analysis of Glu-A3 alleles using
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glua3
PlantBreeding129,574—577(2010)Ó2009BlackwellVerlagGmbH
doi:10.1111/j.1439-0523.2009.01724.x
ShortCommunication
Low-molecular-weightgluteninsindurumwheat:analysisofGlu-A3allelesusingPCRmarkers
SZ3,M.C.GIANIBELLI3,K.R.GALE3andS.RAHMAN3G.IGREJAS1,2,3,4,A.JUHA
s-os-MontesandAltoDouro,5001-801VilaReal,Portugal;DepartmentofGeneticsandBiotechnology,UniversityofTra
s-os-MontesandAltoInstituteforBiotechnologyandBioengineering-CentreofGeneticsandBiotechnology,UniversityofTra
3
Douro,5001-801VilaReal,Portugal;CSIRO,CommonwealthScienti candIndustrialResearchOrganisation,PlantIndustry,GPOBox1600,ACT2601Canberra,Australia;4Correspondingauthor,E-mail:gigrejas@utad.pt
2
1
With1 gureand1table
ReceivedSeptember29,2008/AcceptedSeptember14,2009CommunicatedbyL.Hartl
Abstract
TheaimofthepresentstudywastocomparethedurumandbreadwheatallelesencodedattheGlu-A3locus(representedbyGlu-Ad3andGlu-Aa3,respectively)usingPCRmarkersdesignedforbreadwheatGlu-Aa3allelestodistinguishdi erentdurumalleles.Diversityinlow-molecular-weightgluteninsubunits(LMW-GS)wasanalysedineightcultivarsrepresentingallknownGlu-Ad3allelespresentindurumwheat.SixoftheeightGlu-Ad3allelesofdurumwheatcouldbedistinguishedusingacombinationofsevendi erentprimersetsbasedonLMW-GSsequencesfrombreadwheat.However,allelea(cv.ÔMexicaliÕ),alleleb(cv.ÔLangdonÕ)andallelec(cv.ÔCocoritÕ)ofdurumwheatcouldnotbedistinguishedusinganyofthemarkerstested.Ampliconsofidenticalsizewereproducedforallelesf(cv.ÔClaro noÕ)andalleleg(cv.ÔClarodeBalazoteÕ),usingallexceptoneprimerset(Glu-A3a),indicatinggenesoftheseallelesshareaveryhighdegreeofidentity.ThemarkersdescribedinthisstudyareusefulfortheDNA-basedidenti cationofdurumwheatcultivarsandtheidenti cationoftheGlu-Ad3a,b,c,d,e,f,gandhallelesofdurumwheatforthepurposesofmarker-assistedselection.
Keywords:TriticumturgidumsspdurumDesf.—Triticumaestivum—Glu-Ad3locus—low-molecular-weightgluteninsubunits—PCRmarker—varietyidenti cation—wheatGluteninsarethemajorcomponentofthepolymericstorageproteinsinwheatandaccountforapproximately50%oftheglutenproteinsofwheat ourhavingamajorroleindeterminingdoughcharacteristics(Payneetal.1984a).Thegluteninsubunitsconsistofhigh-molecular-weightgluteninsubunits(HMW-GS)inthemolecularweightrangeof70–90kDaandlow-molecular-weightgluteninsubunits(LMW-GS)inthemolecularweightrange20–45kDa.
TheLMW-GSareclassi edintoB,CandDgroupsaccordingtotheirmobilityinSDS-PAGE(DÕOvidioandMasci2004).LMWgluteninshavealsobeengroupedonthebasisoftheN-terminusaminoacidandthreesubgroupsoftypicalLMW-GShavebeenidenti ed:LMW-s-(serine),LMW-m-(methionine),andLMW-i-type(isoleucine)(TaoandKasarda1989,Lewetal.1992,Mascietal.1995,Cloutieretal.2001).Lewetal.(1992)identi ed39di erentLMWsubunitsinonebreadwheatcultivarbasedontheirproteinN-terminalaminoacidsequences.Ikedaetal.(2002)furthersubdividedtheLMW-GStypesinto12groupsaccordingtothenumberandpositionofthecysteineresidues.ThenumberofgenesforLMWgluteninsin
asinglecultivarhasbeenvariouslyestimatedtobebetween10and40(SabelliandShewry1991,Cassidyetal.1998).
ScoringofLMW-GSalleliccompositionisdi cultduetothenumberofsubunitsexpressedbyasinglecultivarandtheoverlappingmobilityoftheLMW-GSwithsomegliadinsthatareco-extractedwithLMW-GSduringsamplepreparation(Weegelsetal.1996).Inaddition,SDS-PAGEisnotwellsuitedtoroutinehighthroughputanalysis,asisrequiredforselectionoflargenumbersoflinesinabreedingprogrammeandDNAmarkersaremoresuitable(Gale2005).Inapioneeringstudy,Zhangetal.(2004)developedasetofDNAmarkerstodiscriminateallcommonGlu-Aa3allelespresentinbreadwheatandthishasbeenrecentlyfollowedbythedevelopmentofDNAmarkersfortheGlu-B3allelesinhexaploidwheat(Wangetal.2009).
TheLMWgluteninsindurumwheatsareimportantindeterminingpastaquality(Payneetal.1984b,Pognaetal.1990,Porcedduetal.1998)butthedevelopmentofPCRmarkershaslaggedbehindthatofhexaploidwheat.TheaimofthisworkthereforewastocharacterizethedurumreferencetypesbytheapplicationofasetofLMW-GS-speci cPCRmarkersdevelopedpreviouslyforanalysisofGlu-Aa3allelespresentinbreadwheat.
MaterialsandMethods
EachGlu-A3alleleindurumandbreadwheatwasidenti edaccordingtoNieto-Taladrizetal.(1997)andGuptaandShepherd(1990)usingstandardcultivars.Thesuperscriptaanddwereusedtoidentifythebread(aestivum)anddurum(durum)loci,respectively.
Eightdurum(TriticumturgidumsspdurumDef.)wheatcultivarswereusedasstandardsofGlu-A3LMW-GSallelestoanalysetherelevantgenes.ThedurumwheatstandardsdescribedbyNieto-Taladrizetal.(1997)werethefollowing:‘Mexicali’(Glu-Ad3a),‘Langdon’(Glu-Ad3b),‘Cocorit’(Glu-Ad3c),‘Alaga’(Glu-Ad3d),‘Blatfort’(Glu-Ad3e),‘Claro no’(Glu-Ad3f),‘ClarodeBalazote’(Glu-Ad3g)and‘Jiloca’(Glu-Ad3h),andthesewerekindlyprovidedbyMrMichaelMackayfromtheAustralianWinterCerealsCollectioninTamworth,Australia.
Glu-A3i-typeLMW-gluteninmarkers:Mostoftheallelespeci cmarkersusedtocomparebreadanddurumwheatatthegenelevelweredescribedbyZhangetal.(2004)andpublishedPCRcondi-tionswerefollowed.WiththeexceptionofGluA3F1-Glu3R1primers
glua3
PCRmarkersforLMW-GSindurumwheat
thatweredesignedtoamplifytheentireopenreadingframe(ORF),theremainingprimersamplifyshortgenesegmentsbasedonsinglenucleotidepolymorphismsidenti edinthecodingregionsoftheindividuali-typegenes(Zhangetal.2004)fromhexaploidwheat.Otherpublished(Ikedaetal.2002)i-typegenespeci cprimerswereusedtoscreenforfurtherdi erencesintherepetitiveregionofi-typegenes(primersGlu22-Glu13).
Glu-A3m-typeLMW-gluteninmarkers:LMW-GSwiththeN-termi-nalsequenceMDTSCIPGLERorvariantsofMETSCIPGLERhavepreviouslybeenidenti edasm-typegenescontrolledbytheGlu-A3
szandGianibelli2004).LMW-GSgenelocus(Ikedaetal.2002,Juha
sequenceswerealignedandcomparedusingtheGCGprogrampackageonANGIS(.au).Di erenceswereiden-ti edandspeci cprimersweredesignedtoamplifyfragmentsorentirecodingregionsofMETSCIPGLERtypegenes.
DNAextractionandPCRampli cation:Seedsweregerminatedandgrownfor1–2weeksandgenomicDNAwasisolatedfromyoungleavesusingFastDNAÒKit(Q.BIOgene,Carlsbad,CA,USA)follow-ingtheinstructionsofthemanufacturer.PCRwasperformedinaHybaidthermocyclerandreactionswerecarriedoutusingpublished
szetal.2008).conditions(Ikedaetal.2002,Zhangetal.2004,Juha
Sequencingandsequenceanalysis:Sequencingwascarriedoutusing
BigDyechemistryonanABI371machineusingPCRproductsusingprotocolsrecommendedbythemanufacturer.Sequenceswereanaly-sedbytheClustalWprogramavailableathttp://align.genome.jp/using570bpfromthe fthTTTTsequencefoundinalleleausingtheprimerGlu22.Thisregionwaschosenbecauseclearsequenceswereavailablefromallallelesoverthisspan.ThetreewasproducedbytheNeighbour-Joiningmethodwithdistancecalculatedathttp://align.genome.jp/.
575
ResultsandDiscussion
OnlyasmallnumberofLMW-gluteninsequenceshavebeenpublishedsofarfromdurumwheatandtheypartlyoverlapwiththe17di erentgenetypesidenti edinbreadwheat
szandGianibelli2004).Zhangetal.(2004)described(Juha
theuseofasetofmolecularmarkersfortheidenti cationofGlu-Aa3allelesinbreadwheat.
ThesetofprimersGluA3R1-GluA3F1usedbyZhangetal.(2004)fortheampli cationoftheentireORFoftheGlu-Aa3i-typegenes,inbreadwheat,wereusedinthisstudy(Fig.1a,Table1).Allelesa,b,c,d,eandhwererepresentedbyasingleband,whereasadoublebandwasobtainedforallelesfandgindicatingthatatleasttwosequencesofthesametypearepresentinthelinescarryingallelesfandg(Fig.1a).Theseresultswerefurthercon rmedwhenGlu22-Glu13primerswereused,amplifyingtherepetitiveregionofthesametypeofgenes.Thesizeofthefragmentsampli edvariedbetween600and800bp(Fig.1b).Usingbothsetsofprimersalleleewasclearlydi erentiatedfromfandgalleles.However,noPCRproductwasobservedwhenprimersspeci cforthei-typegenesofallelesGlu-A3aabc,Glu-A3aac,Glu-A3aeorGlu-A3af(Zhangetal.2004)frombreadwheatwereusedindurumwheatcultivars.
Usingprimersspeci cforGluA3aainbreadwheat,durumwheatallelesf(‘Claro no’)andg(‘ClarodeBalazote’)producedaPCRfragmentofapproximately400bp(Fig.1c).However,theampliconsizewassmallerthanexpected(585bp)basedontheresultsofthebreadwheat‘ChineseSpring’(allelea).Glu-Ad3allelesfandghadprobablysimilari-typegenesbuttheampliconswereofdi erentsize.
TheprimersetforGluA3adallelefromhexaploidwheatwasthentested(Fig.1d).‘Blatfort’(allelee),‘Claro no’(allelef)and‘ClarodeBalazote’(alleleg)resultedinthesamesizefragmentsasthatproducedfrom‘Suneca’(alleledinbreadwheat).ThuscombinationoftheprimersforGlu-Aa3aandGlu-Aa3dallowedtheidenti cationanddiscriminationofallelesGlu-Ad3e,fandg.
Useofprimersspeci cforGlu-Aa3ginbreadwheatresultedinsimilarproductsforallelesa(Mexicali),b(Langdon),c(Cocorit)andd(Alaga)fromdurumwheat.Theproduct
size
M12345678910M123456789M12345678910
M
12345678910
M123456789
10M12345678910
Fig.1:Separationofampli cationproductsonagarosegels(1–1.5%)usingtheprimersdescribedbelow,nesM,SPP1DNAmarker(360,480,720,980,1160,1390,1510,1860,1950,2810,3590,4840,6110,7350,8510),1Mexicali(a),2Langdon(b)3Cocorit(c),4Alaga(d),5Blatfort(e),6Claro no(f),7ClarodeBalazote(g),8Jiloca(h).(a)ne10iscv.ÔCharaÕ(Glu-Aa3b)usedasbreadwheatcontrol.(b)ne9watercontrol.(c)ne10isTriticumaestivumL.cv.ÔChineseSpringÕ(Glu-A3a).(d)ne10isT.aestivumL.cv.ÔSunecaÕ(Glu-A3d).(e)ne10isT.aestivumL.cv.ÔGlenleaÕ(Glu-A3g).(f)PrimersFNm9-Rm9
glua3
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SZ,GIANIBELLI,GALEandRAHMANIGREJAS,JUHA
Table1:AssociationbetweenwheatGlu-Aa3allele-speci cPCRmarkersandLMW-GSalleles.Foranyprimerpair,productsofidentical
mobilityareindicatedbythesamerepresentation(x,xxorxxx).Ablankspaceinacellindicatesthatnoproductwasdetectedforthatalleleandprimer-paircombination
Primer
Marker
Detects
Expected
Size(bp)Forward
Reverse
Wheats(allele)
MexicaliLangdonCocoritAlagaBlatfortClaro noClarodeJiloca(a)(b)(c)(d)(e)(f)Balazote(g)(h)
xx
xx
xx
xx
xxxxx
xx
xx
xx
x
xx
xxx
xxxxxxxxx
xxxxxxxxxx
xxx
Entireencodingi-type1300–1400region
Repetitivecodingi-type600–800regionGluA3ai-type585GluA3di-type488GluA3gi-type861RepetitiveregionMETSCIPGLER244EntirecodingMETSCIPGLER800–900region
GluA3F1GluA3R1Glu22GluA3F1GluA3dFGluA3gFFNm9FNm8
Glu13GluA3aRGluA3dRGluA3R2Rm9RUNIV
(861bp)wassimilartothatobtainedfrombreadwheatcultivarGlenlea(g)(Fig.1e).Thisallelewasnotpreviouslyidenti edamongthe202breadwheatcultivarsandlinesanalysedbyGuptaandShepherd(1990).However,itappearsonthebasisoftheseresultsthatatleastonepolypeptidecontrolledbytheGlu-Aa3gallelespresentin‘Glenlea’mayalsobepresentinthesedurumlinesrepresentedbyallelesGlu-Ad3a,b,candd.Noproductwasfoundforallelese,f,gandhfromdurumwheatwhentheprimersforGlu-Aa3gfrombreadwheatwereused.Thusallelehfromdurumwheatisonlyampli edbyprimerpairsGluA3R1-GluA3F1andGlu22-Glu13butnotbythosethatrepresentGlu-Aa3a,dorgfromhexaploidwheat.Insummary,usingtheprimersdesignedbyZhangetal.(2004)allelese,f,gandhcouldbedi erentiatedindurumwheat.Inadditionallelesa,b,canddcouldbedistinguishedfromallelese,f,gandhbuttheycouldnotbeidenti edindividually.PrimersreportedbyIkedaetal.(2002)werealsousedforidenti cationoftheGlu-Aa3genesbutnofurtherdiscriminationwasobtained.
szTwonewm-typespeci cprimerpairsdesignedbyJuha
etal.(2008)wereusedtotargetdi erencesbetweenGlu-Ad3allelesindurumwheat.Oneofthemwasdesignedtoamplifyaspeci cpartofagene(FNm9-Rm9)andanother(FNm8-Runiv)wasdesignedtoamplifyentirecodingregions.
WhentheprimersetFNm9-Rm9wasusedwithdurumcultivars,onlytwocultivarscontainingallelesdanderesultedinthegenerationofPCRfragments(Fig.1f).Thusonlyallelesa,bandcremaintobedistinguished.Noneofthemarkerswetestedwereabletodothis.Toobtainfurtherinformationabouttherelationshipbetweenthealleles,sequencingwascarriedoutusingPCRampli cationproductsobtainedwiththeprimersGlu22andGlu13withDNAfromeachofthedurumline.ResultsofClustalWanalysesweredisplayedasanunrootedneighbour-joiningtreewithdistanceandshowedthatthesequencesobtainedfromallelesa,bandcareundistinguishableoverthe570bpanalysedandthatallelesa,b,canddareverycloselyrelated.Allelesh,fandeareincreasinglydivergentandallelegisthemostdi erentfromtheothers.
Tofurthertrytodistinguishallelesa,bandc,sequencingwasalsocarriedoutusingproductsoftheprimersFNm8andRuniv.Againnodistinctioninsequencecouldbeobserved.Theseresultsdemonstratethatalthoughallelebcanbeseparatedfromallelesaandconthebasisofproteinanalysis,thedistinctioncannotbeveri edattheDNAlevel.The
di erencemaypossiblyrepresentaproteinmodi cationthatisdeterminedbyageneatadi erentlocus.Itwouldbeusefultoidentifythestructuralbasisofthedi erencebetweentheproteinproductsofallelebandallelesa/casthismayallowthedevelopmentofsuitableDNAmarkers.Thefactthatmanyoftheseallelesarenotcommonbetweenbreadanddurumwheatmeansthatbreadanddurumwheatcanbeusedasasourceofnewgeneticvariabilityforeachotherandmolecularmarkerscanbeusedtofollowthisintrogression.Acknowledgements
The rstauthorwassupportedbyafellowshipfromtheOECDCo-operativeResearchProgramme:BiologicalResourceManagementforSustainableAgricultureSystems.WewouldliketoacknowledgetheexcellenttechnicalassistanceofNatalieStefanski.WewouldliketothankCurtBrubaker,CSIROPlantIndustryforusefuldiscussions.
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