Buckwheat plants with mutations in the SBE1 gene

Abstract

To obtain buckwheat plants with modified starch properties. [Solution] According to the present invention, a buckwheat plant is provided in which the activity of starch branching enzyme 1 (SBE1) is reduced or deficient.

Description

Technical Field

[0001] The present invention relates to buckwheat plants with reduced or defective starch branching enzyme 1 (SBE1) activity.

Background Art

[0002] Cultivated species of buckwheat plants classified in the Polygonaceae family include buckwheat (Fagopyrum esculentum) (hereinafter referred to as common buckwheat), tartary buckwheat (F. tataricum), and perennial buckwheat (F. cymosum). In Japan, common buckwheat and tartary buckwheat are mainly used as foods.

[0003] Generally, when using grains as foods, ensuring softness after food production is one of the important factors, and starch has a great influence on them. Starch is roughly classified into amylose in which glucose is polymerized linearly and amylopectin with branched chains. In grains, when the chain length of amylopectin becomes shorter, the food remains soft even when cooled, the gelatinization temperature decreases, and the bleeding during freezing and thawing decreases.

[0004] The amylopectin chain length is extended by starch synthase (SSIIa). Rice amylopectin is mainly classified into S-type and L-type depending on the presence or absence of SSIIa activity. S-type amylopectin has a higher ratio of short chains in the amylopectin side chains than L-type amylopectin and a lower starch gelatinization start temperature. For example, japonica rice has S-type amylopectin because it lacks SSIIa and is not likely to become hard even when cooled. Indica rice has L-type amylopectin and is likely to become lumpy when cooled. In addition, it has been reported that buckwheat plants lacking SSIIa activity have a urea breakdown property that is 1.5 times or more that of the wild type, and the gelatinization peak temperature is 95% or less compared to the wild type (Patent Document 1).

[0005] Starch branching enzyme 1 (SBE1) is an enzyme involved in the formation of amylopectin side chains. It is thought that SSIIa extends these side chains. It has been reported that rice varieties into which a functionally deficient rice SBE1 gene has been introduced have a higher proportion of short amylopectin side chains and reduced gluten hardening ability (Non-Patent Literature 1). Furthermore, it has been reported that rice varieties lacking SBE1 activity (SSIIa activity deficient and SBE1 activity deficient) have a starch gelatinization peak temperature measured by differential scanning calorimeter approximately 4°C lower than that of Koshihikari (SSIIa activity deficient) (Non-Patent Literature 2). [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2022-140409 [Non-patent literature]

[0007] [Non-Patent Document 1] Suzuki, Taro. 2019, "Development of 'Aichi Mochi 126,' a glutinous rice variety lacking the activity of starch branching enzyme 1 and exhibiting low mochi hardening properties," Journal of Plant Breeding Science, Vol. 21, 28-34. doi: 10.1270 / jsbbr.18J13 [Non-Patent Document 2] Noriaki Aoki, University of Tsukuba, Doctoral dissertation, 2015, "Breeding and physiological studies on suitability for rice flour bread" https: / / cir.nii.ac.jp / crid / 1910020910756854400 [Overview of the project] [Problems that the invention aims to solve]

[0008] In rice, there are reports that gelatinization temperature decreases in varieties lacking both SSIIa activity and SBE1 activity (Non-Patent Literature 2), but there are no reports for rice varieties lacking only SBE1 activity. To date, there is no knowledge as to whether the starch gelatinization temperature actually changes due to the suppression or deficiency of SBE1 activity in buckwheat.

[0009] To expand the demand for buckwheat, developing buckwheat-containing foods with improved physical properties is crucial. To accommodate diverse consumer preferences and cooking methods, improving the starch properties of buckwheat seeds and flour is essential. In particular, there is a growing need for buckwheat seeds and flour with lower starch gelatinization temperatures. Buckwheat seeds and flour with lower gelatinization temperatures allow for shorter cooking times and a smoother, softer texture, potentially gaining consumer support in terms of both convenience and texture. Furthermore, buckwheat seeds and flour with lower gelatinization temperatures contribute to suppressing hardening and starch retrogradation during refrigerated storage, as well as preventing a decrease in moisture retention. Starch retrogradation is a major cause of hardening and texture deterioration in buckwheat-containing foods during refrigerated storage; however, starch with a lower gelatinization temperature is less prone to recrystallization, thus maintaining a good texture throughout storage. Such improvements are expected to enhance consumer convenience and satisfaction, leading to market expansion for buckwheat-containing foods. Therefore, the object of the present invention is to obtain a buckwheat plant with modified starch properties. [Means for solving the problem]

[0010] The inventors of this invention obtained a buckwheat plant from among buckwheat plants that had undergone mutation treatment, in which a mutation in the SBE1 gene resulted in a lower starch gelatinization temperature compared to the wild type, and thus completed the present invention.

[0011] The present invention provides the following inventions. [1] Buckwheat plants with reduced or deficient starch branching enzyme 1 (SBE1) activity. [2] A buckwheat plant as described in [1], in which reduced or absent SBE1 activity is due to a mutation in the SBE1 gene. [3] The buckwheat plant according to [2], wherein the mutation is a missense mutation, a nonsense mutation, a frameshift mutation, or an in-frame insertion or deletion, a splice site mutation, or a mutation in an expression regulatory region. [4] The wild type of the SBE1 gene having the aforementioned mutation is (1) The SBE1a gene as defined in (i), (ii), or (iii) below: (i) A gene containing the nucleotide sequence shown in Sequence ID No. 6, or (ii) A gene that contains a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in Sequence ID No. 6, and which encodes a protein having SBE1 activity in buckwheat plants, or (iii) A gene encoding a protein having SBE1 activity in a buckwheat plant, comprising the amino acid sequence shown in Sequence ID No. 3, or an amino acid sequence having 90% or more sequence identity with said amino acid sequence. (2) The SBE1b gene as defined in (i), (ii), or (iii) below: (i) A gene containing the nucleotide sequence shown in Sequence ID No. 7, or (ii) A gene that contains a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in Sequence ID No. 7, and which encodes a protein having starch branching enzyme 1 activity in buckwheat plants, or (iii) A gene encoding a protein having SBE1 activity in a buckwheat plant, comprising the amino acid sequence shown in Sequence ID No. 4 or 11, or an amino acid sequence having 90% or more sequence identity with said amino acid sequence. A buckwheat plant described in [2] or [3], which is one of the following. [5] The buckwheat plant described in [4], wherein the mutation is a mutation in which the base at position 4446 in the nucleotide sequence shown in Sequence ID No. 7 is deleted, or a mutation in which the base at position 8194 in the nucleotide sequence shown in Sequence ID No. 7 is replaced from adenine to cytosine. [6] The genus Fagopyrum described in [1]-[5], which is buckwheat (Fagopyrum esculentum), Tartary buckwheat (F. tataricum), perennial buckwheat (F. cymosum), or wild buckwheat (F. homotropicum), or a hybrid of any of these. [7] The plant body of a buckwheat plant as described in any one of the items [1] to [6]. A method for breeding buckwheat plants, including using the plant specimens or their offspring described in [8] [7]. [9] [7] The harvested products, propagating materials, or processed products of the plant described above.

[10] Foods containing raw materials derived from the harvested or processed products of the plants described in [7].

[11] Methods for increasing starch urea disintegration, increasing alkali disintegration of buckwheat grains, or lowering the starch gelatinization temperature in buckwheat plants by reducing or eliminating the activity of at least one of the following proteins: (A) Proteins consisting of the amino acid sequences shown in Sequence ID No. 3, 4, or 11; (B) A protein having an amino acid sequence that is 90% or more identical to the amino acid sequence shown in Sequence ID No. 3, 4, or 11, and that has starch branching enzyme 1 activity in buckwheat plants; (C) A protein having the activity of starch branching enzyme 1 of buckwheat plants, consisting of an amino acid sequence in which 1 to 60 amino acids are deleted, substituted, or added in the amino acid sequence shown in Sequence ID No. 3, 4, or 11. [Brief explanation of the drawing]

[0012] [Figure 1A] Figure 1A shows the estimated cDNA sequence of the wild-type buckwheat SBE1b gene. In the mutant, the cytosine (C) at position 74 of sequence number 2 is deleted. [Figure 1B] Figure 1B shows the putative cDNA sequence in alternative splicing of the wild-type buckwheat SBE1b gene. In the mutant, the adenine (A) at position 712 of sequence number 10 is changed to cytosine (C). [Figure 1C] Figure 1C shows the amino acid sequences of the SBE1b putative translation product (wild type) and the SBE1b putative translation product (mutant = buckwheat UD1). In the mutant, a frameshift occurs due to the deletion of cytosine (C) at position 74 of the putative cDNA sequence of the buckwheat SBE1b gene (SEQ ID NO: 2). As a result, the amino acid residue at position 26 of the amino acid sequence of the buckwheat SBE1b putative translation product is threonine (T) in the wild type (SEQ ID NO: 4), but proline (P) in the mutant (SEQ ID NO: 5). In the mutant (SEQ ID NO: 5), an early stop codon occurs at position 46. [Figure 1D]Figure 1D shows the amino acid sequence of the putative translated product of SBE1b (wild type) when alternative splicing occurred (SEQ ID NO: 11). The amino acid residue at position 238 in the amino acid sequence of the putative translated product of buckwheat SBE1b (SEQ ID NO: 11) is lysine (K) in the wild type, but glutamine (Q) in the mutant type. [Figure 1E] Figure 1E shows the genomic sequence of the wild-type buckwheat SBE1a gene (SEQ ID NO: 6). The bases in the exon regions are shown in bold and underlined. [Figure 1F] Figure 1F shows the genomic sequence of the wild-type buckwheat SBE1b gene (SEQ ID NO: 7). The bases in the exon regions are shown in bold and underlined. In alternative splicing, the ATG present at position 124 in the genomic sequence shown in SEQ ID NO: 7 is utilized as the translation start codon, and the region from positions 4373 to 5168 (shown in italics) is spliced out as an intron, thereby presumably generating the splicing product shown in SEQ ID NO: 10. On the other hand, in the cDNA base sequence shown in SEQ ID NO: 2 (which can be said to be another splicing product), the ATG present at position 4373 serves as the translation start codon. [Figure 2] Figure 2 shows the urea degradability in buckwheat seeds of the wild type "IH3" and the SBE1b mutant type "Sukesuke UD1 strain". [Figure 3] Figure 3 is a graph showing the gene expression levels of the SSIIa1, SSIIa2, SBE1a, and SBE1b genes in maturing seeds. [Figure 4] Figure 4 is a graph showing the change over time in the breaking load of refrigerated stored noodle bands produced from buckwheat flour of the wild-type buckwheat "IH3", the low-amylose buckwheat "Kyushu 12" (deficient only in GBSSa), and the SBE1b mutant buckwheat "Sukesuke UD1 strain".

Mode for Carrying Out the Invention

[0013] The following description of the present invention may be based on representative embodiments and specific examples, but the present invention is not limited to such embodiments. In this specification, positions in a nucleotide sequence are counted with the 5' end of the nucleotide sequence as the 1st position. Positions in an amino acid sequence are counted with the N-terminal amino acid of the sequence as the 1st position.

[0014] [New Buckwheat species] This invention provides buckwheat plants in which starch branching enzyme 1 (SBE1) activity is reduced or deficient. Furthermore, the present invention provides a plant of the genus Buckwheat that satisfies at least one of the following conditions. • Starch urea disintegration is increased compared to the wild type. • The alkalinity of buckwheat grains is increased compared to the wild type. • The starch gelatinization onset temperature is 90% or less compared to the wild type. • The starch gelatinization peak temperature is 85% or less compared to the wild type. • The starch gelatinization onset temperature, as measured by differential scanning calorimeter, is 59.5°C or lower. • The starch gelatinization peak temperature measured by differential scanning calorimeter is 65°C or lower.

[0015] In the examples described below, absorbance obtained by the starch urea disintegration test was used as an indicator for evaluation. As a result, compared to the wild type, the buckwheat plants of the genus Buckwheat in which the SBE1 activity of the present invention was reduced or deficient had an absorbance at least 16 times that of the wild type.

[0016] (Decreased or deficient in starch branching enzyme 1 (SBE1) activity) Starch branching enzyme 1 (SBE1) is an enzyme that synthesizes amylopectin side chains and is a type of enzyme that catalyzes the formation of α-1,6-glucosidic bonds through glycosyltransferase reactions.

[0017] In this invention, reduced or deficient starch branching enzyme 1 (SBE1) activity means that the function of the SBE1 protein in the plant is reduced or not functioning normally compared to the wild type. In this invention, reduced or not functioning normally of the SBE1 protein is not limited to cases where SBE1 activity is reduced by a specific mechanism. Reduced or deficient activity includes not only the suppression of SBE1 enzyme activity but also the suppression of SBE1 gene expression. More specifically, suppression of activity includes not only the inhibition of SBE1 activity but also mutations in the SBE1 gene that prevent the production of active SBE1, reduced SBE1 activity, and mutations in the expression regulatory regions of the SBE1 gene (promoter region, transcription factor binding region, splicing-related region, etc.) that suppress SBE1 production.

[0018] Whether SBE1 activity is reduced or absent can be evaluated by the proportion of short amylopectin side chains (approximately 6-11 degrees of polymerization) compared to the wild type. Furthermore, a higher proportion of short amylopectin side chains compared to the wild type can be evaluated by increased urea disintegration, increased alkali disintegration, a lower gelatinization initiation temperature, and a lower gelatinization peak temperature, as will be described later. The proportion of short amylopectin side chains can also be measured by freeing the side chains through enzymatic debranching (e.g., isoamylase treatment), followed by separation and quantification using HPAEC-PAD (High Performance Anion Exchange Chromatography / Pulsed Amylopectin Peripheral Detection). This method allows for highly accurate quantification of the short chain content.

[0019] As shown in the examples described below, in buckwheat plants, the SBE1a and SBE1b genes are expressed, and the SBE1a and SBE1b proteins are considered to function normally. Buckwheat plants in which these proteins function normally can be said to be wild-type plants. In this specification, when referring to buckwheat plants, SBE1 refers to SBE1a and SBE1b unless otherwise specified. In a particular embodiment, "wild-type buckwheat plant" refers to variety IH3 (developed by the Hokkaido Agricultural Research Center). This variety is available from the National Agriculture and Food Research Organization (Plant Production Science, 20(4), 384-388).

[0020] In buckwheat plants exhibiting reduced or deficient starch branching enzyme 1 (SBE1) activity according to the present invention, the reduction or deficiency of SBE1 activity is attributed to a mutation in the SBE1 gene. This mutation in the SBE1 gene can be obtained through spontaneous mutation or natural selection during breeding, as well as through chemical mutagenesis treatment or by artificial genome editing technology. Chemical mutagenesis treatment is not particularly limited and, for example, can be induced by using an alkylating agent such as ethyl methanesulfonate (EMS) to induce mutations such as base substitutions. On the other hand, artificial genome editing technology is not particularly limited and can be achieved by introducing deletions, substitutions, or insertions into a target base sequence using, for example, CRISPR / Cas systems, TALENs, ZFNs, etc.

[0021] The buckwheat plants of the present invention have a mutation in at least one of the SBE1a gene and the SBE1b gene, thereby suppressing the function of the gene.

[0022] In the above-mentioned buckwheat plant of the present invention, the wild-type SBE1 gene having the aforementioned mutation may be any of the following: (1) The SBE1a gene as defined in (i), (ii), or (iii) below: (i) A gene containing the nucleotide sequence shown in Sequence ID No. 6, or (ii) A gene that contains a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in Sequence ID No. 6, and which encodes a protein having SBE1 activity in buckwheat plants, or (iii) A gene encoding a protein having SBE1 activity in a buckwheat plant, comprising the amino acid sequence shown in Sequence ID No. 3, or an amino acid sequence having 90% or more sequence identity with said amino acid sequence. (2) The SBE1b gene as defined in (i), (ii), or (iii) below: (i) A gene containing the nucleotide sequence shown in Sequence ID No. 7, or (ii) A gene that contains a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in Sequence ID No. 7, and which encodes a protein having starch branching enzyme 1 activity in buckwheat plants, or (iii) A gene encoding a protein having SBE1 activity in a buckwheat plant, comprising the amino acid sequence shown in Sequence ID No. 4 or 11, or an amino acid sequence having 90% or more sequence identity with said amino acid sequence.

[0023] With regard to the present invention, considering polymorphisms of the SBE1 gene among species, varieties, and strains of buckwheat plants, these variations are also included within the scope of the present invention. Therefore, the base identity to SEQ ID NO: 6 or SEQ ID NO: 7 is 90% or higher, and may be 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99%. Furthermore, with regard to the present invention, considering polymorphisms of the SBE1 protein sequence among species, varieties, and strains of buckwheat plants, these variations are also included within the scope of the present invention. Therefore, the amino acid sequence identity to SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 11 is 90% or higher, and may be 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99%.

[0024] The sequence listing of this application shows the sequence of the buckwheat SBE1a gene (wild type) as sequence number 6 and the sequence of the buckwheat SBE1b gene (wild type) as sequence number 7. In addition, the putative cDNA sequence of the buckwheat SBE1a gene (wild type) is shown as sequence number 1, the putative cDNA sequence of the buckwheat SBE1b gene (wild type) is shown as sequence number 2, and the putative cDNA sequence in alternative splicing of the buckwheat SBE1b gene (wild type) is shown as sequence number 10.

[0025] Furthermore, the sequence listing of this application includes the amino acid sequence of buckwheat SBE1a (wild type) as SEQ ID NO: 3, the amino acid sequence of buckwheat SBE1b (wild type) as SEQ ID NO: 4, and the amino acid sequence of buckwheat SBE1b (wild type, alternative splicing product (protein isoform produced by alternative splicing)) as SEQ ID NO: 11. Alternative splicing is a fundamental gene expression regulatory mechanism inherent in buckwheat plants, and this alternative splicing product is considered to be the normal wild-type isoform of the SBE1b gene.

[0026] In one embodiment, mutations in the SBE1 gene are, for example, nucleotide changes due to the substitution, deletion, or insertion of one or more nucleotides compared to the wild-type sequence. Mutations that the buckwheat plants of the present invention may have may include, for example, nonsensical mutations (such as nonsense and missense mutations), frameshift mutations, in-frame insertions or deletions, splice site mutations, and / or mutations in expression regulatory regions such as promoters or enhancers. Nonsense mutations (which result in stop codons) and missense mutations generally produce incomplete and nonfunctional proteins. Frameshift mutations cause a shift in the codon reading frame, resulting in the translation of a protein with a completely different amino acid sequence from the original, often leading to the appearance of early stop codons and the production of incomplete proteins. Splice site mutations generally result in abnormal splicing, the generation of abnormal mRNA, and the production of nonfunctional proteins. Mutations in expression regulatory regions may lead to a decrease in mRNA transcripts compared to the wild-type sequence, thereby reducing protein synthesis. These phenomena are also true for multiple mRNAs resulting from alternative splicing. The mutations in the buckwheat plants of the present invention are not limited to the above mutations, but may be any mutations that lead to a decrease or deficiency of SBE1 activity.

[0027] In one embodiment, the mutation in the buckwheat plant of the present invention is a frameshift mutation, which results in the appearance of an early stop codon and prevents the synthesis of normal SBE1 protein, thus reducing or eliminating SBE1 activity. In another embodiment, the mutation in the buckwheat plant of the present invention is a missense mutation, which prevents the synthesis of normal SBE1 protein through amino acid substitution, thus reducing or eliminating SBE1 activity. In yet another embodiment, the alternative splicing shown in the present invention is an inherent characteristic of buckwheat plants, but even in that case, the missense mutation in the buckwheat plant of the present invention results in the synthesis of abnormal SBE1 protein, thus reducing or eliminating SBE1 activity. As shown in the examples described later, the buckwheat plant "Sugaya UD1 strain" having a mutation in the SBE1 gene has a frameshift mutation in the SBE1b gene, and it is considered that the function of the SBE1b gene is suppressed. Specifically, a frameshift occurs due to the deletion of cytosine (C) at position 74 of the predicted cDNA sequence (SEQ ID NO: 2) of the buckwheat SBE1b gene (position 4446 in the genome sequence (SEQ ID NO: 7)). As a result, the amino acid residue at position 26 of the amino acid sequence of the predicted buckwheat SBE1b translation product is threonine (T) in the wild type (SEQ ID NO: 4), but proline (P) in the mutant (SEQ ID NO: 5), and the subsequent amino acid sequence also differs from the wild type. Furthermore, in the mutant (SEQ ID NO: 5), a stop codon occurs at position 46, and the amino acid length is significantly shorter compared to the wild type. Therefore, it is thought that the activity of the buckwheat SBE1b protein is deficient in the "Su-type UD1 strain." In addition, the "Su-type UD1 strain," a buckwheat plant with a mutation in the SBE1 gene, has a substitution from a basic amino acid residue to a neutral amino acid residue in the amino acid sequence of a key region that is highly conserved in SBE1 orthologs, etc., in the alternative splicing product. It is thought that this substitution significantly reduces or eliminates the activity of the buckwheat SBE1b protein. Specifically, in the mutant, the adenine (A) at position 712 of the putative cDNA (SEQ ID NO: 10) in the alternative splicing of the wild-type buckwheat SBE1b gene (position 8194 in the genome sequence (SEQ ID NO: 7)) is changed to cytosine (C).This mutation causes the amino acid residue at position 238 of the amino acid sequence (SEQ ID NO: 11) of the buckwheat SBE1b predicted translation product to be glutamine (Q) in the mutant, whereas in the wild type it is lysine (K).

[0028] Therefore, the present invention relates to a buckwheat plant in which SBE1 activity is reduced or deficient, (i) A polynucleotide containing a sequence in which the base at position 4446 in the sequence shown in SEQ ID NO: 7 is deleted, or a polynucleotide in which the base at position 8194 in the sequence shown in SEQ ID NO: 7 is changed from adenine to cytosine. (ii) To provide a plant of the genus Buckwheat that has a polynucleotide containing a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in Sequence ID No. 7, in which the nucleotide at position 4446 is deleted, or a polynucleotide in which the nucleotide at position 8194 in the sequence shown in Sequence ID No. 7 is changed from adenine to cytosine. Sequence ID 7 is the sequence of the buckwheat SBE1b gene (wild type). The cytosine at position 4446 in the sequence shown in Sequence ID 7 corresponds to the cytosine at position 74 in the putative cDNA sequence of the buckwheat SBE1b gene (Sequence ID 2). Also, the adenine at position 8194 in the sequence shown in Sequence ID 7 corresponds to the adenine at position 712 in the putative cDNA sequence of the buckwheat SBE1b gene in alternative splicing (Sequence ID 10).

[0029] Buckwheat plants with mutations in the SBE1 gene are not limited to the mutations described above; similar activity deficiency effects may be obtained with other mutations as well. For example, mutations that delete or replace cysteine ​​residues related to the three-dimensional structure of the protein, which greatly affects activity, mutations that delete or replace amino acids in the active site with amino acids of different polarity, or mutations that delete or replace one or several amino acids with other amino acids of different polarity in an amino acid region that is highly conserved among plants are thought to reduce or eliminate SBE1 activity, and similar effects can be expected with such mutations.

[0030] The presence of a gene mutation can be confirmed by methods such as those listed below: decoding the mutated base sequence itself using base sequence analysis methods such as the Sanger assay or next-generation DNA analysis technology; detecting differences in the cleavage status of genomic DNA or PCR amplification fragments using restriction enzymes that recognize the mutated base sequence; detecting differences in the mutated base sequence using technologies such as LGC Genomics' KASP (Kompetitive Allele Specific PCR) genotyping assay or high-resolution melting curve (HRM) analysis; or PCR-SSCP (Single Nucleotide Conformation Polymorphism). Detection may also be performed using a DNA marker linked to the mutated base region.

[0031] Mutations in the SBE1 gene or its expression regulatory region may be carried out by various means. Examples of methods for obtaining buckwheat plants with mutations in the SBE1 gene or its expression regulatory region include selection from mutant strains, genetic engineering, RNA interference, genome editing, artificial genome synthesis, and genome methylation. In a preferred embodiment, selection from mutant strains is used because it allows for more reliable production of the plant.

[0032] The buckwheat plants exhibiting reduced or deficient SBE1 activity according to the present invention may be bred from existing varieties or lines. The buckwheat plants thus obtained can be distinguished from existing varieties or lines by at least one of the following characteristics: a high ratio of short amylopectin side chains, high urea-disintegration and alkali-disintegration properties, and a low gelatinization peak temperature.

[0033] There are no particular limitations on the varieties and lines used as starting materials when acquiring mutant strains, or the varieties and lines used in breeding. Examples of buckwheat varieties include Kitawase Soba, Botan Soba, Kitayuki, Hashikami Wase, Iwate Wase, Mogami Wase, Hashikami Wase, Hitachi Aki Soba, Shinano No. 1, Shinshu Oosoba, Shinano Natsu Soba, Fukui Zairaishu, Kochi Zairaishu, Miyazaki Ootsubu, AOI, KOMA, NARO-FE-1, Akiakane, Gamma no Irodori, Kitano Mashu, Kitamitsuki, Great Ruby, Cobalt no Chikara, Sachiizumi, Sanrutin, and Soba Intermediate Parent No. 1. Examples of buckwheat varieties include Tachiakane, Dewakaori, Toyomusume, Natsumi, Nijiyutaka, Horominori, Miyazaki Ootsubu, Ruchiking, Reranokaori, Natsuyoshi, Aizu no Kaori, Kaida Wase, Miyazaki Wase Kaori, Takamine Ruby, Takamine Ruby 2011, Yamagata BW5, Izumo no Mai, Haru no Ibuki, Hitachi Akisoba, Shin'ei Red, Shinshu Oosoba, Nagano S11, Nagano S8, Shimada Scarlet, Hida No. 1, and Hokkai No. 3. Examples of buckwheat varieties include Shin'ei Yellow, Hokkai T8, Hokkai T9, Hokkai T10, Hokuriku No. 4, Ki no Chikara, Ki no Takara, Ki no Yutaka, Daizen, Shinano Kurotsubu, Daruma Dattan, Aeon no Kisai, Manten Kirari, Nishi no Haruka, and Aeon no Kisai. Many of these can be obtained from seed companies or related organizations.

[0034] This application is the first to disclose that in buckwheat plants, a deficiency in SBE1 activity alters the properties of the starch contained within.

[0035] Starch branching enzyme 1 (SBE1) is an enzyme involved in the formation of amylopectin side chains, and it is thought that SSIIa extends these side chains. In rice, there are reports on varieties lacking both SSIIa and SBE1 activity (e.g., Non-Patent Document 1), but there are no reports on the properties of starch in varieties lacking only SBE1 activity. Therefore, it was impossible to predict what effect a lack of SBE1 activity in buckwheat would have on the properties of starch.

[0036] Furthermore, rice has one SBE1 gene, while buckwheat has two. The two isozymes in buckwheat may have different effects. In addition, rice is a monocotyledonous plant, while buckwheat is a dicotyledonous plant, and they are phylogenetically distant. Therefore, it was impossible to predict how effective the remaining isozymes would be without actually inducing mutations in the isozymes in buckwheat.

[0037] (Urea-disintegrating, alkali-disintegrating, gelatinization temperature) The buckwheat plants of the present invention have improved starch properties. Specifically, compared to conventional buckwheat plants, the proportion of short-chain amylopectin side chains is higher, resulting in increased urea-disintegration and alkali-disintegration properties, as well as a lower gelatinization temperature. Measuring amylopectin chain length requires special equipment and is relatively complicated. However, evaluating urea-disintegration and alkali-disintegration properties does not require special equipment, and if the difference is large, it can be observed with the naked eye, and the procedure is simple.

[0038] In relation to the present invention, when referring to urea disintegration properties of buckwheat plants, unless otherwise specified, it refers to the urea disintegration properties of buckwheat flour (endosperm) obtained by milling buckwheat seeds obtained from the target buckwheat plant using a conventional milling machine. Urea disintegration properties may sometimes be expressed as the degree of urea disintegration in the starch of whole grain flour, but those skilled in the art can perform appropriate calculations for buckwheat flour milled using a milling machine and determine the difference in the degree of urea disintegration properties by comparing it with buckwheat flour distributed on the market. Generally, buckwheat flour is produced by rolling buckwheat (grains) and separating the crushed material with a sieve.

[0039] The urea-disintegrating properties of starch can be quantified by colorimetrically measuring the iodine-starch reaction with starch solubilized in 1-4 M urea from buckwheat flour. More specifically, for example, after removing the hulls from buckwheat seeds, approximately 20 mg of buckwheat flour, ground in a mortar, is weighed into a 0.5 ml tube, 1 ml of 3 M urea is added, and the mixture is stirred at 25°C for 16 hours (200 rpm). 180 μL of iodine solution (0.002% iodine-0.02% potassium iodide solution) is added to 20 μL of the supernatant, and the absorbance at 690 nm is measured using a microplate reader (ThermoScientific: MultiskanFC). At this time, the absolute value of the measurement obtained (absorbance divided by the weight of the buckwheat flour) may be used for evaluation, but it may also be evaluated using a relative value compared to a control buckwheat flour (buckwheat flour obtained from wild-type buckwheat, or commercially available buckwheat flour).

[0040] A high proportion of short-chain amylopectin side chains in starch can be detected as a difference in urea disintegration, that is, a difference in the degree to which amylopectin is solubilized in the presence of urea. In other words, for example, when starch is disintegrated (solubilized) in the presence of 3M urea, short amylopectin is more easily solubilized than long amylopectin. Therefore, the amylopectin chain length can be measured by comparing the soluble fractions after color development using the iodine-starch reaction (Hirokazu Sato, Shinichi Saito, Tomohiko Yoshida. Selection method of glutinous rice varieties based on mochi hardening properties, gelatinization characteristics and urea disintegration properties. Journal of the Crop Science Society of Japan, 74(3), 310-315 (2005) doi:10.1626 / jcs.74.310).

[0041] The alkali-disintegrating properties of buckwheat grains can be determined by observing whether or not starch dissolves after immersing the grains in an alkaline solution for several hours. For example, if hulled buckwheat grains are immersed in a 1.5% potassium hydroxide solution and left to stand at room temperature, the grains are considered highly alkali-disintegrating if starch dissolution is observed, and less alkali-disintegrating if no starch dissolution is observed.

[0042] If the urea-disintegrating properties of buckwheat or the alkali-disintegrating properties of buckwheat grains can be increased, it is possible to keep cooked foods soft, and by modifying their physical properties, it is thought that their use can be expanded to food applications other than noodles.

[0043] In relation to the present invention, when referring to starch gelatinization temperature or gelatinization temperature, unless otherwise specified, it refers to the value (°C) measured for buckwheat flour (endosperm) obtained by milling buckwheat seeds obtained from the target buckwheat plant using a conventional milling machine. The gelatinization temperature can be measured by differential scanning calorimetry (DSC).

[0044] More specifically, for example, after removing the hulls from buckwheat seeds, approximately 10 mg of buckwheat flour is weighed out and placed in a silver pan. Distilled water is added to achieve a starch concentration of 30% (based on dry weight, weight / weight). Using this sealed pan with the added distilled water as a reference, the scan is performed from 25°C to 130°C at a heating rate of 2°C / minute.

[0045] The degree to which the gelatinization temperature of the buckwheat plant of the present invention is reduced is preferably such that it affects the retention of softness after cooking in a food product containing the buckwheat plant as a raw material.

[0046] Specifically, the degree of reduction in the gelatinization peak temperature of the buckwheat plant of the present invention (gelatinization peak temperature for the target buckwheat plant (°C) / gelatinization peak temperature for the wild type (°C) × 100) is 90% or less, preferably 89% or less, more preferably 88% or less, even more preferably 87% or less, 86% or less, and 85% or less. According to the inventors' studies, the gelatinization peak temperature of the buckwheat strain "Su-kei UD1-kei" obtained by the inventors is approximately 85% when compared to wild-type buckwheat.

[0047] Alternatively, the gelatinization peak temperature of the buckwheat plant of the present invention is 65°C or lower, preferably 64°C or lower, and more preferably 63°C or lower. According to the inventors' studies, the gelatinization peak temperature of the buckwheat strain "Su-kei UD1-kei" obtained by the inventors is approximately 62.7°C.

[0048] Specifically, the degree of reduction in the gelatinization onset temperature of the buckwheat plant of the present invention (gelatinization onset temperature for the target buckwheat plant (°C) / gelatinization onset temperature for the wild type (°C) × 100) is 85% or less, preferably 84% or less, more preferably 83% or less, and even more preferably 82% or less. According to the inventors' studies, the gelatinization onset temperature of the buckwheat strain "Su-kei UD1-kei" obtained by the inventors is approximately 81.2% compared to wild-type buckwheat.

[0049] Alternatively, the gelatinization start temperature of the buckwheat plant of the present invention is 59.5°C or lower, preferably 59°C or lower, more preferably 58°C or lower, 57°C or lower, 56°C or lower, or 55°C or lower. According to the inventors' studies, the gelatinization peak temperature of the buckwheat strain "Su-kei UD1-kei" obtained by the inventors is approximately 54.5°C.

[0050] The phenotype of a buckwheat population (variety or line) is not determined by the phenotype of a single individual, but is preferably evaluated by the average of measurements from multiple individuals. Therefore, urea disintegration, alkali disintegration, and gelatinization temperature are also evaluated by the average of measurements from multiple individuals.

[0051] (New buckwheat variety) An example of a buckwheat plant with reduced or deficient starch branching enzyme 1 (SBE1) activity provided by the present invention is the "Su-kei UD1-kei".

[0052] The "Sugae UD1 strain" is self-pollinating and is a strain obtained by the method described in the examples of this specification, and has the following characteristics. • Scientific characteristics (morphological, cultivation characteristics, physiological characteristics, etc.) Taxonomic position: It belongs to the common buckwheat (Fagopyrumesculentum) lineage. It is an annual, upright, branching plant. After sowing, cotyledons unfold, followed by true leaves, and flower clusters develop on the main stem and branches before fruiting. • Origin: Developed through spontaneous mutation breeding at the Kyushu Okinawa Agricultural Research Center. • Cultivation conditions: For the survival confirmation test (germination test conditions), the seeds are immersed in 70% ethanol for 1 minute for sterilization, stirred with a stirrer for 30 minutes in a 1% (effective chlorine concentration) sodium hypochlorite solution, washed 5 times with sterile water, and sown on filter paper soaked in sterile water in a petri dish at 25°C (tolerance range 22-28°C) in the dark. Seedlings that have grown crown roots are judged to have germinated. The germination test will last 12 days. Outdoor weather conditions for the plants are an average daily temperature of 13°C-18°C, a maximum daily temperature of 18°C-25°C, a minimum daily temperature of 10°C-18°C, good drainage, monthly rainfall of 200-400 mm, and more than 100 hours of sunshine per month. • Seed storage method: 5℃

[0053] The "Su-type UD1 strain" is a population in which the mutant SBE1 gene has been fixed to homozygous status. As shown in the examples, the Su-type UD1 strain has increased urea disintegration compared to the wild type, and since this increase in urea disintegration is observed over multiple growing seasons, the Su-type UD1 strain is a stable strain in this trait.

[0054] (Physical properties of buckwheat-containing foods) The inventors have found that buckwheat-containing foods made using buckwheat flour milled from buckwheat plants with reduced or deficient SBE1 activity are less prone to deterioration. In the examples described below, the breaking loads of noodle sheets made from buckwheat flour of wild-type buckwheat "IH3", low-amylose buckwheat "Kyushu No. 12" (only GBSSa is deficient), and SBE1b mutant buckwheat "Sukei UD1" were compared. The breaking loads of wild-type buckwheat "IH3" and low-amylose buckwheat "Kyushu No. 12" increased with the passage of storage time, but the breaking load of SBE1b mutant buckwheat "Sukei UD1" did not increase even after 9 days. Since breaking load is an indicator of hardness, it is thought that a new type of buckwheat-containing food can be developed by using buckwheat flour obtained from buckwheat plants with reduced or deficient SBE1 activity as a raw material.

[0055] [Method for modifying starch from buckwheat plants] The present invention provides a method for increasing starch urea disintegration, increasing alkali disintegration of buckwheat grains, or lowering the starch gelatinization temperature in buckwheat plants by reducing or eliminating the activity of at least one of the following proteins. Here, starch gelatinization temperature refers to the starch gelatinization peak temperature and the starch gelatinization onset temperature. (A) Proteins consisting of the amino acid sequences shown in Sequence ID No. 3, 4, or 11; (B) A protein having an amino acid sequence that is 90% or more identical to the amino acid sequence shown in Sequence ID No. 3, 4, or 11, and that has starch branching enzyme 1 activity in buckwheat plants; (C) A protein having the activity of starch branching enzyme 1 of buckwheat plants, consisting of an amino acid sequence in which 1 to 60 amino acids are deleted, substituted, or added in the amino acid sequence shown in Sequence ID No. 3, 4, or 11.

[0056] Furthermore, with respect to the present invention, considering the polymorphism of SBEI due to differences in species, varieties, and strains, these mutations are also included within the scope of the present invention. Therefore, the amino acid sequence identity to SEQ ID NOs: 3, 4, or 11 is 90% or higher, preferably 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99%. Similarly, the number of deletions, substitutions, or additions in the amino acid sequence is 1 to 60, but may be 1 to 30, 1 to 9, or 1 to 4. If the substitutions are to amino acids with similar properties, there may be even more substitutions, etc. Means for creating polynucleotides or proteins relating to such amino acid sequences are well known to those skilled in the art. With respect to the present invention, the fact that a protein has the starch branching enzyme 1 activity of buckwheat plants can be confirmed, for example, by measuring the amylopectin chain length and observing a high ratio of short amylopectin side chains. The ratio of short-chain amylopectin side chains can be measured by freeing the side chains through enzymatic debranching and then separating and quantifying them using HPAEC-PAD. Furthermore, it can also be confirmed by measuring urea disintegration, alkali disintegration, and starch gelatinization temperature.

[0057] In this invention, when referring to a base sequence (sometimes called a nucleotide sequence) or an amino acid sequence, unless otherwise specified, it means the percentage of matching nucleotides or amino acids shared between two sequences when the two sequences are aligned in the most optimal manner. That is, identity can be calculated as (number of matching positions / total number of positions) × 100, and can be calculated using commercially available algorithms. Such algorithms are incorporated into the NBLAST and XBLAST programs described in Altschuletal., J.Mol.Biol.215(1990)403-410. More specifically, the search and analysis of the identity of base sequences or amino acid sequences can be performed using algorithms or programs well known to those skilled in the art (e.g., BLASTN, BLASTP, BLASTX, ClustalW). When using a program, the parameters can be appropriately set by those skilled in the art, or the default parameters of each program may be used. The specific methods of these analysis methods are also well known to those skilled in the art.

[0058] In this specification, when referring to a high degree of identity with respect to a base sequence or amino acid sequence, unless otherwise specified, it means a sequence identity of at least 70%, preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, even more preferably 95% or more, even more preferably 97.5% or more, and even more preferably 99% or more.

[0059] [Breeding methods, methods for producing buckwheat plants] The present invention also provides a method for breeding novel buckwheat plants using the buckwheat plants of the present invention. The breeding method of the present invention is characterized by using the buckwheat plants of the present invention, their plant bodies, or their progeny as breeding material. The breeding method is not particularly limited and examples include crossing, selection of mutants, backcrossing, genetic recombination, cell fusion, genome editing, etc. In one embodiment of the present invention, breeding can be performed using a mutated SBE1 gene as an indicator. In this case, the mutation in the SBE1 gene can be identified by molecular markers, base sequence analysis, allele identification by PCR, SNP typing, or other molecular biological methods, and individuals possessing the desired allele can be selected.

[0060] The breeding objectives are not particularly limited and include, for example, increased yield, improved ecological characteristics, lodging resistance, limited growth, moisture tolerance, and improved quality (improvement of functional components such as rutin, improvement of proteins, improvement of aroma components, and resistance to pre-harvest sprouting).

[0061] The present invention also provides a method for producing buckwheat plants with relatively high urea-disintegration and alkali-disintegration properties, and buckwheat plants with relatively low gelatinization onset temperature and gelatinization peak temperature. The method for producing buckwheat plants according to the present invention is characterized by comprising the steps of mutating the SBE1 gene of a buckwheat plant, or propagating a buckwheat plant with a mutated SBE1 gene. Various buckwheat varieties and lines can be used as starting materials, but for example, self-pollinating buckwheat varieties (such as Buckwheat Intermediate Parent Line No. 1) can be used.

[0062] [Harvested produce, breeding materials, processed goods, food products] In relation to the present invention, the term "plant" is used to mean a plant body or a part thereof, unless otherwise specified, and "a part thereof" includes, unless otherwise specified, seeds (including germinated seeds and immature seeds), organs or parts thereof (including leaves, roots, stems, flowers, stamens, pistils, and their fragments), plant cultured cells, callus, and protoplasts. Plants include genetically modified plants and transgenic plants. Plants also include harvested products and reproductive materials.

[0063] Unless otherwise specified, "propagation material" refers to all or part of a plant body used for propagation (sometimes called seedlings), such as seeds, seedlings, cells, callus, and sprouts. In this invention, "harvested products" is used in the usual sense, except in special cases, and includes all or part of a plant body that is not used for propagation, such as buckwheat seeds as food ingredients, harvested buckwheat, buckwheat hulls, and bran.

[0064] In relation to the present invention, when referring to a processed product, unless otherwise specified, it means a processed product produced directly from harvested produce, specifically buckwheat grains, buckwheat flour, etc.

[0065] Buckwheat grains or buckwheat flour can be used as ingredients in food products. Examples of such foods include noodles, confectionery (cookies, biscuits, crackers, bolo, snacks, sponge cakes, manju, dango, senbei, arare, okaki, etc.), mochi, bread (e.g., sliced ​​bread, sweet bread, bagels, steamed buns, and butter rolls, etc.), pizza, alcohol, ice cream, candy, chocolate, mixed flours (e.g., fried chicken mix, tempura mix, bread mix, pancake mix, okonomiyaki mix, and takoyaki mix, etc.), and beverages (e.g., buckwheat tea beverages, soups, green juice, smoothies).

[0066] Foods containing raw materials derived from harvested or processed products of buckwheat plants according to the present invention exhibit a smoother and softer texture and reduced hardening over time after cooking, compared to foods made using buckwheat flour or buckwheat grains from wild-type buckwheat plants. Furthermore, they maintain good softness and moisture retention even after refrigeration or freezing. [Examples]

[0067] The present invention will be described more specifically based on the following examples, but the present invention is not limited to these examples. In this specification, unless otherwise specified, "%", "parts", etc., are based on mass, and numerical ranges are described including their endpoints.

[0068] Example 1: Acquisition of a buckwheat plant with an SBE1 gene mutation method Mutation treatment 100g of self-pollinating buckwheat (Fagopyrum esculentum, strain name IH3) seeds were placed in 200mL of ethyl methanesulfonate (0.1%~2.5%) and stirred at 25°C or similar conditions at 150 revolutions per minute. After 2 to 16 hours, the seeds were removed and sown in pots or fields (NARO Kyushu Okinawa Agricultural Research Center (2421 Suya, Koshi City, Kumamoto Prefecture 861-1192) or NARO Hokkaido Agricultural Research Center (4-banchi, Shinsei Minami 9-sen, Memuro-cho, Kasai-gun, Hokkaido 082-0081)). Seeds were obtained from each plant after fruiting. The harvested seeds were sown again in pots or fields, and leaves and seeds were collected for subsequent analysis.

[0069] Urea disintegration test The hulls of fully ripened buckwheat seeds were removed, immersed in 3M urea for 3 days, and then iodine solution was added to observe the color change. Specifically, after removing the hulls from the buckwheat seeds, approximately 20 mg of buckwheat flour, ground in a mortar, was weighed into a 0.5 mL tube, 1 mL of 3M urea was added, and after standing at 25°C, 180 μL of iodine solution (0.002% iodine - 0.02% potassium iodide solution) was added to 20 μL of the supernatant. A bluish-black color was determined to indicate high urea disintegration.

[0070] Gelatinization onset temperature, gelatinization peak temperature, enthalpy The hulls were removed from fully ripened buckwheat seeds, which were then crushed into beads in a plastic tube. The results were then measured using a differential scanning calorimetry (DSC, Seiko Instruments Inc., DSC6100). 10 mg (dry weight) of crushed buckwheat seeds was weighed into a 70 μL silver sealed sample container (Seiko Instruments Inc. for DSC), and distilled water was added to a concentration of 30% (w / w) (dry weight). After sealing the sample container, a sealed sample container containing the same weight of distilled water was used as a blank, and the gelatinization temperature and heat quantity were measured at a heating rate of 2 °C / min from 25 °C to 130 °C. The obtained data was analyzed using the software attached to the differential scanning calorimeter, and the gelatinization temperature and enthalpy change were calculated.

[0071] Enthalpy is the "heat of gelatinization," and a larger value indicates that more heat is required for gelatinization (it is harder to gelatinize). The gelatinization properties of amylopectin molecules are influenced by the length of their side chains (degree of polymerization). As the side chains become longer, the number of hydrogen bonds between them increases, which increases the crystallinity of the amylopectin molecules and requires more thermal energy to gelatinize (break down) the molecules. Therefore, the higher the amylopectin content in starch granules and the longer the side chain length, the more heat of gelatinization is required.

[0072] Genotype determination Genomic DNA was extracted from leaves, and the sequences obtained using a next-generation DNA sequencer (Illumina HiSeq2000) were mapped to a reference genome sequence to investigate mutations. The reference genome was obtained based on information from the Buckwheat Genome Database (BGDB) https: / / buckwheat.kazusa.or.jp / .

[0073] result 32,000 seeds were treated with ethyl methanesulfonate, and 12,000 plants were collected. These were then selected using urea disintegration testing, followed by genotyping, and two lines with mutations in the SBE1 gene were obtained. One of these two lineages is the "Su-type UD1 lineage." The "Su-type UD1 lineage" has a single nucleotide deletion in the exon of the SBE1b gene, resulting in an early stop codon, so its translation product was expected to be shorter than that of the wild type (Figure 1C). Therefore, SBE1b activity may be reduced or absent. Furthermore, RNA sequencing data analysis suggested that at least two translation products of SBE1b may be generated from the same genome through alternative splicing. Due to alternative splicing, in the wild type, it is thought that in addition to the SBE1b putative translation product with the amino acid sequence shown in SEQ ID NO: 4, an SBE1b putative translation product with the amino acid sequence shown in SEQ ID NO: 11 is produced. In the "Su-type UD1 lineage," it is thought that the lysine at position 238 of the amino acid sequence shown in SEQ ID NO: 11 is changed to glutamine. This mutation may be causing reduced or absent SBE1b activity. To fix the mutation in a homozygous state, seeds were sown again, and the process of self-pollination and confirmation of the presence or absence of the gene mutation was repeated. Once the gene mutation was fixed in a homozygous state, the plant was designated as the SBE1 gene mutant buckwheat plant "Sugae UD1 line".

[0074] The SSIIa1, SSIIa2, and SBE1a genes in the "Sugae UD1 strain" were wild-type. The SSIIa1, SSIIa2, SBE1a, and SBE1b genes in the self-pollinating buckwheat strain "IH3" were confirmed to be wild-type. As shown in Figure 2, the "Sugaya UD1 strain" showed increased urea disintegration compared to the wild-type "IH3". As shown in Table 1, compared to the wild-type "IH3," the "Sugae UD1" strain had a starch gelatinization onset temperature approximately 12°C lower, a gelatinization peak temperature approximately 11°C lower, and an enthalpy change of approximately 3 J / g less. A t-test showed a statistically significant difference at the 0.1% level from the mean (N=3). [Table 1]

[0075] In the "Su-type UD1 strain," a mutation in the SBE1b gene leads to a functional defect in the gene's translation product, which is thought to shorten the amylopectin side chains and lower the starch gelatinization initiation temperature. The "Su-type UD1 strain" is a mutation in which only one of the two SBE1 genes is deleted, but the gelatinization peak temperature is reduced by approximately 11°C, which is comparable to the 10°C reduction in gelatinization peak temperature obtained when both SSIIa and SBE1 are deleted in rice. The basis for claiming that the gelatinization peak temperature of "rice lacking SSIIa and SBE1" is 10°C lower compared to "rice with wild-type SSIIa and SBE1" is that there is a report that the gelatinization peak temperature of "rice lacking SSIIa and SBE1" is 4°C lower compared to "rice lacking SSIIa and wild-type SBE1" (Non-Patent Literature 2), and furthermore, there is a report that both the gelatinization onset temperature and peak temperature are about 5°C lower for general japonica rice (Nipponbare; gelatinization onset temperature 60.0°C, gelatinization peak temperature 66.0°C) compared to indica rice (representatively BMBirinari rice; gelatinization onset temperature 65.5°C, gelatinization peak temperature 70.5°C) (Kyoko Tanaka et al., "Comparison of Cooking Characteristics of Myanmar Indica Rice and Japanese Japonica Rice," Journal of Food Science and Technology of Japan, Vol.41, No.L11~17 (2008)). In buckwheat, it is thought that a sufficient gelatinization temperature effect was obtained by simply deleting the SBE1b gene. In cereal starch synthesis, it is thought that SBE1 transfers a short glucose chain to amylose, generating a short amylopectin chain (side chain formation), and that SSIIa then extends this side chain (Reference: Yasunori Nakamura, "Rice as a material for starch synthesis research," Journal of the Japan Society for Biotechnology, 2017, Vol. 95, No. 4). However, since amylopectin is present even in rice lacking SBE1, it is thought that there are other side chain formation mechanisms besides SBE1.

[0076] Example 2: Comparison of gene expression levels method Total RNA was extracted from the endosperm of buckwheat seeds during the maturation process of wild-type buckwheat ("IH3"), converted to cDNA, and then the base sequence was obtained using a next-generation DNA sequencer. The gene transcript quantity was then investigated as TPM (transcripts per million) by mapping it to a reference sequence.

[0077] result In wild-type buckwheat ("IH3"), we confirmed the expression of the SSIIa1, SSIIa2, SBE1a, and SBE1b genes. These four genes are thought to be expressed as functional proteins. We confirmed that in buckwheat, two genes exist at each of four different loci.

[0078] Example 3: Comparison of breaking loads during refrigerated storage of noodle dough method After hulling the fully matured seeds of wild buckwheat "IH3", low-amylose buckwheat "Kyushu No. 12" (lacking only GBSSa), and SBE1b mutant buckwheat "Sukei UD1", the seeds were milled in a mortar and pestle, and water was added to achieve a final moisture content of 83% to produce 2mm thick noodle sheets. The noodle sheets were boiled in boiling water for 10 minutes, cooled in cold water, and then stored in a sealed plastic bag at 4°C for up to 9 days to prevent drying. For the above-mentioned refrigerated noodle sheets, the breaking strength was measured under the following conditions using a physical property testing system (Yamaden Co., Ltd. RE2-3305C-1) on the day of manufacture (no refrigeration, immediately after manufacture), the 3rd day, the 6th day, and the 9th day. • The temperature during measurement was 25℃ • Compressing the noodle sheet (2.0 mm thick) at 0.5 mm / sec

[0079] result The breaking load of the wild-type buckwheat "IH3" and the low-amylose buckwheat "Kyushu No. 12" increased with the passage of storage time, but it did not increase for the SBE1b mutant buckwheat "Sukei UD1". Since fracture load is an indicator of hardness, it is thought that the SBE1b mutant buckwheat "Sukei UD1" has the characteristic of retaining its softness (not hardening easily) when stored in the refrigerator.

[0080] Example 4: Urea disintegration properties by growing season Since weather conditions (temperature, etc.) change with the planting season, the amount of short-chain amylopectin in the maturing seeds may be affected. Therefore, we compared the degree of urea disintegration in each planting season (summer planting in 2024, spring planting in 2025, and summer planting in 2025).

[0081] method: Urea disintegration test: Ripe buckwheat seeds were hulled (N=3), immersed in 3M urea for 5 days, and then iodine solution was added to measure the color change (absorbance at 595 nm). Results: The absorbance of the Suga variety UD1 was higher compared to the wild type. Discussion: The urea disintegration ability of the Su-type UD1 is higher than that of the wild type, even when grown in different seasons. Therefore, it is thought that the trait of starch being converted into short-chain amylopectin in the Su-type UD1 is stably exhibited even when the temperature and other conditions during maturation differ. [Table 2] [Industrial applicability]

[0082] Buckwheat plants in which the starch-forming enzyme of the present invention has been mutated exhibit a lower starch gelatinization temperature compared to the wild type. Therefore, it is believed that by using the technology of the present invention, it will be possible to cultivate buckwheat plants with altered softness in cooked food, thereby meeting consumer needs.

[0083] (Sequence Listing) Sequence ID 1: Buckwheat SBE1a gene, estimated cDNA, wild type Sequence ID No. 2: Buckwheat SBE1b gene, estimated cDNA, wild type Sequence ID 3: Buckwheat SBE1a protein amino acid sequence (wild type) Sequence ID No. 4: Buckwheat SBE1b protein amino acid sequence (wild type) Sequence ID 5: Buckwheat SBE1b protein amino acid sequence variant Sequence ID 6: Buckwheat SBE1a gene, genome, wild type Sequence ID 7: Buckwheat SBE1b gene genome, wild type Sequence ID No. 8: Buckwheat SSIIa1 gene genome, wild type Sequence ID 9: Buckwheat SSIIa2 gene, genome, wild type Sequence ID No. 10: Buckwheat SBE1b gene, presumed cDNA, wild type, alternative splicing product. Sequence ID No. 11: Buckwheat SBE1b protein amino acid sequence, wild type, alternative splicing product.

Claims

1. Buckwheat plants with reduced or deficient starch branching enzyme 1 (SBE1) activity.

2. The buckwheat plant according to claim 1, wherein the reduced or absent SBE1 activity is due to a mutation in the SBE1 gene.

3. The buckwheat plant according to claim 2, wherein the mutation is a missense mutation, a nonsense mutation, a frameshift mutation, or an in-frame insertion or deletion, a splice site mutation, or a mutation in an expression regulatory region.

4. The wild-type SBE1 gene having the aforementioned mutation is (1) The SBE1a gene as defined by (i), (ii), or (iii) below: (i) A gene containing the nucleotide sequence shown in Sequence ID No. 6, or (ii) A gene that contains a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in Sequence ID No. 6, and which encodes a protein having SBE1 activity in buckwheat plants, or (iii) A gene encoding a protein having SBE1 activity in a buckwheat plant, comprising the amino acid sequence shown in Sequence ID No. 3, or an amino acid sequence having 90% or more sequence identity with said amino acid sequence. (2) The SBE1b gene as defined in (i), (ii), or (iii) below: (i) A gene containing the nucleotide sequence shown in Sequence ID No. 7, or (ii) A gene that contains a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in Sequence ID No. 7, and encodes a protein having starch branching enzyme 1 activity in buckwheat plants, or (iii) A gene encoding a protein having SBE1 activity in a buckwheat plant, comprising the amino acid sequence shown in Sequence ID No. 4 or 11, or an amino acid sequence having 90% or more sequence identity with said amino acid sequence. A buckwheat plant according to claim 3, which is any of the following.

5. The buckwheat plant according to claim 4, wherein the mutation is a mutation in which the base at position 4446 in the base sequence shown in Sequence ID No. 7 is deleted, or a mutation in which the base at position 8194 in the base sequence shown in Sequence ID No. 7 is substituted from adenine to cytosine.

6. The buckwheat plant according to claim 1, wherein the buckwheat plant is buckwheat (Fagopyrum esculentum), Tartary buckwheat (F. tataricum), perennial buckwheat (F. cymosum), wild buckwheat (F. homotropum), or a hybrid of any of these.

7. A plant body of a buckwheat plant as described in any one of claims 1 to 6.

8. A method for breeding buckwheat plants, comprising using the plant described in claim 7 or its progeny.

9. A harvested product, propagation material, or processed product of the plant body described in claim 7.

10. A food product comprising raw materials derived from the harvested or processed products of the plant described in claim 7.

11. A method for increasing starch urea disintegration, increasing alkali disintegration of buckwheat grains, or lowering the starch gelatinization temperature in buckwheat plants by reducing or eliminating the activity of at least one of the following proteins: (A) Proteins consisting of the amino acid sequences shown in SEQ ID NOs: 3, 4, or 11; (B) A protein having an amino acid sequence that is 90% or more identical to the amino acid sequence shown in Sequence ID No. 3, 4, or 11, and that has SBE1 activity in buckwheat plants; (C) A protein having SBE1 activity in the buckwheat plant, consisting of an amino acid sequence in which 1 to 60 amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NOs: 3, 4, or 11.

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