View of PAG electrophoregrams of six Finnish potato cultivars

Maataloustieteellinen Aikakauskirja Vol. 57: 147—154, 1985

PAG

of six Finnish potato cultivars

TUULA SONTAG

1

1

1 Department

of

of

SF-00710 HELSINKI

2Finnish State Seed Testing Station, SF-00171 HELSINKI

Abstract. Thepolyacrylamide gel electrophoretic (PAGE)patterns of solubleproteins^and esterasesof six Finnishpotatocultivars (Jaakko,Pito, HankkijanTimo,HankkijanTuomas, HankkijanTanuand Puikula)weredetermined. AllcultivarsarecommonlygrowninFinland.

The PAGE procedure used yielded highly reproducible protein separation and good resolu- tion. Samples studied had specific soluble protein and esterase PAGEpatterns,indicatingthat electrophoregramscan be used for identifying Finnish^potatocultivars. Only twocultivars, HankkijanTanu and HankkijanTuomas,whicharecloserelatives,possessedvery similar PAGE patterns.The electrophoreticpatternof Puikula was verysimilar to that of the Swedish cul- tivar Mandel when compared with the reference presentedinthe literature. Thereforeahypothesis is presented suggesting that these two local cultivars would be representatives^ofthesamecultivar.

Introduction

Inrecent years electrophoresis has proved to be a useful chemotaxonomic tool in the classification ofcultivarsand breeding mate- rial of various organisms. Bymeans of elec- trophoresis a potato variety canbe identified

inacouple of days and needs onlyafew mil- liliters of sap drained from tuber tissue.

However, the identification requires that there are relevant reference electrophoregrams available.

Index tables of electrophoregrams of po- tato cultivars have been publishedto help in research work and control ofcultivars. ^One of the first of these collectionswasthe »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 electrophore- grams of Finnish potatocultivars have been available sofar.

Itwas the aim of thepresent study topro- duce reference dataon electrophoregrams of themostcommonFinnish_potato cultivars to be used inpotato research work and varietal classification.

147

JOURNALOF AGRICULTURAL SCIENCE ^{IN FINLAND}

Table 1. Data of Finnishpotatocultivars examined by polyacrylamide gel electrophoresis.

Potato Breeder Origin Released

onthe market

JAAKKO ^Jo1 Eigenheimer x Goldwdhrung 1951

PITO ^» GoldenWonderxElla 1964

HANKKIJAN TIMO Hja² Fruhnudel x Kalahdin 1975

HANKKIJAN TUOMAS ^» AmylaxHorsa 1975

HANKKIJAN TANU ^» LorixHorsa 1981

PUIKULA Finnish local cultivar

1 Agricultural Research CenterFinland, Institute of Plant Breeding, SF-31600 Jokioinen

2 HankkijaPlant Breeding Institute, SF-04300 Hyrylä

Materials and methods Reagents

Acrylamide (for electrophoresis, 2 X crys- tallized) and Serva blau R-250 (min. 86 ^% purified) wereobtained from Serva Feinbio- chemica; N,N’-methylenebisacrylamide from BDH Chemicals Ltd; TRIS (hydroxymeth- yl)aminomethane 7 —9 and a-naphthyl^acetate from SIGMA Chemical Company; amido black ¹⁰B,ammonium peroxodisulfate, boric acid, bromophenol blue, 3-(dimethylamino)- propionitril, sodium dihydrogen phosphate 1-hydrate, disodium hydrogen phosphate 12-hydrate, sodium disulfite dry, sodium sul- fite anhydrous and trichloroacetic acid were obtained fromE.Merck;fast blue B saltwas obtained from Georg T. Gurr Ltd. All the reagents wereanalytical grade ifnototherwise stated.

Sample preparation

Potato tuberswere obtained from the cul- tivar collection maintained by the Finnish State Seed Testing Station. Tubers werehar- vested in September 1984, and storedat 10°C until analyzed in October.Breeder, origin and the year the cultivar was released ^{on the} market is shown in Table 1.

1ml, ^was pressed with_agarlic squeezer and 20

/d

The resultant slurry was centrifuged at 3000 rpm for ¹⁰minatroom temperatureand the supernatant wasseparated. Supernatants (0.3 ml) werediluted with0.3 ml of buffer solu- tion (30 g saccharose⁺ 10 mg amido black B

10 per 100 ml electrodebuffer, 1:5 dilution).

Sample solutionswere stored frozen in sealed vials until use.

Preparation of gels

The gels for protein and esteraseseparation 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. Recipesof gels and buffers used in potato pro- tein and esterase^PAGE (Maierand ^Wagner 1981).

Reagent Amountrequired

Gel,ml ¹⁰⁰

Acrylamide,^g 5.76

N,N’-Methylenebisacrylamide, g 0.24

Buffer, ml to 100

Catalyst

3-(Dimethylamino)-propionitrile,ml 0.50 Sodiumsulfite, 2^%solution, ml 0.25 Ammonium peroxodisulfate,

2 ^%solution, ml 1.60

For sample preparation four tubers of each potato cultivarwere washed,dried and frozen overnight. After thawing for2 hoursatroom temperature the tuberswere peeled and a 1 cm³piece wascutfrom eachtuber. The juice,

Buffer, ml for proteins for esterases

pH 7.9 pH 8.9

1000 1000

Tris, g 3.79 15.13

Boricacid, ^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 pre- pared simultaneously. Immediately after the catalyst solutionswere addedto the gel solu- tion the solution waspoured 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 storedat4°C until used withina week.

Electrophoresis

The electrophoretic separationwascarried

out in a Pharmacia Gel Electrophoresis Ap- paratus GE-2/4 LS usingaLKB 2103 Power Supply. For each ^run two gels of 140 mm x

180 mmX 1.5 mmwere applied. Each gelac- comodated 14 samples.

The sample solutions (20 /d)wereplaced in

the gel slots witha microliter syringe. Dupli- cate electrophoresis _{runs were} performed of each sample. Voltage was maintainedat 400 V until the marker dye migrated tothe bot- tom of the gel. Each run took2—3 hours.

Buffer temperature was maintainedat 13^— 16°C by tapwater circulation.

Staining

Proteinswere 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 foundto be unnecessary. Esterases were stained as described by^Stegemannand Loeschcke (1976).

Photography

The gelwasplacedon aglass plateon alight box and illuminated from below and photo- graphed with Agfaorto 25 film.

Fig. I. Tray made of acryclic plastic (5 mm thick) for the preparation of gels. Tray dimensions a⁼190 mm, b⁼210mm and c⁼65mm.

Identification

Stegemann and Loeschcke (1976) desig- nated the four main protein bands in the cathodic ^{part of the potato protein} elec- trophoregramasA-, B-, C- and D-bands. By these 4 main bands it is possible to identify 9 different groups of^potato cultivars (Stege- mannand Loeschcke 1976). The final identi- fication ^{of a potato} cultivar is ^based on its anodic protein bandpattern and its esterase bandpattern. As suggested and applied by Maier and ^Wagner(1981) weused 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 weredetermined 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 aband 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 maincathodic bands in the elec- trophoregram ofastandard cultivar Bintjeon 14differentgels. These data indicate that the relative band mobilitieswerehighly reproduc- ible from _onegelto another, i.e., ^{gel polym-} erization and electrophoresisarereproducible (Table 3).

Results and discussion

The electrophoreticpatterns of the soluble proteins and esterases of the six Finnish po- tatocultivars studiedareshown in Fig. 2 and 3. Based on a visual inspection of electro- phoregrams the six cultivars could bedivided intothree groups according totheir 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 group8.

In the anodic partof the gel of these four cultivars the positionand number of protein bands differ from oneanother 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)whichex- plains the similarity of their protein electro- phoregrams. Although these close relatives have much of thesamebandpattern there_are two specific anodic bands thatcan be usedto

Table 3. Anexampleof reproducibility of the electrophoregrams producedinthepresentstudy, expressedasrela- tive mobilities ^(RM)of cathodic protein bands of the Bintje electrophoregramon 14 PA gels.

Cathodic band

A B C D

n 14 14 ^{14 14}

RM 0.1720.274 0.3680.503

S 0.00370.0043 0.00480.0021

P⁼0.95 0.169<ji<0.174 0.271 <,*<0.276 0.365< <0.3710.502^</*<0.504 S⁼Standarddeviation

H⁼Confidenceinterval

distinguish them from each other (Fig. 2).

Each cultivar possessed also a cultivar specific esterase electrophoregram (Fig. 3).

The genetically closely relatedcultivars,^Hank- kijan Tanu and Hankkijan Tuomas,showed onlyaminute difference in theiresterasepat- terns.Hankkijan Tuomas hadanintense band in its esterase pattern (probably a double band) whereas Hankkijan Tanu had only a single band at the _sameposition.

It was of special interest to compare the electrophoreticpattern of the cultivar Puikula with the pattern of the cultivar Mandel pre- sented in the Index of European Potato Va- rieties by ^Stegemannand Loeschcke (1976).

Mandelis alocal cultivar which is grown in the Nordic countries. In Finland the names Puikula and Manteli (Mandel in Swedish)are

usedassynonyms of thesamecultivar. In the comparison the cultivar Puikulawasfoundto have much of the same protein and esterase patterns as Mandel. Figure 4 shows the pro- tein and esterase electrophoregrams of these cultivars. Basedonthis data it would beneces- sary^tomake electrophoresisruns of Puikula and Mandel side by sideon the same gel in oder _to strengthen the hypothesis that these twolocal cultivars_areactually representatives of one and thesame cultivar.

The PAGE procedure used in thepresent study yielded reproducible protein separation and good resolution of protein bands for iden- tifying purposes of Finnish potato cultivars.

The six Finnish_potato cultivars(Jaakko, Pi- to, Hankkijan Timo, ^Hankkijan Tuomas, Hankkijan Tanu and Puikula) could be

Fig. 2. Electrophoregramsof soluble proteins from six Finnish potatocultivars and cultivar Bintje. From left to right: Bintje, HankkijanTimo, HankkijanTimo, Jaakko, Jaakko,HankkijanTuomas,Hankkijan Tuo- mas, HankkijanTanu, HankkijanTanu, Pito, Pito, Puikula,Puikula and Bintje.

readily distinguished by their soluble protein and esterase PAGE patterns. However, two

cultivars, Hankkijan Tanu and Hankkijan Tuomas, whichare closerelatives, ^possessed very similar solubleprotein and esteraseelec- trophoregrams. Special attention should be paidtothe anodic bands of their soluble pro- tein electrophoregrams when identifying either of thesetwo cultivars.

The electrophoreticpatterns ofpotatopro- teins andesterases areinheritedindependent- ly and are stable for more than half a year

when the tubers are stored at4—lo°C after harvest (Stegemann and Schnick 1982). The multiple formsof ^{esterases change even less} than the proteins during sprouting (Stege- mann and Schnick 1982). Esterase electro-

Table 4. ^Groupingof six Finnish potatocultivars by applying the classification system suggested by ^Stege- mannand Loeschcke (1976).

Cultivar Cathodic band formula Group

JAAKKO A B C D 8

PITO B D 2

PUIKULA AB 5

HANKKIJAN TANU A B C D 8

HANKKIJAN TIMO A B C D 8

HANKKIJAN

TUOMAS A B C D 8

phoresis is also aless time-consuming proce- dure than protein electrophoresis. For these reasons a determination from the esterase electrophoregrams would be useful prior to soluble protein electrophoresis.

Fig. 3. Electrophoregramsof esterases from six Finnish potato cultivars and cultivar Bintje. From left to right:

Bintje,Pito,HankkijanTuomas,HankkijanTanu, Pito, Puikula, Jaakko,HankkijanTimo, Bintje,Hank- kijanTuomas, ^{HankkijanTanu} and Bintje.

Conclusions

The procedure applied in this study canbe usedas atool in the identificationof question- able potato samples. The electrophoregrams given should^{be ableto serve as} reference in such work.However, oneshould bear in mind that in order to obtain more accurate proof of the identity ofanunknownpotato sample

References

Maier, G.^& Wagner, K. 1981.Identifizierungöster- reichischer Kartoffelsorten mit Hilfe der Polyacryl- amidgelelectrophorese.Die Bodenkultur32: 314 —325.

Stegemann, H. ^& Loeschcke, V. 1976. Index Euro- päischerKartoffelsorten/Index of European Potato Va- rieties Mitt. Biol. Bundesanst. Land Fortwirlsch.,

SELOSTUS

Kuuden suomalaisen perunalajikkeen PAG-elektroforegrammit

Tuula Sontag

1

1

ja Osmo Ulvinen²

' Helsingin yliopiston elintarvikekemian ja -teknologiantaitos, 00710Helsinki

2 Valtionsiemenlarkastuslailos, 00171Helsinki

Perunalajikkeidenelektoforegrammejakäytetään hy- väksi perunan tutkimus- ja tarkastustoiminnassa. Suoma- laisista perunalajikkeista ei elektroforegrammiaineistoa kuitenkaan ole ollut käytettävissä.

an electrophoretic analysis should also be per- formed with the unknown sample and ^an authentic reference sample side by side^{on a} gel rather than only using the photographic presentation asreferences.

Acknowledgements.The authors are grateful to Mr Jukka Miettinen for excellent laboratory and technical assistance.

Berlin-Dahlem. Heft 168.

&Schnick,D. 1982.Index 1982EuropäischerKartof-

felsorten/Index 1982ofEuropean Potato Varieties/In- dice 1982de Variedades Europeas de Papas. Mitt. Biol.

Bundesanst Land Fortwirtsch., Berlin-Dahlem. Heft 211.

Ms received April 18, 1985

Työn tarkoituksenaoli määrittää yleisimpiensuoma- laisten perunalajikkeiden elektroforeettiset mallit eli elek- troforegrammit.

Kuuden suomalaisen perunalajikkeen (Jaakko, Pito Fig. 4. Soluble proteinand esterase electrophoregraras of Mandel (from Steoemann and ^Loescshke 1976) and

Puikula.

153

HankkijanTimo,HankkijanTuomas, HankkijanTanu jaPuikula) liukoisten proteiinien ja esteraasien elek- troforegrammit määritettiin polyakryyliamidigeelielek- troforeesilla (PAGE).

Käytetyllä PAGE-menetelmällä proteiinienerotusmal- lien toistettavuus oli hyvä. Tutkituilla näytteillä olispe- sifiset liukoisten proteiinien ja esteraasien PAGE-mallit, joitavoidaan käyttää näiden lajikkeiden identifioinnis- sa. Kahden perimmältään lähekkäisen lajikkeen, Hank-

kijan Tanun ja Hankkijan Tuomaksen, elektrofore- grammitolivat hyvin samantyyppiset. Näiden lajikkeiden elektroforegrammeista löytyikuitenkin tietyt vyöhykkeet, joidenperusteellamallitvoidaan erottaa toisistaan. Pui- kulan elektroforegrammi muistutti ruotsalaisen Mandel- lajikkeen kirjallisuudessa esitettyä elektroforegrammia.

Tähänperustuenesitetäänolettamus,ettäPuikula ja Man- del ovat samanlajikkeen edustajia.