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

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/.

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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

576

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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