Method for producing super-flowering wheat

Abstract

To provide wheat with dramatically enhanced chasmogamy.SOLUTION: The inventors discovered that it is possible to improve the chasmogamy of wheat by suppressing the Sof1 gene function, and that the chasmogamy of wheat can be evaluated using suppression of the Sof1 gene function as the index.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for producing and evaluating wheat with improved flowering properties, targeting the Sof1 gene.

Background Art

[0002] The supply of grains in the world is likely to be strained due to various factors such as an increase in food demand due to population growth mainly in developing countries, and a decrease in production due to frequent abnormal weather and water resource constraints. As one of the means to solve this problem, the development of new varieties of grains that can increase agricultural productivity is required.

[0003] Heterosis is a phenomenon in which the production ability of individuals of the first filial generation (F1) hybrids exceeds that of both parents, and has been used in the development of new varieties of various crops. However, in barley, which has extremely high genetic diversity, although the heterosis effect is excellent, the heterosis technology has not matured because of its original self-fertility and low production efficiency of hybrid seeds by cross-pollination.

[0004] Therefore, in order to improve the efficiency of cross-pollination, attempts have been made to identify genes that control the flowering properties of grains. For example, the small lemma of the cleistogamous barley variety is controlled by a single recessive gene, cleistogamy 1 (cly1), on the long arm of chromosome 2H (Non-Patent Document 1), and it has been reported that this gene is an ortholog of the Arabidopsis AP2 transcription factor (Non-Patent Document 2). The flowering-type Cly1 gene (Cly1.a) of barley expands the width and thickness of the lemma, pushes out the outer glume and inner glume from the florets, and mechanically causes flowering (Non-Patent Document 2). The difference between the cleistogamous type cly1.b and the flowering type Cly1.a is caused by a single nucleotide substitution within the specific binding site of microRNA172 (miR172) that encodes the cly1 sequence. In wild species with Cly1.a, cleavage of the cly1 transcript via miR172 is reduced. As a result, the translated CLY1 protein accumulates highly, and the suppressed lemma develops normally (Non-Patent Documents 2 and 3).

[0005] By utilizing the flowering type Cly1.a, it is possible to confer the flowering trait to cleistogamous varieties. However, the flowering trait provided by this gene alone is insufficient to improve the efficiency of hybrid seed production through cross-pollination, and a dramatic improvement in flowering ability is still needed. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Turuspekov, Y. et al., (2004) Theor. Appl. Genet., 109:480-487 [Non-Patent Document 2] Nair, SK et al., (2010) Proceedings of the National Academy of Sciences, 107(1):490-495 [Non-Patent Document 3] Anwar, N. et al., (2018) Annals of Botany, 122(2):251-265 [Overview of the project] [Problems that the invention aims to solve]

[0007] This invention has been made in view of the problems of the prior art, and its purpose is to provide wheat with dramatically improved flowering ability. [Means for solving the problem]

[0008] To achieve the above objective, the inventors first screened for mutants with improved flowering ability by gamma-ray irradiation of a flowering type wild barley line (OUH602). As a result, they succeeded in selecting mutants that exhibited a hyperflowering trait with dramatically improved flowering ability. Next, they crossed the selected mutants with a flowering type cultivated variety (Morex) and performed map-based cloning using the F2 population. As a result, they found that the gene responsible for hyperflowering is located between markers HM7H234200 and HM7H246100 on chromosome 7H, and that this trait is controlled by a single recessive gene (this gene controlling the hyperflowering trait was named "sof1"). Within this candidate region of approximately 3.1 Mb, it was found that there are 43 annotated genes and 5 unannotated pseudogenes.

[0009] Next, to investigate polymorphisms in these genes, whole-genome shotgun sequencing was performed on the mutants and compared with the genome sequence of the wild-type barley line (OUH602). As a result, three homozygous polymorphisms were detected within this target region, two of which were 1 bp insertions located in intergeneric regions, and the remaining one was a 22 bp deletion located within the Horvu_MOREX_7H01G238100 gene.

[0010] To confirm that this gene is sof1, which is responsible for the hyperflowering trait, a mutation was introduced using the CRISPR / Cas9 method, targeting the Sof1 gene (the region near the 22bp deletion mentioned above) of a closed-flowering cultivar (Golden Promise). As a result, a mutation (sof1-2) with a 5bp deletion in the Sof1 gene was obtained, and the effect of the mutation was confirmed as it formed scales that were significantly longer than those of Golden Promise. However, this mutant was influenced by the closed-flowering type cly1.b gene present in Golden Promise and did not form scales large enough to exhibit the hyperflowering trait. Therefore, in order to eliminate the influence of the cly1.b gene, this mutant was crossed with barley varieties Sv73528 and Adorra, which possess the flowering type Cly1.a gene. As a result, the scales of the resulting individuals were significantly longer than those of Sv73528 and Adorra, and exhibited the hyperflowering trait. From the above, it was determined that the sof1 gene is the causative gene for the hyperflowering trait. Furthermore, the inventors searched for homologous genes in other plants and found that a gene corresponding to the barley gene also exists in wheat, and succeeded in obtaining mutants that exhibit the hyperflowering trait.

[0011] Based on the above, the inventors have found that it is possible to improve the flowering ability of wheat by suppressing the function of the Sof1 gene (the wild-type gene corresponding to the mutant sof1 gene), and that it is possible to evaluate the flowering ability of wheat using the suppression of the function of the Sof1 gene as an indicator, thus completing the present invention.

[0012] The present invention includes, in more detail, the following embodiments.

[0013] [1] A method for producing wheat with improved flowering ability, comprising artificially suppressing the function of the endogenous gene (a) or (b) described below in wheat. (a) Endogenous gene encoding a protein consisting of the amino acid sequence described in SEQ ID NO: 2, 5, 8, or 11 (b) Endogenous genes encoding proteins consisting of amino acid sequences having 80% or more homology to the amino acid sequences described in Sequence ID No. 2, 5, 8, or 11. [2] Wheat in which the function of the endogenous gene described in (a) or (b) below is artificially suppressed, thereby improving flowering ability. (a) Endogenous gene encoding a protein consisting of the amino acid sequence described in SEQ ID NO: 2, 5, 8, or 11 (b) Endogenous genes encoding proteins consisting of amino acid sequences having 80% or more homology to the amino acid sequences described in Sequence ID No. 2, 5, 8, or 11. [3] A method for evaluating the flowering ability of wheat, comprising analyzing the base sequence of an endogenous gene or its expression regulatory region in wheat, as described below (a) or (b). (a) Endogenous gene encoding a protein consisting of the amino acid sequence described in SEQ ID NO: 2, 5, 8, or 11 (b) Endogenous genes encoding proteins consisting of amino acid sequences having 80% or more homology to the amino acid sequences described in Sequence ID No. 2, 5, 8, or 11. [4] A method for evaluating the flowering ability of wheat, comprising analyzing the expression of the endogenous gene (a) or (b) below in wheat. (a) Endogenous gene encoding a protein consisting of the amino acid sequence described in SEQ ID NO: 2, 5, 8, or 11 (b) Endogenous genes encoding proteins consisting of amino acid sequences having 80% or more homology to the amino acid sequences described in Sequence ID No. 2, 5, 8, or 11. [5] A method for producing wheat with improved flowering ability, comprising crossing the wheat described in claim 2 with any other wheat. [Effects of the Invention]

[0014] According to the present invention, it becomes possible to produce wheat with a dramatically improved flowering property. The wheat shows a large flowering angle due to the large expansion of its lemma. This trait facilitates the scattering of pollen when introduced into the pollen parent in wheat breeding, while facilitating pollination when introduced into the male-sterile seed parent. This makes it possible to dramatically increase the efficiency of producing hybrid seeds by cross-pollination.

Brief Description of Drawings

[0015] [Figure 1] Photographs of wild-type OUH602 (WT) and mutant 44205 (MT). (A) Spikelets of wild-type OUH602 and (B) mutant 44205. (C) Lemma at the yellow anther stage just before flowering of wild-type OUH602 and (D) mutant 44205. (E) Lemma seen from the front side of the spikelets of wild-type OUH602 and (F) mutant 44205. (G) Lemma seen from the side of the spikelets of wild-type OUH602 and (H) mutant 44205. [Figure 2] Graphs of wild-type OUH602 (WT) and mutant 44205 (MT) (continuation of FIG. 1). (A) Flowering angle, (B) lemma length, (C) lemma thickness, (D) lemma width, (E) number of lemma cells, (F) length of lemma cells. [Figure 3] Diagram showing map-based cloning of sof1. (A) High-resolution genetic map of sof1. The numbers between the markers on the line of the linkage map indicate the number of recombinants covered. (B) Fine mapping of sof1 using a large F2 population (n = 1759). (C) Annotated genes with mutations identified by whole-genome sequencing in a 3.1 Mb physical region. The arrow indicates the direction of transcription of the annotated gene. [Figure 4]Figure showing map-based cloning of sof1 (continuation of Figure 3). (A) Structure of the Sof1 gene. The boxes at both ends represent the 5’ and 3’ untranslated regions (UTRs), the box in between represents the coding exon, and the thin lines represent introns. ATG and TAG indicate the start codon and stop codon, respectively. The dashed line indicates a 22-bp deletion in the mutant. (B) Structure of the SOF1 protein. Each box (positions 1043 - 1334, 1359 - 1473, 1587 - 1661) indicates a domain of SOF1. [Figure 5] Figure showing characterization of Sof1 function by mutagenesis via CRISPR / Cas9. (A) Schematic diagram of the gene structure of the Sof1 candidate gene (Horvu_MOREX_7H01G238100) and mutants generated by CRISPR / Cas9. Exons and introns are indicated by boxes and lines, respectively. The inverted triangles indicate the target sites of the guide RNA (gRNA) within the gene. The lines on the Golden Promise (GP) sequence indicate the target sites of the gRNA and the protospacer adjacent motif (PAM). The dashed lines indicate the deleted bases. (B) Scheme for phenotype identification in F3 plants derived from crosses between the edited T1 lines and two non-closed-flower barley cultivars. The genotypes of each generation are shown within parentheses. [Figure 6] Photograph (top) and graph (bottom) of the scale size of wild-type Golden Promise (GP) and homozygous T2 mutant lines. Each measurement in the graph represents the average of ~9 - 13 scales for statistical analysis. Two-tailed Student’s t-test. ** indicates P < 0.01, and ns indicates no significant difference. [Figure 7] Photograph (top) showing comparison of the flowering angles of florets and photograph (bottom) showing comparison of the scale sizes in two non-closed-flower cultivars and their corresponding F3 plants. [Figure 8] Figure showing phylogenetic analysis of SOF1 homologs in monocots and dicots. The tree was constructed by the neighbor-joining (NJ) method based on an alignment of the polypeptide sequences of SOF1 homologs. The numbers shown on the branches indicate the bootstrap probabilities. [Figure 9] This graph shows the amount of Sof1 transcript in panicle development with vegetative and reproductive organs between wild-type OUH602 (WT) and mutant 44205 (MT). DR (double ridge stage); TM (triple mound stage); GP (glume primordium stage); LP (lemma primordium stage); SP (stamen primordium stage); AP (awn primordium stage); WA (white anther stage); GA (green anther stage); YA (yellow anther stage). DAG (days after germination). Values ​​are mean ± SE (n = 3 independent biological copies). Statistical significance was calculated using the Two-tailed Student's t-test. * indicates P < 0.05, ** indicates P < 0.01, and ns indicates no significant difference. [Figure 10] This graph shows the amount of Sof1 transcript in wild-type wheat varieties (Chinese Spring). [Modes for carrying out the invention]

[0016] <Method for producing wheat with improved flowering ability> In this invention, "flowering ability" refers to the trait in which the flowering angle increases due to the expansion of the scales. The inflorescences of monocotyledonous plants such as wheat, rice, and maize consist of structures called spikelets, each containing one or more florets. Each floret is composed of reproductive organs (stamens and pistils) and is covered by a pair of bract-like organs (glacial and endoglaucoma). The "scales" are located between these reproductive organs and glumes. The scales are generally considered to be equivalent to petals and are located at the base on both sides of the reproductive organs (stamens and pistils). If the scales rapidly expand just before flowering, they mechanically separate the endoglaucoma, making it easier for pollen to be released from the split anthers, and subsequently the stigma becomes capable of cross-pollination (non-cleistogamous pollination). In contrast, if the scales cannot swell, the pollen is hidden within the closed floret, thereby causing self-pollination (cleistogamous pollination).

[0017] In this invention, "improved flowering" means an increase in the flowering angle compared to when the function of the Sof1 gene is not artificially suppressed. The increase in flowering angle is preferably 2° or more, more preferably 5° or more, even more preferably 8° or more, and particularly preferably 10° or more. The flowering angle can be measured as the angle at which a line along the edge of the outer glume (the glume furthest from the rachis) and a line along the edge of the inner glume (the glume closer to the rachis) intersect in the florets of the spike. Note that the flowering angle may vary depending on the wheat species and variety, but for example, the flowering angle of wild barley OUH602 is usually 10.2±2.2°.

[0018] In this invention, the "wheat" targeted for improving flowering ability refers to all plants of the cereal family belonging to the subfamily Poaceae, such as barley, wheat, rye, rye wheat, and oats. It may also be a wild species or a cultivated species. When wheat possesses the closed-flower type cly1 gene, the effect of suppressing the function of the Sof1 gene becomes less likely to occur, therefore, the "wheat" targeted for improving flowering ability is the flowering type C It is preferable to possess the ly1 gene. When the function of the Sof1 gene is suppressed in wheat possessing the closed-flower type cly1 gene, the flowering type subsequently develops. CThe negative effects of the closed-flower type cly1 gene can be eliminated by crossbreeding with wheat plants that possess the ly1 gene, or by modifying the cly1 gene from a closed-flower type to a flowering type through genome editing (for example, a single nucleotide substitution within the specific binding site of miR172).

[0019] The method of the present invention involves artificially suppressing the function of the Sof1 gene (an endogenous gene encoding the SOF1 protein).

[0020] The "Sof1 gene" in this invention is presumed to belong to the SWI / SNF subfamily and encode a chromatin remodeling ATPase protein. Since wheat expressing the mutant protein shows increased scale swelling and improved flowering, it is thought that the wild-type protein (SOF1 protein) has the activity to suppress the swelling of wheat scales.

[0021] The typical cDNA sequence of the barley Sof1 gene is shown as SEQ ID NO: 1, the genomic DNA sequence as SEQ ID NO: 3, and the amino acid sequence of the protein encoded by these DNAs as SEQ ID NO: 2. In addition, the typical cDNA sequences in wheat are shown as SEQ ID NOs: 4, 7, and 10, the genomic DNA sequences as SEQ ID NOs: 6, 9, and 12, and the amino acid sequences of the proteins encoded by these DNAs as SEQ ID NOs: 5, 8, and 11.

[0022] In nature, individual differences can occur in nucleotide sequences, and with the current level of technology, if a specific gene is obtained, a person skilled in the art can use the nucleotide sequence information of that gene to identify a corresponding gene from the same species or other plants. Therefore, the Sof1 gene in this invention includes these homologous genes insofar as suppressing its function improves flowering.

[0023] The amino acid sequence of the protein encoded by a homologous gene usually has high homology to the amino acid sequence of the protein encoded by the particular gene. Here, "high homology" means at least 80%, preferably 85%, more preferably 90%, and even more preferably 95% (for example, 96%, 97%, 98%, 99%).

[0024] Sequence homology can be determined using the BLAST program (Altschul et al., J.Mol.Biol., (1990) 215:403-410). This program is based on the BLAST algorithm by Karlin and Altschul (Karlin, S. & Altschul, SF., (1990) Proc. Natl. Acad. Sci. USA, 87:2264-2268, Karlin, S. & Altschul, SF., (1993) Proc. Natl. Acad. Sci. USA, 90:5873-5877). For example, when analyzing amino acid sequences with BLAST, the parameters might be, for example, score=50 and wordlength=3. Furthermore, when analyzing amino acid sequences using the Gapped BLAST program, the procedure can be carried out as described by Altschul et al. (Altschul, SF. et al., (1997) Nucleic Acids Res., 25:3389-3402). When using BLAST and the Gapped BLAST program, the default parameters of each program should be used. The specific methods for these analyses are publicly known.

[0025] These homologous genes can be obtained, for example, by using hybridization techniques (Southern, EM, (1975) J. Mol. Biol., 98:503) or polymerase chain reaction (PCR) techniques (Saiki, RK et al., (1985) Science, 230:1350-1354, Saiki, RK et al., (1988) Science, 239:487-491).

[0026] In this invention, "suppression of Sof1 gene function" includes both complete suppression (inhibition) and partial suppression of its function. It also includes both suppression of SOF1 protein activity (e.g., activity that suppresses scale swelling) and suppression of Sof1 gene expression (repression of transcription and repression of translation).

[0027] The function of the Sof1 gene can be suppressed, for example, by introducing mutations into the Sof1 gene or its expression regulatory region (e.g., the promoter region). There are no particular restrictions on the mutations introduced into the Sof1 gene as long as they suppress its function; for example, nucleotide substitutions, deletions, additions, and / or insertions are possible, but frameshift mutations, nonsense mutations, and null mutations are preferred.

[0028] There are no particular restrictions on the number of mutations introduced into the Sof1 gene, as long as their function is suppressed; it can be one or multiple mutations (for example, two, three or fewer, five or fewer, ten or fewer, twenty or fewer, thirty or fewer, forty or fewer, fifty or fewer). In fact, in the examples of this application, it has been found that nucleotide deletions in the barley Sof1 gene (22-base deletion [sof1 mutant], 5-base deletion [sof1-2 mutant]) produce mutant proteins with shortened C-terminuses due to frameshift, resulting in improved barley flowering. The SOF1 protein encoded by the Sof1 gene has two functional domains (ATPase domain and SnAC domain) at its N-terminus, but it is also possible to cause loss of function of the SOF1 protein by mutations at the C-terminus where these functional domains are absent. Therefore, the mutations introduced into the Sof1 gene do not need to cause loss of the entire amino acid sequence of the protein encoded by the gene; it is acceptable to introduce mutations into the gene so that only a part of the sequence is lost or altered.

[0029] When a portion of the protein expressed by a gene mutation is deleted, from the viewpoint of suppressing the activity of the SOF1 protein, the deletion is preferably 5% or more of the total, more preferably 10% or more, more preferably 20% or more, more preferably 30% or more, and more preferably 50% or more (for example, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more).

[0030] The introduction of mutations into the Sof1 gene can be achieved by methods known to those skilled in the art. Among the known methods, genome editing is preferred because it allows for the efficient introduction of site-specific mutations.

[0031] Genome editing is a method of modifying target genes using site-specific nucleases. Examples of site-specific nucleases include Cas nucleases (CRISPR / Cas system), transcriptional activation-like effector nucleases (TALENs), and zinc finger nucleases (ZFNs), which have site specificity conferred to them by forming a complex with guide RNA. Various CRISPR / Cas systems can be used, including Class 2 Type II systems (including Cas9 as the Cas), Class 2 Type V systems (including Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas14, etc.), Class 2 Type VI systems (including Cas13a (C2c2), Cas13b, Cas13c, etc.), and Class 1 Type I systems (including Cas3 as the Cas).

[0032] Other methods for introducing mutations into genes include, but are not limited to, physical mutagenesis, the use of chemical mutagens, and the use of transposons.

[0033] Examples of physical mutagenesis methods include gamma-ray irradiation, heavy ion beam (HIB) irradiation, fast neutron irradiation, and ultraviolet irradiation (Hayashi Y. et al., (2007) Cyclotrons and Their Applications, 18th International Conference, 237-239; Kazama, Y. et al., (2008) Plant Biotechnology, 25:113-117).

[0034] Methods using chemical mutagens include, for example, treating seeds with chemical mutagens (Zwar and Chandler, Planta, 1995, Vol. 197, pp. 39-48). There are no particular restrictions on the chemical mutagens, but examples include N-methyl-N-nitrosourea (MNU), ethylmethanesulfate (EMS), N-ethyl-N-nitrosourea (ENU), sodium azide, sodium bisulfite, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyl-N'-nitrosoguanidine (NTG), O-methylhydroxylamine, nitrite, formic acid, and nucleotide analogs.

[0035] One method for introducing transposons into genomic DNA is, for example, T OS Methods include inserting transposons such as 17, T-DNA, etc., into the plant's genomic DNA (Kumar, A. & Hirochika, H., (2001) Trends Plant Sci., 6(3):127-134, Tamara, M. et al., (1999) Trends in Plant Science, 4(3):90-96).

[0036] When using these methods, individuals with mutations in the Sof1 gene are typically selected from a population that has undergone mutation introduction treatment.

[0037] A wheat plant in which a mutation has been introduced into the Sof1 gene may be a heterozygous for the mutated gene. In this case, heterozygotes can be crossed to create F1 plants, and homozygotes for the mutated gene can be selected from these F1 plants. In this invention, the sof1 gene (mutated gene) that causes the hyperflowering trait has been found to be a recessive gene. Therefore, in order to improve the flowering ability of wheat by introducing a mutation into the Sof1 gene, it is preferable for the wheat to retain the mutated Sof1 gene in a homozygous state. Furthermore, wheat in which an unintended mutation has been introduced can have the unintended mutation removed, for example, by backcrossing using wild-type wheat.

[0038] When suppressing the function of the Sof1 gene by repressing its expression, methods targeting the Sof1 gene transcript can be used, in addition to introducing mutations into the expression regulatory region of the Sof1 gene. These methods include using DNA encoding dsRNA (double-stranded RNA, e.g., siRNA) complementary to the Sof1 gene transcript, using DNA encoding antisense RNA complementary to the Sof1 gene transcript (antisense DNA), or using DNA encoding RNA with ribozyme activity that specifically cleaves the Sof1 gene transcript (ribozyme method).

[0039] Furthermore, the suppression of Sof1 gene expression can also be achieved through nucleotide modifications in epigenetic regulation, without the involvement of nucleotide mutations. Examples of epigenetic regulation include DNA methylation and histone chemical modifications (acetylation, methylation, phosphorylation, ubiquitination, etc.).

[0040] In the present invention, treatment to artificially suppress the function of the Sof1 gene can be performed on wheat plants, organs, tissues, or cells, depending on the type of technique used. Examples of organs, tissues, or cells include seeds, microspores, pollen, immature embryos, leaf sections, suspension culture cells, protoplasts, callus, growing points, and young panicles.

[0041] If the molecule used to artificially suppress the function of the Sof1 gene is a DNA-encoded molecule (for example, DNA encoding a site-specific nuclease, DNA encoding a transposon, DNA encoding double-stranded RNA, DNA encoding antisense RNA, DNA encoding ribozyme-active RNA, etc.), it may be introduced into wheat in the form of an insertion into a vector. The vector is not particularly limited as long as it is capable of expressing the inserted DNA in wheat cells. The vector usually contains a promoter for constitutive or inductive expression of the DNA. Examples of promoters for constitutive expression include the barley U3 promoter, the rice ubiquitin promoter, the cauliflower mosaic virus 35S promoter, the rice actin promoter, and the maize ubiquitin promoter. Examples of promoters for inductive expression include promoters known to be expressed by external factors such as treatment with specific compounds, low or high temperatures, drying, ultraviolet irradiation, or infection or invasion by filamentous fungi, bacteria, or viruses.

[0042] Methods known to those skilled in the art can be used to introduce molecules that artificially suppress the function of the Sof1 gene into wheat cells, such as the Agrobacterium method, particle bombardment, polyethylene glycol method, and electroporation.

[0043] If cells with artificially suppressed Sof1 gene function are obtained using the above method, wheat plants with improved flowering ability can be obtained by regenerating wheat plants from these cells. Methods for producing transformed wheat plants include those described by Tingay et al. (Tingay, S. et al., (1997) Plant J., 11:1369-1376), Murray et al. (Murray, F. et al., (2004) Plant Cell Report, 22:397-402), and Travalla et al. (Travalla, S. et al., (2005) Plant Cell Report 23:780-789). Furthermore, transformation and regeneration into plants can be performed using the method described by Tabei et al. (edited by Yutaka Tabei, "Transformation Protocols [Plants]", Kagaku Dojin Co., Ltd., published September 20, 2012).

[0044] Once a plant in which the function of the Sof1 gene has been artificially suppressed is obtained, it is possible to obtain offspring from this plant through sexual or asexual reproduction. Furthermore, it is possible to obtain reproductive materials (e.g., seeds, cuttings, stalks, callus, protoplasts, etc.) from this plant, its offspring, or clones, and mass-produce plant bodies based on these materials. The present invention also provides wheat plants, offspring, clones, and reproductive materials with improved flowering ability obtained in this way.

[0045] <Method for evaluating the flowering ability of wheat> One aspect of the method of the present invention includes analyzing the nucleotide sequence of the Sof1 gene or its expression regulatory region in wheat.

[0046] When analyzing the nucleotide sequence of the Sof1 gene or its regulatory region, amplification products obtained by PCR of the Sof1 gene or its regulatory region can be used. When performing PCR, there are no restrictions on the primers used, as long as they can specifically amplify the Sof1 gene or its regulatory region, and they can be appropriately designed based on the sequence information of the Sof1 gene or its regulatory region.

[0047] The evaluation of flowering ability may include a step of comparing with a "control base sequence." The "control base sequence" is typically the base sequence of a gene or its expression regulatory region encoding the amino acid sequence described in SEQ ID NO: 2 (barley) or the amino acid sequences described in SEQ ID NOs: 5, 8, and 11 (wheat).

[0048] By comparing the nucleotide sequence of the Sof1 gene or its expression regulatory region in the determined test wheat with the nucleotide sequence of the control, it is possible to evaluate whether or not flowering is improved in the test wheat. For example, if there are differences in the nucleotide sequence compared to the control (especially if the appearance of a new stop codon or a frameshift causes a significant change in the molecular weight or amino acid sequence of the encoded protein), the test wheat is considered to have a high probability of being a wheat with improved flowering.

[0049] DNA for sequencing analysis can be prepared using conventional methods, such as the CTAB method. For DNA preparation, not only mature plants but also seeds and young plants can be used. Sequencing can be performed using conventional methods, such as the dideoxy method or the Maxam-Gilbert method. Commercially available sequencing kits and sequencers can be used for sequencing.

[0050] In addition to the direct sequencing described above, whether the nucleotide sequence of the Sof1 gene or its regulatory region in the test wheat differs from that of the control can also be indirectly analyzed using known methods such as PCR-SSCP (single-strand conformation polymorphism).

[0051] Another aspect of the method of the present invention includes analyzing the expression of the Sof1 gene.

[0052] Here, "analysis of gene expression" includes the analysis of the presence and degree of expression (transcription and translation), as well as the analysis of the molecular weight of the expression product.

[0053] The Sof1 gene can be detected at the transcriptional level by conventional methods, such as RT-PCR, Northern blotting, or RNA-seq. The primers used in the PCR are not limited as long as they can specifically amplify the target DNA of the present invention, and can be appropriately designed based on already determined Sof1 gene sequence information. Furthermore, detection at the translational level can be performed by conventional methods, such as Western blotting. The antibody used in Western blotting may be either a polyclonal or monoclonal antibody, and the methods for preparing these antibodies are well known to those skilled in the art.

[0054] Based on the results of gene expression detection, if the expression level of the Sof1 gene in the test wheat is significantly lower than that of varieties that retain the wild-type Sof1 gene (e.g., OUH602 in barley, or Chinese Spring in wheat) (e.g., if the Sof1 gene is not substantially expressed), it is highly likely that the wheat will have improved flowering ability. Furthermore, if the molecular weight of the amplification product or expression product of the Sof1 gene differs significantly from the molecular weight of the expression product of the wild-type Sof1 gene, it is highly likely that the wheat will have improved flowering ability.

[0055] <Method for producing wheat with improved flowering ability> This invention provides a method for producing wheat with improved flowering ability.

[0056] The method of the present invention includes crossing the wheat with improved flowering ability with any other wheat.

[0057] Examples of "any other wheat" to be crossed with wheat that has improved flowering ability include, but are not limited to, wheat varieties in which the function of the Sof1 gene is not suppressed and which do not exhibit the super-flowering trait. From the individuals obtained through crossbreeding, wheat in which the function of the Sof1 gene is suppressed can be selected using the method described above.

[0058] By utilizing the method of the present invention, it becomes possible to appropriately select wheat varieties with improved flowering ability at an early stage, such as at the seed or seedling stage, and to breed varieties possessing such traits in a short period of time. [Examples]

[0059] The present invention will be described more specifically below based on examples, but the present invention is not limited to the following examples.

[0060] A. Materials and Methods (1) Plant materials The non-cleistogamous wild barley strain OUH602 (Hordeum vulgare subsp. spontaneum) was obtained from the Institute of Plant Science and Resources, Okayama University. OUH602 was treated with cobalt 60 during its growth from seedling stage. 60 The plants were treated with low-intensity Co) gamma irradiation (0.24-0.77 Gy per day, 5 days per week). Based on the hyperflowering phenotype, the OUH602 mutant (strain number: 44205) was selected from M3 lines derived from 1600 M2 individuals. For genetic analysis, the OUH602 mutant was crossed with the cultivar "Morex" to create a mapping population. Plant cultivation was carried out in a netted room (natural light and uncontrolled temperature) or an air-conditioned greenhouse (maintained at 24°C), with one plant per 4L pot.

[0061] (2) Evaluation of scale size phenotype and flowering angle Three spikes (with the terminal leaf still attached to the pedicel) were randomly selected from each strain, and florets (at the stage of yellow anthers just before flowering) were collected from the central part of each spike. After removing the glumes from the florets, the scales were photographed using an Axio Zoom v16 microscope (Carl Zeiss, Tokyo, Japan) according to the description in the literature (Non-Patent Literature 2 above). Based on the digital images, the length, thickness, and width of the scales were measured using Makijaku software v1.1 (http: / / lbm.ab.au-tokyo.ac.jp / ~iwata / software / makijaku / ).

[0062] Furthermore, photographs of florets (immediately after flowering: yellow anther stage) from randomly selected spikes of wild-type and mutant varieties were taken. The angle at which a line along the edge of the outer glume (the glume furthest from the rachis) intersects with a line along the edge of the inner glume (the glume closer to the rachis) was measured using Makijaku software v1.1 (http: / / lbm.ab.au-tokyo.ac.jp / ~iwata / software / makijaku / ), and this was defined as the flowering angle.

[0063] (3) Measurement of scale cells After embedding the scales in 30% acrylamide gel, they were cut into 150 μm thick sections using a Linear Slicer PRO10 (Dosaka EM) and stained with 0.01% fluorescent whitening agent 28 (Sigma). Images were acquired using a laser scanning microscope (LSM700, Carl Zeiss) equipped with ZEN 2009 Light Edition CLSM software. For scale cell measurement, the number of cells was counted based on the thickness of the scale, and the cell size was calculated by dividing the scale thickness by the number of cells. An average of 10 samples were measured for each genotype.

[0064] (4) Marker development For SNP genotyping in rough mapping, we developed fluidigm markers based on single nucleotide polymorphisms (SNPs) in the genome sequences of the barley cultivars Sky Golden and Tochinoibuki. For high-resolution gene mapping, we additionally developed CAPS (cleaved amplified polymorphic sequence) markers to identify identified SNPs around the RAD markers FB0278 and FB0088. These specific polymorphic sites were detected by PCR analysis, and the PCR products were sequenced according to the literature (Sakuma, S. et al., (2010) Functional & Integrative Genomics, 10:123-133) and identified by restriction enzyme-based CAPS analysis. A total of 11 CAPS markers were developed by comparing the exome sequences of two strains, Morex (accession number ERR271705) and OUH602 (accession number ERR271737), obtained from the DDBJ database (https: / / ddbj.nig.ac.jp / ). In addition, an InDels (insertion-deletions) marker was developed for a 22bp deletion identified in the whole-genome sequencing of mutant 44205. All developed markers were used to screen strains in the mapping population.

[0065] (5) Genetic map of sof1 The barley F2 population was developed by crossing mutant 44205 with Morex. The size of the scales was investigated by applying the maximization spike culture method (Honda, I. et al., (2005) Physiologia Plantarum, 124:524-531) which involves treating the spikes with 2,4-dichlorophenoxyacetic acid (2,4-D). It is known that non-flowering pollination is perfectly correlated with the size of the scales (non-closed vs. closed flowers) (Nair, SK et al., (2010) Proceedings of the National Academy of Sciences, 107(1): 490-495). When 2,4-D (30 ppm) was added to all closed-flowering pollination types, no flowering was observed. On the other hand, the scales of the flowering pollination types expanded on average to about 1.5 times the width and about 2.3 times the thickness (perpendicular to the glume) compared to before treatment.

[0066] For the initial mapping, 40 plants (20 WT and 20 mutant types) were selected. The genotypes of the 40 F2 plants were determined using ddRAD-seq (Double Digest RAD-seq). In rough mapping, genotyping using the fluidigm marker was performed on 93 F2 plants. Linkage maps were created using AntMap version 1.2 (Iwata, H. & Ninomiya, S. (2006) Breeding Science, 56:371-377). Next, the flowering phenotype of the 93 F2 plants was determined, and their genotypes were determined using the aforementioned CAPS markers. In fine mapping, genotyping of F2 segregation between mutants and Morex was performed using two adjacent markers, HM7H228700 and HM7H254000. The genotypes of recombinants identified with these two markers were determined using other CAPS markers developed within the HM7H228700-HM7H254000 genomic region. To accurately identify the sof1 locus, key F2 recombinants were further genotyped using the InDel marker (HM7H238100-Indel), and their progeny, F3 recombinants (20 individuals), were also genotyped using the InDel marker to confirm the scale size phenotype.

[0067] (6) Identification of mutation sites in genomic regions including sof1 candidate DNA was extracted from fresh seedling leaves of M3 generation plants of mutant 44205 using the DNeasy Plant Extraction Kit (QIAGEN). A DNA library for paired-end sequencing (150 bp) was constructed using the TruSeq DNA PCR-free Sample Preparation Kit (Illumina). DNA sequencing was performed on the Illumina HiSeqXTen platform. The data sequence for wild-type OUH602 was downloaded from the NCBI Short Sequence Read Archive (SRA) database (accession ERX5471675; Sato, K. et al., (2021) Chromosome-scale assembly of wild barley accession “OUH602”. G3 (Bethesda). 27;11(10)). After trimming with Trimmomatic version 0.39 (Bolger, AM. et al., (2014) Bioinformatics, 1;30(15):2114-2120), the wild-type and mutant sequences were aligned to the wild barley OUH602 reference genome (200720_OUH602_pseudomolecules_v1.fasta; Sato, K. et al., (2021) G3(Bethesda), 27;11(10)) using Bowtie2 version 2.3.5.1 (Langmead B. & Salzberg SL., (2012) Nat Methods, 4;9(4):357-359). Variant calling of the genome sequence was performed using SAMtools version 1.10 (Li, H. et al., (2009) Bioinformatics, 15;25(16):2078-2079) (mapping score 30, base score ≥20, phred score threshold 40). Since gamma irradiation is expected to induce both nucleotide deletions and substitutions, coverage depth analysis was used to identify deletions not detected by variant calling. The read depth of the variant at each location was calculated using BCFtools (Li, H. et al., (2011) Bioinformatics, 1;27:2987-2993).Variants were further filtered by applying a threshold of 10 to the read depth. The locations of the variants identified in the sof1 candidate region crossed with gene annotations from the Morex genome (Morex.gff.gz;Jayakodi, M. et al., (2020) Nature, 588(7837):284-289). Variant effects were predicted using SnpEff (Cingolani, P. et al., (2012) Fly(Austin), 6(2):80-92).

[0068] (7) Sequencing of the sof1 gene locus Genomic DNA was extracted from the leaves of seedlings grown for two weeks. The quality and quantity of gDNA were assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc.). For Sanger sequencing, primers used for PCR amplification and sequencing were designed and synthesized using NCBI / Primer-BLAST software (http: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ). The 20kb genomic region of the sof1 locus was split into approximately 5kb overlapping amplicons for a total of five amplicons, and at least 20 sequencing reactions were performed per amplicon. Each amplicon was amplified using the high-fidelity enzyme PrimeSTAR GXL DNA polymerase (TaKaRa) according to the manufacturer's instructions. The amplification reaction, totaling 50 μl, consisted of 100 ng of genomic DNA template, 1× PrimeSTAR GXL buffer, 200 μM dNTPs, 0.3 μM of each primer, and 2.5 units of PrimeSTAR GXL DNA polymerase. The PCR program consisted of 30 cycles of "98°C / 10 sec, 55 or 60°C (primer-dependent) / 15 sec, 68°C / 4-5 sec". The PCR products were purified using the QIAquick PCR purification kit (QIAGEN) and cycle sequencing was performed using Big Dye Terminator v3.1 technology (Applied Biosystem). Each sequencing reaction consisted of an initial denaturation at 96°C / 1 sec, followed by 25 cycles of "96°C / 10 sec, 50°C / 5 sec, 60°C / 4 sec". The reaction products were purified using an Agencourt CleanSEQ system (Beckman) and analyzed using an ABI prism 3130 or 3730xL gene analyzer (Applied Biosystems). Genomic sequences were aligned using Seqman version 7.1.0 (DNASTAR).

[0069] (8) Determination of the genome sequence of the coding region of sof1 Total RNA was extracted from developing spikes in the primordia stage using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. The extracted RNA was treated with RNase-free DNase I (Takara Bio) to remove genomic DNA contamination. RNA was quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc.). Reverse transcription was performed using 1 μg of total RNA from each DNase-treated sample, and first-strand cDNA was synthesized using a SuperScript III kit (Invitrogen) primed with oligo dT. The sof1 coding sequence was amplified and sequenced as five amplicons using designed primers. Each 50 μl volume of PCR reaction contained 20 ng of template, 1 × PrimeSTAR GXL buffer, 200 μM dNTPs, 0.3 μM of each primer, and 2.5 units of PrimeSTAR GXL DNA polymerase. The PCR program consisted of 30 cycles of "98°C / 10 seconds, 55 or 60°C (primer-dependent) / 5 seconds, 68°C / 1-4 minutes". Purification, sequencing, and sorting of the reaction products were performed as described above.

[0070] (9) Genome editing via CRISPR / Cas9 The CRISPR / Cas9 system was used to induce targeted mutagenesis of the barley Sof1 gene. Three guide RNAs (gRNAs, gRNA1:5'-TGCATCCGGGAAGAAAACAG-3' / SEQ ID NO:13, gRNA2:5'-TGCTGCTATGAAGGAACCAG-3' / SEQ ID NO:14, and gRNA4:5'-ACCAGAAGAGAAGAAGGTAG-3' / SEQ ID NO:15) were designed using the WU-CRISPR website (http: / / crisprdb.org / wu-crispr / ; Wong, N. et al., (2015) Genome Biology, 16:218). The complementary oligoDNA pairs for each gRNA sequence were inserted into the BbsI cleavage sites of pHvU3-15 and pHvU3-21 (LC672614 and LC672613, respectively), which were constructed by replacing the rice OsU6 promoter of pU6gRNA-oligo (Abe, F. et al., (2019) Cell Reports, 28:1362-1369) with barley HvU3. Next, the gRNA expression cassette and the pHvU3-15 / 21 scaffold sequences were excised using PacI and AscI. Simultaneously, the target binary vector pZH_gYSA_PubiMMCas9, containing expression cassettes for Cas9 and hygromycin phosphotransferase genes, was digested with the same restriction enzyme to introduce gRNA expression cassettes (Mikami, M. et al., (2015) Plant Molecular Biologuy, 88:561-572). Four constructed vectors, named pZH-HvU3-15:gRNA1, pZH-HvU3-15:gRNA2, pZH-HvU3-15:gRNA4, and pZH-HvU3-21:gRNA2, were used to transform Agrobacterium tumefaciens AGL1 strain. These were then used for barley transformation via Agrobacterium according to the method described in the literature (Hisano, H. & Sato, K., (2016) Scientific Reports, 6:37505).To confirm mutations in the target sequence of the Sof1 gene in regenerated plants, Sanger sequencing of PCR amplicons was performed using specific primer pairs. These included 5'-AGCATTGCACACTAATTCTGG-3' / SEQ ID NO:16 and 5'-AGAAACGATTTCGGGAAGAG-3' / SEQ ID NO:17 (for gRNA1), CGCTCAAGAGGATATTGCTG-3' / SEQ ID NO:18 and 5'-TCACATGATGAAGGTTGCTG-3' / SEQ ID NO:19 (for gRNA2), and 5'-ACTCAAGTCGAACCAGTTGC-3' / SEQ ID NO:20 and 5'-TCACATGATGAAGGTTGCTG-3' / SEQ ID NO:21 (for gRNA4). The conditions for DNA extraction, PCR, and sequencing followed the methods described in the literature (Hisano, H. et al., (2022) Plant Biotechnology Journal, 20:37-46).

[0071] (10) Phylogenetic analysis The amino acid sequences of the proteins were aligned using the default parameters of the ClustalW program implemented in the Molecular Evolutionary Genetics Analysis (MEGA) version 7.0 software package (Kumar, S. et al., (2016) Molecular Biology and Evolution, 33:1870-1874), and a phylogenetic tree was constructed based on neighbor joining (NJ) supported by 1000 bootstrap replications. The gene IDs or symbols of homologs of Arabidopsis and other species shown in the phylogenetic tree were obtained from the Ensembl Plant database (http: / / plants.ensembl.org / Multi / Tools / Blast / ), and the corresponding protein IDs were based on the NCBI (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi) BLASTp search.

[0072] (11) Expression analysis by quantitative PCR analysis Young panicles were collected at 10 developmental stages from the double ridge stage (the first structure formed from the inflorescence meristem after the phase transition from vegetative to reproductive growth, consisting of spikelet ridges and leaf ridges) to flowering, using a stereomicroscope (Axio Zoom v1.6, Zeiss), following the guidelines of the literature (Kirby, EJM & Appleyard, M., (1981) Cereal development guide, Cereal Unit, National Agricultural Centre. Stoneleigh, Kenilworth, Warwickshire, England.). Total RNA was extracted from young panicles, floral organs (including scales, glumes, endoglaucoma, anthers, pistils, glumes, and awns) at the green anther stage, roots and leaves of seedlings 7 days after germination, and leaf sheaths, blades, nodes, and internodes at the awn primordium stage, using TRIzol reagent (Invitrogen).

[0073] Genomic DNA cleavage, quantification, reverse transcription, and cDNA synthesis were performed as described above (see the sof1cDNA sequencing section). A first-strand cDNA derived from 10 ng of total RNA was used as a template for quantitative real-time PCR (qPCR). qPCR was performed according to the protocol using a CFX96 real-time PCR detection system (Bio-Rad) and a THUNDERBIRD SYBR qPCR Mix Kit (Toyobo). qRT-PCR data for each target gene were obtained by performing three replicates (three PCR reactions per replicate) with the biological sample, and the average expression level was shown. The staghorn ostrich tin gene (accession number DN182500) was used as an endogenous control to normalize the expression level of each sample according to the method described in Sakuma, S. et al., (2017) Plant Physiol, 175(4):1720-1731). Relative gene expression was analyzed using the 2-ΔCt method.

[0074] (12) RNA-seq profiling Plants used for RNA sequencing (RNA-seq) were grown in a climate-controlled greenhouse. Panicle tissue was collected from wild-type OUH602 and mutant 44205 at four panicle development stages: vegetative growth stage (VEG), double ridge stage (DR), awn primordium stage (AP), and green anther stage (GA). Six biological replica samples were collected at each of the four stages, with each replica containing bulk samples of 12 shoots from 6 plants at the VEG stage, 10 shoots from 5 plants at the DR stage, 5 panicles from 5 plants at the AP stage, and 3 panicles from 3 plants at the GA stage. All samples were immediately frozen in liquid nitrogen and transferred to -80°C for storage until RNA extraction. RNA from awn primordium stage samples harvested under greenhouse and climate-controlled conditions was also used for RNA-seq. Total RNA was extracted using the RNeasy Mini Kit (QIAGEN), followed by removal of genomic DNA contamination using the RNA-Free DNase Set (QIAGEN). RNA sample quality was assessed using the Agilent 2100 Bioanalyzer instrument (Agilent Technologies). For RNA-seq, strand-specific RNA libraries were constructed from 1 μg of total RNA from each sample using the TruSeq RNA Sample Preparation Kit v2 (Illumina), and analyzed on the Illumina HiSeq X sequencing platform (Illumina) with two lanes (paired-end 150 bp).

[0075] After sequencing, the quality of sequence sequences from all samples was evaluated using FastQC v0.11.8 (http: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / ), and adapter sequences and low-quality sequences were removed using Trimmomatic v0.38 (Bolger, AM. et al., (2014) Bioinformatics, 1;30(15):2114-2120). The remaining reads were mapped to the barley reference genome (Morex_pseudomolecules_v2.fasta;Jayakodi, M. et al., (2020) Nature, 588(7837):284-289) using HISAT2 v2.1.0 (Kim, D. et al., (2015) Nat. Methods, 12:357-360). Based on gene annotation (Morex.gff;Jayakodi, M. et al., (2020) Nature, 588(7837):284-289), reads mapped to genes were counted as expression levels using featureCounts v1.6.3 (Liao, Y. et al., (2014) Bioinformatics, 30(7):923-930), and the counted reads were further converted to total product volume (TPM) per million reads for each locus. Differential gene expression (DEG) analysis (from basal stage samples from air-conditioned and mesh rooms) was performed using the Bioconductor package edgeR in an R environment (Robinson, MD et al., (2010) Bioinformatics, 26(1):139-140). Expression levels were normalized by the trimmed mean (TMM) method of M values, P values ​​were calculated by an exact test based on a negative binomial distribution, and gene expression variability was estimated by the log2 change in edgeR.P-values ​​were adjusted for the false discovery rate (FDR) using the Benjamini-Hochberg method (Benjamini, Y. & Hochberg, Y., (1995) Journal of the Royal Statistical Society: Series B (Methodological) 57(1):289-300). DEGs were considered significant if FDR < 0.05 and log2 > 0.5. All DEG annotations were based on BLASTp search results using the Reciprocal Best Hits (RBH) method against protein databases for Arabidopsis thaliana (TAIR10_-40; http: / / www.arabidopsis.org / ) and rice (IRGSP-1.0_predicted-protein_2020-09-09; https: / / rapdb.dna.affrc.go.jp / ) (10). -8 Based on the E-value cutoff.

[0076] Heatmaps of gene expression data were visualized using the R package pheatmap v1.0.12. Gene expression levels at each stage were measured as the mean TPM from six biological replicas and converted to log2(mean TPM+1) values. Hierarchical clustering was performed using heatmap analysis based on their representation patterns.

[0077] (13) Detection of splicing events BAM files containing mapped reads from RNA-seq data at the double-ridge, rhizome, and green anther stages were merged for each genotype of wild-type OUH602 and mutant 44205 using SAMtools version 1.9. The merged reads for wild-type OUH602 and mutant 44205 were recounted, and GTF files with new gene model annotations for OUH602 and 44205 were created using StringTie version 2.1.4 (Pertea, M. et al., (2015) Nat. Biotechnol, 33(3):290-295). Splice junctions were enabled only if supported by more than 10 mapped reads. The results were visualized using the ggsashimi package integrated into the R environment, following the method described in the literature (Garrido-Martin, D. et al., (2018) 19(1):67).

[0078] (14) Statistical analysis All experimental data are presented as mean ± standard error (SE). Student's t-test was used to compare the phenotypes of wild-type and sof1 mutants, and to examine differences in gene expression levels between samples. Significant effects between samples were assessed by the magnitude of the Two-tailed Student's t-test (P-value < 0.05).

[0079] B. Results (1) Analysis of mutant and wild-type phenotypes When mutant 44205 was compared with the wild-type OUH602, the mutant exhibited a "hyperflowering" phenotype with a greater separation between the outer and inner glumes and a wider flowering angle (Figures 1A and B). Observation of the central spikelet revealed larger scales and much more active cell division in the mutant (Figures 1C-F). Measurement of floret phenotype showed that the mutant exhibited a significantly larger flowering angle compared to the wild-type. That is, the scale size increased and the outer and inner glumes of the mutant were separated (Figures 1G and H, Figures 2A and B) (P<0.01, Two-tailed Student's t-test). The most important difference between the mutant and the wild-type was their scale size (Figure 1H), and this phenotype could only be distinguished between the wild-type and the mutant by scale length (Figures 2B-D). Scale elongation was thought to contribute to the wider flowering angle in the mutant. Furthermore, cell counts in the scales were significantly lower in the mutant compared to the wild type (Figure 2E), corresponding to a marked increase in cell length in the mutant (Figure 2F). These phenotypic results suggest that the elongated scales may be caused by cell proliferation in the mutant.

[0080] (2) Mapping of the sof1 gene locus To investigate the genetic factors responsible for the hyperopening phenotype, a 93-plant F2 segregation was constructed by crossing mutant 44205 with Morex (non-closed-flowering pollination). The F1 plants exhibited normal flowering angles and scale sizes, similar to their parent plant, Morex. The F2 segregation ratio fit a 3:1 Mendelian model (72 normally flowering plants and 21 hyperopening plants; χ²=0.29, P-value>0.05), revealing that hyperopening is controlled by a single recessive gene. This gene was named "sof1 (super open flowering 1)".

[0081] RAD-seq analysis was used in 40 F2 plants to map the sof1 gene. The gene was initially located between C7_77055_96891401 and C7_67011_165769003 on chromosome 7H. Next, rough mapping using 93 F2 individuals confirmed that the sof1 locus was located 3.3 cM apart between the fluidigm markers FB0278 and FB0088 on chromosome 7H. Further genotyping with additional CAPS markers and phenotypic analysis of the above 93 individuals positioned the sof1 locus within a physical region with a genetic distance of 1.1 cM between markers HM7H234200 and HM7H246100 (Figure 3A). According to the latest Morex reference genome (Jayakodi, M. et al., (2020) Nature, 588:284-289), this physical region is approximately 9.2 Mb and contains 118 genes, consisting of 110 annotated genes and 8 unannotated pseudogenes.

[0082] To narrow down the physical region of the sof1 gene, a large F2 population containing 1759 sof1 mutants × Morex was used to screen for recombination events. 99 recombinant plants were identified using markers HM7H228700 and HM7H254000 (Figure 3B). Scale length was measured for 95 recombinant plants, excluding three plants that withered before flowering and one plant that produced smaller floral organs than the others due to a dwarf phenotype. The distribution of scale length fit a 3:1 Mendelian segregation (χ²=0.07, P-value>0.05). The results were consistent with previous data, confirming that hyperflowering is controlled under a single recessive sof1 gene, with sof1 located between markers HM7H234200 and HM7H246100. Further genotyping and evaluation of the phenotypes of 13 key recombinants narrowed the sof1 region to a 0.37 cM interval between markers HM7H234200 and HM7H239100 (Figure 3C). The physical distance was approximately 3.1 Mb and consisted of 43 annotated genes and 5 unannotated pseudogenes between Horvu_MOREX_7H01G234200 and Horvu_MOREX_7H01G246100, as described in the literature (Jayakodi, M. et al., (2020) Nature, 588:284-289).

[0083] (3) Genetic factors of the hyperflowering phenotype To investigate gene polymorphisms within a 3.1 Mb interval between wild-type OUH602 and mutant 44205, whole-genome shotgun sequencing of the mutant was performed and compared to the wild-type genome sequence. Within this target region, only three homozygous polymorphisms were detected between the two genotypes; two 1 bp insertions were located in the intergenetic region, and a 22 bp deletion was located in the intragenetic region. The 22 bp deletion was located at positions 18080 to 18101 of the wild-type Sof1 gene genome (with the transcription start site as 1) and caused a translational frameshift (p.Asp2646fs) in the mutant (Figure 4A, B). Horvu_OUH_7H01G211300 corresponds to Horvu_MOREX_7H01G238100 as a highly reliable gene in the Morex genome and has been annotated as a chromatin remodeling protein (Jayakodi, M. et al., (2020) Nature, 588:284-289). To validate the candidate gene, the genotypes of 13 important F2 recombinants were determined using an In-del marker (HM7H238100-Indel) developed for a 22 bp deletion, and the genotypes and phenotypes of the F3 offspring of three recombinants were subsequently analyzed. The results showed that the In-del marker does not rebind with sof1.

[0084] (4) Target mutagenesis of Sof1 To confirm that the gene corresponding to Sof1 is Horvu_OUH_7H01G211300 in OUH602 and Horvu_MOREX_7H01G238100 in MOREX, additional mutant lines were created in the barley variety Golden Promise using CRISPR / Cas9-mediated mutagenesis. The guide RNA targeted the Horvu_GOLDEN_7H01G199000 gene (near the 22bp deletion in the mutant), which is an ortholog of Horvu_MOREX_7H01G238100 within the Golden Promise genome (Figure 5A). After genotyping and sequencing of the transgenic plants, the resulting mutation yielded independent mutations (sof1-2) with a 5bp deletion. This introduced a stop codon leading to early termination of translation and could be considered a loss-of-function allele (Figure 5A). Golden Promise possesses a cleistogamous allele (cly1.b) at the cly1 locus, resulting in the formation of small scales. However, the genome-edited lines created are derived from this cultivar and therefore retain this cleistogamous allele, potentially hindering the effect of the sof1 mutation. To eliminate the potential negative impact of cly1.b on scale development, the sof1-2 T0 line was crossed with the non-cleistogamous barley varieties Sv73528 and Adorra to create an F3 population with the genotype sof1-2sof1-2Cly1_, and its scale phenotype was determined (Figure 5B). As a result, the scale length of the sof1-2 mutant (T2) was significantly longer compared to Sv73528 and Adorra (Figure 6).

[0085] Compared to Sv73528 and Adorra, the F3 plants exhibited significantly longer scales and florets with a larger flowering angle (Figure 7), although there were no significant differences in scale depth and width. These results suggest that the elongated scales in the sof1-2 mutant are caused by the cleavage of Horvu_GOLDEN_7H01G199000, and that Horvu_MOREX_7H01G238100 is considered the functional gene for Sof1. From these findings, we concluded that the 22bp deletion of Horvu_MOREX_7H01G238100 is responsible for the hyperflowering in the sof1 mutant.

[0086] (5) Structural characteristics of the Sof1 gene Sof1 is the wild-type allele of sof1. The Sof1 gene has a start-stop codon length of 20720 bp (SEQ ID NO: 3) and a full-length CDS of 10326 bp (SEQ ID NO: 1) (Figure 4A), and it encoded a predicted 3441 amino acid protein (SOF1) with two Snf2 family domains (Figure 4B). One is an ATPase domain composed of SNF2_N (the N-terminal domain of the SNF2 family) and Helicase_C (the helicase-conserved C-terminal domain), and the other is a SnAC (Snf2 ATP coupling) domain (Figure 4B). The ATPase domain and SnAC domain are located at the N-terminus of SOF1, but no conserved domains were found in the C-terminal region. Compared to the wild type, the sof1 mutant contains a 22 bp deletion (Figure 4A), causing a frameshift mutation (D2659Efs) that introduces a stop codon at the base corresponding to the 2677th amino acid in the C-terminal region, resulting in a cleaved protein of 2676 amino acids lacking the last 765 amino acids (approximately 22% of the complete SOF1 protein) (Figure 4B). The cleaved protein was thought to lose the biological function of the mutant. SNF2 is an ATP-dependent chromatin remodeling factor that controls many aspects of DNA events, including transcription, replication, homologous recombination, and DNA repair. To investigate the function of SOF1, a phylogenetic analysis of SOF1 homologs was performed. Based on BLASTp searches in plant species, SOF1 homologs were found in monocots and dicots, and higher homologs with high identity to SOF1 were further selected from each taxonomic group for use in phylogenetic analysis. Three other members of the Arabidopsis thaliana SWI / SNF subfamily were included to infer the class to which the homologous protein belongs. The phylogenetic tree showed that all SOF1 homologs are clustered within the SWI / SNF subfamily, suggesting that SOF1 belongs to this SWI / SNF subfamily (SWI / SNF ATPase) and that these sequences are orthologous (Figure 8). SWI / SNF ATPase has the function of activating chromatin and turning on its genes.In this SWI / SNF cluster, SOF1 and the Arabidopsis SPLAYED (SYD) protein were closely related to each other (Figure 8). Arabidopsis SYD acts on several downstream bHLH genes via multiple MADS box genes, suppressing petal enlargement. Therefore, mutations in SYD result in enlarged petals. The grass homologous organ of the petal is the scale. Thus, the gene regulatory system originating from SYD can be one model for scale enlargement (hyperflowering) in barley sof1 mutations. On the other hand, its homolog, HORVU_MOREX_6H01G041600.1, was annotated as an ATP-dependent helicase family protein in the Morex genome. This shows a close relationship with ArabidopsisBRAHMA (BRM), and the NCBI's preserved domain database (Marchler-Baur, A. et al., (2017) Nucleic Acids Res., 4;45(D1):D200-D203) has shown that it contains a potential bromodomain at its C-terminus. This suggests that HORVU_MOREX_6H01G041600.1 is a barley SOF1 paralog. On the other hand, OsSYD (XP_015642458.1) is one of the SYD orthologues based on phylogenetic analysis and has been reported as a SYD orthologue of Oryza sativa ssp japonica (Su, Y. et al., (2006) The Plant Journal, 46(4):685-699, Hu, Y. et al., (2013) Plant physiology and biochemistry, 70:33-42). These results suggest that SOF1 is an ortholog of ArabidopsisSYD. SOF1 (and SYD) are retained in both monocots and dicots, suggesting that these SOF1 orthologs originate from a common ancestor and are evolutionarily conserved (Figure 8). Therefore, Sof1 encodes a chromatin remodeling ATPase protein whose function may be similar to that of ArabidopsisSYD and OsSYD.

[0087] (6) Profiling of Sof1 transcript levels The transcription levels of Sof1 were examined in the entire panicle, in individual reproductive organs across different panicle developmental stages, and in various vegetative organs (Figure 9). qRT-PCR profiling revealed that this gene is widely transcribed throughout the entire panicle developmental stage in both wild-type and mutant strains. In the wild-type, transcription of this gene was detected in six floral organs, leaf sheaths, leaf blades, nodes, and internode vegetative organs at the green anther stage, but was also low in young panicles at the same awn primordium stage. The amount of transcript was significantly lower in young panicles and reproductive organs than in the wild-type (Figure 9). Notably, the transcription level of this gene decreased significantly in mutant strains from awn primordium to flowering, and was also clearly decreased in the scales (P<0.01, Figure 9). Transcript level analysis suggested that this gene may play a role between vegetative growth and reproductive development.

[0088] (7) Predicted transcriptional regulation by Sof1 To elucidate the effects of the 22bp deletion of Sof1 using transcriptome analysis, RNA-seq analysis was performed on wild-type OUH602 and mutant 44205. Analysis of immature spikes (awn primordium stage) from plants grown in an air-conditioned greenhouse (22°C under natural light) and a mesh room revealed a total of 2412 and 1371 differentially expressed genes (DEGs) (FDR < 0.05 and |log2fold change| > 0.5) between the two genotypes under the two growth conditions. Furthermore, 674 common DEGs consisted of 391 (58%) genes whose expression was induced and 283 (42%) genes whose expression was repressed in the mutant compared to the wild type. The decrease in Sof1 expression in the mutant was also confirmed by RNA-seq analysis and was consistent with the Sof1 expression profile obtained by qRT-PCR.

[0089] Among the significantly repressed genes, six flower homeosis genes were detected. These were the B-class gene (AP3-like, Horvu_MOREX_7H01G536500 and PI-like, Horvu_MOREX_1H01G402000), the C-class gene (AG-like, Horvu_MOREX_3H01G171300), the E-class gene (AGL6-like, Horvu_MOREX_6H01G395900 and SEP-like, Horvu_MOREX_7H01G328200), and the SVP-like gene (Horvu_MOREX_4H01G454700). These genes were homologs of rice OsMADS16, OsMADS4, OsMADS3, OsMADS6, and OsMADS6, respectively. In particular, the genes for OsMADS16 (Nagasawa, N. et al., (2003) Development, 130(4):705-18, Yoshida, H. et al., (2007) Plant Biotechnology Journal, 5(6):835-846), OsMADS3 (Yamaguchi, Y. et al., (2006) The Plant Cell, 18(1):15-28, Dreni, L. et al., (2011) The Plant Cell, 23(8):2850-2863), OsMADS6 (Li, H. et al., (2010) Cell research, 20(3):299-313), and OsMADS7 (Cui, B. et al., (2010) Plant J., 61(5):767-781) have been reported to be involved in scale development through rice mutants.

[0090] (8) Estimation of pollination efficiency based on differences in flowering characteristics (15) Estimation of pollination efficiency To estimate the pollination effect, cytoplasmic male-sterile lines of two flowering varieties (Adorra, Sv73528) and one closed-flowering variety (Kawasai-goku) were used. During the flowering period of potted plants, the glumes were physically opened, the awns were fixed with tape, and artificial pollination was performed. The plants were then allowed to mature, and the pollination effect was estimated based on the number of grains. As a result, a significant pollination effect (increase in the number of grains) of more than five times was observed in the flowering varieties Adorra and Sv735108, and the effect was also clear in the closed-flowering variety "Kawasai-goku" (Table 1).

[0091] [Table 1]

[0092] Furthermore, no grains were observed in the ears of the male-sterile lines that did not undergo pollination, confirming that fertilization did not occur through pollen from other individuals other than the pollination treatment. In the flowering type variations of pollinated individuals, a significant increase in the number of grains was observed due to the improvement in the pollination rate.

[0093] (8) Identification and analysis of wheat homologous genes We obtained homologous wheat gene sequences from the genomic information of the wheat variety Chinese Spring. The cDNA sequences of each identified wheat Sof1 gene are shown as SEQ ID NOs: 4, 7, and 10, the genomic DNA sequences as SEQ ID NOs: 6, 9, and 12, and the amino acid sequences of the proteins encoded by these DNAs as SEQ ID NOs: 5, 8, and 11. Similar to barley Sof1, it was found to be strongly expressed in various organs such as roots, leaves, and ears in wheat (Figure 10). Furthermore, using the TILLING method, we successfully selected multiple lines of wheat Sof1 gene mutants in the wheat varieties Kronos and Cadenza. [Industrial applicability]

[0094] As explained above, the present invention makes it possible to produce wheat with improved flowering ability. Since the present invention can greatly increase the efficiency of developing new wheat varieties, it contributes to improving the productivity of grains in the agricultural sector.

Claims

1. A method for producing wheat with improved flowering ability, comprising artificially suppressing the function of the endogenous gene (a) or (b) described below by introducing the following mutation in wheat possessing the flowering type Cly1 gene. (a) Introduce a mutation into the endogenous gene encoding a protein consisting of the amino acid sequence described in Sequence ID No. 2, which causes the deletion of 20% or more of the protein at its C-terminus. (b) Introduce a mutation into an endogenous gene encoding a protein having an amino acid sequence that is 90% or more identical to the amino acid sequence described in Sequence ID No. 2, causing a deletion of 20% or more of the protein at its C-terminus.

2. Wheat that possesses the flowering type Cly1 gene and in which the function of the endogenous gene (a) or (b) below is artificially suppressed by introducing the following mutation, resulting in improved flowering ability. (a) Introduce a mutation into the endogenous gene encoding a protein consisting of the amino acid sequence described in Sequence ID No. 2, which causes the deletion of 20% or more of the protein at its C-terminus. (b) Introduce a mutation into an endogenous gene encoding a protein having an amino acid sequence that is 90% or more identical to the amino acid sequence described in Sequence ID No. 2, causing a deletion of 20% or more of the protein at its C-terminus.

3. A method for evaluating the flowering ability of wheat, comprising analyzing the base sequence of the endogenous gene (a) or (b) below in wheat possessing the flowering type Cly1 gene, and detecting the presence or absence of the following mutation. (a) A mutation in an endogenous gene encoding a protein consisting of the amino acid sequence described in Sequence ID No. 2, which causes the deletion of 20% or more of the protein at its C-terminus. (b) A mutation in an endogenous gene encoding a protein having an amino acid sequence that is 90% or more identical to the amino acid sequence described in Sequence ID No. 2, which causes the deletion of 20% or more of the protein at its C-terminus.

4. A method for producing wheat with improved flowering ability, comprising crossing the wheat described in claim 2 with any other wheat.

Images