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Fagopyrum 23: 17-22 (2006)

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

Sp: Spacinsk a 1
0.6
0.3
'"
;;; 0.2
-i
NO.1
(;
0.0
-0.1
-0.2
- 0.4
_0.5.........
- 1.0 -0.9 -0.8 -0.7 -0.6 -0.5 - 0.4 -0.3 -0.2
Factor 1: 52.53%
0.5
0.4
0.3
0.2
..
0.1
.9
0.0
-0.1
- 0.2
-0.3
-0.4

-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).
On the other hand, the identification of specific protein
band groups with a high index of coincidence (more than
90%) in the different varieties could markedly decrease
the number of tested seeds for electrophoretical analyses.
A more detailed study of detected bands with the high
index of coincidence could also provide further information
about genetic linkages of these bands (alleles) and their
localization on the chromosomes. Varietal stability of
specific protein band frequencies and their coincidences
has to be further verified for their routine application.
In spite of possibilities of single seed analyses as men-
tioned above, the evaluation of bulk seed samples still
remains the most suitable method for the expeditious and
objective identification of buckwheat varieties. Besides
specific band positions, the band intensity also plays an
important role for the identification of buckwheat geno-
types. This fact was also confirmed by Javornik et al.
(1981).
Fig. 4. Differences in protein band positions in bulk seed sam-
ples of tested varieties, J; ' Jana' , P; ' Pyra' , KD; ' Kara-Dag' , N;
Spacinska I'.
22
Cepkova and Dvoracek
culture. Division of Plant Variety Testing. Description of varieties.
Chrungoo, N.K. and Anusuya, 2004. Genetic diversity in accessions of
Himalayan Buckwheats revealed by SDS PAGE of soluble pro-
teins extracted from single seeds and RAPD based DNA finger-
printing. Proc. 9th IntI. Symp. Buckwheat at Prague: 326-335 .
Dolinsek, B., 1980. Studies of polymorphism of electrophoretic protein
patterns in buckwheat. Proc. 1st IntI. Symp. Buckwheat at
Ljubljana: 61-68 .
Dontsonova, T.V. and Z.P. Puasheva, 1979. Study of the comparison of
readily soluble protein components in buckwheat fruits by means
of electrophoresis in polyacrylamide gel. Plant Breed. Abst. 254:
26-29.
Edwardson, S., 1996. Buckwheat: Pseudocereal and nutraceutical. In:
Janick, J. (ed.), Progress in New Crops, pp. 195-207, ASHS
Press, Alexandria, VA.
Eggum, B.O., I. Kreft and B. Javornik, 1980. Chemical composition
and protein quality of buckwheat (Fagopyrum esculentum
Moench). Plant Foods Hum. Nutr. 30: 175-179.
Gardiner, S.E. and M.B. Forde, 1992. Identification of cultivars of
grasses and forage legumes by SDS-PAGE of seed proteins.
Strategies for Cultivars of Cross-Fertlilized and Self-Fertilized
Species. In: Linskens, H.F. and J.F. Jackson (eds.), Seed Analysis,
pp. 49, Speinger Verlag Berlin, Heidelherg, New York.
Iwata, H., K. Imon, Y. Tsumura and R. Ohsawa, 2005. Genetic diversity
among Japanese indigenous common buckwheat (Fagopyrum
esculentum) cultivars as determined from amplified fragment
length polymorphism and simple sequence repeat markers and
quantitative agronomic traits. Genome 48: 367-377 .
Javornik, B., B. Eggum and I. Kreft, 1981. Studies on protein fraction
and protein duality of buckwheat. Genetika 13: 115-121.
Javornik, B. and I. Kreft, 1984. Characterization of buckwheat proteins.
Fagopyrum 4: 30-38.
Kreft, I., V. Skrabanja, S. Ikeda, K. Ikeda and G. Bonafaccia, 1996.
Dietary value of buckwheat. Res. Reports Biochemical Faculty
of Univ. Ljubljana 67: 73-78 .
Kump, B. and B. Javornik, 2002. Genetic diversity and relationships
among cultivated and wild accessions of Tartary buckwheat
(Fagopyrum tataricum Gaernt.) as revealed by RAPD markers.
Genet. Resour. Crop Evol. 49: 565-572.
Laemmli,U.K., 1970. Cleavage of structural proteins during the
assembly of the head ofbacteriophate T4. Nature 227: 680-685 .
Michalova, A., L. Dotlacil and L. Cejka, 1998. Evaluation of common
buckwheat cultivars. Rost. Vyroba 44: 361-368.
Ohnishi, O. and Y. Matsuoka, 1996. Search for the wild ancestor of
buckwheat II. Taxonomy of Fagopyrum (Polygonaceae) species
based on morphology, isozymes and cpDNA variability. Genes
Genet. Syst. 71: 383-390.
Rogl, S. and B. Javornik, 1996. Seedprotein variation for identification of
common buckwheat (Fagophyrum esculentum Moench) cultivars.
Euphytica 87: 111-117.
Shevchuk, T.E., 1985. Storage protein 13S globulin-a genetic marker
in buckwheat breeding. Genetichestie Osnovy selektsii i semen-
ovodstva grechikhi: 115-119.
Tsuji, K. and O. Ohnishi, 2001. Phylogenetic relationships among wild
and cultivated Tartary buckwheat (Fagophyrum tataricum
Geartn.) populations revealed by AFLP analyses. Genes Genet.
Syst. 76: 47-52.
Yasui, Y. and O. Ohnishi, 1998. Phylogenetic relationships among
Fagopyrum species revealed by the nucleotide sequences of the
ITS region of the nuclear rRNA gene. Genes Genet. Syst. 73:
201-210.
Zeller, FJ., H. Weishaeupl and L.K. Hsam, 2004. Identification and
genetics of buckwheat (Fagopyrum) seed storage protein. Proc.
9th IntI. Symp. Buckwheat at Prague: 195-201.

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