cultivar identification and purity testing using acidic page of barley

TECHNICAL BULLETIN 217
ISSN 0070-2315
CULTIVAR IDENTIFICATION AND PURITY TESTING USING
ACIDIC PAGE OF BARLEY STORAGE PROTEINS
Dionysia A. Fasoula, Anna Ilieva and I.M. Ioannides
AGRICULTURAL RESEARCH INSTITUTE
MINISTRY OF AGRICULTURE, NATURAL RESOURCES
AND THE ENVIRONMENT
NICOSIA
CYPRUS
SEPTEMBER 2003
Editor - in Chief
Dr A.P. Mavrogenis, Agricultural Research Institute, Nicosia, Cyprus.
All responsibility for the information in this publication remains with the author(s). The use of
trade names does not imply endorsement of or discrimination against any product by the
Agricultural Research Institute.
2
CULTIVAR IDENTIFICATION AND PURITY TESTING USING
ACIDIC PAGE OF BARLEY STORAGE PROTEINS
Dionysia A. Fasoula, Anna Ilieva and I.M. Ioannides
SUMMARY
A high-resolution system of acidic continuous Polyacrylamide Gel Electrophoresis
(PAGE) was developed in order to analyse the hordein polypeptides of barley. The system was used to study 17 barley cultivars and seven line selections from one cultivar.
Each of the 17 cultivars tested was found to possess a characteristic hordein banding pattern, which enabled the rapid visual assessment of the electropherograms. Electrophoretic patterns of samples from single kernels were also compared with samples
from pooled kernels. In general, single kernels gave identical banding patterns to those
pooled, but with better resolution. On account of the high reproducibility, speed, simplicity and the low cost of the analysis, this method can be easily incorporated in barley
breeding programs to routinely process large numbers of breeding samples for purposes
of barley cultivar identification and genetic purity assessment.
ΠΕΡΙΛΗΨΗ
Στην εργασία αυτή αναπτύχθηκε µέθοδος ηλεκτροφόρησης ακρυλαµιδίου µε
υψηλή αναλυτική δυνατότητα σε συνθήκες χαµηλού pH, για το διαχωρισµό, την
ταυτοποίηση και τον έλεγχο της γενετικής καθαρότητας ποικιλιών κριθαριού.
Εξετάστηκαν 17 ποικιλίες κριθαριού και 7 γραµµές από µία ποικιλία. Όλες οι
ποικιλίες έδωσαν ζώνες διακριτών πολυµορφισµών ως προς τις κύριες
αποθηκευτικές πρωτεΐνες του σπόρου, καθιστώντας δυνατή την ταχεία οπτική
αξιολόγηση του ηλεκτροφερογραφήµατος. Επιπλέον, εξετάστηκαν δείγµατα από
ατοµικούς σπόρους σε σχέση µε δείγµατα από µίγµατα περισσότερων σπόρων
και βρέθηκε ότι ένας σπόρος αρκεί για την εφαρµογή της µεθόδου. Η
επαναληψιµότητα, η ταχύτητα και το χαµηλό κόστος της µεθόδου, την καθιστούν
κατάλληλη για πολλαπλές εφαρµογές αξιολόγησης του απαιτούµενου µεγάλου
αριθµού δειγµάτων ενός βελτιωτικού προγράµµατος.
INTRODUCTION
In the process of crop breeding, there is a
constant need for development of methods
which can reliably assess cultivar identity,
genetic purity and trueness-to-type. Growers
expect high-quality, genetically pure seed that
gives a successful stand establishment. In
addition, when new cultivars are developed
by plant breeders, a limited amount of seed
has to be progressively increased to larger
quantities. During this process, seed must be
monitored to ensure that the genetic purity of
the breeder seed is not compromised
(McDonald, 1998). Traditional methods of
barley cultivar identification, which are based
on visual examination of grain samples, have
certain drawbacks due to the fact that frequently cultivars possess similar kernel characteristics. Thus, visual cultivar identification
based on threshed grain samples is considered
a difficult and generally imperfect process
(Gebre et al., 1986). To overcome these obstacles, various laboratory methods, based on
analysis of seed storage proteins, have been
developed for a number of crops, as seeds are
one of the richest source of plant proteins.
The major storage proteins in the
endosperm of most cereals (with the exception of oats and rice) are the prolamins
(Shewry, 1995). The barley prolamins are
known as hordeins and comprise about 40%
of all the protein found in barley grain
(Shewry et al., 1977). Prolamins are traditionally recognized as a group on the basis of
their solubility in alcohol/water mixtures and
their high levels of glutamine and proline.
Even though some prolamins occur as alcohol-insoluble polymers, all individual prolamin polypeptides are alcohol-soluble in the
3
reduced state (Shewry and Halford, 2002).
Hordeins are customarily classified into
two main groups, referred to as B and C and
two minor groups, D and γ. Groups C, B and
D are encoded by three compound loci,
known as Hor-1 (group C), Hor-2 (group B),
and Hor-3 (group D) on the short (Hor-1 and
-2) or the long (Hor-3) arm of chromosome 5.
The other minor components are possibly
encoded by remote genes on the same chromosome (Shewry and Darlington, 2002).
Each locus has a number of alleles and the
analysis of hordeins is based on the recognition of those alleles from the corresponding
electrophoretically separable proteins. The
two major groups are B and C, accounting for
approximately 95% of the total hordein fraction (Echart-Almeida and Cavalli-Molina,
2001). Hordein groups B and γ belong to sulphur rich (S-rich) prolamins, group C to sulphur poor (S-poor) and group D belongs to
high molecular weight prolamins (Shewry et
al., 1995).
Barley is highly polymorphic regarding
the hordein polypeptide composition. Their
migration pattern on electrophoretic gels is
generally considered independent of environmental influences and can serve as a varietal
characteristic (Shewry et al., 1978; Sozinov
and Poperelya, 1980). This has allowed the
use of hordeins as markers for determining
the distinctiveness of varieties and the identity and purity of seed samples (Shewry et al.,
1979). The chosen method of protein separation affects the patterns of detected polymorphisms, through exploitation of the different
properties of prolamins. Several research
groups have used sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDSPAGE) based on molecular size, isoelectric
focusing (IEF) based on net charge, twodimensional electrophoresis (IEF followed by
SDS-PAGE) and reversed-phase high-pressure liquid chromatography (RP-HPLC)
based on hydrophobicity (Shewry et al.,
1977; Soave and Salamini, 1984; Gebre et al.,
1986; Wilson, 1991; Echart-Almeida and
Cavalli-Molina, 2001). Those methods are
considered rather labour-intensive and expensive for handling the required large numbers
of breeding samples. Recently, a method of
acidic continuous PAGE (Zayakina and Sozinov, 1993) was proposed for studying zein
composition, zein inheritance and organiza4
tion of zein genes on maize chromosomes.
This method allows a high-resolution zein
separation and reveals extraordinarily high
polymorphisms (Zayakina and Sozinov,
2000).
The objective of this study was to explore
the feasibility of adapting the aforementioned
acidic continuous PAGE for zein storage proteins for cultivar identification and genetic
purity testing in barley.
MATERIALS AND METHODS
Plant materials
Seeds from 17 barley cultivars and seven
line selections from one cultivar were
analysed for their hordein electrophoretic patterns (Table 1). The seven lines resulted from
one cycle (field season 2001-2002) of divergent honeycomb selection for yield within the
cultivar Lysi (Fasoula and Fasoula, 2000).
Investigations were carried out both with
samples from pooled kernels and with samples from single kernels.
Acidic continuous PAGE
Hordein extraction and electrophoresis
were carried out according to Zayakina and
Sozinov (1993) with certain modifications.
Hordeins were extracted from embryo-less
ground kernels by 70% ethanol for 1 h at 40
oC. Extraction was performed using 400 µl of
70% ethanol per 100 mg of meal from pooled
samples (2 to 10 kernels) and 120 µl of 70%
ethanol per single kernel. The samples were
centrifuged for 10 min at 5000 g, extracts
were transferred in new tubes, dried at 40 oC
and dissolved in 100 µl of buffer containing 8
M urea, 5% β-mercaptoethanol, 0.1 M acetic
acid and 0.05% pyronin G. Proteins were
incubated at 95 oC for 5 minutes.
Electrophoresis was performed in a vertical slab apparatus (Consort, UK). Polyacrylamide gels (8.5%) contained 7.5 M urea, 13
mM glycine and 0.33 M acetic acid. Polymerisation was initiated by the addition of 0.3
ml TEMED, 55 mg ascorbic acid, 0.5 ml of
0.2% FeSO4x7H2O and 0.3 ml ammonium
persulfate in 100 ml of solution. Twenty-five
µl per sample was loaded on the gel. The running buffer consisted of 0.33 M acetic acid
and 26.6 mM glycine (pH=3.15). Electrophoresis was performed for 30 minutes at
100 V and 20 mA, and then for 5 h at 450 V
and 50 mA. Proteins were fixed and stained
overnight in a solution containing 6%
trichloroacetic acid, 6% acetic acid, 15%
methanol and 0.05% Coomassie Blue R250.
SDS-PAGE
Hordein extraction and electrophoresis
were carried out according to the Hoefer Protein Electrophoresis Applications Guide and
the Community Plant Variety Office guidelines. Extraction solution containing SDS and
a discontinuous system with Tris-glycine
buffer were used. Electrophoresis was performed at 100 V/45 mA until the dye moved
into the running gel and then changed to 300
V/60 mA. Proteins were fixed and stained as
described for acidic PAGE. The molecular
weight standards (Sigma) were albumin
(66000 Da), ovalbumin (45000 Da), glyceraldehyde-3-phosphate dehydrogenase (36000
Da), carbonic anhydrase (29000 Da),
trypsinogen (24000 Da), trypsin inhibitor
(20000 Da) and lactalbumin (14200 Da).
RESULTS AND DISCUSSION
A number of variables affecting the reproducibility of results were investigated at the
beginning of the development of the system.
Reproducibility was found to depend on variations in the ambient temperature at which
electrophoresis was carried out and on the
duration of electrophoresis. Increase in ambient temperature above 25 oC resulted in poor
resolution of polypeptide bands and low
reproducibility of polymorphisms (data not
shown), while maintenance of ambient temperature at 20 oC, resulted in good resolution
and a reproducible number of polypeptide
bands. Freshly prepared hordein extracts gave
better-resolved PAGE patterns than older
extracts (stored at –20 oC for 1 to 2 months).
Older extracts displayed band diffusion, loss
of certain faint bands, and were slightly
coloured. According to Zayakina and Sozinov
(2000), high-resolution zein separation and
extraordinarily high polymorphisms were
achieved using 500 V for 4 to 4.5 h. In the
present study, the use of the above parameters
resulted in poor resolution of hordein banding
patterns and no distinct polymorphisms.
Thus, for the purpose of this study, 450 V for
5 h were used.
Following the standardization procedure,
Table 1. Characteristics of the barley cultivars
Number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Cultivar
Kantara
Kythraia
Trachonas
Arlona
Prasteio
Lysi
Gypsou
Lefkonoiko
Proti
Athenaida
Morocco 628
14-6
13-35
10-17
17-31
19-23
9-23
14-17
Harmal
WI 2291
ER/Apm
Assala-04
Beecher
Rihane-03
Origin
No of Rows
Cyprus
Cyprus
Cyprus
Cyprus
Cyprus
Cyprus
Cyprus
Cyprus
Cyprus
Greece
Morocco
Cyprus
Cyprus
Cyprus
Cyprus
Cyprus
Cyprus
Cyprus
Lebanon/ Icarda
Icarda
Icarda
Lebanon/ Icarda
Lebanon/ Icarda
Icarda
2
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
2
2
2
6
6
6
the method was applied to distinguish seventeen barley cultivars (Table 1) and further
examine seven line selections from cv. Lysi
with respect to their hordein polypeptide
banding patterns (Fig. 1, 2 and 3). Ranges of
16 to 23 polypeptide bands were detected for
each cultivar. These numbers are similar to
those obtained by Shewry et al. (1978) and
Gebre et al. (1986). In the present study,
hordeins were extracted from kernels in the
1
2
3
4
5
6
7
8
Zone 1
Zone 2
Zone 3
Figure 1. Hordein acidic PAGE banding patterns (meal
from 10 kernels) of cultivars: 1) Kantara, 2)
Kythraia, 3) Trachonas, 4) Arlona, 5) Prasteio,
6) Lysi, 7) Gypsou, 8) Lefkonoiko.
5
1
2
3
4
5
6
Zone 1
Zone 2
Zone 3
Figure 2. Hordein acidic PAGE banding patterns of cultivars and lines: 1) Proti, 2) Athenaida, 3)
Morocco-628, 4) 14-6, 5) 13-35, 6) 10-17.
absence of reducing agents, using only 70%
ethanol. Under these conditions, hordeins
maintain their native state, allowing their separation on the basis of different molecular
weight and charge.
To facilitate comparisons, the hordein
bands were divided into three zones, depending on their electrophoretic mobility (Fig. 1,
2, 3, 5, 6 and 7).
Zone 1 High molecular weight bands. Bands
well defined and strongly stained.
Small number of bands
Zone 2 Medium molecular weight bands.
Bands well defined and strongly
stained. Large number of bands
Zone 3 Low molecular weight bands. Bands
diffused and lightly coloured. Small
number of bands
1
2
3
4
5
6
7
8
9
Zone 1
Zone 2
Zone 3
Figure 3. Hordein acidic PAGE banding patterns of
cultivars and lines: 1) Rihane-03, 2) Beecher, 3) Assala-04, 4) ER/Apm, 5) WI 2291, 6)
Harmal, 7) 14-17, 8) 9-23, 9) 19-23.
6
The bands in Zone 2 were easily observable, with distinct differences among cultivars. This distribution of polypeptides into
three groups is based on the Shewry (1993)
classification.
The banding patterns of the examined cultivars and lines are depicted schematically in
Figure 4. In the present study, the acidic
PAGE method allowed a clear discrimination
between two-rowed and six-rowed type cultivars (Fig. 5). As expected from their pedigree,
the seven line selections from cv. Lysi (14-6,
1
2
3
4
5
6
Zone 1
Zone 2
Zone 3
Figure 5. Hordein electrophoretic patterns discriminating between two-rowed and
six-rowed cultivars.
Two-rowed: 1) Harmal, 2) WI 2291, 3) ER/Apm.
Six-rowed: 4) Assala-04, 5) Beecher, 6) Rihane-03.
13-35, 10-17, 17-31, 19-23, 9-23, 14-17)
were not as clearly distinguishable (Fig. 2 and
3). However, two interesting observations
concern the presence of an additional band in
Zone 3 in line 14-17 and the fact that the
selected lines differ slightly in their hordein
banding patterns from the pattern produced
by breeder’s seed of cv. Lysi from the previous year.
The hordein banding patterns were also
studied using single kernels and comparing
samples from five individual kernels with
samples of 2 to 10 pooled kernels for each cultivar. In general, single kernel samples gave
Figure 4. Diagram of a compound-banding pattern of cultivars and lines: 1) Athenaida, 2) Arlona, 3) Proti, 4) Kythraia, 5) Trachonas, 6) Kantara, 7) Lefkonoiko, 8) Prastio, 9) Morocco-628, 10) Gypsou, 11) Lysi, 12) 14-6, 13) 13-35, 14) 1017, 15) 17-31, 16) 19-23, 17) 9-23, 18) 14-17, 19) Harmal, 20) WI 2291, 21) ER/Apm, 22) Assala – 04, 23) Beecher, 24) Rihane-03
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Ia IIa IIIb
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γ
aI – II: Serial number and relative position of bands detected in all samples.
bIII: Hordeins: C - Zone 1, B - Zone 2, γ - Zone 3.
7
1
2
3
4
5
6
7
8
9
10
Zone 1
Zone 2
Zone 3
Figure 6. Hordein acidic PAGE patterns from
pooled (10 kernels) and single kernel
samples.
Lysi: 1-5, -1= pooled, 2-5= single kernel.
Athenaida: 6-10, -6= pooled, 7-10= single kernel.
banding patterns identical to the pooled samples (Fig. 6 and 7), but with better resolution,
thus enabling the use of only one seed per
sample for routine testing.
Using SDS-PAGE, the number of detected
bands increases. Echart-Almeida and CavalliMolina (2001) identified 26 different bands in
14 barley varieties with a range of 10 to 17
bands in each variety. In the present study, the
total number of polypeptide bands detected
on SDS-PAGE gels was even larger (above
40, Fig. 8). This is due to the presence of the
reducing agents SDS and urea in the extraction solution, that results in the loss of the
native state of hordeins, as well as in the
1
2
3
4
5
6
7
8
9
10
Zone 1
Zone 2
Zone 3
Figure 7. Hordein acidic PAGE patterns from pooled
(10 kernels) and single kernel samples.
WI 2291: 1-5, -1= pooled, 2-5= single kernel.
Rihane-03: 6-10, -6= pooled, 7-10= single kernel.
8
extraction of other storage proteins besides
prolamins (Wilson, 1991; Maizel, 1972). The
molecular weight of the polypeptides ranged
from approximately 14200 Da to 66000 Da,
the majority of them between 20000 Da to
66000 Da (Fig. 8). The electrophoregram
showed the presence of bands with higher
molecular weight than 66000 Da. According
to Shewry et al. (1977) and Shewry and
Darligton (2002), C-hordein can be separated
into polypeptides with molecular weight
ranging between 60000 and 72000 Da, Bhordein between 30000 and 60000 Da and γhordein between 13000 and 25000 Da. Overall, the resulting hordein SDS-PAGE electrophoretic patterns are more complicated and
thus, less convenient for purposes of routine
identification of multiple breeding samples.
Da
66000
1
2
3
4
5
6
7
8
9
10
45000
36000
29000
24000
20000
Figure 8. Hordein SDS-PAGE banding patterns of cultivars: 1) Molecular weight markers, 2) Lysi,
3) Gypsou, 4) Prasteio, 5) Kythraia, 6) Trachonas, 7) Arlona, 8) Athenaida, 9) Proti, 10)
Morocco 628.
CONCLUSIONS
The method described in the present study
for acidic continuous PAGE, has the advantages of high reproducibility, simplicity,
speed and low cost. Thus, it can be beneficially incorporated in applied barley breeding
programs for purposes of barley cultivar identification and genetic purity assessment. Traditionally, genetic purity is determined using
various physical traits expressed in the stage
of seed, seedling or mature plant. However,
these tests can be influenced by environmental stress or post-harvest handling operations,
which can mask or alter specific seed and
seedling anatomical and morphological fea-
tures (McDonald, 1998). The acidic continuous PAGE system described here can help circumvent these problems. In comparison, the
more complicated electrophoretic patterns
render the SDS-PAGE a less practical
approach for purposes of applied breeding
programs. However, SDS-PAGE may provide
a finer separation in certain cases where the
results of acidic PAGE are less clear-cut due
to a high degree of similarity between cultivars.
Each of the 17 cultivars tested was found
to possess a characteristic acidic PAGE banding pattern of hordeins. Hordein banding patterns of single kernels were compared to samples from pooled kernels from each cultivar to
test genetic purity. In general, single kernels
gave hordein banding patterns identical to
those pooled, but with better resolution. The
seven line selections within the cultivar Lysi,
were selected from breeder’s seed stock of the
previous year from the stock that was used to
represent cv. Lysi in this study. Interestingly,
the banding patterns from the seven line
selections differed in certain minor bands
from the banding pattern produced by the
newer breeder’s seed stock. There are other
reports in the literature, where differences in
electrophoretic patterns exist between seed
stocks from different sources (Gebre et al.,
1986). Bearing in mind that hordein electrophoresis actually scans the genome for
only a very limited number of loci, it is of
interest that we were able to identify differences among selected lines and in breeder’s
seed depending on year of origin.
In conclusion, the important characteristics of the acidic continuous PAGE system are
extraction in the absence of reducing agents,
high resolution, easy assessment of banding
patterns, and convenience for routine analysis
of large numbers of breeding samples using
single kernels.
ACKNOWLEDGMENTS
The present study was supported by funds
from the INCO-DC FP5 program (ARICY Centre of Excellence) of the European Union
in the framework of the ARI project Breeding
barley for Cyprus conditions with emphasis
on hay quality.
Anna Ilieva, was a visiting scientist to the
Agricultural Research Institute for 9 months.
Her current address is Institute of Forage
Crops, 89 Gen. Vladimir Vazov str., 5800
Pleven, Bulgaria.
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