analysis of Glu-A3 alleles using

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