Teleosts comprise more than half of all vertebrate species and have adapted to a variety of marine and freshwater habitats 1 . Their genome evolution and diversification are important subjects for the understanding of vertebrate evolution. Although draft genome sequences of two pufferfishes have been published 2,3 , analysis of more fish genomes is desirable. Here we report a high-quality draft genome sequence of a small egg-laying freshwater teleost, medaka (Oryzias latipes). Medaka is native to East Asia and an excellent model system for a wide range of biology, including ecotoxicology, carcinogenesis, sex determination 4-6 and developmental genetics 7 . In the assembled medaka genome (700 megabases), which is less than half of the zebrafish genome, we predicted 20,141 genes, including 2,900 new genes, using 59-end serial analysis of gene expression tag information. We found single nucleotide polymorphisms (SNPs) at an average rate of 3.42% between the two inbred strains derived from two regional populations; this is the highest SNP rate seen in any vertebrate species. Analyses based on the dense SNP information show a strict genetic separation of 4 million years (Myr) between the two populations, and suggest that differential selective pressures acted on specific gene categories. Four-way comparisons with the human, pufferfish (Tetraodon), zebrafish and medaka genomes revealed that eight major interchromosomal rearrangements took place in a remarkably short period of 50 Myr after the whole-genome duplication event in the teleost ancestor and afterwards, intriguingly, the medaka genome preserved its ancestral karyotype for more than 300 Myr.We applied the whole-genome shotgun approach to an inbred strain, , derived from the southern Japanese population, as the main target. A total of 13.8 million reads amounting to approximately 10.6-fold genome coverage were obtained from the shotgun plasmid, fosmid and bacterial artificial chromosome (BAC) libraries. A newly developed RAMEN assembler was used to process the shotgun reads to generate contigs and scaffolds. The N50 values (50% of nucleotides in an assembly are in scaffolds-or contigs-longer than or equal to the N50 value) are ,1.41 megabases (Mb) for scaffolds and ,9.8 kilobases (Kb) for contigs. The total length of the contigs reached 700.4 Mb, which, from now on, we refer to as the medaka genome size.To construct ultracontigs, the scaffolds were integrated with the medaka genetic map by using SNP markers. For this purpose, we further obtained about 2.8-fold coverage of shotgun reads from another inbred strain HNI (refs 9, 10), which is derived from the northern Japanese population. The reads were assembled by RAMEN to scaffolds covering 648 Mb. Aligning the HNI contigs with the HdrR genome using BLASTZ 11 , we identified 16.4 million SNPs as well as 1.40 million insertions and 1.45 million deletions in non-repetitive regions (Supplementary Table 2). We selected 2,401 SNPs and genetically mapped them onto medaka chromosomes using a backcross panel between the...
SummaryCilia/flagella are highly conserved organelles that play diverse roles in cell motility and sensing extracellular signals. Motility defects in cilia/flagella often result in primary ciliary dyskinesia (PCD). However, the mechanisms underlying cilia formation and function, and in particular the cytoplasmic assembly of dyneins that power ciliary motility, are only poorly understood. Here we report a novel gene, kintoun (ktu), involved in this cytoplasmic process. This gene was first identified in a medaka mutant, and found to be mutated in PCD patients from two affected families as well as in the pf13 mutant of Chlamydomonas. In the absence of Ktu/PF13, both outer and inner dynein arms are missing or defective in the axoneme, leading to a loss of motility. Biochemical and immunohistochemical studies show that Ktu/PF13 is one of the long-sought proteins involved in pre-assembly of dynein arm complexes in the cytoplasm before intraflagellar transport loads them for the ciliary compartment.
We have generated transgenic medaka (teleost, Oryzias latipes), which allow us to monitor germ cells by green fluorescent protein (GFP) fluorescence in live specimens. Two medaka strains, himedaka (orange-red variety) and inbred QurtE, were used. The transgenic lines were achieved by microinjection of a construct containing the putative promoter region and 3 region of the medaka vasa gene (olvas). The intensity of GFP fluorescence increases dramatically in primordial germ cells (PGCs) located in the ventrolateral region of the posterior intestine around stage 25 (the onset of blood circulation). Whole-mount in situ hybridization and monitoring of ectopically located cells by GFP fluorescence suggested that (i) the increase in zygotic olvas expression occurs after PGC specification and (ii) PGCs can maintain their cell characteristics ectopically after stages 20 -25. Around the day of hatching, the QurtE strain clearly exhibits sexual dimorphisms in the number of GFP fluorescent germ cells, a finding consistent with the appearance of leucophores, a sex-specific marker of QurtE. The GFP expression persists throughout the later stages in the mature ovary and testis. Thus, these transgenic medaka represent a live vertebrate model to investigate how germ cells migrate to form sexually dimorphic gonads, as well as a potential assay system for environmental substances that may affect gonad development. The use of a transgenic construct as a selective marker to efficiently isolate germ-line-transmitting founders during embryogenesis is also discussed.
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