AU2010255180B2 - Method for selection of barley species for fermented malt beverage, and fermented malt beverage - Google Patents

Abstract

A selection method for selecting a barley species for a fermented malt beverage having excellent head-retaining properties, which comprises: identifying the genotype of at least one DNA marker that is specified by the alignment of (a) the nucleotide sequence for a region around Haruna-type barley protein Z4 genetic locus with (b) the nucleotide sequence for a region around Copeland-type barley protein Z4 genetic locus in barley species to be tested; and selecting a barley species of which the genotype corresponds with the Copeland-type genotype as a barley species having excellent head-retaining properties.

Description

FP10-0256-00 DESCRIPTION METHOD FOR SELECTION OF BARLEY SPECIES FOR FERMENTED MALT BEVERAGE, AND FERMENTED MALT BEVERAGE 5 Technical Field [0001] The present invention relates to a method for selection of barley species for a fermented malt beverage, and to a fermented malt beverage. Background Art 10 [0002] Foam on fermented malt beverages such as beer consists of aggregates of carbon dioxide gas bubbles surrounded by a thin liquid film, and it acts as a covering to prevent escape of gas and alteration of flavor, while also enhancing the aroma. The foam stability is therefore an important factor determining the quality of fermented malt beverages 15 such as beer. [0003] The degree of foam stability depends on a variety of factors. One of the factors is thought to be the involvement of proteins, and proteins such as protein Z (Z4, Z7) and LTP-1 have hitherto been considered to be proteins related to foam stability (see Non-patent 20 documents 1-3, for example). [0004] Protein Z4 has been reported to be involved in foam stability (Non-patent document 4), but it has also been suggested that it is not directly involved in foam stability (Non-patent document 5). Therefore, there is a room for argument in the relationship between 25 protein Z4 and foam stability. On the other hand, a Restriction Fragment Length Polymorphism (RFLP) marker for the seed protein Z4 1 C:\NRPrtb\DCC\REC\4821561_I.DOC-16M/2113 -2 content has already been developed (Non-patent document 6). Citation List Non-Patent Literature [0005] [Non-patent document 1] J. Agric. Food. Chem., Vol. 56, p.1458-146 4 , 2008 [Non-patent document 2] J. Agric. Food. Chem., Vol. 56, p.8664-8671, 2008 [Non-patent document 3] J. Am. Soc. Brew. Chem., Vol. 60, p.47-57, 2002 [Non-patent document 4] J. Inst. Brew., Vol. 105, p.171-1777, 1999 [Non-patent document 5] Proc. Congr. Eur. Brew. Conv., Vol. 22, p.561 568, 1989 [Non-patent document 6] Breed. Sci., Vol. 49, p.69-74, 1999 Summary of Invention [0006] For breeding of barley for foamed malt beverages, it is essential and important for the traits of barley with excellent foam stability to be reliably transmitted to progeny. In recent years, in the field of crop breeding, screening techniques for crops with desired traits have been developed in a variety of traits, which employ DNA markers for the traits. However, no effective DNA marker for foam stability has yet been reported. [0007] Non-patent document 6 has disclosed an RFLP marker for protein Z4 content in barley seed. However, RFLP is a method based on restriction enzyme cleavage and Southern hybridization of genomic DNA. Therefore, the procedure of RFLP is cumbersome, and a long C \NRPonbRDCCREC\I4821561_ .DOC-16/01/2013 -3 period of time is necessary for discrimination of genotypes, which the screening method is not suitable for processing of a large number of specimens. [0008] In one or more aspects the invention may advantageously provide a selection method that allows more reliable selection of barley species for fermented malt beverages with excellent foam stability by an easy procedure, allowing discrimination of genotypes in a short period of time and permitting processing of a large number of specimens. Means for Solving the Problems [0009] The invention provides a selection method for selection of barley species for a fermented malt beverage with excellent foam stability, which comprises: identifying the genotype of at least one DNA marker specified by alignment of the nucleotide sequence of a region around the Haruna type barley protein Z4 gene locus and the nucleotide sequence of a region around the Copeland-type barley protein Z4 gene locus in a barley species specimen, and isolating a barley species specimen having the genotype identical to the Copeland-type genotype as a barley species with excellent foam stability. [0010] Here, "Haruna-type" is a barley species wherein the genotype of the DNA marker present in the region around protein Z4 gene locus matches the genotype of the same DNA marker in Haruna Nijo species. An example of a nucleotide sequence of the region around "Haruna type" protein Z4 gene locus is the nucleotide sequence listed as SEQ ID NO: 1. [0011] Similarly, "Copeland-type" is a barley species wherein the FP1O-0256-00 genotype of the DNA marker present in the region around protein Z4 gene locus matches the genotype of the same DNA marker in Copeland species. An example of a nucleotide sequence of the region around "Copeland-type" protein Z4 gene locus is the nucleotide sequence listed 5 as SEQ ID NO: 2. [0012] By alignment of the nucleotide sequence of the region around the Haruna-type barley protein Z4 gene locus and the nucleotide sequence of the region around the Copeland-type barley protein Z4 gene locus, it is possible to identify nucleotide positions where the two 10 nucleotide sequences do not match. The nucleotide positions identified as such are specified as DNA markers. Several DNA markers are present in the region around barley protein Z4 gene locus, and the genotype of at least one of the DNA markers in the barley species specimen is identified and a barley species specimen matching 15 the Copeland-type genotype is selected. [0013] It is sufficient if at least one of the genotypes identified in the barley species specimen matches the Copeland-type genotype. [0014] In the selection method of the invention, a novel DNA marker correlated with barley protein Z4 content is utilized for selection of a 20 barley species specimen based on its genotype, thereby allowing more reliable selection of a barley species specimen with a high protein Z4 content. [0015] The present inventors have confirmed that a significant correlation exists between protein Z4 content and foam stability in beer, 25 as described in detail below (see Fig. 1), and have found that protein Z4 content can serve as an effective marker for foam stability. The 4 FP10-0256-00 selection method therefore allows selection of barley species specimens with excellent foam stability. [0016] The foam stability of a fermented malt beverage can be assessed, for example, based on the time (seconds) required for the foam height to 5 collapse 30 mm after the fermented malt beverage at 20'C has been poured into a standard glass with a foam dispenser (this is the NIBEM value). [0017] DNA markers specified by alignment of the nucleotide sequences listed as SEQ ID NO: 1 and SEQ ID NO: 2 are examples of 10 such DNA markers. For example, they include the nucleotide positions corresponding to the 12th, 14th, 46th, 240th, 286-288th, 866th or 952nd nucleotides of the nucleotide sequence listed as SEQ ID NO: 1 (hereunder referred to as M1, M2, M3, M4, M5, M6 and M7, in that order). 15 [0018] The invention provides a method, which comprises (1) amplifying a polynucleotide including at least one of the aforementioned DNA markers by PCR using DNA of a barley species specimen as template, (2) identifying the nucleobase type of at least one of the DNA markers by using the polynucleotide, and (3) selecting a 20 barley species whose identified genotype matches the Copeland-type genotype as a barley species with excellent foam stability. [0019] This selection method allows a large number of specimens to be processed, since it allows the genotype of a DNA marker to be identified in a short period of time by a basic and relatively easy 25 procedure comprising a step of extracting DNA from barley, a step of amplifying a polynucleotide including the DNA marker by PCR, and a 5 FP10-0256-00 step of analyzing the sequence of the polynucleotide. [0020] The invention further provides a method, which comprises (1) amplifying a polynucleotide including at least one of the aforementioned DNA markers by PCR using DNA of a barley species 5 specimen as template, (2) reacting the polynucleotide with a restriction enzyme, (3) identifying the genotype by whether or not the cleavage of the polynucleotide occurs by the restriction enzyme at the DNA marker position, and (4) selecting a barley species whose identified genotype matches the Copeland genotype as a barley species with excellent foam 10 stability. [0021] In this selection method, it is possible to identify the genotype of a DNA marker by a basic and relatively easy procedure comprising a step of extracting DNA from barley species, a step of amplifying a polynucleotide including at least one DNA marker by PCR, a step of 15 reacting it with a restriction enzyme, and a step of detecting the DNA fragment size by agarose gel electrophoresis. Also, the time required for the step of detecting the DNA fragment size is short, making it possible to process a large number of specimens. Furthermore, since decoding of the nucleotide sequence is not necessary, it is possible to 20 minimize cost for identification of the genotype. [0022] The invention still further provides a barley species derived from a progeny line of a barley species selected by the selection method described above. [0023] The invention still further provides a method for producing a 25 fermented malt beverage comprising at least a mashing step and a fermentation step, wherein the barley species used in the mashing step is 6 C:\NRPorbtbDCC\RECB2156 1. DOC-16A)1/2013 -7 a barley species selected by the selection method of the invention. The invention still further provides a fermented malt beverage that is obtained by the production method described above. [0024] A barley species selected by the selection method of the invention has a high protein Z4 content and a fermented malt beverage produced from the barley species has excellent foam stability. Effect of the Invention [0025] According to the invention it is possible to more reliably select barley species for fermented malt beverages with excellent foam stability. In addition, in selection of barley species for fermented malt beverages with excellent foam stability, it is possible to identify genotypes in a short period of time by an easy procedure, to allow processing of a large number of specimens. Brief Description of the Drawings [0026] Fig. 1 is a graph showing the relationship between protein Z4 content and NIBEM value for beer. Fig. 2 is an alignment diagram for the nucleotide sequences of the region around the Haruna-type and the Copeland-type barley protein Z4 gene locus specified by SEQ ID NO: 1 and SEQ ID NO: 2, and the nucleotide sequence of the region around the Kendall-type barley protein Z4 gene locus specified by SEQ ID NO: 3. The DNA markers specified by the alignment are indicated by arrows. The block outlined nucleotide sequences are the nucleotide sequences corresponding to the initiation codon (ATG) and the stop codon (TAA) of protein Z4. Fig. 3 is a photograph for visualization of the electrophoresis pattern FP10-0256-00 obtained by amplification of polynucleotide comprising the DNA marker M3 by PCR, reaction with restriction enzyme AccI, and then agarose gel electrophoresis and ethidium bromide staining. The letter H or C over each lane indicates that the genotype of the barley species 5 specimen matches the Haruna-type genotype or the Copeland-type genotype. Fig. 4 is a graph comparing average protein Z4 contents of known barley species selected as the Haruna-type and the Copeland-type by the method shown in Fig. 3. 10 Fig. 5 is a graph comparing average protein Z4 contents of Mikamo Golden/Harrington barley doubled haploid lines, selected as the Haruna type and the Copeland-type by the method shown in Fig. 3. Fig. 6 is a graph comparing average protein Z4 contents of barley species selected as the Haruna-type and the Copeland-type by 15 identification of the nucleobase types of DNA markers Ml, M2, M3, M4, M6 and M7. Fig. 7 is a graph comparing average protein Z4 contents of barley species selected as the Haruna-type and the Copeland-type by identification of the nucleobase type of DNA marker M5. 20 Fig. 8 is a marker map of the 4H chromosome containing the protein Z4 gene locus. Fig. 9 is a graph comparing average protein Z4 contents of Mikamo Golden/Harrington barley doubled haploid lines, selected as the Haruna type and the Copeland-type by the RFLP marker MWG2033Dra. 25 Embodiments for Carrying Out the Invention [0027] Embodiments for carrying out the invention will now be 8 FP10-0256-00 explained in further detail. [0028] The invention provides a selection method for selection of barley species for a fermented malt beverage with excellent foam stability, wherein: 5 identifying the genotype of at least one DNA marker specified by alignment of the nucleotide sequence of a region around the Haruna type barley protein Z4 gene locus and the nucleotide sequence of a region around the Copeland-type barley protein Z4 gene locus in a barley species specimen, and selecting a barley species specimen having 10 the genotype identical to the Copeland-type genotype as a barley species with excellent foam stability. [0029] One embodiment of the invention is a selection method for selection of barley species for a fermented malt beverage with excellent foam stability, the selection method comprising: 15 a step of identifying the genotype of at least one DNA marker in a barley species specimen from among DNA markers specified by alignment of the nucleotide sequence of a region around the Haruna type barley protein Z4 gene locus and the nucleotide sequence of a region around the Copeland-type barley protein Z4 gene locus, and 20 a step of selecting a barley species specimen whose identified genotype matches the genotype of the Copeland-type DNA marker, as a barley species for a fermented malt beverage with excellent foam stability. [0030] In the present invention, the region around barley protein Z4 25 gene locus comprises not only the exons and introns of barley protein Z4 but also the DNA region associated with transcriptional control, and 9 FP1O-0256-00 its neighboring regions. The region around barley protein Z4 gene locus may be a range within 25 cM, preferably within 5 cM and more preferably within 1 cM, upstream of the ATG sequence which corresponds to the initiation codon. It may also be a range within 25 5 cM, preferably within 5 cM and more preferably within 1 cM, downstream from the TAA sequence which corresponds to the stop codon. [0031] A "cM" (centimorgan) is a unit representing the distance between genes on a chromosome as determined by a genetic method, 10 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. [0032] Specifically, the probability of recombination is no greater than 5% within 5 cM from the protein Z4 gene locus while the probability is 15 no greater than 1% within 1 cM, and therefore correlation between the DNA marker and protein Z4 content is maintained with a high degree of probability. Within this range, therefore, a barley species specimen with a high protein Z4 content can be more reliably selected in a statistically significant manner. 20 [0033] The nucleotide sequence of the region around barley protein Z4 gene locus can be obtained, for example, by using genomic DNA extracted from a barley species as template for amplification of a DNA fragment from the region around barley protein Z4 gene locus by PCR, purifying the DNA fragment if necessary, and performing sequence 25 analysis using the DNA fragment as template to determine the nucleotide sequence. 10 FP10-0256-00 [0034] The genomic DNA used may be extracted from any portion of barley, and barley leaves, stems, roots, seeds or the like may be used as the genomic DNA source. The method of extracting the genomic DNA is not particularly restricted, and any commonly employed method 5 may generally be applied. A commercially available kit may also be suitably used. [0035] The PCR primers may be designed based on nucleotide sequence information upon procuring a genomic nucleotide sequence corresponding to the region around barley protein Z4 gene locus from a 10 database such as NCBI, Gene Bank or the like. The parameters such as the primer lengths, nucleotide sequences and GC contents 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 DNA purification and sequencing may be accomplished by methods 15 commonly employed in the technical field. [0036] By the alignment of the nucleotide sequence of a region around the Haruna-type barley protein Z4 gene locus and the nucleotide sequence of a region around the Copeland-type barley protein Z4 gene locus, according to the invention, it is possible to identify nucleotide 20 positions where the two nucleotide sequences do not match. The nucleotide positions identified as such are specified as DNA markers. [0037] The nucleotide sequence of a region around the Haruna-type barley protein Z4 gene locus can be determined by the method described above, typically using genomic DNA extracted from a barley 25 Haruna Nijo species. Genomic DNA extracted from a barley species other than Haruna Nijo may also be used, examples of such barley 11 FP1O-0256-00 species including, but not being limited to, Mikamo-Golden, Ryofu, Ryoun, Hokuiku 41, Lofty Nijo, Schooner, Flanklin and Barke. [0038] The nucleotide sequence of a region around the Copeland-type barley protein Z4 gene locus can be determined by the method 5 described above, typically using genomic DNA extracted from a barley Copeland species. Genomic DNA extracted from a barley species other than Copeland may also be used, examples of such barley species including, but not being limited to, AC Metcalfe, Harrington, Flagship and Gairdner. 10 [0039] Alignment, according to the invention, means the alignment of nucleotide sequences with appropriate gaps between them to line up the corresponding nucleotide sequence sections. Multiple alignment of 3 or more nucleotide sequences will also be referred to as alignment. The gap positions inserted for optimal alignment are defined, according 15 to the invention, as DNA markers, i.e. nucleotide positions where the nucleotide sequences do not match. [0040] A known alignment program may be utilized for the alignment. For example, Clustal W, Clustal X or the like may be suitably used. [0041] Examples of DNA markers for the invention include the 20 nucleotide positions corresponding to the 12th, 14th, 46th, 240th, 286 288th, 866th and 952nd nucleotides of the nucleotide sequence specified by SEQ ID NO: 1 (Ml, M2, M3, M4, M5, M6 and M7). [0042] The invention provides a method, which comprises (1) amplifying a polynucleotide including at least one of the 25 aforementioned DNA markers by PCR using DNA of a barley species specimen as template, (2) identifying the nucleobase type of at least one 12 FP10-0256-00 of the DNA markers by using the polynucleotide, and (3) selecting a barley species whose identified genotype matches the Copeland-type genotype as a barley species with excellent foam stability. [0043] The method of extracting the genomic DNA from the barley 5 species specimen is not particularly restricted, and any commonly employed method may generally be applied. A commercially available kit may also be suitably used. While the genomic 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 10 consideration of selection at the breeding stage. By extracting from leaves, it is possible to select barley with preferred traits at an earlier stage. [0044] When a polynucleotide comprising at least one DNA marker is to be amplified by PCR from the genomic DNA, the PCR primers may 15 be designed based on the nucleotide sequence of the region around barley protein Z4 gene locus, determined during specifying the DNA marker. The length of the polynucleotide to be amplified with the primers may be 20-30,000 bp. In consideration of amplification efficiency in PCR and ease of handling during analysis of the amplified 20 polynucleotide, the upper limit is preferably 10,000 bp, more preferably 3000 bp and even more preferably 2000 bp. Similarly, the lower limit is preferably 100 bp, more preferably 300 bp and even more preferably 500 bp. A polynucleotide having such a length can be amplified by appropriate selection of the type of polymerase used for the PCR among 25 polymerases well known by those skilled in the art. [0045] The method for identifying the nucleobase type of the DNA 13 FP10-0256-00 marker using the polynucleotide may be a method of decoding of the nucleotide sequence by sequence analysis. [0046] The identified nucleobase type of the DNA marker is compared with the nucleobase type of the same DNA marker of the Haruna-type 5 and the Copeland-type, and a barley species specimen is selected that matches the Copeland-type nucleobase type. This allows selection of a barley species with excellent foam stability. [0047] The nucleobase type comparison may be carried out for a single DNA marker, or it may be carried out for two or more DNA markers. 10 When comparison has been made for two or more DNA markers, a barley species is selected in which at least one of the DNA markers matches the Copeland-type. This also allows selection of a barley species with excellent foam stability. [0048] The invention further provides a method, which comprises (1) 15 amplifying a polynucleotide including at least one of the aforementioned DNA markers by PCR using DNA of a barley species specimen as template, (2) reacting the polynucleotide with a restriction enzyme, (3) identifying whether or not cleavage of the polynucleotide occurs by the restriction enzyme at the DNA marker position, and (4) 20 selecting a barley species whose identified genotype matches the Copeland-type genotype as a barley species with excellent foam stability. [0049] A nucleotide sequence may be formed that can be recognized by a restriction enzyme at the DNA marker position, for either the Haruna 25 type or the Copeland-type, depending on the genotype of the DNA marker. In such cases, a polynucleotide comprising the DNA marker 14 FP10-0256-00 may be amplified by PCR and then the genotype can be identified by judging whether or not cleavage occurs by the restriction enzyme. [0050] The method of extracting the genomic DNA from the barley species specimen, and the portion from which the genomic DNA is 5 extracted, are the same as described above. [0051] Design of the PCR primers for the step of amplifying the polynucleotide comprising at least one DNA marker by PCR, is also the same as described above. There are no particular restrictions on the length of the polynucleotide to be amplified by the primers, but 10 considering that the restriction fragments are to be detected with fragment sizes in later steps, the upper limit is preferably 5000 bp, more preferably 3000 bp and even more preferably 2000 bp. Similarly, the lower limit is preferably 200 bp, more preferably 300 bp and even more preferably 500 bp. A polynucleotide having such a length can be 15 amplified by appropriate selection of the type of polymerase used for the PCR among polymerases well known by those skilled in the art. [0052] The reaction with the restriction enzyme may be carried out with a known optimum buffer at the optimum reaction temperature. The step of detecting the DNA fragment size by agarose gel electrophoresis 20 may also be carried out by a method commonly employed by those skilled in the art. [0053] As a result of detecting the DNA fragment sizes by agarose gel electrophoresis, it is possible to judge whether or not cleavage has occurred with the restriction enzyme at the DNA marker position, based 25 on the number of DNA fragments and the DNA fragment sizes. This may be defined as the genotype, and a barley species specimen 15 FP10-0256-00 matching the Copeland-type genotype may be selected. [0054] The invention still further provides a barley species derived from a progeny line of a barley species selected by the selection method described above. 5 [0055] The barley species selected by the aforementioned selection method has a genomic DNA nucleotide sequence that matches the Copeland-type, and breeding between these barley species results in, with high probability, progeny line barley species that are also the Copeland-types, with a high protein Z4 content. They are therefore 10 useful as barley species for fermented malt beverages with excellent foam stability. [0056] 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 15 a barley species selected by the selection method of the invention, or a barley species derived from a progeny line of a barley species selected by the selection method of the invention. The invention still further provides a fermented malt beverage that is obtainable by the production method described above. 20 [0057] It is possible to obtain malt by malting a barley species selected according to the invention, or a barley species derived from a progeny line of a barley species selected by the selection method of the invention. The method of malting is not particularly restricted, and any known method may be used. Specifically, for example, steeping 25 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 16 FP10-0256-00 malt. [0058] The mashing step is a step in which raw materials 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 5 from the saccharified malt or barley is obtained. In the mashing step, in addition to malt obtained from a barley species selected according to the invention or a barley species derived from a progeny line of a barley species selected by the selection method of the invention, there may even be used the barley species itself selected according to the 10 invention, or barley species itself derived from a progeny line of a barley species selected by the selection method of the invention. 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. 15 [0059] 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 here may be, for example, Saccharomyces cerevisiae or Saccharomyces uvarum. 20 [0060] The fermented malt beverage may be the beverage that may be produced by fermentation using malt as a portion of the raw materials, 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 25 also fermented malt beverages since similar production methods are used as for beer. 17 FP1O-0256-00 Examples [0061] The present invention will now be explained in more specific detail based on examples, with the understanding that the invention is in no way limited to the examples. 5 [0062] (Preliminary test: Relationship between beer protein Z4 content and NIBEM value) A total of 42 test beers were used as samples, being brewed from malt of 11 different varieties (Haruna Nijo, Amagi Nijo, Mikamo-Golden, Nitta Nijo 23, Ryofu, Ryoun, Hokuiku 41, CDC Kendall, CDC 10 Copeland, CDC Reserve, Lofty Nijo), in a 400 L pilot plant. [0063] [Measurement of protein Z4 concentration in test beers] The protein Z4 concentration of the beer was measured by the sandwich ELISA method. Rabbit anti-protein Z4 antibody, prepared using purified barley protein Z4 as antigen, was used as the primary antibody, 15 and peroxidase-conjugated anti-protein Z4-Fab' was used as the secondary antibody. After applying 100 pL of primary antibody to a 96-well plate, it was allowed to stand overnight at 4*C. After blocking with casein solution, 100 p.L of each appropriately diluted beer sample was added, reaction was conducted at room temperature for 2 hours, and 20 then washed 3 times with TBS (20 mM Tris-HCl, 150 mM NaCl, 0.05% Tween20, pH 7.5). Secondary antibody was added next, reaction was conducted for 2 hours, and washing was performed 3 times with TBS. Detection was performed by development with a POD colorimetric kit (Sumitomo Bakelite Co., Ltd.), followed by measurement of the 25 absorbance at 492 nm. A calibration curve was drawn for the absorbance of a purified barley protein Z4 standard solution of known 18 FP10-0256-00 concentration, and the protein Z4 concentration of the sample was calculated based on the relative absorbance of the sample. The barley protein Z4 standard solution used was obtained by purifying protein Z4 from barley, freeze-drying it, and then dissolving a weighed portion 5 thereof in Phosphate Buffered Saline (PBS). [0064] [Measurement of NIBEM value of test beer] The NIBEM value measurement was carried out using an INPACK2000 NIBEM-T apparatus by Haffmans BV and a standard glass for NIBEM value measurement. Specifically, each test beer was brought to 20"C 10 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. [0065] [Relationship between protein Z4 concentration and NIBEM value] 15 A significant correlation was found between protein Z4 concentration and NIBEM value (Fig. 1). 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 20 related to foam stability (J. Am. Soc. Brew. Chem., Vol. 60, p.47-57, 2002). For the analysis there were used 42 samples with 11 malt varieties, and the variation in malt KI or beer BU was also large, as shown in Table 1. Since a significant correlation was exhibited between protein Z4 and NIBEM value in the parent population which 25 had large variation in KI or BU, this demonstrated that protein Z4 serves as an effective marker for the NIBEM value. 19 FP10-0256-00 [0066] [Table 1] Table 1. Summary of test beer sample analysis Malt KI Beer BU NIBEM Protein Z4 (sec.) (g/mL) Average 46.0 21.7 250 8.61 S.D. 3.7 2.1 17 3.70 Maximum 54.0 25.3 285 17.49 Minimum 39.4 16.3 219 2.42 [0067] (Example 1: Identification of DNA marker) [Nucleotide sequence analysis] 5 Genomic DNA was extracted by the following method from Haruna Nijo, CDC Copeland and CDC Kendall barley leaves. After adding extraction buffer (200 mM Tris-HCl, 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. An equal amount of isopropanol was added to 10 the supernatant following centrifugal separation for precipitating the DNA, and after centrifugal separation, 70% ethanol was added to the precipitate, and centrifugation was performed again. The produced DNA precipitate was dissolved in sterilized water and used as template for PCR. Nucleotide sequence information for protein Z4 was 15 acquired from the NCBI database, sense primers and antisense primers were designed to include the gene region and the region upstream from the translation initiation site, and PCR was conducted using a Premix Taq (Ex Taq Version) by Takara Bio, Inc. for amplification of the DNA fragment. In the PCR, the target sequence was amplified as 2 20 fragments using a combination of sense primer 1 and antisense primer 1 and a combination of sense primer 2 and antisense primer 2. The sizes 20 FP10-0256-00 of the amplification products were 2.0 and 1.7 kbp, respectively. The primer sequences were as follows. Sense primer 1 (SEQ ID NO: 4); 5'-GGAGTATATGAGGGCTCGCG-3' 5 Antisense primer 1 (SEQ ID NO: 5); 5'-CCCTTCGCGTAAGGAAGCTT-3' Sense primer 2 (SEQ ID NO: 6); 5'-GGTGGTGCAATTTGTGCTCGC-3' Antisense primer 2 (SEQ ID NO: 7); 10 5'-CTTTGAAACCGCGGTCTGTAC-3' [0068] Each amplification product was separated by agarose gel electrophoresis, the DNA fragment of the target size was cut out of each gel, and the DNA fragment was purified with a QIAquick Gel Extraction Kit by Qiagen. The sequence of the DNA fragment was 15 then determined. Sequence analysis was performed by consignment to Sigma Corp. The determined Haruna Nijo, CDC Copeland and CDC Kendall barley nucleotide sequences are listed as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively. [0069] [Nucleotide sequence alignment analysis] 20 Nucleotide sequence alignment analysis was performed with a GENETYX Ver.8 (Genetyx Corporation) using the nucleotide sequences listed as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3. As shown in Fig. 2, seven positions (9 nucleotides) were identified as nucleotide positions with non-matching nucleotide sequences, within a region of 25 1853 bp length from the position 318 bp upstream from the start codon of the protein Z4 gene to the position of the stop codon of the protein Z4 21 FP10-0256-00 gene. [0070] The seven nucleotide positions were the nucleotide positions corresponding to the 12th, 14th, 46th, 240th, 286-288th, 866th and 952nd nucleotides, respectively, of the nucleotide sequence specified by 5 SEQ ID NO: 1. These were designated as DNA markers Ml, M2, M3, M4, M5, M6 and M7, respectively. [0071] (Example 2: Verification of selection effect for known barley species using DNA marker M3) Using the 16 barley varieties CDC Kendall, AC Metcalfe, Harrington, 10 CDC Copeland, Ryoun, Ryofu, Hokuiku 41, Haruna Nijo, Lofty Nijo, Schooner, Flagship, Flanklin, Gairdner, Barke, Braemer and Scarlett for analysis, the aforementioned DNA marker M3 was used to identify the genotype, in order to verify whether barley species with high protein Z4 contents can be selected based on the identified genotype. 15 [0072] [Identification of DNA marker M3 genotype] Genomic DNA was extracted from the aforementioned 16 varieties, PCR was conducted using the genomic DNA as template, and an approximately 2000 bp amplified DNA fragment was obtained containing the DNA marker M3. The primers used were those listed as 20 SEQ ID NO: 4 and SEQ ID NO: 5. [0073] Restriction enzyme AccI (product of Takara Bio, Inc.) was added to the obtained PCR product, and reaction was conducted overnight at 37"C. The reaction product was separated by 1.5% agarose gel electrophoresis, and the electrophoresis pattern was 25 visualized by ethidium bromide staining (Fig. 3). In the agarose gel electrophoresis image shown in Fig. 3, samples from 22 FP10-0256-00 the barley species CDC Kendall, AC Metcalfe, Harrington, CDC Copeland, Ryofu, Ryoun, Hokuiku 41, Haruna Nijo, Lofty Nijo, Schooner, Flagship, Flanklin, Gairdner, Barke, Braemer and Scarlett are loaded into each lane, from left to right. 5 The DNA marker M3 is present on the restriction enzyme AccI recognition site (GTAGAC). CDC Copeland has the nucleotide sequence "ATAGAC" in this region, and therefore the nucleotide sequence is not cleaved by AccI. While Haruna Nijo has the sequence "GTAGAC", and therefore the sequence is cleaved by AccI, producing 10 approximately 1200 bp and 800 bp DNA fragments. The analysis results indicated that, of the 16 varieties, 8 had the same electrophoresis pattern as Haruna Nijo, and 8 had the same pattern as CDC Copeland. These were classified as Haruna-type and Copeland-type. [0074] [Quantitation of protein Z4 content by ELISA] 15 Protein Z4 was quantitated by ELISA among the 16 varieties of barley seeds. ELISA was conducted by the following direct adsorption method, using rabbit anti-protein Z4 antibody with the purified barley protein Z4 as antigen. 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 20 of Phosphate Buffer Saline (PBS) containing 0.28% DTT (Wako Pure Chemical Industries, Ltd.) 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, 100 pL of each appropriately 25 diluted barley seed protein extract was applied to a 4 repeat-load 96 well plate and allowed to stand overnight at 4*C. After blocking with 23 FP10-0256-00 casein solution, the purified anti-protein Z4 antibody was applied with a protein A column, reaction was conducted at room temperature for 2 hours, and then rinsing was performed 3 times with TBS. Next, the secondary antibody (goat anti-rabbit IgG-AP, Santa Cruz 5 Biotechnology), diluted 1000-fold, was added, reaction was conducted at room temperature for 2 hours, and rinsing was performed 3 times with TBS. Development was carried out by adding 150 VL of a 1 mg/mL disodium p-nitrophenylphosphate hexahydrate (10% diethanolamine) (Wako Pure Chemical Industries, Ltd.) solution, and at 10 the stage where a moderate degree of development was seen, 50 pL of 3N NaOH was added to suspend the reaction, and the absorbance at 405 nm was measured. A calibration curve was drawn for the absorbance of a purified barley protein Z4 standard solution of known concentration, and the protein Z4 concentration of the sample was 15 calculated based on the relative absorbance of the sample. The barley protein Z4 standard solution used was obtained by purifying protein Z4 from barley, freeze-drying it, and then dissolving a weighed portion thereof in Phosphate Buffered Saline (PBS). [0075] [Comparison of Haruna-type and Copeland-type protein Z4 20 contents] The mean values were calculated for the Copeland-type and Haruna type protein Z4 contents, which had been divided into groups based on the genotype of the DNA marker M3, and these were compared (Fig. 4). The results were as follows: 25 Copeland-type: 23.5 ±4.37 ng/pg-protein Haruna-type: 16.5 ±2.83 ng/pg-protein, 24 FP10-0256-00 and statistical testing by a t-test revealed that the Copeland-type had a significantly higher protein Z4 content than the Haruna-type, with a critical rate of 1%. [0076] (Example 3: Verification of selection effect for doubled haploid 5 line barley species using DNA marker M3) In order to verify the effect of the marker in a different population, a Mikamo-Golden/Harrington doubled haploid barley line was used. Protein Z4 content was quantitated and genotype of DNA marker M3 was identified by the same procedure as Example 2. These were 10 classified into Copeland-type and Haruna-type according to the identified genotype, and the mean value of each protein Z4 content was calculated and compared (Fig. 5). [0077] The results were as follows: Copeland-type: 29.4 +19.85 ng/tg-protein 15 Haruna-type: 11.6 +4.64 ng/pg-protein, and statistical testing by a t-test revealed that the Copeland-type had a significantly higher protein Z4 content than the Haruna-type, with a critical rate of 1%. Thus, the results of Example 2 and Example 3 demonstrated that this DNA marker M3 can be effectively used as a 20 DNA marker for selection of a barley species with a high protein Z4 content. [0078] (Example 4: Verification of selection effect for known barley species using DNA markers Ml, M2, M3, M4, M6 and M7) The 14 major varieties used as barley for beer brewing were used to 25 evaluate the effectiveness of other DNA markers. The 14 varieties used for verification were Haruna Nijo, Ryofu, Ryoun, Hokuiku 41, 25 FP10-0256-00 Lofty Nijo, Schooner, Flanklin, Barke, CDC Kendall, AC Metcalfe, CDC Copeland, Harrington, Flagship and Gairdner. [0079] [Nucleotide sequence analysis] The same method as in Example 1 was used for extraction of genomic 5 DNA from the 14 varieties and PCR using the genomic DNA as template, to amplify DNA fragments containing all of the DNA markers M1-M7, and the nucleobase types of the DNA markers were identified by sequence analysis. [0080] [Nucleotide sequence comparison] 10 The genotype of each of the DNA markers of the 14 varieties was compared with the Haruna-type and Copeland-type genotypes. As a result, the 8 varieties Haruna Nijo, Ryofu, Ryoun, Hokuiku 41, Lofty Nijo, Schooner, Flanklin and Barke were classified as Haruna-types while the 6 varieties CDC Kendall, AC Metcalfe, CDC Copeland, 15 Harrington, Flagship and Gairdner were classified as Copeland-types (Table 2), based on the genotypes of the DNA markers Ml, M2, M3, M4, M6 and M7. [0081] [Table 2] 26 FP10-0256-00 Table 2: Genotype classification for DNA markers M1 to M7 in 14 major barley varieties MI M2 M3 M4 M5 M6 M7 Haruna Nijo H H H H H H H Ryofu H H H H H H H Ryoun H H H H H H H Hokuiku 41 H H H H H H H Lofty Nijo H H H H H H H Schooner H H H H H H H Flanklin H H H H H H H Barke H H H H H H H CDC Kendall C C C C H C C AC Metcalfe C C C C C C C CDC Copeland C C C C C C C Harrington C C C C C C C Flagship C C C C C C C Gairdner C C C C C C C C: Copeland-type, H: Haruna-type [0082] [Quantitation of protein Z4 content by ELISA] The protein Z4 contents of seeds of the 14 varieties were quantitated by the same method as described in Example 2. 5 [0083] The mean values of the Copeland-type and Haruna-type protein Z4 contents were calculated and compared (Fig. 6). The results were as follows: Copeland-type: 25.2 ±3.57 ng/pg-protein Haruna-type: 16.5 ±2.83 ng/pg-protein, 10 and statistical testing by a t-test revealed that the Copeland-type had a significantly higher protein Z4 content than the Haruna-type, with a critical rate of 1%. The results demonstrated that these DNA markers M1, M2, M3, M4, M6 and M7 can be effectively used as DNA markers 27 FP10-0256-00 for selection of barley species with high protein Z4 contents. [0084] Based on these results, it will be self-evident to a person skilled in the art that any DNA marker not mentioned in the examples, that can be located in the same manner as these DNA markers Ml, M2, M3, M4, 5 M6 and M7 when alignment analysis has been conducted with the Haruna-type and Copeland-type nucleotide sequences in the vicinity of the barley protein Z4 gene, can be effectively utilized as a DNA marker for selection of a barley species with excellent foam stability. [0085] (Example 5: Verification of selection effect for known barley 10 species using DNA marker M5) The 14 major varieties used as barley for beer brewing were used to evaluate the effectiveness of other DNA markers. The 14 varieties used for verification were Haruna Nijo, Ryofu, Ryoun, Hokuiku 41, Lofty Nijo, Schooner, Flanklin, Barke, CDC Kendall, AC Metcalfe, 15 CDC Copeland, Harrington, Flagship and Gairdner. [0086] [Nucleotide sequence analysis] In the same manner as Example 4, genomic DNA was extracted from the aforementioned 14 varieties and PCR was conducted using the genomic DNA as template, to amplify DNA fragments containing all of 20 the DNA markers M1-M7, and the nucleotide types of the DNA markers were identified by sequence analysis. [0087] [Nucleotide sequence comparison] The genotype of each of the DNA markers of the 14 varieties was compared with the Haruna and Copeland genotypes. As a result, the 9 25 varieties Haruna Nijo, Ryofu, Ryoun, Hokuiku 41, Lofty Nijo, Schooner, Flanklin, Barke and CDC Kendall were classified as Haruna 28 FP10-0256-00 types while the 5 varieties AC Metcalfe, CDC Copeland, Harrington, Flagship and Gairdner were classified as Copeland-types (Table 2), based on the genotype of the DNA marker M5. [0088] [Quantitation of protein Z4 content by ELISA] 5 The protein Z4 contents of seeds of the 14 varieties were quantitated by the same method as described in Example 2. [0089] The mean values of the Copeland-type and Haruna-type protein Z4 contents were calculated and compared (Fig. 7). The results were as follows: 10 Copeland-type: 24.9 ±3.94 ng/pg-protein Haruna-type: 17.6 ±4.19 ng/pg-protein, and statistical testing by a t-test revealed that the Copeland-type had a significantly higher protein Z4 content than the Haruna-type, with a critical rate of 1%. The results demonstrated that this DNA marker M5 15 can be effectively used as a DNA marker for selection of a barley species with a high protein Z4 content. [0090] In Example 4, the genotypes were identified by the DNA markers MI, M2, M3, M4, M6 and M7 and barley species matching the Copeland-type genotype were selected, resulting in selection of 6 20 varieties. Also, in Example 5, the genotypes were identified by the DNA marker M5 and barley species matching the Copeland-type genotype were selected, with no Kendall varieties being selected among the 6 varieties. These examples demonstrate that varieties with high protein Z4 contents can be selected even with the DNA marker M5, 25 whose genotype matched in 5 of the 6 barley varieties selected as Copeland-types by the DNA markers Ml, M2, M3, M4, M6 and M7. 29 FP10-0256-00 [0091] (Reference Example: Selection of known barley species by RFLP marker MWG2033Dra) The range of DNA markers that can be utilized as DNA markers for the invention in the region around protein Z4 gene locus was tested. 5 [0092] Fig. 8 shows a marker map for the 4H chromosome of the barley genome, created based on the RFLP marker. The protein Z4 gene is located at 77.6 cM from the position of the RFLP marker BamyXba as the origin. In this reference example, the RFLP marker MWG2033Dra located at 99.2 cM was utilized for classification of the Mikamo 10 Golden/Harrington doubled haploid barley line, and comparison of the Haruna-type and Copeland-type protein Z4 contents. As a result of judging the genotype, the Mikamo-Golden variety was classified as Haruna-type with all of the DNA markers M1-M7 (data not shown). [0093] [Genotype screening] 15 Using leaves of Mikamo-Golden/Harrington doubled haploid barley line as specimen, genomic DNA was extracted by the CTAB method (Murray, M.G. and Thompson, W.F., 1980, Nucleic Acids Res., Vol. 8, p.4321-4325). Restriction enzyme DraI (Takara Bio, Inc.) was added to 5 ptg of each genomic DNA, and reaction was conducted overnight at 20 37 0 C. The reaction product was separated by 0.7% agarose gel electrophoresis and transferred to a nylon membrane by the alkaline transfer method (Reed, K.C. and D.A. Mann, 1985, Nucleic Acids Res., Vol. 13, p.

7 2 0 7

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72 2 1). A probe DNA fragment that had been cloned in p-Bluescript was amplified by PCR with M13 primer, and then a DIG 25 high prime kit (product of Boehringer Mannheim) was used for labeling reaction with the PCR product as template, to obtain a DIG-labeled 30 FP10-0256-00 probe. Hybridization and signal detection were accomplished according to the Boehringer DIG procedure. [0094] [Genotype classification] Barley species were classified into 2 types based on the detected band 5 pattern, with the barley species group to which Mikamo-Golden was classified being designated as Haruna-type, and the barley species group to which Harrington was classified being designated as Copeland-type. [0095] The mean values of the Copeland-type and Haruna-type protein Z4 contents were calculated and compared (Fig. 9). The results were 10 as follows: Copeland-type: 27.1 ±20.00 ng/tg-protein Haruna-type: 19.2 ±15.65 ng/pg-protein, and statistical testing by a t-test revealed that the Copeland-type had a significantly higher protein Z4 content than the Haruna-type, with a 15 critical rate of 5%. [0096] When the mean values and variation in the protein Z4 contents of the reference example and Examples 2-5 were compared, a trend was seen toward lower selection precision of barley species with greater distance between the DNA marker and the protein Z4 gene. In the 20 reference example, however, the RFLP marker MWG2033Dra was usable as a DNA marker of the invention despite its distance of 21.6 cM from the protein Z4 gene. Therefore, a DNA marker is suitable for use according to the invention so long as the distance from the protein Z4 gene is within this range. 25 Sequence Listing Text [0097] SEQ ID NO: 4-7: Synthetic primers 31 C \NRPorbl\DCC\REC\4S2156Il.DOC.16/01/2013 - 31A [0098] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. [0099] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Claims (13)

1. A selection method for selection of barley species for a fermented malt beverage with excellent foam stability, which comprises: identifying the genotype of at least one DNA marker specified by alignment of (a) the nucleotide sequence of a region around the Haruna-type barley protein Z4 gene locus and (b) the nucleotide sequence of a region around the Copeland-type barley protein Z4 gene locus in a barley species specimen, and isolating a barley species specimen having the genotype identical to the Copeland-type genotype as a barley species with excellent foam stability.

2. The selection method according to claim 1, wherein the nucleotide sequences of (a) and (b) are the nucleotide sequences specified by SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

3. The selection method according to claim I or claim 2, wherein the DNA marker is the nucleotide position corresponding to the I2th, 14th, 46th, 240th, 286-288th, 866th, or 952nd nucleotide of the nucleotide sequence specified by SEQ ID NO: 1.

4. The selection method according to claim 1 or claim 2 wherein the DNA marker is the nucleotide position corresponding to the 46th nucleotide of the nucleotide sequence specified by SEQ ID NO: 1.

5. The selection method according to any one of claims I to 4, wherein identification of the genotype comprises: amplifying a polynucleotide including at least one of the DNA markers by PCR using DNA of a barley species specimen as template, and C NRPortb\DCC\REC\482156 ILDOC-16/01/2013 -33 identifying the nucleobase type of at least one of the DNA markers by using the polynucleotide.

6. The selection method according to any one of claims I to 4, wherein identification of the genotype comprises: amplifying a polynucleotide including at least one of the DNA markers by PCR using DNA of a barley species specimen as a template, reacting the polynucleotide with a restriction enzyme, and identifying whether or not cleavage of the polynucleotide occurs by the restriction enzyme at the DNA marker position.

7. The selection method according to claim 1 substantially as hereinbefore described with reference to any one of the examples and/or figures.

8. A barley species derived from a progeny line of a barley species isolated by the selection method according to any one of claims I to 7.

9. The barley species according to claim 8 substantially as hereinbefore described with reference to any one of the examples and/or figures.

10. 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 by the selection method according to any one of claims I to 7, or a barley species according to claim 8 or claim 9.

11. The method according to claim 10 substantially as hereinbefore described with reference to any one of the examples and/or figures. C.\NRPonbNDCC\REC\4521561 I DOC- 16/)1/2013 -34

12. A fermented malt beverage that is obtained by the production method according to claim 10 or claim 11.

13. The fermented malt beverage according to claim 12 substantially as hereinbefore described with reference to any one of the examples and/or figures.