全 文 ：GeneticDiversityinWildPopulationsofPoacynumhendersonifromInland
AridRegionsofNorthwestChina
ZHAOJin-feng1 , ZHANGWei-ming1, 2 , PENGXue-mei1 , TANGZe-zi1 , GUGong-ping2 , LUChang-mei1＊
CollegeofLifeSciences, NanjingNormalUniversityNanjing210046NanjingInstituteforComprehensiveUtilizationofWildPlants
Abstract　[Objective] StudyonthegeneticdiversityinwildpopulationsofPoacynumhendersoni.[ Method] RandomamplifiedpolymorphicDNA
(RAPD)techniquewasemployedtoanalyzethegeneticdiversityinfivewildpopulationsofP.hendersonisampledfromXinjiang, GansuandQinghai
provinces.[ Result] Totaly165clearandrepeatablebandsweregeneratedinRAPDreactionbyusing20primersscreenedfrom80primers, ofwhich110
werepolymorphic, accountingfor66.67%.Atspecieslevel, Neisgenediversityindex(H), Shannonsinformationindex(I)andgeneticdiferentiationcoeficient(Gst)were0.220 5, 0.304 7and0.908 2, respectively.P.hendersoniigermplasmresourcesshareahighlevelofgeneticdiversity, andgenetic
diferentiationmainlyexistsamongthepopulations.ResultsfromgeneticdistancesandclusteranalysisshowedthatrelationshipsamongP.hendersoniipop-
ulationsweretosomeextentrelatedwiththeirgeographicalandclimaticcharacters.[ Conclusion] ThisstudysuggeststhattheconservationofP.henderso-
nishouldfocusontheprotectionofmanypopulations, particularlytheQinghaipopulation.
Keywords　Poacynumhendersoni;Wildpopulation;Geneticdiversity;RAPD
Received:September12, 2008　　Accepted:September26, 2008
SupportedbyNationalScienceandTechnologyResearchItemin9th
FiveYearPlan(2004BA502B10);JiangsuProvincialHi-techIndustriali-
zationDevelopmentProject(BG2006318).
＊Correspondingauthor.E-mail:luchangmei@njnu.edu.cn
　　 Poacynum hendersoni(Hook.f.)Woodson, also
caledapocynum, isanannualherbaceousplantthatbelongs
toPoacynumBailofApocynaceae.Withdevelopedrootsys-
tem, P.hendersoni isendowed withextremelystrong
droughttolerance, coldtolerance, saline-alkalitoleranceand
galetolerance, andcangrowintheenvironmentwithbaren
soilthatthecommonplantscanhardlylive.InChina, P.
hendersonimainlydistributesintheinlandaridareasatthe
westofE104°, includingTarimBasininXinjiang, HexiCor-
ridorinGansu, Qaidam BasininQinghai.Anothertwo
plants, ApocynumvenetumandPoacynumpictum, alsoshare
thenameofapocynum.Duetothehighmedicinalvalue, A.
venetumhasbeenregardedasmedicineformorethanathou-
sandyearsandembodiedbypharmacopoeia;however, theo-
ver-exploitationinrecentyearshasrestrictedtheutilization
ofthisresource.Meanwhile, thelessamountindicatesthe
limitedexploitationprospectiveofP.pictumresource.With
thesharpfalofA.venetum, P.hendersoniasitssubstitute
hasbeengradualyunderstood.Moreimportantly, P.hend-
ersonigrowsinnorthwestsaline-alkalidesert, soitistheex-
celentspeciesforsandcontrol, aforestationandsaline-alka-
lilandtransformation.Protectionandutilizationofwildre-
sourcesofP.hendersonihavebeengradualystrengthened,
largescaleartificialplantationbasesinsomeregionsarebe-
ingestablished.ForutilizingP.hendersonisustainablyand
exertingitseconomicandecologicalvaluetothelargestex-
tent, itisessentialtorevealthepresentresourcesituationof
P.hendersoniinChinaatgeneticslevel.However, thereis
litlereportinvolvinginthegeneticdiversityandgenetic
structureofP.hendersoni.
RAPDtechniquehasbeenprovedtobeasimpleandef-
ficientmethodinstudyingthegeneticdiversityandgenetic
structureofplantpopulations[ 1] .Thus, weanalyzedthege-
neticdiversityandgeneticstructureoffivewildpopulations
ofP.hendersonibyusingRAPDmarkers, aimingtoprovide
theoreticalbasisandcountermeasuresfortheprotection, cul-
tivationandstandardizationplantationofP.hendersoniin
inlandaridarea.
MaterialsandMethods
Experimentalmaterials
P.hendersonimaterialsfortest(Table1)weresam-
pledbyScientificExpeditionTeamforInvestigatingtheA.
venetumResourcesinChina2005 organizedbyNanjingInsti-
tuteforComprehensiveUtilizationofWildPlants.Because
theP.hendersoniplantsmainlyreproduceviavegetative
propagationundernaturalconditions, foravoidingtherepeat-
edcolectionofsampleswithidenticalgeneticcompositions,
thedistanceofanytwosamplesusedthisstudyismorethan
1km.Thesampledleavespreservedaftersilicageldryness
andnaturaldryneswereidentifiedbyresearchfelowQIAN
Xue-sheandresearchfelowXIAOZheng-chunfromNanjing
InstituteforComprehensiveUtilizationofWildPlants.
Table1　Originsofthesamplesusedinthisstudy
No. Populationcode Origin Samplenumber
1 XJB Bachu, Xinjiang 5
2 XJK Korla, Xinjiang 5
3 GSY Yumen, Gansu 5
4 GSD Dunhuang, Gansu 5
5 QHG Germu, Qinghai 5
Experimentalmethods
IsolationanddetectionofgenomicDNA　Extractionof
genomicDNAwasreferedtothepreviousreportofourre-
searchgroup[ 2] .ThequalityandpurityoftheextractedDNA
wereobservedby0.8% agarosegelelectrophoresiswith
ethidiumbromide.
PrimerscreeningandPCRamplification　The80 RAPD
primersfortestwerepurchasedformShanghaiSangonBiolog-
AgriculturalScience＆Technology, 2008, 9(4):39-43
Copyright 2008, InformationInstituteofHAAS.Alrightsreserved. AgriculturalBiotechnology
icalEngineeringTechnology＆ServicesCo., Ltd.andnum-
beredwithA01-A20, B01 -B20, C01 -C20 andD01 -
D20.ThePCRreactioncomponentsinvolvedwerepurchased
fromShanghaiBiocolorBioscience＆ TechnologyCompany.
Onesamplefromeachpopulationwasrespectivelyusedas
templateforoptimizingreactionsystemandscreeningprim-
ers.Finalythereactionsystemwasdeterminedas:MgCl2
2.5 mmol/L, dNTPs5 pmol/L, templateDNA30 ng, Taq
DNApolymerase1.5 U, primer10pmol/L, ddH2Oupto25μl.Cyclingconditionswere4 minat94 ℃ forpredenatur-
ation, 42 cyclesof45sat94℃, 30sat36℃, 1 minat72
℃ andextensionat72 ℃ for7 min.PCRproductwasob-
servedunderUVafterelectrophoresisona1.5%agarosegel
withethidiumbromideandphotographedunderautomated
imagedocumentationsystem(UVPGDS-8000).
Analysisofstatisticaldata　Basedontheelectrophoresis
patern, atthecorespondingposition, thereisorisn t
band(includingpoorband)wouldberespectivelymarked
withsign(1)and(0), yieldingthedualmatrixdata.The
indices, suchasNeisgenediversityindex(H), Shannons
informationindex(I), geneticdiferentiationcoeficient
(Gst), geneflows(Nm)andgeneticdistance, wereana-
lyzedbyusingPopgen32 software;geneticsimilarityand
clusteranalysiswererevealedbyNTSYS-PC(version2.10
e)andUPGMAmethods, respectively.Thediferencesin
specificbandsamong5 populationswereusedasidentifica-
tionbasis.
ResultsandAnalysis
GeneticdiversityandgeneticdiferentiationinP.hend-
ersoni
Twenty10 bpRAPDprimerssuitableforP.hendersoni
werescreenedfrom80 testedprimers.Usingthesescreened
primers, totaly165 clearandrepeatablebandsweregenera-
tedinRAPDreactionwithgenomicDNAfrom5 diferentP.
hendersoniresources, ofwhich110 werepolymorphic, ac-
countingfor66.67% (Table2), suggestingthehighgenetic
diversitylevelofP.hendersoni.
Theanalyticresultsofpercentageofpolymorphicbands
(PPB), Nei sindex(H)andShannon sindex(I)were
listedinTable3.PPBsineachpopulationarebetween
13.46% and15.12%, andboththeHandIwerelowerthan
1.0, indicatingthelowgeneticdiversitylevelwithineach
wildpopulationofP.hendersoni.Comparatively, QHGpop-
ulationshowedthelowestgeneticdiversitywithpolymorphic
percentageof12.79%, Nei sindex(H)of0.021 1 and
Shannon sindex(I)of0.034 2.Atspecieslevel, gross
Nei sindex(H)andShannon sindex(I)ofP.henderso-
niwere0.220 5 and0.304 7, respectively.Additionaly,
geneticdiferentiationcoeficient(Gst)reached0.908 2, the
resultindicatesthat90.82%ofgeneticdiferentiationsarea-
mongpopulations, just9.18% ofgeneticdiferentiationsare
withinpopulation.Geneflows(Nm)calculatedfromGstwas
just0.050 5, indicatingthelowgeneflowrateamongwild
populationsofP.hendersoni.
Geneticdistanceandclusterresults
AsshowninlowertriangleofTable4, thegeneticdis-
tancevariedbetween0.248 3 and0.620 3, thegeneticdis-
tancesamongfourpopulationslyinginXinjiangandGansu
weresimilarandsharetheaverageof0.356 4, ofwhichthe
geneticdistancebetweenXJK andGSY wasthelowest
(0.248 3);whilethegeneticdistancesbetweenQinghai
populationandother4 populationswerelargeandsharedthe
averageof0.588 2, ofwhichthelargestgeneticdistance
(0.620 3)wasobservedbetweenQinghaipopulationand
Gansupopulation.
Table2　Sequencesof20screenedprimersandtheiramplificationre-
sults
Primer
code
Nucleotide
sequence
Totalampli-
fiedbands
Polymorphic
bands
Percentageofpoly-
morphicbands∥%
A02 TGCCGAGCTG 7 5 71.43
A09 GGGTAACGCC 12 10 83.33
A10 GTGATCGCAG 7 4 57.14
A14 TCTGTGCTGG 10 8 80.00
A17 GACCGCTTGT 6 4 66.67
A18 AGGTGACCGT 7 6 85.71
B01 GTTTCGCTCC 11 6 66.67
B02 TGATCCCTGG 6 4 66.67
B03 CATCCCCCTG 9 7 77.78
B04 GGACTGGAGT 6 3 50.00
B05 TGCGCCCTTC 9 4 44.44
B08 GTCCACACGG 8 6 75.00
B11 GTAGACCCGT 7 5 71.43
C06 GAACGGACTC 9 4 44.44
C09 CTCACCGTCC 10 8 80.00
C10 TGTCTGGGTG 9 7 77.78
C15 GACGGATCAG 4 3 75.00
D16 AGGGCGTAAG 9 6 66.67
D18 GAGAGCCAAC 8 4 50.00
D20 ACCCGGTCAC 11 6 54.55
Total 165 110 66.67
Table3　GeneticdiversityindifferentpopulationsofP.hendersoni
Population
code
Total
bands
Polymorphic
bands
Percentage
ofpolymorphic
bands∥%
Polymorphic
index
Shannon s
index
XJB 120 17 14.17 0.030 7 0.042 2
XJK 110 15 13.64 0.027 3 0.038 8
GSY 113 16 14.16 0.028 0 0.040 5
GSD 104 14 13.46 0.023 9 0.038 0
QHG 86 11 12.79 0.021 1 0.034 2
Table4　Thegeneticdistancesandgeneticsimilaritiesamong5 wild
populationsofP.hendersoni
Populationcode 1 2 3 4 5
XJB 0.687 6 0.689 0 0.638 0 0.543 2
XJK 0.374 5 0.780 1 0.737 0 0.581 2
GSY 0.372 5 0.248 3 0.678 2 0.560 2
GSD 0.449 4 0.305 2 0.388 4 0.537 8
QHG 0.610 2 0.542 6 0.579 5 0.620 3
Lowertriangleanduppertrianglerepresentgeneticdistanceandgeneticsimi-
larity, respectively.
　　ThegeneticsimilaritiesanalyzedbyusingNTSYS-PC
software(uppertriangle)andthephylogenetictree(Fig.1)
showedthat5 testedpopulationsweregroupedintotwoclas-
sesat0.05 coeficient.Qinghaipopulationsinglyfelintoa
clusterbecauseofitslowersimilaritywithotherfourpopula-
tions(Ⅰ), andXJK, GSY, GSDandXJBwereinturn
40 AgriculturalScience＆TechnologyVol.9, No.4, 2008
groupedinanothercluster(Ⅱ).Thephylogenetictreecanre-
flectthegeneticrelationshipsamongfivepopulationsintui-
tively.
Fig.1　ThephylogenetictreeoffivetestedpopulationsofP.
hendersonibyNTSYS-PC
Posibilityofidentifyingthepopulationsatmolecular
level
Mostofthetwentyscreenedprimersperformedabundant
polymorphismamongthe5 wildpopulationsofP.henderso-
ni, andshowedsomediferencesamongindividualswithin
eachpopulation.However, therewasanexceptionofprimer
A18 thatshowedidenticalamplificationpaternfordiferent
individualswithinapopulationandvariousamplificationpat-
ternfordiferentpopulations(Fig.2).Whetherthisprimer
couldbeasatoolforidentifyingthewildpopulationsofP.
hendersoni, furtherstudiesontheextendedpopulationsand
individualsamplesshouldbecariedoutforconfirmation.
M:marker;XJB, XJK, GSY, GSDandQHGrepresentpopulation
codesmentionedinTable1.
Fig.2　TheamplificationresultsoffivetestedpopulationsofP.
hendersonibyusingprimerA18
Discussion
SimilarityanalysisofdifferentwildpopulationsofP.
hendersoni
Fromthegeographicalposition, QHGisfarfromother
fourpopulationssampledfromXinjiangandGansu, thesimi-
laritybetweenQHGandotherpopulationswaslowestonthe
phylogenetictree, indicatingthecorelationbetweensimilar-
ityandgeographicalposition.Atgeographicalposition, XJK
liesneartheXJB, whileGSYliesnearGSD, however, XJK
showslargersimilaritywithtwoGansupopulationsandless
similaritywithXJB, indicatingthatthesimilaritiesamong
populationsarenotabsolutelyrelatedwithgeographicaldis-
tance.QHGliesintheQaidamBasinwithaltitudemorethan
2 700 mshowinglesrainfal, lowtemperature, severesalin-
ization, whileotherfourpopulationslieinTarimBasinin
XinjiangandHexiCoridorinGansuwithdriedclimate[ 3] .
Thismaybeimportantreasonresultinginthelowersimilarity
betweenQHGandotherfourpopulations.
GeneticdiversityandgeneticdiferentiationlevelofP.
hendersoni
Statisticalanalysesshowthatthefactorsinfluencingge-
neticvariationatpopulationlevelareinturn:breedingsys-
tem>distributionrange>lifeform>classificationlocation
>seeddiseminationmechanism[ 4] .Breedingsystemisthe
mostimportantfactorsinfluencinggeneticdiversityinpopu-
lations.Thegeneticvariationlevelinthespecieswithvege-
tativepropagationislowerthanthosewithsexualreproduc-
tion, grosgeneticdiferentiationmainlyexistsamongpopu-
lations[ 5] .P.hendersoniplantscanproducealotofseeds,
buttheydependonvegetativepropagationviarhizomeinnat-
uralconditions.Thus, withinpopulation, boththegenetic
diversityandgeneticdiferentiationwerelow, andthegenet-
ic diferentiation mainly occurred among populations
(90.82%).Thestresconditions, suchasdroughtandtor-
ridity, promotetheformationofasystemofplantanti-adver-
sitymechanismandabundantadversityresistancegenes, fur-
therenhancegeneticdiferentiationandincreasegeneticad-
versity[ 6-7] .Thevariousadversityconditionsresultinthe
preservationandevolutionofcorrespondinggenesunderdif-
ferentselectivestresses.Accordingly, atspecieslevel, per-
centageofpolymorphicbands(PPB), Nei sindex(H)and
Shannon sindex(I)ofP.hendersonilieinthemiddling
level[ 8-10] .
UtilizationstrategiesforwildresourcesofP.hendersoni
Forthehighergeneticdiversity, P.hendersonicould
beasanexcelentplantspeciesappliedinrestorationand
constructionofecologicalenvironmentinaridandsaline-al-
kaliregion.However, thegeneticdiferentiationmainlyoc-
cursamongpopulations, eachpopulationhasitsowngenetic
characteristics;thustheextinctofeachpopulationwould
leadtothedisappearanceofitsgeneticinformation.Inaddi-
tion, thesecondarymetabolites(suchasquercetinandfla-
vonoids)andfibrecontentsarediferentamongvariousP.
hendersonispecies[ 11-12] .Therefore, protectionofP.hend-
ersonispecies, especialytheQHG, shouldbeintensively
enforcedalongwithexploitationandutilizationofP.hender-
soniresources.Thisisnotsolelythemorespecificgenetic
informationofQHGthanotherpopulations, butthesopoor
climateandecologyofthispopulationthatitishardlytore-
storeoncedestroyed.Inrecentyears, investigationofapocy-
numresourceshowedthat, P.hendersonihasgradated, the
plantheightwasjust0.2 -0.5 m, whiletheplantdistance
reached0.8-3 m.Ifthekeyprotectionisstilnotconduc-
ted, theresourceswildieoutandtheecologicalenvironment
inGermuwilsurelycontinuetoaggravate.
References
[ 1] LIUJ, ZHANGCH.ApplicationandprospectsofRAPDtechniquein
Chinesemateriamedica[ J].HeilongjiangMedicineAndPharmacy,
2006, 29(6):92-93.(inChinese).
[ 2] ZHANGWM, WANGML, PENGXM, etal.IsolationofgenomicDNA
41ZHAOJin-fengetal.GeneticDiversityinWildPopulationsofPoacynumhendersonifromInlandAridRegionsofNorthwestChina
fromdriedleavesofthreekindsofLuobuma[J] .JournalofNanjingNor-
malUniversity:NaturalScienceEdition, 2007, 30(1):102-105.(in
Chinese).
[ 3] SHAOW, HURL, QIANXS.GeographicaldivisionofApocynumfiber
distributionareainChina[J].ChineseWildPlantResources, 2000, 19(4):20-22.(inChinese).
[ 4] LIQM, ZHAOJL.GeneticdiversityofPhylanthusemblicapopulationsin
dry-hotvaleysinYunnan[J] .BiodiversityScience, 2007, 15(1):84
-91.(inChinese).
[ 5] GRANTV.Theevolutionaryproces:Acriticalstudyofevolutionarytheo-
ry[M].2nded.NewYork:ColumbiaUniversityPress, 1991.
[ 6] RICARDOTC, O CONNELLMA.Geneticdiversityofdrought-responsive
genesinpopulationsofthedesertforageDactylisglomerata[ J].Plant
Science, 2005, 168:1327-1335.
[ 7] CECCARELLIS, GRANDOS.Droughtasachalengefortheplantbreed-er[J].PlantGrowthRegulation, 1996, 20:149-155.
[ 8] PANQJ, LIZH, WANGY, etal.RAPDanalysisonthegeneticdiversity
ofwildandcultivatedpopulationsofMuselalasiocarpa[J].ForestRe-
search, 2007, 20(5):668-672.(inChinese).
[ 9] WANGYQ, HUANGLQ, SHAOAJ, etal.GeneticdiversityofEucom-
miaulmoidesbyRAPDanalysis[ J] .ChinaJournalofChineseMateria
Medica, 2006, 31(19):1583-1586.(inChinese).
[ 10] ZHANGJY, YUANQH, WANGYR, etal.RAPDanalysisongenetic
diversityofwildpopulationofLespedezaMichx.[J] .ActaAgrectirSini-ca, 2006, 14(3):214-218.(inChinese).
[ 11] MAM, HONGCL, ANSQ, etal.Seasonal, spatial, andinter-specific
variationinquercetininApocynumvenetumandPoacynumhendersoni,
Chinesetraditionalherbalteas[J] .AgriFoodChem, 2003, 51:2390-
2393.
[ 12] GUOJY, MAM.Spatialvariationincontentofquercetinandtotalfla-
vonoidsofPoacynumhendersoni[ J].JournalofShiheziUniversity:Natu-
ralScience, 2007, 25(1):9-11.(inChinese).
[ 13] LANBX, LILH, WANGH.GeneticdiversityofAgropyronmongolicumKengpopulations[J].ScientiaAgriculturaSinica, 2005, 3:468-474.
(inChinese).
[ 14] YANWH, XUZ, SHIWG, etal.Studyofseedproteinbandpaterndi-
versityof40wildpopulationsofLespedeza[ J].PrataculturalScience,
2007, 5:58-63.(inChinese).
[ 15] JIEJJ.ExtractionanddeterminationofcontentofflavonoidsinApocynum
venetumleaves[ J].ChineseJournalofHospitalPharmacy, 2006, 12:
1490.(inChinese).
[ 16] LUXY, ZHANGC, ZHAOHQ, etal.Determinationofcontentsoftotal
flavonoidsandquercetininTrigonelafoenum-graecumL.atdifentgrowingareas[J].JournalofShenyangPharmaceuticalUniversity, 2004,
6:430-432.(inChinese).
[ 17] LIJ, ZHANGYX, LISP.ApplicationofRAPDtechniqueinthestudy
ongeneticdiversityofinsect[J].JournalofAnhuiAgriculturalSci-
ences, 2007, 35(6):47-48, 79.(inChinese).
[ 18] LIUJF, XIAOWF, FENGX.ApplicationofRAPDtechniqueinre-
searchofgeneticdiversityinrareandendangeredplants[ J] .Scientia
SilvaeSinicae, 2004, 3:156-161.(inChinese).
[ 19] SHIKM, ZHOUYF.AninprovedmethodofextractingArtemisiaabrota-numgenomicDNA[ J] .AgriculturalScience＆Technology, 2008, 9
(2):38-40.
[ 20] ZENGK, ZHANGXQ.Studyongeneticdiversityofgermplasmresources
ofLoliumL.[J].JournalofAnhuiAgriculturalSciences, 2007, 35
(11):3252-3254.(inChinese).
中国西北内陆干旱区大叶白麻野生居群遗传多样性研究
赵金凤 1 ,张卫明1, 2 ,彭雪梅1 ,唐泽紫 1 ,顾龚平 2 ,陆长梅 1＊　
(1.南京师范大学生命科学学院 ,江苏南京 210046;2.南京野生植物综合利用研究院 ,江苏南京 210042)
　　利用 RAPD分子技术对大叶白麻 5个野生居群多个样本进
行遗传多样性分析 ,以期为干旱区大叶白麻的保护 、繁育栽培和
标准化种植提供理论依据。
1　材料与方法
1.1　材料　试材均由南京野生植物综合利用研究院组织的
2005年全国罗布麻资源科学考察队采集(表 1)。由于大叶白麻
在自然条件下主要进行无性繁殖 ,为避免对同一植株遗传组成
完全相同的不同基株的重复采集 ,不同样本间的距离至少在 1
km以上 。叶片经硅胶干燥或自然干燥保存 ,并由南京野生植物综
合利用研究院钱学射研究员与肖正春研究员鉴定 。
　　表 1 样本来源
编号 居群代号 来源 样本数
1 XJB 新疆巴楚 5
2 XJK 新疆库尔勒 5
3 GSY 甘肃玉门 5
4 GSD 甘肃敦煌 5
5 QHG 青海格尔木 5
1.2　方法
1.2.1　基因组 DNA提取与检测 。大叶白麻基因组 DNA的提
取采用该课题组已建立的罗布麻总 DNA提取方法。所提取
DNA经 0.8%琼脂糖凝胶电泳及溴化乙锭(EB)染色后 ,观察其
质量和纯度 。
1.2.2　引物筛选与 PCR扩增 。试验所用 80条 RAPD引物购自
上海生工生物工程技术服务有限公司 ,分别标记为 A01 ～ A20,
B01 ～B20, C01 ～ C20 , D01 ～ D20。所用 PCR反应缓冲液、Taq酶
及 dNTP等购自上海申能博彩生物有限公司 。反应在 PTC-200
扩增仪(美国 BIO-RAD公司)上进行。从 5个居群中各选 1个
样本的 DNA作为模板 ,进行 RAPD体系优化和引物筛选 。最终
确定 PCR反应体系如下:总体积为 25 μl, 其中 MgCl2 2.5
mmol/L, dNTP5pmol/L,模板 DNA30ng, TaqDNA聚合酶 1.5
U,随机引物 10 pmol/L。反应程序如下:94℃预变性 4 min;94
℃变性 45s, 36℃复性 30 s, 72℃延伸 1min,循环 42次;最后 72
℃延伸 7min,反应结束后 4℃保存 。PCR产物经 1.5%的琼脂糖
凝胶电泳检测 ,凝胶成像系统(UVPGDS-8000)进行图谱采集。
1.2.3　数据统计分析。大叶白麻 5个居群经不同引物扩增所
得条带 ,按琼脂糖凝胶电泳图谱同一位置上 DNA条带的有无 ,
将电泳结果转化为二元数据矩阵 ,有带的(包括弱带)赋值为 1 ,
无带的赋值为 0。运用 Popgen32软件分析大叶白麻 5个居群的
Nei s基因多样性指数(H)、Shannon s信息指数(I)、居群间基
因分化系数 Gst及基因流 Nm和遗传距离等;运用 NTSYS-PC
(version2.10e)分析居群间遗传相似度;运用 UPGMA方法对各
居群进行聚类分析。根据 5个居群特征性条带的存在和缺失 ,
作为分子鉴别的依据 。
2　结果与分析
2.1　大叶白麻的遗传多样性及遗传分化　从 80条 10 bp随机
引物中筛选出 20条重复性好且多态性丰富的引物 ,对大叶白麻
5个居群的样本进行了 RAPD扩增 ,共获得 165条扩增条带 ,其
中 55条带(约占 33.33%)在白麻 5个居群的样本中都存在 ,其
余 110条为多态性带 ,多态性程度为 66.67%(表 2),说明大叶
白麻具有的遗传多样性水平较高 。
42 AgriculturalScience＆TechnologyVol.9, No.4, 2008
　　表 2 20条有效引物序列及其扩增结果
引物代号 序列 扩增总位点数
多态
位点数
多态位点
百分率∥%
A02 TGCCGAGCTG 7 5 71.43
A09 GGGTAACGCC 12 10 83.33
A10 GTGATCGCAG 7 4 57.14A14 TCTGTGCTGG 10 8 80.00
A17 GACCGCTTGT 6 4 66.67
A18 AGGTGACCGT 7 6 85.71
B01 GTTTCGCTCC 11 6 66.67
B02 TGATCCCTGG 6 4 66.67
B03 CATCCCCCTG 9 7 77.78
B04 GGACTGGAGT 6 3 50.00
B05 TGCGCCCTTC 9 4 44.44
B08 GTCCACACGG 8 6 75.00
B11 GTAGACCCGT 7 5 71.43
C06 GAACGGACTC 9 4 44.44
C09 CTCACCGTCC 10 8 80.00
C10 TGTCTGGGTG 9 7 77.78
C15 GACGGATCAG 4 3 75.00
D16 AGGGCGTAAG 9 6 66.67
D18 GAGAGCCAAC 8 4 50.00
D20 ACCCGGTCAC 11 6 54.55
总计 165 110 66.67
　　各居群的多态位点百分率(PPB)为 13.46% ～ 15.12%,且
Nei s指数和 Shannon s指数均小于 0.1,表明大叶白麻各个野
生居群内部遗传多样性水平都很低;对 5个野生居群而言 ,青海
格尔木 (QHG)居群的多态百分比 (12.79%)、Nei s指数
(0.021 1)和 Shannon s指数(0.034 2)在 5个居群中遗传多样
性最低 。在物种水平上 ,大叶白麻总的 Nei s基因多样性指数
为 0.220 5, Shannon s信息指数为 0.304 7。此外 ,居群间遗传
分化系数(Gst)高达 0.908 2,表明大叶白麻种内的遗传分化有
90.82%发生在居群间 ,只有 9.18%发生在居群内部个体之间。
由 Gst得到居群间的基因流(Nm)只有 0.050 5,可见大叶白麻野
生居群之间基因交流很小(表 3)。
　　表 3 大叶白麻不同居群遗传多样性
居群
代号
总条
带数
多态性
条带数
多态百
分率∥%
多样性
指数
Shannon s
信息指数
XJB 120 17 14.17 0.030 7 0.042 2
XJK 110 15 13.64 0.027 3 0.038 8
GSY 113 16 14.16 0.028 0 0.040 5
GSD 104 14 13.46 0.023 9 0.038 0
QHG 86 11 12.79 0.021 1 0.034 2
2.2　遗传距离和聚类分析　5个居群间的遗传距离(表 4对角
线下方)变化范围为 0.248 3 ～ 0.620 3,分布于新疆和甘肃的 4
个居群间的遗传距离值相当 ,平均值为 0.356 4 ,其中新疆库尔
勒与甘肃玉门居群的遗传距离最小(0.248 3);而青海居群与其
他 4个居群间的遗传距离都较大 ,平均值为 0.588 2 ,其中青海
居群与甘肃敦煌居群间的遗传距离最大 ,达 0.620 3。
　　表 4 大叶白麻居群间的遗传距离和遗传相似度
居群代号 1 2 3 4 5
XJB 0.687 6 0.689 0 0.638 0 0.543 2
XJK 0.374 5 0.780 1 0.737 0 0.581 2
GSY 0.372 5 0.248 3 0.678 2 0.560 2
GSD 0.449 4 0.305 2 0.388 4 0.537 8
QHG 0.610 2 0.542 6 0.579 5 0.620 3
　注:对角线下方为遗传距离 ,对角线上方为遗传相似度。
　　大叶白麻 5个居群在相似度(表 4对角线上方)为 0.55处
分为 2个大支 ,其中青海居群因与其他 4个居群间相似度较低
而单独构成 1支(II), I中 ,新疆库尔勒居群与甘肃玉门 、敦煌以
及新疆巴楚居群依次在不同的相似度处聚类 (图见第 41页
Fig.1:大叶白麻 5个居群 NTSYS-PC聚类树)。
2.3　居群间分子鉴别的可能性　筛选出的 20条引物对大叶白
麻 5个野生居群的扩增条带多态性都较丰富 ,且居群内不同个
体间多有少量差异 ,且引物 A18对每个居群内的 5个个体的扩
增图谱相同 ,且居群间互不相同(图见第 41页 Fig.2:引物 A18
对大叶白麻 5个居群 RAPD扩增结果 ,图中 M为 Marker;XJB、
XJK、GSY、GSD、QHG为居群代号),是否该引物可发展为大叶白
麻野生居群鉴别的手段 ,还需要扩大居群及样本的个体数做进
一步分析。
3　讨论
3.1　大叶白麻野生居群间的相似度分析　在地理位置上 ,青海
格尔木与新疆和甘肃的 4个采样地相距较远 ,聚类分析图也显
示青海格尔木居群与其他 4个居群间的相似度相对较小 ,这显
示各居群间的相似度和各居群间的地理位置有一定的相关性;
但新疆的库尔勒在地理位置上与巴楚靠近 ,甘肃的玉门与敦煌
靠近 ,而在聚类图上新疆库尔勒居群却与 2个甘肃居群间的相
似度较大 ,而与新疆巴楚居群间的相似度略小 ,说明大叶白麻各
居群间的相似度与地理距离间并不完全相关 。进一步分析各居
群所在地的气候特征发现 ,青海格尔木地区位处海拔超过 2 700
m以上的柴达木盆地 ,此地不仅降水少 ,而且气温低 、土壤盐渍
化严重 ,属于干旱 、高盐碱、高寒地带 ,而其余 4个居群位处塔里
木盆地和河西走廊地区 , 此处虽然极度干燥 , 但由于海拔仅
1 000余米 ,气温相对较高 ,土地盐碱化也相对不重 ,属于典型的
干旱区 。这可能是青海格尔木居群和其他 4个居群相似度较小
的更重要原因 。
3.2　大叶白麻遗传多样性和遗传分化水平分析 　统计学检验
表明 ,在居群水平上影响遗传变异大小的因素依次为:繁育系统
>分布范围 >生活型 >分类地位 >种子散播机制。繁育系统是
影响居群遗传多样性的最重要因素 。无性繁殖物种的居群内遗
传变异水平低于有性繁殖物种 ,总的遗传分化主要存在于居群
间。大叶白麻虽然能产生大量的种子 ,但在野生状态下是靠地
下根茎进行营养繁殖 ,因而 ,其居群内遗传多样性水平很低(表
3),遗传分化很小(9.18%),物种水平的遗传分化主要发生在居
群间(90.82%)。 Ricardo等认为干旱、炎热等逆境条件能够促
使植物形成一整套抗逆机制和丰富的抗性基因 ,从而能增大物
种遗传分化 ,增加遗传多样性 。炎热 、寒冷及高盐碱等逆境胁迫
致使不同居群在不同选择压力下各自保留和进化相应基因 ,因
此在物种水平上 ,大叶白麻遗传多样性水平并不低 ,其多态百分
率(66.67%)、Nei s基因多样性指数(0.220 5)和 Shannon s信
息指数(0.304 7)在大多数已被检植物中均处于中等水平。
3.3　大叶白麻野生资源开发利用策略　大叶白麻具有相对较
高的遗传多样性 ,可作为绿化荒滩 、治沙造林 、改造盐碱地的优
良植物应用于干旱与盐碱地区的生态环境修复和建设中。但由
于大叶白麻的遗传分化主要发生在居群间 ,因而每个居群都有
一定的遗传特殊性 ,任何一个居群的绝灭都会导致一部分遗传
信息的消失 ,从而导致大叶白麻总体遗传多样性水平降低 。此
外 ,不同居群的大叶白麻在次生代谢产物(如槲皮素、总黄酮等)
含量和纤维质量上存在一定的差异 。因此 ,在开发利用大叶白
麻资源的同时 ,要注重保护大叶白麻的各个居群 ,其中尤其需要
加大对青海格尔木居群的保护 。这不仅因为格尔木居群相较其
他居群来说具有更特殊的遗传信息 ,更重要的是该地区气候恶劣 ,
生态脆弱 ,既存麻田系多年演变形成 ,一旦破坏 ,就不易恢复。
基金项目　“十五”国家科技攻关计划(2004BA502B10);江苏省高新技
术研究计划(BG2006318)。
作者简介　赵金凤(1982-),女,山西大同人, 硕士研究生 ,研究方向:
植物资源。 ＊通讯作者。
收稿日期　2008-09-12　　修回日期　2008-09-26
43ZHAOJin-fengetal.GeneticDiversityinWildPopulationsofPoacynumhendersonifromInlandAridRegionsofNorthwestChina