Ultra-chasmogamous wheat, method for producing ultra-chasmogamous wheat, and method for evaluating ultra-chasmogamous wheat

Abstract

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.

Description

Super-flowering wheat and methods for producing and evaluating same

[0001] The present invention relates to wheat and barley with improved flowering ability, which is targeted to the Sof1 gene, and methods for producing and evaluating the same.

[0002] The world's grain supply is at risk of becoming tight due to factors such as increased food demand caused by population growth, primarily in developing countries, as well as reduced production due to frequent extreme weather events and water resource constraints. One means of resolving this problem is the development of new grain varieties that can increase agricultural productivity.

[0003] Heterosis is a phenomenon in which the productivity of first generation (F1) hybrids exceeds that of their parents, and has been utilized to develop new varieties of various crops. However, despite the excellent effects of heterosis in barley, which has extremely high genetic diversity, the technology for heterosis has not matured because barley is inherently self-pollinating and the efficiency of hybrid seed production through cross-pollination is low.

[0004] Therefore, efforts have been made to identify genes that control the flowering habit of grains in order to improve the efficiency of cross-pollination. For example, the small lodicules of cleistogamy barley varieties are controlled by a single recessive gene, cleistogamy 1 (cly1), located on the long arm of chromosome 2H (Non-Patent Document 1), which has been reported to be an ortholog of the Arabidopsis AP2 transcription factor (Non-Patent Document 2). The barley flowering-type Cly1 gene (Cly1.a) increases the width and thickness of the lodicules, pushing the lemma and glume outward from within the floret, thereby mechanically inducing flowering (Non-Patent Document 2). The difference between cleistogamy cly1.b and flowering Cly1.a is due to a single base substitution in the specific binding site of microRNA172 (miR172), which encodes the cly1 sequence; wild-type species with Cly1.a exhibit reduced cleavage of the cly1 transcript via miR172. As a result, the translated CLY1 protein accumulates in high amounts, and the lodicules, which had been suppressed, develop normally (Non-Patent Documents 2 and 3).

[0005] By utilizing the flowering-type Cly1.a, it is possible to impart the trait of flowering to cleistogamy varieties. However, the flowering ability conferred by this gene alone is insufficient to improve the efficiency of hybrid seed production by cross-pollination, and a dramatic improvement in flowering ability is still required.

[0006] Turuspekov, Y. et al. , (2004) Theor. Appl. Genet. , 109:480-487Nair, S. K. et al. , (2010) Proceedings of the National Academy of Sciences, 107(1):490-495 Anwar, N. et al. , (2018) Annals of Botany, 122(2):251-265

[0007] The present invention has been made in view of the problems associated with the prior art, and an object of the present invention is to provide wheat and barley with dramatically improved flowering ability, as well as methods for producing and evaluating the same.

[0008] To achieve this objective, the inventors first irradiated a flowering wild barley line (OUH602) with gamma rays to screen for mutants with improved flowering habit. As a result, they successfully selected mutants exhibiting dramatically improved flowering habit, the trait of super-flowering. The selected mutants were then crossed with a flowering cultivar (Morex), and map-based cloning was performed using the F2 population. As a result, they found that the gene responsible for super-flowering is located on chromosome 7H between markers HM7H234200 and HM7H246100, and that this trait is controlled by a single recessive gene (the gene controlling this super-flowering trait was named "sof1"). Within this approximately 3.1 Mb candidate region, 43 annotated genes and 5 unannotated pseudogenes were identified.

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

[0010] To confirm that this gene is sof1, which causes the super-flowering trait, the Sof1 gene (region near the 22 bp deletion) of a cleistogamy cultivar (Golden Promise) was targeted, and a mutation was introduced using the CRISPR / Cas9 method. As a result, a mutation (sof1-2) with a 5 bp deletion in the Sof1 gene was obtained, and lodicules significantly longer than those of Golden Promise were formed, confirming the effect of the mutation. However, this mutant was affected by the cleistogamy cly1.b gene of Golden Promise, and did not form lodicules large enough to exhibit the super-flowering trait. Therefore, with the aim of eliminating the influence of the cly1.b gene, this mutant was crossed with barley cultivars Sv73528 and Adolra, which have the flowering Cly1.a gene. As a result, the lodicules of the obtained individuals were significantly longer than those of Sv73528 and Adorra, indicating the trait of super-flowering. From the above, it was revealed that the sof1 gene is the gene responsible for the trait of super-flowering. Furthermore, the inventors searched for homologous genes in other plants and found the presence of a gene corresponding to the barley gene in wheat, and succeeded in obtaining a mutant exhibiting the trait of super-flowering.

[0011] Based on the above, the inventors discovered 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, thereby completing the present invention.

[0012] More specifically, the present invention includes the following aspects.

[0013] [1] A method for producing wheat with improved flowering ability, the method comprising artificially suppressing the function of the following endogenous gene (a) or (b) in wheat: (a) an endogenous gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, 5, 8 or 11; (b) an endogenous gene encoding a protein consisting of an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO: 2, 5, 8 or 11. [2] Wheat with improved flowering ability, in which the function of the following endogenous gene (a) or (b) has been artificially suppressed: (a) an endogenous gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, 5, 8 or 11; (b) an endogenous gene encoding a protein consisting of an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO: 2, 5, 8 or 11. [3] A method for evaluating the flowering ability of wheat, the method comprising analyzing the nucleotide sequence of the following endogenous gene (a) or (b) or its expression control region in wheat: (a) an endogenous gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, 5, 8 or 11; (b) an endogenous gene encoding a protein consisting of an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO: 2, 5, 8 or 11. [4] A method for evaluating the flowering ability of wheat, comprising analyzing the expression of the following endogenous gene (a) or (b) in wheat: (a) an endogenous gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, 5, 8 or 11; or (b) an endogenous gene encoding a protein consisting of an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO: 2, 5, 8 or 11. [5] A method for producing wheat with improved flowering ability, comprising crossing the wheat of claim 2 with any other wheat.

[0014] According to the present invention, it is possible to produce wheat with dramatically improved flowering ability. The wheat exhibits a large flowering angle due to the large expansion of its lodicules. When this trait is introduced into a pollen parent in wheat breeding, it facilitates pollen dispersal, while when introduced into a male-sterile seed parent, it facilitates pollination. This makes it possible to dramatically increase the efficiency of hybrid seed production by cross-pollination.

[0015] Photographs of wild-type OUH602 (WT) and mutant 44205 (MT). Spikelets of (A) wild-type OUH602 and (B) mutant 44205. Lodicules of (C) wild-type OUH602 and (D) mutant 44205 at the yellow anther stage just before anthesis. Lodicules of (E) wild-type OUH602 and (F) mutant 44205 viewed from the front of the spikelet. Lodicules of (G) wild-type OUH602 and (H) mutant 44205 viewed from the lateral side of the spikelet. Graphs (continuation of Figure 1) of wild-type OUH602 (WT) and mutant 44205 (MT). (A) Anthesis angle, (B) lodicule length, (C) lodicule thickness, (D) lodicule width, (E) lodicule cell number, and (F) lodicule cell length. Figure 3: Map-based cloning of sof1. (A) High-resolution genetic map of sof1. Numbers between markers above the linkage map lines 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 over a 3.1 Mb physical region. Arrows indicate the direction of transcription of the annotated genes. Diagram (continuation of Figure 3) showing map-based cloning of sof1. (A) Structure of the Sof1 gene. Boxes at either end represent the 5' and 3' untranslated regions (UTRs), boxes between them represent coding exons, and thin lines represent introns. ATG and TAG represent the start and stop codons, respectively. The dashed line indicates the 22-bp deletion in the mutant. (B) Structure of the SOF1 protein. Each box (positions 1043-1334, 1359-1473, and 1587-1661) represents a domain of SOF1. Figure 1 shows characterization of Sof1 function by CRISPR / Cas9-mediated mutagenesis. (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. Inverted triangles indicate guide RNA (gRNA) target sites within the gene. Lines on the Golden Promise (GP) sequence indicate the gRNA target site and protospacer adjacent motif (PAM). Dashed lines indicate deleted bases.(B) Scheme of phenotypic identification in F3 plants derived from a cross between an edited T1 line and two non-cleistogamous barley cultivars. The target genotype for each generation is indicated in parentheses. Photographs (top) and a graph of lodicule size (bottom) of the wild-type Golden Promise (GP) and homozygous T2 mutant lines. Each measurement in the graph represents the average of 9 to 13 lodicules for statistical analysis. Two-tailed Student's t-test. ** indicates P<0.01, and ns indicates no significant difference. Photographs (top) and (bottom) showing a comparison of the anthesis angle of florets and lodicule size in two non-cleistogamous cultivars and their corresponding F3 plants. Phylogenetic analysis of SOF1 homologs in monocots and dicots. The tree was constructed by the neighbor-joining (NJ) method based on the alignment of the polypeptide sequences of SOF1 homologs. The numbers on the branches indicate the bootstrap probability. Figure 1 is a graph showing the transcript abundance of Sof1 in developing panicles 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); stage); GA (Green anther stage) stage); YA (yellow anther stage); DAG (days after germination). Values ​​are means ± SE (n = 3 independent biological replicates). Statistical significance was calculated by two-tailed Student's t-test. * indicates P < 0.05, ** indicates P < 0.01, and ns indicates no significant difference. This is a graph showing the transcript abundance of Sof1 in a wild-type wheat cultivar (Chinese Spring).Photographs showing the anthesis angles of wild-type OUH602 (left) and the hyper-flowering OUH602 mutant (hereafter referred to as "mutant 44205" unless otherwise specified) (right). The measured anthesis angles are indicated in the photographs.

[0016] <Method for Producing Wheat with Improved Flowering Ability> In the present invention, "flowering ability" refers to a trait in which the flowering angle increases due to the expansion of lodicules. The inflorescences of monocotyledonous plants, such as wheat, rice, and corn, consist of a structure called a spikelet, which contains one or more florets. Each floret is composed of reproductive organs (stamens and pistils) and is covered by a pair of bract-like organs (lemma and lemma). "Licodicules" are located between these reproductive organs and glumes. Lodicules are generally considered to correspond to petals and are located at the base of the reproductive organs (stamens and pistils) on both sides. Rapid expansion of lodicules just before anthesis mechanically separates the lemma and lemma, facilitating the release of pollen from dehiscent anthers, which then allow the stigma to cross-pollinate (cleistogamy). In contrast, when lodicules are unable to expand, pollen is hidden within the closed florets, thereby causing self-pollination (cleistogamy).

[0017] In the present invention, "improved flowering ability" means that the flowering angle is increased 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 lemma (the glume farther from the rachis) intersects with a line along the edge of the inner lemma (the glume closer to the rachis) in the florets of the panicle. Note that the flowering angle may vary depending on the species and variety of wheat; for example, the flowering angle of wild-type barley OUH602 is usually 10.2±2.2° ( FIG. 11 (left)).

[0018] In the present invention, "wheat" as the target for improving flowering ability refers to all plants of the wheat family belonging to the Poaceae subfamily, including barley, wheat, rye, triticale, and oats. Wild species or cultivated species may be used. When wheat possesses a cleistogamy cly1 gene, the effect of suppressing the function of the Sof1 gene is less likely to be observed. Therefore, it is preferable that the "wheat" as the target for improving flowering ability possesses a flowering cly1 gene. When the function of the Sof1 gene is suppressed in wheat possessing a cleistogamy cly1 gene, the negative effects of the cleistogamy cly1 gene can be eliminated by subsequently crossing the wheat with a wheat possessing a flowering cly1 gene or by modifying the cly1 gene from cleistogamy to flowering by genome editing (e.g., by substituting a single base 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 that encodes the SOF1 protein).

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

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

[0022] In nature, individual differences in nucleotide sequences may occur, and with the current state of the art, if a specific gene is obtained, a person skilled in the art can use the nucleotide sequence information of that gene to identify corresponding genes in the same or other plants. Therefore, the Sof1 gene of the present invention includes these homologous genes as long as suppression of its function improves flowering ability.

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

[0024] Sequence homology can be determined using the BLAST program (Altschul et al., J. Mol. Biol., (1990) 215:403-410), which is based on the BLAST algorithm by Karlin and Altschul (Karlin, S. & Altschul, S. F., (1990) Proc. Natl. Acad. Sci. USA, 87:2264-2268, Karlin, S. & Altschul, S. F., (1993) Proc. Natl. Acad. Sci. USA, 90:5873-5877). For example, when analyzing an amino acid sequence using BLAST, the parameters are, for example, score = 50 and wordlength = 3. Furthermore, when analyzing an amino acid sequence using the Gapped BLAST program, it can be performed as described by Altschul et al. (Altschul, S. F. et al., (1997) Nucleic Acids Res., 25:3389-3402). When using the BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific techniques for these analysis methods are known.

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

[0026] In the present invention, "suppression of the function of the Sof1 gene" includes both complete and partial suppression of the function, as well as suppression of the activity of the SOF1 protein (e.g., the activity of suppressing scale expansion) and suppression of the expression of the Sof1 gene (suppression of transcription and suppression of translation).

[0027] The function of the Sof1 gene can be suppressed, for example, by introducing a mutation into the Sof1 gene or its expression control region (e.g., promoter region). The mutation to be introduced into the Sof1 gene is not particularly limited as long as it suppresses its function, and examples thereof include nucleotide substitution, deletion, addition, and / or insertion, although frameshift mutation, nonsense mutation, and null mutation are preferred.

[0028] The number of mutations introduced into the Sof1 gene is not particularly limited as long as it suppresses its function, and may be one or more (e.g., 2, 3 or less, 5 or less, 10 or less, 20 or less, 30 or less, 40 or less, 50 or less). Indeed, in the Examples of the present application, it has been found that nucleotide deletions in the barley Sof1 gene (22-base deletion [sof1 mutant], 5-base deletion [sof1-2 mutant]) result in a mutant protein shortened at the C-terminus due to frameshift, resulting in improved flowering ability in barley. The SOF1 protein encoded by the Sof1 gene contains two functional domains (ATPase domain and SnAC domain) on its N-terminus, but it is also possible to lose the function of the SOF1 protein by mutations on the C-terminus, where these functional domains are not present. Therefore, the mutation introduced into the Sof1 gene does not need to result in the loss of the entire amino acid sequence of the protein encoded by the gene, and may be introduced into the gene so that only a portion of the amino acid sequence is lost or changed.

[0029] When a portion of a protein expressed by a gene mutation is deleted, 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 (e.g., 60% or more, 70% or more, 80% or more, 90% or more, 95% or more) from the viewpoint of suppressing the activity of the SOF1 protein.

[0030] Those skilled in the art can introduce a mutation into the Sof1 gene by known methods. Among known methods, genome editing is preferred because it allows efficient site-specific introduction of mutations.

[0031] Genome editing is a method of modifying a target gene using a site-specific nuclease. Examples of site-specific nucleases include Cas nucleases (CRISPR / Cas systems) that are given site specificity by forming a complex with a guide RNA, transcription activation-like effector nucleases (TALENs), and zinc finger nucleases (ZFNs). As the CRISPR / Cas system, various systems can be used, such as a class 2 type II system (including Cas9 as Cas), a class 2 type V system (including Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas14, etc. as Cas), a class 2 type VI system (including Cas13a (C2c2), Cas13b, Cas13c, etc. as Cas), and a class 1 type I system (including Cas3 as Cas).

[0032] Other methods for introducing mutations into genes include, but are not limited to, physical mutagenesis, methods using chemical mutagens, and methods using 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] An example of a method using a chemical mutagen is treating seeds with a chemical mutagen (Zwar and Chandler, Planta, 1995, vol. 197, pp. 39-48). There are no particular limitations on the chemical mutagen, and examples include N-methyl-N-nitrosourea (MNU), ethyl methanesulfonate (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, nitrous acid, formic acid, and nucleotide analogs.

[0035] Methods for introducing transposons into genomic DNA include, for example, ＯＳ Examples of such methods include inserting transposons such as 17, T-DNA, or the like into the genomic DNA of a plant (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 these methods are used, individuals having a mutation in the Sof1 gene are usually selected from a group of individuals that have been subjected to a mutagenesis treatment.

[0037] Wheat into which a mutation has been introduced into the Sof1 gene may be a heterozygote for the mutant gene. In this case, heterozygotes can be crossed with each other to create an F1 plant, and a homozygote for the mutant gene can be selected from the F1 plant. In the present invention, the sof1 gene (mutant gene) responsible for the hyper-flowering 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 that the wheat retains the mutated Sof1 gene in a homozygous state. Note that, for wheat into which an unintended mutation has been introduced, the unintended mutation can be removed, for example, by backcrossing with wild-type wheat.

[0038] When suppressing the function of the Sof1 gene by suppressing its expression, in addition to introducing a mutation into the expression control region of the Sof1 gene, methods that target the transcription product of the Sof1 gene can also be used, such as a method using DNA encoding dsRNA (double-stranded RNA, for example, siRNA) complementary to the transcription product of the Sof1 gene, a method using DNA encoding antisense RNA (antisense DNA) complementary to the transcription product of the Sof1 gene, or a method using DNA encoding RNA with ribozyme activity that specifically cleaves the transcription product of the Sof1 gene (ribozyme method).

[0039] In addition, the expression of the Sof1 gene can be suppressed by nucleotide modification through epigenetic regulation, which does not involve nucleotide mutation. Examples of epigenetic regulation include DNA methylation and chemical modifications of histones (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 segments, suspension-cultured cells, protoplasts, calluses, meristems, and young panicles.

[0041] When the molecule for artificially suppressing the function of the Sof1 gene is encoded by DNA (e.g., DNA encoding a site-specific nuclease, DNA encoding a transposon, DNA encoding a double-stranded RNA, DNA encoding an antisense RNA, DNA encoding an RNA with ribozyme activity, etc.), it may be introduced into wheat in the form of a vector inserted therein. There are no particular limitations on the vector, as long as it is capable of expressing the inserted DNA in wheat cells. The vector typically contains a promoter for constitutive or inducible 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 inducible expression include promoters known to be expressed in response to external factors such as treatment with specific compounds, low temperature, high temperature, drought, ultraviolet radiation, and infection or invasion by filamentous fungi, bacteria, or viruses.

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

[0043] When cells in which the function of the Sof1 gene has been artificially suppressed are obtained by the above method, wheat plants with improved flowering ability can be obtained by regenerating wheat plants from the cells. Techniques 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). Transformation and regeneration into plants can also be performed using the method described in Tabei et al. (Tabei, Yutaka, ed., "Transformation Protocol [Plant Edition]", 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 progeny from the plant through sexual or asexual reproduction. Furthermore, propagation materials (e.g., seeds, cuttings, stumps, callus, protoplasts, etc.) can be obtained from the plant, its progeny, or clones, and used to mass-produce plants. The present invention also provides the thus-obtained wheat plants, progeny, clones, and propagation materials with improved flowering ability.

[0045] <Method for Evaluating Flowering Ability of Wheat> One embodiment of the method of the present invention comprises analyzing the nucleotide sequence of the Sof1 gene or its expression control region in wheat.

[0046] When analyzing the nucleotide sequence of the Sof1 gene or its expression control region, an amplification product obtained by amplifying the Sof1 gene or its expression control region by PCR can be used. When PCR is performed, there are no limitations on the primers used as long as they can specifically amplify the Sof1 gene or its expression control region, and they can be appropriately designed based on the sequence information of the Sof1 gene or its expression control region.

[0047] Evaluation of flowering ability may include a step of comparing with a "control nucleotide sequence." The "control nucleotide sequence" is typically the nucleotide sequence of a gene encoding the amino acid sequence set forth in SEQ ID NO: 2 (barley) or the amino acid sequence set forth in SEQ ID NO: 5, 8, or 11 (wheat), or the expression control region thereof.

[0048] By comparing the determined nucleotide sequence of the Sof1 gene or its expression control region in the test wheat with the nucleotide sequence of the control, it is possible to evaluate whether the test wheat has improved flowering ability. For example, if there is a difference in the nucleotide sequence compared to the nucleotide sequence of the control (particularly if there is a significant change in the molecular weight or amino acid sequence of the encoded protein due to the appearance of a new stop codon or a frameshift), the test wheat is evaluated as having a high probability of being a wheat with improved flowering ability.

[0049] DNA for analyzing the base sequence can be prepared by conventional methods, such as the CTAB method. Wheat for DNA preparation can be obtained not only from mature plants, but also from seeds or young plants. The base sequence can be determined by conventional methods, such as the dideoxy method or the Maxam-Gilbert method. Commercially available sequencing kits and sequencers can be used to determine the base sequence.

[0050] In addition to the above-mentioned direct determination of the base sequence, whether or not the base sequence of the Sof1 gene or its expression control region in the test wheat differs from that of the control can also be analyzed indirectly by a known method such as PCR-SSCP (single-strand conformation polymorphism).

[0051] Another embodiment of the method of the present invention comprises analyzing the expression of the Sof1 gene.

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

[0053] Detection of the Sof1 gene at the transcription level can be carried out by conventional methods, such as RT-PCR, Northern blotting, or RNA-seq. The primers used in carrying out 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 the sequence information of the Sof1 gene that has already been determined. Detection at the translation level can be carried out by conventional methods, such as Western blotting. The antibody used in Western blotting may be a polyclonal or monoclonal antibody, and methods for preparing these antibodies are well known to those skilled in the art.

[0054] If the result of detecting gene expression shows that the expression level of the Sof1 gene in the test wheat is significantly lower than that of a variety that retains 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), the wheat is evaluated as having a high probability of having improved flowering ability. Furthermore, if the molecular weight of the amplification product or expression product of the Sof1 gene is significantly different from the molecular weight of the expression product of the wild-type Sof1 gene, the wheat is evaluated as having a high probability of having improved flowering ability.

[0055] <Method for Producing Wheat with Improved Flowering Ability> The present invention provides a method for producing wheat with improved flowering ability.

[0056] The method of the present invention comprises crossing the above-described wheat having improved flowering ability with any other wheat.

[0057] Examples of "any other wheat" to be crossed with the wheat having 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 trait of hyper-flowering. From the individuals obtained by crossing, wheat in which the function of the Sof1 gene is suppressed can be selected by the above-mentioned method.

[0058] By using the method of the present invention, it is possible to appropriately select wheat varieties with improved flowering ability at an early stage, such as seeds or seedlings, and to develop varieties with these traits in a short period of time.

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

[0060] A. Materials and Methods (1) Plant Materials OUH602, a non-cleistogamous wild barley line (Hordeum vulgare subsp. spontaneum), was obtained from the Plant Science Resources Institute, Okayama University. OUH602 was treated with cobalt-60 ( ６０ The plants were treated with chronic gamma irradiation (0.24-0.77 Gy per day, 5 days per week) using Co. The OUH602 mutant (line number: 44205) was selected from M3 lines derived from 1600 M2 individuals based on its super-flowering phenotype. For genetic analysis, the OUH602 mutant was crossed with the cultivar "Morex" to create a mapping population. Plants were grown in a screened greenhouse (natural light, not temperature-controlled) or an air-conditioned greenhouse (maintained at 24°C) with one plant per 4 L pot.

[0061] (2) Evaluation of lodicule size phenotype and flowering angle Three spikes (with flag leaves still attached to the pedicel) per line were randomly selected, and florets (yellow anther stage just before flowering) were collected from the center of each spike. After removing the lemma from the florets, the lodicules were photographed using an Axio Zoom v16 microscope (Carl Zeiss, Tokyo, Japan) as described in the literature (Non-Patent Document 2). Based on the digital images, the length, thickness, and width of the lodicules were measured using Makijaku software v1.1 (http: / / lbm.ab.a.u-tokyo.ac.jp / ~iwata / software / makijaku / ).

[0062] In addition, photographs of randomly selected panicle florets (immediately after flowering: yellow anther stage) were taken for the wild type and mutant, and the angle formed by a line along the edge of the lemma (the one farthest from the rachis) and a line along the edge of the lemma (the one nearer from the rachis) was measured using Makijaku software v1.1 (http: / / lbm.ab.a.u-tokyo.ac.jp / ~iwata / software / makijaku / ), and this was taken as the flowering angle.

[0063] (3) Measurement of lodicule cells. After embedding the lodicules in 30% acrylamide gel, sections 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 taken using a laser scanning microscope (LSM700, Carl Zeiss) equipped with ZEN 2009 Light Edition CLSM software. For measurement of lodicule cells, the number of cells was counted based on the lodicule thickness, and cell size was calculated by dividing the lodicule 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, Fluidigm markers were developed based on single nucleotide polymorphisms (SNPs) in the genome sequences of barley cultivars Sky Golden and Tochinoibuki. For high-resolution genetic mapping, additional cleaved amplified polymorphic sequence (CAPS) markers were developed around RAD markers FB0278 and FB0088 to identify the identified SNPs. These specific polymorphic sites were detected by PCR analysis, and the PCR products were sequenced and identified by restriction enzyme-based CAPS analysis according to the literature (Sakuma, S. et al., (2010) Functional & Integrative Genomics, 10:123-133). A total of 11 CAPS markers were developed by comparing two exome sequences of the Morex cultivar (accession number ERR271705) and OUH602 (accession number ERR271737) obtained from the DDBJ database (https: / / ddbj.nig.ac.jp / ). In addition, InDels (insertion-deletions) markers were developed for a 22-bp deletion identified in the whole-genome sequence of mutant 44205. All developed markers were used to screen the lineages of the mapping population.

[0065] (5) Genetic map of sof1. A barley F2 population was developed by crossing mutant 44205 with Morex. The size of lodicules was investigated using the maximized spike culture method (Honda, I. et al., (2005) Physiologiae Plantarum, 124:524-531), in which panicles were treated with 2,4-dichlorophenoxyacetic acid (2,4-D). It is known that the size of the lodicules (non-cleistogamous and cleistogamous) is completely consistent with the non-cleistogamous and cleistogamous varieties (Nair, S. K. et al., (2010) Proceedings of the National Academy of Sciences, 107(1): 490-495). Addition of 2,4-D (30 ppm) to all cleistogamous lines did not result in any flowering. On the other hand, the lodicules of the anthego-pollinated lines expanded, on average, by approximately 1.5 times in width and approximately 2.3 times in thickness (perpendicular to the lemma) compared to before treatment.

[0066] For the initial mapping, 40 plants (20 WT and 20 mutant types) were selected. Forty F2 plants were genotyped using ddRAD-seq (Double Digest RAD-seq). For rough mapping, 93 F2 plants were genotyped using Fluidigm markers. A linkage map was 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 genotyped using the CAPS markers described previously. For fine mapping, F2 segregation between the mutant and Morex was genotyped using two flanking markers, HM7H228700 and HM7H254000. Recombinants identified using these two markers were genotyped using other CAPS markers developed within the HM7H228700-HM7H254000 genomic region. To accurately identify the sof1 locus, key F2 recombinants were further genotyped using an InDel marker (HM7H238100-Indel). Their progeny, F3 recombinants (20 individuals), were also genotyped using the InDel marker to confirm the lodicule size phenotype.

[0067] (6) Identification of mutation sites in the genomic region containing the 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 wild-type OUH602 data sequence 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, A. M. et al., (2014) Bioinformatics, 1; 30(15): 2114-2120), 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), Germany) using Bowtie2 version 2.3.5.1 (Langmead B. & Salzberg S. L., (2012) Nat Methods, 4; 9(4): 357-359). 27;11(10)). 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). Because 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 variants at each position was calculated by 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 variants identified in the sof1 candidate region were intersected with the gene annotation of 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) Genomic Sequencing of the sof1 Locus. Genomic DNA was extracted from leaves of 2-week-old seedlings. 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 20-kb genomic region of the sof1 locus was divided into approximately 5-kb 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 50-μl amplification reaction consisted of 100 ng of genomic DNA template, 1x 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 for 10 seconds, 55 or 60°C (primer-dependent) for 15 seconds, and 68°C for 4–5 minutes. PCR products were purified using a QIAquick PCR Purification Kit (QIAGEN) and cycle-sequenced using Big Dye Terminator v3.1 technology (Applied Biosystems). Each sequencing reaction consisted of an initial denaturation at 96°C for 1 minute, followed by 25 cycles of 96°C for 10 seconds, 50°C for 5 seconds, and 60°C for 4 minutes. Reaction products were purified using an Agencourt CleanSEQ system (Beckman) and analyzed on an ABI Prism 3130 or 3730xL genetic analyzer (Applied Biosystems). Genomic sequences were aligned using Seqman version 7.1.0 (DNASTAR).

[0069] (8) Genomic Sequencing of the sof1 Coding Region Total RNA was extracted from developing spikes at the awn primordium 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 DNase-treated total RNA from each sample, and first-strand cDNA was synthesized using an oligo-dT-primed SuperScript III kit (Invitrogen). The sof1 coding sequence was amplified and sequenced as five amplicons using designed primers. Each 50 μl 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 for 10 seconds, 55 or 60°C (primer-dependent) for 5 seconds, and 68°C for 1 to 4 minutes. Purification, sequencing, and alignment of reaction products were as described above.

[0070] (9) Genome editing by CRISPR / Cas9-mediated method. The CRISPR / Cas9 system was used for targeted mutagenesis of the Sof1 gene in barley. 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). A complementary oligo DNA pair for each gRNA sequence was inserted into the BbsI cleavage site 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 scaffold sequence of pHvU3-15 / 21 were excised with PacI and AscI. At the same time, the target binary vector pZH_gYSA_PubiMMCas9, which contained expression cassettes for Cas9 and the hygromycin phosphotransferase gene, was digested with the same restriction enzymes to introduce the gRNA expression cassette (Mikami, M. et al., (2015) Plant Molecular Biology, 88:561-572). The four constructed vectors, designated pZH-HvU3-15:gRNA1, pZH-HvU3-15:gRNA2, pZH-HvU3-15:gRNA4, and pZH-HvU3-21:gRNA2, were transformed into Agrobacterium tumefaciens AGL1 strain. They were used for Agrobacterium-mediated transformation of barley according to the method described in the literature (Hisano, H. & Sato, K., (2016) Scientific Reports, 6:37505).To confirm the mutation of the target sequence of the Sof1 gene in the regenerated plants, Sanger sequencing of the PCR amplicons was performed using specific primer pairs: 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), 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 were as described in the literature (Hisano, H. et al., (2022) Plant Biotechnology Journal, 20:37-46).

[0071] (10) Phylogenetic Analysis. Protein amino acid sequences 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 1,000 bootstrap replicates. Gene IDs or symbols for homologs in Arabidopsis and other species shown in the phylogenetic tree were obtained from the Ensembl Plant database (http: / / plants.ensembl.org / Multi / Tools / Blast / ), and corresponding protein IDs were based on NCBI (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi) BLASTp searches.

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

[0073] Genomic DNA digestion, quantification, reverse transcription, and cDNA synthesis were performed as described above (see the sof1 cDNA sequence section). First-strand cDNA derived from 10 ng of total RNA was used as a template for quantitative real-time PCR (qPCR). qPCR was performed using a CFX96 Real-Time PCR Detection System (Bio-Rad) and a THUNDERBIRD SYBR qPCR Mix Kit (Toyobo) according to the protocol. qRT-PCR data for each target gene represent the average expression level of triplicate PCR reactions (three replicates per replicate) for each biological sample. The barley actin gene (accession number DN182500) was used as an endogenous control to normalize the expression level of each sample according to the method described in the literature (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 an air-conditioned greenhouse. Young panicle tissue was collected from wild-type OUH602 and mutant 44205 at four stages of panicle development: vegetative growth stage (VEG), double retraction stage (DR), awn primordium stage (AP), and green anther stage (GA). Six biological replicate samples were collected at each of the four stages. Each replicate included bulk samples: 12 shoots from six plants at the VEG stage, 10 shoots from five plants at the DR stage, five young panicles from five plants at the AP stage, and three young panicles from three 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 samples harvested under screened conditions and in an air-conditioned greenhouse 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 an 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 a two-lane (paired-end 150 bp) Illumina HiSeq X sequencing platform (Illumina).

[0075] After sequencing, the quality of sequences from all samples was assessed 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, A. M. 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 annotations (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 transcript abundance per million reads (TPM) for each locus. Differential gene expression (DEG) analysis (awn primordium stage samples from the air-conditioned greenhouse and screened greenhouse) was performed in the R environment using the Bioconductor package edgeR (Robinson, M. D. et al., (2010) Bioinformatics, 26(1):139-140). Expression levels were normalized by the trimmed mean M (TMM) method, P values ​​were calculated using an exact test based on the negative binomial distribution, and variations in gene expression were estimated by log2-fold change in edgeR.P values ​​were adjusted by 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. Annotations of all DEGs were performed using BLASTp search results using the Reciprocal Best Hits (RBH) method against the Arabidopsis (TAIR10_-40; http: / / www.arabidopsis.org / ) and rice (IRGSP-1.0_predicted-protein_2020-09-09; https: / / rapdb.dna.affrc.go.jp / ) protein databases ( 10 －８ The E-value cutoff was based on the following:

[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 replicates and transformed into log2(mean TPM + 1) values. Hierarchical clustering was performed on the heatmap analysis based on their expression patterns.

[0077] (13) Detection of splicing events. BAM files containing mapped reads from RNA-seq data at the double-bulge, awn primordium, and green anther stages were merged for each genotype: wild-type OUH602 and mutant 44205, using SAMtools version 1.9. Merged reads for wild-type OUH602 and mutant 44205 were recounted, and new gene model annotation GTF files for OUH602 and mutant 44205 were generated using StringTie version 2.1.4 (Pertea, M. et al., (2015) Nat. Biotechnol. 33(3):290-295). Splice junctions were considered valid only if they were supported by more than 10 mapped reads. The results were visualized using the ggsashimi package integrated into the R environment according to 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 a two-tailed Student's t-test (P value < 0.05).

[0079] B. Results (1) Phenotypic Analysis of Mutants and Wild-Types. Mutant 44205 was compared with wild-type OUH602, demonstrating a "hyperflowering" phenotype with a wider separation between the lemma and lemma and a wider flowering angle (Fig. 1A and B). Observation of the central spikelet revealed larger lodicules and significantly more active cell division in the mutant (Fig. 1C-F). Measurement of the floret phenotype revealed that the mutant exhibited a significantly larger flowering angle than the wild type. That is, the size of the lodicules was increased, and the lemma and lemma were separated in the mutant (Fig. 1G and H, Fig. 2A and B) (P < 0.01, Two-tailed Student's t-test). The most significant difference between the mutant and wild-type was the size of their lodicules (Fig. 1H). This phenotype could be distinguished between the wild type and mutant only by the length of the lodicule (Fig. 2B-D). The elongation of lodicules was thought to result in a wider flowering angle in the mutant. Furthermore, measurement of lodicule cells revealed that the cell number was significantly lower in the mutant than in the wild type (Fig. 2E), corresponding to the marked increase in cell length in the mutant (Fig. 2F). These phenotypic results suggested that the elongated lodicules may be caused by cell proliferation in the mutant.

[0080] (2) Mapping of the sof1 Locus To investigate the genetic factors responsible for the super-flowering phenotype, we constructed an F2 segregating population of 93 individuals by crossing mutant 44205 with Morex (non-cleistogamy). The F1 plants exhibited normal flowering angle and lodicule size, similar to their parent Morex. The F2 segregation ratio conformed to a 3:1 Mendelian model (72 normal flowering plants vs. 21 super-flowering plants; χ2 = 0.29, P value > 0.05), indicating that super-flowering is controlled by a single recessive gene. This gene was named "sof1 (super open flowering 1)."

[0081] To map the sof1 gene, RAD-seq analysis was used on 40 F2 plants. The gene was initially located on chromosome 7H between C7_77055_96891401 and C7_67011_165769003. Next, coarse mapping using 93 F2 plants confirmed that the sof1 locus was located on chromosome 7H, in a 3.3-cM interval, between Fluidigm markers FB0278 and FB0088. Further genotyping with additional CAPS markers and phenotypic analysis of these 93 plants localized 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, we screened for recombination events using a large F2 population containing 1,759 sof1 mutant x Morex individuals. We identified 99 recombinant plants using markers HM7H228700 and HM7H254000 (Figure 3B). We measured lodicule length for 95 recombinant plants, excluding three plants that died before flowering and one plant that produced smaller floral organs than the other plants due to a dwarf phenotype. The distribution of lodicule lengths conformed to a 3:1 Mendelian segregation (χ2 = 0.07, P value > 0.05). Our results were consistent with previous data, confirming that hyperflowering is controlled by a single recessive sof1 gene and that sof1 is located between markers HM7H234200 and HM7H246100. Further genotyping and evaluation of the phenotypes of 13 significant 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, consisting of 43 annotated genes and 5 unannotated pseudogenes between markers Horvu_MOREX_7H01G234200 and Horvu_MOREX_7H01G246100, as described in the literature (Jayakodi, M. et al., (2020) Nature, 588:284-289).

[0083] (3) Genetic Contributors to the Hyper-Flowering Phenotype To investigate polymorphisms in the 3.1 Mb interval between wild-type OUH602 and mutant 44205, we performed whole-genome shotgun sequencing of the mutant and compared it with the wild-type genome sequence. Within this target region, only three homozygous polymorphisms were detected between the two genotypes: two 1-bp insertions in intergenic regions and a 22-bp deletion in an intragenic region. The 22-bp deletion was located between positions 18080 and 18101 of the wild-type Sof1 gene (based on the transcription start site of position 1) and resulted in a translational frameshift (p.Asp2646fs) in the mutant (Figure 4A and B). Horvu_OUH_7H01G211300 corresponds to Horvu_MOREX_7H01G238100, a high-confidence gene in the Morex genome, and was annotated as a chromatin remodeling protein-like (Jayakodi, M. et al., (2020) Nature, 588:284-289). To validate the candidate gene, an indel marker (HM7H238100-Indel) developed for the 22-bp deletion was used to genotype 13 significant F2 recombinants, followed by genotyping and phenotyping of the F3 progeny of three recombinants. The results showed that the indel marker did not recombine with sof1.

[0084] (4) Targeted Mutagenesis of Sof1 To confirm that the genes corresponding to Sof1 are Horvu_OUH_7H01G211300 in OUH602 and Horvu_MOREX_7H01G238100 in MOREX, we used CRISPR / Cas9-mediated mutagenesis to generate additional mutant lines in the barley cultivar Golden Promise. The guide RNA target site was the Horvu_GOLDEN_7H01G199000 gene (near the 22-bp deletion in the mutant), an ortholog of Horvu_MOREX_7H01G238100 in the Golden Promise genome (Figure 5A). After genotyping and sequencing of the transgenic plants, an independent mutation (sof1-2) with a 5-bp deletion was identified. This introduced a stop codon leading to premature termination of translation and could be considered a loss-of-function allele (Figure 5A). Golden Promise retains a cleistogamy allele (cly1.b) at the cly1 locus, which results in small lodicules. However, the genome-edited lines derived from this cultivar retain this cleistogamy allele, potentially preventing the effect of the sof1 mutation. Therefore, to eliminate any potential negative effects of cly1.b on lodicule development, the sof1-2 TO line was crossed with non-cleistogamy barley strains Sv73528 and Adorra to generate an F3 population with the genotype sof1-2sof1-2Cly1_, and the lodicule phenotype was determined (Figure 5B). As a result, the lodicule length of the sof1-2 mutant (T2) was significantly longer than that of Sv73528 and Adorra (Fig. 6).

[0085] Compared with Sv73528 and Adorra, F3 plants exhibited florets with significantly longer lodicules and a larger flowering angle (Figure 7), but the depth and width of the lodicules were not significantly different. These results indicate that the elongated lodicules of the sof1-2 mutant are caused by the disruption of Horvu_GOLDEN_7H01G199000 and that Horvu_MOREX_7H01G238100 is considered to be the functional gene for Sof1. Based on these findings, we conclude that the 22-bp deletion of Horvu_MOREX_7H01G238100 is responsible for the hyper-flowering of the sof1 mutant.

[0086] (5) Structural Characteristics of the Sof1 Gene. Sof1 is the wild-type allele of sof1. The Sof1 gene is 20,720 bp from the start codon to the stop codon (SEQ ID NO: 3) and has a full-length CDS of 10,326 bp (SEQ ID NO: 1) (Figure 4A). It encodes a predicted 3,441-amino acid protein (SOF1) with two Snf2 family domains (Figure 4B). One is an ATPase domain consisting of SNF2_N (the N-terminal domain of the SNF2 family) and Helicase_C (the helicase-conserved C-terminal domain), and the other is an Snf2 ATP coupling (SnAC) domain (Figure 4B). The ATPase and SnAC domains are located at the N-terminus of SOF1, but no conserved domains are found in the C-terminal region. Compared to the wild-type, the sof1 mutant contains a 22-bp deletion (Figure 4A), resulting in a frameshift mutation (D2659Efs) that introduces a stop codon at the 2677th amino acid in the C-terminal region, resulting in a truncated protein of 2676 amino acids lacking the last 765 amino acids (approximately 22% of the complete SOF1 protein) (Figure 4B). The truncated protein was considered to have lost its biological function. SNF2 is an ATP-dependent chromatin remodeling factor that regulates many aspects of DNA events, including transcription, replication, homologous recombination, and DNA repair. To investigate the function of SOF1, we performed a phylogenetic analysis of SOF1 homologs. Based on a BLASTp search of plant species, SOF1 homologs were found in both monocots and dicots, and the top homologs with high identity to SOF1 from each taxon were further selected for phylogenetic analysis. The other three Arabidopsis SWI / SNF subfamily members were included to infer the class to which the homologous proteins belong. The phylogenetic tree showed that all SOF1 homologs clustered within the SWI / SNF subfamily, suggesting that SOF1 belongs to this SWI / SNF subfamily (SWI / SNF ATPase) and that these sequences are orthologous (Fig. 8). SWI / SNF ATPases function by activating chromatin and turning on genes.In this SWI / SNF cluster, SOF1 and the Arabidopsis SPLAYED (SYD) protein were closely related to each other (Figure 8). Arabidopsis SYD acts via multiple MADS-box genes on several downstream bHLH genes, suppressing petal enlargement. Therefore, SYD mutations result in enlarged petals. The grass homolog of petals is the lodicule. Therefore, the gene regulatory system emanating from SYD can be used as a model for the enlarged lodicule (hyperflowering) in barley sof1 mutants. Meanwhile, the homolog HORVU_MOREX_6H01G041600.1 has been annotated as an ATP-dependent helicase family protein in the Morex genome. It showed a close relationship to Arabidopsis BRAHMA (BRM) and contained a potential bromodomain at the C-terminus according to the NCBI Conserved Domain Database (Marchler-Baur, A. et al., (2017) Nucleic Acids Res., 4;45(D1):D200-D203). HORVU_MOREX_6H01G041600.1 was suggested to be a barley SOF1 paralog. On the other hand, OsSYD (XP_015642458.1) is one of the SYD orthologs based on phylogenetic analysis and has been reported to be the SYD ortholog 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 Arabidopsis SYD. SOF1 (and SYD) are conserved in both monocotyledonous and dicotyledonous plants, suggesting that these SOF1 orthologs are derived from a common ancestor and are evolutionarily conserved (Fig. 8). Thus, Sof1 encodes a chromatin-remodeling ATPase protein whose function may be similar to that of Arabidopsis SYD and OsSYD.

[0087] (6) Transcript Abundance Profiling of Sof1 The transcript abundance of Sof1 was examined in the whole panicle, individual reproductive organs across panicle developmental stages, and various vegetative organs (Figure 9). qRT-PCR profiling revealed that this gene was widely transcribed throughout panicle development in both the wild type and mutant. In the wild type, transcription of this gene was detectable in six floral organs, including leaf sheaths, leaf blades, nodes, and internodes, at the green anther stage, but was also low in young panicles at the same awn primordium stage. Transcript abundance in the mutant was significantly lower than that in the wild type in young panicles and reproductive organs (Figure 9). Notably, the transcript level of this gene was significantly reduced in the mutant from the awn primordium stage to flowering, and was also significantly reduced in lodicules (P < 0.01, Figure 9). Transcript abundance analysis suggested that this gene may play a role during vegetative and reproductive development.

[0088] (7) Putative Transcriptional Regulation by Sof1. To characterize the effects of the 22-bp deletion of Sof1 on the transcriptome, we performed RNA-seq analysis on wild-type OUH602 and mutant 44205. Analysis of immature panicles (awn primordium stage) from plants grown in an air-conditioned greenhouse (22°C under natural light) and a screened greenhouse revealed a total of 2,412 and 1,371 differentially expressed genes (DEGs) (FDR < 0.05 and |log2fold change | > 0.5) between the genotypes under the two growth conditions, respectively. Furthermore, the 674 common DEGs consisted of 391 (58%) upregulated genes and 283 (42%) downregulated genes in the mutant compared to the wild type. The reduction in Sof1 expression in the mutants was also confirmed by RNA-seq analysis, which was consistent with the results of the Sof1 expression profile by qRT-PCR.

[0089] Among the significantly downregulated genes, six floral homeotic genes were detected: B-class genes (AP3-like, Horvu_MOREX_7H01G536500 and PI-like, Horvu_MOREX_1H01G402000), C-class genes (AG-like, Horvu_MOREX_3H01G171300), E-class genes (AGL6-like, Horvu_MOREX_6H01G171300), and PI-like genes (Horvu_MOREX_1H01G402000). These genes were homologous to rice OsMADS16, OsMADS4, OsMADS3, OsMADS6, and OsMADS6, respectively. In particular, 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 genes (Cui, B. et al., (2010) Plant J., 61(5):767-781) have been reported to affect lodicule development through rice mutants.

[0090] (8) Estimation of pollination efficiency due to differences in flowering habit (15) Estimation of pollination efficiency To estimate pollination efficiency, cytoplasmic male sterile lines of two flowering varieties (Adorra, Sv73528) and one cleistogamous variety (Kawasai Goku) were used. Potted plants were physically opened at the flowering stage, the awns were fixed with tape, and then artificially pollinated and allowed to ripen. Pollination efficiency was estimated based on the number of grains set. As a result, a significant pollination efficiency (increase in grain set) of more than five times was observed in the flowering varieties Adorra and Sv735108, and the effect was also clear in the cleistogamous variety Kawasai Goku (Table 1).

[0091]

[0092] Furthermore, no grains were observed in the panicles of the non-pollinating male sterile lines, confirming that fertilization was not caused by pollen from other individuals other than the pollination treatment. A significant increase in the number of grains set due to the improved pollination rate was observed in the flowering type mutants of pollinating individuals.

[0093] (8) Identification and Analysis of Wheat Homologous Genes Wheat homologous gene sequences were obtained from the genome information of the wheat cultivar Chinese Spring. The cDNA nucleotide sequences of the identified wheat Sof1 genes are shown in SEQ ID NOs: 4, 7, and 10, the genomic DNA nucleotide sequences are shown in SEQ ID NOs: 6, 9, and 12, and the amino acid sequences of the proteins encoded by these DNAs are shown in SEQ ID NOs: 5, 8, and 11. Similar to barley Sof1, it was found that wheat Sof1 genes are strongly expressed in various organs, such as roots, leaves, and panicles ( Figure 10 ). Furthermore, multiple wheat Sof1 gene mutants were successfully selected using the tiling method in the wheat cultivars Kronos and Cadenza.

[0094] As described above, according to the present invention, it is possible to produce wheat with improved flowering ability. The present invention can greatly improve the efficiency of developing new wheat varieties, thereby contributing to improving grain productivity in the agricultural field.

Claims

1. A method for producing wheat with improved flowering ability, comprising artificially suppressing the function of the following endogenous gene (a) or (b) in wheat: (a) an endogenous gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, 5, 8 or 11; or (b) an endogenous gene encoding a protein consisting of an amino acid sequence having 80% or more homology to the amino acid sequence set forth in SEQ ID NO: 2, 5, 8 or 11.

2. Wheat with improved flowering ability, in which the function of the following endogenous gene (a) or (b) has been artificially suppressed: (a) an endogenous gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, 5, 8, or 11; or (b) an endogenous gene encoding a protein consisting of an amino acid sequence having 80% or more homology to the amino acid sequence set forth in SEQ ID NO: 2, 5, 8, or 11.

3. A method for evaluating the flowering habit of wheat, comprising analyzing the nucleotide sequence of an endogenous wheat gene (a) or (b) below, or its expression control region: (a) an endogenous wheat gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, 5, 8, or 11; or (b) an endogenous wheat gene encoding a protein consisting of an amino acid sequence having 80% or more homology to the amino acid sequence set forth in SEQ ID NO: 2, 5, 8, or 11.

4. A method for evaluating the flowering ability of wheat, comprising analyzing the expression of the following endogenous gene (a) or (b) in wheat: (a) an endogenous gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, 5, 8 or 11; or (b) an endogenous gene encoding a protein consisting of an amino acid sequence having 80% or more homology to the amino acid sequence set forth in SEQ ID NO: 2, 5, 8 or 11.

5. A method for producing wheat with improved flowering ability, comprising crossing the wheat of claim 2 with any other wheat.