EA045156B1 - CEREAL PLANTS WITH IMPROVED CELL WALL PROPERTIES - Google Patents

Description

Technical field of the invention

The present invention relates to barley plants having improved cell wall properties. In particular, the invention relates to barley plants having cell wall properties suitable for the production of barley-based beverages, such as beer. In addition, the invention further relates to methods for producing barley-based beverages, as well as products obtained from barley plants according to the invention.

State of the art

In commercial malting processes, barley grains are germinated, or malted, under controlled conditions that allow partial mobilization of starch and starch endosperm protein reserves over a period of 4 to 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 its moisture content to about 40 to 45% w/w so that the subsequent stage of endosperm mobilization occurs more quickly. During soaking, the water is drained once to re-aerate the grain. This stage is known as an air rest and is considered necessary primarily because the grain submerged in water becomes starved of oxygen after about 16 hours. After an air rest of about 8 hours, the grain is immersed again in water to complete the treatment by soaking for another 8 hours. - an hour period or a series of repeated soakings. The two-step soaking process to increase the moisture content of dry grain to 40% or higher takes approximately 32 hours in total.

The soaked grain is scattered for germination, during which enzymes secreted from the aleurone and scutellar epithelial cells, together with some enzymes already present in the starchy endosperm cells, decompose the cell walls, starch and protein. Malthouse typically aims to induce high levels of enzymes that degrade cell wall polysaccharides in barley grain, particularly (1,3;1,4)-β-glucans and arabinoxylans. Incompletely degraded (1,3;1,4)-glucans can be particularly troublesome for brewers because they can be extracted from the malt in soluble forms, which form highly viscous aqueous solutions that slow down brewery filtration processes and contribute to unwanted cloudiness in finished beer. Additionally, brewing at high levels of (1,3;1,4)-β-glucans has been shown to negatively impact malt extract levels. Thus, low levels of soluble (1,3;1,4)-β-glucans represent an important parameter of malt quality, which is why high levels of (1,3;1,4)-β-glucanase enzymes remain important indicators of malt quality . In addition, the maltster strives to quickly stimulate the synthesis in grains of as many enzymes as possible that destroy starch. Starch-degrading enzymes, which include α- and β-amylases, enzymes that break down starch molecules at branch sites (eg, limiting dextrinase), and α-glucosidases, partially depolymerize grain starch reserves into monosaccharides, oligosaccharides, and glucose. The products of starch depolymerization are subsequently used by yeast cells as a carbon source and fermented into beer alcohol.

As noted above, the germination process usually takes about 5 days. After controlled germination stages, the wet malt is dried, reducing the moisture content from about 40% to 4 to 5%. This drying process, called oven drying, is very energy intensive and represents a large portion of production costs. The entire process, including oven drying, usually takes 6 to 7 days.

At the brewery, kiln-dried malt is crushed to break down the grain, and the resulting contents are extracted with hot water in a process known as mashing. The extracted material includes partially digested starch, protein and cell wall molecules as described above, and these are further digested by endogenous grain enzymes that have been extracted from the malt. At this stage, some brewers add additional and usually cheaper carbon sources (additives) to support subsequent yeast fermentation and reduce malt costs. These additives may be barley, rice, wheat or other unsprouted grain flours, but their addition may require the concomitant addition of hydrolytic enzymes since there are not enough endogenous enzymes in the malt to break down the additive components. Added enzymes are usually obtained from crude and relatively cheap extracts of fungal and/or bacterial cultures. The addition of exogenous enzymes is not permitted in some countries, especially when the beer must be produced under highly regulated conditions.

Further degradation of starch and other endosperm components extracted in hot water occurs in a process known as saccharification. After mashing, the extracts are filtered, often in a lauter tun, and cooled. The extract may be boiled in the presence of hops or hop extracts, with yeast cultures added after it has cooled to ferment the released sugars into alcohol. The beer produced in this way is usually aged and filtered before bottling. Beer can also be carbonated with carbon dioxide before bottling.

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Barley is the most popular grain crop used for beer production. It is the large amount of starch contained in its kernels that has made barley a very attractive raw material for the brewing industry. To ensure the full potential of barley grains is utilized in brewing, it is essential to ensure optimal degradation of the cell wall structures enclosing the starch granules. In a simplified representation of its architecture, the barley kernel consists primarily of starchy endosperm surrounded by an aleurone layer. The cell walls of the aleurone and endosperm of the barley kernel are mainly composed of non-starch polysaccharides (NSP). The starchy endosperm consists of 75% (1-3,1-4)-β-glucans (BGL) and 20% arabinoxylan (ACh), while the aleurone consists of 71% ACh and 26 % from BGL. Barley BGLs are unbranched, long linear chains of glucose residues linked by both β-(1-3) and β-(1-4) bonds. Barley ACh consists of a molecular backbone of D-xylanopyranosyl linked by β-(1-4) bonds, randomly connected to L-arabinofuranose by α-(1-2) and α-(1-3) bonds.

The essence of the invention

As stated above, one of the stages of beer production that requires a lot of time and energy is malting. The rate-limiting step in the malting procedure is the reduction of (1,3;1,4)-β-glucans in the germinating barley kernels to acceptably low levels. Accordingly, there is a need to provide materials and methods that can reduce the time required for malting. In particular, there is a need for barley plants with low levels of (1,3;1,4)-β-glucans. However, barley plants that completely lack (1,3;1,4)-β-glucans are characterized by reduced height, vigor, and yield (to approximately 70% of control) (Taketa et al., 2012). In fact, Taketa et al. concludes that as agronomic performance is reduced, the utility of bgl mutants for malting may not be very great.... Hu et al. (2014) describe the barley mutant m351 containing very low levels of mixed linkage (1-3,1-4) β-glucans (<1.6%). However, the m351 mutant was characterized by reduced grain hardness, as evidenced by a more than fourfold increase in grain breakage compared to the parent plant, and sensitivity to salts, which led to poorer germination under 400 mM salt conditions.

Accordingly, there is a need for barley plants with low levels of (1,3;1,4)-β-glucans, which at the same time have good agronomic characteristics and grain hardness.

Barley (1,3;1,4)-β-glucans contain cellotriosyl (DP3) and cellotetraosyl (DP4) residues in ratios that typically range from 2.5 to 4. The DP3/DP4 ratio influences the properties of (1 ,3;1,4)-β-glucans.

In one embodiment, the invention provides barley plants having low levels of (1,3;1,4)-β-glucans with a DP3/DP4 ratio comparable to the DP3/DP4 ratio of wild-type barley. Such barley plants may be of agronomic importance and have barley grains with a reduced tendency to break. One of the technical problems solved by the present invention is the provision of barley plants having low levels of (1,3;1,4)-β-glucans, while the barley plants at the same time have acceptable agronomic traits, acceptable frequency of grain destruction (e.g., less than 2-fold compared to a wild-type plant that does not have the CsIF6 mutation but otherwise has the same genotype) and in addition a DP3/DP4 ratio similar to that of wild-type barley.

In one embodiment, the invention relates to barley plants having low levels of (1,3;1,4)-β-glucans with a high or low DP3/DP4 ratio. Such barley plants may have agronomically acceptable grains, and they may have higher levels of insoluble (1,3;1,4)-β-glucans. Insoluble (1,3;1,4)-β-glucans can potentially be removed during the brewing process, and are thus preferred in some embodiments. Burton & Fincher 2014 suggested that the solubility of (1,3;1,4)-glucan molecules can be predicted from the DP3:DP4 ratio, and that high and low ratios may result in more insoluble aggregates.

Jobling et al. (2015) describe the expression of CSLF in an artificial system in the tobacco leaf, and describe that one amino acid (Ile757) in the fourth transmembrane domain of CSLF controls the DP3:DP4 ratio, but do not describe that modification of this domain and the DP3:DP4 ratio correlate with the amount of ( 1,3;1,4)-β-glucans.

One of the technical problems solved by the present invention is the provision of barley plants having low levels of (1,3;1,4)-β-glucans, while the barley plants at the same time have acceptable agronomic traits and, in addition, a beneficial ratio DP3:DP4.

In one aspect, the invention relates to barley plants bearing mutations of individual amino acids in transmembrane domains. The invention unexpectedly demonstrates that such barley plants contain low levels of (1,3;1,4)-β-glucans (typically in the range of 1.7 to 5%), are viable, agronomically acceptable and have yields comparable to other varieties barley. Such barley plants are particularly useful for cereal-based beverage production processes

- 2 045156 with reduced germination time.

In addition, the present invention relates to methods and apparatus for providing barley plants with a finely controlled content of (1,3;1,4)-β-glucans. Thus, the barley plants of the invention not only have a low content of (1,3;1,4)-β-glucans, but also make it possible to control the DP3:DP4 ratio of these barley plants.

In one aspect, the present invention relates to a barley plant or part thereof, wherein the kernels of said barley plant have a reduced content of (1,3;1,4)-β-glucans, and wherein said barley plant carries a mutation in the CsIF6 gene, wherein said mutated gene CsIF6 encodes a mutant CsIF6 polypeptide, wherein the mutant CsIF6 is the CsIF6 of SEQ ID NO: 1 or SEQ ID NO: 3 except that the mutant CsIF6 contains a single amino acid substitution in the membrane-localized domain of CsIF6. wherein said substitution is a substitution of a non-polar amino acid for a charged amino acid or a substitution of a polar amino acid for a non-polar amino acid, wherein the membrane-localized CsIF6 domains consist of amino acids 109 to 128, 137 to 158, 700 to 731, 741 to 758, 835 to 857 and 864 to 882 of SEQ ID NO: 1 or SEQ ID NO: 3.

It is also an aspect of the invention to provide plant products prepared from said barley plant, as well as methods for preparing plant products from said barley plant. The barley plants of the present invention may preferably be used to prepare plant products, e.g., wort, with reduced viscosity compared to wort prepared from the kernels of a barley plant carrying the wild type CsIF6 gene, but otherwise having the same barley plant genotype as the plants barley according to the invention.

Description of drawings

In fig. Figure 1 shows the content of (1,3;1,4)-β-glucans (abbreviated β-glucan or BGL) in mutants identified either by Digital Mutation Identification (DMI) (Mut1, 2, 3 and 4 with the corresponding standard 1), or sequencing (Mut5 and the corresponding standard). Columns show ±standard deviation (SD).

In fig. Figure 2 shows the DP3:DP4 ratio in mutants identified either by DMI (Mut1, 2, 3, 4 with the corresponding reference 1) or by sequencing (Mut5 and the corresponding reference).

In fig. Figure 3 shows a model of the CsIF6 protein embedded in the plasma membrane. These mutations are DMI mutants.

In fig. Figure 4 shows an example of equipment used to prepare green malt. The equipment contains a tank (2) in which the grains can be immersed in an aqueous solution and continuously aerated. The equipment contains an inlet for grains of grain crops (1), a reservoir, for example, a soaking reservoir (2); gas inlets, for example through porous sinter stones (3); a pump, for example an air pump (4); outlet for grain grains (5); grain pump (6); equipment for finely crushing grains of grain crops, for example, a mill (7); inlet (8); a container, for example a mash container (9), and; outlet (10).

In fig. 5 shows the activities of α-amylase (a), β-amylase (b) and free limiting dextrinase (c) measured in grains of mutant 2 (mutant) and in grains of the Paustian variety (reference) during malting.

In fig. Figure 6 shows the content of (1,3;1,4)-β-glucans in the grains of mutant 2 (mutant) and the Paustian variety (standard) in ripened barley grains (day 0) and in green malt on days 1, 2, 3, 4 , 5, 6 days, as well as in roasted malt (7 day).

In fig. Figure 7 shows the viscosity (mPa-s) of filtered wort CW-3a, Paustian, CW-4 and Quench.

Detailed description

Definitions.

As used herein, the indefinite article a can mean one or more, depending on the context in which it is used.

As used herein, the term additive refers to sources of carbon-rich raw materials added during the preparation of beer. The additive may be unsprouted cereal grains, which can be ground together with sprouted grains prepared according to the invention. The additive may also be syrup, sugar or the like.

As used herein, the term amino acid refers to a proteinogenic amino acid. Preferably, the proteinogenic amino acids are one of the 20 amino acids encoded by the standard genetic code. One- and three-letter codes according to IUPAC nomenclature are used to name amino acids.

The term amino acid corresponding to X is used herein to describe the amino acids of a given polypeptide (eg, a CsIF6 mutant polypeptide) relative to the amino acids of a reference polypeptide (eg, CsIF6 of SEQ ID NO: 1). After aligning said polypeptide with a reference polypeptide, an amino acid matches X if it is in the same position as X in said alignment.

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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. This leads to the appearance of branch points in α-1-4-linked glucose chains, resulting in the formation of clusters of parallel chains at regular intervals along the axis of the molecule.

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

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

As used herein, the term barley flour refers to ground barley kernels.

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

As used herein, the term charged amino acid refers to amino acids with electrically charged side chains. Preferably, the charged amino acid is selected from the group consisting of Arg, His, Lys, Asp and Glu. The negatively charged amino acids are preferably selected from the group consisting of Asp and Glu.

As used herein, the term sprout refers to the embryonic growing embryo that is visible during the germination phase of a cereal grain. Term α-amylase

As used herein, the term DP refers to the degree of polymerization and indicates the number of α-1,4-linked glucose units in the side chains of amylopectin. Thus, as an example, DP3 refers to the side chains of amylopectin, consisting of a sequence of three α1,4-linked glucose units. Similarly, DP-4 refers to the side chains of amylopectin, consisting of a sequence of four α-1,4-linked glucose units. The term DP3:DP4 (1,3;1,4)-β-glucan ratio, as used herein, refers to the ratio of amylopectin side chains, consisting of a sequence of three α-1,4-linked glucose units, and amylopectin side chains , consisting of a sequence of four α-1,4-linked glucose units, in the indicated (1,3;1,4)-β-glucans. The DP3:DP4 ratio can be determined by digestion of (1,3;1,4)-β-glucans with lichenase followed by quantification of the released DP3 and DP4 oligomers, for example, using HPAEC-PAD. In particular, the DP3:DP4 ratio can be determined as described in Example 3.

By encoding or encoded in the context of the specified nucleic acid is meant the inclusion of information for translation into the specified protein. A nucleic acid or polynucleotide encoding a protein may contain untranslated sequences, such as introns, in the translated regions of the nucleic acid, or may not have such intervening untranslated sequences, such as in cDNA. The information with which a protein is encoded is determined by the use of codons.

As used herein, the term expression in the context of nucleic acids should be understood as the transcription and accumulation of mRNA. 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 production of a polypeptide chain; it includes the regions preceding the coding region and following the coding region (promoter and terminator). In addition, plant genes typically consist of exons interrupted by nitrons.

As used herein, the term sprouted grain refers to a grain that has a visible sprout and a visible stem.

As used herein, the term germination initiation refers to the point in time when barley grains with less than 15% water content are contacted with sufficient water to initiate germination.

As used herein, the term β-glucanase refers to enzymes capable of depolymerizing cereal β-glucan. Accordingly, unless otherwise specified, the term β-glucanase refers to an endo- or exoenzyme or mixture thereof characterized by (1,3;1,4)-β- and/or (1,4)-β-glucanase activity.

The content of (1,3;1,4)-β-glucans as used herein can be determined by any applicable method. Preferably, the content of (1,3;1,4)-β-glucans is determined as the sum of the content of Glc-e-(1^4)-Glc-e-(1^3)-Glc (DP3) oligomers Glc-e-(1^4)-Glc-e-(1^4)-Glc-e-(1^3)-Glc (DP4). The content of oligomers DP3 and DP4 can, for example, be determined by lichenase digestion of (1,3;1,4)-β-glucans followed by quantitative determination, for example, high performance anion exchange chromatography with pulsed amperometric detection ( HPAEC-PAD)). Preferably, the content of (1,3;1,4)-β-glucans is determined as described below in Example 3.

The term (1,3;1,4)-β-glucan synthase, as used herein, should be considered to mean any protein that catalyzes the synthesis of (1,3;1,4)-β-glucans and, optionally, catalyzes the polymerization of glucopyranosyl monomers. For example, (1,3;1,4)-β-glucan synthase may be a polypeptide encoded by the CsIF gene or a functional homologue thereof.

The term kernel is defined to include the grain karyopsis, also referred to as the inner seed. In addition, the core may contain a lemma and a paleu. In most barley varieties, the lemma and palea adhere to the karyopsis and are part of the kernel after threshing. However, naked varieties of barley are also found. In them, karyopsis is free from lemma and palea and is washed out freely, as in wheat. The terms kernel and grain are used interchangeably here.

As used herein, the term malting refers to the controlled germination of cereal grains (particularly barley grains) occurring under controlled environmental conditions. In some embodiments, malting may further include the step of drying said germinated cereal grains, such as oven drying.

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

As used herein, the term kiln-dried malt refers to sprouted grain grains that have been kiln-dried. In general, these cereal grains germinated under controlled environmental conditions. In some embodiments, the kiln-dried malt is ground kiln-dried malt.

Mashing involves incubating ground malt (such as green malt or kiln-dried malt) and/or unsprouted cereal grains in water. Mashing is preferably carried out at a certain temperature(s) and in a certain volume of water.

Mutations include deletions, insertions, substitutions, transversions, and point mutations in the coding and noncoding regions of the gene. Deletions can be of an entire gene or just part of a gene. Point mutations may involve single base pair changes and may result in premature stop codons, frameshift mutations, splice site mutations, or amino acid substitutions. The gene containing the mutation may be referred to as a mutant gene. If the specified mutant gene encodes a polypeptide with a sequence different from the wild type, the specified polypeptide may be referred to as a mutant polypeptide.

The term milled refers to material (such as barley kernels or malt) that has been finely ground, such as by cutting, grinding, crushing or crushing. Barley kernels can be ground wet, for example using a chopper or wet grinder. Ground barley kernels or ground malt are ground sufficiently finely to make the material suitable for aqueous extracts. Ground barley kernels or malt cannot be regenerated into a whole plant by essentially biological means.

As used herein, the term nonpolar amino acid refers to amino acids with hydrophobic side chains. Preferably, the non-polar amino acid is selected from the group consisting of Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp and Gly, more preferably from the group consisting of Ala, Val, Ile, Leu, Met, Phe, Trp and Gly .

The term plant product refers to the product obtained by processing a plant or plant material. Such a plant product may be, for example, green malt, kiln-dried malt, wort, fermented or non-fermented beverage, food product or feed product.

As used herein, the term polar amino acid refers to amino acids with polar, uncharged side chains. Preferably, the polar amino acid is selected from the group consisting of Ser, Thr, Asn and Gln.

As used herein, the term progeny means a plant that is directly or indirectly the progeny of a given plant. Thus, offspring is not limited to direct descendants, but also includes descendants through numerous generations. Typically, the offspring of a barley plant that carries a specific mutation will also carry that specific mutation. Thus, the progeny of a barley plant that carries a specific mutation in the CsIF6 gene also carries this specific mutation.

As used herein, the term sequence identity refers to %

- 5,045,156 identical amino acids or nucleotides between the candidate sequence and the reference sequence after alignment. Thus, a candidate sequence having 80% amino acid identity with a reference sequence requires that, after alignment, 80% of the amino acids in the candidate sequence are 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, PMD: 23671338 and the default parameters it offers.Clustal Omega software is available on the EMBL-EBI website at https://www.ebi.ac.uk/Tools/msa/clustalo/. When using this program With default settings, the mature (bioactive) part of the query and the reference polypeptide are aligned.The number of completely conserved residues is counted and divided by the length of the reference polypeptide.The MUSCLE or MAFFT algorithms can be used to align nucleotide sequences. Sequence identities can be calculated in a similar manner as indicated for amino acid sequences. Thus, the sequence identity provided herein is calculated over the entire length of the reference sequence.

As used herein, the term starch refers to the composition of one or both of the discrete macromolecules amylose and amylopectin.

As used herein, the term soaking refers to the process of increasing the water content of the cereal kernel.

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

As used herein, the term wild-type CsIF6 refers to a gene encoding a polypeptide of the sequence SEQ ID NO: 1 or SEQ ID NO: 3.

Enzymatic activities of cereal grains in the context of the present description refer to the activities measured in flour prepared from the specified type of grain. For example, 10 U/g β-amylase activity per gram of cereal grain refers to the specified β-amylase activity (10 U) measured in an aqueous extract obtained from 1 g of flour (dry matter) from said grain. α-Amylase activity was determined using K-CERA 01/12 (protocol and kit available from Megazyme, Ireland). β-Amylase activity was determined using K-BETASE (protocol and kit available from Megazyme, Ireland). Limiting dextrinase activity was determined using T-LDZ1OOO (protocol and kit available from Megazyme, Ireland).

The volume of gas indicated in this document refers to the volume of the specified gas at 1 atm and 20°C.

The volume of O ₂ as defined herein refers to the volume of O ₂ at 1 atm and 20°C. In embodiments of the invention where O2 is contained in a mixture of gases, then the total volume of the gas mixture can be determined, and the volume of O2 can be calculated as the percentage of the total volume that is O2. For example, atmospheric air contains 21% O ₂ . Thus, the volume of O ₂ in ambient air used in this document is 21% of the total volume of ambient 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 typically produced by mashing, optionally followed by sparging, the process of extracting residual sugars and other compounds from the spent grain after mashing with hot water. Sparging is typically done in a settling drum, mash screen, or other device to ensure that the recovered water is separated from the spent grain. The wort prepared after mashing is usually called the first wort, while the wort prepared after sparging is usually called the second wort. If not specified, the term wort may be first wort, second wort, or a combination thereof. In conventional beer production, the wort is boiled together with hops. Wort without hops may also be referred to as sweet wort, while wort brewed with hops may be referred to as boiled wort or simply wort.

CsIF6.

The present invention provides a barley plant or a portion thereof, wherein the core of said barley plant has a reduced content of (1,3;1,4)-β-glucans and wherein said barley plant carries a mutation in a CsIF6 gene, wherein said mutated CsIF6 gene encodes a mutant CsIF6 polypeptide .

The complete coding sequence of the wild-type cellulose synthase-like gene CsIF6 (CsIF6) from the barley cultivar Sloop Hordeum vulgare is provided here as SEQ ID NO: 2.

The sequence sequence of the wild-type cellulose synthase-like polypeptide CsIF6 from the barley cultivar Sloop Hordeum vulgare is presented here as SEQ ID NO: 1. In addition to the sequence presented here as SEQ ID NO: 1, the wild-type CsIF6 polypeptide can also carry the A590T polymorphism (Taketa et al. (2012)). Accordingly, the wild-type CsIF6 polypeptide is also

- 6045156 may have the sequence shown herein as SEQ ID NO: 3.

Despite considerable research efforts, the specific functions of individual CsI genes are largely unknown. The CsI genes were classified into eight groups, designated CsIA to CsIH. In accordance with the present invention, it has been discovered that (1,3;1,4)-β-glucan synthases are encoded by members of the CsIF gene family.

A barley plant carrying a mutation in the CsIF6 gene.

The present invention provides a barley plant or part thereof, wherein the core of said barley plant has a reduced content of (1,3;1,4)-β-glucans, wherein said barley plant carries a mutation in a CsIF6 gene, and wherein said mutated CsIF6 gene encodes a mutant CsIF6 polypeptide . The mutation in the CsIF6 gene may be any of the mutations described herein, however, in preferred embodiments of the present invention, the mutation is a point mutation in the coding region of the CsIF6 gene.

The mutant CsIF6 polypeptide encoded by said mutant CsIF6 gene may be any mutant CsIF6 polypeptide, however, preferably said mutant CsIF6 polypeptide contains a single amino acid substitution.

In particular, a mutant CsIF6 polypeptide may involve a single amino acid substitution in the membrane-localized domain of CsIF6. The mutant CsIF6 polypeptide may preferably include one or more of the following substitutions in the membrane-localized domain:

substitution of a non-polar amino acid with a charged amino acid; and replacing a polar amino acid with a non-polar amino acid.

As used herein, the terms substitution of amino acid XX for amino acid YY or substitution of amino acid XX for amino acid YY refers to amino acid XX in the reference sequence (typically the wild-type CsIF6 sequence) being replaced by amino acid YY.

In one embodiment of the invention, the mutant CsIF6 polypeptide may include a single amino acid substitution in one of the membrane-localized domains of CsIF6. The membrane-localized domains of CsIF6 are shown herein in Table. 3.

In one embodiment of the invention, the mutant CsIF6 polypeptide may include the replacement of a non-polar amino acid with a charged amino acid in one or more of the following membrane-localized domains of CsIF6: amino acids 109 to 128, 137 to 158, 700 to 731, 741 to 758, 835 to 857 and 864 to 882 in SEQ ID NO: 1 or SEQ ID NO: 3.

In one embodiment of the invention, the barley plant carries a mutation in the CsIF6 gene, resulting in a mutant CsIF6 gene encoding a mutant CsIF6 polypeptide comprising a substitution, wherein the substitution may be a substitution of a non-polar amino acid with a charged amino acid, in one or more of the following membrane-localized CsIF6 domains: amino acids 109 to 128, 137 to 158, 700 to 731, 741 to 758, 835 to 857 and 864 to 882 sequences SEQ ID NO: 1 or SEQ ID NO: 3.

In one embodiment, the mutant CsIF6 polypeptide may be CsIF6 of the sequence SEQ ID NO: 1 or SEQ ID NO: 3 except that the mutant CsIF6 contains a single amino acid substitution in the transmembrane domain consisting of amino acids 835 to 857 of CsIF6, where this substitution is the replacement of a non-polar amino acid with a charged amino acid. Said non-polar amino acid may, for example, be any of amino acids 835 to 857, which are underlined in the table. 3. For example, the substitution may be a substitution of a non-polar amino acid with a negatively charged amino acid.

For example, a mutant CsIF6 polypeptide may be CsIF6 of SEQ ID NO: 1 or SEQ ID NO: 3 except that the mutant CsIF6 contains a substitution of amino acid 847 for a charged amino acid. For example, the substitution may be a substitution of glycine (G) at position 847 with a negatively charged amino acid, such as Glu or Asp.

Preferably, the mutant CsIF6 polypeptide may be CsIF6 of SEQ ID NO: 1 or SEQ ID NO: 3 except that the mutant CsIF6 contains a substitution at amino acid 847, wherein the substitution is a substitution of glycine (G) at position 847 with glutamic acid. (E).

In one embodiment, the mutant CsIF6 polypeptide may be CsIF6 of the sequence SEQ ID NO: 1 or SEQ ID NO: 3 except that the mutant CsIF6 contains a single amino acid substitution in the transmembrane domain consisting of amino acids 741 to 758 of CsIF6, wherein a substitution is the replacement of a nonpolar amino acid with a charged amino acid. Said non-polar amino acid may, for example, be any of amino acids 741 to 758, which are underlined in the table. 3. For example, the substitution may be a substitution of a non-polar amino acid with a negatively charged amino acid.

For example, a mutant CsIF6 polypeptide may be CsIF6 of SEQ ID NO: 1 or SEQ ID NO: 3 except that the mutant CsIF6 contains a substitution of amino acid 748 for a charged amino acid. For example, the substitution may be a substitution of glycine (G) at position 748 with a negatively charged amino acid such as Glu or Asp. Preferably, the mutant CsIF6 polypeptide may be CsIF6 of the sequence SEQ ID NO: 1 or SEQ ID NO: 3 except that the mutant CsIF6 contains a substitution at amino acid 748, wherein said substitution is a substitution of glycine (G) at position 748 with aspartic acid ( D). Thus, a barley plant may carry a mutated HvCsIF6 gene encoding a mutant HvCsIF6 protein containing a Gly^Asp mutation at amino acid 748 in SEQ ID NO: 1.

In one embodiment of the invention, a barley plant may carry a Gly^Asp mutation at nucleotide 2243 in the coding sequence of the HvCsIF6 gene (SEQ ID NO: 2).

In one embodiment, the mutant CsIF6 polypeptide may include a single amino acid substitution in any membrane-localized domain of the CsIF6 polypeptide. For example, the substitution may be a substitution of a polar amino acid for a nonpolar amino acid in one or more of the following membrane-localized domains of CsIF6: amino acids 109 to 128, 137 to 158, 700 to 731, 741 to 758, 835 to 857, and 864 to 882 in SEQ ID NO: 1 or SEQ ID NO: 3.

The mutant CsIF6 polypeptide may be CsIF6 of the sequence SEQ ID NO: 1 or SEQ ID NO: 3 except that the mutant CsIF6 contains a single amino acid substitution in the transmembrane domain consisting of amino acids 700 to 731 of CsIF6, wherein said substitution is replacement of a polar amino acid with a non-polar amino acid. Said polar amino acid may, for example, be any of amino acids 700 to 731, as highlighted in Table. 3.

For example, a mutant CsIF6 polypeptide may be CsIF6 of SEQ ID NO: 1 or SEQ ID NO: 3 except that the mutant CsIF6 contains a substitution of amino acid 709 for a nonpolar amino acid.

Preferably, the mutant CsIF6 polypeptide may be CsIF6 of SEQ ID NO: 1 or SEQ ID NO: 3 except that the mutant CsIF6 contains a substitution at amino acid 709, wherein said substitution is a substitution of threonine (T) at position 709 for isoleucine (I ).

For the purposes of this patent application, seeds of a barley plant (Hordeum vulgare), designated mutant 2, have been deposited with NCIMB Ltd. Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA Scotland in accordance with the provisions of the Treaty of Budapest. Barley plant mutant 2 was deposited on November 12, 2018 and received NCIMB accession number 43273.

In one embodiment, the barley plant of the present invention is a barley plant (Hordeum vulgare) deposited on October 12, 2018 in NCIMB under NCIMB number 43273, and is referred to as mutant 2; or his offspring. Thus, the barley plant of the present invention may be a barley plant mutant 2 deposited with NCIMB on October 12, 2018 and having NCIMB accession number 43273, or any barley plant progeny thereof, where the barley plant carries the Gly^Asp nucleotide 2243 mutation the coding sequence of the HvCsIF6 gene (SEQ ID NO: 2) and/or wherein the HvCsIF6 gene from said barley plant encodes a mutant HvCsIF6 protein containing a Gly^Asp mutation at amino acid 748 in SEQ ID NO: 1.

Interestingly, a barley plant carrying a mutation in the CsIF6 gene resulting in a mutant CsIF6 gene encoding a mutant CsIF6 polypeptide carrying a mutation beyond the membrane-localized amino acids (G732D) did not show any significant reduction in (1.3; 1,4)-β-glucans. In barley plants containing a mutation in the CsIF6 gene resulting in a premature stop codon (T676Stop), levels of (1,3;1,4)-β-glucans were extremely low and such barley plants did not have good agronomic properties. Thus, such plants were characterized by significantly reduced fertility and plant growth.

Barley plant.

The barley plant of the invention may be any plant of the species Hordeum vulgare, including any breeding line, variety or variety of variety.

Wild barley, Hordeum vulgare ssp. spontaneum, is considered the progenitor of modern cultivated forms of barley. Domesticated but heterogeneous mixtures of barley are called local barley varieties. Currently, most local varieties have been replaced in advanced farming by pure linear varieties. Compared to local varieties, modern barley varieties have many improved properties (Nevo, 1992; Pelger et al., 1992).

For the purposes of the present invention, the term barley plant includes any barley plant, such as landraces of barley or modern varieties of barley. Thus, the invention relates to any barley plant containing a mutation in the CsIF6 gene.

However, preferred barley plants for use in the present invention are modern barley varieties or pure lines. The barley variety used in the present invention may be selected, for example, from the group consisting of

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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, Century, Kym, Asahi 5, KOU A, Swan Hals, Kanto Nakate Gold, Hakata No. 2, Kirin - choku No. 1, Kanto late Variety Gold, Fuji Nijo, New Golden, SatukioNijo, SeijoNo. 17, Akagi Nijo, Azuma Golden, Amagi Nijpo, Nishino Gold, Misato golden, Haruna Nijo, Scarlett, Rosalina and Jersey, preferably from the group consisting of

HarunaNijo, Sebastian, Quench, Celeste, Lux, Prestige, Saloon, Neruda and Power, preferably from a 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, Century, Kym, Asahi 5, KOU A, Swan Hals, Kanto Nakate Gold, Hakata No. 2, Kirin choku No. 1, Kanto late Variety 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 can be in any suitable form. For example, the barley plant of the 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 bud, a kernel, a sprouted kernel, a malt kernel (eg, in the form of green malt or kiln-dried malt), milled malt kernel, crushed kernel, or the like.

The barley plant parts may be any suitable part of the plant, such as kernels, buds, leaves, stems, roots, flowers, or fractions thereof. The fraction may be, for example, part of a kernel, embryo, leaf, stem, root or flower. The barley plant parts may also be a homogenate fraction or a crushed barley plant or kernel fraction.

In one embodiment of the 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. In other embodiments, however, the barley plant parts may be viable cells that are unable to mature into a whole barley plant, i.e. cells that are not reproductive material.

Characteristics of barley plants carrying a mutation in CsIF6.

The invention provides barley plants carrying a mutation in the CsIF6 gene. One of the main advantages of such barley plants is that the kernels of said barley plant have a reduced content of (1,3;1,4)-β-glucans.

One advantage provided by barley plants carrying a mutation in the CsIF6 gene and having a reduced content of (1,3;1,4)-β-glucans is that the lower content of (1,3;1,4)-β-glucans in grain can be beneficial in the brewing process as it can help ensure the optimal amount of Sugars is available for yeast fermentation at the end of the mash and therefore reduce the overall cost of raw materials. Another advantage provided by barley plants carrying a mutation in the CsIF6 gene and having a reduced content of (1,3;1,4)-β-glucans is that a barley plant with a low content of (1,3;1,4)-β -glucans can improve industrial procedures such as filterability. In addition, such barley plants are especially useful for energy-saving malting processes. While reducing (1,3;1,4)-β-glucans can be beneficial, too low levels of (1,3;1,4)-β-glucans can also be problematic and may lead to poor agronomic performance. properties.

Thus, in one embodiment, a barley plant carrying a mutation in the CsIF6 gene may be a barley plant with kernels having a (1,3;1,4)-β-glucan content ranging from 1 to 3% total dry matter mass of kernels.

Barley plants of the invention carrying a mutation in the CsIF6 gene and having kernels with a reduced content of (1,3;1,4)-β-glucans are additionally characterized by good agronomic qualities, such as agronomic qualities comparable to the wild type.

The barley plant may be a barley plant with kernels having a (1,3;1,4)-β-glucan content in the range of 1 to 5% of the total dry matter weight of the kernels, for example, from 1 to 3% of the total dry matter weight of the kernels , such as from 1.3 to 4% of the total dry matter weight of the kernels, for example from 1.3 to 3% of the total dry matter weight of the kernels, preferably from 1.3 to 2% of the total dry matter weight of the kernels.

In one embodiment, the barley plant may be a barley plant carrying a mutation in the CsIF6 gene and encoding a mutant CsIF6 polypeptide, wherein the CsIF6 polypeptide is a CsIF6 polypeptide of the sequence SEQ ID NO: 1 or SEQ ID NO: 3 except that the mutant CsIF6 contains a single amino acid substitution in one or more of the following membrane-localized domains of CsIF6: amino acids 109 to 128, 137 to 158, 700 to 731, 741 to 758, 835 to 857, and 864 to 882 in SEQ ID NO : 1 or SEQ ID NO: 3, wherein the substitution is a substitution of a non-polar amino acid for a charged amino acid or a substitution of a polar amino acid for a non-polar amino acid and wherein the kernels of said barley plant may have a (1,3;1,4)-β-glucan content in the range from 1.0 to 3.0% of the total dry matter weight of the kernels, for example, the content of (1,3;1,4)-β-glucans in the range from 1.0 to 3.0% of the total dry matter weight of the kernels, such as content of (1.3;1.4)-β-glucans in the range from 1.0 to 2.5% of the total mass of dry matter of the kernels, preferably the content of (1.3;1.4)-β-glucans in the range from 1 .0 to 2.0% of the total dry matter weight of the kernels, for example, the content of (1.3;1.4)-glucans in the range of 1.3 to 3.0% of the total dry matter weight of the kernels, such as the content of ( 1,3;1,4)-β-glucans in the range from 1.5 to 3.0% of the total dry matter weight of the kernels, preferably the content of (1,3;1,4)-β-glucans in the range from 1.7 up to 3.0% of the total mass of dry matter of kernels.

In one embodiment, the barley plant may be a barley plant carrying a mutation in the CsIF6 gene and encoding a mutant CsIF6 polypeptide, wherein the CsIF6 polypeptide is a CsIF6 polypeptide of the sequence SEQ ID NO: 1 or SEQ ID NO: 3 except that the mutant CsIF6 contains a substitution at amino acid 847, wherein said substitution is a substitution of glycine (G) for glutamic acid (E), and the kernels of said barley plant may have a (1,3;1,4)-β-glucan content ranging from 1.0 to 3.0% of the total dry matter weight of the kernels, for example, the content of (1,3;1,4)-β-glucans in the range from 1.0 to 3.0% of the total dry matter weight of the kernels, such as the content of (1,3 ;1,4)-β-glucans in the range from 1.0 to 2.5% of the total dry matter weight of the kernels, preferably the content of (1,3;1,4)-β-glucans in the range from 1.0 to 2, 0% of the total dry matter weight of the kernels, for example, the content of (1,3;1,4)-β-glucans in the range of 1.3 to 3.0% of the total dry matter weight of the kernels, such as the content of (1,3;1 ,4)-β-glucans in the range from 1.5 to 3.0% of the total dry matter weight of the kernels, preferably the content of (1,3;1,4)-β-glucans in the range from 1.7 to 3.0% total dry matter mass of kernels.

In one embodiment, the barley plant may be a barley plant carrying a mutation in the CsIF6 gene and encoding a mutant CsIF6 polypeptide, wherein the CsIF6 polypeptide is a CsIF6 polypeptide of the sequence SEQ ID NO: 1 or SEQ ID NO: 3 except that the mutant CsIF6 contains a substitution at amino acid 748, wherein said substitution is a substitution of aspartic acid (D) for glycine (G), and the kernels of said barley plant may have a (1,3;1,4)-β-glucan content ranging from 1.0 to 3.0 of the total dry matter weight of the kernels, for example, the content of (1.3;1.4)-β-glucans in the range from 1.0 to 3.0% of the total dry matter weight of the kernels, such as the content of (1.3; 1,4)-β-glucans in the range from 1.0 to 2.5% of the total dry matter weight of the kernels, preferably the content of (1,3;1,4)-β-glucans in the range from 1.0 to 2.0 % of the total dry matter weight of the kernels, for example, the content of (1,3;1,4)-β-glucans in the range from 1.3 to 3.0% of the total dry matter weight of the kernels, such as the content of (1,3;1, 4)-β-glucans in the range from 1.5 to 3.0% of the total weight of the dry matter of the kernels, preferably the content of (1.3; 1,4)-β-glucans in the range from 1.7 to 3.0% of the total mass of dry matter of kernels.

In one embodiment, the barley plant may be a barley plant carrying a mutation in the CsIF6 gene and encoding a mutant CsIF6 polypeptide, wherein the CsIF6 polypeptide is a CsIF6 polypeptide of the sequence SEQ ID NO: 1 or SEQ ID NO: 3 except that the mutant CsIF6 contains a substitution of amino acid 709, wherein said substitution is a substitution of isoleucine (I) for threonine (T), and the kernels of said barley plant may have a (1,3;1,4)-β-glucan content ranging from 1.0 to 3 .0% of the total dry matter weight of the kernels, for example, the content of (1.3;1.4)-β-glucans in the range from 1.0 to 3.0% of the total dry matter weight of the kernels, such as the content of (1.3; 1,4)-β-glucans in the range from 1.0 to 2.5% of the total dry matter weight of the kernels, preferably the content of (1,3;1,4)-β-glucans in the range from 1.0 to 2.0 % of the total dry matter mass of kernels, for example, the content of (1.3;1.4)-glucans in the range from 1.0 to 1.7% of the total dry matter mass of the kernels, for example, the content of (1. 3;1,4)-β-glucans in the range from 1.2 to 3.0% of the total dry matter weight of the kernels, such as (1,3;1,4)-β-glucans content in the range from 1.5 to 3.0% of the total dry matter weight of the kernels, preferably the content of (1,3;1,4)-β-glucans in the range from 1.3 to 3.0% of the total dry matter weight of the kernels.

In one embodiment of the invention, the barley plant may have the (1,3;1,4)β-glucan content in the kernels reduced to no more than 70%, preferably no more than 60%, for example no more than 55% of the ( 1,3;1,4)-β-glucans in the kernels of barley containing the wild-type CsIF6 gene, but otherwise having the same genotype.

The barley plant may be a barley plant having a (1,3;1,4)-β-glucan content in the kernels reduced to at least 30% and not more than 60%, preferably at least 40% and not more than 60% , for example at least 40% and not more than 55% of the (1,3;1,4)-β-glucan content in the kernels of barley containing the wild-type CsIF6 gene, but otherwise having the same genotype.

In one embodiment, the barley plant may be a barley plant having a (1,3;1,4)-β-glucan content in the kernels reduced to at least 30% and no more than 60%, preferably at least 40% and not more than 60%, for example, at least 40% and not more than 55% of the (1,3;1,4)-β-glucan content in the kernels of the Paustian barley plant.

In one embodiment, the barley plant may be a barley plant carrying a mutation in the CsIF6 gene and encoding a mutant CsIF6 polypeptide, wherein the CsIF6 polypeptide is a CsIF6 polypeptide of the sequence SEQ ID NO: 1 or SEQ ID NO: 3 except that the mutant CsIF6 contains a substitution of amino acid 847, wherein said substitution is a substitution of glycine (G) for glutamic acid (E), and the kernels of said barley plant may have a (1,3;1,4)-β-glucan content reduced to at least 30 % and not more than 60%, for example at least 40% and not more than 60% of the content of (1,3;1,4)-β-glucans in the kernels of barley containing the wild-type CsIF6 gene, but otherwise having that the same genotype.

In one embodiment, the barley plant may be a barley plant carrying a mutation in the CsIF6 gene and encoding a mutant CsIF6 polypeptide, wherein the CsIF6 polypeptide is a CsIF6 polypeptide of the sequence SEQ ID NO: 1 or SEQ ID NO: 3 except that the mutant CsIF6 contains a substitution of amino acid 748, wherein said substitution is a substitution of glycine (G) for aspartic acid (D), and the kernels of said barley plant may have a (1,3;1,4)-β-glucan content reduced to at least 30 % and not more than 60%, for example at least 40% and not more than 60% of the content of (1,3;1,4)-β-glucans in the kernels of barley containing the wild-type CsIF6 gene, but otherwise having the same genotype.

In one embodiment, the barley plant may be a barley plant carrying a mutation in the CsIF6 gene and encoding a mutant CsIF6 polypeptide, wherein the CsIF6 polypeptide is a CsIF6 polypeptide of the sequence SEQ ID NO: 1 or SEQ ID NO: 3 except that the mutant CsIF6 contains a substitution of amino acid 709, wherein said substitution is a substitution of threonine (T) for isoleucine (I), and the kernels of said barley plant may have a (1,3;1,4)-β-glucan content reduced to at least 30% and not more than 60%, for example at least 40% and not more than 60% of the (1,3;1,4)-β-glucan content in the kernels of barley containing the wild-type CsIF6 gene, but otherwise having that the same genotype.

Barley plants of the invention carrying mutations in the CsIF6 gene and encoding a mutated CsIF6 polypeptide, and having kernels with a reduced content of (1,3;1,4)-β-glucans, can further be characterized by kernels with a given DP3:DP4 ratio, for example a DP3 ratio :DP4 is lower than the DP3:DP4 ratio for a wild-type plant, or higher than the DP3:DP4 ratio for a wild-type plant, or similar to the DP3:DP4 ratio for a wild-type plant.

Depending on various factors, different DP3:DP4 may be desirable.

Wild-type barley plants typically have a DP3:DP4 ratio ranging from 2.5 to 4.

In one embodiment, the barley plants of the invention contain (1,3;1,4)-β-glucan in the kernels having a DP3:DP4 ratio of no more than 2.5, for example no more than 2.2, for example ranging from 1.0 to 2.2.

In one embodiment, the barley plant may be a barley plant carrying a mutation in the CsIF6 gene and encoding a mutant CsIF6 polypeptide, wherein the CsIF6 polypeptide is a CsIF6 polypeptide of the sequence SEQ ID NO: 1 or SEQ ID NO: 3 except that the mutant CsIF6 contains a substitution of amino acid 847, wherein said substitution is a substitution of glycine (G) for glutamic acid (E), and the kernels of said barley plant may have a DP3:DP4 ratio of no more than 2.5, such as no more than 2.2, for example , ranging from 1.0 to 2.2.

In one embodiment, the kernels of said barley plant may have the ratio

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DP3:DP4 in the range from 2.5 to 4.

In one embodiment, the barley plant may be a barley plant carrying a mutation in the CsIF6 gene and encoding a mutant CsIF6 polypeptide, wherein the CsIF6 polypeptide is a CsIF6 polypeptide of the sequence SEQ ID NO: 1 or SEQ ID NO: 3 except that the mutant CsIF6 contains a substitution at amino acid 748, wherein said substitution is a substitution of glycine (G) for aspartic acid (D), and the kernels of said barley plant may have a DP3:DP4 ratio ranging from 2.5 to 4.

In one embodiment, the kernels of said barley plant may have a DP3:DP4 ratio of at least 3.5, such as at least 4.0, such as at least 4.5, such as in the range of 4 to 6.

In one embodiment, the barley plant may be a barley plant carrying a mutation in the CsIF6 gene and encoding a mutant CsIF6 polypeptide, wherein the CsIF6 polypeptide is a CsIF6 polypeptide of the sequence SEQ ID NO: 1 or SEQ ID NO: 3 except that the mutant CsIF6 contains a substitution of amino acid 709, wherein said substitution is a substitution of threonine (T) for isoleucine (I), and the kernels of said barley plant may have a DP3:DP4 ratio of at least 3.5, such as at least 4.0, for example at least 4.5, for example in the range from 4 to 6.

(1,3;1,4)-β-glucan is a component of the cell wall, and decreased levels of (1,3;1,4)-β-glucan can affect barley plants. For example, a decrease in the level of (1,3;1,4)-β-glucans and/or a change in the DP3/DP4 ratio of (1,3;1,4)-β-glucans can lead to brittle barley grains with a high percentage of broken grains .

In one embodiment, it is preferred that the barley plants of the invention have kernels of acceptable grain hardness. Thus, in one embodiment, barley plants may have kernels with a broken grain rate of less than 5%, such as less than 3%, when determined as described in Example 6 below.

In particular, it is preferable that the frequency of broken grains after threshing grains of barley plants according to the invention is no more than 3 times higher, more preferably no more than 2 times higher, than the frequency of broken grains after threshing grains of barley plants that do not carry a mutation in the gene CsIF6, but otherwise identical. For example, barley plants carrying mutations in the CsIF6 gene encoding a mutant HvCsIF6 protein containing a mutation (e.g., Gly^Asp) at amino acid 748 in SEQ ID NO: 1 or SEQ ID NO: 3 have a frequency of broken grains after grain threshing, which is no more than 3 times higher, more preferably no more than 2 times higher, than the frequency of broken grains after threshing grains of a barley plant that does not carry the specified mutation, but is otherwise identical. The frequency of broken grains is preferably determined as described in Example 6 below.

Barley plants containing more than one mutation.

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

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

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

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

In addition to one or more of the mutations described above, a barley plant may also contain any of the mutations resulting in increased alpha-amylase activity described in the co-pending application entitled Barley with increased hydrolytic enzyme activity. assigned to the same applicant and with the same

- 12 045156 with the same filing date as this application.

Plant products.

The present invention also relates to plant products obtained from a barley plant with a reduced content of (1,3;1,4)-β-glucans and carrying a mutation in the CsIF6 gene, or its progeny, where the specified mutated CsIF6 gene encodes a mutant CsIF6 polypeptide, for example , any of the barley plants described herein.

The plant product can be any product made from the barley plant, such as food, feed or drink. Thus, the plant product may be any of the beverages described below in the Beverage and Method of Production section below. The plant product may also be an aqueous extract of a barley plant and/or malt prepared from the grains of said barley plant, for example the plant product may be a wort. Said aqueous extract may, for example, be prepared as described below under 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, Kiln-Kiln Malt and Methods for Making The Same, or malt-based product such as malt beverages. Although malt is primarily used in the beverage industry, it can also be used in other industrial processes, for example as a source of enzymes in the baking industry or in the food industry as a flavoring and coloring agent, for example in the form of malt or malt flour or indirectly, like malt syrup, etc. Thus, the herbal product of the invention may be any of the above-mentioned products.

In one embodiment of the invention, the plant product is barley flour, i.e. barley flour prepared from the grains of the barley plant according to the invention.

In another aspect, the plant products of the 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 invention also relates to malt obtained from a barley plant carrying a mutation in the CsIF6 gene, for example, any of the barley plants described herein. The malt may be green malt or kiln-dried malt made from barley grains from a barley plant carrying a mutation in the CsIF6 gene or its progeny. The mutation may be any of the mutations in the CsIF6 gene described above herein.

Green malt can be prepared by malting, i.e. by germinating grain crops under controlled environmental conditions. Typically, said sprouting may include a step of soaking the barley kernels, followed by a sprouting step. Soaking and sprouting can also be done 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 kernels at elevated temperatures. Thus, kiln-dried malt can be prepared by subjecting green malt to a kiln-drying stage.

Thus, in one embodiment, a malting method may include the steps of (a) providing kernels to a barley plant, in particular, a barley plant carrying a mutation in the CsIF6 gene;

(b) soaking said barley kernels;

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

Sprouted barley grains can be produced by a method comprising the steps of (a) providing kernels of a barley plant, in particular a barley plant carrying a mutation in the CsIF6 gene;

(b) soaking said barley kernels;

(c) germinating said barley kernel.

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

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

Soaking can be performed by any conventional method known to one skilled in the art. One non-limiting example includes holding at temperatures ranging from 10 to 25°C with alternating dry and wet conditions. For example, during soaking, barley kernels can be incubated under humid conditions for 30 minutes to 3 hours, and then incubated under dry conditions for 30 minutes.

- 13 045156 up to 3 hours and, if necessary, repeat the specified incubation scheme in the range from 2 to 5 times. The final water content after soaking may be, for example, 40 to 50%, for example 40 to 45%.

The barley plants provided in the present invention are characterized by the presence of a mutation in the CsIF6 gene and thus encoding a mutant CsIF6 polypeptide. One of the main advantages of such barley plants is that the kernels have a reduced content of (1,3;1,4)-β-glucans and an adequate DP3:DP4 ratio. The grains of such barley plants with a reduced content of (1,3;1,4)-β-glucans can successfully germinate during a short germination process. Examples of applicable short germination processes are described in international patent application PCT/EP2O17/O65498, which is incorporated herein by reference. One example of a useful short germination process is a germination process involving a step where barley grains are incubated in an aqueous solution, typically with aeration, and the entire germination process is carried out in no more than 72 hours. In fact, during germination (1.3; 1,4)-β-glucans are usually at least partially hydrolyzed by enzymes such as β-glucanase. Hydrolytic enzymes specific for (1,3;1,4)-β-glucans are usually present in small quantities at the beginning of the germination process. The presence of hydrolytic enzymes specific for (1,3;1,4)-β-glucans increases with the continuation of the germination process. A germination process carried out for no more than 72 hours generally does not provide sufficient time for (1,3;1,4)-glucans to be hydrolyzed to the desired low level if the (1,3;1,4)-g content -glucans are high from the start. However, the barley plants of the invention already have low levels of (1,3;1,4)-β-glucans to begin with and thus hydrolysis of (1,3;1,4)-β-glucans during germination is less critical.

As described above, germination may include the step of incubating grains of a barley plant carrying a mutation in the CsIF6 gene in an aqueous solution under aeration. The barley grains may be incubated in said aqueous solution for a sufficient time to allow germination of most of the said barley grains. The barley grains may also be incubated in said aqueous solution for a period of time sufficient to obtain a water content of at least 35%, preferably at least 37%, for example in the range of 35 to 60%. Typically, the barley grains are incubated in the aqueous solution for at least 20 hours, such as at least 24 hours. Typically, the grains are incubated in said aqueous solution for no more than 72 hours, such as no more than 60 hours, such as no more than 48 hours. Thus, in some embodiments, the barley grains are incubated in said aqueous solution for 20 to 72 hours, e.g., 20 to 60 hours, e.g., 20 to 48 hours, e.g., 20 to 30 hours, e.g. range from 22 to 26 hours.

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

Said barley grains are often incubated in said aqueous solution while O ₂ is passed through the aqueous solution. Said O ₂ may be added to said aqueous solution in the form of pure O ₂ . However, often the specified O ₂ is contained in the gas mixture. In one embodiment, said O2 is contained in ambient air.

Typically 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 O ₂ pass through said aqueous solution per kg barley grains per hour. The weight of these barley grains is the weight of dry matter. For example, in the range from 2 to 100 l, for example in the range from 2 to 75 l, for example in the range from 2 to 50 l, for example in the range from 4 to 100 l, for example in the range from 4 to 75 l, for example in the range from 4 to 50 l, for example in the range from 6 to 100 l, for example in the range from 6 to 75 l, for example in the range from 6 to 50 l O ₂ passes through the specified mixture of aqueous solution/barley grains per kg of barley grains (dry matter weight ) at one o'clock.

As noted above, often atmospheric air is passed through the aqueous solution. Thus, the method may include 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 of atmospheric air through said aqueous solution per kg of barley grains per hour. The weight of these barley grains is the weight of dry matter. For example, in the range from 10 to 500 l, for example in the range from 10 to 375 l, for example in the range from 10 to 250 l, for example in the range from 20 to 500 l, for example in the range from 20 to 375 l, for example in the range from 20 to 250 l, for example in the range from 30 to 500 l, for example in the range from 30 to 375 l, for example in the range from 30 to 250 l, atmospheric air is passed through the specified aqueous solution per kg of barley grains (dry matter weight) per hour .

In some embodiments, the germination step includes

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

b) at least one step of incubating said barley kernels in air.

In some embodiments, after incubating the barley grains in said aqueous solution

- 14 045156 the water content of barley is at least 20%, preferably at least 30%, for example in the range from 30 to 60%, for example in the range from 30 to 50%, for example in the range from to 60%, for example in the range from 30 to 50%.

During said stage of incubation of said barley grains in air, at least 2 liters of O ₂ per kg dry matter weight of barley can be passed through said barley kernels per hour. For example, during incubation in air, the same amount of O ₂ can be passed through the barley kernels as during incubation in the specified aqueous solution, as described above.

Sprouted barley kernels produced by this process 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 the weight of wet and dry barley grains is considered water, and the water content is represented as the weight of water divided by the total weight of the barley grains (wet barley grains). The water content in % is therefore % w/w. (by weight).

Barley grain can be incubated at any applicable temperature, however, it may be preferable that the incubation be carried out at a temperature high enough to allow a rapid increase in water content.

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

Germination of grains can also be carried out by any conventional method known to one skilled in the art. One non-limiting example includes germination at a temperature in the range of 10 to 25°C, optionally with a temperature variation in the range of 1 to 4 days.

As mentioned above, in some embodiments of the invention, sprouted barley grains (ie, green malt) can be oven dried. In some embodiments, it is preferable that the green malt is not kiln dried. In particular, it is preferred that when green malt is produced by germination, including the step of incubating said barley grains in an aqueous solution under aeration, then the green malt is not oven dried.

If green malt is kiln dried, this may be done at conventional temperatures, such as at least 75°C, for example in the range of 80 to 90°C, for example in the range of 80 to 85°C. Thus, malt, for example, can be produced by any of the methods described by Hough et al. (1982). However, any other suitable method for producing malt can also be used with 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 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, milled, kiln-dried malt or milled green malt. Milled malt and its flour contain chemical components of malt and dead cells that do not have the ability to germinate again.

In some embodiments, the barley is hulled barley, and the method includes the step of removing at least a portion of said hull before incubating said kernels in an aqueous solution. Hulled cereal grains can be processed to remove the hull by subjecting the cereal grain to a physical treatment that removes the hull. Said physical treatment may be selected, for example, from the group consisting of polishing, grinding, peeling and smoothing. Preferably, physical processing results in loss of the shell. Shell loss can be defined as total mass loss. Thus, physical processing preferably results in a loss in the range of 1 to 4%, for example, a loss in the range of 1.5 to 3.0% of the total weight of the crop.

Aqueous extract and methods for its preparation.

The invention provides barley-based drinks, as well as methods for preparing barley-based drinks, where the barley plant or its progeny carries a mutation in the CsIF6 gene. The invention also provides aqueous extracts of barley plant kernels carrying a mutation in the CsIF6 gene. Said aqueous extract may, for example, be prepared from green malt or kiln-dried malt.

Often, methods of preparing the beverage include the step of preparing an aqueous extract of barley plant grains of the invention and/or malt obtained from barley plants of the invention.

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

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

- 15 045156

a) obtaining the kernels of a barley plant, wherein said barley plant has a reduced content of (1,3;1,4)-β-glucans and carries a mutation in the CsIF6 gene, as described herein;

b) subjecting the barley kernels to a germination stage, thereby obtaining germinated kernels, said germination stage comprising incubating said kernels in an aqueous solution for no more than 72 hours;

c) finely crushing said sprouted grains, while said sprouted kernels have a water content of at least 20%, provided that said barley kernels do not have a water content below 20 at any time between stages b) and c);

d) preparing an aqueous extract of said crushed germinated kernels, thereby obtaining an aqueous extract of barley.

The germination stage is described in detail in the above section Green malt, kiln-dried malt and the method of their production.

Typically, said mash solution may be water, such as tap water, to which one or more additional components may be added. Additional components may be present in the aqueous solution from the very beginning, or they may be added during the preparation of the aqueous extract. Said additional components 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 subsequently during the process.

Said enzymes may, for example, be 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-glucanase 1,4-β-glucanases), xylanases (eg endo- or exo-1,4-xylanases, arabinofuranosidases or ferulic acid esterases), glucoamylases and proteases.

In one embodiment, no or only a limited amount of β-glucanase is added to said mash solution.

Said additive components, preferably food grade, may also be a salt, for example CaCl ₂ , or an acid, for example H ₃ PO ₄ .

The aqueous extract is typically prepared by incubating barley flour, green malt flour and/or kiln-dried malt flour in a mash solution at one or more predetermined temperatures. Said target temperature may also be referred to herein as the mash temperature. Said mashing temperatures may be, for example, normal temperatures used for mashing. The mash temperature is typically 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 to form an aqueous extract.

The mash temperature(s) is typically a temperature(s) in the range of 30 to 90°C, for example in the range of 40 to 85°C, for example in the range of 50 to 85°C. Mashing temperatures can be selected according to the type of barley used. In particular, a relatively low mash temperature may be used, for example a temperature in the range of 50 to 60°C. Often the mash incubation includes a final step of heating to a higher temperature, for example a temperature in the range of 75 to 80°C.

After incubation in an aqueous solution, such as a mash vessel, the aqueous solution may be transferred to another container, such as a lauter tun, and held for additional time at elevated temperature.

Non-limiting examples of applicable mashing protocols can be found in the malting literature, such as Hough et al. (higher).

Mashing (i.e. incubating barley flour, green malt flour and/or kiln-dried malt flour in a mashing solution) can occur in the presence of additives, which are considered to include any source of carbohydrates other than malt or sprouted barley grains such as, but not limited to, barley, barley syrups or corn or rice - either as whole grains 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 while the wort is heating). The additive handling requirements at the brewery depend on the condition and type of additive used.

After incubation in the mash solution, the aqueous extract can usually be separated, for example by filtration, into the aqueous extract and residual undissolved solids, the latter also referred to as spent grain. Filtration can be carried out, for example, in a filtration tank. Alternatively, the filtration may be gate filtration

- 16 045156 filter. The aqueous extract thus obtained can also be designated as the first wort. Additional liquid, such as water, can be added to the spent grains during a process also referred to as sparging. After bubbling and filtering, you can get a second wort. Further wort can be prepared by repeating the procedure. Thus, the aqueous extract may be a wort, such as a first wort, a second wort, an additional wort, or a combination thereof.

The method for preparing an aqueous extract in one embodiment can be performed using any of the devices described in international patent application PCT/EP2O17/O65498, for example, any of the devices described therein on p. 20-22. A non-limiting example of a useful device is shown herein in FIG. 4.

Incompletely degraded (1,3;1,4)-glucans can be particularly troublesome to brewers because they can be extracted from the malt in soluble forms, which form highly viscous aqueous solutions that slow filtration processes in the brewery. Interestingly, the barley plants of the invention have low levels of (1,3;1,4)-β-glucans, and thus wort prepared from such barley plants typically has low viscosity. In one embodiment, the invention relates to wort obtained from a barley plant carrying a mutation in the CsIF6 gene, wherein the wort has a lower viscosity compared to wort obtained from a barley plant carrying the wild type CsIF6 gene, but otherwise having the same genotype barley plants disclosed herein. Said wort is usually obtained by extracting barley grains and/or malt.

In one embodiment, the invention relates to wort, wherein said wort is obtained from a barley plant carrying any of the mutations in the CsIF6 gene described herein, wherein said wort has a viscosity in the range of 0.5 to 1.0 mPa-s below, for example, in the range of 0.5 to 0.8 mPa-s lower than the viscosity of wort obtained in the same way from a barley plant carrying the wild-type CsIF6 gene, but otherwise having the same genotype. In one embodiment, the invention relates to a wort, wherein said wort is obtained from a barley plant carrying any of the mutations in the CsIF6 gene described herein, and wherein said wort has a viscosity in the range of 1.7 to 2.5 mPa-s, such as in the range from 1.8 to 2.5 mPa-s, for example from 2.0 to 2.5 mPa-s, such as from 2.1 to 2.5 mPa-s, for example from 1.8 up to 2.2 mPa-s, such as about 2 mPa-s, for example, about 2.1 mPa-s, such as about 2.2 mPa-s. In particular, a barley plant may carry a mutated HvCsIF6 gene encoding a mutant HvCsIF6 protein containing a mutation (e.g., Gly^Asp) at amino acid 748 in SEQ ID NO: 1.

In one embodiment, the invention relates to wort, wherein said wort is obtained from malt obtained from a barley plant carrying any of the mutations in the CsIF6 gene described herein, wherein said wort has a viscosity of no more than 2.2 mPas.

The viscosity of wort made from standard malt prepared by soaking for 1 to 2 days and sprouting for 5 to 7 days followed by oven drying is about 2 mPa-s.

Drink and method of its production.

The present invention also relates to barley-based beverages and methods for producing such beverages, wherein the barley plant or its progeny has a reduced level of (1,3;1,4)-β-glucans and carries a mutation in the CsIF6 gene. The invention also relates to barley-based drinks prepared from barley plants carrying a mutation in the CsIF6 gene or their progeny.

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 can be selected, for example, from the group consisting of altbier, Amber ale, Barley wine, Berliner Weisse, Biere de Garde, Bitter, Blonde Ale, Bock, Brown ale,

California Common, Cream Ale, Dortmunder Export, Doppelbock, Dunkel, Dunkelweizen,

Eisbock, Fruit lambic, Golden Ale, Gose, Gueuze, Hefeweizen, Helles, India pale ale, Kolsch, Lambic, Light ale, Maibock, Malt liquor, Mild, Marzenbier, Old ale, Oud bruin, Pale ale, Pilsener, Porter, Red ale, Roggenbier, Saison, Scotch ale, Steam beer, Stout, Schwarzbier, lager, Witbier, Weissbier and Weizenbock.

Said distilled alcohol may be any distilled alcohol. In particular, the distilled alcohol may be barley based, 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 a malt.

The beverage may, for example, be prepared by a method comprising the steps of:

(i) providing barley plant kernels according to the invention and/or green malt and/or dried in

- 17 045156 kilns of malt prepared from the kernels of a barley plant according to the invention;

(ii) preparing an aqueous extract from said kernels and/or said malt, for example as described herein above in the section on preparing an aqueous extract;

(iii) processing 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 worts 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 to 120 minutes.

Step (iii) may contain

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

b) cooling the aqueous extract;

c) fermenting said aqueous extract with yeast to produce a fermented beverage.

Step (iii) may in particular comprise fermenting said aqueous extract, for example by fermenting wort. Thus, the drink can be prepared by fermenting the water extract with yeast.

Once the aqueous extract is prepared, it can be processed into beer by any method, including conventional brewing methods. Unlimited descriptions of examples of suitable brewing methods can be found, for example, in the publications of Hough et al. (1982). Numerous, regularly updated methods for analyzing barley and beer products are available, such as, but not limited to, American Association of Cereal Chemists (1995), American Society of Brewing Chemists (1992) , European Brewery Convention (1998) and 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 beer production method can be used 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 whirlpool bath. One or more additional compounds may be added to the aqueous extract, for example, one or more 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 may be fermented for any suitable period of time, typically ranging from 1 to 20 days, for example 1 to 10 days. Fermentation is carried out at any applicable temperature, for example at a temperature in the range from 10 to 20°C. The methods may also include adding 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. Non-limiting examples of proline-specific endoprotease include Brewer's Clarex, available from DSM. In other embodiments, exogenous enzymes are not added during the 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 if there is no further decrease in %R.

Subsequently, the beer can be further processed, for example, cooled. It can also be filtered and/or lagered, a process that develops a pleasant aroma and reduces the yeast taste. Additives may also be added. In addition, CO2 can be added. Finally, the beer may be pasteurized and/or filtered before packaging (eg, transferred to containers or kegs, bottled, or canned). Beer can also be pasteurized using standard methods.

Additional connections.

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

Functional ingredients can be any ingredient added to achieve a given function. Preferably, the functional ingredient makes the drink healthier. 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 may also be CO ₂ . In particular, CO ₂ can be added to produce a carbonated drink.

The flavor compound used in the present invention may be any suitable flavor compound. The flavor compound may be selected, for example, from the group consisting of flavors, herbal extracts, herbal concentrates, plant parts, and herbal infusions. In particular, the flavor compounds may be hops.

Method for obtaining a barley plant with a mutation in CsIF6.

Barley plants carrying a mutation in CsIF6, for example, any of the specific mutations described herein, can be obtained by any applicable method.

For example, such barley plants can be produced by a process comprising the steps of subjecting a plurality of barley plants or barley kernels to random mutagenesis, for example, by irradiation or chemical treatment, for example by treatment with sodium azide;

identification of barley plants or barley kernels carrying a mutation in CsIF6.

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

In particular, barley plants carrying a particular mutation in CsIF6 can be obtained and identified essentially as described in international patent application PCT/EP2O17/O65516, using primers and probes designed to identify a mutation in the CsIF6 gene. Examples of primers and probes useful for identifying a barley plant carrying a mutation in the CsIF6 gene, resulting in the gene encoding a mutant CsIF6 protein carrying one of the G847E or G748D mutations, are presented in Table. 2. Those skilled in the art, based on general knowledge and/or guidance provided in international patent application PCT/EP2O17/O65516, which is incorporated herein by reference, will be able to develop suitable primers and probes for identifying other mutants.

Barley plants carrying a mutation in the CsIF6 gene can also be produced using various site-directed mutatogenesis methods, which, for example, can be designed based on the CsIF6 gene sequence presented herein. In one embodiment of the invention, a barley plant is produced using any of CRISPR, TALEN, zinc finger, meganuclease and DNA-cutting antibiotic, as described in WO 2017/138986. In one embodiment, a barley plant is produced using CRISPR/cas9 technology, for example, using Cas9 Guide RNA nuclease. This can be done as described in Lawrenson et al., Genome Biology (2015), 16:258, DOI: 10.1186/sl30 59-015-0826-7, except that the single guide RNA sequence is designed based on those provided in this gene sequence document. In one embodiment of the invention, a barley plant is produced using a combination of both TALEN and CRISPR/cas9 methods, for example using the Cas9 RNA guide nuclease. This can be done 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 provided herein.

In one embodiment of the invention, a cereal plant is produced using homology-directed repair, a combination of a DNA cutting nuclease and a donor DNA fragment. This can be done 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 The -cutting nuclease is designed based on the gene sequences provided herein, and the donor DNA fragment is designed based on the coding sequence of the mutated cereal variant provided herein.

In one embodiment of the invention, the object is to provide agronomically useful barley plants carrying a mutation in the CsIF6 gene. In addition to the mutation in the CsIF6 gene, there are other factors that may also be considered when developing a commercial barley variety useful for malting and/or brewing and/or as a beverage base, such as yield and kernel size, and other parameters that refer to malting performance or brewing performance. Since many, if not all, of the relevant traits have been shown to be under genetic control, the present invention also relates to modern, homozygous, high-yielding malting varieties that can be obtained from crosses with the barley plants disclosed herein. A specialist barley breeder will be able to select and grow barley plants that, when crossed with other barley plants, will result in superior varieties. Alternatively, the plant breeder may use the plants of the present invention for further mutagenesis to produce new varieties carrying additional mutations in addition to the CsIF6 gene mutation.

The invention also includes barley plants carrying a mutation in the CsIF6 gene, obtained by plant breeding methods, including methods of selfing, backcrossing, crossing with populations, and the like. Backcrossing methods can be used with the present invention to introduce a CsIF6 gene mutation into another variety.

In one embodiment, the invention relates to the progeny of a barley plant deposited on 12-11-2018 with NCIMB under NCIMB registration number 43273 and referred to as mutant 2. Said progeny can be produced by any applicable method, including, but not limited to, selfing, recurrent interbreeding or interbreeding with other populations. In particular, said progeny may also carry a G^A mutation at nucleotide 2243 in the coding sequence of the HvCsIF6 gene (SEQ ID NO: 2).

A way to speed up the plant breeding process is to initially propagate the generated mutants through the use of tissue culture and regeneration techniques. Thus, another aspect of the present invention is to create cells that, when grown and differentiated, produce barley plants carrying a mutation in the CsIF6 gene. For example, breeding may involve traditional crossbreeding, the preparation of fertile plants derived from anthers, or the use of microspore culture.

In one embodiment, the barley plant of the invention was not obtained solely using an essentially biological method. The progeny of a barley plant obtained by a technical method is considered herein as not exclusively obtained by an essentially biological method, since the parent plant is obtained by a technical method.

In one embodiment, the barley plant carries a mutation in the CsIF6 gene, where the mutation is induced by chemical and/or physical means.

In one embodiment, the barley plant was produced by a process including an induced mutagenesis step, or said plant is a progeny of a plant produced by a process 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 plant produced by a process comprising the following step: mutagenesis of the barley plant or part thereof, for example using a chemical mutagenizing agent such as NaN ₃

Selection of barley plants carrying one of the above-mentioned mutations in the CsIF6 gene.

In one embodiment, said mutation of the CsIF6 gene results in a mutant CsIF6 gene encoding a mutant CsIF6 polypeptide, wherein said mutant CsIF6 polypeptide is CsIF6 of the sequence SEQ ID NO: 1 or SEQ ID NO: 3 except that the mutant CsIF6 contains replacing amino acid 847 with a charged amino acid, such as glutamic acid. In particular, said mutant CsIF6 gene may carry a G^A mutation at nucleotide 2243 in the coding sequence of the HvCsIF6 gene (SEQ ID NO:2).

Items.

The invention may be further defined by the following points.

1. A barley plant or part thereof, where the kernels of said barley plant have a reduced content of (1,3;1,4)-β-glucans, and where said barley plant carries a mutation in the CsIF6 gene, where the specified mutated CsIF6 gene encodes a mutant CsIF6 polypeptide wherein said mutant CsIF6 is the CsIF6 of SEQ ID NO: 1 except that the mutant CsIF6 contains a single amino acid substitution in the membrane-localized domain of CsIF6, wherein said substitution is a substitution of a non-polar amino acid for a charged amino acid or a substitution of a polar amino acid for a non-polar amino acid amino acid, wherein the membrane-localized CsIF6 domains consist of amino acids 109 to 128, 137 to 158, 700 to 731, 741 to 758, 835 to 857, and 864 to 882 of SEQ ID NO: 1 or SEQ ID NO : 3.

2. A barley plant or part thereof, where the kernels of said barley plant have a reduced content of (1,3;1,4)-β-glucans and where said barley plant carries a mutation in the CsIF6 gene, where said mutated CsIF6 gene encodes a mutant CsIF6 polypeptide, wherein said mutant CsIF6 is the CsIF6 of SEQ ID NO: 1 except that the mutant CsIF6 contains one amino acid substitution in the membrane-localized domain in CsIF6, wherein said substitution is a substitution of a non-polar amino acid for a charged amino acid or a substitution of a polar amino acid for a non-polar amino acid wherein the membrane-localized domain is selected from the group consisting of membrane-localized CsIF6 domains consisting of amino acids 835 to 857 or amino acids 700 to 731 or amino acids 741 to 775 in SEQ ID NO: 1 or SEQ ID NO: 3.

3. The barley plant according to any one of the preceding claims, wherein said barley plant has a (1,3;1,4)-β-glucan content in the range of 1 to 5% of the total dry matter weight of the kernels, for example, from 1,3 to 3 % of the total dry matter weight of the kernels, preferably from 1.3 to 2% of the total dry matter weight of the kernels.

4. The barley plant according to any one of the preceding claims, wherein the kernels of said barley plant have a (1,3;1,4)-β-glucan content of at least 30% and not more than 60%, preferably not less than 40% and no more than 60% of the content of (1,3;1,4)-β-glucans of the barley plant, non-

- 20 045156 existing wild-type CsIF6 gene, but otherwise having the same genotype.

5. The barley plant according to any one of the preceding claims, wherein the kernels of said barley plant have a (1,3;1,4)-β-glucan content of at least 30% and not more than 60%, preferably at least 40% and not more than 60% of the content of (1,3;1,4)-β-glucans of a barley plant carrying the wild-type CsIF6 gene, but otherwise having the same genotype.

6. The barley plant according to any of the previous paragraphs, where the barley plant contains grains with a frequency of broken grains after threshing that is no more than 3 times higher than the frequency of broken grains after threshing grains of a barley plant that does not carry a mutation in the CsIF6a gene, but otherwise has the same genotype.

7. The barley plant according to any of the preceding claims, wherein the barley plant contains grains with a broken grain frequency after threshing that is no more than 2 times higher than the frequency of broken grains after threshing grains of a barley plant that does not carry a mutation in the CsIF6a gene, but otherwise has the same genotype.

8. The barley plant of any one of the preceding claims, wherein the substitution is a substitution of a non-polar amino acid for a charged amino acid.

9. The barley plant of any one of the preceding claims, wherein said mutant CsIF6 polypeptide is CsIF6 of the sequence SEQ ID NO: 1 or SEQ ID NO: 3 except that the mutant CsIF6 contains a single amino acid substitution in a transmembrane domain consisting of amino acids from 835 to 857 in CsIF6, where the substitution is a substitution of a non-polar amino acid for a charged amino acid.

10. The barley plant of any one of the preceding claims, wherein said mutant CsIF6 polypeptide is CsIF6 of SEQ ID NO: 1 or SEQ ID NO: 3 except that the mutant CsIF6 contains a substitution of amino acid 847 for a charged amino acid.

11. The barley plant of any one of the preceding claims, wherein said mutant CsIF6 polypeptide is CsIF6 of SEQ ID NO: 1 or SEQ ID NO: 3 except that the mutant CsIF6 contains a substitution at amino acid 847, wherein said substitution is a glycine substitution (G) to glutamic acid (E).

12. The barley plant according to any one of the preceding claims, wherein said barley plant carries a mutation in the CsIF6 gene, wherein said mutated CsIF6 gene encodes a mutant CsIF6 polypeptide of the sequence SEQ ID NO: 27.

13. The barley plant according to any one of the preceding claims, wherein said barley plant carries a G^A mutation at nucleotide 2243 in the coding sequence of the HvCsIF6 gene.

14. The barley plant according to claim 13, wherein the coding sequence of the HvCsIF6 gene is SEQ ID NO:2.

15. The barley plant of any one of the preceding claims, wherein the barley plant is mutant 2 deposited with NCIMB under NCIMB accession number 43273, or a progeny thereof.

16. A barley plant or its progeny or parts thereof, wherein the genome of said plant contains the barley plant CsIF6 gene mutant 2, deposited with NCIMB under NCIMB accession number 43273.

17. The barley plant according to any one of the preceding claims, wherein the kernels of said barley plant have a β-glucan content ranging from 1.7 to 5.0% of the total dry matter weight of the kernels.

18. The barley plant according to any of the previous claims, wherein the kernels of said barley plant have a DP3:DP4 ratio of no more than 2.5, for example no more than 2.1, for example in the range of 1.0 to 2.1.

19. The barley plant of any one of the preceding claims, wherein said mutant CsIF6 polypeptide is CsIF6 with the sequence SEQ ID NO: 1 or SEQ ID NO: 3 except that the mutant CsIF6 contains a single amino acid substitution in a transmembrane domain consisting of amino acids from 741 to 758 in CsIF6, where the substitution is a substitution of a non-polar amino acid for a charged amino acid.

20. The barley plant according to any one of claims 1 to 8 and claim 19, wherein said mutant CsIF6 polypeptide is CsIF6 with the sequence SEQ ID NO: 1 or SEQ ID NO: 3 except that the mutant CsIF6 contains a replacement of amino acid 748 by charged amino acid.

21. The barley plant according to any one of claims 1-8 and 19, 20, wherein said mutant CsIF6 polypeptide is CsIF6 with the sequence SEQ ID NO: 1 or SEQ ID NO: 3 except that the mutant CsIF6 contains a substitution of amino acid 748, wherein said substitution is a substitution of aspartic acid (D) for glycine (G).

22. The barley plant according to any one of claims 1-8 and 19-21, wherein the kernels of said barley plant have a β-glucan content ranging from 1.7 to 3% of the total dry matter weight of the kernels.

23. The barley plant according to any one of claims 1-8 and 19-22, wherein the kernels of said barley plant have a DP3:DP4 ratio ranging from 2.5 to 4.

24. The barley plant according to any one of claims 1 to 8, wherein said mutant CsIF6 polypeptide is

- 21 045156 is CsIF6 with the sequence SEQ ID NO: 1 or SEQ ID NO: 3 except that the mutant CsIF6 contains a single amino acid substitution in the transmembrane domain consisting of amino acids 700 to 731 of CsIF6, wherein said substitution represents a substitution polar amino acid to non-polar amino acid.

25. The barley plant according to any one of claims 1 to 8 and 24, wherein said mutant CsIF6 polypeptide is CsIF6 with the sequence SEQ ID NO: 1 or SEQ ID NO: 3 except that the mutant CsIF6 contains a single amino acid substitution located in membrane domain consisting of amino acids 700 to 711 of CsIF6, wherein said substitution is a substitution of a polar amino acid for a non-polar amino acid.

26. The barley plant according to any one of claims 1-8 and 24, 25, wherein said mutant CsIF6 polypeptide is CsIF6 with the sequence SEQ ID NO: 1 or SEQ ID NO: 3 except that the mutant CsIF6 contains a substitution of amino acid 709 by non-polar amino acid.

27. The barley plant according to any one of claims 1-8 and 24-26, wherein said mutant CsIF6 polypeptide is CsIF6 with the sequence SEQ ID NO: 1 or SEQ ID NO: 3 except that the mutant CsIF6 contains a substitution of amino acid 709, wherein said substitution is a substitution of isoleucine (I) for threonine (T).

28. The barley plant according to any one of claims 1-8 and 24-27, wherein the kernels of said barley plant have a β-glucan content ranging from 1.3 to 3% of the total dry matter weight of the kernels.

29. The barley plant according to any one of claims 1-8 and 24-28, wherein the kernels of said barley plant have a DP3:DP4 ratio of at least 3.5, such as at least 4.0, for example at least 4.5, such as in the range from 4 to 6.

30. The barley plant of any of the previous paragraphs or its progeny, where said barley plant was not obtained exclusively by biological means.

31. The barley plant according to any of the preceding paragraphs or its progeny, where the barley plant is obtained by a method including the following steps: mutagenesis of barley plants or parts thereof, for example, using a chemical mutagenizing agent, selection of barley plants carrying any of the above-mentioned mutations in the CsIF6 gene.

32. A plant product containing a barley plant according to any of the previous paragraphs, or a part thereof.

33. The plant product of claim 32, wherein the plant product is green malt or kiln-dried malt obtained from the kernels of said barley plant.

34. The plant product of claim 32, wherein the plant product is a wort prepared from the kernels of said barley plant and/or from green malt or kiln-dried malt, containing processed kernel(s) of said barley plant.

35. The plant product according to any one of claims 32 to 34, wherein the plant product is a wort and wherein said wort has a viscosity of not more than 2.5 mPa-s, preferably not more than 2.2 mPa-s.

36. The plant product of claim 32, wherein the plant product is a beverage prepared from said barley plant parts thereof.

37. The beverage according to claim 32, wherein said beverage is obtained from grains of said barley plant and/or from malt containing processed kernel(s) of said barley plant.

38. The drink according to any one of claims 36, 37, where the drink is beer.

39. A method for preparing green malt, said method comprising the steps

a) providing barley plant kernels according to any one of claims 1 to 31;

b) soaking said kernels;

c) germination of soaked kernels under specified conditions.

40. A method for preparing kiln-dried malt, said method comprising the steps

a) providing barley plant kernels according to any one of claims 1 to 31;

b) soaking said kernels;

c) germination of soaked kernels under specified conditions;

d) drying said germinated kernels.

41. A method for producing a drink, wherein said method includes the stages

a) Providing barley plant kernels according to any one of claims 1 to 31 and/or malt in accordance with claim 33;

b) Preparation of an aqueous extract of said kernels and/or said malt;

c) processing said aqueous extract into a beverage.

42. A method for producing an aqueous extract, wherein said method includes the stages

a) providing barley plant kernels according to any one of claims 1 to 31;

b) subjecting the barley kernels to a germination stage, thereby obtaining germinated kernels, said germination stage comprising incubating said kernels in an aqueous solution for no more than 72 hours;

c) finely crushing said sprouted kernels, while said sprouted kernels

- 22 045156 have a water content of at least 20%, provided that said barley kernels do not have a water content below 20% at any time between stages b) and c);

d) preparing an aqueous extract of said crushed germinated kernels, thereby obtaining an aqueous extract of barley.

43. A method for producing an aqueous extract, wherein said method includes the steps of:

a) providing barley plant kernels according to any one of claims 1 to 31;

b) subjecting the barley kernels to a germination stage, thereby obtaining germinated kernels, said germination stage comprising incubating said kernels in an aqueous solution for no more than 72 hours;

c) finely crushing said sprouted grains while said sprouted kernels have a water content of at least 20%, provided that said barley kernels do not have a water content below 20% at any time after germination and until the sprouted kernels are precisely separated;

d) preparing an aqueous extract of said crushed germinated kernels, thereby obtaining an aqueous extract of barley.

44. The method according to any one of claims 42 and 43, wherein the germination step (b) includes incubating said kernels in an aqueous solution until the water content of the kernels is at least 30%, while passing at least at least 2 l O ₂ per kg of dry matter weight of barley grains per hour.

45. The method according to any one of claims 42-44, where the barley kernels are immersed in an aqueous solution during the entire germination stage.

46. The method according to any one of claims 42-45, where the germination step comprises

a) at least one stage of incubating said kernels in an aqueous solution, where at least 2 liters of O2 per kg dry matter weight of barley grains per hour are passed through said aqueous solution; And

b) at least one step of incubating said barley kernels in air.

47. Method according to any one of claims 42-46, where at least 3 l, more preferably at least 4 l, even more preferably at least 5 l, even more preferably at least 6 l O ₂ per kg weight dry matter of barley grains is passed through the specified aqueous solution per hour.

48. The method according to any one of claims 42-47, where the specified O ₂ is contained in a gas mixture, and the gas mixture is atmospheric air.

49. The method according to any one of claims 42-48, where the entire germination stage does not exceed 72 hours, more preferably does not exceed 60 hours, even more preferably does not exceed 54 hours.

50. The method according to any one of claims 42 to 49, wherein the barley is hulled barley, and the method includes the step of removing at least a portion of said hull before incubating said kernels in an aqueous solution.

51. A method for producing a drink, wherein said method includes the stages

a) preparing an aqueous extract by the method according to any one of claims 42-50;

b) processing said extract into a beverage.

52. The method according to claim 51, where stage b) contains the stages

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

b) cooling the aqueous extract;

c) fermenting said aqueous extract with yeast to produce a fermented beverage.

53. The method according to any one of claims 51 to 52, wherein the beverage is beer.

54. A method for producing barley plants with a β-glucan content in the range from 1 to 5% dry weight of the total number of kernels, the method comprising the stages

a) providing barley kernels; And

b) subjecting said barley kernels to random mutagenesis, thereby introducing a mutation into the CsIF6 gene, wherein said mutated CsIF6 gene encodes a mutant CsIF6 polypeptide, wherein said mutant CsIF6 is CsIF6 with the sequence SEQ ID NO: 1 or SEQ ID NO: 3 except that the CsIF6 mutant contains a single amino acid substitution in the CsIF6 transmembrane domain, where the substitution is a substitution of a non-polar amino acid with a charged amino acid or a substitution of a polar amino acid with a non-polar amino acid, where the CsIF6 transmembrane domain consists of amino acids 109 to 128, 137 to 158, 700 to 731, 741 to 758, 835 to 857 and 864 to 882; And

c) selection of barley kernels or its progeny carrying the mutated CsIF6 gene.

55. The method of claim 54, wherein the barley plant or mutation is as defined in any one of claims 1 to 31.

56. The barley plant according to any one of claims 1 to 31, wherein the barley plant contains a mutation in one or more additional genes, for example one or more of the following mutations:

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

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

LOX-2;

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

Sequences >SEQ ID NO: 1, cellulose synthase-like CsIF6 sequence (based on GenBank NCBI number: EU267181.1).

MAPAVAGGGRVRSNEPVAAAAAAPAASGKPCVCGFQVCACTGSAAVASAASSLDMDIVAMGQIGAVNDESWVGVE LGEDGETDESGAAVDDRPVFRTEKIKGVLLHPYRVLIFVRLIAFTLFVIWRISHKNPDAMWLWVTSICGEFWFGF SWLLDQLPKLNPINRVPDLAVLRQRFDRPDGTSTLPGLDIFVTTADPIKEPILSTANSV LSILAADYPVDRNTCY VSDDSGMLLTYEALAESSKFATLWVPFCRKHGIEPRGPESYFELKSHPYMGRAQDEFVNDRRRVRKEYDEFKARI NSLEHDIKQRNDGYNAAIAHSQGVPRPTWMADGTQWEGTWVDASENHRRGDHAGIVLVLLNHPSHRRQTGPPASA DNPLDLSGVDVRLPMLVYVSREKRPGHDHQKKA GAMNALTRASALLSNSPFILNLDCDHYINNSQALRAGICFMV GRDSDTVAFVQFPQRFEGVDPTDLYANHNRIFFDGTLRALDGMQGPIYVGTGCLFRRITVYGFDPPRINVGGPCF PRLAGLFAKTKYEKPGLEMTTAKAKAAPVPAKGKHGFLPLPKKTYGKSDAFVDTIPRASHPSPYAAAAEGIVADE ATIVEAVNVTAAAFEKK TGWGKEIGWVYDTVTEDWTGYRMHIKGWRSRYCSIYPHAFIGTAPINLTERLFQVLR WSTGSLEIFFSKNNPLFGSTYLHPLQRVAYINITTYPFTAIFLIFYTTVPALSFVTGHFIVQRPTTMFYVYLGIV LSTLLVIAVLEVKWAGVTVFEWFRNGQFWMTASCSAYLAAVCQVLTKVIFRRDISFKLTSKLPSG DEKKDPYADL YWRWTPLMITPIIIIFVNIIGSAVAFAKVLDGEWTHWLKVAGGVFFNFWVLFHLYPFAKGILGKHGKTPVWLV WWAFTFVITAVLYINIPHMHTSGGKHTTVHGHHGKKLVDTGLYGWLH >SEQ ID NO: 2, Hordeum vulgare cellulose synthase-like CsIF6 (CsIF6), full cds ( based on GenBank NCBI number: EU267181.1).

ATGGCGCCAGCGGTGGCCGGAGGGGGCCGCGTGCGGAGCAATGAGCCGGTTGCTGCTGCTGCCGCCGCGCCGGCG GCCAGCGGCAAGCCCTGCGTGTGCGGCTTCCAGGTTTGCGCCTGCACGGGGTCGGCCGCGGTGGCCTCCGCCGCC TCGTCGCTGGACATGGACATCGTGGCCATGGGGCAGATCGGCGCCGTCAACGACGAGAGCTGGGTGGGCGTGGAG CTCGGCGAAGATG GCGAGACCGACGAAAGCGGTGCCGCCGTTGACGACCGCCCCGTATTCCGCACCGAGAAGATC AAGGGTGTCCTCCTCCACCCCTACCGGGTGCTGATTTTCGTTCGTCTGATCGCCTTCACGCTGTTCGTGATCTGG CGTATCTCCCACAAGAACCCAGACGCGATGTGGCTGTGGGTGACATCCATCTGCGGCGAGTTCTGGTTCGGTTTC TCGTGGCTGCTAGATCAGCTGCCCAAG CTGAACCCCATCAACCGCGTGCCGGACCTGGCGGTGCTGCGGCAGCGC TTCGACCGCCCCGACGGCACCTCCACGCTCCCGGGGCTGGACATCTTCGTCACCACGGCCGACCCCATCAAGGAG CCCATCCTCTCCACCGCCAACTCGGTGCTCTCCATCCTGGCCGCCGACTACCCCGTGGACCGCAACACATGCTAC GTCTCCGACGACAGTGGCATGCTGCTCACCTACGAGGCCCTGGCAGAG TCCTCCAAGTTCGCCACGCTCTGGGTG CCCTTCTGCCGCAAGCACGGGATCGAGCCCAGGGGTCCGGAGAGCTACTTCGAGCTCAAGTCACACCCTTACATG GGGAGAGCCCAGGACGAGTTCGTCAACGACCGCCGCCGCGTTCGCAAGGAGTACGACGAGTTCAAGGCCAGGATC AACAGCCTGGAGCATGACATCAAGCAGCGCAACGACGGGTACAACGCCGCCATTGCCCA CAGCCAAGGCGTGCCC CGGCCCACCTGGATGGCGGACGGCACCCAGTGGGAGGGCACATGGGTCGACGCCTCCGAGAACCACCGCAGGGGC GACCACGCCGGCATCGTACTGGTGCTGCTGAACCACCCGAGCCACCGCCGGCAGACGGGCCCGCCGGCGAGCGCT GACAACCCACTGGACTTGAGCGGCGTGGATGTGCGTCTCCCCATGCTGGTGTACGTGTCCCGTGAGAAGCG CCCC GGGCACGACCACCAGAAGAAGGCCGGTGCCATGAACGCGCTTACCCGCGCCTCGGCGCTGCTCTCCAACTCCCCC TTCATCCTCAACCTCGACTGCGATCATTACATCAACAACTCCCAGGCCCTTCGCGCCGGCATCTGCTTCATGGTG GGACGGGACAGCGACACGGTTGCCTTCGTCCAGTTCCCGCAGCGCTTCGAGGGCGTCGACCCCACCGACCTCTAC GCCAACCACA ACCGCATCTTCTTCGACGGCACCCTCCGTGCCCTGGACGGCATGCAGGGCCCCATCTACGTCGGC ACTGGGTGTCTCTTCCGCCGCATCACCGTCTACGGCTTCGACCCGCCGAGGATCAACGTCGGCGGTCCCTGCTTC CCCAGGCTCGCCGGGCTCTTCGCCAAGACCAAGTACGAGAAGCCCGGGCTCGAGATGACCACGGCCAAGGCCAAG GCCGCGCCCGTGCCCGCCAAGGGTA AGCACGGCTTCTTGCCACTGCCCAAGAAGACGTACGGCAAGTCGGACGCC TTCGTGGACACCATCCCGCGCGTCGCACCCGTCGCCCTACGCCGCGGCGGCTGAGGGGATCGTGGCCGACGAG GCGACCATCGTCGAGGCGGTGAACGTGACGGCCGCCGCGTTCGAGAAGAAGACCGGCTGGGGCAAAGAGATCGGC TGGGTGTACGACACCGTCACGGAGGACGTGGTCAC CGGCTACCGGATGCATATCAAGGGGTGGCGGTCACGCTAC TGCTCCATCTACCCACACGCCTTCATCGGCACCGCCCCCATCAACCTCACGGAGAGGCTCTTCCAGGTGCTCCGC TGGTCCACGGGATCCCTCGAGATCTTCTTCTCCAAGAACAACCCGCTCTTCGGCAGCACATACCTCCACCCGCTG CAGCGCGTCGCCTACATCAACATCACCACTTACCCCTTCACCGCCATCTTCCTCATCT TCTACACCACCGTGCCG GCGCTATCCTTCGTCACCGGCCACTTCATCGTGCAGCGCCCGACCACCATGTTCTACGTCTACCTGGGCATCGTG CTATCCACGCTGCTCGTCATCGCCGTGCTGGAGGTCAAGTGGGCCGGGGTCACAGTCTTCGAGTGGTTCAGGAAC GGCCAGTTCTGGATGACAGCAAGTTGCTCCGCCTACCTCGCCGCCGTCTGCCAGGTGCTGACCAA GGTGATATTC CGGCGGGACATCTCCTTCAAGCTCACATCCAAGCTACCCTCGGGACGAGAAGAAGGACCCCTACGCCGACCTC TACGTGGTGCGCTGGACGCCGCTCATGATTACACCCATCATCATCTTCGTCAACATCATCGGATCCGCCGTG GCCTTCGCCAAGGTTCTCGACGGCGAGTGGACGCACTGGCTCAAGGTCGCCGGCGGCGTCTTCTTCAACTTCTGG GTGCTCTT CCACCTCTACCCCTTCGCCAAGGGCATCCTGGGGAAGCACGAAAGACGCCAGTCGTGGTGCTCGTC TGGTGGGCATTCACCTTCGTCATCACCGCCGTGCTCTACATCAACATCCCCCACATGCATACCTCGGGAGGCAAG CACACAACGGTGCATGGTCACCATGGCAAGAAGTTGGTCGACACAGGGCTCTATGGCTGGCTCCATTGA

- 24 045156 >SEQ ID NO: 3, sequence of cellulose synthase-like CsIF6 (containing the A590T polymorphism).

MAPAVAGGGRVRSNEPVAAAAAAPAASGKPCVCGFQVCACTGSAAVASAASSLDMDIVAMGQIGAVNDESWVGVE

LGEDGETDESGAAVDDRPVFRTEKIKGVLLHPYRVLIFVRLIAFTLFVIWRISHKNPDAMWLWVTSICGEFWFGF

SWLLDQLPKLNPINRVPDLAVLRQRFDRPDGTSTLPGLDIFVTTADPIKEPILSTANSVLSILAADYPVDRNTCY

VSDDSGMLLTYEALAESSKFATLWVPFCRKHGIEPRGPESYFELKSHPYMGRAQDEFVNDRRRVRKEYDEFKARI

NSLEHDIKQRNDGYNAAIAHSQGVPRPTWMADGTQWEGTWVDASENHRRGDHAGIVLVLLNHPSHRRQTGPPASA

DNPLDLSGVDVRLPMLVYVSREKRPGHDHQKKAGAMNALTRASALLSNSPFILNLDCDHYINNSQALRAGICFMV

GRDSDTVAFVQFPQRFEGVDPTDLYANHNRIFFDGTLRALDGMQGPIYVGTGCLFRRITVYGFDPPRINVGGPCF

PRLAGLFAKTKYEKPGLEMTTAKAKAAPVPAKGKHGFLPLPKKTYGKSDAFVDTIPRASHPSPYTAAAEGIVADE

ATIVEAVNVTAAAFEKKTGWGKEIGWVYDTVTEDWTGYRMHIKGWRSRYCSIYPHAFIGTAPINLTERLFQVLR

WSTGSLEIFFSKNNPLFGSTYLHPLQRVAYINITTYPFTAIFLIFYTTVPALSFVTGHFIVQRPTTMFYVYLGIV

LSTLLVIAVLEVKWAGVTVFEWFRNGQFWMTASCSAYLAAVCQVLTKVIFRRDISFKLTSKLPSGDEKKDPYADL

YWRWTPLMITPI111FVNIIGSAVAFAKVLDGEWTHWLKVAGGVFFNFWVLFHLYPFAKGILGKHGKTPVWLV

WWAFTFVITAVLYINIPHMHTSGGKHTTVHGHHGKKLVDTGLYGWLH >SEQ ID NO: 27, Cell Wall mutant 2 (CW3a) based on SEQ ID NO: 1, cellulose synthase-like CsIF6 sequence (based on GenBank number NCBI:EU267181.1).

MAPAVAGGGRVRSNEPVAAAAAAPAASGKPCVCGFQVCACTGSAAVASAASSLDMDIVAMGQIGAVNDESWVGVE

LGEDGETDESGAAVDDRPVFRTEKIKGVLLHPYRVLIFVRLIAFTLFVIWRISHKNPDAMWLWVTSICGEFWFGF

SWLLDQLPKLNPINRVPDLAVLRQRFDRPDGTSTLPGLDIFVTTADPIKEPILSTANSVLSILAADYPVDRNTCY

VSDDSGMLLTYEALAESSKFATLWVPFCRKHGIEPRGPESYFELKSHPYMGRAQDEFVNDRRRVRKEYDEFKARI

NSLEHDIKQRNDGYNAAIAHSQGVPRPTWMADGTQWEGTWVDASENHRRGDHAGIVLVLLNHPSHRRQTGPPASA

DNPLDLSGVDVRLPMLVYVSREKRPGHDHQKKAGAMNALTRASALLSNSPFILNLDCDHYINNSQALRAGICFMV

GRDSDTVAFVQFPQRFEGVDPTDLYANHNRIFFDGTLRALDGMQGPIYVGTGCLFRRITVYGFDPPRINVGGPCF

PRLAGLFAKTKYEKPGLEMTTAKAKAAPVPAKGKHGFLPLPKKTYGKSDAFVDTIPRASHPSPYAAAAEGIVADE

ATIVEAVNVTAAAFEKKTGWGKEIGWVYDTVTEDWTGYRMHIKGWRSRYCSIYPHAFIGTAPINLTERLFQVLR

WSTGSLEIFFSKNNPLFGSTYLHPLQRVAYINITTYPFTAIFLIFYTTVPALSFVTGHFIVQRPTTMFYVYLDIV

LSTLLVIAVLEVKWAGVTVFEWFRNGQFWMTASCSAYLAAVCQVLTKVIFRRDISFKLTSKLPSGDEKKDPYADL

YWRWTPLMITPIIIIFVNIIGSAVAFAKVLDGEWTHWLKVAGGVFFNFWVLFHLYPFAKGILGKHGKTPVWLV

WWAFTFVITAVLYINIPHMHTSGGKHTTVHGHHGKKLVDTGLYGWLH

Examples

Example 1.

Development of probes for digital identification of mutants.

The coding sequence of the HvCsIF6 gene with accession number AB621332 was obtained from the National Center for Biotechnology Information (NCBI). This sequence was used for a BLAST search on the website: http://webblast.ipk-gatersleben.de/barley/. HvCsIF6 gene and transcript sequences with accession number MLOC_57200 were selected using: http://plants.ensembl.org/Hordeum_vulgare/. Digital mutant identification probes were designed based on the DNA and protein coding sequence MLOC_57200.2.

Example 2.

Screening of barley mutants with specific mutations in the cellulose synthase-like CsIF6 gene.

A total of 5 mutants were identified with a specific mutation leading to the substitution of an amino acid residue in cellulose synthase-like CsIF6 (HvCsIF6) (Table 1).

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Table 1

Identified mutants

Mutant No. Nucleotide change in coding sequence (SEQ ID NO: 2) Amino acid change in protein (SEQ ID NO: 1) Mutant 1 G^A (2540) Gly^Glu (847) Mutant 2 G^A (2243) Gly^Asp (748) Mutant 3 G^A (2028) Trp^Stop (676) Mutant 4 G^A (2195) Gly^Asp (732) Mutant 5 C^T (2126) Thrill (709)

Mutant 1, mutant 2, mutant 3 and mutant 4 were identified using the ddPCR screening method essentially as described in international patent application PCT/EP2O17/O65516. More specifically, a pool of randomly mutagenized barley grains was prepared, followed by the preparation of an ordered library, as described in international patent application PCT/EP2O17/065516 in WS1 and WS2 on p. 66-69, as well as in Examples 1 to 2. Mutant 1, Mutant 2, Mutant 3 and Mutant 4 were identified and selected as described in international patent application PCT/EP2O17/O65516 in WS3 and WS4 on pages 67-72 , as well as in examples from 3 to 15, using the primers and probes indicated in table. 2 below. Specifically, screening was performed essentially as described in international patent application PCT/EP2017/065516 in WS3 and Examples 3 to 7, using the primers and probes listed in Table 1. 2 below. Individual barley grains carrying the gene mutation were identified essentially as described in international patent application PCT/EP2O17/O65516 in WS4 (pages 69-72) and in examples 8 to 15, using the primers and probes specified in table . 2 below. Primers and probes that were designed specifically to identify specific mutants at the HvCsIF6 locus are described in Table. 2.

table 2

Primers and probes designed for specific mutants

Mutant Target specific forward primer Target specific reverse primer Mutant-specific detection probe labeled with 6carboxyfluore coupling (FAM) Hexachlorofluorescein (HEX)-labeled reference-specific detection probe Mut 1 TASSASSAT CATCATCAT ST (SEQ Ш NO:4) CGAGAACCT TGGCGA (SEQ IDNO:5) ATCATCGAAT CCGCCG (SEQ IDNO:6) CATCATCGGAT CCGCC (SEQ Ш NO: 7) Mut 2 CCGACCAC CATGTTCT (SEQ ID NO:8) GGCGATGAC GAGCAG (SEQ IDNO:9) CTACCTGGAC ATCGTGC (SEQ ID NO: 10) CTACCTGGGCA TCGTG (SEQ Ш NO: И) Mut 3 ACTGCTCCA TCTACCCAC (SEQ ID NO: 12) GTATGTGCTG CCGAAGAG (SEQ ID NO: 13) TGCTCCGCTG ATCCA (SEQ ID NO: 14) TCCGCTGGTCC ACG (SEQ Ш NO:15) Mut 4 CACCACCG TGCCG (SEQ ID NO: 16) CATGGTGGT CGGGC (SEQ ID NO: 17) CGTCACCGAC CACTTC (SEQ ID NO: 18) TCACCGGCCAC TTCA (SEQ Ш NO: 19)

Mutant 5 was identified using direct sequencing of 6000 mutagenized M3 barley mutants using specific primers (forward primer

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5'-ACTGCTCCATCTACCCACAC-3'; reverse primer 5'-GATGACGAAGGTGAATGCCC-3') _for amplification of parts of the HvCsIF6 locus.

Mutant 1, Mutant 2, Mutant 3, Mutant 4 and Mutant 5 are also referred to herein as

Mut1, Mut2, Mut3, Mut4 and Mut5 respectively.

To visualize the location of mutations, the Swissprot program (swissmodel.expasy.org/) was used to model the CsIF6 protein. First, the entire protein sequence is loaded into the model building feature, and then the model is calculated based on the available data. The CsIF6 protein sequence is recognized to have an embedded sequence structure resembling the cellulose synthase A subunit. The program then uses the recognized sequence (polypeptide position 109-883) to build the model. At this stage, the SwissProt program does not recognize that the protein is membrane bound, although the helices are located on one side of the model. The literature indicates that the CsIF6 protein is membrane associated. Therefore, the QMEANBrane function in SwissProt was used for further verification. This simulation program uses the PDB file covering position 109-883 created during the first simulation and then creates a new structure that models the membrane and a selected part of the CsIF6 protein together. The two dotted gray planes indicate a two-layer membrane. All three mutations that provide low content of (1,3;1,4)-β-glucans are located in helical structures embedded in the membrane (T709I, G748D, G847E), while the mutation that does not affect the content of (1,3; 1,4)β-glucans (G732D), is located in the outward-facing linker sequence connecting the two transmembrane domains (Fig. 3). The membrane-localized amino acids in CsIF6 are listed in Table. 3.

Table 3

Localization of membrane-localized amino acid sequences in the CsIF6 protein

Position AA in SEQ ID NO: 1 or SEQ ID NO: 3 Sequence AA Functionality 109-128 RVLIFVRLIAFTLFVIWRIS Transmembrane region (SEQIDNO:20) 137-158 LWVTSICGEFWFGFSWLLDQLP Transmembrane region (SEQIDNO:21) 700-711 LQRVAYINITTY Part of the transmembrane region (SEQ Ш NO:22) 712-714 PTF Helical linker in the membrane giving rise to a kink 715-731 AIFLIFYTTVPALS FVT Part of the transmembrane region (SEQ GO NO:23) 741-758 TMFYVYLGIVLSTLLVIA Transmembrane region (SEQIDNO:24) 835-857 IT PIIIIFVNIIGSAVAFAKVLD Transmembrane region (SEQIDNO:25) 864-882 LKVAGGVFFNFWVLFHLYPF Transmembrane region (SEQIDNO:26)

Example 3.

(1,3;1,4)-β-glucan content and DP3:DP4 ratio of mature mutant grains. The mutants identified as described in Example 1 were grown to maturity and homozygous grains were sown in rows. When mature, the plants were harvested and the grains used for propagation in small plots of a few square meters. Mutants 1, 2, 4 and 5 all performed well in the field, producing yields similar to the standards. Accordingly, these mutants have acceptable agronomic traits. The reference was the Paustian variety for mutants 1, 2, 3 and 4, and the Quench variety for mutant 5). Mutant 3 showed significantly reduced fertility and was therefore not included in the yield trials.

Table 4 shows the average yield of at least four plots measuring 7.5 m ² . Barley was planted in Nr Aaby in 2017.

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Table 4

Mutant No. Mutant Output (kg) SD Mut1 CW2 6.0 0.2 Mut2 CW3 6.2 o, s Mut5 CW4 5.9 0.2 Paustian 6.2 0.2 Mut 4 CW7 6.0 0.5 Quench 6.1 0.2

The collected grains were analyzed for total (1,3;1,4)-β-glucans content and the DP3:DP4 ratio was determined. Ten ml of barley grains were ground in a Retch cyclone mill. All samples were analyzed in triplicate. 20 mg of flour was poured into 2 ml Eppendorf tubes, heated for 2 hours at 100°C in an oven, then cooled to room temperature. A total of 500 μl of 50% aqueous methanol was added and the sample was shaken at 1400 rpm for 1 h. After centrifugation at 16,000 g for 10 min, the supernatant was discarded and the sample was dried overnight. Then, 400 μl of 20 mM NaHPO ₄ at pH 6.5 C/ml lichenase (Megazyme, International, Ireland) was added to 10 mg of flour and incubated at 50°C for 2.5 hours. The sample was centrifuged at 16,000 g for 10 min and the supernatant was filtered through 0.45 µm filters, and the oligomers Glc-e-(1^4)-Glc-3-(1^3)-Glc (DP3) and Glc-e-(1^4) were released )-Glc-e-(1^4)-Glc-e-(1^3)-Glc (DP4) was quantified by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Oligomers Glc-e-(1^4)-Glc-e-(1^3)-Glc (DP3) and Glc-e-(1^4)-Glc-e-(1^4)-Glc-3- (1^3)-Glc(DP4) was quantified with HPAEC-PAD using a Dionex ICS 5000+ DC system equipped with a 4 μm CA-10 2x250 mm column and precolumn. The run conditions were 0.4 mL/min, column temperature 40°C, isocratic eluent 100 mM NaOH for 15 min. The standard for quantitation was produced by digesting 1 U/ml lichenase (Megazyme International, Ireland) with known amounts of medium-viscosity (1,3;1,4)-β-glucans (Megazyme International, Ireland) in 20 mM NaHPO4, pH 6.5, assuming equal molar ratio of PAD responses between DP3 and DP4. The total content of (1,3;1,4)-β-glucans was considered as the sum of the content of DP3 and DP4 oligomers.

Results. The contents of (1,3;1,4)-β-glucans in mutants 1, 2 and 5 were all in the range from 1.4 to 2%, while mutant 3 had almost no (1-3;1-4)- β-glucan (Fig. 1 and Table 6). Examination of the DP3:DP4 ratios of mutant 1 showed a lower ratio than the references, mutant 2 was similar to the references, the ratio of mutants 3 and 4 was slightly higher than the reference, and mutant 5 showed a much higher DP3:DP4 ratio (Fig. 2 and table 6).

Example 4.

The content of (1,3;1,4)-β-glucans of mutant grains during accelerated germination of grains of mutants 1, 2 and 4, as well as the standard (the standard for comparing mutants 1, 2, 3 and 4 was the Paustian variety and the Quanch variety for the mutant 5) were subjected to a one-step soaking and germination in a tank, essentially as described in example 1 of the international patent application PCT/EP2O17/O65498. Samples of germinating grains were collected after 24 hours and 48 hours.

All samples were analyzed using Comprehensive Microarray Polymer Profiling (CoMPP) (Moller et al., 2007). Each sample was analyzed in triplicate of 20 mg, sequentially extracted with 1 ml of H2O at 85°C for 1 hour at 1000 rpm and 1 ml of 4 M NaOH with 0.1% by volume NaBH ₄ at 20°C for 2 h at 1000 rpm. Each extraction was carried out in 2 ml Eppendorf tubes with a 3 mm glass bead, followed by centrifugation for 10 min at 10,000 g, and the supernatant was collected.

Extracts were mixed 50/50 with matrix inkjet printing buffer (55.2% glycerol, 44% water, 0.8% Triton X-100) and applied to a 0.45 μm nitrocellulose membrane (Whatman, Maidstone, UK). using Arrayjet Sprint (Arrayjey, Roslin, UK). Each sample was printed with four technical replicates and 3 fivefold dilutions and assayed as described in (Pedersen et al., 2012). Arrays were scanned using a flatbed scanner (CanoScan 9000 Markll, Canon, S0borg, Denmark) at 2400 dpi and quantified using an Array-Pro Analyzer 6.3 (Media Cybernetics, Rockville, MD, USA). For each sample, the average value was calculated based on four dilutions and four technical replicates, resulting in 16 measurements for each sample replicate signal value. The largest value in the data set was set to 100 and the rest were adjusted accordingly. The data is presented in table. 5 shows the average of triplicate samples.

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Table 5

Content of (1,3;1,4)-β-glucans in barley mutants and standards

% Glucan - 24 h % Glucan - 48 h Mutant 1 59 55 Mutant 2 84 69 Mutant 4 100 95 Standard 1 98 85

The highest value at 24 h was set to 100, and the others were adjusted accordingly.

The analysis shows that mutants 1 and 2 have lower (1,3;1,4)-β-glucans content after 24 and 48 h during germination in an air tank compared to the standard, and mutant 4 serves to confirm the lower content of (1,3;1,4)-β-glucans inherent in mutants 1 and 2.

Example 5.

Analysis of grain strength.

Mutants 1, 2, 3, 4 and 5, as well as reference lines of cultivar Paustian and cultivar Quench, were grown in the field in 7.5 m ² plots. They were collected using a Wintersteiger Classic breeding combine (Wintersteiger (WINTERSTEIGER AG, Johann-Michael-Dimmelstrasse 94910 Ried im Innkreis, Austria). Further grain purification was carried out on a Pfeuffer sample cleaner, model SLN4 (Pfeuffer GmbH, FlugplatzstraOe, 70, 97318, Kitzingen , Germany) using a 2.5 mm sieve. Broken grains were counted in four randomly selected samples weighing approximately 10 g and the number of broken grains was calculated relative to weight. The number of broken grains after threshing (Wintersteiger test) and before screening is an indicator of grain strength. The level of broken grains after threshing was calculated as the fold increase in broken grains in the mutant plants relative to the wild-type reference plants (reference plants are listed in Table 6a).

Table 6a

Summary of characteristics of mutant grains

Patent record Betaglucan in flour(%) Mass/mass, DP3/DP4 ratio % betaglucan from standard Broken grains (%) Mut 1 1.7 2.0 50 4.4 Mut 2 1.9 h,o 54 2.7 Mut 3 0.1 3.7 2 ND Mut 4 3 3 3.4 94 1.2 Standard 1 - Variety Paustian 3.5 2.9 100 1.6 Mut 5 1.4 4.7 44 3.2 Standard 2 - Quench variety 3.2 2.8 100 2.1

A similar determination of the percentage of broken kernels (the level of damage after threshing) was carried out on 50 g samples of barley plants grown in New Zealand in 2016-2017. (Mut1; Mut2; Mut4; Mut5) and in Denmark 2018 (Mut2). The frequency of broken kernels after threshing (level of damage after threshing) was determined as described above and compared with the frequency for reference wild-type barley plants (cv. Paustian for Mut1, Mut2 and Mut4 and cv. Quench for Mut5). The fold increase compared to reference wild-type barley plants is shown in Table. 6b below.

Table 6b

Barley line Mutation compared to SEQ GO NO: 1 Multiple increase in the number of broken grains 2017 Multiple increase in the number of broken grains 2018 Mut 1 G847E 2.7 Mut 2 G748D 1.7 1.9 Mut 4 G732D 0.8 Mut 5 T709I 1.5

Interestingly, Hu et al., 2014 disclose a barley line (m351) carrying a mutation in the CsIF6 gene, resulting in the A849T mutation of the CsIF6 protein. This barley line has a 4.2-fold increase in the level of broken grains compared to the wild-type control.

Example 6. Hydrolytic enzymatic activity in malt samples.

Barley kernels of mutant 2 and standard (Paustian variety) were processed in triplicate, 50 g each

- 29 045156 sample. Mutant 2 was as described in example 2 above. Dried barley kernels placed in stainless steel glasses were micromalted according to standard methods. Briefly, barley kernels were forced into fresh water in fresh water at 15°C for about 7 h on the first day, 3 h on the second day and 1 h on the third day to achieve a water content of 35, 40 and 45%, respectively, in at the end of each soak. After each soak, the stainless steel beakers containing the samples were transferred to a germination box at 15°C and stored there until the next stage of the process. After the final soak water drainage, samples were kept for 120 h (days 3 to 7) in germination boxes equilibrated to 45% water and sprayed with water to overcome water loss through respiration.

At the end of the germination process (day 7), the samples were dried in a stainless steel oven in a Termaks incubator for 21 hours using the following temperature program: 25-55°C (2°C/h), 55-85°C (4 °C/h), 85°C for 1.5 hours.

The germination and oven drying steps were performed with a recirculating air flow of 80% fresh air.

At the end of each micromalting day, samples were collected for analysis in triplicate and lyophilized for 48 h.

Prior to enzymatic activity analysis, malt samples were ground using a standard Foss Cyclotech mill equipped with a tungsten carbide grinding ring (Foss 10004463), a nickel-plated impeller (Foss 1000 2666) and a 1 mm outlet screen (Foss 10001989). All measurements of the enzymatic activity of barley malt were carried out within 48 hours after grinding the dry sample. Alpha-amylase activity assays were performed using the Ceralpha kit (K-CERA) from Megazyme using standard laboratory equipment. Amylase assays were done according to the manufacturer's protocol (K-CERA 01/12). Calculation of amylase activity was based on the formula in the Megazyme protocol (K-CERA 01/12). Beta-amylase activity assays were performed using the Betamyl (K-BETA3) kit from Megazyme using standard laboratory equipment. Amylase assays were done according to the manufacturer's protocol (K-BETA3 10/10). Calculation of beta-amylase activity was based on the formula in the Megazyme protocol (K-BETA3 10/10). Limiting dextrinase activity assays were performed using the pullulanase/limiting dextrinase (PullG6 method) assay kit (K-PullG6) from Megazyme using standard laboratory equipment. Limiting dextrinase assays were performed according to the manufacturer's protocol (K-PullG6 05/17). Calculation of limiting dextrinase activity was based on the formula in the Megazyme protocol (K-PullG6 05/17).

Results.

Total enzymatic activities measured for α-amylase, β-amylase, and free limiting dextrinase followed the same pattern in mutant 2 and the barley reference line (Fig. 5). Surprisingly, limiting dextrinase activity appeared to be slightly higher in the mutant starting at day 3, although limiting dextrinase gene expression was slightly lower.

Example 7. Content of (1,3;1,4)-glucans.

Sprouted grains obtained as described in example 6 were analyzed for the total content of (1,3;1,4)-β-glucans. Ten ml of barley grains were ground in a Retch cyclone mill. All samples were analyzed in triplicate. Twenty mg of flour was poured into 2 ml Eppendorf tubes, heated for 2 hours at 100°C in an oven, followed by cooling to room temperature. A total of 500 μl of 50% aqueous methanol was added and the samples were shaken at 1400 rpm for 1 h. After centrifugation at 16000xg for 10 min, the supernatant was discarded and the samples were dried overnight. Then, 400 μl of 20 mM NaHPO ₄ at pH 6.5 s / ml lichenase (Megazyme, International, Ireland) was added to 10 mg of flour and incubated at 50 ° C for 2.5 hours. The sample was centrifuged at 16000xg for 10 min the supernatant was filtered through 0.45 μm filters, and the oligomers Glc-e-(1^4)-Glc-e-(1^3)-Glc (DP3) and Glc-e-(1^4)Glc were released -e-(1^4)-Glc-P-(1^3)-Glc (DP4) was quantified by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Oligomers Glc-β(1^4)-Glc-e-(1^3)-Glc (DP3) and Glc-e-(1^4)-Glc-e-(1^4)-Glc-e-( 1^3)-Glc (DP4) was quantified with HPAEC-PAD using a Dionex ICS 5000+ DC system equipped with a 4 μm CA-10 2x250 mm column and a pre-column. The run conditions were 0.4 mL/min, column temperature 40°C, isocratic eluent 100 mM NaOH for 15 min. The quantitation standard was produced by digestion of 1 U/ml lichenase (Megazyme International, Ireland) with known amounts of medium-viscosity (1.3;1.4)-β-glucans (Megazyme International, Ireland) in 20 mM NaHPO ₄ , pH 6.5 , assuming an equal molar ratio of PAD responses between DP3 and DP4. The total content of (1,3;1,4)-β-glucans was calculated as the sum of DP3 and DP4 oligomers.

Results.

The content of (1,3;1,4)-β-glucans in mutant 2 (mutant) and in the Paustian variety (reference) was controlled in barley and in green malt on days 1, 2, 3, 4, 5, 6 of the malting procedure ( Fig. 6), as well as in dried

- 30 045156 malt ovens (day 7 in Fig. 6). During the entire malting process, the content of (1,3;1,4)-β-glucans in mutant 2 was only 50% of the reference value, and on the 5th day the content of (1,3;1,4)-β-glucans in the mutant was similar to the kiln-dried reference malt.

Example 8: Viscosity.

Malting.

Barley samples were malted in duplicate using 50 g of barley per sample cup. Barley samples were soaked by forced placement in water at a temperature of 15°C in a closed chamber with a sample grid. The soaking procedure was carried out for the first three days so that at the end of each soak the water content was 35, 40 and 45% respectively. Samples were stored for the next three days at 15°C in germination boxes and equilibrated to 45% water and adjusted by spraying with water to overcome water loss through respiration. After these six days, the samples were oven dried for 21 hours in a Termaks incubator and processed using a manual root removal system. Green malt was produced within three days; after the third day of malting, the samples were freeze-dried to 4% water content.

Mashing.

Before mashing, the samples were ground to a fine powder. 70 g of dry matter was mixed at a water:grit ratio of 5:1 and mashed in a Lochner mash according to the following mash program: 10 min at 52°C, 50 min at 65°C and 5 min at 78°C, spaced by temperature change by 1°/min. Samples were collected during the mash program.

After mashing, the wort is filtered through a paper filter MN 614 _1/4 , diameter 320 mm (MachereyNagel). The rheological behavior of the filtered wort is measured on a RheolabQC rotary rheometer (Anton Paar GmbH) with a Peltier temperature support system (C-PTD 180/AIR/QC) and dual-zone measuring systems (C-DG42/SS, Anton Paar GmbH).

Results.

The viscosity of the 3-day wort (barley after 3 days of malting) of the micromalted Mut2 and Mut5 mutants was determined and compared with the reference (3-day wort of micromalted wild-type grains of the Paustian variety and the Quench variety). The viscosity is significantly lower for wort prepared from any mutant compared to the standards, as shown in table. 7 and fig. 6. Plato were comparable in worts prepared from mutants or references.

The viscosity of wort made from standard malt prepared by steeping for 1 to 2 days and sprouting for 5 to 7 days followed by oven drying is about 2 mPa s.

Table 7

Mut 2 Paustian Mut 5 Quench Plateau 13.1 13.3 13.3 13.3 (1-3;1-4)-βglucan (BTG) (mg/l) 2.44 ± 0.02 5.50 ±0.02 2.78 ±0.01 4.91 ± 0.22 Viscosity (mPa*s) 2.01 ± 0.02 2.95 ± 0.01 2.15 ±0.05 2.71 ± 0.05

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Claims (7)

Links Burton RA and Fincher GB (2014). Evolution and development of cell walls in cereal grains. Front Plant Sci. 5:456. Jobling SA (2015). Membrane pore architecture of the CslF6 protein controls (l-3,l-4)-bglucan structure. Sci. Adv. l:el500069. Holme IB, Wendt T, Gil-Humanes J, Deleuran LC, Staker CG, Voytas DF and BrinchPedersen H, (2017). Evaluation of the mature grain phytase candidate HvPAPhya gene in barley (Hordeum vulgare L.) using CRISPR/Cas9 and TALENs. Plant Mol Biol 95:111–121; (DOI: 10.1007/sl 1103-017-0640-6). Hu G, Burton C, Hong Z and Jackson E (2014). A Mutayop of the cellulose-synthase-like (CslF6) gene in barley (Hordeum vulgare L.) partially affects the b-glucan content in grains. Journal of Cereal Science 59, 189-195. Lawrenson T, Shorinola O, Stacey N, Li C, Ostergaard L, Patron N, Uauy C, Harwood W. Induction of targeted, heritable MyTations in barley and Brassica oleracea using RNA-guided Cas9 nuclease. Genome Biol. 2015 Nov 30;16:258. doi:10.1186/sl3059-015-0826-7. Taketa S, Yuo T, Tonooka T, Tsumuraya Y, Inagaki Y, Haruyama N, Larroque O and Jobling SA (2012). Functional characterization of barley betaglucanless mutant demonstrates a unique role for CslF6 in (l,3;l,4)-3-D-glucan biosynthesis. J Exp Bot. 63(1):381-92. CLAIM 1. A barley plant or part thereof, wherein said barley plant carries a mutation in the CsIF6 gene, wherein said mutated CsIF6 gene encodes a mutant CsIF6 polypeptide, wherein said mutant CsIF6 is CsIF6 with the sequence SEQ ID NO: 1 or SEQ ID NO: 3 except that the mutant CsIF6 contains a substitution of amino acid 748 from SEQ ID NO: 1 or SEQ ID NO: 3 to a charged amino acid, or a substitution of amino acid 709 from SEQ ID NO: 1 or SEQ ID NO: 3 to a nonpolar amino acid, or a substitution of amino acid 847 from SEQ ID NO: 1 or SEQ ID NO: 3 per charged amino acid, wherein the charged amino acid is selected from the group consisting of Arg, His, Lys, Asp and Glu, wherein the kernels of said barley plant have a reduced content (1.3;1.4 )-β-glucans compared to barley kernels containing the wild-type CsIF6 gene but otherwise having the same genotype. 2. The barley plant according to claim 1, wherein said barley plant has a content of (1,3;1,4)-β-glucans in the range of 1 to 5% of the total dry matter weight of the kernels, for example from 1.3 to 3% of the total weight of dry matter of kernels, preferably from 1.3 to 2% of the total weight of dry matter of kernels. 3. The barley plant according to any one of claims 1 and 2, wherein the kernels of said barley plant have a (1,3;1,4)-β-glucan content of at least 30% and not more than 60%, preferably at least at least 40% and no more than 60% of the content of (1,3;1,4)-β-glucans of a barley plant carrying the wild-type CsIF6 gene, but otherwise having the same genotype. 4. The barley plant according to any one of claims 1 to 3, wherein said mutant CsIF6 polypeptide is CsIF6 with the sequence SEQ ID NO: 1 or SEQ ID NO: 3 except that the mutant CsIF6 contains a substitution at amino acid 847, wherein said substitution represents is the replacement of glycine (G) with glutamic acid (E). 5. The barley plant according to any one of claims 1 to 4, wherein the kernels of said barley plant have a DP3:DP4 ratio of no more than 2.5, such as no more than 2.1, for example in the range of 1.0 to 2, 1. 6. The barley plant according to any of the previous paragraphs, where the barley plant contains grains having a frequency of broken grains after threshing that is not more than 2 times higher than the frequency of broken grains after threshing grains of a barley plant that does not carry a mutation in the CsIF6 gene, but otherwise having the same genotype. 7. The barley plant according to any one of claims 1 to 3, wherein said mutant CsIF6 polypeptide is CsIF6 with the sequence SEQ ID NO: 1 or SEQ ID NO: 3 except that the mutant CsIF6 contains a substitution at amino acid 748, wherein said substitution represents is a substitution of glycine (G) for aspartic acid (D). -