Buckwheat plants with reduced amylose content

Abstract

This invention provides a low-amylose buckwheat plant. By reducing the amylose content, it also improves the texture of foods using buckwheat plants and the workability during food manufacturing. [Solution] A buckwheat plant is provided that satisfies at least one of the following conditions: The amylose content is at least 8.0% lower than that of the wild type. The amylose content is 26% or less. Such a buckwheat plant can be obtained by mutating the gene for granule-bound starch synthase (GBSS).

Description

Technical Field

[0001] The present invention relates to buckwheat plants with modified starch characteristics (amylose content).

Background Art

[0002] Cultivated species of buckwheat plants classified in the Polygonaceae family include buckwheat (Fagopyrum esculentum), tartary buckwheat (F. tataricum), and perennial buckwheat (F. cymosum). In Japan, buckwheat and tartary buckwheat are mainly used as food. The use of perennial buckwheat is less, but it has strong waterlogging resistance and is expected to be used in the future. In addition, the use of the self-compatible buckwheat wild species (F. homotropicum) has also started. Furthermore, the use of hybrids of any of these varieties, for example, the hybrid of tartary buckwheat and perennial buckwheat (F. giganteum), the hybrid of common buckwheat and wild species, etc. is also starting to be used.

[0003] When using buckwheat grains, texture and workability during food production (such as the cohesion and elongation of dough) are 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 branches. In grains, when the amylose content is low, the so-called sticky feeling becomes stronger and the dough tends to cohere.

[0004] In rice and wheat, it has been shown that the texture is greatly improved by a decrease in the amylose content of a few percent. The amylose content of buckwheat is higher than that of current major varieties of rice and wheat, and in whole grain flour, it is approximately 20 to 30% (Non-Patent Document 1). In Japan, buckwheat is generally used as noodles, but as described above, due to the high amylose content, the cohesion of the dough is poor and the texture variations are limited. Also, in Russia, buckwheat is used as grains (kasha), but due to the high amylose content, starch easily ages over time after cooking, and the texture deteriorates (becomes hard and brittle).

[0005] Amylose is believed to be synthesized by granule-bound starch synthase (GBSS). For example, in rice, wheat, and barley, it has been shown that suppressing GBSS expression reduces the amylose content, and complete deficiency results in waxy rice.

[0006] One technique being considered to reduce the amylose content of buckwheat is to suppress or knock out the expression of GBSS in buckwheat (Patent Documents 1-6). However, there is no knowledge as to whether suppressing or knocking out GBSS expression actually reduces the amylose content in buckwheat.

[0007] Furthermore, there is a report on a low-amylose buckwheat strain (Non-Patent Literature 2), but the expression of GBSS in that strain is unknown, and it has been lost due to the inability to collect seeds, making it impossible to reproduce. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Special Publication 2001-516575 [Patent Document 2] Special Publication No. 2001-519664 [Patent Document 3] Special Publication No. 2002-525029 [Patent Document 4] Special Publication No. 2003-507034 (Patent No. 4871466) [Patent Document 5] Special Publication No. 2005-508166 [Patent Document 6] Japanese Patent Publication No. 2012-223137 [Non-patent literature]

[0009] [Non-Patent Document 1] Suzuki T, Noda T, Morishita T, Ishiguro K, Otsuka S, Andrea B. (2019) Present status and future perspectives of breeding for buckwheat quality. Breed. Sci. 70: 48-66. [Non-Patent Document 2] Gregori, M. and Kreft I. (2012) Breakable starch granules in a low-amylose buckwheat (Fagopyrum esculentum Moench) mutant. J. Food Agric. Environ. 10: 258-262. [Disclosure of the Invention] [Problems that the invention aims to solve]

[0010] Reducing the amylose content of buckwheat can improve its texture and workability during food manufacturing, and it is believed that modifying its physical properties could expand its use to food applications other than noodles. While there are technologies to modify the physical properties of buckwheat noodles by adding wheat flour or modified starch as auxiliary ingredients, the degree of improvement is not sufficient. Furthermore, the unique aroma of buckwheat is an important quality characteristic, but since the proportion of buckwheat flour in the food decreases relatively due to the addition of auxiliary ingredients, there is a need to reduce the amylose content of the buckwheat itself. [Means for solving the problem]

[0011] The inventors have now obtained a buckwheat plant from which a mutation in the GBSS gene has occurred, resulting in a lower amylose content compared to the wild type, from among buckwheat plants that have undergone mutation treatment, and have completed the present invention.

[0012] The present invention provides the following: [1] A plant of the genus Buckwheat that satisfies at least one of the following conditions: • Amylose content is at least 8.0% lower compared to the wild type. • The amylose content is 26% or less. [2] A buckwheat plant described in 1, having a mutation in the active starch synthase (GBSS) gene. [3] A buckwheat plant as described in 2, wherein the mutation is selected from the group consisting of splicing mutations, stop codon insertion mutations, and amino acid substitution mutations. [4] A buckwheat plant described in any one of items 1-3, wherein GBSS is GBSSa. [5] A plant of the genus Buckwheat having any of the following polynucleotides: (a) A polynucleotide consisting of sequence number 2. (b) A polynucleotide having 80% or more sequence identity with the polynucleotide consisting of the sequence of SEQ ID NO: 2, wherein the nucleotide corresponding to position 1846 of SEQ ID NO: 2 is not A. [6] A plant of the genus Fagopyrum described in any one of items 1 to 5, wherein the plant is buckwheat (Fagopyrum esculentum), Tartary buckwheat (F. tataricum), perennial buckwheat (F. cymosum), 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 items 1 to 6. [8] A method for breeding buckwheat plants, including using the plant described in 7 or its progeny. [9] The harvested products, propagating materials, or processed products of the plant described in 7.

[10] A method for reducing the amylose content of buckwheat plants by inhibiting the activity of any of the following proteins: (A) Sequence ID: A protein consisting of one amino acid sequence from 8 to 13; (B) A protein consisting of an amino acid sequence having 90% or more identity with one of the amino acid sequences 8-13, and having the function of controlling the amylose content of buckwheat plants; A protein consisting of an amino acid sequence in which 1 to 60 amino acids are deleted, substituted or added in any one of the amino acid sequences of SEQ ID NOs: 8 to 13, and having a function of controlling the amylose content of buckwheat plants.

[11] The method according to 10, wherein suppressing the activity of the protein is suppressing the activity of any of the following proteins: (A') A protein consisting of any one of the amino acid sequences of SEQ ID NOs: 8, 10, and 12; (B') A protein consisting of an amino acid sequence having 90% or more identity with any one of the amino acid sequences of SEQ ID NOs: 8, 10, and 12, and having a function of controlling the amylose content of buckwheat plants; (C') A protein consisting of an amino acid sequence in which 1 to 60 amino acids are deleted, substituted or added in any one of the amino acid sequences of SEQ ID NOs: 8, 10, and 12, and having a function of controlling the amylose content of buckwheat plants. [Advantages of the Invention]

[0013] With the buckwheat plant having a reduced amylose content of the present invention, buckwheat grains and flour with improved texture and workability during food production can be obtained without blending auxiliary raw materials such as wheat flour and modified starch.

[0014] The buckwheat grains and flour obtained from the buckwheat plant having a reduced amylose content of the present invention can be widely used in various foods by taking advantage of their glutinousness. [Brief Description of the Drawings]

[0015] [Figure 1] Accumulation of GBSSa and b proteins in the splicing mutant line "18N de 6" [Figure 2] Splicing of the GBSSa gene mutant buckwheat plant "18N de 6" [Figure 3] Degree of reduction in amylose content due to GBSSa splicing mutation ***: There is a significant difference at the 0.1% level in the average value in the t-test. [Figure 4] Base sequence of the GBSSa gene (mutant) (Sequence ID: 2) ATG (circled in the figure): Start codon, TAA (circled in the figure): Stop codon, GT, AG: Splicing signal sequence, Shaded area: Part of the exon that is translated into protein, T (circled in the figure): Mutant base in GBSS gene mutant buckwheat plant ("18N de 6") [Figure 5-1] Year-to-year difference in amylose content: A: Spring sown in 2020, B and C: Summer sown in 2020, *: Significant difference at the 5% level between the mean values ​​of wild type and GBSSa-deficient lines (t-test) (same in Figures 5-1 and 5-2) [Figure 5-2] The year-to-year difference in amylose content: D-1 and D-2's "wild type" is a self-pollinated line that is resistant to shattering and sprouting in the early stages of buckwheat growth. The "GBSSa-deficient" line is a line (F4) obtained by crossing the wild type (self-pollinated line that is resistant to shattering and sprouting in the early stages of buckwheat growth) with the GBSSa-deficient line 18N De6, and selecting only self-pollinated plants with GBSSa deficiency and shattering / sprouting-resistant traits in the grandchild generation. The "wild type" of D-3 and D-4 is the IH3 line, and the "GBSSa-deficient" line is the GBSSa-deficient line 18N De6. Since D-1 and D-3 were not grown in isolation, they may have been pollinated by pollen from the surrounding wild-type buckwheat. In other words, the amylose content of the GBSSa-deficient line may appear higher than it actually is. Note that the explanation for D-1 to D-4 also applies to E-1 to E-4 in the following figure. Significant differences were observed between the mean values ​​of wild-type and GBSSa-deficient strains at the 1% and 0.1% levels (t-test). Measurements were taken using a MEGAZYME kit. [Figure 5-3] Year-to-year differences in amylose content: Significant differences were observed between the mean values ​​of wild-type and GBSSa-deficient lines at the *:5%, **:1%, and ***:0.1% levels (t-test). [Figure 6] Year-to-year differences in GBSSa protein accumulation: The mutant line 18N-de6 lacks GBSSa protein accumulation regardless of sowing time. However, the amylose content was not reduced in the 2020 spring sowing. [Figure 7] Genotype determination using KASP: KASP (registered trademark) allows for easy determination of genotypes. [Modes for carrying out the invention]

[0016] [New Buckwheat species] The present invention provides a novel buckwheat plant that satisfies at least one of the following conditions. • Amylose content is at least 8.0% lower compared to the wild type. • The amylose content is 26% or less.

[0017] The genus Fagopyrum refers to plants belonging to the genus Fagopyrum. The genus Fagopyrum includes buckwheat (Fagopyrum esculentum), Tartary buckwheat (Fagopyrum tataricum), perennial buckwheat (Fagopyrum cymosum), cypress buckwheat (Fagopyrum dibotrys), wild buckwheat relatives (Fagopyrum homotropicum), hybrids of Tartary buckwheat and perennial buckwheat (Fagopyrum giganteum and Fagopyrum tatari-cymosum), and other Fagopyrum species such as Fagopyrum megaspartanium, Fagopyrum pillus, Fagopyrum macrocarpum, Fagopyrum callianthum, Fagopyrum urophyllum, Fagopyrum gilesii, Fagopyrum statice, Fagopyrum leptopodum, Fagopyrum gracilipes, Fagopyrum capillatum, Fagopyrum pleioramosum, Fagopyrum lineare, and Fagopyrum rubifolium.

[0018] The species of the genus Buckwheat according to the present invention are not particularly limited, but preferred species include buckwheat, Tartary buckwheat, perennial buckwheat, or wild buckwheat, or hybrids of any of them (for example, F. giganteum, a hybrid of Tartary buckwheat and perennial buckwheat), and a more preferred species is buckwheat.

[0019] The buckwheat plants of the present invention may be bred from existing varieties or lines. The buckwheat plants obtained in this manner are distinguishable from existing varieties or lines by at least a lower amylose content. There are no particular limitations on the varieties or lines used in breeding. Examples of buckwheat varieties include: Kitawase buckwheat, Botan buckwheat, Kitayuki, Hashikami Wase, Iwate Wase, Mogami Wase, Hashikami Wase, Hitachi Aki buckwheat, Shinano No. 1, Shinshu Oosoba, Shinano Natsu buckwheat, 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. Examples of varieties of buckwheat include Nakanaka Motono No. 1, 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 BW No. 5, 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 varieties of buckwheat 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 carriers.

[0020] (Low-amylose) The buckwheat plants of the present invention have a lower amylose content compared to conventional buckwheat plants.

[0021] In relation to the present invention, when referring to amylose content, unless otherwise specified, it refers to the percentage (by mass) of amylose in the starch of buckwheat flour (endosperm) obtained by milling buckwheat seeds in a conventional milling machine. In some cases, the amylose content may be expressed as the percentage of amylose in the starch of whole grain flour, but those skilled in the art can determine the amylose content by performing appropriate calculations for buckwheat flour milled in a milling machine as appropriate. Generally, buckwheat flour is produced by rolling buckwheat (seeds) and separating the crushed material with a sieve.

[0022] For example, if the starch content of buckwheat flour milled with a milling machine is 85.7% and the starch content of whole grain flour is 65.8% (as in the case of Koto in Izydorczyk, MS and D. Head (2010) Characterization and potential uses of functional buckwheat fractions obtained by roller milling of new Canadian buckwheat genotypes. The European Journal of Plant Science and Biotechnology 4: 71-81), the amylose content of buckwheat flour milled with the milling machine referred to in this application can be determined by multiplying the amylose content of the starch in the whole grain flour by 85.7 / 65.8.

[0023] The amount of amylose in starch can be quantified by colorimetric analysis of the iodine-starch reaction with starch solubilized with dimethyl sulfoxide.

[0024] For example, it can be quantified using an external standard method such as the one described below. Weigh approximately 30 mg of buckwheat flour, add 1 ml of dimethyl sulfoxide (DMSO), and infuse at 75°C for 16 hours. Dilute the resulting supernatant 1000-fold with desalted water. Add 20 μL of iodine solution (0.02% iodine - 0.2% potassium iodide solution) to 180 μL of the diluted solution, and measure the absorbance at 690 nm. Commercially available potato amylose can be used as a standard sample to create a calibration curve.

[0025] The degree of amylose reduction in the buckwheat plant according to the present invention is preferably such that it affects the texture and workability during food production in a food product containing the buckwheat plant as a raw material.

[0026] Specifically, the amylose content of the buckwheat plants of the present invention is preferably 26% or less, preferably 25% or less, more preferably 24% or less, and even more preferably 23% or less. According to the inventors' studies, the amylose content of wild-type buckwheat is 22.6% when based on whole grain flour (see Examples section), and 29.4% (=22.6*85.7 / 65.8) when milled using a milling machine. Furthermore, the buckwheat strain 18Nde6 obtained by the inventors has an amylose content of 18.6% when based on whole grain flour, and 24.3% (=18.6*85.7 / 65.8) when milled using a milling machine.

[0027] Furthermore, the amylose content of the buckwheat plants of the present invention is lower than that of the wild type from which the buckwheat plants are derived. In relation to the present invention, when the decrease in amylose content compared to the wild type is expressed numerically, unless otherwise specified, the value is calculated using the formula: Amylose content of the wild type - Amylose content of the mutant type.

[0028] The amylose content of the buckwheat plant of the present invention is preferably low, specifically at least 8.0%, preferably 9.0% or more, more preferably 10.0% or more, and even more preferably 11.0% or more.

[0029] In wheat, it is known that a deficiency in Wx-B1 reduces the amylose content by several percent, resulting in a moderate stickiness that makes it suitable for noodles. A report on wheat (M. Yamamori & NT Quynh, Differential effects of Wx-A1, -B1 and -D1 protein deficiencies on apparent amylose content and starch pasting properties in common wheat. Theoretical and Applied Genetics volume 100, pages 32-38 (2000)) showed that when the wild type (Type 1) mutated into the Wx-B1 deficient type (Type 3), the amylose content decreased. Although there is year-to-year variation, on average, a decrease of 7.7% in amylose content occurs. Therefore, it is thought that if the buckwheat plants of the present invention also experience a similar decrease in amylose content, the desired effect can be expected.

[0030] It should be noted that in some years, the actual amylose content of the buckwheat plants of the present invention may not decrease. In wheat, it has been reported that there are years in which the amylose content does not decrease when one GBSS isozyme is missing, but even taking that into account, it is still useful and is becoming widespread. Even if the actual amylose content of a buckwheat plant does not decrease, if a decrease in amylose content is observed in other years compared to conventional buckwheat plants, it is included in the buckwheat plants of the present invention.

[0031] (Inhibition of GBSS activity) A reduction in amylose content can be achieved by suppressing the activity of starch synthase (GBSS). In this invention, when GBSS is referred to in relation to buckwheat plants, it refers to GBSSa and GBSSb unless otherwise specified.

[0032] In relation to the present invention, when we refer to suppressing GBSS activity, we are not limited to cases where GBSS activity is suppressed by a specific mechanism. Activity suppression includes not only the suppression of GBSS enzyme activity but also the suppression of GBSS gene expression. More specifically, activity suppression includes not only the inhibition of GBSS activity but also mutations in the GBSS gene that prevent the production of active GBSS, and mutations in the GBSS gene's expression regulatory region (promoter region, transcription factor binding region, etc.) that suppress GBSS production.

[0033] This invention is the first to disclose that buckwheat plants can become low-amylose due to a mutation in GBSS.

[0034] Whether a mutation in GBSS leads to low amylose content in a particular plant cannot be determined without actually measuring the amylose content in the mutated plant. For example, in wheat, which possesses multiple GBSS, it has been reported that deleting one GBSS does not result in a decrease in amylose content, or the decrease is negligible. More specifically, we present data on amylose content for each of the three GBSS deficiency types: Wx-A1, -B1, and -D1. We note that in the case of Wx-A1 deficiency, the amylose content does not decrease significantly, while Wx-B1 deficiency has a greater impact (M. Yamamori et al., 2000, cited above). Furthermore, the amylose content does not decrease significantly in Wx-A1 deficiency mutations (M. Yamamori, T. Nakamura, TR Endo & T. Nagamine. Waxy protein deficiency and chromosomal location of coding genes in common wheat. Theoretical and Applied Genetics volume 89, pages 179-184 (1994)). Finally, in Kanto 82, which lacks Wx-A1, there is no significant difference compared to the comparative variety "Chinese Spring (CS)" (Araki, E, Miura, Hideho, Watanabe, N, Sawada, Souhei. Variation for amylose content in wheat cultivars). The following has been reported: "carrying different null alleles at the Wx loci." (Obihiro University of Agriculture and Veterinary Medicine Academic Research Report. Natural Sciences, 21(1): 9-15).

[0035] Furthermore, some GBSS molecules function in the leaves. If GBSS that function in the leaves are deficient, photosynthetic products cannot be accumulated, which can lead to death or severe growth disorders. Therefore, prior to this application, there was a concern that buckwheat plants with mutated GBSS molecules might not be able to be produced as plants because the GBSS molecules might be common to both the leaves and seeds.

[0036] Furthermore, most of the cereals in which GBSS mutations have been reported are grasses, which are monocots. Specifically, these include rice, wheat, barley, maize, sorghum, millet, foxtail millet, and adlay. Among dicots, only amaranth of the Amaranthaceae family has been reported (Kenji Fukunaga. Origin of waxy cereals from a genetic point of view: From cultural history to genetic history of waxy cereals. Prefectural University of Hiroshima, Shobara, Hiroshima 727-0023, Japan). Prior to this application, the results of GBSS mutations in buckwheat plants, which belong to the Polygonaceae family and are also dicots, were unpredictable, and there were concerns that they might be lethal.

[0037] (Mutations in the GBSS gene or its regulatory region) One means of suppressing GBSS activity is mutation in the GBSS gene or its expression regulatory region. In one preferred embodiment, a buckwheat plant has a mutation in at least one of the GBSSa gene and the GBSSb gene. Preferably, the mutation is in the GBSSa gene. The sequence listing shows, as SEQ ID NO: 1, the sequence of the buckwheat GBSSa (wild type) gene; as SEQ ID NO: 2, the sequence of the buckwheat GBSSa (mutant type) gene obtained by the present inventors; as SEQ ID NO: 3, the buckwheat GBSSb gene; as SEQ ID NO: 4, the sequence of the Tartary buckwheat GBSSa cDNA; as SEQ ID NO: 5, the sequence of the Tartary buckwheat GBSSb cDNA; as SEQ ID NO: 6, the sequence of the perennial buckwheat GBSSa cDNA; and as SEQ ID NO: 7, the sequence of the perennial buckwheat GBSSb cDNA.

[0038] In one preferred embodiment, the mutations include mutations involving amino acid mutations (amino acid substitutions, stop codon insertions), splicing mutations (splicing signal mutations, genomic mutations involving splicing mutations due to intron mutations), and mutations in untranslated regions (including nucleic acid methylation) that result in changes in the expression level of gene transcripts. A specific example is a gene mutation in which a mutation occurs in the splicing signal site of GBSSa, a stop codon is inserted into the gene transcript, the accumulation of GBSS protein decreases, and as a result the amylose content decreases.

[0039] In one preferred embodiment, buckwheat plants have a mutation in GBSSa as shown in SEQ ID NO: 2, specifically the substitution of the nucleotide corresponding to position 1846 in SEQ ID NO: 2 with a nucleotide other than A. However, similar effects may be obtained with mutations at different positions. For example, mutations that delete or substitute cysteine ​​residues related to the three-dimensional structure of the protein, which greatly affects activity, mutations that delete or substitute amino acids in the active site with amino acids of different polarity, or mutations that delete or substitute 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 suppress GBSS activity, and similar effects can be expected with such mutations. On the other hand, mutations that only slightly shorten the C-terminus of GBSS allow GBSS to exhibit its original activity, resulting in no or low effect. However, buckwheat plants that have a polynucleotide with high identity (e.g., 80% or more) with the polynucleotide consisting of the sequence of SEQ ID NO: 2, and in which the nucleotide corresponding to position 1846 in SEQ ID NO: 2 is not A, can be expected to produce similar effects.

[0040] The presence of a gene mutation can be confirmed, for example, by the following methods. Methods for detecting the mutation include decoding the mutated base sequence itself using base sequence analysis methods such as the Sanger method 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 base sequence, detecting differences in the base sequence using techniques 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 portion. In addition to the GBSS protein investigation methods described in the examples above, methods for measuring activity and immunochemical methods (immunoblotting, immunochromatography, etc.) can be used to determine the deletion status of the GBSS protein.

[0041] The presence of a mutation in the GBSS gene can also be confirmed by the expression of GBSS protein in seeds. Specifically, when the protein is extracted from seeds and its expression is investigated by electrophoresis, it can be seen that GBSS protein accumulation is either lacking or the amount of GBSS protein is lower than in the wild type in plants with GBSS gene mutations. According to our research, buckwheat plants with a mutation in the GBSS gene did not experience a decrease in amylose content depending on the year of cultivation, but GBSS protein accumulation was lacking. From this, it can be said that GBSS gene mutations can be expressed regardless of the cultivation environment.

[0042] In the buckwheat plants of the present invention, mutations in the GBSS gene or its expression regulatory region may be carried out by various means. Examples of methods for obtaining buckwheat plants in which the GBSS gene or its expression regulatory region has been mutated include selection from mutant strains, genetic recombination, RNA interference, genome editing, artificial genome synthesis, and genome methylation.

[0043] In a preferred embodiment, a method of selection from mutant strains is used because it allows for more reliable production of plant bodies. While there are prior arts related to suppressing or knocking out GBSS expression (see Patent Documents 1-5 and Non-Patent Document 1), these have not yet led to the production of plant bodies.

[0044] (New buckwheat variety) An example of a buckwheat plant according to the present invention is 18N-de6. 18N-de6 is a strain obtained by the method described in the examples of this specification and has the following characteristics. • Scientific properties (morphological, cultivation characteristics, physiological characteristics, etc.) Taxonomic position: It belongs to the same lineage as common buckwheat (Fagopyrum esculentum). This is an annual, upright, branching plant. After sowing, the cotyledons unfold, followed by the development of true leaves. Flower clusters then appear on the main stem and branches, and finally, fruit is produced. • Origin: Developed through spontaneous mutation breeding at the Hokkaido 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 developed 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℃

[0045] [Method for reducing the amylose content of buckwheat plants] The present invention provides a method for reducing the amylose content of buckwheat plants by suppressing the activity of any of the following proteins. (A) Sequence ID: A protein consisting of one amino acid sequence from 8 to 13; (B) A protein consisting of an amino acid sequence having 90% or more identity with one of the amino acid sequences 8-13, and having the function of controlling the amylose content of buckwheat plants; (C) Sequence ID: A polynucleotide that codes for an amino acid sequence in which 1 to 60 amino acids are deleted, substituted, or added in any one of the amino acid sequences 8 to 13, and which has the function of controlling the amylose content of buckwheat plants.

[0046] In a preferred embodiment of the method for reducing the amylose content of buckwheat plants according to the present invention, the activity of any of the following proteins is suppressed. (A') Sequence ID: A protein consisting of one of the amino acid sequences 8, 10, and 12; (B') Sequence ID: A protein consisting of an amino acid sequence having 90% or more identity with one of the amino acid sequences 8, 10, and 12, and having the function of controlling the amylose content of buckwheat plants; (C') Sequence ID: A polynucleotide that encodes an amino acid sequence in which 1 to 60 amino acids are deleted, substituted, or added in any one of the amino acid sequences 8, 10, and 12, and has the function of controlling the amylose content of buckwheat plants.

[0047] Note that the sequence listing includes the amino acid sequence of buckwheat GBSSa as SEQ ID NO: 8, the amino acid sequence of buckwheat GBSSb as SEQ ID NO: 9, the amino acid sequence of Tartary buckwheat GBSSa as SEQ ID NO: 10, the amino acid sequence of Tartary buckwheat GBSSb as SEQ ID NO: 11, SEQ ID NO: 12 The amino acid sequence of GBSSa in perennial buckwheat, sequence number: 13 The amino acid sequence of GBSSb from perennial buckwheat is shown.

[0048] In relation to the present invention, when referring to "hybridizing polynucleotides under stringent conditions," unless otherwise specified, the hybridization conditions for any polynucleotide can be appropriately selected according to the polynucleotide to be obtained, in accordance with the descriptions in Molecular Cloning. A Laboratory Manual. 2nd ed. (Sambrook et al., Cold Spring Harbor Laboratory Press) and Hybridization of Nucleic Acid Immobilization on Solid Supports (ANALYTICAL BIOCHEMISTRY 138, 267-284 (1984)). For example, to obtain DNA with 85% or more identity, hybridization can be performed at 40°C in the presence of a 2x concentration SSC solution and 50% formamide, followed by washing the filter at 55°C using a 0.1x concentration SSC solution (the composition of the 1x concentration SSC solution is 150 mM sodium chloride and 15 mM sodium citrate). Furthermore, to obtain DNA with more than 90% identity, hybridization should be performed at 55°C in the presence of a 2x concentration SSC solution and 50% formamide, followed by washing the filter at 60°C with a 0.1x concentration SSC solution.

[0049] Furthermore, with respect to the present invention, when referring to an amino acid sequence in which "one or more amino acids are substituted, deleted, inserted, and / or added," the number of amino acids substituted, deleted, inserted, and / or added is not particularly limited in any protein as long as the protein consisting of that amino acid sequence has the desired function, unless otherwise specified. However, it is generally around 1 to 120, 1 to 60, 1 to 30, 1 to 9, or 1 to 4 amino acids, or even more substitutions are possible if the substitutions are with amino acids of similar properties. Means for preparing polynucleotides or proteins relating to such amino acid sequences are well known to those skilled in the art.

[0050] 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 Altschul et al., 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.

[0051] 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.

[0052] [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 offspring as breeding material. The breeding method is not particularly limited and examples include crossbreeding, selection of mutants, backcrossing, genetic modification, cell fusion, genome editing, etc.

[0053] 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).

[0054] The present invention also provides a method for producing low-amylose buckwheat plants. The method for producing buckwheat plants according to the present invention comprises the steps of mutating the GBSS gene of a buckwheat plant, or propagating a buckwheat plant with a mutated GBSS gene. It is characterized by including.

[0055] [Harvested produce, breeding material, or processed 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.

[0056] 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.

[0057] 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.

[0058] 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, etc.).

[0059] The present invention will now be described in more detail with reference to examples, but the present invention is not limited thereto. [Examples]

[0060] [1. Acquisition of buckwheat plants with GBSS gene mutations] To induce mutation, 100g of 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 2-16 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 genomic DNA was prepared from leaves and seeds to determine the genomic DNA sequence related to the GBSS gene. Genomic DNA preparation was carried out using commercially available genome extraction kits.

[0061] The genomic DNA sequence of the GBSS gene was amplified by PCR using DNA primers that specifically amplify the gene. The method was based on Katsu, K., Suzuki, T., Fujino, K., Morishita, T. and Noda, T. (2019). Development of a DNA marker for variety discrimination specific to 'Manten-Kirari' based on an NGS-RNA sequence in Tartary buckwheat (Fagopyrum tataricum). Food Chem., 295, 51-57. The resulting amplified DNA fragments were sequenced using the Sanger assay or by outsourcing to an analytical company. The determined base sequences were compared with wild-type base sequences, specifically Fes_sc0002521.1.g000007.aua.1 and Fes_sc0005258.1.g000004.aua.1 obtained from the Buckwheat Genome Database (BGDB) (http: / / buckwheat.kazusa.or.jp / index.html), to identify plants with base sequence mutations. Next, to fix the mutation in a homozygous state, the seeds were sown again, and seed collection by self-pollination and confirmation of the presence or absence of the gene mutation were repeated. Once the gene mutation was fixed in a homozygous state, the plants were designated as GBSS gene mutant buckwheat plants. The results are shown in the table below.

[0062] [Table 1]

[0063] [2. Confirmation of GBSS protein accumulation in acquired GBSS gene mutant buckwheat plants] The seeds of the plant in question were ground in a mortar and pestle for 2 minutes, and the proteins from the prepared buckwheat flour were subjected to 2D electrophoresis. 2D electrophoresis was outsourced to an analytical company (APPLIED BIOMICS, https: / / www.appliedbiomics.com / Services / 2d-dige.html). Specifically, seed proteins were solubilized in a urea-containing solution, labeled with a fluorescent dye, separated by isoelectric focusing and SDS-PAGE, and each protein was visualized. The accumulation of GBSS proteins was compared by comparing images of the plant and wild-type plants. GBSS proteins were identified by digesting protein spots excised from the electrophoresis gel with trypsin, measuring the precise mass of the peptide by TOFMS, and then comparing the precise mass with a database (MASCOT search).

[0064] The results are shown in Figure 1. In the "18N De6" population, seed proteins from wild-type (AA) and SVA mutant (aa) strains were subjected to 2D-DIGE and MASCOT analysis before gene fixation. Specifically, wild-type (AA) and SVA mutant (aa) seed proteins were labeled with two different fluorescent dyes (green and red), mixed, and subjected to 2D electrophoresis. Spots expected to represent the target protein were excised, and after trypsin digestion, precise mass analysis was performed by TOFMS. The protein was then identified by MASCOT analysis. (Database: buckwheat).

[0065] Yellow spots indicate similar levels of protein accumulation in the wild-type and GBSSa mutant. Green indicates lower protein accumulation in the GBSSa mutant compared to the wild-type. The signal intensity of GBSSa in the mutant (aa) was low, similar to the background level, and therefore it was determined to be deficient.

[0066] Estimated molecular weight and isoelectric point (PI) from cDNA (after removal of plastid migration site) • GBSSa: 58.27KD, PI=5.70 • GBSSb: 60.44KD, PI=5.52

[0067] [3. Mutation sites on the genome of acquired GBSS gene mutant buckwheat plants] The sequences of the wild-type GBSSa gene and the mutant GBSSa gene obtained in this study ("18N de6") are shown as Sequence ID 1 and Sequence ID 2 in the sequence listing, and in Figure 4, respectively.

[0068] [4. Splicing mutations in buckwheat plants obtained from GBSS gene mutations] To investigate the effects of the above-mentioned genomic base mutations on gene transcript splicing, total RNA from ripening seeds was prepared using a commercially available kit, and cDNA was obtained by RT-PCR for buckwheat GBSSa. The method was based on M. Takagi, et al., Characterization of DNA polymerase from Pyrococcus sp. strain KOD1 and its application to PCR. Appl. Environ. Microbiol., 63: 4504-4510 (1997). Subsequently, the base sequence was determined using the Sanger method.

[0069] The splicing mutations in the wild-type and mutant ("18Nde6") GBSSa gene are shown in Figure 2. While the wild-type GBSS has a length of 607 amino acid residues, the mutant ("18Nde6") GBSSa has a length of 226 amino acid residues, and translation terminates prematurely.

[0070] [5. Degree of decrease in amylose content of acquired GBSS gene mutant buckwheat plants ("18Nde6")] To investigate the effect of the above-mentioned splicing mutation on amylose content, we examined the amylose content of strains homozygous for the splicing mutation and strains homozygous for the wild type (unmuted) from a buckwheat population containing both mutants and wild types, prior to trait fixation of "18N De6". Amylose content was determined by colorimetric quantification of the iodine-starch reaction with starch solubilized with dimethyl sulfoxide.

[0071] Specifically, approximately 30 mg of buckwheat flour was weighed into a 2 ml tube, 1 ml of dimethyl sulfoxide (DMSO) was added, and the mixture was centrifuged at 75°C for 16 hours (200 rpm). The supernatant was diluted 1000-fold with desalted water, and 20 μL of iodine solution (0.02% iodine - 0.2% potassium iodide solution) was added to 180 μL of the diluted solution. The absorbance at 690 nm was measured using a microplate reader (Thermo Scientific: Multiskan FC). A standard straight line was prepared using potato amylose (Sigma-Aldrich), and the amylose content in the sample was measured using the external standard method.

[0072] The results are shown in Figure 3. Compared to the wild type, the splicing mutation resulted in a 17.7% (1-(18.6 / 22.6)=0.177) decrease in amylose content. A t-test showed a statistically significant difference at the 0.1% level from the mean (N=154).

[0073] [6.Summary] From these results, it can be seen that by introducing mutations into the GBSS gene, it is possible to create buckwheat plants with reduced amylose content, resulting in changes related to improved soil properties.

[0074] [7. Annual differences in the degree of decrease in amylose content between buckwheat plants with GBSSa gene mutations and wild-type buckwheat.] Wild-type buckwheat line IH3 and GBSSa gene mutant buckwheat line 18Nde6 were cultivated at the Kyushu Okinawa Agricultural Research Center of the National Agriculture and Food Research Organization (NARO) in spring 2020, summer 2020, and spring 2021. The amylose content of whole grain flour from GBSS gene mutant plants was measured using a kit from MEGAZYME. The actual measurement procedure is described in the booklet from Nippon Biocon Co., Ltd. This was done in accordance with (K-AMYL (0618).pdf (biocon.co.jp)) (Non-Patent Document 3).

[0075] The results are shown in Figures 5-1 to 5-3. In the spring sowing of 2020, the amylose content of GBSSa gene mutant buckwheat plants (GBSSa-deficient lines) did not decrease compared to the wild type (A). However, in the summer sowing of 2020 (B) and the spring sowing of 2021 (D-4), the amylose content of GBSSa gene mutant buckwheat plants (GBSSa-deficient lines) decreased compared to the wild type. In wheat, it has been reported that there are years in which the amylose content does not decrease when one GBSS isozyme is deficient, but even taking that into account, it is still useful and is therefore becoming more widespread.

[0076] In addition, the amylose content in processed starch (C) was measured for the summer-sown cultivation in 2020, and the amylose content per buckwheat flour (E-4) was measured for the spring-sown cultivation in 2021. The method was as described in section 5, "Degree of Amylose Content Reduction in Acquired GBSS Gene Mutant Buckwheat Plants ("18N De6")." In these cases as well, the GBSSa gene mutant buckwheat plants (GBSSa-deficient lines) showed a lower amylose content compared to the wild type.

[0077] Furthermore, in lines in which the GBSSa-deficient gene was introduced by crossbreeding into cross-pollinating populations (D-1, E-1) and self-pollinating lines (D-2, E-2), the GBSSa gene mutant buckwheat plants (GBSSa-deficient lines) showed a lower amylose content compared to the wild type. This indicates that the deficiency of the GBSSa gene is an important factor in reducing the amylose content of buckwheat.

[0078] Furthermore, since buckwheat is pollinated by flowering, cross-pollinating buckwheat lines will cross-pollinate with other buckwheat plants if they are present in the vicinity. Although self-pollinating buckwheat lines have a lower rate of cross-pollination with other pollen compared to cross-pollinating buckwheat lines, their cross-pollination rate is still around 10% (Non-patent Literature 4, 5). When comparing self-pollinating buckwheat lines grown in isolation (D-3, E-3) with those grown in a situation where other buckwheat lines are present in the vicinity without isolation (D-4, E-4), it was found that even without isolation cultivation, GBSSa gene mutant buckwheat plants (GBSSa-deficient lines) had a lower amylose content compared to the wild type. This is due to the advantage of self-pollinating lines having a low cross-pollination rate, indicating that self-pollinating lines can obtain the effect of lower amylose content due to the GBSSa-deficient gene even without isolation cultivation.

[0079] The table below summarizes the amylose content and the rate of decrease in variants of amylose content for summer-sown crops in 2020 and spring-sown crops in 2021.

[0080] [Table 2]

[0081] [8. Detection of year-to-year differences in GBSSa protein in GBSS gene mutant buckwheat plants] Proteins were extracted from the seeds of GBSS gene mutant plants (GBSSa-deficient line (lineage name: "18Nde6") and wild-type plants (lineage name: "IH3"), and the accumulation of GBSSa protein was investigated by two-dimensional electrophoresis. The actual procedure followed Non-Patent Literature 6.

[0082] The results are shown in Figure 6. In plants with the GBSSa gene mutation, the accumulation of GBSSa protein was lacking regardless of the year of cultivation. This indicates that the gene mutation is expressed regardless of the cultivation environment.

[0083] [9. Detection of mutation sites using DNA markers in acquired GBSSa gene mutant buckwheat plants] Genomic DNA was extracted from the leaves of six GBSSa gene mutant plants, six wild-type plants, and two heterozygous plants resulting from crossing them, using a commercially available genomic DNA extraction kit. Genotyping was then performed using LGC Genomics' KASP® (Kompetitive Allele Specific PCR) genotyping assay, with the aforementioned gene mutation sites as indicators. The actual procedure followed the instructions on the following website. SNP / InDel Analysis PCR Reagent KASP™ Genotyping Assay | LGC Genomics Limited | Primetech Co., Ltd. (primetech.co.jp)

[0084] After performing the above procedure, the genotype was determined from the fluorescence dye intensity ratio corresponding to each genotype.

[0085] The results are shown in Figure 7. Since the GBSSa gene mutant plants, heterozygous plants, and wild-type plants separated into different locations, it is believed that genotype differentiation was possible.

[0086] [References cited in section 10. Examples] Non-patent document 3: Yun, SH & Matheson, NK (1990). Estimation of Amylose Content of Starches after Precipitation of Amylopectin by Concanavalin-A. Starch / Starke, 42, 302-305. Non-patent document 4: Hayashi, H., Wang, Y., & Campbell, CG (2004). Gene flow in selfpollinating buckwheat. Proceedings of the 9th international symposium on buckwheat, pp. 355-359. Non-patent document 5: Mukasa, Y., Suzuki, T., & Honda, Y. (2006). The degree of outcrossing in open pollinated self-pollinating buckwheat. Report of the Hokkaido Branch, Jpn. Soc. Breeding and Hokkaido Branch. Crop Science Society of Japan, 47, 65-66. Non-patent document 6: Klose J., From 2-D electrophoresis to proteomics, Electrophoresis, vol. 30, no. S1, S142-S149 (2009) [Industrial applicability]

[0087] The buckwheat plants in which the starch synthase of the present invention has been mutated have a lower amylose content compared to the wild type. Therefore, it is believed that the technology of the present invention will enable the cultivation of buckwheat plants with altered dough properties, thereby meeting consumer needs. [Sequence Listing Free Text]

[0088] Sequence ID: 1 Buckwheat GBSSa (wild type) gene Sequence ID:2 Buckwheat GBSSa (mutant) gene Sequence ID: 3 Buckwheat GBSSb gene Sequence ID: 4 Tartary buckwheat GBSSa cDNA Sequence ID: 5 Tartary buckwheat GBSSb cDNA Sequence ID: 6 Perennial buckwheat GBSSa cDNA Sequence ID: 7 Perennial buckwheat GBSSb cDNA Sequence ID: 8 Buckwheat GBSSa Sequence ID: 9 Buckwheat GBSSb Sequence ID: 10 Tartary buckwheat GBSSa Sequence ID: 11 Tartary buckwheat GBSSb Sequence ID: 12 Perennial buckwheat GBSSa Accession number: 13, Perennial buckwheat GBSSb (Sequence listing) <210> 1 <211> 3778 <212> DNA <213> Fagopyrum esculentum <220> <223> Fagopyrum esculentum, GBSSa wild type gene <400> 1 ttgatttatt tgttatatag catactaatt acttactttg tgttactcta tgtaggcatt 60 ttgcaatgac gtcactcaca atcacaagta gcttgcctgc agtgagcacc gtagcaggag 120 ccacatcttc aagcaaggca atgttcttgc aaaatagtct caaaagcact gcagggctcc 180 gacctttcaa cgacgtcgat atgctcaaat gcggggccaa ctcaaataac gcaccttcca 240 agttaccaac aatggaaccc aagagatccg caaataattc tgtggaatca agagttatcc 300 gatgtgggat gaacttggtc tttgtaggag ctgaggtagc tccatggagc aaaaccggtg 360 gccttggaga cgttcttgcc ggcctcccgc ctgcaatggc ggtatgtcat taatcctacc 420 atcttattgt taattggttt taggatagaa ctcattgagt ttatgcgttg aactggagtc 480 taacgtgtgt gacctcgtga ggatcaggcg aatgggcacc gagtgatgac agtctccccg 540 cgttatgatc agtaccaaga cgcttgggac acaagcgtgc aagctgaggt aaaagtggga 600 gataagacgg aaactgtgcg atttttcac tcttataagc gcggggtcga tcgcgtcttt 660 gttgatcatc ctttgttctt agccaaggta tagtatacat cttcgcatta taacgttacc 720 tgcaagattt gagatgaatt gtttgattaa tttacgttac gtgtatgata tcgaaggtat 780 ggggaaaaac tggattcaaa atctatggtc caagagccgg cgaagattat gaagacaacc 840 aacttcggtt tagcctctta gcacaggtca atataacgtg tataagatca tagtatatac 900 atagtttatc ttttatatcc gataggtata agacgggagg gtatgcacta ctaggggtga 960 gcaaaaatat ccgatttgac taatccgaag atccggttcg aaaaaatccg aatccgaaga 1020 tatccgacaa ttggtgatcc gatctgatcc agacatattt ttcggatcag atatggatcg 1080 gggtgtggag aaaaagtga gatcggatcg gatagatccg actagtctaa gaataatac 1140 ccggatcgga tcttcggatc agatatctgt ccggataaaa ctgtcggata tccgcatcct 1200 ctcggaacgg atagattcga ctattctaag aaaatacc cggatcggat cggatcaaat 1260 ctttggatcg gatatgtc aggataaac tgtcagatat ccggttctc tcggatcaga 1320 ttcgaatcgg acttttctaa gattgtcgga tcggatatcc ggaaatgccc atccctatgc 1380 actaggcacc caactttc gggttccaga gtagtagcac taagactt tggttcacgt 1440 gttatcatat tgtcgatgtg aaacattttc acactcaca ctccctcaa gtgcaatat 1500 catggctgac acttgaccca aaaccttaag gccggctag tcaacggac tggatacat 1560 attttcgcta atcacagcccc aaacccgtga tcgactccctc attatcatgt taatttatt 1620 tttatactgg gatgagacat tttcacattt ggacaataac father catgttttta fatherggttg tgttgcatgg atttcaggca gctttggag caccaaggat tcttaatctc agcaacaagt tcttttcagg accttatggt ttgtgcatat gatttaagta ttgtacttat gattgttttg ttctactaat actaatcata tgcatatatg tacgtaggtg aggatgttgt atttatcgcc aatgattggc acactgcact gctgccatgc tacctgaaaa ccatgtacaa atcgagagga father atgcaaaggt father ttcatatcta ctgtcactct tcctcttaat atctaggtta ttaagatgct ttgttgtgat tctaggtggc gttttgcatt 2100. 2100. 2100. 2100. 2100. 2100. 2100. 2100. 2100. 2100 gaagattac ttagatcttt caaatttcaa gacgggtatt ctattctaaa atcaattaac fathers gttgctgaat tcatgataat tacataaatt fathers atacgccggt ggaagggcct aagatcaact ggatgcaagc aggaatactc gaatccgaca aggttgtgac 2280 tgtgagcccg tattatgcgg aagagctgat ttcgggtgca gaaagaggag tcgagttaga 2340 cactgtgatt cgatcaaccg gaattacagg gatcttgaat ggcatggaca aacaagagtg 2400 gaatccttta actgataaac acatcactta tcaatatgat gtttcaaatg taatgcataa 2460 gtcgtcttta attaaaacaa tatgcttaat ataaaagtgt tgaaatactg ataaatctaa 2520 cacttatttt gtgattttag gtgatggagg caaaggcttt gaacaaggaa gccttgcaag 2580 ctgaggtggg attgcctgtt gataggaaca tacctcttat aggattcatt ggaaggctag 2640 aggagcaaaa gggttcggat attctcgtcg aatcaattcc tcaatgcatc aatgagaacg 2700 tccagcttct tgttttggta agcttaatga attgaaactt gtttgatcta ataagggtca 2760 atctctttgc tgctattaaa aaaaaaacat gttgtggcct ataaagggaa ccggaaagaa 2820 gaaattcgag aaagaaatcg aagaactcga aaatctgtat ccaaacaagg ttagaggagt 2880 gaccaagttt aacccagccc tagctcacaa gatcattgca ggggctgatt tcatgctcat 2940 accaagccga ttcgagccat gcgggcttat ccaactacat gccatgcttt atggaacggt 3000 acgacgttat agaacaagtc tatcatataa ggttggtggg ttatttcacg gattctaatt 3060 ttgcatcaca attttgttag gttcctatgt gtgcctcaac tggaggactt gttgactgcg 3120 tcaaagaagg cattacagga ttccacatgg gccgattcgg agcccaggta tatagttata 3180 aattgaattt gcttatgtta aataattttg tgagtatttt agttggtgac tgaacaaagt 3240 cttgatttgt aacagtgcaa agctgtagat gcggctgatg ttgcagctgt gacatcgact 3300 gtgaggaggg cagttgcagt gcatggtaca cctgctatga gagacatgat tctcaattgc 3360 atgtatcaag acctttcatg gaaggcaagt cctattgaat ttcccttttc gtttcggttt 3420 tgacctgaac taacttcgaa acgtacttgc atgaatgtaa acaatgaata tacaggagcc 3480 tgcaaggaaa tgggaagaga tgctacttgg aatgcaggtg gctgggagcg ggcctgggat 3540 caagggcgaa gaaattgcgc cgttagctgc ggaaaacatc gccactccat aatcaggagg 3600 gttcatgtac ctgcagaaca gtataaggtg tatgactgta tgtcattact agcatgacct 3660 tatatatctt gtcaatgcta ctatattgtg aaccgaatat ggatctatct tccatatctg 3720 ttgcatgaat aaagttcaca tctttgtttg tcaagtatct atttgttca acaacagt 3778 <210> 2 <211> 3778 <212> DNA <213> Fagopyrum esculentum <220> <223> Fagopyrum esculentum , GBSSa mutant gene <400> 2 ttgatttatt tgttatatag catactaatt acttactttg tgttactcta tgtaggcatt 60 ttgcaatgac gtcactcaca atcacaagta gcttgcctgc agtgagcacc gtagcaggag 120 ccacatcttc aagcaaggca atgttcttgc aaaatagtct caaaagcact gcagggctcc 180 gaccttcaa cgacgtcgat atgctcaaat gcggggccaa ctcaaataac gcaccttcca 240 agttaccaac aatggaaccc aagagatccg caaatattc tgtggaatca agagttatcc 300 gatgtgggat gaacttggtc tttgtaggag ctgaggtagc tccatggagc aaaaccggtg 360 gccttggaga cgttcttgcc ggcctcccgc ctgcaatggc ggtatgtcat taatcctacc 420 atcttattgt taattggttt taggatagaa ctcattgagt ttatgcgttg aactggagtc 480 taacgtgtgt gacctcgtga ggatcaggcg aatgggcacc gagtgatgac agtctccccg 540 cgttatgatc agtaccaaga cgcttgggac acaagcgtgc aagctgaggt aaaagtggga 600 gataagacgg aaactgtgcg atttttcac tcttataagc gcggggtcga tcgcgtcttt 660 gttgatcatc ctttgttctt agccaaggta tagtatacat cttcgcatta taacgttacc 720 tgcagattt gagatgaatt gtttgattta tttacgttac gtgtatgata tcgaaggtat 780 ggggaaaaac tggattcaa atctatggtc caagagccgg cgagattat gagacaacc 840 aacttcggtt tagcctctta gcacaggtca ataacgtg tataagatca tagtatatac 900 atagtttatc ttttatatcc gataggtata agacgggagg gtatgcacta ctaggggtga 960 gcaaaataat ccgatttgac taatccgaag atccggttcg aaaaaatccg aatccgaaga 1020 tatccgacaa ttggtgatcc gatctgatcc agacatattt ttcggatcag atatggatcg 1080 gggtgtggag aaaaagtga gatcggatcg gatagatccg actagtctaa gaataatac 1140 ccggatcgga tcttcggatc agatatctgt ccggataaaa ctgtcggata tccgcatcct 1200 ctcggaacgg atagattcga ctattctaag aaaatacc cggatcggat cggatcaaat 1260 ctttggatcg gatatgtc aggataaac tgtcagatat ccggttctc tcggatcaga 1320 ttcgaatcgg acttttctaa gattgtcgga tcggatatcc ggaaatgccc atccctatgc actaggcacc caagactttc gggttccaga gtagtagcac taagacttat tggttcacgt gttatcatat tgtcgatgtg aaacattttc acactcaaca ctcccctcaa gtgcaaatat catggctgac acttgaccca aaaccttaag gccggcttag tcaaacggac tggataacat attttcgcta atcacagccc aaacccgtga tcgactcctc attatcatgt taatttattt tttatactgg gatgagacat tttcacattt ggacaataac father catgttttta fatherggttg tgttgcatgg atttcaggca gctttggag caccaaggat tcttaatctc agcaacaagt tcttttcagg accttatggt ttgtgcatat gatttaagta ttgtacttat gattgttttg ttctactaat actaatcata tgcatatatg tacgttggtg aggatgttgt atttatcgcc aatgattggc acactgcact gctgccatgc tacctgaaaa ccatgtacaa atcgagagga atatacaaca atgcaaaggt ataccaatct ttcatatcta ctgtcactct 1980 tcctcttaat atctaggtta ttaagatgct ttgttgtgat tctaggtggc gttttgcatt 2040 cacaatatct cttaccaagg acgatttgcc cgatctgact acaattctct caatttgccc 2100 gaagaattac ttagatcttt caaatttcaa gacgggtatt ctattctaaa atcaattaac 2160 aataagtaat gttgctgaat tcatgataat tacataaatt aacaggtatg atacgccggt 2220 ggaagggcct aagatcaact ggatgcaagc aggaatactc gaatccgaca aggttgtgac 2280 tgtgagcccg tattatgcgg aagagctgat ttcgggtgca gaaagaggag tcgagttaga 2340 cactgtgatt cgatcaaccg gaattacagg gatcttgaat ggcatggaca aacaagagtg 2400 gaatccttta actgataaac acatcactta tcaatatgat gtttcaaatg taatgcataa 2460 gtcgtcttta attaaaacaa tatgcttaat ataaaagtgt tgaaatactg ataaatctaa 2520 cacttatttt gtgattttag gtgatggagg caaaggcttt gaacaaggaa gccttgcaag 2580 ctgaggtggg attgcctgtt gataggaaca tacctcttat aggattcatt ggaaggctag 2640 aggagcaaaa gggttcggat attctcgtcg aatcaattcc tcaatgcatc aatgagaacg 2700 tccagcttct tgttttggta agcttaatga attgaaactt gtttgatcta ataagggtca 2760 atctctttgc tgctattaaa aaaaaaacat gttgtggcct ataaagggaa ccggaaagaa 2820 gaaattcgag aaagaaatcg aagaactcga aaatctgtat ccaaacaagg ttagaggagt 2880 gaccaagttt aacccagccc tagctcacaa gatcattgca ggggctgatt tcatgctcat 2940 accaagccga ttcgagccat gcgggcttat ccaactacat gccatgcttt atggaacggt 3000 acgacgttat agaacaagtc tatcatataa ggttggtggg ttatttcacg gattctaatt 3060 ttgcatcaca attttgttag gttcctatgt gtgcctcaac tggaggactt gttgactgcg 3120 tcaaagaagg cattacagga ttccacatgg gccgattcgg agcccaggta tatagtata 3180 aattgaattt gcttatgtta aataatttg tgagtatttt agttggtgac tgaacaaagt 3240 cttgatttgt aacagtgcaa agctgtagat gcggctgatg ttgcagctgt gacatcgact 3300 gtgaggaggg cagttgcagt gcatggtaca cctgctatga gagacatgat tctcaattgc 3360 atgtatcaag acctttcatg gaaggcaagt cctattgaat ttccctttc gtttcggttt 3420 tgacctgaac taacttcgaa acgtacttgc atgaatgtaa acaatgaata tacaggagcc 3480 tgcaaggaaa tgggaagaga tgctacttgg aatgcaggtg gctgggagcg ggcctgggat 3540 caagggcgaa gaaattgcgc cgttagctgc ggaaaacatc gccactccat aatcaggagg 3600 gttcatgtac ctgcagaaca gtataaggtg tatgactgta tgtcattact agcatgacct 3660 tatatatctt gtcaatgcta ctatattgtg aaccgaatat ggatctatct tccatatctg 3720 ttgcatgaat aaagttcaca tctttgtttg tcaagtatct atttgttca acaacagt 3778 <210> 3 <211> 3681 <212> DNA <213> Fagopyrum esculentum <220> <223> Fagopyrum esculentum, GBSSb gene <400> 3 gtacgatcat ttgttgtaag agactaagaa agtgatcttg gagttttagt tcaggcaagt 60 ttctattttt cttaagagtt ttacaaagtt taattagcat atgagttttt gaccagttgt 120 tactctgtgc aggctcatca atggcgtctc taataccgc tgtgtgtatg gcaagcagcg 180 gaggaaccac aactggctct gatgctagca aggttgtgtc gttgcaaaat ggtttaagaa 240 accacacggg gcttcgtcct ctcaacagag ttgacatgct gaaatggaga gccaacactt 300 ccaaattacc gagaatggaa gagaagagag ttgttacgag tcccagcttt aaaaaagggg 360 ttattcagtg tggaatgaac ttggtctttg tctcagctga ggtggctcct tggagcaaaa 420 ctggtggcct tggggatgtc cttgctggcc tcccaccagc catggcggta ttttattaat 480 cttattagta actagtacca agttcttgta ctatgccttt ccagcactct aaaatttttg 540 atttgcttgt ttatgttcag gccaatgggc accgagtcat gacaatctcc cctcggtatg 600 atcagtacca agatgcatgg gatacaagcg tggaggctga ggtgaaaata ttcacaattt 660 tgcctgctta tatactctaa gaaatcaatt tcaagaatca ataagcactt tttaaatgct 720 taattgtgtt agtgacaatt atcgattgat acaactatga taccctacag gtaaaagtgg 780 gagataagac ggaaactgtg cgctttttcc actcttacaa acgtggggtt gatcgtgtct 840 tcgtggatca tccattgttc ctagctaagg tatgaggaat attagtaatg attctcaaat 900 caagtgtttg atttggtgtt taacatctgc tatttacatt atgggtgttg taggtatggg 960 ggaaaactgg attccaaatc tatggtccaa gagctggtga agattacgaa gacaaccaac 1020 ttcgattcag tgtcttatct caggtgagga tgattgattc acttgcatca aatctaggtg 1080 tgatcaaact aagatcatat gaaaagcctt ttggctagct tatccttaag taacccttaa 1140 ttagctatca ttcgttagaa aaacaaaaaa taaagcttat ctagctgagt ttacttgtat 1200 agttggctct atataacata attgccaata gattaacctc tcttttgaaa gcacaattga 1260 aaataaaaac aaactaaatt catgttatct gcctgcactg tgtgaattca ggcagcattg 1320 gaggctccaa ggattctaaa tctcaacaac aaatttttct caggaccata tggtttgtaa 1380 ctttcttctc aaaccaaata aatgataact ttgtttacta attaaatcgt atatttgtaa 1440 atatgtatac tagttacttg gtatgtagat tgtaatttat ctaacgcttg agtaatgtca 1500 tctgtatgac tttaggagag gatgttttgt ttgtcgcaaa cgattggcac tctgctctcc 1560 taccatgcta cctgaagtcc atgtacaaat caaggggaat ctataagaat gcaaaggtaa 1620 tacttgcagg ctctctaact ttcctttttc tacgaatccc agctattcat tcttcattcg 1680 ttttactttc aggttgcatt ctgcatccac aacatttctt atcaaggccg atttgcccca 1740 actgacttca aagctctcaa tttacccaat gaatttctga agtcgtttga attttttgat 1800 gggtattgcc ctcacgtccc aacttctctt ctttacatag aatgcaagct ttcttgtata 1860 tttgaggtgc attaagatca catacgtgat acatgtgtat taacgtgtaa acaggtatga 1920 cacgcctatc acaggggaca aaatcaactg gatgcaagcc ggaatactag agtccgacaa 1980 ggttgtgacc gtgagcccat attatgctga agagttggtt tcgggtgctg aaagaggtgt 2040 ggaactagaa aaaatcattc gaaaaactgg agtaattggt atcctaaatg gaatggataa 2100 acaagagtgg gatccttcca gtgacaagtt catcaattac aaatatgatg tttcaactgt 2160 aagtatcatc taaatacaca taaatttctt catctgacta tgttgcttaa tctgaagaaa 2220 tcttttaggt gatggaggca aagccactca taaaggaatc ccttcaagcc gagttgggct 2280 tgcccgttga caggaacatt ccacttatag gattcattgg aaggttggaa gagcaaaagg 2340 gttcggatat tcttgttgaa tccatcccag aatttatcaa tgagaatgtc caaattgtta 2400 tcttggtagg gaaactagat tctttttctt gatctttaac cttgattctt cgatcatgtg 2460 tttcaacttt gaatttatga catggccttt ttcaggggac tggaaagaag cgatttgaga 2520 aacaaatcga agagctcgag gccctttatc ccgacaaggc tagaggagta gcagcattca 2580 acccatccat ggctcacaag atcaatgctg gtgttgactt tatgttaatc cctagtcggt 2640 ttgagccatg tgggcttacc caactacacg ctatgctcta tggaacggta ctatgtccaa 2700 aatctcttat aacaaattaa gcatttgatc ctcttttcga tttttctcac ttctaatctg 2760 attgatgtgt aatgtcctta ggttccaatt tgtgcttcaa ctggtgggct tgtggattgt 2820 gtaaaagaag gcgtcacagg gtttcacatg ggcaagttca gcgccgaggt acgtacacaa 2880 ctttagtcgg aatttttctt tttctttttc tttttttaaa atcaacgatt gattttatta 2940 tgaaacaatc aaaccgagtg cactaaagcg aaccctagac acgttaccag tccgcctcct 3000 actcgcattg cttccgggaa cctgtaggtt gtaaacccaa gaaaatgctt tccacggaat 3060 tataaccatt gctccgaagt tcgaattttg acgagttatg acatatgaga tgttttttca 3120 ttgagcttgg ggatgcattc ccatcgagtc cgggaggctc atatgtggta cggagcgccg 3180 aaaccgaatt caacgttcaa attcaattgc tagaattttg tacggaatag gagtggggcg 3240 cattcctggt gcgtttggga gtcgcatttc tagtggaagt gggaggcgga ttcggatcgg 3300 aatggaaata acggtttcaa tctataaatg ttgttatttg tatgattagt gcaaagccgt 3360 ggatccaact gacgtggcgg cagtcacaag tactgtgagg aaagcggtag ctctacatgg 3420 taccccagcc atgcaagaga tgatcctcaa ctgcatgtat caagacctta cgtggaaggc 3480 aagtcattcg atctttcgtt tctgttttca tagtaacaat aggagattga tttgtaaaac 3540 ttaaatgcat gaacaatgaa acaggagcca gcaaagaaat gggaggagat gctactaggg 3600 ttacaggtgg cggggagcgg acctgggatt gagggggaag aaatcgcccc gcttgcagtg 3660 gaaaatgttg ctgctccttg a 3681 <210> 4 <211> 1827 <212> DNA <213> Fagopyrum tataricum <220> <223> Fagopyrum tataricum , GBSSa cDNA <400> 4 atgacgtcac tcacaatcac aagtagcttg cctgcagtga gcaccgtagc aggagccaca 60 tcttcaagca aggcaatgtt cttgcaaaat agtctcaaaa gcactgcagg gctccgacct 120 ttcaacgacg tcgatatgct caaatgcggg gccaactcaa ataacgcacc ttccaagtta 180 ccaacaatgg aacccaagag atccgcaaat aattctgcac aacctggggt tatccgatgt 240 gggatgaact tggttttcgt aggagctgag gtagctccct ggagcaaaac cggtggcctt 300 ggagacgtc ttgctggcct cccgcctgca atggcagcga atggacacag agtgatgaca 360 atctccccgc gttatgatca gtatcaagac gcatgggaca caagcgtgca agctgaggta 420 aaagtgggag ataagacgga aactgtgcga ttttttcact cttataagcg tggtgttgat 480 cgcatctttg tagatcatcc tttgttctta gccaaggtat ggggaaaaac tggattcaaa 540 atctatggtc caagagcagg agaagattat gaagacaacc aacttcggtt cagcctctta 600 gcacaggcag ctttggaagc accaaggatt cttaatctga gcaacaaatt cttttcagga 660 ccttatggtg aggatgttgt attcatcgct aatgattggc acactgcact gctgccatgc 720 tacctgaaaa ccatgtacaa atcgagagga atctacaaca atgcaaaggt ggcgttttgc 780 attcacaata tctcttatca aggacgatt gcccaatccg attttatttc tctcaatttg 840 cctgaagaat tacttagatc tttcgaattt cagacggat atgatacgcc ggtggaaggg 900 cctaagatca actggatgca agcaggaata ctcgaatccg acaggttgt gaccgtgagc 960 ccgtatttg ctgaagagct gatttcgggt gcagaaagg gtgtcgagtt agacaaagtg 1020 attggatcaa ccggaattac agggatcttg aatggcatgg acaacaga gtggaatcct 1080 ttaactgata aacatatcac ttaccatat gatgtttcaa atgtgga ggcaaggct 1140 ttgaacagg aagcattgca agctgaggtg ggattgccag ttgatagaa catacctt 1200 ataggattca ttggaggct agagagcaa aagggttcgg atattctcgt cgaatccatt 1260 cctcaatgca tcaatgagaa cgtccagtttt ctcgttctgg gaaccggaaa gaagaaatttc 1320 gagaaaaaaa tcgaagaact cgaaaacctg tatccaaca agattagagg agtgaccaaa 1380 tttaacccag ccttagctca caagatcatt gcaggggctg atttcatgct cattccaagc 1440 cgattcgagc catgcgggct tatccaacta catgccatgc tttatggaac agttcctatg 1500 tgtgcctcaa ctggaggact tgttgactgt gtgaaagaag gcattacagg attccacatg 1560 ggccgattcg gagcccagtg caaagctgta gatccagctg atgttgcagc tgtgacatcg 1620 actgtgagga gggcagttgc agtgcatggt acacctgcta tgagagacat gattctcaac 1680 tgcatgtatc aagacctttc atggaaggag cctgcaaaga aatgggaaga gatgttactt 1740 ggaatgcagg tggcagggag cgggtctggg ttcaagggcg aagaaattgc gccactagct 1800 gcggaaaaca tcgccactcc ataataa 1827 <210> 5 <211> 1818 <212> DNA <213> Fagopyrum tataricum <220> <223> Fagopyrum tataricum , GBSSb cDNA <400> 5 atggcgtctc tgaataccgc tgtgtgtatg gcaagcagtg gaggaactac aactggctct 60 gatgctagta aggttgtgtt gttgcaaaat ggtttaagaa accacacggg gcttcgtcct 120 ctcaacagag ttgacatgct gaaatggaga gccaacactt ccaaattacc gagaatggaa 180 gagaagagag ttgttacgag tcccagcttc aaaaaagggg ttattcagtg tggaatgaac 240 ttggtatttg ttcggctga ggtggctcct tggagcaaaa ctggtggtct tggggatgtc 300 cttgctggcc tcccaccagc catggcggcc aatgggcacc gagtcatgac aatctccccct 360 cggtatgatc agtaccaaga tgcatgggat acaagtgtag aagccgaggt aaaagtggga 420 gataagacgg aaactgtgcg ttttttccat tcttacaaac gtggggttga tcgtgtcttc 480 gtggatcatc cattgttcct agctaaggta tgggggaaaa ctggattcca aatctatggt 540 ccaagagctg gtgaagatta cgaagacaac caacttcgat tcagcgtctt atctcaggca 600 gcattggagg ctccaaggat tctaaatctc agcaacaaat ttttctcagg accgtatgga 660 gaggatgttt tgtttgttgc aaacgattgg cactctgctc tcctaccatg ctacctaaaa 720 tccatgtaca aatcaagagg aatctacaag aatgcaaagg tcgcattctg catccacaac 780 atttcttatc aaggacgatt tgccccaact gacttcggag ctctcaattt gcccaatgaa 840 tttctgaagt cttttgaatt ttttgatggg tatgatacgc ctatcacagg ggacaaaatc 900 aactggatgc aagccggaat actagaatcc gacaaggttg tgactgtgag cccatattat 960 gctgaagagt tggtttcagg tgctgaaaga ggtgtggaac tagaaaaaat aattcgaaaa 1020 actggagtaa ttggtatcct aaatggaatg gataagcaag aatgggatcc ttctagtgac 1080 aagttcatca attacaaata tgatgtttca actgtgatgg aggcaaagcc cctcataaag 1140 gaatcccttc aagccgagtt gggcttgccc gttgacagga atattcctct tataggattc 1200 attggaaggt tggaagagca aaagggttcg gatattcttg ttgaatccat cccagaattt 1260 atcaatgaga atgtccaaat tgttatcttg gggactggaa agaagcggtt tgagaaacaa 1320 atcgaagagc ttgaggctct ttatcccaac aaggctagag gagtagcagc attcaatcca 1380 tccatggctc acaagatcaa tgctggtgtt gactttatgt taatccctag tcggtttgag 1440 ccatgcgggc ttacccaact gcacgctatg ctctatggaa cgattccaat ttgtgcttca 1500 actggtgggc ttgtggattg cgtaaaagaa ggcatcacag ggtttcatat gggcaagttc 1560 aacgccgagt gcaaagccgt ggatccagct gacgtggcgg cagtcacaag tactgtgaga 1620 aaagcggtaa ccctacatgg tactccagcc atgcgagaga tgatcctcaa ctgcatgtat 1680 caagacctta catggaagga gccagcaaag aaatgggagg agatgctatt agggttacag 1740 gtggtgggga gcggacccgg gattgagggg gaagaaattg ccccgctggc aatggaaaat 1800 gttgctgctc cttgatga 1818 <210> 6 <211> 1824 <212> DNA <213> Fagopyrum cymosum <220> <223> Fagopyrum cymosum, GBSSa cDNA <400> 6 atgacgtcac tcacgatcac aagtagcttg cctgcagtga gcaccgcagg aggagccaca 60 tcttcaagca aggcaatgtt cttgcaaaat agtctcaaaa gtactgcagg gctccgacct 120 ctcaacgacg tcgacatgct caaatgcagg gcgaactcga ataacgcacc ttccaagtta 180 ccaacaatgg aagtcaagag atctgcaaat aattatgcgg aacctggggt tatccgatgt 240 gggatgaact tggtcttcgt aggagctgag gtggctccat ggagcaaaac cggtggcctt 300 ggagacgttc ttgccggcct cccgcctgca atggcggcga atggacaccg agtgatgaca 360 atctccccgc gttatgatca gtaccaagac gcatgggaca caagcgtgca agctgaggta 420 aaagtgggag ataagactga aactgtgcga ttttttcact cttataagcg cggcgttgat 480 cgcatctttg tcgatcatcc tttgttctta gccaaggtat ggggaaaaac cggattcaaa 540 atctatggtc caagagccgg agaagattat gaagacaacc aacttcggtt cagcctctta 600 gcacaggcag ctttggaagc accaaggatt cttaatctga gcaacaaatt cttttcagga 660 ccttatggtg aggatgttgt attcatcgct aatgattggc acactgcact gctgccatgc 720 tacctgaaa ccatgtacaa atcaagagga atctacaaca atgcaaaggt ggcgttttgc 780 attcacaata tctcttacca aggacgattt gcccaatccg attttatttc tctcaatttg 840 cccgaagaat tacttagatc ttcgaattt caagacgggt atgatacgcc ggtggaaggg 900 cctaagatca actggatgca agcaggaata ctcgaatccg acaaggttgt gaccgtgagc 960 ccgtattatg ctgaagagct gattcgggt gcagaaagag gtgtcgagtt agacaaagtg 1020 attcgatcaa ccggaattac agggatctta aatggcatgg acaaacaaga gtggaatcct 1080 ttaactgata aacatatcac ttaccaatat gatgtttcaa atgtgatgga ggcaaaggct 1140 ttgaataagg aagccttgca agctgaggtg ggattgcctg ttgataggaa catacctctt 1200 ataggattca ttggaaggct agaagagcaa aagggttcgg atattctcgt cgaatccatt 1260 cctcaatgca tcaatgagaa cgtccagttt ctcgttctgg gaaccggaaa gaagaaattc 1320 gagaaaa tcgaagaact cgaaaacctg tatccaaaca agattagagg agtgaccaag 1380 tttaacccag ccttagctca caagatcatt gcaggggctg atttcatgct cattcccagc 1440 cgattcgagc catgtgggct tatccaacta catgccatgc tttatggaac ggttcctatg 1500 tgtgcctcaa ctggaggact tgttgactgc gtgaaagag gcattacagg attccacatg 1560 ggccgattcg gagcccagtg caaagcagta gatcctgctg atgttgcagc tgtgacatcg 1620 actgtgagga gggcagtagc agtgcatggt acacctgcta tgagagacat gattctcaat 1680 tgcatgtatc agaccttc atggaggag cctgcagga aatgggaaga gatgttactt 1740 ggaatgcagg tggcaggg cgggcctggg tcaagggcg aagaattgc gccgctagct 1800 1824 - 1824 <210> 7 <211> 1815 <212> DNA <213> Phagopyrum cymosum <220> <223> Phagopyrum cymosum , GBSSb cDNA <400> 7 atggcgtctc taaataccgc tgtgtgtatg gcaagcagcg gaggaaccac aactggctct 60 gatgctagca aggttgtgtc gttgcaaat gttttaagaa accacacggg gcttcgtcct 120 ctcacagag ttgacatgct gaaatggaga gccaactt ccaattacc gagaatggaa 180 gagagagag tgttackggtgctchttt aaaaagggg tgtgtg tgtgtg tgtgtg 240 tggtctttg tctcagctga ggtggctct tggagcaaa ctggtggct tggtgtc 300 cttgctggcc tcccaccagc catggcggcc aatgggcacc gagtcatgac aatctccccct 360 cggtatgatc agtaccaaga tgcatgggat acaagcgtgg aggctgaggt aaaagtggga 420 gataagacgg aaactgtgcg ctttttccac tcttacaaac gtggggttga tcgtgtcttc 480 gtggatcatc cattgttcct agctaaggta tgggggaaaa ctggattcca aatctatggt 540 ccaagagctg gtgaagatta cgaagacaac caacttcgat tcagtgtctt atctcaggca 600 gcattggagg ctccaaggat tctaaatctc aacaacaaat ttttctcagg accatatgga 660 gaggatgttt tgtttgtcgc aaacgattgg cactctgctc tcctaccatg ctacctgaag 720 tccatgtaca aatcaagggg aatctataag aatgcaaagg ttgcattctg catccacaac 780 atttcttatc aaggccgatt tgccccaact gacttcaaag ctctcaattt acccaatgaa 840 tttctgaagt cgtttgaatt ttttgatggg tatgacacgc ctatcacagg ggacaaaatc 900 aactggatgc aagccggaat actagagtcc gacaaggttg tgaccgtgag cccatattat 960 gctgaagagt tggtttcggg tgctgaaaga ggtgtggaac tagaaaaaat cattcgaaaa 1020 actggagtaa ttggtatcct aaatggaatg gataaacaag agtgggatcc ttccagtgac 1080 aagttcatca attacaaata tgatgtttca actgtgatgg aggcaaagcc actcataaag 1140 gaatcccttc aagccgagtt gggcttgccc gttgacagga acattccact tataggattc 1200 attggaaggt tggaagagca aaagggttcg gatattcttg ttgaatccat cccagaattt 1260 atcaatgaga atgtccaaat tgttatcttg gggactggaa agaagcgatt tgagaaacaa 1320 atcgaagagc tcgaggccct ttatcccgac aaggctagag gagtagcagc attcaaccca 1380 tccatggctc acaagatcaa tgctggtgtt gactttatgt taatccctag tcggtttgag 1440 ccatgtgggc ttacccaact acacgctatg ctctatggaa cggttccaat ttgtgcttca 1500 actggtgggc ttgtggattg tgtaaaagaa ggcgtcacag ggtttcacat gggcaagttc 1560 agcgccgagt gcaaagccgt ggatccaact gacgtggcgg cagtcacaag tactgtgagg 1620 aaagcggtag ctctacatgg taccccagcc atgcaagaga tgatcctcaa ctgcatgtat 1680 caagacctta cgtggaagga gccagcaaag aaatgggagg agatgctact agggttacag 1740 gtggcgggga gcggacctgg gattgagggg gaagaaatcg ccccgcttgc agtggaaaat 1800 gttgctgctc cttga 1815 <210> 8 <211> 607 <212> PRT <213> Fagopyrum esculentum <220> <223> Fagopyrum esculentum, GBSSa <400> 8 Put Thr Ser Leu Thr Ile Thr Ser Ser Leu Pro Ala Val Ser Thr Val 1 5 10 15 Here Gly Here Thr Dear Dear Dear Lys Here Put Phe Leu Gln Asn Dear Leu 20 25 30 Lys Ser Thr Ala Gly Leu Arg Pro Phe Asn Asp Val Asp Met Leu Lys 35 40 45 Cys Arg Ala Asn Ser Asn Asn Ala Pro Ser Lys Leu Pro Thr Met Glu 50 55 60 Pro Lys Arg Ser Ala Asn Asn Ser Val Glu Ser Arg Val Ile Arg Cys 65 70 75 80 Gly Met Asn Leu Val Phe Val Gly Ala Glu Val Ala Pro Trp Ser Lys 85 90 95 Thr Gly Gly Leu Gly Asp Val Leu Ala Gly Leu Pro Pro Ala Met Ala 100 105 110 Ala Asn Gly His Arg Val Met Thr Val Ser Pro Arg Tyr Asp Gln Tyr 115 120 125 Gln Asp Ala Trp Asp Thr Ser Val Gln Ala Glu Val Lys Val Gly Asp 130 135 140 Lys Thr Glu Thr Val Arg Phe Phe His Ser Tyr Lys Arg Gly Val Asp 145 150 155 160 Arg Val Phe Val Asp His Pro Leu Phe Leu Ala Lys Val Trp Gly Lys 165 170 175 Thr Gly Phe Lys Ile Tyr Gly Pro Arg Ala Gly Glu Asp Tyr Glu Asp 180 185 190 Asn Gln Leu Arg Phe Ser Leu Leu Ala Gln Ala Ala Leu Glu Ala Pro 195 200 205 Arg Ile Leu Asn Leu Ser Asn Lys Phe Phe Ser Gly Pro Tyr Gly Glu 210 215 220 Asp Val Val Phe Ile Ala Asn Asp Trp His Thr Ala Leu Leu Pro Cys 225 230 235 240 Tyr Leu Lys Thr Met Tyr Lys Ser Arg Gly Ile Tyr Asn Asn Ala Lys 245 250 255 Val Ala Phe Cys Ile His Asn Ile Ser Tyr Gln Gly Arg Phe Ala Arg 260 265 270 Ser Asp Tyr Asn Ser Leu Asn Leu Pro Glu Glu Leu Leu Arg Ser Phe 275 280 285 Lys Phe Gln Asp Gly Tyr Asp Thr Pro Val Glu Gly Pro Lys Ile Asn 290 295 300 Trp Met Gln Ala Gly Ile Leu Glu Ser Asp Lys Val Val Thr Val Ser 305 310 315 320 Pro Tyr Tyr Ala Glu Glu Leu Ile Ser Gly Ala Glu Arg Gly Val Glu 325 330 335 Leu Asp Thr Val Ile Arg Ser Thr Gly Ile Thr Gly Ile Leu Asn Gly 340 345 350 Met Asp Lys Gln Glu Trp Asn Pro Leu Thr Asp Lys His Ile Thr Tyr 355 360 365 Gln Tyr Asp Val Ser Asn Val Met Glu Ala Lys Ala Leu Asn Lys Glu 370 375 380 Ala Leu Gln Ala Glu Val Gly Leu Pro Val Asp Arg Asn Ile Pro Leu 385 390 395 400 Ile Gly Phe Ile Gly Arg Leu Glu Glu Gln Lys Gly Ser Asp Ile Leu 405 410 415 Val Glu Ser Ile Pro Gln Cys Ile Asn Glu Asn Val Gln Leu Leu Val 420 425 430 Leu Gly Thr Gly Lys Lys Lys Phe Glu Lys Glu Ile Glu Glu Leu Glu 435 440 445 Asn Leu Tyr Pro Asn Lys Val Arg Gly Val Thr Lys Phe Asn Pro Ala 450 455 460 Leu Ala His Lys Ile Ile Ala Gly Ala Asp Phe Met Leu Ile Pro Ser 465 470 475 480 Arg Phe Glu Pro Cys Gly Leu Ile Gln Leu His Ala Met Leu Tyr Gly 485 490 495 Thr Val Pro Met Cys Ala Ser Thr Gly Gly Leu Val Asp Cys Val Lys 500 505 510 Glu Gly Ile Thr Gly Phe His Met Gly Arg Phe Gly Ala Gln Cys Lys 515 520 525 Ala Val Asp Ala Ala Asp Val Ala Ala Val Thr Ser Thr Val Arg Arg 530 535 540 Ala Val Ala Val His Gly Thr Pro Ala Met Arg Asp Met Ile Leu Asn 545 550 555 560 Cys Met Tyr Gln Asp Leu Ser Trp Lys Glu Pro Ala Arg Lys Trp Glu 565 570 575 Glu Met Leu Leu Gly Met Gln Val Ala Gly Ser Gly Pro Gly Ile Lys 580 585 590 Gly Glu Glu Ile Ala Pro Leu Ala Ala Glu Asn Ile Ala Thr Pro 595 600 605 <210> 9 <211> 604 <212> PRT <213> Fagopyrum esculentum <220> <223> Fagopyrum esculentum, GBSSb <400> 9 Met Ala Ser Leu Asn Thr Ala Val Cys Met Ala Ser Ser Gly Gly Thr 1 5 10 15 Thr Thr Gly Ser Asp Ala Ser Lys Val Val Ser Leu Gln Asn Gly Leu 20 25 30 Arg Asn His Thr Gly Leu Arg Pro Leu Asn Arg Val Asp Met Leu Lys 35 40 45 Trp Arg Ala Asn Thr Ser Lys Leu Pro Arg Met Glu Glu Lys Arg Val 50 55 60 Val Thr Ser Pro Ser Phe Lys Lys Gly Val Ile Gln Cys Gly Met Asn 65 70 75 80 Leu Val Phe Val Ser Ala Glu Val Ala Pro Trp Ser Lys Thr Gly Gly 85 90 95 Leu Gly Asp Val Leu Ala Gly Leu Pro Pro Ala Met Ala Ala Asn Gly 100 105 110 His Arg Val Met Thr Ile Ser Pro Arg Tyr Asp Gln Tyr Gln Asp Ala 115 120 125 Trp Asp Thr Ser Val Glu Ala Glu Val Lys Val Gly Asp Lys Thr Glu 130 135 140 Thr Val Arg Phe Phe His Ser Tyr Lys Arg Gly Val Asp Arg Val Phe 145 150 155 160 Val Asp His Pro Leu Phe Leu Ala Lys Val Trp Gly Lys Thr Gly Phe 165 170 175 Gln Ile Tyr Gly Pro Arg Ala Gly Glu Asp Tyr Glu Asp Asn Gln Leu 180 185 190 Arg Phe Ser Val Leu Ser Gln Ala Ala Leu Glu Ala Pro Arg Ile Leu 195 200 205 Asn Leu Asn Asn Lys Phe Phe Ser Gly Pro Tyr Gly Glu Asp Val Leu 210 215 220 Phe Val Ala Asn Asp Trp His Ser Ala Leu Leu Pro Cys Tyr Leu Lys 225 230 235 240 Ser Met Tyr Lys Ser Arg Gly Ile Tyr Lys Asn Ala Lys Val Ala Phe 245 250 255 Cys Ile His Asn Ile Ser Tyr Gln Gly Arg Phe Ala Pro Thr Asp Phe 260 265 270 Lys Ala Leu Asn Leu Pro Asn Glu Phe Leu Lys Ser Phe Glu Phe Phe 275 280 285 Asp Gly Tyr Asp Thr Pro Ile Thr Gly Asp Lys Ile Asn Trp Met Gln 290 295 300 Ala Gly Ile Leu Glu Ser Asp Lys Val Val Thr Val Ser Pro Tyr Tyr 305 310 315 320 Ala Glu Glu Leu Val Ser Gly Ala Glu Arg Gly Val Glu Leu Glu Lys 325 330 335 Ile Ile Arg Lys Thr Gly Val Ile Gly Ile Leu Asn Gly Met Asp Lys 340 345 350 Gln Glu Trp Asn Pro Ser Ser Asp Lys Phe Ile Asn Tyr Lys Tyr Asp 355 360 365 Val Ser Thr Val Met Glu Ala Lys Pro Leu Ile Lys Glu Ser Leu Gln 370 375 380 Ala Glu Leu Gly Leu Pro Val Asp Arg Asn Ile Pro Leu Ile Gly Phe 385 390 395 400 Ile Gly Arg Leu Glu Glu Gln Lys Gly Ser Asp Ile Leu Val Glu Ser 405 410 415 Ile Pro Glu Phe Ile Asn Glu Asn Val Gln Ile Val Ile Leu Gly Thr 420 425 430 Gly Lys Lys Arg Phe Glu Lys Gln Ile Glu Asp Leu Glu Ala Leu Tyr 435 440 445 Pro Asp Lys Ala Arg Gly Val Ala Ala Phe Asn Pro Ser Met Ala His 450 455 460 Lys Ile Asn Ala Gly Val Asp Phe Met Leu Ile Pro Ser Arg Phe Glu 465 470 475 480 Pro Cys Gly Leu Thr Gln Leu His Ala Met Leu Tyr Gly Thr Ile Pro 485 490 495 Ile Cys Ala Ser Thr Gly Gly Leu Val Asp Cys Val Lys Glu Gly Ile 500 505 510 Thr Gly Phe Gln Met Gly Lys Phe Ser Ala Glu Cys Lys Ala Val Asp 515 520 525 Pro Thr Asp Val Ala Ala Val Thr Arg Thr Val Arg Lys Ala Val Gly 530 535 540 Leu His Gly Thr Pro Ala Met Arg Glu Met Ile Leu Asn Cys Met Tyr 545 550 555 560 Gln Asp Leu Thr Trp Lys Glu Pro Ala Lys Lys Trp Glu Glu Met Leu 565 570 575 Leu Gly Leu Gln Val Ala Gly Ser Gly Pro Gly Ile Glu Gly Glu Glu 580 585 590 Ile Ala Pro Leu Ala Val Glu Asn Val Ala Ala Pro 595 600 <210> 10 <211> 607 <212> PRT <213> Fagopyrum tataricum <220> <223> Fagopyrum tataricum, GBSSa <400> 10 Met Thr Ser Leu Thr Ile Thr Ser Ser Leu Pro Ala Val Ser Thr Val 1 5 10 15 Ala Gly Ala Thr Ser Ser Ser Lys Ala Met Phe Leu Gln Asn Ser Leu 20 25 30 Lys Ser Thr Ala Gly Leu Arg Pro Phe Asn Asp Val Asp Met Leu Lys 35 40 45 Cys Gly Ala Asn Ser Asn Asn Ala Pro Ser Lys Leu Pro Thr Met Glu 50 55 60 Pro Lys Arg Ser Ala Asn Asn Ser Ala Gln Pro Gly Val Ile Arg Cys 65 70 75 80 Gly Met Asn Leu Val Phe Val Gly Ala Glu Val Ala Pro Trp Ser Lys 85 90 95 Thr Gly Gly Leu Gly Asp Val Leu Ala Gly Leu Pro Pro Ala Met Ala 100 105 110 Ala Asn Gly His Arg Val Met Thr Ile Ser Pro Arg Tyr Asp Gln Tyr 115 120 125 Gln Asp Ala Trp Asp Thr Ser Val Gln Ala Glu Val Lys Val Gly Asp 130 135 140 Lys Thr Glu Thr Val Arg Phe Phe His Ser Tyr Lys Arg Gly Val Asp 145 150 155 160 Arg Ile Phe Val Asp His Pro Leu Phe Leu Ala Lys Val Trp Gly Lys 165 170 175 Thr Gly Phe Lys Ile Tyr Gly Pro Arg Ala Gly Glu Asp Tyr Glu Asp 180 185 190 Asn Gln Leu Arg Phe Ser Leu Leu Ala Gln Ala Ala Leu Glu Ala Pro 195 200 205 Arg Ile Leu Asn Leu Ser Asn Lys Phe Phe Ser Gly Pro Tyr Gly Glu 210 215 220 Asp Val Val Phe Ile Ala Asn Asp Trp His Thr Ala Leu Leu Pro Cys 225 230 235 240 Tyr Leu Lys Thr Met Tyr Lys Ser Arg Gly Ile Tyr Asn Asn Ala Lys 245 250 255 Val Ala Phe Cys Ile His Asn Ile Ser Tyr Gln Gly Arg Phe Ala Gln 260 265 270 Ser Asp Phe Ile Ser Leu Asn Leu Pro Glu Glu Leu Leu Arg Ser Phe 275 280 285 Glu Phe Gln Asp Gly Tyr Asp Thr Pro Val Glu Gly Pro Lys Ile Asn 290 295 300 Trp Met Gln Ala Gly Ile Leu Glu Ser Asp Lys Val Val Thr Val Ser 305 310 315 320 Pro Tyr Tyr Ala Glu Glu Leu Ile Ser Gly Ala Glu Arg Gly Val Glu 325 330 335 Leu Asp Lys Val Ile Gly Ser Thr Gly Ile Thr Gly Ile Leu Asn Gly 340 345 350 Met Asp Lys Gln Glu Trp Asn Pro Leu Thr Asp Lys His Ile Thr Tyr 355 360 365 Gln Tyr Asp Val Ser Asn Val Met Glu Ala Lys Ala Leu Asn Lys Glu 370 375 380 Ala Leu Gln Ala Glu Val Gly Leu Pro Val Asp Arg Asn Ile Pro Leu 385 390 395 400 Ile Gly Phe Ile Gly Arg Leu Glu Glu Gln Lys Gly Ser Asp Ile Leu 405 410 415 Val Glu Ser Ile Pro Gln Cys Ile Asn Glu Asn Val Gln Phe Leu Val 420 425 430 Leu Gly Thr Gly Lys Lys Lys Phe Glu Lys Glu Ile Glu Glu Leu Glu 435 440 445 Asn Leu Tyr Pro Asn Lys Ile Arg Gly Val Thr Lys Phe Asn Pro Ala 450 455 460 Leu Ala His Lys Ile Ile Ala Gly Ala Asp Phe Met Leu Ile Pro Ser 465 470 475 480 Arg Phe Glu Pro Cys Gly Leu Ile Gln Leu His Ala Met Leu Tyr Gly 485 490 495 Thr Val Pro Met Cys Ala Ser Thr Gly Gly Leu Val Asp Cys Val Lys 500 505 510 Glu Gly Ile Thr Gly Phe His Met Gly Arg Phe Gly Ala Gln Cys Lys 515 520 525 Ala Val Asp Pro Ala Asp Val Ala Ala Val Thr Ser Thr Val Arg Arg 530 535 540 Ala Val Ala Val His Gly Thr Pro Ala Met Arg Asp Met Ile Leu Asn 545 550 555 560 Cys Met Tyr Gln Asp Leu Ser Trp Lys Glu Pro Ala Lys Lys Trp Glu 565 570 575 Glu Met Leu Leu Gly Met Gln Val Ala Gly Ser Gly Ser Gly Phe Lys 580 585 590 Gly Glu Glu Ile Ala Pro Leu Ala Ala Glu Asn Ile Ala Thr Pro 595 600 605 <210> 11 <211> 604 <212> PRT <213> Fagopyrum tataricum <220> <223> Fagopyrum tataricum, GBSSb <400> 11 Met Ala Ser Leu Asn Thr Ala Val Cys Met Ala Ser Ser Gly Gly Thr 1 5 10 15 Thr Thr Gly Ser Asp Ala Ser Lys Val Val Leu Leu Gln Asn Gly Leu 20 25 30 Arg Asn His Thr Gly Leu Arg Pro Leu Asn Arg Val Asp Met Leu Lys 35 40 45 Trp Arg Ala Asn Thr Ser Lys Leu Pro Arg Met Glu Glu Lys Arg Val 50 55 60 Val Thr Ser Pro Ser Phe Lys Lys Gly Val Ile Gln Cys Gly Met Asn 65 70 75 80 Leu Val Phe Val Ser Ala Glu Val Ala Pro Trp Ser Lys Thr Gly Gly 85 90 95 Leu Gly Asp Val Leu Ala Gly Leu Pro Pro Ala Met Ala Ala Asn Gly 100 105 110 His Arg Val Met Thr Ile Ser Pro Arg Tyr Asp Gln Tyr Gln Asp Ala 115 120 125 Trp Asp Thr Ser Val Glu Ala Glu Val Lys Val Gly Asp Lys Thr Glu 130 135 140 Thr Val Arg Phe Phe His Ser Tyr Lys Arg Gly Val Asp Arg Val Phe 145 150 155 160 Val Asp His Pro Leu Phe Leu Ala Lys Val Trp Gly Lys Thr Gly Phe 165 170 175 Gln Ile Tyr Gly Pro Arg Ala Gly Glu Asp Tyr Glu Asp Asn Gln Leu 180 185 190 Arg Phe Ser Val Leu Ser Gln Ala Ala Leu Glu Ala Pro Arg Ile Leu 195 200 205 Asn Leu Ser Asn Lys Phe Phe Ser Gly Pro Tyr Gly Glu Asp Val Leu 210 215 220 Phe Val Ala Asn Asp Trp His Ser Ala Leu Leu Pro Cys Tyr Leu Lys 225 230 235 240 Ser Met Tyr Lys Ser Arg Gly Ile Tyr Lys Asn Ala Lys Val Ala Phe 245 250 255 Cys Ile His Asn Ile Ser Tyr Gln Gly Arg Phe Ala Pro Thr Asp Phe 260 265 270 Gly Ala Leu Asn Leu Pro Asn Glu Phe Leu Lys Ser Phe Glu Phe Phe 275 280 285 Asp Gly Tyr Asp Thr Pro Ile Thr Gly Asp Lys Ile Asn Trp Met Gln 290 295 300 Ala Gly Ile Leu Glu Ser Asp Lys Val Val Thr Val Ser Pro Tyr Tyr 305 310 315 320 Ala Glu Glu Leu Val Ser Gly Ala Glu Arg Gly Val Glu Leu Glu Lys 325 330 335 Ile Ile Arg Lys Thr Gly Val Ile Gly Ile Leu Asn Gly Met Asp Lys 340 345 350 Gln Glu Trp Asp Pro Ser Ser Asp Lys Phe Ile Asn Tyr Lys Tyr Asp 355 360 365 Val Ser Thr Val Met Glu Ala Lys Pro Leu Ile Lys Glu Ser Leu Gln 370 375 380 Ala Glu Leu Gly Leu Pro Val Asp Arg Asn Ile Pro Leu Ile Gly Phe 385 390 395 400 Ile Gly Arg Leu Glu Glu Gln Lys Gly Ser Asp Ile Leu Val Glu Ser 405 410 415 Ile Pro Glu Phe Ile Asn Glu Asn Val Gln Ile Val Ile Leu Gly Thr 420 425 430 Gly Lys Lys Arg Phe Glu Lys Gln Ile Glu Glu Leu Glu Ala Leu Tyr 435 440 445 Pro Asn Lys Ala Arg Gly Val Ala Ala Phe Asn Pro Ser Met Ala His 450 455 460 Lys Ile Asn Ala Gly Val Asp Phe Met Leu Ile Pro Ser Arg Phe Glu 465 470 475 480 Pro Cys Gly Leu Thr Gln Leu His Ala Met Leu Tyr Gly Thr Ile Pro 485 490 495 Ile Cys Ala Ser Thr Gly Gly Leu Val Asp Cys Val Lys Glu Gly Ile 500 505 510 Thr Gly Phe His Met Gly Lys Phe Asn Ala Glu Cys Lys Ala Val Asp 515 520 525 Pro Ala Asp Val Ala Ala Val Thr Ser Thr Val Arg Lys Ala Val Thr 530 535 540 Leu His Gly Thr Pro Ala Met Arg Glu Met Ile Leu Asn Cys Met Tyr 545 550 555 560 Gln Asp Leu Thr Trp Lys Glu Pro Ala Lys Lys Trp Glu Glu Met Leu 565 570 575 Leu Gly Leu Gln Val Val Gly Ser Gly Pro Gly Ile Glu Gly Glu Glu 580 585 590 Ile Ala Pro Leu Ala Met Glu Asn Val Ala Ala Pro 595 600 <210> 12 <211> 607 <212> PRT <213> Fagopyrum cymosum <220> <223> Fagopyrum cymosum, GBSSa <400> 12 Met Thr Ser Leu Thr Ile Thr Ser Ser Leu Pro Ala Val Ser Thr Ala 1 5 10 15 Gly Gly Ala Thr Ser Ser Ser Lys Ala Met Phe Leu Gln Asn Ser Leu 20 25 30 Lys Ser Thr Ala Gly Leu Arg Pro Leu Asn Asp Val Asp Met Leu Lys 35 40 45 Cys Arg Ala Asn Ser Asn Asn Ala Pro Ser Lys Leu Pro Thr Met Glu 50 55 60 Val Lys Arg Ser Ala Asn Asn Tyr Ala Glu Pro Gly Val Ile Arg Cys 65 70 75 80 Gly Met Asn Leu Val Phe Val Gly Ala Glu Val Ala Pro Trp Ser Lys 85 90 95 Thr Gly Gly Leu Gly Asp Val Leu Ala Gly Leu Pro Pro Ala Met Ala 100 105 110 Ala Asn Gly His Arg Val Met Thr Ile Ser Pro Arg Tyr Asp Gln Tyr 115 120 125 Gln Asp Ala Trp Asp Thr Ser Val Gln Ala Glu Val Lys Val Gly Asp 130 135 140 Lys Thr Glu Thr Val Arg Phe Phe His Ser Tyr Lys Arg Gly Val Asp 145 150 155 160 Arg Ile Phe Val Asp His Pro Leu Phe Leu Ala Lys Val Trp Gly Lys 165 170 175 Thr Gly Phe Lys Ile Tyr Gly Pro Arg Ala Gly Glu Asp Tyr Glu Asp 180 185 190 Asn Gln Leu Arg Phe Ser Leu Leu Ala Gln Ala Ala Leu Glu Ala Pro 195 200 205 Arg Ile Leu Asn Leu Ser Asn Lys Phe Phe Ser Gly Pro Tyr Gly Glu 210 215 220 Asp Val Val Phe Ile Ala Asn Asp Trp His Thr Ala Leu Leu Pro Cys 225 230 235 240 Tyr Leu Lys Thr Met Tyr Lys Ser Arg Gly Ile Tyr Asn Asn Ala Lys 245 250 255 Val Ala Phe Cys Ile His Asn Ile Ser Tyr Gln Gly Arg Phe Ala Gln 260 265 270 Ser Asp Phe Ile Ser Leu Asn Leu Pro Glu Glu Leu Leu Arg Ser Phe 275 280 285 Glu Phe Gln Asp Gly Tyr Asp Thr Pro Val Glu Gly Pro Lys Ile Asn 290 295 300 Trp Met Gln Ala Gly Ile Leu Glu Ser Asp Lys Val Val Thr Val Ser 305 310 315 320 Pro Tyr Tyr Ala Glu Glu Leu Ile Ser Gly Ala Glu Arg Gly Val Glu 325 330 335 Leu Asp Lys Val Ile Arg Ser Thr Gly Ile Thr Gly Ile Leu Asn Gly 340 345 350 Met Asp Lys Gln Glu Trp Asn Pro Leu Thr Asp Lys His Ile Thr Tyr 355 360 365 Gln Tyr Asp Val Ser Asn Val Met Glu Ala Lys Ala Leu Asn Lys Glu 370 375 380 Ala Leu Gln Ala Glu Val Gly Leu Pro Val Asp Arg Asn Ile Pro Leu 385 390 395 400 Ile Gly Phe Ile Gly Arg Leu Glu Glu Gln Lys Gly Ser Asp Ile Leu 405 410 415 Val Glu Ser Ile Pro Gln Cys Ile Asn Glu Asn Val Gln Phe Leu Val 420 425 430 Leu Gly Thr Gly Lys Lys Lys Phe Glu Lys Glu Ile Glu Glu Leu Glu 435 440 445 Asn Leu Tyr Pro Asn Lys Ile Arg Gly Val Thr Lys Phe Asn Pro Ala 450 455 460 Leu Ala His Lys Ile Ile Ala Gly Ala Asp Phe Met Leu Ile Pro Ser 465 470 475 480 Arg Phe Glu Pro Cys Gly Leu Ile Gln Leu His Ala Met Leu Tyr Gly 485 490 495 Thr Val Pro Met Cys Ala Ser Thr Gly Gly Leu Val Asp Cys Val Lys 500 505 510 Glu Gly Ile Thr Gly Phe His Met Gly Arg Phe Gly Ala Gln Cys Lys 515 520 525 Ala Val Asp Pro Ala Asp Val Ala Ala Val Thr Ser Thr Val Arg Arg 530 535 540 Ala Val Ala Val His Gly Thr Pro Ala Met Arg Asp Met Ile Leu Asn 545 550 555 560 Cys Met Tyr Gln Asp Leu Ser Trp Lys Glu Pro Ala Arg Lys Trp Glu 565 570 575 Glu Met Leu Leu Gly Met Gln Val Ala Gly Ser Gly Pro Gly Phe Lys 580 585 590 Gly Glu Glu Ile Ala Pro Leu Ala Ala Glu Asn Ile Pro Thr Pro 595 600 605 <210> 13 <211> 604 <212> PRT <213> Fagopyrum cymosum <220> <223> Fagopyrum cymosum, GBSSb <400> 13 Met Ala Ser Leu Asn Thr Ala Val Cys Met Ala Ser Ser Gly Gly Thr 1 5 10 15 Thr Thr Gly Ser Asp Ala Ser Lys Val Val Ser Leu Gln Asn Gly Leu 20 25 30 Arg Asn His Thr Gly Leu Arg Pro Leu Asn Arg Val Asp Met Leu Lys 35 40 45 Trp Arg Ala Asn Thr Ser Lys Leu Pro Arg Met Glu Glu Lys Arg Val 50 55 60 Val Thr Ser Pro Ser Phe Lys Lys Gly Val Ile Gln Cys Gly Met Asn 65 70 75 80 Leu Val Phe Val Ser Ala Glu Val Ala Pro Trp Ser Lys Thr Gly Gly 85 90 95 Leu Gly Asp Val Leu Ala Gly Leu Pro Pro Ala Met Ala Ala Asn Gly 100 105 110 His Arg Val Met Thr Ile Ser Pro Arg Tyr Asp Gln Tyr Gln Asp Ala 115 120 125 Trp Asp Thr Ser Val Glu Ala Glu Val Lys Val Gly Asp Lys Thr Glu 130 135 140 Thr Val Arg Phe Phe His Ser Tyr Lys Arg Gly Val Asp Arg Val Phe 145 150 155 160 Val Asp His Pro Leu Phe Leu Ala Lys Val Trp Gly Lys Thr Gly Phe 165 170 175 Gln Ile Tyr Gly Pro Arg Ala Gly Glu Asp Tyr Glu Asp Asn Gln Leu 180 185 190 Arg Phe Ser Val Leu Ser Gln Ala Ala Leu Glu Ala Pro Arg Ile Leu 195 200 205 Asn Leu Asn Asn Lys Phe Phe Ser Gly Pro Tyr Gly Glu Asp Val Leu 210 215 220 Phe Val Ala Asn Asp Trp His Ser Ala Leu Leu Pro Cys Tyr Leu Lys 225 230 235 240 Ser Met Tyr Lys Ser Arg Gly Ile Tyr Lys Asn Ala Lys Val Ala Phe 245 250 255 Cys Ile His Asn Ile Ser Tyr Gln Gly Arg Phe Ala Pro Thr Asp Phe 260 265 270 Lys Ala Leu Asn Leu Pro Asn Glu Phe Leu Lys Ser Phe Glu Phe Phe 275 280 285 Asp Gly Tyr Asp Thr Pro Ile Thr Gly Asp Lys Ile Asn Trp Met Gln 290 295 300 Ala Gly Ile Leu Glu Ser Asp Lys Val Val Thr Val Ser Pro Tyr Tyr 305 310 315 320 Ala Glu Glu Leu Val Ser Gly Ala Glu Arg Gly Val Glu Leu Glu Lys 325 330 335 Ile Ile Arg Lys Thr Gly Val Ile Gly Ile Leu Asn Gly Met Asp Lys 340 345 350 Gln Glu Trp Asp Pro Ser Ser Asp Lys Phe Ile Asn Tyr Lys Tyr Asp 355 360 365 Val Ser Thr Val Met Glu Ala Lys Pro Leu Ile Lys Glu Ser Leu Gln 370 375 380 Ala Glu Leu Gly Leu Pro Val Asp Arg Asn Ile Pro Leu Ile Gly Phe 385 390 395 400 Ile Gly Arg Leu Glu Glu Gln Lys Gly Ser Asp Ile Leu Val Glu Ser 405 410 415 Ile Pro Glu Phe Ile Asn Glu Asn Val Gln Ile Val Ile Leu Gly Thr 420 425 430 Gly Lys Lys Arg Phe Glu Lys Gln Ile Glu Glu Leu Glu Ala Leu Tyr 435 440 445 Pro Asp Lys Ala Arg Gly Val Ala Ala Phe Asn Pro Ser Met Ala His 450 455 460 Lys Ile Asn Ala Gly Val Asp Phe Met Leu Ile Pro Ser Arg Phe Glu 465 470 475 480 Pro Cys Gly Leu Thr Gln Leu His Ala Met Leu Tyr Gly Thr Val Pro 485 490 495 Ile Cys Ala Ser Thr Gly Gly Leu Val Asp Cys Val Lys Glu Gly Val 500 505 510 Thr Gly Phe His Met Gly Lys Phe Ser Ala Glu Cys Lys Ala Val Asp 515 520 525 Pro Thr Asp Val Ala Ala Val Thr Ser Thr Val Arg Lys Ala Val Ala 530 535 540 Leu His Gly Thr Pro Ala Met Gln Glu Met Ile Leu Asn Cys Met Tyr 545 550 555 560 Gln Asp Leu Thr Trp Lys Glu Pro Ala Lys Lys Trp Glu Glu Met Leu 565 570 575 Leu Gly Leu Gln Val Ala Gly Ser Gly Pro Gly Ile Glu Gly Glu Glu 580 585 590 Ile Ala Pro Leu Ala Val Glu Asn Val Ala Ala Pro 595 600

Claims

1. A plant of the genus Buckwheat that satisfies at least one of the following conditions. - Amylose content is at least 8.0% lower compared to the wild type. - The amylose content is 26% or less.

2. A buckwheat plant according to claim 1, having a mutation in the active starch synthase (GBSS) gene.

3. The buckwheat plant according to claim 2, wherein the mutation is selected from the group consisting of splicing mutations, stop codon insertion mutations, and amino acid substitution mutations.

4. A buckwheat plant according to any one of claims 1 to 3, wherein GBSS is GBSSa.

5. A plant of the genus Buckwheat that contains any of the following polynucleotides. (a) Sequence ID: A polynucleotide consisting of sequence 2. (b) A polynucleotide having 80% or more sequence identity with the polynucleotide consisting of sequence number 2, wherein the nucleotide corresponding to position 1846 of sequence number 2 is not A.

6. A plant of the genus Fagopyrum according to any one of claims 1 to 5, wherein the plant of the genus Fagopyrum 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. A plant body of a buckwheat plant according to 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 method for reducing the amylose content of buckwheat plants by inhibiting the activity of one of the following proteins: (A) Sequence ID: A protein consisting of one amino acid sequence from 8 to 13; (B) Sequence ID: A protein consisting of an amino acid sequence that has 90% or more identity with any one of the amino acid sequences 8 to 13, and which has the function of controlling the amylose content of buckwheat plants; (C) Sequence ID: A protein consisting of an amino acid sequence in which 1 to 60 amino acids are deleted, substituted, or added in any one of the amino acid sequences 8 to 13, and which has the function of controlling the amylose content of buckwheat plants.

11. A method according to claim 10, wherein inhibiting the activity of a protein means inhibiting the activity of any of the following proteins: (A') Sequence ID: A protein consisting of one of the amino acid sequences 8, 10, and 12; (B') Sequence ID: A protein consisting of an amino acid sequence having 90% or more identity with one of the amino acid sequences 8, 10, and 12, and having the function of controlling the amylose content of buckwheat plants; (C') Sequence ID: A protein consisting of an amino acid sequence in which 1 to 60 amino acids are deleted, substituted, or added in any one of the amino acid sequences 8, 10, and 12, and which has the function of controlling the amylose content of buckwheat plants.

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