analysis of Glu-A3 alleles using(5)

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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.

References

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Cloutier,S.,C.Rampitsch,G.A.Penner,andO.M.Lukow,2001:CloningandexpressionofaLMW-igluteningene.J.CerealSci.33,143—154.

DÕOvidio,R.,andS.Masci,2004:Thelow-molecularweightgluteninsubunitsofwheatgluten.J.CerealSci.39,321—339.

Gale,K.R.,2005:DiagnosticDNAmarkersforqualitytraitsinwheat.J.CerealSci.41,181—192.

Gupta,R.B.,andK.W.Shepherd,1990:Two-stepone-dimensionalSDS-PAGEanalysisofLMWsubunitsofglutelin.1.Variationandgeneticcontrolofthesubunitsinhexaploidwheats.Theor.Appl.Genet.80,65—74.

Ikeda,T.M.,T.Nagamine,H.Fukuoka,andH.Yano,2002:Identi cationofnewlow-molecularweightgluteninsubunitgenesinwheat.Theor.Appl.Genet.104,680—687.

sz,A.,andM.C.Gianibelli,2004:Informationhiddeninthelow-Juha

molecular-weightgluteningenesequences.In: andra,S.Masci,andR.DÕOvidio(eds),TheGlutenProteins,62—65.RSC,CambridgeUK.

Lew,E.J.L.,D.D.Kuzmicky,andD.D.Kasarda,1992:Characterizationoflow-molecular-weightgluteninsubunitsbyreversed-phasehigh-performanceliquidchromatography,sodiumdodecylsulphate-polyacrylamidegelelectrophoresis,andN-termi-nalaminoacidsequencing.CerealChem.69,508—515.

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