Copyright Ó 2005 by the Genetics Society of America
DOI: 10.1534/genetics.105.042051
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.
T
HE tiger pufferfish, Takifugu rubripes (fugu), was proposed as a genomic model because of its compact
genome size, which is about eight times smaller than that
of the human genome (Brenner et al. 1993). Since fishes
have a body plan and physiological systems similar to
mammals, the compact genome of fugu can help to discover genes and gene regulatory regions in the human
genome and serve as a reference to understand the evolution of vertebrate genomes and karyotype (Grutzner
et al. 1999; Hedges and Kumar 2002). Three years ago,
a ‘‘draft’’ sequence of fugu was generated purely by a
whole-genome shotgun strategy (Aparicio et al. 2002).
The draft sequence comprises of 12,381 scaffolds .2 kb
and efforts are currently underway to obtain the complete sequence of the genome. Since fugu has 22 pairs of
chromosomes (Miyaki et al. 1995), the ultimate objective is to merge the draft sequence scaffolds into 22
‘‘superscaffolds’’ corresponding to the 22 chromosomes.
However, the linkage relationship of the scaffolds is
currently unknown due to the lack of a genetic linkage
map or a genome-wide physical framework for this fish.
This has restricted the use of the fugu genome sequence
for global comparison with other vertebrate genomes to
address questions related to genome architecture and
1
These authors contributed equally to this work.
Corresponding author: Fisheries Laboratory, The University of Tokyo,
2971-4 Maisaka, Shizuoka 431-0214, Japan.
E-mail: [email protected]
2
Genetics 171: 227–238 (September 2005)
evolution of vertebrate chromosomes. Thus, it is recognized that a genetic linkage map is essential for completing the genome sequence of fugu and using it in
comparative genomic studies.
In addition to fugu, genomes of two other teleosts,
the zebrafish (Danio rerio) and medaka (Oryzias latipes),
are being sequenced. These two species are excellent
genetic models. Fugu and medaka are grouped together
under the common superorder Acanthopterygii and are
phylogenetically closer to each other than to zebrafish,
which are classified under the more basal superorder
Ostariophysi (Nelson 1994). Molecular clock analyses
of the mitochondrial sequences have suggested that the
common ancestors of the pufferfish and zebrafish diverged 280 million years ago (Kumazawa et al. 1999).
Although there are no estimates for the divergence time
of the medaka and pufferfish lineages, since the fossil
evidence shows that fishes of the order Tetraodontiformes have been around for 95 million years (Tyler
and Sorbini 1996), it is assumed that the two lineages
diverged .95 million years ago.
Excellent gene maps of zebrafish and medaka have
been generated, using 1500 and 800 gene or EST
sequences, respectively (Woods et al. 2000; Naruse et al.
2004). Recently, the draft genome sequence of another
pufferfish, the spotted green pufferfish, Tetraodon nigroviridis, has been generated ( Jaillon et al. 2004). A partial
physical map of the Tetraodon genome has been constructed by anchoring 64.4% of the genome assembly to
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W. Kai et al.
its 21 chromosomes ( Jaillon et al. 2004). The Tetraodon
lineage has been estimated to have diverged from the
fugu lineage 18–30 million years ago (CrnogoracJurcevic et al. 1997).
Comparisons of gene maps of zebrafish, medaka, and
Tetraodon with humans have helped to infer the number and content of the chromosomes of the last common ancestor of teleosts and mammals (Woods et al.
2000; Jaillon et al. 2004; Naruse et al. 2004). However,
there has been no attempt to compare the extent of
genome reorganization between the teleost species and
the mammalian species. A genetic linkage map of fugu
would be useful for such comparative studies among
teleost lineages and between teleost and mammalian
lineages and would help to understand the process of
teleost genome evolution.
In addition to comparative genomics, genetic linkage
maps are invaluable in forward genetic analysis for the
identification of gene loci responsible for genetic traits.
Pufferfishes belonging to the genus Takifugu are mainly
distributed in coastal regions of East Asia and include
.20 species (Matsuura 1990). Interestingly, hybrids
produced by artificial fertilization of eight Takifugu
interspecies crosses, including fugu and Takifugu niphobles, were found viable (Fujita 1967; Miyaki 1992).
Fugu and T. niphobles show marked differences in body
size, body color pattern, body shape, the number of
meristic skeletons, temperature preference, parasite
resistance, and behavior (Uno 1955; Ogawa 1991).
Given the the ability to generate fertile crosses between
Takifugu species, the availability of the draft genome
sequence of fugu provides an unprecedented opportunity to understand the genetic basis of the evolution of
natural species of these vertebrates. Availability of a
genetic map of fugu would greatly facilitate such studies.
Here we report a genetic linkage map for fugu, the
first map for a pufferfish. The map consists of genetic
markers physically linked to the assembled genome sequences, allowing us to establish a long-range relationship of the scaffold sequences and to compare gene
maps between fugu and other vertebrates. Comparison
of parental maps showed sex-specific differences in recombination ratio. Comparison of gene maps revealed a
tendency for highly conserved synteny among teleost
genomes as well as a lineage-specific rearrangement in
the pufferfish lineages. This genetic map will serve as a
foundation for obtaining the complete sequence of the
fugu genome, a basis for comparison of the genome map
with those of other vertebrates, and an important tool for
the future genome-wide analysis of phenotypic differences observed within and between Takifugu species.
MATERIALS AND METHODS
Mapping population: Wild female and male fugu were
caught in the Sea of Japan at Toyama Bay and in the Pacific
Ocean off Shizuoka prefecture, respectively. A mapping
population was obtained by in vitro crossing (Fujita 1967;
Matsuyama et al. 1997). A total of 64 full-sib progeny from the
single cross were used for developing a linkage map.
DNA extraction: Muscle tissue (50–100 mg) or whole bodies
of juveniles (100 mg each) were preserved in 400 ml of TNESurea buffer (10 mm Tris-HCl, pH 7.5, 125 mm NaCl, 10 mm
EDTA, 1% sodium dodecyl sulfate, 8 m urea) at room temperature for months. Genomic DNA was extracted from the
preserved samples using standard phenol-chloroform technique with slight modifications (Asahida et al. 1996). Approximately 3–15 mg of purified DNA was obtained from each
preserved sample.
Microsatellite marker: The CA repeat is the most common
microsatellite interspersed in the genomes of vertebrates,
including fugu (Aparicio et al. 2002). We first identified 160
CA-repeat loci by querying (CA)50 sequence against the fugu
draft sequence data on the website at the Joint Genome Institute (http://www.jgi.gov/fugu/index.html). An additional
40 loci were then chosen by scanning the fugu scaffolds
starting from the largest scaffold (no. 1). To increase the
map density and compare the linkage relationships of orthologs among fugu, medaka, and zebrafish, we searched the CArepeat loci physically linked to fugu orthologs of medaka and
zebrafish genes that have been mapped to their respective
maps (see Sequence comparison below) and identified 152 loci.
The analysis was carried out on the August 2002 freeze data set
for the fugu genome. Primers flanking the CA repeats were
designed and used for PCR to find heterozygous markers in
one or both parents. Construction of initial male and female
genetic maps of fugu using 192 microsatellite markers and
their consolidation identified 23 linkage groups. Comparison
of this map with gene maps of medaka and Tetraodon
indicated that two of the fugu linkage groups might be merged
into one linkage group (LG2). We therefore increased the
density of microsatellite markers on LG2 to obtain adequate
markers for merging the two linkage groups. Details of primer
sequences are shown in supplementary Table S1 at http://
www.genetics.org/supplemental/ as well as on our website at
http://park.itc.u-tokyo.ac.jp/suijitsu/. Primer sequences have
also been deposited in DDBJ/EMBL/GenBank with accession
nos. AB213693–AB214112.
Genotyping: PCR was performed in 20-ml reaction volume
of 100 mm Tris-HCl (pH 8.3) containing 0.5 m KCl, 2.5 mm
dNTPs, 0.5 mm of each primer, Taq DNA polymerase (Takara,
Tokyo, Japan), and 5 ng of genomic DNA. The following PCR
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
extension period at 59° for 3 min. The PCR products were heat
denatured with formamide loading buffer and fractionated on
a denaturing 6% acrylamide gel (Long-Ranger, ABI, Columbia,
MD) with 8 m urea. The gels were stained with SYBR gold and
visualized with a fluorescent imaging system (FLA3000,
FUJIFILM).
Genotypic classification: The progeny data are subdivided
into two independent data sets that contain the meiotic segregation data from each parent. Ancestry-unknown markers
in the three-generation pedigree were analyzed within a phaseknown model of inbred pedigree and converted to ancestryknown markers following the method of Sewell et al. (1999).
Map construction: The map was constructed using MAPMAKER (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
visual analysis of the distribution of marker genotypes in the
reference mapping panel.
Sequence comparison: Fugu orthologs of medaka, zebrafish, and human genes were identified by BLAST searching
these sequences against the fugu draft sequence data with a
Genetic Map for the Tiger Pufferfish
cutoff E-value of e10. For the search, we used 819 gene and EST
sequences for medaka and their human orthologs and 792
gene and EST sequences for zebrafish and their human orthologs reported by Naruse et al. (2004) and Woods et al. (2000).
In cases where the searches hit two or more scaffolds with a lessthan-threefold difference in the E-value, we did not assign any
orthology. In particular, to identify fugu orthologs to medaka
and zebrafish genes, we chose 239 gene and EST sequences
whose orthologs have been predicted between medaka and
zebrafish (Naruse et al. 2000) and BLAST searched their
sequences (a maximum E-value of e10) against the fugu database. Fugu ortholog was predicted if both medaka and zebrafish sequences showed the highest similarity to the same fugu
gene. In cases where the searches hit two or more scaffolds,
fugu orthologs were identified on the basis of phylogenetic
analysis. For the phylogenetic analysis, fugu protein sequences
identified in the BLAST search were aligned with medaka,
zebrafish, and human gene family members and phylogenetic
analysis was carried out using the program Clustal-X (Thompson
et al. 1997) and the neighbor-joining method (Galtier et al.
1996). Medaka and zebrafish mapping information and orthologous relationships among medaka, zebrafish, and human were
obtained from Naruse et al. (2004) and Woods et al. (2000), who
compiled the latest peer-reviewed medaka and zebrafish maps
available. Medaka and zebrafish gene and EST sequences
were obtained from DDBJ (http://getentry.ddbj.nig.ac.jp) or
the Zebrafish Information Network (http://www.zfin.org) using
accession numbers listed in Naruse et al. (2004) or Woods et al.
(2000). Map position of human and mouse orthologs were
determined using the NCBI human genome resources (http://
www.ncbi.nlm.nih.gov/genome/guide/human/). Fugu genes
on the same scaffolds are represented as a single locus in our
analysis to prevent an overestimation of the extent of conserved
synteny. To find putative orthologous segments of the fugu
scaffolds in the ‘‘partial physical map’’ of Tetraodon, at least three
DNA sequences of 10 kb were chosen from the 59-end, middle,
and 39-end regions of the fugu scaffolds and searched for
BLAT (Kent 2002) sequence similarity (a minimum score of
2000) against the Tetraodon draft sequence data (http://www.
genoscope.cns.fr/). When the fugu scaffold size was ,30 kb, the
entire scaffold sequence was used for the search.
Nomenclature: All loci identified are simple sequence
length polymorphisms and are named in accordance with
laboratory rules, e.g., f35, where ‘‘f’’ denotes both fugu and the
fisheries laboratory at the University of Tokyo, and the number
indicates the assay number. Ultimately, once the map is
completed and physically anchored to each chromosome,
each marker should receive a locus name following the system
used for the dog, mouse, pig, and rat maps.
RESULTS
Markers: To obtain informative markers for linkage
analysis in the progeny from the cross between noninbred parents, we examined the segregation types of
the CA-repeat locus in both parents and four progeny.
PCR reactions successfully amplified 376 loci in a total of
389 primer pairs that were designed on the basis of the
fugu draft genome sequence. The father and mother
were heterozygous for 184 (48.9%) and 188 (50%) of
these markers, respectively. Among them, 160 markers
were heterozygous in both parents with ab 3 cd or ab 3
ac type of segregation and allowed the identification of
homologous pairs of linkage groups of the male and
229
female parents. In total, 212 markers (56.4%) were
informative for segregation analyses in the mapping
population. An additional 60 progeny were scored for
these markers to construct the linkage map.
Linkage map: The male map consists of 24 linkage
groups with 169 microsatellite loci that are physically
linked to multi-gene-sized DNA sequences assembled in
the fugu draft genome database (Table 1). Among the
informative markers in male meiosis, 91.8% of the markers showed detectable linkage to another marker. The
map spans 697.1 cM and the average spacing between
two markers is 4.1 cM. The sizes of linkage groups range
from 0 to 75 cM (mean 29 cM). The number of markers
per linkage group varied from 3 to 17, with an average of
7 markers per group.
The female map consists of 29 linkage groups with
171 microsatellite markers. Among the informative
markers in female meiosis, 90.9% of the markers tested
showed detectable linkage to another marker. The maps
span 1213.5 cM and the average distance between two
markers is 7.1 cM. The sizes of linkage groups range
from 4.7 to 117.4 cM (mean 41.8 cM). The number of
markers per linkage group varied from 2 to 13, with an
average of 5.8 markers per group.
By using heterozygous markers in both parents, we
identified homologous pairs of linkage groups of the
male and female parents and arranged these maps in 22
linkage groups, designated LG1–LG22 (Figure 1 and
Table 1). The resulting map consists of 200 markers
physically linked to the assembled genome sequences
ranging from 1,145,486 to 1447 bp. The total length of
genomic sequences mapped is 39.2 Mb and contains
4452 gene loci (http://www.jgi.gov/fugu/index.html).
Seven pairs of markers that are linked in the male map
remain unlinked in the female map whereas two pairs of
markers in the female map remain unlinked in the male
map (Figure 1 and Table 1). This could be explained by
differences in recombination ratio between sexes as
seen in most of the linkage groups. For example, while
f330 and f39 are linked in the male map of LG1, they are
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
described in the map for the other sex when marker
density increases.
Differences in recombination rate between male and
female: To estimate the relative rates of recombination
between parents, we chose 104 pairs of adjacent markers
heterozygous for both parents and compared sexspecific recombination rates. Figure 2 shows a comparison of male and female recombination ratio between
the common intervals flanked by the paired markers
from all the linkage groups. The number of intervals
that shows a higher recombination ratio in females is
larger than that in males. On the other hand, the number of intervals that shows expansion in the male map
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W. Kai et al.
TABLE 1
Number of markers, genetic length, and total scaffold size for each linkage group
Male
LG
No. of
markers
Total scaffold
size (bp)
No. of gene
models
LG1
17
3,284,077
394
LG2
LG3
LG4
LG5
17
12
12
14
3,003,930
2,521,477
2,344,834
1,844,110
481
231
233
279
LG6
LG7
7
10
1,543,293
1,743,445
190
197
LG8
LG9
LG10
9
8
12
1,005,816
1,526,000
2,414,664
LG11
8
LG12
LG13
LG14
LG15
Female
No. of
markers
Length
(cM)
LG1
16
64.8
110
146
299
LG2
LG3
LG4
LG5-M1
LG5-M2
LG6
LG7-M1
LG7-M2
LG8
LG9
LG10
16
10
11
4
6
7
2
6
7
7
9
75
31.4
38.8
3.2
11.1
56.5
11.7
22.5
34.6
21.2
49.8
1,695,911
135
LG11
7
35.3
12
5
7
8
3,690,823
1,403,550
582,240
2,018,465
451
65
40
186
LG12
LG13
LG14
LG15
10
5
5
5
20.5
11
12.6
6.3
LG16
LG17
5
8
645,346
906,885
76
139
LG16
LG17
4
7
42
43.3
LG18
LG19
LG20
LG21
LG22
3
5
6
6
9
992,949
1,286,233
1,409,226
2,031,407
1,302,580
92
136
111
254
207
LG18
LG19
LG20
LG21
LG22
2
5
6
5
7
0
36.7
22.1
22.6
24.1
Total
200
39,197,261
4452
169
697.1
LG
relative to the female map is small but not negligible.
Summing up the length of the common interval for each
linkage group on both the male and female maps gave a
total length of 507.1 and 1100.8 cM, respectively. These
results suggest that overall recombination is suppressed
in male meiosis compared to female meiosis.
Comparison with T. nigroviridis: To compare the
extent of conserved synteny between the two pufferfish
genomes, we assigned 200 of the fugu scaffolds to putative orthologous segments of the Tetraodon genome.
Among them, 152 segments have been anchored to
Tetraodon chromosomes on its ‘‘partial physical map,’’
whereas 48 segments have not been anchored. This is
consistent with the limited anchoring of the Tetraodon
genome sequences to chromosomes (64.4%). In Figure 3,
putative orthologous segments have been arrayed ac-
LG
No. of
markers
Length
(cM)
LG1-F1
LG1-F2
LG1-F3
LG2
LG3
LG4
LG5-F1
LG5-F2
LG6
LG7
3
11
2
13
11
11
4
8
7
10
12.6
75.2
21.2
117.4
96
86.1
17.4
65.8
76.2
69
LG8
LG9
LG10-F1
LG10-F2
LG11-F1
LG11-F2
LG12
LG13
LG14
LG15-F1
LG15-F2
LG16
LG17-F1
LG17-F2
LG18
LG19
LG20
LG21
LG22
7
8
7
4
2
5
12
4
5
4
3
3
4
3
3
3
5
3
5
68.9
63.4
38.5
19.3
23.7
24
46.4
43.1
42.6
31.9
4.7
34.9
20.9
7.9
27
23.2
22.5
22.4
11.3
171
1213.5
cording to linkage groups of fugu and chromosomes of
Tetraodon. The display shows that each of the fugu
linkage groups shares putative orthologous segments
with one Tetraodon chromosome except for LG4, LG8,
LG10, and LG14. In the fugu linkage group LG10, six
scaffolds (scaffold 4, 146, 429, 446, 1117, and 2289) were
assigned to putative orthologous segments on Tetraodon chromosome 6, whereas two other scaffolds, scaffold 1628 and scaffold 572, showed similarity over large
regions to Tetraodon chromosomes 18 and 17, respectively. (Figure 3 and supplementary Table S2 at http://
www.genetics.org/supplemental/). This suggests that
there were interchromosomal translocations of segments of the ancestral sequences of scaffolds 1628 and
572 in either the fugu or the Tetraodon lineage after the
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,
and LG14. Thus, of the 152 orthologous segments, only
6 segments indicate possible interchromosomal rearrangement events after the divergence of the two species
of pufferfishes. This shows that most regions of the
chromosomes have been conserved during the 18–30
million years of divergent evolution of the two lineages.
Figure 3 also indicates that each of Tetraodon chromosomes 1, 3, 17, 18, and 20 shows conserved synteny with
two to three linkage groups of fugu. The disruption of
the one-to-one relationship of the Tetraodon chromosomes to fugu linkage groups may be due to interchromosomal translocations that include recent fusion of
chromosomes in the Tetraodon lineage or fission in the
fugu lineage after the divergence of the two pufferfish
species. This could be related to the presence of an
additional pair of chromosomes in fugu (n ¼ 22) compared to Tetraodon (n ¼ 21).
Conservation of synteny with other teleosts: To compare the extent of conserved synteny between the fugu
and medaka genomes, we identified fugu orthologs to
medaka genes chosen from a medaka linkage map
(Naruse et al. 2004) and found that 108 of them were
present in the fugu map. In the array of orthologs assigned on the basis of the linkage groups for each species, we were able to identify the cluster of orthologous
gene pairs that are located on conserved syntenic segments in fugu and medaka (Figure 4A). Among 108
putative orthologs, 94 orthologs fell in 22 conserved
synteny segments containing two or more genes between the two species. Furthermore, 18 pairs of linkage
groups shared conserved syntenic segments containing
at least three orthologous gene pairs. These correlations
imply that these pairs of linkage groups are derived from
a common ancestral chromosome. Of the 101 genes
from these 18 fugu linkage groups, 86 genes (85.1%) are
assigned to orthologous linkage groups in medaka.
These results suggest that the syntenic relationship of
a significant fraction of the genome has been remarkably conserved in the fugu and medaka lineages. Using a
similar strategy, we compared syntenic relationships
between the fugu and zebrafish genomes by using 88
Genetic Map for the Tiger Pufferfish
Figure 2.—Differences in recombination ratio between
male and female. The common intervals flanked by adjacent
markers from the 22 linkage groups are compared.
pairs of orthologous genes (Figure 4C). Among them,
61 orthologs fell in 22 conserved syntenic segments,
each containing two or more orthologous genes. Indeed, 9 of the conserved syntenic segments contain
three or more orthologous gene pairs between the
linkage groups of the two fishes (Figure 4C).
To compare the degree of conserved synteny between
the teleost and mammalian genomes, we defined
conserved syntenic segments using 105 quadruplets of
orthologous genes from our data set of fugu, medaka,
human, and mouse (Figure 4, A and B). This analysis
revealed that whereas only 14 orthologous gene pairs
did not fall in conserved syntenic segments between
fugu and medaka (Figure 4A), 28 orthologous gene
pairs between the human and mouse did not fall in conserved syntenic segments (Figure 4B). Since the evolutionary divergence period between fugu and medaka is
similar or even longer than that of human and mouse,
this result suggests that syntenic relationships are more
conserved in the teleost lineage than in the mammalian
lineage. We did similar analysis using 86 quadruplets of
orthologous genes for fugu, zebrafish, human, and
mouse and defined syntenic segments in the teleost
and mammalian genomes. In this analysis, 26 and 32
orthologous genes for fugu and zebrafish, and the two
mammals, respectively, did not fall in conserved syntenic
segments (Figure 4D). Such a similar degree of conserved synteny between the fugu and the zebrafish and
the human and mouse, despite the longer divergence
period between the two fishes, further supports a lower
frequency of interchromosomal rearrangements in the
teleost lineage than in the mammalian lineage.
Hox clusters: In vertebrates, Hox genes are present in
tight clusters of up to 14 genes and these clusters have
been often used as landmarks for comparison of gene
233
Figure 3.—Summary of the number of orthologous genome regions in fugu and T. nigroviridis. The linkage groups
of fugu are arrayed as columns and the chromosomes of Tetraodon as rows. Genome sequences that have not been mapped in Tetraodon are displayed in the last row (UN,
unknown). Numbers in boxes indicate the number of orthologous genome sequences. Details of mapped sequences are
listed in supplementary Table S2 at http://www.genetics.
org/supplemental/.
maps (Amores et al. 1998). We mapped the Hox clusters
of fugu to our genetic linkage map and compared the
location of Hox clusters in the gene maps of fugu,
Tetraodon, medaka, and zebrafish. To determine the
position of Hox clusters in the fugu map, we first identified nine scaffolds that contained members of six Hox
clusters (scaffold 47 for HoxAa, scaffold 330 and 5310 for
HoxAb, scaffold 1439 and 706 for HoxBa, scaffold 1245
and 2182 for HoxBb, scaffold 93 for HoxCa, and scaffold
214 for HoxDa; also see supplementary Table S2 at
http://www.genetics.org/supplemental/) by BLAST
search and obtained genetic markers from these sequences. A polymorphic marker for the HoxDb cluster
could not be obtained because we could identify only
one scaffold that contained a member of the HoxDb
cluster (scaffold 19,911 for HoxDb9) in the fugu database
and this scaffold contained no CA-repeat locus. The six
markers physically linked to the six Hox clusters (HoxAa,
HoxAb, HoxBa, HoxBb, HoxCa, HoxDa) were then mapped to the linkage groups of fugu (Table 2). Unexpectedly, the six markers mapped to only five linkage groups,
with two Hox clusters, HoxBb and HoxDa, being located in
the same linkage group, LG1. To determine whether the
genetic events that led to the two Hox clusters being
located to the same linkage group in fugu involved any
of the flanking genomic regions, we compared the
syntenic relationship of the Hox clusters and their known
genetically linked genes such as CYCS, POU3F3, PSMC5,
CRP, HBOA, ACO2, and TEF among fugu, medaka, and
zebrafish (Figure 5A). The comparison revealed that
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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
together with their linked non-Hox genes were located in
the same linkage group in fugu, the two syntenic clusters
were assigned to different linkage groups in both
medaka and zebrafish, suggesting that the two Hox
clusters and their flanking regions were translocated
into one linkage group in an ancestor of fugu after it
diverged from the medaka lineage. We also identified
the location of Hox clusters in the ‘‘partial physical map’’
of Tetraodon by BLAT searching with the fugu scaffold
sequences containing Hox genes and found that both
Tetraodon HoxBb and HoxDa clusters are localized on
chromosome 2 (Table 2). This suggests that the translocation event occurred before the separation of the
fugu and Tetraodon lineages.
DISCUSSION
Genetic map and genetics: We have constructed the
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
HoxAa
HoxAb
HoxBa
HoxBb
HoxCa
HoxCb
HoxDa
HoxDb
LG19
LG16
LG3
LG12
LG23
LG11
LG9
LG11
LG16
LG8
LG19
LG7
Tetraodon
Chromosome
Chromosome
Unmappeda
Chromosome
Chromosome
Fugu
21 LG12
8 LG7
LG5
2 LG1
9 LG3
LG21 Chromosome 2 LG1
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 Tetraodon 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 genomewide genetic analysis of fugu. In our male and female
maps, .94% of the markers are linked to other markers,
suggesting a high probability that the markers may be
linked to genetic traits. Such a map would be useful for
the initial low-resolution genetic mapping of traits.
Although we cannot expect that all markers that we
used will be informative in other crosses—e.g., interspecific 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
development of additional markers that are in close
proximity to the noninformative markers by searching
the genome sequence database.
The large collection of microsatellite markers will be
also useful for aquaculture purposes, because fugu is
becoming one of the most economically important
fish for aquaculture (Statistics and Information
Department 2001). These markers will be essential
for the identification of loci responsible for commercially desirable traits in different strains of this fish.
Molecular identification of such loci combined with
the technology for marker-assisted selective breeding
would be useful for establishing commercially better
strains of fugu. The collection of markers is also critical
for characterizing genetic background of wild and cultured fugu to maintain heterozygosity of stock and
for tracking parentage.
Sex-specific difference in recombination ratio:
Among mammals, human, dog, pig, and mouse show
Figure 5.—(A) Comparative linkage map of HoxBb and
HoxDa clusters and their flanking regions in fugu, medaka,
and zebrafish. Genes are named according to the nomenclature for the orthologous genes of human or zebrafish. The
zebrafish and medaka genes for which there is no mapping
information are not shown in the linkage map. Orthologous
genes are linked by dashed lines. (B) Genomic organization
of HoxBb and HoxDa clusters in pufferfishes (fugu, Tetraodon,
and Spheroides) (p) and zebrafish (z). Hox paralog group is
shown along the top.
reduced chromosomal recombination frequency in
males, while cattle do not show such sex-specific differences (Dib et al. 1996; Dietrich et al. 1996; Marklund
et al. 1996; Neff et al. 1999). Among teleost fishes, an
overall lower recombination ratio has been identified in
males of trout and zebrafish (Sakamoto et al. 2000;
Singer et al. 2002). The recombination around the
centromere region is preferentially lower in male
meiosis than in female meiosis in these fishes. In
addition, detailed studies in zebrafish have revealed
that the recombination in the region near the telomere
is preferentially lower in female meiosis than in male
meiosis (Singer et al. 2002). Our analysis of the fugu
genetic map shows that males have an overall reduction
in recombination ratio relative to females. While a large
number of the common intervals between the male map
and female map show expansion in the female map, some
intervals also show expansion in the male map. This
tendency is similar to that observed in trout and zebrafish (Sakamoto et al. 2000; Singer et al. 2002). Since
236
W. Kai et al.
the male-specific reduction in the recombination ratio
seems to exist in diverse species of teleosts such as trout,
zebrafish, and fugu, it is likely that this feature is shared
by all teleosts.
This finding has practical significance in facilitating
efficient experimental design in the genome-wide
linkage analysis for species differences among Takifugu
species (Singer et al. 2002). A decreased rate of recombination in males is an advantage for mapping genetic
traits in initial low-resolution analyses, especially when
analyzing quantitative trait loci (Glazier et al. 2002).
For example, if the phenotype shows complete or partial
dominance in F1 hybrids between two Takifugu species,
it is adequate to use a mapping population produced
from a cross between male hybrid and a female wild
species for primary mapping. On the other hand, it
would be necessary to use a higher frequency of recombination in females for fine mapping of these loci.
Comparison of genomes: Comparisons of the genetic
map of fugu with that of other teleosts revealed that, as
expected, the synteny between the two pufferfishes,
fugu and Tetraodon, is highly conserved. However, we
also found highly conserved synteny between fugu and
medaka, which shared a common ancestor .95 million
years ago. Our comparisons of the teleost maps and
human and mouse maps showed that syntenic relationships are more conserved in the teleost fish lineage than
in the mammalian lineage. For example, even though
the divergence period between fugu and zebrafish is
about threefold longer than that between the human
and mouse, a similar degree of conserved synteny was
found between the two fish lineages and between the
two mammalian lineages. By comparing the complete
physical map of human and the ‘‘partial physical map’’
of Tetraodon, Jaillon et al. (2004) reconstructed the
karyotype of the last common ancestor of the teleost fish
and mammals. Their model suggests a lower frequency
of major interchromosomal exchanges in the teleost
lineage than in the human lineage. Naruse et al. (2004)
also inferred the ancestral karyotype using the medaka,
zebrafish, and human maps. Their result suggested that
karyotype evolution in teleosts occurred mainly by
inversion and not by fusion or fission of chromosomes
that is frequent in mammalian lineages. Our results
independently demonstrate the lower frequency of
interchromosomal exchanges in the teleost lineage and
provide further support to the models proposed by
Jaillon et al. (2004) and Naruse et al. (2004). We also
showed that the extent of conserved synteny between
fugu and zebrafish is less than that between fugu and
medaka. This result is consistent with the phylogenetic
positions of the three fish species, i.e., zebrafish being
basal to both fugu and medaka. However, this can also
have other explanations. The larger genome size of
zebrafish compared to fugu and medaka (Venkatesh
et al. 2000) implies that the genome of this teleost
contains a significant proportion of transposable ele-
ments. Accumulation of transposable elements might
have favored a higher frequency of interchromosomal
rearrangements in the zebrafish (Grutzner et al. 1999).
Hox clusters: In vertebrates Hox genes occur in tight
clusters of up to 14 paralogous genes. They play an
important role in anterior/posterior patterning in development, including skeletal complexity of animals.
The expression domains of the genes in a cluster are
colinear with the position of the genes in the cluster. In
all tetrapods studied so far, four clusters of Hox genes
(HoxA, HoxB, HoxC, and HoxD) have been identified.
The clusters are located on different chromosomes
(Scott 1992; Ladjali-Mohammedi et al. 2001). These
four clusters are presumed to have arisen by two rounds
of whole-genome or segmental duplication during the
evolution of early vertebrates (Larhammar et al. 2002).
The distribution of four Hox clusters on four chromosomes is often sighted as evidence for whole-genome (or
whole-chromosome) duplications. Although extensive
interchromosomal rearrangements are suggested in the
evolution of amniote chromosomes (Bourque et al.
2004), all amniotes so far examined have retained the
presumptive ancient organization of Hox clusters and
chromosomes; that is, a single Hox cluster is located
on each chromosome. In contrast to tetrapods, teleosts
such as zebrafish, medaka, and pufferfishes (fugu, Spheroides, and Tetraodon) contain up to seven Hox clusters
(Amores et al. 1998, 2004; Naruse et al. 2000). The
additional Hox clusters in teleosts have been proposed to
be the result of a whole-genome duplication (Amores
et al. 1998) that occurred early during the evolution of the
ray-finned fish (Christoffels et al. 2004). Presumably,
one of the duplicate Hox clusters was lost following the
whole-genome duplication. In the medaka and zebrafish,
in which Hox clusters have been mapped to the genetic
linkage groups, the seven clusters are located in different
linkage groups. The genomic position of the Hox clusters
in the pufferfish was not known until now. In our study,
we unexpectedly found that two Hox clusters, HoxBb and
HoxDa, along with their flanking genes are located in the
same linkage group in fugu as well as in Tetraodon
(Figure 5A, Table 2). Since these two Hox clusters are
located in different linkage groups in the zebrafish and
medaka, it seems likely that the two clusters were colocalized to the same chromosome through interchromosomal rearrangements, most likely through fusion of two
chromosomes. Each of the pufferfish HoxBb and HoxDa
clusters lacks one of the genes present in the zebrafish
HoxB8b and HoxD13a (see Figure 5B). Although a detailed gene content of the medaka Hox clusters has not
been analyzed, the overall organization of Hox clusters in
medaka has been reported to be more similar to that in
pufferfishes than in zebrafish (Amores et al. 2004) (see
Table 2). For example, while two HoxD clusters, HoxDa
and HoxDb, are present in medaka and pufferfishes, zebrafish have a single HoxDa cluster but two HoxC clusters
(HoxCa and HoxCb). Thus, it is assumed that the contents
Genetic Map for the Tiger Pufferfish
of the HoxBb and HoxDa clusters in medaka are more
similar to those in pufferfishes than to those in zebrafish. Pufferfish are the only known vertebrate group in
which two Hox clusters are located on the same chromosome. It is not known if this has any influence on the
expression pattern of the genes on these two clusters. A
comparative study of the expression patterns of genes in
these clusters in medaka and pufferfish should shed light
on this.
Takifugu species as model systems for studies of the
genetic basis of phenotypic diversity: Teleost fishes
such as cichlid and stickleback have recently been
proposed as model vertebrate species for identifying
the genetic variation that is responsible for phenotypic
differences among related species. Viable crosses between closely related species of these fishes can be
generated under laboratory conditions, and forward
genetic analysis can be conducted (Cresko et al. 2004;
Kocher 2004). Although many loci have been mapped
onto genetic linkage maps of these teleost species,
further progress in molecular identification of the locus
has been hindered by lack of whole-genome sequences
of these fishes. We propose that the fishes belonging to
the genus Takifugu are also a good model system to
study the genetic basis of phenotypic evolution in
nature. A major drawback in using Takifugu fishes as a
genetic model system was thought to be the difficulty of
maintaining and breeding this marine species. This difficulty has, however, been largely overcome by developing suitable technology for controlled breeding under
laboratory conditions (Miyaki et al. 1996; Matsuyama
et al. 1997; Takaoka et al. 1998). The embryos of fugu
have also been demonstrated to be amenable for molecular genetic investigations (Suzuki et al. 2002; Amores
et al. 2004). More importantly, Takifugu can produce
fertile progeny when crossed with closely related species
(Miyaki 1992) and a draft sequence of the wholegenome of fugu has been generated. Thus, Takifugu
species can be very useful tools to study the genetic basis
of phenotypic variation between closely related species
and to understand the process of speciation in vertebrates. The genetic map of fugu constructed by us and
its further enrichment with additional genetic markers
should greatly facilitate these types of studies.
We thank Kiyoshiro Kumasaka and Kazumitsu Honda (Nisshin
Marinetech) for obtaining fish embryos; Kiyoshi Naruse, Osame
Tabeta, and Yusuke Yamanoue for helpful discussion; and Kiyoshi
Furukawa for technical advice. Hidekazu Suzuki and Naoki Mizuno
provided technical support. This work was partially supported by
grants from the Ministry of Education, Culture, Sports, Science and
Technology of Japan. B.V. is supported by Singapore’s Agency for
Science, Technology and Research.
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Communicating editor: N. Takahata