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. 220.127.116.11; Malate Dehydrogenase MDH, E. C. 18.104.22.168 and Polyphenoloxidase PPO, 22.214.171.124) 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)).
MATERIALS AND METHODS
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).
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.
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).
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).
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
RESULTS AND DISCUSSION
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).
(1.) Agar, G., 1996. Isoenzymes variation of Vicia canescens populations. Ph. D. Dissertation. Univ. of Ataturk, Graduate School of Natural and Applied Science, Erzurum, Turkey.
(2.) Ainsworth, C. C., Miller,T.E. and Gale, M.D. 1987. [alpha]-Amylase and [beta]-Amylase homoeoloci in species related to wheat. Genet. Res., Camb., 49: 93-103.
(3.) Beaver, J. A., Iezzoni, A. F. and Ramm, C. W., 1995. Isoenzyme diversity in sour,--sweet and ground cherry. Theor. Appl. Genet. 90 : 847-852.
(4.) Bilgen, G. and Celen, A.E. 1991. Possibilies of using isoenzymes electrophoresis technique in alfalfa breeding. Second Rangelands and Forage Crops Congress of Turkey, 28-31 May 1991, Izmir: p. 532-539.
(5.) Bosch, A., Figueiras, Ana M., Gonzales-Jean, M. T., and Benito C.,1986. Leaf peroxidases--A biochemical marker for the group 2 chromosomes in the Triticianae. Genet. Res., 47:103-107.
(6.) Cabrita, L. F., Aksoy, U., Hepaksoy, S. and Leitao, J. M., 2001. Suitability of isoenzymes, RAPD and AFLP marker to assess genetic differences and related among fig (Ficus carita L.) clones. Sci. Hort. 87 : 261-273.
(7.) Chaisurisri, K. and El-Kassaby, Y.A. 1993. Genetic control of isoenzymes in Sitka Spruce. J. of Heredity., 84 : 206-211.
(8.) Drefahl, S. and Buschbeck, R. 1991. Gene localization of Aspartate Aminotransferese and Endopeptidase izozymes in Wheat and Rye using developmental and organ-specific patterns. Plant Breed., 107 : 218-225.
(9.) Du Cros, D. L. and Wrigley,W.1979.Improved electrophoreic methods for identifing cereal varieties. J. Sci. Food. Agric., 30: 785-794.
(10.) Felsenstein, J., 1993.PHYLIP (Phylogenyinference package) version 3.5c. Department of Genetics, University of Washington, Seattle WA.
(11.) Haddioouni, A. and Baaziz, M. 2001. Genetic diversity of natural populations of Atriplex halimus L. in Morocco: An isoenzymes-based overview. Euphytica 121:99-106.
(12.) Hoffman, W., Mudra, A. and Plarre, W. 1985. Lehrbuch der Zuchtung Landwintschaftlicher Kulturpflanzen. Verlag Paul Parey, Berlin und Hamburg: pp. 170-172.
(13.) Ilbi, H. and Eser, B., 1995. Identification of batanical variety and cultivars in Brassica oloracea species on the basis electrophoretic patterns of seed proteins. Workshop "Biotechnology and Plant Breeding" 17-19 April 1995, Gebze/Kocaeli, Turkey :p 89-96.
(14.) Keskin, S., Asal, S. and Kavuncu, O., 1995. Genetic analysis of gliadin pattern in hybrids of some Turkish hexaploid wheat varieties. Workshop "Biotechnology and Plant Breeding" 17-19 April 1995, Gebze/Kocaeli, Turkey : p. 45-51.
(15.) Koller , O. L. and Zeller, F. J. 1976. The homoeologous relationships of rye chromosomes. Genet. Res. 28:177-188.
(16.) Koksel, H., Atli, A. and Ozkaya, H. 1992. The utilization of marker proteins in the quality selection of durum wheat breeding programs. Tr. J. of Agric. and Forest, 17: 531-536.
(17.) Kun, E. 1988. Cool Season Cereals. Publication No: 1032. Univ. of Ankara, Ankara/Turkey : p. 253-263.
(18.) Liu, E. H. 1973. A simple method for determining the relative activities of individual peroxidase isoenzymes in a tissue extract. Anal. Biochem., 56 : 149-154.
(19.) Markert, C. L. and Moller, F. 1959. Multiple forms of enzymes. Proc. Nat. Acad. Sci., 45:753-763.
(20.) Orcen, N., Firat, A.E. and Sekin, S. 1995. Research on identification of wheat lines possessing different vernalisation genes by PAGE method. Workshop "Biotechnology and Plant Breeding" 17-19 April 1995, Gebze/Kocaeli, Turkey : p. 111-118.
(21.) Ozer, I. and Sagsoz, S.1991. Obtaining artificial tetraploids of perennial rye (Secale montanum Guss.) and the comparison of their certain cytological and morphological characteristics. Second Rangelands and Forage Crops Congress of Turkey, 28-31 May 1991, Izmir, : p. 594-602.
(22.) Persson, K. and Von Bothmer, R., 2000. Assessing the allozyme variation in cultivars and Swedish landreces of rye (Secale cereale L.). Hereditas, 132 : 7-17.
(23.) Reberdinos, L. and Perez de le Vega, M. 1988. Gene duplication in the structural dene for a glutamat oxaloacetate transaminase zone (GOT1) in Secale. The J. of Heredity, 79: 78-80.
(24.) Salinas, J. and Benito, C. 1984. Cromosomal Location of Peroxidase structural genes in Rye (Secale cereale L.). Z.Pflanzenzuchtg., 93 : 291-308.
(25.) Salinas, J. and Benito, C. 1985a. Esterase isoenzymes in rye-charecterisation, genetic control and chromosomal location. Theor. Appl. Genet., 71:136-140.
(26.) Salinas, J. and Benito, C. 1985b. Cromosomal Location of Malat dehidrogenase structural genes in Rye (Secale cereale L.). Z.Pflanzenzuchtg., 94: 208-217.
(27.) Samuel, R., Pinsker W. and Ehrendorfer, F. 1990. Allozyme polymorphism in diploid and polyploid population of Galium. Heredity. 65:369-378.
(28.) Seidel, A. 1989. Isoenzyme als biochemische und genetische marker zur identifizierung von weizen choromozomen in roggen-cytoplasmatischen. Roggen-Weizen-Additionen, Diss. Math. Nath. Fak., Hochsch. Gustrow.
(29.) Sencer, H. A. and Hawkes, J.G.1980. On the origin of cultivated rye. Biolog. J. the Linear Society, 13: 99-313.
(30.) Sesli, M., Erdogan, M. and Demir, I. 1995. Research on identification of some cereal varieties by fingerprints of glutenin and albumin proteins. Workshop "Biotechnology and Plant Breeding" 17-19 April 1995, Gebze/Kocaeli, Turkey : p. 61-71.
(31.) Seyhan, G., Bilgen, G., Demir, I. and De Joung , D.W.1995. Identification of wheat grain protein (glutenin and gliadin) by electrophoresis. Workshop "Biotechnology and Plant Breeding" 17-19 April 1995, Gebze/Kocaeli, Turkey : p. 81-88.
(32.) Shewry, P. R., Parmar, S. and Miflin, B.J. 1983. Extraction, seperation and polymorphism of the prolamin storage proteins (Secalins) of rye. Cereal Chem., 60: 1-6.
(33.) Simonsen, V. and Heneen , W. K. 1995. Genetic variation within and among different cultivars and landraces of Brassica campestris L. and B. olaracea L. based on isoenzymes. Theor. Appl. Genet. 91:346-352.
(34.) Skiebe, K. and Selinger, P. 1990. Isoenzymes and their importance for breeding autopolyploids. Plant Breed., 105: 106-111.
(35.) Senel, G. and Kadioglu, A. 1992. An electrophoretical study on some enzymes and proteins of four Iris L. species. Doga Tr. J. Bot., 16: 1-6.
(36.) Tanyolac, B., Bilgen,G., Demir,I., and De Joung, D.W. 1995. A research on identification of wheat varieties by polyacrilamide gel electrophoresis of gliadin and glutenin proteins. Workshop "Biotechnology and Plant Breeding" 17-19 April 1995, Gebze/Kocaeli, Turkey : p. 52-60.
(37.) Tosun, M., Akgun, I., Taspinar, M.S. and Kanli, M. 2002. Determination of variations for some enzymes in orchardgrass (Dactylis glomerata l.) ecotypes. Acta Agric. Scan. Sec. B-Soil and Plant Sci.
(38.) William, M. and Mujeeb-Kazi , A. 1992. Isoenzyme and cytological Markers of some Psathyrostachys juncea accessions. Theor. Appl. Genet., 84: 528-534.
(39.) Zhebentyayeva, T. N. and Sivolap, Y. M., 2000. Genetic diversity of apricot determined by isoenzymeand RAPD analyses. Acta. Hort. 538:525-529.
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). PRX S. Cereale S. montanum Rf Diploid Tetraploid Diploid Tetraploid 0,040 0,050 -- -- -- -- 0,060 0,080 0,090 -- -- -- -- 0,100 0,110 -- -- -- -- 0,130 0,135 0,140 -- -- 0,160 0,170 0,180 -- -- -- 0,190 0,230 0,240 0,250 -- -- 0,270 0,280 0,290 -- -- 0,300 0,320 -- -- -- 0,340 0,350 0,360 -- -- -- 0,370 0,380 0,400 0,430 -- -- 0,440 0,460 0,480 -- 0,520 -- -- 0,540 0,570 -- -- 0,590 -- 0,610 MDH S. Cereale S. montanum Rf Diploid Tetraploid Diploid Tetraploid 0,040 0,050 0,060 0,080 0,090 0,100 0,110 0,130 0,135 0,140 -- 0,160 0,170 0,180 0,190 -- -- -- -- 0,230 -- -- -- 0,240 -- -- 0,250 0,270 -- -- -- -- 0,280 -- -- -- 0,290 0,300 -- -- -- 0,320 0,340 -- -- -- -- 0,350 0,360 -- 0,370 -- -- -- -- 0,380 -- -- -- -- 0,400 0,430 0,440 0,460 -- 0,480 0,520 -- -- 0,540 0,570 0,590 0,610
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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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