全 文 ：广 西 植 物 Guihaia Jan．２０１４,３４(１):３４－３７ http://journal．gxzw．gxib．cn
DOI:１０．３９６９/j．issn．１０００Ｇ３１４２．２０１４．０１．００７
卢然然,杨振艳,陶程程,等．中国西南地区特有水生植物海菜花微卫星引物开发[J]．广西植物,２０１４,３４(１):３４－３７
LuRR,YangZY,TaoCC,etal．MicrosatelitesprimerdevelopmentforOteliaacuminata (Hydrocharitaceae),asubmergedmacrophyteendemicto
southwesternChina[J]．Guihaia,２０１４,３４(１):３４－３７
MicrosatelitesprimerdevelopmentforOteliaacuminata
(Hydrocharitaceae),asubmergedmacrophyte
endemictosouthwesternChina
LURanＧRan１,２,YANGZhenＧYan１,TAOChengＧCheng１,２,
CHENShaoＧTian１,JIYunＧHeng１∗
(１．KeyLaboratoryofBiodiversityandBiogeography,KunmingInstituteofBotany,ChineseAcademyofSciences,
Kunming６５０２０１,China;２．UniversityofChineseAcademyofSciences,Beijing１０００４９,China)
Abstract:Oteliaacuminata(Gagnepain)Dandy(Hydrocharitaceae),isasubmergedmonocotendemictosouthwestＧ
ernChina．UsingthefastisolationbyAFLPofsequencescontainingrepeats(FIASCO)protocol,ninepolymorphic
microsatelitelociwereidentifiedbythegenotypingoffortyＧfiveindividualsfromthreenaturalpopulations．ThenumＧ
berofaleles(NA)perlocuswithinpopulationsvariedfromonetothree．Theobservedandexpectedheterozygosities
rangedfrom０．０００to０．９３３and０．０００to０．６０５,respectively．Thesemicrosateliteprimerscanbeusedinfuturestudies
onthephylogeographyandecologicalgeneticsofO．acuminata．
Keywords:Hydrocharitaceae;Oteliaacuminata;polymorphic;microsatelite
GLCNumber:Q９４９ DocumentCode:A ArticleID:１０００Ｇ３１４２(２０１４)０１Ｇ００３４Ｇ０４
中国西南地区特有水生植物海菜花微卫星引物开发
卢然然１,２,杨振艳１,陶程程１,２,陈绍田１,纪运恒１∗
(１．中国科学院昆明植物研究所 生物多样性与生物地理学重点实验室,昆明６５０２０１;２．中国科学院大学,北京１０００４９)
摘 要:水鳖科(Hydrocharitaceae)海菜花(Otteliaacuminata)是中国西南地区特有的水生单子叶植物.基
于AFLP技术的磁珠富集快速分离技术(FastIsolationbyAFLPofSequencesContainingRepeats,FIASＧ
CO),共筛选出９对多态性引物并对３个居群４５个个体进行分析.结果表明:三个居群的等位基因数目为
１~３个,观测杂合度从０．０００~０．９３３,期望杂合度从０．０００~０．６０５.这些筛选出的微卫星引物将用于海菜花
后续的谱系地理学和生态遗传学研究.
关键词:水鳖科;海菜花;多态;微卫星
Oteliaacuminata (Hydrocharitaceae),asubＧ
merged monocotendemicto southwestern China
(Guizhou,Sichuan,Yunnan,andGuangxiprovinces),
isscatteredintheplateaufreshwaterlakes,ponds,and
streamsamongthedrainageareasoftheUpperYanＧ
gtze,Pearl,Mekong,andSalweenRivers(Li,１９８１)．
Thisplantisdioecious,anduseshydrochoryforseeds
dispersal(Jiangetal．,２０１０)．O．acuminataisanecoＧ
nomicalyandecologicalyimportantplant．TheinfloＧ
rescenceisafamoustraditionalvegetableinYunnan．
收稿日期:２０１３Ｇ０５Ｇ０９ 修回日期:２０１３Ｇ０８Ｇ２３
基金项目:国家高技术研究发展计划项目(２０１０AA０６Z３０１);国家自然科学基金(３１０７０２９７,３１０７０１９８).
作者简介:卢然然(１９８８Ｇ),女(蒙古族),内蒙古赤峰人,硕士研究生,植物学专业,(EＧmail)luranran＠mail．kib．ac．cn.
∗通讯作者:纪运恒,副研究员,硕士生导师,从事植物地理学、植物分类学以及植物保护生物学等研究,(EＧmail)jiyh＠mail．kib．ac．cn.
Duetoitsextremesusceptivitytothewaterpolution,
theplanthasbeenusedasanindicatortomonitorwaＧ
terdeteriorationinplateaulakes(Li,１９８１)．Forthe
pastfewyears,becauseoftherapidlossofthenatural
populations,O．acuminatahasbecomeanendangered
plantandbeenlistedintheChinesePlantRedBook
(Fu,１９９２)．Moreover,thesexratioofO．acuminatais
notconstantlyequal１∶１innaturalpopulations,with
thevariationfrom maleＧtofemaleＧbiased,andanenＧ
tirelymalepopulations(propagationbybulbswithin
themalespathe)werealsodocumented(Li,１９８１)．
Therefore,thisplantprovidesusanidealsystemtoinＧ
vestigatetheimpactsofdrainageonthegeneticdiverＧ
gence,andtheecologicalgeneticsofsexratiosinnatuＧ
ralpopulations．
Recently,studiesonclassifyingthepopulations,
morphologicalvarietiesandtheevolutionprocessofO．
acuminatawerereported(Heetal．,１９９１;Zhaietal．,
２０１０;Longetal．,２０１０),andalthesewilbegood
contextforfurtherinvestigationonitsecologicalgenetＧ
icsandphylogeography．OurobjectiveherewastodeＧ
velopasetofnewmicrosatelitesforO．acuminata,in
ordertofacilitatethefurtherresearchonitspatternof
geneticdiversity．
１ Materialandmethods
１．１Plantmaterials
TheplantmaterialsofO．acuminatawerecolectＧ
edfrom Yunnan,GuizhouandGuangxi,respectively．
Thesepopulationsincludedeach１５individualsfrom
theHeilongtanSpring(２６°３５′N,１００°１１′E,Xinhua
Vilage,JianchuanCounty,YunnanProvince),theCaoＧ
haiLake(２６°５１′N,１０４°１６′E,WeiningCounty,
GuizhouProvince),andtheJiangxiwanpopulation(２５°
０６′N,１０９°４４′E,YongfuCounty,GuangxiProvince)
(Table１)
１．２DNAExtraction
TotalgenomicDNAwasextractedfromsilicaＧgelＧ
driedleavesfolowingtheCTABprotocoldescribedby
Doyle(１９９１)．
１．３IsolationofMicrosateliteLoci
WeusedthefastisolationbyAFLPofsequences
containingrepeats(FIASCO)protocol(Zaneetal．,２００２)
Table１ SamplinglocationsforOtteliaacuminata(Hydrocharitaceae)
Population VoucherNo． Colectiondate Longitudeandlatitudeofsamplinglocation Elevation(m) Habitat
HeilongtanSpring LYJ００４ Sep１７,２０１０ ２６°３５′９″N,１００°１１′２２″E ２１９８ Spring
Caohai JH００９ Aug６,２００９ ２６°５１′７″N,１０４°１６′３″E ２１６０ Plateaufreshwaterlake
Jiangxiwan LYW００５ Mar１３,２０１１ ２５°０６′N,１０９°４４′E ２３３ Stream
todevelopmicrosatelitemarkersforO．acuminata．
GenomicDNAweredigestedwithMseＧIrestrictionenＧ
zyme(NewEnglandBiolabs,Ipswich,Massachusetts,
USA)at３７℃for３handthenligatedthefragments
totheMseＧIadaptorpairs(５′ＧTACTCAGGACTCATＧ
３′/５′ＧGACGATGAGTCCTGAGＧ３′)withT４DNAligＧ
ase(Fermentas,Burlington,Ontario,Canada)at３７℃
for２h．Theseproductswereamplifiedaccordingtothe
folowingprotocol:３minat９５℃,２０cyclesof３０sat
９４℃,１minat５３℃,１minat７２℃,andafinalexＧ
tensioncycleof７minat７２℃．ThePCRproducts
weredetectedbyagaroseelectrophoresisandthose
fragmentsranged２００Ｇ８００bpwerepurifiedwithan
agarosegelextractionkit(Sangon,Shanghai,China)．
ThepurifiedDNA wasenriched with(AG)１５ and
(AC)１５ biotinylated microsateliteprobes．Thenwe
isolatedthefragmentswhichcontainingmicrosatelite
repeatsbymagnesphere．ThesefragmentswererecovＧ
eredandamplified with MseＧIＧN primer(５′ＧGATＧ
GAGTCCTGAGTAANＧ３′)withthesamePCRproＧ
grammentionedabove,butwith３０cyclesandalast
extensioncycleof８minat７２℃here．
Theproductswerepurifiedandthenclonedinto
thepGEMＧTvector(Promega,Madison,Wisconsin,
USA),andtransformedintoTrans１ＧT１PhageResistＧ
antChemicalyCompetentCels(Quanshijin,Beijing,
China)．ClonescontainingtheinsertwereselectedacＧ
cordingtoamethodofblueＧwhitescreening,andculti
vatedinanincubatorat３７℃for１２h,thendetected
byPCRwiththeprimerpairs(AG)１０/(AC)１０and
５３１期 卢然然等:中国西南地区特有水生植物海菜花微卫星引物开发
Table２ CharacterizationoftwentymicrosateliteprimersforOteliaacuminata
Locus Primersequence(５′Ｇ３′) Repeatmotif Expectedlength(bp) Ta(℃) GenBankaccessionNo．
oa０２ FＧATTCGACCGTACTGTACTCTG (AG)１５ １４４ ５６ JN８６２９７１
RＧGGTAGCCCTTGCCTTTT
oa０３ FＧGAGGACGGTCGGATATTGT (CT)８ １８５ ５６ JN８６２９７２
RＧAATGACCTCCAGTCTTTGC
oa０７∗ FＧGACCTCAGGGCCTTCACTTT (GA)１０(TCC)４ １９０ ５７ JN８６２９７３
RＧTTGGAGGATTGGCACGA
oa１２∗ FＧCATCTGAGAATGGCTTGG (CA)３CC(CA)５ ２７９ ５７ JN８６２９７４
RＧCCGAATTGGAGCCTGTA
oa１３ FＧGCGGTGAATAGAGGGTGAA (GT)６ ２５６ ５６ JN８６２９７５
RＧGCTAGGATAATGACTGCCAAC
oa１５ FＧAGTACACGGGACTCACAAA (CA)５ ２３０ ５６ JN８６２９７６
RＧTAGCTTGGATTAGCAGGAG
oa２２∗ FＧGGCACCATAACTGGACTAAA (GT)５ １５０ ５９ JN８６２９７７
RＧTATCAGCGAGCGGGATT
oa２３∗ FＧTGGTGAATCGGGAGTTTGT (GT)５ １５７ ５９ JN８６２９７８
RＧAAGGAGGAGATGGATACGAGA
oa２５ FＧTACAGCGGTCATCGTTTG (CA)６ １４２ ５７ JN８６２９７９
RＧAGCGTGAATTAGCAGGAG
oa３０ FＧTTACATCTGTTGTCGCCTCG (TC)９ ２００ ５５ JN８６２９８０
RＧGAAATACGCCATTTGCTCCT
oa３５ FＧCATGTGGACCATTGGATTTG (TC)７ ２４５ ６０ JN８６２９８１
RＧAAGCACCGAAGAAGCGTAG
oa３６∗ FＧCCCTTGTCTTCGCTGGTTT (GAG)２(GA)２(AGGAG)２ ２６０ ５３ JN８６２９８２
RＧCACCTCCATCATCCTCACTTC
oa３７∗ FＧTGAGTGCGTGAGTGAGTCGA (TG)３(GT)３G(CT)２(TG)２ １１０ ５６ JN８６２９８３
RＧCACCTTCTCCGTTTCATTTC
oa４４ FＧAGGTAGCCCTAGCATTTGA (CT)５ ２４６ ５５ JN８６２９８４
RＧATCTCCTGGTCTCGTCTCAC
oa６３∗ FＧGCCCCTTCCTGAGCATCTG (CA)３CC(CA)５ １０４ ５０ JN８６２９８５
RＧCCCCGAATTGGAGCCTGTA
oa６６ FＧTTGCTGGACCATGAAGACC (CA)６ ２６６ ５２ JN８６２９８６
RＧGCGTGAATTAGCGGGAGAT
oa７０∗ FＧCGGTGAATAGAGGGTGAAG (GT)６ ２５４ ５５ JN８６２９８７
RＧCTAGGATAATGACTGCCAAC
oa７２ FＧGGACCATGAAGACCGAGGAT (CA)６ ２２３ ４８ JN８６２９８８
RＧTGAATCGAGTGCGGAGCGT
oa７３∗ FＧGAATTTGAGGACGGATTTG (TG)１０ １３４ ５５ JN８６２９８９
RＧTTCCAGCACTCACAATGTTT
oa７５ FＧGAGATCGAGATAACCAAGTC (GT)５A(TG)４ ３０３ ５０ JN８６２９９０
RＧTACAAAGAAAGACGACCAT
Ta:PCRannealingtemperature;∗indicatingpolymorphisms．
M１３F(５′ＧGTAAACGACGGCCAGＧ３′),and(AG)１０/
(AC)１０and M１３R(５′ＧGATGAGTCCTGAGTAANＧ
３′),respectively．
Atotalof２８７positivecolonieswereselectedtobe
sequenced．Inthosecolonies,１１１sequencescontaining
therepeatregionwereidentifiedbytheSSRITsoftＧ
ware(http://www．gramene．org/db/searches/ssrＧ
tool/)．ThesoftwarePrimerPremier５．０(PREMIER
BiosoftInternational,PaloAlto,California,USA)was
usedtodesignprimers,and８３pairsofprimerswere
designedandsynthetizedbyBGI(Shenzhen,GuangＧ
dong,China)．
１．４DetectionofPolymorphism
Polymorphismof microsatelites was detected
among４５individualsofO．acuminatafromthreenatＧ
uralpopulations．PCRamplificationwasperformedina
reactionvolumeof２５μL,containing１μLofgenomic
DNA,２．５μLof１０×PCRbufer,０．５μLofdNTP(２．５
μmoleach),０．５μLofeachprimer(１０μmol/L),and
０．３UofTaqDNApolymerase(Takara,Dalian,LiaonＧ
ing,China)．PCRamplificationswereperformedby３
minat９３℃;folowedby３５cyclesof３０sat９３℃,３０
sattheoptimizedannealingtemperature(Table２),and
３０sat７２℃;thenafinalstepfor７minat７２℃．The
PCRproductswereseparatedbythe８％sodiumdodeＧ
cylsulfatepolyacrylamidegel(SDSＧPAGE)．Andthe
fragmentsizesweredeterminedbyastandard２５Ｇbp
DNAladder(２５－５００bp)．
６３ 广 西 植 物 ３４卷
１．５DataAnalysis
GeneticsstatisticswereanalyzedusingGENEPOP
Version３．４software(RaymondandRousset,１９９５)to
computealelenumbers(NA),expectedheterozygosiＧ
ties(HE),observedheterozygosities(HO),anddeviaＧ
tionsfromtheHardyＧWeinbergequilibrium(HWE)．
２ Resultsanddiscussion
Foratotalof２０microsatelitesisolated,ninedisＧ
playedpolymorphism．DetailsofthesepolymorphicmicＧ
rosateliteswerelistedinTable２．Thenumberofaleles
perlocusvariedfromonetothreewithinpopulations．
AndthevaluesofHOandHEforHeilongtanSpring
populationrangedfrom０．０００to０．４００andfrom０．０００to
０．６０５,respectively．ThevaluesofHOandHEforCaohai
populationrangedfrom０．０００to０．９３３and０．０００to
０．６０５,respectively．AndthevaluesofHOandHEfor
Jiangxiwanpopulationrangedfrom０．０００to０．７３３and
０．０００to０．５１５,respectively(Table３)．HWEtestsreＧ
vealedthatfourloci(oa１２,oa２２,oa２３andoa７０)hadsigＧ
nificantlydeviatedfromtheequilibrium(P＜０．００１)．
Thisresultshowed muchlow polymorphism
withinthesethreepopulationsandrelativelyhighpolyＧ
morphismamongthem,likelyowingtothehomozyＧ
gotesaccountingforalargeproportionofindividualsof
thesethreepopulations,whichseemstoberesulted
fromthegeographicisolation．EachofthesethreepopＧ
ulationscomesfromadiferentriversystem．JianLake
locatesintheMekongRiver;Caohailocatesinthe
Table３ Geneticdiversityofninepolymorphicmicrosatelites
Locus
Heilongtanspring
NA Ho HE
Caohai
NA Ho HE
Jiangxiwan
NA Ho HE
oa０７ ２ ０．０６７ ０．１８６ ２ ０．２００ ０．４８１ １ ０．０００ ０．０００
oa１２ ２ ０．０００ ０．３３１∗ ２ ０．０６７ ０．０６７ １ ０．０００ ０．０００
oa２２ ２ ０．４００ ０．４６０ ３ ０．２６７ ０．６０５ ２ ０．０００ ０．１２９∗
oa２３ ３ ０．２００ ０．６０５∗ ２ ０．９３３ ０．５１５ ２ ０．７３３ ０．５０８
oa３６ １ ０．０００ ０．０００ ２ ０．２００ ０．４８１ １ ０．０００ ０．０００
oa３７ １ ０．０００ ０．０００ １ ０．０００ ０．０００ ２ ０．１３３ ０．２３９
oa６３ １ ０．０００ ０．０００ ２ ０．０６７ ０．１８６ ２ ０．０６７ ０．２８７
oa７０ ２ ０．０００ ０．１２９∗ ２ ０．０６７ ０．４３５∗ ２ ０．１３３ ０．５１５
oa７３ ２ ０．１３３ ０．１２９ ２ ０．１３３ ０．４９７ １ ０．０００ ０．０００
NA:thenumberofaleles;Ho:observedheterozygosity;HE:expectedheterozygosity;∗statisticaldeviationfromHardyＧWeinbergequilibrium(HWE)(P＜０．００１)．
ChinＧshaRiver,whichistheupperreachesoftheYanＧ
gtzeRiver;andJiangxiwanlocatesinthePearlRivers．
Thedistributionofaquatichabitatsmayinfluencethe
patternsofgeneticdiferentiationamongpopulations
(Spencer,１９９３)．Theselakesarediscontinuousand
longＧtermisolated,andO．acuminatahasbeenadapted
totherespectivelakehabitatsandproducedsomeenＧ
demicvariations(Li,１９８１),whichwerefixedineach
populationowingtothelackofhydrochorytofacilitate
seedflowamongpopulations．Therefore,ourresults
seemtoindicategeneticisolationbywatersystemin
O．acuminata．
３ Conclusions
Wereportedtheninepolymorphicmicrosatelite
locidevelopedinO．acuminata．Theywilfacilitatethe
studiesonthephylogeographyofthespecies,which
wilimproveknowledgeontheimpactsofdrainageon
geneticdiferentiationofaquaticplants．Furthermore,
theywilbeusefultoolsinfurtherstudiesonecological
geneticsofsexratiosofitsnaturalpopulation．
Acknowledgements Theauthorsaregratefulto
DrsYingYang,YaowuXing,TaoSu,andZhilingDao
fortheirhelpinsamplecolection．
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(下转第８３页Continueonpage８３)
７３１期 卢然然等:中国西南地区特有水生植物海菜花微卫星引物开发