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Elrcrrophorrsis 1991, 12. 3311-337

5 References [ l ] Cooke, R. J . , Adv. Electrophoresis 1988, 2, 171-261. [2] Cooke, R. J . , Elecfrophoresi.~1984, 5, 59-72. [3] Shah,A.A. and Stegemann, H., Nachrichtenhl. Deut. Pflanzenschutzd. 1986,38, 100-103. 141 Zacharius, R. M., Zel1,T. E., Morrison, J. H . and Woodlock, J., A n d . Biochem. 1969, 30, 148-150. [5] Eckhardt,A. E.,Hayes,C.E.and Goldstein,I.J.,Anal.Biochem. 1976, 73, 192-197. [6] Burridge, K., Proc. Narl. Acad. Sci. USA 1976, 73, 4457-4461. [7] Furlan, M., Perret, B. A. and Beck, E. A , , Anal. Biochem. 1979, 96, 208-214. [8] Avigad, G., Anal. Biochem. 1978, 86, 443-449. [9] Wood, J . G. and Sarinana, F. O., Anal. Biochem. 1975, 69,320-322. [lo] Towbin, H., Staehelin,T., and Gordon, J . , Proc. Natl. Acad. Sci. USA 1979, 76, 4350-4354. [ l l ] Gershoni, J. M., Bayer, E. A. and Wilchek, M., Anal. Biochem. 1985, 146,59-63. [12] Bartles, J. R. and Hubbard, A . L., Anal. Biochem. 1984,140,282-292.

Walter Weiss Wilhelm Postel Angelika Gorg Lehrstuhl fiir Allgemeine Lebensmitteltechnologie, Technische Universitat Miinchen, Freising-Weihenstephan

[13] Glass,W. F.,Briggs,R. C.and Hnilica.1. S.,Anal. U/ochc.m.1981,115, 2 19-224. [I41 Hawkes, R., Anal. Biochem. 1982, 123, 143-146. [15] Clegg, J. C. S . , Anal. Biochem. 1982, /27,389-394. [16] Rohringer, R. and Holden,D. W.,Anal. Biochcm. 1985, /44,118-127. [17] Gorg, A., Postel,W.,Giinther, S . and Weser,J., in: Dunn, M. J. (Ed.), Electrophoi-esis ‘86, VCH Verlagsgesellschaft, Weinheim 1986, pp. 435449. [I81 Laemmli, U. K., Nature 1970, 227. 680-685. [19] Gorg,A.,Postel,W.,Weser,J.,Westermeier, R. and Ek.K.,LKBApplicnrion Nore 348, LKB, Bromma (Sweden) 1987. [20] Blum, H., Beier, H. and Gross, H. J., Elecrrophoresis 1987, 8,93-99. [21] Kyhse-Andersen, J.,J.Biochem. Bioph.ys. Methods 1984, 10,203-209. [22] Hancock, K. and Tsang,V. C . W., Anal. Biochem. 1983,133,157-162. [23] Osborne, T. B., The Proteins of the Wheat Kernel, Carnegie Inst. Washington Publ. 84, Judd and Detweiler, Washington, USA, 1907. [24] Taketa, K., Electrophoresis 1987, 8, 409-414. [25] Schott, K. J . , Neuhoff,V., Nessel, B., Potter, U. and Schroter, J., Electrophoresis 1984, 5, 77-83. [26] Dulaney, J. T., Anal. Biochem. 1979, 99, 254-267.

Barley cultivar discrimination: 11. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing with immobilized pH gradients Isoelectric focusing performed with immobilized pH gradients was found superior to other commonly used electrophoretic methods for discrimination of 55 European winter and spring barley cultivars. Hordeins, the alcohol-soluble proteins, yielded 32 different patterns, allowing identification of 22 cultivars and classification of the remaining ones into ten groups of two to eight cultivars each. Only 21 different hordein patterns were observed using horizontal sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by silver staining. Twelve cultivars exhibited unique hordein patterns, the remaining nine groups contained 2-1 1 cultivars. Resolution of isoelectric focusing with immobilized pH gradients was further enhanced in some cases when the patterns of urea/dithiothreitolsoluble proteins were used instead of the hordein patterns. However, evaluation was more complicated because of the larger number of protein bands detected.

1 Introduction Barley (Hordeum vulgare L.) is the fourth most extensively grown cereal on the globe, and in some countries of the cool, temperate zone its production exceeds even that of wheat [l].Barley is generally grown in the form of tworowed or six-rowed cultivars. In Germany, two-rowed

Correspondence: Priv. Doz. Dr. A. Gorg, Institut fur Allgemeine Lebensmitteltechnologie, Technische Universitat Munchen-Weihenstephan, DW-8050 Freisiog-Weihenstephan, Germany Abbreviations: Bis, N,N-methylenebisacrylamide; CV., cultivar; DTT, dithiothreitol; IEF,isoelectric focusing; IPG,immobilized p H gradient; M,, relative molecular mass; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; TEMED, N,N,N,N-tetramethylethylenedianiine; Tris, Tris(hydroxymethy1)aminomethane

0VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991

spring barley cultivars are used for malting and brewing, whereas two- and six-rowed winter barleys are mainly grown for animal feeding. Because of the different processing properties of the cultivars, their correct identification is important to the malting and brewing industries. Although barley cultivars are often identified by grain morphological characteristics (e. g. rachilla hair length, aleurone color), many cultivars cannot be distinguished by this means. For this reason, supplementary techniques for identification are needed. One of the most powerful techniques is electrophoresis of the aqueous alcohol-soluble endosperrn proteins, the prolamins (known as hordeins), and, to a lesser extent, electrophoresis of albumins and globulins. Identification of cultivars by protein electrophoresis is possible because the electrophoretic patterns of storage proteins are cultivar-specific and independent of environmental conditions [l, 21. Usually, electrophoresis of hordeins is per-

13ecfruphorrsis 1YY1, 12, 330-337

formed by using polyacrylamide gel electrophoresis (PAGE) at acidic pH [3-81 or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) [9-121. Some authors have described the separation of hordeins by starch gel electrophoresis [13] or isoelectric focusing (IEF) [13161. Others have used multiple forms of enzymes for cultivar identification [17-191. Although electrophoretic separation techniques have greatly facilitated cultivar identification, a significant number of barley cultivars still remains indistinguishable. Therefore, the present study was undertaken to investigate whether better discrimination between cultivars than by the conventional techniques can be achieved by applying the most powerful one-dimensional electrophoretic technique with a maximum resolution of ApZ= 0.001 [20]), namely IEF with immobilized pH gradients (IEF-IPG) [21, 221. The results obtained with IEF-IPG of hordeins were compared to the results obtained with a standard method of barley cultivar discrimination, SDS-PAGE of hordeins followed by silver staining. Additionally, we tested several extraction solvents (aqueous alcohols, urea-dithiothreitol buffer) to evaluate which one was best suited for barley cultivar discrimination by IEF-IPG.

Barley cultivar di~criininationby SDS-PAGE and IEF with IPG

33 1

2.2.2 Extraction of hordeins For the extraction of barley prolamins (hordeins), individual kernels, or groups of five kernels of each variety, were crushed with a hammer until they passed through a 1 mm sieve, transferred into tared 1.5 mL microfuge tubes (Eppendorf, Hamburg, Germany), and weighed. A volume of aqueous 55%v/v2-propanol, equal to fourtimes the weight of the sample, was added. The suspension was mixed several times with the help of a vortex mixer and extracted for 2 h or overnight at room temperature, followed by centrifugation for 20 min (15"C, 20 000 g). The supernatants were removed with the help of a disposable pipette and in most cases used immediately. However, storage at -80°C for up to several weeks did not alter the resulting hordein patterns. In preliminary experiments we had also tested 30 O/o v/v 2-chloroethanol and 70 O/o v/v ethanol (with or without addition of reducing agents) as possible solvents for hordein extraction. For IEF-IPG, extraction supernatants were applied onto the gel without further treatment. For SDS-PAGE, extraction supernatants were diluted fivefold with SDS sample buffer (25 mM Tris-HC1 pH 8.8, lo/, w/v SDS, 0.2% w/v DTT, 10°/o w/v glycerol and a trace of Bromophenol Blue), heated in a boiling water bath for 5 min, then cooled to room temperature, and, after adding 0.5 O/o w/v dithiothreitol, centrifuged (10 min, 15", 20000 g ) .

2 Materials and methods 2.1 Apparatus and chemicals

2.2.3 Extraction of urea-DTT soluble-barley seed proteins

Equipment for IEF and horizontal SDS-PAGE (Ultrophor, Multiphor 11, gradient mixer, power supply), Immobilines and GelBond PAG film were from Pharmacia-LKB (Bromma, Sweden). Acrylamide (2 X crystallized), N , W niethylenebisacrylamide (Bis), ammonium peroxodisulfate, N,N,N',N'-tetramethylethylenediamine (TEMED), Coomassie Brilliant Blue G-250 (Serva Blue G), glycine, 2-mercaptoethanol and SDS were from Serva (Heidelberg, Germany). Tris(hydroxymethy1)aminomethane (Tris), dithiothreitol (DTT) and 2-propanol were from Sigma (Miinchen, Germany). Urea, silver nitrate, glycerol, and all other chemicals, all of analytical grade, were from Merck (Darmstadt, Germany).

Seed samples were crushed with a hammer. Then ureaDTT buffer (4 M urea, 0.5% w/v DTT), equal to five times the weight of the sample, was added. Extraction of barley seed proteins was carried out for two hours at room temperature; afterwards, the suspension was centrifuged (20 min, 15 ', 20 000 g ) and the supernatant was used for IEF-IPG immediately or stored at -80°C.

2.2 Sample preparation

2.3 Electrophoresis The electrophoretic separations (IEF-IPG and SDSPAGE) were performed on horizontal systems according to Gorg etal. [23]. All experiments were performed at least in duplicate.

2.2.1 Seed material

2.3.1 Gel casting

Samples of 55 European barley (Hordeum vulgare L.) cultivars were obtained from G. Giinzel (Weihenstephan, Germany). These cultivars are listed in Table 1.

All gels were 0.5 mm thick and cast on GelBond PAG film. For SDS-PAGE the GelBond PAG film was washed 4 X 15 min with deionized water prior to use in order to minimize spot-streaking when silver staining was performed [24]. The mold used for gel casting consisted of two glass plates, one covered with the prewashed GelBond PAG film, and a U-frame 0.5 mm thick between the glass plates. The gels were cast from the top according to Gorg etal. [25].Sample application slots were cast into the gel by applying 2 or 7 mm wide strips of Dymo@tape (0.25 mm thick) to the glass plate which bears the U-frame. Spacers in the strips were cut off with a razor blade to obtain 7 X 7 mm and 2 X7 mm pieces for IEF-IPG and SDS-PAGE gels, respectively. Up to 24 different samples were applied onto each gel. Sample application strips were not used because they tended to leak when 2-propanol sample solutions were applied.

Table 1. List of barley cultivars 1. Two-rowed spring barley cultivars Apex, Aramir, Arena, Aura, Berenice, Birka, Candice, Carina, Ceres, Cerise, Cytris, Europa, Gerlinde, Gimpel, Golf, Grit, Gunhild, Harry, Helena, Ingrid, Koral, Kym, Luna, M a r k s , Maris, Otter, Menuet, Perle, Roland, Steina, Severa, Tipper, Trumpf, Ultra, Ursel, Villa, Welam 2. Two- or six-rowed winter barley cultivars Alpha, Diana, Franka, Gerbel, Gloria, Hasso, Igri, Ma, Isabell, Mammut, Marylin, Robusta, Rebekka, Sonate, Sonja, Tapir, Triton, Viola, Vogelsanger Gold

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2.3.1.1 IEF gels with IPG

IPG gels with lineargradients, pH 4-9(4 O/o C, size 250 X 120 X 0.5 mm) were cast according to the recipes of Bjellqvist etal. [21].The two starter solutions (10 mL each) were prepared according to Gianazza etal. [26] (Table 2). For detailed information see Righetti [22]and Gorg et al. [27].Briefly, 2 mL of the dense solution (pH 4) was pipetted into the precooled mold to form a pH plateau at the anodic side. Then the gradient was cast by mixing 7 mL of dense (pH 4) and 7 mL of light (pH 9) solution in a gradient mixer. The mold was kept at room temperature for 15 min; the gel was then polymerized for 1 h at 50 "C. After polymerization the gel was cooled in a refrigerator, removed from the mold and washed 6 X 10 min with deionized water to remove any unpolymerized material. Then the gel was soaked in 2% w/v glycerol (30 min) and dried at room temperature overnight. The dry gels were stored in a sealed plastic bag at -20 "C for a period up to several months. Prior to use, the dry IPG gels were rehydrated to their original thickness (0.5 mm) in a reswelling cassette. The rehydration solution consisted of 8 M urea and 15% w/v glycerol. Rehydration time was 6 h, but for practical reasons overnight rehydration was performed in some cases. 2.3.1.2 SDS-pore gradient gels

SDS gels (250 X 125 X 0.5 mm on plastic backing) contained a resolving gel with a linear acrylamide gradient from 12-15% T, 4% C, 375 mM Tris-HC1, pH 8.8, and 0.1% w/v SDS and a stacking gel of 4% T, 4% C, 375 mM Tris-HC1, pH 8.8, and 0.1% w/v SDS (Table 3). For gel casting, the stacklhble 2. Solutions for casting IEF-IPG pH 4-9 gels (4% T, 4 % c ) : Reagents

0.2

M

Immobilines 3.6 4.6 6.2 7.0 8.5 9.3

Acrylamide/Bis (28N1.2) Glycerol (100Yo) Deionized water TEMED (100%) Persulfate 140%)

Heavy solution PH 4

Light solution PH 9

(PL)

(WL)

553 157 155 15 167 147

98 283 240 197 47 442

1.3 mL

1.3 mL

2.5 g 5.5 mL 6 WL 10 UL

Table 3. Solutions for casting SDS pore-gradient gels (12-15% T, 4% C) Reagents

Stacking gel

T = 4 'Yo Tris-HC1 (1.5 M, pH 8.8, 0.4% SDS) AcrylamideIBis (28.U1.2) Glycerol (100%) Deionized water TEMED (100%) Persulfate (40%)

Resolving gel Heavy Light solution solution T = 12% T = 15%

2.5 mL

2.5 mL

2.5 mL

1.3 mL

4.0 mL

5.0 m L

3.15 g 3.2 mL 3W L 10 p L

2.5 g 1.5 mL 3pL 10pL

2.5 mL 3pL 10pL

-

12, 330-337

ing gel solution (5 mL) was pipetted into the precooled mold, followed by a linear acrylamide gradient prepared from 5.5 mL of dense acrylamide solution and 5.5 mL of the light acrylamide solution. After pouring the gradient, the mold was left for 15 rnin at room temperature to allow adequate leveling of the density gradient before polymerization at 50°C for 30 min. Because there was no pH discontinuity between stacking gel and resolving gel, the cast gels could be stored in a refrigerator for at least one week without loss of resolution. 2.3.2 Running conditions 2.3.2.1 IEF-IPG

The rehydrated IPG gel (acidic end at the anode) was placed onto the cooling block (15 "C). The electrode strips were soaked with 10 mM glutamic acid (anode) and 10 mM lysine (cathode). Carbon dioxide was eliminated from the electrophoresis chamber by paper wicks soaked in 5 N NaOH. Samples (10 WL)were applied near the anode into the precast application slots. To obtain reproducible protein patterns, it was essential to start focusing at low voltages; otherwise, protein bands were heavily distorted and sample entry was diminished. Maximum settings were 150 V, 1 mA, 2.5 W for 1 h, followed by 300 V, 1 mA, 2.5 W for another hour. Then voltage was increased to 5000 V (at 2 mA, 2.5 W).Usually focusing was finished after 6 h at 5000 V, but could also be continued overnight without changes in the protein patterns. After focusing, the gel was immediately stained with Coomassie Brilliant Blue G-250 in perchloric acid according to Neuhoff etal. [28]. 2.3.2.2 SDS-PAGE

Horizontal SDS-PAGE was performed as described in detail previously [25,29].The electrode buffer was 25 mM Tris, 192 mMglycine,pH8.5,and0.1%w/vSDS.Samplesof2pL were applied into the precast application slots with the aid of a disposable pipette. Electrophoresis was performed at a maximum voltage of 200 V for 75 min at 10 "C until the Bromophenol Blue tracking dye had migrated into the resolving gel. Then maximum voltage was increased to 600 V (30 mA, 30 W maximum setting) and electrophoresis was continued for another 2 h. Upon completion of electrophoresis, the gels were fixed in methanol/acetic acid/water (4/1/5) for at least 30 rnin and then silver-stained. Alternatively, the gel could also be stored in the fixative for up to 24 h before silver staining, without any loss in resolution by band diffusion. 2.3.3 Staining of the gels 2.3.3.1 Coomassie staining

IEF gels were stained with the colloidal dye Coomassie Brilliant Blue G-250 in 1.5'Yo v/v perchloric acid according to Neuhoff etal. [28], with minor modifications. First, 250 mg of Coomassie Brilliant Blue G-250 were dissolved in 250 mL of deionized water using a magnetic stirrer (30 min). Then 5.5 mL of perchloric acid (70%) were added and

Llertrophoresis 1991, 12, 330-337

stirring was continued for another 30 min. The IEF gel was stained with the unfiltered dye suspension, by gentle shaking for 90 min, and destained with 1.5% perchloric acid under vigorous shaking for 20 min, until an only slightly colored background was obtained. For short-time storage (up to several days) the gel was placed in 20% wlv ammonium sulfate, which had the additional effect that the protein bands were intensified. For long-term storage, the gel was washed several times with water, then soaked in 2% wlv glycerol for 30 min and dried at room temperature.

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333

patterns obtained by single-seed extraction were compared to those obtained from flour. Ethanol extraction revealed typical hordein patterns but proved less suitable because the amount of protein extracted was relatively low compared to the other solvents, resulting in faint patterns. Extraction with 2-chloroethanol yielded more protein but the best results were obtained with 2-propanol. The protein amount extracted with the latter was as high as with 2-chloroethanol, but protein bands were usually sharper (Fig. 1). 2-Propanol is nontoxic, which is an additional advantage compared to 2-chloroethanol.

2.3.3.2 Silver staining SDS-PAGE gels were either stained by the procedure of Heukeshoven and Dernick 1301 or according to Oakely etal. [31] with some modifications (Table 4). The silver-stained gels were stored wet in a sealed plastic bag. Alternatively, they could be dried with the help of a gel dryer.

3 Results and discussion

Table 4. Modified silver staining procedure according to Oakely etal. [28]"'

Steps 1 Fix 2 Wash 3 Sensitize 4 Wash 5 Wash 6 Stain

3.1 Extraction of barley seed proteins 3.1.1 Extraction of hordeins Hordeins, the major storage proteins of barley grain endosperm, are only soluble in aqueous alcohols and urea- or SDS buffers. We tested three different alcohols (with or without addition of DTT) with regard to their ability to extract hordeins: 70% v l v ethanol, 30% vlv 2-chloroethanol, and 55 O/o vlv 2-propanol. Different extraction times and temperatures were investigated. Additionally, the hordein

7 Wash 8 Develop

9 stop 10 Wash

Reagents

Time

40% Methanol/lO% acetic acid Deionized water 10% Glutardialdehyde Deionized water 20% Ethanol 0.2% Silver nitrate 0.25% Ammonia 0.2% Sodium hydroxide 20% Ethanol 20% Ethanol 0.04% Formaldehyde 0.012°/o Citric acid 20% Ethanol I % Ethylenedianiinetetraacetic acid (Titriplex 111) Deionized water

> 30 min 2 X 5 min 15 min 2 X 10 min I5 min 20 min

2 X 5 min 1x30s 1X 2-3 min

5 min 3 x 1 0 min

a) Staining and developing solutions (reagents 6 and 8) have to be prepared fresh; steps 6-8 should be carried out in the dark.

Figure I . IEF-IPG pH 4-9 of barley prolamins (hordeins). (a) Hordeins extracted with 30% 2-chloroethanol; (b) Hordeins extracted with 5 5 % 2-propanol. Samples: (1) Helena, ( 2 ) Ultra, (3) Aramir, (4) Roland, ( 5 ) Berenice, ( 6 ) Ceres, (7) Robusta, (8) M a r k s , (9) Gunhild, (10) Welam, (11) Cytris, (12) Birka, (13) Maris Otter, (14) Ingrid, (15) Tipper, (16) Steina, (17) Arena, (18) Villa, (19) Trumpf, (20) Perle.

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Extraction of grains at room temperature for 2 h gave the same hordein patterns as overnight extraction or extraction for2 h at 50°C. Single-seed analysis within one cultivar usually resulted in uniform hordein band patterns (Fig. 21, identical to flour samples, but some exceptions were observed. Variations in hordein bands of single seeds may be due to different genotypes within one cultivar, exhibiting one or more additional (or fewer) bands. This phenomenon, known as hordein biotypes, has already been described by several authors [ 8,321. Cooke et al. [33] found 14 hordein biotypes in 191 cultivars investigated. We were not able to study this aspect in detail because of insufficient quantities of grain samples. However, the existence of biotypes does not exclude the application of hordein electrophoresis for cultivar discrimination if the biotypes are recognized and catalogued [32]. When the hordeins were extracted by aqueous alcohols and a reducing agent (DTT), the resulting IEFpattern showed a greater number of protein bands than without DTT (results not shown). On the other hand, evaluation was more timeconsuming due to the more complicated hordein patterns, whereas the number of differences detected between the cultivars tested was not significantly increased. On the whole, 2-propanol without a reducing agent seemed to be the best extraction solvent for hordeins for routine use and was therefore chosen as the standard extraction solvent for the following study.

I?. 33(1-337

3.2 Protein staining 3.2.1 Coomassie staining of proteins on IEF-IPG gels Conventional staining methods, e. g . Coomassie Brilliant Blue in alcohol/acetic acid, are not suitable for staining alcohol-soluble proteins because the intensity of the color of the protein bands decreases during the destaining step [16]. For this reason, Gunther etal. [I61 recommended the staining of hordeins with Coomassie Brilliant Blue G-250 according to Neuhoff etal. [28] because in this procedure no alcohol is utilized in the staining or destaining solutions. This method is fast and sensitive, and it has the advantage that no fixing or washing step with alcohol is necessary and that destaining time is short (about 20 min). Hordein bands are alreadyvisible after staining for 15 min, and after90 min proteins are completely stained, with low background staining. Total time for staining and destaining is less than 2 h. Summing up, the colloidal Coomassie Brilliant Blue G-250 staining technique [28] is easy to perform, sensitive,and especially useful in cases requiring fast results.

3.1.2 Extraction of urea-DTT-soluble barley seed proteins A solution of 4 M urea and 0.5% DTT was also an excellent extraction solvent and on IEF-IPG a great number of wellresolved protein bands was observed (Fig. 3). In some cases, better differentiation between cultivars was achieved using the urea-DTT-soluble proteins instead of hordeins. For example, the cultivars “Helena” and “Aramir”, which could be differentiated by their hordein patterns only with great difficulty, were easily discriminated on the basis of their urea-DTT patterns (Fig. 3, marked by arrows). Because of increased resolution, evaluation was more complicated. Therefore, we recommend the use of the urea-DTT solvent only in those cases were relatively closely related cultivars have to be discriminated; otherwise 2-propanol seems to be preferable.

Figure 3. IEF-IPG p H 4-8 ofbarley seed proteins extracted with 4 M urea and 0.5% DTT. Samples: (1) Helena, (2) Ultra, (3) Aramir, (4) Roland, (5) Berenice, ( 6 ) Ceres, (7) Robusta, (8) Marlies, (9) Gunhild,

(10) Welam, (11) Cytris, (12) Birka, (13) Maris Otter, (14) Ingrid, (15) Tipper, (16) Steina.

Figure 2. IEF-IPG pH 4-9 of hordeins. Multiple single-seed analysis. Samples: (a) Ultra, (b) Steina, (c) Marylin, (d) Alpha

lii?cfmphor?rir 1991. 12, 330-337

3.2.2 Silver staining of proteins on SDS-PAGE gels Silver staining is one of the most sensitive methods for the detection of electrophoretically separated proteins but it is complicated and laborious. The more convenient Coomassie staining methods are often prefered when the sensitivity achieved with these techniques is adequate. This is usually the case with low-percentage acrylamide gels such as IEF gels where the staining and destaining steps do not require an excessively long time. However, for high percentage acrylamide gels such as SDS-pore gradient gels, Coomassie staining techniques (especially the colloidal ones) are less suitable because of the long staining and destaining times, necessary for obtaining fully stained protein bands with a low background. In these cases, silver staining is superior to Coomassie Brilliant Blue staining methods. Another advantage of silver staining is that considerably smaller amounts of protein need to be loaded onto the gel, in

Barley cullivar discrimination by SDS-PAGE and IEF witti IPG

335

comparison to Coomassie staining, with resultant sharp protein bands and n o need for alkylation of proteins. Because of the high sensitivity of silver staining, only a small portion of one barley kernel is required for SDS-PAGE of hordeins, leaving the kernel intact for additional tests in combination with hordein electrophoresis. Less than 1 Yo of the extractable hordein from one kernel has to be applied onto the SDS gel to give well-stained protein bands. The procedures of Heukeshoven and Dernick [30] and Oakely etal. [31], were both found to give good results, but in our hands the diamine-silver procedure of Oakely was more sensitive (Fig. 4). This was particularly evident when looking at the C hordein bands, insufficiently stained by the Heukeshoven and Dernick method, but with resultant sharper B hordein bands due to their lower sensitivity on application of equal amounts of protein.

F/gure 4. Horizontal SDS-PAGE (12-15% T) of barley prolamins (hordeins). Hordeins were extracted with 55% 2-propanol and dissolved in SDS sample buffer. (a) Silver staining according to Oakely etal. [31]. (b) Silver staining according to Heukeshoven and Dernick [30]. Samples: (1) Gerlinde, (2) Grit, (3) Roland, (4) Gerbel, (5) Ceres, (6) Cytris, (7) Villa, (8) Golf, (9) Marlies, (10) Sonja, (11) Diana, (12) Franka, (13) Triton, (14) Mammut, (15) Severa, (16) Gimpel, (17) Carina, (18) Koral, (19) Kym, (20) Luna, (21) Aura.

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3.3 Barley cultivar discrimination

3.3.2 Cultivar discrimination by SDS-PAGE of hordeins

3.3.1 Cultivar discrimination by IEF-IPG of hordeins

Hordeins have been divided into four groups (A, B, C, and D) on the basis of their molecular weights M,[34]. The A hordeins ( M , lo0 000) were only insufficiently extracted with propanol (or did not stain well with silver nitrate) and were not used for cultivar discrimination. Of the 55 cultivars studied with SDS-PAGE, 19 different B hordein and 12 different C hordein patterns were obtained. The com bined B and C hordein patterns gave only 21 different hordein patterns (Table 6), less than one might have expected. Reasons for this lie in a genetic linkage between the two hordein loci coding for the B and C hordeins (see below). Twelve barley cultivars were identified uniquely, whereas the remaining 43 cultivars were classified into nine groups containing two to eleven cultivars. Distinguishing winter barleys from spring barley cultivars was always possible because they differed in the B and (especially) C hordein patterns.

By IEF-IPG, hordeins were fractionated into about 30 different bands. Of the 55 cultivars examined, the economically most important ones in the “German list of cultivars”, 32 different hordein band patterns (subgroups) were obtained, which were classified into 16 main groups (A-Q) of variable size (Table 5). Main groups usually differed from each other in at least several bands, whereas subgroups showed differences in only one or two bands. Differences in the relative band intensities were used in only a few cases, to distinguish cultivars (“Aramir”group) when the realtive band intensities differed to agreat extent (weak uersus distinct band). Twenty two cultivars could be distinguished uniquely; the remaining cultivars formed ten groups containing two to eight cultivars (Table 5). Two- and six-rowed feed winter barleys could usually be distinguished from two-rowed malting barley cultivars, which is of economic importance to the malting and brewing industries. The only exception observed is cv. “Robusta”, a sixrowed winter barley that shows the same hordein pattern as cv. “Berenice” (a two-rowed spring barley cultivar); however, this is of no importance in practice because cv. “Berenice” is not of economic importance.

Table 5. Classification of55 barley cultivars due to hordein band patterns as obtained by IEF-IPG Main group A

B

C D

E F

G H I

K L M N 0 P

Q

Hordein pattern

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

Cultivar Aramir, Perle, Ultra, Ursel Gerlinde. Grit Helena Bunhild, Ingrid, Maris Otter, Marlies, Roland Gerenice, Robusta Ceres Steina Tipper Alpha, Gerbel Cytris Welam Birka, Harry Villa Arena, Trumpf Candice, Cerise, Golf, Menuet Marlies b Apex Gloria, Igri, Isabell, Marylin, Rebekka Sonja, Sonate, Viola Tapir Hasso Irla Diana Franka, Mammut, Vogelsanger Gold Triton Europa Severa Carina Koral Kym Gimpel Luna Aura

3.3.3 Comparison between different electrophoretic methods From a total of 55 barley cultivars examined, 32 types of hordein band patterns could be distinguished by IEF-IPG. This is a marked improvement in resolution over that achieved by SDS-PAGE (21 different hordein patterns observed). At present, IEF-IPG is the one-dimensional eIectrophoresis method with the highest resolution, which may additionally be increased by applying narrow or ultranarrow pH gradients (only pH 2-3 units wide). When using narrower pH gradients, several cultivars could be distinguished that exhibit identical hordein patterns in the broad pH

Table 6. Classification of55 barleycultivars due to combined B and C hordein patterns as obtained by SDS-PAGE Hordein pattern ~

Cul tivar ~~

1 2

3

11 12 13 14 15 16 17 18 19 20 21

~~

~

Aramir, Gerlinde, Helena, Perle, Ultra, Ursel Grit Berenice, Gunhild, Iris, Marlies, Maris Otter, Roland, Steina, Tipper Alpha, Gerbel Ceres Birka, Cytris, Harry, Welam Arena, Villa, Trumpf Apex, Candice, Cerise, Golf, Menuet Robusta Gloria, Hasso, Igri, Irla, Isabelt, Marylin, Rebekka, Sonate, Sonja, Tapir, Viola Diana Franka Triton Mammut, Vogelsanger Gold Europa, Severa Gimpel Carina Koral Kym Luna Aura

t-lpctrophowsis

1991, l2, 330-337

range from 4-9 [16]. However, one disadvantage of IEFIPG that should be mentioned is the long focusing time (about 8 h), which allows only a relatively small number of cultivars to be examined in one day. However, some remedies are possible: Sample capacity can be increased by reducing the width of the application slots, so that up to 50 samples can be applied on a single gel and the long focusing time can be reduced by shortening the separation distance 1351, although in this case some loss of resolution might occur.

3.3.4 Cultivar discrimination by combination of different electrophoretic methods Despite the fact that electrophoretic methods allow a number of barley cultivars to be distinguished, the degree of discrimination between cultivars is not as high as with crops such as wheat. There are several reasons for this [l]: First, barley is a diploid, whereas wheat is a hexaploid species; the triplication of the chromosomes in wheat increases the number of prolamin bands to be observed by electrophoresis. Secondly, most modern barley cultivars can be traced back to just a few ancesters and are therefore genetically closely related. Thirdly, the genes coding for the B and C hordeins are located on the same arm of chromosome 5, thus limiting the possibilities of recombination. Although some of the cultivars that are indistinguishable by one-dimensional electrophoretic methods can be subdivided further by using two-dimensional techniques, there are several cultivars that cannot be differentiated by their two-dimensional hordein patterns [36]. However, by using extracting solvents other than alcohols such as urea/DTT/ NonidetP-40, a greater number of proteins is solubilized, revealing a higher diversity in two-dimensional patterns, which allows an improved discrimination between barley cultivars (Gorg etal. in preparation).

4 Concluding remarks The possibility of identifying 55 European winter and spring barley cultivars by their IEF-IPG and SDS-PAGE patterns of hordeins was studied with the following results. IEF-IPG, which was superiorto SDS-PAGE, yielded 32 different hordein band patterns, whereas SDS-PAGE exhibited only 21 different patterns. Neither the IEF-IPG nor the SDS-PAGE method could distinguish all barley cultivars examined. Better discrimination may be achieved either by using protein fractions other than hordeins, in combination with high resolution two-dimensional electrophoresis, or with more specific stains (such as enzyme or glycoprotein staining) in addition to hordein electrophoresis. Received November 23. 1990

Barley cultivar discrimination by SDS-PAGE and I E F with IPG

337

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