全 文 ：DNAExtractionandISSRPrimerScreeningof
Cameliachekiangoleosa
XIEYun1, 2 , LIJi-yuan1＊ , FANZheng-qi1 , ZHUGao-pu1 , ZHOUXing-wen1
1.ResearchInstituteofSubtropicalForestry, ChineseAcademyofForestry, Fuyang311400;2.ZhejiangAgricultureandForestryUniversity,
Linan311300
Abstract　[ Objective] TheaimwastoscreenoutsimplemethodsofDNAextractionandefectiveISSRprimerssuitabletoalgermplasmmateri-
alsofCameliachekiangoleosa.[ Method] ThemodifiedCTABmethodwasusedintheextractionofthegenomicDNA, 50 ISSRprimersfrom
otherCameliaplantswereusedintheISSR-PCRamplificationfor20germplasmmaterialsfrom 10populationsinFujian, Zhejiang, Jiangxiand
AnhuiProvince.[ Result] PureDNAcouldbeobtainedrapidlybyusingtheimprovedCTABmethod, andthe20selectedefectiveprimershad
richpolymorphism, clearbandsandgoodrepeatability.337DNAbandswereobtained, ofwhich281 bandswerepolymorphic, accountingfor
83.4% ofthetotalamplifiedbands.And16.85bandscouldbeamplifiedwithaprimer, averagely.[ Conclusion] Theselected20primerscould
beefectivelyappliedtoISSRanalysisofthegermplasmresourcesofC.chekiangoleosainZhejiang.
Keywords　Cameliachekiangoleosa;ISSRmarker;Primerscreening
Received:May13, 2011　　Accepted:May31, 2011
SupportedbyProjectofZhejiangProvincialDepartmentofScience
andTechnology(2010C32043).
＊Correspondingauthor.E-mail:jiyuan li@126.com
　　Inter-simpleSequenceRepeat(ISSR)molecularmarker
isanewmarkertechniquedevelopedonthebasisofSSRand
hasbeenappliedinstudiesaboutthegermplasmidentifica-
tion, geneticmapping, genemappingandgeneticdiversitya-
nalysisinavarietyofplantsandanimals.TheproductsofIS-
SRshowricherpolymorphismthanRFLP, SSRandRAPD;in
addition, ISSRhasgoodrepeatabilitybecauseofitslonger
primers.ISSRapplicationinplantssuchasCameliajaponi-
ca[1] , teatree[2-3] , CamelianitidissimaChi[ 4] , Cameliaoleif-
era[ 5] , Cameliaazalea[ 6] hasbeenreported, butnoreporta-
boutCameliachekiangoleosaHuinZhejiangProvincehas
beenfound.
C.chekiangoleosaisanevergreenwoodyornamental,
oilplantinCamelia, Theaceae, withlarger, colorfulflower,
andthushasgreatornamentalandeconomicvalues.Itisan
endemicspeciesinChina, widelydistributedinZhejiang,
Jiangxi, Fujian, Anhuiandotherprovinces.Currently, there-
searchesonC.chekiangoleosaaresomefewer[ 7] , mainly
limitedtofatyacidanalysis[ 8] , introduction[ 9] , resistance[ 10]
andvariationofmajorphenotypiccharactersandsoon[ 11] ;in
addition, classificationofthespeciesisstilcontroversial[ 12] .
Sofar, noreportonthegeneticdiversityofgermplasm re-
sourceandISSRmolecularmarkeretc.ofC.chekiangoleosa
hasbeenfound.Inpresentstudy, 20 germplasmmaterials
from10 populations(twomaterialswerecolectedfromeach
population)wereselectedforscreeningidealISSRprimers,
soastoprovidereferenceforthefurtherresearchongenetic
diversityofC.chekiangoleosagermplasmresources.
MaterialsandMethods
Materials
10 populationsofC.chekiangoleosawerecolectedfrom
10countiesoffourprovinces.Atotalof20 materials(twoma-
terialsfrom eachpopulation)wereusedforISSRprimer
screening(Table1).PrimersY1 -Y50 wereselectedbyre-
ferringtoISSRstudiesaboutotherCameliaplants[ 1-6] , com-
posedofuniversalprimersanddesignedprimers(Table2),
synthesizedbySangonBiotech(Shanghai)Co., Ltd.Taq
polymerase, dNTPandDNAmarkerwerepurchasedfrom
TaKaRabranchcompanyinNanjing.
Methods
GenomicDNAextractionanddetection　ThegenomicDNA
offrozentenderleavesofC.chekiangoleosawasextractedby
usingtheimprovedCTABmethod.First, appropriateamount
offrozenleafwasplacedinice-coldmortar, groundintofine
powderinliquidnitrogenbyaddingappropriateamountof
PVP, transferredinto5 mlcentrifugetubecontaining2 ml2×
CTABextractionsolution(2% CTAB, 100.0 mmol/LTris-
HC1, 20.0 mmol/LEDTA1.4 mo1/LNaCl, pH8.0, 2%
β-mercaptoethanol, preheatingat65 ℃), gentlymixedprior
toincubationin65℃waterbathfor1hwithmixingonceever-
y15 min.Then, thetubewastakenoutofthewaterbathand
cooledtoroomtemperature, addedwith2 mlofchloroform:
isoamylalcohol(24∶1)beforemixinggentlyupsidedown,
centrifugedat8 000 r/minfor15 min.Subsequently, thesu-
pernatantwascarefulytransferredintoanew5 mltube, add-
edwithanequalvolumeofchloroform:isoamylalcohol
(24∶1), centrifugedat8 000 r/minfor15 min.Then, 1.0 ml
ofthesupernatantwastransferredcarefulyintotwo1.5 ml
centrifugetubes(0.5 mlineachtube).After1.0mlofetha-
nolprecooledat-20 ℃ wasaddedtoeachtube, thetubes
wereplacedat-20 ℃ for2 htoprecipitategenomicDNA,
andthencentrifugedat12 000r/minfor10min.Thesuperna-
tantwasremovedandDNAprecipitationwaswashedwith
75%ethanoltwiceandwithdehydratedalcoholonce, centri-
fugedat10 000r/minfor5min.Afterthesupernatantwasre-
moved, theresultingDNAwasair-driedanddissolvedinap-
propriateamountofTEbuffer, preservedin-20℃ refrigera-
tor.TheintegrityofDNAwasdetectedthrough0.8%agarose
gelelectrophoresis.5 μlDNAsolutionmixedwith2 μl0.25%
bromophenolbluewasaddedassampleinto0.8% agarose
gelin1×TBEbuferforhorizontalelectrophoresis(electro-
phoreticapparatustypeDYY-80, horizontalelectrophoresis
AgriculturalScience＆Technology, 2011, 12(6):825-828
Copyright 2011, InformationInstituteofHAAS.Alrightsreserved. AgriculturalBiotechnology
DOI :10.16175/j.cnki.1009-4229.2011.06.029
tankDYCP-33A), byusingtheknownconcentrationofDNA
solutionascontrol.Theelectrophoresiswasconductedat
voltage100 Vfor30 min.Afterelectrophoresis, thegelwas
stainedwithethidium bromide(EB), observedandphoto-
graphedwithautomaticgelimageanalysissystem(Shanghai
PeiqingTechnologyCo., Ltd.).Accordingtotheresultthe
DNAsolutionwasdilutedtoconcentrationof30 ng/μland
usedforlaterproceduresinthisstudy.
Table1　40 germplasmmaterialsofC.chekiangoleosausedforISSRandtheirorigins
Number Origins Number Origins
ZTT1-ZTT2 HuadingshanMountaininTiantaiCounty, ZhejiangProvince AYX1-AYX2 HengheVilage, BangzhenTown, YuexiCountyinDabieshanMountain, AnhuiProvince
ZQY1-ZQY2 BaishanzuNatureReserveinQingyuanCounty,ZhejiangProvince FJIR1-FJN2 YingtaoMountaininMinjiangyuanNatureReserveofJianningCounty, FujianProvince
ZKH1-ZKH2 GutianshanNatureReserveinKaihuaCountyinZhejiangProvince FXP1-FXP2 Badouqiu Vilage, ShuimenTownship ofXiapuCounty
ZJIR1-ZJN2 ShangbiaoForestFarm inJingnanTownshipofJingningCounty, ZhejiangProvince JDX1-JDX2 ShibamuduanVilageofXingangshanTown, Dex-ingCity, JiangxiProvince
ZJY1-ZJY2 DayangshanForestFarm inJinyunCounty, Zhe-jiangProvince JJX1-JJX2 XiajiulonginJiulongVilage, SanwanTownship,YongxinCounty, JiangxiProvince
Table2　Primersandtheirsequencesinthetest
Primernumber Primersequence Primernumber Primersequence Primernumber Primersequence
UBC-808 (AG)8C UBC-835 (AG)8YC UBC-873 (GACA)4
UBC-810 (GA)8T UBC-836 (AG)8YG UBC-881 (GGGTG)3UBC-811 (GA)8C UBC-840 (GA)8YT IR1 G(AC)8
UBC-812 (GA)8A UBC-841 (GA)8YC IR2 (GGAGA)3
UBC-813 (CT)8T UBC-843 (CT)8RA IR3 (GA)8CTUBC-814 (CT)8A UBC-844 (CT)8RC IR4 (CAA)6
UBC-815 (CT)8G UBC-845 (CT)8RG IR5 (CAG)8TUBC-816 (CA)8T UBC-846 (CA)8RT IR6 (CAA)8G
UBC-817 (CA)8A UBC-847 (CA)8RC IR7 (GT)8GG
UBC-818 (CA)8G UBC-848 (CA)8RG IR8 (AC)8TGUBC-820 (GT)8C UBC-851 (GT)8YG IR9 (AC)8CG
UBC-821 (GT)8T UBC-853 (TC)8RT IR10 (CT)8GGUBC-823 (TC)8C UBC-854 (TC)8RG IR11 (CAG)6T
UBC-825 (AC)8T UBC-855 (AC)8YT IR12 (CAA)6G
UBC-826 (AC)8C UBC-860 (TG)8RA IR13 (GACA)4TUBC-827 (AC)8G UBC-866 (CTC)6 IR14 (CT)8C
UBC-834 (AG)8YT UBC-868 (GAA)6
Theboldareselectedprimers.
PCRreactionsystem andamplificationprocedures　The
totalvolumeofPCRreactionsystemwas20 μlcontaining2μl
of10×bufer, 30ngofDNAtemplate, 2.0mmol/LofMg2+,
300.0 μmol/LofdNTP, 0.8 μmol/Lprimers, and2.5 UTaq
DNApolymerase.ThePCRwasstartedwithpredenaturingat
94℃ for5 min, folowedby40 cyclesofdenaturingat94 ℃
for45s, annealingatgradienttemperaturesfor50 s, andex-
tensionat72 ℃for90 s;theamplificationwascompletedby
holdingthereactionmixtureat72 ℃ for7 mintoalowcom-
pleteextensionofPCRproducts.TheresultingPCRproduct
waspreservedat4 ℃.ThePCRamplificationwasconducted
intheGeneAmpPCRSystem9700PCRinstrument(Applied
Biosystems, USA). 10 μlPCR productwasseparated
throughelectrophoresison1.2 mg/Lofagarosegelcontai-
ningEBat100Vfor2 h, byusing2 000 bpDNAladderasa
control.Theelectrophoresisresultswereobserved, analyzed
andphotographedundertheAlphaImagerimagingsystem
(U.S.AlphaInnotechCorporation).
Selectionofprimersandtestofannealingtemperature
gradient　Accordingtothemeltingtemperature(Tm)ofIS-
SRprimers, fourgradienttemperaturesweredesignedfor
PCRamplification.TheprimerswiththesimilarTm values
wereusedinthesamerangeofannealingtemperaturegradi-
ent, andannealingtemperaturegradientwassetintherange
ofTm±5 ℃.Theprimersgivingclearbands, stableresult,
highpolymorphismandlessnon-specificbandsweredistin-
guishedfromthosecouldpresentsomebandsbutnotgood
resultevenafterannealingtemperaturescreening.Thenthe
annealingtemperatureoftheprimersselectedweretestedat
12 gradientsbyusingrandomtemplates.
ResultsandAnalysis
DNAqualitytesting
ThegenomicDNAextractedfromC.chekiangoleosawas
whiteflocculentorwaterwhiteprecipitate, andpresentedonly
uniformbandthroughelectrophoresis, indicatingthattheex-
tractedDNAwaswithgoodintegrityandthattheprotein, poly-
saccharide, phenolsandotherimpuritiesweremostlyre-
moved.DNA concentrationwasdeterminedbyultraviolet
spectrophotometry, andresultshowedthattheOD260 /OD280 of
theDNAsolutionwasabout1.80, whichcouldmeetrequire-
mentsofISSR-PCR.
Screeninganddeterminationofannealingtemperatureof
theprimers
Atotalof20 primerswhichcouldpresentabundantpoly-
morphismandclearbandswerescreenedout.Theamplifica-
tionresultofprimersIR13, UBC-835, UBC-841, UBC-873
andUBC-815 inpresenceoftemplateZKH1 wasshownin
Fig.1.ForprimerIR13, morePCRproductbandswereob-
tainedatannealingtemperature51.0 and47.8 ℃, butthe
826 AgriculturalScience＆TechnologyVol.12, No.6, 2011
lowerannealingtemperaturemadethebackgrounddarker, so
51℃ wasthepreferedannealingtemperature.PrimerUBC-
835 producedmorenon-specificbandsandbrighterback-
groundatannealingtemperature47.8and54.9 ℃, indicating
thatmorenon-specificbandscouldbeproducedatloweran-
nealingtemperature, and58.4 ℃ wasthebetterannealing
temperature.ThePCRproductsofprimersUBC-841, UBC-
873 andUBC-815 presentedlessandfuzzybandsathighan-
nealingtemperature, butmoreandclearbandsatlowertem-
perature, andthebestresultat47.8 ℃.Thus, 20 primers
werescreened, folowedbyannealingtemperaturetestat12
gradients.Aftertheannealingtemperaturewereselected, IS-
SR-PCRamplificationwascarriedoutbyusingNo.JYX1-25
asthetemplate, andtheresultswereshowninTable3.
Table3　Thesequenceofprimersandtheirpolymorphism
Primers Sequences Annealingtemperature℃
Amplifiedbands Polymorphicbands
Percentageofpolymorphism% Primers Sequences
Annealingtemperature℃
Amplifiedbands Polymorphicbands Percentageofpolymorphism∥%
UBC-811 (GA)8C 48.7 13 10 76.9 UBC-855 (AC)8 YT 53.2 15 13 86.7
UBC-816 (CA)8 T 50 17 14 82.4 UBC-868 (GAA)6 49.4 16 13 81.3
UBC-817 (CA)8 A 55 16 13 81.3 UBC-873 (GACA)4 51 14 12 85.7
UBC-825 (AC)8 T 53.2 20 17 85.0 UBC-881 (GGGTG)3 52 18 15 83.3
UBC-827 (AC)8 G 51.6 21 18 85.7 IR2 (GGAGA)3 53.2 18 16 88.9
UBC-834 (AG)8YT 49.7 17 14 82.4 IR3 (GA)8CT 50.2 20 17 85.0
UBC-835 (AG)8YC 53.2 15 13 86.7 IR4 (CAA)6 49.4 22 18 81.8
UBC-844 (CT)8RC 50.2 13 11 84.6 IR8 (AC)8 TG 52 22 17 77.3
UBC-845 (CT)8RG 51.6 14 12 85.7 IR9 (AC)8 CG 49.4 15 12 80
UBC-846 (CA)8 RT 49.4 15 12 80.0 IR13 (GACA)4T 52 16 14 87.5
M:DL2000DNAMarker;1-4:IR13;5-8:UBC835;9-12:UBC841;13-16:UBC873;17-20:UBC815.Annealingtemperatureof
theprimerswere58.4 , 54.9, 51and47.8℃, respectively.
Fig.1　Primersandtheircorespondingannealingtemperature
M:DL2000DNAMarker;1-25:ISSR-PCRamplifiedmapof33germplasmmaterialsofC.chekiangoleosa
Fig.2　TheamplifiedresultsofprimerUBC-846
AmplificationresultsofISSR-PCRanddatastatistics
ThestatisticalresultsofISSRamplifiedbands(Table3)
indicatedthat13-22 bands(16.85 bandsinaverage)could
beamplifiedwithaprimer.Atotalof337amplifiedbandswere
producedbyusingthe20 ISSRprimers, ofwhich281 bands
werepolymorphic, accountingfro83.4%ofthetotalamplified
bands.Althe20 primerscouldproduceclearDNAfinger-
print, andtheDNAfragmentswereabout100 -2 000 bpin
length, withrichpolymorphisms.Fig.2 showstheISSR-PCR
amplificationfingerprintofgermplasmNo.JYX1-25byusing
primerUBC-846.
Conclusions
The20 ISSRprimerssuitableforC.chekiangoleosawere
selectedinpresentstudy.Atotalof286 DNAbandsweream-
plifiedbyusingthe20 primers, ofwhich80.77% werepoly-
morphic.Thestudywilprovidefoundationfortheapplication
ofISSRtechniquetorapidandaccuratelyanalyzethegenetic
diversityofC.chekiangoleosa.
827XIEYunetal.DNAExtractionandISSRPrimerScreeningofCameliachekiangoleosa
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浙江红山茶总 DNA提取及 ISSR-PCR引物筛选(摘要)
谢云 1, 2 ,李纪元1＊ ,范正琪 1 ,朱高浦 1 ,周兴文 1　(1.中国林业科学研究院亚热带林业研究所,浙江富阳 311400;2.浙江农林大
学天目学院 ,浙江临安 311300)
[目的 ]筛选出简易的DNA提取方法及适合于所有浙江红山茶种质材料进行ISSR分析的有效引物。
[方法 ]采用改良的 CTAB法提取基因组DNA,参考其他山茶科植物的 50个 ISSR引物 ,对来自浙江 、福建、江西、安徽 10个居群中共 20份浙
江红山茶种质材料进行了PCR扩增。
[结果 ]改进的CTAB法可简单、快速地提取到高纯度DNA产物;筛选出的 20条引物多态性丰富 、条带清晰且可重复性良好。共扩增出 337
条 DNA谱带 ,其中 281条为多态性带 ,占总扩增带数的 83.4%,平均每个引物扩增出 16.85条谱带。
[结论 ]所筛选的 20条引物可有效应用于浙江红山茶种质资源材料的ISSR分析。
关键词　浙江红山茶;ISSR标记;引物筛选
基金项目　浙江省科技厅项目(2010C32043)。
作者简介　谢云(1968-),女 ,湖南衡阳人,副教授,博士研究生 ,从事园林植物教学和科研工作 , E-mail:xieyun@zafu.edu.cn。 ＊通讯作者,研究员 ,
博士 ,博士生导师 ,从事茶花分子育种研究 , E-mail:jiyuan li@ 126.com。
收稿日期　 2011-05-13　　修回日期　 2011-05-31
(Frompage808)
生豆浆基因组 DNA不同提取方法的比较研究(摘要)
徐伟丽1 ,杜明 1 ,马莺 1＊ ,李启明 2 ,汪家琦2　
(1.哈尔滨工业大学食品科学与工程学院 ,黑龙江哈尔滨 150090;2.新希望乳业控股有限公司技术中心,四川成都 610023)
[目的 ]探讨适宜生豆浆的DNA提取方法。
[方法 ]以市售豆浆为材料 ,分别采用热解法、异丙醇沉淀法、CTAB法 、SDS法 、高盐低pH和异硫氰酸胍法以及它们的改良方法提取基因组
DNA,并比较了以上方法的提取效果。
[结果 ]除异丙醇沉淀法外 ,其他方法提取的基因组DNA均可满足PCR检测要求。同时 ,综合考虑基因组DNA的纯度和浓度 ,生豆浆基因组
DNA提取方法的优劣依次为:改进高盐低pH法 、高盐低pH法 、改进CTAB法、改进异丙醇沉淀法、异硫氰酸胍法和改进热解法。
[结论 ]这几种豆粉基因组DNA提取方法均具有操作简单、耗时短、利于快速检测的优点。
关键词　生豆浆;基因组DNA;提取;聚合酶链式反应
基金项目　四川省应用基础研究项目(2009JY0101)。
作者简介　徐伟丽(1977 -),女 ,黑龙江牡丹江人,讲师 ,博士 ,从事食品生物技术和食品安全领域研究 , E-mail:weilixu698@163.com。 ＊通讯作者 ,
教授 ,博士生导师 ,从事食品安全与检测研究, E-mail:maying@hit.edu.cn。
收稿日期　 2011-03-16　　修回日期　 2011-05-31
828 AgriculturalScience＆TechnologyVol.12, No.6, 2011