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[A Synthesis of the Molecular Phylogeny and the Compara-tive Genomics in Fishes]Organized by Kiyoshi Naruse1 and Shigeki Yasumasu2

1Department of Biological Sciences, Graduate School of Science, University of Tokyo, 2Life Science In-stitute, Sophia University

Phylogenetic Relationships of Actinopterygian Fishes: An Esti-mation from Extensive Analysis of Their Complete Mitocho-ndiral GenomesMutsumi Nishida1 and Masaki Miya2

1Ocean Research Institute, University of Tokyo, 2Natural History Museum and Institute, Chiba

From the phylogenetic viewpoint, living members of the Teleostomi (~“Osteichthys”) are composed of bothsarcopterygians (lobe-finned fish; including lungfishes, coelacanths, and tetrapods) and actinopterygians (ray-finned fish; including most other fishes such as medaka, fugu, etc.). As well as the former, the latter forms ahuge group of organisms, having more than 27,000 species. Though a number of comparative anatomical studieshave been conducted in attempts to resolve their inter- and intra-relationships using modern cladistic methodol-ogy, there remains much controversy over the higher-level relationships of actinopterygians. Considering the highspecies diversity and their ancient origin (>200 million years ago), it is no wonder that comparative anatomicalapproaches have faced difficulties in uncovering the higher-level relationships of them. The same is true for mo-lecular phylogenetic studies, which usually have employed shorter sequences (mostly <1 kbp) based on limitedtaxonomic representation. Adequate resolution of higher-level relationships in any organisms should requirelonger DNA sequences from many taxa.The recent development of our PCR-based approach for determination of the complete DNA sequences of fishmitochondrial genomes (mitogenomes; ~16-17 kbp) has made possible to obtain a number of such sequencesfrom a wide variety of taxa at a reasonable amount of expense of time and cost. Using this approach, we havebeen aiming to establish a reliable phylogenetic framework of actinopterygian fishes. We estimated the inter-rela-tionships of major lineages of “basal” actinopterygians (Polypteriformes, Acipenseriformes, Semionotiformes, andAmiiformes) with the Teleost as the most “derived” actinopterygians. We then resolved the inter-relationships ofmajor lineages of “basal” teleosts (Osteoglossomorpha, Elopomorpha, Clupeomorpha, Ostariophysi, andProtacanthopterygii), and subsequently examined detailed intra-relationships of each of these groups in a seriesof studies.The Neoteleostei, representing “derived” teleosts, is the most diversified group of all vertebrates, currently com-prising over 15,000 extant species. Our mitogenomic analysis has suggested that many of major groups aboveordinal level as currently defined in neoteleots appeared to be nonmonophyletic. Our taxonomic sampling, none-theless, appears still insufficient for drawing explicit conclusions. Therefore, we are now analyzing mitogenomedata from more than 300 fish species mainly from higher teleosts. The preliminary resultant trees were well re-solved and local phylogenies agreed well with those from previous mitogenomic analyses.

Evolution of Hatching Enzyme Genes in TeleostMari Kawaguchi1, Shigeki Yasumasu1, Junya Hiroi2 and Ichiro Iuchi11Life Science Institute, Sophia University, 2Department of Anatomy, St Marianna University

At hatching of teleost embryos, hatching enzyme is secreted from the embryos to digest their envelopes. Thehatching enzyme of medaka Oryzias latipes is composed of two enzymes, MHCE and MLCE. Both enzymes be-long to the astacin family metallo-proteases. Their genomic gene structures are quite different, that is, MHCEgenes are intron-less genes, MLCE gene comprises 8 exons and 7 introns. Generally, the exon-intron structureof homologous genes is conserved beyond animal species. However, the exon-intron structure of Japanese eelHCE (EHCE) (8-exon-7-intron) was similar to that of the MLCE gene, not the MHCE gene. To clarify how such

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intron loss had occurred during the evolutionary processes of HCE genes, we determined the exon-intron struc-tures of the HCE genes cloned from various fish species such as Indo-Pacific tarpon Megalops cyprinoids(TpHCE), Japanese anchovy Engraulis japonicus (AcHCE), milkfish Chanos chanos (MfHCE), zebrafish Daniorerio (ZHCE), catfish Silurus arotus (CHCE), neon tetra Paracheirodon innesi (NeHCE), electric eel Electrophoruselectricus (EeHCE), masu salmon Oncorhynchus masou (MsHCE), redfin pickerel Esox americanus americanus(PkHCE), Ayu Plecoglossus altivelis altivelis (AyHCE), Alcock’s boafish Stomias nebulosus (BfHCE), slickheadAlepocephalus longirostris (ShHCE), Fundulus Fundulus heteroclitus (FHCE), flatfish Paralichthys olivaceus(FlHCE) and Fugu Takifugu rubripes (FgHCE). The structure of TpHCE gene (7 introns) was the same as that ofEHCE gene. Compared with the EHCE gene, the gene of AcHCE, MfHCE and ShHCE lost one intron. ZHCEgene lost 3 introns, while CHCE, NeHCE and EeHCE genes lost 4 introns. The HCE genes of all other examinedfishes belonging to Euteleostei lost all the introns. Recently, using mitochondrial DNA, the detailed phylogenetictree of teleostean fishes has been constructed by Nishida et al. According to the tree, Elopomorpha fishes (nointron loss) such as Japanese eel and Indo-Pacific tarpon first branch off from the ancestor, second Otocephala(Ostariophysi + Clupeomorpha) fishes (1 to 4 intron loss) such as Japanese anchovy, milkfish, slickhead,zebrafish, catfish, neon tetra and electric eel, and finally Euteleostei fishes (7 intron loss). Especially withinOtocephala, the HCE genes of the first branched clade comprising Japanese anchovy, milkfish and slickhead lost1 intron, that of the second clade Cypriniformes (zebrafish) lost 3 introns, and those of the clade consisting ofcatfish, neon tetra and electric eel lost 4 introns. Thus, the number of introns of HCE gene in Otocephala de-creased in a phylogeny-dependent, step-by-step manner. On the other hand, the exon-intron structures of LCEgenes were well conserved among fishes such as Ayu (Osmeriformes), medaka (Beloniformes), Fundulus(Cypridontiformes) and Fugu (Tetradontiformes). The results suggest that only the HCE genes lost their intronsduring the evolutionary process of teleosts.

Draft Genome Sequence of Medaka (Orizias latipes) and Its Use-ful ApplicationsMasahiro Kasahara1, Shin Sasaki1, Yoichiro Nakatani1, Wei Qu1, Ahsan Budrul1,Tomoyuki Yamada1, Yukinobu Nagayasu1, Koichiro Doi1, Kiyoshi Naruse2, TakanoriNarita3, Tadasu Shin-I3, Tomoko Jindo2, Shin-ichi Hashimoto4, Koji Matsushima4, YokoKuroki5, Asao Fujiyama5, Hiroyuki Takeda2, Shinichi Morishita1 and Yuji Kohara3

1Department of Computational Biology, Graduate School of Frontier Sciences, University of Tokyo, 2De-partment of Biological Sciences, Graduate School of Science, University of Tokyo, 3Center for GeneticResource Information, National Institute of Genetics, 4Department of Molecular Preventive Medicine,School of Medicine, University of Tokyo, 5National Institute of Informatics

The sequencing project of medaka has reached its later phase. Whole genome shotgun (WGS) approach wasadopted to the project as well as in other projects sequencing teleost fish. However, WGS approach was oncecriticized for its limited capability to reconstruct original genome sequences. We will explain from technical pointof view why we could generate a high-quality draft genome sequence of medaka. We will give detailed algorithmto assemble the genome in order to illustrate non-repetitive sequences were well reconstructed in the WGS as-sembly.We will also introduce unique resources of the medaka WGS project including gene prediction by 5’-SAGE (Se-rial Analysis of Gene Expression) and high density polymorphism map between HdrR/HNI strains.Current ab initio gene prediction (e.g. GenScan) often outputs many false positives, and it often misses the initialexons. We exploited 5’-SAGE tags to improve prediction by GenScan. For every transcription start site, a genestructure is predicted by GenScan and the initial exon is added by heuristic algorithm if it is missed by GenScan.This algorithm is named TransS, which predicted over 16,000 genes. RT-PCR analysis suggests that the speci-ficity of the prediction is over 92%.High density polymorphism map between HdrR/HNI strains is one of the most important resources for positionalcloning. HdrR inbred strain from southern population of medaka is used for WGS sequencing, while HNI inbredstrain from northern population is also shotgun sequenced to 2.5×depth. Although they can be interbred to pro-duce fully fertile hybrids, there is 3% difference between in their nucleotides. SNPs are distributed all across thegenome, enabling us to design SNP marker to virtually any position of the genome.Comparative analysis revealed that there is almost one-to-one correspondence between tetraodon genome and

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medaka genome. The typical size of syntenic block reaches several megabases. Interchromosomal rearrange-ments were much less observed than intrachromosomal rearrangements. These results suggest the possibilitythat the draft sequence of medaka can provide a framework for determining standard genome structure of teleostfish.

A Genetic Linkage Map for the Tiger Pufferfish, Takifugu rubripes:Comparative Genomics and Evolution of Teleost GenomesKiyoshi Kikuchi, Wataru Kai, Masashi Fujita, Hiroaki Suetake and Yuzuru SuzukiFisheries Laboratory, The University of Tokyo

The tiger pufferfish, Takifugu rubripes (fugu) was proposed as a genomic model because of its compact genome.Three years ago, the genome of fugu has been sequenced to the draft level and annotated to identify all thegenes (Aparicio et al., 2002). However, the assembly of the draft genome sequence is highly fragmented due tothe lack of a genetic or a physical map. To determine the long-range linkage relationship of the sequences, wehave constructed the first genetic linkage map for fugu (Kai et al., 2005). The maps for the male and femalespanning 697.1 and 1,213.5 cM, respectively, were arranged into 22 linkage groups by markers heterozygous inboth parents. The resulting map consists of 200 microsatellite loci physically linked to genome sequences span-ning 39.2 Mb in total and contains ~4,000 predicted genes.Recently the draft genome sequence of another pufferfish, Tetraodon nigroviridis, has been generated and 64.4%of the genome assembly was anchored on its chromosome (Jaillon et al., 2004). Moreover, gene maps of twoother teleosts, medaka and zebrafish have been constructed, using hundreds of gene or EST sequences (Naruseet al., 2004; Woods et al., 2000). Availability of gene maps of those teleosts and the fugu map constructed by usprovides an opportunity to compare the extent of genome reorganization between the teleost species and themammalian species. The comparative analysis suggests that syntenic relationship is more conserved in the te-leost lineage than in the mammalian lineage. We also compared the genomic location of Hox gene clusters thatare often used as landmarks for comparison of the vertebrate genomes and found a pufferfish lineage specificrearrangement of the genome resulting in co-localization of two Hox clusters in one linkage group.Besides comparative genomics, genetic linkage maps are invaluable in forward genetic analysis for the identifica-tion of gene loci responsible for genetic traits. Pufferfishes belonging to genus Takifugu are mainly distributed incoastal regions of East Asia and include more than 20 species. These fishes show marked differences in bodysize, body color pattern, the number of meristic skeletons, parasite resistance and behavior. Interestingly, hybridsproduced by eight Takifugu inter-species crosses, including fugu, were found viable (Fujita, 1967; Miyaki, 1992).Given the ability to generate fertile crosses between species, the availability of the draft genome sequence offugu provides an unprecedented opportunity to understand the genetic basis of the evolution of phenotypic diver-sity in nature. Our genetic map of fugu would greatly facilitate such studies.

REFERENCESAparicio S et al. (2002) Science 297: 1301–1310Fujita S (1967) Jpn J Michurin Biology 3: 5–11Jaillon O et al. (2004) Nature 431: 946–957Kai W et al. (2005) Genetics 171: 227–238Miyaki K (1992) PhD Thesis, Nagasaki UniversityNaruse K et al. (2004) Genome Res 14: 820–828Woods IG et al. (2000) Genome Res 10: 1903–1914