome Res. (2002) 12, 1418-1427.
•D.E. Neafsey et S.R. Palumbi, Genome Res. (2003) 13, 821-830.
Puffer fish

Scientific classification
Kingdom: Animalia
Phylum: Chordata
Class: Actinopterygii
Order: Tetraodontiformes
Family: Tetraodontidae
Genus: Tetraodon
Species: nigroviridis