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Role of perennial rye (Secale montanum Guss.) on the evolution of cultivated rye (Secale cereale L.).


Rye (Secale cereale L.) is the only species of cultivated species at the Secale. The annual forms of Secale montanum Guss. together with various levels of brittle spike characters had extensively involved in wheat and barley fields during the period of time in which the plant were firstly cultivated. It is possible to think that rye (Secale cereale L.) developed from the plants not showing brittle spike, which had bigger kernels than the wild forms (Sencer and Hawkes, 1980; Kun, 1988).

Turkey is located in the primer genus center of perennial rye and it is agreed that the geographic origin of rye is accepted to be the area around the Mount Ararat and Van Lake. In a previous study was performed on the wild forms of perennial rye collected from the natural population and by the colchicine application artificial autotetraploids were formed (Ozer and Sagsoz, 1991).

Previously, morphologic characters were used in studies concerning identification of species and genetical variation in plant populations. But recently, genetical variations are being commonly measured in protein level by electrophoretic methods.

Electrophoresis is a process that proteins and the others biomacromolecules are forced to migrate through an introduced molecular sieving medium by the influence of an electric current (Market ve Moller 1959).

The proteins in which gene expression can be directly translated act as fingerprints. Electrophoretic band patterns are used as a genetically marker isoenzymes are commonly used as biochemical markers for gene localization and identification of distinct chromosomes (Seidel, 1989; Skiebe ve Selinger, 1990; Drefahl ve Buschbeck, 1991, Ilbi and Eser, 1995; Seyhan et al., 1995).

Izoenzyme patterns which are genotypic characters have been largely studied on the identification of biotype, provenance research hybrid zones and mating systems, to discriminate between morphologic breeding lines, to investigate kingship, origin and ancestors of plant (Du Cros and Wrigley, 1979; Shewry et al., 1983; Salinas and Benito, 1985a; Koksel et al., 1992; Keskin et al., 1995; Tanyolac et al. 1995). Furthermore, electrophoretic band pattern can be used for biosystematics and population genetics (Bilgen and Celen, 1991; Tosun et al., 2002).

This article reports an analysis of inheritance and genetic relationships based on segregation of three enzyme systems (Peroxidase PRX, E. C.; Malate Dehydrogenase MDH, E. C. and Polyphenoloxidase PPO, in four rye plants (Diploid (2n=14) S. cereale L. tetraploid S. cereale L. (2n=28), wild form of S. montanum Guss. (2n=14) and artificial autotetraploid S. montanum Guss.(2n=28)).


The materials used in this study were diploid (2n=14) S. cereale L. tetraploid S. cereale L. (2n=28) accessions of University of Ataturk, Agricultural Faculty, Departments of Fields Crops. Diploid (2n=14) form of perennial rye(Secale montanum Guss.) was collected from primer genus center of this plant and autotetraploids (2n=28). Secale montanum Guss. was formed artificially by the colchicine application in a previous study (Ozer and Sagsoz, 1991).

Analyses were carried out on 21-day-old seedling leaves from seeds germinated and grown under greenhouse conditions (Salinas and Benito, 1985b).

Protein Extraction:

Individual samples were extracted in 0.05 M sodium phosphate (pH 6.5) buffer for peroxidase (PRX) and polyphenol oxidase (PPO). Malate dehydrogenase (MDH) was extracted with 0.2 M Tris-HCL buffer (pH 7.5) (William and Mujeeb-Kazi, 1992). Extractions were made at + 4 [degrees]C for 1-hour period. Samples were filtered and centrifuged at 22.075 g for 15 min. at [+ or -] 0 [degrees]C. The supernatant was stored at + 4 [degrees]C for electrophoresis.

Gel Electrophoresis:

The Polyacrylamide Gel Electrophoresis (PAGE) process was conducted in a OWL dual vertical slab gel apparatus. Resuling gels were 12.5 (h) x 20 (w) x 0.75 (t) cm and contained 7 % acrylamide, while the stacking gels were 2.5 (h) x 20 (w) x 0.75 (t) cm and contained 2.5 % acrylamide. Reservoir buffer contained 0.025 M Tris and 0.133 M Glycine at a pH 8.3 (Ferguson and Grabe, 1986). Protein supernatant was placed in the stacking gel sample wells, followed by 20 [micro]l of reservoir buffer containing bromophenol blue, which served as the tracking dye.

Electrophoresis was done at 2-4 [degrees]C for 1/2 hour at a constant voltage of 80 V and followed by 4.5-5 hours at a voltage of 150 V until the tracking dye was approximately 1 cm from the gel bottom (Agar, 1996).

Staining Procedure:

The staining solution for MDH contained: 100 ml 0.2 M Tris-HCI (pH 7.5), 3 ml 1.0 M D-L malate (pH 7.5), 12 mg [beta]-nicotine with adenine dinucleotide (NAD), 15 mg 3-(4-5-dimetylhiazol 2.yl) 2.5-diphenyl tetrazolium bromide (MTT), 2 mg phenozin metasulfate (PMS). Gels were stained at + 37[degrees]C in the dark until the bands developed. After the bands became visible, the gel was rinsed with distilled water and stored in 3 % acetic acid solution (William and Mujeeb-Kazi, 1992).

The staining solution for PRX enzyme consisted of 20 ml 0.6 % hydrogen peroxide, 20 ml stock benzidine (2 g benzidine in 18 ml acetic acid was solved by heating slowly). Than 72 ml dd H2O was added), 70.4 mg ascorbic acid and 60 ml distilled water. The gels were stained at room temperature until the bands developed. After the bands become visible the reaction was halted with 10 % acetic acid solution and left in 3 % acetic acid solution (Liu, 1973).

The staining solution for PPO contained 0.3 M dihidroxyphenyl alanin (DOPA) (DOPA resolved 1 % KOH) and 0.1 M sodium phosphate buffer. Gels were stained at 37 [degrees]C in the dark until the bands developed (Senel and Kadioglu, 1992).

Statistical Analyses:

The isoenzymal band data were evaluated and the description of the phylogeny by the program of DROWGRAM from PHYLIP package (Felsenstein, 1993).

The band patterns were compared to each other and the similarity index was calculated according to formula written below;

S.I. = Similar Band Number/Similar Bank Number + Non Similar Band Number x 100


In this study, the evolutionary relationship of S. cereale L. (diploid and tetraploid) and S. montanum Guss. (diploid and tetraploid) were investigated view of 3-enzyme system (PRX, MDH and PPO) using of 21-day seedling at these species. It was previously stated by many authors that the electrophoretic isoenzyme band patterns could be used to determine evolutionary relationships between species and in origin tracing studies (Du Cros and Wrigley, 1979; Shewry et al., 1983; Koksel et al., 1992; Keskin et al., 1995; Tanyolac et al., 1995).

In order to determine genetic kinship of species, the isoenzyme band patterns was used as molecular markers in this study. For this purpose, the relative mobility value of bands (Rf) was taken into consideration (Chaisurisri and El-Kassaby, 1993; Ilbi and Eser, 1995; Orcen et al., 1995; Sesli et al 1995, Seyhan et al 1995).

The 3-enzyme systems, which were studied in all groups formed bands both in the anodal (+ pole) and catodal (-pole) regions (Figure. 1. a, b ,c). Similarly, Salinas and Benito (1984) investigated chromosomal locations of structural genes of PRX enzymes in rye and they stated that PRX enzymes produced band formation in both poles. Bosch et al,(1986), determined anodal and catodal isoenzyme bans of rye leaf PRX enzyme. The whole study material produced anodal and catodal bands for MDH enzyme. Salinas and Benito (1985b) stated that rye MDH electrophoresis produced anodal and catodal isoenzyme bands. Nevertheless, Persson and Von Bortmer (2000), determined that rye MDH isoenzymes were localized in both zones. The PPO enzyme that was investigated in this study also produced bands in both zones. But, we couldn't find information about PPO enzyme in the literature.

The plants were divided into different groups according to ploidy levels and 2 species were compared in the same ploidi level. Skiebe and Selinger (1990), declared that with the change of ploidy levels of rye plant, allelic differences occurred by the increase of gene locus and allelic interactions as a result of multiplication of chromosome number. Samuel et. al, (1990), studied isoenzymes polymorphism in 15 enzyme system of Galium (Galium austriecum and G. pumilum agg.) population. These investigators determined that although there were similar isoenzymes band existed in plants, alteration of ploidy levels lead to multiple allelic existence and isoenzyme polymorphisms. Similarly, Beaver et. al., (1995) found isoenzyme band differences occurred according to change of ploidy levels.

The results of this study were presented by comparing the S. cereale L. and S. montanum Guss. species in the same ploidy level. Diploid plants formed 10 PRX bands in both 2 species. Five of these bands we determined as companion bands. Three bands formed from diploid S. cereale L. and 2 band formed from diploid S. montanum Guss. were recorded as polymorphic bands. 14 bands formed in tetraploids. Six of these bands were found in both species. Three polymorphic bands occurred in S. cereale L. and 5 polymorphic bands occurred in S. montanum Guss. Similarly, Salinas and Benito (1984) studied PRX enzyme for different purpose in these 2 species and they determined that a lot of companion bands were formed by these two species.

In the comparison made from the view of MDH enzyme; it was determined that diploid plants produce a total of 10 bands. Apart from these bands; diploid S. cereale L. formed 1, S. montanum Guss. formed 2 bands. Number of similar bands was 7. The tetraploid plants formed total of bands and 6 of these bands were companion bands. Tetraploid S. cereale L. plants formed 3, S. montanum Guss. formed 4 polymorphic bands. A lot of companion bands were formed by MDH enzyme in S. cereale L. and S. montanum Guss. species. This finding was also supported by researches of different investigators (Salinas and Benito 1985b; Kain and Von Bothner, 2000).

Diploid plants formed total of 16 PPO bands. 8 of these bands were found in both species. On the other hand, 3 of these bands was found only in S. cereale L. and other 5 bands was found only in S. montanum Guss. Total of 21 PPO bands formed in tetraploid plants and companion band number was 8. S. cereale L. formed 4 polymorphic and S. montanum Guss. formed 9 polymorphic bands.

It existence of many companion bands of these 2 species in other researches carried on for different enzyme systems were determined (Glutamate oxoloacetate transaminase, a and [beta] amylase) (Ainsworth et al, 1987; Rebordinos and Perez de la Vega, 1988).

Isoenzymes band data were evaluated and genetically relationships of these two species were determined. Similarity Index (%) and phylogenic tree were given in figure 2. a. and figure 2. b. The two species showed genetically similarity as 70.60 % on diploid level and 68.24 % on tetraploid level. Number of investigators noticed that this method could be used in order to determine genetically similarity and kinship relations (Simonsen and Heneen, 1995; Zhebentyayeva and Sivolap, 2000; Cabrita et al., 2001).


Taking in to consideration of the formation of bands of these two species in both diploid and tetraploid levels and phylogenic tree and Similarity Index, it can be concluded that S. cereale L. and S. montanum Guss. species were genetically relative species and S. montanum Guss. probably played genitor role on the evolution of S. cereale L. Similarly, various investigators who made isoenzymes electrophoresis with different enzyme systems stated the presence of number of companion isoenzyme bands of two species and point out that these two species were close relatives (Ainsworth et al., 1987; Rebordinos and Perez de la Vega, 1988). Also, there are other different study carried on with different methods, determined that these two species could be relatives (Koller and Zeller, 1976; Sencer and Hawkes, 1980; Kun, 1988).


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Mahmut Sinan Taspinar* (1), Guleray Agar (2) and Sevim SAGSOZ (1)

(1) University of Ataturk, Faculty of Agriculture, Department of Field Crops, Erzurum, Turkey
Chart 1. Similarly Index (%)

 A B C D

A --
B 88,46 --
C 70,60 53,33 --
D 58,57 68,24 74,29 --

A) Diploid S. cereale, B) Tetraploid
S. cereale C) Diploid S. montanum
D) Tetraploid S. montanum

Figure 1. Band patterns for PRX, MDH and PPO enzymes (-- Band seen on
the gel).


 S. Cereale S. montanum

Rf Diploid Tetraploid Diploid Tetraploid

0,050 -- -- -- --
0,090 -- -- -- --
0,110 -- -- -- --
0,140 -- --
0,180 -- -- --
0,250 -- --
0,290 -- --
0,320 -- -- --
0,360 -- -- --
0,430 -- --
0,480 --
0,520 -- --
0,570 -- --
0,590 --


 S. Cereale S. montanum

Rf Diploid Tetraploid Diploid Tetraploid

0,140 --
0,190 -- -- -- --
0,230 -- -- --
0,240 -- --
0,270 -- -- -- --
0,280 -- -- --
0,300 -- -- --
0,340 -- -- -- --
0,360 --
0,370 -- -- -- --
0,380 -- -- -- --
0,460 --
0,520 -- --
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Author:Taspinar, Mahmut Sinan; Agar, Guleray; Sagsoz, Sevim
Publication:Bulletin of Pure & Applied Sciences-Botany
Date:Jan 1, 2006
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