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2 Develop. Growth Differ. (2008) 50, S157 S166 doi: /j X x Review Draft genome of the medaka fish: A comprehensive resource for medaka developmental genetics and vertebrate evolutionary biology Hiroyuki Takeda* Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo , Japan The medaka Oryzias latipes is a small egg-laying freshwater teleost, and has become an excellent model system for developmental genetics and evolutionary biology. The medaka genome is relatively small in size, ~800 Mb, and the genome sequencing project was recently completed by Japanese research groups, providing a high-quality draft genome sequence of the inbred Hd-rR strain of medaka. In this review, I present an overview of the medaka genome project including genome resources, followed by specific findings obtained with the medaka draft genome. In particular, I focus on the analysis that was done by taking advantage of the medaka system, such as the sex chromosome differentiation and the regional history of medaka species using single nucleotide polymorphisms as genomic markers. Key words: mutant, sex chromosome, SNP, speciation. Introduction The medaka Oryzias latipes is a model vertebrate of increasing interest in developmental and evolutionarily biology (Ishikawa 2000; Wittbrodt et al. 2002). In addition to common shared features with zebrafish, the medaka has several advantages, such as a smaller genome (approximately 800 Mb, half the size of the zebrafish genome), the existence of polymorphic and highly fertile inbred strains and the fact that they are growth permissive at a wide range of temperatures during embryonic development. For decades, the medaka was an important test system in ecotoxicology and carcinogenesis studies (Patyna et al. 1999). Furthermore, medaka research has a long and outstanding history in Japan (reviewed in (Shima & Mitani 2004)), especially in the field of sex determination, leading to the first demonstration in any species of Y-linked inheritance (Aida 1921), the first successful sex-reversal in vertebrates (Yamamoto 1958) and the *Author to whom all correspondence should be addressed. htakeda@biol.s.u-tokyo.ac.jp Received 7 December 2007; accepted 28 December Journal compilation 2008 Japanese Society of Developmental Biologists identification of the male-determining gene, Dmy, the first non-mammalian equivalent of SRY (Matsuda et al. 2002). Recently, large-scale mutagenesis projects were conducted by several groups in Japan and delivered a vastly expanded pool of medaka mutant stocks (Furutani-Seiki et al. 2004). Indeed, some medaka mutations appear to have unique phenotypes, demonstrating the utility of multiple teleost genetic models (Morinaga et al. 2007; Yokoi et al. 2007). Over the past decade, most model organisms, including humans and mice, have had their genomes completely sequenced and even non-model organisms are currently being sequenced or are on a waiting list for future genome sequencing projects. The last common ancestor of both medaka and zebrafish lived more than Ma, and medaka are a much more closely related to fugu ( Myr apart) than to zebrafish (Yamanoue et al. 2006; Fig. 1). The complete draft sequence of fugu (Aparicio et al. 2002; Jaillon et al. 2004) had a large impact on medaka genomics, as medaka and fugu are evolutionarily close to each other. In 2000, the zebrafish genome sequencing project began at the Sanger Centre and sequencing data continues to be updated frequently on the database web site ( pre.ensembl.org/danio_rerio/). Rapid completion of the genomic sequence of medaka was a crucial step for the rapid movement from mutant phenotypes to

3 S158 H. Takeda Fig. 1. Phylogenetic tree and teleost genome evolution. The date we adopt for whole genome duplication (WGD) and lineage divergence is based on molecular clock estimates (Yamanoue et al. 2006). gene functions, and was expected to greatly benefit fish comparative genomics, bridging the gap between these evolutionarily distant fish species. Under these circumstances the medaka genome project started in 2002 and was successfully completed in 2006, providing a high-quality draft genome with a vast number of reliable genetic markers (Kasahara et al. 2007). It greatly facilitates positional cloning of medaka developmental mutants and impacts on comparative genomics which are underway in many laboratories over the world. In this review, I will give an overview of the medaka draft genome and describe some unique aspects of the medaka genome which were found during the course of the project. Overview of the medaka draft genome The medaka genome project started in late 2002 as a collaborative work of three core laboratories led by H. Takeda (University of Tokyo, Japan), S. Morishita (University of Tokyo, Japan) and Y. Kohara (National Institute of Genetics [NIG], Japan), and was conducted under the support of the Grants-in-Aid for Scientific Research in Priority Area Genome Science from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Sequencing was carried out at the Academia Sequencing Centre of NIG. There are two main approaches for sequencing large, complex genomes such as medaka: shotgun sequencing of the entire genome (whole-genome shotgun; WGS) and shotgun sequencing of bacterial artificial chromosome (BAC) clones or contigs aligned in a physical map. In the medaka genome project, we adopted the WGS approach because it has the advantage of both simplicity and rapid early coverage of the whole genome. Fortunately, the medaka genome is relatively simple with minimum polymorphism (inbred strain), and the WGS approach worked very well together with the integration of detailed map information and pair-mate sequence data from large inserts in BAC and fosmid vectors. We chose the inbred strain Hd-rR as the main target for sequencing because it is derived from the southern Japanese population from which most medaka mutants are derived. Since Hd-rR underwent inbreeding for more than 50 generations, the genome of this strain is largely homozygous. For sequencing, a total of 13.8 million reads from the shotgun libraries and fosmid and BAC libraries were obtained, amounting to an approximately fold redundancy in sequence coverage. In addition, we obtained about a 2.8-fold redundancy in sequence coverage from another inbred strain, HNI, that is derived from the northern Japanese population. RAMEN assembler, a computer program that we recently developed to assemble paired-end sequences, was used to process all of the medaka shotgun reads to generate initial scaffolds. Comparative analysis with the genomes of the Hd-rR and HNI strains revealed more than 16 million single nucleotide polymorphisms (SNP; see also below). This enabled us to develop a high density SNP map in which about 2400 SNP were mapped genetically using an Hd-rR/HNI back-cross panel with 93 or 368 individuals. Where possible, at least one SNP marker was selected in each Hd-rR scaffold of greater than 60 kb. This dense and accurate genetic map was used for aligning generated scaffolds. The newly

4 Draft genome of the medaka fish S159 developed assembler as well as the SNP genetic map were the key to the high-quality draft genome of the medaka. In summary, anchored nucleotides cover about 90% of the assembled genome, and the total length of the contigs is Mb with 99.6% sequence accuracy. The N50 ultracontig size became ~5.1 Mb (excluding gaps), which means that half of the nucleotides were within ultracontigs greater than ~5.1 Mb in size. Considering the relatively lower coverage of the preceding Tetraodon genome (65%), the medaka genome project has achieved the highest quality among fish genomes. According to the Ensembl database ( released in July 2007, the zebrafish genome also reached a coverage of 88.6%. Thus, as of the end of 2007, the medaka and zebrafish genomes are of a high quality comparable to sequenced mammalian genomes such as the human, mouse, rat, and dog genomes. Sex-chromosome differentiation in medaka Since we sequenced the genome of an inbred strain, essentially no polymorphism between allelic regions was found except for sequencing errors. However, we observed unusual haplotypes in linkage group 1 (LG1), a sex chromosome in the medaka. Like mammals, medaka has an XX-XY sex-determining system (Aida 1921), but the differentiation of its sex chromosomes seems primitive: LG1 (33.7 Mb) inserted with a 250 kb Y-specific region that contains a male-determining gene, Dmy (dmrt1b) (Matsuda et al. 2002; Kondo et al. 2003; Kondo et al. 2004), serves as a Y chromosome, while LG1 without it as an X chromosome (Fig. 2a). In fact, they are not clearly distinguishable sex chromosomes. Thus, although we sequenced male genomic DNA, it was difficult to distinguish whether the sequence reads of LG1 were derived from the X or Y chromosome. However, our RAMEN assembler detected polymorphisms that were arranged in a haplotype manner in a region extending over 3.5 Mb around the Y-specific region (Fig. 2b,c). This directly demonstrates the local suppression of crossing-over near the male-determining region, which has long been known in medaka genetic recombination tests (Kondo et al. 2001). On the other hand, the region that is far from the sex-determining locus in LG1 is highly homozygous, indicating free crossingover. These results indicate that the inserted maledetermining region has an impact on the recipient LG1 with respect to restriction of recombination, but at present, the effect is limited to about 10% of its length, 3.5 Mb. It is widely accepted that proto-x and proto-y that fail to cross over with the sex-determining locus are consequently genetically isolated from one another, and will diverge over evolutionary time (Charlesworth 2004). The genome sequencing project of the medaka therefore reveals for the first time the suppression of recombination between the primitive sex chromosomes at the sequence level; the medaka Y chromosome is, so to speak, at an initial step toward sexual differentiation at the chromosomal level. Similar local suppression of crossing-over near the sex-determining regions was also reported in other fish such as the stickleback (Peichel et al. 2004). Further comparative analysis of the medaka Y chromosome may yield important insights into the evolution of vertebrate sex chromosomes. Polymorphisms between two inbred strains of medaka, Hd-rR and HNI As mentioned above, we identified 16.4 million SNP, 1.40 million insertions and 1.45 million deletions by aligning the HNI contigs with the Hd-rR genome (Fig. 3a). The genome-wide SNP rate between the two inbred strains is 3.42%, which is, to our knowledge, the highest SNP rate seen in any vertebrate species. Figure 3b is an example of the distribution pattern of such polymorphisms over a 150 kb region harboring the HoxAa cluster. As expected, SNP show a roughly uniform distribution at the average frequency of 3.4%, but locally, there are some fluctuations with a tendency for the rate to be lowered in the coding regions (1.8%). Furthermore, the distribution patterns of small insertions/deletions (In/Dels), where >50% of such In/Dels are one or two nucleotides in length, are similar to the SNP across the genome but their frequency is 5 7-fold lower than the SNP frequency. Interestingly, the relative frequency of In/Dels to SNP is 0.173, which is higher than those in human and chimpanzee (0.145; Chimpanzee Sequencing and Analysis Consortium 2005) and chicken strains (0.091; Wong et al. 2004). This tendency, together with the recent whole genome duplication (WGD) event, may contribute to the genome diversity that enables the rapid adaptive radiation of teleost species. The two medaka inbred strains, Hd-rR and HNI, are estimated to have diverged about 4 Ma (Takehana et al. 2003). Recent study using the mitochondrial cytochrome b gene has elucidated the detailed phylogenetic relation among four genetically different wild populations (Northern Japanese, Southern Japanese, East Korean, and China-West Korean) as well as Japanese local groups (Takehana et al. 2003; Fig. 3d). Despite the undoubted accumulation of genetic variations, they all can mate and produce healthy and

5 S160 H. Takeda Fig. 2. Sex chromosome differentiation in medaka. (a) Sex chromosomes in medaka. The Y-specific region (250 kb) itself is derived from the duplication of a fragment from linkage group (LG) 9 containing dmrt1. One of the duplicated fragments was inserted into LG1 10 Ma, and its dmrt1 became the male-determining gene, Dmy. (b) An example of haplotypes near the Y-specific region. (c) Haplotypes are observed in a 3.5 Mb region surrounding the Y-specific region. SNP, single nucleotide polymorphism. (b, c) courtesy of Dr Masahiro Kasahara (University of Tokyo). fertile offspring (i.e. they are still the same species, Oryzias latipes). The massive SNP resources and living stock of medaka regional strains enabled us to perform a genome-wide SNP analysis for the history of medaka wild populations during regional diversification in Japan. For this, we sequenced 96 randomly selected loci (four loci for each chromosome) amplified by polymerase chain reaction (PCR) from seven selected medaka strains with different regional histories (Fig. 3c). We performed in-depth comparative analysis using 47 loci which produced fragments of expected sizes in all strains examined. For simplicity, we focused on 475 SNP sites identified between Hd-rR and HNI in these 47 loci (approximately 24.9 kb in total). We first examined what proportion of these SNP sites is polymorphic or fixed within each wild population by comparison with the sequences of each sister strain (Hd-rR and Nago in the southern population, and HNI, Niigata, and Kaga in the northern population). Nago and Kaga strains were chosen because they are most distantly related to Hd-rR and HNI within each population, respectively. The analysis revealed that 130 (27%) and 28 (5.8%) of 475 SNP sites were found to be polymorphic in the southern and northern populations, respectively, and one mutation happened to be shared by both populations. The remaining 318 (475 [ ]) Hd-rR/HNI SNP are thus preserved or fixed between the southern and northern populations (common SNP in Figure 3c). These common SNP were also introduced independently in each lineage during and/or after separation from the ancestor of the northern and southern populations. We then examined the mutation events that had created

6 Draft genome of the medaka fish S161 Fig. 3. Genetic variations between two medaka strains. (a) The two inbred lines, Hd-rR and HNI, from southern and northern Japanese populations. (b) Distribution of single nucleotide polymorphisms (SNP) and insertions/deletions in the region of HoxAa cluster. (c) Phylogenetic analysis of the SNP identified between Hd-rR and HNI during regional diversification. (d) Collection sites of medaka strains in Japan. these common SNP in each lineage by comparison with the consensus sequence of Korea-Taiwan-China medaka strains, which are the same species O. latipes, as an outgroup. This analysis assigned 120 and 185 mutation events to the southern and northern lineages, respectively, but failed to determine 13 events due to a second mutation found in the consensus sequence (three alleles at a single site). Overall, the total numbers of mutations introduced for 4 Myr are 250 ( ) for the Hd-rR and 213 (185+28) for the HNI strain, indicating that almost the same levels of mutations accumulated in the history of the two strains. The above analysis demonstrated the characteristic features of wild medaka populations in Japan. The lower levels of polymorphism in the northern lineage support their rapid and recent expansion from a small population (i.e. a bottleneck effect), which has been

7 S162 H. Takeda Fig. 4. Medaka genes. (a) Breakdown of medaka gene homologues in other species. (b) Dots represent pairs of medians of K a/k s ratios in the northern-southern medaka strains and the human-chimpanzee lineage for individual Gene Ontology categories. Rapidly and slowly evolving gene ontology categories in the medaka species relative to the hominid lineage are colored yellow and blue, while the others are pink. Remarkable categories are highlighted by circles. (c) Global distribution of duplicated genes in the medaka genome. Paralogue pairs are 1:1 reciprocal best matches with an aligned portion greater than 30%. suggested by the previous phylogenetic study using the mitochondrial cytochrome b gene (Takehana et al. 2003). More importantly, the high ratio of common SNP (>65%) as well as few shared-polymorphic sites indicate a strict genetic separation between the two medaka populations for 4 Myr without major species differentiation (or speciation). This makes the medaka a unique model for the study of genetic variations and speciation in vertebrates. Medaka genes and their evolution Medaka gene catalogue Having sequences in hand, the next important step was, of course, a construction of a medaka gene catalogue. In the human genome project, genes were predicted mainly based on the information obtained from expressed sequence tags (EST) and full-length cdna. However, such information was very limited in medaka, and we had no choice but to collect expression data ourselves. For this, we adopted a 5 -end serial analysis of gene expression (5 SAGE). A large collection of 5 SAGE tags, each tag representing bases of the 5 -end of an mrna, allowed us to globally identify transcriptional start sites (TSS) and the frequency of individual mrna because we used un-normalized cdna sources. Indeed Hashimoto et al. (2004) showed that this method detected TSS in the human genome with a 99% degree of accuracy (Hashimoto et al. 2004). In the medaka genome project, we collected SAGE tags in total from a mixture of cdna of 0 7 day old medaka embryos and adult body tissues. Combination of the tag information (evidence-based) and Genscan with our newly developed algorithm (an in silico method) finally produced non-redundant sets of predicted genes. As expected, some genes have a single TSS, whereas others have multiple TSS mostly within 500 base regions, possibly reflecting the levels of expression. The predicted genes were then compared with those of human, Tetraodon, Takifugu, zebrafish, chicken, mouse and so on, to find their homologues under the condition TBLASTX E-vale < E-04. The comparative analysis demonstrated that about half of them are shared by all animals examined, that is to say, core genes and that 80% are found in at least one of those animals (Fig. 4a). Rapidly and slowly evolving gene categories in the medaka species For the northern-southern medaka lineage, the average SNP rate is 3.42% and the average K a/k s ratio (where K a is the number of non-synonymous substitutions and K s, the number of synonymous substitutions) of 8889 medaka predicted genes (qualified by a certain criteria) is These figures are significantly higher than those for the human-chimpanzee lineage (1.23% for SNP and 0.23 for K a/k s) (Chimpanzee Sequencing and Analysis Consortium 2005), which of course has experienced major speciation for more than 6 Myr. The high degree of intra-species variation led us to examine a genome-wide comparison of the evolutionary rate in protein-coding genes between the northern-southern medaka and human-chimpanzee lineages, expecting insight into the relationship between genetic variations and species differentiation. First we calculated the median K a/k s ratio of each functional category of genes, based on the Gene Ontology (GO) classification. A large-scale comparison was then conducted between the northernsouthern medaka and human-chimpanzee lineages. The results revealed the following interesting tendencies for rapidly and slowly evolving gene categories in the medaka species relative to the hominid lineage. In this analysis, we looked at specific categories referred by the previous analyses among mammalian species because they have identified immunity, host defense, reproduction (sexual reproduction, gamatogenesis etc.) and olfaction as a rapidly evolving category, while intracellular signaling, neurogenesis and neurophysiology (e.g. synaptic transmission) as a slowly evolving one (Chimpanzee Sequencing and Analysis Consortium 2005). The rapidly evolving categories are thought to be involved in adaptation to environment and sexual separation, both of which are essential processes during and/or after speciation. Intriguingly, these rapidly evolving categories are not evident in the medaka lineage, and they are mostly ranked as intermediate (Fig. 4b). In particular, the reduced evolutionary rate in the reproduction- and sex-related categories might explain why the two medaka strains can mate and produce fertile offspring even after a long period of geographical and genetic separation. The difference in sex-determining systems between the medaka and mammals could affect the

8 Draft genome of the medaka fish S163

9 S164 H. Takeda evolutionary rate on genes with reproduction-related functions. Furthermore, the level of sex-chromosome differentiation, which is a minimum in medaka, could contribute to this difference. In contrast, mammalian slowly evolving neural-related categories exhibit relaxed constraints in the medaka lineage (Fig. 4b), and this tendency may reflect less complicated neural circuits in fish, or potentiate the adaptation ability of the fish nervous system to a variety of living environment. It is generally accepted that the extent of phenotypic variation between organisms is not strictly related to the degree of sequence variation. This holds true in the case of species differentiation. The two medaka polymorphic strains, Hd-rR and HNI, remain in the same species, although their sequence difference is much greater than that observed between human and chimpanzee and between the mouse species Mus musculus and Mus spretus. Our comparative analysis suggests that the differential selective pressures act on specific categories in a lineage-specific manner, which may contribute to a pattern of evolution, for example adaptation with or without speciation. The genome sequences of polymorphic medaka inbred lines provide unique resources for the analysis of vertebrate gene evolution. Genome evolution The availability of genome sequences has opened up new avenues to the study of important evolutionary questions. In fish, whole-genome duplication (WGD) and subsequent asymmetric changes in duplicated genes were thought to play an important role in genome evolution. The recent Tetraodon draft genome sequence data provided some conclusive answers that WGD did occur in the fish lineage after its divergence from the tetrapods. With the high-quality medaka draft genome we draw more precise pictures. First, we made pairwise comparisons among the predicted medaka genes and identified 1730 pairs of duplicated genes (paralogues). Figure 4c, in which these 1730 pairs are mapped into linkage groups, shows their global distribution pattern on the medaka chromosomes. The distribution pattern strongly suggests the occurrence of WGD in the past, because most of the chromosomes share a significant number of common paralogues with one or two other chromosomes. We then conducted large-scale four-way comparisons of the medaka, human, zebrafish and Tetraodon genomes to construct a scenario for teleost genome evolution. Since detailed methods and results are described elsewhere (Kasahara et al. 2007), I focus on the early events that are depicted in Figure 1. The ancestral karyotype of teleosts was reconstructed to be thirteen, and our scenario shows eight major interchromosomal rearrangements that took place for a remarkably short period of ~50 Myr between the WGD event and the divergence of zebrafish from medaka and Tetraodon. After the divergence, however, no major inter-chromosomal rearrangements occurred in the medaka genome for ~320 Myr. This reconstruction was achieved by our newly developed method as well as by using the sufficient number of medaka genes provided by the high-quality draft genome (Kasahara et al. 2007). The high-quality medaka genome also contributed to the analysis of vertebrate genome evolution (Nakatani et al. 2007). Genomic resource database Since the medaka genome project was launched in 2002, substantial genomic resources of medaka have been opened to the public through the University of Tokyo Genome Browser Medaka (UTGB/medaka) database at: (Ahsan et al. 2007). This database provides basic genomic information, such as predicted genes, EST, guanine/ cytosine (GC) content, repeats and comparative genomics, as well as unique data resources including: (i) 2473 genetic markers and experimentally confirmed PCR primers that amplify these markers; (ii) BAC and fosmid end sequences that amount to 15- and 11.1-fold clone coverage of the entire genome, respectively, and were used for draft genome assembly; (iii) SNP, and insertions/deletions detected between the two medaka inbred strain genomes; and (iv) SAGE tags that identified TSS on the genome. For researchers working on gene function using medaka, the information on BAC and fosmid clones covering the genes of interest would be particularly useful. An example is shown in Figure 5, in which the predicted gene, # , is covered by 10 BAC clones. Importantly, those BAC clones are available at the National Bioresource Project (NBRP) medaka: genome/top.jsp Perspective The high-quality medaka draft genome is providing an important reference for various ray-finned fishes that have yet to be sequenced. Cichlids and stickleback, which are new emerging model systems for understanding the genetic basis of vertebrate speciation and evolution, are evolutionally closer to medaka than

10 Draft genome of the medaka fish S165 Fig. 5. Snapshot of UTGB/medaka. Note the information on end-pairs of bacterial artificial chromosome and fosmid clones aligned in the genome. zebrafish. The same is true for many commercially important fish species, including tuna, flounder, sea bream and fugu (Miya et al. 2003). Furthermore, there are many close relatives of medaka that are indigenous to East to South-East Asia (Takehana et al. 2005), many of which are now maintained by the NBRP medaka. Together with the medaka draft genome, a low-coverage sequence of the genomes of these fish species would shed further light on the mechanisms underlying speciation and diversity, which have yet to be fully addressed at the genome sequence level. Finally, the medaka draft genome is the first high-quality fish genome with numerous SNP markers. This comprehensive genomic information available online is accelerating quantitative trait loci analyses of interesting traits in individual strains and positional cloning of many developmental mutant genes, leading to the elucidation of novel genes and pathways that have crucial functions in vertebrate development and in human disease. Acknowledgment The medaka genome project was jointly conducted by nearly 40 researchers, including seven principal investigators in Japanese laboratories of the University of Tokyo, National Institute of Genetics, National Institute of Informatics, RIKEN, Keio University, and Niigata University. Here I would like to thank again all these people for their devoted contribution to the project. Conflict of Interest No conflict of interest has been declared by H. Takeda. References Ahsan, B., Kobayashi, D., Yamada, T. et al UTGB/ medaka: genomic resource database for medaka biology. Nucleic Acids Res. 36, D Aida, T On the inheritance of color in a fresh-water fish, APLOCHEILUS LATIPES temmick and schlegel, with special reference to sex-linked inheritance. Genetics 6, Aparicio, S., Chapman, J., Stupka, E. et al Whole-genome shotgun assembly and analysis of the genome of Fugu rubripes. Science 297, Charlesworth, B Sex determination: primitive Y chromosomes in fish. Curr. Biol. 14, R745 R747.

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