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Evolution of different Y chromosomes in two medaka species, Oryzias

dancena and O. latipes

Yusuke Takehana,*,1,2 Diana Demiyah,*,1 Kiyoshi Naruse, † Satoshi Hamaguchi* and Mitsuru

Sakaizumi*

*Graduate School of Science and Technology, Niigata University, Ikarashi, Niigata 950-2181,

Japan and †Department of Biological Science, School of Science, University of Tokyo,

Bunkyo-ku, Tokyo, 113-0033, Japan

1These authors contributed equally to this work.

Genetics: Published Articles Ahead of Print, published on December 28, 2006 as 10.1534/genetics.106.068247

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Running head: Sex chromosome evolution in Oryzias

Key words: DMY, sex-determining gene, and sex chromosome evolution

2Corresponding author: Yusuke Takehana, Department of Environmental Science, Faculty of

Science, Niigata University, 8050 Ikarashi-2, Niigata, 950-2181, Japan. Tel: +81-25-262-

6368; Fax: +81-25-262-6817; E-mail: yusuke@env.sc.niigata-u.ac.jp

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ABSTRACT

Although the sex-determining gene DMY has been identified on the Y chromosome in the

medaka (Oryzias latipes), this gene is absent in the most Oryzias species, suggesting that

closely related species have different sex-determining genes. Here, we investigated the sex

determination mechanism in Oryzias dancena, which do not possess the DMY gene. Since

heteromorphic sex chromosomes have not been reported in this species, a progeny test of sex-

reversed individuals produced by hormone treatment was performed. Sex-reversed males

yielded all-female progeny, indicating that O. dancena has an XX/XY sex determination

system. To uncover the cryptic sex chromosomes, sex-linked DNA markers were screened

using expressed sequence tags (ESTs) established in O. latipes. Linkage analysis of isolated

sex-linked ESTs showed a conserved synteny between the sex chromosomes in O. dancena

and an autosome in O. latipes. Fluorescence in situ hybridization (FISH) analysis of these

markers confirmed that sex chromosomes of these species are not homologous. These

findings strongly suggest an independent origin of sex chromosomes in O. dancena and O.

latipes. Further analysis of the sex-determining region in O. dancena should provide a key

insight into the evolution of sex determination mechanisms in vertebrates.

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INTRODUCTION

Almost all vertebrates have different sex, males and females. Despite such universal

occurrence, sex can be determined by a variety of different mechanisms. Among the major

vertebrate groups, highly divergent genetic and environmental factors control sex

determination. These include a male-heterogamety (XX/XY system typified by mammals), a

female-heterogamety (ZZ/ZW system in birds and snakes), as well as environmental sex

determination systems (such as a temperature-dependent sex determination in alligators). In

mammals, the sex-determining gene, SRY/Sry has been identified on the Y chromosome

(Gubbay et al. 1990; Sinclair et al. 1990). However, no equivalent genes have been found in

non-mammalian vertebrates until recently.

In the medaka Oryzias latipes, which has an XX/XY sex determination system, DMY was

identified as the Y-specific sex-determining gene (Matsuda et al. 2002; Matsuda 2005). This

gene encodes a putative transcription factor containing a DM domain, which was originally

described as a DNA-binding motif found in two proteins, DSX in Drosophila melanogaster

and MAB-3 in Caenorhabditis elegans (Raymond et al. 1998). Vertebrates have several DM

domain-containing genes and one of these, DMRT1 (DM-related transcription factor 1) has

been implicated in male sexual development in mammals, birds, reptiles and fishes (Raymond

et al. 1999; Smith et al. 1999; De Grandi et al. 2000; Guan et al. 2000; Kettlewell et al. 2000;

Marchand et al. 2000). The cDNA sequences of the medaka DMY and DMRT1 showed a high

similarity (about 80%) and DMY appears to have arisen through a gene duplication event of

an autosomal DMRT1 gene (Matsuda et al. 2002; Nanda et al. 2002).

In contrast to the widespread distribution of Sry in mammals, the DMY has not been

detected, even in closely related species. Although one sister species of O. latipes has the

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DMY gene on a homologous Y chromosome (Matsuda et al. 2003), the gene has not been

found in other Oryzias species (Kondo et al. 2003). Fishes in the genus Oryzias have been

divided into three monophyletic species groups, the latipes, javanicus and celebensis groups

(Takehana et al. 2005) and a recent phylogenetic analysis of DMY and DMRT1 genes from

Oryzias species suggested that duplication of DMRT1 (generating DMY gene) appears to have

occurred within the latipes group lineage (Kondo et al. 2004). These findings suggested that

Oryzias fishes in other species groups (javanicus and celebensis groups) must have different

sex-determining genes. Accordingly, comparisons between closely related medaka fishes with

different sex determination mechanisms should be important in understanding how these

diverse developmental mechanisms evolve.

In the present study, as a first step to identify a sex-determining gene in other fish species,

we investigated the sex determination system and sex chromosomes in Oryzias dancena, a

member of the javanicus group (Takehana et al. 2005). Because a previous cytogenetic study

has not reported heteromorphic sex chromosomes in this species (as O. melastigma) (Uwa et

al. 1983), a genetic analysis of sex-reversed individuals was performed to demonstrate

whether this species has an XX/XY system of sex determination. Moreover, we took

advantage of genomic tools established in O. latipes to isolate sex-linked DNA markers, map

the sex-determining locus and identify the sex chromosomes.

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MATERIALS AND METHODS

Fish: All fish used in this study were supplied from a sub-center (Niigata University) of

National BioResource Project (medaka) in Japan. Wild stocks of Oryzias dancena were

originally collected at Chidambaram (CB), India, in 1981 and at Phuket (PK), Thailand, in

1988 (for detail, see Takehana et al. 2005). Fish were maintained in aquaria under an artificial

photoperiod of 14L:10D at 27±2 ºC.

Sex steroid treatments for sex reversal and progeny test: The sex-reversal experiment

was performed as previously described by Hamaguchi et al., (2004). Briefly, fertilized eggs of

O. dancena (CB) were incubated in water containing estradiol-17ß (E2; Sigma) at 0.01, 0.04

and 0.2 µg/ml, or methylteststerone (MT; Sigma) at 0.001, 0.005 and 0.025 µg/ml. Hatched

fry were transferred to normal tap water and fed on a commercial pet-food diet until sexual

maturation. Sex of the treated fish was judged from secondary sex characteristics and treated

fish were subsequently mated with normal fish. Sexing of F1 progeny from the mating was

carried out by histological cross-sections of fry gonads, sampled at 20 days after hatching.

Genetic crosses: By crossing CB female and (CB female×PK male) F1 male, 45 backcross

progeny were obtained for genotyping. Phenotypic sex was determined by secondary sex

characteristics of adult fish and reconfirmed by visual examination of the gonads. We fixed

adult fish in 100% ethanol and isolated their genomic DNA from some muscle tissue using

the PI-50 isolation system (Kurabo).

Isolation of sex-linked DNA markers and linkage analysis: We searched for sex-linked

markers using expressed sequence tag (EST) markers mapped to each of the O. latipes

chromosome (linkage group; LG) (Naruse et al. 2004). ESTs were amplified using previously

published primers designed for O. latipes (supplemental Table 1 at

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http://www.genetics.org/supplemental/). PCR genotyping for two loci, BJ014360 and

BJ732639, was performed using primers that we designed based on the EST sequences

(http://mbase.bioweb.ne.jp/~dclust/medaka_top.html) and the draft genome sequence

(http://dolphin.lab.nig.ac.jp/medaka/) of O. latipes. PCR amplification of each EST marker

was performed in a total volume of 10 µl for 3min at 95 ºC followed by 35 cycles of 10 s at

95 ºC, 30 s at 55-60 ºC, 60 s at 72 ºC, with a final elongation step of 3 min at 72 ºC.

Restriction fragment length polymorphisms (RFLPs) in PCR-amplified fragments of the

parents (CB female and PK male) were analyzed by polyacrylamide gel electrophoresis. For

those markers that showed a polymorphic pattern between the parents, F1 and backcross

progeny were genotyped, and assessed as to whether such polymorphisms segregated with

phenotypic sex. Using these isolated sex-linked ESTs, a sex linkage map of Oryzias dancena

was constructed.

Fluorescence in situ hybridization analysis with fosmid and BAC clones: A fosmid

genome library of Oryzias dancena was constructed from a F1 male between CB and PK

using the Copy Control Fosmid Library Production kit (Epicentre). Fosmid clones containing

sex-linked ESTs were screened by colony hybridization. A fosmid clone Od38_01 (containing

BJ014360) was used as a probe. A bacterial artificial chromosome (BAC) genomic library,

constructed from Hd-rR strain of O. latipes (Matsuda et al. 2001), was also screened and three

clones, Md0173J11 (containing SL1), Md0172B19 (containing DMRT1) and Md0172I07

(containing OLd17.11a), isolated. These BAC clones were located on the sex chromosomes

(LG 1) and on autosomes (LG 9 and LG 10) in O. latipes, respectively.

Metaphase cells from cultured caudal fins were prepared by standard cytogenetic methods

(Uwa and Ojima 1981; Matsuda et al. 1998). Fluorescence in situ hybridization (FISH) was

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performed as described (Matsuda and Chapman 1995). Probe DNAs of the genomic clones

were labeled separately by nick translation using biotin-16dUTP (Roche) or digoxigenin-11-

dUTP (Roche). For two-color hybridization, equal amounts of labeled probes and a 500-fold

amount of autoclaved genomic DNA of O. latipes were mixed with hybridization solution and

preannealed for 30 min at 37 ºC. After overnight hybridization, probes were detected with

avidin-FITC (Roche) and rhodamine-labeled anti-digoxigenin antibodies (Roche). Slides were

counterstained with 4,6-diamidino-2-phenylindole (DAPI) and examined under a Nikon

Eclipse 80i microscope using three filters (UV-1A, B-2A and G-2A). Images were captured

with a DXM1200C digital camera (Nikon).

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RESULTS

Progeny test of sex-reversed fish: The sex ratios of the hormone-treated fish were shown in

Table 1. The sex ratio of MT-treated group was biased toward male, suggesting that MT

induced sex reversal from female to male. In the groups reared under normal conditions, the

sex ratio was almost 1:1 (data not shown). Therefore, the MT-treated group was expected to

include sex-reversed males whose genetic sex is female (XX or ZW). On the other hand, the

sex ratio of the E2-treated groups did not deviate toward females, suggesting that E2 could

not reverse the sex of the fish from male to female at concentrations of 0.01–0.04 µg/ml. In

addition, all fish died at concentration of 0.2 µg/ml E2 before or just after hatching (data not

shown).

Of 20 mature males obtained by the 0.025 µg/ml MT treatment, 11 were randomly selected

and subjected to a progeny test by matings with normal females. The sex ratio of the offspring

from each mating is shown in Table 2. As a result, five of 11 males from the MT-treated

group yielded all-female progeny, demonstrating that these males were sex-reversed XX

males. This result reveals that Oryzias dancena has a male-heterogametic (XX/XY) sex

determination system.

Sex linkage map of Oryzias dancena: To isolate sex-linked DNA markers in Oryzias

dancena, we searched for RFLPs between the parents using ESTs established in O. latipes,

and genotyped F1 and backcross progeny. We screened 397 ESTs and identified 65

polymorphic markers. Linkage analysis showed that eight of these 65 markers segregated

with the phenotypic sex. The female parent was homozygous, and the male parent and/or F1

male were heterozygous in these markers. In the backcross progeny, males had the paternal

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genotype, while females had the maternal genotype, confirming an XX/XY sex determination

system (Figure 1).

Using eight isolated EST markers, we constructed a sex linkage map of O. dancena, and

showed that the sex-determining (SD) locus was mapped to the same position with an EST,

BJ014360 (Figure 2). Based on the draft genome sequence data of O. latipes

(http://dolphin.lab.nig.ac.jp/medaka/), all eight ESTs were located on LG 10 in O. latipes.

Comparison of gene order between the O. dancena sex linkage map and the physical map of

O. latipes LG 10 indicated a conserved synteny between the two chromosomes. These results

suggest that the sex chromosomes in O. dancena are homologous to an autosome (LG 10) of

O. latipes, whose sex chromosomes are LG 1.

In this sex linkage map, the total map length was about 23 cM in male meiosis.

Corresponding region of O. latipes has a similar length (24.4 cM)

(http://dolphin.lab.nig.ac.jp/medaka/), suggesting that recombination along the sex

chromosomes is not suppressed in O. dancena. Indeed, we obtained 4/45 (8.9%) recombinants

between MF01SSA032H09 and BJ732639, which flanked the SD locus. Based on the draft

genome sequence, this region was calculated to be about 3.0 megabase (Mb) in O. latipes.

These findings suggest a high recombination frequency around the sex-determining region in

O. dancena.

Identification of sex chromosomes by FISH mapping: Karyotype of Oryzias dancena

consists of 24 acrocentric chromosome pairs with no heteromorphic sex chromosomes (Uwa

et al. 1983). To identify the sex chromosomes in O. dancena, we conducted a FISH analysis

of male chromosomes using a fosmid clone containing BJ014360 as a probe, which is tightly

linked to the SD locus. Clear hybridization signals were observed on the middle position of an

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acrocentric chromosome pair, identifying the cryptic sex chromosomes (Figure 3A). We also

found sex chromosome localization of the O. latipes BAC clone (Md0172I07) containing

another sex-linked EST, OLd17.11a. Two-color FISH indicated that the BJ014360 signal was

located more proximal to the centromere than the OLd17.11a signal on the sex chromosomes

(Figure 3B). Morphological difference between X and Y chromosomes was not observed in

this study, indicating that O. dancena has homomorphic sex chromosomes.

On the other hand, the BAC clone (Md0173J11) containing an O. latipes sex-chromosomal

marker (SL1) was not located on the sex chromosomes in O. dancena (Figure 3C), confirming

that sex chromosomes were different between these two species. Furthermore, the

hybridization signals of DMRT1-positive BAC (Md0172B19) were detected only on a pair of

autosomes (Figure 3D). In O. latipes, previous FISH analyses revealed the presence of

DMRT1 at the tip of a pair of autosomes, as well as at one of the sex chromosome pair

(namely the DMY gene on the Y chromosomes) (Kondo et al. 2004, 2006). Although we

found the similar FISH pattern to that of autosomal DMRT1 in O. latipes, the additional Y-

chromosomal location (corresponding to the DMY) was not observed in O. dancena. Hence,

these results strongly suggest that Y chromosomes of O. dancena and O. latipes are not

homologous.

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DISCUSSIONS

Oryzias dancena has an XX/XY sex determination system: It has been demonstrated that

Oryzias dancena in the javanicus species group has a male-heterogametic (XX/XY) sex

determination system, based on sex ratios in the progeny of sex-reversed fish and segregation

patterns of sex-linked DNA markers. A previous study showed that all four fishes in the

latipes group also have an XX/XY sex determination system (Hamaguchi et al. 2004) (Figure

4). Two of these four species, O. latipes and O. curvinotus, have the common sex-determining

gene, DMY, on the homologous Y chromosomes (Matsuda et al. 2002, 2003). On the other

hand, the gene has not been detected in the remaining two species, O. luzonensis and O.

mekongensis (Kondo et al. 2003, 2004). Based on these findings with a phylogenetic analysis

of DMY and DMRT1, Kondo et al. (2004) suggested that the DMY gene appears to have

occurred in the common ancestor of O. latipes, O. curvinotus and O. luzonensis. Therefore,

the DMY gene is considered to be recently lost in O. luzonensis, and originally absent in O.

mekongensis and in other Oryzias species, such as O. dancena. This suggests that different

sex-determining genes have evolved in Oryzias species, although the male-heterogamety is

likely to be common in the genus.

Identification of sex chromosomes in O. dancena: Oryzias species with no DMY gene

must have different sex chromosomes from those in O. latipes; however, no sex chromosomes

have been identified in these species so far. In this study, we screened sex-linked markers of

O. dancena using ESTs established in O. latipes. As a result, most of ESTs primers worked

well in O. dancena, and we could successfully isolate eight sex-linked EST markers. Linkage

analysis showed that these ESTs are located on an autosome (LG 10) in O. latipes with the

same gene order, indicating a conserved synteny between sex chromosomes in O. dancena

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and an autosome of O. latipes. In addition, FISH analysis confirmed that sex chromosomes in

O. dancena and O. latipes were not homologous. These results indicated an independent

origin of X/Y chromosomes in these two species, suggesting that a novel sex-determining

gene is located on the sex chromosomes in O. dancena.

The lack of conservation of sex chromosomes among closely related fish species is

probably common. Although all salmonid fishes have a male-heterogametic system, closely

related species have evolved different sex chromosomes, as evidenced by a comparative

linkage analysis (Woram et al. 2003) and FISH analyses (Phillip et al. 2001, 2005). In

sticklebacks, Gasterosteus aculeatus and G. wheatlandi also appear to have different X/Y sex

chromosomes, and Apeltes quadracus has heteromorphic Z/W sex chromosomes (Peichel et

al. 2004). These studies suggest that fishes may use a variety of sex-determining gene.

Sex-determining region in O. dancena: Linkage analysis mapped the SD locus between

MF01SSA032H09 and BJ732639, and showed that BJ014360 is tightly linked to the SD locus

with no recombinants (0/45). This suggests that a single chromosomal region controls the sex

in O. dancena. FISH analysis indicated that the sex-determining region is located on the

middle of one acrocentric chromosome pair, in accordance with the linkage analysis.

Therefore, only this region should be different between X and Y chromosomes.

Standard models for sex chromosome evolution hypothesized that the first step is the

occurrence of a novel single locus on an autosome, in which heterozygosity leads to the

development of one sex and homozygosity to the other sex, thereby establishing a proto-sex

chromosome system. Heteromorphic sex chromosomes are considered to arise through the

recombination isolation between such homologous sex chromosomes. This suppression of

recombination maintained one chromosome (such as Y) in a constant heterozygous state in

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one sex and the subsequent degeneration process spreads the sex-specific region over almost

the entire chromosome, as in mammals (reviewed in Graves 2006).

Our present results indicate that sex chromosomes in O. dancena are at an early stage of

evolution, as in O. latipes. First, phenotypic sex was easily converted by sex hormones, and

the resultant sex-reversed fish were fully fertile. Furthermore, YY males have been obtained

from estrogen-induced XY females (Y. Takehana and D. Demiyah, unpublished), indicating

viability of YY individuals. Second, sex chromosomal crossing-over occurred over almost the

entire length of the chromosome, indicating that this recombining section is considered to be a

pseudoautosomal region. In addition, the region between MF01SSA032H09 and BJ732639,

which flanked the SD locus in O. dancena was calculated to be 3.0 Mb in O. latipes, based on

the draft genome sequence. This suggests that the Y-specific region of O. dancena is likely to

be very small. Finally, FISH analysis demonstrated that the Y chromosome is not

cytogenetically distinguished from X, although the sex determination system in the species is

male-heterogametic. Taken together, these findings suggest that there are no functional

difference between X and Y sex chromosomes, other than the sex-determining gene.

Only two sex-determining genes, Sry and DMY, have been identified in vertebrates so far.

In the present study, we showed that Oryzias dancena should have a novel sex-determining

gene on the sex chromosomes homologous to an autosome (LG 10) in O. latipes. One

approach to identify sex-determining genes is positional cloning. This method has been

successfully used to isolate human and medaka sex-determining genes. Two major conditions

are required for using this method: a firm genetic sex determination and a feasibility of

genetic mapping for the SD locus (Matsuda 2005). Oryzias dancena has a strict genetic sex

determination (XX/XY system) and a high recombination frequency around the SD locus,

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satisfying both requirements. In addition, O. dancena and O. latipes share a similar character

of sex chromosomes, a very small Y-specific region, suggesting that this method is expected

to be effective also in O. dancena. Construction of a high-resolution recombination map

around the SD locus and chromosome walking to the SD locus, are necessary steps to isolate

the sex-determining gene using the positional cloning method. This is currently in progress.

Comparative analyses of sex-determining genes and sex chromosomes among Oryzias species

will provide an opportunity to unravel the evolution of sex determination mechanisms in

vertebrates.

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ACKNOWLEDGEMENTS

We thank Dr. Yoichi Matsuda, Dr. Takahiro Murakami (Hokkaido University) and Dr.

Masaru Matsuda (National Institute of Basic Biology) for their helpful technical advice on

FISH analysis and Dr. Wichian Magtoon (Srinakharinwirot University) for his generous help

in the collection of materials. This work was supported in part by a Grant-in-Aid for Scientific

Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan

(16370094) to M.S.

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FIGIRE LEGENDS

Figure 1. Linkage analysis of EST markers in Oryzias dancena. Electrophoretic patterns of

MF01SSA032H09 PCR product digested with HinfI (A) and BJ014360 PCR product digested

with HhaI (B). Genomic DNA from female (F) and male (M) fish was used as templates for

PCR. Abbreviations: P, parents (CB female and PK male); F1, F1 progeny from the parents;

BC1, backcross progeny. Note that the digested bands (arrowheads) segregated perfectly with

BC1 males.

Figure 2. Comparison of gene order between a sex linkage map in Oryzias dancena and a

physical map of LG 10 in O. latipes. Lines between the compared chromosomes connect

positions of orthologous gene pairs in these two species. The distances between franking

markers are shown in physical length (left) and in centimorgans (cM) (right). Map positions

for genes and distances in O. dancena were derived from this study; those in O. latipes were

obtained from the draft genome sequence data (http://dolphin.lab.nig.ac.jp/medaka/).

Figure 3. FISH analysis of male metaphase chromosomes in Oryzias dancena using O.

dancena fosmid and O. latipes BAC clones. (A) FISH mapping of a fosmid clone (Od38_01)

containing a sex-linked EST, BJ014360. A specific hybridization signal (red) was located on

a homomorphic acrocentric chromosome pair. (B) Gene ordering of the sex-linked ESTs,

BJ014360 (fosmid Od38_01) and OLd17.11a (BAC Md0172I07), on the sex chromosomes.

The locations of the markers are visualized as red and green signals, respectively. Arrowheads

indicate the centromere positions. (C) Chromosomal location of the O. latipes sex-

chromosomal marker SL1 (BAC Md0173J11, green) and the O. dancena sex-chromosomal

- 22 -

marker BJ014360 (fosmid Od38_01, red). (D) Chromosomal location of the DMRT1 gene

(BAC Md0172B19, green) with the BJ014360 fosmid, Od38_01 (red).

Figure 4. Phylogenetic relationships and sex determination mechanisms in Oryzias fishes. The

phylogenetic information was taken from Takehana et al. (2005). Data of sex determination

mechanisms in O. latipes, O. curvinotus, O. luzonensis and O. mekongensis were obtained

from Matsuda et al. (2002, 2003) and Hamaguchi et al. (2004).

Concentration (µg/ml) Male Female Total

Methylteststerone

0.001 20 2 22

0.005 30 5 35

0.025 20 0 20

Estradiol-17β

0.01 10 12 22

0.04 10 9 19

Sex ratios of the hormone-treated fish.

Table 1

Mating Male Female Total Remark

#1 0 8 8 Sex-reversed XX male

#2 18 18 36

#3 17 16 33

#4 9 14 23

#5 16 21 37

#6 15 13 28

#7 0 43 43 Sex-reversed XX male

#8 14 19 33

#9 0 47 47 Sex-reversed XX male

#10 0 47 47 Sex-reversed XX male

#11 0 57 57 Sex-reversed XX male

Sex ratio of the offspring of mating between a methylteststerone-treated male and a normal female.

Table 2

900800700600500

400300

200

100

bp

bp

MF M M M M M M MF FFFFFF

MF M M M M M M MF FFFFFF

F1

F1P

P BC1

BC1A

B

OLc31.06aMF01SSA044G11MF01SSA032H09

BJ014360BJ732639

OLb25.11eAU168326

OLd17.11a

OLc31.06aMF01SSA044G11MF01SSA032H09

BJ014360

BJ732639OLb25.11e

AU168326

OLd17.11a

SD

O. latipesLG 10

O. dancenasex linkage map

5 Mb

7.3

2.3

2.3

11

O. latipesO. curvinotus

O. dancena

O. mekongensis

O. luzonensis

latipes group

javanicus group

XX/XYXX/XY

XX/XY

XX/XY

XX/XY

Sex de

termina

tion

system

LG 1LG 1

LG 10unknown

unknown

Sex ch

romoso

me

(O. lat

ipes LG

)

DMYDMY

unknown

unknownunknown

Sex-de

terminin

g

gene