Variation of seed storage protein profiles within spring wheat varieties
by Mark Edward McLendon
A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in
Agronomy
Montana State University
© Copyright by Mark Edward McLendon (1991)
Abstract:
Bread-making capability of wheat (Triticum aestivum L.) is dependent on the seed storage proteins
which consist of gliadins and glutenins. We have analyzed 25 widely grown or historically significant
hexaploid spring wheat cultivars for intravarietal differences of gliadin and glutenin proteins. The
objective of this study was to improve the quality of existing spring wheat cultivars by selecting for
seed storage proteins and to test the effect of particular proteins on bread-making capability of spring
wheat.
Twelve of the cultivars showed plant-to-plant variation in either their gliadin or glutenin makeup as
revealed by SDS-PAGE. In 1990, a randomized split plot experiment with three replications was
planted to determine if the protein variants were due to contamination or a biotype that naturally occurs
within a cultivar, and to obtain enough seed for quality testing. Based on agronomic data, visual
comparisons and gel profile ratios, we determined that 5 cultivars out of the original 25 contained
biotypes.
A quality analysis was then conducted to compare bread-making capability of the biotypes with the
original cultivar they were selected from. Very few significant differences were noted for quality
between the biotypes and the control cultivar except in Wampum, which differed in HMW glutenin
proteins. One of the biotypes, which contained a HMW glutenin subunit known to confer good quality,
showed increased mixing and peak time, both indicators of dough strength.
A possible explanation for the lack of quality differences among biotypes is that our experiment was
too small to detect any. We did not have enough seed to do the standard baking tests, which can cause
problems with proper mixing of dough. A larger field experiment has been set up for 1991 which will
give us a better indication of quality for the biotypes. t
VARIATION OF SEED STORAGE PROTEIN PROFILES
WITHIN SPRING WHEAT VARIETIES
by
Mark Edward McLendon
A thesis submitted in partial fulfillment
of the requirements for the degree
of
Master of Science
in
'\
Agronomy
(!
MONTANA STATE UNIVERSITY
Bozeman, Montana
September, 1991
ii
APPROVAL
of a thesis submitted by
Mark Edward McLendon
This thesis has been read by each member of the thesis
committee and has been found to be satisfactory regarding
content, English usage, format, citations, bibliographic
style, and consistency, and is ready for submission to the
College of Graduate Studies.
Date
Chairperson, Graduate Committee
Approved foi
Approved for the College of Graduate Studies
r\
IT
Date
/
GraduatevDean
iii
STATEMENT OF PERMISSION TO USE
In presenting this thesis in partial fulfillment of the
requirements for a master's degree at Montana state
University, I agree that the Library shall make it available
to borrowers under rules of the Library.
Brief quotations
from this thesis are allowable without special permission,
provided that accurate acknowledgement of source is made.
Permission for extensive quotation from or reproduction
of this thesis may be granted by my major professor, or in
his absence, by the Dean of Libraries when, in the opinion
of either, the proposed use of the material is for scholarly
purposes.
Any copying or use of the material in this thesis
for financial gain shall not be allowed without my written
permission.
Signature
Date
Z
iv
ACKNOWLEDGMENTS
I wish to express my
sincere gratitude to the
following people:
/
To the members of my committee: Dr. Luther Talbert, Dr.
Charles McGuire, and Dr. Jack Martin for their advice and
time.
Special thanks to Dr. Talbert for being patient and for
challenging me to better myself.
I am indebted for this.
Susan banning for her excellent assistance in the field
and with statistical analysis.
Glenn Magyar for laboratory advice and help.
Special thanks to Robert Dunn for his friendship,
guidance, encouragement, and being a decent fishing partner.
Special thanks to Darcy Wright for being a special
friend, and for her understanding and patience.
Tim Majerus for being a great friend during my academic
career.
My father and mother, and the rest of my family, for
their support and believing in me.
V
TABLE OF CONTENTS
Page
LIST OF TABLES
.....................................
LIST OF FIGURES
. . ................
ABSTRACT
.........................................
INTRODUCTION
vi
Vli
viii
. . ........................................ I
REVIEW OF LITERATURE
.................................
4
Protein Composition
................................... 4
Genetics of the EndospermProteins . . . . . . . . . .
5
Allelic Variation
...............................
10
HMW Glutenin and Quality
.. . ....................
10
Gliadin and Quality
.............................
13
LMW Glutenin and Q u a l i t y .........................
14
Evidence of B i o t y p e s .............
16
MATERIALS AND METHODS
Cultivar Selection
Protein Extraction
...................
...............................
.................
Electrophoresis of Proteins
.....................
Selection of Biotypes
...........................
Experimental Design
. . . . .....................
Quality A n a l y s i s ............................. . .
Statistical Analysis ...............
RESULTS AND DISCUSSION
.............................
Identification of StorageProtein Biotypes .........
Quality Evaluation of Biotypes ...................
18
18
18
19
19
20
20
22
23
23
28
S U M M A R Y .............................................
39
LITERATURE CITED
41
APPENDIX
...................................
....................................... . .
47
vi
LIST OF TABLES
Table
1.
Page
Partial listing of allele designations used in
our study for the GLU-Al. GLU-Bl. and GLU-Dl.
loci as recommend by Payne and Lawrence (1983) . .
9
2.
Agronomic comparisons between storage protein
variants and the original cultivars................. 24
3.
Quality data for storage protein biotypes........... 29
4.
HMW subunits for Borah biotypes.................. 30
5.
HMW subunits for Fortuna biotypes................ 30
6.
HMW subunits for Sheridan biotypes..............
31
7.
HMW subunits for Thatcher biotypes..............
31
8.
HMW subunits for Wampum biotypes..........
32
9.
Solutions for seed storage protein analysis.
. .
48
10.
Analysis of variance for protein variants.' . . .
49
11.
Analysis of variance for quality data of
biotypes
49
vii
LIST OF FIGURES
Figure
I.
2.
3.
4.
Page
HMW glutenin variant subunits. This is a partial
list of subunits from Payne and Lawrence (1983)
showing only standard cultivars (Table I) that
we used to mark our variant subunits. To number
these subunits they were electrophoresed beside
the standard cultivars and lined up with the
corresponding subunit............................
8
Gel profiles of Borah (top) and Fortuna (bottom)
with selected biotypes pictured on the right,
labelled A and B and control variety (mixture
of both types) on the left................. ..
36
Gel profiles of Sheridan (top) and Thatcher
(bottom) with selected biotypes labelled either
A or B. The control variety, with both types, is
pictured on the left.................... .
37
Gel profiles of Wampum, with selected biotypes
pictured on the right, labelled A,B, and D
respectively. The control variety (with the
three types) is on the left. * Apparent
biotype that was missed in the original
selection of variants............................ 38
viii
ABSTRACT
Bread-making capability of wheat fTriticum aestivum L.)
is dependent on the seed storage proteins which consist of
gliadins and glutenins. We have analyzed 25 widely grown or
historically significant hexaploid spring wheat cultivars
for intravarietal differences of gliadin and glutenin
proteins. The objective of this study was to improve the
quality of existing spring wheat cultivars by selecting for
seed storage proteins and to test the effect of particular
proteins on bread-making capability of spring wheat.
Twelve of the cultivars showed plant-to-plant variation
in either their gliadin or glutenin makeup as revealed by
SDS-PAGE. In 1990, a randomized split plot experiment with
three replications was planted to determine if the protein
variants were due to contamination or a biotype that
naturally occurs within a cultivar, and to obtain enough
seed for quality testing. Based on agronomic data, visual
comparisons and gel profile ratios, we determined that 5
cultivars out of the original 25 contained biotypes.
A quality analysis was then conducted to compare
bread-making capability of the biotypes with the original
cultivar they were selected from. Very few significant
differences were noted for quality between the biotypes and
the control cultivar except in Wampum, which differed in HMW
glutenin proteins. One of the biotypes, which contained a
HMW glutenin subunit known to confer good quality, showed
increased mixing and peak time, both indicators of dough
strength.
A possible explanation for the lack of quality
differences among biotypes is that our experiment was too
small to detect any. We did not have enough seed to do the
standard baking tests, which can cause problems with proper
mixing of dough. A larger field experiment has been set up
for 1991 which will give us a better indication of quality
for the biotypes.
I
INTRODUCTION
Wheat provides man more protein than any other source.
A kernel of wheat contains 9% to 16% protein, most of which
is found in the endosperm in the form of storage proteins
classified as prolamines.
Prolamines play a major role in
the type of end-use product produced from a particular
cultivar of wheat.
The prolamines consist of two types,
gliadins and glutenins, that to a large extent determine the
quality of wheat.
They give bread dough made from flour the
characteristics of extensibility and elasticity.
The
balance between these two properties is genetically
determined and can vary greatly between cultivars.
Cultivars that produce a strongly elastic dough with some
extensibility are used for bread, while varieties producing
dough with high extensibility are used for cookies.
The gliadins and glutenins have been studied
extensively with the goal of manipulating the quality of
wheat.
A better understanding of how these proteins perform
I
in food products could provide plant breeders with a useful
tool to implement into a program.
Sodium Dodecyl Sulphate -
Polyacrylamide Gel Electrophoresis (SDS-PAGE) has been the
most widely used method to study these proteins.
This
technique separates proteins on the basis of molecular
weight into a series of subunits that can be visualized on a
gel.
Some evidence suggests that these subunits play a key
(
2
role in the functional properties of the protein.
Thus, a
question of interest regards whether quality can be improved
by selecting for specific subunits.
Spring wheat is an important economic crop in Montana,
grown on 1,134,000 ha and producing 3,786,156,500 kg in
1990, mainly in the northeastern and northcentral parts of
the state.
Superior end-use properties are an important
factor in the marketability of the crop.
Montana has a dry,
semi-arid climate that is conducive to producing a high
quality product.
In 1989, 90% of the spring wheat produced
in the state was shipped to the Pacific Northwest and then
exported to Pacific Rim countries.
Montana grown wheat is
preferred because of its high quality.
We have found, using SDS-PAGE, that several spring
wheat varieties are composed of 2-3 apparent biotypes based
on seed storage proteins.
These protein variants could have
two explanations, either an actual biotype inherent within
the variety, or a contamination from some unknown source.
This provided an opportunity to test the variants, determine
whether they were contaminant or biotypes, and then to
establish pure lines for any actual biotypes.
These
biotypes should be genetically similar for all traits except
seed storage proteins.
The purpose of this research was to
determine if the biotypes had different end-use properties
compared to that of the original cultivar.
This allowed us
to test the value of specific subunits, and may also allow
3
derivation of superior wheat cultivar by establishing purelines of the storage protein biotypes.
4
REVIEW OF LITERATURE
Protein Composition
Wheat proteins are divided into four classes: albumins,
globulins, gliadins, and glutenins (Osborne 1907).
The
albumins and globulins are mainly enzymes, rich in essential
amino acids, but do not affect bread-baking properties.
Hoseney et al. (1969) found that addition of these proteins
to wheat flours showed no affect on the baking
characteristics of the flours.
Two types of storage proteins are normally recognized
in the endosperm, gliadin and glutenin, which together make
up gluten.
Gluten gives wheat dough its unique cohesive and
elastic properties which allow it to be used for baking
bread (Wall 1979).
These proteins are synthesized on the
endoplasmic reticulum and then deposited in protein bodies
and provide a source of nitrogen for germinating seeds
(Shewry and Milfin 1985).
Originally, they were defined on
the basis of solubility in alcohol, but now, are normally
classified by their state of aggregation in dissociating
media.
Glutenins are considered to be aggregated storage
proteins while gliadins are non-aggregated proteins
occurring in free polypeptide chains (Jackson et al., 1983).
Protein quality differences between cultivars is largely a
function of different combinations of seed storage proteins
(Payne et al., 1984).
5
Gliadin proteins are small molecules, approximately
35,000 molecular weight, and consist of a complex mixture of
polypeptides.
Fractionations of these polypeptides
according to charge by SDS-PAGE causes separation into four
groups: a, /
8 , 7 , w (Wall 1979) .
A hexaploid wheat genotype
will consist of about 25 major gliadin polypeptides and as
many minor components (Wrigley and Shepherd 1973).
Glutenins are large molecules made from different
subunits that are linked by disulfide bonds (Wall 1979).
When exposed to a reducing agent, such as 2-mercaptoethanol,
the disulfide bonds break and glutenin molecules dissociate
into several subunits that are classified into two groups,
high molecular weight (HMW) and low molecular weight (LMW)
subunits (Payne and Corfield 1979).
Storage proteins of
wheat flour typically consist of 50% gliadin, 10% HMW
glutenin subunits, and 40% LMW glutenin subunits.
Endosperm proteins are also useful for varietal
identification.
Jones et al. (1982) studied gliadin
profiles obtained by SDS-PAGE of 88 wheat cultivars grown in
the U.S. and found that 76 out of 88 showed unique profiles.
Cultivars that had identical profiles were found to have
very similar genetic backgrounds.
Genetics of the Endosperm Proteins
Wheat (Triticum aestivum L.) is a hexaploid species
that contains three genomes of seven pairs of chromosomes
6
each.
The genomes are designated A, B, and D .
Each one of
the 21 chromosome pairs is given a number, I to 7 , followed
by the appropriate genome designation (Payne 1987).
Use of
chromosome mutants has been exploited by cytogeneticists in
genetic studies of wheat.
Nullisomi1C and tetrasomic lines
of Chinese Spring have been particularly helpful in studying
storage proteins (Sears 1966).
Genes that control high
molecular weight (HMW) glutenin subunits are located on the
long arm of chromosomes IA, IB, and ID (Payne 1987).
Loci
that contain these glutenin genes are together called Glu-I .
while individually labelled as Glu-Al. Glu-Bl. and Glu-Dl.
Each genome in hexaploid wheat contains two HMW subunits,
broken into x and y types based on higher or lower molecular
weight, respectively.
A cultivar will have six glutenin
genes but will only synthesize three to five subunits,
presumably because of gene inactivations.
Gene locus Glu-Al
contributes one subunit whereas the other two loci normally,
but not always, contribute a pair of subunits (Payne et al.,
1981a; Payne et al., 1982; Anderson and Greene 1989).
Payne and Lawrence (1983) devised a standard labelling
scheme for the HMW glutenin subunits using Chinese Spring
and 15 other cultivars as standards (Fig. I, Table I).
These standards consist of all subunits known to exist,
except for several new ones found in landraces.
Subunits
from other cultivars can be compared to these standards and
labelled with the appropriate locus followed by a small
7
letter.
For example, cultivars that contain the 2 + 1 2
subunits from the D locus are defined as being Glu-Dla.
while cultivars containing the 5 + 1 0 subunits are labelled ■
as containing Glu-Dld (Table I).
Gliadin proteins are synthesized by genes located on
chromosomes of homological groups one and six.
Genes that
control w gliadins, and 7 gliadins occur on the short arms
of chromosome IA, IB, ID, and are collectively known as Gli1.
Genes for a and j8 gliadins are on the short arm of
chromosomes GA, GE, GD, and are collectively known as Gli-2
(Payne et al., 1982; Payne 1987).
Evidence suggests that
gliadins are inherited in groups or specific blocks of
components.
Little recombination has been observed between
the allelic variants of the blocks, suggesting the existence
of clusters that are closely linked (Baker and Bushuk 1978;
Sozinov and Poperelya 1980;
Payne et al., 1984).
Metakovsky et al., 1985, 1986;
A few gliadin coding genes exist that
are removed from the main cluster.
These genes have been
identified in a number of cultivars, including Chinese
Spring (Branlard 1983; Galili and Feldman 1984; Metakovsky
et al.,1986).
Low molecular weight (LMW) subunits of glutenin are
encoded by genes on chromosomes IA, IB, and ID (Payne 1987).
Genes for LMW subunits are designated Glu-3. and have been
shown to be very closely linked with the Gli-I locus (Singh
and Shepherd 1988).
Strong associations between the two
8
ID
IB
IB
ID
— 2
— 7
——8
— 12
— 2*
Gene Locus Glu-Al
a
b
c
ID — 2
IB — 7
IB — 8
ID — 12
——6
— 7
a
ID — 2
“"”2
— 7
— 8
b
— 7
_
— 9
e
--8
d
— 4
--3
Gene Locus Glu-Bl
--5
IB — 7
IB — 8
ID — 12
Gene Locus Glu-Dl
— 12 — 12 — 12 " 10
a
tr
Chinese Spring
HMW subunits
(Standard)
b
e
d
tr
HMW glutenin standard subunits based
on Payne (1983).
Figure I. - HMW glutenin variant subunits. This is a
partial list of subunits from Payne and Lawrence (1983)
showing only standard cultivars (Table I) that we used to
mark our variant subunits. To number these subunits, they
were electrophoresed beside the standard cultivars and lined
up with the corresponding subunit.
9
Table I.
Locus
GlU-Al
Partial listing of allele designations used in our
study for the Glu-Al. Glu-Bl. and Glu-Dl loci as
recommended by Payne and Lawrence (1983).
Allele
Designation
Number
Designation
a
b
I
2*
Hope
Bezostaya-I
Chinese Spring
C
GlU-Bl
a
b
C
d
Glu-Dl
a
b
C
d
Variety
Standard
7
7 + 8
7 + 9
6 + 8
Flinor
Chinese Spring
Bezostaya-I
Hope
+
+
+
+
Chinese Spring
Hobbit
Champlein
Hope
2
3
4
5
12
12
12
10
HMW glutenin subunits are designated first by the locus
which they appear in, followed by the allele designation.
A cultivar will contain one variant type from each of the
loci. For example, Hope contains the following subunits:
I = Glu-Ala. 6 + 8 = Glu-Bld. 5 + 10 = Glu-Dld. Allele
Glu-Alc produces a non-detectable subunit and is considered
to be null.
10
have been noted (Payne et al., 1984; Autran and Galterio
1989).
LMW glutenins have been difficult to study because,
upon extraction in an SDS buffer and subsequent reduction,
they have a similar mobility to gliadins using SDS-PAGE
(Payne et al., 1984).
This has been overcome by a two-step,
one dimensional method that allows LMW subunits to be
visualized free from other proteins (Singh and Shepherd
1985, 1988).
Allelic Variation
Although only nine major gene loci code for seed
storage proteins, considerable allelic variation still
exists between these proteins (Payne 1987).
When 300 wheat
cultivars were analyzed for HMW glutenin variation, 3
alleles for Glu-Al were identified, 11 alleles for Glu-Bl.
and 5 alleles for Glu-Dl (Payne and Lawrence 1983).
Abundant allelic variation also exists for gliadins
(Metakovsky et al., 1984).
Thus, the total number of
,possible combinations is large, approaching 500 million.
This allelic variation is the basis for studying quality
differences between wheat cultivars.
HMW Glutenin and Quality
Payne et al. (1979, 1981a) were the first to report an
association between HMW subunits and flour quality,
particularly glutenin subunits 5 + 1 0 and 2 + 1 2 ,
which are
11
associated with resistance to dough extension and dough
weakness, respectively.
These results have been
substantiated by several other studies using different
methods and genetic materials (Payne et al., 1984, 1987b;
Branlard and Dardevet 1985; Lawrence et al., 1987, 1988;
Mansur et al., 1990).
Branlard and Dafdevet (1985) found that HMW subunits
2*, 5 + 10, and 7 + 9
were positively correlated with gluten
strength and tenacity, while subunits I, 1 3 + 1 6 ,
17+18
were positively correlated with dough extensibility.
Lawrence et al. (1987) examined biotypes derived from within
a variety and found the greatest contrasts were found
between pairs of biotypes differing at the Glu-Dl locus for
5 + 1 0 and 2 + 1 2 .
Lawrence et al. (1988) also studied
lines that were deficient in glutenin subunits expressed by
the three loci and found that the loss of 5 + 10 from Glu-Dl
and 17 + 18 from Glu-Bl had greater effects than the loss of
subunit I at Glu-Al locus.
Overall, most studies support the idea that the 2*
subunit at locus Glu-Al is superior to subunit I, and at
locus Glu-Dl. 5 + 1 0 subunits are superior to 2 + 12 for
wheat flour quality.
However, the Glu-Bl locus has shown no
clear correlations, with some work showing positive results
and others showing none (Anderson and Greene 1989).
et al. (1984) concluded bands 7 + 8 ,
13+16,
were equivalent in quality contributions.
Payne
and 17 + 18
Branlard and
12
Dardevet (1985) found positive correlations for dough
quality in alleles 7 + 9 ,
1 3 + 1 6 , and 17 + 18.
al. (1987) rated the 7 + 8
quality and 7 + 9
Grama et
allele as having the highest
with the lowest.
Dougherty et al. (1990)
used two dimensional electrophoresis to study baking quality
factors.
They found that two protein spots, considered to
be HMW glutenins coded by chromosome IB, had negative
correlations for mixograph mixing time.
Protein spots
thought tq be associated with chromosome ID showed no
significant relationships with loaf volume or mixing time.
Flavell et al. (1989) studied the amino acid sequence
of subunits 10 and 12 and found relatively few differences
between them.
The nonrepetitive regions at the N and C
termini were identical, suggesting that differences in dough
property between the two are from an array of 6 to 9 residue,
repeats found in the central segments of the proteins.
A
possibility they suggested was that subunit 10 had more of
the amino acid repeat units, leading to longer regions of
predicted /3 turns' which could give better elastic dough
properties.
Cysteine residues, which form the intermolecular
disulfide linkages, are thought to be important in dough
structure (Flavell et al., 1989).
Anderson et al. (1989)
found that subunit 5 had an additional cysteine residue when
compared to subunit 2.
Subunit 2*, which is associated with
good quality, also lacked the cysteine residue.
Another
13
difference was that a Glu-Al subunit had an increased charge
density while one Glu-Bl subunit had a different repeat
motif.
No conclusive evidence has been found to show that
these factors directly affect quality, but they may be areas
worthy of further study.
Gliadin and Quality
The presence of certain 7 -gliadins have been shown to
be correlated to flour quality (Damidaux et al., 1978;
Kosmolak et al., 1980; du Cros et al., 1983).
Durum wheat,
which lacks the D chromosome and contains no Glu-Dl alleles,
has been studied extensively.
Most of the allelic variation
of subunits is determined by the Gli-Bl locus which codes
for the 7 -gliadin (Autran and Galterio 1989).
Gliadin
subunit 7-45 (nomenclature of Bushuk and Zillman, 1978) is
associated with increased elasticity and dough strength,
while gliadin subunit 7-42 is associated with poor
viscoelastic properties and dough weakness (Kosmolak et al.,
1980; du Cross et al., 1982).
Branlard and Dardevet (1985) tested seventy wheat
cultivars with different genetic backgrounds and quality
characteristics using several quality tests.
They found
eighteen bands correlated with at least one aspect of
quality.
Ten correlations were positive and 11 correlations
were negative.
14
Campbell et al. (1987) noted that gliadin bands 58, 59,
60, and 67, presumably coded by the group six chromosomes
(Gli-2), were strongly associated with dough resistance.
These bands have received little attention, probably due to
poor resolution by most electrophoretic procedures.
LMW Glutenin and Quality
With the advent of a two-step, 1-dimensional SDS-PAGE
procedure (Singh and Shepherd 1985, 1988), it was revealed
that LMW glutenin had extensive variation in both bread
wheats and durum wheats (Gupta and Shepherd 1987, 1988).
In
the first step of the technique proteins are extracted in
hot ethanol and then electorphoresed at a high voltage
without exposing the proteins to a reducing agent.
This
allows gliadins to migrate down the gel and leave HMW and
LMW glutenins at the top.
The top first cm, containing two
types of glutenins is cut off, reduced, and applied on top
of a second polyacrylamide gel and electrophoresed.
This
allows visualization of LMW glutenins free from the presence
of the gliadin proteins.
Since the evidence suggests that LMW glutenin are
closely linked to the Gli-I locus (Singh and Shepherd 1988),
which encodes gliadin bands 45 and 42 in durum wheat, these
glutenins could be the cause of quality differences noted
(Autran et al., 1987; Gupta et al., 1989; Pogna et al.,
1988).
The first evidence of this was given by Moonen et
15
al., (1982), who used the SDS-sedimentation test to screen
wheat varieties and found the Gli-I locus positivelycorrelated with bread-baking.
Sedimentation tests measure
quality as a function of glutenin not gliadin, thus
suggesting LMW subunits were involved.
More recent studies
have shown that allelic variation of LMW subunits are
significantly correlated to flour quality in both hexaploid
and durum wheats (Payne et al. , 1984; Autran et al 1-987;
Gupta et al., 1989).
Pogna et al. (1988) studied a durum
wheat variety that contained only gliadin band 42 and LMW
subunits and found that this line had quality equal to high
quality varieties containing gliadin band 45 and LMW
subunits.
This showed that the LMW subunits were likely
responsible for the increased quality in the durum variety
instead of gliadin band 45.
Gupta et al. (1989) showed that
a specific Glu-3 band (Glu-A3itO was positively correlated
with dough resistance and extensibility at two different
protein levels (7% and 14%) and had a larger affect on
quality than the Glu-Alb HMW allele.
Payne et al. (1987a)
found that a different LMW glutenin subunit, Glu-A3a had
positive effects on bread quality as measured by the
sedimentation test and extensometer.
Thus, evidence
suggests that LMW glutenins are more responsible for quality
differences than gliadins and need to be studied more
extensively.
16
Evidence of Biotvoes
A biotype is defined as a naturally occurring
variation, like storage proteins, found within a cultivar
that is genetically similar for all other traits.
Lawrence
et al. (1987) found a number of Australian wheat cultivars
that contained two or three storage protein biotypes.
This
gave them the opportunity to establish pure lines within
cultivars that were likely to. be genetically similar, except
for seed storage protein composition, and to compare their
qualities.
Of ten cultivars found to contain biotypes, all
showed a difference in HMW glutenin subunits but, except in
one case, were identical for gliadin makeup.
Their findings
showed that the main difference in quality occurred at the
Glu-Dl locus with the 5 + 10 subunit conferring greater
dough resistance than the 2 + 1 2
subunit.
Only small
differences were noted between biotypes at the Glu-Al and
Glu-Bl loci.
Gupta and Shepherd (1990) used the two step-one
dimensional SDS-PAGE technique to analyze LMW glutenin
subunit composition of 222 hexaploid wheats.
They found
that thirteen out of the 222 cultivars studied consisted of
two biotypes with regards to LMW subunits.
Five of the
biotypes were grown to maturity and were found to have
similar or identical plant morphological characteristics and
thus were determined to be actual biotypes and not
contamination due to seed mixture or outcrossing.
17
In conjunction with our spring wheat work, we examined
I
eighteen common North American winter wheat varieties using
single kernel analysis with SDS-PAGE for within cultivar
variation of seed storage proteins.
It was determined that
six cultivars consisted of two apparent biotypes each. . Data
is from gel profiles only, agronomic and visual inspections
have not been done.
Four cultivars differed in their HMW
glutenin subunits while two showed a difference in the
gliadin or LMW glutenin profile (McLendon and Kisha,
unpublished).
18
MATERIALS AND METHODS
Cultivar Selection
Twenty-five spring wheat cultivars were chosen on the
basis of being commonly grown or having historical
significance to Montana.
The cultivars used in the study
were Alex, Amidon, Borah, Butte, Coteau, Era, Fortuna,
Glenman, Lew, Len, Marquis, MT 8402 (Hi-Line), MT 8561,
Pondera, Newana, Norana, Olaf, Oslo, Protor, Rambo,
Sheridan, Thatcher, Tioga, Waldron, and Wampum.
Seed was
obtained from stocks maintained by the spring wheat breeding
program at Montana State University.
Protein Extraction
Twenty seeds were selected at random from each cultivar,
surface sterilized, cut in half, and the embryo portion
stored at 4 o c.
Seed storage proteins were extracted from
the brush end of the seed based on the method of Laemmli
(1970).
Individual endosperm portions were crushed with a
pestle, placed in an eppendorf tube, and suspended in 500 ul
of 70% ethanol.
Samples were vortexed, incubated at 55o C
for one h, spun for two min at IOK on a eppendorf ipicrocentrifuge, and 250 ul of supernatent was removed.
Supernatents were then placed at 4 o c for one h, dried in a
centrifuge under vacuum, and reduced in cracking buffer.
19
Recipes for all reagents are in the Appendix (Table 9).
Samples were then placed at 4 o C until loading time.
Electrophoresis of Proteins
Protein samples were fractionated using single kernel
analysis by SDS-PAGE, based on the method of Laemmli (1970),
and improved by Payne et al. (1981a).
Electrophoresis was
through acrylamide gels consisting of a 6% stacking gel and
a 12% resolving gel, each one centimeter thick.
After
boiling, 20//l from each sample was loaded into individual
gel slots and then electrophoresed for 16 h at 80 v.
Each
gel contained 20 individual seeds selected from a cultivar.
Gels were stained for two h in coomasie blue, destained
until protein bands were visible, and dried down on 3mm
Whatman paper under vacuum.
Selection of Biotvnes
Gels were visually examined for variation in the
gliadin or glutenin protein subunits within a cultivar.
If
variants were noted within a cultivar, then embryos were
matched to the correct gel profile.
Embryos were placed in
an eppendorf tube holder containing a wet kimwipe and
allowed to germinate.
Seedlings were transplanted
individually to pots in the greenhouse where two generations
were grown.
Seeds from like profiles within a cultivar were
20
bulked from each variant, giving enough seed for a field
experiment.
Experimental Design
Variants were planted at Bozeman in the spring of 1990.
The design was a randomized split plot experiment with three
replicates, with cultivars as main plots and variants as
subplots.
Subplots consisted of variants planted in plots
of one row, and a control consisting of the original
cultivar.
Seed was planted in 3.04 m rows, 2.63 g/m and
later trimmed to 2.43 m at harvest.
Data collected
throughout the growing season included heading date (days),
plant height (cm), stem solidness (5-25) with 5 being hollow
and 25 being solid, and yield (kg/ha).
Biotypes were also
visually compared to the control for morphological
characteristics.
Plots were harvested in August for baking
quality analysis.
Quality Analysis
Baking analysis was determined with the help of
personnel in the Cereal Quality Laboratory, Montana State
University, Bozeman, Montana.
Grain samples were first
tempered overnight and then milled with a Buhler Laboratory
mill (American Association of Cereal Chemists (AACC), 1983
method 26-10).
Grain protein was determined using Near
Infrared Reflectance (NIR) (Williams, 1979).
Approved
21
methods of the AACC were used for the straight-dough method
of baking quality analysis, modified to a 25g sample (AACC,
1983 method IO-IOb).
The following data were recorded for
each flour sample.
- Grain protein (%)
- Water absorption (%) - the amount of water estimated
from the mixograph curve.
- Peak Time (converted from cm - paper units) - the
time from the first addition of water to the doughs
maximum consistency.
- Peak height (converted from cm - paper units) distance in mixograph units when curve reaches
maximum height.
- Bake Absorption (ml) - The amount of water added to
prepare flour for baking.
- Loaf volume - measured using the rape seed
displacement
- Crumb score - determined on a scale from I to 10
(I = poor and 10 = excellent) by visual comparison
with standard loaves (baked from commercial flour
Mello Judith).
- Mixing time (minutes) - the time needed to bring the
dough to optimum consistency.
22
Statistical Analysis
Data were subjected to analysis of variance for each
measured variable.
The main interest was to compare
variants and control within each cultivar rather than
comparing cultivar means.
Variants and control within a
cultivar were compared using the Least Significant
Difference (LSD).
The subplot error mean square and degrees
of freedom was used in computing the LSD.
Analysis of
variance tables are listed in the appendix (Tables 10 and
11) .
23
RESULTS AND DISCUSSION
Identification of Storage Protein Biotvoes
Of the twenty-five cultivars that were analyzed with
SDS-PAGE, twelve showed within cultivar variation for
storage proteins.
These twelve cultivars were analyzed to
determine if protein variants were actually biotypes or an
unknown source of contamination.
A biotype is defined as a
naturally occurring variant within the cultivar for storage
proteins.
However, the possibility existed that variants
were a result of contamination that occurred from
outcrossing or a different seed source.
Each variant was
compared to the original cultivar for morphological
characters, both visually and agronomically, and protein
profiles from polyacrylamide gels were compared using SDSPAGE (Table 2).
If a profile occurred only once in 20 seeds
examined from a cultivar during electrophoresis, it was
suspected to be a contamination.
Profiles that occurred
multiple times were candidates to be actual biotypes.
The
following description of the twelve cultivars refers to
Table 2.
The cultivar Borah was determined to consist of two
storage protein biotypes.
Visual comparisons and agronomic
data measured in the field showed no differences between
biotypes and the control cultivar.
Biotype profiles
24
Table 2. Agronomic comparisons between storage protein
_________ variants and the original cultivar.
Biotypes
Heading
Date
days
Stem
Solid
5-25
Yield
kg/ha
1000
K e rnel
Weight g
# Seeds
/Biotype
Borah
A
B
C
186.0 A
186.0 A
185.7 A
10.3 A
10.7 A
11.3 A
2324 A
2291 A
2143 A
30.0 A
30.7 A
29.8 A
18/30
12/30
Butte
A
B
C
186.0 A
187.0 B
186.3 AB
7.2 A
18.9 B
7.8 A
2438 A
2002 A
2197 A
31.3 A
31.4 A
30.5 A
19/20
1/20
Fortune
A
B
C
D
188.0
187.7
187.7
188.8
20.4
22.3
17.5
15.7
1404 A
1552.A
1747 AB
2264 B
41 . 0
38.4
39.7
30.8
19/35
15/35
Lew
A
B
C
189.0 A
189.3 A
185.7 A
9.8 A
15.4 B
15.1 B
3232 B
2298 A
2264 A
33.4 A
33.2 A
35.1 A
1/20
19/20
MT8561
A
B
C
190.7 A
190.7 A
190.7 A
8.1 A
8.3 A
9.3 A
2345 A
2345 A
2177 A
28.6 A
27.8 A
28.6 A
1/20
19/20
Norana
A
B
189.0 A
188.0 A
9.5 A
9.8 A
2977 B
2271 A
31.2 A
30.4 A
4/20
16/20
Olaf
A
B
C
188.0 A
188.3 A
187.7 A
9.1 A
9.3 A
8.7 A
2499 A
2459 A
2177 A
36.2 A
35.3 A
34.9 A
4/20
16/20
Pondera
A
B
C
D
189.3
186.0
186.3
187.0
7.4
8.4
8.4
7.1
2600
2177
2278
2163
30.5
27.9
29.3
30.7
2/20
17/20
A
A
A
A
B
A
A
A
.
BC
C
AB
A
A
A
A
A
A
A
A
A
C
B
BC
A
B
A
AB
B
1/35
-
1/20
S h e ridan A
B
C
189.7 A
189.7 A
189.0 A
8.4 A
7.5 A
7.9 A
3003 B
2204 A
1854 A
32.0 A
31.6 A
30.9 A
22/32
10/32
Thatcher A
B
C
188.0 A
188.0 A
187.0 A
8.8 A
6.4 A
6*7 A
2305 A
2103 A
1968 A
26.6 A
25.6 A
25.7 A
22/33
11/33
Unknown
A
B
C
185.0 A
185.0 A
185.7 A
10.1 A
8.8 A
9.9 A
2506 A
2277 A
2378 A
31.4 A
31.6 A
32.2 A
8/20
12/20
Wampum
A
B
C
D
189.7
189.7
189.7
190.0
2855
2660
3184
2586
33.5
31.9
31.6
32.7
8/34
12/34
A
A
A
A
7.4
9.6
7.0
8.1
A
A
A
A
AB
AB
B
A
A
A
A
A
8/34
Means are compared within individual cells. Means followed
by the same letter are not significantly different at the
0.05 level by LSD. C = control variety. Stem solidness 5 = hollow and 25 = solid.
25
showed up on a polyacrylamide gel in a ratio of 12 to 8 for
biotypes A and B, respectively.
The cultivar Butte contained no biotypes.
We suspected
that Butte contained a contaminant from the gel profile
ratio of 19 to I for the two variants.
and agronomic data confirmed this.
Visual comparison
The B variant was
awnless compared to the normal Butte cultivar, which is
awned.
A large difference was noted for stem solidness
(18.9 - B, 7.8 - Control).
The Butte B variant was
determined to be due to contamination and the other variant,
Butte A, is the actual variety.
Fortuna had three variant profiles that showed up on a
gel in a ratio of 11 A, 8 B, to I for D (suspected
contaminant).
Visual inspection in the field showed that A
and B were morphologically similar to the control while D
had a lighter head color.
interpret.
The agronomic data was harder to
Variant B showed no significant differences for
the control, but A differed for stem solidness and D
differed for yield.
A difference also occurred between the
A and B variants for kernel weight.
Variant D was
determined to be a contamination based on the gel profile
ratios, the lighter head color, and yield difference.
To
further test that A and B were true biotypes we obtained
certified Fortune seed from the Montana seed certification
program.
This seed showed the same two variants close to
the same ratio observed previously.
Based on this
26
information, Fortuna was determined to consist of two
biotypes.
The cultivar Lew was determined to contain no biotypes.
The ratio for the gel profile was 19 A to I for B .
When
grown in the field, variant B had a large club head compared
to a slender, smaller head for the control.
Large
differences also occurred in stem solidness and yield.
Variant B looks like a type of white wheat and is a
contaminant in the cultivar Lew.
The results from MT8561 are difficult to interpret.
The gel profiles showed a ratio of I A to 19 B, suggesting a
contaminant, but visual inspection and agronomic data showed
no differences when compared to the control.
The original
selections were made from a gel with poor resolution.
A
different gel, using seed from three generations later,
showed that A, B and the control cultivar had identical
profiles, suggesting the original selection was a mistake.
Either MT8561 has two biotypes, one in a very low ratio, or
it contains no biotypes.
Data suggests the latter to be
true.
The cultivar Norana contained no biotypes. Even though
the original selections showed up in a ratio of 3 A to 17 B ,
a polyacrylamide gel using material three generations later
failed to show variant B .
Variant B also showed a large
difference in yield compared to A.
27
The cultivar Olaf contains no biotypes or contaminants.
No differences were noted with the visual inspection or
agronomic data between the two selections.
The original gel
profiles showed a ratio of 3 A to 17 B , but the gel was
poorly resolved.
A gel done three generations later showed
no variation between variant A, B, or the control.
The
selection which was made from a poorly resolved gel, and
thought to be a variant, was actually the same as the other
profiles.
Pondera contained no biotypes.
The gel profile showed
a ratio of 2 A, 17 B , and I D, suggesting two contaminants .
present at low ratios.
Significant differences were also
noted for A and D for several agronomic characteristics.
A
gel consisting of a different seed source, certified
Pondera, showed only the B profile.
Variants A and D were
contaminants while B was the actual original variety.
Sheridan contained two biotypes.
The gel profiles had
a ratio of 13 for the A variant and 7 for B .
No differences
were observed either for visual inspection or agronomic
data, except that variant A out-yielded the control by 1142
kg/ha.
This could be due to a genetic factor that links
yield to the protein variation.
It will be interesting to
see if this observation holds true next year with a larger
field experiment.
The cultivar Wampum was determined to have three
biotypes with a possible fourth.
The gel ratios were, 5 for
28
the A variant, 8 for B, and 5 for the D variant.
A fourth
variant, which was present twice in the original gel but was
missed, showed up in approximately 27% (10/40) of the
profiles in a gel of the control Wampum done three
generations later.
An unknown cultivar was determined to have two
biotypes.
The cultivar was originally thought to be Len,
but when it was electrophoresed next to certified Len on a
gel, its profile was different.
The cultivar was
mislabelled and its true identity is unknown.
ratios were, 8 for the A variant and 12 for B.
Gel profile
No
differences were noted for visual inspection or in agronomic
data.
In summary, 12 of the 25 cultivars we studied
contained variants.
Of these 12 cultivars, 5 were
determined to consist of biotypes.
Quality Evaluation of Biotvoes
The following observations are based on data from the
quality analysis done on biotypes (Table 3).
The cultivar Borah contains two biotypes (Figure 2),
which consist of the gluten subunits shown in Table 4.
Biotypes had the same HMW glutenin subunits but varied in
their gliadin or LMW glutenin makeup.
Neither biotype
showed significant differences for any quality factors
measured.
Table
3.
Quality data
Biotype
for storage p r o t e i n biotypes.
Protein
%
Water
Absorption
%
Peak
Height
cm
Peak
Time
cm
Bake
Absorption
ml
Mix
Time
min.
Loaf
Volume
Crumb
Score
CC
Borah
A
B
C
14.0 A
14.1 A
14.4 A
70.2 A
69.9 A
69.4 A
7.2 A
7.2 A
7.0 A
3.2 A
3.4 A
3.3 A
73.5 A
74.0 A
73.3 A
5.0 A
4.8 A
4.9 A
260.0 A
265.0 A
253.0 A
4.3 A
3.7 A
3.7 A
Fortune
A
B
C
D
17.1
17.3
16.2
15.3
75.4
75.4
73.6
73.6
8.9
8.9
8.3
8.3
2.9
2.8
3.1
2.9
78.3
78.4
78.1
78.3
5.1
5.0
4.3
4.7
270.0
263.3
261.7
271.7
3.7
3.0
3.3
3.0
C
C
B
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
Sheridan A
B
C
16.0 A
15.9 A
16.0 A
69.3 A
70.3 A
70.7 A
7.0 A
7.3 A
7.4 A
2.9 A
2.9 A
3.2 A
75.8 A
76.3 A
76.1 A
5.4 A
5.2 A
5.2 A
246.7 A
263.3 A
253.3 A
3.3 A
3.7 A
4.3 A
Thatcher A
B
C
15.2 A
15.1 A
15.2 A
69.5 A
69.3 A
69.4 A
7.0 A
6.9 A
7.0 A
3.5 A
3.2 A
3.3 A
70.7 A
70.9 A
70.6 A
5.5 A
5.0 A
5.0 A
270.0 A
261.7 A
273.3 A
4.3 A
4.3 A
4.0 A
Wampum A
B
C
D
13.2
13.0
12.6
13.8
69.6
69.5
70.5
69.2
7.1
7.0
7.5
6.9
3.3
3.4
3.6
4.5
70.3
69.2
70.9
69.8
5.7
5.6
5.7
6.7
275.0
271.7
265.0
255.0
4.3
4.7
4.0
4.0
AB
A
A
B
A
A
A
A
A
A
A
A
A
A
A
B
A
A
A
A
A
A
A
B
A
A
A
A
Means are compared within individual cells only. Means followed by the same letter are not
significantly different at the 0.05 level by LSD.
A
A
A
A
30
Table
4.
HMW subunits
for B orah biotypes.
Variety
HMW Subunits I/
Borah A
Glu-Alb (2*)
Glu-Blc ( 7 + 9 )
Glu-Dld (5 + 10)
Gliadin/LMW Variation
Yes
Borah B
Same as above
Yes
I / Based on method of Payne and Lawrence (1983).
Fortuna contains two biotypes (Figure 2), which contain
glutenin subunits shown in Table 5.
The biotypes had the
same HMW glutenin subunit composition, but differed in
either gliadin or LMW glutenin makeup.
Table 5.
HMW subunits for Fortuna biotypes
Variety
Fortuna
HMW Subunits I/
A
Glu-Alb (2*)
Glu-Bla ( 7 + 8 )
Glu-Dla (5 +10)
Gliadin/LMW Variation
Yes
Fortuna B
Same as above
Yes
I/ Based on met hod of Payne and Lawrence (1983).
No differences were noted between the two biotypes for
any quality factors except that both A and B showed a higher
protein concentration than the control cultivar (Table 3).
This difference corresponds with the yield data, where both
types had a lower yield (201-268 kg/ha), though it was not
significantly different from the control (P < 0.05).
, Sheridan contains two biotypes (Figure 3), which show
variation in HMW glutenin profile, but not the gliadin/LMW
glutenin profile (Table 6).
No significant differences were
noted for any quality factors measured (Table 3).
Biotype A
31
also had the same level of protein as the control, though it
had a much higher yield (1142 kg/ha).
Table 6.
HMW subunits for Sheridan biotypes.
Variety
HMW Subunits I/
Sheridan A
Glu-Bld ( 6 + 8 )
Glu-Dlc (5 + 10)
Gliadin/LMW Variation
No
Sheridan B
I/
Glu-Blb ( 7 + 8 )
No
Glu-Dlc (5 + 10)
Based on met hod of Payne and Lawrence (1983).
Thatcher contained two biotypes (Figure 3), which show
no difference for the HMW glutenin profile but do vary in
the gliadin/LMW glutenin profile (Table 7).
No significant
differences were noted for quality between the biotypes or
the control for any factors measured (Table 3).
Table 7.
HMW glutenin subunits for Thatcher.
Variety
HMW Subunits I/
Thatcher A
Glu-Alb (2*)
Glu-Blc ( 7 + 9 )
Glu-Dld (5 + 10)
Gliadin/LMW Variation
Yes
Thatcher B
Same as above
Yes
I / Based on method of Payne and Lawrence(1983).
Wampum contained three biotypes with a possible fourth
that was not selected for (Figure 4).
Three biotypes
differed in their HMW glutenin profiles but not for the
gliadin/LMW glutenin profile (Table 8).
32
Table
8.
HMW glutenin subunits
for Wampum biotypes.
Variety
HMW Subunits I/
Wampum A
Glu-Blc (7 + 9)
Glu-Dla (2 + 12)
No
Wampum B
Glu-Ala (I)
Glu-Blc ( 7 + 9 )
Glu-Dla (2 + 12)
No
Gliadin/LMW Variation
Wampum D
T7
Glu-Ala (I)
No
Glu-Blc ( 7 + 9 )
Glu-Dla (5 + 10)
Based on method of Payne and Lawrence (1983).
Wampum D was the only biotype to show differences for
quality factors analyzed.
Biotype D had an increased peak
time and mix time when compared to the control and other two
biotypes.
This indicates that D has superior dough
strength.
The D biotype contains high quality 5 + 10
subunits while the other two biotypes have poorer quality 2
+ 12 subunits (Payne et al., 1979, 1981a, 1987b; Branlard
and Dardevet 1985; Lawrence et al., 1987, 1988; Mansur et
al., 1990).
studies.
This finding is consistent with previous
Biotype D also had a significantly higher protein
level than the control.
This has not been reported before
in other studies involving biotypes.
Of the three cultivars (Borah, Fortuna, and Thatcher)
which showed variation in either their gliadin or LMW
glutenin proteins but not in HMW glutenins, none showed
significant differences for quality factors examined.
The
two step-one dimensional electrophoresis has not been done
yet to determine whether these three biotypes differ in
33
their gliadin or LMW glutenin makeup.
Several possible
explanations for this result are, first, the variation in
profiles for these biotypes is due to gliadins, which may
have no effect on quality.
This has been suggested in
several other studies in which data have shown that LMW
glutenins affected quality, though gliadins did not (Moonen
et al., 1982;
Pogna et al., 1987).
A strong possibility
however exists that at least one of the biotypes differs in
its LMW glutenin makeup and that this difference had no
effect on quality.
Second, this experiment may have lacked
precision to detect differences among biotypes.
The
equipment in the cereal quality lab is set up to do 100 gram
samples but we had only 25 grams of flour.
Steps were taken
to minimize problems that might arise due to this
discrepancy, but the risk is higher of getting poor mixing,
resulting in dough that is not developed properly, which can
affect measurement of quality factors.
Of the two cultivars which varied for HMW glutenin
subunits but not for gliadins, one showed quality
differences and the other did not.
Wampum was discussed
earlier and did show a quality difference between biotypes.
The Sheridan biotypes varied at the Glu-Bl locus but showed
no differences.
explanations.
biotypes, 6 + 8
Again, there are several possible
First, the difference between the two
compared to 7 + 8 subunits, has no affect on
bread baking quality.
Other studies have found conflicting
34
results concerning the Glu-Bl locus.
rated subunits 7 + 8 ,
Payne et al. (1984)
1 3 + 1 6 , and 17 + 18 equivalent and
Branlard and Dardevet (1985) found positive correlations for
7+9,
13+16,
rated the 7 + 8
and 17 + 18.
However, Gramma et al. (1987)
allele highest for quality and 7 + 9
the
lowest while Dougherty (1990), using two dimensional
electrophoresis, found protein spots thought to be from the
IB locus that had negative effects.
A second possibility
for the lack of observed quality differences is that our
experiment may not have the precision necessary to detect
any.
One rather surprising finding from this experiment is
the lack of purity we found in the seed stocks maintained by
our spring wheat breeding program.
This shows how useful
SDS-PAGE can be for identifying sources of contamination in
a breeding program.
The Lew A variant, determined to be a
contaminant in this experiment, has become a problem in
several certified spring wheat fields in the northeastern
part of the state.
By being able to identify and then
visualize this contaminant in the field, the seed
certification program can now determine its source and take
steps to purify Lew.
Another example is Len, which was originally thought to
contain two biotypes.
However, when electrophoresis was
performed with our seed source evaluated next to certified
Len, the profiles did not match.
Our seed source, thought
35
to be Len for at least the last five years, is actually
something else.
We have been unable to identify the seed
source, but it is definitely not Len.
This suggests that it
would be worth while for plant breeders to check their seed
stocks for proper labelling.
By checking only 25 varieties
maintained by our breeding program with SDS-PAGE, two
serious mistakes were identified that have been now
corrected.
36
BORAH
BtOTYPE
A A A
FORTUNA
B B B
BIOTYPE
A A A A B B B
Figure 2. Gel profiles of Borah (top) and Fortuna (bottom)
with selected biotypes pictured on the right, labelled A and
B , and control variety (mixture of both types) on the left.
37
SHERIDAN
BIOTYPE
A A A B B B
Figure 3. Gel profiles of Sheridan (top) and Thatcher
(bottom). The Sheridan biotypes are on the right with the
control variety (mixture of both types) on the left. The
Thatcher picture is reversed with biotypes on the left.
38
Figure 4. Gel profiles of Wampum, with selected biotypes
pictured on the right, labelled A, B , and D respectively.
The control variety (with the three types) is on the left.
* Apparent biotype that was missed in the original selection
of variants.
39
SUMMARY
Our experiment allowed identification of spring wheat
cultivars that were heterogeneous for gluten proteins.
Analysis of biotypes showed no effect on quality when
compared to the original cultivar, except with biotypes from
Wampum.
A much larger experiment consisting of 4 replicates
with 4 rows has been set up for the storage protein
biotypes.
This should allow more precise quality analysis
and thus give a better indication of effects biotypes have
on quality.
We can then draw better conclusions on affects
of variation in HMW glutenins and gliadins/LMW glutenins
found in biotypes.
Our initial work however, has produced
several interesting findings that are worth further study.
If biotype D in Wampum produces the same results.found
in this study then it could be a superior cultivar to the
original variety.
It would have better quality
characteristics and a higher protein content.
However, our
data suggest that it may also yield less (up to 615 kg/ha)
than regular Wampum.
Does the increase in protein content
and quality offset the lower yield?
Similarly, while Fortuha biotypes A and B did not show
any quality effects, each had a significantly higher protein
level than the control without a significant reduction in
yield (335 kg/ha < for A, 201 kg/ha < for B).
If this trend
holds true, a selection for biotype B would produce a
40
cultivar with an increased protein level but otherwise very
similar to normal Fortuna.
The Sheridan A biotype will also be interesting to
watch.
Even if a larger experiment shows up no quality
differences for the Glu-Bl locus, the yield advantage for
biotype A may be worth selecting for.
Again, a cultivar
could be produced with a much higher yield than the original
cultivar, but with similar protein content and other
important agronomic characteristics.
While our data did not show storage protein biotypes as
having a great effect on quality, the possibility exists
they still do.
A larger experiment, producing more seed
should give more accurate quality data. The possibility also
exists that by evaluating variants found within cultivars, a
breeder may find a biotype that is superior to the original
variety for a desirable trait, like yield or protein
content, but is otherwise similar for all other traits.
\
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APPENDIX
48
Table 9. Solutions for seed storage protein analysis
Resolvina Gel
32.0 ml resolving gel stock
25.0 ml resolving gel buffer
22.0 ml distilled water
8.0 ml 10% SDS
H O ul 10% ammonium persulfate
Resolvina Gel Stock
33.3 g acrylamide
.268 g bisacrylamide
To 100 ml with water
Store at 4*
Stackina Gel
4.0 ml stacking gel stock
5.0 ml stacking gel buffer
10.6 ml distilled water
.2 ml 10% SDS
180 ul APS
Stackina Gel Stock
30.0 g acrylamide
.8 g bisacrylamide
To 100ml with water
Store at 4*
Resolvina Gel Buffer
18.1 g Tris
To 100 ml with water
pH to 8.8 with cone. HCL
Stackina Gel Buffer
6.0 g Tris
To 100 ml with water
pH to 6.8 with cone. HCL
Crackina Buffer
2.0 ml 100% glycerol
2.5 ml stacking buffer
2.0 ml 10% SDS
1.3 ml sterile water
.5 ml 2% bpb* & XC* dye
.2 ml beta-mercaptoethanol
Tank
28.8
6.0
2.0
To 2
Stain
200 ml methanol
740 ml distilled water
60 ml acetic acid
Destain
1000 ml methanol
2680 ml distilled water
320 ml acetic acid
*
*
bpb - Bromo Phenol Blue
XC - Xylene Cyanole
'
Buffer
g glycine
g Tris
g SDS
liters with d-H20
Table
10.
Source
Block
Analysis
d.f.
2
of v a r i a n c e
Heading
date
1.56*1/
for
ag r o n o m i c traits.
Plant
Stem
Yield
Height____ Solid_______ ._______
8.85*
Kernel
Protein
Weight__________
5.3
7384.2*
1.45
C).30
120.7*
11570.1*
95.71*
8.15*
Cultivar
11
24.80*
70.51*
Block x
Cultivar
22
1.15*
3.23*
3.2
2210.5*
2.21
().30
Variant x
Cultivar
27
1.21*
1.44
18.4*
4421.1*
8.51*
(5.16*
2.1
1033.4
1.76
1.28
0.38
54
Error
I/ Mean squares for individual traits.
*
Significant at 0.05 level.
Table 11.
Source
Analysis of variance for quality data including biotypes only.
Mix
Loaf
Bake
Peak
Peak
Water
Time
Volume
Absorp.
Time
Height
Absorp.
d.f.
334.2
1.9*
33.9*
0.51*
290.4
3.7*
0.26
0.5
0.35
146.7
0.3
0.15
0.8
0.17
167.2
0.4
52.2*
5.30*
1.05*
135.0*
20.3*
2.08*
0.54*
1.4
0.16
4
Block x
Cultivar
8
12
2.43*
8.2*
Cultivar
Variant x
- Cultivar
2.5*
1.00*
106.5*1/
0.17
1.6
24
Error
I/ Mean squares for individual traits.
*
Significant at the 0.05 level.
Crumb
Score
2789.7*
10.80*
"2
Block
K 5.09
0.43
,RSjcBV
E eS d
UTICA/OMAHA
NE.