Genes Genet. Syst. (2007) 82, p. 135–144 The genome size evolution of medaka (Oryzias latipes) and fugu (Takifugu rubripes) Shuichiro Imai1,2, Takashi Sasaki2, Atsushi Shimizu2, Shuichi Asakawa2, Hiroshi Hori1 and Nobuyoshi Shimizu2* 1Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan 2Department of Molecular Biology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan (Received 10 January 2007, accepted 29 January 2007) Evolution of the genome size in eukaryotes is often affected by changes in the noncoding sequences, for which insertions and deletions (indels) of small nucle- otide sequences and amplification of repetitive elements are considered responsi- ble. In this study, we compared the genomic DNA sequences of two kinds of fish, medaka (Oryzias latipes) and fugu (Takifugu rubripes), which show two-fold diff- erence in the genome size (800 Mb vs. 400 Mb). We selected a contiguous DNA sequence of 790 kb from the medaka chromosome LG22 (linkage group 22), and made a precise comparison with the sequence (387 kb) of the corresponding region of Takifugu. The sequence of 178 kb in total was aligned common between two fishes, and the remaining sequences (612 kb for medaka and 209 kb for fugu) were found abundant in various repetitive elements including many types of unclassi- fied low copy repeats, all of which accounted for more than a half (54%) of the genome size difference. Furthermore, we identified a significant difference in the length ratio of the unaligned sequences that locate between the aligned sequences (USBAS), particularly after eliminating known repetitive elements. These USBAS with no repetitive elements (USBAS-nr) located within the intron and intergenic region. These results strongly indicated that amplification of repeti- tive elements and compilation of indels are major driving forces to facilitate changes in the genome size. Key words: genome size evolution, indels, medaka, repetitive element, Takifugu remained unsolved. INTRODUCTION In contrast to biological traits, the genomic DNA sequ- The genome size of the organism is highly diverse ences of various species can be directly compared to find- among species (Gregory, 2005), and the genome size out what types of nucleotide sequences have increased or diversity is found even between very closely related spe- decreased during evolutionary time span. In the eukary- cies (Wendel and Cronn, 2003; Hickey and Clements, ote genome, the amount of coding sequences is much 2005; Boulesteix et al., 2006). This phenomenon is clas- smaller than the non-coding sequences, and hence the lat- sically referred to as “C-value paradox” (Thomas, 1971), ter should have exerted greater influence on the genome which represents the discrepancy between the amount of size change. In general, changes in the non-coding sequ- genome DNA and developmental complexity of the organ- ences occur mainly by insertions and deletions (indels) of ism. Previous studies attempted to clarify this paradox small nucleotide sequences or amplification of repetitive by focusing on the adaptive significance of the relation- elements. In fact, the small indels were considered as a ship between genome size and biological traits such as major driving force of genome size evolution (Petrov et al., cell size, metabolic rate, and longevity (Chipman et al., 1996; Petrov, 2002a) and the rate of DNA loss through 2001; Griffith et al., 2003; Cavalier-Smith, 2005; Hughes accumulation of small deletions was emphasized as a and Piontkivska, 2005). However, little or no causal major driving force for the genome to shrink (Petrov, links were found with biological traits and the paradox 2001; Petrov, 2002b). However, those studies utilized somewhat limited sequences such as transposons and Edited by Yoko Satta pseudogenes to investigate indel bias, and hence the * Corresponding author. E-mail: [email protected] information was insufficient to clarify the process how 136 S. IMAI et al. genomic architecture changed along with genome size al., 2002) to evaluate the basis for genome size difference evolution. and the genomic architecture. Recently, amplification of the repetitive elements has received more attention as another driving force (Kidwell, MATETIALS AND METHODS 2002; Neafsey and Palumbi, 2003; Boulesteix et al., 2006) because repetitive elements occupy a significant portion Sequencing Strategy and Assembly For the medaka of the eukaryote genome, as evidenced for human (Inter- LG22 DNA sequence, we filled sequence gaps in BAC national Human Genome Sequencing Consortium, 2001) clones and determined a contiguous sequence for precise and other organisms (SanMiguel et al., 1996). As an comparison of genomic DNA sequences. For the present exception, pufferfishes contain minute amounts of repeti- study, we selected a particular 1 Mb sequence consisting tive elements, having the smallest genome (~400 Mb) of five BAC clones (Md0172F16, Md0159H14, Md0170- among vertebrates (Crollius et al., 2000; Aparicio et al., F19, Md0147C05, and Md0200E16) from the Medaka 2002). BAC Library (Matsuda et al., 2001). These clones were However, it is still unknown how genome sizes expand sequenced with a 3730xl DNA Analyzer and a 3100 or shrink by changing the amounts of small indels and Genetic Analyzer (Applied Biosystems) as described pre- repetitive elements. One way to answer this question is viously (Kawasaki et al., 1997). DNA sequence assembly to directly compare DNA sequences among appropriate was performed using the Phred/Phrap/Consed program species. Thanks to the genome sequencing projects, (Ewing and Green, 1998; Ewing et al., 1998; Gordon et al., enormous amounts of genomic DNA sequences are now 1998) and sequence gaps were filled by primer walking. available for various species, especially mammals (Tho- mas et al., 2003; Chapman et al., 2004). However, there Sequence analysis The coding sequence was analyzed are no significant differences in the genome sizes among with BLASTN, (Altschul et al., 1990) against nr database mammalian species (human 3.4 Gb, chimpanzee 3.7 Gb, in NCBI and the medaka EST database (Naruse et al., mouse 3.3 Gb, rat 3.0 Gb, and cow 3.6 Gb; calculated from 2004; The TIGR Gene Index Databases, The Institute the data in Animal Genome Size Database at http:// for Genomic Research, Rockville, MD 20850 http://www. www.genomesize.com (Gregory, 2005)). Interestingly, tigr.org/tdb/tgi; Heinz Himmelbauer unpublished data). the situation is different in fish species. Two puffer- GENSCAN was utilized for gene prediction (Burge and fishes, Takifugu rubripes (Aparicio et al., 2002) and Tet- Karlin, 1997). To determine orthologous genes between raodon nigroviridis (Jaillon et al., 2004) have almost medaka and human, whose genome is annotated most equal size of genome (400 Mb), however medaka has 2- precisely in the sequenced vertebrate species so far, puta- fold bigger genome (800 Mb) and zebrafish has 4-fold big- tive genes predicted by GENSCAN were analyzed by ger genome (1700 Mb), showing the genome size diversity. BLASTP against human genes in the public database. Furthermore, substantial amounts of DNA sequences are Genomic structures of medaka genes identified with available for these fishes and this was considered advan- human orthologous genes were determined by Wise2 tageous for investigating the genome size evolution. For (available at http://www.ebi.ac.uk/Wise2/). Finally, the meaningful comparison, it is essential to select proper exons were determined by the est2genome program (Mott, species that have high degree of homology in the genomic 1997) and exon-intron boundaries of each medaka gene DNA sequences. It may not be feasible to compare geno- were confirmed with the DOTTER program (Sonnhammer mic DNA sequences between fish and mammals because and Durbin, 1995). To mask repetitive elements in the these two lineages show low degree of homology except medaka genome sequence, we developed a Medaka Repeat coding sequences and regulatory elements (Goode et al., Database (ver.1.0 available at http://biol1.bio. nagoya-u. 2003; Thomas et al., 2003). On the contrary, medaka ac.jp:8000/). For this, we utilized fish repetitive elements and pufferfishes exhibit high degree of sequence homol- (T. rubripes, T. nigroviridis, Lepidiolamprologus elon- ogy despite 2-fold genome size difference. Thus, we con- gates, and Danio rerio) from the public database giri sidered medaka and Takifugu as an ideal combination to (http://www.girinst.org/~server/repbase.html) and repeti- evaluate effects of indels and repetitive elements on the tive elements previously found (Naruse et al., 1992, Koga genome size evolution. Ohtsuka et al. (2004) compared et al,. 2002, Matsuo and Nonaka 2004) and newly found the 229 kb medaka sequence with Takifugu, human and in 19 Mb of the medaka genome sequence of LG22. The mouse, however, it was a gene-poor region and analytical 6914 entries of repetitive elements were classified into 6 methods used were not sufficient to analyze amplification categories (“LTR”, “LINE”, “SINE”, “DNA transposon”, of repetitive elements and compilation of indels. “Simple Repeat and Low Complexity”, and “Unclassified”) In this study, we utilized approximately 1 Mb genomic based on their structures or the homology with the known DNA sequence of medaka chromosome LG22 (Sasaki et repetitive elements. Using this database, repetitive ele- al., 2004; Shimizu et al., 2006; Sasaki et al., 2007) and the ments were identified with the RepeatMasker2 program corresponding sequence of Takifugu genome (Aparicio et (Smit, A. F. A. and Green, P. RepeatMasker at http:// Genome size evolution of medaka and Takifugu 137 www.repeatmasker.org). Takifugu. Those gaps were found only in the USBASs of Takifugu and could not be evaluated for precise length. Takifugu genome sequence The Takifugu genome Thus, prior to analyses, we excluded pairs of USBASs sequence corresponding to the medaka 1 Mb-sequence with gaps in Takifugu from both species to allow genomic was basically searched from the database at the Joint sequences to be compared between species as accurately Genome Institute (JGI), Takifugu rubripes ver.