PAG electrophoregrams of wheat cultivars grown in Finland

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 obtained 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 independent of growth conditions (Wrigley 1970,   ZiLLMANand Bushuk 1979).Bushuk and Zillman (1978) developed a gliadin PAGE meth- od for wheat which has also been recommended as a reference method for wheat cultivar 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- 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.

Samples
Samples of 14 wheat 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).

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.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 then centrifuged 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 into 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 intensities (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 application) to the centre of the band.The migration distance of the given band divided by 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 subjectively 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 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 Canadian cultivar 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 60 Co-mutant of Ruso, which explains the similarity of their gliadin patterns.
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)

Discussion
Analysis of the gliadin electrophoregrams serves as a useful tool for research especially in wheat breeding, where accurate identification 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 composition.However, the electrophoretic pro- cedure described here has found recent application 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 deviations 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 specific.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.

Table 1 .
Data of the spring and winter wheat cultivars grown in Finland and examined in the present study.

Table 2 .
Recipes for gel and tank buffer solutions.