A Genetic Linkage Map for the Tiger Pufferfish, Takifugu Rubripes

A Genetic Linkage Map for the Tiger Pufferﬁsh, Takifugu rubripes

Wataru Kai,*,1 Kiyoshi Kikuchi,*,1,2 Masashi Fujita,* Hiroaki Suetake,* Atushi Fujiwara,† Yasutoshi Yoshiura,† Mitsuru Ototake,† Byrappa Venkatesh,‡ Kadoo Miyaki§ and Yuzuru Suzuki* *Fisheries Laboratory, The University of Tokyo, Maisaka, Shizuoka 431-0214, Japan, †National Research Institute of Aquaculture, Fisheries Research Agency, Tamaki, Mie 519-0423, Japan, ‡Institute of Molecular and Cell Biology, Singapore 138673, Republic of Singapore and §Nagasaki Prefectural Institute of Fisheries, Taira, Nagasaki 851-2213, Japan Manuscript received February 15, 2005 Accepted for publication June 13, 2005

ABSTRACT The compact genome of the tiger pufferfish, Takifugu rubripes (fugu), has been sequenced to the ‘‘draft’’ level and annotated to identify all the genes. However, the assembly of the draft genome sequence is highly fragmented due to the lack of a genetic or a physical map. To determine the long-range linkage relationship of the sequences, we have constructed the first genetic linkage map for fugu. The maps for the male and female spanning 697.1 and 1213.5 cM, respectively, were arranged into 22 linkage groups by markers heterozygous in both parents. The resulting map consists of 200 microsatellite loci physically linked to genome sequences spanning 39 Mb in total. Comparisons of the genome maps of fugu, other teleosts, and mammals suggest that syntenic relationship is more conserved in the teleost lineage than in the mammalian lineage. Map comparisons also show a pufferfish lineage-specific rearrangement of the genome resulting in colocalization of two Hox gene clusters in one linkage group. This map provides a foundation for development of a complete physical map, a basis for comparison of long-range linkage of genes with other vertebrates, and a resource for mapping loci responsible for phenotypic differences among Takifugu species.

HE tiger pufferfish, Takifugurubripes (fugu), was pro- evolution of vertebrate chromosomes. Thus, it is recog- Tposed as a genomic model because of its compact nized that a genetic linkage map is essential for com- genome size, which is about eight times smaller than that pleting the genome sequence of fugu and using it in of the human genome (Brenner et al. 1993). Since fishes comparative genomic studies. have a body plan and physiological systems similar to In addition to fugu, genomes of two other teleosts, mammals, the compact genome of fugu can help to dis- the zebrafish (Danio rerio) and medaka (Oryzias latipes), cover genes and gene regulatory regions in the human are being sequenced. These two species are excellent genome and serve asareferencetounderstandtheevolu- genetic models. Fugu and medaka are grouped together tion of vertebrate genomes and karyotype (Grutzner under the common superorder Acanthopterygii and are et al. 1999; Hedges and Kumar 2002). Three years ago, phylogenetically closer to each other than to zebrafish, a ‘‘draft’’ sequence of fugu was generated purely by a which are classified under the more basal superorder whole-genome shotgun strategy (Aparicio et al. 2002). Ostariophysi (Nelson 1994). Molecular clock analyses The draft sequence comprises of 12,381 scaffolds .2kb of the mitochondrial sequences have suggested that the and efforts are currently underway to obtain the com- common ancestors of the pufferfish and zebrafish di- plete sequence of the genome. Since fugu has 22 pairs of verged 280 million years ago (Kumazawa et al. 1999). chromosomes (Miyaki et al. 1995), the ultimate objec- Although there are no estimates for the divergence time tive is to merge the draft sequence scaffolds into 22 of the medaka and pufferfish lineages, since the fossil ‘‘superscaffolds’’corresponding to the 22 chromosomes. evidence shows that fishes of the order Tetraodonti- However, the linkage relationship of the scaffolds is formes have been around for 95 million years (Tyler currently unknown due to the lack of a genetic linkage and Sorbini 1996), it is assumed that the two lineages map or a genome-wide physical framework for this fish. diverged .95 million years ago. This has restricted the use of the fugu genome sequence Excellent gene maps of zebrafish and medaka have for global comparison with other vertebrate genomes to been generated, using 1500 and 800 gene or EST address questions related to genome architecture and sequences, respectively (Woods et al. 2000; Naruse et al. 2004). Recently, the draft genome sequence of another pufferfish, the spotted green pufferfish, Tetraodon nigro- 1 These authors contributed equally to this work. viridis, has been generated ( Jaillon et al. 2004). A partial 2Corresponding author: Fisheries Laboratory, The University of Tokyo, 2971-4 Maisaka, Shizuoka 431-0214, Japan. physical map of the Tetraodon genome has been con- E-mail: [email protected] structed by anchoring 64.4% of the genome assembly to

Genetics 171: 227–238 (September 2005) 228 W. Kai et al. its 21 chromosomes ( Jaillon et al. 2004). The Tetraodon population was obtained by in vitro crossing (Fujita 1967; lineage has been estimated to have diverged from the Matsuyama et al. 1997). A total of 64 full-sib progeny from the fugu lineage 18–30 million years ago (Crnogorac- single cross were used for developing a linkage map. urcevic DNA extraction: Muscle tissue (50–100 mg) or whole bodies J et al. 1997). of juveniles (100 mg each) were preserved in 400 ml of TNES- Comparisons of gene maps of zebrafish, medaka, and urea buffer (10 mm Tris-HCl, pH 7.5, 125 mm NaCl, 10 mm Tetraodon with humans have helped to infer the num- EDTA, 1% sodium dodecyl sulfate, 8 m urea) at room tem- ber and content of the chromosomes of the last com- perature for months. Genomic DNA was extracted from the oods preserved samples using standard phenol-chloroform tech- mon ancestor of teleosts and mammals (W et al. sahida aillon aruse nique with slight modifications (A et al. 1996). Approx- 2000; J et al. 2004; N et al. 2004). However, imately 3–15 mg of purified DNA was obtained from each there has been no attempt to compare the extent of preserved sample. genome reorganization between the teleost species and Microsatellite marker: The CA repeat is the most common the mammalian species. A genetic linkage map of fugu microsatellite interspersed in the genomes of vertebrates, paricio would be useful for such comparative studies among including fugu (A et al. 2002). We first identified 160 CA-repeat loci by querying (CA)50 sequence against the fugu teleost lineages and between teleost and mammalian draft sequence data on the website at the Joint Genome In- lineages and would help to understand the process of stitute (http://www.jgi.gov/fugu/index.html). An additional teleost genome evolution. 40 loci were then chosen by scanning the fugu scaffolds In addition to comparative genomics, genetic linkage starting from the largest scaffold (no. 1). To increase the maps are invaluable in forward genetic analysis for the map density and compare the linkage relationships of orthologs among fugu, medaka, and zebrafish, we searched the CA- identification of gene loci responsible for genetic traits. repeat loci physically linked to fugu orthologs of medaka and Pufferfishes belonging to the genus Takifugu are mainly zebrafish genes that have been mapped to their respective distributed in coastal regions of East Asia and include maps (see Sequence comparison below) and identified 152 loci. .20 species (Matsuura 1990). Interestingly, hybrids The analysis was carried out on the August 2002 freeze data set produced by artificial fertilization of eight Takifugu for the fugu genome. Primers flanking the CA repeats were designed and used for PCR to find heterozygous markers in interspecies crosses, including fugu and Takifugu nipho- ujita iyaki one or both parents. Construction of initial male and female bles, were found viable (F 1967; M 1992). genetic maps of fugu using 192 microsatellite markers and Fugu and T. niphobles show marked differences in body their consolidation identified 23 linkage groups. Comparison size, body color pattern, body shape, the number of of this map with gene maps of medaka and Tetraodon meristic skeletons, temperature preference, parasite indicated that two of the fugu linkage groups might be merged resistance, and behavior (Uno 1955; Ogawa 1991). into one linkage group (LG2). We therefore increased the density of microsatellite markers on LG2 to obtain adequate Given the the ability to generate fertile crosses between markers for merging the two linkage groups. Details of primer Takifugu species, the availability of the draft genome sequences are shown in supplementary Table S1 at http:// sequence of fugu provides an unprecedented opportu- www.genetics.org/supplemental/ as well as on our website at nity to understand the genetic basis of the evolution of http://park.itc.u-tokyo.ac.jp/suijitsu/. Primer sequences have natural species of these vertebrates. Availability of a also been deposited in DDBJ/EMBL/GenBank with accession nos. AB213693–AB214112. genetic map of fugu would greatly facilitate such studies. Genotyping: PCR was performed in 20-ml reaction volume Here we report a genetic linkage map for fugu, the of 100 mm Tris-HCl (pH 8.3) containing 0.5 m KCl, 2.5 mm first map for a pufferfish. The map consists of genetic dNTPs, 0.5 mm of each primer, Taq DNA polymerase (Takara, markers physically linked to the assembled genome se- Tokyo, Japan), and 5 ng of genomic DNA. The following PCR quences, allowing us to establish a long-range relation- protocol was used: initial denaturation at 94° for 1 min, 35 cycles of 94° for 30 sec, and 59° for 2 min, followed by a final ship of the scaffold sequences and to compare gene extension period at 59° for 3 min. The PCR products were heat maps between fugu and other vertebrates. Comparison denatured with formamide loading buffer and fractionated on of parental maps showed sex-specific differences in re- a denaturing 6% acrylamide gel (Long-Ranger, ABI, Columbia, combination ratio. Comparison of gene maps revealed a MD) with 8 m urea. The gels were stained with SYBR gold and tendency for highly conserved synteny among teleost visualized with a fluorescent imaging system (FLA3000, FUJIFILM). genomes as well as a lineage-specific rearrangement in Genotypic classification: The progeny data are subdivided the pufferfish lineages. This genetic map will serve as a into two independent data sets that contain the meiotic se- foundation for obtaining the complete sequence of the gregation data from each parent. Ancestry-unknown markers fugu genome, a basis for comparison of the genome map in the three-generation pedigree were analyzed within a phase- with those of other vertebrates, and an important tool for known model of inbred pedigree and converted to ancestry- known markers following the method of Sewell et al. (1999). the future genome-wide analysis of phenotypic differ- Map construction: The map was constructed using MAP- ences observed within and between Takifugu species. MAKER (Lander et al. 1987). Linkage groups were determined using a two-point analysis. Local order was established by multipoint analysis. Marker orders were modified after MATERIALS AND METHODS visual analysis of the distribution of marker genotypes in the reference mapping panel. Mapping population: Wild female and male fugu were Sequence comparison: Fugu orthologs of medaka, zebra- caught in the Sea of Japan at Toyama Bay and in the Pacific fish, and human genes were identified by BLAST searching Ocean off Shizuoka prefecture, respectively. A mapping these sequences against the fugu draft sequence data with a Genetic Map for the Tiger Pufferfish 229 cutoff E-value of e10. For the search, we used 819 gene and EST female parents. In total, 212 markers (56.4%) were sequences for medaka and their human orthologs and 792 informative for segregation analyses in the mapping gene and EST sequences for zebrafish and their human ortho- population. An additional 60 progeny were scored for logs reported by Naruse et al. (2004) and Woods et al. (2000). In cases where the searches hit two or more scaffolds with a less- these markers to construct the linkage map. than-threefold difference in the E-value, we did not assign any Linkage map: The male map consists of 24 linkage orthology. In particular, to identify fugu orthologs to medaka groups with 169 microsatellite loci that are physically and zebrafish genes, we chose 239 gene and EST sequences linked to multi-gene-sized DNA sequences assembled in whose orthologs have been predicted between medaka and the fugu draft genome database (Table 1). Among the zebrafish (Naruse et al. 2000) and BLAST searched their sequences (a maximum E-value of e10) against the fugu data- informative markers in male meiosis, 91.8% of the mark- base. Fugu ortholog was predicted if both medaka and zebra- ers showed detectable linkage to another marker. The fish sequences showed the highest similarity to the same fugu map spans 697.1 cM and the average spacing between gene. In cases where the searches hit two or more scaffolds, two markers is 4.1 cM. The sizes of linkage groups range fugu orthologs were identified on the basis of phylogenetic from 0 to 75 cM (mean 29 cM). The number of markers analysis. For the phylogenetic analysis, fugu protein sequences identified in the BLAST search were aligned with medaka, per linkage group varied from 3 to 17, with an average of zebrafish, and human gene family members and phylogenetic 7 markers per group. analysis was carried out using the program Clustal-X (Thompson The female map consists of 29 linkage groups with et al. 1997) and the neighbor-joining method (Galtier et al. 171 microsatellite markers. Among the informative 1996). Medaka and zebrafish mapping information and orthol- markers in female meiosis, 90.9% of the markers tested ogous relationships among medaka, zebrafish, and human were obtained from Naruse et al. (2004) and Woods et al. (2000), who showed detectable linkage to another marker. The maps compiled the latest peer-reviewed medaka and zebrafish maps span 1213.5 cM and the average distance between two available. Medaka and zebrafish gene and EST sequences markers is 7.1 cM. The sizes of linkage groups range were obtained from DDBJ (http://getentry.ddbj.nig.ac.jp) or from 4.7 to 117.4 cM (mean 41.8 cM). The number of the Zebrafish Information Network (http://www.zfin.org) using markers per linkage group varied from 2 to 13, with an accession numbers listed in Naruse et al. (2004) or Woods et al. (2000). Map position of human and mouse orthologs were average of 5.8 markers per group. determined using the NCBI human genome resources (http:// By using heterozygous markers in both parents, we www.ncbi.nlm.nih.gov/genome/guide/human/). Fugu genes identified homologous pairs of linkage groups of the on the same scaffolds are represented as a single locus in our male and female parents and arranged these maps in 22 analysis to prevent an overestimation of the extent of conserved linkage groups, designated LG1–LG22 (Figure 1 and synteny. To find putative orthologous segments of the fugu scaffolds in the‘‘partial physical map’’ofTetraodon, at leastthree Table 1). The resulting map consists of 200 markers DNA sequences of 10 kb were chosen from the 59-end, middle, physically linked to the assembled genome sequences and 39-end regions of the fugu scaffolds and searched for ranging from 1,145,486 to 1447 bp. The total length of BLAT (Kent 2002) sequence similarity (a minimum score of genomic sequences mapped is 39.2 Mb and contains 2000) against the Tetraodon draft sequence data (http://www. 4452 gene loci (http://www.jgi.gov/fugu/index.html). genoscope.cns.fr/). When the fugu scaffold size was ,30 kb, the entire scaffold sequence was used for the search. Seven pairs of markers that are linked in the male map Nomenclature: All loci identified are simple sequence remain unlinked in the female map whereas two pairs of length polymorphisms and are named in accordance with markers in the female map remain unlinked in the male laboratory rules, e.g., f35, where ‘‘f’’ denotes both fugu and the map (Figure 1 and Table 1). This could be explained by fisheries laboratory at the University of Tokyo, and the number differences in recombination ratio between sexes as indicates the assay number. Ultimately, once the map is completed and physically anchored to each chromosome, seen in most of the linkage groups. For example, while each marker should receive a locus name following the system f330 and f39 are linked in the male map of LG1, they are used for the dog, mouse, pig, and rat maps. not linked in the female map. In the adjacent intervals flanked by f330 and f167, female recombination rates are much higher than those observed in males. We expect that the pairs of unlinked markers will be linked as RESULTS described in the map for the other sex when marker Markers: To obtain informative markers for linkage density increases. analysis in the progeny from the cross between non- Differences in recombination rate between male and inbred parents, we examined the segregation types of female: To estimate the relative rates of recombination the CA-repeat locus in both parents and four progeny. between parents, we chose 104 pairs of adjacent markers PCR reactions successfully amplified 376 loci in a total of heterozygous for both parents and compared sex- 389 primer pairs that were designed on the basis of the specific recombination rates. Figure 2 shows a compar- fugu draft genome sequence. The father and mother ison of male and female recombination ratio between were heterozygous for 184 (48.9%) and 188 (50%) of the common intervals flanked by the paired markers these markers, respectively. Among them, 160 markers from all the linkage groups. The number of intervals were heterozygous in both parents with ab 3 cd or ab 3 that shows a higher recombination ratio in females is ac type of segregation and allowed the identification of larger than that in males. On the other hand, the num- homologous pairs of linkage groups of the male and ber of intervals that shows expansion in the male map 230 W. Kai et al.

TABLE 1 Number of markers, genetic length, and total scaffold size for each linkage group

Male Female No. of Total scaffold No. of gene No. of Length No. of Length LG markers size (bp) models LG markers (cM) LG markers (cM) LG1 17 3,284,077 394 LG1 16 64.8 LG1-F1 3 12.6 LG1-F2 11 75.2 LG1-F3 2 21.2 LG2 17 3,003,930 481 LG2 16 75 LG2 13 117.4 LG3 12 2,521,477 231 LG3 10 31.4 LG3 11 96 LG4 12 2,344,834 233 LG4 11 38.8 LG4 11 86.1 LG5 14 1,844,110 279 LG5-M1 4 3.2 LG5-F1 4 17.4 LG5-M2 6 11.1 LG5-F2 8 65.8 LG6 7 1,543,293 190 LG6 7 56.5 LG6 7 76.2 LG7 10 1,743,445 197 LG7-M1 2 11.7 LG7 10 69 LG7-M2 6 22.5 LG8 9 1,005,816 110 LG8 7 34.6 LG8 7 68.9 LG9 8 1,526,000 146 LG9 7 21.2 LG9 8 63.4 LG10 12 2,414,664 299 LG10 9 49.8 LG10-F1 7 38.5 LG10-F2 4 19.3 LG11 8 1,695,911 135 LG11 7 35.3 LG11-F1 2 23.7 LG11-F2 5 24 LG12 12 3,690,823 451 LG12 10 20.5 LG12 12 46.4 LG13 5 1,403,550 65 LG13 5 11 LG13 4 43.1 LG14 7 582,240 40 LG14 5 12.6 LG14 5 42.6 LG15 8 2,018,465 186 LG15 5 6.3 LG15-F1 4 31.9 LG15-F2 3 4.7 LG16 5 645,346 76 LG16 4 42 LG16 3 34.9 LG17 8 906,885 139 LG17 7 43.3 LG17-F1 4 20.9 LG17-F2 3 7.9 LG18 3 992,949 92 LG18 2 0 LG18 3 27 LG19 5 1,286,233 136 LG19 5 36.7 LG19 3 23.2 LG20 6 1,409,226 111 LG20 6 22.1 LG20 5 22.5 LG21 6 2,031,407 254 LG21 5 22.6 LG21 3 22.4 LG22 9 1,302,580 207 LG22 7 24.1 LG22 5 11.3

Total 200 39,197,261 4452 169 697.1 171 1213.5

relative to the female map is small but not negligible. cording to linkage groups of fugu and chromosomes of Summing up the length of the common interval for each Tetraodon. The display shows that each of the fugu linkage group on both the male and female maps gave a linkage groups shares putative orthologous segments total length of 507.1 and 1100.8 cM, respectively. These with one Tetraodon chromosome except for LG4, LG8, results suggest that overall recombination is suppressed LG10, and LG14. In the fugu linkage group LG10, six in male meiosis compared to female meiosis. scaffolds (scaffold 4, 146, 429, 446, 1117, and 2289) were Comparison with T. nigroviridis: To compare the assigned to putative orthologous segments on Tetrao- extent of conserved synteny between the two pufferﬁsh don chromosome 6, whereas two other scaffolds, scaf- genomes, we assigned 200 of the fugu scaffolds to puta- fold 1628 and scaffold 572, showed similarity over large tive orthologous segments of the Tetraodon genome. regions to Tetraodon chromosomes 18 and 17, respec- Among them, 152 segments have been anchored to tively. (Figure 3 and supplementary Table S2 at http:// Tetraodon chromosomes on its ‘‘partial physical map,’’ www.genetics.org/supplemental/). This suggests that whereas 48 segments have not been anchored. This is there were interchromosomal translocations of seg- consistent with the limited anchoring of the Tetraodon ments of the ancestral sequences of scaffolds 1628 and genome sequences to chromosomes (64.4%). In Figure 3, 572 in either the fugu or the Tetraodon lineage after the putative orthologous segments have been arrayed ac- divergence of the two species. Similar rearrangements

< Figure 1.—Male and female genetic linkage maps of fugu. Allelic bridges are indicated by lines connecting male (left) and female (right) linkage maps. Genetic distances between adjacent markers are shown (Kosambi mapping function). Genetic Map for the Tiger Pufferﬁsh 231 232 W. Kai et al.

Figure 1.—Continued. were also evident in the fugu linkage groups LG4, LG8, medaka genes chosen from a medaka linkage map and LG14. Thus, of the 152 orthologous segments, only (Naruse et al. 2004) and found that 108 of them were 6 segments indicate possible interchromosomal rear- present in the fugu map. In the array of orthologs as- rangement events after the divergence of the two species signed on the basis of the linkage groups for each spe- of pufferfishes. This shows that most regions of the cies, we were able to identify the cluster of orthologous chromosomes have been conserved during the 18–30 gene pairs that are located on conserved syntenic seg- million years of divergent evolution of the two lineages. ments in fugu and medaka (Figure 4A). Among 108 Figure 3 also indicates that each of Tetraodon chromo- putative orthologs, 94 orthologs fell in 22 conserved somes 1, 3, 17, 18, and 20 shows conserved synteny with synteny segments containing two or more genes be- two to three linkage groups of fugu. The disruption of tween the two species. Furthermore, 18 pairs of linkage the one-to-one relationship of the Tetraodon chromo- groups shared conserved syntenic segments containing somes to fugu linkage groups may be due to interchro- at least three orthologous gene pairs. These correlations mosomal translocations that include recent fusion of imply that these pairs of linkage groups are derived from chromosomes in the Tetraodon lineage or fission in the a common ancestral chromosome. Of the 101 genes fugu lineage after the divergence of the two pufferfish from these 18 fugu linkage groups, 86 genes (85.1%) are species. This could be related to the presence of an assigned to orthologous linkage groups in medaka. additional pair of chromosomes in fugu (n ¼ 22) com- These results suggest that the syntenic relationship of pared to Tetraodon (n ¼ 21). a significant fraction of the genome has been remark- Conservation of synteny with other teleosts: To com- ably conserved in the fugu and medaka lineages. Using a pare the extent of conserved synteny between the fugu similar strategy, we compared syntenic relationships and medaka genomes, we identified fugu orthologs to between the fugu and zebrafish genomes by using 88 Genetic Map for the Tiger Pufferfish 233

Figure 2.—Differences in recombination ratio between Figure 3.—Summary of the number of orthologous ge- male and female. The common intervals flanked by adjacent nome regions in fugu and T. nigroviridis. The linkage groups markers from the 22 linkage groups are compared. of fugu are arrayed as columns and the chromosomes of Tet- raodon as rows. Genome sequences that have not been mapped in Tetraodon are displayed in the last row (UN, pairs of orthologous genes (Figure 4C). Among them, unknown). Numbers in boxes indicate the number of orthol- 61 orthologs fell in 22 conserved syntenic segments, ogous genome sequences. Details of mapped sequences are each containing two or more orthologous genes. In- listed in supplementary Table S2 at http://www.genetics. deed, 9 of the conserved syntenic segments contain org/supplemental/. three or more orthologous gene pairs between the linkage groups of the two fishes (Figure 4C). maps (Amores et al. 1998). We mapped the Hox clusters To compare the degree of conserved synteny between of fugu to our genetic linkage map and compared the the teleost and mammalian genomes, we defined location of Hox clusters in the gene maps of fugu, conserved syntenic segments using 105 quadruplets of Tetraodon, medaka, and zebrafish. To determine the orthologous genes from our data set of fugu, medaka, position of Hox clusters in the fugu map, we first iden- human, and mouse (Figure 4, A and B). This analysis tified nine scaffolds that contained members of six Hox revealed that whereas only 14 orthologous gene pairs clusters (scaffold 47 for HoxAa, scaffold 330 and 5310 for did not fall in conserved syntenic segments between HoxAb, scaffold 1439 and 706 for HoxBa, scaffold 1245 fugu and medaka (Figure 4A), 28 orthologous gene and 2182 for HoxBb, scaffold 93 for HoxCa, and scaffold pairs between the human and mouse did not fall in con- 214 for HoxDa; also see supplementary Table S2 at served syntenic segments (Figure 4B). Since the evolu- http://www.genetics.org/supplemental/) by BLAST tionary divergence period between fugu and medaka is search and obtained genetic markers from these se- similar or even longer than that of human and mouse, quences. A polymorphic marker for the HoxDb cluster this result suggests that syntenic relationships are more could not be obtained because we could identify only conserved in the teleost lineage than in the mammalian one scaffold that contained a member of the HoxDb lineage. We did similar analysis using 86 quadruplets of cluster (scaffold 19,911 for HoxDb9) in the fugu database orthologous genes for fugu, zebrafish, human, and and this scaffold contained no CA-repeat locus. The six mouse and defined syntenic segments in the teleost markers physically linked to the six Hox clusters (HoxAa, and mammalian genomes. In this analysis, 26 and 32 HoxAb, HoxBa, HoxBb, HoxCa, HoxDa) were then map- orthologous genes for fugu and zebrafish, and the two ped to the linkage groups of fugu (Table 2). Unexpect- mammals, respectively, did not fall in conserved syntenic edly, the six markers mapped to only five linkage groups, segments (Figure 4D). Such a similar degree of con- with two Hox clusters, HoxBb and HoxDa, being located in served synteny between the fugu and the zebrafish and the same linkage group, LG1. To determine whether the the human and mouse, despite the longer divergence genetic events that led to the two Hox clusters being period between the two fishes, further supports a lower located to the same linkage group in fugu involved any frequency of interchromosomal rearrangements in the of the flanking genomic regions, we compared the teleost lineage than in the mammalian lineage. syntenic relationship of the Hox clusters and their known Hox clusters: In vertebrates, Hox genes are present in genetically linked genes such as CYCS, POU3F3, PSMC5, tight clusters of up to 14 genes and these clusters have CRP, HBOA, ACO2, and TEF among fugu, medaka, and been often used as landmarks for comparison of gene zebrafish (Figure 5A). The comparison revealed that 234 W. Kai et al.

Figure 4.—Conservation of synteny among fugu, medaka, zebrafish, human, and mouse. The putative orthologous genes from two species are arrayed according to linkage group or chromosome for each species. Numbers in boxes indicate the number of orthologous gene pairs. The boxes containing more than three orthologous gene pairs are shaded. (A) Fugu-medaka comparison; (B) human-mouse comparison based on genes orthologous to data set in A; (C) fugu-zebrafish comparison; (D) human-mouse comparison based on genes orthologous to data set in C. (*) Three of the orthologs in comparisons between A and B and two of the orthologs in comparisons between C and D, respectively, have been excluded as we failed to identify their mouse orthologs. Details of the orthologous genes are listed in supplementary Table S2 at http://www.genetics.org/supplemental/. whereas the syntenic segments of both HoxBb and HoxDa sequences containing Hox genes and found that both together with their linked non-Hox genes were located in Tetraodon HoxBb and HoxDa clusters are localized on the same linkage group in fugu, the two syntenic clusters chromosome 2 (Table 2). This suggests that the trans- were assigned to different linkage groups in both location event occurred before the separation of the medaka and zebrafish, suggesting that the two Hox fugu and Tetraodon lineages. clusters and their flanking regions were translocated into one linkage group in an ancestor of fugu after it DISCUSSION diverged from the medaka lineage. We also identified the location of Hox clusters in the ‘‘partial physical map’’ Genetic map and genetics: We have constructed the of Tetraodon by BLAT searching with the fugu scaffold male and female genetic maps for fugu and arranged Genetic Map for the Tiger Pufferfish 235

TABLE 2 Linkage groups containing Hox clusters in zebraﬁsh, medaka, T. nigroviridis, and fugu

Hox cluster Zebrafish Medaka Tetraodon Fugu HoxAa LG19 LG11 Chromosome 21 LG12 HoxAb LG16 LG16 Chromosome 8 LG7 HoxBa LG3 LG8 Unmappeda LG5 HoxBb LG12 LG19 Chromosome 2 LG1 HoxCa LG23 LG7 Chromosome 9 LG3 HoxCb LG11 HoxDa LG9 LG21 Chromosome 2 LG1 HoxDb LG15 Chromosome 17b Unmapped Data on zebrafish and medaka are from Wood et al. (2000) and Naruse et al. (2004), respectively. a Tetraodon HoxBa genes were identified by BLAT search using fugu HoxBa sequence as query. Their chromosomal locations were not assigned in the Tetraodon map. b HoxDb genes of fugu and Spheroides nephalus were characterized by Amores et al. (2004). Their putative orthologs in Tetrao- don have been labeled as HoxCb in Jaillon et al. (2004). them in 22 linkage groups in which 94.3% of the markers are linked to at least one other marker. While much effort has been concentrated on using the fugu as a ‘‘genome model,’’ less attention has been paid to the potential of this fish as a ‘‘genetic model.’’ One of the purposes of this study is to construct a tool for genome- Figure 5.—(A) Comparative linkage map of HoxBb and wide genetic analysis of fugu. In our male and female HoxDa clusters and their flanking regions in fugu, medaka, and zebrafish. Genes are named according to the nomencla- maps, .94% of the markers are linked to other markers, ture for the orthologous genes of human or zebrafish. The suggesting a high probability that the markers may be zebrafish and medaka genes for which there is no mapping linked to genetic traits. Such a map would be useful for information are not shown in the linkage map. Orthologous the initial low-resolution genetic mapping of traits. genes are linked by dashed lines. (B) Genomic organization Although we cannot expect that all markers that we of HoxBb and HoxDa clusters in pufferfishes (fugu, Tetraodon, and Spheroides) (p) and zebrafish (z). Hox paralog group is used will be informative in other crosses—e.g., interspe- shown along the top. cific crossing of fugu and closely related species—the high level of polymorphic information content of the CA repeats in wild individuals will allow us rapid reduced chromosomal recombination frequency in development of additional markers that are in close males, while cattle do not show such sex-specific differ- proximity to the noninformative markers by searching ences (Dib et al. 1996; Dietrich et al. 1996; Marklund the genome sequence database. et al. 1996; Neff et al. 1999). Among teleost fishes, an The large collection of microsatellite markers will be overall lower recombination ratio has been identified in also useful for aquaculture purposes, because fugu is males of trout and zebrafish (Sakamoto et al. 2000; becoming one of the most economically important Singer et al. 2002). The recombination around the fish for aquaculture (Statistics and Information centromere region is preferentially lower in male Department 2001). These markers will be essential meiosis than in female meiosis in these fishes. In for the identification of loci responsible for commer- addition, detailed studies in zebrafish have revealed cially desirable traits in different strains of this fish. that the recombination in the region near the telomere Molecular identification of such loci combined with is preferentially lower in female meiosis than in male the technology for marker-assisted selective breeding meiosis (Singer et al. 2002). Our analysis of the fugu would be useful for establishing commercially better genetic map shows that males have an overall reduction strains of fugu. The collection of markers is also critical in recombination ratio relative to females. While a large for characterizing genetic background of wild and cul- number of the common intervals between the male map tured fugu to maintain heterozygosity of stock and and female map show expansion in the female map, some for tracking parentage. intervals also show expansion in the male map. This Sex-specific difference in recombination ratio: tendency is similar to that observed in trout and zebra- Among mammals, human, dog, pig, and mouse show fish (Sakamoto et al. 2000; Singer et al. 2002). Since 236 W. Kai et al. the male-specific reduction in the recombination ratio ments. Accumulation of transposable elements might seems to exist in diverse species of teleosts such as trout, have favored a higher frequency of interchromosomal zebrafish, and fugu, it is likely that this feature is shared rearrangements in the zebrafish (Grutzner et al. 1999). by all teleosts. Hox clusters: In vertebrates Hox genes occur in tight This finding has practical significance in facilitating clusters of up to 14 paralogous genes. They play an efficient experimental design in the genome-wide important role in anterior/posterior patterning in de- linkage analysis for species differences among Takifugu velopment, including skeletal complexity of animals. species (Singer et al. 2002). A decreased rate of recom- The expression domains of the genes in a cluster are bination in males is an advantage for mapping genetic colinear with the position of the genes in the cluster. In traits in initial low-resolution analyses, especially when all tetrapods studied so far, four clusters of Hox genes analyzing quantitative trait loci (Glazier et al. 2002). (HoxA, HoxB, HoxC, and HoxD) have been identified. For example, if the phenotype shows complete or partial The clusters are located on different chromosomes cott adjali ohammedi dominance in F1 hybrids between two Takifugu species, (S 1992; L -M et al. 2001). These it is adequate to use a mapping population produced four clusters are presumed to have arisen by two rounds from a cross between male hybrid and a female wild of whole-genome or segmental duplication during the species for primary mapping. On the other hand, it evolution of early vertebrates (Larhammar et al. 2002). would be necessary to use a higher frequency of re- The distribution of four Hox clusters on four chromo- combination in females for fine mapping of these loci. somes is often sighted as evidence for whole-genome (or Comparison of genomes: Comparisons of the genetic whole-chromosome) duplications. Although extensive map of fugu with that of other teleosts revealed that, as interchromosomal rearrangements are suggested in the expected, the synteny between the two pufferfishes, evolution of amniote chromosomes (Bourque et al. fugu and Tetraodon, is highly conserved. However, we 2004), all amniotes so far examined have retained the also found highly conserved synteny between fugu and presumptive ancient organization of Hox clusters and medaka, which shared a common ancestor .95 million chromosomes; that is, a single Hox cluster is located years ago. Our comparisons of the teleost maps and on each chromosome. In contrast to tetrapods, teleosts human and mouse maps showed that syntenic relation- such as zebrafish, medaka, and pufferfishes (fugu, Spher- ships are more conserved in the teleost fish lineage than oides, and Tetraodon) contain up to seven Hox clusters in the mammalian lineage. For example, even though (Amores et al. 1998, 2004; Naruse et al. 2000). The the divergence period between fugu and zebrafish is additional Hox clusters in teleosts have been proposed to about threefold longer than that between the human be the result of a whole-genome duplication (Amores and mouse, a similar degree of conserved synteny was et al. 1998) that occurred early during the evolution of the found between the two fish lineages and between the ray-finned fish (Christoffels et al. 2004). Presumably, two mammalian lineages. By comparing the complete one of the duplicate Hox clusters was lost following the physical map of human and the ‘‘partial physical map’’ whole-genome duplication. In the medaka and zebrafish, of Tetraodon, Jaillon et al. (2004) reconstructed the in which Hox clusters have been mapped to the genetic karyotype of the last common ancestor of the teleost fish linkage groups, the seven clusters are located in different and mammals. Their model suggests a lower frequency linkage groups. The genomic position of the Hox clusters of major interchromosomal exchanges in the teleost in the pufferfish was not known until now. In our study, lineage than in the human lineage. Naruse et al. (2004) we unexpectedly found that two Hox clusters, HoxBb and also inferred the ancestral karyotype using the medaka, HoxDa, along with their flanking genes are located in the zebrafish, and human maps. Their result suggested that same linkage group in fugu as well as in Tetraodon karyotype evolution in teleosts occurred mainly by (Figure 5A, Table 2). Since these two Hox clusters are inversion and not by fusion or fission of chromosomes located in different linkage groups in the zebrafish and that is frequent in mammalian lineages. Our results medaka, it seems likely that the two clusters were colo- independently demonstrate the lower frequency of calized to the same chromosome through interchromo- interchromosomal exchanges in the teleost lineage and somal rearrangements, most likely through fusion of two provide further support to the models proposed by chromosomes. Each of the pufferfish HoxBb and HoxDa Jaillon et al. (2004) and Naruse et al. (2004). We also clusters lacks one of the genes present in the zebrafish showed that the extent of conserved synteny between HoxB8b and HoxD13a (see Figure 5B). Although a de- fugu and zebrafish is less than that between fugu and tailed gene content of the medaka Hox clusters has not medaka. This result is consistent with the phylogenetic been analyzed, the overall organization of Hox clusters in positions of the three fish species, i.e., zebrafish being medaka has been reported to be more similar to that in basal to both fugu and medaka. However, this can also pufferfishes than in zebrafish (Amores et al. 2004) (see have other explanations. The larger genome size of Table 2). For example, while two HoxD clusters, HoxDa zebrafish compared to fugu and medaka (Venkatesh and HoxDb, are present inmedakaand pufferfishes, zebra- et al. 2000) implies that the genome of this teleost fish have a single HoxDa cluster but two HoxC clusters contains a significant proportion of transposable ele- (HoxCa and HoxCb). Thus, it is assumed that the contents Genetic Map for the Tiger Pufferfish 237 of the HoxBb and HoxDa clusters in medaka are more Amores, A., T. 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