Seed protein polymorphism of four common buckwheat varieties
registered in the Czech Republic Petra CEPKOVA-1 and Vaclav DvoRAcEK2 I Department of Tropical and Subtropical Crops and Agroforestry, Institute ofTropics and Subtropics, Czech University ofAgriculture Prague, Kamycka /29, 16521 Prague 6 - Suchdol, Czech Republic 2 Department of Gene Bank, Division of Genetics and Plant Breeding, Research Institute of Crop Production, Drnovskd 507, 16/06 Prague 6 - Ruzyne, Czech Republic Received May 1,2006; accepted in revised form October 8, 2006 Key words: Buckwheat, Fagopyrum esculentum Moench, SDS-PAGE, seed protein polymorphism ABSTRACT Four buckwheat varieties 'Pyra', 'Spacinska I', 'Kara-Dag' and 'Jana' registered in the Czech Republic were evaluated by discontinuous SDS-PAGE electrophoresis. Bulk samples and individually 250 single seeds from each variety were analyzed. In the four registered buckwheat varieties 36 significant band positions were described. The highest poly- morphism of buckwheat proteins was represented by bands with a molecular weight of 30-60 kDa. No single specific bands for the tested varieties were detected. The designated bands showed a similar frequency and coincidence in all varieties . In spite of this fact, varietal differences in frequency and coincidence of some designated bands were found among the tested varieties. Significant differences in protein band frequencies and their coincidences could serve as a supplemental identification criterion, especially in such cases when the electrophoretical analyses of bulked samples are not sufficient. INTRODUCTION Buckwheat (Fagopyrum esculentum Moench, Polygo- naceae) is a crop characterized by excellent agronomic and nutritional traits (Edwardson, 1996). The comeback of interest in buckwheat as an alternative crop is based on its high nutritional value (Javornik, 1986; Bonafaccia and Kreft , 1994). Eggum et al. (1981) reported that the crude protein content varied in buckwheat varieties (12% on the average) and its level was comparable with wheat. Albumins and globulins are the main fractions of buck- wheat proteins and minor fractions are represented by prolamins and glutelins (Javornik and Kreft, 1984). The proportion and quality of proteins, characterized by a high and balanced content of the essential amino acids (above all lysine, methionine and cysteine) , high content of rutin and other polyphenolic compounds, trace elements, and fibre make buckwheat a valuable food crop, suitable for a healthy diet and for people suffering from diabetes or coeliac disease (Kreft et aI., 1996; Michalova et aI., 1998). Strict allogamy of common buckwheat leads to a high intra-varietal variability which causes many breeding and legislative problems. One of the most serious problems is the difficulty in the identification of contemporary varieties, their registration and legal protection. Identification prin- ciples of four registered buckwheat vaneties ('Pyra', ' Kara-Dag' , 'Spacinska l ' and ' l ana' ) in the Czech Republic are still based on 12 to 22 morphological and phenological characteristics which were obtained in one locality during 3 testing years before registration. Differ- ent extent of characteristics, different testing periods and only one testing locality for each variety can hardly create a reliable system for their clear distinguishability and identification. This reality is confirmed by the fact that none of these varieties is legally protected at present and their registrations will terminate after expiration of a ten years registry period (CISTA, 2005). Application of direct genetic analyses in buckwheat varieties, based on protein or DNA polymorphism, seems to be suitable tool for reliable variety identification and an effective breeding process. Genetic interpretation of protein polymorphism in buck- wheat varieties has not been well established. Some studies on the protein electrophoretic analysis were per- formed (Dontsonova and Puasheva, 1979; Dolinsek, 1980; Shevchuk, 1985). Storage protein polymorphism was used as a genetic marker for cultivar identification in studies by Zeller et ai. (2004), Rogl and Javornik (1996) and Bonafaccia and Kreft (1994). The characterization of protein fractions was carried out on a diploid buckwheat variety 'Siva' (Javornik and Kreft , 1984). Many studies Correspondence author: Phone: +420-224382182, Fax: +420-224381829, E-mail: cepkova@itsz.czu.cz Abbreviations: M-molecular weight marker, 1', P- 'Pyra ', KD- 'Kara-Dag' , ]- ']ana', CZ -Czech Republic, SK-Slovakia, UKR- Ukraine, PCA - principal components analysis, SDS-PAGE - sodium dodecyl sulphate polyacrylamide gel electrophoresis method, UPGMA- Unweighted Pair Group Method with Arithmetic Mean. 17 18 Cepkova and Dvoracek e.g. Ohnishi and Matsuoka (1996), Yasui and Ohnishi (1998), Tsuji and Ohnishi (2001), Kump and Javornik (2002) and Iwata et al. (2005) have been carried at the molecular level using RAPD, SSR and AFLP techniques. However these were mainly used in taxonomical studies ofthe genus Fagopyrum. The aim of the present study was to describe the genetic diversity of four registered buckwheat varieties and possibilities of their identification by means of seed protein polymorphism evaluated under SDS-PAGE con- ditions. MATERIAL AND METHODS The following common buckwheat varieties registered in the Czech Republic were studied: P - 'Pyra' (CZ), registered in 1990; N - 'Spacinska I' (SK), registered in 2002; KD - 'Kara-Dag' (UKR), registered in 2001 and J - ' Jana' (UKR), registered in 1997. The samples were obtained from the Seed Collection of Prague Gene Bank and were used for electrophoretic analyses of seed proteins. 250 grains of each variety were individually analyzed by discontinuous SDS-PAGE according to the procedure of Laemmli (1970). Electrophoregrams of bulk seed samples (a mixture of at least 300 ground grains from each variety) were obtained in the same way. This strategy was recommended by Gardiner and Forde (1992) for the evaluation of cross-fertilized species. The electrophoretic patterns were digitalized and eval- uated by specialized software Bioprofil ID++ (Vilber Lourmat, Paris, France). Similarity matrixes were calculated by means of Nei and Li' s coefficient of genetic similarity and dendrograms were constructed by the UPGMA method. The graphical presentation of variability in single seed proteins of each variety was done by the statistical method PCA (software Statistica 7.0 CZ). The evaluation of statistical differences in band fre- quencies was carried out by chi suare test (software Statistica 7.0 CZ). The index of the band pair coincidence was calculated separately for each variety according to the following equation: Xij/(Abs (Ni-Xij)+Abs (Nj- Xij)), where Xij is the number of coincidences of the i-th and j-th band in 250 individually evaluated seeds. Ni and Nj are frequencies of the i-th and j-th band. The matrix of these coefficients was used for the construction of the final dendrogram. RESULTS AND DISCUSSION A total of 36 band positions were identified in the four registered buckwheat varieties in both single seed and in the bulk samples. Protein banding profiles, under the conditions of SDS-PAGE were in the range of molecular weights from 30-205 kDa. These band descriptions enabled the evaluation of wide protein polymorphism in single seeds. An example of the band profile (36 designated bands) of ' Pyra' variety is presented in Fig. 1. The high- est band polymorphism and simultaneously the most intensive bands of buckwheat proteins were situated in the middle part of the gel with molecular weights from 30-60 kDa (bands 12-30). No specific bands (with the frequency of occurrence of more than 50%) were found by comparison of the electrophoretic spectra of 250 single seeds from each variety and almost all designated bands showed similar frequencies of occurrence and intensities in all four varieties. The following evaluation was focused on the frequencies of designated bands in each variety. The occurrences of evaluated bands are shown in Table 1. The frequency of individual bands was found to be very variable. Groups of bands with very low occurrence or zero occurrence in some varieties were found (bands 12', 14', 20' and 26"). In contrast to these bands, other bands with 100% frequency were also described (bands 7, 12, 14, 23, 26 and 27). The occurrence of identical genotypes which were found in the population of particular bands exhibited a low frequency from 1 to 3%. Although all four varieties showed a similar trend of band frequency, some statisti- cally significant differences in the band frequency were detected (e.g. bands 0; I; 8; 9; 9*; 11; 13; 16; 24; 25 and 29). Each of these band frequencies enabled the divi- sion of the tested varieties into at least three statistically " Q .:< Fig. 1. Description of important (polymorphic) band positions in a bulk seed sample of the buckwheat variety ' Pyra' . " Additionally designated bands which were found in seed population and which were situated in very close position to original band designed by the same number. Seed protein polymorphism in Czech varieties 19 Table I. Differences in the frequencies of bands in evaluated similar intravarietal variability. Only the variety ' Pyra' buckwheat varieties (The bands with specific andstatistically sig- (Fig. 2-3) showed a different genetic shift of intravarietal nificant frequencies at leastfor3 varieties areboldly emphasized) variability (axis x: -0.8--0.5; axis y: 0.1-0.4) in com- Band! 'lana' 'Pyra' ' Kara-Dag' 'Spacinska I' parison with the other varieties, whose tested genotypes Variety Frequency Frequency Frequency Frequency were situated in the lower area of PCA graphs (axis x: 0 33' 17' 2 b 53 d -0.9- -0.6; axis y: -0.1--0.3) (Figs. 2-1,2-2 and 2-4 ). I 78' 50 c 9 d 1I4 b The calculated similarity dendrogram is refered to peA 2 25' 16' 22' 27' analyzes and can describe the total (mean) dissimilarity 3 15' b 9 b 26 a 7 b among the varieties (Fig. 3). In accordance with the PCA 4 65 b 54 b 34' 74 b graphs a closer linkage was found between the three 5 160' 171" 192' 192' varieties 'SpaCinska 1', 'Kara-Dag ' and 'lana' whereas 6 182 ab 156 b 213' 211' ' Pyra' variety created a more individual cluster. 7 240" 230" 250' 246' The evaluation of coincidences of designated bands of 8 124' 179' 240 b 223 bc tested varieties was mainly focused on finding of the 9 75 b 31c 161a 146' potential differences between varieties. The bands with a 9* 85' 31' 3 b 84' high frequency of occurrence in the tested seed samples 10 175' 9P 222' 203a logically showed the high index of coincidence. The high 11 160 b 118' 243 a 211' values of coincidence were not found in bands with low 12 250" 244' 246' 250' or medium frequency of occurrence. The highest number of 12' 0" 18 b 16 b 0' bands with the closest coincidence (more than 0.80) was 13 2' 36 b 2 a 14' determined in the buckwheat variety 'Kara-Dag' (14 bands). 14 247' 226' 247' 229' On the other hand, the variety 'Pyra' was characterized 14* 14' 3 b loa b ll a by only nine bands with a similar level of coincidence. 15 225' 218 a 222' 207 a These bands with a specific coincidence reflect contempo- 16 57'b 39 b 97' 36 b rary genetic differences among the tested varieties. After 17 182 a 124 b 207' 188 a several years of verifying these results it could be another 18 49 b 40 b 110' 94 a important criterion for buckwheat variety identification. 19 91' 109' 90' 85 a All four tested varieties were characterized by common 20 112- 59 b 139' 116' bands with a high index of coincidence (bands 7, 12, 14, 20* 0" 0" 0" 2' 23, 26, 27 and 28). Each variety also had other specific 21 202' 226' 228' 212 a bands with a high index of coincidence ('Kara-Dag' : 8, 22 127' b 103 b 157' 152 a 10,11,21,15,6 and 17; 'Pyra': 15 and 21; 'SpaCinska I' : 23 241" 243' 246 a 240" 8,10 and 11; 'lana': 6; 10, 11 and 15). 24 114' ss- 182 h 72' The high similarity of the tested varieties was also con- 25 26 ab 41 b ' 53 c 23' firmed by the protein electrophoretic evaluation of the 26 243' 244' 249' 231" bulk seed samples (Figs. 4 and 6). No specific differences 26" 7 a Ob Ib 3 ab in electrophoretic spectra were found in two varieties 27 232- 240' 242' 235' ' SpaCinska I' and 'Kara-Dag'. The most significant dif- 28 224 a 218' 226' 215' ference in the protein profile was determined in band 29 236' 84' 197'b 156 b number 22 (Fig. 4). This band was very intensive in the 30 202' 68 b 96 bc 122 c variety 'Pyra' in contrast to the other varieties where this Different letters in the sameroware statistically significant at p band exhibited a low expression. More significant differ- lessthan0.05. ences in the band intensity of the bulk seed samples were found in 'Pyra' variety only (bands 15 and 26). There were other specific differences in the bands with low significant categories (Table 1). intensity and electrophoretic mobility (band positions 0, 1 The high intravarietal variability of the presence of 36 and 2) (Fig. 5). We did not confirm linear relationship designated bands (protein components) was represented between the frequency of bands in single seeds and their in 4 identical graphs plotted by means of principal com- occurrence in bulk samples. The detection ability of spe- ponents analysis (PCA) (Figs. 2-1-2-4). Each graph cific protein band in protein profile of bulk sample was shows the evaluation of all four tested varieties (4x250 not only dependent on its frequency in the population but seed samples) and only one of the tested varieties was also on its total concentration. always emphasized in each graph. Clusters based on the The detected high intravarietal polymorphism of seed protein spectra of the evaluated seeds confirmed high and proteins in buckwheat confirmed the well-known fact of 20 Cepkova and Dvoracek Figure 2 -1 (Kara-Dag) Figure 2-2 (Spacinska 1) PCA analysis - Kara-Daq peA analysis - Spacinska 1
-1.0 - 0.9 -0.8 -0.7 -0.6 -0.5 -0.4 - 0. 3 - 0.2 Factor 1: 52.53% Figure 2-3 (Pyra) Figure 2-4 (Jana) peA analysis - Pyra peA analys is - Jana ........ -1 .0 -0.9 -0.8 - 0.7 - 0.6 - 0.5 - 0.4 - 0.3 -0.2 Factor 1: 52.53% '.. -0.4 - 0.3 - 0.2 -0.2 - 0.3 -0.4 ........ - 1.0 - 0.9 - 0.8 - 0.7 - 0. 6 - 0.5 Factor 1: 52.53% 0.3 0.2 0.1 OJ 0.0 If-D.1 ... ... -0.2 - 0.3 -0. 4 Fig. 2. Individual genetic variability of singleseedsin testedvarities (Results of PCAanalysis). - Nei&Li's Coefficient- UPGMA - posed of individual genotypes mutually crossing with each other, and creating an almost ideal panmictic population. The history of the origin (development) of these four registered varieties and their further development are not known well. At the level of seed storage protein poly- morphism it is possible to express the theory that detection of minimal differences may be attributed to their com- mon historical evolution and close relationship between the regions of their cultivation. The identified differences of protein polymorphism in the tested varieties could be caused by random selection during their distribution to other areas, by influences of different soil and climatic conditions in these areas or by mutual crossing with indigenous local populations. The two characteristics (band frequency and band co- incidence) were obtained by the protein electrophoretic evaluation of individual seeds. The detected significant differences of band frequencies among the varieties have, in this case, less importance for the identification of varieties in comparison with band coincidence. The reason is a higher dependence of a band with lower frequency on the number of tested seeds . In our case, the low number of identical genotypes (1-3% in each buckwheat variety) found in the tested buckwheat varieties indicated that the seed sample size was still insufficient for objective lana I 0.85 0,9 0,95 i I 0,7 0,75 0,8 buckwheat's strict allogamy . Similar results have been published by Slovene and Indian authors (Javomik et aI., 1981; Chrugoo and Anusuya, 2004). Bonafacia and Kreft (1994) observed higher variability within Italian varieties than among them. The high intravarietal polymorphism in buckwheat was also described by Zeller et al. (2004). It is possible to suppose that buckwheat varieties are com- Fig. 3. Similarity dendrogram constructed as the sumof intra- varietal polymorphism of singleseedsof buckwheat varieties. Kara-Dag Seed protein polymorphism in Czech varieties 21 Pyra Fig. 6. Similarity dendrogram in bulk seed samples of tested 20 varieties. 205 116 kDa 0.90 1 ACKNOWLEDGEMENTS CONCLUSIONS REFERENCES o 1 2 This study was supported by Grant 521104/P031 of Grant Agency of the CR. It is possible to assume that in the future an increasing interest in buckwheat consumption in the Czech Republic and potential registration of new buckwheat varieties will require the strict verification of new buckwheat varieties and also for their legal protection. The evaluation of seed protein polymorphism under the conditions of SDS-PAGE appears to be an applicable tool for characterization of buckwheat varieties (populations) . In the present study no specific bands were observed for each variety. Detailed analyses of single seeds confirmed a high intravarietal genetic variability of tested varieties and whereas the inter-varietal variability was lower. These results in similarity were probably caused by of their com- mon origin and historical evolution. Significant differ- ences in protein band frequencies and their coincidences could serve as useful identification criterion, especially in such cases when the analysis of bulked samples was not sufficient (e.g. ' Spacinska I' versus 'Kara-Dag' in our study). Bonafaccia, G. and I. Kreft , 1994. Technological and qualitative charac- teristics of food products made with buckwheat. Fagopyrum 14: 35-42. CISTA, 2005. Central Institute for Supervising and Testing in Agri- Fig. 5. Detail of the top part of gel with detected protein band differences (bulk samples) . See Fig. 4 for abbreviations. Jana Spacinska 1 1 29 Kara-Dag 1 1-- --.... 24 M KD N p J 0 205 1 2 116 97 84 66 55 45 22 36 evaluation of band frequencies, although a similar seed number of other cross-fertilized crops is frequently used and recommended for electrophoretic analyses (Gardiner and Forde, 1992). 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