Medaka LG22 Project

Medaka as a model organism for functional genomics

Medaka as a model organism for functional genomics

Fish provide more than 50% of vertebrate classes, with a particularly deep and broad phylogeny (Nelson 1994). So using diverse fish species are particularly good to identify conserved, as well as species-specific, genetic and molecular mechanisms that underlie development and evolution, such as the mechanisms and processes that trigger adaptive radiation, the multiplication of a single ancestral species into a variety of functionally different species, and ecological adaptation related to speciation.

Medaka is a small freshwater fish native to Asia that is found primarily in Japan, but also in Korea and China. This fish has been used widely as an experimental animal because of its relatively short life cycle, high fecundity, transparent egg chorion, small size and so on. It provides an important test system for environmental research, cancer research, and developmental biology (for review, see Wittbrodt, Shima and Schartl, 2002 and see the Medakafish homepage http://biol1.bio.nagoya-u.ac.jp:8000/ for an overview of techniques.). Furthermore, most experimental tools for gene function analysis can be applied to both zebrafish and medaka.

Medaka in genome research

Large scale mutagenesis project

Shima and Shimada (1991) established a multi-locus tester strain that is homozygous at several recessive loci and is used to detect induced recessive mutations in the germ line. The germ cell mutagenesis protocols they established turned out to be useful for mutagenesis not only in medaka but also in zebrafish. Loosli et al. (2000) report the first systematic mutagenesis approach to isolate embryonic-lethal developmental mutants in medaka, and recently, the ERATO project by Hisato Kondo and Makoto Furutani-Seiki started a large scale genome-wide screening by ENU mutagenesis which is comparable to that done using zebrafish ( http://www.dsp.jst.go.jp/MedGenIn/index.html ). Surprisingly, many mutant phenotypes found by similar morphological screening are species-specific. So both spontaneous and induced medaka mutants should provide new insight into the function of genes during development (Shima et al. 2003).

Genome mapping

A medaka linkage map was first described by Aida (1921). He demonstrated that the male determining factor (Y) was linked with the gene that controls carotinoid deposition in xanthophores (R). Since Aida's study, over 60 visible mutants have been isolated and analyzed by allele sharing and allelic association. (Tomita 1975; Tomita 1983). The first multipoint linkage map including 170 loci and 28 linkage groups was reported using RAPD fingerprints and allozyme analysis (Kubota et al. 1995; Wada et al. 1995). 638 markers (489 AFLPs, 28 RAPDs, 34 IRSs, 78 ESTs, four STSs and four phenotypic markers) were mapped to 24 linkage groups corresponding to the haploid number of medaka chromosomes (Naruse et al. 2000). Recently, some linkage groups were identified by chromosomal FISH analysis using mapped ESTs (Matsuda et al. 1998 , Miyake et al. unpublished). About 100,000 EST (expressed sequence tag) sequences of medaka have been sequenced and deposited in the public database. It was found that cultured cell lines could be a good source for cDNA libraries to increase the variation of cDNAs, since they could provide different cDNA species from various tissues. ESTs with high similarity to known vertebrate genes are a good source for the mapping. To assign about 800 loci encoding expressed genes to each linkage group, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis is used. The current row mapping data and BLAST results for medaka ESTs can be viewed by Mbase, ( http://mbase.bioweb.ne.jp/~dclust/ml_base.html ).

Genome sequencing

An important genome resource, gridded BAC (bacterial artificial chromosome) libraries, have been established from the southern and the northern inbred strains of medaka by cooperation of Shimizu Group (Keio Univ.) (Matsuda et al. 2001; Kondo S. et al. 2001). They are essential resources for both genome sequencing and fine mapping.

As a research group with international collaboration, the Medaka Genome Initiative (MGI) is based on genetic and physical mapping resources. Laboratories that are part of this initiative collaborate to physically and computationally interconnect the resources (the MGI home page: http://www.dsp.jst.go.jp/MedGenIn/index.html ).

And recently, the NBRP medaka genome project did whole-genome shotgun sequencing of the medaka genomic DNA under “Genome Analyses”, a subproject of the National Bioresource Project by the Ministry of Education, Culture, Sports, Sciences and Technology (MEXT) in a fiscal year 2002. The decoded genome was obtained by one million sequencing and 726 Mbp reads. This size is considered to be 0.9 sequence coverage of the whole medaka genome (http://shigen.lab.nig.ac.jp/medaka/genome/indexen.html ).

This whole genome shotgun sequencing project is continued by Kohara (NIG) group, and they will sequence clones as many as 6x coverage and 95% of whole genome will be sequenced. It will give most of gene information. And to finish the assembly of WGS data, new genomic assembler is developed by Morishita (University of Tokyo).

Medaka CBC genome project: Overview

Keio group (Lab head: N. Shimizu, Keio Univ.) proposed the new strategy which is the Clone by Clone (CBC) strategy using BAC clone combined with WGS strategy and is based on the experience in which Keio group participated in the Human Genome Project. Combining data generated by two separate CBC and WGS strategies should be of great benefit. To do this, we have developed a novel method named as HTSL (high throughput shotgun library) to rapidly generate a large number of shotgun clones from individual BAC clones. By sequencing these HTSL clones with 2-3X redundancy, we will be able to correlate most of overlapping WGS scaffolds with each BAC clone. Since total sequence data will become ~8X genome coverage and each BAC clone is arranged as a contig, scaffolds will be easily extended and contiguous genomic sequence of substantial size will be generated. Armed with this novel strategy, we have begun sequencing of a single medaka chromosome of linkage group 22 (LG22) under Priority Area #813 supported by MEXT as a first trial case. There are about 20 mapped ESTs in LG22 which could be used as anchor points for this project. The location of BAC contig could be confirmed by chromosomal FISH analysis. The international collaborations in the framework of the MGI will provide the next generation genome project with integrated medaka genome database within a few years. It will provide us with new insights how gene functions evolved in vertebrates, and how they can be experimentally examined in model systems.

The strategy for Medaka LG22 sequencing

As a first step of the Medaka genome sequencing, we try to sequence the medaka LG.22 (Medaka LG.22 project). A BAC library derived from a medaka inbred strain, Hd-rR strain, is used in the analyses. BAC clone contigs are constructed by Heinz Himmelbauer (MPIMG) using information of EST markers mapped on LG22 by Hiroshi Mitani (Univ.Tokyo). Radiation hybrid mapping are done by Makoto Furutani-Seiki and Hisato Kondoh and chromosomal FISH analysis is done by Manfred Schartl and Indrajit Nanda (Univ. Wuerzburg) to confirm the contig data. The BAC end sequencing project is also carried out by Shimizu group (Keio Univ.), and it is performed by searching the contiguity BAC clone using BAC end sequence data. These selected BAC clones are sequenced by the shotgun method which is corresponding to 6X. We deduced that 5 ~ 20 gaps will be remained, but the contig direction and the gap size will be confirmed by PCR at the first step of LG22 project.