j.1439-0523.2006.01173.x

Detection of HMW glutenin subunit variations among205cultivated emmer accessions(Triticum turgidum ssp.dicoccum)Q.Y.Li1,3,Y.M.Yan1,3,A.L.Wang1,X.L.An1,Y.Z.Zhang1,S.L.K.Hsam2and F.J.Zeller2,31Key Laboratory of Genetics and Biotechnology,College of Life Science,Capital Normal University,Beijing,China;2Division of Plant Breeding and Applied Genetics,Technical University of Munich,D-85350Freising-Weihenstephan,Germany; 3Corresponding authors,E-mail:zeller@zwz.tum.de;yanym@With3figures and1tableReceived March14,2005/Accepted July22,2005Communicated by W.E.WeberAbstractThe high molecular weight glutenin subunits(HMW-GS)encoded by Glu-1loci among205accessions of cultivated emmer wheat(Triticum turgidum ssp.dicoccum Schrank)collected from different regions of Europe and China were separated and characterized by SDS-PAGE in combination with two-dimensional gel electrophoresis(A-PA-GE·SDS-PAGE)and acidic capillary electrophoresis.High genetic polymorphisms in HMW-GS compositions were found.A total of40 alleles(6for Glu-A1and34for Glu-B1)and62subunit combinations (genotypes)were detected,some of which were not previously described.At Glu-A1locus,two novel alleles,designated Glu-A1x coding for the subunit1A·1.1and Glu-A1y coding for the subunit 1A·2.1¢were found while seven new subunits(1B·17*,1B·6¢, 1B·13¢,1B·20*,1By9*,1By14.1and1By8.1)and20novel alleles at Glu-B1locus were detected.In particular,some additional protein components were detected,which probably were1Ay subunits encoded by Glu-A1locus.The introduction of both Ax and Ay subunits from tetraploid wheats into hexaploid wheats may increase the genetic variability of gluten genes and consequently improveflour technolo-gical properties.Key words:Triticum dicoccum—acidic capillary electrophor-esis—high molecular weight glutenin subunits—SDS-PAGE —two-dimensional electrophoresisThe high molecular weight glutenin subunits(HMW-GS) encoded by the complex Glu-1loci present on the long arm of group1homoeologous chromosomes are the major determinants of important endosperm proteins contributing to the baking quality of wheatflour(Payne et al.1984). They form polymers through disulphide bonds and give dough strength(Shewry et al.1992).Variation in the compositions of HMW glutenin subunits has been shown to be responsible for differences in bread-making quality of genotypes(Payne et al.1979).Tetraploid emmer(Triticum turgidum ssp.dicoccum Sch-rank,2n¼4x¼28,AABB),is a primitive hulled wheat. Currently,it is cultivated only in limited areas of some European countries,where it is mainly used both for feeding livestock and human consumption(Galterio et al.1994; Pflu ger et al.2001).Some investigations showed that there exists considerable allelic variation for HMW-GS in dicoccum wheat(Vallega and Waines1987;Galterio et al.1994; Piergiovanni and Blanco1999;Pflu ger et al.2001;Yan et al. 2003a).As the genetic base of modern wheat cultivars is generally narrow,the primitive emmer wheat is anticipated as rich resources of useful genes for wheat quality improvement.The aim of this study is to detect the HMW glutenin subunit compositions of an extensive collection of cultivated emmer wheat from European and Chinese gene pools by SDS-PAGE, two-dimensional polyacrylamide gel electrophoresis(A-PA-GE·SDS-PAGE)and acidic capillary electrophoresis(A-CE) methods.Materials and MethodsPlant materials:A total of205cultivated emmer accessions (T.dicoccum Schrank)were studied,which were from GenBanks of Braunschweig and Gatersleben,Germany and National germ-plasm pool of China.SDS-PAGE:The HMW-GS extraction from single kernels was based on Singh et al.(1991)with some modifications.First,gliadins were removed with70%ethanol and55%(v/v)isopropanol at65°C for three times,and then glutenins were extracted with the method described by Yan et al.(2003a).SDS-PAGE was performed on a vertical electrophoresis unit(Ho fer,Freiburg,Germany;Bio-Rad, Hercules,CA,USA).Discontinuous electrophoresis was conducted with15%running gel and4%stacking gel.2-DE(A-PAGE·SDS-PAGE):Two-dimensional electrophoresis (2-DE)of HMW-GS was carried out according to Yan et al. (2003a).A-PAGE based on Morel(1994)wasfirst performed followed by SDS-PAGE.After thefirst-dimension A-PAGE analysis,the gels were cut into single strips and incubated for30min at room temperature in the equilibration solution containing10%glycerol, 2%SDS and0.0625M Tris–HCl at pH6.8.The equilibrated gel strips were placed on top of the second-dimension SDS-PAGE gel(15%). Electrophoresis was performed on Bio-Rad Mini Cell(PROTEAN3, Bio-Rad)at10mA for20min followed by25mA for4h at room temperature.Acidic capillary electrophoresis:(A-CE)The sample preparation and acidic capillary electrophoresis(A-CE)of HMW-GS were based on the methods of Yan et al.(2004).Flour(about10–20mg)from a single kernel was accurately weighted and wasfirst extracted four times with 50%propanol for removing monomeric gliadins,and then glutenins were reduced and extracted with50%propanol+1%Dithiothreitol (DTT;10l l/mg)for30min at65°C with regular stirring.After centrifuging for10min at10000g,HMW-GS was precipitated from glutenin extracts with the addition of acetone to afinal concentration of40%.Precipitated HMW-GS were redissolved in25%acetonitrile (ACN)+0.1%trifluoroacetic acid(TFA)(w/v).The capillary electrophoretic separations of the HMW-GS were carried out using the BioFocus3000instrument(Bio-Rad,New York,Plant Breeding125,120—124(2006)Ó2006The AuthorsJournal compilationÓ2006Blackwell Verlag,BerlinNY,USA)at40°C and12.5kV with0.1M phosphate-glycine buffer, pH2.5,containing20%ACN and0.05%hydroxypropyl-methylcel-lulose with an uncoated fused-silica capillary of25.5cm separation length and50l m internal diameter.HMW glutenin subunits were detected by UV absorbance at200nm.All samples were injected at 10kV for8s.After each separation,capillaries were rinsed with1M phosphoric acid for2min and then with separation buffer for2min. HMW glutenin subunit identification,nomenclature and analysis:The identification of HMW-GS and alleles were based on the methods of Payne and Lawrence(1983)and Yan et al.(2003b).The new HMW-GS that were not previously detected were numerically named according to their relative mobilities and the allele designation was based on McIntosh et al.(2003).HMW-GS identification by A-CE was achieved through comparison of single and mixture samples employing standard cultivars and the results were obtained from various gel electrophoresis.The genetic diversity at Glu-A1and Glu-B1loci was calculated onthe basis of Nei(1973):H¼1)R p2i (H is Nei’s genetic variationindex and p i is the frequency of a particular allele at that locus).ResultsThe HMW glutenin subunit compositions and their frequen-cies identified from205cultivated emmer accessions are listed in Table1.Some typical HMW-GS detected were showed in Fig.1.At the Glu-A1locus,six alleles were identified and Glu-A1a, Glu-A1c and Glu-A1b were three most frequent alleles with 57.56%,40%and29%,respectively.The subunit1A·2.1*,first identified in European spelts and cultivated emmer wheat by Yan et al.(2003a),was located between1A·1and 1A·2*and occurred at a frequency of11%.Comparing with the previous investigations,two novel subunits1A·1.1 and1A·2¢were detected and designated as Glu-A1x and Glu-A1y,respectively.The subunit1.1was between subunit1 and 2.1*and was present infive accessions(PI355465, PIPI355480,PI94682,NGB7201and TT826)at a frequency of 2.44%.Two accessions(TRI14165/91and PI352333) possessed the subunit2.1¢that moved slightly slower than the subunit2but slightly faster than the subunit2*.At the Glu-B1locus,a total of34alleles were detected,and 20alleles and seven subunits had not been described previ-ously.The allele Glu-B1bh coding for13+22*identified by Yan et al.(2003a)was the most frequent(19.51%).The second most frequent allele was Glu-Bb(17.07%)coding for subunit 7+8while another allele with a little higher frequency (7.32%)was Glu-Bbe coding for subunits6.1+22.1.This allele as well as Glu-B1bh was also frequently present in central European spelt wheats(Yan et al.2003a;An et al.2005).The other11alleles(Glu-B1d,Glu-B1bf,Glu-B1ac,Glu-B1ao,Glu-B1c,Glu-B1a,Glu-B1as,Glu-B1aj,Glu-B1an,Glu-B1g and Glu-B1h)that were identified previously in emmer,durum and hexaploid Triticum species(McIntosh et al.2003)appeared at lower frequencies in this study.As shown in Table1,a total of20novel alleles were found and designated as new alleles from Glu-B1bk to Glu-B1cd based on the nomenclature of McIntosh et al.(2003). The subunits17*and9*encoded by Glu-B1bk appeared in 12accessions(5.85%),which moved slightly faster than subunits17and9,respectively(Fig.1).Nine accessions (PI377653,PI377661,PI94659,HTRI14734/87,HTRI16796/ 93,TT¢175,TT36601,TT36602and TT36604)possessed allele Glu-B1bl(14.1+22*),in which subunit14.1was slightly faster than subunit14.Seven accessions (ATRI13482/84,HTRI17200/94,TT260,TT807,TT42068, TT43527and TT43528)contained the allele Glu-B1bm coding for subunit6¢+22.1while another seven accessions (PI94680,ATRI4303/74,ATRI5329/74,TT42059,TT42062, TT42071and TT42077)possessed Glu-B1bn allele coding for subunit13¢+22*.The subunits6¢and13¢moved slightly slower than subunits6and13,respectively.For the remaining two novel subunits detected,the subunit20*in TT236and TT692apparently moved faster than subunit20Table1:HMW glutenin subunit and allele compositions and their frequencies in205cultivated emmer accessions(Triticum turgidum ssp. dicoccum Schrank)Loci Subunits AllelesFrequencyLoci Subunits AllelesFrequency No%No%Glu-A11a11857.56Glu-B1Null+22.1bq3 1.46 Null c4019.516+22*br3 1.462*b2914.157+8.1bs3 1.462.1*w11 5.376¢+null bt20.981.1x52.4420*+null bu20.982.1¢y20.9820*+8bv20.98 H10.60714.1+22.1bw20.98 Glu-B113+22*bh4019.51 6.1+16bx20.98 7+8b3517.077+null a20.986.1+22.1be157.3213+null as20.9817*+9*bk12 5.85Null+8aj10.496+8d12 5.85Null+16by10.496.1+null bf10 4.88Null+9*bz10.4914.1+22*bl9 4.39 6.1+8ca10.496¢+22.1bm7 3.42 6.1+8.1cb10.4913¢+22*bn7 3.4213+8cc10.496.1+22*bo6 2.9314+22*cd10.496+16ac6 2.936+null an10.497+16ao5 2.4413+19g10.497+9c5 2.4414+15h10.4914+8bp3 1.46H10.9081H-Nei’s genetic variation index.Detection of HMW glutenin subunit variations in cultivated emmer121while the subunit 8.1in TT261and TRI16883/93was located between subunits 8and 9.For the frequencies of different HMW-GS compositions at Glu-1loci among 205cultivated emmer genotypes,a total of 62combinations were detected.Two major genotypes 1,13+22*and 1,7+8were the most frequent among all accessions,at 31.2%and 25.4%,respectively.For the remaining 60genotypes,most of them were present at lower frequencies,generally less than 5%.According to the fre-quency of a particular allele at that locus,the genetic diversity at Glu-A1and Glu-B1loci was calculated and estimated by Nei’s genetic variation index H .As shown in Table 1,H -values of Glu-A1and Glu-B1loci were rather high and up to 0.607and 0.908,respectively.As arrowed in Fig.1,most of the cultivated emmer accessions showed an additional band or a major band with a minor band on the SDS-PAGE gel except for the identified HMW-GS.In general,these bands were present with higher mobilities and located at similar positions with 1By and 1Dy subunits.These additional protein components may be 1Ay subunits coded by Glu-A1locus (Waines and Payne 1987)or 1Bz subunits controlled by chromosome 1B (Holt et al.1981)or induced by sample preparation (see Discussion later).The genetic and functional properties of these protein subunits are not known and therefore further identification is needed.In this investigation,the new subunits and alleles detected by SDS-PAGE were further identified by two-dimensional gel electrophoresis (A-PAGE ·SDS-PAGE)and A-CE.Several typical HMW glutenin subunits fractionated by 2-DE were showed in Fig.2.It was obvious that all subunits analysed were well separated and most of them showed a single spot on the 2-DE gel.However,almost all 1By subunits were separated into a major dot and a minor dot.A-CE patterns of some novel subunits were showed in Fig.3.Both 1A and 1B subunits eluted in 5–12min and their migration rank was similar to that of A-PAGE.For instance,in contrast to SDS-PAGE,the subunit 1A ·1eluted earlier than the subunits 1Ax 2*and 2.1*.The migration time of all 1By subunits was faster than that of 1Bx subunits.Furthermore,some additional minor peaks separated by A-CE were also present,which1.61SG -W M L 1.411.221*22311.2*1*71*9.116′.221*02838T T 528T T 081T T 632T T 062T T 49T T M H G -W S122L I ,Y A N ,W A N G ,A N ,Z H A N G ,H S A M and Z E L L E Rprobably correspond to1Ay or1Bz subunits as shown above or produced by post-translational modification of HMW-GS.DiscussionNowadays,the genetic variability of wheat cultivars is decreasing as a consequence of the genetic erosion of cultivated hexaploid wheats.Therefore,it is important to search for new contributing genes for wheat improvement.Previous investi-gations showed that wild and closely related Triticum species of cultivated wheats,such as Aegilops tauschii(2n¼2x¼14, DD),wild emmer(T.turgidum var.dicoccoides)and cultivated emmer(T.turgidum var.dicoccum)possess considerable allelic variations in HMW-GS composition(Yan et al.2003a,b; Pflu ger et al.2001;Levy et al.1988;Vallega and Waines1987). These rich gene resources may play an important role in wheat quality improvement.In this study,we detected the allelic variations at Glu-A1 and Glu-B1loci in205cultivated emmer accessions from German and Chinese GenBanks and these germplasms were not previously investigated for their genetic diversity of gluten compositions.According to our experience,separation method was highly important for HMW-GS identification. To obtain higher resolution of HMW-GS fractionation,an improved SDS-PAGE method combined with two-dimen-sional polyacrylamide gel electrophoresis(A-PAGE·SDS-PAGE)and A-CE were used.The results obtained showed rather high polymorphism in HMW-GS variation,especially for Glu-B1locus.Of40HMW-GS alleles and62genotypes detected,22novel alleles and nine new subunits at Glu-1loci were found.The high genetic polymorphism detected suggests that the cultivated emmer germplasms collected in German and Chinese GenBanks may provide more genetic resources for wheat improvement.The genetic diversity revealed in this work was much higher than that in previous reports(Pflu ger et al.2001;Vallega and Waines1987),in which only13and23alleles at two Glu-1loci were detected in97and167accessions from Spain and USA gene pools, respectively.This may be because of the limited accessions used in their work or the emmer germplasms in this study possessed per se higher genetic diversity.It is still possible that our improved SDS-PAGE combined with2-DE and A-CE methods can reveal more allelic variations because of their higher resolution for glutenin fractionation(Yan et al. 2003a,b,c).It was previously reported that many of the HMW-GS bands detected by SDS-PAGE separated into several components when fractionated by IEF·SDS-PAGE(Holt et al.1981),A-PAGE·SDS-PAGE(An et al.2005)and A-CE(Yan et al. 2003c,2004).As shown in this study,some single bands separated by SDS-PAGE,especially for1By subunits were generally isolated into a major and a minor spot or peak by A-PAGE·SDS-PAGE and A-CE(indicated in Figs2and3). Although the reason for the multiplicity of glutenin subunits is not clear,it is most likely that these multiple components are the results of post-translational modification of the subunits.By far, glycosylation and phosphotyrosine of some HMW-GS have been found(Tilley et al.1993;Tilley and Schofield1995; Lauriere et al.1996)and their roles forflour quality have been unknown.However,HMW-GS generally showed more com-ponents under acidic condition,such as A-PAGE and A-CE. Furthermore,some of the minor spots separated by IEF·SDS-PAGE appeared to be more acidic than the major components (Holt et al.1981).In general,the presence of urea and TFA during preparation of gluten samples for A-PAGE,A-CE and2-DE analyses could produce more acidic protein components (Holt et al.1981;Yan et al.2003c,2004).Therefore,it cannot be excluded that the multiple components of HMW-GS were artefacts of sample preparation.As shown in Fig.1,many accessions possessed an additional band or a major band with a minor band.Most of them were in the positions of1By and1Dy subunits with higher mobilities.Holt et al.(1981)identified a third group of HMW-GS coded by chromosome1B and designated1Bz subunits by IEF·SDS-PAGE and NEPHGE·SDS-PAGE in the common wheat(Triticum aestivum L.).They deduced that the1By and1Bz subunits were the products of the same gene or they were coded by adjacent genes on chromosome1B. Thus,the additional bands detected may be the1Bz subunits. It is known that the Ay subunit genes are silent in common wheat.However,some investigations demonstrated that the 1Ay subunit gene expressed in wild emmer(T.dicoccoides)and diploid wheats(Triticum boeoticum and Triticum urartu)as well as in some cultivated hexaploid wheat lines(Waines and Payne1987;Levy et al.1988;Margiotta et al.1996).There-fore,it is most likely that the additional main band detected in cultivated emmer accessions was1Ay subunit while the minor band was induced by sample preparation,but further identi-fication is needed.Interestingly,the presence of both x-and y-type subunits at the Glu-A1locus may produce superior quality when compared to the lines that only possessx-type Detection of HMW glutenin subunit variations in cultivated emmer123subunit1or2*(Johansson et al.1993).Hence,cultivated emmer is expected to be valuable as a genetic resource for quality improvement of cultivated hexaploid wheat. AcknowledgementsThis research wasfinancially supported by a DAAD Scholarship, Germany and a research grant(no.JY-03-A-13)from the National Special Project on Transgenic Plants and Industrialization Research of China,respectively.ReferencesAn,X.L.,Y.M.Yan,Y.H.Xiao,Q.Y.Li,S.L.K.Hsam,and F.J.Zeller,2005:Genetic diversity of European spelt wheat (Triticum spelta L.)revealed by glutenin subunit variations at Glu-1 and Glu-3loci.Euphytica.DOI:10.1007/s10681-005-9002-6. Galterio,G.,M.Cappelloni,E.Desiderio,and N.E.Pogna,1994: Genetic,technological and nutritional characteristics of three Italian populations ofÔÔfarrumÕÕ(Triticum turgidum ssp.dicoccum).J.Genet. Breed.48,391—398.Holt,L.M.,R.Astin,and P.I.Payne,1981:Structure and genetical studies on the high-molecular subunits of wheat glutenin.Part2: relative isoelectric points determined by two-dimensional fraction-ation in polyacrylamide gels.Theor.Appl.Genet.60,237—243. Johansson,E.,P.Henriksson,G.Svensson,and W.K.Heneen,1993: Detection chromosome location and evaluation of the functional value of a novel high Mr glutenin subunit found in Swedish wheats. J.Cereal.Sci.17,237—245.Lauriere,M.,I.Bouchez,C.Doyen,and L.Eynard,1996:Identifi-cation of glycosylated forms of wheat storage proteins using two-dimensional electrophoresis and blotting.Electrophoresis17, 497—501.Levy,A.A.,G.Galili,and M.Feldman,1988:Polymorphism and genetic control of high molecular weight glutenin subunits in wild tetraploid wheat Triticum turgidum var.dicoccoides.Heredity61, 63—72.Margiotta,B.,M.Urbano,G.Golaprico,E.Buonucore,R.D’Ovidio, and fiandra,1996:Detection of y-type subunit at the Glu-A1 locus in some Swedish bread wheat lines.J.Cereal.Sci.23, 203—211.McIntosh,R.A.,Y.Yamazaki,K.M.Devos,J.Dubcovsky,W.J. Rogers,and R.Appels,2003:Catalogue of gene symbols for wheat. /ggpages/wgc.Morel,M.H.,1994:Acid-polyacrylamide gel electrophoresis of wheat glutenins:a new tool for the separation of high-and low molecular-weight subunits.Cereal Chem.7,238—242.Nei,M.,1973:Analysis of gene diversity in subdivided populations. A70,3321—3323.Payne,P.I.and wrence,1983:Catalogue of alleles for the complex gene loci Glu-A1,Glu-B1and Glu-D1which code for high-molecular weight subunits of glutenin in hexaploid wheat.Cereal mun.11,29—35.Payne,P.I.,K.G.Corfield,and J.A.Blackmann,1979:Identification of high-molecular-weight subunit of glutenin whose presence corre-lates with bread-making quality in wheats of related pedigree. Theor.Appl.Genet.55,153—159.Payne,P.I.,L.M.Holt,E.A.Jackson,and w,1984:Wheat storage proteins:their genetics and their potential for manipulation by plant breeding.Philos.Trans.R.Soc.Lond.B Biol.Sci.304, 359—371.Pflu ger,L.A.,L.M.Martın,and J.B.Alvarez,2001:Variation in the HMW and LMW glutenin subunits from Spanish accessions of emmer wheat(Triticum turgidum ssp.dicoccum Schrank).Theor. Appl.Genet.102,767—772.Piergiovanni,A.R.,and A.Blanco,1999:Variation of HMW glutenin and c-gliadin subunits in selected accessions of Triticum dicoccum (Schrank)and T.spelta(L.).Cereal mun.27,205—211. Singh,N.K.,K.W.Shepherd,and G.B.Cornish,1991:A simplified SDS-PAGE procedure for separating LMW subunits of glutenin. J.Cereal Sci.14,203—208.Shewry,P.R.,N.G.Halford,and A.S.Tatham,1992:High-molecular weight subunits of wheat glutenin.J.Cereal Sci.15, 105—120.Tilley,K.A.,and J.D.Schofield,1995:Detection of phosphotyrosine in the high Mr subunits of wheat glutenin.J.Cereal Sci.22,17—19. Tilley,K.A.,G.L.Lookhart,R.C.Hoseney,and T.P.Mawhinney, 1993:Evidence for glycosylation of the high molecular weight glutenin subunits2,7,8,and12from Chinese Spring and TAM105 wheats.Cereal Chem.70,602—606.Vallega,V.,and J.G.Waines,1987:High-molecular-weight glutenin subunit variation in Triticum turgidum var.dicoccum.Theor.Appl. Genet.74,706—710.Waines,J.G.,and P.I.Payne,1987:Electrophoretic analysis of the high molecular-weight glutenin subunits of Triticum monococcum, T.urartu,and the A genome of bread wheat(T.aestivum).Theor. Appl.Genet.74,71—76.Yan,Y.,S.L.K.Hsam,J.Yu,Y.Jiang,I.Ohtsuka,and F.J.Zeller, 2003a:HMW and LMW glutenin alleles among putative tetraploid and hexaploid European spelt wheat(Triticum spelta L.)progeni-tors.Theor.Appl.Genet.107,1321—1330.Yan,Y.,S.L.K Hsam,J.Yu,Y.Jiang,and F.J.Zeller,2003b:Allelic variation of the HMW glutenin subunits in Aegilops tauschii accessions detected by sodium dodecyl sulphate(SDS-PAGE),acid polyacrlamide gel(A-PAGE)and capillary electrophoresis.Euphy-tica130,377—385.Yan,Y.,J.Z.Yu,Y.Jiang,Y.Hu,M.Cai,S.L.K.Hsam,and F.J.Zeller,2003c:Capillary electrophoresis separation of high molecular weight glutenin subunits in bread wheat(Triticum aestivum)and related species with phosphate-based buffers.Elec-trophoresis24,1429—1436.Yan,Y.,Y.Jiang,M.Sun,J.Yu,Y.Xiao,J.Zheng,Y.Hu,M.Cai,Y. Li,S.L.K Hsam,and F.J.Zeller,2004:Rapid identification of HMW glutenin subunits from different hexaploid wheat species by acidic capillary electrophoresis.Cereal Chem.81,561—566.124L I,Y A N,W A N G,A N,Z H A N G,H S A M and Z E L L E R。