EA043494B1 - BARLEY WITH INCREASED HYDROLYTIC ENZYME ACTIVITY - Google Patents

Description

BACKGROUND OF THE INVENTION

The present invention relates to barley plants with increased activity of hydrolytic enzymes. In particular, the barley plants of the present invention may exhibit increased hydrolytic enzyme activity during the early stages of germination.

BACKGROUND OF THE INVENTION

In industrial malting processes, barley grains are germinated or malted under controlled conditions that allow partial mobilization of starch and starchy endosperm protein reserves over a period of 4-6 days. The malting process is usually initiated by immersing dry barley grains in water. This process is known as soaking, where the goal is not only to clean the grain, but also to increase the moisture content to approximately 40% (w/w) so that the subsequent stage of mobilizing the endosperm contents occurs more quickly. During soaking, the water is drained once to ensure re-aeration of the grain. This stage is known as an air rest and is considered necessary primarily because the immersed grain becomes oxygen depleted after approximately 16 hours. After an air pause of approximately 8 hours, the grain is re-immersed in water to complete the treatment by soaking for a further 8 hours or through a series of re-soaking steps. The two-step soaking process to increase the moisture content of dry grain to 40% or higher takes a total of approximately 32 hours.

The soaked grain is laid out for germination, during which enzymes secreted by the aleurone layer cells and scutellum epithelial cells, along with those already existing in the starchy endosperm cells, break down the cell walls, starch and protein. Under normal germination conditions, the phytohormone gibberellic acid (GA) is believed to be synthesized in the node region, or other area of the embryo, from where it diffuses along the movement of water.

The maltster's task is usually to quickly induce the synthesis of as many starch-degrading enzymes in the grains as possible. Under many malting conditions, GA can be added to speed up the process of enzyme release from the aleurone layer. Starch-degrading enzymes, which include α- and β-amylases, starch-degrading debranching enzymes (eg, limiting dextrinase), and α-glucosidases, partially depolymerize grain starch reserves to monosaccharides, oligosaccharides, and glucose. The products of starch depolymerization are subsequently consumed by yeast cells as a carbon source and fermented to form ethanol in beer.

A-amylases play a key role in the breakdown of starch in the endosperm. In cereal plants, the expression of α-amylases is strictly regulated. In wild-type barley, there is very little expression of α-amylase genes during endosperm growth and maturation, consistent with extensive starch accumulation during this period. During germination, α-amylase activity increases. Precise regulation of α-amylase activity is important because aberrant α-amylase activity can have serious consequences for plant health. For example, certain varieties of barley may experience premature germination. This may be associated with shoot and root growth, high α-amylase production and drying of grain at ripeness (Green et al., 1997). It has also been found that α-amylase activity is often increased in dried grains compared to normal grains (Green et al., 1997). In the case of barley cv. Himalaya carrying the sln1 mutation was also consistently observed to have both premature germination and high α-amylase production (Green et al., 1997). Overexpression of α-amylase genes in developing rice endosperm results in the formation of varying degrees of chalky grains, i.e. grains containing immature loosely packed starch granules (Nakata et al., 2017).

Maltsters also try to induce the production of high levels of enzymes that break down cell wall polysaccharides in the barley grain, particularly (1,3;1,4)-β-glucans and arabinoxylans. Incompletely degraded (1,3; 1,4)-glucans can be particularly problematic for brewers as they can be extracted from the malt in soluble forms, creating highly viscous aqueous solutions that slow down brewery filtration processes and promote the formation of unwanted turbidity in the final beer. Thus, low levels of soluble (1,3;1,4)-β-glucan are an important parameter of malting quality, while high levels of (1,3;1,4)-β-glucanases enzymes remain important indicators of malt quality.

As noted above, the germination process usually takes approximately 4-5 days. Once the controlled germination stages are complete, the wet malt is dried, changing the moisture content from approximately 40% to 4-5%. This drying process, called kiln drying, is very energy intensive and represents a major cost in production. The entire process, including oven drying, usually takes 6-7 days.

At the brewery, kiln-dried malt is ground to open up the grain, and the resulting contents are extracted using hot water in a process known as mashing. Extracted material includes partially degraded starch, protein and molecules

- 1 043494 cell wall, as described above, and the following are destroyed by the action of endogenous grain enzymes that have been extracted from the malt. At this stage, some brewers add additional, and usually cheaper, carbon sources (additives) to aid the subsequent yeast fermentation process and offset higher malt costs. These additives may be barley, rice, wheat flour or other unsprouted cereal flours, but their addition may require the concomitant addition of hydrolytic enzymes, since the amount of endogenous enzymes in the malt is insufficient to break down the additive components. Added enzymes are usually obtained from crude and relatively inexpensive extracts from fungal and/or bacterial cultures. The addition of exogenous enzymes is not permitted in some countries, in which, in particular, beer must be produced under strictly regulated conditions.

Additional destruction of starch and other endosperm components extracted in hot water occurs through a process known as saccharification. After mashing, the extracts are filtered, often in a lauter tun, and cooled. The extract can be boiled in the presence of hops or hop extracts, and after cooling, yeast cultures are added to ferment the released sugars to form ethanol. The beer thus obtained is subjected to maturation and filtration before bottling. Also, before bottling, beer can be saturated with carbon dioxide.

Brief Disclosure of the Present Invention

As stated above, one of the most time- and energy-intensive stages of beer production is malting. The rate-limiting step in the malting procedure is the synthesis of sufficient starch-degrading enzymes in the grains. Therefore, there is a need to provide materials and methods by which the amount of time required for malting can be reduced. In particular, there is a need for methods that allow cereal grains containing sufficient amounts of starch-degrading enzymes to germinate soon after germination has been initiated.

However, as noted above, high α-amylase activity is associated with undesirable effects on plant fitness. In particular, high α-amylase activity may be associated with reduced grain starch content and/or preharvest sprouting. Preharvest germination is a phenomenon in which physiologically mature grain kernels on a plant begin to behave like a germinating seed rather than a starchy grain. Pre-harvest sprouting is characterized by premature germination of grains before harvest, followed by reduced seed viability and final value. Thus, pre-harvest germination creates conditions under which barley loses seed viability relatively quickly, and since germination is necessary for the malting process, its occurrence is highly undesirable when malting barley crops. Preharvest germination is most often associated with the amount of precipitation and prolonged wet weather after the grain has fully ripened. α-amylase activity correlates with pre-emergence and premature germination and can be used as an indicator of damage due to germination (Schwarz et al., 2004).

Thus, there is a need for barley plants that have high levels of starch-degrading enzyme activity soon after initiation of germination, but which also have high starch grains that produce high yields and/or have low pre-harvest germination rates.

The present invention relates to barley plants with high levels of starch-degrading enzyme activity shortly after the initiation of germination, in particular, said barley plants are characterized by high levels of α-amylase and limiting dextrinase activity. Such barley plants are particularly suitable for processes for producing cereal grain-based beverages with reduced germination times.

One of the technical problems solved by the present invention is to provide barley plants with high activity of α-amylase and limiting dextrinase soon after the initiation of germination, for example, already 72 hours or even 48 hours after the initiation of germination, and barley plants at that time At the same time, they are characterized by acceptable agronomic characteristics. In one aspect, the present invention relates to barley plants carrying a mutation in the HRT gene. According to the present invention, it has surprisingly been demonstrated that barley plants with loss of HRT function are viable, have good agronomic properties and have yield levels comparable to those of other barley varieties. At the same time, such barley plants are characterized by high activity of α-amylase and limiting dextrinase already 48 hours after the initiation of germination. Another technical problem solved by the present invention is to provide barley plants with a sufficient degree of germination even in low oxygen conditions. Interestingly, the use of barley plants with loss of HRT function also allows one to solve this technical problem.

Raventos et al., 1998 described that the full-length protein product of the barley Hv REPRESSOR OF TRANSCRIPTION (HvHRT) gene functions as a transcriptional repressor at a number of different promoters when tested using plant cells after transient expression. Transient expression typically produces expression levels that are much higher than natural expression levels and, therefore, it is not possible to conclusively determine protein function using transient expression assays. Under such artificial conditions, transient expression of HRT results in a reduced level of expression of the reporter gene, which is under the control of different α-amylase gene promoters (Amy2 and Amy1), as well as a reduced level of expression from the constitutive CaMV 35S promoter (Raventos et al. 1998). Thus, according to Raventos et al., HRT, a protein with three DNA-binding domains, can bind to and influence expression from different promoter sequences. A loss-of-function HRT mutant may exhibit aberrant transcription from multiple genes. In addition, Raventos et al. describe that HRT mRNA accumulates in immature tissues, such as immature seeds, immature embryos, and immature endosperm/aleurone layer. In contrast, HRT mRNA is almost undetectable in the layers of germinating seeds. Based on this, HRT could potentially be involved in ensuring that α-amylase is not expressed at certain time points during barley development at which high α-amylase activity is undesirable. High α-amylase activity during plant development is undesirable because it can interfere with proper grain filling, can cause reduced grain starch content and lead to pre-harvest sprouting. The present invention demonstrates that such barley mutants are healthy and viable. Moreover, although Raventos et al. describe that HRT mRNA is almost undetectable in the layers of germinating seeds, in accordance with the present invention it has been observed that loss of HRT function leads to increased α-amylase activity in germinating seeds.

The effect of a loss-of-function mutation on a putative repressor is generally difficult to predict. First of all, whether a repressor is actually a repressor in vivo cannot be definitively predicted from transient expression studies. In addition, a repressor mutation can affect multiple genes, and thus the effect cannot be predicted. In addition, it is also impossible to predict whether a mutation will lead to increased expression levels of target genes. For example, KGM has been described to mediate specific repression of alpha-amylase gene promoter activity at the level of GAMYB function during transient transfection (Woodger et al., 2003). However, two different mutations in the KGM-encoding gene, one of which was a mutation causing the introduction of a premature stop codon, did not lead to increased α-amylase activity.

The present invention further describes the identification of the barley HBL12 (HomeoBoxLike12) gene. The barley gene is also referred to herein as HvHBL12. Mascher et al., 2017, described the sequencing of the barley genome. Additionally, Mascher et al., 2017, provide a transcriptome of a model cv. barley Morex, containing a total of 123,875 identified sequences. From these data, the present inventors have discovered that HvHBL12 is expressed in several tissues, including the germ tissue of germinating grains.

Arabidopsis thaliana contains a gene with limited sequence identity to HvHBL12. Thus, the Arabidopsis thaliana ATHB12 (Arabidopsis thaliana homeobox 12) gene has approximately 70% DNA sequence identity with HvHBL12. ATHB12 is a class I homeodomain leucine zipper (HD-Zip) transcription factor discovered and studied in the model plant Arabidopsis thaliana. Valdes et al. (2012) described that ATNB12 expression is induced by the hormone ABA. Son et al. (2010) described that the ATNB12 gene product represses the GA 20-oxidase 1 (GA20oxl) gene involved in the GA biosynthesis pathway in Arabidopsis stems. Arabidopsis thaliana plants with the ATNB12 loss-of-function mutation have a longer stem. Neither Valdes et al., 2012 nor Son et al., 2010 discuss the role of ATNB12 during early germination.

As noted above, one of the technical problems solved by the present invention is to provide barley plants with high α-amylase and limiting dextrinase activity soon after the initiation of germination, for example, as early as 72 hours or even as early as 48 hours after the initiation of germination, and barley plants at the same time are characterized by acceptable agronomic traits. In one aspect, the present invention relates to barley plants carrying a mutation in the HvHBL12 gene. The present invention surprisingly demonstrates that HvHBL12 loss-of-function barley plants are viable, have adequate agronomic properties, have stem lengths comparable to wild-type barley, and have yields comparable to other barley varieties. At the same time, such barley plants are characterized by high activity of α-amylase and limiting dextrinase already 48 hours after the initiation of germination. Another technical challenge

- 3 043494 solved by the present invention is to provide barley plants with lower sensitivity to oxygen content and hypoxic conditions during grain germination. Interestingly, the use of barley plants with loss of HvHBL12 function also addresses this technical challenge.

Zou et al., 2008, suggest that protein complexes modulate Amy32b expression and that the relative amounts of repressor or activator complexes regulate expression. Zou et al., 2008 also suggest that HvWRKY38 and BPBF may act as transcriptional repressors. However, these assumptions are based on data obtained under completely artificial conditions using the GUS reporter gene under the control of the Amy32b promoter. Moreover, according to Zou et al., 2008, it is not possible to predict what relative amounts of repressor or activator will result in modulation of Amy32b expression. As noted above, the effect of a putative repressor loss-of-function mutation is generally difficult to predict.

As noted above, one of the technical problems solved by the present invention is to provide barley plants with high α-amylase and limiting dextrinase activity shortly after initiation of germination, while at the same time barley plants are characterized by acceptable agronomic traits. In one aspect, the present invention relates to barley plants carrying a mutation in the WRKY38 gene.

Thus, according to one aspect, the present invention relates to barley plants or parts thereof with high α-amylase activity, wherein said barley plant:

carries a mutation in the HvHRT gene causing loss of HvHRT function; and/or carries at least one α-amylase gene containing a mutant α-amylase gene promoter providing a mutation in the GARE box; and/or carries at least four α-amylase genes containing a GARE box with the sequence TAACAAA; and/or carries at least one α-amylase gene in the amy2 cluster containing a mutant α-amylase gene promoter involving a mutation in the GARE box, or which has the sequence TAACAAA; and/or carries a mutation in the HvHBL12 gene causing loss of HvHBL12 function; and/or carries at least four α-amylase genes containing an α-amylase gene promoter containing a non-standard tandem repeat W-box, wherein said non-standard tandem repeat W-box contains the sequence (TGAC(C) _n (X) _m TTGACC) wherein one or more of the particular nucleotides have been replaced or deleted, and wherein X can be any nucleotide, n is 0 or 1, and m is an integer in the range 0-20; and/or carries at least one α-amylase gene in the amy2 cluster containing an α-amylase gene promoter containing a non-standard tandem repeat W-box;

carries a mutation in the WRKY38 gene, causing loss of WRKY38 function.

Description of drawings

In fig. 1 shows the % of sprouted grains cv. Paustian (wt) and HENZ-2 mutant barley after performing a standard germination test in the presence of either 4 ml (left columns) or 8 ml (right columns) water.

In fig. Figure 2 shows the grain water content for cv grains. Paustian (wt) and the HENZ-2 mutant of barley after exposure for 24 hours and 48 hours in water with an air flow of 90 l/h per kg of dry weight of barley grains.

In fig. Figure 3 shows the gene expression levels for α-amylase mRNA encoded by the α-amylase genes of the amy1_1 cluster (2 columns on the left), the amy1_2 cluster (2 middle columns) and BGL2A+BGL2B mRNA (2 columns on the right - designated BGL2A), determined using ddPCR at cv. Paustian (wt) and the HENZ-2 mutant of barley after exposure for 48 hours in water with an air flow of 90 l/h per kg of dry weight of barley grains.

In fig. Figure 4 shows α-amylase activity (Fig. 4A), β-amylase activity (Fig. 4B), and limiting dextrinase activity (Fig. 4C) in cv. Paustian (wt) and the HENZ-2 mutant of barley after exposure for 24 hours and 48 hours in water with an air flow of 90 l/h per kg of dry weight of barley grains.

In fig. Figure 5 shows α-amylase activity (Figure 5A), β-amylase activity (Figure 5B), and limit dextrinase activity (Figure 5C) in cv grains. Paustian (wt) and HENZ-2 mutant barley, which were dehusked and kept for 24 hours in water (24 hours) or for 24 hours in water and 24 hours in air (48 hours) with an air flow of 90 l/h at kg of dry weight of barley grains during the specified aging.

In fig. 6 shows the % of sprouted grains cv. hulless barley 1 (wt) and mutant barley HENZ-10 after performing a standard germination test in the presence of either 4 ml (left columns) or 8 ml (right columns) water.

In fig. Figure 7 shows the gene expression levels for α-amylase mRNA encoded by the α-amylase genes of the amy1_1 cluster, amy1_2 cluster, limiting dextrinase (LD) mRNA, AGL97 mRNA, BGL2A mRNA, and BGL2B mRNA determined by ddPCR in cv. naked barley 1 (wt) and mutant HENZ-10 barley-4 043494 me after 48 hours of exposure in water.

In fig. Figure 8 shows the gene expression levels for α-amylase mRNA encoded by the α-amylase genes of the amy1_1 cluster, the amy1_2 cluster, limiting dextrinase (LD) mRNA, and BGL2A mRNA in cv. naked barley 1 (wt) and mutant barley HENZ-10 after exposure to water with an air flow of 90 l/h per kg dry weight of barley grains, determined using ddPCR.

In fig. 9 shows α-amylase activity (Figure 9A), β-amylase activity (Figure 9B) and limiting dextrinase activity (Figure 9C) in hulless barley 1 (wt) and HENZ-10 mutant barley after incubation for 24 hours and 48 hours in water with an air flow of 90 l/h per kg of dry weight of barley grains.

In fig. 10 shows the plant height of naked barley 1 (wt) and the barley mutant HENZ-10 after cultivation in the field under the same conditions.

In fig. Figure 11 shows the results of an alignment of parts of the α-amylase gene promoter containing regulatory boxes from different α-amylase genes of the amy1_1 and amy1_2 clusters, as well as from the amy1_1 mutant HENZ-43 (Fig. 11A), as well as from the α-amylase genes of the amy2 cluster (Fig. 11B). The HENZ-43 mutant mutation is marked with #.

In fig. Figure 12 shows an example of equipment suitable for producing green malt. The equipment includes a vat (2) in which the grains can be immersed in an aqueous solution and continuously aerated. The equipment includes an inlet for barley grains (1), a vat, for example a key vat (2); gas inlets, such as sintered stainless steel aeration stones (3); a pump, for example an air pump (4); outlet for barley grains (5); pump for pumping grain (6); equipment for fine grinding of barley grains, for example a mill (7); inlet (8); a container, such as a mash container (9), and an outlet (10).

In fig. 13 shows the activity of α-amylase in barley grains germinating in a state immersed in water with an air flow of 9 l/h, 48 hours and 72 hours after the start of germination. In fig. 13A shows the α-amylase activity of the barley HENZ-2 mutant and control homozygous barley plants (WT), while FIG. 13B shows the α-amylase activity of barley mutant HENZ-10 and wild-type (wt) barley 1 hulless.

In fig. 14 shows α-amylase activity in barley grains germinating submerged in water without air flow, 24 hours, 48 hours and 72 hours after the onset of germination in the HENZ-10 mutant barley and wild-type (wt) hulless barley 1.

In fig. 15 shows α-amylase activity in barley grains germinating submerged in water without air flow, 72 hours after the start of germination. On the top panel of Fig. 15 shows the α-amylase activity of the barley HENZ-2a mutant and wild-type barley cv. Planet, in the middle panel of fig. 15 shows the α-amylase activity of the barley HENZ-54 mutant and wild-type barley cv. Planet and in the bottom panel of Fig. 15 shows the α-amylase activity of the barley HENZ-43 mutant and wild-type barley cv. Planet.

In fig. 16 shows gene expression levels for α-amylase mRNA encoded by the α-amylase cluster genes amy1_1 (FIG. 16A), the amy1_2 gene (FIG. 16B), and the amy2 cluster genes (FIG. 16C) determined by RT ddPCR in germinating grains of the mutant HENZ-2 barley and control homozygous barley plants (WT) 0, 12 h, 24 h and 48 h after the start of germination.

In fig. 17 shows gene expression levels after 72 hours for α-amylase mRNA encoded by the α-amylase genes of the amy1_1 cluster, determined by RT ddPCR in germinating grains of the barley HENZ-2a mutant and wild-type barley cv Planet (FIG. 17A), the HENZ- mutant. 54 barley and wild-type barley cv Planet (Fig. 17B) and the barley mutant HENZ-43 and wild-type barley cv Planet (Fig. 17C).

In fig. 18 shows gene expression levels after 72 hours of germination for α-amylase mRNA encoded by the α-amylase genes of the amy1_2 cluster (FIGS. 18A, 18B and 18C) and the amy2 cluster (FIG. 18D) determined by RT ddPCR in germinating grains of the HENZ mutant -2a barley and wild-type barley cv Planet (Fig. 18A), the HENZ-54 mutant barley and wild-type barley cv Planet (Fig. 18B), and the HENZ43 mutant barley and wild-type barley cv Planet (Figs. 18C and 18D).

In fig. 19 shows the expression levels after 72 hours of limiting dextrinase mRNA determined by RT ddPCR in germinating grains of barley HENZ-2a mutant and wild-type barley cv Planet (top panel), barley mutant HENZ-54 and wild-type barley cv Planet (middle panel) and barley HENZ-43 mutant and wild-type barley cv Planet (lower panel).

In fig. 20 shows gene expression levels after 72 hours for the Bgl2 gene determined by RT ddPCR in germinating grains of barley HENZ-2a mutant and wild-type cv Planet barley (FIG. 20A), barley HENZ-54 mutant and wild-type cv Planet barley (FIG. 20B) and the barley HENZ-43 mutant and wild-type barley cv Planet (Fig. 20C).

- 5 043494

Detailed Disclosure of the Present Invention

Definitions.

As used herein, the singular form may mean one or more, depending on the context in which it is used.

As used herein, the term α-amylase refers to an enzyme with α-amylase activity. In particular, the α-amylase according to the present invention is an enzyme capable of catalyzing the endohydrolysis of (1^4)-α-glycosidic linkages in polysaccharides containing three or more (1^4)-α-linked D-glucose units. α-Amylase activity can be determined using K-CERA 01/12 (protocol and kit available from Megazyme, Ireland).

The term additive as used herein refers to high carbon raw material sources added during the production of beer. The additive may be an unsprouted cereal grain, which can be ground together with the sprouted grains obtained in accordance with the present invention. The additive may also be syrup, sugar, etc.

The term about as used herein in relation to numerical values preferably means ±10%, more preferably ±5%, even more preferably ±1%.

The term amino acid as used herein refers to a proteinogenic amino acid. Preferably, the proteinogenic amino acids are one of the 20 amino acids encoded in accordance with the standard genetic code. Amino acids are named using one- and three-letter codes according to IUPAC.

The expression amino acid corresponding to X is used herein to describe the amino acids of a given polypeptide (e.g., an HRT, HBL12, or WRKY38 mutant polypeptide) relative to the amino acids of a reference polypeptide (e.g., any of the polypeptides of SEQ ID NO: 2, 6, 11, or 12). According to the alignment of the specified polypeptide and the reference polypeptide, an amino acid matches X if it is in the same position as X in the specified alignment.

The term amylose refers to homopolymers of a-D-glucose. Amylose has a linear molecular structure because its glucose units are almost exclusively linked by α-1-4 glycosidic bonds.

The term amylopectin refers to homopolymers of a-D-glucose. Amylopectin molecules contain frequent α-1-6-glycosidic bonds. They introduce branch points into α-1-4-linked glucose chains, resulting in clusters of parallel chains found at regular intervals along the molecular axis.

The term barley flour as used herein refers to the ground kernels of barley grain.

The term β-amylase refers to an enzyme that catalyzes the hydrolysis of (1^4)-alpha-glycosidic bonds in polysaccharides to remove successive maltose units from the non-reducing ends of the chains. β-Amylase activity can be determined using K-BETA3 (protocol and kit available from Megazyme, Ireland).

The term shoot as used herein refers to the growing bud of the embryo visible during the germination phase of the cereal grain.

As used herein, the term high yield refers to a yield comparable to that of high-yielding barley varieties. In particular, high yield may be a yield that is at least 95%, such as at least 98%, such as at least 100% of the yield of the barley plant cv. Planet, grown under similar conditions.

The term barley, in the context of the process of making barley-based beverages such as beer, particularly when used to describe the malting process, means the kernels of barley grains. In all other cases, unless otherwise specified, barley means the barley plant (Hordeum vulgare, L.), including any line of cross, or cultivar, or variety, while a barley plant part may be any part of the barley plant, such as any tissue or cells.

A cereal plant, as defined herein, is a member of the Poaceae family of plants cultivated primarily for their starchy seeds or grain kernels. Cereal plants include, but are not limited to, barley (Hordeum), wheat (Triticum), rice (Oryza), maize (Zea), rye (Secale), oats (Avena), sorghum (Sorghum) and triticale, a hybrid of rye and wheat.

By encoding or encoded in the context of a specified nucleic acid is meant to contain information for translation into a specific protein. A protein-encoding nucleic acid or polynucleotide may contain untranslated sequences, such as introns, within the translated regions of the nucleic acid, or may lack intervening untranslated sequences, such as in cDNA. The information in which a protein is encoded is determined by the use of codons.

In the context of this document, expression in relation to nucleic acids should be understood as transcription and production of mRNA. The term expression, as used in the context of proteins, refers to the translation of mRNA into a polypeptide.

The term gene means a segment of DNA involved in the formation of a polypeptide chain; it includes regions upstream and downstream of the coding region (promoter and terminator).

In addition, plant genes typically consist of exons interrupted by introns.

The term germination initiation as used herein refers to the point in time at which barley grains with less than 15% water content are brought into contact with sufficient water to initiate germination.

The term malting as used herein refers to the controlled germination of cereal kernels (in particular barley kernels) occurring under controlled environmental conditions. In some embodiments, malting may also include the step of drying said germinated cereal kernels, for example, by oven drying.

The term sprouted grain as used herein refers to grain that has developed a visible sprout and a visible stem.

The term green malt as used herein refers to sprouted cereal kernels that have not been kilned. In general, these cereal grain kernels were germinated under controlled environmental conditions. In some embodiments, the green malt is ground green malt.

The term kiln-dried malt as used herein refers to sprouted cereal grain kernels that have been dried by kiln drying. In general, these cereal grain kernels were germinated under controlled environmental conditions. In some embodiments, the kiln-dried malt is ground kiln-dried malt.

The term limit dextrinase as used herein refers to an enzyme capable of catalyzing the hydrolysis of (1^6)-alpha-D-glycosidic bonds in the alpha and beta limit dextrins of amylopectin and glycogen, amylopectin and pullulan. In particular, limiting dextrinase may be an enzyme classified as EC 3.2.1.142. Limiting dextrinase activity was determined using T-LDZ1000 (protocol and kit available from Megazyme, Ireland).

Mashing is the soaking of ground malt (such as green malt or kiln-dried malt) and/or unsprouted cereal kernels in water. Mashing is preferably carried out at a specific temperature(s) and in a specific volume of water.

The term ground refers to material (such as barley kernels or malt) that has been finely ground, such as by cutting, grinding, grinding or crushing. Barley kernels can be ground wet using, for example, a crusher or a wet mill. The ground barley kernels or ground malt are sufficiently finely ground to render the material suitable for the preparation of aqueous extracts. Ground barley kernels or ground malt cannot be regenerated into an intact plant by most biological methods.

Mutations include deletions, insertions, substitutions, transversions, and point mutations in the coding and noncoding regions of the gene. Deletions can affect an entire gene or just a portion of a gene. Point mutations may involve changes in a single base pair and may result in premature stop codons, frameshift mutations, splice site mutations, or amino acid substitutions. The gene providing the mutation may be called a mutant gene. If the specified mutant gene encodes a polypeptide with a sequence different from that of the wild type, the specified polypeptide may be called a mutant polypeptide. A mutant polypeptide may be described as having a mutation if it contains an amino acid sequence that differs from that of the wild type. The notation XnnnY indicates that the amino acid or nucleotide X at position nnn has been replaced by Y. Thus, for example, XnnnStop indicates that the codon encoding amino acid X at position nnn has been replaced with a stop codon.

The term plant product means a product obtained by processing a plant or plant material. Thus, said plant product may be, for example, green malt, kiln-dried malt, wort, a fermented or non-fermented beverage, a food product or a feed product.

The term progeny as used herein means a plant that is directly or indirectly a descendant of a given plant. Thus, offspring is not limited to a direct descendant, but also includes a descendant in a number of multiple generations. In general, the offspring of a barley plant that carries a particular mutation will also carry that particular mutation.

The term sequence identity as used herein refers to the % identical amino acids or nucleotides between a candidate sequence and a reference sequence resulting from an alignment. Thus, in the case of a candidate sequence having 80% amino acid identity with the reference sequence,

- 7 043494 requires that the alignment results in 80% of the amino acids in the candidate sequence being identical to the corresponding amino acids in the reference sequence. Identity in accordance with the present invention is determined using computer analysis, such as, without limitation, the Clustal Omega computer alignment program for polypeptide sequence alignment (Sievers et al. (2011 October 11) Molecular Systems Biology 7:539, PMID: 21988835; Li et al., (2015 April 06) Nucleic Acids Research 43 (W1):W580-4 PMID: 25845596; McWilliam et al., (2013 May 13) Nucleic Acids Research 41 (Web Server issue):W597-600

PMID: 23671338, and the default parameters proposed in it. Clustal Omega software is available from EMBL-EBI at https://www.ebi.ac.uk/Tools/msa/clustalo/. Using such a program with default settings, the mature (biologically active) part of the given and reference polypeptide is aligned. The number of completely conserved residues is counted and divided by the length of the reference polypeptide. To align nucleotide sequences, you can use the MUSCLE or MAFFT algorithms. Sequence identity scores can be calculated in the same manner as indicated for amino acid sequences. Thus, sequence identity, as provided herein, is calculated over the entire length of the reference sequence.

The term soaking as used herein refers to the process of increasing the water content in the kernel of a cereal grain.

The term starch as used herein refers to a compound of one or both of the discrete macromolecules amylose and amylopectin.

The term splice site as used herein refers to consensus sequences that act as splice signals for the splicing process. A splice site mutation is a genetic mutation that causes insertions, deletions or changes in a number of nucleotides at a particular site where splicing occurs during the splicing process, i.e. processing of precursor messenger RNA to form mature messenger RNA (mRNA). Splice site consensus sequences that mediate exon recognition are typically located at the very ends of introns.

The term stop codon as used herein refers to a triplet of nucleotides within the genetic code that, when contained within an mRNA, results in translation termination. The term stop codon as used herein also refers to a triplet of nucleotides within a gene encoding a stop codon in mRNA. A stop codon in DNA is usually characterized by one of the following sequences: TAG, TAA or TGA.

The term grain water content as used herein refers to the % H ₂ O w/w in said grain.

Cereal enzyme activity as used herein refers to the activity measured in flour obtained from a specific type of grain. For example, 10 U/g α-amylase activity per gram of cereal grain refers to the specified α-amylase activity (10 U) measured in the aqueous extract obtained from 1 g of flour (dry matter) from the specified cereal.

The volume of gas as specified herein refers to the volume of said gas at 1 atm and 20°C.

The volume of O2 as stated herein refers to the volume of O2 at 1 atm and 20°C. In certain embodiments of the present invention in which O ₂ is present in a gas mixture, the total volume of the gas mixture can be determined and the volume of O ₂ can then be calculated as the percentage of the total volume formed by O ₂ . As an example, atmospheric air contains 21% O ₂ . Thus, the volume of O2 in atmospheric air in the context of this document is 21% of the total volume of atmospheric air.

By the term wort is meant a liquid extract of malt and/or cereal kernels, such as ground malt and/or ground cereal kernels, and optionally additional additives. Wort is generally produced by mashing, optionally followed by sparging the spent grains, a process that extracts residual sugars and other compounds from the spent grains after mashing using hot water. Washing of spent grains is usually performed in a launder tun, mash strainer, or other device to ensure that the recovered water is separated from the spent grains.

The wort obtained after mashing is usually called first wort, while the wort obtained after sparging is usually called second wort. If not specified, the term wort may mean the first wort, the second wort, or a combination of both. During traditional beer production, the wort is boiled along with hops. Wort without hops may also be called sweet wort, while wort boiled with hops may be called boiled wort or simply wort.

A-amylase and barley plants carrying a mutation in the α-amylase gene promoter

The term α-amylase as used herein refers to an enzyme that is capable of catalyzing the hydrolysis of alpha bonds in alpha-linked polysaccharides such as starch. A- 8 043494 amylase is generally an enzyme classified as EC 3.2.1.1. A-amylase is also known as alpha-amylase.

Cereal plants may contain more than one gene encoding α-amylase. Therefore, a barley plant according to the present invention may contain more than one gene encoding α-amylase. Often, genes encoding α-amylase can form gene clusters.

The A-amylase according to the present invention may, in particular, be a polypeptide having the sequence accession number HORVU6Hr1G078330.1, HORVU6Hr1G078360.1, HORVU6Hr1G078420.1, HORVU6Hr1G080790.1, HORVU7Hr1G091150.1, HORV U7Hr1G091240.1 or HORVU7Hr1G091250.3, or a functional homolog thereof sharing at least 90%, such as at least 95% sequence identity, wherein said accession numbers are accession numbers from the barley genome sequencing project published under the authorship of Mascher et al. , 2017. Sequences are also available in the BARLEX database: https://apex.ipk-gatersleben.de/apex/f?p=284:10.

Thus, a barley plant according to the present invention may contain at least 1, preferably at least 2, such as at least 3, such as 3 α-amylase gene clusters. In particular, a barley plant may contain an amy1_1 cluster, an amy1_2 cluster, and an amy2 cluster. The amy1_2 cluster often contains only one gene, but is still referred to herein as the amy1_2 cluster for the sake of simplicity. In addition to the above clusters, the barley plant may contain additional α-amylase genes/clusters (eg, amy 3, amy4_1 and amy4_2 genes). Each of these clusters may contain one or more α-amylase genes.

Example α-amylase gene sequences are available from the Barley Genome Sequencing Project, the results of which are published under

Name by Mascher 2017 Gene ID amylla HORVU6HrlG078330.1 amyl lb HORVU6HrlG078360.1 amyl 1c HORVU6HrlG078420.1 amyl Id HORVU0HrlG032700.1 amyl le HORVU0HrlG032850.5 amyl 2 HORVU6HrlG080790.1 amy2 1 HORVU7HrlG091150.1 amy2 2 HORVU7HrlG091240.1 amy2 3 HORVU7HrlG091250.3 amy3 HORVU5HrlG068350.1 amy4 1 HORVU2HrlG071710.5 amy4 2 HORVU3HrlG067620.1

Mascher et al., 2017:

Each α-amylase gene typically contains a promoter region (herein referred to as the α-amylase gene promoter), a coding region, and one or more introns. As used herein, an α-amylase gene promoter contains or consists of a 1000 nucleotide sequence, such as 800 nucleotides immediately upstream of the start codon (ATG) of the α-amylase gene coding sequence. The exact copy sequences of individual genes within each cluster and between different barley varieties in the α-amylase gene promoter regions can vary significantly, but certain regulatory boxes are conserved among many α-amylase genes. In particular, these regulatory boxes may be conserved among the α-amylase genes of the amy1_1 cluster, the amy1_2 cluster, and the amy2 cluster. In fig. 11 shows the results of an alignment of parts of the α-amylase gene promoter containing the indicated regulatory boxes from different α-amylase genes of the amy1_1 and amy1_2 clusters (Fig. 11A), as well as from the α-amylase genes of the amy2 cluster (Fig. 11B).

In particular, one or more of the barley plant α-amylase genes of the present invention may contain a GARE box with the sequence TAACARA, wherein R is G or A. In particular, this is true in the case of the α-amylase genes of the amy1_1 cluster, the amy1_2 cluster or amy2 cluster. In general, each α-amylase gene promoter contains no more than one GARE box. The α-amylase genes of the amy3 and amy4 clusters often do not contain a GARE box.

According to one embodiment of the present invention, it is preferred that the barley plant contains one or more α-amylase genes containing α-amylase gene promoters,

- 9 043494 containing a mutated GARE box. In other words, one or more α-amylase gene promoters may not contain a wild-type GARE box, but instead they may contain a mutant GARE box where one or more of the TAACARA nucleotides have been either replaced or deleted. Preferably, said mutant GARE box is a GARE box in which only one of the TAACARA nucleotides has either been replaced or deleted. In particular, a barley plant may contain one or more amy1_1 cluster, amy1_2 cluster, or amy2 cluster α-amylase genes containing α-amylase gene promoters containing a mutated GARE box.

According to another embodiment, it is preferred that the barley plant of the present invention contains one or more α-amylase genes, preferably at least 4, such as at least 5, such as at least 6 α-amylase genes, such as , at least 7 α-amylase genes containing α-amylase gene promoters containing one GAREbox with the sequence TAACAAA. In particular, said α-amylase genes may be α-amylase genes of the amy1_1 cluster, the amy1_2 cluster, or the amy2 cluster. Said α-amylase gene may also contain a PYR box. The term PYR box as used herein refers to the sequence CMTTTT where M represents C or A.

In addition, one or more of the barley plant α-amylase genes of the present invention may contain a tandemly repeating W box. A standard tandemly repeated W box has the sequence (TGAC(C) _n (X) _m TTGACC), with each X individually being any nucleotide, n being 0 or 1, and m being an integer in the range 0-20. It should be understood that within any given tandemly repeated W box, the individual X's may be the same or different nucleotides. In general, each α-amylase gene promoter contains only one tandemly repeating W box.

According to one embodiment of the present invention, it is preferred that the barley plant contains one or more α-amylase genes containing α-amylase gene promoters containing a non-standard tandem repeat W box. In other words, one or more α-amylase gene promoters may not contain a standard tandem repeat W box, but instead they may contain a non-standard tandem repeat W box, wherein one or more of the nucleotides of the standard tandem repeat W box (TGAC(C) ) _n (X) _m TTGACC) were either replaced or removed. It should be understood that the one or more nucleotides that have been replaced or deleted are one or more of the specific nucleotides of a standard tandem repeat W box (ie, not any of the Xs).

In one embodiment, the barley plant of the present invention contains one or more α-amylase genes, such as at least 4, such as at least 5, such as at least 6, such as at least 7 genes α-amylases containing α-amylase gene promoters containing a non-standard tandem repeating W-box individually selected from the group consisting of the following sequences:

(TGACR(X)mYTGRCC), wherein R is either G or A, Y is either C or T, and m is an integer in the range 0-20; or (TGACR(X) _m TTGACC), wherein R is either G or A and m is an integer in the range 0-20; or (TGACR(X) _m TTGAC), wherein R is either G or A and m is an integer in the range 0-20; or (TGAC(C)n(X)mYTGRCC), wherein R is either G or A, and Y is either C or T, n is 0 or 1, and m is an integer in the range 0-20; or (TGAC(C)n(X)mCTGRCC), wherein R is either G or A, n is 0 or 1, and m is an integer in the range 0-20;

(TGAC(C)n(X)mYTGGCC), and wherein Y is either C or T, n is 0 or 1 and m is an integer in the range 0-20; or (TGAC(C) _n (X) _m CTGACC), where n is 0 or 1 and m is an integer in the range 0-20;

(TGAC(C) _n (X) _m TTGGCC), and wherein n is 0 or 1 and m is an integer in the range 0-20 (TGAC(C) _n (X) _m TTGATC), wherein n is 0 or 1 and m is an integer in the range 0-20;

As mentioned above, m may be an integer in the range of 0-20, preferably m is an integer in the range of 0-10, even more preferably m is an integer in the range of 0-6.

In one embodiment, a barley plant of the present invention contains one or more α-amylase genes, for example, at least 5 α-amylase genes containing α-amylase gene promoters containing a non-standard tandem repeat W-box individually selected from the non-standard tandem repeat W-boxes with the following sequences

- 10 043494 mi:

TGACGGTCGTATTGACC (SEQ Ш NO:31);

TGACAGTGGTATTGGCC (SEQ ID NO:32);

TGACAGTGGTACTGGCC (SEQ ID NO:33);

GTGACAGTGGTATTGGCC (SEQ ID NO:34);

TGACGGTCGTATTGATC (SEQ ID NO:3 5);

TGACCGTCGTATTGATC (SEQ ID NO:36); And

TTGACTTGATC (SEQ ID NO:37).

From the database search and promoter resequencing results presented herein, it is apparent that the α-amylase promoters of the α-amylase genes of the amy1_1, amy1_2, and amy2 clusters contain GARE box sequences. In particular, the α-amylase genes of the amy2 cluster in wild-type barley contain a GARE box with the sequence TAACAGA.

The amy1_1 cluster may contain 1-5 α-amylase genes, but not all α-amylase genes are necessarily expressed. Thus, in some barley plants (e.g., cv. Barke or its progeny), the amy1_1 cluster contains at least three major copies, whereas other barley plants (e.g., cv. Planet or its progeny) may contain three α-amylase genes in the amy1_1 cluster, of which only two can be expressed. For example, some α-amylase gene promoters lack a PYR box and are therefore not thought to be expressed. The A-amylase according to the present invention may be an α-amylase encoded by the amy1_1 cluster gene. For example, the α-amylase may be a polypeptide having the sequence accession number AAA98790.1 or BAK03603.1, or a functional homolog thereof, sharing at least 90%, such as at least 95% sequence identity, with it, wherein Accession numbers listed are accession numbers from the NCBI database. A-amylase may also be a polypeptide having the sequence accession number HORVU6Hr1G078330.1, HORVU6Hr1G078360.1, HORVU6Hr1G078420.1,

HORVU0Hr1G032700.1 or HORVU0Hr1G032850.5, or a functional homolog thereof sharing at least 90%, such as at least 95% sequence identity, wherein said accession numbers are accession numbers from the BARLEX database (https:// apex.ipk-gatersleben.de/apex/f?p=284:10) and published under the authorship of Mascher et al., 2017.

The barley plant of the present invention contains the amy1_1 cluster, wherein at least one of the α-amylase gene promoters, preferably all of the α-amylase gene promoters, contains:

only a mutated GARE box, and one of the nucleotides of the GARE box (TAACARA) was either replaced or deleted; and/or

GARE box with sequence TAACAAA; and/or a non-standard tandem repeat W box only, wherein one or more of the specific nucleotides of the standard tandem repeat W box (TGAC(C) _n (X) _m TTGACC) have either been replaced or deleted, for example, said non-standard tandem repeat W box The -box may have any of the non-standard tandem repeat W-box sequences described herein above.

In addition, these α-amylase gene promoters may contain a PYR box.

The term "mutated GARE box only" as used herein refers to said promoter containing only one GARE box, said GARE box being mutated, i.e. this said promoter does not contain a wild-type GARE box in addition to said mutated GARE box. Likewise, the term non-standard tandem repeat W box only as used herein refers to said promoter containing only one tandem repeat W box, wherein said tandem repeat W box is a non-standard tandem repeat W box, i.e. this said promoter does not contain a standard tandem repeating W box in addition to said non-standard tandem repeating W box.

As can be seen from the present invention, a GARE box with the sequence TAACAAA has a lower binding capacity for HRT compared to that of a GARE box with the sequence TAACAGA. Likewise, WRKY38 is believed to have lower binding affinity to the non-standard tandem repeat W box compared to that to the standard tandem repeat W box. In an international panel of 96 malting barley varieties (2-row spring barley, 2-row winter barley, 6-row winter barley), six unique amy1_1 clusters can be found. From this panel, barley plants with the highest α-amylase activity always have the amy1_1 cluster containing at least three α-amylase genes containing an α-amylase gene promoter containing a GARE box with the sequence TAACAAA. Unfortunately, the barley plants tested in the panel with

- 11 043494 the highest α-amylase activity was not characterized by the highest yield. In one embodiment, the present invention relates to barley plants with both high yield and high α-amylase activity.

In one embodiment, the barley plant contains an amy1_1 cluster, wherein at least one of the α-amylase gene promoters contains a non-standard tandem repeat W box containing the sequence TTGATC. For example, a barley plant may contain an amyl_l cluster, wherein at least one of the α-amylase gene promoters contains a non-standard tandem repeat W box containing the sequence CTGACGGTCGTATTGATC (SEQ ID NO: 72). In particular, the barley plant may contain an α-amylase gene promoter containing the sequence shown in FIG. 11A as HENZ-43 amy1_1. Said barley plant may be, for example, HENZ-43 or its progeny. For example, the barley plant may be the barley plant identified as described in Example 13A or its progeny.

The amy1_2 cluster typically contains only one α-amylase gene. The amy1_2 cluster in the barley genome is usually closely related to the amy1_1 cluster. The α-amylase according to the present invention may be an α-amylase encoded by the amy1_2 cluster gene. For example, the α-amylase may be a polypeptide having the sequence accession number AAA98615.1, or a functional homolog thereof sharing at least 90%, such as at least 95% sequence identity, wherein said accession numbers are accession numbers from the NCBI database. A-amylase may also be a polypeptide having the sequence accession number HORVU6Hr1G080790.1, or a functional homologue thereof, sharing at least 90%, such as at least 95% sequence identity therewith, said accession numbers being accessed from the BARLEX database (https://apex.ipk-gatersleben.de/apex/f?p=284:10) and are described in Mascher et al., 2017.

Often, the α-amylase gene of the amy1_2 cluster contains an α-amylase gene promoter containing a GAREbox with the sequence TAACAAA. The barley plant of the present invention may contain an amy1_2 cluster containing an α-amylase gene with an α-amylase gene promoter containing:

only a mutated GARE box, and one of the nucleotides of the GARE box (TAACARA) was either replaced or deleted; and/or

GARE box with sequence TAACAAA; and/or a non-standard tandem repeat W box only, wherein one or more of the specific nucleotides of the standard tandem repeat W box (TGAC(C) _n (X) _m TTGACC) have either been replaced or deleted, for example, said non-standard tandem repeat W box The -box may have any of the non-standard tandem repeat W-box sequences described herein above.

The amy2 cluster may consist of three expressed copies of the α-amylase gene. For example, this is true in the case of cv. Morex. In wild-type barley, each α-amylase gene of the amy2 cluster may contain a GARE box with the sequence TAACAGA.

The A-amylase according to the present invention may be an α-amylase encoded by the amy2 cluster gene. For example, α-amylase may be a polypeptide having the sequence accession number HORVU7Hr1G091150.1, HORVU7Hr1G091240.1,

HORVU7Hr1G091250.3, or a functional homologue thereof, sharing with it at least 85%, such as at least 90% sequence identity, wherein the accession numbers indicated are accession numbers from the BARLEX database (https://apex.ipkgatersleben. de/apex/f?p=284:10) and are described in Mascher et al., 2017.

Alternatively, barley plants of the present invention may provide an amy2 cluster containing one or more α-amylase genes with α-amylase gene promoters containing:

only a mutated GARE box, and one or more of the TAACARA nucleotides have been either replaced or deleted,

GARE box with the sequence TAACAAA, a non-standard tandem repeat W box only, wherein one or more of the specific nucleotides of the standard tandem repeat W box (TGAC(C) _n (X) _m TTGACC) have either been replaced or deleted, e.g. the non-standard tandem repeat W-box may have any of the non-standard tandem repeat W-box sequences described herein above.

In particular, a barley plant may comprise an amy2 cluster containing one or more α-amylase genes with α-amylase gene promoters containing a mutated tandem repeat W box containing the sequence TTGACTTGACC, wherein at least one nucleotide has been replaced or deleted.

HRT.

The full-length protein product of the barley Hv transcriptional repressor gene (HvHRT) functions as a transcriptional repressor at a number of different promoters when tested using plant cells after transient expression. Under such completely artificial conditions, transient expression of HRT results in a reduced level of expression of the reporter gene, which is under the control of different α-amylase gene promoters (Amy2 and Amy1), as well as a reduced level of expression from the constitutive CaMV 35S promoter. Repression of the Amy2 promoter was found to be stronger in this system (Raventos et al. 1998). HRT binds to and blocks the so-called GARE(GA response element)-6okcom, which is also the binding site for the putative activator of α-amylase gene expression, HvGAMyb (Gubler et al. 1995).

The coding sequence of barley wild type HRT is provided herein as SEQ ID NO: 1. For HvHRT CDS sequences AK362734.1 (cv. Haruna Nijo), AK252040.1 (Haruna Nijo), HORVU2Hr1G035630.1 (cv. Morex), AJ001317 .1 (Himalaya) subjected to multiple sequence alignment, and manually assembled sequences in the case of cv. Bowman and Barke derived from genomic DNA contigs in IBSC2012 (available in the BARLEX database https://apex.ipk-gatersleben.de/apex/f?p=284:10), 100% sequence identity was demonstrated between the CDS sequences . The protein sequence of wild-type barley HRT is provided herein as SEQ ID NO: 2. For the HvHRT protein sequences AK362734.1 (cv. Haruna Nijo), AK252040.1 (Haruna Nijo), HORVU2Hr1G035630.1 (cv. Morex), AJ001317. 1/CAA04677.1 (Himalaya), subjected to multiple sequence alignment, and manually assembled and translated sequences in the case of cv. Bowman and Barke derived from genomic DNA contigs in IBSC2012, 100% sequence identity between protein sequences was demonstrated. The HvHRT protein contains two putative nuclear localization signals (NLS). Thus, the putative NLS is amino acids 276-292 SEQ ID NO: 2 (Arg276-Arg292) and amino acids 527-530 SEQ ID NO: 2 (Arg527-Arg530) (Raventos et al., 1998). In addition, HvHRT contains three putative zinc finger DNA-binding domains (also referred to herein as HRTdb) containing the following consensus sequence: VCG _X4 DG _X2 C _X2 C _X3 PV _X2 RKRC _X2 HKG _X R, where X may be any amino acid. Thus, the putative DNA binding domains are amino acids 302-331 of SEQ ID NO: 2, amino acids 463-491 of SEQ ID NO: 2, and amino acids 509-539 of SEQ ID NO: 2.

A barley plant carrying a mutation in the HRT gene.

In one embodiment, the present invention relates to a barley plant carrying a mutation in the HRT gene causing loss of HRT function, and in particular, complete loss of HRT function. Loss of HRT function can be determined by determining the level of HRT expression at either the mRNA or protein level. In one embodiment, a barley plant is considered to have loss of HRT function if the barley plant contains less than 50%, preferably less than 25%, and even more preferably less than 10% mutant HvHRT mRNA or wild-type HvHRT mRNA compared to the level of HvHRT mRNA in the barley plant , containing the wild-type HvHRT gene but otherwise having the same genotype. A barley plant may be considered to have complete loss of HRT function if the barley plant contains less than 5%, preferably less than 1%, mutant HvHRT mRNA or wild-type HvHRT mRNA compared to that of a barley plant containing the wild-type HvHRT gene but otherwise having the same genotype. Said mutant HvHRT is an mRNA encoded by a mutated HvHRT gene carrying a mutation in the mRNA coding region. HvHRT mRNA is RNA encoding a polypeptide of SEQ ID NO: 2 or a functional homologue thereof, and a wild-type HvHRT gene is a gene encoding a polypeptide of SEQ ID NO: 2 or a functional homologue thereof. Said functional homologue preferably has at least 95% sequence identity to SEQ ID NO: 2. In one embodiment, a barley plant with complete loss of HRT function may not contain mutant HvHRT mRNA or wild-type HvHRT mRNA in detectable amounts when determined by standard quantitative assay. RT-PCR.

In one embodiment, a barley plant is considered to have loss of HRT function if the barley plant contains less than 50%, preferably less than 25%, and even more preferably less than 10% mutant HvHRT protein or wild-type HvHRT protein compared to the level of HvHRT protein in the barley plant , containing the wild-type HvHRT gene but otherwise having the same genotype. A barley plant may be considered to have complete loss of HRT function if the barley plant contains less than 5%, preferably less than 1%, of mutant HvHRT protein or wild-type HvHRT protein compared to that of a barley plant containing the wild-type HvHRT gene but otherwise having the same genotype. The HvHRT mutant protein is a polypeptide encoded by a mutated HvHRT gene carrying a mutation in the coding region. The HvHRT protein is a polypeptide of SEQ ID NO: 2 or a functional homologue thereof, and the wild-type HvHRT gene is a gene encoding a polypeptide of SEQ ID NO: 2 or a functional homologue thereof. Said functional homolog preferably has at least 95% sequence identity to SEQ ID NO: 2. In one embodiment, a barley plant with complete loss of HRT function may not contain mutant HvHRT protein or wild-type HvHRT protein in detectable amounts when detected using standard Western blotting.

In one embodiment, a barley plant is considered to have loss of HRT function if an increased level of expression can be observed from a promoter containing a GARE box. Thus, loss of HRT function can be determined using transient transfection with a reporter construct containing a reporter gene (eg, luciferase) under the control of the α-amylase gene promoter from the Amy2 cluster, for example, the construct described in Raventos et al., 1998, p. 23314 in Transient Gene Expression Assays in Onion Epidermal and Barley Aleurone Cells.

An increase in luciferase activity of at least 10%, such as at least 25%, following transfection of said reporter construct into cells of a given barley plant, compared with that of transfecting said reporter construct into cells of a barley plant containing the wild-type HvHRT gene, but in otherwise having the same genotype is an indication that the barley plant in question is characterized by loss of HRT function.

In one embodiment, a barley plant is considered to have a loss of HRT function if said barley plant carries a mutation resulting in an HRT gene encoding a mutant HvHRT protein lacking one or more of the following domains:

NLS1: R276-R292 SEQ ID NO:2 NLS2: R527-R530 SEQ ID NO:2 HRTdbl: V302-R331 SEQ ID NO:2 HRTdb2: L463-E491 SEQIDNO:2 HRTdb3: V509-A539 SEQ ID NO:2

A barley plant carrying a mutation in the HRT gene causing loss of HRT function may carry different types of mutations, such as any of the mutations described in this section of this document.

In one embodiment, the barley plant carries a mutation in the promoter region of the HvHRT gene or in the intron of the HvHRT gene causing aberrant transcription of HvHRT mRNA and/or aberrant translation of HvHRT protein. Such barley plants may, in particular, be characterized by reduced levels of HvHRT mRNA, as described in this section herein above, and/or reduced levels of HvHRT protein, as described in this section herein above.

In one embodiment, a barley plant of the present invention carries a mutation resulting in a deletion of the HvHRT gene. The genomic sequence of the HvHRT gene differs among barley varieties, but the coding region is highly conserved. Examples of barley HvHRT gene genomic sequences include NCBI accession ID# AJ001317.1. Additionally, most of the genomic sequence of the HvHRT gene can be found in morex_contig_368180 and morex_contig_1570244 (cv. Morex) (IBSC, 2012), however these contigs are not overlapping and part of the intronic sequence is missing. The HRT coding sequence of certain wild-type barley plants is provided herein as SEQ ID NO: 1.

In one embodiment, the barley plant of the present invention carries a mutation resulting in a mutant HvHRT gene encoding a mutant HvHRT protein. In one embodiment, the mutation may be a mutation resulting in a premature stop codon. In another embodiment, the mutation is a mutation in a splice site of the HvHRT gene. This mutation may cause aberrant splicing of HvHRT mRNA.

In one embodiment, the barley plant of the present invention carries a mutation resulting in the formation of a mutant HvHRT gene encoding a mutant HvHRT protein lacking at least the amino acids corresponding to amino acids 527-530 of SEQ ID NO: 2. It should be understood that in A mutant HvHRT lacking at least amino acids XX-YY may also lack amino acids other than amino acids XX-YY.

In one embodiment, the barley plant may contain a mutant HvHRT gene encoding a mutant HvHRT protein that lacks at least amino acids 463-491 of SEQ ID NO: 2, for example, said mutant HvHRT protein may lack at least amino acids 463-491 and amino acids 527-530 SEQ ID NO: 2.

In one embodiment, the barley plant may contain a mutant HvHRT gene encoding a mutant HvHRT protein that is missing at least amino acids

- 14 043494

509-539 SEQ ID NO: 2.

In one embodiment, a barley plant of the present invention may contain a mutant HvHRT gene encoding a mutant HvHRT protein lacking at least 21 of the most C-terminal amino acids, such as at least 39 of the most C-terminal amino acids, such as , e.g., at least the 85 most C-terminal amino acids, e.g., the at least 100 most C-terminal amino acids of SEQ ID NO: 2. For example, a barley plant may contain a mutant HvHRT gene encoding a mutant HvHRT protein, in which is missing at least 118 amino acids closest to the C-terminus of SEQ ID NO: 2.

In one embodiment, a barley plant of the present invention may contain a mutant HvHRT gene encoding a truncated HvHRT protein containing an N-terminal HvHRT fragment containing no more than 526 N-terminal amino acids of SEQ ID NO: 2, for example, no more than 508 N-terminal amino acids SEQ ID NO: 2, such as no more than 462 N-terminal amino acids of SEQ ID NO: 2, preferably no more than 431 N-terminal amino acids of SEQ ID NO: 2.

In one embodiment, a barley plant of the present invention may contain a mutant HvHRT gene carrying a premature stop codon among any of codons 1-527, such as among any of codons 1-509, such as among any of codons 1-463, for example, among any of codons 1-431. For example, a barley plant may contain a mutant HvHRT gene carrying a premature stop codon at codon position 431. The codons are numbered according to SEQ ID NO: 1, starting at the 5' end, with one codon comprising 3 nucleotides.

In one embodiment, the barley plant of the present invention contains a mutant HvHRT gene encoding a mutant HvHRT protein with the W431stop mutation of SEQ ID NO: 2. In one preferred embodiment, the barley plant of the present invention contains a mutant HvHRT gene encoding a polypeptide of SEQ ID NO: 4. In particular, said barley plant may contain a G^A mutation at nucleotide 1293 of the HvHRT coding sequence SEQ ID NO: 1. For example, the barley plant may contain a mutant HvHRT gene containing the coding sequence SEQ ID NO: 3.

In one embodiment, a barley plant of the present invention contains a mutant HvHRT gene encoding a mutant HvHRT protein with the W170stop mutation SEQ ID NO: 2. For example, a barley plant may contain a mutant HvHRT gene carrying a premature stop codon at codon position 170 (codon numbering is provided in the reference to SEQ ID NO: 1, as explained above). In particular, said barley plant may include a G^A mutation at nucleotide 510 of the HvHRT coding sequence SEQ ID NO: 1. Said barley plant may be, for example, HENZ-53 or its progeny. For example, the barley plant may be the barley plant identified as described in Example 14 or its progeny.

In one embodiment, a barley plant of the present invention contains a mutant HvHRT gene encoding a mutant HvHRT protein with the W371stop mutation SEQ ID NO: 2. For example, a barley plant may contain a mutant HvHRT gene carrying a premature stop codon at codon position 371 (codon numbering is provided in the reference to SEQ ID NO: 1, as explained above). In particular, said barley plant may include a G^A mutation at nucleotide 1113 of the HvHRT coding sequence SEQ ID NO: 1. Said barley plant may be, for example, HENZ-54 or its progeny. For example, the barley plant may be the barley plant identified as described in Example 14 or its progeny.

For the purposes of this patent application, seeds of a barley plant (Hordeum vulgare), designated HENZ-2, have been deposited with NCIMB Ltd. Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA, Scotland, in accordance with the provisions of the Budapest Treaty for the recognition of the deposit of microorganisms for the purposes of patent proceedings. The barley plant HENZ-2 was deposited on November 12, 2018 and received NCIMB accession number 43270.

In one embodiment, the barley plant of the present invention is a barley plant (Hordeum vulgare) deposited on November 12, 2018 in NCIMB under accession number NCIMB 43270 and named HENZ-2; or his offspring. Thus, the barley plant of the present invention may be a HENZ-2 barley plant deposited in NCIMB on November 12, 2018 and having NCIMB accession number 43270, or any progeny of a barley plant, wherein the progeny of a barley plant carries the G^A mutation at nucleotide 1293 of the coding HvHRT sequence SEQ ID NO: 1, and/or wherein the HvHRT gene of said barley plant encodes a mutant HvHRT protein comprising the W431stop mutation SEQ ID NO: 2.

HvHBL12.

The barley HvHBL12 gene has not been previously described. The coding sequence of the HvHBL12 gene of the barley variety Haruna Nijo is provided herein as SEQ ID NO: 5. The sequence of the HvHBL12 protein encoded by this sequence is provided herein as SEQ ID NO: 6. An additional coding sequence can be obtained from Haruna Nijo, but such a sequence carries a stop codon at codon position 216. The authors of the present invention believe

- 15 043494 it is a non-functional copy. Two HvHBL12 sequences were submitted to the NCBI database under accession numbers AK376953.1 and AK361212.1. These sequences encode proteins with different polymorphisms compared to SEQ ID NO: 6, including the following: N141D, M142V and E184D. All of polypeptide SEQ ID NO: 6, as well as polypeptides of SEQ ID NO: 6 with any of the N141D, M142V and E184D polymorphisms, are herein considered wild-type HvHBL12 proteins.

The genomic sequence of the HvHBL12 gene may differ among different barley varieties. Examples of genomic sequences of the barley HvHBL12 gene can be found in morex_contig_56855 (cv. Morex) (IBSC, 2012) and sequence accession numbers AK376953.1 and AK361212.1 in the NCBI database (partial sequences). However, the start of the coding sequence listed in the NCBI database is likely incorrect. The coding sequence of the HvHBL12 gene also differs slightly among different barley varieties. Preferably, the coding sequence of a functional HvHBL12 gene has the sequence represented herein by SEQ ID NO: 5, or a sequence sharing at least 90%, preferably at least 95%, sequence identity therewith, said sequence encoding a polypeptide of SEQ ID NO: 6 or its functional homologue, characterized by at least 95% sequence identity with it. Examples of barley HvHBL12 gene coding sequences include the sequence shown herein as SEQ ID NO: 5 or the sequence accession number HORVU5Hr1G081090.1 (Mascher et al., 2017).

Using blast searches of protein sequences using the HvHBL12 protein sequence, HvHBL12 was shown to have some degree of homology to homeodomain and leucine zipper proteins.

Amino acids 26-79 of SEQ ID NO: 6 constitute a putative homeobox-encoded domain, while amino acids 81-122 of SEQ ID NO: 6 constitute a putative homeodomain-associated leucine zipper.

A barley plant carrying a mutation in the HvHBL12 gene.

In one embodiment, the present invention relates to a barley plant carrying a mutation in the HvHBL12 gene causing loss of HvHBL12 function, and in particular, complete loss of HvHBL12 function. Loss of HvHBL12 function can be determined by determining the expression level of HvHBL12 at either the mRNA or protein level. In one embodiment, a barley plant is considered to have loss of HvHBL12 function if the barley plant contains less than 50%, preferably less than 25%, and even more preferably less than 10% mutant HvHBL12 mRNA or wild-type HvHBL12 mRNA compared to the level of HvHBL12 mRNA in the barley plant , containing the wild-type HvHBL12 gene, but otherwise having the same genotype. A barley plant may be considered to have complete loss of HvHBL12 function if the barley plant contains less than 5%, preferably less than 1%, mutant HvHBL12 mRNA or wild-type HvHBL12 mRNA compared to that of a barley plant containing the wild-type HvHBL12 gene but otherwise having the same genotype. Said mutant HvHBL12 is an mRNA encoded by a mutated HvHBL12 gene carrying a mutation in the mRNA coding region. The HvHBL12 mRNA is an RNA encoding a polypeptide of SEQ ID NO: 6 or a functional homologue thereof, and the wild-type HvHBL12 gene is a gene encoding a polypeptide of SEQ ID NO: 6 or a functional homologue thereof. Said functional homolog preferably has at least 95% sequence identity to SEQ ID NO: 6. In one embodiment, a barley plant with complete loss of HvHBL12 function may not contain mutant HvHBL12 mRNA or wild-type HvHBL12 mRNA in detectable amounts when determined by standard quantitative assay. RT-PCR.

In one embodiment, a barley plant is considered to have loss of HvHBL12 function if the barley plant contains less than 50%, preferably less than 25%, and even more preferably less than 10% mutant HvHBL12 protein or wild-type HvHBL12 protein compared to the level of HvHBL12 protein in the barley plant , containing the wild-type HvHBL12 gene, but otherwise having the same genotype. A barley plant may be considered to have complete loss of HvHBL12 function if the barley plant contains less than 5%, preferably less than 1%, of mutant HvHBL12 protein or wild-type HvHBL12 protein compared to that of a barley plant containing the wild-type HvHBL12 gene but otherwise having the same genotype. The HvHBL12 mutant protein is a polypeptide encoded by a mutated HvHBL12 gene carrying a mutation in the coding region. The HvHBL12 protein is a polypeptide of SEQ ID NO: 6 or a functional homologue thereof, and the wild-type HvHBL12 gene is a gene encoding a polypeptide of SEQ ID NO: 6 or a functional homologue thereof. Said functional homologue preferably has at least 95% sequence identity to SEQ ID NO: 6. In one embodiment, a barley plant with complete loss of HvHBL12 function may not contain mutant HvHBL12 protein or wild-type HvHBL12 protein in detectable amounts when detected. using standard Western blotting.

In one embodiment, a barley plant is considered to have a loss of HvHBL12 function if said barley plant carries a mutation resulting in an HvHBL12 gene encoding a mutant HvHBL12 protein lacking one or more of the following domains:

homeobox encoded domain: amino acids 26-79 SEQ ID NO: 6;

homeodomain-associated leucine zipper: amino acids 81-122 SEQ ID NO: 6;

C-terminal portion: amino acids 228-250 SEQ ID NO: 6.

A barley plant carrying a loss-of-function mutation in the HvHBL12 gene may carry different types of mutations, such as any of the mutations described in this section of this document.

In one embodiment, the barley plant carries a mutation in the promoter region of the HvHBL12 gene or in the intron of the HvHBL12 gene causing aberrant transcription of HvHBL12 mRNA and/or aberrant translation of HvHBL12 protein. Such barley plants may, in particular, be characterized by reduced levels of HvHBL12 mRNA, as described in this section herein above, and/or reduced levels of HvHBL12 protein, as described in this section herein above.

In one embodiment, a barley plant of the present invention carries a mutation resulting in a deletion of the HvHBL12 gene.

In one embodiment, the barley plant of the present invention carries a mutation resulting in a mutant HvHBL12 gene encoding a mutant HvHBL12 protein. In one embodiment, the mutation may be a mutation resulting in a premature stop codon. In another embodiment, the mutation is a mutation in the splice site of the HvHBL12 gene. This mutation may cause aberrant splicing of HvHBL12 mRNA.

In one embodiment, a barley plant of the present invention carries a mutation resulting in the formation of a mutant HvHBL12 gene encoding a mutant HvHBL12 protein lacking one or more amino acids of SEQ ID NO: 6, or a functional homologue thereof having at least the same 95% sequence identity.

In one embodiment, the barley plant may contain a mutant HvHBL12 gene encoding a mutant HvHBL12 protein lacking at least:

amino acids 26-79 SEQ ID NO: 6 or SEQ ID NO: 6, carrying one or more of the N141D, M142V or E184D polymorphisms; or amino acids 81-122 of SEQ ID NO: 6 or SEQ ID NO: 6, carrying one or more of the N141D, M142V or E184D polymorphisms; or amino acids 228-250 of SEQ ID NO: 6 or SEQ ID NO: 6 carrying one or more of the N141D, M142V or E184D polymorphisms.

It should be understood that a mutant HvHBL12 lacking at least amino acids XX-YY may also lack amino acids other than amino acids XX-YY.

In one embodiment, a barley plant of the present invention may contain a mutant HvHBL12 gene encoding a mutant HvHBL12 protein that is missing at least 5 most C-terminal amino acids, such as at least 10 most C-terminal amino acids, such as , for example, at least 15 amino acids closest to the C-terminus, for example, at least 20 amino acids closest to the C-terminus of SEQ ID NO: 6, or a functional homologue thereof having at least 95% sequence identity with it. For example, a barley plant may contain a mutant HvHBL12 gene encoding a mutant HvHBL12 protein lacking at least the C-terminal 22 amino acids of SEQ ID NO: 6, or a functional homologue thereof that has at least 95% sequence identity with it. .

In one embodiment, a barley plant of the present invention may contain a mutant HvHBL12 gene encoding a truncated HvHBL12 protein containing an N-terminal fragment of HvHBL12 containing no more than 245 N-terminal amino acids of SEQ ID NO: 6, or a functional homologue thereof sharing the same at least 95% sequence identity, such as no more than 240 N-terminal amino acids of SEQ ID NO: 6, or a functional homologue thereof having at least 95% sequence identity therewith, such as no more than 235 N-terminal amino acids of SEQ ID NO: 6, or a functional homologue thereof sharing at least 95% sequence identity with it, preferably no more than the N-terminal 227 amino acids of SEQ ID NO: 6, or a functional homolog thereof sharing at least 95% sequence identity with it.

In one embodiment, a barley plant of the present invention may contain a mutant HvHBL12 gene carrying a premature stop codon among any of codons 1-17 043494

245, for example, among any of codons 1-240, such as among any of codons 1-235, for example, among any of codons 1-228. For example, a barley plant may contain a mutant HvHBL12 gene that carries a premature stop codon at codon position 228. The codons are numbered according to SEQ ID NO: 5, starting at the 5' end, with one codon comprising 3 nucleotides.

In one embodiment, the barley plant of the present invention contains a mutant HvHBL12 gene encoding a mutant HvHBL12 protein with the W228stop mutation SEQ ID NO: 6. In one preferred embodiment, the barley plant of the present invention contains a mutant HvHBL12 gene encoding a polypeptide SEQ ID NO: 9 or SEQ ID NO: 9, carrying one or more of the N141D, M142V or E184D polymorphisms. In particular, said barley plant may contain a G^A mutation at nucleotide 684 of the HvHBL12 coding sequence SEQ ID NO: 5. For example, the barley plant may contain a mutant HvHBL12 gene containing the coding sequence SEQ ID NO: 8.

The functional homologue of SEQ ID NO: 6 referred to in this section may be, in particular, SEQ ID NO: 6 carrying one or more of the following polymorphisms: N141D, M142V and E184.

For the purposes of this patent application, seeds of a barley plant (Hordeum vulgare), designated HENZ-10, have been deposited with NCIMB Ltd. Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA, Scotland, in accordance with the provisions of the Budapest Treaty for the recognition of the deposit of microorganisms for the purposes of patent proceedings. Barley plant HENZ-10 was deposited on November 12, 2018 and received NCIMB accession number 43271.

In one embodiment, the barley plant of the present invention is a barley plant (Hordeum vulgare) deposited on November 12, 2018 in NCIMB under NCIMB accession number 43271 and named HENZ-10; or his offspring. Thus, the barley plant of the present invention may be a HENZ-10 barley plant deposited in NCIMB on November 12, 2018 and having NCIMB accession number 43271, or any progeny of a barley plant, wherein the barley plant carries the G^A mutation at nucleotide 684 of the coding sequence HvHBL12 SEQ ID NO: 5, and/or wherein the HvHBL12 gene of said barley plant encodes a mutant HvHBL12 protein comprising the W228stop mutation SEQ ID NO: 6.

HvWRKY38

The full-length protein product of the barley HvWRKY38 gene is thought to act as a transcriptional repressor by binding to tandemly repeated W-boxes. HvWRKY38 is a member of group II WRKY transcription factors, which contain only one WRKY domain. Zou et al., 2008, suggested that HvWRKY38 and BPBF may act as transcriptional repressors of Amy32b.

One of the barley wild-type WRKY38 coding sequences is provided herein as SEQ ID NO: 10. For HvWRKY38 CDS sequences derived from sequence accession numbers AJ536667.1 (cv. Ingrid), AK360269.1 (cv. Haruna Nijo), AY541586.1 (cv. Nure), MLOC_60890.1 (cv. Morex) subjected to multiple sequence alignment, and sequences from contig_242376 cv. Bowman (IBSC, 2012) demonstrated very high sequence identity between CDS sequences with 2 nucleotide polymorphisms 159 and 292 SEQ ID NO: 10. The protein sequence of wild-type barley HvWRKY38 is reported herein as SEQ ID NO: 11 or SEQ ID NO: 12. As a result of multiple sequence alignments, the HvWRKY38 protein sequences translated from the above genomic sequences were All HvWRKY38 proteins are demonstrated to have the sequence shown herein as SEQ ID NO: 11 or SEQ ID NO: 12, wherein amino acid 98 can be either Val or Met. Both of these sequences are considered herein to be wild-type HvWRKY38 sequences. The HvWRKY38 protein contains a WRKY domain spanning amino acids 200206 SEQ ID NO: 11 or SEQ ID NO: 12, the conserved hydrophobic residues Leu63, Val70, Leu77, Val84 and Leu91 in the leucine zipper motif SEQ ID NO: 11 or SEQ ID NO: 12 and a putative leucine zipper-like motif (C _X4 -5C _X2 2-2 ₃ H _X1 H), including Cys220, Cys226, His250 and His252 SEQ ID NO: 11 or SEQ ID NO: 12.

The genomic sequence of the WRKY38 gene may differ among different barley varieties. An example of genomic sequences of the barley WRKY38 gene is the sequence with accession number AY541586.1 in the NCBI database, as well as morex_contig_44877 (cv. Morex) (IBSC, 2012) and bowman_contig_242376 (cv. Bowman), available from the BARLEX database (https:// apex.ipkgatersleben.de/apex/f?p=284:10)).

A barley plant carrying a mutation in the WRKY38 gene.

In one embodiment, the present invention relates to a barley plant carrying a mutation in the HvWRKY38 gene causing loss of HvWRKY38 function, and in particular, complete loss of HvWRKY38 function. Loss of HvWRKY38 function can be determined by determining the expression level of HvWRKY38 at either the mRNA or protein level. In one embodiment, a barley plant is considered to have loss of HvWRKY38 function if the barley plant contains less than 50%, preferably less than 25%, and even more preferably less than 10% mutant HvWRKY38 mRNA or wild-type HvWRKY38 mRNA compared to the level of HvWRKY38 mRNA in the barley plant , containing the wild-type HvWRKY38 gene, but otherwise having the same genotype. A barley plant may be considered to have complete loss of HvWRKY38 function if the barley plant contains less than 5%, preferably less than 1%, mutant HvWRKY38 mRNA or wild-type HvWRKY38 mRNA compared to that of a barley plant containing the wild-type HvWRKY38 gene but otherwise having the same genotype. Said mutant HvWRKY38 is an mRNA encoded by a mutated HvWRKY38 gene carrying a mutation in the mRNA coding region. The HvWRKY38 mRNA is an RNA encoding a polypeptide of SEQ ID NO: 11 or SEQ ID NO: 12 or a functional homolog thereof, and the wild-type HvWRKY38 gene is a gene encoding a polypeptide SEQ ID NO: 11 or SEQ ID NO: 12 or a functional homologue thereof . Said functional homolog preferably has at least 95% sequence identity to SEQ ID NO: 11 or SEQ ID NO: 12. In one embodiment, a barley plant with complete loss of HvWRKY38 function may not contain mutant HvWRKY38 mRNA or wild-type HvWRKY38 mRNA in detectable amounts when determined using standard quantitative RT-PCR.

In one embodiment, a barley plant is considered to have loss of HvWRKY38 function if the barley plant contains less than 50%, preferably less than 25%, and even more preferably less than 10% mutant HvWRKY38 protein or wild-type HvWRKY38 protein compared to the level of HvWRKY38 protein in the barley plant , containing the wild-type HvWRKY38 gene, but otherwise having the same genotype. A barley plant may be considered to have complete loss of HvWRKY38 function if the barley plant contains less than 5%, preferably less than 1%, of mutant HvWRKY38 protein or wild-type HvWRKY38 protein compared to that of a barley plant containing the wild-type HvWRKY38 gene but otherwise having the same genotype. This HvWRKY38 mutant protein is a polypeptide encoded by a mutated HvWRKY38 gene carrying a mutation in the coding region. The HvWRKY38 protein is a polypeptide of SEQ ID NO: 11 or SEQ ID NO: 12 or a functional homologue thereof, and the wild-type HvWRKY38 gene is a gene encoding a polypeptide of SEQ ID NO: 11 or SEQ ID NO: 12 or a functional homologue thereof. Said functional homolog preferably has at least 95% sequence identity to SEQ ID NO: 11 or SEQ ID NO: 12. In one embodiment, a barley plant with complete loss of HvWRKY38 function may not contain mutant HvWRKY38 protein or wild-type HvWRKY38 protein in detectable amounts when detected using standard Western blotting.

In one embodiment, a barley plant is considered to have a loss of HvWRKY38 function if increased levels of expression can be observed from a promoter containing a tandemly repeated W box. Thus, loss of HvWRKY38 function can be determined using transient transfection with a reporter construct containing a reporter gene (eg, luciferase) under the control of an α-amylase gene promoter, such as the Amy2-1 gene. Such a construct can be prepared in the same manner as those described in Raventos et al., 1998, on page 23314 in Transient Gene Expression Assays in Onion Epidermal and Barley Aleurone Cells. An increase in luciferase activity of at least 10%, such as at least 25%, following transfection of said reporter construct into cells of a given barley plant, compared with that of transfecting said reporter construct into cells of a barley plant with the wild-type HvWRKY38 gene, but in otherwise having the same genotype is an indication that the barley plant in question is characterized by loss of HvWRKY38 function.

In one embodiment, a barley plant is considered to have a loss of HvWRKY38 function if said barley plant carries a mutation resulting in an HvWRKY38 gene encoding a mutant HvWRKY38 protein lacking one or more of the following amino acids:

amino acids 200-206 SEQ ID NO: 11 or SEQ ID NO: 12;

amino acid 63 (Leu) SEQ ID NO: 11 or SEQ ID NO: 12;

amino acid 70 (Val) SEQ ID NO: 11 or SEQ ID NO: 12;

amino acid 77 (Leu) SEQ ID NO: 11 or SEQ ID NO: 12;

amino acid 84 (Val) SEQ ID NO: 11 or SEQ ID NO: 12;

amino acid 91 (Leu) SEQ ID NO: 11 or SEQ ID NO: 12;

amino acid 220 (Cys) SEQ ID NO: 11 or SEQ ID NO: 12;

amino acid 226 (Cys) SEQ ID NO: 11 or SEQ ID NO: 12;

amino acid 250 (His) SEQ ID NO: 11 or SEQ ID NO: 12;

amino acid 252 (His) SEQ ID NO: 11 or SEQ ID NO: 12.

A barley plant carrying a loss-of-function mutation in the HvWRKY38 gene

- 19 043494

HvWRKY38 may carry different types of mutations, such as any of the mutations described in this section of this document.

In one embodiment, the barley plant carries a mutation in the promoter region of the HvWRKY38 gene or in the intron of the HvWRKY38 gene, causing aberrant transcription of HvWRKY38 mRNA and/or aberrant translation of the HvWRKY38 protein. Such barley plants may, in particular, be characterized by reduced levels of HvWRKY38 mRNA, as described in this section herein above, and/or reduced levels of HvWRKY38 protein, as described in this section herein above.

In one embodiment, a barley plant of the present invention carries a mutation resulting in a deletion of the HvWRKY38 gene.

In one embodiment, the barley plant of the present invention carries a mutation resulting in a mutant HvWRKY38 gene encoding a mutant HvWRKY38 protein. In one embodiment, the mutation may be a mutation resulting in a premature stop codon. In another embodiment, the mutation is a splice site mutation of the HvWRKY38 gene. This mutation may cause aberrant splicing of HvWRKY38 mRNA.

In one embodiment, the barley plant of the present invention carries a mutation resulting in the formation of a mutant HvWRKY38 gene encoding a mutant HvWRKY38 protein lacking at least one of the amino acids corresponding to amino acids 200-206, 220, 226, 250 and/or 252 SEQ ID NO: 11 or SEQ ID NO: 12. It is understood that a mutant HvWRKY38 lacking at least amino acids XX-YY may also lack amino acids other than amino acids XX-YY.

In one embodiment, the barley plant may contain a mutant HvWRKY38 gene encoding a mutant HvWRKY38 protein lacking at least amino acids 200-206 of SEQ ID NO: 11 or SEQ ID NO: 12. In one embodiment, the barley plant may contain a mutant HvWRKY38 gene , encoding a mutant HvWRKY38 protein lacking at least one of amino acids 220, 226, 250 and/or 252 of SEQ ID NO: 11 or SEQ ID NO: 12.

In one embodiment, the barley plant may contain a mutant HvWRKY38 gene encoding a mutant HvWRKY38 protein lacking at least one of amino acids 63, 70, 77, 84, and/or 91 of SEQ ID NO: 11 or SEQ ID NO: 12.

In one embodiment, a barley plant of the present invention may contain a mutant HvWRKY38 gene encoding a mutant HvWRKY38 protein lacking at least 102 most C-terminal amino acids, such as at least 104 most C-terminal amino acids, such as , e.g., at least the 128 most C-terminal amino acids, e.g., the at least 134 most C-terminal amino acids of SEQ ID NO: 11 or SEQ ID NO: 12. For example, a barley plant may contain a mutant HvWRKY38 gene encoding a mutant an HvWRKY38 protein lacking at least the C-terminal 154 amino acids of SEQ ID NO: 11 or SEQ ID NO: 12.

In one embodiment, a barley plant of the present invention may contain a mutant HvWRKY38 gene encoding a truncated HvWRKY38 protein containing an N-terminal fragment of HvWRKY38 containing no more than 251 N-terminal amino acids of SEQ ID NO: 11 or SEQ ID NO: 12, for example, no more than 249 N-terminal amino acids SEQ ID NO: 11 or SEQ ID NO: 12, such as not more than 225 N-terminal amino acids SEQ ID NO: 11 or SEQ ID NO: 12, such as not more than 219 N-terminal amino acids SEQ ID NO: 11 or SEQ ID NO: 12, preferably no more than 199 N-terminal amino acids of SEQ ID NO: 11 or SEQ ID NO: 12.

In one embodiment, a barley plant of the present invention may contain a mutant HvWRKY38 gene carrying a premature stop codon among any of codons 1252, for example, among any of codons 1-250, such as among any of codons 1-226, for example, among any of codons 1-220, preferably among any of codons 1-200. For example, a barley plant may contain a mutant HvWRKY38 gene that carries a premature stop codon at codon position 200. The codons are numbered according to SEQ ID NO: 10, starting at the 5' end, with one codon comprising 3 nucleotides.

In one embodiment, the barley plant of the present invention contains a mutant HvWRKY38 gene encoding a mutant HvWRKY38 protein with the W200stop mutation of SEQ ID NO: 11 or 12. In one preferred embodiment, the barley plant of the present invention contains a mutant HvWRKY38 gene encoding a polypeptide SEQ ID NO: 14 or SEQ ID NO: 14, wherein amino acid 98 is Met. In particular, said barley plant may contain a G^A mutation at nucleotide 600 of the HvWRKY38 coding sequence of SEQ ID NO: 10. For example, the barley plant may contain a mutant HvWRKY38 gene containing the coding sequence of SEQ ID NO: 13.

Barley plant.

- 20 043494

A barley plant carrying a mutation in one or more α-amylase gene promoters, in the gene

The HRT, in the HBL12 gene and/or in the WRKY38 gene, in accordance with the present invention, can be any plant of the species Hordeum vulgare, including any cross line, or cultivar, or variety.

Wild barley, Hordeum vulgare ssp. spontaneum, is considered the progenitor of currently existing cultivated forms of barley. Cultivated but heterogeneous combinations of barley are called local barley varieties. Currently, most local varieties in advanced farming have been replaced by varieties that are pure lines. Compared to local varieties, modern barley varieties have a number of improved properties (Nevo, 1992; Pelger et al., 1992).

In the present invention, the term barley plant includes any barley plant, such as local varieties of barley or modern varieties of barley. Thus, the present invention relates to any barley plant containing a mutation in one or more of the α-amylase gene promoters, the HRT gene, the HBL12 gene and/or the WRKY38 gene, such as any of the mutations described herein.

However, preferred barley plants for use with the present invention are modern barley varieties or pure lines. The barley variety to be used in connection with the present invention, for example, can be selected from the group consisting of Paustian, Sebastian, Quench, Celeste, Lux, Prestige, Saloon, Neruda, Harrington, Klages, Manley, Schooner, Stirling, Clipper, Franklin, Alexis, Blenheim, Ariel, Lenka, Maresi, Steffi, Gimpel, Cheri, Krona, Camargue, Chariot, Derkado, Prisma, Union, Beka, Kym, Asahi 5, KOU A, Swan Hals, Kanto Nakate Gold, Hakata No. 2, Kirin Choku No. 1, late-ripening variety Kanto Gold, Fuji Nijo, New Golden, Satukio Nijo, Seijo No. 17, Akagi Nijo, Azuma Golden, Amagi Nijpo, Nishino Gold, Misato golden, Haruna Nijo, Scarlett, Rosalina and Jersey, preferably from the group consisting of Haruna Nijo, Sebastian, Quench, Celeste, Lux, Prestige, Saloon, Neruda and Power , preferably from the group consisting of Paustian, Harrington, Klages, Manley, Schooner, Stirling, Clipper, Franklin, Alexis, Blenheim, Ariel, Lenka, Maresi, Steffi, Gimpel, Cheri, Krona, Camargue, Chariot, Derkado, Prisma, Union , Beka, Kym, Asahi 5, KOU A, Swan Hals, Kanto Nakate Gold, Hakata No. 2, Kirin Choku No. 1, late-ripening variety Kanto Gold, Fuji Nijo, New Golden, Satukio Nijo, Seijo No. 17, Akagi Nijo, Azuma Golden, Amagi Nijpo, Nishino Gold, Misato golden, Haruna Nijo, Scarlett and Jersey, preferably from the group consisting of Paustian, Haruna Nijo, Sebastian, Tangent, Lux, Prestige, Saloon, Neruda, Power, Quench , NFC Tipple, Barke, Class, Vintage, Applaus, Bowie, Broadway, Champ, Chanson, Charles, Chimbon, Cosmopolitan, Crossway, Dragoon, Ellinor, Embrace, Etoile, Evergreen, Flair, Highway, KWS Beckie, KWS Cantton, KWS Coralie , KWS Fantex, KWS Irina, KWS Josie, KWS Kellie, LG Diablo, LG Figaro, LG Nabuco, LG Tomahawk, Laureate, Laurikka, Lauxana, Luther, Odyssey, Ovation, Prospect, RGT Elysium, RGT Observer, RGT Planet, Rotator, Sarbi, Scholar, Subway and Golden Promise.

The barley plant may be in any suitable form. For example, the barley plant of the present invention may be a viable barley plant, a dried plant, a homogenized plant, or a ground barley kernel. The plant may be a mature plant, a germ, a grain kernel, a sprouted grain kernel, a malted grain kernel (for example, in the form of green malt or kiln-dried malt), ground malted grain kernel, ground grain kernel, etc.

The barley plant parts may be any suitable part of the plant, such as kernels, germs, leaves, stems, roots, flowers, or fragments thereof. The fragment may be, for example, a segment of a grain kernel, embryo, leaf, stem, root or flower. The barley plant parts may also be a homogenate fraction or a fraction of ground barley plant or ground grain kernel.

According to one embodiment of the present invention, the barley plant parts may be cells of said barley plant, such as viable cells that can be propagated in vitro in tissue culture. However, in other embodiments, the barley plant parts may be viable cells that are not capable of developing into a whole barley plant, i.e. cells that are not material for reproduction.

Characteristics of barley plants according to the present invention

The present invention relates to barley plants carrying a mutation in one or more of the α-amylase gene promoters, the HRT gene, the HBL12 gene and/or the WRKY38 gene. One of the main benefits of such barley plants is the increase in α-amylase activity. In particular, these barley plants may be characterized by an increase in α-amylase activity in the grain kernels during the early stages of germination.

The activity of α-amylase in barley plants is clearly regulated. During germination, α-amylase activity promotes the conversion of starch to sugar. However, at other times during plant development, such as during grain filling, α-amylase activity is undesirable because it may result in decreased grain filling, decreased starch content, grain drying out, and/or preharvest sprouting.

Moreover, the barley plants of the present invention are also characterized by having adequate agronomic qualities, such as agronomic qualities comparable to those of wild type barley. Thus, for example, it may be preferable for barley plants to have high yields, for example yields comparable to those of modern high-yielding varieties such as cv. Planet.

Therefore, one aspect of the present invention is to provide barley plants that have increased α-amylase activity during germination, but which also have adequate plant fitness.

In particular, barley plants of the present invention carrying any of the mutations described herein can have a yield that is at least 90% of the yield of a barley plant not carrying the mutation but otherwise having the same genotype. Barley plants of the present invention carrying a mutation in the HvHRT gene may, for example, have a yield that is at least 90%, preferably at least 95% of the yield of a barley plant containing the wild type HvHRT gene, but otherwise having the same genotype. Barley plants of the present invention carrying a mutation in the HvHBL12 gene may, for example, have a yield that is at least 90% of the yield of a barley plant containing the wild-type HvHBL12 gene but otherwise having the same genotype.

Thus, barley plants according to the present invention may have a 1000 grain weight (TKW) of at least 38 g, such as at least 40 g. In particular, barley plants according to the present invention bearing any of those described in mutations herein may be characterized by a TKW that is at least 90% of the TKW of a barley plant not carrying the mutation but otherwise having the same genotype. Barley plants of the present invention carrying a mutation in the HvHRT gene may, for example, have a TKW that is at least 90%, such as at least 95% of the TKW of a barley plant containing the wild-type HvHRT gene, but otherwise having the same genotype. Barley plants of the present invention carrying a mutation in the HvHBL12 gene may, for example, have a TKW that is at least 90%, such as at least 95% of the TKW of a barley plant containing the wild-type HvHBL12 gene, but otherwise having the same genotype.

Barley plants according to the present invention may have a starch content of at least 55% w/w, such as at least 60% w/w. The percentage is expressed as % starch dry weight of the total grain dry weight. In particular, barley plants of the present invention carrying any of the mutations described herein may have a starch content that is at least 90% of the starch content of a barley plant not carrying the mutation but otherwise having the same genotype. Barley plants of the present invention carrying a mutation in the HvHRT gene may, for example, have a starch content that is at least 90%, such as at least 95% of the starch content of a barley plant containing the wild-type HvHRT gene, but otherwise having the same genotype. Barley plants of the present invention carrying a mutation in the HvHBL12 gene may, for example, have a starch content that is at least 90%, such as at least 95% of the starch content of a barley plant containing the wild-type HvHBL12 gene, but otherwise having the same genotype.

Barley plants according to the present invention may have a protein content of at least 9.5% w/w. The percentage is expressed as % protein dry weight of the total dry weight of the grain. In particular, barley plants of the present invention carrying any of the mutations described herein may have a protein content that is at least 90% of the protein content of a barley plant not carrying the mutation but otherwise having the same genotype. Barley plants of the present invention carrying a mutation in the HvHRT gene may, for example, have a protein content that is at least 90%, such as at least 95% of the protein content of a barley plant containing the wild-type HvHRT gene, but otherwise having the same genotype. Barley plants of the present invention carrying a mutation in the HvHBL12 gene may, for example, have a protein content that is at least 90%, such as at least 95% of the protein content of a barley plant containing the wild-type HvHBL12 gene, but otherwise having the same genotype.

Barley plants of the present invention carrying any of the mutations described herein can have a height that is at least 90% of the height of a barley plant not carrying the mutation but otherwise having the same genotype. Barley plants of the present invention carrying a mutation in the HvHRT gene may, for example, have a height that is at least 90%, such as at least 95% of the height of a barley plant containing the wild type HvHRT gene , but otherwise having the same genotype. Barley plants of the present invention carrying a mutation in the HvHBL12 gene may, for example, have a height that is at least 90%, such as at least 95% of the height of a barley plant containing the wild-type HvHBL12 gene, but otherwise having the same genotype.

Reduced gibberellic acid levels in barley plants have been described to be associated with higher head counts. Thus, the barley sdw1/denso mutant is characterized by a reduced level of expression of genes involved in GA biosynthesis and an increased number of ears per unit area (Jia et al., 2011). Therefore, there is a risk that barley plants with elevated GA levels will have a reduced number of heads. However, the barley plants of the present invention preferably have approximately the same number of ears as wild-type barley plants. Thus, barley plants of the present invention carrying any of the mutations described herein may have a number of ears/m ² that is at least 90% of the number of ears/m ² of a barley plant not carrying the mutation, but otherwise having the same genotype. Barley plants of the present invention carrying a mutation in the HvHRT gene may, for example, have a number of ears/m ² that is at least 90%, such as at least 95% of the number of ears/m ² of a barley plant containing the HvHRT gene is wild type, but otherwise has the same genotype. Barley plants of the present invention carrying a mutation in the HvHBL12 gene may, for example, have a number of ears/m ² that is at least 90%, such as at least 95% of the number of ears/m ² of a barley plant containing the HvHBL12 gene is wild type, but otherwise has the same genotype.

Barley plants of the present invention carrying any of the mutations described herein are preferably not pre-harvest germinated. In particular, the barley plants of the present invention are characterized by a percentage of germination of kernels collected from barley plants that have been regularly sprayed with water for 20 days that is the same as or greater than the percentage of germination of a barley plant of the same species that has not been sprayed and collected in ripeness. In particular, barley plants in accordance with the present invention are preferably not subjected to pre-harvest germination when determined by the method described herein below in Example 3B.

In one embodiment, the barley plant of the present invention has an α-amylase activity of at least 16 U/g 48 hours after initiation of germination.

In one embodiment, the barley plant of the present invention has an α-amylase activity of at least 4 U/g after 48 hours and/or at least 7 U/g after 72 hours after initiation of germination, in the case where said barley plants were germinated by immersion in water with an air flow of 9 l/h. This, for example, may be true in the case of barley plants carrying a mutation in the HvHRT gene.

In one embodiment, the barley plant of the present invention has an α-amylase activity of at least 15 U/g after 48 hours and/or at least 20 U/g after 72 hours after initiation of germination, in the case where said barley plants were germinated by immersion in water with an air flow of 9 l/h. This, for example, may be true in the case of barley plants carrying a mutation in the HvHBL12 gene.

In one embodiment, the barley plant of the present invention has an α-amylase activity of at least 6 U/g 72 hours after initiation of germination when said barley plants were germinated by immersion in water without air flow. This, for example, may be true in the case of barley plants carrying a mutation in the HvHRT gene.

In one embodiment, the barley plant of the present invention has an α-amylase activity of at least 4 U/g after 48 hours and/or at least 14 U/g after 72 hours after initiation of germination, in the case where said barley plants were germinated by immersion in water without air flow. This may, for example, be true in the case of barley plants carrying a mutation in the HvHBL12 gene, or barley plants carrying at least four α-amylase genes containing an α-amylase gene promoter containing a non-standard tandem repeating W-box.

In one embodiment, the barley plant of the present invention has an α-amylase activity 48 hours after initiation of germination that is at least 105%, such as at least 110%, such as at least 120%, of the plant's α-amylase activity barley that does not carry the specified mutation, but otherwise has the same genotype.

In one embodiment, the barley plant of the present invention has an α-amylase activity 72 hours after initiation of germination that is at least 120%, such as at least 150%, such as at least 170% α-23 activity 043494 amylase in a barley plant that does not carry the indicated mutation, but otherwise has the same genotype.

This may be particularly true in the case of barley plants carrying a mutation in HvHRT, such as any of the mutations described herein.

In one embodiment, the barley plant of the present invention has an α-amylase activity 72 hours after initiation of germination that is at least 120%, such as at least 150%, such as at least 170% of the barley plant α-amylase activity , which does not carry the indicated mutation, but otherwise has the same genotype, in the case of germination when immersed in water without aeration. This may particularly be true in the case of barley plants carrying a mutation in HvHBL12, such as any of the mutations described herein.

In one embodiment, the barley plant of the present invention has an α-amylase activity 72 hours after initiation of germination that is at least 150%, such as at least 200%, such as at least 300%, of the barley plant α-amylase activity , which does not carry the indicated mutation, but otherwise has the same genotype, in the case of germination when immersed in water without aeration. This may particularly be true in the case of barley plants carrying at least four α-amylase genes containing an α-amylase gene promoter containing a non-standard tandem repeating W-box, such as any of those described herein.

In one embodiment, the barley plant of the present invention is a naked barley plant, and wherein said barley plant has an α-amylase activity of at least 140 U/g, such as at least 150 U/g, such as at least 160 U/g, such as at least 170 U/g 48 hours after initiation of germination. In one embodiment, the barley plant is a naked barley plant, and wherein said barley plant has an α-amylase activity of at least 100 U/g, such as at least 110 U/g 48 hours after initiation of germination.

In one embodiment, the barley plant is a naked barley plant and has a limiting dextrinase activity of at least 30 mU/g, such as at least 35 mU/g, such as at least 40 mU/g 48 hours after initiation of germination. In one embodiment, the barley plant of the present invention has a limiting dextrinase activity of at least 20 mU/g 48 hours after initiation of germination.

In one embodiment, the barley plant is a filmy barley plant that has an α-amylase activity of at least 140 U/g, such as at least 150 U/g, such as at least 160 U/g, such as eg at least 170 U/g 48 hours after initiation of germination, provided that at least part of the film has been removed from the barley grain kernels before initiation of germination. In one embodiment, the barley plant is a filmy barley plant that has an α-amylase activity of at least 100 U/g, such as at least 110 U/g 48 hours after initiation of germination, provided that at least part of the film was removed from the kernels of barley grains before germination was initiated. In one embodiment, the barley plant is a filmy barley plant that has a limiting dextrinase activity of at least 30 mU/g, such as at least 35 mU/g, such as at least 40 mU/g after 48 hours after initiation of germination, provided that at least part of the film has been removed from the kernels of barley grains before initiation of germination. In one embodiment, the barley plant is a filmy barley plant that has a limiting dextrinase activity of at least 20 mU/g, provided that at least a portion of the film has been removed from the barley grain kernels before germination is initiated. For example, said film can be removed by mechanical processing. Said film may be removed by mechanical treatment in a manner, for example resulting in a reduction in the total weight of the barley kernels by at least 2%, such as by at least 3%, for example by a value in the range of 3-6% .

In one embodiment, the barley plants of the present invention may have an improved ability to germinate under stressful conditions. Thus, in one embodiment, at least 60%, preferably at least 70%, e.g., at least 75% of the kernels of said barley plant germinate under high water conditions, e.g., as determined by the method described in Example 3C .

Barley plants containing more than one mutation.

The present invention also relates to barley plants having more than one mutation. Therefore, the barley plants of the present invention may contain one or more of the following mutations:

- 24 043494 mutation in one or more α-amylase gene promoters, for example, any of the mutations described herein above under A-amylase and barley plants carrying a mutation in the α-amylase gene promoter, a mutation in the HvHRT gene, for example, any of the mutations described herein above under Barley plant carrying a mutation in the HRT gene, mutation in the HvHBL12 gene, for example, any of the mutations described herein above under Barley plant carrying a mutation in the HvHBL12 gene, mutation in the HvWRKY38, for example, any of the mutations described herein above under Barley Plant Carrying a Mutation in the WRKY38 Gene.

In addition to the mutations described herein, barley plants may also harbor one or more additional mutations. Therefore, a barley plant may harbor one or more of the following mutations.

In addition to one or more of the mutations described above, the barley plant may also harbor a mutation in the LOX-1 encoding gene, resulting in a complete loss of functional LOX-1. Said mutation, for example, may be any of the mutations described in international patent application WO 2005/087934. For example, a barley plant may contain a LOX-1 encoding gene that provides a premature stop codon, wherein said codon corresponds to base nos. 3572-3574 of SEQ ID NO: 2 of WO 2005/087934, or a splice site mutation, wherein said mutation corresponds to base no. 2311 SEQ ID NO: 6 or SEQ ID NO: 2 of WO 2005/087934.

In addition to one or more of the mutations described above, the barley plant may also harbor a mutation in the LOX-2 encoding gene, resulting in a complete loss of functional LOX-2. Said mutation, for example, may be any of the mutations described in international patent application WO 2010/075860. For example, a barley plant may contain a gene encoding LOX-2 that has a mutation at nucleotide position 2689 of SEQ ID NO: 1 of WO 2010/075860 causing a premature stop codon.

In addition to one or more of the mutations described above, the barley plant may also harbor a mutation in the MMT-encoding gene, resulting in a complete loss of functional MMT. Said mutation, for example, may be any of the mutations described in international patent application WO 2010/063288. For example, a barley plant may contain an MMT-encoding gene comprising the G^A mutation at base No. 3076 of SEQ ID NO: 3 of WO 2010/063288, or an MMT-encoding gene comprising the G^A mutation at base No. 1462 of SEQ ID NO: 16 of WO 2010/063288.

In addition to one or more of the mutations described above, the barley plant may also include a mutation in the CslF6 encoding gene, wherein the mutant gene encodes a mutant CslF6 protein with reduced CslF6 activity. Said mutation may be, for example, any of the mutations described in a related application entitled Cereal plants with improved cell wall properties, owned by the same applicant and having the same filing date as this application. For example, a barley plant may contain a CslF6-encoding gene encoding a CslF6 mutant comprising the G847E mutation, or the G748D mutation, or the T709I mutation of SEQ ID NO: 1 or SEQ ID NO: 3 of the related application. SEQ ID NO: 1 of the identified related application has GenBank NCBI accession number: EU267181.1.

Products of plant origin

The present invention also relates to plant products obtained from a barley plant carrying a mutation in one or more of the α-amylase gene promoters, the HRT gene, the HBL12 gene and/or the WRKY38 gene, for example, any of the barley plants described herein .

The plant product may be any product derived from the barley plant, such as a food, feed or beverage. Thus, the plant product may be any of the beverages described herein below in the Beverage and Method of Preparation section. The plant product may also be an aqueous extract of a barley plant and/or malt derived from the kernels of said barley plant, for example the plant product may be a wort. This aqueous extract can be obtained, for example, as described herein below in the section Aqueous extract and methods for its preparation.

In one embodiment, the plant product may be malt, such as green malt or kiln-dried malt, such as any of the malts described herein below under Green Malt, Malt and Methods for Making Them, or Malt-Based Products , such as malt-based drinks. Although the primary purpose of green malt and/or kiln-dried malt is to produce a beverage, it can also be

- 25 043494 used in other production processes, for example as a source of enzymes in the baking industry or in the food industry as a flavoring agent and coloring agent, for example in the form of malt or malt flour, or indirectly in the form of malt syrup, etc. e. Thus, the herbal product according to the present invention may be any of the above-mentioned products.

According to another aspect, the plant products of the present invention contain or even consist of a syrup, such as barley syrup or barley malt syrup. The plant product may also be barley or malt extract. Thus, the plant product may be a wort.

Green malt, kiln-dried malt and methods for their production.

The present invention also relates to malt obtained from a barley plant carrying a mutation in one or more of the α-amylase gene promoters, the HRT gene, the HBL12 gene and/or the WRKY38 gene, for example, any of the barley plants described herein. The malt may be green malt or kiln-dried malt obtained from barley grains of a barley plant carrying a mutation in one or more of the α-amylase gene promoters, the HRT gene, the HBL12 gene and/or the WRKY38 gene. Said mutation may be any of the mutations described herein above.

Green malt can be obtained by malting, i.e. by germinating cereal grains under controlled environmental conditions. Typically, said germination may involve a step of soaking the barley grain kernels followed by a germination step. Soaking and germination can also be carried out simultaneously or partially simultaneously. In some embodiments, the production of malt may include the step of drying the sprouted grains. Said drying step may preferably be oven drying of the germinated grain kernels at elevated temperatures. Thus, kiln-dried malt can be produced by subjecting green malt to a kiln-drying step.

Thus, in one embodiment, the malting process may comprise the steps of:

(a) providing grain kernels of a barley plant, in particular a barley plant carrying a mutation in one or more of the α-amylase gene promoters, the HRT gene, the HBL12 gene and/or the WRKY38 gene;

(b) soaking said barley grain kernels;

(b) germinating said barley grain kernel; and (c) drying said germinated barley grain kernels, preferably by oven drying.

Sprouted barley grains can be obtained using a method involving the following stages:

(a) providing grain kernels of a barley plant, in particular a barley plant carrying a mutation in one or more of the α-amylase gene promoters, the HRT gene, the HBL12 gene and/or the WRKY38 gene;

(b) soaking said barley grain kernels;

(b) germinating said barley grain kernel.

The soaking and germination steps can be carried out sequentially, simultaneously or partially simultaneously.

In one preferred embodiment, soaking and sprouting are carried out simultaneously in a sprouting process that involves keeping the barley grains in an aqueous solution, usually with aeration, for no more than 72 hours.

Soaking can be carried out using any standard method known to one skilled in the art. One non-limiting example includes soaking at temperatures in the range of 10-25°C with alternating dry and wet conditions. During soaking, for example, the barley kernels may be kept wet for a period of time ranging from 30 minutes to 3 hours, followed by a dry condition for a time ranging from 30 minutes to 3 hours, and optionally repeating the said conditioning pattern. in the range from 2 to 5 times. The final water content after soaking may, for example, be in the range of 40 to 50%, for example in the range of 40-45%.

The barley plants of the present invention are characterized in that they carry a mutation in one or more of the α-amylase gene promoters, the HRT gene, the HBL12 gene and/or the WRKY38 gene. One of the main advantages of such barley plants is the increased activity of hydrolytic enzymes such as α-amylase and limiting dextrinase during germination. The grains of such barley plants can be successfully germinated during a short germination method, since one of the goals of malting is to induce sufficient hydrolytic enzyme activity. Interestingly, the barley plants of the present invention are characterized by a certain level of hydrolytic enzyme activity in the early stages of germination, which allows short germination methods to be used.

Examples of suitable short germination methods are described in the international patent application

- 26 043494

PCT/EP2017/065498, which is incorporated herein by reference. One example of a suitable short sprouting method is a sprouting method that involves a step in which the barley grains are kept in an aqueous solution, usually with aeration, and the entire sprouting process is completed in no more than 72 hours.

As described above, germination may include the step of keeping grains of a barley plant carrying a mutation in one or more of the α-amylase gene promoters, the HRT gene, the HBL12 gene and/or the WRKY38 gene in an aqueous solution with aeration. Barley grains can be kept in said aqueous solution for a sufficient period of time to ensure germination of most of said barley grains. Barley grains can also be kept in said aqueous solution for a sufficient period of time to obtain a water content of at least 35%, preferably at least 37%, for example in the range of 35-60%. Typically the barley grains are kept in the aqueous solution for at least 20 hours, such as at least 24 hours. Typically the grains are kept in said aqueous solution for no more than 72 hours, such as no more than 60 hours, for example for no more than 48 hours. Thus, in some embodiments, the barley grains are incubated in said aqueous solution for a time in the range of 20-72 hours, such as for a time in the range of 20-60 hours, e.g. in the range of 20-48 hours, for example, for a time in the range of 20-30 hours, such as for a time in the range of 22-26 hours.

It may be preferable that the barley grains are completely covered with said aqueous solution throughout the entire aging process.

Said barley grains are often kept in said aqueous solution, with O ₂ being passed through the aqueous solution. Said _O2 may be added to said aqueous solution as pure O2. However, this O2 is often contained in a mixture of gases. According to one embodiment, said O2 is contained in atmospheric air.

In general, at least 2 L, preferably at least 3 L, more preferably at least 4 L, even more preferably at least 5 L, even more preferably at least 6 L of O2 per kg of barley grains are passed through said aqueous solution per hour. The weight of the indicated barley grains is the dry weight. For example, O ₂ is passed through a mixture of said aqueous solution/barley grain in an amount in the range of 2-100 L, for example in the range of 2-75 L, such as in the range of 2-50 L, for example in the range of 4- 100 l, e.g. in the range 4-75 l, as in the range 4-50 l, e.g. in the range 6-100 l, e.g. in the range 6-75 l, as in the range 6-50 l per kg of barley grains (dry weight) in h.

As noted above, it is often found in atmospheric air, which is passed through an aqueous solution. Thus, the method may involve passing at least 10 L, preferably at least 15 L, more preferably at least 20 L, even more preferably at least 25 L, even more preferably at least 30 L, through said aqueous solution. air per kg of barley grains per hour. The weight of the indicated barley grains is the dry weight. For example, atmospheric air is passed through said aqueous solution in an amount in the range of 10-500 L, for example in the range of 10-375 L, such as in the range of 10-250 L, for example in the range of 20-500 L, for example , in the range of 20-375 l, such as in the range of 20-250 l, for example in the range of 30-500 l, for example in the range of 30-375 l, such as in the range of 30-250 l per kg of grains barley (dry weight) in h.

In some embodiments, the germination step comprises:

a) at least one step of keeping said kernels in an aqueous solution, wherein at least 2 liters of O ₂ per kg dry weight of barley kernels per hour are passed through said aqueous solution; And

b) at least one stage of keeping said barley grain kernels in air.

In some embodiments, after incubating the barley grains in said aqueous solution, the barley grains have a water content of at least 20%, preferably at least 30%, such as in the range of 30-60%, such as in the range of 30-50%, for example, in the range of 30-60%, as in the range of 30-50%.

During said step of exposing said barley kernels to air, at least 2 liters of O ₂ per kg dry weight of barley kernels per hour may be passed through said barley kernels. For example, a similar amount of O ₂ may be passed through the barley kernels during exposure in air, as during exposure in the specified aqueous solution, as described above.

The sprouted barley kernels obtained by this method are also referred to herein as green malt.

The water content of barley grains can be determined by determining the weight of the barley grains, then drying said barley grains and determining the weight of the dried barley grains. The difference in weight between wet and dry barley grains is due to water, and the water content is obtained as the weight of water divided by the total weight of the barley grains (wet barley grains). Thus, the water content represented as % is w/w %.

The barley grain can be kept at any suitable temperature, but it may be preferable that the conditioning be carried out at a temperature high enough to allow a rapid increase in water content.

In particular, according to embodiments of the present invention in which barley grains are kept at a temperature in the range of 20-30°C, said barley grains can be kept for a time in the range of 20-48 hours.

Germination of grains can also be carried out using any standard method known to a person skilled in the art. One non-limiting example involves germination at a temperature in the range of 10-25°C, optionally varying the temperature at intervals in the range of 14 days.

As mentioned above, in some embodiments of the present invention, sprouted barley grains (ie, green malt) can be oven dried. In some embodiments, it is preferable that the green malt is not oven dried. In particular, it is preferable that if the green malt is produced by germination, which involves the step of keeping said barley grains in an aqueous solution with aeration, the green malt is not oven dried.

If green malt is kiln dried, it can be done at standard temperatures, such as at least 75°C, for example in the range of 80-90°C, such as in the range of 80-85°C. Thus, malt, for example, can be produced using any of the methods described by Hough et al. (1982). However, any other suitable method for producing malt may also be used for purposes of the present invention, such as methods for producing specialty malt, including, but not limited to, methods for roasting malt.

Kiln-dried malt and green malt can be further processed, for example by grinding. Thus, the plant product of the present invention may be any type of malt, such as raw malt or ground malt such as flour. Thus, the plant product may be, for example, ground, kiln-dried malt or ground green malt. Ground malt and malt flour contain chemical components of malt and dead cells that have lost the ability to germinate again.

In some embodiments, the barley is chaffed barley, and the method includes the step of removing at least a portion of said film before exposing said kernels to an aqueous solution. Filmy cereal grains can be processed to remove the film by subjecting the cereal grains to mechanical processing to remove the film. Said mechanical treatment, for example, may be selected from the group consisting of polishing, abrasive peeling, peeling and grinding. Preferably, mechanical processing results in loss of film. Film loss can be defined as loss relative to total weight. Thus, mechanical processing preferably results in a loss of 1-4%, such as a loss of 1.5-3.0% of the total weight of the cereal grains.

Aqueous extract and methods for its preparation.

The present invention relates to barley-based beverages, as well as methods for producing them, wherein the barley plant carries a mutation in one or more of the α-amylase gene promoters, the HRT gene, the HBL12 gene and/or the WRKY38 gene, for example, any of those described in mutations herein. The present invention also relates to aqueous extracts of grain kernels of barley plants carrying a mutation in one or more of the α-amylase gene promoters, the HRT gene, the HBL12 gene and/or the WRKY38 gene. Said aqueous extract can be obtained, for example, from green malt or kiln-dried malt.

Often, methods for producing a beverage include the step of obtaining an aqueous extract of barley plant kernels of the present invention and/or malts obtained from barley plants of the present invention.

The aqueous extract can typically be prepared by soaking barley flour, green malt flour and/or kiln-dried malt flour in water or an aqueous solution. This aqueous solution is also referred to herein as a mash solution. In particular, the aqueous extract can be obtained by mashing.

The present invention also relates to a method for producing an aqueous extract, said method comprising the steps of:

a) providing grain kernels of a barley plant, wherein said barley plant carries a mutation in one or more of the α-amylase gene promoters, the HRT gene, the HBL12 gene and/or the WRKY38 gene, for example, any of the mutations described herein;

b) subjecting barley grain kernels to the germination stage, thereby obtaining sprouted grain kernels, wherein said germination stage involves keeping said grain kernels in an aqueous solution for no more than 72 hours;

c) fine grinding of said sprouted grain kernels, wherein said sprouted kernels

- 28 043494 kernels are characterized by a water content of at least 20%, with the proviso that said barley grain kernels are not characterized by a water content below 20 at any time between stages b) and c);

d) obtaining an aqueous extract of said ground sprouted grain kernels, thereby obtaining an aqueous extract of barley.

The germination stage is described in more detail above in the section Green malt, kiln-dried malt and methods for producing them.

Typically, said mash solution may be water, such as tap water, to which one or more additional agents may be added. Additional agents may be present in the aqueous solution from the very beginning, or they may be added during the process of obtaining the aqueous extract. Said additional agents may be enzymes. Thus, the mash solution may contain one or more enzymes. These enzymes can be added to the aqueous solution from the very beginning or later in the process.

Said enzymes may be, for example, one or more hydrolytic enzymes. Suitable enzymes include lipases, starch-degrading enzymes (eg amylases), glucanases [preferably (1-4)- and/or (1,3;1,4)-β-glucanases], and/or xylanases (such as arabinoxylanases) , and/or proteases or enzyme mixtures containing one or more of the above enzymes, for example Cereflo, Ultraflo or Ondea Pro (Novozymes). For example, the aqueous solution may contain one or more hydrolytic enzymes selected from the group consisting of α-amylase, β-amylase, limit dextrinase, pullulanase, β-glucanase (for example, endo-(1,3;1,4)-glucanase or endo-1,4-β-glucanases), xylanases (eg endo- or exo-1,4-xylanases, arabinofuranosidases or ferulic acid esterases), glucoamylases and proteases.

In one embodiment, only limited amounts of α-amylase, or none at all, are added to said mash solution.

In one embodiment, only limited or no amounts of limiting dextrinase and pullulanase are added to said mash solution.

Said additional agents, preferably for food purposes, may also be a salt, for example CaCl ₂ , or an acid, for example H ₃ PO ₄ .

An aqueous extract is typically produced by soaking barley flour, green malt flour and/or kiln-dried malt flour in a mash solution at one or more specified temperatures. The specified target temperature may also be referred to herein as the mash temperature. Said mashing temperatures may be, for example, standard temperatures used for mashing. The mash temperature is usually either kept constant (isothermal mashing) or gradually increased, e.g. incrementally. In either case, the soluble substances in the barley grains and/or malt are released into said mash liquor, resulting in an aqueous extract.

The mash temperature(s) is typically a temperature(s) in the range of 30-90°C, such as in the range of 40-85°C, such as in the range of 50-85°C. Often the mash soak includes a final heating step to a higher temperature, such as a temperature in the 75-80°C range.

After soaking in an aqueous solution, such as a mash tun, the aqueous solution can be transferred to another container, such as a lauter tun, and held for an additional period of time at an elevated temperature.

Non-limiting examples of suitable mash protocols can be found in the brewing literature, such as Hough et al. (higher).

Mashing (i.e., aging barley flour, green malt flour, and/or kiln-dried malt flour in a mash liquor) may occur in the presence of additives, which are considered to provide any carbohydrate source other than malt or sprouted grains barley, such as, without limitation, barley, barley syrups or maize, or rice, either in the form of whole grain kernels or in the form of processed products such as cereals, syrups or starches. All of the above additives can be used primarily as an additional source of extract (syrups are usually dosed during the heating of the wort). The additive handling requirements at the brewery depend on the condition and type of additive used.

After soaking in the mash liquor, the aqueous extract can usually be separated, for example by filtration, separating the aqueous extract and residual undissolved solids, the latter also called spent grain. Filtration can be carried out, for example, in a filtration tank. Alternatively, the filtration may be filtering through a filter to separate the mash. The aqueous extract thus obtained may also be called the first wort. In a process also called rinsing the spent grain, additional liquid, such as water, can be added to the spent grain. After washing the spent grains and filtering, a second wort can be obtained. Additional wort fractions can be obtained by repeating the procedure.

- 29 043494

Thus, the aqueous extract may be a wort, such as a first wort, a second wort, an additional wort, or a combination thereof.

According to one embodiment, the method for producing an aqueous extract can be carried out using any of the devices described in international patent application PCT/EP2017/065498, for example, any of the devices described therein on pages 20-22. A non-limiting example of a suitable device is shown herein in FIG. 12.

The drink and how to get it

The present invention also relates to barley-based beverages and methods for producing such beverages, wherein the beverages are obtained from a barley plant carrying a mutation in one or more of the α-amylase gene promoters, the HRT gene, the HBL12 gene and/or the WRKY38 gene, for example, any of the mutations described herein.

Said beverage may be barley-based alcoholic beverages or barley-based soft drinks. Barley-based alcoholic beverages can be, for example, beer or distilled alcohol.

Said beer may be any type of beer, such as lager or ale. Thus, the beer, for example, may be selected from the group consisting of the following: Altbier, Amber Ale, Barley Wine, Berliner Weisse, Bière de Garde, Bitters, Blonde Ale, Bock, Brown Ale, California Regular Beer , butter ale, Dortmunder, Doppelbock, Dunkel, Dunkelweizen, Eisbock, Fruit lambic, Golden ale, Gose, Guez, Hefeweizen, Helles, Indian pale ale, Kelyp, Lambic, pale ale, Maibock, malt liqueur, Mild, March beer, Old Ale, Flanders Brown Ale, Pale Ale, Pilsner, Porter, Red Ale, Rye Beer, Seasonal Beer, Scotch Ale, Steam Beer, Stout, Black Beer, Lager, Belgian White Beer, Wheat Beer and Wheat Bock.

Said distilled alcohol may be any type of distilled alcohol. In particular, the alcohol obtained by distillation can be obtained from barley, for example barley malt. Non-limiting examples of such distilled spirits include whiskey and vodka.

The beverage may be a non-alcoholic beverage, such as a barley-based soft drink, such as a non-alcoholic beer or a non-alcoholic malt beverage such as Maltina.

For example, the beverage can be produced using a process comprising the steps of:

(i) providing barley plant kernels in accordance with the present invention, and/or green malt and/or kiln-dried malt obtained from barley plant kernels in accordance with the present invention, (ii) obtaining an aqueous extract of said grain kernels, and /or said green malt, and/or said kiln-dried malt, for example as described herein above in the section Preparation of an aqueous extract, (iii) processing of said aqueous extract into a beverage.

The aqueous extract can be boiled with or without hops, after which it can be called boiled wort. The first, second and additional wort can be combined and then boiled. The aqueous extract can be boiled for any suitable period of time, for example, in the range of 60 minutes to 120 minutes. Stage (iii) may include:

a) heating said aqueous extract, optionally in the presence of hops or hop extract;

b) cooling the aqueous extract;

c) fermenting said aqueous extract using yeast to thereby obtain a fermented beverage.

Step (iii) in particular involves fermenting said aqueous extract, for example by fermenting wort. Thus, the drink can be obtained by fermenting the aqueous extract using yeast.

Once the aqueous extract is obtained, it can be processed into beer using any method, including traditional brewing methods. Unlimited descriptions of examples of suitable brewing methods can be found, for example, in publications by Hough et al. (1982). Numerous regularly updated methods for the analysis of barley and beer products are offered by, for example, but not limited to, the American Association of Cereal Chemists (1995), the American Society of Brewing Chemists (1992), the European Brewing Convention (1998) and the Institute of Brewing (1997). It is recognized that many specific procedures are used for a given brewery, with the most significant differences being local consumer preferences. Any such method for producing beer can be used in connection with the present invention.

The first stage of producing beer from an aqueous extract preferably involves boiling said aqueous extract as described above herein, followed by a cooling phase and optionally a vortex rest. One or more additional compounds may be added to the aqueous extract, such as one or more of the additional compounds described below under Additional Compounds. After cooling, the aqueous extract can be transferred to fermentation tanks containing yeast, for example brewer's yeast such as S. pastorianus or S. cerevisiae. The aqueous extract can be fermented for any suitable period of time, usually for a period of time in the range of 1-20 days, such as 1-10 days. Fermentation is carried out at any suitable temperature, for example at a temperature in the range of 10-20°C. The methods may also include the addition of one or more enzymes, for example, one or more enzymes may be added to the wort before or during fermentation. In particular, said enzyme may be a proline-specific endoprotease. A non-limiting example of a proline-specific endoprotease is Brewer's Clarex, available from DSM. In other embodiments, no exogenous enzymes are added during these methods.

During the fermentation process, which lasts several days, sugar is converted into alcohol and CO ₂ , at the same time as some aromatic substances are formed. Fermentation can be stopped at any desired time, for example immediately after there is no further reduction in %P.

The beer can then be further processed, for example chilled. It can also be filtered and/or lagered, a process that produces a pleasant aroma and reduces yeasty flavors and odors. You can also add additives. In addition, CO2 can be added. Finally, beer can be pasteurized and/or filtered before it is packaged (for example, before being transferred to containers or barrels, bottled or canned). Beer can also be pasteurized using standard methods.

Additional connections

The methods of the present invention may include the step of adding one or more additional compounds. Said additional compounds may be, for example, a food flavoring agent, a preservative, a functional ingredient, a coloring agent, a sweetener, a pH adjusting agent, or a salt. The pH adjusting agent may be, for example, a buffer or an acid such as phosphoric acid.

Functional ingredients can be any ingredient added to provide a specific function. Preferably, the functional ingredient makes the drink more dietary. Non-limiting examples of functional ingredients include vitamins or minerals.

The preservative may be any food preservative, for example it may be benzoic acid, sorbic acid, sorbates (eg potassium sorbate), sulfites and/or salts thereof.

The additional compound can also be CO2. In particular, CO2 can be added to produce a carbonated beverage.

The flavoring agent used in connection with the present invention may be any suitable flavoring agent.

The flavoring agent, for example, may be selected from the group consisting of flavors, herbal extracts, herbal concentrates, plant parts and herbal infusions. In particular, the flavoring agents may be hops.

A method for producing a barley plant carrying a mutation in one or more promoters of the α-amylase genes, the HRT gene, the HBL12 gene and/or the WRKY38 gene

Barley plants carrying a mutation in one or more of the α-amylase gene promoters, the HRT gene, the HBL12 gene and/or the WRKY38 gene, for example, any of the mutations described herein, can be produced by any suitable method.

For example, such barley plants can be produced using a process comprising the steps of:

exposing a plurality of barley plants or barley grain kernels to non-specific mutagenesis, for example by irradiation or chemical treatment, for example treatment with sodium azide;

identifying barley plants or barley grain kernels carrying the desired mutation (eg, a mutation in one or more α-amylase gene promoters, the HRT gene, the HBL12 gene and/or the WRKY38 gene).

Such methods may also include one or more steps of propagating said barley plants/barley kernels to produce a plurality of barley plants/kernels, each of which carries random mutations.

In particular, barley plants carrying a particular mutation can be obtained and identified essentially as described in international patent application PCT/EP2017/065516 using primers and probes designed to identify the desired mutation. A non-limiting example of primers and probes useful for identifying a barley plant carrying a mutation in the α-amylase gene promoter is presented in Example 13A. Non-limiting example of primers and probes suitable for

- 31 043494 identification of a barley plant carrying a mutation in the HRT gene is presented in example 2A. Two additional non-limiting examples of primers and probes useful for identifying a barley plant carrying a mutation in the HRT gene are presented in Example 14. A non-limiting example of primers and probes useful for identifying a barley plant carrying a mutation in the HBL12 gene is presented in Example 7A. A non-limiting example of primers and probes useful for identifying a barley plant carrying a mutation in the WRKY38 gene is presented in Example 12A. Those skilled in the art will be able to design suitable primers and probes to identify other mutants using prior knowledge in the various fields and/or guidance provided in international patent application PCT/EP2017/065516, which is incorporated herein by reference.

Barley plants carrying a mutation in one or more of the α-amylase gene promoters, the HRT gene, the HBL12 gene and/or the WRKY38 gene can also be produced using various site-directed mutagenesis methods, which, for example, can be developed based on the sequences of the promoters of the α-amylase genes, the HRT gene, the HBL12 gene and/or the WRKY38 gene presented herein. In one embodiment, a barley plant is produced using any of the CRISPR, TALEN, zinc finger nuclease, meganuclease, and DNA cutting antibiotic methods as described in WO 2017/138986. In one embodiment, a barley plant is produced using a CRISPR/cas9 technique, for example using an RNA-guided Cas9 nuclease. This technique can be performed as described in Lawrenson et al., Genome Biology (2015) 16:258; DOI 10.1186/s13059-015-0826-7, except that the single guide RNA sequence is designed based on the gene sequences presented herein. In one embodiment, a barley plant is produced using a combination of both TALEN and CRISPR/cas9 techniques, for example, using a Cas9 RNA-guided nuclease. This technique can be performed as described in Holme et al., Plant Mol Biol (2017) 95:111–121; DOI: 10.1007/s11103-017-0640-6, except that the TALEN and single guide RNA sequence are designed based on the gene sequences presented herein.

In one embodiment, a cereal plant is produced using a homologous repair process, a combination of a DNA cutting nuclease and a donor DNA fragment. This technique can be performed as described in Sun et al., Molecular Plant (2016) 9:628–631; DOI: https://doi.org/10.1016/j.molp.2016.01.001, except that the DNA cutting nuclease is designed based on the gene sequences presented herein and the donor DNA fragment is designed based on the coding sequence of the mutated cereal variant presented herein.

According to one embodiment of the present invention, the object is to provide agronomically useful barley plants carrying a mutation in one or more of the α-amylase gene promoters, the HRT gene, the HBL12 gene and/or the WRKY38 gene. In addition to mutations in one or more of the α-amylase gene promoters, the HRT gene, the HBL12 gene, and/or the WRKY38 gene, there are additional factors that may also be considered in the development of a commercial barley variety suitable for malting and/or brewing, and/or as a base for beverages, for example, grain yield and kernel size, as well as other parameters that relate to malting performance or brewing performance. Since many, if not all, significant traits have been shown to be genetically determined, the present invention also relates to modern homozygous high-yielding brewing varieties that can be obtained by crossing with the barley plants disclosed herein. A skilled barley breeder will be able to select and grow barley plants that, when crossed with other barley plants, will produce improved varieties. Alternatively, the plant breeder may use the plants of the present invention for additional mutagenesis to produce new varieties carrying additional mutations in addition to mutations in one or more of the α-amylase gene promoters, the HRT gene, the HBL12 gene and/or the WRKY38 gene .

The present invention also includes barley plants carrying a mutation in one or more of the α-amylase gene promoters, the HRT gene, the HBL12 gene and/or the WRKY38 gene, resulting from a plant breeding method, including selfing, backcrossing, and population crossing methods. etc. Backcrossing methods can be used in connection with the present invention to introduce into another variety a mutation in one or more of the α-amylase gene promoters, the HRT gene, the HBL12 gene and/or the WRKY38 gene.

In one embodiment, the present invention relates to a progeny of a barley plant deposited on November 12, 2018 in NCIMB under NCIMB accession number 43270 and named HENZ-2, or a progeny of a barley plant deposited on November 12, 2018 in NCIMB under accession number NCIMB 43271 and named HENZ -10.

A method for accelerating the process of plant selection involves initial propagation obtained

- 32 043494 new mutants through the use of tissue culture and regeneration techniques. Thus, another aspect of the present invention is to provide cells that subsequently grow and differentiate into barley plants bearing a mutation in one or more of the α-amylase gene promoters, the HRT gene, the HBL12 gene, and/or the WRKY38 gene. For example, breeding may involve traditional crossings, the production of fertile plants by anther culture, or the use of microspore culture.

In one embodiment, the barley plant of the present invention was not produced solely through an essentially biological process. The progeny of a process-produced barley plant is herein considered to be one that was not produced solely through an essentially biological process because the parent plant was produced by a process.

In one embodiment, the barley plant carries any of the mutations described herein, and said mutation was induced by chemical means and/or physical factors.

In one embodiment, the barley plant was produced by a process including an induced mutagenesis step, or the plant is a progeny of a plant produced by a method including an induced mutagenesis step. Thus, the barley plant may be a barley plant produced by a process comprising the following steps, or a progeny of a plant produced by a process comprising the following steps:

mutagenesis of barley plants or parts thereof, for example, using a chemical mutagen such as NaN ₃ , selection of barley plants carrying any of the mutations described herein.

Sequences

SEQ ID NO:1 Coding sequence of the HvHRT gene ID# AK362734 (cv. Haruna Nijo) ATGCCTGCGGTCGCCGCTGCCAGATTGAAGCGGGAGGAC TGCCCCCGCACCAAACACGATTCCCTCTTCTCCCCATGGA AGGTTCTTGTCGGGCCGTCGGACTGGGAGGACCACTCCGC CGGCAAGGAGGGGGTCCAGAGGTATCACACACGCAACCT CCCGGACAACTTCCCTGGCCTCTACGAGCTGGGCGTTGCA AGGCCTTCCTATGATGGTGTCAGGCTCGCAGAAATCG AT CAGTTGTCGTCGTGGTGGTATACCTCGGGCAGGCCGATAA TGTCAGGGCGAGGCTCCAGCAGTACGGGCGGACAGGTC ACACCTGGACACCGGGAATCCGTTGGCTGCTGTCTGTAAA GCTGAGATGAACGCGCTCACGGCAGGACCTGGATTGTTCA GGGAAGTCTTCTCCAGAGGCTACTCTATGATGTTTCGATG

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TGCGCTGATGGGTTCCAAAAAGGCAGCTGAGAAGACTGA AGGTCAGCTACTGGGAGTATTTGATTATGCATGGAATAAA CTGCAGAATGGTGCGTGTCGTCGCGAAGAAATACTGCTCA AGTTAGAACAGGGAAGCAATAGATTATCTTTGCTTAGCAG AGTCCGGCACTTAAAACAGAGGGTGTTTGGAGAGAAAGC AGGTATAAAGATTAACAGCAGTGGGTCTGTTGAGATTT CA TCTAGCAGTATGAAAAATATGCTTCCAAGAGTCCGTACGT TTGTCGGCTTCAGGCCTCGTTTGGTTAACTCTGGCGACGA TTTAAACGAGGCAAGTGATATTCACCGAAAATGCACACCT CAGGCCAATACTGCTGGTAAACAAGCACATAGAAGGTCT GAAGGATACAAGGTGAAAAAAGATCGATGTTATTAAACGG CGAACTGCACCGATAAGAAGCCGAAGCTGTTTGTGGA G A ATGCAAAGGAGCCATCTTGGCAAAGGAACAGCTTCAAAG CAAATGAGATGAAAATCGGAAGCTCCTACAGAAGATG AAGCATATGGAACCTCCCATGCAGAATCTCAGTTCCACGA AGATGAGCCTTGTGGAAGGAAGTGGTTTGAGCGGCTCAA AGCACAGAAATCAGCCAACGCACCATCGTCGAGAGGCCA AGGATGTCAGCCAAGAAGCAAACAACGACGCATCAGC CTTATG TGGAGTAGTGACAGATAATGGATACTGCAAACTG GAACCGGTGATAGGAAGGGAAAGATGCGAGGAGCACAG AGGAATTGAGGTCACTGGTGCGTCATCGGCACCATGTTCC GGAAGGTCGGTATTGCCATCTGTCTGTGGAGCTCGGGCAT CCGATGGTTCACCTTGCAAGAATCAGCCAATCGCAAGGA GGAAGAGATGTGCGTTGCACAAAGGTCAAAGAGCGTGCT GCGCCTCCGC GCCATCAGTCAAA SEQ ID NO:2 Protein subsequence HvHRT MPAVAAARLKREDCPRTKHDSLFSPWKVLVGPSDWEDHSA GKEGVQRYHTRNLPDNFPGLYELGVARPSYDGVRARRNRS VVVVVVYLGQADNVRARLQQYGRTGSHLDTGNPLAAVCK AEMNALTAGPGLFREVFSRGYSMMFRCALMGSKKAAEKTE

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Sh# AK362734 (cv. Haruna Nijo) STC Q VAIPTVESIPQQT ANPSKR DQAWQTSADQSKNLSTNAKEPSWQRNSFKANEMKIGEAPT EDEAYGTSHAESQFHEDEPCGRKWFERLKAQKS ANAP S SRG QGCQPREANNDASALCGVVTDNGYCKLEPVIGRERCEEHRG IEVTGASSAPCSGRSVLPSVCGARASDGSPCKNQPIARRKRC ALHKGQRACCASAPSVK * SEQ ID NO:3 Coding sequence of the HvHRT gene of the barley HENZ-2 mutant. Substitution of G for A results in the appearance of the W431stop protein mutation ATGCCTGCGGTCGCCGCTGCCAGATTGAAGCGGGAGGAC TGCCCCCGCACCAAACACGATTCCCTCTTCTCCCCATGGA AGGTTCTTGTCGGGCCGTCGGACTGGGAGGACCACTCCGC CGGCAAGGAGGGGGTCCAGAGGTATCACACACGCAACCT CCCGGACAACTTCCCTGGCCTCTACGAGCTGGGCGTTGCA AGGCCTTCCTATGATGGTGTCAGGCTCGCAGAAATCG AT CAGTTGTCGTCGTGGTGGTATACCTCGGGCAGGCCGATAA TGTCAGGGCGAGGCTCCAGCAGTACGGGCGGACAGGTC ACACCTGGACACCGGGAATCCGTTGGCTGCTGTCTGTAAA GCTGAGATGAACGCGCTCACGGCAGGACCTGGATTGTTCA GGGAAGTCTTCTCCAGAGGCTACTCTATGATGTTTCGATG TGCGCTGATGGGTTCCAAAAAGGCAGCTGA GAAGACTGA AGGTCAGCTACTGGGAGTATTTGATTATGCATGGAATAAA CTGCAGAATGGTGCGTGTCGTCGCGAAGAAATACTGCTCA AGTTAGAACAGGGAAGCAATAGATTATCTTTGCTTAGCAG AGTCCGGCACTTAAAACAGAGGGTGTTTGGAGAGAAAGC AGGTATAAAAGATTAACAGCAGTGGGTCTGTTGAGATTTCA TCTAGCAGTATGAAAAATATGCTTCCAAGAG TCCGTACGT TTGTCGGCTTCAGGCCTCGTTTGGTTAACTCTGGCGACGA TTTAAACGAGGCAAGTGATATTCACCGAAAATGCACACCT CAGGCCAATACTGCTGGTAAACAAGCACATAGAAGGTCT GAAGGATACAAGGTGAAAAAGATCGATGTTATTAAACGG CGAACTGCACCGATAAGAGAAGCCGAAGCTGTTTGTGGA GTAATGCTAGAAGATGGTTCTTCTTGTTTGGAGG ATCCAA TGGAAGGAAGGAAGGGTGTGAGTTGCACAAAGGTAGAA

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A AGCATATGGAACCTCCCATGCAGAATCTCAGTTCCACGA AGATGAGCCTTGTGGAAGGAAGTGHTTTGAGCGGCTCAA AGCACAGAAATCAGCCAACGCACCATCGTCGAGAGGCCA AGGATGTCAGCCAAGAGAAGCAAACAACGACGCATCAGC CTTATGTGGAGTAGTGACAGATAATGGATACTGCAAACTG GAACCGGTGATAGGAAGGGAAAGATGCGAGGAGCACAG AGGAA TTGAGGTCACTGGTGCGTCATCGGCACCATGTTCC GGAAGGTCGGTATTGCCATCTGTCTGTGGAGCTCGGGCAT CCGATGGTTCACCTTGCAAGAATCAGCCAATCGCAAGGA GGAAGAGATGTGCGTTGCACAAAGGTCAAAGAGCGTGCT GCGCCTCCGCGCCATCAGTCAAATAA SEQ ID NO:4 Sequence of the barley HENZ-2 mutant HvHRT mutant. MPAVAAARLKREDCPRTKHDSLFSPWKVLVGPSDWEDHSA GKEGVQRYHTRNLPDNFPGLYELGVARPSYDGVRARRNRS VVVVVVYLGQADNVRARLQQYGRTGSHLDTGNPLAAVCK AEMNALTAGPGLFREVFSRGYSMMFRCALMGSKKAAEKTE GQLLGVFDYAWNKLQNGACRREEILLKLEQGSNRLSLLSRV RHLKQRVFGEKAGIKINS SGS VEIS S S SMKNMLPRVRTF VGF RPRLVNSGDDLNEASDIHRKCTPQANTAGKQAHRRSEGYKV KKIDVIKRRTAPIREAEAVCGVMLEDGSSCLEDPMEGRKRCE LHKGRRVRVAYSRKVS S S S STCQ VAIPTVESIPQQT ANPSKR DQAWQTSADQSKNLSTN AKEPSWQRNSFKANEMKIGEAPT EDEAYGTSHAESQFHEDEPCGRK* SEQ ID NO:5 Coding sequence of the HvHBL12 gene Ш# AK376953 (cv. Haruna Nij o) ATGGAGCAGGGGGAGGAGGACGGGGACTGGATGATGGA GCCGGCGTCGGGGAAGAAGGGCGGGGTGATGATCGACAG GAAGAAGCGCTTCAGCGAGGAGCAGATCAAGTCGCTCGA GTCCATGTTCGCCACGCAGACCAAGCTGGAGCCCCGCCAG AAGCTGCAGCTGGCCCGGGAGCTCGGCCTGCAGCCGCGC CAGGTCGCCATCTGGTTCCAGAACAAGCGCGCGCGCTGG

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AAGTCCAAGCAGCTCGAGCGCCAGTACGCCGCGCTCCGG GACGACTACGACGCCCTCCTCTCCAGCTACGACCAGCTCA AGAAGGACAAGCAAGCGCTCGTCAACCAGCTGGAGAAGC TAGCAGAGATGCTGCGGGAGCCGGGCGGGGCCAAGTGCG GAGATAATGCCGGCGCTGCTGACAGGGACAACATGCGCC TGGCCGTGGCCGGCATGAGCATGAAGGACGAGTTCGC GG ACGCTGCCGGGGCCAGCAAGCTCTACTCGGCGTCTGCCGA GGGCTGCGGCGGCAGCGGCAAGCTCTCCCTTCGGCGAG GAGGATGACGACGCGGGCCTCTTCCTCCGGCCCTCGCTGC AGCTGCCAACCGCGCACGACGGCGGCTTCACGGCGTCGG GGCCGGCCGAGTACCAGCAGCAGTCGCCGTCGTCGTTCCC GTTCCACTCGAGCTGGCCGTCGTCCGCGACGGAG CAGACC TGCAGCAGCTCCCAATGGTGGGAATTCGAGTCCCCGAGCG AGTAA SEQ ID NO:6 MEQGEEDGDWMMEPASGKKGGVMIDRKKRFSEEQIKSLES Protein MFATQTKLEPRQKLQLARELGLQPRQVAIWFQNKRARWKS subsequence KQLERQYAALRDDYDALLSSYDQLKKDKQALVNQLEKLAE HvHBL12 MLREPGGAKCGDNAGAADRDNMRLAVAGMSMKDEFADA Ш# AK362734 (cv. AGASKLYSASAEGCGGSGKLSLFGEEDDDAGLFLRPSLQLPT Haruna Nijo) AHDGGFT ASGP AE YQQQ SP S SFPFHS S WP S S ATEQTC S S SQW WEFESPSE* SEQ ID NO:7 GAGACGGGAGACCCGGCTACGCATGCACGCCACCGCGCT Subsequence CCATTGGCCGCCCCGTTGCCATCACCGCGCCCATCGCTCC genomic DNA ID# ATCCCCCGATTAAACTACTCCATATCGCTAGTAAGCAGAA morex_contig_5 68 5 5 GCAGAATCGATCCATCACACCAAGCTAGCTAGCCTCCTAG (exons are designated CTCGCTCGCTCGCCCGCACACCCGCGATCCATTCTGCTTCT italic and bold TCCCCTTCCTTCCCACTCCGGATCAGGTGCATGACCACCG font) GCGAGACCTAGCTAGGTAGGTAGGGAGGGAGGGAGGG4T GGAGCAGGGGGAGGAGGACGGGGACTGGATGATGGAGCC GGCGTCGGGGAA GAA GGGCGGGGTGA TGA TCGA CA GGAA GAA GCGCTTCA GCGA GGA GCA GA TCAA GTCGCTA GA GTCC ATGTTCGCCACGCAGACCAAGCTGGAGCCCCGCCAGAAGC TGCAGCTGGCC CGGGAGCTCGGCCTGCAGCCGCGCCAGGT CGCCATCTGGTTCCAGAACAAGCGCGCGCGCTGGAAGTCC AAGCAGCTCGAGCGCCAGTACGCCGCGCTTCGGGACGACT

- 37 043494

ACGACGCCCTCCTCTCCAGCTACGACCAGCTCAAGAAGGA CAAGCAAGCGCTCGTCAACCAGGTATATACTCCTATGTCTG TCTGTCTGTGCTACGTACCGTGTGTTTCTCCGTGCTCTCCG CTCGGTGGCGTGGAGCTCGTGGTGCCTCTGGCTAATGCAT GGTCGACGGGTTTCTTGCCTTGCGTGTCCGTGCAGCTG'G'A GAAGCTAGCAGAGATGCTGCGGG AGCCGGGCGGGGCCAA GTGCGGA GA TAA TGCCGGCGCTGCTGA CA GGGA CGA CGTG CGCCTGGCCGTGGCCGGCATGAGCATGAAGGACGAGTTCG CGGACGCTGCCGGGGCCAGCAAGCTCTACTCGGCGTCTGC CGA GGGCTGCGGCGGCA GCGGCAA GCTCTCCCTCTTCGGC GAGGACGATGACGACGCGGGCCTCTTCCTCCGGCCCTCGC TGC A GCTGCCAA CCGCGCA CGA CGGCGGCTTCA CGGCGTC GGGGCCGGCCGAGTACCAGCAGCAGTCGCCGTCGTCGTTC CCGTTCCA CTCGA GCTGGCCGTCGTCCGCGA CGGA GCA GA CCTGCA GCA GCTCCCAA TGGTGGGAA TTCGA GTCCCCGA G CGAGTAAGTAGAGCCATCGGTCAAGCACCATGCAAGGAA TCGCCGACGTGATCGACCATGCAA CAGATCAGTGTTCCTA ACACAGAGCACTATACTGCCGATCGAATCCGTGGAGAAG ACGACGGCGCGATCGATCATATGCAACCGAAGATGGTGG TGTCAAGTGTGTACATAGCTCGAAACCCAGGTCTGTCCAG TCCAGTACGTCCAGGCAGCCTCTTCCTTCTCAATCAGCAG TCAGCACGCCATTTTTCTTCACCCTCTTCCTCTTTAAGAAT CACTTGCTCTTGTCAATTACCTGCCACACCGTGTAATCCAC AGGGAAACTAGTCACAAAACCAAATTATAGAGACCGATC TTCAGATGCAAGTGCATGCAACTATTTAGC SEQ ID NO:8 Coding sequence of the barley HENZ-10 mutant HvHBL12 gene. The sequence shows a substitution of G for A, thus encoding a mutant ATGGAGCAGGGGGAGGAGGACGGGGACTGGATGATGGA GCCGGCGTCGGGGAAGAAGGGCGGGGTGATGATCGACAG GAAGAAGCGCTTCAGCGAGGAGCAGATCAAGTCGCTCGA GTCCATGTTCGCCACGCAGACCAAGCTGGAGCCCCGCCAG AAGCTGCAGCTGGCCCGGGAGCTCGGCCTGCAGCCGCGC CAGGTCGCCATCTGGTTCCAGAACAAGCGCGCGCGCTGG AAG TCCAAGCAGCTCGAGCGCCAGTACGCCGCGCTCCGG GACGACTACGACGCCCTCCTCTCCAGCTACGACCAGCTCA AGAAGGACAAGCAAGCGCTCGTCAACCAGCTGGAGAAGC TAGCAGAGATGCTGCGGGAGCCGGGCGGGGCCAAGTGCG

- 38 043494

protein (W228Stop). Based on Sh# AK376953 (cv. Haruna Nijo) GAGATAATGCCGGCGCTGCTGACAGGGACAACATGCGCC TGGCCGTGGCCGGCATGAGCATGAAGGACGAGTTCGCGG ACGCTGCCGGGGCCAGCAAGCTCTACTCGGCGTCTGCCGA GGGCTGCGGCGGCAGCGGCAAGCTCTCCCTCTTCGGCGAG GAGGATGACGACGCGGGCCTCTTCCTCCGGCCCTCGCTGC AGCTGCCAACCGCGCACGACGGCGGCTTCACGGCGTC GG GGCCGGCCGAGTACCAGCAGCAGTCGCCGTCGTCGTTCCC GTTCCACTCGAGCTGACCGTCGTCCGCGACGGAGCAGACC TGCAGCAGCTCCCAATGGTGGGAATTCGAGTCCCCGAGCG AGTAA SEQ ID NO:9 Protein sequence of the mutant HvHBL12 from the barley HENZ-10 mutant. MEQGEEDGDWMMEPASGKKGGVMIDRKKRFSEEQIKSLES MFATQTKLEPRQKLQLARELGLQPRQVAIWFQNKRARWKS KQLERQYAALRDDYDALLSSYDQLKKDKQALVNQLEKLAE MLREPGGAKCGDNAGAADRDNMRLAVAGMSMKDEFADA AGASKLYSASAEGCGGSGKLSLFGEED DDAGLFLRPSLQLPT AHDGGFT ASGP AE YQQQ SP S SFPFHS S * SEQ ID NO: 10 Coding sequence reHaWRKY38 Accession number AJ536667.1 Barley cv. Ingrid ATGGATCCATGGATGGGCAGCCAGCCATCCCTGAGCCTCG ACCTGCACGTCGGCCTACCGCCGATGGGGCACCCGCACCA CCACCAGAGCCAATACCAGGCGCCGCCGATGATCGCGCT GGCCAAGCCCAAGATCCTCGTGGAGGAGAACTTCATGCC ACTCAAGAAGGACCCTGAGGTTGCGGTTCTTGAGTCGGAG CTACAGCGGGTGAGCGAGGAGAACCGGCGGCTGGGCGAG A TGCTCAGGGAGGTGGCCTCCAAGTACGAGGCCCTGCAG GGCCAGTTCACCGACGTGGTCACGGCCGGCGGCAACAAC AACCACTACCACAACCAGCCGTCCTCCGCGTCGGAGGGC GGGTCGGTGTCGCCGTCGAGGAAGCGCAAGAGCGAGGAG AGCCTCGGCACGCCGCCACCGTCGCATACTCAGCAGCAGC ACTATGCCGCCGGCCTCGCGTACGCGTGGCGCCCGGAC CA GGCGGAGTGCACGTCCGGCGAGCCGTGCAAGCGCATCCG GGAGGAGTGCAAGCCCGTCATCTCCAAGCGCTACGTCCAC GCCGACCCCTCCGACCTCAGCCTGGTGGTGAAGGACGGGT ACCAATGGCGCAAGTACGGGCAGAAGGTGACCAAGGACA ACCCATGCCCCAGAGCCTACTTCCGGTGCTCCTTCGCCCC CGGCTGCCCCGTCAAGAAGAAGGTGCAGAGGAGCGCCGA GGACAAGACCATACTCGTGGCGACGTACGAGGGCGAGCA

- 39 043494

CAACCACACCCAGCCCCCGCCGTCGCAGCCGCAGCAGCA GAACGACGGCTCCGGCGCCGGCAAGAACGCCGGGAACGG GAAGCCGCCCCAGGCGCCGGCCACGCCTCACCACCCGCA GCAGCAGCACAAGCAGGAAGCGGCAGCGGTCGTCGTCAG CGGCGAATCGGCCGCGGCGGCGTCCGAGCTGATCCGGCG CAACCTGGCGGAGCAGATGGCCATGACGCTGACGAGGGA CCCCAGCTTCAAGGCGGCGCTGGTCACCGCGCTCTCCGGC CGGATCCTCGAGCTCTCGCCGACCAGGGACATCAATTAA SEQ ID NO: 11 Protein subsequence WRKY38 MDPWMGSQPSLSLDLHVGLPPMGHPHHHQSQYQAPPMIAL AKPKILVEENFMPLKKDPEVAVLESELQRVSEENRRLGEML REVASKYEALQGQFTDVVTAGGNNNHYHNQPSSASEGGSV SPSRKRKSEESLGTPPPSHTQQQHYAAGLAYAVAPDQAECTS GEPCKRIREECKPVISKRYVHADPSDLSLV VKDGYQWRKYG QKVTKDNPCPRAYFRCSFAPGCPVKKKVQRSAEDKTILVAT YEGEHNHTQPPPSQPQQQNDGSGAGKNAGNGKPPQAPATP HHPQQQHKQEAAAVVVSGESAAAASELIRRNLAEQMAMTL TRDPSFKAALVTALSGRILELSPTRDIN* SEQ ID NO: 12 Protein subsequence WRKY38 MDPWMGSQPSLSLDLHVGLPPMGHPHHHQSQYQAPPMIAL AKPKILVEENFMPLKKDPEVAVLESELQRVSEENRRLGEML REVASKYEALQGQFTDMVTAGGNNNHYHNQPSSASEGGSV SPSRKRKSEESLGTPPPSHTQQQHYAAGLAYAVAPDQAECTS GEPCKRIREECKPVISKRYVHADPSDLSLV VKDGYQWRKYG QKVTKDNPCPRAYFRCSFAPGCPVKKKVQRSAEDKTILVAT YEGEHNHTQPPPSQPQQQNDGSGAGKNAGNGKPPQAPATP HHPQQQHKQEAAAVVVSGESAAAASELIRRNLAEQMAMTL TRDPSFKAALVTALSGRILELSPTRDIN SEQ ID NO: 13 Coding sequence of WRKY38 HENZ-50 mutant gene Based on sequences with accession number AJ536667.1 ATGGATCCATGGATGGGCAGCCAGCCATCCCTGAGCCTCG ACCTGCACGTCGGCCTACCGCCGATGGGGCACCCGCACCA CCACCAGAGCCAATACCAGGCGCCGCCGATGATCGCGCT GGCCAAGCCCAAGATCCTCGTGGAGGAGAACTTCATGCC ACTCAAGAAGGACCCTGAGGTTGCGGTTCTTGAGTCGGAG CTACAGCGGGTGAGCGAGGAGAACCGGCGGCTGGGCGAG A TGCTCAGGGAGGTGGCCTCCAAGTACGAGGCCCTGCAG GGCCAGTTCACCGACGTGGTCACGGCCGGCGGCAACAAC AACCACTACCACAACCAGCCGTCCTCCGCGTCGGAGGGC

- 40 043494

Barley cv. Ingrid. The sequence shows a change from G to A, thus encoding a mutant protein (W200Stop). GGGTCGGTGTCGCCGTCGAGGAAGCGCAAGAGCGAGGAG AGCCTCGGCACGCCGCCACCGTCGCATACTCAGCAGCAGC ACTAGCCGCCGGCCTCGCGTACGCGGTGGCGCCGGACCA GGCGGAGTGCACGTCCGGCGAGCCGTGCAAGCGCATCCG GGAGGAGTGCAAGCCCGTCATCTCCAAGCGCTACGTCCAC GCCGACCCCTCCGACCTCAGCCTGGTGGTGAAGGA GAA GCCGCCCCAGGCGCCGGCCACGCCTCACCACCCGCA GCAGCAGCACAAGCAGGAAGCGGCAGCGGTCGTCGTCAG CGGCGAATCGGCCGCGGCGGCGTCCGAGCTGATCCGGCG CAACCTGGCGGAGCAGATGGCCATGACGCTGACGAGGGA CCCCAGCTTCAAGGCGGCGCTGGTCACCGCGCTCTCCGGC CGGATCCTCGAGCTCTCGCCGACCAGGGACATCAATTAA SEQ ID NO: 14 Protein sequence of mutant WRKY38 from barley mutant HENZ-50. MDPWMGSQPSLSLDLHVGLPPMGHPHHHQSQYQAPPMIAL AKPKILVEENFMPLKKDPEVAVLESELQRVSEENRRLGEML REVASKYEALQGQFTDVVTAGGNNNHYHNQPSSASEGGSV SPSRKRKSEESLGTPPPSHTQQQHYAAGLAYAVAPDQAECTS GEPCKRIREECKPVISKRYVHADPSDLSLV VKDGYQ*

Signs

The present invention, for example, can be defined in the following paragraphs.

1. A barley plant or part thereof with high α-amylase activity, wherein said barley plant:

carries a mutation in the HvHRT gene causing loss of HvHRT function; and/or carries at least one α-amylase gene containing a mutant α-amylase gene promoter providing a mutation in the GARE box; and/or carries at least four α-amylase genes containing a GARE box with the sequence TAACAAA; and/or carries at least one α-amylase gene in the amy2 cluster containing a mutant α-amylase gene promoter involving a mutation in the GARE box, or which has the sequence TAACAAA; and/or carries a mutation in the HvHBL12 gene causing loss of HvHBL12 function; and/or carries at least four α-amylase genes containing an α-amylase gene promoter containing a non-standard tandem repeat W-box, wherein said non-standard tandem repeat W-box contains the sequence (TGAC(C)n(X) _m TTGACC) wherein one or more of the particular nucleotides have been replaced or deleted, and wherein X can be any nucleotide, n is 0 or 1, and m is an integer in the range 0-20; and/or carries at least one α-amylase gene in the amy2 cluster containing an α-amylase gene promoter containing a non-standard tandemly repeated W-box;

carries a mutation in the WRKY38 gene, causing loss of WRKY38 function.

2. A barley plant or part thereof, where said barley plant:

a) carries a mutation in the HvHRT gene resulting in the formation of a mutant HvHRT gene encoding a mutant HvHRT protein lacking one or more of the amino acids of SEQ ID NO: 2, or a mutation resulting in a deletion of at least the coding region of the gene HvHRT, wherein the coding region of the HvHRT gene encodes the polypeptide SEQ ID NO: 2; and/or

b) carries at least one α-amylase gene containing a mutant promoter of the α-amylase gene, providing a mutation in the GARE box; and/or

c) carries at least four α-amylase genes containing a GARE box with the sequence TAACAAA; and/or

d) carries at least one α-amylase gene in the amy2 cluster containing a mutant promoter

- 41 043494 α-amylase gene, involving a mutation in the GARE box, or which has the sequence

TAACAAA; and/or

e) carries a mutation in the HvHBL12 gene resulting in the formation of a mutant HvHBL12 gene encoding a mutant HvHBL12 protein lacking one or more of the amino acids of SEQ ID NO: 6, or a mutation resulting in a deletion of at least the coding region of the gene HvHBL12, wherein the coding region of the HvHBL12 gene encodes the polypeptide SEQ ID NO: 6; and/or

f) carries at least four α-amylase genes containing an α-amylase gene promoter containing a non-standard tandem repeat W-box, wherein said non-standard tandem repeat W-box contains the sequence (TGAC(C) _n (X)mTTGACC), wherein one or more of the particular nucleotides have been replaced or deleted, and wherein X can be any nucleotide, n is 0 or 1, and m is an integer in the range 0-20; and/or

g) carries at least one α-amylase gene in the amy2 cluster containing an α-amylase gene promoter containing a non-standard tandemly repeated W-box; and/or

h) carries a mutation in the WRKY38 gene resulting in the formation of a mutant WRKY38 gene encoding a mutant WRKY38 protein lacking one or more of the amino acids present in both SEQ ID NO: 11 and SEQ ID NO: 12, or a mutation resulting in a deletion of at least a coding region of the WRKY38 gene, wherein the coding region of the WRKY38 gene encodes a polypeptide of SEQ ID NO: 11 or SEQ ID NO: 12.

3. A barley plant according to any of the preceding paragraphs, wherein said barley plant carries a mutation in the HvHRT gene.

4. Barley plant in accordance with paragraph 3, where the mutation is:

a mutation resulting in the formation of a mutant HvHRT gene encoding a mutant HvHRT protein lacking at least amino acids corresponding to amino acids 527-530 of SEQ ID NO: 2, a mutation resulting in a deletion of the HvHRT gene.

5. A barley plant in accordance with any of paragraphs 3-4, where the mutation mediates the introduction of a premature stop codon in the HvHRT gene.

6. A barley plant according to any of paragraphs 3-5, wherein the mutation is a mutation in the splice site of the HvHRT gene.

7. The barley plant according to any one of claims 3 to 6, wherein the barley plant contains a mutant HvHRT gene encoding a mutant HvHRT protein lacking at least amino acids 463 to 491 of SEQ ID NO: 2.

8. The barley plant according to any one of claims 3 to 7, wherein the barley plant contains a mutant HvHRT gene encoding a mutant HvHRT protein lacking at least amino acids 509 to 539 of SEQ ID NO: 2.

9. The barley plant according to any one of claims 3 to 8, wherein the barley plant contains a mutant HvHRT gene encoding a mutant HvHRT protein lacking at least 21 of the closest amino acids to the C-terminus, e.g., at least 39 of the closest to the C-terminus of amino acids, such as at least 85 of the amino acids closest to the C-terminus, such as at least 100 of the amino acids closest to the C-terminus of SEQ ID NO: 2.

10. The barley plant according to any one of claims 3 to 9, wherein the barley plant contains a mutant HvHRT gene encoding a mutant HvHRT protein lacking at least the 118 most C-terminal amino acids of SEQ ID NO: 2.

11. The barley plant according to any of paragraphs 3 to 10, wherein the barley plant contains a mutant HvHRT gene encoding a truncated HvHRT protein containing an N-terminal HvHRT fragment containing no more than 526 N-terminal amino acids SEQ ID NO: 2, for example, no more than 508 N-terminal amino acids of SEQ ID NO: 2, such as no more than 462 N-terminal amino acids of SEQ ID NO: 2, preferably no more than 431 N-terminal amino acids of SEQ ID NO: 2.

12. The barley plant according to any one of paragraphs 3-11, wherein the barley plant contains a mutant HvHRT gene carrying a premature stop codon among any of codons 1-431.

13. The barley plant according to any one of paragraphs 3 to 12, wherein the barley plant contains a mutant HvHRT gene carrying a premature stop codon at 431 SEQ ID NO: 1.

14. The barley plant according to any one of claims 3 to 13, wherein the barley plant contains a mutant HvHRT gene encoding a mutant HvHRT protein, wherein said mutant HvHRT protein carries the W431stop mutation SEQ ID NO: 2.

15. The barley plant in accordance with any of paragraphs 3-14, where the barley plant contains a mutant HvHRT gene, providing for the G^A mutation at nucleotide 1293 of the HvHRT coding sequence SEQ ID NO: 1.

16. A barley plant as defined in any of paragraphs 3 to 15, wherein the barley plant is a barley plant deposited with NCIMB under NCIMB accession number 43270, or a progeny thereof.

- 42 043494

17. The barley plant according to any one of paragraphs 3 to 12, wherein the barley plant contains a mutant HvHRT gene carrying a premature stop codon at 170 SEQ ID NO: 1.

18. The barley plant according to any one of claims 3-12, wherein the barley plant contains a mutant HvHRT gene encoding a mutant HvHRT protein, wherein said mutant protein

HvHRT carries the W170stop mutation SEQ ID NO: 2.

19. A barley plant in accordance with any of paragraphs 3-12, where the barley plant contains a mutant HvHRT gene, providing a G^A mutation at nucleotide 510 of the HvHRT coding sequence SEQ ID NO: 1.

20. The barley plant according to any one of paragraphs 3 to 12, wherein the barley plant contains a mutant HvHRT gene carrying a premature stop codon at 371 SEQ ID NO: 1.

21. The barley plant according to any one of paragraphs 3 to 12, wherein the barley plant contains a mutant HvHRT gene encoding a mutant HvHRT protein, wherein said mutant HvHRT protein carries the W371stop mutation SEQ ID NO: 2.

22. A barley plant in accordance with any of paragraphs 3-12, where the barley plant contains a mutant HvHRT gene, providing a G^A mutation at nucleotide 1113 of the HvHRT coding sequence SEQ ID NO: 1.

23. The barley plant according to any one of paragraphs 3-22, wherein the barley plant contains a mutant HvHRT gene encoding a polypeptide of SEQ ID NO: 4.

24. The barley plant according to any one of paragraphs 3 to 23, wherein the barley plant contains a mutant HvHRT gene containing the coding sequence of SEQ ID NO: 3.

25. A barley plant according to any of paragraphs 3-24, wherein the barley plant contains less than 10% mutant HvHRT mRNA or wild-type HvHRT mRNA compared to that of a barley plant containing the wild-type HvHRT gene but otherwise having the same genotype wherein the HvHRT mRNA is an RNA encoding a polypeptide of SEQ ID NO: 2 or a functional homologue thereof, and the wild-type HvHRT gene is a gene encoding a polypeptide of SEQ ID NO: 2 or a functional homologue thereof, wherein said functional homologue is characterized by at least 95 % sequence identity to SEQ ID NO: 2.

26. A barley plant according to any one of paragraphs 2-25, wherein the mutation in the HvHRT gene is a mutation that causes complete loss of HvHRT function.

27. A barley plant according to any of the preceding claims, wherein the barley plant contains at least one α-amylase gene comprising a mutant α-amylase gene promoter involving a mutation in the GARE box, wherein one of the TAACARA nucleotides has either been replaced or deleted

28. A barley plant according to any of the preceding claims, wherein the barley plant contains at least two α-amylase genes, such as at least three α-amylase genes, such as at least 4 α-amylase genes containing the indicated mutant α-amylase gene promoter.

29. A barley plant according to any of the preceding claims, wherein said barley plant contains at least five α-amylase genes containing a GARE box with the sequence TAACAAA.

30. A barley plant according to any of the preceding claims, wherein said barley plant contains at least six, such as at least seven α-amylase genes containing a GARE box with the sequence TAACAAA.

31. A barley plant as defined in any of the preceding paragraphs, wherein the barley plant is a high yielding variety of spring barley.

32. A barley plant according to any of the preceding claims, wherein said barley plant contains at least one, such as at least two, for example at least three α-amylase genes in the amy2 cluster containing a GARE box with sequence TAACAAA.

33. A barley plant according to any one of paragraphs 27-32, wherein said barley plant further carries a mutation in the HvHRT gene, wherein said mutation is a mutation according to any one of paragraphs 3-26.

34. The barley plant according to any one of paragraphs 3-33, wherein the barley plant has an α-amylase activity of at least 16 U/g 48 hours after initiation of germination.

35. The barley plant according to any one of paragraphs 3 to 34, wherein the barley plant has an α-amylase activity of at least 140 U/g, such as at least 150 U/g, such as at least 160 U/g, such as at least 170 U/g 48 hours after initiation of germination, provided that said barley plant is either naked or at least part of the film has been removed before initiation of germination.

36. The barley plant according to any one of paragraphs 3-35, wherein the barley plant has a limiting dextrinase activity of at least 30 mU/g, such as at least 35 mU/g, such as at least 40 mU /g 48 hours after germination initiation,

- 43 043494, provided that the specified barley plant is either naked or at least part of the film has been removed.

37. A barley plant according to any of claims 1-2, wherein said barley plant carries a mutation in the HvHBL12 gene.

38. A barley plant in accordance with paragraph 37, where the mutation is:

a) a mutation resulting in the formation of a mutant HvHBL12 gene encoding a mutant HvHBL12 protein lacking one or more amino acids of SEQ ID NO: 6, or a functional homologue thereof having at least 95% sequence identity therewith; or

b) a mutation resulting in deletion of the HvHBL12 gene.

39. A barley plant according to any of paragraphs 37-38, wherein the mutation mediates the introduction of a premature stop codon in the HvHBL12 gene.

40. A barley plant according to any one of paragraphs 37-39, wherein the mutation is a mutation in the splice site of the HvHBL12 gene.

41. The barley plant according to any one of paragraphs 37-40, wherein the barley plant contains a mutant HvHBL12 gene encoding a mutant HvHBL12 protein lacking at least 10 amino acids closest to the C-terminus, such as at least 20 the amino acids closest to the C-terminus of SEQ ID NO: 6, or a functional homologue thereof having at least 95% sequence identity with it.

42. The barley plant according to any one of paragraphs 37-41, wherein the barley plant contains a mutant HvHBL12 gene encoding a mutant HvHBL12 protein lacking at least the 22 most proximal amino acids of SEQ ID NO: 6, or a functional one thereof a homolog sharing at least 95% sequence identity with it.

43. The barley plant in accordance with any of paragraphs 37-42, where the barley plant contains a mutant HvHBL12 gene encoding a truncated HvHBL12 protein containing an N-terminal fragment of HvHBL12 containing no more than 228 N-terminal amino acids of SEQ ID NO: 6, or its a functional homolog sharing at least 95% sequence identity with it.

44. The barley plant according to any one of paragraphs 37-43, wherein the barley plant contains a mutant HvHBL12 gene carrying a premature stop codon among any of codons 1-228.

45. The barley plant according to any one of paragraphs 37-44, wherein the barley plant contains a mutant HvHBL12 gene carrying a premature stop codon at 228 SEQ ID NO:5.

46. The barley plant according to any one of paragraphs 37 to 45, wherein the barley plant contains a mutant HvHBL12 gene encoding a mutant HvHBL12 protein, wherein said mutant HvHBL12 protein carries the W228stop mutation SEQ ID NO: 6.

47. The barley plant in accordance with any of paragraphs 37-46, where the barley plant contains a mutant HvHBL12 gene providing a G^A mutation at nucleotide 684 of the HvHBL12 coding sequence SEQ ID NO: 5.

48. A barley plant as defined in any of paragraphs 37 to 47, wherein the barley plant is a barley plant deposited with NCIMB under NCIMB accession number 43271, or a progeny thereof.

49. The barley plant according to any one of paragraphs 37-48, wherein the barley plant contains a mutant HvHBL12 gene encoding a polypeptide of SEQ ID NO: 9 or SEQ ID NO: 9 carrying one or more of the N141D, M142V or E184D polymorphisms.

50. The barley plant according to any one of paragraphs 37-49, wherein the barley plant contains a mutant HvHBL12 gene containing the coding sequence of SEQ ID NO: 8.

51. A barley plant according to any one of paragraphs 37-50, wherein the barley plant contains less than 10% HvHBL12 mRNA compared to that of a barley plant containing the wild-type HvHBL12 gene but otherwise having the same genotype, wherein the HvHBL12 mRNA is An RNA encoding a polypeptide of SEQ ID NO: 6 or a functional homologue thereof, and the wild-type HvHBL12 gene is a gene encoding a polypeptide of SEQ ID NO: 6 or a functional homolog thereof, wherein said functional homologue has at least 95% sequence identity with SEQ ID NO: 6.

52. The barley plant of any one of paragraphs 37 to 51, wherein the mutation in the HvHBL12 gene is a mutation that causes complete loss of HvHBL12 function.

53. The barley plant in accordance with any of paragraphs 37-52, wherein said barley plant further carries a mutation in the HvHRT gene, wherein said mutation is a mutation in accordance with any of paragraphs 3-26, and/or said barley further contains at least at least one α-amylase gene according to any of paragraphs 27-32.

54. A barley plant according to any of claims 1-2, wherein said barley plant carries a mutation in the HvWRKY38 gene.

55. A barley plant in accordance with paragraph 54, where the mutation is:

- 44 043494

a) a mutation resulting in the formation of a mutant HvWRKY38 gene encoding a mutant HvWRKY38 protein lacking at least one of amino acids 200-206,

220, 226, 250 and/or 252 SEQ ID NO: 11 or SEQ ID NO: 12;

b) a mutation resulting in deletion of the HvWRKY38 gene.

56. A barley plant according to any of paragraphs 54-55, wherein the mutation mediates the introduction of a premature stop codon in the HvWRKY38 gene.

57. A barley plant according to any one of paragraphs 54-56, wherein the mutation is a mutation in the splice site of the HvWRKY38 gene.

58. The barley plant according to any one of paragraphs 54-57, wherein the barley plant contains a mutant HvWRKY38 gene encoding a mutant HvWRKY38 protein lacking at least amino acids 200-206 of SEQ ID NO: 11 or SEQ ID NO: 12.

59. The barley plant according to any one of claims 54-58, wherein the barley plant contains a mutant HvWRKY38 gene encoding a mutant HvWRKY38 protein lacking at least 102 of the amino acids closest to the C-terminus, e.g., at least 104 of the closest to the C-terminus of an amino acid, such as at least the 128 most C-terminal amino acids, such as at least the 134 most C-terminal amino acids of SEQ ID NO: 11 or SEQ ID NO: 12.

60. The barley plant of any one of claims 54 to 59, wherein the barley plant contains a mutant HvWRKY38 gene encoding a mutant HvWRKY38 protein lacking at least the 154 most C-terminal amino acids of SEQ ID NO: 11 or SEQ ID NO : 12.

61. The barley plant in accordance with any of paragraphs 54-60, where the barley plant contains a mutant HvWRKY38 gene encoding a truncated HvWRKY38 protein containing an N-terminal fragment of HvWRKY38 containing no more than 251 N-terminal amino acids of SEQ ID NO: 11 or SEQ ID NO: 12, e.g., not more than 249 N-terminal amino acids of SEQ ID NO: 11 or SEQ ID NO: 12, such as, e.g., not more than 225 N-terminal amino acids of SEQ ID NO: 11 or SEQ ID NO: 12, e.g. no more than 219 N-terminal amino acids of SEQ ID NO: 11 or SEQ ID NO: 12, preferably no more than 199 N-terminal amino acids of SEQ ID NO: 11 or SEQ ID NO: 12.

62. The barley plant according to any one of paragraphs 54-61, wherein the barley plant contains a mutant HvWRKY38 gene carrying a premature stop codon among any of codons 1-200.

63. The barley plant according to any one of paragraphs 54-62, wherein the barley plant contains a mutant HvWRKY38 gene carrying a premature stop codon among any of the codons 1-200 of SEQ ID NO: 10.

64. The barley plant according to any one of paragraphs 54-63, wherein the barley plant contains a mutant HvWRKY38 gene carrying a premature stop codon at 200 SEQ ID NO: 10.

65. The barley plant according to any one of paragraphs 54-64, wherein the barley plant contains a mutant HvWRKY38 gene encoding a mutant HvWRKY38 protein, wherein said mutant HvWRKY38 protein carries the W200stop mutation of SEQ ID NO: 11 or 12.

66. The barley plant in accordance with any of paragraphs 54-65, where the barley plant contains a mutant gene HvWRKY38, providing a mutation G^A at nucleotide 600 of the coding sequence HvWRKY38 SEQ ID NO: 10.

67. The barley plant according to any one of paragraphs 54-66, wherein the barley plant contains a mutant HvWRKY38 gene encoding a polypeptide of SEQ ID NO: 14 or a polypeptide of SEQ ID NO: 14 in which amino acid 98 is Met.

68. The barley plant according to any one of paragraphs 54-67, wherein the barley plant contains a mutant HvWRKY38 gene containing the coding sequence of SEQ ID NO: 13.

69. A barley plant according to any of paragraphs 54-68, wherein the barley plant contains less than 10% mutant HvWRKY38 mRNA or wild-type HvWRKY38 mRNA compared to that of a barley plant containing the wild-type HvWRKY38 gene but otherwise having the same genotype wherein the HvWRKY38 mRNA is an RNA encoding a polypeptide of SEQ ID NO: 11 or 12 or a functional homologue thereof, and the wild-type HvWRKY38 gene is a gene encoding a polypeptide SEQ ID NO: 11 or 12 or a functional homologue thereof, wherein said functional homolog is characterized by at least 95% sequence identity to SEQ ID NO: 11 or 12.

70. The barley plant of any one of claims 54 to 69, wherein the mutation in the HvWRKY38 gene is a mutation that causes complete loss of function of HvWRKY38.

71. The barley plant in accordance with any of paragraphs 54-70, wherein said barley plant further carries a mutation in the HvHRT gene, wherein said mutation is a mutation in accordance with any of paragraphs 3-26, and/or said barley further contains at least at least one α-amylase gene according to any one of paragraphs 27-32, and/or said barley plant further carries a mutation in the HvHBL12 gene, wherein said mutation is a mutation according to any one of paragraphs 37-52.

- 45 043494

72. The barley plant according to any of the preceding claims, wherein the barley plant contains at least five α-amylase genes comprising an α-amylase gene promoter containing a non-standard tandemly repeated W-box.

73. The barley plant according to any of the preceding claims, wherein the barley plant contains at least six, such as at least seven α-amylase genes comprising an α-amylase gene promoter containing a non-standard tandem repeating W-box.

74. A barley plant as defined in any of the preceding paragraphs, wherein the barley plant is a high-yielding variety of spring barley.

75. A barley plant according to any of the preceding claims, wherein said barley plant contains at least one, such as at least two, such as at least three α-amylase genes in the amy2 cluster containing a non-standard tandem repeating W -boxing.

76. A barley plant according to any of the preceding paragraphs, wherein the non-standard tandem repeating W-boxes are individually selected from the group consisting of:

(TGACR(X)mYTGRCC);

(TGACR(X)mTTGACC);

(TGACR(X)mTTGAC);

(TGAC(C) _n (X)mYTGRCC);

(TGAC(C) _n (X)mCTGRCC);

(TGAC(C) _n (X)mYTGGCC);

(TGAC(C) _n (X)mCTGACC);

(TGAC(C) _n (X)mTTGGCC); and (TGAC(C) _n (X)mTTGATC), wherein R is either G or A, Y is either C or T, n is 0 or 1, and m is an integer in the range 0-20.

77. A barley plant according to any of the previous paragraphs, where m is an integer in the range 0-10.

78. A barley plant according to any of the preceding paragraphs, wherein m is an integer in the range 0-6.

79. A barley plant according to any of the preceding paragraphs, wherein one or more α-amylase genes contain a non-standard tandemly repeated W-box individually selected from the following sequences:

TGACGGTCGTATTGACC;

TGACAGTGGTATTGGCC;

TGACAGTGGTACTGGCC;

GTGAC AGTGGT ATTGGCC;

TGACGGTCGTATTGATC;

TGACCGTCGTATTGATC; And

TTGACTTGATC.

80. The barley plant according to any of the preceding claims, wherein the barley plant contains the amy1_1 cluster, wherein at least one of the α-amylase gene promoters contains a non-standard tandem repeat W-box containing the sequence TTGATC.

81. The barley plant according to any of the preceding claims, wherein the barley plant contains the amy1_1 cluster, wherein at least one of the α-amylase gene promoters contains a non-standard tandem repeat W-box containing the sequence CTGACGGTCGTATTGATC (SEQ ID NO:72),

82. A barley plant according to any of the preceding claims, wherein the barley plant contains an amy1_1 cluster containing the sequence shown as HENZ-43 amy1_1 in FIG. 11A.

83. The barley plant according to any one of paragraphs 37 to 82, wherein the barley plant has an α-amylase activity of at least 100 U/g, such as at least 110 U/g 48 hours after initiation of germination, provided that said barley plant is either naked or at least a portion of the membrane has been removed before germination is initiated.

84. A barley plant in accordance with any of paragraphs 37 to 83, wherein the barley plant has a limiting dextrinase activity of at least 20 mU/g 48 hours after initiation of germination, provided that said barley plant is either naked or at least part of the film was removed before the initiation of said germination.

85. A barley plant according to any of the preceding claims, wherein the barley plant has an α-amylase activity 48 hours after initiation of germination that is at least 105%, such as at least 110%, such as at least 120%, like, on-

- 46 043494 example, at least 150%, for example at least 170%, of α-amylase activity in a barley plant that does not carry the specified mutation, but otherwise has the same genotype.

86. A barley plant according to any of the preceding paragraphs, wherein the barley plant has a yield that is at least 90% of the yield of a barley plant not carrying the mutation but otherwise having the same genotype.

87. A barley plant according to any of the preceding paragraphs, wherein the barley plant has a TKW of at least 38 g, such as at least 40 g.

88. The barley plant according to any of the preceding claims, wherein the barley plant has a starch content of at least 55% w/w, such as at least 60% w/w.

89. A barley plant according to any of the preceding paragraphs, wherein the barley plant has a protein content of at least 9.5% w/w.

90. A barley plant according to any of the preceding paragraphs, wherein the barley plant has a height that is at least 90% of the height of a barley plant not carrying the mutation but otherwise having the same genotype.

91. A barley plant according to any of the preceding paragraphs, wherein the barley plant is characterized by a number of ears/m ² that is at least 90% of the number of ears/m ² of a barley plant not carrying the specified mutation, but otherwise having the same genotype .

92. Barley plant in accordance with any of the previous paragraphs, where the barley plant is not subjected to pre-harvest germination.

93. Barley plants in accordance with any of the previous paragraphs, where the kernels of the specified barley plant, when collected from barley plants that have been regularly sprayed with water for 20 days, are characterized by a percentage of germination that is the same as or greater than the percentage of germination of the kernels of the specified plant barley that has not been subjected to the specified spraying and collected at ripeness.

94. A barley plant in accordance with any of the preceding paragraphs, wherein said part of the barley plant is the kernels of the grains.

95. A barley plant according to any of the preceding paragraphs or its progeny, where the barley plant was not obtained solely through an essentially biological process.

96. A barley plant according to any of the preceding paragraphs, where the barley plant has been produced by a process comprising the following steps or is a progeny of a plant produced by a process comprising the following steps:

mutagenesis of barley plants or parts thereof, for example, using a chemical mutagen such as NaN ₃ , selection of barley plants carrying any of the mutations described herein.

97. A barley plant according to any of the preceding paragraphs, wherein the barley plant contains at least two, such as at least three, of the following mutations or properties:

a) carries a mutation in the HvHRT gene causing loss of HvHRT function; and/or

b) carries at least one α-amylase gene containing a mutant promoter of the α-amylase gene, providing a mutation in the GARE box; and/or

c) carries at least four α-amylase genes containing a GARE box with the sequence TAACAAA; and/or

d) carries at least one α-amylase gene in the amy2 cluster containing a mutant α-amylase gene promoter involving a mutation in the GARE box, or which has the sequence TAACAAA; and/or

e) carries a mutation in the HvHBL12 gene, causing loss of HvHBL12 function; and/or

f) carries at least four α-amylase genes containing an α-amylase gene promoter containing a non-standard tandem repeat W-box, wherein said non-standard tandem repeat W-box contains the sequence (TGAC(C) _n (X) _m TTGACC), wherein one or more of the particular nucleotides have been replaced or deleted, and wherein X can be any nucleotide, n is 0 or 1, and m is an integer in the range of 0-20; and/or

g) carries at least one α-amylase gene in the amy2 cluster containing an α-amylase gene promoter containing a non-standard tandemly repeated W-box;

h) carries a mutation in the WRKY38 gene, causing loss of WRKY38 function.

98. A barley plant according to any of the preceding paragraphs, wherein the barley plant contains at least two, such as at least three, of the following mutations:

a) a mutation in one or more α-amylase gene promoters, for example, any of the mutations described herein above under A-amylase and barley plants carrying a mutation in the α-amylase gene promoter,

b) a mutation in the HvHRT gene, for example, any of the mutations described herein above in

- 47 043494 section Barley plant carrying a mutation in the HRT gene,

c) a mutation in the HvHBL12 gene, for example, any of the mutations described herein above in the section Barley plant carrying a mutation in the HvHBL12 gene,

d) a mutation in the HvWRKY38 gene, for example, any of the mutations described herein above under Barley plant carrying a mutation in the WRKY38 gene.

99. A barley plant according to any of the preceding paragraphs, wherein the barley plant contains a mutation in one or more additional genes, such as one or more of the following mutations:

a) a mutation in the gene encoding LOX-1, resulting in complete loss of functional LOX-1,

b) a mutation in the gene encoding LOX-2, resulting in complete loss of functional LOX-2,

c) a mutation in the MMT-encoding gene resulting in complete loss of functional MMT,

d) a mutation in a gene encoding CslF6, wherein said mutant gene encodes a mutant CslF6 protein with reduced CslF6 activity.

100. A product of plant origin, comprising or derived from a barley plant in accordance with any of the preceding paragraphs or a part thereof.

101. A vegetable product according to paragraph 100, wherein the vegetable product is selected from the group consisting of barley flour, green malt and kiln-dried malt.

102. A plant product according to any one of paragraphs 100-101, wherein the plant product is green malt or kiln-dried malt comprising processed kernel(s) of grain(s) of said barley plant.

103. A plant product as defined in paragraphs 100-102, where the plant product is ground green malt or ground kiln-dried malt.

104. Product of vegetable origin in accordance with paragraphs 100-101, where the product of vegetable origin is barley flour.

105. Product of plant origin in accordance with paragraph 100, where the product of plant origin is a wort obtained from the kernels of the specified barley plant and/or from green malt or kiln-dried malt, providing processed kernel(s) of the grain (-en) of the specified barley plant.

106. A product of plant origin in accordance with paragraph 100, where the product of plant origin is a drink obtained from the specified barley plant or parts thereof.

107. The beverage according to paragraph 106, wherein said beverage is derived from the kernel(s) of said barley plant and/or from green malt or kiln-dried malt comprising processed kernel(s) of said barley plant.

108. A beverage as defined in any of paragraphs 106-107, wherein the beverage is beer.

109. A method for producing green malt, wherein said method involves the following steps:

(i) providing barley plant kernels in accordance with any of paragraphs 1 to 99;

(ii) soaking said grain kernels;

(iii) germinating soaked grain kernels under specified conditions.

110. A method for producing kiln-dried malt, said method comprising the steps of:

(i) providing barley plant kernels in accordance with any of paragraphs 1 to 99;

(ii) soaking said grain kernels;

(iii) germinating soaked grain kernels under specified conditions;

(iv) drying said sprouted grain kernels.

111. A method for producing a drink, wherein said method involves the following steps:

(i) providing barley plant kernels in accordance with any of paragraphs 1-99 and/or green malt or kiln-dried malt in accordance with paragraphs 102-103 (ii) obtaining an aqueous extract of said grain kernels and/or said malt (iii) ) processing said aqueous extract into a drink.

112. A method for producing an aqueous extract, wherein said method involves the following steps:

a) providing barley plant grains in accordance with any of paragraphs 1 to 99;

b) subjecting the barley grains to a germination step, thereby obtaining germinated grains, wherein said germination step involves keeping said grains in an aqueous solution until the grains have a water content of at least 30%, and passing through said aqueous solution at least 2 l O ₂ per kg dry weight of barley grains per hour;

- 48 043494

c) finely grinding said sprouted grains, wherein said sprouted grains have a water content of at least 20%, with the proviso that said barley grains do not have a water content below 20 at any time between steps b) and c);

d) obtaining an aqueous extract of said finely ground sprouted grains, thereby obtaining an aqueous extract of barley.

113. The method in accordance with paragraph 112, in which the barley grains are immersed in an aqueous solution during the entire germination stage.

114. The method in accordance with any of paragraphs 112-113, in which the germination stage includes:

ia) at least one step of keeping said grains in an aqueous solution, wherein at least 2 liters of O ₂ per kg dry weight of barley grains per hour are passed through said aqueous solution; and ii b) at least one step of exposing said barley grains to air.

115. A method according to any one of paragraphs 112-114, wherein at least 3 L, more preferably at least 4 L, even more preferably at least 5 L, even more preferably at least 6 L are passed through said aqueous solution l O2 per kg dry weight of barley grains per hour.

116. The method according to any one of paragraphs 112-115, wherein said O2 is contained in a gas mixture, the gas mixture being atmospheric air.

117. The method according to any one of paragraphs 112-116, wherein the entire germination step does not exceed 72 hours, more preferably does not exceed 60 hours, even more preferably does not exceed 54 hours.

118. The method according to any one of paragraphs 112-117, wherein the barley is chaffed barley, and the method comprises the step of removing at least a portion of said film before keeping said grains in an aqueous solution.

119. A method for producing a drink, wherein said method involves the following steps:

(i) obtaining an aqueous extract using a method in accordance with any of paragraphs 112-118;

(ii) processing said extract into a beverage.

120. The method according to paragraph 119, wherein step (iii) comprises the steps:

a. heating said aqueous extract, optionally in the presence of hops or hop extract;

b. cooling the aqueous extract;

c. fermenting said aqueous extract using yeast to thereby obtain a fermented beverage.

121. The method according to any of paragraphs 119-120, wherein the beverage is beer.

122. A method for producing barley plants with high α-amylase activity, the method comprising the steps of:

a) providing barley grain kernels; And

b) carrying out non-specific mutagenesis of said barley grain kernels, thereby inducing at least one of the following mutations in at least one barley grain kernel:

mutation in the HvHRT gene;

mutation in one or more α-amylase gene promoters mutation in the HvHBLH gene mutation in the WRKY38 gene

c) selecting barley grain kernels or their progeny carrying at least one of the specified mutations.

123. The method according to paragraph 122, wherein the mutation is as defined in one of paragraphs 3-26, 27-32, 37-52, 54-70 and 72-82.

Examples

The present invention is further illustrated by the following examples, which, however, should not be construed as limiting the present invention.

Example 1.

The barley HvHRT gene is represented in the 2012 version of the barley genome as a CDS under ID# MLOC_51005 (cv. Morex) or ID# AK362734 (cv. Haruna Nijo) and as genomic DNA in morex_contig_368180 (cv. Morex) (IBSC, 2012). According to the 2017 version of the barley genome (Mascher et al., 2017), HvHRT is represented as a CDS and protein sequence under ID# HORVU2Hr1G035630. According to the 2017 version of the barley genome, HvHRT is located on chromosome 2H at position 150902998 bp. by 150911087 p.o. According to the 2017 version of the barley genome, HvHRT is annotated as transcription effector 2. According to the 2012 version of the barley genome, HvHRT consists of three exons. The original sequence has been submitted to NCBI under ID# AJ001317.1.

A barley mutant called HENZ-2 was identified and isolated as described in Examples 2

- 49 043494 below. HENZ-2 carries a nucleotide substitution in the barley HvHRT gene, resulting in a premature stop codon in the coding sequence. The coding sequence of the HRT gene of the mutant HENZ-2 is presented herein as SEQ ID NO: 3, while the sequence of the mutant protein encoded by the mutant HRT gene is presented as SEQ ID NO: 4.

The mutation results in an amino acid change replacing tryptophan 431 with a stop codon (W431Stop). The plant from which the mutant was created is cv. Paustian. The original isolated mutant was heterozygous for the polymorphism, but it was later propagated to produce homozygous mutants.

Example 2A.

DdPCR screening for barley mutants with specific mutations in the Hordeum vulgare transcriptional repressor gene, HRT.

A barley plant carrying a specific mutation in the HvHRT gene was identified using the methods described in international patent application PCT/EP2017/065516.

DdPCR analysis.

A unique ddPCR assay was designed specifically to distinguish between the mutant and wild-type alleles of HvHRT at nucleotide position 1293 in the wild-type coding sequence SEQ ID NO: 1. As stated above, several barley HvHRT genetic sequences are available, and the ddPCR assay used in this example was based on the cv genomic sequence. Himalaya from GenBank NCBI with number: AJ001317.1. The mutant detection probe was complementary to the coding sequence containing base A at nucleotide position 1293. The reference detection probe was complementary to the coding sequence containing base G at nucleotide position 1293. To amplify the genomic sequence surrounding nucleotide 1293 in the coding sequence, two flanking probes were designed primer.

The following primers and probes were designed specifically for the HvHRT locus: target-specific forward primer (SEQ ID NO: 15): 5'-CACGAAGATGAGCCTTG-3'; target specific reverse primer (SEQ ID NO: 16): 5'-TTGGCTGATTTCTGTGC-3'; mutant-specific detection probe (SEQ ID NO: 17): 5'-AAGTGATTTGAGCGGCT-3' - labeled with 6-carboxyfluorescein (FAM);

reference-specific detector probe (SEQ ID NO: 18): 5'-AGGAAGTGGTTTGAGCG-3' - hexachlorofluorescein (HEX)-labeled.

A pool of barley grains subjected to nonspecific mutagenesis was obtained, followed by the production of an ordered library, as described in international patent application PCT/EP2017/065516 in WS1, WS2 and WS3 on pages 66-69, as well as in examples 1-7. The barley variety used to obtain said library of non-specifically mutagenized sequences was barley cv. Paustian.

Determining whether a library sample contains mutated grains.

The next step was to determine whether the library contained the desired mutated grains. Screening was carried out essentially as described in international patent application PCT/EP2017/065516 in WS3 and Examples 3-7, with the following details:

Screening was carried out against a total of 376 subpools (designated GLP#1 to GLP#376), together representing approximately 120,000 mutated barley plants.

One 5 μL gDNA sample was prepared from each subpool (designated GT#1-GT#376). GT#1GT#94 gDNA samples were added to individual wells of a microtiter plate, each well also containing 17 μl of PCR reaction mixture, and mixed thoroughly by pipetting.

The PCR microtiter plate was loaded into a QX200 droplet reader (Bio-Rad) for droplet analysis. The obtained data were analyzed using QuantaSoft software (version v1.7, Bio-Rad). The threshold value was determined using a 2D plot, set to 3700 and 2500 for amplitude amplification in the case of channel 1 and channel 2, respectively. Comparison of individual values for relative abundance showed that, with respect to mutant detection, gDNA (GT#64) produced higher levels of signal than any other sample. The relative abundance of gDNA (GT#64) was 0.062% versus 0.012%, with the latter value representing the average relative abundance for all of the 94 gDNA samples tested.

Search for individual grain(s) characterized by a mutation of interest

Individual barley grains carrying the gene mutation were identified primarily as described in international patent application PCT/EP2017/065516 in WS4 (pages 69-72) and in examples 8-15, including the following details in sequential order:

1. Based on the results of analysis of gDNA derived from GT#1-GT#94 using an HvHRT-specific ddPCR assay, it was highly predicted that 4500 GLP#64 grains [corresponding to the positive gDNA sample (GT#64)] would include one or

- 50 043494 several grains with a gene mutation of interest.

2. FGLP#64 was obtained by sequentially selecting 96x12 grain samples from GLP#64. Each aliquot of 12 grains was placed on a piece of paper to be weighed, and then sequentially fixed with tweezers, while using a router (Marathon-3, Saeyang Microtech) equipped with a 1.6 mm drill to drill a small hole in the endosperm with a depth of 2-3 mm. The rotating motion caused the flour to move from the endosperm to the top of the grain and the surrounding weighing paper. Samples with 12 drilled grains were placed in individual 2-ml microtiter plate wells to obtain a secondary subpool of drilled grains of PDGLP#64 barley. 96 flour samples, each flour from 12 drilled barley grains, were transferred into separate wells on a 1.5 ml microtiter plate (PFGLP#64), maintaining a sample numbering system that coincided with the drilled grains.

3. PFGLP#64 was then subjected to gDNA extraction using a semi-automated DNA extraction procedure as detailed in the instructions for the NucleoSpin 96 Plant II kit (Macherey-Nagel). Therefore, each microtiter plate well contained gDNA from 12 grain flour.

4. The gDNA obtained from PFGLP#64 was analyzed as described above. Data were analyzed using QuantaSoft software (version v1.7, Bio-Rad). The threshold value was determined using a 2-dimensional plot and was set to 3700 for amplitude in the case of channel 1 and 2500 for amplitude in the case of channel 2. Comparison of individual values for relative abundance revealed that one PFGLP#64 well contained the mutant grain. Well C04 showed a relative prevalence of 3.92%, indicating the presence of a heterozygous mutant PDGLP#64.

5. All 12 grains from well C04 PDGLP#64 were germinated. Leaf material from 12 seedlings was collected and subjected to DNA extraction using REDExtract (Sigma Aldrich). gDNA obtained from leaf samples was analyzed as described above. Data were analyzed using QuantaSoft software (version v1.7, Bio-Rad). The threshold value was determined using a 2D plot and was set to 3700 for amplitude in the case of channel 1 and 2500 for amplitude in the case of channel 2. One seedling obtained from well C04 PDGLP#64 showed a relative prevalence of 45%, confirming the presence heterozygous mutant. Seeds from the specified plant were multiplied and the progeny were crossed to produce homozygous plants. Homozygous plants were propagated to increase the amount of material. Plants homozygous for the mutation were designated HENZ-2.

Therefore, HENZ-2 barley plants carry the G^A mutation at nucleotide 1293 of the HvHRT coding sequence SEQ ID NO: 1. Thus, HENZ-2 carries a mutant HvHRT gene encoding a mutant HvHRT protein, providing the W431stop mutation SEQ ID NO: 2.

Example 2B

The barley HENZ-2a mutant was prepared essentially as described in Example 2A, except that the barley variety used to produce the non-specifically mutagenized sequence library was barley cv. Planet. The same primers and probes were used.

HENZ-2a also carries a G^A mutation at nucleotide 1293 of the HvHRT SEQ ID NO: 1 coding sequence. Thus, HENZ-2a also carries a mutant HvHRT gene encoding a mutant HvHRT protein, providing a W431stop mutation SEQ ID NO: 2.

Thus, while HENZ-2 may be considered to have a genetic background of cv. Paustian, HENZ-2a can be considered to have a genetic background cv. Planet.

Example 3A.

HENZ-2 mutant barley plants, as well as control homozygous barley plants, were propagated in the field. Control homozygous plants were obtained from crosses of the heterozygous mutant described in Example 2A, and control homozygous plants contain the wild-type HvHRT gene encoding HvHRT SEQ ID NO: 2. Therefore, control homozygous plants are predicted to be identical to HENZ-2, except that they do not carry mutations in the HvHRT gene.

HENZ-2 mutant barley plants, as well as control homozygous barley plants, were propagated in a field in New Zealand (1 plot) and on the island of Funen (5 separate plots). A 3.6-fold excess of TKW was sown according to standard procedure per 10 m ² . Plots were treated with 150 kg nitrogen per hectare and fungicide treatments were carried out according to standard agricultural procedures. In New Zealand, each plot of land was approximately 2 ^m2 , while on the island of Funen, each plot of land was approximately 7.5 ^m2 . Plots were treated in duplicate and HENZ-2 and control were cultivated close together. To allow comparison, HENZ-2 and the control were grown on plots of the same size. Productivity was determined by collecting barley plants at maturity and determining the weight of grain kernels per plot of land. The grain kernel weight for 1000 grain kernels (TKW) was determined by weighing a given number of grain kernels and calculating the weight for 1000 grain kernels. The starch content (starch) and protein content (protein) of barley grain kernels were determined using a

- 51 043494 near-infrared using a Foss Tecator Infratec 1241 grain analyzer in accordance with the manufacturer's instructions (Foss, Denmark). Starch content and protein content are presented as % weight of dry starch or protein of the total dry weight of the grain kernels. Average plant height was determined by measuring plant height for selected plants from the bottom (beginning of the first internode, just above the roots) to the top of the ear (the barley ear was held upright). The number of ears per ^m2 was determined by counting the number of ears on plants from a given area and calculating the number of ears per ^m2 .

The results are shown in table. 1 below.

Table 1

Productivity Kg/land TKW g Starch % w/w Protein % w/w Height plants cm Ears/m ² HENZ-2 New Zealand 1.7 40.7 62.3 11.6 ND ND Control New Zealand 1.6 42.6 62.3 I,6 ND ND HENZ-2 Funen Island 5.6 53.8 62.6 I.5 61.8 1033.6 Control Funen Island 5.4 53.5 62.3 I,7 61.3 1068.8

As can be seen from the data in table. 1, surprisingly there is no significant difference in yield, TKW, starch content, protein content, plant height and number of ears/ ^m2 between the HENZ-2 mutant and control plants.

Example 3B.

Pre-harvest germination is highly undesirable as it results in immediate loss of seed viability. A pre-harvest germination experiment was conducted to evaluate the extent of pre-harvest germination for the barley HENZ-2 mutant. As controls, preharvest germination of wild-type barley cv. Paustian and cv. Flagship. Barley cv. Flagship is known to have high levels of pre-harvest germination. The experiment was carried out as follows. HENZ-2, Paustian and Flagship were grown in the field on separate plots of land close to each other. When the grain was ripe, a portion of each plot was harvested (harvest 1). The remaining areas of the plot were irrigated by spraying water onto the plants for 10 minutes per hour for 12 hours a day for 10 days, then a portion of each area of the plot was harvested (harvest 2). The remaining area of the plot was subjected to a similar irrigation scheme for another 10 days and then harvested (harvest 3). The day after harvest, germination tests began for all three harvests. Germination tests were carried out by placing 100 grain kernels on filter paper in a 9.5 cm Petri dish and adding either 3 ml or 5 ml of water. The percentage of germination of grain kernels was calculated 3 days after aging and is presented as % germinated grain kernels. Barley varieties with reduced germination percentages from Harvest 1 to Harvests 2 and 3 are considered to have undergone preharvest sprouting, while barley varieties with the same or increased germination percentages from Harvest 1 to Harvests 2 and 3 are not subject to preharvest sprouting. The result is shown in table. 2.

table 2

1. harvest 2. harvest 3.harvest G.R. (%) 3 ml G.R. (%) 5 ml G.R. (%) 3 ml G.R. (%) 5 ml G.R. (%) 3 ml G.R. (%) 5 ml Various* 3 ml Various* 5 ml HENZ-2 72 79 98 98 98 98 21 16

- 52 043494

REP1 HENZ-2 REP2 79 83 78 95 95 95 Paustian RE P I 67 80 60 97 97 98 24 17 Paustian REP2 73 77 94 90 90 92 Flagship REP1 95 96 74 91 81 81 -18 -18 Flagship REP2 99 99 64 85 78 79

G.R. denotes the percentage of germination in %.

Diff.* denotes the difference between the average germination percentage for the first and third harvests as a percentage.

Both HENZ-2 and cv. Paustian were characterized by an increase in germination percentage from the first to the third harvest, and thus none of these barley plants experienced preharvest germination. Also for HENZ-2 and cv. Paustian did not observe a significant difference in the increase in germination percentage from the first to the third harvest. In contrast, cv. Flagship had a significant reduction in germination percentage and thus, as expected, preharvest germination occurred.

Example 3C.

Material in the form of grains from mutant barley plants HENZ-2 and from barley plants cv. Paustian grown in the field under identical conditions were germinated using a standard germination test:

grains were sorted by size using a Pfeuffer grain sorter > grain sizes of 2.5 and 2.8 mm were used,

100 whole grains were placed on 2 pieces of Whatman paper (grade 1, 85 mm, catalog number 1001-085) in 90 mm diameter Petri dishes and added 4 ml of distilled water.

Another 100 whole grains were placed on 2 pieces of Whatman paper (Grade 1, 85 mm, catalog number 1001-085) in 90 mm diameter Petri dishes and added 8 ml of distilled water (high water conditions) Petri dishes were sealed and stored in the dark chamber covered with a damp cloth at 20°C

Petri dishes with a diameter of 4 ml were checked after 24 hours, 48 hours and 72 hours, germinating grains (appearance of the primary root) were collected from the Petri dish and the number of sprouted grains was noted

Petri dishes with a diameter of 8 ml were checked after 72 hours, the germinating grain (appearance of the primary root) was collected from the Petri dish and the number of sprouted grains was noted

The results after 72 hours are shown in FIG. 1. Grain germination from wild type and HENZ-2 barley in 4 ml of water was 99.5%. This indicates that the material was not at rest. When tested for sensitivity to high water content (8 ml water), HENZ-2 had significantly higher percentage germination rates (80% compared to 40% for wild type barley). This indicates that HENZ-2 performs significantly better under water and oxygen stress, as observed in a malt bed or air flow vat as described in Example 4.

Example 4.

Grain material from barley HENZ-2 mutants and from barley plants cv. Paustian grown in a field under identical conditions were germinated in a vat of water, essentially as described in example 1 of international patent application PCT/EP2017/065498, with the following details: 200 g barley grains (dry weight) were kept for 48 h in vat and covered with water containing 1 mm GA, while the grains were exposed to an air flow of 90 l/h per kg dry weight of barley grains. The water content of the grain was measured during germination after 24 hours and 48 hours, and the results are shown in FIG. 2. HENZ-2 in both cases had a higher grain water content compared to that of wild-type barley, demonstrating improved germination properties.

The expression level of the genes α-amylases (amy1_1 and amy1_2) and β-glucanases 2A and 2B (BGL2A) was measured in the same material after 48 h using ddPCR (Biorad) with primers and probes specific for these genes. The general gene expression ddPCR protocol described in Example 20 was used. The primers and probe used to determine the expression level of BGL2A also mediate the amplification and detection of BGL2B, and thus the combined expression of BGL2A and BGL2B was determined (denoted BGL2A in FIG. .3). The results are shown in Fig. 3. In the case of HENZ-2 material, a significantly higher level of expression of all tested genes was shown, indicating improved germination properties. The BGL2A sequence is available under accession number HORVU7Hr1G120450.1.

The activities of α-amylase, β-amylase and limiting dextrinase were measured in the same material after 24 h and after 48 h using standard Megazyme assays as follows.

Preparation of samples.

- 53 043494

Before enzyme activity analysis, samples of germinated grains were ground using a standard Foss Cyclotech mill (Foss, Denmark) equipped with a tungsten carbide grinding ring (Foss 10004463), a nickel-coated paddle wheel (Foss 1000 2666) and a 1 mm outlet sieve (Foss 10001989). All measurements of enzymatic activity in sprouted barley grains were performed within 48 hours after sample milling.

α-amylase activity

The α-amylase activity of sprouted grains was determined using flour prepared as described above in the Sample Preparation section. For α-amylase activity assays, the Ceralpha kit from Megazyme was used using standard laboratory equipment. Assays were performed according to the manufacturer's protocol (K-CERA 01/12), including calculation of α-amylase activity.

β-amylase activity.

In the case of measuring beta-amylase activity of sprouted grains, flour was prepared as described above in the Sample Preparation section. To perform β-amylase activity assays, the recommendations provided with the Betamyl kit from Megazyme (K-BETA3) were followed.

Activity of limiting dextrinase.

To measure limiting dextrinase activity in sprouted grains, flour was prepared as described above in the Sample Preparation section. Limit dextrinase activity was determined using the Limit Dextrizyme T-LDZ1000 kit from Megazyme. Assays, including activity measurements, were performed according to the manufacturer's protocol (T-LDZ1000 07/9).

The results are shown in Fig. 4. In the case of the HENZ-2 material, a significantly higher activity of all enzymes tested was shown, indicating improved germination properties (exception: the sample for measuring α-amylase after 24 hours, which in any case was generally characterized by poor results).

Example 5.

Grain material from barley HENZ-2 mutants and from barley plants cv. Paustian grown in a field under the same conditions were germinated in a vat of water, essentially as described in example 9 of international patent application PCT/EP2017/065498, with the following details: 200 g of barley grains were shelled for 1 min as described in Example 8 of international patent application PCT/EP2017/065498. Then the hulled barley grains were transferred to a vat and covered with 500 ml of water containing 0.01% H ₂ O ₂ , defoamer-204, 1 mM GA3, and kept for 24 hours with aeration at an air flow of 90 l/h. This corresponds to 18 liters of air per hour per 100 ml of water. Excess water was drained and the grains were kept for 24 hours with aeration at an air flow of 90 l/h). The activities of α-amylase, β-amylase and limiting dextrinase were measured 24 hours and 48 hours after germination using standard Megazyme assays as described in Example 4. In addition, the activity of these enzymes was determined in malt produced in accordance with the EBC 19 standard (obtained from Institute Francais De La Brasserie Et De La Malterie (IFBM), France).

The results are shown in Fig. 5. In the case of HENZ-2 material, significantly higher activity of all enzymes tested was shown, indicating improved germination properties. The activity levels measured for HENZ-2 even exceeded the EBC 19 standard.

Example 6.

A barley mutant called HENZ-10 was identified and isolated as described below. HENZ10 carries a nucleotide substitution in the barley HvHBL12 gene, resulting in a premature stop codon during translation of the HBL12 protein. The amino acid substitution is the substitution of tryptophan 228 with a stop codon (W228Stop). The plant from which the mutant was created is a variety of hulless barley (also referred to herein as hulless barley 1). Hulless barley plants generally exhibit higher α-amylase activity during germination compared to that of chaffy barley varieties. The coding sequence of the mutant HvHBL12 gene is presented herein as SEQ ID NO: 8, while the sequence of the mutant protein encoded by the mutant HvHBL12 gene is presented as SEQ ID NO: 9.

The original isolated mutant barley plant was homozygous for the mutation.

Example 7A.

DdPCR screening for barley mutants with specific mutations in the HvHBL12 gene.

The barley plant carrying a particular mutation was identified using the methods described in international patent application PCT/EP2017/065516.

DdPCR analysis.

A unique ddPCR assay was designed specifically to distinguish the mutant allele from the wild-type allele of HvHBL12 at nucleotide position 684 in the wild-type coding sequence (GenBank NCBI accession number: JX878491.1). There are slightly different HvHBL12 sequences, and the ddPCR assay was developed based on the NCBI GenBank sequence number:

- 54 043494

JX878491.1. The mutant detection probe was complementary to the coding sequence containing base A at nucleotide position 684 (corresponding to nucleotide 684 of SEQ ID NO: 5). The reference detection probe was complementary to the coding sequence containing the G base at nucleotide position 684. Two flanking primers were designed to amplify the genomic sequence surrounding nucleotide 684 in the coding sequence.

The following primers and probes were designed specifically for the HvHBL12 locus:

target specific forward primer (SEQ ID NO: 19): 5'-GTCGTCGTTCCCGTT-3';

target specific reverse primer (SEQ ID NO: 20): 5'-CTGCAGGTCTGCTCC-3';

mutant-specific detection probe (SEQ ID NO: 21): 5'-ACTCGAGCTGACCGT-3' - labeled with 6-carboxyfluorescein (FAM);

reference-specific detector probe (SEQ ID NO: 22): 5'-CTCGAGCTGGCCGT-3' - hexachlorofluorescein (HEX)-labeled.

A pool of barley grains subjected to nonspecific mutagenesis was obtained, followed by the production of an ordered library, as described in international patent application PCT/EP2017/065516 in WS1 and WS2 on pages 66-67, as well as in examples 1-2. The barley variety used to obtain this library of non-specifically mutagenized sequences was hulless barley type 1.

Determining whether a library sample contains mutated grains.

The next step was to determine whether the library contained the desired mutated grains. Screening was carried out essentially as described in international patent application PCT/EP2017/065516 in WS3 (pages 67-69) and in examples 3-7, with the following details:

Screening was carried out against a total of 376 subpools (designated GLP#1 to GLP#376), representing approximately 120,000 mutated barley plants.

One 5 μL gDNA sample was prepared from each subpool (designated GT#1-GT#376). GT#283GT#376 gDNA samples were added to individual wells of a microtiter plate, each well also containing 17 μl of PCR reaction mixture, and mixed thoroughly by pipetting.

The PCR microtiter plate was loaded into a QX200 droplet reader (Bio-Rad) for droplet analysis. The obtained data were analyzed using QuantaSoft software (version v1.7, Bio-Rad). The threshold value was determined using a 2D plot, set to 3500 and 1500 for amplitude amplification in the case of channel 1 and channel 2, respectively. Comparison of individual values for relative abundance showed that, with respect to mutant detection, gDNA (GT#291) produced higher signal levels than any other sample. The relative abundance of gDNA (GT#291) was 0.105% versus 0.0094%, with the latter value corresponding to the average relative abundance for all of the 94 gDNA samples tested.

Search for individual grain(s) characterized by a mutation of interest.

Individual barley grains carrying the gene mutation were identified primarily as described in international patent application PCT/EP2017/065516 in WS4 (pages 69-72) and in examples 8-15, including the following details in sequential order:

6. Based on the results of analysis of gDNA derived from GT#283-GT#376 using an HvHBL12-specific ddPCR assay, it was highly likely that 4500 GLP#291 grains [corresponding to the positive gDNA sample (GT#291)] would include one or several grains with a gene mutation of interest.

7. FGLP#291 was obtained by sequentially selecting 96x12 grain samples from GLP#291. Each aliquot of 12 grains was placed on a piece of paper to be weighed, and then sequentially fixed with tweezers, while using a router (Marathon-3, Saeyang Microtech) equipped with a 1.6 mm drill to drill a small hole in the endosperm with a depth of 2-3 mm. The rotating motion caused the flour to move from the endosperm to the top of the grain and the surrounding weighing paper. Samples with 12 drilled grains were placed in individual 2-ml microtiter plate wells to obtain a secondary subpool of drilled grains of PDGLP#291 barley. 96 flour samples, each made from 12 drilled barley grains, were transferred into separate wells on a 1.5 ml microtiter plate (PFGLP#291), maintaining the sample numbering system matching the drilled grains.

8. PFGLP#291 was then subjected to gDNA extraction using a semi-automated DNA extraction procedure as detailed in the instructions for the NucleoSpin 96 Plant II kit (Macherey-Nagel). Therefore, each microtiter plate well contained gDNA from 12 grain flour.

9. DNA obtained from PFGLP#291 g was analyzed as described above. Data were analyzed using QuantaSoft software (version v1.7, Bio-Rad). The threshold value was determined using a 2-dimensional plot and was set to 4000 for amplitude in the case of channel 1 and 1500 for amplitude in the case of channel 2. Three separate samples were identified in the microtiter plate.

- 55 043494 wells (E03, E11, G01), which showed relative abundances of 12.5%, 4.42% and 7.3%, in all cases indicating the presence of three separate mutants in 3 independent wells

PDGLP#291.

10. All 12 grains from hole G01 PDGLP#291 were germinated. Leaf material from 12 seedlings was collected and subjected to DNA extraction using REDExtract (Sigma Aldrich). gDNA obtained from leaf samples was analyzed as described above. Data were analyzed using QuntaSoft a software (version v1.7, Bio-Rad). The threshold value was determined using a 2-dimensional plot and was set to 6000 for amplitude in the case of channel 1 and 2000 for amplitude in the case of channel 2. For one seedling obtained from well H01 PDGLP#291, a relative prevalence of 96.9% was shown, which confirms the presence of a homozygous mutant. This plant was propagated to increase the amount of material. Plants homozygous for the mutation were designated HENZ-10.

Therefore, HENZ-10 barley plants contain a G>A mutation at nucleotide 684 of the HvHBL12 coding sequence (SEQ ID NO: 5). Thus, HENZ-10 contains a HvHBL12 gene containing the coding sequence of SEQ ID NO: 8. Thus, HENZ-10 carries a mutant HvHBL12 gene encoding a mutant HvHBL12 protein containing the W228stop mutation.

Example 7B.

The barley HENZ-61 mutant was prepared essentially as described in Example 7A, except that the barley variety used to generate the non-specifically mutagenized sequence library was barley cv. Planet. The same primers and probes were used.

HENZ-61 also contains a HvHBL12 gene containing the coding sequence of SEQ ID NO: 8. Thus, HENZ-61 also carries a mutant HvHBL12 gene encoding a mutant HvHBL12 protein containing the W228stop mutation.

Thus, while HENZ-10 can be considered to be based on hulless barley 1, HENZ-61 can be considered to be based on cv. Planet.

Example 8A.

Mutant barley plants HENZ-10 and hulless barley plants 1 were cultivated on plots of land under the same conditions. Various agronomic traits were determined, including average plant height. The HENZ-10 mutant barley plants appeared healthy by visual inspection. Average plant height was determined by measuring plant height for selected plants per plot for five separate field plots from the bottom (beginning of the first internode, just above the roots) to the top of the ear (the barley ear was held upright). The results are shown in Fig. 10. Interestingly, no differences in plant height were observed between the HENZ mutant barley plants and the hulless barley 1 parent plants.

Mutant barley plants HENZ-10, as well as hulless barley plants 1, were propagated in a field in New Zealand (1 plot of land) and on the island of Funen (5 separate plots of land). Reproduction, land sizes, etc. were essentially as described in Example 3A above. Yield, grain kernel weight per 1000 grain kernels (TKW), starch content (starch) (as % dry starch of total dry weight of grain kernels), protein content (protein) (as % dry protein of total dry weight of grain kernels) , plant height and number of ears per m ² were determined as described in Example 3A above.

Table 3

Productivity Kg/land TKW G Starch % w/w Protein % w/w Height plants cm Ears/m ² HENZ-10 New Zealand 1.9 42.2 65.1 12.1 ND ND Hulless barley 1 New Zealand 1.8 43.3 65.8 I,4 ND ND HENZ-10 Funen Island 4.8 46.3 62.8 12.9 70.6 995.2 Hulless barley 1 Funen Island 5.2 48.2 63.9 12.1 70.2 1030.4

As can be seen from the data in table. 1, there is surprisingly no significant difference in yield, TKW, starch content, protein content, or plant height between the HENZ-10 mutant and control plants.

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Example 8B.

HENZ-61 mutant barley plants, as well as wild-type barley plants cv. Planet, propagated in the field on separate but nearby plots of land. Yield, grain kernel weight per 1000 grain kernels (TKW), starch content (starch), protein content (protein), plant height and number of ears per m ² were determined as described in Example 3A above.

HENZ-61 mutant barley plants were expected to have yield, grain kernel weight per 1000 kernels (TKW), starch content (starch), protein content (protein), plant height and/or number of ears per m ² similar to those of cv. Planet.

Example 9.

Grain material from mutant barley plants HENZ-10 and from hulless barley plants 1, grown in the field under the same conditions, was germinated in a standard germination test in Petri dishes as follows:

grains were sorted by size using a Pfeuffer grain sorter > grain sizes of 2.5 and 2.8 mm were used,

100 whole grains were placed on 2 pieces of Whatman paper (grade 1, 85 mm, catalog number 1001-085) in Petri dishes with a diameter of 90 mm and added 4 ml of distilled water,

Another 100 whole grains were placed on 2 pieces of Whatman paper (Grade 1, 85 mm, catalog number 1001-085) in 90 mm Petri dishes and added 8 ml of distilled water.

Petri dishes were sealed and stored in a dark chamber covered with a damp cloth at 20°C

Petri dishes with a diameter of 4 ml were checked after 24 hours, 48 hours and 72 hours, germinating grains (appearance of the primary root) were collected from the Petri dish and the number of sprouted grains was noted

Petri dishes with a diameter of 8 ml were checked after 72 hours, the germinating grain (appearance of the primary root) was collected from the Petri dish and the number of sprouted grains was noted.

The results after 72 hours are shown in FIG. 6. The percentage of grain germination from wild type 1 hulless barley and HENZ-10 in 4 ml of water was 98 and 97.5%, respectively. This indicates that the material was not at rest. When tested for sensitivity to high water content (8 ml water), HENZ-10 had significantly higher germination percentages (100% compared to 89% for wild type barley). This indicates that HENZ-10 performs better under water and oxygen stress, conditions that may occur in a malt bed or in a vat with air flow, as described in Example 4.

Example 10.

Material in the form of grains from barley mutants HENZ-10 and from hulless barley plants 1, grown in the field under the same conditions, was germinated in an Erlenmeyer flask filled with water on a shaker for 48 hours at room temperature. Gene expression levels of α-amylases (amy1_1 and amy1_2), limiting dextrinase (LD), α-glucosidase 97 (AGL97), and β-glucanases 2A and 2B (BGL2A and BGL2B) were measured after 48 h using ddPCR specific for these genes. primers and probes. The results are shown in Fig. 7. The BGL2B sequence is available under number HORVUlHr1G057680.1. The HENZ-10 material showed increased expression levels of all genes tested, indicating improved germination properties compared to its wild-type hulless barley parent plant 1. It should be noted that in hulless barley 1 changes in the endosperm begin earlier than those in many wild-type varieties of chaffy barley, as evidenced, for example, by the early high activity of α-amylase. Therefore, it is noteworthy that HENZ-10 showed increased expression levels of all genes tested compared with that of hulless barley 1. Thus, although hulless barley 1 already exhibits early high α-amylase activity, HENZ-10 characterized by even higher α-amylase activity.

Example 11.

Grain material from barley mutants HENZ-10 and from hulless barley plants 1, grown in the field under the same conditions, was germinated in a vat of water, essentially as described in example 1 of international patent application PCT/EP2017/065498, with the following details : 200 g barley grains (dry weight) were kept in a vat for 48 h and covered with water containing 1 mM GA, while the grains were exposed to an air flow of 90 l/h. The expression levels of α-amylase (amy1_1 and amy1_2), limiting dextrinase (LD), and β-glucanase 2A (BGL2A) genes were measured after 48 h by ddPCR using gene-specific primers and probes. The general gene expression ddPCR protocol described in Example 20 was used. The results are shown in FIG. 8. HENZ-10 material showed significantly higher expression levels of all genes tested, indicating improved germination properties compared to wild type 1 hulless barley.

α-Amylase, β-amylase and limiting dextrinase activities were measured in the same material after 24 hours and after 48 hours using standard Megazyme assays as described in Example 4. The results are shown in FIG. 9. In the case of HENZ-10 material, higher activity was shown

- 57 043494 of all enzymes tested, indicating improved germination properties.

Example 12A.

A barley plant called HENZ-50 was identified and isolated essentially as described in Example 2A above. HENZ-50 carries a nucleotide substitution in the barley WKRY38 gene, resulting in a gene containing a premature stop codon in the coding sequence. Thus, HENZ-50 carries a G^A mutation at nucleotide 600 of the HvWRKY38 coding sequence (SEQ ID NO: 10). The coding sequence of barley WRKY38 cv. Ingrid is shown herein as SEQ ID NO: 10. Two different wild-type WRKY38 polypeptide sequences are shown as SEQ ID NO: 11 and SEQ ID NO: 12. The barley plant HENZ-50 carries a point mutation causing a premature stop codon ( W200Stop) in the barley HvWRKY38 gene. The mutation is located at the beginning of the signature sequence WRKYGQK (aa 200-206 SEQ ID NO: 11 or 12) of the WKRY motif. The HvWRKY38 HENZ-50 mutant gene encodes the HvWRKY38 mutant protein with the sequence SEQ ID NO: 14.

The HENZ-50 mutant barley plant was identified in the M2 mutant barley population cv. Planet, obtained by non-specific mutagenesis, mainly as described in example 2A. HENZ-50 was identified and isolated essentially as described herein in Example 2A above, except that the following primers and probes were used for ddPCR analysis:

Target Specific Forward Primer (SEQ ID NO: 23)

5'-CTCAGCCTGGTGGTG-3'

Target Specific Reverse Primer (SEQ ID NO: 24)

5'-TGTCCTTGGTCACCTTC-3'

Mutant Specific Detector Probe (SEQ ID NO: 25) 5'-CCAATGACGCAAGTACG-3' - FAM Labeled

Reference-specific detector probe (SEQ ID NO: 26) 5'-TACCAATGGCGCAAGTA-3' - HEX labeled.

Example 12B.

Barley HENZ-50 mutant and barley cv. Wild-type Planet was grown in a greenhouse in Denmark under identical conditions. 100 grains of each were germinated in 250 mL Erlenmeyer flasks filled with 100 mL deionized water, covered with parafilm, and placed on a shaker at 250 rpm. During germination, the grains are constantly immersed in H ₂ O. No air other than the air in the flask is supplied.

α-Amylase activity was measured 72 hours after the start of germination (i.e., from the moment the grains were immersed in water) using a standard Megazyme assay as described in Example 4.

HENZ-50 was expected to have significantly higher α-amylase activity compared to that of cv. Planet.

Example 13A.

A barley plant called HENZ-43 was identified and isolated essentially as described in Example 2A above. HENZ-43 carries a nucleotide substitution in the promoter of the barley α-amylase gene of the amy1_1 cluster. The mutation results in a mutation of the tandem repeat W box, resulting in the formation of a non-standard tandem repeat W box of the following sequence: TGACGGTCGTATTGATC (SEQ ID NO: 35). In other words, in HENZ-43, the wild-type TTGACC sequence of the amy1_1 gene was modified to become TTGATC. In fig. 11A shows the results of an alignment of partial sequences of various wild-type α-amylase gene promoters and a partial sequence of the barley α-amylase gene promoter of the amy1_1 mutant HENZ-43 cluster.

The HENZ-43 mutant barley plant was identified and isolated essentially as described in Example 2A, except that the barley variety used to generate the non-specifically mutagenized sequence library was barley cv. Planet, and with the exception that the following primers and probes were used for ddPCR analysis:

Target Specific Forward Primer (SEQ ID NO: 27)

5' -AAACAGAGGTTGAGGATAAC-3'

Target Specific Reverse Primer (SEQ ID NO: 28)

5'-GCCTTCGCCTTCCAT-3'

Mutant Specific Detection Probe (SEQ ID NO: 29)

5'-AGGCACCGATCAATACG-3' - tagged FAM

Reference-specific detector probe (SEQ ID NO: 30) 5'-AAGGCACCGGTCAATAC-3' - HEX labeled.

Example 13B.

HENZ-43 mutant barley plants, as well as wild-type barley plants cv. Planet, propagated in the field on separate but nearby plots of land. Yield, grain kernel weight per 1000 grain kernels (TKW), starch content (starch), protein content (protein), plant height and number of ears per m ² were determined as described in Example 3A above.

HENZ-43 mutant barley plants were expected to have yield, grain kernel weight per 1000 kernels (TKW), starch content (starch), protein content (protein), plant height and/or number of ears per m ² similar to those of cv. Planet.

Example 14.

Two barley plants named HENZ-53 and HENZ-54 were identified and isolated essentially as described in Example 2A above.

HENZ-53 carries a G^A mutation at nucleotide 510 of the HvHRT coding sequence (SEQ ID NO: 1). Thus, HENZ-53 carries a mutant HvHRT gene encoding a mutant HvHRT protein containing the W170Stop mutation.

The HENZ-53 mutant barley plant was identified and isolated substantially as described herein in Example 2A above, except that the barley variety used to produce said library of non-specifically mutagenized sequences was Planet barley, and except that The following primers and probes were used for ddPCR analysis:

Target Specific Forward Primer (SEQ ID NO: 73)

5'-GTCAGCTACTGGGAGTATT-3'

Target Specific Reverse Primer (SEQ ID NO: 74)

5'-TCTTCGCGACGACAC-3'

Mutant Specific Detection Probe (SEQ ID NO: 75)

5'-TGCATGAAATAAACTGCAGA-3' - FAM™ labeled

Reference-specific detector probe (SEQ ID NO: 76) 5'-TGCATGGAATAAACTGCAG-3' - HEX™ labeled.

HENZ-54 carries a G^A mutation at nucleotide 1113 of the HvHRT coding sequence (SEQ ID NO: 1). Thus, HENZ-54 carries a mutant HvHRT gene encoding a mutant HvHRT protein containing the W371Stop mutation.

The HENZ-54 mutant barley plant was identified and isolated substantially as described herein in Example 2A above, except that the barley variety used to produce said library of non-specifically mutagenized sequences was Planet barley, and except that The following primers and probes were used for ddPCR analysis:

Target Specific Forward Primer (SEQ ID NO: 77)

5'-TGCTAATCCAAGCAAACG-3'

Target Specific Reverse Primer (SEQ ID NO: 78)

5'-TTTGTGGACAGATTTTTGGA-3'

Mutant Specific Detection Probe (SEQ ID NO: 79)

5'-AGCCTGACAAACCAGTG-3' - FAM™ labeled

Reference-Specific Detector Probe (SEQ ID NO: 80)

5'-AAGCCTGGCAAACCAG-3' - HEX™ labeled

Example 15.

Barley HENZ-2 mutant, wild-type barley cv. Paustian, HENZ-10 mutant barley and wild-type hulless barley 1 were grown in a field in New Zealand under identical conditions. 100 grains from each barley were germinated in 250 ml Erlenmeyer flasks filled with 100 ml deionized water, covered with parafilm, placed on a shaker at 250 rpm. During germination, the grains were constantly immersed in H ₂ O. Air was passed through the water, with an air flow of 9 l/h per 100 ml of water. The grains were kept in water with aeration for 48 hours or 72 hours.

α-Amylase activity was measured 48 hours and 72 hours after the start of germination (i.e. from the moment the grains were immersed in water) using a standard Megazyme assay as described in example 4.

The results are shown in Figures 13A and 13B. In fig. 13A shows α-amylase activity of HENZ-2 and cv. Paustian (wt). In fig. 13B shows α-amylase activity of HENZ-10 and hulless barley 1 (wt). Both HENZ-2 and HENZ-10 had increased α-amylase activity compared with that of their respective wild-type controls.

Example 16.

The barley HENZ-10 mutant and wild-type hulless barley 1 were grown in a field in New Zealand under identical conditions. 100 grains of each barley were germinated in flasks

Erlenmeyer 250 ml filled with 100 ml deionized water, covered with parafilm, placed on a shaker at 250 rpm. During germination, grains are constantly immersed in H2O. No air other than the air in the flask was supplied.

α-Amylase activity was measured 24 hours, 48 hours and 72 hours after the start of germination (i.e. from the moment the grains were immersed in water) using a standard Megazyme assay as described in example 4.

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The results are shown in Fig. 14. In FIG. 14 shows the α-amylase activity of HENZ-10 and hulless barley 1 (wt). HENZ-10 is characterized by significantly increased α-amylase activity, particularly after 72 h.

Example 17.

Barley HENZ-2a mutant, barley HENZ-54 mutant, barley HENZ-43 mutant and wild-type barley cv. Planet was grown in a greenhouse in Denmark under identical conditions. 100 grains of each barley were germinated in 250 ml Erlenmeyer flasks filled with 100 ml deionized water, covered with Parafilm, placed on a shaker at 250 rpm. During germination, the grains are constantly immersed in H ₂ O. No air other than the air in the flask is supplied.

α-Amylase activity was measured 72 hours after the start of germination (i.e., from the moment the grains were immersed in water) using a standard Megazyme assay as described in Example 4.

The results are shown in Fig. 15, which shows the α-amylase activity of HENZ-2a in the top panel, HENZ-54 in the middle panel, and HENZ-43 in the bottom panel. HENZ-2a, HENZ-54 and HENZ43 all have significantly increased α-amylase activity.

Example 18.

The grains were obtained from the HENZ-2 mutant barley and from the control homozygous barley plants described in example 3A, grown under the same conditions.

grains per line were swollen for 24 hours in a conical flask at 20°C (reaching a water content of ~40%). The swollen grains were transferred to a Petri dish (2 ml H2O) and left to germinate. Samples were collected 0, 12, 24 and 48 hours after completion of swelling, i.e. from the moment the grains are transferred to the Petri dish. Three biological replicates per time point were prepared in separate Petri dishes. cDNA was synthesized from 300 ng of total RNA and then diluted 10-fold. Three technical cDNA replicates were prepared per sample (i.e., a total of 9 replicates per line per treatment).

The mRNA expression levels of the amy1_1, amy1_2 and amy2 genes were determined by ddPCR using the following primers and probes. The general gene expression ddPCR protocol described in Example 20 was used. All analyzes were performed on the 3' end of the cDNA sequence in question.__________________________________________________________________________

Target Straight primer 5’-3’ Reverse primer 5'-3' Probe 5’-3’ amyl-1 GACTGGGGGCCTGAAG (SEQIDNO:81) GTGCCGGGTCCTGAC (SEQ ID NO:82) AGATCGATCGCCTGGTG TC (SEQ ID NO:83) atu 1-2 AGATCGATCGTCTGGT G (SEQ ID NO:84) TCCATGATCTGCAGC TTG (SEQ ID NO:85) TCAATCAGGACCCGACA GG (SEQ ID NO:86) atu2 CGAGCTCAAGGAGTGG (SEQ ID NO:87) CGTCGATGTACACCT TG (SEQIDNO:88) AAGAGCGACCTCGGCTT C (SEQ ID NO:89)

The results are shown in Fig. 16. In the figure, the control is designated as WT. HENZ-2 was characterized by increased transcript levels for all analyzed amy genes compared to the control. Amy1 expression appeared to be triggered earlier than amy2 expression.

Example 19.

Material used was in the form of grains from the barley HENZ-2a mutant, the barley mutant HENZ-54, the barley mutant HENZ-43 and the wild-type barley cv. Planet, grown in a greenhouse in Denmark under the same conditions. 100 grains were germinated in 250 ml Erlenmeyer flasks filled with 100 ml H2O, covered with parafilm, placed on a shaker at 250 rpm. During germination, the grains are constantly immersed in H ₂ O. No air other than the air in the flask is supplied.

The gene expression levels of α-amylases (amy1_1, amy1_2, and amy2), limiting dextrinase (LD), and the expression levels of both β-glucanases 2A and 2B (together referred to as bgl2) were measured after 12 h by ddPCR using the primers and probes indicated below. Both β-glucanases 2A and 2B can be amplified and detected using the same primers and probes._________

Target Straight primer 5’-3’ Reverse primer 5'-3' Probe 5’-3’ amyl-1 GACTGGGGGCCTGAAG (SEQIDNO:81) GTGCCGGGTCCTGAC (SEQ ID NO:82) AGATCGATCGCCTGGTGT C (SEQ ID NO:83)

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amyl-2 AGATCGATCGTCTGGT G (SEQ ID NO:84) TCCATGATCTGCAGCT TG(SEQIDNO:85) TCAATCAGGACCCGACA GG (SEQ ID NO:86) amy2 CGAGCTCAAGGAGTG G (SEQ ID NO:87) CGTCGATGTACACCTT G(SEQ ID NO:88) AAGAGCGACCTCGGCTT C (SEQ ID NO:89) Bgl2 TACCAGAACCTGTTCG AC (SEQ ID NO:90) ACACCACCAGCTTCAC (SEQIDNO:91) ACCGTGGACGCCTTCTAC (SEQ ID NO:92) LD CTTCGATGGGGTTTTGA AC (SEQ ID NO:93) CCGATTTCCTCACCAA AG (SEQ ID NO:94) CCTGTGCAGGTGAATTCA TCA (SEQIDNO:95)

The results are shown in Fig. 17-20. HENZ-2a, HENZ-54 and HENZ-43 all had increased expression levels of amy1_1, amy1_2 and amy2, limiting dextrinase (LD) and bgl2 genes.

Example 20.

Gene expression levels were determined by ddPCR using standard methods. More specifically, RNA was generally extracted from freeze-dried cereal flour (~50 mg) using first the Trizol kit protocol (Qiagen #15596026), followed by purification using the Aurum™ Total RNA Mini Total RNA Isolation Kit (Bio-Rad # 7326820). cDNA was synthesized using the iScript™ cDNA Synthesis Kit from Bio-Rad (#1708891).

For ddPCR analysis, each sample was mixed with primers, probe(s) (FAM or HEX labeled), ddPCR reaction component, ddPCR Supermix (no dUTP), and water (BioRad). The samples were mixed before drop formation (Bio-Rad QX200™ Automated Drop Generator #1864101). The plate with drops was incubated in a thermal cycler.

The presence of FAM- and/or HEX-positive droplets was detected on a Bio-Rad QX200™ Droplet Reader and analyzed using Bio-Rad Quantasoft software.

Bibliography

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3. Gubler, F., Kalla, R., Roberts, J. K., & Jacobsen, J. V. (1995). Gibberellin-regulated expression of a myb gene in barley aleurone cells: evidence for Myb transactivation of a high-pl alpha-amylase gene promoter. The Plant Cell, 7(11), 1879-1891.

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6. Mascher, M., Gundlach, H., Himmelbach, A., Beier, S., Twardziok, S. 0., Wicker, T., ... & Bayer, M. (2017). A chromosome conformation capture ordered sequence of the barley genome. Nature, 544(7651), 427-433. Nakata M, Fukamatsu Y, Miyashita T, Hakata M, Kimura R, Nakata Y, Kuroda M, Yamaguchi T and Yamakawa H (2017) High Temperature-Induced Expression of Rice-Amylases in Developing Endosperm Produces Chalky Grains. Front. Plant Sci. 8:2089. doi: 10.3389/fpls.2017.02089

7. Raventos, D., Skriver, K., Schlein, M., Kamahl, K., Rogers, S. W., Rogers, J. C., & Mundy, J. (1998). HRT, a novel zinc finger, transcriptional repressor from barley. Journal of Biological Chemistry, 273(36), 23313-23320. Schwarz et al., 2004, J. Am. Soc. Brew. Chern. 62(4):147-154

8. Son, 0., Hur, Y. S., Kim, Y. K., Lee, H. J., Kim, S., Kim, M. R.,... & Park, J. (2010). ATHB12, an ABA-inducible homeodomain-leucine zipper (HD-Zip) protein of Arabidopsis, negatively regulates the growth of the inflorescence stem by decreasing the expression of a gibberellin 20-oxidase gene. Plant and Cell Physiology, 57(9), 1537-1547.

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List of sequences <110> Carlsberg A/S <120> Barley with increased activity of hydrolytic enzymes <130> P4598PC00 <150> 17210964.7 <151> 2017-12-28 <160> 95 <170> PatentIn version 3.5 <210> 1 <211 > 1644 <212> DNA <213> Hordeum vulgare <400> 1

atgcctgcgg tcgccgctgc cagattgaag cgggaggact gccccccgcac caaacacgat 60 tccctcttct ccccatggaa ggttcttgtc gggccgtcgg actgggga ccactccgcc 120 ggcaaggagg gggtccagag gtatcacaca cgcaacctcc cggacactt ccctggcctc 180

- 62 043494

tacgagctgg gcgttgcaag gccttcctat gatggtgtca gggctcgcag aaatcgatca 240 gttgtcgtcg tggtggtata cctcggggcag gccgataatg tcagggcgag gctccagcag 300 tacggggcgga caggtcaca cctggacacc gggaatccgt tggctgctgt ctgtaaagct 360 gagatgaacg cgctcacggc aggacctgga ttgttcaggg aagtcttctc cagaggctac 420 tctatgatgt ttcgatgtgc gctgatgggt tccaaaaagg cagctgagaa gactgaaggt 480 cagctactgg gagtatttga ttatgcatgg aataaactgc agaatggtgc gtgtcgtcgc 540 gaagaaatac tgctcaagtt agaacaggga agcaatagat tatctttgct tagcagagtc 600 cggcacttaa aacagaggt gtttggagag aaagcaggta taaagattaa cagcagtggg 660 tctgttgaga tttcatctag cagtatgaaa aatatgcttc caagagtccg tacgtttgtc 720 ggcttcaggc ctcgtttggt taactctggc gacgatttaa acgaggcaag tgatattcac 780 cgaaaatgca cacctcaggc caatactgct ggtaaacaag cacatagaag gtctgaagga 840 tacaaggtga aaaagatcga tgttattaaa cggcgaactg caccgataag agaagccgaa 900 gctgtttgtg gagtaatgct agaagatggt tcttcttgtt tggaggatcc aatggaagga 960 aggaagaggt gtgagttgca caaaggtaga agagtcagag tggcatacag tcgcaaagta 1020 tcctcttcta gctccacttg ccaagttgct attccaactg ttgaatccat acctcaacaa 1080 actngctaatc caagcaaacg agatcaagcc tggcaaacca gtgcagacca atccaaaaat 1140 ctgtccacaa atgcaaagga gccatcttgg caaaggaaca gcttcaaagc aaatgagatg 1200 aaaatcggag aagctcctac agaagatgaa gcatatggaa cctcccatgc agaatctcag 1260 ttccacgaag atgagccttg tggaaggaag tggtttgagc ggctcaaagc acagaaatca 1320 gccaacgcac catcgtcgag aggccaagga tgtcagccaa gagaagcaaa caacgacgca 1380 tcagccttat gtggagtagt gacagataat ggatactgca aactggaacc ggtgatagga 1440 agggaaagat gcgaggagca cagaggaatt gaggtcactg gtgcgtcatc ggcaccatgt 1500 tccggaaggt cggtattgcc atctgtctgt ggagctcggg catccgatgg ttcaccttgc 1560 aagaatcagc caatcgcaag gaggaagaga tgtgcgttgc acaaaggtca aagagcgtgc 1620 tgcgcctccg cgccacatcagt caaa 1644

<210>2 <211>548 <212> PROTEIN <213> Hordeum vulgare <400>2

Met Pro Ala Val Ala Ala Ala Arg Leu Lys Arg Glu Asp Cys Pro Arg

- 63 043494

Thr Lys His Asp Ser 20

Leu Phe Ser Pro Trp

Lys Val Leu Val Gly 30

Ser Asp Trp Glu Asp 35

His Ser Ala Gly Lys 40

Glu Gly Val Gln Arg 45

His Thr Arg Asn Leu 50

Pro Asp Asn Phe Pro 55

Gly Leu Tyr Glu Leu 60

Val Ala Arg Pro Ser 65

Tyr Asp Gly Val Arg 70

Ala Arg Arg Asn Arg 75

Val Val Val Val Val

Val Tyr Leu Gly Gln 90

Ala Asp Asn Val Arg 95

Arg Leu Gln Gln Tyr

100

Gly Arg Thr Gly Ser

105

His Leu Asp Thr Gly

110

Pro Leu Ala Ala Val

115

Cys Lys Ala Glu Met 120

Asn Ala Leu Thr Ala

125

Pro Gly Leu Phe Arg 130

Glu Val Phe Ser Arg

135

Gly Tyr Ser Met Met 140

Arg Cys Ala Leu Met 145

Gly Ser Lys Lys Ala 150

Ala Glu Lys Thr Glu 155

Gln Leu Leu Gly Val

165

Phe Asp Tyr Ala Trp

170

Asn Lys Leu Gln Asn

175

Ala Cys Arg Arg Glu

180

Glu Ile Leu Leu Lys

185

Leu Glu Gln Gly Ser

190

Arg Leu Ser Leu Leu 195

Ser Arg Val Arg His 200

Leu Lys Gln Arg Val

205

Gly Glu Lys Ala Gly 210

Ile Lys Ile Asn Ser

215

Ser Gly Ser Val Glu

220

Ser Ser Ser Ser Met 225

Lys Asn Met Leu Pro 230

Arg Val Arg Thr Phe 235

Gly Phe Arg Pro Arg

245

Leu Val Asn Ser Gly

250

Asp Asp Leu Asn Glu

255

Ser Asp Ile His Arg

260

Lys Cys Thr Pro Gln

265

Ala Asn Thr Ala Gly

270

Pro

Tyr

Gly

Ser 80

Ala

Asn

Gly

Phe

Gly 160

Gly

Asn

Phe

Ile

Val 240

Ala

Lys

- 64 043494

Gln Ala His Arg Arg

275

Ser Glu Gly Tyr Lys

280

Val Lys Lys Ile Asp

285

Ile Lys Arg Arg Thr

290

Ala Pro Ile Arg Glu

295

Ala Glu Ala Val Cys 300

Val Met Leu Glu Asp 305

Gly Ser Ser Cys Leu 310

Glu Asp Pro Met Glu 315

Arg Lys Arg Cys Glu

325

Leu His Lys Gly Arg

330

Arg Val Arg Val Ala

335

Ser Arg Lys Val Ser

340

Ser Ser Ser Ser Thr

345

Cys Gln Val Ala Ile

350

Thr Val Glu Ser Ile

355

Pro Gln Gln Thr Ala

360

Asn Pro Ser Lys Arg 365

Gln Ala Trp Gln Thr

370

Ser Ala Asp Gln Ser

375

Lys Asn Leu Ser Thr

380

Ala Lys Glu Pro Ser 385

Trp Gln Arg Asn Ser 390

Phe Lys Ala Asn Glu 395

Lys Ile Gly Glu Ala

405

Pro Thr Glu Asp Glu

410

Ala Tyr Gly Thr Ser

415

Ala Glu Ser Gln Phe

420

His Glu Asp Glu Pro

425

Cys Gly Arg Lys Trp

430

Glu Arg Leu Lys Ala

435

Gln Lys Ser Ala Asn 440

Ala Pro Ser Ser Arg 445

Gln Gly Cys Gln Pro 450

Arg Glu Ala Asn Asn 455

Asp Ala Ser Ala Leu 460

Gly Val Val Thr Asp 465

Asn Gly Tyr Cys Lys 470

Leu Glu Pro Val Ile 475

Arg Glu Arg Cys Glu

485

Glu His Arg Gly Ile

490

Glu Val Thr Gly Ala

495

Ser Ala Pro Cys Ser

500

Gly Arg Ser Val Leu

505

Pro Ser Val Cys Gly

510

Arg Ala Ser Asp Gly 515

Ser Pro Cys Lys Asn 520

Gln Pro Ile Ala Arg

525

Val

Gly

Gly 320

Tyr

Pro

Asp

Asn

Met 400

His

Phe

Gly

Cys

Gly 480

Ser

Ala

Arg

- 65 043494

Lys Arg Cys Ala Leu His Lys Gly Gln Arg Ala Cys Cys Ala Ser Ala

530 535540

Pro Ser Val Lys 545 <210>3 <211>1647 <212> DNA <213> Artificial Sequence <220>

<223> Coding sequence of the HvHRT gene of the barley HENZ-2 mutant <400>3 atgcctgcgg tcgccgctgc cagattgaag cgggaggact gcccccgcac caaacacgat60 tccctcttct ccccatggaa ggttcttgtc gggccgtcgg actgggagga ccactccgcc120 ggcaaggagg gggt ccagag gtatcacaca cgcaacctcc cggacaactt ccctggcctc180 tacgagctgg gcgttgcaag gccttcctat gatggtgtca gggctcgcag aaatcgatca240 gttgtcgtcg tggtggtata cctcgggcag gccgataatg tcagggcgag gctccagcag300 tac gggcgga cagggtcaca cctggacacc gggaatccgt tggctgctgt ctgtaaagct360 gagatgaacg cgctcacggc aggacctgga ttgttcaggg aagtcttctc cagaggctac420 tctatgatgt ttcgatgtgc gctgatgggt tccaaaaagg cagctgagaa gactgaaggt480 cagctactgg gagtatttga ttatgcatgg a ataaactgc agaatggtgc gtgtcgtcgc540 gaagaaatac tgctcaagtt agaacaggga agcaatagat tatctttgct tagcagagtc600 cggcacttaa aacagagggt gtttggagag aaagcaggta taaagattaa cagcagtggg660 tctgttgaga tttcatctag cag tatgaaa aatatgcttc caagagtccg tacgtttgtc720 ggcttcaggc ctcgtttggt taactctggc gacgatttaa acgaggcaag tgatattcac780 cgaaaatgca cacctcaggc caatactgct ggtaaacaag cacatagaag gtctgaagga840 tacaaggtga aaaagatcga tgttattaaa cggcgaactg caccgataag agaagccgaa900 gctgtttgtg gagtaatgct agaagatggt tcttcttgtt tggaggatcc aatggaagga960 aggaagaggt gtgagttgca caaaggtaga agagtcagag tggcatacag tcgca aagta1020 tcctcttcta gctccacttg ccaagttgct attccaactg ttgaatccat acctcaacaa1080 actgctaatc caagcaaacg agatcaagcc tggcaaacca gtgcagacca atccaaaaat1140 ctgtccacaa atgcaaagga gccatcttgg caaaggaaca gcttcaaagc aa atgagatg1200 aaaatcggag aagctcctac agaagatgaa gcatatggaa cctcccatgc agaatctcag1260 ttccacgaag atgagccttg tggaaggaag tgatttgagc ggctcaaagc acagaaatca1320

- 66 043494 gccaacgcac tcagccttat agggaaagat tccggaaggt aagaatcagc tgcgcctccg catcgtcgag gtggagtagt gcgaggagca cggtattgcc caatcgcaag cgccatcagt aggccaagga gacagataat cagaggaatt atctgtctgt gaggaagaga caaataa tg tcagccaa ggatactgca gaggtcactg ggagctcggg tgtgcgttgc gagaagcaaa aactggaacc gtgcgtcatc catccgatgg acaaaggtca caacgacgca ggtgatagga ggcaccatgt ttcaccttgc aagagcgtgc

1380

1440

1500

1560

1620

1647 <210> 4 <211> 430 <212> PROTEIN <213> Artificial sequence <220>

<223> Sequence of the barley HENZ-2 mutant HvHRT mutant <400> 4

Met Pro 1

Ala Val

Ala Ala 5

Ala Arg

Leu Lys 10

Arg Glu

Asp Cys

Thr Lys

His Asp

Ser Leu

Phe Ser

Pro Trp 25

Lys Val

Leu Val

Ser Asp

Trp Glu 35

Asp His

Ser Ala

Gly Lys

Glu Gly

Val Gln 45

His Thr

Arg Asn

Leu Pro

Asp Asn 55

Phe Pro

Gly Leu 60

Tyr Glu

Val Ala 65

Arg Pro

Ser Tyr

Asp Gly

Val Arg

Ala Arg 75

Arg Asn

Val Val

Val Val

Val Val 85

Tyr Leu

Gly Gln 90

Ala Asp

Asn Val

Arg Leu

Gln Gln

100

Tyr Gly

Arg Thr

Gly Ser 105

His Leu

Asp Thr

110

ProLeu

Ala Ala 115

Val Cys

Lys Ala

120

GluMet

Asn Ala

Leu Thr 125

Pro Gly

130

Leu Phe

Arg Glu

Val Phe 135

Ser Arg

Gly Tyr

140

SerMet

Arg Cys 145

Ala Leu

Met Gly

150

Ser Lys

Lys Ala

Ala Glu 155

Lys Thr

Pro Arg 15

Gly Pro

Arg Tyr

Leu Gly

Arg Ser 80

Arg Ala 95

Gly Asn

Ala Gly

Met Phe

Glu Gly

160

- 67 043494

Gln Leu Leu Gly Val

165

Phe Asp Tyr Ala Trp

170

Asn Lys Leu Gln Asn

175

Ala Cys Arg Arg Glu

180

Glu Ile Leu Leu Lys

185

Leu Glu Gln Gly Ser

190

Arg Leu Ser Leu Leu 195

Ser Arg Val Arg His 200

Leu Lys Gln Arg Val

205

Gly Glu Lys Ala Gly 210

Ile Lys Ile Asn Ser

215

Ser Gly Ser Val Glu

220

Ser Ser Ser Ser Met 225

Lys Asn Met Leu Pro 230

Arg Val Arg Thr Phe 235

Gly Phe Arg Pro Arg

245

Leu Val Asn Ser Gly

250

Asp Asp Leu Asn Glu

255

Ser Asp Ile His Arg

260

Lys Cys Thr Pro Gln

265

Ala Asn Thr Ala Gly

270

Gln Ala His Arg Arg

275

Ser Glu Gly Tyr Lys

280

Val Lys Lys Ile Asp

285

Ile Lys Arg Arg Thr

290

Ala Pro Ile Arg Glu

295

Ala Glu Ala Val Cys 300

Val Met Leu Glu Asp 305

Gly Ser Ser Cys Leu 310

Glu Asp Pro Met Glu 315

Arg Lys Arg Cys Glu

325

Leu His Lys Gly Arg

330

Arg Val Arg Val Ala

335

Ser Arg Lys Val Ser

340

Ser Ser Ser Ser Thr

345

Cys Gln Val Ala Ile

350

Thr Val Glu Ser Ile

355

Pro Gln Gln Thr Ala

360

Asn Pro Ser Lys Arg 365

Gln Ala Trp Gln Thr

370

Ser Ala Asp Gln Ser

375

Lys Asn Leu Ser Thr

380

Ala Lys Glu Pro Ser 385

Trp Gln Arg Asn Ser 390

Phe Lys Ala Asn Glu 395

Lys Ile Gly Glu Ala

405

Pro Thr Glu Asp Glu

410

Ala Tyr Gly Thr Ser

415

Gly

Asn

Phe

Ile

Val 240

Ala

Lys

Val

Gly

Gly 320

Tyr

Pro

Asp

Asn

Met 400

His

- 68 043494

Ala Glu Ser Gln Phe His Glu Asp Glu Pro Cys Gly Arg Lys

420 425 430 <210> 5 <211> 753 <212> DNA <213> Hordeum vulgare <400> 5

atggagcagg ggggagga cggggactgg atgatggagc cggcgtcggg gaagaagggc ggggtgatga tcgacaggaa gaagcgcttc agcgaggagc agatcaagtc gctcgagtcc atgttcgcca cgcagaccaa gctggagccc cgccagaagc tgcagctggc ccggggagctc ggcctgcagc cgcgccaggt cgccatctgg ttccagaaca agcgcgcgcg ctggaagtcc aagcagctcg agcgccagta cgccgcgctc cgggacgact acgacgccct cctctccagc tacgaccagc tcaagaagga caagcaagcg ctcgtcaacc agctggagaa gctagcagag atgctgcggg agccggggcgg ggccaagtgc ggagataatg ccggcgctgc tgacagggac aacatgcgcc tggccgtggc cggcatgagc atgaaggacg agttcgcgga cgctgccggg gccagcaagc tctactcggc gtctgccgag ggctgcggcg gcagcggcaa gctctccctc ttcggcgagg aggatgacga cgcgggcctc ttcctccggc cctcgctgca gctgccaacc gcgcacgacg gcggcttcac ggcgtcgggg ccggccgagt accagcagca gtcgccgtcg tcgttcccgt tccactcgag ctggccgtcg tccgcgacgg agcagacctg cagcagctcc caatggtggg aattcgagtc cccgagcgag taa

120

180

240

300

360

420

480

540

600

660

720

753 <210>6 <211>250 <212> PROTEIN <213> Hordeum vulgare <400>6

Met Glu 1

Gln

Gly Glu Glu Asp 5

Gly Lys

Lys

Gly Gly Val Met 20

Gly Asp Trp Met Met Glu Pro Ala Ser 10 15

Ile Asp Arg Lys Lys Arg Phe Ser Glu 25 30

Glu Gln

Glu Pro

Arg Gln 65

Ile Lys 35

Arg Gln

Val Ala

Ser Leu

Glu Ser

Met Phe

Ala Thr

Lys Leu

Gln Leu 55

Ala Arg

Glu Leu

Ile Trp

Phe Gln

Asn Lys

Arg Ala 75

Gln Thr 45

Gly Leu

Arg Trp

Lys Leu

Glin Pro

Lys Ser 80

- 69 043494

Lys Gln

Leu Glu

Leu Leu

Ser Ser

100

Asn Gln

Leu Glu 115

Lys Cys

130

Gly Asp

Arg Gln 85

Tyr Asp

Lys Leu

Asn Ala

Tyr Ala

Gln Leu

Ala Glu

120

Gly Ala 135

Ala Val 145

Ala Gly

Met Ser

150

Met Lys

Ala Ser

Lys Leu

Tyr Ser 165

Ala Ser

Lys Leu

Ser Leu

180

Phe Gly

Glu Glu

Arg Pro

Ser Leu 195

Gln Leu

Pro Thr

200

Ala Leu

Arg Asp

Asp Tyr

Lys Lys 105

Met Leu

Ala Asp

Asp Glu

Ala Glu

170

Asp Asp 185

Ala His

Asp Lys

Arg Glu

Arg Asp

140

Phe Ala 155

Gly Cys

Asp Ala

Asp Gly

Gln Ala

110

Pro Gly 125

Asn Met

Asp Ala

Gly Gly

Gly Leu

190

Gly Phe 205

Ser Gly

210

Pro Ala

Glu Tyr

Gln Gln

215

Gln Ser

Pro Ser

220

Ser Phe

His Ser 225

Ser Trp

Pro Ser

230

Ser Ala

Thr Glu

Gln Thr

235

Cys Ser

Asp Ala 95

Leu Val

Gly Ala

Arg Leu

Ala Gly

160

Ser Gly 175

Phe Leu

Thr Ala

Pro Phe

Ser Ser

240

Gln Trp

Trp Glu

Phe Glu 245

Ser Pro

Ser Glu

250

1546

DNA

Hordeum vulgare <210>

<211>

<212> <213>

<400> 7 gagacgggag tcaccgcgcc cagaatcgat ccgcgatcca gcgagaccta ggactggatg acccggctac catcgctcca ccatcacacc ttctgcttct gctaggtagg atggagccgg gcatgcacgc tcccccgatt aagctagcta tccccttcct tagggagga cgtcggggaa caccgcg ctc aaactactcc gcctcctagc tcccactccg gggagggatg gaagggcggg cattggccgc atatcgctag tcgctcgctc gatcaggtgc gagcaggggg gtgatgatcg cccgttgcca taagcagaag gcccgcacac atgaccaccg aggaggacgg acaggaagaa

120

180

240

300

360

- 70 043494

gcgcttcagc gaggagcaga tcaagtcgct agagtccatg ttcgccacgc agaccaagct 420 ggagccccgc cagaagctgc agctggcccg ggagctcggc ctgcagccgc gccaggtcgc 480 catctggttc cagaacaagc gcgcgcgctg gaagtccaag cagctcgagc gccagtacgc 540 cgcgcttcgg gacgactacg acgccctcct ctccagctac gaccagctca agaaggacaa 600 gcaagcgctc gtcaaccagg tatatactcc tatgtctgtc tgtctgtgct acgtaccgtg 660 tgtttctccg tgctctccgc tcggtggcgt ggagctcgtg gtgcctctgg ctaatgcatg 720 gtcgacgggt ttcttgcctt gcgtgtccgt gcagctggag aagctagcag agatgctgcg 780 ggagccggggc ggggccaagt gcggagataa tgccggcgct gctgacaggg acgacgtgcg 840 cctggccgtg gccggcatga gcatgaagga cgagttcgcg gacgctgccg gggccagcaa 900 gctctactcg gcgtctgccg agggctgcgg cggcagcggc aagctctccc tcttcggcga 960 ggacgatgac gacgcgggcc tcttcctccg gccctcgctg cagctgccaa ccgcgcacga 1020 cggcggcttc acggcgtcgg ggccggccga gtaccagcag cagtcgccgt cgtcgttccc 1080 gttccactcg agctggccgt cgtccgcgac gggagcagacc tgcagcagct cccaatggtg 1140 ggaattcgag tccccgagcg agtaagtaga gccatcggtc aagcaccatg caaggaatcg 1200 ccgacgtgat cgaccatgca acagatcagt gttcctaaca cagagacta tactgccgat 1260 cgaatccgtg gagaagacga cggcgcgatc gatcatatgc aaccgaagat ggtggtgtca 1320 agtgtgtaca tagctcgaaa cccaggtctg tccagtccag tacgtccagg cagcctcttc 1380 cttctcaatc agcagtcagc acgccatttt tcttcaccct cttcctcttt aagaatcact 1440 tgctcttgtc aattacctgc cacaccgtgt aatccacagg gaaactagtc acaaaaccaa 1500 attatagaga ccgatcttca gatgcaagtg catgcaacta tttagc 1546

<210> 8 <211> 753 <212> DNA <213> Artificial sequence <220>

<223> Coding sequence of the mutant HvHBL12 gene of the barley HENZ-10 mutant <400>8 atggagcagg gggaggagga cggggactgg atgatggagc cggcgtcggg gaagaagggc60 ggggtgatga tcgacaggaa gaagcgcttc agcgaggagc agatcaagtc gctcgagtcc120 atgtt cgcca cgcagaccaa gctggagccc cgccagaagc tgcagctggc ccgggagctc180 ggcctgcagc cgcgccaggt cgccatctgg ttccagaaca agcgcgcgcg ctggaagtcc240 aagcagctcg agcgccagta cgccgcgctc cgggacgact acgacgccct cctctcca gc300 tacgaccagc tcaagaagga caagcaagcg ctcgtcaacc agctggagaa gctagcagag360

- 71 043494

atgctgcggg agccggggcgg ggccaagtgc ggagataatg ccggcgctgc tgacaggac 420 aacatgcgcc tggccgtggc cggcatgagc atgaaggacg agttcgcgga cgctgccggg 480 gccagcaagc tctactcggc gtctgccgag ggctgcggcg gcagcggcaa gctctccctc 540 ttcggcgagg aggatgacga cgcgggcctc ttcctccggc cctcgctgca gctgccaacc 600 gcgcacgacg gcggcttcac ggcgtcgggg ccggccgagt accagcagca gtcgccgtcg 660 tcgttcccgt tccactcgag ctgaccgtcg tccgcgacgg agcagacctg cagcagctcc 720 caatggtggg aattcgagtc cccgagcgag taa 753

<210> 9 <211> 227 <212> PROTEIN <213> Artificial sequence <220>

<223> Coding sequence of the mutant gene HvHBL12 of the barley HENZ10 mutant <400> 9

Met Glu 1

Gln

Gly Glu Glu Asp 5

Gly Lys

Lys

Gly Gly Val Met 20

Gly Asp Trp Met Met Glu Pro Ala Ser 10 15

Ile Asp Arg Lys Lys Arg Phe Ser Glu 25 30

Glu Gln Ile Lys Ser Leu Glu Ser Met Phe Ala Thr Gln Thr Lys Leu 35 4045

Glu Pro Arg Gln Lys Leu Gln Leu Ala Arg Glu Leu Gly Leu Gln Pro 50 5560

Arg Gln Val Ala Ile Trp Phe Gln Asn Lys Arg Ala Arg Trp Lys Ser 65 70 7580

Lys Gln Leu Glu Arg Gln Tyr Ala Ala Leu Arg Asp Asp Tyr Asp Ala 85 9095

Leu Leu Ser Ser Tyr Asp Gln Leu Lys Lys Asp Lys Gln Ala Leu Val

100 105110

Asn Gln Leu Glu Lys Leu Ala Glu Met Leu Arg Glu Pro Gly Gly Ala

115 120125

Lys Cys Gly Asp Asn Ala Gly Ala Ala Asp Arg Asp Asn Met Arg Leu

130 135140

- 72 043494

Ala Val Ala Gly Met Ser Met Lys

145 150

Asp Glu Phe Ala Asp Ala Ala Gly

155 160

Ala Ser Lys Leu Tyr Ser Ala Ser

165

Ala Glu Gly Cys Gly Gly Ser Gly

170 175

Lys Leu Ser Leu Phe Gly Glu Glu 180

Asp Asp Asp Ala Gly Leu Phe Leu

185 190

Arg Pro Ser Leu Gln Leu Pro Thr

195 200

Ala His Asp Gly Gly Phe Thr Ala

205

Ser Gly Pro Ala Glu Tyr Gln Gln

210 215

Gln Ser Pro Ser Ser Phe Pro Phe

220

His Ser Ser

225 <210> 10 <211> 1062 <212> DNA <213> Hordeum vulgare

<400> 10 atggatccat ggatggggcag ccagccacatcc ctgagcctcg acctgcacgt cggcctaccg 60 ccgatggggc acccgcacca ccaccagagc caataccagg cgccgccgat gatcgcgctg 120 gccaagccca agatcctcgt ggaggagaac ttcatgccac tcaagaagga ccctgaggtt 180 gcggttcttg agtcggagct acaggggtg agcgaggaga accggcggct gggcgagatg 240 ctcagggagg tggcctccaa gtacgaggcc ctgcaggggcc agttcaccga cgtggtcacg 300 gccggcggca acaacaacca ctaccacaac cagccgtcct ccgcgtcgga gggcgggtcg 360 gtgtcgccgt cgaggaagcg caagagcgag gagagcctcg gcacgccgcc accgtcgcat 420 actcagcagc agcactatgc cgccggcctc gcgtacgcgg tggcgccgga ccaggcggag 480 tgcacgtccg gcgagccgtg caagcgcatc cggggaggt gcaagcccgt catctccaag 540 cgctacgtcc acgccgaccc ctccgacctc agcctggtgg tgaaggacgg gtaccaatgg 600 cgcaagtacg ggcagaaggt gaccaaggac aacccatgcc ccagagccta cttccggtgc 660 tccttcgccc ccggctgccc cgtcaagaag aaggtgcaga ggagcgccga ggacaagacc 720 atactcgtgg cgacgtacga gggcgagcac aaccacacc agcccccgcc gtcgcagccg 780 cagcagcaga acgacggctc cggcgccggc aagaacgccg ggaacgggaa gccgccccag 840 gcgccggcca cgcctcacca cccgcagcag cagcacaagc aggaagcggc agcggtcgtc 900 gtcagcggcg aatcggccgc ggcggcgtcc gagctgatcc ggcgcaacct ggcggagcag 960 atggccatga cgctgacgag ggaccccagc ttcaaggcgg cgctggtcac cgcgctctcc 1020

- 73 043494 ggccggatcc tcgagctctc gccgaccagg gacatcaatt aa

1062 <210>11 <211>353 <212> PROTEIN <213> Hordeum vulgare <400>11

Met Asp Pro Trp Met Gly Ser Gln 1 5

Pro Ser Leu Ser Leu Asp Leu His

15

Val Gly Leu Pro Pro Met Gly His 20

Pro His His His Gln Ser Gln Tyr 25 30

Gln Ala Pro Pro Met Ile Ala Leu

40

Ala Lys Pro Lys Ile Leu Val Glu 45

Glu Asn Phe Met Pro Leu Lys Lys

55

Asp Pro Glu Val Ala Val Leu Glu 60

Ser Glu Leu Gln Arg Val Ser Glu 65 70

Glu Asn Arg Arg Leu Gly Glu Met

80

Leu Arg Glu Val Ala Ser Lys Tyr 85

Glu Ala Leu Gln Gly Gln Phe Thr

95

Asp Val Val Thr Ala Gly Gly Asn

100

Asn Asn His Tyr His Asn Gln Pro

105 110

Ser Ser Ala Ser Glu Gly Gly Ser

115 120

Val Ser Pro Ser Arg Lys Arg Lys

125

Ser Glu Glu Ser Leu Gly Thr Pro

130 135

Pro Pro Ser His Thr Gln Gln Gln

140

His Tyr Ala Ala Gly Leu Ala Tyr

145 150

Ala Val Ala Pro Asp Gln Ala Glu

155 160

Cys Thr Ser Gly Glu Pro Cys Lys

165

Arg Ile Arg Glu Glu Cys Lys Pro

170 175

Val Ile Ser Lys Arg Tyr Val His

180

Ala Asp Pro Ser Asp Leu Ser Leu

185 190

Val Val Lys Asp Gly Tyr Gln Trp

195 200

Arg Lys Tyr Gly Gln Lys Val Thr

205

- 74 043494

Lys Asp Asn Pro Cys Pro Arg Ala Tyr

210 215

Gly Cys Pro Val Lys Lys Lys Val Gln

225 230

Ile Leu Val Ala Thr Tyr Glu Gly Glu

245

Pro Ser Gln Pro Gln Gln Gln Asn Asp

260 265

Ala Gly Asn Gly Lys Pro Pro Gln Ala

275 280

Gln Gln Gln His Lys Gln Glu Ala Ala

290 295

Ser Ala Ala Ala Ala Ser Glu Leu Ile

305 310

Met Ala Met Thr Leu Thr Arg Asp Pro

325

Thr Ala Leu Ser Gly Arg Ile Leu Glu

340 345

Asn <210>12 <211>353 <212> PROTEIN <213> Hordeum vulgare <400>12

Met Asp Pro Trp Met Gly Ser Gln Pro 15

Val Gly Leu Pro Pro Met Gly His Pro 20 25

Gln Ala Pro Pro Met Ile Ala Leu Ala

40

Glu Asn Phe Met Pro Leu Lys Lys Asp

55

Arg Cys Ser Phe Ala Pro 220

Ser Ala Glu Asp Lys Thr

235 240

Asn His Thr Gln Pro Pro

255

Ser Gly Ala Gly Lys Asn

270

Ala Thr Pro His His Pro

285

Val Val Val Ser Gly Glu

300

Arg Asn Leu Ala Glu Gln

315 320

Phe Lys Ala Ala Leu Val

335

Ser Pro Thr Arg Asp Ile

350

Leu Ser Leu Asp Leu His

His His Gln Ser Gln Tyr 30

Pro Lys Ile Leu Val Glu 45

Glu Val Ala Val Leu Glu

- 75 043494

Ser 65

Leu

Asp

Ser

Ser

His 145

Cys

Val

Val

Lys

Gly 225

Ile

Pro

Ala

Gln

Ser 305

Glu Leu

Gln Arg

Arg Glu

Val Ala

Met Val

Thr Ala 100

Ser Ala

115

Ser Glu

Val Ser 70

Ser Lys

Gly Gly

Gly Gly

Glu Glu

Asn Arg

Arg Leu

Gly Glu

Glu Glu 130

Tyr Ala

Thr Ser

Ile Ser

Val Lys

195

Asp Asn 210

Cys Pro

Leu Val

Ser Gln

Gly Asn

275

Gln Gln

290

Ala Ala

Ser Leu

Gly Thr

135

Ala Gly

Gly Glu

165

Lys Arg 180

Asp Gly

Pro Cys

Val Lys

Ala Thr

245

Pro Gln 260

Gly Lys

His Lys

Ala Ala

Leu Ala 150

Pro Cys

Tyr Val

Tyr Gln

Pro Arg

215

Lys Lys 230

Tyr Glu

Gln Gln

Pro Pro

Gln Glu

295

Ser Glu

310

Tyr Glu

Asn Asn 105

Ser Val 120

Pro Pro

Tyr Ala

Lys Arg

Ala Leu 90

Asn His

Ser Pro

Pro Ser

Val Ala

155

Ile Arg 170

Gln Gly

Gln Phe

Tyr His

Ser Arg

125

His Thr 140

Pro Asp

Glu Glu

Asn Gln

110

Lys Arg

Gln Gln

Gln Ala

Cys Lys

175

His Ala

185

Trp Arg 200

Ala Tyr

Val Gln

Gly Glu

Asn Asp

265

Gln Ala

280

Ala Ala

Leu Ile

Asp Pro

Lys Tyr

Phe Arg

Arg Ser

235

His Asn 250

Gly Ser

Pro Ala

Ala Val

Arg Arg

315

Ser Asp

Gly Gln

205

Cys Ser 220

Ala Glu

His Thr

Gly Ala

Thr Pro

285

Val Val 300

Asn Leu

Leu Ser 190

Lys Val

Phe Ala

Asp Lys

Glin Pro

255

Gly Lys 270

His His

Ser Gly

Ala Glu

Met 80

Thr

Pro

Lys

Gln

Glu 160

Pro

Leu

Thr

Pro

Thr 240

Pro

Asn

Pro

Glu

Gln 320

- 76 043494

Met Ala Met Thr Leu Thr Arg Asp Pro Ser Phe Lys Ala Ala Leu Val

325

330

335

Thr Ala Leu Ser Gly Arg Ile Leu Glu Leu Ser Pro Thr Arg Asp Ile

340

345

350

Asn <210> 13 <211> 1062 <212> DNA <213> Artificial sequence <220>

<223> Coding sequence of the mutant gene WRKY38 HENZ-50 <400>13 atggatccat ggatgggcag ccagccatcc ctgagcctcg acctgcacgt cggcctaccg60 ccgatggggc acccgcacca ccaccagagc caataccagg cgccgccgat gatcgcgctg120 gccaagccca agatcct cgt ggaggagaac ttcatgccac tcaagaagga ccctgaggtt180 gcggttcttg agtcggagct acagcgggtg agcgaggaga accggcggct gggcgagatg240 ctcagggagg tggcctccaa gtacgaggcc ctgcagggcc agttcaccga cgtggtcacg300 gccggcggca a caacaacca ctaccacaac cagccgtcct ccgcgtcgga gggcgggtcg360 gtgtcgccgt cgaggaagcg caagagcgag gagagcctcg gcacgccgcc accgtcgcat420 actcagcagc agcactatgc cgccggcctc gcgtacgcgg tggcgccgga ccaggcggag480 tgcacgtccg gcgagccgtg caagcgcat c cgggaggagt gcaagcccgt catctccaag540 cgctacgtcc acgccgaccc ctccgacctc agcctggtgg tgaaggacgg gtaccaatga600 cgcaagtacg ggcagaaggt gaccaaggac aacccatgcc ccagagccta cttccggtgc660 tccttcgccc ccggctgccc cgtca agaag aaggtgcaga ggagcgccga ggacaagacc720 atactcgtgg cgacgtacga gggcgagcac aaccacaccc agcccccgcc gtcgcagccg780 cagcagcaga acgacggctc cggcgccggc aagaacgccg ggaacgggaa gccgccccag840 gcgccggcca cgcctcacca cccgcagcag cagcacaagc aggaagcggc agcggtcgtc900 gtcagcggcg aatcggccgc ggcggcgtcc gagctgatcc ggcgcaacct ggcggagcag960 atggccatga cgctgacgag ggaccccagc ttcaaggcgg cgctggtcac cgcgctct cc1020 ggccggatcc tcgagctctc gccgaccagg gacatcaatt aa1062 <210>

<211>

<212>

<213>

199

PROTEIN

Artificial sequence

- 77 043494 <220>

<223> Protein sequence of the barley HENZ-50 mutant WRKY38 <400> 14

Met Asp Pro Trp 1

Met Gly Ser Gln 5

Pro Ser Leu Ser 10

Leu Asp Leu His 15

Val Gly Leu Pro 20

Pro Met Gly His

Pro His His His 25

Gln Ser Gln Tyr 30

Gln Ala Pro Pro

Met Ile Ala Leu

Ala Lys Pro Lys

Ile Leu Val Glu 45

Glu Asn Phe Met

Pro Leu Lys Lys 55

Asp Pro Glu Val 60

Ala Val Leu Glu

Ser Glu Leu Gln 65

Arg Val Ser Glu 70

Glu Asn Arg Arg 75

Leu Gly Glu Met 80

Leu Arg Glu Val

Ala Ser Lys Tyr 85

Glu Ala Leu Gln

Gly Gln Phe Thr 95

Asp Val Val Thr

100

Ala Gly Gly Asn

Asn Asn His Tyr 105

His Asn Gln Pro

110

Ser Ser Ala Ser

115

Glu Gly Gly Ser

120

Val Ser Pro Ser

Arg Lys Arg Lys 125

Ser Glu Glu Ser

130

Leu Gly Thr Pro

135

Pro Pro Ser His

140

Thr Gln Gln Gln

His Tyr Ala Ala 145

Gly Leu Ala Tyr 150

Ala Val Ala Pro

155

Asp Gln Ala Glu

160

Cys Thr Ser Gly

Glu Pro Cys Lys 165

Arg Ile Arg Glu

170

Glu Cys Lys Pro

175

Val Ile Ser Lys

180

Arg Tyr Val His

Ala Asp Pro Ser 185

Asp Leu Ser Leu 190

Val Val Lys Asp Gly Tyr Gln 195 <210> 15 <211> 17 <212> DNA <213> Artificial sequence

- 78 043494 <220>

<223> Primer <400>

cacgaagatg agccttg <210> 16 <211> 17 <212> DNA <213> Artificial sequence <220>

<223> Primer <400>16 ttggctgatt tctgtgc17 <210>17 <211>17 <212> DNA <213> Artificial sequence <220>

<223> Probe <400>

aagtgatttg agcggct <210> 18 <211> 17 <212> DNA <213> Artificial sequence <220>

<223> Probe <400>18 aggaagtggt ttgagcg17 <210>19 <211>15 <212> DNA <213> Artificial sequence <220>

<223> Primer <400>

gtcgtcgttc ccgtt <210>

<211>

<212>

<213>

DNA

Artificial sequence <220>

<223>

Primer

- 79 043494 <400>20 ctgcaggtct gctcc <210>21 <211>15 <212> DNA <213> Artificial sequence <220>

<223> Probe <400>21 actcgagctg accgt15 <210>22 <211>14 <212> DNA <213> Artificial sequence <220>

<223> Probe <400>

ctcgagctgg ccgt <210> 23 <211> 15 <212> DNA <213> Artificial sequence <220>

<223> Primer <400>23 ctcagcctgg tggtg15 <210>24 <211>17 <212> DNA <213> Artificial sequence <220>

<223> Primer <400>

tgtccttggt caccttc <210> 25 <211> 17 <212> DNA <213> Artificial sequence <220>

<223> Probe <400> 25 ccaatgacgc aagtacg 17

- 80 043494 <210> 26 <211> 17 <212> DNA <213> Artificial sequence <220>

<223> Probe <400>26 taccaatggc gcaagta17 <210>27 <211>20 <212> DNA <213> Artificial sequence <220>

<223> Primer <400>

aaacagaggt tgaggataac <210> 28 <211> 15 <212> DNA <213> Artificial sequence <220>

<223> Primer <400>28 gccttcgcct tccat15 <210>29 <211>17 <212> DNA <213> Artificial sequence <220>

<223> Probe <400>

aggcaccgat caatacg <210> 30 <211> 17 <212> DNA <213> Artificial sequence <220>

< 223> Probe < 400> 30 aaggcaccgg tcaatac 17 <210>

<211>

- 81 043494 <212> DNA <213> Artificial sequence <220>

<223> Non-standard tandem repeat W-box <400> 31 tgacggtcgt attgacc 17 <210> 32 <211> 17 <212> DNA <213> Artificial sequence <220>

<223> Non-standard tandem repeat W-box <400> 32 tgacagtggt attggcc <210> 33 <211> 17 <212> DNA <213> Artificial sequence <220>

<223> Non-standard tandem repeat W-box <400> 33 tgacagtggt actggcc <210> 34 <211> 18 <212> DNA <213> Artificial sequence <220>

<223> Non-standard tandem repeat W-box <400> 34 gtgacagtgg tattggcc <210> 35 <211> 17 <212> DNA <213> Artificial sequence <220>

<223> Non-standard tandem repeat W-box <400> 35 tgacggtcgt attgatc <210> 36 <211> 17 <212> DNA <213> Artificial sequence

- 82 043494 <220>

<223> Custom <400>

tandem repeating

W-box tgaccgtcgt attgatc <210> 37 <211> 11 <212> DNA <213> Artificial sequence <220>

<223> Non-standard tandem repeat W-box <400>37 ttgacttgat c11 <210>38 <211>12 <212> DNA <213> Artificial sequence <220>

<223> W-box <220>

< 221> misc_feature < 222> (5)..(5) < 223> C, or nucleotide missing <220>

< 221> misc_feature < 222> (6)..(6) <223> X(n), where X can be any nucleotide and n is an integer from 0 to 20 <400>38 tgacnnttga cc <210 >39 <211>12 <212> DNA <213> Artificial sequence <220>

<223> W-box <220>

< 221> misc_feature < 222> (5)..(5) < 223> G or A <220>

< 221> misc_feature < 222> (6)..(6) < 223> X(n), where X can be any nucleotide and n is an integer from 0 to 20

- 83 043494 <220>

< 221> misc_feature < 222> (7)..(7) <223> X(n), where X can be either C or T, n or 1 <400>39 tgacnnntgr cc <210>40 <211> 12 <212> DNA <213> Artificial sequence <220>

<223> W-box <220>

< 221> misc_feature < 222> (5)..(5) < 223> either G or A <220>

< 221> misc_feature < 222> (6)..(6) <223> X(n), where X can be any nucleotide and is an integer from 0 to 20 <400>40 tgacnnttga cc <210>41 < 211>11 <212> DNA <213> Artificial sequence <220>

<223> W-box <220>

< 221> misc_feature < 222> (5)..(5) < 223> G or A <220>

< 221> misc_feature < 222> (6)..(6) <223> X(n), where X can be any nucleotide and is an integer from 0 to 20 <400>41 tgacnnttga c equals 0 n represents 12 n representing11 <210>42 <211>12 <212> DNA <213> Artificial sequence

- 84 043494 <220>

<223> W-box <220>

< 221> misc_feature < 222> (5)..(5) < 223> X = C, or no nucleotide <220>

< 221> misc_feature < 222> (6)..(6) < 223> X(n), where X can be any nucleotide and n is an integer from 0 to 20 <220>

< 221> misc_feature < 222> (7)..(7) < 223> C or T <220>

< 221> misc_feature < 222> (10)..(10) < 223> G or A <400>

tgacnnntgn cc <210> 43 <211> 12 <212> DNA <213> Artificial sequence <220>

<223> W-box <220>

< 221> misc_feature < 222> (5)..(5) < 223> C, or nucleotide missing <220>

< 221> misc_feature < 222> (6)..(6) < 223> X(n), where X can be any nucleotide and n is an integer from 0 to 20 <220>

< 221> misc_feature < 222> (10)..(10) < 223> G or A <400>

tgacnnctgn cc <210>

<211>

<212>

<213>

DNA

Artificial sequence

- 85 043494 <220>

<223> W-box <220>

< 221> misc_feature < 222> (5)..(5) < 223> C, or nucleotide missing <220>

< 221> misc_feature < 222> (6)..(6) < 223> X(n), where X can be any nucleotide and n is an integer from 0 to 20 <220>

< 221> misc_feature < 222> (7)..(7) < 223> C or T <400>

tgacnnntgg cc <210> 45 <211> 12 <212> DNA <213> Artificial sequence <220>

<223> W-box <220>

< 221> misc_feature < 222> (5)..(5) < 223> C, or nucleotide missing <220>

< 221> misc_feature < 222> (6)..(6) < 223> X(n), where X can be any nucleotide and n is an integer from 0 to 20 <400> 45 tgacnnctga cc <210 >

<211>

<212>

<213>

DNA

Artificial sequence <220>

<223>

W-box <220>

<221>

<222>

<223>

misc_feature (5) . . (5)

C, or nucleotide missing <220>

<221>

misc_feature

- 86 043494 <222> (6)..(6) <223> X(n), where X can be any nucleotide and n is an integer from 0 to 20 <400>46 tgacnnttgg cc <210>47 < 211>12 <212>DNA <213>Artificial sequence <220>

< 223> W-box < 220>

< 221> misc_feature < 222> (5)..(5) < 223> C, or nucleotide missing < 220>

< 221> misc_feature < 222> (6)..(6) < 223> X(n), where X can be any nucleotide and n is an integer from 0 to 20 <400>47 tgacnnttga tc < 210 >48 <211>53 <212> DNA <213> Artificial sequence <220>

<223> (Amy6-4) <220>

<221> misc_feature <222> (1)..(53) <223> (Amy6-4) <400>48 taactgacgg tcgtattgac cggtgccttc ttatggaagg cgaaggctgc ctc53 <210>49 <211>53 <212> DNA <213> Artificial sequence <220>

<223> amy1_1a <220>

<221> misc_feature <222> (1)..(53) <223> amy1_1a

- 87 043494 <400> 49 taactgacgg tcgtattgac cggtgccttc ttatggaagg cgaaggctgc ctc <210> 50 <211> 53 <212> DNA <213> Artificial sequence <220>

<223> amy1_1b <220>

<221> misc_feature <222> (1).. (53) <223> amy1_1b <400>50 taactgacgg tcgtattgac cggtgccttc ttatggaagg cgaaggctgc ctc53 <210>51 <211>53 <212> DNA <213> Artificial sequence <220>

<223> amy1_1c <220>

<221> misc_feature <222> (1)..(53) <223> amy1_1c <400>

taactgacgg tcgtattgac cagtgccttc ttatggaagg cgaaggctgc ctc <210> 52 <211> 29 <212> DNA <213> Artificial sequence <220>

<223>Amy46 <220>

<221> misc_feature <222> (1)..(29) <223> Amy46 <400>52 taactgacag tcgtactggc cggtgcctt29 <210>53 <211>29 <212> DNA <213> Artificial sequence

- 88 043494 <220>

<223> amy1_2 <220>

<221> misc_feature <222> (1)..(29) <223> amy1_2 <400>53 taagtgacag tggtattggc cggtgcctt <210>54 <211>53 <212> DNA <213> Artificial sequence <220>

<223> Amy6-4 <220>

<221> misc_feature <222> (1)..(53) <223> Amy6-4 <400>54 catctacatc acttgggcat tgaatcgcct tttgagctca ccgtaccggc cga53 <210>55 <211>53 <212> DNA <213> Artificial sequence <220 >

<223> amy1_1a <220>

<221> misc_feature <222> (1)..(53) <223> amy1_1a <400>55 catctacatc acttgggcat tgaatcgcct tttgagctca ccgtaccggc cga53 <210>56 <211>53 <212> DNA <213> Artificial sequence <220>

<223> amy1_1b <220>

<221> misc_feature <222> (1)..(53) <223> amy1_1b

- 89 043494 <400>56 catctacatc acttgggcat tgaatcgcct tttgagctca ccgtaccggc cga <210>57 <211>53 <212> DNA <213> Artificial sequence <220>

<223> amy1_1c <220>

< 221> misc_feature < 222> (1).. (53) < 223> amy1_1c < 400>57 catctccatc acttgggcat tgaatcgcct tttgagctca ccgcaccggc cga53 < 210>58 < 211>53 < 212> DNA < 213> Artificial sequence < 220>

<223>Amy46 <220>

<221> misc_feature <222> (1)..(53) <223> Amy46 <400>

cttgtcgaag gctggatcca tcagtcgcct tttgagctca ccgcaccggc cga <210> 59 <211> 53 <212> DNA <213> Artificial sequence <220>

<223> amy1_2 <220>

< 221> misc_feature < 222> (1)..(53) < 223> amy1_2 < 400>59 ctcatcgaag ccggtgctca tcattcgcct tttgagctca ccgcaccggc cga53 < 210>60 < 211>29 < 212> DNA < 213> Artificial sequence

- 90 043494 <220>

<223> Amy6-4 <220>

< 221> misc_feature < 222> (1)..(29) < 223> Amy6-4 < 400>60 taacaaactc cggccgacat atccactgg29 < 210>61 < 211>29 < 212> DNA < 213> Artificial sequence <220>

<223> amy1_1a <220>

< 221> misc_feature < 222> (1)..(29) < 223> amy1_1a <400>

taacaaactc cggccgacat atccactgg <210> 62 <211> 29 <212> DNA <213> Artificial sequence <220>

<223> amy1_1b <220>

< 221> misc_feature < 222> (1)..(29) < 223> amy1_1b < 400>62 taacaaactc cggccgacat atccactgg29 < 210>63 < 211>29 < 212> DNA < 213> Artificial sequence <220>

<223> amy1_1c <220>

<221> misc_feature <222> (1)..(29) <223> amy1_1c

- 91 043494 <400>63 taacaaactc cggccaacat atccactgg <210>64 <211>29 <212> DNA <213> Artificial sequence <220>

<223>Amy46 <220>

< 221> misc_feature < 222> (1)..(29) < 223> Amy46 < 400>64 taacaaactc cggccgacat atccatcga29 < 210>65 < 211>29 < 212> DNA < 213> Artificial sequence <220>

<223> amy1_2 <220>

<221> misc_feature <222> (1)..(29) <223> amy1_2 <400>

taacaaactc cggccgacat atccatcga <210> 66 <211> 42 <212> DNA <213> Artificial sequence <220>

<223> amy2_1 <220>

< 221> misc_feature < 222> (1)..(42) < 223> amy2_1 < 400>66 ggaatttgtg ccggcccgga ttgacttgac catcatctgt tg42 < 210>67 < 211>43 < 212> DNA <213> Artificial sequence

- 92 043494 <220>

<223> amy2_2 <220>

<221> misc_feature <222> (1)..(43) <223> amy2_2 <400> 67 ggaggctgtg ccaacccagc ttgacttgac catcaccagt tac <210> 68 <211> 48 <212> DNA <213> Artificial sequence <220>

<223> amy2_3 <220>

<221> misc_feature <222> (1)..(48) <223> amy2_3 <400> 68 ggaacttgtg ccaccccgga ttgacttgac cgtcatcggc tccggatg <210> 69 <211> 36 <212> DNA <213> Artificial sequence <220>

<223> amy2_1 <220>

<221> misc_feature <222> (1)..(36) <223> amy2_1 <400>69 caccttttct cgtaacagag tctggtatcc atgcag <210>70 <211>36 <212> DNA <213> Artificial sequence <220>

<223> amy2_2 <220>

<221> misc_feature <222> (1)..(36) <223> amy2_2

- 93 043494 <400>70 tccattttc cataacagag gccggtaccc atgcat <210>71 <211>36 <212> DNA <213> Artificial sequence <220>

<223> amy2_3 <220>

< 221> misc_feature < 222> (1).. (36) < 223> amy2_3 < 400>71 caccttttat cgtaacagag tccggtatcc atgcag36 < 210>72 < 211>18 < 212> DNA < 213> Artificial sequence <220>

<223> cluster amy1_1 <220>

< 221> misc_feature < 222> (1)..(18) < 223> amy1_1 <400>

ctgacggtcg tattgatc <210> 73 <211> 19 <212> DNA <213> Artificial sequence <220>

<223> Target-specific forward primer for HENZ-53 <220>

< 221> misc_feature < 222> (1)..(19) < 223> HENZ-53 < 400>73 gtcagctact gggagtatt19 < 210>74 < 211>15 < 212> DNA < 213> Artificial sequence

- 94 043494 <220>

<223> Target-specific reverse primer for HENZ-53 <220>

<221> misc_feature <222> (1)..(15) <223> Reverse primer for HENZ-53 <400>74 tcttcgcgac gacac15 <210>75 <211>20 <212> DNA <213> Artificial sequence <220>

<223> Mutant-specific probe for HENZ-53 <220>

< 221> misc_feature < 222> (1)..(20) <223> Mutant-specific probe for HENZ-53 <400>

tgcatgaaat aaactgcaga <210> 76 <211> 19 <212> DNA <213> Artificial sequence <220>

<223> Reference-specific detector probe for HENZ-53 <220>

<221> misc_feature <222> (1)..(19) <223> Reference-specific detector probe for HENZ-53 <400>76 tgcatggaat aaactgcag19 <210>77 <211>18 <212> DNA <213> Artificial sequence <220>

<223> Target-specific forward primer for HENZ-54 <220>

< 221> misc_feature < 222> (1)..(18) < 223> Target-specific forward primer for HENZ-54

- 95 043494 <400>77 tgctaatcca agcaaacg <210>78 <211>20 <212> DNA <213> Artificial sequence <220>

<223> Target-specific reverse primer for HENZ-54 <220>

< 221> misc_feature < 222> (1)..(20) < 223> Target-specific reverse primer for HENZ-54 <400>78 tttgtggaca gatttttgga20 < 210>79 <211>17 < 212> DNA < 213> Artificial sequence <220>

<223>Mutant-specific detection probe for HENZ-54 <220>

< 221> misc_feature < 222> (1)..(17) < 223> Reference-specific detector probe for HENZ-54 <400>79 agcctgacaa accagtg < 210>80 <211>16 < 212> DNA < 213> Artificial sequence <220>

<223> Reference-specific detector probe for HENZ-54 <220>

<221> misc_feature <222> (1)..(16) <223> Reference-specific detector probe for HENZ-54 <400>80 aagcctggca aaccag <210>81 <211>15 <212> DNA <213> Artificial sequence

- 96 043494 <220>

<223> Direct primer for amy1-1 <220>

<221> misc_feature < 222> (1)..(15) < 223> Primer < 400> 81 gactggggcc tgaag <210> 82 <211> 15 < 212> DNA < 213> Artificial sequence <220>

<223> Reverse primer for amy1-1 <220>

< 221> misc_feature < 222> (1)..(15) < 223> Primer < 400> 82 gtgccgggtc ctgac <210> 83 <211> 19 < 212> DNA < 213> Artificial sequence <220>

<223> Probe for amy1-1 <220>

<221> misc_feature < 222> (1)..(19) < 223> Probe < 400> 83 agatcgatcg cctggtgtc <210> 84 <211> 17 < 212> DNA < 213> Artificial sequence <220>

<223> Direct primer for amy1-2 <220>

<221> misc_feature <222> (1)..(17) <223> Primer

- 97 043494 <400>84 agatcgatcg tctggtg <210>85 <211>18 <212> DNA <213> Artificial sequence <220>

<223> Reverse sequence for amy-1-2 <220>

< 221> misc_feature < 222> (1)..(18) < 223> Primer < 400>85 tccatgatct gcagcttg18 < 210>86 < 211>19 < 212> DNA < 213> Artificial sequence <220>

<223> Probe for amy1-2 <220>

< 221> misc_feature < 222> (1)..(19) < 223> Probe <400>

tcaatcagga cccgacagg <210> 87 <211> 16 <212> DNA <213> Artificial sequence <220>

<223> Direct primer for amy2 <220>

< 221> misc_feature < 222> (1)..(16) < 223> Primer < 400>87 cgagctcaag gagtgg16 < 210>88 < 211>17 < 212> DNA < 213> Artificial sequence

- 98 043494 <220>

<223> Reverse primer for amy2 <220>

< 221> misc_feature < 222> (1) .. (17) < 223> Primer < 400>88 cgtcgatgta caccttg17 < 210>89 < 211>18 < 212> DNA < 213> Artificial sequence <220>

<223> Probe for amy2 <220>

< 221> misc_feature < 222> (1)..(18) < 223> Probe <400>

aagagcgacc tcggcttc <210> 90 <211> 18 <212> DNA <213> Artificial sequence <220>

<223> Forward primer for Bgl2 <220>

<221> misc_feature <222> (1)..(18) <223> Primer <400>90 taccagaacc tgttcgac18 <210>91 <211>16 <212> DNA <213> Artificial sequence <220>

<223> Reverse primer for Bgl2 <220>

< 221> misc_feature < 222> (1)..(16) < 223> Primer

- 99 043494 <400> 91 acaccaccag cttcac <210> 92 <211> 18 <212> DNA <213> Artificial sequence <220>

<223> Bgl2 probe <220>

<221> misc_feature < 222> (1)..(18) < 223> Probe < 400> 92 accgtggacg ccttctac <210> 93 <211> 18 < 212> DNA < 213> Artificial sequence <220>

<223> Direct primer for LD <220>

<221> misc_feature <222> (1)..(18) <223> Primer <400> 93 cttcgatggg gtttgaac <210> 94 <211> 18 <212> DNA <213> Artificial sequence <220>

<223> Reverse primer for LD <220>

< 221> misc_feature < 222> (1)..(18) < 223> Primer < 400> 94 ccgatttcct caccaaag <210> 95 <211> 21 <212> DNA <213> Artificial sequence

- 100 -

Claims (11)

<220> <223> LD probe <220> < 221> misc_feature < 222> (1)..(21) < 223> Probe < 400> 95 cctgtgcagg tgaattcatc a CLAIM 1. A barley plant or part thereof, wherein the barley plant has been produced by a process including an induced mutagenesis step, or said plant is a progeny of a plant produced by a method including an induced mutagenesis step, and wherein said barley plant carries a mutation in the HvHRT gene resulting in the formation of a mutant HvHRT gene encoding a mutant HvHRT protein lacking at least the C-terminal 118 amino acids of SEQ ID NO: 2, or a mutation resulting in a deletion of at least the coding region of the HvHRT gene , wherein the coding region of the HvHRT gene encodes the polypeptide SEQ ID NO: 2. 2. The barley plant according to claim 1, where the barley plant contains a mutant HvHRT gene encoding a truncated HvHRT protein containing an N-terminal HvHRT fragment containing no more than 431 N-terminal amino acids SEQ ID NO: 2. 3. The barley plant according to claim 1, wherein said barley plant contains a mutant HvHRT gene carrying a premature stop codon among any of codons 1-431. 4. The barley plant according to any of the previous claims, wherein the barley plant contains a mutant HvHRT gene carrying a premature stop codon at 431 SEQ ID NO: 1. 5. The barley plant according to any one of claims 1 to 3, wherein the barley plant contains a mutant HvHRT gene carrying a premature stop codon at 371 SEQ ID NO: 1. 6. The barley plant according to any one of claims 1-3, where the barley plant contains a mutant HvHRT gene, providing for the G^A mutation at nucleotide 1293 of the HvHRT coding sequence SEQ ID NO: 1. 7. The barley plant according to any one of claims 1-3, where the barley plant contains a mutant HvHRT gene, providing for the G^A mutation at nucleotide 1113 of the HvHRT coding sequence SEQ ID NO: 1. 8. The barley plant according to any of the preceding claims, wherein the barley plant has an α-amylase activity of at least 100 U/g, such as at least 110 U/g 48 hours after initiation of germination, provided that said barley plant is either naked or at least part of the membrane has been removed before germination is initiated. 9. The barley plant according to any of the preceding claims, wherein the barley plant has a limiting dextrinase activity of at least 20 mU/g 48 hours after initiation of germination, provided that said barley plant is either naked or at least part of the film was removed before said germination was initiated. 10. The barley plant according to any of the preceding claims, wherein the barley plant contains a mutation in one or more additional genes, such as one or more of the following mutations: a) a mutation in the gene encoding LOX-1, resulting in complete loss of functional LOX-1; b) a mutation in the gene encoding LOX-2, resulting in complete loss of functional LOX-2; c) a mutation in the gene encoding MMT, resulting in complete loss of functional MMT; d) a mutation in a gene encoding CslF6, wherein said mutant gene encodes a mutant CslF6 protein with reduced CslF6 activity. 11. A plant-based beverage product obtained from the barley plant according to any of the preceding claims, wherein said plant-based product is selected from - 101 -