CA2767145C - Method for selection of barley species based on protein z7 content, and fermented malt beverage - Google Patents

Method for selection of barley species based on protein z7 content, and fermented malt beverage Download PDF

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CA2767145C
CA2767145C CA2767145A CA2767145A CA2767145C CA 2767145 C CA2767145 C CA 2767145C CA 2767145 A CA2767145 A CA 2767145A CA 2767145 A CA2767145 A CA 2767145A CA 2767145 C CA2767145 C CA 2767145C
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Takashi Iimure
Makoto Kihara
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Sapporo Breweries Ltd
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    • C12Q2600/13—Plant traits
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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    • C12Q2600/156—Polymorphic or mutational markers

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Abstract

The invention provides a selection method, 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 selecting a barley species specimen having the genotype identical to the Haruna-type genotype as a barley species with high protein Z7 content, or 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.

Description

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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 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, [Non-patent document 2] J. Agric. Food. Chem., Vol. 56, p.1458-1464, [Non-patent document 3] J. Agric. Food. Chem., Vol. 56, p.8664-86'71, [Non-patent document 4] J. Am. Soc. Brew. Chem., Vol. 60, p.47-57, [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 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 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 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 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.

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 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 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 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 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
=
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 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
[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 =
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 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 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 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 =
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 =
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 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 =
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 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 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 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

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 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 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.
[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 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 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 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 , 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.
[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 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 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) 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 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 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 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 B arke-type 0 X 401 251 Haruna-type 0 0 401 251
[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 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 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-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 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.

[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 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 =
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 <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

Claims (9)

CLAIMS:
1. 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.
2. 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 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 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.
3. The method according to claim 1 or claim 2, wherein identification of the at least one genotype is 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.
4. The method according to any one of claims 1 to 3, wherein the polymorphic marker A is the nucleotide position corresponding to the 62nd, 93-94th, 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 specified by SEQ ID NO: 1.
5. The method according to any one of claims 1 to 4, wherein the polymorphic marker B is the nucleotide position corresponding to the 260th, 262nd, 305-306th, 343rd, 378th, 386th or 422nd nucleotide of the nucleotide sequence specified by SEQ
ID NO: 1.
6. The method according to claim 3, wherein identification of the at least one genotype is performed based on the number and/or sizes of fragments obtained by digesting a polynucleotide including at least one of the selection polymorphic markers with one or more restriction enzymes having a recognition sequence containing at least one of the selection polymorphic markers.
7. The method according to claim 6, wherein the polynucleotide includes selection polymorphic markers corresponding to the 253rd and 343rd nucleotides of the nucleotide sequence specified by SEQ ID NO: 1, and the restriction enzyme is BglII and/or Hinfl.
8. The method according to claim 6 or claim 7, wherein the polynucleotide is amplified by PCR from genomic DNA of the barley species specimen, using a primer pair having the nucleotide sequences specified by SEQ ID NO: 8 and SEQ ID NO: 9, and the restriction enzyme is BglII and/or HinfI.
9. A kit for selection of barley species based on protein Z7 content, comprising a primer pair having the nucleotide sequences specified by SEQ ID NO: 8 and SEQ
ID NO: 9.
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