5 Maataloustieteellinen Aikakauskirja Vol. 57: 271—277, 1985 PAG electrophoregrams of wheat cultivars grown in Finland TUULA SONTAG and HANNU SALOVAARA Department of Food Chemistry and Technology, University of Helsinki, SF-00710 HELSINKI, Finland Abstract. The polyacrylamidegel electrophoretic (PAGE) patterns of gliadins of9 spring wheat cultivars (Apu, Drabant, Taava, Tapio, Ulla, Kadett, Luja, Ruso and Tähti) and of 5 winter wheat cultivars (Aura, Ilves, Linna, Nisu and Vakka) were determined. Most of the samples studied had specific gliadin PAGE patterns, indicating that electrophoregrams ob- tained with the procedure employed here can be used for identifying wheat cultivars grown in Finland. Only two cultivars, Taava and Ruso, which are close relatives, possessed similar PAGE patterns. The procedure uses a commercial vertical electrophoresis apparatus and thin gels. Up to 28 samples could be electrophoresed in three hours and analyzed after staining. The procedure can be applied in the identification of wheat cultivars currently grown in Finland. Index words: Wheat, gliadins, electrophoresis, cultivar identification. Introduction Polyacrylamide gel electrophoresis (PAGE) is extensively applied to wheat cultivar iden- tification in breeding programs and other con- nections where accurate information on the identification of a small wheat sample is needed. The procedure separates the alcohol soluble proteins, or gliadins, of wheat endo- sperm into protein bands of different electro- phoretic mobilities and intensities. Cultivars have band patterns (electrophoregrams) that are characteristic of the genotype and indepen- dent of growth conditions (Wrigley 1970, ZiLLMANand Bushuk 1979). Bushuk and Zill- man (1978) developed a gliadin PAGE meth- od for wheat which has also been recom- mended as a reference method for wheat cul- tivar identification by the International Asso- ciation for Cereal Science and Technology (ICC) (Anon. 1984). The interest of institutes and industry towards the identification of wheat cultivars by electrophoresis has increased in Finland. Therefore the need for reference data on wheat electrophoregrams of the cultivars grown in Finland has become evident. It was the aim of the present study to produce ref- 271 JOURNAL OF AGRICULTURAL SCIENCE IN FINLAND https://www.c-info.fi/en/info/?token=IY96PSnSFE5NmlPj.V9-bnzEya1XHDdEjR0iJ3w.SfcnTK9eYIdZg4GhrqYf8oK-2OqnpvPEp72QGyJ433pMhTtMinEQUFeTvsYPauKE4L1P2OA34JJ2VG-9FxEp3Apeju-jqbBr-HWicg0uxfYtw9KD6pqtWhCFvrYN061FduETuNEtJkoS_-5mBrOTi_PXLg6Efi-iWOPkqJUzaPdXHo94stnKHDscGSoDfw Table 1. Data of the spring and winter wheat cultivars grown in Finland and examined in the present study. Name of cultivar Breeder Origin Put on the market Spring wheats Apu Jo 1 Garnet/Pika 1949 Drabant WW! cl 12633/Ring 1972 Hankkija’s Taava Hja 3 “Co mutant from Ruso 1978 Hankkija’s Tapio Hja Hja c3929/Kolibri 1979 Hankkija’s Ulla Hja Tammi/Hja a4431 1975 Kadett WW Kolibri/Pompe 1981 Luja Jo Svenno//Hopea/Tammi 1981 Ruso Hja Reward/Pika//free poll. % 1967 Tähti Jo Kärn//Aurora/Pika 1972 Winter wheats Aura Jo Ertus/Vakka 1975 Hankkija’s lives Hja Hja b356/Vakka 1984 Linna Hja Panu/Hja 04819/VVirtus 1965 Nisu Jo Varma/Kehra 1966 Vakka Jo Varma/Kehra 1953 I = Agricultural Research Centre, Department of Plant Breeding, SF—3l6OO Jokioinen 2 = W.Weibul AB, Sweden 3 = Hankkija Plant Breeding Institute, SF-04300 Hyrylä erence data on the PAG electrophoregrams of spring and winter wheat cultivars currently grown in Finland. Materials and methods Samples Samples of 14wheat cultivars were obtained from the cultivar collection maintained by the Finnish State Seed Testing Station. Complete name, breeder, pedigree and the year the cul- tivar had been released on the market are shown in Table 1. As a reference sample served Canadian cultivar Marquis, which was received from Dr. W. Bushuk (University of Manitoba, Canada). Reagents Acrylamide (for electrophoresis, 2 x crystallized) and Serva blau R-250 (C.I. 42660) were obtained from Serva Feinbio- chemica; aluminium lactate was obtained from Fluka; N,N’-methylene-bisacrylamide from BDH Chemicals Ltd; ammoniumperox- odisulphate, L( -I- )-ascorbic acid, ferrosul- phate (FeSG4 x 7H 2G), methyl green and trichloroacetic acid were obtained from E.Merck; lactic acid was obtained from the Pharmacy of University of Helsinki. All the reagents were analytical grade. Apparatus The electrophoresis apparatus was a Phar- macia Gel Electrophoresis Apparatus GE-2/4 LS (Sweden) with a LKB 2103 Power Supply (Sweden). Preparation of the gels The gel solution was prepared by the recipe of Maier and Wagner (1980) (Table 2). The gels (140 X 180 X 1.5 mm) were polymerized as described previously (Sontagcl al. 1985). Before use they were kept overnight at 4°C. The gels were usable I—2 weeks after prepa- ration. 272 Table 2. Recipes for gel and tank buffer solutions. Solution Amount required Gel, 1000 ml Acrylamide, g 60.0 N,N’-Methylene bisacrylamide, g 3.0 Ascorbic acid, g 1.0 FeS04 x7 H2 O, g 0.046 Aluminium lactate, g 2.5 Lactic acid, ml to pH 3.2 Catalyst, 100 ml Ammonium persulphate, g 1.4 Tank buffer, 1000 ml Aluminium lactate, g 2.5 Lactic acid, ml to pH 3.2 Sample preparation Whole wheat kernels were ground by hand using a mortar and pestle. The ground grain was extracted with three times its weight of 70 ®7o aqueous ethanol in a stoppered centri- fuge tube. The tube was allowed to stand at room temperature for 1 h with occasional mixing on a vortex mixer and thencentrifuged for 10 min at 3500 X rpm. The supernatant was removed to a vial (1.5 ml) and diluted with two times its volume of sample buffer. The buffer was made of tank buffer with 30 % saccharose to increase the density of the pro- tein solution and 1 % methyl green to serve as a marker dye and to show how the sample layered in the gel slot. Gliadin extracts were stored at 4°C in sealed vials until use. Freezing of the gliadin extracts seemed to cause great losses in band intensities of the anodic bands. Electrophoresis The gliadin extracts (10 /d) were loaded in- to the 14 gel slots with microliter syringe. The gliadin extract of the cultivar Marquis was placed in each gel to serve as a reference sam- ple. Electrophoresis was performed at 400 V for about 3 h and buffer temperature was main- tained at 16°C by tapwater circulation. Elec- trophoresis was allowed to proceed until the second (purple) marker dye band of methyl green had migrated to exactly 0.5 cm form the end of the gel. During this time the albumins and globulins that the protein extract also con- tains run off the gel leaving only the gliadins on the gel (Khan 1982). Staining and photography The gel was stained overnight (16 h) in a so- lution of 5 ml of an 94 % ethanol solution of 1 % Serva blau R-250 diluted with 200 ml of 12 % trichloroacetic acid. After staining the gel was rinsed with water and photographed. The stained gel was placed on a glass plate on a light box and illuminated from below and photographed with Agfaortho 25 film. Determination of mobilities and intensities The relative mobilities (Rm) and relative in- tensities (Ri) of gliadin bands were evaluated by the procedure of Bushuk and Zillman (1978). Rm values were determined by mea- suring the migration distance of the gliadin band from the origin (point of sample appli- cation) to the centre of the band. The migra- tion distance of the given band dividedby the migration distance of a specific band, termed band 50 (Fig. 1), of the cultivar Marquis on the same gel and the result multiplied by 50 gave the band intensities were rated subjec- tively by visual examination of the band staining intensities by a number from 1 to 5, with 5 representing the most intensely stained and 1 the most weakly stained bands. Reproducibility Duplicate electrophoresis of each of the four replicate samples was performed to en- sure visual similarity of the electrophoresis formula of replicates. Reproducibility of the electrophoretic procedure used in this study was determined by measuring the relative mo- bilities of three bands in the electrophoregram of the standard cultivar Marquis (Fig. 1) on 273 8 different gels. The coefficients of variation for the relative band mobilities of band A (Rm = 17), band B (Rm = 45) and band C (Rm = 68) were 2.9 %, 1.2 % and 4.0 %, respectively. Results Electrophoregrams of the wheat cultivars analyzed in this study are shown in Fig. 1 and 2. Electrophoregram formulas for these wheat cultivars expressed by relative band mobility and band intensity are given in Table 3. All the cultivars studied could be identified by their electrophoregrams. The most distinct differences between the gliadin patterns of these cultivars are found in the anodic part of their electrophoregrams. The spring wheat cultivars studied could be divided into three groups on the base of the pattern of their anodic bands. Three of the cultivars, i.e. Tähti, Ruso and Taava were dis- tinguished by an anodic band pattern charac- teristic also of the Canadiancultivar Marquis which was used as a reference. However, in spite of this similarity there was a distinct dif- ference in the cathodic region of the bands between cultivars Tähti, Ruso and Marquis, making identification possible (Fig. 1). Taa- va had exactly the same gliadin pattern as Ruso. These two cultivars are close relatives, Taava being 60Co-mutant of Ruso, which explains the similarity of their gliadin patterns. Five cultivars, i.e. Apu, Drabant, Tapio, Kadett and Luja, had fewer anodic bands than Ruso, Taava and Tähti (Fig. 1). Distinct dif- ferences were seen between the anodic band patterns of these five cultivars permitting pre- cise identification. One of the spring wheat cultivars, i.e. Ulla, had a strong anodic band Fig. I. Electrophoregrams of gliadins from 9 spring wheat cultivars grown in Finland and reference cultivar Marquis. From left to right: Marquis, Tähti, Ruso, Luja, Kadett, Tapio, Drabant, Marquis, Ulla, Taava, Apu, Taa- va. Marquis. 274 275 Table 3. Electrophoretic formulas of gliadins of 14 wheat cultivars grown in Finland. Mobility of bands relative to Marquis standard band and relative intensities of bands (scale 1 to 5) Spring wheats Marquis 17(4), 20(3), 22(3), 25(3), 28(1), 31(2), 34(2), 45(5), 46(5), 50(5), 53(2), 55(3), 57(2), 59(3), 63(4), 65(3), 68(3), 71(4), 80(2), 82(3) Apu 17(4), 20(3), 31(3), 40(1), 45(5), 47(4), 50(2), 53(3), 55(2), 59(4), 63(4), 65(1), 68(3), 71(3), 80(2) Drabant 15(2), 17(4), 21(2), 25(4), 38(2), 45(5), 46(5), 49(3), 50(3), 53(2), 56(3), 59(4), 63(4), 65(3), 68(3), 71(4), 80(2), 82(1) Kadett 15(2), 17(4), 21(2), 25(1), 28(2), 35(3), 45(5), 46(5), 49(3), 50(3), 53(2), 56(4), 59(4), 63(4), 65(3), 68(3), 71(4), 80(2), 82(1) Luja 17(4), 20(3), 26(1), 30(3), 34(3), 35(2), 45(5), 46(5), 48(4), 50(3), 53(3), 55(3), 57(3), 59(4), 63(4), 68(2), 71(4), 80(2) Ruso 17(4), 20(3), 22(3), 25(4), 28(1), 31(2), 36(2), 38(2), 40(5), 46(5), 48(3), 50(5), 53(3), 55(3), 57(3), 59(3), 63(4), 65(1), 68(3), 71(4), 80(3) laava 17(4), 20(3), 22(3), 25(4), 28(1), 31(2), 36(2), 38(2), 40(5), 46(5), 48(3), 50(5), 53(3), 55(3), 57(3), 59(3), 63(4), 65(1), 68(3), 71(4), 80(3) Tapio 17(4), 19(4), 23(1), 26(1), 30(2), 34(3), 40(1), 45(5), 48(4), 53(3), 55(3), 55(3), 57(3), 59(4), 62(4), 65(2), 71(4), 80(2) Tähti 15(1), 17(4), 20(3), 22(3), 25(4), 28(1), 31(2), 36(2), 38(2), 40(5), 46(5), 48(3), 50(5), 53(3), 55(3), 57(3), 59(3), 63(4), 65(4), 67(3), 68(3), 71(4), 80(3), 85(3) Ulla 14(3), 17(3), 19(3), 23(2), 24(1), 26(2), 30(3), 34(3), 35(2), 45(5), 46(5), 50(3), 53(2), 55(3), 57(3), 59(3), 63(3), 65(2), 68(3), 71(4), 80(3), 82(1) Winter wheats Aura 15(2), 17(4), 21(2), 28(1), 30(1), 32(2), 40(2), 45(5), 46(5), 49(3), 50(3), 53(2), 55(4), 57(4), 59(3), 62(4), 65(3), 69(2), 71(3), 77(2) Ilves 17(5), 20(3), 22(3), 25(4), 28(2), 30(2), 31(1), 34(3), 45(5), 46(5), 49(3), 50(5), 53(3), 55(4), 59(4), 60(4), 62(4), 65(3), 69(2), 71(4), 77(4), 80(2), 82(2) Linna 15(2), 17(5), 21(2), 30(1), 31(3), 35(1), 40(2), 45(5), 46(5), 49(3), 50(2), 53(5), 55(4), 57(3), 60(4), 62(4), 65(2), 69(3), 71(4), 74(3), 77(3), 80(2), 82(2) Nisu 15(4), 17(4), 21(2), 25(3), 28(1), 30(2), 35(1), 38(3), 45(5), 46(5), 49(3), 53(5), 55(3), 57(3), 60(4), 62(4), 65(3), 69(2), 71(4), 77(3), 80(2) Vakka 15(2), 17(4), 21(2), 30(2), 31(2), 34(3), 45(5), 46(5), 49(3), 53(5), 55(4), 57(4), 60(4), 62(4), 65(3), 69(3), 71(4), 77(3) pattern which could not be detected in any other spring wheat cultivar studied. The winter wheat cultivars also had charac- teristic band patterns by which they were iden- tifiable. The four cultivars Vakka, Nisu, Lin- na and Aura shared three anodic bands in similar positions (Fig. 2). However, differ- ences in the rest of the anodic bands facilitated identification. The fifth winter wheat cultivar studied, lives, showed an anodic pattern with distinct differences as compared to the rest of the winter wheat cultivars. A considerable part of the electrophoregram obtained from lives had close similarity with spring wheat cultivars Ruso and Tähti (Fig. 1 and 2). However, li- ves was clearly characterized by the location of its bands in the central part of the elec- trophoregram. Discussion Analysis of the gliadin electrophoregrams serves as a useful tool for research especially in wheat breeding, where accurate identifica- tion of breeding lines is needed. The electro- phoretic method has several serious short- comings in other applications where rapid cul- tivar identification of commercial parcels is desirable. The small sample size may be an ad- vantage in plant breeding work but it is sub- ject to many problems when cargoes of wheat should be characterized for their cultivar com- position. However, the electrophoretic pro- cedure described here has found recent appli- cation in the wheat processing industry for the verification of certain lots of wheat of sus- pected variety. The procedure used in this study is a modification of the reference meth- od deviced by the International Association for Cereal Science and Technology (Anon. 1984). In the present study a commercially available apparatus was used. Further devia- tions in the method were a smaller gel size and the use of a greater number of samples on one gel. It should be realized that the ICC standard procedure is primarily not developed for rou- tine work but rather to serve as a general ref- erence method applicable in situations where a comparability of data is needed. Like the functional quality of wheat protein, the gliadin electrophoretic pattern is cultivar spe- cific. Although a part of the functional quality of wheat proteins is associated with gliadins, evidence for a systematic relationship has not been found between the breadmaking quality of wheat cultivars and the electrophoretic pat- tern of gliadins. Instead, a relationship may exist between the band patterns obtained from the glutenin fraction of the cultivars in a sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) and the bread- making quality of wheat cultivars (Payne et al. 1979, 1981 and Moonen et al. 1982, 1984). Our subsequent work will be concentrated on possible relationships between SDS-PAGE band patterns and the functional properties of wheat cultivars grown in Finland. Acknowledgements. The authors are grateful to Mr Osmo Ulvinen in the Finnish Seed Testing Station for the wheat cultivar samples. The work was financed in part by the Grain Research Committee, Finland and the Finn- ish cultural Foundation. References Anon. 1984. ICC Draft Standard No. 143. Wheat cul- tivar identification bypolyacrylamide gel electrophoresis of the gliadin proteins. Bushuk, W. & Zillman, R.R. 1978.Wheat cultivar iden- tification by gliadin electrophoregrams. I. Apparatus, method and nomenclature. Can. J. Plant Sci. 58: 505—515. Khan, 1982. Polyacrylamide gel electrophoresis of wheat gluten proteins. Bakers Dig. 56, 5: 14—19. Moonen, J.H.E., Scheepstra, A. & Graveland, A. 1982. Use of the SDS-sedimentation test and SDS poly- acrylamide gel electrophoresis for screening breeder’s samples of wheat for bread-making quality. Euphytica 31: 677—690. —, Scheepstra, A. & Graveland, A. 1984. Genetische Aspecte des Glutenins im Zusammenhang mit der Back- Fig. 2. Electrophoregrams of gliadins from 5 winter wheat cultivars grown in Finland and reference cultivar Marquis From left to right: Marquis, Vakka, Nisu, Linna, Ilves, Aura and Marquis. 276 fähigkeit deutscher Weizensorten. Die Muhle u. Misch- futtertechnik. 10: 124—126. Payne, P. J.,Corfield, K.G. & Blackman, J.A. 1979. Identification of a high-molecular-weight subunit of glutenin whose presence correlates with bread-making quality in wheats of related pedigree. Theor. Appi. Genet. 55: 153—159. —, Corfield, K. G., Holt, L.M. & Blackman, J.A. 1981. Correlations between the inheritance of certain high-molecular-weight subunits of glutenin and bread- making quality in progenies of six crosses of bread wheat. J. Sci. Food Agric. 32: 51—60. Sontao, T., Salovaara, H. & Ulvinen, O. 1985. PAG electrophoregrams of six Finnish potato cultivars. J. Agric. Sci. Finl. 57: 147—153. Wrioley, C.W. 1970. Protein mapping by combined gel electrofocusing and electrophoresis: Application to the study of genotypic variations in wheat gliadins. Bio- chem. Genet. 4; 509—516. Zillman,R.R. & Bushuk, W. 1979. Wheat cultivar iden- tification by gliadin electrophoregrams 11. Effects of environmental and experimental factors on the gliadin electrophoregram. Can. J. Plant Sci. 59; 281 —286. Ms received October 4, 1985 SELOSTUS Suomessa viljeltyjen vehnälajikkeiden PAG-elektroforegrammit Tuula Sontag ja Hannu Salovaara Helsingin yliopiston elintarvikekemian ja -teknologian laitos, 00710 Helsinki Polyakryyliamidigeelielektroforeesilla (PAGE) määri- tettiin vehnän gliadiinien elektroforegrammit 14 Suomessa viljeltävästä vehnälajikkeesta. Tutkimuksessa oli 9 kevät- vehnälajiketta (Apu, Drabant, Hankkijan Taava, Hank- kijan Tapio, Hankkijan Ulla, Kadett, Luja ja Ruso) ja 5 syysvehnälajiketta(Aura, Hankkijan Ilves, Linna, Nisu ja Vakka). Kaikilla tutkituilla lajikkeilla oli tyypillinen gliadiinien elektroforegrammi, lukuunottamatta kahta pe- rimältään lähekkäistä lajiketta, Laavaa ja Rusoa, joilla oli samanlaiset gliadiinien elektroforegrammit. Tutki- muksessa käytetyllä vertikaalisella elektroforeesilaitteel- la kyettiin yhdellä ajokerralla määrittämään 28 gliadii- ninäytettä. Hlektroforeesiajo kesti kolme tuntia ja vär- jäys 12 tuntia. Menetelmää voi käyttää suomalaisten veh- nälajikkeiden aitoustutkimuksessa. 277