C opyright 2007 by the G enetics Society of A mericaD O I: 10.1534 / ge netics.106.068247

Evolution o f D i f f erent Y C hromosomes in Two Medaka Species,Oryzias dancena and O . latipes

Yusuke Takehana,*,1,2 D iana D emiyah,*,1 K iyoshi N ar use,†

Satoshi H amaguchi* and Mitsur u 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

Man uscript received N ovember 14, 2006Accepted for publication D ecember 19, 2006

A B S T R A C TAlth ough the sex-determining gene DMY has been identified on the Y ch romosome in the medaka

( Oryzias latipes) , this gene is absent in most O ryzias species, suggesting that closely related species havedifferent sex-determining genes. H ere, we investigated the sex-determination mechanism in O . dancena,which does n ot possess the DMY gene. Since heteromorp hic sex ch romosomes have not been reported inthis species, a progeny test of sex-reversed in dividuals produced by hormone treatment was per formed .Sex-reversed males yielded all-female progeny, in dicating that O . dancena has an X X / X Y sex-determinatio nsystem . To u ncover the cryptic sex ch romosomes, sex-lin ked D N A markers were screened using expressedsequence tags ( ESTs) established in O . latipes. L in kage analysis of isolated sex-lin ked ESTs showed aco nserved synteny between the sex chromosomes in O . dancena an d an autosome in O . latipes. Fluorescencein situ hybridization ( F IS H ) analysis of these markers co n fi rmed that sex chromosomes of these species aren ot h omologous. T hese fi n dings strongly suggest an in depen dent origin of sex ch romosomes in O . dancenaan d O . latipes. F urther analysis of the sex-determining region in O . dancena should provide crucial insightsinto the evolutio n of sex-determinatio n mechanisms in vertebrates.

A L M O ST all vertebrates have different sexes, malesan d females. D espite such u niversal occurrence,

sex can be determined by a variety of different mech-anisms. A mong th e major vertebrate grou ps, highlydivergent gen etic an d enviro n men tal factors controlsex determi natio n . T hese inclu de male heterogamety( X X / X Y system typified by mam mals) an d female het-erogamety ( Z Z / Z W system in birds an d snakes) , as wellas environ men tal sex determinatio n systems (such asa tem perature-depen de nt sex determinatio n in alli-gators) . I n mam mals, th e sex-determi ning gene, SRY /Sry has been iden tified o n the Y ch romosome ( G ubba yet al. 1990; Si n c l a i r et al. 1990) . H owever, n o equivalentgenes have been fou n d in n o n mam malian vertebrates,u nti l recently.

I n the medaka, Oryzias latipes, which has an X X / X Ysex determinatio n system , DMY was identified as theY-specifi c sex-determining gene ( M a t su d a et al. 2002;M a t su d a 2005) . T his gen e encodes a p utative tran-scriptio n factor co ntai ning a D M domain , which wasoriginally described as a D N A-bin ding motif fou n d intwo proteins, DS X in Drosophila melanogaster an d M A B-3in Caenorhabditis elegans (Ra ymo n d et al. 1998) . Verte-

brates have several D M domai n-containing genes an done of these, DMR T1 ( D M-related transcription factor1) has been im plicated in male sexual develop men t inmam mals, birds, reptiles, an d fishes (Ra ymo n d et al.1999; Smi t h et al. 1999; D e G r a n d i et al. 2000; G u a net al. 2000; Ke t t l e w e l l et al. 2000; M a r c h a n d et al.2000) . T he c D N A sequences of the medaka DMY an dDMR T1 showed a high similarity ( 80% ) an d DMYappears to have arisen through a gene d uplication eventof an autosomal DMR T1 gene ( M a t su d a et al. 2002;N a n d a et al. 2002) .

I n co ntrast to the widespread distribution of Sry inmam mals, the DMY gene has not been detected , even inclosely related species. Although one sister species ofO . latipes has the DMY gene on a homologous Y chro-mosome ( M a t su d a et al. 2003) , the gen e has not beenfou n d in other O ryzias species ( K o n d o et al. 2003) .Fishes in the gen us O ryzias have been divided into threemonop hyletic species groups, the latipes, javanicus, an dcelebensis groups ( T a ke h a n a et al. 2005) an d a recentp hylogen etic analysis of DMY an d DMR T1 genes fromO ryzias species suggested that duplication of the DMR T1gene (gen erating the DMY gene) appears to have oc-curred within the latipes group lineage ( K o n d o et al.2004) . T hese fi n dings suggested that O ryzias fishes inother species groups ( javanicus and celebensis groups)must have different sex-determining genes. Accordingly,comparisons between closely related medaka fishes with

1 T hese authors contributed equally to this work.2Corresponding author: Department of E nvironmental Science, Faculty

of Science, Niigata U niversity, 8050 Ikarashi-2, N iigata, 950-2181, Japan.E-mail: yusuke@env.sc.niigata-u.ac.jp

G en etics 175: 1335–1340 ( March 2007)

differen t sex-determi natio n mechan isms sh ould be im-portant in u n derstan ding h ow these diverse develop-mental mechan isms evolve.

I n this study, as a fi rst step to identify a sex-deter-mining gene in other fish species, we investigated thesex-determination system an d sex chromosomes in O .dancena, a member of the javanicus grou p ( T a ke h a n aet al. 2005) . Because a previous cytogen etic study hasnot reported heteromorp hic sex ch romosomes in th isspecies (as O . melastigma) ( U w a et al. 1983) , a geneticanalysis of sex-reversed in divid uals was per formed todemonstrate whether this species has an X X / X Y systemof sex determinatio n . Moreover, we took advantage ofgenomic tools established in O . latipes to isolate sex-lin ked D N A markers, map the sex-determining locus,an d identify th e sex ch romosomes.

M A T E R I A L S A N D M E T H O D S

F ish: All fish used in this study were sup plied from asubcenter ( N iigata U niversity) of the N atio nal BioResourceProject ( medaka) in Japan . Wild stocks of O . dancena wereoriginally collected at C hidambaram ( C B ) , I n dia, in 1981 an dat Ph uket (PK ) , T hailan d , in 1988 (for details, see T a ke h a n aet al. 2005) . Fish were maintained in aquaria u n der an artificialp hotoperiod of 14 hr light:10 h r dark at 27 6 2 .

Sex steroid treatments f or sex reversal and progeny testing:T he sex-reversal experiment was per formed as previously de-scribed by H ama g u c h i et al. (2004) . Brie fly, fertilized eggs ofO . dancena ( C B ) were incubated in water containing estradiol-17b ( E2; Sigma, St. L ouis) at 0.01, 0.04, an d 0.2 mg / ml ormethyltestosterone ( M T; Sigma) at 0.001, 0.005, an d 0.025mg / ml. H atched fry were transferred to normal tap water an dfed on a com mercial pet-food diet u ntil sexual maturatio n . Sexof the treated fish was ju dged from seco n dary sex character-istics an d treated fish were subsequently mated with normalfish . Sexing of F1 progeny from the mating was carried out byhistological cross-sections of fry go nads, sam pled at 20 daysafter hatching.

G enetic crosses: By crossing a C B female an d ( C B female 3PK male) an F1 male, 45 backcross progeny were obtained forgen otyping. Phen otypic sex was determined by secon dary sexcharacteristics of adult fish an d recon firmed by visual exam-ination of the go nads. We fi xed ad ult fish in 100% ethan ol an disolated their gen omic D N A from some m uscle tissue using thePI-50 isolatio n system ( K urabo) .

Isolation o f sex-linked D N A markers and linkage analysis:We searched for sex-lin ked markers using expressed sequencetag ( EST ) markers mapped to each of the O . latipes chromo-somes [lin kage grou p ( L G ) ] ( N a r use et al. 2004) . ESTs weream plified using previously p ublished primers designed forO . latipes (su pplemental Table 1 at http: // www.genetics.org /sup plemental / ) . PC R gen otyping for two loci, BJ014360 an dBJ732639, was per formed using primers that we designed o nthe basis of the EST sequences ( http: // mbase.bioweb.ne.jp / dclust / medaka_top .html) an d the draft gen ome sequence

( http: // dolp hin .lab.nig.ac.jp / medaka / ) of O . latipes. PC R am-plification of each EST marker was per formed in a totalvolu me of 10 ml for 3 min at 95 followed by 35 cycles of 10 secat 95 , 30 sec at 55 –60 , 60 sec at 72 , with a fi nal elo ngatio nstep of 3 min at 72 . Restrictio n fragment length polymor-p hisms (RF LPs) in PC R-am plified fragments of the parents( C B female an d PK male) were analyzed by polyacrylamide gelelectrop h oresis. For those markers showing a polymorp hic

patter n between the parents, F1 an d backcross progeny weregenotyped an d assessed as to whether such polymorp hismssegregated with p henotypic sex. U sing these isolated sex-lin ked ESTs, a sex-lin kage map of O . dancena was constructed .

F luorescence in situ hybridization analysis with f osmid andB A C clones: A fosmid gen ome library of O . dancena wasco nstructed from an F1 male between C B an d PK using theC opy C o ntrol Fosmid L ibrary Production kit ( E picentre,Madiso n , WI ) . Fosmid clo nes containing sex-lin ked ESTs werescreened by colo ny hybridizatio n . A fosmid clo ne O d38_01(containing BJ014360) was used as a probe. A bacterial ar-tificial ch romosome ( B A C ) genomic library, constructed fromthe H d-rR strain of O . latipes ( M a t su d a et al. 2001) , was alsoscreened an d three clones, M d0173J11 (containing SL1) ,M d0172B19 (containing DMR T1) , an d M d0172I07 (co ntain-ing O Ld17.11a ) , were isolated . T hese B A C clones were located ,respectively, o n the sex ch romosomes ( L G 1) an d on auto-somes ( L G 9 an d L G 10) in O . latipes.

Metaphase cells from cultured caudal fi ns were prepared bystandard cytogenetic methods ( U w a and O j ima 1981; Ma t su d aet al. 1998) . Fluorescence in situ hybridizatio n ( F IS H ) was per-formed as described ( M a t su d a an d C h apma n 1995) . ProbeD N As of the genomic clones were labeled separately by nicktranslatio n using biotin-16d U T P (Roche, I n dianapolis) ordigoxigenin-11-d U T P (Roche) . For two-color hybridizatio n ,equal amou nts of labeled probes an d a 500-fold amou nt ofautoclaved gen omic D N A of O . latipes were mixed with hybridi-zation solutio n an d prean nealed for 30 min at 37 . After over-night hybridizatio n , probes were detected with avidin–F I T C(Roche) an d rh odamine-labeled anti-digoxigenin antibodies(Roche) . Slides were cou nterstained with 49,6-diamidino-2-p henylin dole ( D API ) an d examined u n der a N ikon E clipse 80imicroscope using three filters ( U V-1A , B-2A , an d G-2A ) .Images were captured with a D X M1200C digital camera ( N ikon,Tokyo ) .

R E S U L T S

Progeny testing o f sex-reversed fish: Sex ratios of th ehormo n e-treated fish are shown in Table 1. T he sex ra-tio of the M T-treated group was biased toward male,suggesting that M T in duced sex reversal from female tomale. I n the groups reared u n der normal co n ditions,the sex ratio was almost 1:1 ( data n ot shown ) . T here-fore, the M T-treated group was expected to include sex-reversed males whose genetic sex is female ( X X or ZW ) .O n the other han d , th e sex ratio of the E2-treatedgroups did not deviate toward females, suggesting thatE2 could not reverse the sex of th e fish from male tofemale at conce ntratio ns of 0.01–0.04 mg / ml. I n addi-tion , all fish died at a concentration of 0.2 mg / ml E2before or just after hatch ing ( data not sh own ) .

O f 20 mature males obtained by the 0.025 mg / ml M Ttreatme nt, 11 were ran domly selected an d subjected toa progeny test by matings with nor mal females. T he sexratio of the offspring from each mating is shown inTable 2. As a result, 5 of 11 males from the M T-treatedgroup yielded all-female progeny, demonstrating thatthese males were sex-reversed X X males. T his resultreveals th at O . dancena has a male-heterogametic ( X X /X Y) sex-determi nation system .

Sex-linkage map o f O . dancena: To isolate sex-lin kedD N A markers in O . dancena, we searched for RF LPs

1336 Y. Takehana et al.

between the parents using ESTs established in O . latipesan d genotyped F1 an d backcross progeny. We scree ned397 ESTs an d identified 65 polymorp hic markers. L in k-age analysis showed th at 8 of th ese 65 markers segre-gated with the p hen otypic sex. T he female parent wash omozygo us, an d th e male parent an d / or F1 male wereheterozygous in th ese markers. I n the backcross prog-eny, males had the pater nal gen otype, while femaleshad th e mater nal gen otype, co n fi rming an X X / X Y sexdetermination system ( Figure 1) .

U sing eight isolated EST markers, we co nstructeda sex-lin kage map of O . dancena an d sh owed th at thesex-determi ning (SD) locus was mapped to the sameposition wit h an EST, BJ014360 ( Figure 2) . O n the basisof the draft genome sequence data of O . latipes ( http: //dolp hin .lab.n ig.ac.jp / medaka / ) , all eigh t ESTs werelocated on L G 10 in O . latipes. C om pariso n of geneorder between the O . dancena sex-lin kage map an d thep hysical map of O . latipes L G 10 in dicated a conservedsynteny between the two ch romosomes. T hese resultssuggest that the sex ch romosomes in O . dancena arehomologous to an autosome ( L G 10) of O . latipes, wh osesex chromosomes are L G 1.

I n this sex-l in kage map , the total map length was 23 cM in male meiosis. T he correspo n ding region of

O . latipes has a similar length (24.4 cM ) ( http: // dolp hin .lab.n ig.ac.jp / medaka / ) , suggesti ng that recombinatio n

along the sex ch romosomes is not suppressed in O .dancena. I n deed , we obtained 4 / 45 (8.9%) recombi-nants between MF01SSA032 H 09 an d BJ732639, wh ichfl an ked th e SD locus. O n th e basis of the draft genome

T A B L E 1

Sex ratio o f the hor mone-treated fish

C oncentratio n (mg / ml) Male Female Total

Methyltestostero ne0.001 20 2 220.005 30 5 350.025 20 0 20

Estradiol-17b0.01 10 12 220.04 10 9 19

T A B L E 2

Sex ratio of the offspring of mating between a methyltestosterone-treated male and a nor mal f emale

Mating n o. Male Female Total Remark

1 0 8 8 Sex-reversed X X male2 18 18 363 17 16 334 9 14 235 16 21 376 15 13 287 0 43 43 Sex-reversed X X male8 14 19 339 0 47 47 Sex-reversed X X male10 0 47 47 Sex-reversed X X male11 0 57 57 Sex-reversed X X male

F i g u r e 1.—L in kage analysis of EST markers in Oryziasdancena. E lectrop h oretic patterns of MF01SSA032 H 09 PCRproduct digested with H infI ( A ) an d BJ014360 PC R productdigested with H haI ( B ) are sh own . G enomic D N A from female( F ) an d male ( M ) fish was used as tem plates for PCR. Abbre-viatio ns: P, parents ( C B female an d PK male); F1, F1 progenyfrom the parents; B C1, backcross progeny. N ote that thedigested ban ds (arrowheads) segregated per fectly with B C1males.

F i g u r e 2.—C om pariso n of gene order between a sex-linkage map in Oryzias dancena and a physical map of L G 10in O . latipes. L ines between the com pared chromosomes con-nect positions of orth ologous gene pairs in these two species.T he distances between fran king markers are shown in p hysi-cal length (left) an d in centimorgans (right) . Map positionsfor genes an d distances in O . dancena were derived from thisstudy; those in O . latipes were obtained from the draft genomesequence data ( http: // dolp hin .lab.nig.ac.jp / medaka / ) .

Sex C h romosome Evolutio n in O ryzias 1337

sequence, this regio n was calculated to be 3.0 Mb in O .latipes. T hese fi n dings suggest a high recombination fre-quency aroun d the sex-determining regio n in O . dancena.

I denti fi cation o f sex chromosomes by F IS H map-ping: T he karyotype of O . dancena co nsists of 24 acro-centric chromosome pairs with n o heterom orp hic sexch romosomes ( U w a et al. 1983) . To iden tify the sexch romosomes in O . dancena, we co n d ucted a F IS H anal-ysis of male ch romosomes using a fosm id clo n e con-taining BJ014360 as a probe, which is tightly lin ked tothe SD locus. C lear hybridizatio n signals were observedon th e mid dle positio n of an acrocen tric ch romosomepair, iden tifying the cryptic sex ch romosomes ( Figu re3A ) . We also fou n d sex ch romosome localizatio n of theO . latipes B A C clo n e ( M d0172I07) co ntai ning anothersex-lin ked EST, O Ld17.11a. Two-color F IS H in dicatedthat the BJ014360 signal was located more proximal tothe centro mere th an the O Ld17.11a signal o n th e sexchromosomes ( Figure 3B ) . Morp h ological differencesbetween the X an d Y ch romosomes were n ot observedin this study, in dicati ng that O . dancena has h omomor-p hic sex chromosomes.

O n the oth er han d , th e B A C clon e ( M d0173J11) , con-taini ng an O . latipes sex-chromosomal marker (SL1) , wasn ot located on the sex chromosomes in O . dancena ( Fig-ure 3C ) , con firming that sex chromosomes are differen tbetween these two species. F urther more, the hybridiza-tion signals of the DMR T1-positive B A C ( M d0172B19)were detected only on a pair of autosom es ( Figure 3D ) .I n O . latipes, previous F IS H analyses revealed theprese nce of DMR T1 at the tip of a pair of autosomes,as well as at o ne of th e sex chromosome pair ( namely th eDMY gene on th e Y chromosomes) ( K o n d o et al. 2004,2006) . Although we fou n d a similar F IS H patter n to thatof autosomal DMR T1 in O . latipes, the additio n al Y-chromosomal location (corresp o n ding to the DMY) wasnot observed in O . dancena. H ence, th ese results stro nglysuggest th at Y chromosomes of O . dancena an d O . latipesare not homologous.

D I S C U SS I O N

O . dancena has an X X / X Y sex-deter mination system:It has been demo nstrated th at, o n the basis of sex ratiosin th e progeny of sex-reversed fish an d segregationpatter ns of sex-lin ked D N A markers, O . dancena in th ejavanicus species group has a male-heterogametic ( X X /X Y) sex-determin ation system . A previous study showedthat all four fishes in the latipes group also have anX X / X Y sex-determi nation system ( H ama g u c h i et al.2004) ( Figure 4). Two of these four species, O. latipes andO . curvinotus, have th e com mon sex-determin ing gene,DMY, on the homologous Y ch romosomes ( M a t su d aet al. 2002, 2003) . O n the other han d , the gene has n otbeen detected in the oth er two species, O . luzonensis an dO . mekongensis ( K o n d o et al. 2003, 2004) . O n the basis ofthese fi n dings with a p hylogen etic analysis of DMY an dDMR T1, K o n d o et al. (2004) suggested th at th e DMYgene appears to have occurred in th e com mon ancestorof O . latipes, O . curvinotus, an d O . luzonensis. T herefore,the DMY gene is considered to be recently lost in O .luzonensis an d originally abse nt in O . mekongensis an d inother O ryzias species, such as O . dancena. T his suggeststhat different sex-determin ing genes have evolved in

F i g u r e 3.—F IS H analysis of male metap hase ch romosomesin Oryzias dancena using O . dancena fosmid an d O . latipes B A Cclo nes. ( A ) F IS H map ping of a fosmid clo ne ( O d38_01) co n-taining a sex-lin ked EST, BJ014360. A specific hybridizatio nsignal (red ) was located o n a h omomorp hic acrocentric ch ro-mosome pair. ( B ) G ene ordering of the sex-lin ked ESTs,BJ014360 (fosmid O d38_01) and OLd17.11a (BAC Md0172I07),o n the sex chromosomes. T he locatio ns of the markers arevisualized as red an d green signals, respectively. Arrowheadsin dicate the centromere positio ns. ( C ) C hromosomal loca-tio n of the O . latipes sex-ch romosomal marker SL1 ( B A CM d0173J11, green ) an d the O . dancena sex-ch romosomalmarker BJ014360 (fosmid O d38_01, red ) . ( D ) C hromosomallocatio n of the DMR T1 gene ( B A C M d0172B19, green ) withthe BJ014360 fosmid , O d38_01 (red ) .

F i g u r e 4.—Phylogenetic relationships and sex-determinationmechanisms in O ryzias fishes. T he p hylogenetic informationwas taken from T a ke h a n a et al. (2005) . D ata of sex-determi-natio n mechanisms in O . latipes, O . curvinotus, O . luzonensis,and O. mekongensis were obtained from Ma t su d a et al. (2002,2003) an d H ama g u c h i et al. (2004) .

1338 Y. Takehana et al.

O ryzias species, alth ough male heterogamety is likely tobe com mo n in th e gen us.

I denti fi cation o f sex chromosomes in O . dancena:O ryzias species with n o DMY gene m ust have differentsex chromosomes from th ose in O . latipes; h owever, nosex chromosomes have been identified in these speciesso far. I n this study, we scree ned sex-l in ked markers of O .dancena using ESTs established in O . latipes. As a result,most ESTs primers worked well in O . dancena, an d wecould successfully isolate eight sex-lin ked EST markers.L in kage analysis sh owed that these ESTs are located o nan autosome ( L G 10) in O . latipes with the same geneorder, in dicating a co nserved synteny between sex ch ro-mosomes in O . dancena an d an autosome of O . latipes. I nad ditio n , F IS H analysis co n fi rmed th at sex chromo-somes in O . dancena an d O . latipes were n ot h omologous.T hese results in dicated an in depen de nt origin of X / Ych romosomes in th ese two species, suggesti ng th at an ovel sex-determi ning gen e is located o n the sex chro-mosomes in O . dancena.

T he lack of conservatio n of sex ch romosomes amo ngclosely related fish species is probably com mon . Al-th ough all salmo nid fishes have a male heterogameticsystem , closely related species have evolved differentsex chromosomes, as evidenced by a com parative lin k-age analysis (W o r am et al. 2003) an d F IS H analyses(P h i l l ips et al. 2001, 2005) . I n sticklebacks, Gasterosteusaculeatus an d G. wheatlandi also ap pear to have differentX / Y sex chromosomes, an d Apeltes quadracus has hetero-morp hic Z / W sex ch romosomes (Pe i c h e l et al. 2004) .T hese stu dies suggest th at fishes may use a variety ofsex-determi ning genes.

Sex-deter mining region in O . dancena: L in kage anal-ysis mapped the SD locus between MF01SSA032 H 09and BJ732639 and showed that BJ014360 is tightly linkedto the SD locus wit h n o recombinan ts (0 / 45) . T his sug-gests that a single ch romosomal regio n co ntrols sexin O . dancena. F IS H analysis in dicated that th e sex-determining regio n is located o n the mid dle of oneacrocentric chromosome pair, in accordance with thelin kage analysis. T herefore, o nly th is regio n should bedifferent between X an d Y ch romosomes.

Stan dard models of sex ch romosome evolutio n hy-pothesized that the fi rst step is th e occurrence of a novelsingle locus o n an autosom e, in which heterozygosityleads to the develop men t of o ne sex an d h omozygosityto the other sex, thereby establish ing a protosex chro-mosome system . H eteromorp h ic sex ch romosomes areco nsidered to arise th rough the recombination isola-tio n between such h omologous sex ch romosomes. T hissuppressio n of recombinatio n main tained o ne chromo-some (such as Y) in a co nstan t heterozygous state in o nesex an d the subsequent degeneratio n process spreadsth e sex-specific regio n over almost the entire chromo-some, as in mam mals (reviewed in G r a ves 2006) .

O ur results in dicate th at sex ch romosomes in O .dancena are at an early stage of evolutio n , as in O . latipes.

First, p he notypic sex was easily converted by sex hor-mones, an d the resultant sex-reversed fish were fullyfertile. F urtherm ore, YY males have been obtainedfrom estroge n-in duced X Y females (Y. T a ke h a n a an dD . D emi y a h , u n published results) , in dicating viabilityof YY in dividuals. Seco n d , sex-ch romoso mal crossingover occurred over almost the entire length of the chro-mosome, in dicating that this recombini ng sectio n isconsidered to be a pseudoautosomal region . In addition,the region between MF01SSA032 H 09 an d BJ732639,which flanked the SD locus in O. dancena, was calculatedto be 3.0 Mb in O. latipes, on the basis of the draft genomesequence. T his suggests th at the Y-specifi c region ofO . dancena is likely to be very small. Finally, F IS H analy-sis demonstrated that th e Y chromosome is n ot cyto-genetical ly distinguished from the X , although thesex-determi nation system in the species is male het-erogametic. Taken together, these fi n dings suggest thatthere are no functional differences between X and Y sexchromosomes, other than the sex-determin ing gene.

O n ly two sex-determining gen es, Sry an d DMY, havebeen identified in vertebrates so far. I n this study, wehave shown that O . dancena should have a novel sex-determi ning gene o n the sex chromosomes homolo-gous to an autosome ( L G 10) in O. latipes. O ne approachto identify sex-determining genes is positional clon-ing. T his method has been successfully used to isolateh u man an d medaka sex-determining genes. Two majorcon ditions are required for using this method: a firmgenetic sex determinatio n an d a feasibility of geneticmapping for the SD locus ( M a t su d a 2005) . O . dancenahas a strict genetic sex determination ( X X / XY system)an d a high recombinatio n frequency arou n d th e SDlocus, satisfyi ng both requiremen ts. I n addition , O .dancena an d O . latipes share a similar ch aracter of sexchromosomes, a very small Y-specific region , suggestingthat th is method will also be effective in O . dancena.C onstruction of a high-resolutio n recombinatio n maparound the SD locus and chromosome walking to the SDlocus are necessary steps to isolate the sex-determininggene using the positio nal clo ning method . T his is cur-rently in progress. C om parative analyses of sex-deter-mining gen es an d sex chromosomes amo ng O ryziasspecies will prove useful in u n raveling the evolutio n ofsex-determi nation mechanisms in vertebrates.

We than k Yoichi Matsuda, Takahiro M urakami ( H okkaido U niver-sity) , an d Masaru Matsuda ( N atio nal I nstitute for Basic Biology) fortheir helpful tech nical advice o n F IS H analysis an d Wichian Magtoon(Srinak harinwirot U niversity) for his generous help in the collectionof materials. T his work was sup ported in part by a grant-in-aid forScientific Research from the Ministry of E ducatio n , C ulture, Sports,Science, an d Tech nology of Japan (16370094) to M .S.

L I T E R A T U R E C I T E D

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Sex C h romosome Evolutio n in O ryzias 1339

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C om m u nicating editor: S. Yo k o y ama

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