PAG electrophoregrams of six Finnish potato cultivars

The polyacrylamide gel electrophoretic (PAGE) patterns of soluble proteins and esterases of six Finnish potato cultivars (Jaakko, Pito, Hankkijan Timo, Hankkijan Tuomas, Hankkijan Tanu and Puikula) were determined. All cultivars are commonly grown in Finland. The PAGE procedure used yielded highly reproducible protein separation and good resolution. Samples studied had specific soluble protein and esterase PAGE patterns, indicating that electrophoregrams can be used for identifying Finnish potato cultivars. Only two cultivars, Hankkijan Tanu and Hankkijan Tuomas, which are close relatives, possessed very similar PAGE patterns. The electrophoretic pattern of Puikula was very similar to that of the Swedish cultivar Mandel when compared with the reference presented in the literature. Therefore a hypothesis is presented suggesting that these two local cultivars would be representatives of the same cultivar.


Introduction
In recent years electrophoresis has proved to be a useful chemotaxonomic tool in the classification of cultivars and breeding mate- rial of various organisms.By means of elec- trophoresis a potato variety can be identified in a couple of days and needs only a few milliliters of sap drained from tuber tissue.However, the identification requires that there are relevant reference electrophoregrams available.
Index tables of electrophoregrams of potato cultivars have been published to help in research work and control of cultivars.One of the first of these collections was the »Index Index words: Potato, polyacrylamide gel electrophoresis, cultivar identification, protein, esterase of European Potato Varieties» by Stegemann  and Loeschcke (1976).Their extensive work included electrophoregrams of 530 European potato varieties from almost all European countries.Later many workers have produced additional electrophoretic data on specific potato cultivars (e.g., Maier and Wagner  1981).However, no data on electrophoregrams of Finnish potato cultivars have been available so far.
It was the aim of the present study to produce reference data on electrophoregrams of the most common Finnish potato cultivars to be used in potato research work and varietal classification.

Sample preparation
Potato tubers were obtained from the cultivar collection maintained by the Finnish State Seed Testing Station.Tubers were har- vested in September 1984, and stored at 10°C until analyzed in October.Breeder, origin and the year the cultivar was released on the market is shown in Table 1.
1 ml, was pressed with a garlic squeezer and 20 /d of sulphite solution (1.0 g Na 2 SG 3 + 0.75 g Na 2 S 2 O s to 20 ml H 2 G) was added.The resultant slurry was centrifuged at 3000 rpm for 10 min at room temperature and the supernatant was separated.Supernatants (0.3 ml) were diluted with 0.3 ml of buffer solu- tion (30 g saccharose + 10 mg amido black B 10 per 100 ml electrode buffer, 1:5 dilution).
Sample solutions were stored frozen in sealed vials until use.

Preparation of gels
The gels for protein and esterase separation were prepared by the method of Stegeman and Loeschcke (1976) as applied by Maier and Wagner (1981).Details of the recipes used are given in Table 2.
Table 2. Recipes of gels and buffers used in potato pro- tein and esterase PAGE (Maier and Wagner 1981).

Reagent
Amount required Gel, ml 100 Acrylamide, g 5.76 N,N'-Methylenebisacrylamide, g 0.24 Buffer, ml to 100 Catalyst 3-(Dimethylamino)-propionitrile, ml 0.50 Sodium sulfite, 2 % solution, ml 0.25 Ammonium peroxodisulfate, 2 % solution, ml 1.60 For sample preparation four tubers of each potato cultivar were washed, dried and frozen overnight.After thawing for 2 hours at room temperature the tubers were peeled and a 1 cm 3 piece was cut from each tuber.The juice, Buffer, ml for proteins for esterases pH 7.9 pH 8.9 1000 1000 Tris, g 3.79 15.13 Boric acid, g 4.56 1.15 The gels were polymerized in a tray con- structed of acrylic plastic according to the measurements shown in Fig. 1.Up to 8 gels (140 mm x 180 mm x 1.5 mm) could be prepared simultaneously.Immediately after the catalyst solutions were added to the gel solu- tion the solution was poured quickly into the gel tray through the inlet tubing, avoiding entry of air bubbles.After an hour the gels were removed from the tray, wrapped in household polyethylene film and stored at 4°C until used within a week.

Electrophoresis
The electrophoretic separation was carried out in a Pharmacia Gel Electrophoresis Apparatus GE-2/4 LS using a LKB 2103 Power Supply.For each run two gels of 140 mm x 180 mm X 1.5 mm were applied.Each gel ac- comodated 14 samples.
The sample solutions (20 /d) were placed in the gel slots with a microliter syringe.Duplicate electrophoresis runs were performed of each sample.Voltage was maintained at 400 V until the marker dye migrated to the bot- tom of the gel.Each run took 2-3 hours.Buffer temperature was maintained at 13 - 16°C by tapwater circulation.

Staining
Proteins were stained by immersing the gel overnight in 200 ml of 12 °/o TCA + 5 ml of Serva blau R 250 (1 °7o ethanol solution).De- staging found to be unnecessary.Esterases were stained as described by Stegemann and  Loeschcke (1976).

Photography
The gel was placed on a glass plate on a light box and illuminated from below and photographed with Agfaorto 25 film.

Identification
Stegemann and Loeschcke (1976) designated the four main protein bands in the cathodic part of the potato protein electrophoregram as A-, B-, C-and D-bands.By these 4 main bands it is possible to identify 9 different groups of potato cultivars (Stege- mann and Loeschcke 1976).The final identi- fication of a potato cultivar is based on its anodic protein band pattern and its esterase band pattern.As suggested and applied by Maier and Wagner (1981) we used the cul- tivar Bintje as an internal standard on each gel plate.

Determination of relative mobilities of protein bands
The mobilities and band positions of cathodic protein bands were determined from the photographic enlargement by measuring the distance from the origin (inner edge of sample slot) to the center of the band.The migration distance of a band divided by the migration distance of the reference band F (Fig. 2) equals the relative mobility of the band.

Reproducibility
Duplicate electrophoretic runs of the four replicate samples were performed to ensure visual similarity of the electrophoresis formula of the replicates.Reproducibility of the elec- trophoretic procedure used in this study was determined by the measuring relative mobili- ties of four main cathodic bands in the electrophoregram of a standard cultivar Bintje on 14 different gels.These data indicate that the relative band mobilities were highly reproducible from one gel to another, i.e., gel polym- erization and electrophoresis are reproducible (Table 3).

Results and discussion
The electrophoretic patterns of the soluble proteins and esterases of the six Finnish potato cultivars studied are shown in Fig. 2 and 3. Based on a visual inspection of electrophoregrams the six cultivars could be divided in to three groups according to their four main cathodic bands of soluble proteins (Table 4).The cultivars Puikula and Pito were clearly differentiated from the other four cultivars by their main cathodic protein bands.In the nine- group systematics suggested by Stegemann and Loeschcke(l976) Puikula would go into group 5 and Pito into group 2, whereas the four other cultivars go into group 8.
In the anodic part of the gel of these four cultivars the position and number of protein bands differ from one another and therefore serve as characteristic fingerprints for their precise identification (Fig. 2).Two of these four potato cultivars, Hankkijan Tanu and Hankkijan Tuomas, have a common parent (Horsa) in their pedigrees (Table 1) which ex- plains the similarity of their protein electro- phoregrams.Although these close relatives have much of the same band pattern there are two specific anodic bands that can be used to Table 3.An example of reproducibility of the electrophoregrams produced in the present study, expressed as rela- tive mobilities (RM) of cathodic protein bands of the Bintje electrophoregram on 14 PA gels.distinguish them from each other (Fig. 2).
The genetically closely related cultivars, Hank- kijan Tanu and Hankkijan Tuomas, showed only a minute difference in their esterase pat- terns.Hankkijan Tuomas had an intense band in its esterase pattern (probably a double band) whereas Hankkijan Tanu had only a single band at the same position.
It was of special interest to compare the electrophoretic pattern of the cultivar Puikula with the pattern of the cultivar Mandel presented in the Index of European Potato Va- rieties by Stegemann and Loeschcke (1976).Mandel is a local cultivar which is grown in the Nordic countries.In Finland the names Puikula and Manteli (Mandel in Swedish) are used as synonyms of the same cultivar.In the comparison the cultivar Puikula was found to have much of the same protein and esterase patterns as Mandel.Figure 4 shows the pro- tein and esterase electrophoregrams of these cultivars.Based on this data it would be necessary to make electrophoresis runs of Puikula and Mandel side by side on the same gel in oder to strengthen the hypothesis that these two local cultivars are actually representatives of one and the same cultivar.
The PAGE procedure used in the present study yielded reproducible protein separation and good resolution of protein bands for iden- tifying purposes of Finnish potato cultivars.

Fig
Fig. I. Tray made of acryclic plastic (5 mm thick) for the preparation of gels.Tray dimensions a = 190 mm, b = 210 mm and c = 65 mm.
Data of Finnish potato cultivars examined by polyacrylamide gel electrophoresis.