Note: Descriptions are shown in the official language in which they were submitted.
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DESCRIPTION
Title of Invention
METHOD FOR SELECTION OF BARLEY SPECIES BASED ON
PROTEIN Z7 CONTENT, AND FERMENTED MALT BEVERAGE
Technical Field
[0001] The present invention relates to a selection method for selection
of barley species based on protein Z7 content, and to a fermented malt
beverage.
Background Art
[0002] Protein Z7, together with protein Z4, protein Zx and others,
belongs to the serine protease inhibitor (serpin) subfamily of barley.
These proteins belonging to the serpin subfamily are believed to be
involved in the mechanism of biological defense when a barley species
has been infected with mold or the like. However, much regarding this
function is still poorly understood (Non-patent document 1).
[0003] A relationship between protein Z and beer foam stability has
also been suggested. Foam stability is an important quality for beer,
and therefore a large body of research results has been published on the
factors relating to foam stability. A variety of factors are involved in
determining the degree of foam stability, one of which is thought to be
the proteins themselves, and proteins such as protein Z (protein Z4,
protein Z7) and lipid transfer protein 1 (LTP1) have hitherto been
considered to be proteins involved in foam stability (Non-patent
documents 2 to 4).
[0004] Almost no research has been conducted to date on the
relationship between protein Z7 and beer foam stability, with only a
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very few reports thereof (Patent document 1 and Non-patent document
5).
[0005] Patent document 1 describes that a correlation exists between
protein Z7 concentration in barley seeds, malt, malt liquor, fermented
malt beverages or the pre-fermentation or fermenting raw liquor of
fermented malt beverages, and the NIBEM value, which is an indicator
of foam stability, and it describes a method for judging foam stability
quality based on protein Z7 concentration.
[0006] On the other hand, Non-patent document 5 describes, as a result
of examining the relationship between malt protein Z7 contents and
foam stability, that no significant correlation was found between protein
Z7 and foam stability, and therefore a different conclusion is drawn than
that of Patent document 1 mentioned above.
Citation List
Patent literature
[0007] [Patent document 1] Japanese Unexamined Patent Application
Publication No. 2008-249704
Non-Patent Literature
[0008] [Non-patent document 1] FEBS Letters, Vol. 394, p.165-168,
1996
[Non-patent document 2] J. Agric. Food. Chem., Vol. 56, p.1458-1464,
2008
[Non-patent document 3] J. Agric. Food. Chem., Vol. 56, p.8664-86'71,
2008
[Non-patent document 4] J. Am. Soc. Brew. Chem., Vol. 60, p.47-57,
2002
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[Non-patent document 5] J. Inst. Brew., Vol. 105, p.171-1'7'7, 1999
Summary of Invention
Problems to be Solved by the Invention
[0009] While protein Z7 has been implicated in industrially useful
functions, such as being involved in body defense mechanisms in barley
infection and in beer foam stability, it cannot be said to have been
sufficiently researched to date. In order to correctly analyze the
influence of protein Z7 content on barley traits, it is necessary to
establish a method of accurately discriminating barley with high protein
Z7 contents and/or barley with low protein Z7 contents.
[0010] The method of judging beer foam stability described in Patent
document 1 is based on measurement of protein Z7 concentration by
protein levels. While this allows temperature, weather and artificial
factors to be excluded in comparison to methods based on measurement
of NIBEM values, it is subject to variations in protein levels. When
applied to breeding of barley suitable for beer with excellent foam
stability, it is necessary to carry out screening of hundreds of barley
individuals especially nearer the initial stage of barley breeding, but the
method described in Patent document 1 is not suited for such treatment
of large number of specimens.
[0011] Advances are being made in molecular screening techniques for
breeding of a variety of crops, in addition to barley. Among such
molecular screening techniques, selection by DNA markers based on
DNA polymorphisms is a topic of much active research and
development because it allows selection at the initial generations of
breeding, the operability is excellent, and analysis is possible by using
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leaves. However, no DNA markers are known as indicators for barley
protein Z7 content.
[0012] It is an object of the present invention to provide a selection
method for selection of barley species that is easy to operate, that allows
processing of multiple specimens in a short period of time, that allows
accurate selection of barley species based on protein Z7 content, and
that is suited for application to barley breeding.
[0013] It is another object of the invention to provide a method for
producing a fermented malt beverage using as raw material a barley
species that has been selected based on protein Z7 content, and to the
fermented malt beverage.
Means for Solving the Problems
[0014] The invention provides a selection method for selection of
barley species based on protein Z7 content, which comprises:
identifying at least one genotype of each of polymorphic marker
A and polymorphic marker B in a barley species specimen, wherein the
polymorphic marker A is specified by multiple alignment of the
nucleotide sequence of a region around the Haruna-type barley protein
Z7 gene locus and the nucleotide sequence of a region around the
Kendall-type barley protein Z7 gene locus, wherein the polymorphic
marker B is specified by multiple alignment of the nucleotide sequence
of a region around the Haruna-type barley protein Z7 gene locus and the
nucleotide sequence of a region around the Barke-type barley protein Z7
gene locus, and
carrying out the following (i) and/or (ii):
(i) selecting a barley species specimen having the genotype identical to
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the Haruna-type genotype as a barley species with high protein Z7
content,
(ii) selecting a barley species specimen having the genotype identical to
the Kendall-type or Barke-type genotype as a barley species with low
protein Z7 content.
[0015] Herein, "Haruna-type" refers to a barley species wherein the
genotype of a selection polymorphic marker selected from the group
consisting of the polymorphic marker A and the polymorphic marker B
present in the region around the barley protein Z7 gene locus, matches
the genotype of Haruna Nijo barley. An example of a nucleotide
sequence of the region around the barley protein Z7 gene locus of
"Haruna-type" is the nucleotide sequence listed as SEQ ID NO: 1.
[0016] Similarly, herein, "Kendall-type" and "Barke-type" refer to
barley species wherein the genotype of the aforementioned selection
polymorphic marker in the region around the barley protein Z7 gene
locus matches the genotypes of CDC Kendall barley or Barke barley,
respectively. Examples of nucleotide sequences of the region around
the barley protein Z7 gene locus of "Kendall-type" and "Barke-type" are
the nucleotide sequences listed as SEQ ID NO: 2 and SEQ ID NO: 3.
[0017] By multiple alignment of the nucleotide sequence of the region
around "Haruna-type" barley protein Z7 gene locus and the nucleotide
sequence of the region around "Kendall-type" barley protein Z7 gene
locus, it is possible to identify nucleotide positions where the two
nucleotide sequences do not match, as a polymorphic marker A.
Similarly, by multiple alignment of the nucleotide sequence of the
region around "Haruna-type" barley protein Z7 gene locus and the
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nucleotide sequence of the region around "Barke-type" barley protein
Z7 gene locus, it is possible to identify nucleotide positions where the
two nucleotide sequences do not match, as a polymorphic marker B.
[0018] The genotypes of one or more of the polymorphic markers A are
identified and the genotypes of one or more of the polymorphic markers
B are identified, in a barley species specimen. By then selecting a
barley species specimen in which the identified genotypes match the
Haruna-type genotype, it is possible to select barley species with high
protein Z7 content. By selecting a barley species specimen in which
the identified genotypes match the Kendall-type or Barke-type
genotype, it is possible to select barley species with low protein Z7
content.
[0019] The selection method utilizes novel selection polymorphic
markers that correlate with barley protein Z7 content in barley tissue,
and therefore allows accurate selection of a barley species specimen
based on protein Z7 content. Furthermore, since molecular biological
methods can be employed to identify genotype, the selection method
has excellent operability and a large number of specimens can be
processed in a short period of time.
[0020] The invention also provides a selection method for selection of
barley species with low protein Z7 content, which comprises:
identifying at least one genotype of polymorphic marker A and
polymorphic marker B in a barley species specimen, wherein the
polymorphic marker A is specified by multiple alignment of the
nucleotide sequence of a region around the Haruna-type barley protein
Z7 gene locus and the nucleotide sequence of a region around the
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Kendall-type barley protein Z7 gene locus, wherein the polymorphic
marker B is specified by multiple alignment of the nucleotide sequence
of a region around the Haruna-type barley protein Z7 gene locus and the
nucleotide sequence of a region around the Barke-type barley protein Z7
gene locus, and
carrying out the following (iii) or (iv):
(iii) selecting a barley species specimen having the genotype of the
polymorphic marker A identical to the Kendall-type genotype as a
barley species with low protein Z7 content,
(iv) selecting a barley species specimen having the genotype of the
polymorphic marker B identical to the Barke-type genotype as a barley
species with low protein Z7 content.
[0021] At least one genotype of the polymorphic marker A and the
polymorphic marker B is identified in a barley species specimen. By
selecting a barley species specimen in which the identified polymorphic
marker A genotype matches the Kendall-type or selecting a barley
species specimen in which the identified polymorphic marker B
genotype matches the Barke-type genotype, it is possible to select a
barley species with low protein Z7 content.
[0022] Identification of the genotype is preferably performed with a
polynucleotide containing at least one selection polymorphic marker
selected from the group consisting of polymorphic marker A and
polymorphic marker B, amplified by PCR using genomic DNA of the
barley species specimen as template.
[0023] Because the polynucleotide duplicates the nucleotide sequence
of the genomic DNA, it is preferred for genotype identification.
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Furthermore, since only a small amount of plant tissue sample is
necessary for analysis, it is particularly suitable for selection at the
initial stage of breeding. In addition, since a large amount of the
nucleotide sequence that is to be analyzed is present in the sample,
compared to identification of genotype from actual genomic DNA that
comprises a large amount of DNA that is not to be analyzed, it is
possible to more easily identify the genotype. Furthermore, since the
genotype of a selection polymorphic marker can be identified in a short
period of time by a basic and easy procedure including a step of
extracting DNA from a barley species specimen, a step of amplifying a
polynucleotide containing at least one selection polymorphic marker by
PCR, and a step of identifying the nucleobase type of the selection
polymorphic marker, it is even more suited for processing of a large
number of specimens.
[0024] The polymorphic markers A may be the nucleotide positions
corresponding to the 62nd, 93-94th (representing a gap), 94th, 96th,
98th, 113th, 116th, 123rd, 148th, 151st, 153rd, 156th, 159th, 160-186th,
217th, 231st, 239th, 246-247th, 253rd, 305-306th, 378th or 422nd
nucleotides of the nucleotide sequence listed as SEQ ID NO: 1.
Similarly, the polymorphic markers B may be the nucleotide positions
corresponding to the 260th, 262nd, 305-306th, 343rd, 378th, 386th or
422nd nucleotides of the nucleotide sequence listed as SEQ ID NO: 1.
[0025] Identification of the genotype is preferably performed based on
the number and/or sizes of fragments obtained by digesting a
polynucleotide containing at least one of the aforementioned selection
polymorphic markers with one or more restriction enzymes containing
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at least one of the aforementioned selection polymorphic markers in the
recognition sequence.
[0026] In this case, the step of identifying the nucleobase type of the
selection polymorphic marker may be carried out by a basic and simple
procedure of restriction enzyme digestion and detection of the number
and/or sizes of the digested fragments. Also, since the genotype of the
selection polymorphic marker can be identified in a short period of time,
the method is suited for processing of a large number of specimens.
Furthermore, it is possible to minimize cost for identification of the
genotype.
[0027] The restriction enzyme comprising at least one of the
aforementioned selection polymorphic markers in the recognition
sequence may be BglII or Hinfl, which are restriction enzymes
comprising a selection polymorphic marker corresponding to the 253rd
and 343rd nucleotides of the nucleotide sequence listed as SEQ ID NO:
1 in their recognition sequences. Also, a primer pair comprising the
nucleotide sequences listed as SEQ ID NO: 8 and SEQ ID NO: 9 may
be used in PCR for amplification of a polynucleotide comprising a
selection polymorphic marker corresponding to the 253rd and 343rd
nucleotides.
[0028] The invention further provides a kit for selection of barley
species based on protein Z7 content, comprising a primer pair having
the nucleotide sequences listed as SEQ ID NO: 8 and SEQ ID NO: 9.
[0029] By using the primer pair having the nucleotide sequences listed
as SEQ ID NO: 8 and SEQ ID NO: 9, it is possible to efficiently
perform amplification of a polynucleotide comprising a selection
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polymorphic marker corresponding to the 253rd and 343rd nucleotides,
by PCR.
[0030] The invention still further provides a barley species of a progeny
line that can be obtained by cross-breeding of barley species that have
been selected as barley species with a low protein Z7 content by the
selection method described above.
[0031] The barley species selected by the selection method is a barley
species for which the genotype of the selection polymorphic marker is
"Kendall-type" or "Barke-type", and in the case of cross-breeding
between selected barley species, the genotype of the barley species of
the progeny line is almost certainly the same as the genotype of the
parent barley species. Consequently, the traits relating to protein Z7
content are inherited to the progeny line.
[0032] The invention further provides a method for producing a
fermented malt beverage comprising at least a mashing step and a
fermentation step, wherein the barley species used in the mashing step is
a barley species selected as a barley species specimen matching the
Kendall-type or Barke-type genotype by the selection method described
above, and/or a barley species of a progeny line that can be obtained by
cross-breeding the barley species. The invention still further provides
a fermented malt beverage that can be obtained by the production
method described above.
[0033] As explained hereunder, a correlation exists between beer
protein Z7 content and NIBEM value. Barley species selected as a
barley species specimen matching the Kendall-type or Barke-type
genotype by the selection method of the invention have low protein Z7
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contents, and therefore fermented malt beverages produced from raw materials
derived from
these barley species can exhibit excellent foam stability.
[0033A] Specific aspects of the invention include:
- a method for selection of barley species based on protein Z7 content, which
comprises: identifying at least one genotype of each of polymorphic marker A
and
polymorphic marker B in a barley species specimen by performing sequencing, by
determining whether or not a restriction enzyme recognition sequence is
present or absent, or
by performing hybridization using a perfect match probe or mismatch probe,
wherein the
polymorphic marker A is specified by multiple alignment of the nucleotide
sequence of a
region around the Haruna-type barley protein Z7 gene locus and the nucleotide
sequence of a
region around the Kendall-type barley protein Z7 gene locus, wherein the
polymorphic
marker B is specified by multiple alignment of the nucleotide sequence of a
region around the
Haruna-type barley protein Z7 gene locus and the nucleotide sequence of a
region around the
Barke-type barley protein Z7 gene locus, and carrying out the following (i)
and/or (ii):
(i) isolating a barley species specimen having the genotype identical to the
Haruna-type
genotype as a barley species with high protein Z7 content, (ii) isolating a
barley species
specimen having the genotype identical to the Kendall-type or Barke-type
genotype as a
barley species with low protein Z7 content, wherein the region around the
Haruna-type barley
protein Z7 gene locus, the region around the Barke-type barley protein Z7 gene
locus, and the
region around the Kendall-type barley protein Z7 gene locus are regions
ranging from 5 cM
upstream of the ATG sequence corresponding to the initiation codon of barley
protein Z7
gene to 5 cM downstream from the TAA sequence corresponding to the stop codon
of barley
protein Z7 gene; and
- a method for selection of barley species with low protein Z7 content, which
comprises: identifying at least one genotype of polymorphic marker A and
polymorphic
marker B in a barley species specimen by performing sequencing, by determining
whether or
not a restriction enzyme recognition sequence is present or absent, or by
performing
hybridization using a perfect match probe or mismatch probe, wherein the
polymorphic
marker A is specified by multiple alignment of the nucleotide sequence of a
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region around the Haruna-type barley protein Z7 gene locus and the nucleotide
sequence of a
region around the Kendall-type barley protein Z7 gene locus, wherein the
polymorphic marker
B is specified by multiple alignment of the nucleotide sequence of a region
around the
Haruna-type barley protein Z7 gene locus and the nucleotide sequence of a
region around the
Barke-type barley protein Z7 gene locus, and carrying out the following (iii)
or (iv):
(iii) isolating a barley species specimen having the genotype of the
polymorphic marker A
identical to the Kendall-type genotype as a barley species with low protein Z7
content,
(iv) isolating a barley species specimen having the genotype of the
polymorphic marker B
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identical to the Barke-type genotype as a barley species with low protein Z7
content, wherein
the region around the Haruna-type barley protein Z7 gene locus, the region
around the Barke-
type barley protein Z7 gene locus, and the region around the Kendall-type
barley protein Z7
gene locus are regions ranging from 5 cM upstream of the ATG sequence
corresponding to
the initiation codon of barley protein Z7 gene to 5 cM downstream from the TAA
sequence
corresponding to the stop codon of barley protein Z7 gene.
Advantageous Effects of Invention
[0034] The selection method of the invention has excellent operability, is
easy to carry out,
allows processing of a large number of specimens in a short period of time,
and allows
accurate selection of barley species based on protein Z7 content, in the
initial stage of
breeding.
[0035] Moreover, the selection method of the invention allows selection of
barley species
with excellent foam stability based on protein Z7 content, and the selected
barley species or
barley species of a progeny line that can be obtained by cross-breeding
therewith, may be
used as raw materials to produce fermented malt beverage with excellent foam
stability.
[0036] In breeding of barley for a fermented malt beverage, the bred variety
must have high
quality in all aspects including the barley, malt and fermented malt beverage
(for example,
beer). However, because consistent sample volumes are necessary for evaluation
of beer
quality, it is only currently possible to conduct evaluation in the latter
stages of the breeding
process. In regard to foam stability, for example, the quality of foam
stability is judged in a
brewing test, but an age period of 10 years is usually required until the
brewing test.
1 1 a
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Therefore, the selection method of the invention, that allows selection of
barley species
according to their properties relating to beer quality (foam stability) in the
initial stage of the
breeding, is extremely effective.
Brief Description of Drawings
1 1 b
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[0037] Fig. 1 shows a multiple alignment of nucleotide sequences of a
region around the protein Z7 gene locus.
Fig. 2 is a photograph showing the results of agarose gel electrophoresis
of a polynucleotide containing a selection polymorphic marker
amplified by PCR and digested with a restriction enzyme.
Fig. 3 is a set of graphs showing the results of measuring mean protein
Z7 contents for barley species belonging to genotype 1 and genotype 2,
selected from barley species produce of 2000, 2004 and 2008, using the
type 2 selection polymorphic marker of Table 1.
Fig. 4 is a set of graphs showing the results of measuring mean protein
Z7 contents for barley species belonging to genotype 1 and genotype 2,
selected from barley species produce of 2000, 2004 and 2008, using the
type 3 selection polymorphic marker of Table 1.
Fig. 5 is a set of graphs showing the results of measuring mean protein
Z7 contents for barley species belonging to Haruna-type, Kendall-type
or Barke-type, selected from barley species produce of 2000, 2004 and
2008, by the selection method of the invention.
Fig. 6 is a graph showing the relationship between beer protein Z7
content and NIBEM value.
Embodiments for Carrying Out the Invention
[0038] The present invention provides a selection method for selection
of barley species with high or low protein Z7 content. In the selection
method, at first, polymorphic marker A specified by multiple alignment
of the nucleotide sequences of a region around the Haruna-type and
Kendall-type barley protein Z7 gene loci, and polymorphic marker B
specified by multiple alignment of the nucleotide sequences of a region
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around the Haruna-type and Barke-type barley protein Z7 gene loci are
obtained. One or more genotypes of a barley species specimen are
identified for each of polymorphic marker A and polymorphic marker B,
and a barley species specimen having the genotype identical to the
Haruna-type genotype is selected as barley species with high protein Z7
content, or a barley species specimen having the genotype identical to
the Kendall-type or Barke-type genotype is selected as barley species
with low protein Z7 content.
[0039] The region around the barley protein Z7 gene locus comprise not
only the regions including the exons and introns of the barley protein Z7
gene, but also the region including the DNA sequences involved in
transcriptional control, and its neighboring regions. Specifically, the
regions around the barley protein Z7 gene locus may be a range within 5
cM, preferably a range within 1 cM, more preferably a range within
0.01 cM and even more preferably a range within 0.0001 cM, upstream
of the ATG sequence corresponding to the initiation codon. It may
also be a range within 5 cM, preferably a range within 1 cM, more
preferably a range within 0.01 cM and even more preferably a range
within 0.0001 cM, downstream from the TAG sequence corresponding
to the stop codon.
[0040] A "cM" (centimorgan) is a unit representing the distance
between genes on a chromosome as determined by a genetic method,
where 1 M is the distance in which an average of 1 crossover takes
place between homologous chromosomes for each meiotic division, and
1 cM is 1/100 of that distance.
[0041] Specifically, the probability of recombination is no greater than
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5% within 5 cM from the protein Z7 gene locus while the probability is
no greater than 1% within 1 cM, the probability is no greater than
0.01% within 0.01 cM, and the probability is no greater than 0.0001%
within 0.0001 cM, and therefore within these ranges, correlation
between the selection polymorphic marker and protein Z7 content is
maintained with a high degree of probability. If the selection
polymorphic marker is set to be within this range, therefore, a barley
species specimen can be selected based on protein Z7 content, in a
statistically significant manner.
[0042] The nucleotide sequence information for a region around the
barley protein Z7 gene locus can be obtained, for example, by
amplifying a polynucleotide from the region around the barley protein
Z7 gene locus by PCR using DNA extracted from barley species as
template, purifying the amplified polynucleotide if necessary, and
determining the polynucleotide nucleotide sequence by sequence
analysis.
[0043] The DNA used may be extracted from any portion of barley, and
barley leaves, stems, roots, seeds or the like may be used as the DNA
source. The method of extracting the DNA from such tissue may be
any method commonly used for extraction of plant DNA. A
commercially available DNA extraction kit may also be suitably used.
[0044] The nucleotide sequences of the primer pair to be used for PCR
with the extracted DNA as template may be designed based on
nucleotide sequence information, upon procuring a genomic (partial)
nucleotide sequence corresponding to the region around the barley
protein Z7 gene locus from a database such as NCBI, Gene Bank or the
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like. The parameters such as the nucleotide sequence lengths,
nucleotide sequence and GC content of each primer may be determined
as appropriate, within the range of ordinary trial and error by a person
skilled in the art. Also, the PCR method, amplified polynucleotide
purification and sequence analysis may be methods generally employed
in the technical field, and may be carried out according to common
methods.
[0045] In addition, by designing a specific primer for the protein Z7
gene sequence based on the database-registered (partial) nucleotide
sequence, and combining it with random primers for Thermal
Asymmetric Interlaced (TAIL) PCR, it is possible to amplify a
polynucleotide corresponding to a neighboring region with an unknown
nucleotide sequence. Sequence analysis of the amplified
polynucleotide allows acquisition of nucleotide sequence information
that has not been registered in a database. The method of acquiring the
neighboring region with an unknown nucleotide sequence may be, for
example, a method such as Inverse PCR, instead of TALL PCR.
[0046] The polymorphic marker A of the invention can be specified by
multiple alignment between a nucleotide sequence of a region around
the Haruna-type barley protein Z7 gene locus and a nucleotide sequence
of a region around the Kendall-type barley protein Z7 gene locus. The
polymorphic marker B of the invention can be specified by multiple
alignment between a nucleotide sequence of a region around the
Haruna-type barley protein Z7 gene locus and a nucleotide sequence of
a region around the Barke-type barley protein Z7 gene locus.
[0047] The nucleotide sequence of the region around the Haruna-type
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barley protein Z7 gene locus can typically be obtained from a Haruna
Nijo barley species by the aforementioned method for determining
nucleotide sequence information. A barley species other than Haruna
Nijo may also be used, such barley species including, but not limited to,
Sakitama Nijo, Tone Nijo, Nasu Nijo, Kinuyutaka, Mikamo-Golden,
Ryofu, Ryoun, Commander, Keel, Myogi Nijo, Asaka Gold, Misato
Golden, Hokkaido Silvery, Harrington, CDC Reserve, CDC Copeland,
CDC Meredith, CDC Aurora Nijo, CDC Select and Gairdner species.
[0048] The nucleotide sequence of the region around the Kendall-type
barley protein Z7 gene locus can typically be obtained from a CDC
Kendall barley species by the aforementioned method for determining
nucleotide sequence information. Barley species other than CDC
Kendall species may also be used, such barley species including, but not
limited to, Hoshimasari, Betzes, AC Metcalf, CDC Polar Star, Newdale,
SloopSA, Scarlett, Cellar, Prior, Chevallier, Hanna, Golden Melon,
Amagi Nijo, Akagi Nijo, Seijo #1, Asahi #5, Clipper, Schooner,
Franklin, Lofty Nijo, Baudin and Tirnori species.
[0049] The nucleotide sequence of the region around the Barke-type
barley protein Z7 gene locus can typically be obtained from a Barke
barley species by the aforementioned method for determining nucleotide
sequence information. A barley species other than Barke may also be
used, such barley species including, but not limited to, Braemar, Optic,
Triumph, Alexis, Sebastian and Power species.
[0050] Multiple alignment, according to the invention, means the
alignment of nucleotide sequences with appropriate gaps between them
to line up the corresponding nucleotide sequence sections, in order to
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allow comparison between the nucleotide sequences. Herein,
alignment of two nucleotide sequences will also be referred as "multiple
alignment". A known multiple alignment program may be utilized for
the multiple alignment. For example, Clustal W, Clustal X or the like
may be suitably used.
[0051] Multiple alignment allows nucleotide positions with non-
matching nucleobases to be identified. The identified nucleotide
positions may be used as selection polymorphic markers. The gap
positions inserted for optimal alignment are defined, according to the
invention, as selection polymorphic markers, i.e. nucleotide positions
where the nucleobases do not match.
[0052] An example of a multiple alignment is shown in Fig. 1. It
shows multiple alignment of nucleotide sequences of the regions around
the protein Z7 gene loci of Haruna Nijo (indicated as Harun; SEQ ID
NO: 1), Harrington (indicated as Harri), CDC Copeland (indicated as
Copel), CDC Kendall (indicated as Kenda; SEQ ID NO: 2) and Barke
(indicated as Barke; SEQ ID NO: 3) barley.
[0053] Based on Fig. 1, it is possible to identify nucleotide positions
corresponding to the 62nd, 93-94th (representing a gap), 94th, 96th,
98th, 113th, 116th, 123rd, 148th, 151st, 153rd, 156th, 159th, 160-186th,
217th, 231st, 239th, 246-247th, 253rd, 305-306th, 378th or 422nd
nucleotide of the nucleotide sequence listed as SEQ ID NO: 1, as
examples for the polymorphic marker A. Similarly, it is possible to
identify nucleotide positions corresponding to the 260th, 262nd, 305-
306th, 343rd, 378th, 386th or 422nd nucleotides of the nucleotide
sequence listed as SEQ ID NO: 1, as examples for the polymorphic
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marker B.
[0054] The term "corresponding", used for, for example, the nucleotide
position corresponding to the 422nd nucleotide, means either the
nucleotide position is aligned or possible aligned to that nucleotide row
by the multiple alignment.
[0055] Identification of the genotype of the selection polymorphic
marker in a barley species specimen may be performed, for example, by
determining the nucleobase type of the nucleotide position of the
ss ee ee oc tt oo nn ppool y:moor pr phi ico mina ra rk ek re, r o s b y
presenter. d e tm g whether
ininDeterminat oionr nooft the e
nucleobase type can be performed, for example, by determining the
nucleobase type by sequence analysis, by determining the nucleobase
type by the presence or absence of a restriction enzyme recognition
sequence, or by determining the nucleobase type by hybridization using
a perfect match probe or mismatch probe.
[0056] The selection polymorphic markers can be classified into 3 types
(Table 1). Type 1 selection polymorphic markers are those wherein the
Haruna-type genotype (genotype 1), and Kendall-type and Barke-type
genotype (genotype 2) are different, and the Kendall-type and Barke-
type genotype (genotype 2) are identical. That is, the Type 1 selection
polymorphic marker can be used as polymorphic marker A or as
polymorphic marker B. Type 2 selection polymorphic markers are
those wherein the Haruna-type and Barke-type genotype (genotype 1)
are identical, and only the Kendall-type genotype (genotype 2) differs
from others. That is, the Type 2 selection polymorphic marker can be
used only as polymorphic marker A. Type 3 selection polymorphic
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markers are those wherein the Haruna-type and Kendall-type genotype
(genotype 1) are identical, and only the Barke-type genotype (genotype
2) differs from others. That is, the Type 3 selection polymorphic
marker can be used only as polymorphic marker B.
[Table 1]
Type Genotype 1 Genotype 2 Polymorphic marker
1 H K B May be used as A or B
2 H B K May be used as A
3 H K B May be used as
The symbols in the genotype 1 and genotype 2 columns of Table 1 are
H: Haruna-type, K: Kendall-type and B: Barke-type, respectively.
[0057] By selecting a barley species specimen based on the genotype of
polymorphic marker A, it is possible to classify the barley species
specimen as Haruna-type or Kendall-type. In this case, however, a
Barke-type barley species specimen may be classified as either Haruna-
type or Kendall-type. Similarly, by selecting a barley species
specimen based on the genotype of polymorphic marker B, it is possible
to classify the barley species specimen as Haruna-type or Barke-type,
while a Kendall-type barley species specimen may be classified as
either Haruna-type or Barke-type.
[0058] Therefore, by identifying at least one genotype for polymorphic
marker A and at least one genotype for polymorphic marker B in a
barley species specimen, and comparing the identified genotypes with
the Haruna-type, Kendall-type and Barke-type genotypes, it is possible
to classify the barley species specimen into Haruna-type, Kendall-type
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=
or Barke-type. That is, a barley species specimen can be accurately
classified into barley species with high protein Z7 content (identical to
the Haruna-type) and barley species with low protein Z7 content
(identical to the Kendall-type or Barke-type), based on genotype.
[0059] In one embodiment, identification of one or more genotypes for
a barley species specimen for each of polymorphic marker A and
polymorphic marker B may be performed by identifying the genotype
of at least one selection polymorphic marker of Type 1 shown in Table
1, or identifying the genotype of at least one of each of Type 2 and Type
3 shown in Table 1. Since the selection polymorphic marker of Type 1
is polymorphic marker A and is also polymorphic marker B,
identification of the genotype of at least one allows identification of the
genotype of one or more of each of polymorphic marker A and
polymorphic marker B.
[0060] For selection of only a barley species specimen with low protein
Z7 content, on the other hand, it is sufficient to identify the genotype of
one or more of either or both polymorphic marker A and polymorphic
marker B. When the identified polymorphic marker A genotype in a
barley species specimen is identical to the Kendall-type genotype, or
when the identified polymorphic marker B genotype is identical to the
Barke-type genotype, the barley species specimen may be selected as a
barley species with low protein Z7 content.
[0061] The protein Z7 content sometimes differs within the same
variety, depending on the harvesting year of the barley species. Since
it is therefore difficult to perform judgment with absolute numerical
values when comparing the protein Z7 content in barley species of
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different harvesting years, the comparison is preferably made with the
protein Z7 content as the ratio to total protein in the seeds, with respect
to Haruna Nijo barley, as a typical barley species with high protein Z7
content. In this case, a barley species with high protein Z7 content is a
barley species with a numerical value of, for example, 0.50 or greater,
preferably 0.60 or greater and more preferably 0.70 or greater with
respect to the protein Z7 content of Haruna Nijo barley. On the other
hand, a barley species with low protein Z7 content is a barley species
with a protein Z7 content ratio to total protein in the seeds of no greater
than 0.50, preferably no greater than 0.40 and more preferably no
greater than 0.30, with respect to Haruna Nijo barley.
[0062] The method of extracting the DNA from a barley species
specimen may generally be a CTAB method or a method described
below, which is commonly used for extraction of DNA from plant
tissue. A commercially available kit may also be suitably used.
While the DNA may be extracted from the leaves, stems, roots, seeds or
other part of the barley species specimen, it is preferably extracted from
leaves in consideration of selection at the breeding stage. By
extraction of leaves, it is possible to select barley with preferred traits at
an earlier stage.
[0063] When a polynucleotide comprising the aforementioned selection
polymorphic marker is to be amplified by PCR using DNA extracted
from a barley species specimen, the PCR primer pair may be designed
based on the nucleotide sequence of the region around the barley protein
Z7 gene locus, determined in the course of identifying the selection
polymorphic marker. The length of the polynucleotide to be amplified
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with the primer pair may be 20-30,000 bp. In consideration of
amplification efficiency in PCR and ease of handling during analysis of
the amplified polynucleotide, the upper limit is preferably 10,000 bp,
more preferably 3000 bp and even more preferably 2000 bp. Also, the
lower limit is preferably 100 bp, more preferably 200 bp and even more
preferably 300 bp. A polynucleotide having such a length can be
amplified by appropriate selection of the type of polymerase used for
the PCR, among polymerases well known by those skilled in the art.
[0064] The method for identifying the nucleobase type of the selection
polymorphic marker using the polynucleotide may be a method of
decoding of the nucleotide sequence by sequence analysis.
[0065] Identification of the genotype may be performed by digesting
the polynucleotide with one or more restriction enzymes containing the
selection polymorphic marker in the recognition sequence, and
analyzing the number and/or sizes of the obtained fragments.
[0066] When the selection polymorphic marker is present in the
recognition sequence of the restriction enzyme, it is possible to identify
the genotype based on whether or not the polynucleotide is cleaved by
the restriction enzyme, depending on the genotype of the selection
polymorphic marker. Whether or not the polynucleotide has been
cleaved by the restriction enzyme can be determined based on the
number and/or sizes of the fragments by size fractionation of the
polynucleotide that has been digested by the restriction enzyme.
[0067] Design of the primer pair to be used in PCR, for PCR
amplification of the polynucleotide comprising the selection
polymorphic marker that is present in the recognition sequence of the
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restriction enzyme, may be performed according to a common method,
as described above. The length of the polynucleotide to be amplified
by the primer pair is preferably a maximum of 5000 bp, more preferably
3000 bp and even more preferably 2000 bp, considering that the
fragments are to be detected by size fractionation after having been
digested with the restriction enzyme. It is also preferably a minimum
of 100 bp, more preferably 200 bp and even more preferably 300 bp.
A polynucleotide having such a length can be amplified by appropriate
selection of the type of polymerase used for the PCR, among
polymerases well known by those skilled in the art.
[0068] The digestion reaction with the restriction enzyme may be
carried out with buffer that is optimal for each restriction enzyme, at the
optimal reaction temperature. A suitable method for detecting the
restriction enzyme-digested fragments by size fractionation is agarose
gel electrophoresis, which is commonly employed by those skilled in
the art. Also, HPLC using an appropriate known column allows
detection of the fragments by size fractionation.
[0069] As an example of a method of identifying genotype based on
whether or not the polynucleotide is cleaved by a restriction enzyme,
there may be mentioned a method utilizing selection polymorphic
markers corresponding to the 253rd and 343rd nucleotides of the
nucleotide sequence listed as SEQ ID NO: 1, the selection polymorphic
markers being present in the BglII and HinfI recognition sequences.
More specifically, a polynucleotide is amplified by PCR using DNA
extracted from a barley species specimen as template and using a primer
pair having the nucleotide sequences listed as SEQ ID NO: 8 and SEQ
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ID NO: 9, and the amplified polynucleotide is digested with restriction
enzyme BglII and restriction enzyme Hinff. Upon size fractionation of
the digested fragments by 4.0% (w/v) agarose gel electrophoresis,
Haruna-type barley species specimen produce 3 fragments with sizes of
251 bp, 91 bp and 59 bp. On the other hand, Kendall-type barley
species specimen produce 2 fragments with sizes of 389 bp and 59 bp,
and Barke-type barley species specimen produce 2 fragments with sizes
of 251 bp and 150 bp.
[0070] One embodiment of the invention is a kit comprising a primer
pair having the nucleotide sequences listed as SEQ ID NO: 8 and SEQ
ID NO: 9. For the selection method described above, this kit may be
suitably used as a mode of amplifying a polynucleotide comprising the
selection polymorphic marker by PCR.
[0071] The kit may also be one further comprising a restriction enzyme.
Also included may be a kit for extraction of DNA from barley species
tissue.
[0072] The invention further provides barley species of a progeny line
that can be obtained by cross-breeding of barley species that have been
selected as barley species with a low protein Z7 content by the selection
method described above. The progeny line may be obtained by cross-
breeding between Kendall-type and Kendall-type barley species or
between Barke-type and Barke-type barley species, or by cross-breeding
between Kendall-type and Barke-type barley species, among the barley
species selected as barley species with low protein Z7 content. This
can yield a barley species of a progeny line with low protein Z7 content,
from barley species selected as barley species with low protein Z7
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content.
[0073] The barley species selected by the selection method described
above may be used in a method for producing a fermented malt
beverage comprising at least a mashing step and a fermentation step.
The barley species used in the mashing step is preferably a barley
species selected as a barley species with low protein Z7 content by the
aforementioned selection method, or a barley species which is a
progeny line thereof.
[0074] Malt may also be obtained by malting of a barley species
selected as a barley species with low protein Z7 content by the selection
method, or a barley species which is a progeny line thereof. The
malting may be performed by a commonly employed method.
Specifically, for example, steeping may be carried out until the
percentage of steeping is 40%-45%, followed by germinating at 10-
20 C for 3-6 days and roasting, to obtain malt.
[0075] The mashing step is a step in which a raw material containing
malt or barley is mixed with mashing water, the obtained mixture is
heated for saccharification of the malt or barley, and the malt liquor
from the saccharified malt or barley is obtained. In the mashing step,
it is possible to use not only malt obtained from the barley species
selected as a barley species with low protein Z7 content by the selection
method, or a barley species which is a progeny line thereof, but also the
barley species themselves. Additional raw materials that may be
added include auxiliary materials such as corn starch, corn grits, rice or
saccharides, in addition to the malt or barley.
[0076] The fermentation step is a step in which hops are added to the
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malt liquor, and yeast is added to the boiled and cooled cold malt liquor
for fermentation to obtain a fermented malt beverage (intermediate)
product. The yeast used in the step may be, for example,
Saccharomyces pastorianus, Saccharomyces cerevisiae, Saccharomyces
uvarum, or the like.
[0077] The fermented malt beverage may be the beverage that may be
produced by fermentation using malt as a portion of the raw material,
without particular limitation to degree of usage ratio of malt for
production. Specific examples include beer and low-malt beer
(Happoshu). Non-alcoholic beer or non-alcoholic low-malt beer are
also fermented malt beverages since similar production methods are
used as for beer.
[0078] As explained hereunder, a negative correlation has been
confirmed between protein Z7 content and NMEM value, and by using
as raw material a barley species selected as a barley species with low
protein Z7 content by the aforementioned selection method, or a barley
species which is a progeny line thereof, it is possible to produce a
fermented malt beverage with excellent foam stability.
Examples
[0079] The present invention will now be explained in greater detail
based on examples, with the understanding that these examples are not
limitative on the invention.
[0080] (Example 1: Determining unknown sequence of the region
around the protein Z7 gene locus)
Five barley species: Haruna Nijo, CDC Copeland, Harrington, CDC
Kendall and Barke, were used in the following example.
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[0081] [Extraction of DNA]
DNA was extracted from each of the barley species mentioned above by
the following method, using leaves as the DNA source. After adding
extraction buffer (200 mM Tris-HC1, 250 mM NaCl, 25 mM EDTA, pH
7.5) and zirconia balls to the leaves and shaking the mixture, it was kept
at 60 C for 30 minutes. Following centrifugal separation, an
equivalent amount of isopropanol was added to the obtained supernatant
for precipitating the DNA. This was centrifuged, 70% ethanol was
added to the obtained precipitate, and centrifugal separation was
repeated. The obtained DNA precipitate was dissolved in sterilized
water and the DNA solution was used as template for PCR.
[0082] [Nucleotide sequence analysis of protein Z7 gene and its
upstream region]
TAIL PCR was used to determine the 5'-flanking unknown sequence of
protein Z7 from the translation initiation codon (ATG). TAIL PCR is a
method for obtaining an amplification product comprising an unknown
sequence adjacent to a known sequence, and it employs a pair which is a
random primer with a random nucleotide sequence and a primer with a
nucleotide sequence capable of specifically binding to the known
sequence, with variable control of a low annealing temperature (44 C)
and a high annealing temperature (68 C) to inhibit amplification of the
nonspecific product, such that a product comprising the unknown
sequence adjacent to the known sequence is preferentially amplified (for
example, Plant PCR Experimental Protocols, p.'73-79, Shujunsha
Publishing, 1995). The following primers were used for the TAIL
PCR. Specific primers 1-3 were designed with reference to nucleotide
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sequence information for the protein Z7 gene in the NCBI database
(NCBI accession No.X95277).
[0083] Random primer (SEQ ID NO: 4);
5'-GTNCGA (G/C) (A/T)CANA (A/T)GTT-3'
Specific primer 1 (SEQ ID NO: 5);
5'-CGTTGGTGGCAGCAGACTCGGGG-3'
Specific primer 2 (SEQ ID NO: 6);
5'-GGTCGGAGGAGATGGCGGAGGCG-3'
Specific primer 3 (SEQ ID NO: 7);
5'-GGTCGGTGGTGAGGGTGGTTGCCA-3'
[0084] Specific primers 1-3 are designed to be "nested", and nested
PCR was utilized to obtain a polynucleotide with the 5'-flanking
unknown sequence from the translation initiation codon (ATG) of
protein Z7.
[0085] First, the random primer and specific primer 1 were used, with
DNA extracted from the barley species as template, for a first PCR
according to the following PCR program.
After keeping at 94 C for 1 minute, it was further kept at 95 C for 1
minute. Next, 5 cycles were carried out, where 1 cycle was heat
denaturation at 94 C for 1 minute, annealing at 65 C for 1 minute and
extension reaction at 72 C for 3 minutes, and then 1 cycle was carried
out, where the 1 cycle was heat denaturation at 94 C for 1 minute,
annealing at 30 C for 3 minutes and extension reaction at 72 C for 3
minutes. Next, 15 cycles were carried out, where 1 cycle consisted of
9 steps: heat denaturation at 94 C for 30 seconds, annealing at 68 C for
1 minute, extension reaction at 72 C for 3 minutes, heat denaturation at
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94 C for 30 seconds, annealing at 68 C for 1 minute, extension reaction
at 72 C for 3 minutes, heat denaturation at 94 C for 30 seconds,
annealing at 44 C for 1 minute, and extension reaction at 72 C for 3
minutes. Finally, extension reaction was conducted at 72 C for 5
minutes.
[0086] Second PCR was then conducted according to the following
PCR program, using the random primer and specific primer 2, with the
PCR product obtained in the first PCR as template.
Here, 13 cycles were carried out, where 1 cycle consisted of 9 steps:
heat denaturation at 94 C for 30 seconds, annealing at 68 C for 1
minute, extension reaction at 72 C for 3 minutes, heat denaturation at
94 C for 30 seconds, annealing at 68 C for 1 minute, extension reaction
at 72 C for 3 minutes, heat denaturation at 94 C for 30 seconds,
annealing at 44 C for 1 minute, and extension reaction at 72 C for 3
minutes. Finally, extension reaction was conducted at 72 C for 5
minutes.
[0087] Third PCR was then conducted according to the same PCR
program as the second PCR, using the random primer and specific
primer 3, with the second PCR product as template. This PCR
amplification product was analyzed by agarose gel electrophoresis, and
the detected amplified polynucleotide was recovered from the gel. A
continuous unknown sequence region extending across 290-337 bp
upstream from the translation initiation codon (ATG) of the protein Z7
gene was amplified as a polynucleotide by the TAIL PCR method
described above. The recovered amplified polynucleotide was used as
template for sequence analysis after purification using a QIAquick Gel
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Extraction kit (Qiagen, cat.No.28706), to determine the nucleotide
sequence. Sequence analysis was performed utilizing the Data
Analysis Service of Sigma Corp. The Haruna Nijo, CDC Kendall and
Barke nucleotide sequences are listed as SEQ ID NO: 1, SEQ ID NO: 2
and SEQ ID NO: 3, respectively.
[0088] [Multiple alignment of protein Z7 gene nucleotide sequences]
Multiple alignment was performed with the determined nucleotide
sequences of Hanma Nijo, CDC Copeland, Harrington, CDC Kendall
and Barke species (Fig. 1). A GENETYX Ver.8 (Genetyx
Corporation) alignment analysis tool was used for the multiple
alignment. Rows with "*" symbols under them indicate nucleotide
positions where the nucleobase types of all of the 5 barley species was
identical, and rows without "*" symbols indicate nucleotide positions
where the nucleobase type differed in any of the 5 barley species.
[0089] [Polymorphic markers]
As a result of the multiple alignment shown in Fig. 1, a total of 26
nucleotide positions where the nucleobase types did not match between
the 5 barley species, were existed and these were identified as
polymorphic markers. Specifically, these are the nucleotide positions
corresponding to the 62nd nucleotide, 93-94th nucleotides
(corresponding to a gap in Fig. 1), 94th nucleotide, 96th nucleotide,
98th nucleotide, 113th nucleotide, 116th nucleotide, 123rd nucleotide,
148th nucleotide, 151st nucleotide, 153rd nucleotide, 156th nucleotide,
159th nucleotide, 160-186th nucleotides, 217th nucleotide, 231st
nucleotide, 239th nucleotide, 246-247th nucleotides, 253rd nucleotide,
260th nucleotide, 262nd nucleotide, 305-306th nucleotides, 343rd
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,
nucleotide, 378th nucleotide, 386th nucleotide and 422nd nucleotide, of
the nucleotide sequence listed as SEQ ID NO: 1.
[0090] The Haruna Nijo, CDC Copeland and Harrington species had
identical genotypes for all of the polymorphic markers.
[0091] The 26 identified polymorphic markers were classified (Table 2)
according to the classification in Table 1.
[Table 2]
Type Genotype 1 Genotype 2 Polymorphic marker
No.62 2 H B K May be used as A
No.93-94 2 H B K May be used as A
No.94 2 H B K May be used as A
No.96 2 H B K May be used as A
No.98 2 H B K May be used as A
No.113 2 H B K May be used as A
No.116 2 H B K May be used as A
No.123 2 H B K May be used as A
No.148 2 H B K May be used as A
No.151 2 H B K May be used as A
No.153 2 H B K May be used as A
No.156 2 H B K May be used as A
No.159 2 H B K May be used as A
No.160-186 2 H B K May be used as A
No.217 2 H B K May be used as A
No.231 2 H B K May be used as A
No.239 2 H B K May be used as A
No.246-247 2 H B K May be used as A
No.253 2 H B K May be used as A
No.260 3 HK B May be used as B
No.262 3 HK B May be used as B
No.305-306 1 H KB May be used as A or B
No.343 3 HK B May be used as B
No.378 1 H KB May be used as A or B
No.386 3 I-IK B May be used as B
No.422 1 H KB May be used as A or B
The symbols in the genotype 1 and genotype 2 columns of Table 2
represent the genotypes of H: Haruna-type, K: Kendall-type and B:
Barke-type, respectively.
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[0092] (Example 2: Construction of CAPS marker)
If the genotypes of the selection polymorphic markers can be
determined by whether or not they are cleaved by a specific restriction
enzyme, then they are useful as CAPS (Cleaved Amplified Polymorphic
Sequence) markers. Of the aforementioned polymorphic markers,
barley species of genotype 1 (Table 2) have a recognition sequence for
restriction enzyme BglII (AGATCT) at the nucleotide position
corresponding to the 253rd nucleotide of the nucleotide sequence listed
as SEQ ID NO: 1. Barley species of genotype 1 (Table 2) also have a
recognition sequence for restriction enzyme HinfI (GANTC) at the
nucleotide position corresponding to the 343rd nucleotide. These
polymorphic markers were used to construct CAPS markers.
[0093] [Polymorphic marker corresponding to 253rd nucleotide of
nucleotide sequence listed as SEQ ID NO: 1]
By PCR amplification of a polynucleotide comprising the polymorphic
marker corresponding to the 253rd nucleotide of the nucleotide
sequence listed as SEQ ID NO: 1, and digestion of the PCR product
with restriction enzyme BglII, it is possible to identify the genotype
based on cleavage. Cleavage of the PCR product indicates a barley
species having genotype 1 (Table 2), while lack of cleavage of the PCR
product indicates a barley species having genotype 2 (Table 2).
[0094] DNA was extracted by the same method as Example 1, for 23
barley varieties (Haruna Nijo, Myogi Nijo, Satsuki Nijo, Golden Melon,
Akagi Nijo, Ryofu, Ryoun, Hoshimasari, CDC Kendall, AC Metcalf,
Harrington, CDC Copeland, SloopSA, Schooner, Clipper, Franldin,
Barke, Scarlett, Betzes, Braemar, Triumph, Hanna and Prior). This
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DNA was used as template for PCR using primers CAPS1 and CAPS2.
Approximately 0.4 kbp PCR products were obtained for each. The
PCR products were digested with BglII, and subjected to electrophoresis
after digestion. In the samples with cleavage of the PCR products by
BglII, two DNA fragments were detected with sizes of approximately
250 bp and 150 bp. This allows classification into barley species with
cleavage of the PCR products by BglII, and barley species without
cleavage.
[0095] CAPS1 (SEQ ID NO: 8);
5'-GGTCACATGACGTGTATTAATCTCC-3'
CAPS2 (SEQ ID NO: 9);
5'-CGTTGGTGGCAGCAGACTCGGGG-3'
[0096] [Quantitation of protein Z7 contents]
Barley seeds cultivated in fields of Sapporo Breweries Ltd. in Gunma
Prefecture during 2000, 2004 and 2008 were ground with a mill, and the
protein Z7 contents were quantitated by ELISA. The ELISA method
was sandwich ELISA using a protein Z7-specific antibody provided by
Prof. Evans of the University of Tasmania. A 50 mg portion of barley
seeds that had been crushed with a mill was taken into a 2 mL screw-
capped tube, and 1 mL of Phosphate Buffer Saline (PBS) containing
0.28% Dithiothreitol (DTT) was added and the mixture was shaken
overnight. The centrifugation supernatant of the solution was used as
the barley seed protein extract. After quantitating the protein
concentration by the Bradford method, it was supplied for ELISA.
ELISA of the protein Z7 was conducted according to the method of
Evans et al. (Non-patent document 5). The protein Z7 content was
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expressed as (ng/pg-protein), considering variations in seed protein
content. The measurement results for the protein Z7 contents are
shown in Table 3.
[0097] [Table 3]
Protein Z7 (ng/tig protein)
2000 produce 2004 produce 2008 produce
Hamm Nijo 27.6 30.6 41.2
Myogi Nijo 22.9 20.7 30.1
Satsuki Nijo 26.1 26.6 NA
Golden Melon 7.4 7.9 13.8
Akagi Nijo 7.0 8.2 NA
Ryofu 20.3 21.6 30.1
Ryouun 25.9 23.5 35.9
Hoshimasari 6.6 6.0 8.1
CDC Kendall 5.9 6.3 15.4
AC Metcalfe 11.7 11.0 7.0
Harrington 22.0 20.5 21.1
CDC Copeland 18.3 13.2 15.7
SloopSA 5.6 6.3 NA
Schooner 7.0 6.9 8.0
Clipper 7.3 8.0 NA
Franklin 14.4 14.9 NA
Barke 9.3 12.1 14.0
Scarlett 6.8 11.6 8.5
Betzes 11.5 6.0 5.9
Braemer 5.4 5.6 6.8
Triumph 7.2 NA 11.3
Hanna 6.2 6.9 7.9
Prior 15.0 14.8 13.3
NA: Not Available
[0098] The effectiveness of this CAPS marker was verified by
comparing the average value of the protein Z7 content for barley species
whose PCR product was cleaved by BglII (genotype 1) with barley
species that was not cleaved (genotype 2) (Fig. 3). As a result, the
barley species of genotype 1 had significantly higher protein Z7
contents than the barley species of genotype 2, with a critical rate of
1%, for all years including 2000, 2004 and 2008.
Average values for protein Z7 content (2000)
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Genotype 1; 18.51 8.26 ng/m-protein
Genotype 2; 8.66 3.30 ng/m-protein
Average values for protein Z7 content (2004)
Genotype 1; 19.37 7.79 ng/m-protein
Genotype 2; 8.83 3.20 ng/m-protein
Average values for protein Z7 content (2008)
Genotype 1; 22.90 11.90 ng/R-protein
Genotype 2; 9.76 3.44 ng/pg-protein
[0099] However, the phenomenon was observed that not all of the
protein Z7 contents of the selected barley species were high, even
selecting a barley species specimen belonging to genotype 1 whose
PCR product was cleaved with BglII. That is, the polymorphic marker
corresponding to the 253rd nucleotide of the nucleotide sequence listed
as SEQ ID NO: 1 was not sufficient as a selection marker for reliable
classification of a barley species specimen into barley species with high
and barley species with low protein Z7 contents.
[0100] [Polymorphic marker corresponding to 343rd nucleotide of
nucleotide sequence listed as SEQ ID NO: 1]
By PCR amplification of a polynucleotide comprising the polymorphic
marker corresponding to the 343rd nucleotide of the nucleotide
sequence listed as SEQ ID NO: 1, and digestion of the PCR product
with restriction enzyme HinfI, it is possible to identify the genotype
based on PCR cleavage. Cleavage of the PCR product indicates a
barley species having genotype 1 (Table 2), while lack of cleavage of
the PCR product indicates a barley species having genotype 2 (Table 2).
[0101] DNA was extracted by the same method as Example 1, for 23
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barley varieties (Haruna Nijo, Myogi Nijo, Satsuki Nijo, Golden Melon,
Akagi Nijo, Ryofu, Ryoun, Hoshimasari, CDC Kendall, AC Metcalf,
Harrington, CDC Copeland, SloopSA, Schooner, Clipper, Franklin,
Barke, Scarlett, Betzes, Braemar, Triumph, Hanna and Prior). This
DNA was used as template for PCR using primers CAPS1 and CAPS2.
Approximately 0.4 kbp PCR products were obtained for each. The
PCR products were digested with Hinfl, and subjected to
electrophoresis after digestion. In the samples with cleavage of the
PCR products by Hinfl, two DNA fragments were detected with sizes of
approximately 350 bp and 60 bp. This allows classification into barley
species with cleavage of the PCR products by Hinfl, and barley species
without cleavage.
[0102] The protein Z7 contents were measured in the manner described
above. The average values of the protein Z7 contents for barley
species whose PCR product was cleaved by Hinfl (genotype 1) and
barley species in which it was not cleaved (genotype 2) were compared
(Fig. 4). As a result, no statistically significant difference was found
for any of the years including 2000, 2004 and 2008, despite searching
for a tendency toward a higher average value for the protein Z7 content
in the barley species of genotype 1 compared to the barley species of
genotype 2.
Average values for protein Z7 content (2000)
Genotype 1; 13.79 7.86 ng/pg-protein
Genotype 2; 7.31 1.98 ng/Itg-protein
Average values for protein Z7 content (2004)
Genotype 1; 13.57 7.74 ng/pg-protein
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Genotype 2; 8.83 4.56 ng/pg-protein
Average values for protein Z7 content (2008)
Genotype 1; 17.45 11.54 ng/ptg-protein
Genotype 2; 10.72 3.63 ng/pg-protein
[0103] The results of Example 2 indicate that, of the polymorphic
markers identified in Example 1, the polymorphic markers
corresponding to the 253rd and 343rd nucleotides of the nucleotide
sequence listed as SEQ ID NO: 1, when either was used alone, were not
sufficient as selection markers for accurate classification of a barley
species specimen into barley species with high and barley species with
low protein Z7 contents, based on the presence or absence of restriction
enzyme cleavage.
[0104] (Example 3: Construction of CAPS markers utilizing multiple
polymorphic markers)
It is expected that if products of PCR with primers CAPS1 and CAP S2
are digested with two different restriction enzymes, BglII and Hinfl, this
would allow classification into 3 types with different resulting DNA
fragment numbers and sizes (Table 4).
[0105] This DNA was extracted from the barley species specimen in the
same manner as Example 2, and the DNA was used as template for PCR
using primers CAPS1 and CAPS2. The PCR products were digested
with 2 restriction enzymes (BglII and Hinfl). The results of 4.0%
(w/v) agarose gel electrophoresis of the DNA fragments after restriction
enzyme digestion are shown in Fig. 2. Barley species in which an
approximately 400 bp band was detected were designated as Kendall-
type, barley species in which approximately 250 bp and approximately
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90 bp bands were detected were designated as Haruna-type, and barley
species in which approximately 250 bp and approximately 150 bp bands
were detected were designated as Barke-type. These results indicated
that it is possible to classify barley protein Z7 genotypes into 3 types,
Kendall-type, Haruna-type or Barke-type, with these CAPS markers.
[Table 4]
BglII HinfI PCR product Fragment lengths
obtained by digestion
cleavage cleavage length (bp) with restriction
enzymes (bp)
Kendall-type x 0 448 389
59
B arke-type 0 X 401 251
150
Haruna-type 0 0 401 251
91
59
[0106] (Example 4: Verification of effect of selecting barley species
based on protein Z7 content, by CAPS markers utilizing multiple
polymorphic markers)
The genotypes of CAPS markers were identified as explained in
Example 3, for 23 barley varieties (Haruna Nijo, Myogi Nijo, Satsulci
Nijo, Golden Melon, Akagi Nijo, Ryofu, Ryoun, Hoshimasari, CDC
Kendall, AC Metcalf, Harrington, CDC Copeland, SloopSA, Schooner,
Clipper, Franldin, Barke, Scarlett, Betzes, Braemar, Triumph, Hanna
and Prior), and they were classified into 3 types: Kendall-type, Haruna-
type or Barke-type. The relationship between genotype and protein Z7
content was investigated.
[0107] [Comparison between genotype classification and protein Z7
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content]
As demonstrated in Example 3, the effectiveness of the CAPS markers
was verified by identifying the CAPS marker genotypes and comparing
the average values of the protein Z7 contents of each of the barley
species classified as Kendall-type, Haruna-type or Barke-type (Fig. 5).
The Haruna-type barley species had significantly higher protein Z7
contents than the Kendall-type barley species and Barke-type barley
species, at a critical rate of 1%, for all years including 2000, 2004 and
2008 (Fig. 5).
[0108] Average values for protein Z7 content (2000)
Haruna-type barley species; 23.30+3.37 ng/ g-protein
Kendall-type Barley species; 8.66+3.30 ng/ g-protein
Barke-type Barley species; 7.30+1.98 ng/pg-protein
Average values for protein Z7 content (2004)
Haruna-type barley species; 22.38+5.47 ng/pg-protein
Kendall-type Barley species; 8.83+3.20 ng/ g-protein
Barke-type Barley species; 8.80+4.56 ng/ g-protein
Average values for protein Z7 content (2008)
Haruna-type barley species; 28.99+9.37 ng/ g-protein
Kendall-type Barley species; 9.76+3.44 ng/n-protein
Barke-type Barley species; 10.70+3.63 ng/pg-protein
[0109] As a result of classification of the barley species specimen into
the 3 types, Haruna-type, Kendall-type and Barke-type, as explained
above, Haruna-type barley species specimen were reliably identified as
barley species with high protein Z7 content while Kendall-type and
Barke-type barley species specimen were reliably identified as barley
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species with low protein Z7 content, and therefore a sufficient selection
effect was exhibited.
[0110] Thus, the CAPS markers are effective as selection markers for
reliably classifying a barley species specimen into barley species with
high and barley species with low protein Z7 content, based on
identifying the genotypes of 2 polymorphic markers corresponding to
the 253rd and 343rd nucleotides of the nucleotide sequence listed as
SEQ ID NO: 1.
[0111] Also, the results of Example 4 demonstrate that a barley species
specimen whose genotype is identical to the Haruna-type can be
selected as barley species with high protein Z7 content, and that a barley
species specimen whose genotype is identical to the Kendall-type or
Barke-type can be selected as barley species with low protein Z7
content. That is, based on the knowledge obtained in Example 4, even
when the genotype of only one of the 2 polymorphic markers
corresponding to the 253rd and 343rd nucleotides of the nucleotide
sequence listed as SEQ ID NO: 1 has been identified, it is possible to
select a barley species specimen with low protein Z7 content, by
selecting a barley species specimen whose PCR product is not cleaved
by BglII as a barley species matching the Kendall-type, or selecting a
barley species specimen whose PCR product is not cleaved by Hinff as
a barley species matching the Barke-type.
[0112] (Example 5: Relationship between beer protein Z7 content and
NLBEM value)
A total of 42 test beers were used as samples, being ordinarily brewed
from malt of 11 different varieties (Haruna Nijo, Amagi Nijo, Mikamo-
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Golden, Nitta Nijo 21, Ryofu, Ryoun, Holcuilcu 41, CDC Kendall, CDC
Copeland, CDC Reserve, Lofty Nijo), in a 400 L pilot plant (Table 5).
[Table 5]
(Table 5. Summary of analysis of test beer samples)
Malt KI Beer BU NIBEM Protein Z7
(sec.) (ug/mL)
Mean 46.0 21.7 250 6.88
S.D. 3.7 2.1 17 5.01
Maximum 54.0 25.3 285 17.06
Minimum 39.4 16.3 219 1.02
[0113] [Measurement of protein Z7 concentration in test beer]
Quantitation of protein Z7 in the beer was performed by ELISA
described above.
[0114] [Measurement of NIBEM value of test beer]
The NIBEM value measurement was carried out using an INPACK2000
NIBEM-T apparatus by Haffinans BV and a standard glass for NIBEM
value measurement. Specifically, each test beer was brought to 20 C
and poured into a standard glass using carbon dioxide gas in a foam
dispenser, and measurement was performed by using the NIBEM-T
apparatus to follow collapse of the height of the produced foam.
[0115] [Relationship between beer protein Z7 concentration and
NIBEM value]
The protein Z7 content of ordinary malt beer was measured, and the
relationship with NIBEM value was examined (Fig. 6). A significant
negative correlation was observed between protein Z7 concentration and
NIBEM value. On the other hand, the Kolbach index (KI), which is
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one indicator of the degree of decomposition of protein in the malting
step, and the BU value, which is associated with iso-a acid in hops and
is an indicator of bitterness, are considered to be closely related to foam
stability (J. Am. Soc. Brew. Chem., Vol. 60, p.47-57, 2002). For the
analysis there were used 42 beer samples with 11 malt varieties, and the
variation in malt KI or beer BU was also large, as shown in Table 1. A
close relationship between malt KI or BU and foam stability has been
reported, but since a significant correlation was exhibited between
protein Z7 content and NIBEM value in the sample population which
had large variation in KI or BU, this suggested that protein Z7 serves as
an effective marker for the NlBEM value.
[0116] Among the few reports that have been hitherto published
discussing protein Z7 and foam stability, Evans et al. examined malt
protein Z7 content and reported no significant correlation with foam
stability (Non-patent document 5). It is well known that the sample
population used is extremely important for statistical analysis. The
ratio between minimum and maximum values for protein Z7 in the
sample population used by Evans et al. was 4.81, which is much lower
than the ratio between minimum and maximum values in the samples
used for this experiment (16.73). It is possible that the sample
population used by Evans et al. was not sufficient to elucidate the
relationship between protein Z7 and foam stability.
[0117] In other words, it was demonstrated that the selection method of
the invention, wherein barley species are selected based on protein Z7
content utilizing the selection polymorphic markers described above, is
also useful for breeding of barley with excellent foam stability.
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[Sequence Listing Free Text]
[0118] SEQ rD NO: 1; Haruna-type
SEQ ID NO: 2; Kendall-type
SEQ ID NO: 3; Barke-type
SEQ ID NO: 4-9: Synthetic primers
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SEQUENCE LISTING IN ELECTRONIC FORM
In accordance with Section 111(1) of the Patent Rules, this description
contains a sequence listing in electronic form in ASCII text format (file:
78233-52 SEQ 17-03-2012 vl.txt).
A copy of the sequence listing in electronic form is available from the
Canadian Intellectual Property Office.
The sequences in the sequence listing in electronic form are reproduced in
the following table.
SEQUENCE TABLE
<110> SAPPORO BREWERIES CO LTD.
<120> A selection method of the barley class based on protein Z7 contents and
malt fermented drink thereof.
<130> G20100006W0
<160> 9
<170> PatentIn version 3.1
<210> 1
<211> 424
<212> DNA
<213> Hordeum sp.
<400> 1
tggtcacatg acgtgtatta atctccacat aactccaatg ttttagagaa ttcagacgtg 60
ttccagaagt tggtggctct ccgagatata tacctttgtt gtgagataca tatccttctc 120
aaattgcttg tacgctgtct tttggcctgc acactccaga taactccaag ttctgaccta 180
aacgcacctg ttaaatatcc acccaacacc catgcacact gtacttccca gtcattccgt 240
cttcttctct cagatctaag caaaaagtca gacgaaccac aaccaccgcc atggcaacca 300
ccctcaccac cgacctccgc ctctccatcg cgcaccaaac ccgattcggc ctccgcctcg 360
cctccgccat ctcctccgac cccgagtctg ctgccaccaa cgttgctttc tccccggtct 420
cgct 424
<210> 2
<211> 471
<212> DNA
<213> Hordeum sp.
<400> 2
tggtcacatg acgtgtatta atctccacat aactccaatg ttttagagaa ttcagacgtg 60
tgccagaagt tggtggctct ccgagatata tacgatcgat ttcttccgag aaccagatga 120
cgcgacggtg actaattaaa gggcacctaa aagccacttt tatttgcctt tatattgtga 180
gatacatagc cctctcaagt tgcttgtacg ctgtcttttg gccggcgctc tgcaccctgt 240
taaatatcca cccaacaccc atgcaaactg tacttcccat tcattccctc ttcttctcaa 300
atctaagcaa aaagtcagac gaaccacaac caccgccatg gcaaccaccc ttgccaccga 360
43a
CA 02767145 2012-03-22
=
cctccgcctc tccatcgcgc accaaacccg attcggcctc cgcctcgcct ccgccatctc 420
ctccaacccc gagtctgctg ccaccaacgt tgctttctcc ccggtctcac t 471
<210> 3
<211> 424
<212> DNA
<213> Hordeum sp.
<400> 3
tggtcacatg acgtgtatta atctccacat aactccaatg ttttagagaa ttcagacgtg 60
ttccagaagt tggtggctct ccgagatata tacctttgtt gtgagataca tatccttctc 120
aaattgcttg tacgctgtct tttggcctgc acactccaga taactccaag ttctgaccta 180
aacgcacctg ttaaatatcc acccaacacc catgcacact gtacttccca gtcattccgt 240
cttcttctct cagatctaac ctaaaagtca gacgaaccac aaccaccgcc atggcaacca 300
cccttgccac cgacctccgc ctctccatcg cgcaccaaac ccaattcggc ctccgcctcg 360
cctccgccat ctcctccaac cccgactctg ctgccaccaa cgtcgctttc tccccggtct 420
cact 424
<210> 4
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Random primer
<220>
<221> misc_feature
<222> 3
<223> Any nucleotide
<220>
<221> misc feature
<222> 7
<223> strong interactions, 3H-bonds
<220>
<221> miscjeature
<222> 8
<223> weak interactions, 2H-bonds
<220>
<221> misc_feature
<222> 11
<223> Any nucleotide
<220>
<221> misc_feature
<222> 13
<223> weak interactions, 2H-bonds
<400> 4
gtncgaswca nawgtt 16
4 3b
CA 02767145 2012-03-22
<210> 5
<211> 23
<212> DNA
<213> Artificial sequence
<220>
<223> specific primer 1
<400> 5
cgttggtggc agcagactcg ggg 23
<210> 6
<211> 23
<212> DNA
<213> Artificial sequence
<220>
<223> Specific primer 2
<400> 6
ggtcggagga gatggcggag gcg 23
<210> 7
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223> Specific primer 3
<400> 7
ggtcggtggt gagggtggtt gcca 24
<210> 8
<211> 25
<212> DNA
<213> Artificial sequence
<220>
<223> CAPS1 primer
<400> 8
ggtcacatga cgtgtattaa tctcc 25
<210> 9
<211> 23
<212> DNA
<213> Artificial sequence
<220>
<223> CAPS2 primer
<400> 9
cgttggtggc agcagactcg ggg 23
43c
