analysis of Glu-A3 alleles using(3)

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

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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

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