全 文 ：OptimizationoftheConditionsofSRAPMolecular
MarkerUsedinAnalysisofFagopyrumtataricum
WANGYao-wen1 , XIANan1 , HANRui-xia1 , LIYan-qin1＊ , WANGAn-hu2 , CAIGuang-ze2
1.KeyLaboratoryofChemicalBiologyandMolecularEngineeringoftheMinistryofEducation, InstituteofBiotechnology, ShanxiUniversity,
Taiyuan030006;2.XichangColege, Xichang615013
Abstract　[ Objective] TheaimwastoinvestigatetheoptimalconditionsofSRAPmolecularmarkerusedintheanalysisonFagopyrumtataricum
(L.)Gaertn.[Method] SRAP-PCRamplificationsystemonFagopyrumtataricumwasoptimizedbyinteractiveorthogonaldesignL27(313)in5elements(Mg2+ , dNTP, TaqDNApolymerase, templateDNAandprimer)at3levels.Andthenon-denaturinganddenaturingPAGEdetectionmethodswerecompared.ThecomparativetestofDYCZ-24FandDYCZ-20Celectrophoresisoperatingsystemswascariedout.[Result] The
effectsoffoursingle-factor(Mg2+ , dNTP, TaqDNApolymeraseandprimer)andtwointeractions(Mg2+ ×dNTP, Mg2+ ×TaqDNApolymer-
ase)ontartarybuckwheatSRAP-PCRweresignificant.Anoptimalreactionsystemwasestablishedcontaining1.5mmol/LMg2+ , 0.2mmol/L
dNTP, 1.5uTaqDNApolymerase, 40ngDNA, 0.25μmol/Lprimerand2μl10×bufer.Sevensamplesoftartarybuckwheatwereamplifiedu-
singthissystem, andelectrophoresisresultsshowedclearbands, highlevelofpolymorphismandgoodreproducibility.ThePCRproductswere
testedbydenaturingandnon-denaturingPAGE, andtheresultsshowedthatthenon-denaturingPAGE, DYCZ-24Foperatingsystemwasmore
suitableforSRAPanalysis.[Conclusion] ThisstudyestablishedafoundationfortheconstructionofSRAPgeneticmapoftartarybuckwheat.
Keywords　Fagopyrumtataricum;SRAP-PCR;Interactiveorthogonaldesign;DenaturingPAGE;Non-DenaturingPAGE
Received:September6, 2010　　Accepted:November30, 2010
Supported by NationalNaturalScience Foundation ofChina
(30771310).
＊Correspondingauthor.E-mail:yanqin@sxu.edu.cn
　　Fagopyrumtataricum(L.)Gaertnisoneofmedicineand
foodresourcesoriginatedinChina.Itisnotonlynutritionalybal-
anced, butalsocontainsmedicinalingredientsothercropsdo
nothave, whichcanefectivelyinhibitglucosidaseandamylase
activity[1-2] andplaysaroleinpreventionofdiabetes, lowering
bloodsugar, regulatingbloodlipidsandbloodpressure[3-5] .
Sequence-relatedAmplifiedPolymorphism(SRAP), pro-
ducedbyDepartmentofVegetableCrops, CaliforniaUniversi-
tyofU.S.in2001, isanovelPCR-basedmolecularmarker
technique.Itsmechanismisasfolows:basedontheexons
regionofgenesisrichinG, Cwhileintronsregionisrichin
A, T, primersaredesignedtoamplifytheopenreading
frames(ORFs), andthenthepolymorphismwilbeproduced
duetodiferentintervalsbetweenintronsandpromoterofdif-
ferentspecies[ 6] .Thismethodissimple, rapidwithlowcost
andabundantpolymorphisms, etc., thusbeingwidelyusedin
geneticmapconstructionandQTLanalysis[ 7-9] , geneticdi-
versityanalysis[ 10-12] , Heterosisprediction[13] , speciesidenti-
ficationandotherfields.BudakHetal[ 14] usedthebuffalo
grassasthematerialstofourkindsofmolecularmarkertech-
nologiesSRAP, RAPD, SSR andISSR, andtheresult
showedthatthepolymorphism anddiferentiationabilityof
SRAPwerehigherthanotherthreemarkers.SRAPisaPCR-
basedmolecularmarkertechnology, whichiseasiertobeaf-
fectedbyanumberoffactorsinreactionconditions, there-
fore, theestablishmentandoptimizationoftheSRAP-PCR
systemarenecessarytoensuretheaccuracyandrepeatability
ofSRAP-PCR.Orthogonaldesignisrecognizedasamethod
withabalanceddispersion, comprehensivecomparableand
scalable, cleareffectandothercharacteristics.Thismethodnot
onlyreducesthescaleoftheexperimentwithoutmakingtoo
muchlossofinformation, butalsoovercomesthedisadvantages
ofsinglefactortest, thusquicklyfindingtheoptimallevelcombi-
nation[15] .Inthisstudy, SRAP-PCRamplificationsystem on
Fagopyrum tataricum wasoptimizedbyinteractiveorthogonal
design, andthenon-denaturingPAGEanddenaturingPAGE
detectionmethodwerecomparedsoastoprovidebasisforthe
constructionofSRAPgeneticmapofF.tataricum.
MaterialsandMethods
Materials
Dianning-1 (femaleparent), wildbuckwheat(malepar-
ent), Jiujiangbuckwheat(femaleparent), threecopiesofF1
generationofDianning-1 ×wildbuckwheat, andF1 genera-
tionofJiujiangbuckwheat×wildbuckwheatwereprovidedby
XichangColegeandcolectedinLiangshanofSichuanProv-
ince.Aftertheleaveswerecolected, theywereplacedin-
80 ℃refrigeratorforpreparation.
Reagentsandinstruments
50, 100 bpLadderDNAMarkerandTaqDNApolymer-
asewerepurchasedfromTransGenBiotech;10 ×PCRbuf-
er, dNTPandMgCl2 werepurchasedfrom TaKaRaCo.,Ltd.;otherreagentswereofanalyticalgrade.PTC-200 type
PCRinstrument(BIO-RADCompany), DYCZ-20CandDY-
CZ-24Felectrophoresistank(BeijingLiuyiInstrumentFacto-
ry), JP-3000sxelectrophoresis instrument(Polyscience
CompanyofUSA), nucleicacidproteinanalyzer(Eppen-
dorf), gelimagingsystem(BIO-RADCompany).
Primers
PrimersweresynthesizedbyBeijingAokeBiotechnology
Company, andthesequenceswereshowninTable1.
Methods
DNAextractionanddetection　ThegenomicDNAofBuck-
wheatwasextractedbyusingplantgenomicDNAextraction
kit.NucleicacidproteinanalyzerwasusedtodetecttheDNA
concentrationandA260 /A280 value, and0.8% agarosegele-lectrophoresiswasutilizedtodetectDNAintegrityandRNAdi-
gestionsituation.
AgriculturalBiotechnologyAgriculturalScience＆Technology, 2010, 11(9-10):32-36, 179Copyright 2010, InformationInstituteofHAAS.Alrightsreserved.
Table1　Sequenceofprimers
Name Sequenceofprimers(5′※3′)
F--me1 TGAGTCCAAACCGGATA
R--em1 GACTGCGTACGAATTAAT
R--em2 GACTGCGTACGAATTTGC
R--em3 GACTGCGTACGAATTGAC
OrthogonaltestonPCRreactionsystem　Orthogonalde-
signL27 (313)infivefactorsatthreelevelswasperformed,
andtheinteractionsbetweenMg2+ anddNTP, Mg2+ andTaq
polymerasewereanalyzed.Factorsandlevelswereshownin
Table2, andthedesignwasshowninTable3, theexperi-
mentwasrepeatedtwice.ThePCRreactionsystemwas20
μl, containing2 μlof10 ×bufer(withoutMg2+), theDNAof
wildbuckwheatwasusedasthetemplate, andtheusedprim-
ercombinationwasme1/em1.
Table2　Thefactorsandtheirlevelsinorthogonaldesign
Code Factor Level
1 2 3
A Mg2+∥mmol/L 1.00 1.50 2.00
B dNTP∥mmol/L 0.20 0.25 0.30
C TaqDNApolymerase∥U/20μl 0.50 1.00 1.50
D DNAtemplate∥ng/20μl 20.00 30.00 40.00
E Primer∥μmol/L 0.25 0.50 0.75
Table3　StatisticalresultsandorthogonaldesignwithL27(313)
Code Mg2+(A)mmol/L
dNTP(B)
mmol/L
TaqDNApolymerase(C)
U/20 μl
Template(D)
ng/20 μl
Primer(E)
μmol/L
Testresult
BandⅠ BandⅡ
1 1 0.2 0.5 20 0.25 8 8
2 1 0.2 1 30 0.5 7 8
3 1 0.2 1.5 40 0.75 6 5
4 1 0.25 0.5 30 0.5 10 10
5 1 0.25 1 40 0.75 9 9
6 1 0.25 1.5 20 0.25 10 10
7 1 0.3 0.5 40 0.75 9 8
8 1 0.3 1 20 0.25 8 7
9 1 0.3 1.5 30 0.5 8 9
10 1.5 0.2 0.5 20 0.75 5 6
11 1.5 0.2 1 30 0.25 8 7
12 1.5 0.2 1.5 40 0.5 3 3
13 1.5 0.25 0.5 30 0.25 7 6
14 1.5 0.25 1 40 0.50 8 9
15 1.5 0.25 1.5 20 0.75 5 4
16 1.5 0.3 0.5 40 0.5 8 9
17 1.5 0.3 1 20 0.75 8 7
18 1.5 0.3 1.5 30 0.25 9 8
19 2 0.2 0.5 20 0.5 5 4
20 2 0.2 1 30 0.75 6 7
21 2 0.2 1.5 40 0.25 8 7
22 2 0.25 0.5 30 0.75 6 5
23 2 0.25 1 40 0.25 10 10
24 2 0.25 1.5 20 0.5 5 5
25 2 0.30 0.5 40 0.25 8 7
26 2 0.30 1.0 20 0.5 10 10
27 2 0.30 1.5 30 0.75 5 5k1 =K1 /3 8.278 6.167 7.167 6.944 8.111k2 =K2 /3 6.667 7.667 8.222 7.278 7.278k3 =K3 /3 6.833 7.944 6.389 7.556 6.389R 1.611 1.778 1.833 0.611 1.722
PCRapplicationandelectrophoresisdetection　Amplifica-
tionprocedurewasaccordingtorenaturationtemperature-
switchingmethod.Andtheamplificationprocedurewasasfol-
lows:denaturationat94 ℃for5 min;5cyclesofdenaturation
at94℃ for1 min, annealingat35 ℃ for1 min, extensionat
72℃ for1 min;35 cyclesofdenaturationat94 ℃ for1 min,
annealingat50 ℃for1 min, extensionat72℃ for1 min;fi-
nalextensionwasat72 ℃ for6 min, andthentheproducts
waspreservedat4 ℃.PCRproductsweredetectedby10%
non-denaturingPAGE, aftersilverstaining, thestatistical
analysiswasusedtogaintheoptimalSRAP-PCRreaction
systemofbuckwheat.
Non-denaturingPAGEanddenaturingPAGE　① Non-de-
naturingPAGE:containing2.5 μlofPCRproductsand6 ×
loadingbufer(98% glycerol, 10 mmol/LEDTApH 8.0,
0.25% bromophenolblue, 0.25% xyleneblue), andthe300
Vconstantvoltageand1 ×TBEwereperformedforelectro-
phoresistilthexylenebluewasoutofgel.② Denaturing
PAGE:containing2.5 μlofPCRproducts(camefrom the
samegroupwiththesamplesusedfornon-denaturingPAGE)
and6 ×loadingbufer(98% formamide, 10 mmol/LEDTA
pH8.0, 0.25%bromophenolblue, 0.25% xyleneblue).Af-
terthesamplesweredenaturingat95 ℃ for5 min, theywere
quicklyplacedoniceforcooling, andthen30 W ofconstant
powerand1 ×TBEwereperformedforelectrophoresistilthe
xylenebluewasoutofgel.
Theelectrophoresiscarriedoutbytwotypesofelectro-
phoresistank　TheaddedsamplesofDYCZ-20Ctypeof
electrophoresistankwas6μl, whilethatofDYCZ-24Ftypeof
electrophoresistankwas2.5μl, andthe300 Vconstantvolt-
ageand1 ×TBEwereperformedforelectrophoresistilthe
xylenebluewasoutofgel.
ResultsandAnalysis
SRAP-PCRorthogonaltestanalysis
Resultsandrangeanalysisoforthogonaltest　ThePCR
productsofeachtreatmentweredetectedby10% non-dena-
33WANGYao-wenetal.OptimizationoftheConditionsofSRAPMolecularMarkerUsedinAnalysisofFagopyrumtataricum
turingPAGE(Fig.1).Thenumberofamplifiedbandswas
usedforthevaluationofeachtreatmentandtherangeanaly-
sis.ItcouldbeconcludedfromtheRvalueinTable3thatTaq
polymorase, dNTP, primersandMg2+ hadgreaterimpactson
buckwheatSRAP-PCRreaction, whiletheeffectofDNAtem-
platewasrelativelysmal;thecorrespondingcombination
A1B3 C2D3E1 ofthehighestaveragevaluekofeachfactorwas
theoptimalcombinationscreenedbyrangeanalysis.
M:D2000 DNAMarker;1-27:Codesoforthogonaltest.
Fig.1　ElectrophoresispaternofPCRproductsoftheorthogonaltest
Varianceanalysis　Rangeanalysisdoesnotconsiderthein-
teractionsamongfactorsandtheexperimentalerrorscannot
beestimated.Therefore, DPSsoftwarewasusedinthisstudy
forvarianceanalysisofdata.Itwasrevealedthattheefectof
fivefactorsonSRAP-PCRreactionofbuckwheatwasconsistent
withtheresultofrangeanalysis;theinteractionsofMg2+ ×Taq
polymeraseandMg2+ ×dNTPshowedsignificanteffecton
SRAP-PCRreaction.Therefore, theorthogonaldesignassis-
tantsoftwarewasusedtocalculatetheaveragenumberof
bandsundertheinteractionsbetweenMg2+ andTaqpolymer-
ase, dNTP.TheFvalueofMg2+ ×Taqpolymerase>Fval-
ueofMg2+ ×dNTP, sotheinteractionbetweenMg2+ andTaq
polymeraseshouldbefirstconsidered.Afterthelevelsof
Mg2+ andTaqpolymeraseweredetermined, thedNTPlevel
couldbedeterminedaccordingtotheselectedMg2+ level(Ta-
ble4).AndDuncanmethodwasusedforthemultiplecom-
parisonanalysisonotherfactors, andtheresultwasshownin
Table5.
Table4　AveragenumberofbandswithinteractionamongMg2+ ,
TaqDNApolymeraseanddNTP
Mg2+(A) TaqDNApolymerase(C)
1 2 3
dNTP(B)
1 2 3
1 7.833 6.833 5.833 7.000 5.333 6.167
2 8.000 7.833 8.833 9.667 6.500 6.833
3 8.000 5.333 5.833 8.167 8.167 7.500
　　Table4 revealedthatatlevel2ofMg2+ andlevel3ofTaq
DNApolymerase, theaveragenumberofbandswerethe
highest;whileatlevel2 ofMg2+ andlevel1 ofdNTP, theav-
eragenumberofbandswerethehighest.Itcouldbeconclu-
dedfrom Table5 thatatlevel3 oftemplateandlevel1 of
primer, theaveragenumberofbandswasthehighest.There-
fore, theoptimalSRAP-PCR reactionofbuckwheatwas
A2 B1C3D3 E1 , thatwas, inthe20 μlofsystem, theMg2+ was
1.5 mmol/L, dNTPwas0.2 mmol/L, TaqDNApolymerase
was1.5 U, DNAtemplatewas40 ng, primerswere0.25
μmol/Land10 ×buferwas2 μl.
Repeatabilityverificationoftheoptimizationsystem　The
DNAofsevencopiesofmaterialswasusedasthetemplate,
andthreepairsofprimerswereusedfortherepeatabilityveri-
ficationoftheoptimizationsystem.Fig.2 (10% non-denatu-
ringPAGEpatern)showedmanyclearbandswithnodisper-
sionandtailingphenomenon;itwaseasytofoundthatthehy-
bridshadthesignaturebandsoftheparents.
Table5　TestingsignificanceofdifferenceatdiferentlevelsbyDun-
can
Level AveragenumberofbandsDNAtemplate Primer
1 6.944bB 8.111aA
2 7.278abAB 7.278bB
3 7.556aA 6.389cC
Diferentcapitalandlowercaselettersstandforsignificantdifference
at0.01and0.05, respectively.
Comparisonofnon-denaturinganddenaturingPAGE　
Non-denaturingPAGEanddenaturingPAGEarebothusedin
thedetectionofSRAP-PCRproducts[ 16-17] .Inthisstudy,
comparisonresultsofthesetwomethods(thesamplesoftwo
methodswerefromthesamegroupwiththesameamount)
wereshowninFig.2and3.Itwasfoundthatthebackground
ofmapofdenaturingPAGEwasdeepwithfuzzybands, es-
pecialy, thelargerfragmentscouldnotbeseparatedwel;the
mapsofM1 andM2 thesetwomarkersinthenon-denaturing
PAGEwereconsistentwiththestandardmaps, whilethatin
thedenaturingPAGEcouldnotcorrespondent.
Comparisonoftwotypesofelectrophoresistank
DYCZ-24FandDYCZ-20Cthesetwotypesofelectropho-
resistankwereusedtocarryout10% non-denaturingPAGE
onPCRproductsfromthesamebatch(Fig.2, 4).There-
sultsshowedthatalthoughtheaddedsamplesofFig.4 was
1.4 timesmorethanFig.2, themapswerealsounclear;and
thenumberofbandsweresignificantlylessthanthecorre-
spondinglanesinFig.2, thismightberelatedtothepoorsil-
verstainingeffectcausedbythinnerofgels.
ConclusionsandDiscussions
(1)Byusingasetofnormalizedorthogonaltableinor-
thogonaldesign, thecombinationofeachfactorandlevelcan
bematchedandarrangedreasonably, thusgreatlyreducing
thenumberoftestsandprovidingmoreinformation[ 18] .There-
fore, theorthogonaldesigncansolvetheproblemsofsingle
factortest.Generaly, therearetwoanalysismethodsforan-
alyzingtheresultoforthogonaldesign:rangeanalysismethod
(visualanalysis)andvarianceanalysis.Althoughtherange
34 AgriculturalScience＆TechnologyVol.11, No.9-10, 2010
analysisissimpleandclear, andtheoptimalconditioncanbe
obtainedthroughthevisualcomparison, itdoesnottakeinto
accounttheexperimentalerorandcannotgiveaccurateesti-
matesontheinfluencedegreeofeachfactor;inaddition, it
cannotanalyzetheinteractionsamongfactors.Therefore,
thisstudyhadusedvarianceanalysistomakeuptheabove
shortcomingsofrangeanalysismethod.Asforthefactorshad
significantefectsontheexperiments, theoptimallevelofthis
35WANGYao-wenetal.OptimizationoftheConditionsofSRAPMolecularMarkerUsedinAnalysisofFagopyrumtataricum
factorwasselected;whileasforthefactorshadnotsignificant
efectsontheexperiments, thelevelswereselectedbasedon
thelawofsavingtimeandcostinglower.
　　(2)Asfortheanalysisontheelectrophoresispaterns,
someresearchescalculatedandanalyzedbasedontheback-
groundofpaterns, thenumberofbandsandthebrightnessof
bands[19] ;whilesomeresearcherscalculatedaccordingtothe
numberofbands[ 20] .Theformerhastakingintoaccountof
comprehensivefactorswithstrongsubjectivity;whilethelater
considersless, butthenumberofbandsareexact, thusbeing
moresuitablefortheanalysisonmultiplebandsinthesame
laneunderthesamebackgroundconditions.Therefore, the
numberofamplifiedbandswasusedinthisstudyfortheas-
signmentofeachtreatment.
(3)Non-denaturingPAGEanddenaturingPAGEare
bothusedinthedetectionofSRAP-PCRproducts.Therefore,
thecomparison ofnon-denaturingPAGE and denaturing
PAGEwascarriedoutinthisstudy.Theresultshowedthat
thedenaturingPAGEshowedunclearbands, andthemapsof
M1 andM2 thesetwomarkersinthedenaturingPAGEcould
notcorrespondentwiththestandardpatterns, thusgreatlyaf-
fectingtheinterpretationofthebands, sothattheerrorwould
beproduced.Intheoperatedprocess, theureawouldbee-
ducedform thesampleapplicationholeofthedenaturing
PAGEgel.Ifitcouldnotbewashedclear, thesampleswould
notbeinasameplane, thusresultinginerrors.Thegelmak-
ingofnon-denaturingPAGEissimple, thesampleswouldnot
bedenatured, andthesampleapplicationcouldbequickly
managed;aftertheelectrophoresis, theamplifiedbandswere
clearwithhigheraccuracyofbandpaternanalysis, sothere-
sultsweremorereliable.
(4)DYCZ-24FandDYCZ-20Cwerebothpurchased
fromBeijingLiuyiInstrumentFactory, andthediferencesbe-
tweenthemwere:①DYCZ-20Cwasoperatedwiththesharks
toothcomb, sothesamplewaseasytobedriftedtoother
sides, resultinginmutualcontaminations;whileDYCZ-24F
wasoperatedwithgreatwaltoothcomb, sothesampleswere
separatedbythegelsamongdifferentholes.② Repel-Si-
laneandBind-Silane(bothweretoxicagents)wereneeded
forDYCZ-20C, whileDYCZ-24Fwasnot.③ Thegelthick-
nessofDYCZ-20Cwas0.4 mm, andthegelwasnotabso-
lutelysmooth, resultinginpoorsilverstainingeffectinsome
areas;whilethegelthicknessofDYCZ-24Fwas1 mm, so
theaboveproblemswilbenothappened.④ ThegelofDY-
CZ-20Cwasthinandmustbesticktotheglass, andthede-
velopersolutionwasstronglyalkaline, sothelong-termofgel
soakingwouldleadtoexcessivelossandfragmentation;while
thegelofDYCZ-24Fwassothickthancouldbepeeledof
fromtheglass, sotheoperationofDYCZ-24Fwassimple,
shortandefective.⑤ ThesamplevolumeofDYCZ-20Cwas
2.4 timeshigherthanDYCZ-24F, buttheelectrophoresispat-
ternsofDYCZ-20Cwerenotclearwithfewerbands.⑥There
wasnocoolingsysteminDYCZ-20C;whileDYCZ-24Fhada
water-cyclingsystem, thuscouldcontroltheelectrophoresis
temperature.Therefore, itcouldbeconcludedthattheDYCZ-
24FwasmoresuitablefortheanalysisofSRAPproductsand
hadgreateraccuracythanDYCZ-20C.
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Responsibleeditor:YINJian-li Responsibleproofreader:WUXiao-yan
提高玉米育种效率的途径
张 旭 1 ,刘晓萍 2 ,郝学景 1 ,李立公1 ,魏 春1 ,何光荣 1 ,马永良 1＊ 　
(1.中国农业大学农学院 ,北京 100193;2.全国农业展览馆 ,北京 100125)
摘要　结合国内外玉米育种文献 ,对提高玉米育种效率的可能途径进行了探讨。结果表明 ,通过对玉米育种目标和策略的总结和反思 ,对育种技
术和方法的改进和优化 ,以及对试验设计和实施过程的调整与简化 ,将大大降低玉米育种的成本投入 ,提高玉米育种的工作效率。
关键词　玉米;育种方法;改进途径
基金项目　国家科技支撑计划项目(2007BAD69B05)。
作者简介　张旭(1967-),男 ,安徽亳州人,副研究员 ,博士 ,从事玉米育种研究, E-mail:zhangxu cau@126.com。 ＊通讯作者 ,教授。
收稿日期　 2010-11-03　　修回日期　 2010-12-03
(上接第 36页)
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2008, 28(6):693-697.
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al.OptimizationforISSR-PCRsystem ofFreesiarefractaKlat
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反应体系)[ J] .BuletinofBotanicalResearch(植物研究), 2008,
28(4):402-407.
Responsibleeditor:LITing-ting Responsibleproofreader:WUXiao-yan
SRAP分子标记用于苦荞分析中的条件优化(摘要)
王耀文 1 ,夏楠 1 ,韩瑞霞 1 ,李艳琴1＊ ,王安虎 2 ,蔡光泽 2　
(1.山西大学生物技术研究所 ,化学生物学与分子工程教育部重点实验室 ,山西太原 030006;2.西昌学院 ,四川西昌 615013)
[目的 ]研究SRAP分子标记用于苦荞分析中的条件优化。
[方法]采用交互正交设计L27(313)对苦荞 SRAP-PCR反应体系进行了 5因素(Mg2+、dNTP、Taq酶、模板 DNA和引物)3水平优化筛选 ,比较
了非变性和变性PAGE检测方法 ,并进行了DYCZ-24F与DYCZ-20C2种电泳操作系统的对比试验。
[结果] 4个单因素(Mg2+ 、dNTP、Taq酶和引物)和 2个交互作用(Mg2+ ×dNTP、Mg2+ ×Taq酶)对苦荞SRAP-PCR有极显著影响;最佳反应体
系为:20μl总体积 , Mg2+ 1.5mmol/L, dNTP0.2mmol/L, Taq酶 1.5U,模板DNA40ng,引物 0.25μmol/L, 10×缓冲液 2μl。运用该体系对 7
份苦荞材料进行扩增 ,电泳结果显示扩增条带清晰 、多态性高 、重复性好。将扩增产物进行非变性与变性PAGE和 2种电泳操作系统检测 ,结
果显示 ,非变性PAGE、DYCZ-24F操作系统更适合于SRAP的分析。
[结论 ]该研究为今后构建苦荞SRAP遗传图谱奠定了基础。
关键词　苦荞;SRAP-PCR;交互正交设计;变性 PAGE;非变性 PAGE
基金项目　国家自然科学基金项目(30771310)。
作者简介　王耀文(1983 -),男 ,山西吕梁人 ,在读硕士 ,研究方向:植物分子遗传。 ＊通讯作者,学士 ,副教授 ,从事植物 、微生物分子生物学研究, E-
mail:yanqin@sxu.edu.cn。
收稿日期　 2010-09-06　　修回日期　 2010-11-30
179ZHANGXuetal.TheWaystoImprovetheEficiencyofMaizeBreeding