Method for producing shatter-resistant rice, shatter-resistant rice, and method for evaluating rice shattering

Abstract

To provide a method for producing non-shattering rice that reduces the shattering tendency of rice and can yield the strains of rice that prevent the seeds (hulls) from detaching from the ears or have moderate levels of shattering tendency.SOLUTION: A method for producing non-shattering rice includes the step of artificially impairing the functions of a first endogenous gene of rice, where the first endogenous gene is a gene encoding a specific amino acid sequence or an ortholog thereof.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for producing shatter-resistant rice, shatter-resistant rice, and a method for evaluating rice shattering habit. [Background technology]

[0002] When rice has high shedding ability, i.e., the grains are easily detached from the ears, problems such as reduced yield due to shedding during harvesting, rice that falls into the paddy field causing leakage of unripened rice, and contamination of different varieties occur. On the other hand, when rice has low shedding ability, i.e., the grains are difficult to detach from the ears, the burden of threshing increases and there is a risk of affecting work efficiency. For this reason, it is desirable for rice to have shedding ability suited to each cultivation and harvesting method. For example, when using manual threshing, varieties with a slightly high shedding ability are preferred, when using a method in which rice bundles harvested with a binder or the like are hung to dry, and when using a harvesting method with a combine harvester, varieties with shedding ability intermediate between the above two methods tend to be preferred.

[0003] Conventionally, as a technique for obtaining rice with a desired shedding habit, for example, a method is known in which rice that is easy to shedding is crossed with a rice to be improved, and a method for evaluating shedding habit by squeezing the harvested ears is used to select a line with an ideal degree of shedding habit from the progeny. (Kazuo Okubo et al., "Practicality of individual testing for evaluating rice shedding habit by squeezing ears," Journal of the Crop Science Society of Japan, 2016, 85(2), pp.188-192 (Non-Patent Document 1)). However, the method of crossbreeding rice has problems such as the need for numerous crossbreeding combinations to obtain rice with ideal shedding habit, the need for a large field area to select from among them, and the need for a great deal of time and effort. In addition, the method of evaluating shedding habit has problems such as the difficulty of evaluating a small number of individuals and the difficulty of evaluating the degree of shedding habit in multiple stages (for example, shedding habit is slightly high, medium, slightly low, etc.).

[0004] In addition, it is known that rice grain shedding occurs mainly due to the formation and collapse of the abscission layer at the base of the glume. Most Japanese rice varieties (Japonica rice, Oryza sativa) that are widespread in Japan have low grain shedding tendency, i.e., are difficult to shedding, due to a lack of function of the abscission layer formation-promoting gene qSH1. However, it is known that many Indica rice varieties (Indica rice, Oryza sativa) have high or moderate grain shedding tendency (Konishi et al., Science, 2006, 312, p. 1392-1396 (Non-Patent Document 2)).

[0005] For this reason, it has been reported that, for example, by crossing a Japanese rice variety having a defective qSH1 gene with the rice to be improved, and using a DNA marker for the qSH1 gene, rice with low grain shedding tendency at an early stage of growth can be selected (JP Patent Publication No. 2003-52381 (Patent Document 1); Okubo Kazuo et al., "Development of near-isogenic lines for evaluating grain shedding tendency in Japanese rice and genetic analysis of the degree of grain shedding," Journal of the Crop Science Society of Japan, 2017, 86(4), pp. 358-366 (Non-Patent Document 3)). It has also been reported that rice with low grain shedding can be created by targeting the causative genes SHAT1 (Zhou et al., The Plant Cell 24, 2012, p. 1034-1048 (Non-Patent Document 4)), SSH1 (Jiang et al., The Plant Cell, 31, 2019, p. 17-36 (Non-Patent Document 5)), and OsSh1 (Feng Li et al., Scientific Reports, 2020, 10:14936 (Non-Patent Document 6)), which were found in mutants of Indica rice varieties with low grain shedding, and suppressing their expression using RNA interference (RNAi) or causing functional deletion by genome editing. However, using the DNA marker selection methods, expression suppression methods, and genome editing methods that use the qSH1 gene, SHAT1 gene, SSH1 gene, and OsSh1 gene, it was difficult to fine-tune the shedding tendency, for example, to improve rice with high shedding tendency (easy shedding) to rice with moderate shedding tendency, or to improve rice with moderate shedding tendency to rice with slightly lower shedding tendency (harder to shedding).

[0006] In addition, "Oonari," an Indica rice variety, is a variety with moderate shedding tendency selected from mutant individuals induced by gamma irradiation of the highly shedding variety "Takanari." The present inventors have reported that the shedding tendency of "Oonari" is related to the Sh13 locus on the second chromosome (Feng Li et al., Mol Breeding, 2019, 39(3):36 (Non-Patent Document 7)). However, such genes alone are not yet sufficient to identify genes involved in the regulation of rice shedding tendency, especially fine regulation. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] JP 2003-52381 A [Non-patent literature]

[0008] [Non-Patent Document 1] Kazuo Okubo et al., "Practicality of individual testing for evaluating rice shedding by squeezing the ears," Journal of the Crop Science Society of Japan, 2016, 85(2), pp.188-192 [Non-Patent Document 2] Konishi et al., Science, 2006, 312, pp.1392-1396 [Non-Patent Document 3] Kazuo Okubo et al., "Development of near-isogenic lines for evaluation of grain shattering in Japonica rice and genetic analysis of the degree of grain shattering," Journal of the Crop Science Society of Japan, 2017, 86(4), pp.358-366 [Non-Patent Document 4] Zhou et al., The Plant Cell 24, 2012, p.1034-1048 [Non-Patent Document 5] Jiang et al., The Plant Cell, 31, 2019, p.17-36 [Non-Patent Document 6] Feng Li et al., Scientific Reports, 2020, 10:14936 [Non-Patent Document 7] Feng Li et al., Mol Breeding, 2019, 39(3):36 Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention has been made in consideration of the problems associated with the above-mentioned conventional technologies, and aims to provide a novel method for producing rice with low shedding tendency, which reduces the shedding tendency of rice and can produce rice in which rice seeds (husks) are difficult to detach from the rice ear or rice with moderate shedding tendency, a novel rice with low shedding tendency, and a method for evaluating rice shedding tendency that can evaluate the shedding tendency of rice. [Means for solving the problem]

[0010] The present inventors have conducted intensive research to achieve the above-mentioned object, and first, from mutants obtained by irradiating the conventional variety "Oonari" with moderate grain shedding tendency with gamma rays, a mutant with a grain shedding tendency lower than "Oonari" but higher than the conventional variety "Nipponbare" with low grain shedding tendency was selected and developed into a line called "Oonari Difficult to Shedding." Next, in order to search for novel factors involved in rice grain shedding tendency, genome analysis was performed between "Oonari" and "Oonari Difficult to Shedding," and only a single-base deletion in the gene Os10g0446100 and an associated frameshift mutation were extracted as a mutation accompanied by a loss-of-function amino acid mutation in "Oonari Difficult to Shedding" (loss-of-function mutation). Furthermore, when genome editing was used to introduce frameshift mutations accompanied by functional loss-of-amino acid mutations into the Os10g0446100 gene of “Oonari” and the conventional variety “Hokuriku 193,” which has moderate grain shedding tendency, it was confirmed that the shedding tendency was reduced and the variety became resistant to shedding.

[0011] Based on these findings, the present inventors have found that the gene Os10g0446100 is a factor involved in rice shedding, and have designated this gene as the "Sh14 gene". Furthermore, they have found that when the function of the Sh14 gene is deleted in rice, the shedding habit is significantly reduced compared to before the deletion of the function. However, the reduction in the shedding habit is not as drastic as when the functions of the SHAT1 gene, SSH1 gene, OsSh1 gene, etc., which have been known in the past, are deleted. Therefore, it has been found that by deleting the function of the Sh14 gene, it is possible to finely adjust the shedding habit of rice, and for example, it is possible to obtain rice with a medium shedding habit. In addition, they have found that it is possible to evaluate the shedding habit of rice using the deletion of the function of the Sh14 gene as an indicator, and have completed the present invention.

[0012] Therefore, the present invention provides the following aspects. [1] The method includes a step of artificially deleting the function of a first endogenous gene of rice, A method for producing shatter-resistant rice, wherein the first endogenous gene is a gene encoding the amino acid sequence shown in SEQ ID NO:1 or an orthologue thereof. [2] A method for producing shatter-resistant rice described in [1], comprising a step of artificially introducing a loss-of-function mutation into rice so that the first endogenous gene has the loss-of-function mutation in homozygous form. [3] the function of the first endogenous gene is deleted, Rice, wherein the first endogenous gene is a gene encoding the amino acid sequence shown in SEQ ID NO:1 or an orthologue thereof. [4] The rice according to [3], wherein the first endogenous gene has a loss-of-function mutation in a homozygous form. [5] The method includes an evaluation step of evaluating the rice grain shattering habit using the absence of a function of the first endogenous gene as an indicator, A method for evaluating rice grain shattering habit, wherein the first endogenous gene is a gene encoding the amino acid sequence shown in SEQ ID NO:1 or an orthologue thereof. [6] The method for evaluating rice grain shedding habit described in [5], wherein the evaluation step is a step of evaluating the shedding habit of rice using as an indicator whether the first endogenous gene has a homozygous functional loss-type mutated gene. Effect of the Invention

[0013] According to the present invention, it is possible to provide a novel method for producing rice with low shedding tendency, which can reduce the shedding tendency of rice and obtain rice in which rice seeds (husks) are difficult to detach from the rice ear or rice with moderate shedding tendency, a rice with low shedding tendency, and a method for evaluating rice shedding tendency that can evaluate the shedding tendency of rice. Therefore, according to the present invention, it is also possible to provide a novel method for improving rice shedding tendency, which makes it more difficult for rice seeds to detach from the rice ear. [Brief description of the drawings]

[0014] [Figure 1] 1 is a graph showing the results (bending stress) of bending tests for the varieties "Kasalath", "Takanari", "Oonari", "Oonari Shattering Difficulty", and "Nihonbare" in Test Example 1 (2). [Diagram 2] 1 is a graph showing the results (breakage rate) of the breakage test for the varieties "Kasalath", "Takanari", "Oonari", "Oonari Shattering Difficulty", and "Nihonbare" in Test Example 1 (3). [Diagram 3] This is a graph showing the results (shattering rate) of a field grain shedding evaluation test for ``Oonari'' and ``Oonari difficult to shedding'', as well as lines with the genotype ``Sh14W'' and lines with the genotype ``Sh14m'' in Test Example 3 (4). [Figure 4] 1 is a schematic diagram of the CRISPR-Cas9 system of Test Example 4(1). (a) is a schematic diagram showing the target site of gRNA in the map of the genomic region of gene Os10g0446100, and (b) is a schematic diagram of a plasmid vector expressing the CRISPR-Cas9 system including a sequence encoding the OsCas9 protein (OsCas9) and a sequence encoding the gRNA (Guide RNA common). [Diagram 5]1 is a graph showing the results (bending stress) of bending tests for "Hokuriku No. 193" and the genome-edited lines "H-Cas9-1" and "H-Cas9-2" in Test Example 4(1). [Figure 6] This is a graph showing the results (bending stress) of bending tests for ``Oonari'' and ``Oonari difficult to shatter'', as well as genome-edited lines ``O-Cas9-1'', ``O-Cas9-2'', and ``O-Cas9-3'' in Test Example 4 (2). [Figure 7] 1 is a graph showing the results (percentage of shedding) of a field grain shedding evaluation test for "Sh13WSh14W," "Sh13mSh14W," "Sh13WSh14m," and "Sh13mSh14m" in Test Example 5(2). [Figure 8] Graph (a) showing the relative expression level of SHAT1, graph (b) showing the relative expression level of SH5, graph (c) showing the relative expression level of qSH1, and graph (d) showing the relative expression level of OsSh1 in Test Example 6. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The present invention will be described in detail below based on preferred embodiments thereof.

[0016] [Rice] In the present invention, "rice" refers to a plant belonging to the genus Oryza in the family Poaceae. Such rice is not particularly limited, and examples thereof include Oryza sativa, Oryza brachyantha, Oryza nivara, Oryza punctata, Oryza glaberrima, Oryza glumipatula, Oryza meridionalis, and Oryza barthii, each of which also includes subspecies thereof. In addition, crosses between these species may be used. Among these, Oryza sativa is preferred as the rice according to the present invention.

[0017] For example, in Oryza sativa, Indica and Japonica subspecies are known, and in Japan, for example, various varieties such as Kasalath, Takanari, Oonari, Nipponbare, Hokuriku 193, and Mochidawara are known. However, there is no particular limitation on the rice varieties of the present invention, and the varieties may be lines in the process of breeding or testing, or crosses between these varieties (including lines).

[0018] [First endogenous gene (Sh14 gene)] The present inventors have discovered that the first endogenous gene involved in rice grain shattering is a gene encoding the amino acid sequence shown in SEQ ID NO: 1. In this specification, the first endogenous gene is sometimes also referred to as "Sh14 gene".

[0019] Therefore, the first endogenous gene according to the present invention is typically "(1a) a gene encoding the amino acid sequence shown in SEQ ID NO: 1". A typical nucleotide sequence of the first endogenous gene according to the present invention is shown in SEQ ID NO: 2. The amino acid sequence described in SEQ ID NO: 1 is that of rice (Oryza sativa) with the cultivar name "Oonari" developed by the National Agriculture and Food Research Organization. The nucleotide sequence described in SEQ ID NO: 2 indicates a coding sequence (CDS) excluding an intron in the gene encoding the amino acid sequence shown in SEQ ID NO: 1 in "Oonari". The gene encoding the amino acid sequence shown in SEQ ID NO: 1 is known to be located on the 10th chromosome of rice as "Os10g0446100", and typically consists of two exons via one intron, as shown in (a) of FIG. 4 below.

[0020] In addition, since the nucleotide sequence of endogenous rice genes can change due to genetic mutations in nature, crossbreeding, etc., the first endogenous gene of the present invention also includes endogenous genes that are orthologous to the gene (1a) above.

[0021] In the present invention, "ortholog" includes genes that have the same function among different species, as well as genes that have the same function among different varieties, strains, and individuals of the same species. More specifically, in the present invention, "an endogenous gene that is orthologous to the gene (1a)" refers to "a gene that encodes an amino acid sequence that has the closest identity to the amino acid sequence shown in SEQ ID NO: 1" in a certain rice plant. The sequence of such a gene can be obtained, for example, by obtaining the nucleotide sequence of the entire genomic DNA of the target rice plant and searching using the BLAST program (Basic Local Alignment Search Tool at the National Center for Biological Information; Altschul et al., J. Mol. Biol., 215, 1990, pp. 403-410) or the like, using the amino acid sequence of SEQ ID NO: 1 as a query. The nucleotide sequence of the whole genomic DNA of the target rice can be determined and obtained by an appropriate known method (e.g., whole genome sequencing, etc.), or can be obtained from databases such as GenBank (NCBI), RAP-DB (National Agriculture and Food Research Organization), Rice Genome Annotation Project (NSF), etc. In addition, the "endogenous gene orthologous to the gene (1a)" according to the present invention can also be obtained from a database of gene orthologs such as EggNOG.

[0022] More specifically, an orthologue of the gene encoding the amino acid sequence shown in SEQ ID NO: 1 according to the present invention can be, for example, "(1b) a gene encoding an amino acid sequence in which one or more amino acids have been substituted, deleted, inserted, and / or added in the amino acid sequence shown in SEQ ID NO: 1." Here, "an amino acid sequence in which amino acids have been substituted, deleted, inserted, and / or added" refers to an amino acid sequence in which amino acid residues have been substituted, deleted, inserted, or added, or an amino acid sequence in which two or more of these have been combined. In addition, "multiple" preferably refers to an integer of, for example, 100, 80, 70, 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2. The term "one or multiple amino acids" refers preferably to 1 to 50 amino acids, 1 to 40 amino acids, 1 to 30 amino acids, 1 to 20 amino acids, or 1 to 10 amino acids, more preferably 1 to 5 amino acids, even more preferably 1 to 3 amino acids, and particularly preferably 2 amino acids or less.

[0023] Furthermore, the amino acid sequence encoded by the first endogenous gene is conserved with high homology among rice species or varieties. For example, the homology of the amino acid sequence encoded by the first endogenous gene between the known varieties "Oonari" and "Hokuriku 193" is 100%, and the homology of the amino acid sequence encoded by the first endogenous gene between "Oonari" and "Nipponbare" is 99%.

[0024] Therefore, specific examples of orthologues of the gene encoding the amino acid sequence shown in SEQ ID NO: 1 according to the present invention include "(1c) a gene encoding an amino acid sequence having 70% or more homology (preferably identity) with the amino acid sequence shown in SEQ ID NO: 1". The homology or identity of the amino acid sequence according to the present invention can be determined, for example, using the BLAST program. The homology (preferably identity) of the amino acid sequence is typically preferably 70% or more, more preferably 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more (for example, 96% or more, 97% or more, 98% or more, or 99% or more).

[0025] [Functional loss of the first endogenous gene] The present inventors have found that by targeting the first endogenous gene (Sh14 gene) of rice and deleting its function, the rice can be made less susceptible to shattering, i.e., the rice can be made less susceptible to shattering than before the function of the Sh14 gene is deleted. In the present invention, the deletion of the function of the Sh14 gene (i.e., the deletion of the function of the gene encoding the amino acid sequence shown in SEQ ID NO: 1 or its orthologue) means that a mutation accompanied by a loss-of-function type amino acid mutation is made in the Sh14 gene in a homozygous form, and / or the expression of the Sh14 gene is reduced. The present inventors have found that the Sh14 gene is involved in the control pathway of abscission layer formation. Although the specific function of the amino acid sequence (protein) encoded by the Sh14 gene has not yet been fully elucidated, the present inventors have found that the rice can be made less susceptible to shattering by deleting the function of the Sh14 gene, i.e., the function of encoding and expressing at least the correct amino acid sequence (e.g., the amino acid sequence shown in SEQ ID NO: 1). Therefore, it can be said that the "function of the Sh14 gene" in the present invention includes at least the function of improving (and maintaining) grain shattering habit (the same applies below).

[0026] (Mutation in the Sh14 gene with loss-of-function amino acid mutation) In the present invention, a "functionally impaired amino acid mutation" is not particularly limited as long as it is an amino acid mutation that impairs the function of improving grain shedding, but examples include mutations in the Sh14 gene that involve amino acid substitution, deletion, insertion and / or addition in all or most of the amino acid sequence encoded by the Sh14 gene.

[0027] Mutations in the Sh14 gene involving amino acid substitution, deletion, insertion and / or addition in all or a majority of the amino acid sequence encoded by the Sh14 gene (sometimes referred to simply as "functional loss-of-function mutations" in the present specification) include, for example, missense mutations, nonsense mutations, frameshift mutations, and combinations thereof, caused by the substitution, deletion, insertion and / or addition of one or more bases in the nucleotide sequence of the Sh14 gene, and also include the substitution and / or deletion of multiple or all bases caused by gene knockout, etc.

[0028] In a preferred loss-of-function amino acid mutation that more fully reduces the function of the Sh14 gene, the number of substituted, deleted, inserted and / or added (preferably substituted or deleted) amino acids is 10% or more of the total amino acids encoded by the Sh14 gene (the amino acid sequence set forth in SEQ ID NO: 1 or a corresponding amino acid sequence thereto (preferably the amino acid sequence set forth in SEQ ID NO: 1: total number of amino acids: 433)), more preferably 20% or more, even more preferably 30% or more, even more preferably 50% or more, and particularly preferably 80% or more. Furthermore, such loss-of-function amino acid mutations particularly preferably include substitution, deletion, insertion and / or addition in at least all or part of the WRC domain of the amino acid sequence encoded by the Sh14 gene, and more preferably include substitution or deletion of at least all or part of the WRC domain.

[0029] In the present invention, the WRC domain of the amino acid sequence encoded by the Sh14 gene refers to the region from Ala at position 193 to Asp at position 235 in the amino acid sequence shown in SEQ ID NO: 1, and the region corresponding to said region. SEQ ID NO: 3 shows a typical sequence of the WRC domain (the region from Ala at position 193 to Asp at position 235 in the amino acid sequence shown in SEQ ID NO: 1).

[0030] In the present invention, an amino acid sequence or region that "corresponds to" an amino acid sequence or region refers to an amino acid sequence or region that is aligned in the same order as a control amino acid sequence or region (e.g., the amino acid sequence set forth in SEQ ID NO: 1 or the amino acid sequence set forth in SEQ ID NO: 3) when amino acid sequences are aligned using amino acid sequence analysis software (GENETYX-MAC, Sequencher, etc.) or ClustalW, etc. (e.g., parameters: default values ​​(i.e., initial settings)).

[0031] More specifically, the substitution or deletion of all or part of the WRC domain is preferably a substitution, insertion or deletion (preferably a substitution or deletion) of 20% or more of the amino acids in the WRC domain (43 amino acids in SEQ ID NO:3), and more preferably a substitution, insertion or deletion (preferably a substitution or deletion) of 50% or more, or even 80% or more of the amino acids.

[0032] In rice, which is made up of diploid cells, one endogenous Sh14 gene (first endogenous gene) is present, and two alleles exist. When the nucleotide sequences of the two alleles are identical to each other, the genotype is called "homozygous type", and when the nucleotide sequences of the two alleles are different from each other, the genotype is called "heterozygous type". In the present invention, in order for the function of the Sh14 gene to be lost by a loss-of-function mutation in the Sh14 gene, it is necessary for the Sh14 gene to have a loss-of-function mutated gene in a homozygous type.

[0033] (Method for detecting mutations in the Sh14 gene) In the present invention, the method for detecting loss-of-function mutations in the rice Sh14 gene is not particularly limited, and examples thereof include the following methods. Note that in the present invention, "detection of mutations" of a gene means, in principle, detection of mutations in genomic DNA, but since mutations in the genomic DNA are reflected in base changes in transcription products or amino acid changes in translation products, it also includes detection of the changes in these transcription products or translation products (i.e., indirect detection at the transcription or translation level).

[0034] One embodiment of the method for detecting loss-of-function mutations in the Sh14 gene includes a method for detecting mutations by directly determining the nucleotide sequence of the gene region of the Sh14 gene and comparing it with a control. In the present invention, the "gene region of the Sh14 gene" means a certain region on genomic DNA containing the Sh14 gene, and may include, in addition to the translation region, the expression control region of the Sh14 gene (e.g., promoter region, enhancer region), the intervening region (exon), the 5'-terminal untranslated region of the Sh14 gene, etc.

[0035] In this embodiment, a nucleic acid sample is first prepared from the target rice. Examples of the nucleic acid sample include a genomic DNA sample, an mRNA sample, and a cDNA sample prepared by reverse transcription from mRNA. There are no particular limitations on the method for extracting genomic DNA or mRNA from the rice, and the method for preparing cDNA, and each method may be appropriately adopted using a known method or a method similar thereto. Next, DNA containing the gene region of the Sh14 gene is isolated, and the nucleotide sequence of the isolated nucleic acid is determined. The isolation of the nucleic acid can be performed, for example, by PCR using genomic DNA or mRNA as a template, using a pair of oligonucleotide primers designed to sandwich all or a part (preferably all) of the gene region of the Sh14 gene. The nucleotide sequence of the isolated nucleic acid can be determined by a method known to those skilled in the art, such as the Maxam-Gilbert method or the Sanger method. In addition, a DNA library may be created from the prepared genomic DNA sample or cDNA sample, and the nucleotide sequence of the entire genomic DNA may be determined using a next-generation sequencer or the like.

[0036] When the determined nucleotide sequence of the nucleic acid (preferably DNA or cDNA) is different from that of the control, it can be determined that the gene region of the Sh14 gene is mutated in the subject rice. Depending on the type of mutation in the gene region of the Sh14 gene, it is determined whether the mutation is a loss-of-function mutation. Examples of the loss-of-function mutation include missense mutation, nonsense mutation, frameshift mutation, and combinations thereof, and preferred embodiments thereof are as described above. For example, missense mutation, nonsense mutation, frameshift mutation, or combinations thereof in which the number of amino acids substituted, deleted, inserted, and / or added is 20% or more, 50% or more, or 80% or more of the total amino acids encoded by the Sh14 gene are preferred.

[0037] In the production method or method for reducing or improving grain shattering habit of the present invention, the "reference" in this case may be the nucleotide sequence of the rice Sh14 gene before the step of artificially deleting the function of the Sh14 gene is carried out. When detecting a mutation in the Sh14 gene in the rice, evaluation method, or screening method of the present invention, the "reference" may be the nucleotide sequence of the Sh14 gene (i.e., the above gene (1a) and an endogenous gene orthologous to the gene (1a)) obtained from a public database (e.g., Genbank (NCBI), RAP-DB, Rice Genome Annotation Project, etc.), and the nucleotide sequence set forth in SEQ ID NO: 2 is preferred.

[0038] In another embodiment of the method for detecting loss-of-function mutations in the Sh14 gene, for example, a DNA sample is prepared from the target rice plant in the same manner as described above, and the DNA sample is subjected to the following method: a method using restriction fragment length polymorphism (RFLP) or a PCR-RFLP method in which DNA containing all or a part (preferably all) of the gene region of the Sh14 gene is amplified using the DNA sample as a template, and the size of the DNA fragment of the amplified product by restriction enzyme is compared with a control; PCR-SSCP (single-strand conformation polymorphism) method in which the amplified products are dissociated into single-stranded DNA, separated on a non-denaturing gel, and their mobility on the gel is compared with that of a control; denaturant gradient gel electrophoresis (DGGE) in which the amplification products are separated on a gel containing a gradient of a DNA denaturant and their mobility on the gel is compared with that of a control; a MALDI-TOF / MS method in which the DNA sample is used as a template to carry out a ddNTP primer extension reaction using "an oligonucleotide primer having a base on the 3' side of all or a part (preferably all) of the bases in the gene region of the Sh14 gene and a nucleotide sequence complementary to the nucleotide sequence on the 3' side of the base" and the genotype determined by mass measurement is compared with a control; a pyrosequencing method in which the amplification product is dissociated into single strands, only one strand is separated, and an extension reaction is carried out for each base from the vicinity of all or a part (preferably all) of the bases in the gene region of the Sh14 gene, and the pyrophosphate generated during the extension reaction is measured and compared with a control; The AcycloPrime method uses the amplification product as a template in the presence of fluorescently labeled nucleotides to carry out a single-base extension reaction with "an oligonucleotide primer having a nucleotide sequence complementary to the nucleotide sequence on the 3' side of one base of all or part (preferably all) of the bases in the gene region of the Sh14 gene and the nucleotide sequence on the 3' side of the nucleotide" and measures the polarization degree of the fluorescence and compares it with a control; the SNuPE method determines the type of base used in the single-base extension reaction and compares it with a control. etc.

[0039] In the production method or the method for reducing or improving grain shattering habit of the present invention, the "reference" in these methods includes the result of a DNA sample extracted from rice before the step of artificially deleting the function of the Sh14 gene. In addition, in the case of detecting a mutation in the Sh14 gene in the rice, evaluation method, or screening method of the present invention, the result of a standard DNA sample synthesized based on the nucleotide sequence of the Sh14 gene obtained from the public database or the nucleotide sequence set forth in SEQ ID NO: 2 can be included. In each detection method, if the detection result is different from the control, it can be determined that the gene region of the Sh14 gene is mutated in the target rice. Depending on the type of mutation in the gene region of the Sh14 gene at this time, it is determined whether the mutation is the loss-of-function mutation. The loss-of-function mutation is as described above, including its preferred embodiments.

[0040] Furthermore, in the present invention, in order for the function of the Sh14 gene to be lost by a loss-of-function mutation in the Sh14 gene, it is necessary for the Sh14 gene to have a loss-of-function mutation in homozygous form. That is, when the target rice plant has a loss-of-function mutation in the Sh14 gene in homozygous form, it can be determined that loss of function of the Sh14 gene has been detected.

[0041] Whether or not the target rice plant has a homozygous loss-of-function mutation in the Sh14 gene can be confirmed by a conventionally known method or a method similar thereto, for example, a method of determining the genotype by the RELP (Restriction Fragment Length Polymorphism) method, the RAPD (Random Amplified Polymorphic DNA) method, the CAPS (Cleaved Amplified Polymorphic Sequences) method, etc., using a region containing the loss-of-function mutation confirmed by each of the above detection methods as a marker.

[0042] (Decreased expression of Sh14 gene and its detection method) The loss of function of the Sh14 gene according to the present invention may be confirmed by a decrease in expression of the Sh14 gene. For example, a method for detecting a decrease in expression of the Sh14 gene includes detecting the expression level of mRNA encoded by the Sh14 gene at the transcription level and confirming that the expression level is lower than that of a control.

[0043] In this case, for example, in the production method or method for reducing or improving grain shattering habit of the present invention, the control can be the expression level of mRNA in rice before the step of artificially deleting the function of the Sh14 gene. In the case of the rice, evaluation method, or screening method of the present invention, the control can be the expression level of mRNA in rice known to be free of the function of the Sh14 gene, for example, the expression level of mRNA in "Oonari."

[0044] As a method for detecting the expression level of mRNA encoded by the Sh14 gene at the transcription level, a known method can be appropriately selected and used. For example, first, mRNA is extracted from the target rice (e.g., young panicle), and a cDNA sample is prepared by reverse transcription from the mRNA. There are no particular limitations on the method for extracting mRNA from the rice and the method for preparing the cDNA sample, and known methods or methods equivalent thereto can be appropriately adopted. Next, the expression at the transcription level can be detected by detecting the amplified product by an amplification reaction using an oligonucleotide primer capable of amplifying a part or all (preferably all) of the Sh14 gene using the cDNA sample as a template. Examples of such methods include RT-PCR. Those skilled in the art can design suitable oligonucleotide primers in a conventional manner based on the nucleotide sequence of the Sh14 gene available from the public database (preferably the nucleotide sequence set forth in SEQ ID NO: 2).

[0045] If the amount of the amplification product is less than that of the control or if the amplification product is not confirmed, it can be determined that the expression level of mRNA encoded by the Sh14 gene in the rice is low or not expressed, i.e., the function of the Sh14 gene is missing.

[0046] It is also known in the art that one of the factors in the reduced expression of a gene is hypermethylation of the promoter. Therefore, as another embodiment of the method for detecting the reduced expression of the Sh14 gene, for example, a method for detecting the expression of the Sh14 gene at the translational level using the methylation of the promoter of the Sh14 gene as an indicator can be adopted. For the detection of the methylation of the promoter, for example, a method for directly detecting the change in the nucleotide sequence after bisulfite treatment, which has the activity of converting methylated cytosine to uracil, by nucleotide sequencing, or a method for indirectly detecting the change by using a restriction endonuclease that can recognize (cut) the nucleotide sequence before bisulfite treatment but cannot recognize (cut) the nucleotide sequence after bisulfite treatment can be used. Depending on the degree of the methylation, it can be determined that the expression at the translational level is not detected, that is, that the function of the Sh14 gene is lost.

[0047] In addition, as a method for detecting decreased expression of the Sh14 gene, a method can be used in which expression at the translation level of the Sh14 gene is detected using, for example, substitution, deletion, insertion, and / or addition of one or more bases in the nucleotide sequence of the promoter region of the Sh14 gene as an indicator.

[0048] In the present invention, the method for detecting "functional loss of the first endogenous gene (Sh14 gene)" in rice may be any one of the above detection methods, or a combination of two or more of them. Among these, from the viewpoints of accuracy and simplicity, a method for detecting functional loss of the Sh14 gene by detecting that the target rice has a homozygous mutation accompanied by a functional loss-type amino acid mutation in the Sh14 gene (functional loss-type mutation) is preferred, and a method for detecting the functional loss-type mutation by directly determining the nucleotide sequence of the Sh14 gene and comparing it with a control is more preferred.

[0049] [Loss of function or overexpression of a second endogenous gene] In the present invention, in order to further adjust the rice shedding habit obtained by the production method or the method for reducing / improving shedding habit, or as an additional indicator for evaluating rice shedding habit, the functional deficiency of the first endogenous gene may be further combined with the functional deficiency or overexpression of another endogenous gene of rice (collectively referred to in this specification as a "second endogenous gene").

[0050] Such a second endogenous gene may be, for example, one or a combination of two or more of the genes that have been reported to be involved in the control (particularly fine-tuning) of rice grain shattering, and an example of such a gene is the Sh13 gene described below.

[0051] The present inventors have demonstrated that in the "Oonari" variety, which is a variety with moderate shedding tendency, a tandem duplication mutation was induced in a 6.3 kbp DNA fragment containing the microRNA gene Osa-miR172d that suppresses shedding tendency, and that this resulted in a significant increase in the expression level of Osa-miR172d compared to the "Takanari" variety, which is a variety with easy shedding (Non-Patent Document 7). Therefore, it is presumed that the locus containing Osa-miR172d contains a candidate gene that causes "Oonari" to have moderate shedding tendency, and the present inventors have designated this as the "Sh13 gene."

[0052] <Method for producing rice that is difficult to shed, method for reducing shedding property, and method for improving shedding property> The method for producing shatter-resistant rice of the present invention is a method including a step of artificially deleting the function of the rice Sh14 gene (sometimes referred to as "the production method of the present invention" in this specification). According to the production method of the present invention, by artificially deleting the function of the rice Sh14 gene, it is possible to reduce the shattering property of the rice and obtain shatter-resistant rice with reduced shattering property. Therefore, the present invention also provides a method for reducing the shattering property of rice including a step of artificially deleting the function of the rice Sh14 gene (sometimes referred to as "method for reducing shattering property" in this specification) and a method for improving the shattering property of rice including a step of artificially deleting the function of the Sh14 gene (sometimes referred to as "method for improving shattering property" in this specification).

[0053] [Shedding property] In rice, the "panicle" which is an organ extending from the head node to the end of the panicle generally has 8 to 11 nodes on the main axis (rachis), and each node has a primary rachis. Secondary rachis branches extend from the base of the primary rachis branch, and rachis twigs extend from each node of the primary rachis branch and secondary rachis branch, and the spikelet is at the end of the secondary rachis. The spikelet forms seeds after flowering, and when it matures, it is also called "husk." In the present invention, "shattering" of rice refers to the detachment of rice seeds (i.e., "husks") from rachis twigs. It is known that rice shedding occurs mainly due to the formation and collapse of the abscission layer at the base of the glume. In the present invention, "shattering habit" of rice refers to a trait indicating the ease or difficulty of shedding, and those which are relatively easy to shedding are called "easy shedding habit" and those which are relatively difficult to shedding are called "hard to shedding habit."

[0054] In the present invention, "hard to shatter" also includes rice with moderate shattering ability. Furthermore, in the production method of the present invention, if the shattering ability is lower (harder to shatter) than before the function of the first endogenous gene is artificially deleted, it can be said to be "hard to shatter". According to the present invention, it is possible to fine-tune the shattering ability depending on the target rice, for example, to improve rice with high shattering ability (easy to shatter) into rice with moderate shattering ability that is more difficult to shatter, or to improve rice with moderate shattering ability into rice with slightly lower shattering ability (slightly more difficult to shatter).

[0055] More specifically, the shedding property can be confirmed and evaluated by at least one type of shedding property test selected from the group consisting of a bending test, a breaking test, and a field shedding property evaluation test.

[0056] That is, in the bending test, for example, first, ears in which the whole grains have turned yellow are harvested, stored at 4°C and 20% humidity, and dried for 2 weeks to obtain ear samples. Next, the stalks are fixed vertically (longitudinal direction), and the grains are pulled horizontally to measure the maximum tensile strength when the grains are detached from the stalks, and this is defined as the bending stress [gf]. Preferably, measurements are performed for 96 or more grains per test rice species, and the average value is obtained. The larger the bending stress or its average value, the lower the grain shedding property, i.e., the more it can be confirmed and evaluated as "hard to shedding", and the smaller the bending stress, the higher the grain shedding property, i.e., the more easily it can be confirmed and evaluated as "easy to shedding". In the present invention, the bending stress in rice when referring to "hard to shedding" may be moderate and is not strictly limited, but for example, the average value of the bending stress for 96 or more grains is 35gf or more, 45gf or more, or 55gf or more.

[0057] In the breaking test, for example, first, from the panicle sample prepared in the same manner as in the bending test, the unhulled rice with the rachis attached is cut out with scissors, the unhulled rice is fixed with the rachis facing upward (vertical direction), and the rear end of the tweezers is slowly pressed horizontally (laterally) at a point 3 mm from the base of the glume of the rachis, and it is observed whether the rachis breaks. Preferably, 24 grains or more and 4 or more individuals are tested per panicle for each rice variety to be tested, for a total of 96 or more unhulled rice. The rate at which the rachis breaks in one panicle (number of unhulled rice with broken rachis / number of tested unhulled rice x 100) is taken as the breaking rate (%), and the average value of 4 or more individuals (panicles) is preferably obtained. It can be confirmed and evaluated that the smaller the breaking rate (or the average value), the more difficult the grain to shatter, and the larger the breaking rate, the more easily the grain is shattered. In the present invention, the breaking rate of rice referred to as "resistant to shattering" may be moderate and is not strictly limited, but examples of the average breaking rate for 4 or more individuals (total of 96 or more grains) include 10% or less, 5% or less, and 2% or less.

[0058] The field shedding evaluation test is carried out in a field on a day when it is not raining for two days before the test. In the test, for example, ears in which all the rice grains have turned yellow are not harvested, but the tips of three ears in the order of the length of the culms in the plant are aligned and tightly gripped once in one hand, and the palm is opened, and the number of rice grains that have detached from the ears is taken as the number of shedding per plant. Note that even if the rice grains have detached, the number of rice grains that are sterile, diseased, or have stalks attached is not included in the number of shedding. Preferably, six or more plants are tested for each type of rice to be tested, and the average value is obtained. In addition, the number of rice grains within the gripping width of one hand is counted six times, and the average value is taken as the number of rice grains gripped at one time. The rate at which rice grains have detached (number of shedding grains / number of rice grains gripped at one time x 100) is taken as the shedding rate (%), and it can be confirmed and evaluated that the smaller the shedding rate, the more difficult the shedding, and the larger the shedding rate, the easier the shedding. In the present invention, the shedding rate in rice that is referred to as "resistant to shedding" may be moderate and is not strictly limited, but examples of the average shedding rate in six or more plants include 5% or less, 3% or less, and 1% or less.

[0059] [Function loss process] The production method or method for reducing or improving grain shattering characteristic of the present invention includes a step of artificially deleting the function of the rice Sh14 gene (first endogenous gene) (sometimes referred to as the "first function deleting step" in this specification). The production method or method for reducing or improving grain shattering characteristic of the present invention may further include a step of artificially deleting the function of a second endogenous gene of rice or a step of artificially overexpressing the second endogenous gene (sometimes referred to as the "second gene manipulation step" in this specification). In this case, the first function deleting step and the second gene manipulation step may be performed in either order, or simultaneously.

[0060] The rice to be subjected to the production method or the method for reducing or improving grain shedding habit of the present invention is not particularly limited, but from the viewpoint of more effectively exerting the effects of the present invention, it is preferably rice having high or high possibility of shedding habit, and more preferably rice in which the function of the Sh14 gene is not deleted. Rice having high or high possibility of shedding habit can be confirmed, for example, by the above-mentioned grain shedding habit test or the rice screening method of the present invention described below.

[0061] Examples of the method for artificially deleting the function of the Sh14 gene in the target rice plant include a method for homozygously introducing a mutation accompanied by a loss-of-function amino acid mutation into the Sh14 gene and / or a method for reducing the expression of the Sh14 gene. Examples of the mutation accompanied by a loss-of-function amino acid mutation and reduced expression are as described above, including preferred embodiments thereof. As the method for artificially deleting the function of the Sh14 gene according to the present invention, a conventionally known method or a method equivalent thereto can be appropriately adopted depending on the embodiment.

[0062] For example, methods for homozygously introducing a mutation involving a loss-of-function amino acid mutation into the Sh14 gene include artificial gene modification methods such as gene knockout method; site-specific mutagenesis methods such as gene targeting using homologous recombination, Kunkel method, and SOE-PCR method; genome editing methods using site-specific nucleases (meganucleases, zinc finger nucleases, TALEN, PPR, CRISPR-Cas, etc.); and methods of inducing mutations by gamma ray irradiation, etc., and combinations of two or more of these may be used.

[0063] For example, methods for reducing the expression of the Sh14 gene include artificial gene expression suppression methods such as knockdown method, methylation of the promoter, suppression of expression by expression of antisense RNA (van der Krol AR et al., Nature 333:866, 1988), and suppression of expression by RNA interference (RNAi) using double-stranded RNA having a sequence corresponding to the Sh14 gene, and two or more of these may be combined. Also, they may be appropriately combined with the gene modification method.

[0064] Furthermore, as a method for artificially deleting the function of the second endogenous gene or artificially overexpressing the second endogenous gene, a conventionally known method or a method similar thereto can be appropriately adopted depending on the genes.

[0065] In the manufacturing method or method for reducing or improving shedding ability of the present invention, the shedding ability needs to be reduced at least compared to the rice before the first functional deficiency step, and the reduction in shedding ability can be confirmed, for example, by a change in at least one of the above-mentioned shedding ability tests, i.e., the bending stress measured in the bending stress measurement test, the breaking rate calculated in the breaking test, and the shedding rate calculated in the field shedding ability evaluation test, in a direction indicating greater difficulty in shedding than the rice before the first functional deficiency step.

[0066] In addition, the manufacturing method or method for reducing / improving grain shedding tendency of the present invention may further include a confirmation step of confirming that the rice that has been subjected to the first function loss step has indeed become resistant to shedding, for example by carrying out at least one of the above-mentioned grain shedding tests.

[0067] <Rice that is resistant to shattering> The present invention also provides rice in which the function of the Sh14 gene is deleted, i.e., rice in which the function of a first endogenous gene is deleted, and the first endogenous gene is a gene encoding the amino acid sequence shown in SEQ ID NO:1 or an orthologue thereof (sometimes referred to herein simply as "the rice of the present invention").

[0068] Therefore, the rice of the present invention can be a shatter-resistant rice. The preferred degree of shatter resistance can be, for example, the criteria listed in the above-mentioned grain shatter resistance test, i.e., the bending stress measured in the bending stress measurement test, the breaking rate calculated in the breaking test, and the shedding rate calculated in the field grain shatter resistance evaluation test. In addition, in the present invention, whether or not the function of the rice Sh14 gene is lost can be confirmed by, for example, the above-mentioned method for detecting a mutation in the Sh14 gene or the method for detecting reduced expression of the Sh14 gene.

[0069] The rice of the present invention is obtained by the above-mentioned production method of the present invention, and the function of the Sh14 gene is artificially deleted. The rice of the present invention may further have a second endogenous gene that is deleted in function or overexpressed. In the present invention, whether or not the second endogenous gene of rice is deleted in function or overexpressed can be confirmed by an appropriate known method.

[0070] In addition, the rice of the present invention may have mutations other than the above-mentioned mutation in the Sh14 gene accompanied by a loss-of-function amino acid mutation and the mutation in the second endogenous gene accompanied by a loss-of-function amino acid mutation, within a range that does not inhibit the effects of the present invention.

[0071] <Rice evaluation and screening methods> As described above, rice in which the Sh14 gene function is deleted exhibits shattering resistance, and therefore the present invention provides a method for evaluating the shattering resistance of rice (sometimes referred to as "the evaluation method of the present invention" in this specification) that includes an evaluation step of evaluating the shattering resistance of rice using the deletion of the Sh14 gene function as an indicator. The present invention also provides a screening method for shattering-resistant rice (sometimes referred to as "the screening method of the present invention" in this specification) that includes a selection step of selecting rice using the deletion of the Sh14 gene function as an indicator.

[0072] The evaluation method and screening method of the present invention include a method for evaluating or screening rice as being resistant to shattering, as well as a method for evaluating or screening rice as being highly likely to be resistant to shattering. In addition, the evaluation method and screening method of the present invention may be, for example, a method for evaluating or screening rice at a level where a medium degree of shattering tendency can be expected.

[0073] In the evaluation and selection steps, for example, at least one of the detection methods listed above as the method for detecting a mutation in the Sh14 gene and the method for detecting reduced expression of the Sh14 gene is used to determine whether or not the Sh14 gene is functionally deficient (i.e., whether or not a mutation involving a functionally deficient amino acid mutation in the Sh14 gene is detected in a homozygous form, and / or whether or not reduced expression of the Sh14 gene is detected), and rice in which a functional deficiency of the Sh14 gene is detected is evaluated or selected as rice with shattering resistance (including rice with moderate shattering resistance) or rice with a high possibility of being shattering resistance. The determination method, including its preferred embodiments, is as described for each of the detection methods above.

[0074] In addition, the evaluation method and screening method of the present invention may further include a confirmation step of confirming that the evaluated or screened rice is indeed resistant to shedding, for example by conducting the above-mentioned grain shedding test. EXAMPLES

[0075] The present invention will be described in more detail below based on examples and comparative examples, but the present invention is not limited to the following examples.

[0076] (Test Example 1) Creation of shatter-resistant rice (Oonari shatter-resistant) and shattering test (1) The following four existing rice varieties: Kasalath: Indian rice, Oryza sativa, a variety known for its high shedding tendency (easy shedding of grains), Takanari: Indian rice, Oryza sativa, a variety known to have a lower shedding tendency than Kasalath, but a higher shedding tendency (easiness to shed grains), Oonari: A variety developed by selecting a mutant with moderate grain shattering habit from the mutants obtained by gamma irradiation of the indica rice, Oryza sativa, Takanari. Nipponbare: Japanese rice, Oryza sativa, a variety known for its low grain shedding tendency (difficulty in shedding), and, Oonari resistant to shedding: A lineage developed by selecting mutants from the Oonari variety, an Indian rice variety, Oryza sativa, obtained by irradiating the variety with gamma rays, that have a lower shedding tendency than Oonari (harder to shedding than Oonari), but a higher tendency than Nipponbare (easier to shedding than Nipponbare). was used in the following tests.

[0077] Samples of rice panicles were collected in the following manner. Four weeks after sowing, each rice variety (Kasalath, Takanari, Oonari, Nipponbare, Oonari resistant to shattering) was transplanted into paddy fields in two rows (14 cm between rows, 20 cm between plants) with one plant per plant. The spacing between each variety and line was 28 cm. Approximately five months after transplanting, when the grains in the panicle had turned yellow, a panicle with good growth was harvested from each rice plant, placed in a paper envelope, and dried in a refrigerator at 4°C and 20% humidity for two weeks to prepare the panicle sample.

[0078] (2) Bending test The bending test was carried out in accordance with the method described in Feng Li et al., Mol Breeding (2019) 39:36 (Non-Patent Document 7). That is, for the panicle sample obtained in (1) above, the stalk was fixed vertically (longitudinal direction) and the rice grain was pulled horizontally. The maximum tensile strength at which the rice grain detached from the stalk was measured using a tensile tester (AL-107-03A, Alpha Machine Co., Ltd.) equipped with a digital force gauge (FGP-0.5, Nidec-Shimpo Corporation), and the bending stress (gf) was recorded. For the evaluation of one variety / line, the bending stress of a total of 96 grains of rice grains was measured for each of four individuals (one panicle per individual (plant) and 24 grains per panicle), and the average value was recorded as the bending stress (gf) for that variety / line.

[0079] The results are shown in Figure 1. In Figure 1, the white circles indicate the bending stress (average value of 96 kernels) for each variety and line. When the varieties and lines were compared using the Wilcoxon test, the p-values ​​for all five varieties and lines (Kasalath, Takanari, Oonari, Nipponbare, and Oonari difficult to shatter) (between A and E in Figure 1) were less than 0.01, confirming that there are significant differences in bending stress between varieties and lines with different shattering tendencies.

[0080] (3) Bending test The breaking test was also carried out according to the method described in Non-Patent Document 7. That is, for the panicle sample obtained in (1) above, the unhulled rice with the rachis attached was cut out with scissors, the unhulled rice was fixed with the rachis facing upward (vertical direction), and the rear end of the tweezers was slowly pressed horizontally (lateral direction) at a point 3 mm from the base of the glume of the rachis to confirm whether the rachis broke. For the evaluation of one variety / line, one panicle per individual (plant) and 24 grains per panicle, for a total of 96 grains, was tested for four individuals (panicles), and the proportion of broken rachis per panicle (24 grains) (number of grains with broken rachis / 24×100) was taken as the breaking rate (%). The average of the breaking rates per four individuals was taken as the breaking rate (%) of the variety / line.

[0081] The breakage rate (average value per four individuals) for each variety / line is shown in Figure 2. In Figure 2, error bars indicate standard deviation. Comparisons between each variety / line were performed using Tukey's test. The p-values ​​were all less than 0.05 between "Kasalath," "Takanari," and "Oonari" (between A and C in Figure 2), and between each of these varieties and "Nipponbare" and "Oonari Shatter-resistant" (between A and C and D in Figure 2). This confirmed that there were significant differences in the breakage rate at least among the four varieties / lines, "Kasalath," "Takanari," "Oonari," and "Oonari Shatter-resistant."

[0082] From the above (2) and (3), it was confirmed that “Oonari Difficult to Shatter” is a rice variety that is difficult to shatter, with lower shedding tendency than “Oonari.”

[0083] (Test Example 2) Detection of gene mutations by genome sequencing (1) Acquisition of sequence data Thirty days after sowing, leaves of "Oonari" and "Oonari shatsuran" were collected and genomic DNA was extracted using the DNeasy Plant Maxi Kit (Qiagen). A DNA library was prepared from the obtained genomic DNA and sequence analysis was performed using the Illumina HiSeq X Ten system by the 2 × 150 bp paired-end method.

[0084] (2) Analysis of sequence data Analysis of the sequence data was carried out in accordance with the method described in Non-Patent Document 6. That is, for the sequence data obtained in (1) above, genome mapping was performed using the Burrows-Wheeler Alignment Tool (BWA) against the "Nipponbare" genome sequence, which was used as the reference genome sequence, and mutations were identified using the Genome Analysis Toolkit (GATK), Pindel, and Manta. Furthermore, for "Oonari," only homozygous mutations of "Oonari shattering resistance" were extracted and annotations were added using SnpEff.

[0085] In "Oonari resistant to shattering," there were 64 homozygous mutation sites from "Oonari" in the whole genome. Of these mutations, only one mutation accompanied by a loss-of-function amino acid mutation (loss-of-function mutation). This mutation was a frameshift mutation caused by the deletion of one base (T at the 107th position in the nucleotide sequence described in SEQ ID NO: 2) in the first exon of the gene Os10g0446100 on chromosome 10. Therefore, the amino acid sequence encoded by this has 92% of the amino acids following the amino acid corresponding to the codon containing the deleted base (F at the 36th position in the amino acid sequence described in SEQ ID NO: 1) substituted or deleted from the amino acid sequence encoded by the Oonari gene Os10g0446100.

[0086] (Test Example 3) Analysis of genotype (1) Design of CAPS marker First, based on the nucleotide sequence information of the above mutation site in gene Os10g0446100 (Sh14 gene) detected in Test Example 2(2), a sequence capable of detecting only the above mutation (loss-of-function mutation) by TfiI restriction enzyme treatment was set as a target sequence, i.e., a Cleaved Amplified Polymorphic Sequence (CAPS) marker. The nucleotide sequence of the set CAPS marker is "5'-GATTC", and the region between G and A is cleaved by TfiI restriction enzyme. Furthermore, a primer set capable of amplifying a fragment containing the set CAPS marker (CAPS_F primer (nucleotide sequence: SEQ ID NO: 4) and CAPS_R primer (nucleotide sequence: SEQ ID NO: 5)) was designed.

[0087] (2) Field shedding evaluation test The field shedding evaluation test for the analysis of genotypes was performed as follows. First, each line or variety of rice (each line of the F3 population, Oonari, and Oonari shedding-resistant) was sown, and 4 weeks later, 10 individuals per variety or line were transplanted into a paddy field in a row with regular row planting (row spacing 30 cm, plant spacing 15 cm) so that each plant had one stalk. Approximately 5 months after transplantation, when the grains in the entire panicle had turned yellow, the test was performed on a day when it had not rained for 2 days prior to the transplant. The test was performed on 6 well-growing plants out of the 10 individuals (plants), excluding the plants at both ends, by lining up the tips of 3 panicles in order of the length of the culm within each plant, firmly gripping them once in one hand, opening the palm, and counting the number of grains that had detached from the panicle as the number of shed grains per plant (however, sterility, diseased, or stalk-attached rice grains were not included in the number of detached grains). The number of rice grains within the gripping width of one hand was counted six times, and the average was taken as the number of rice grains gripped at one time. All tests were performed by the same investigator. The rate at which rice grains detached per plant was calculated from the number of shed rice grains per plant and the number of rice grains gripped at one time per plant (average of six repetitions) (number of shed rice grains / number of rice grains gripped at one time x 100), and this was taken as the shed rice rate (%) per plant. The average of these shed rice rates per six plants was taken as the shed rice rate (%) for that line or variety.

[0088] (3) Mating type analysis of F3 population First, an F2 population was created by crossing "Oonari" with "Oonari shattering-resistant", and the F2 individuals were self-seeded to create an F3 population. Genomic DNA was extracted from young leaves obtained by sowing the F3 individuals, and PCR was performed using the obtained genomic DNA as a template and the primer set designed in (1) above.

[0089] Next, the obtained amplification product (fragment containing the CAPS marker) was cleaved with TfiI restriction enzyme, and the cleaved and uncleaved amplification products were separated by agarose gel electrophoresis. Lines in which only the uncleaved amplification product was confirmed were determined to have a homozygous genetic mutation, lines in which only the cleaved amplification product was confirmed were determined to have no genetic mutation, and lines in which both amplification products were confirmed were determined to have a heterozygous genetic mutation. Hereinafter, genes without the mutation (loss-of-function mutation) detected in the above Test Example 2(2) are referred to as wild type (W), and genes with the same mutation are referred to as mutant type (m). Lines in the F3 population whose genotype of the Sh14 gene is homozygous for the wild type are referred to as "WW" or "Sh14 W " and strains that are homozygous for the mutant genotype are indicated as "mm" or "Sh14 m " is displayed.

[0090] The above (2) field shedding evaluation test was carried out for the lines with the genotype "WW" and the lines with the genotype "mm". When the obtained shedding rate was compared with the genotype, the shedding rate was significantly lower for the "mm" genotype than for the "WW" genotype. Therefore, it was confirmed that the homozygous form of the above-mentioned Sh14 gene mutation reduces shedding rate (becomes less susceptible to shedding).

[0091] (4) Grain shedding test of F3 population Among the F3 populations described in (3) above, 32 lines with the genotype "WW" and 23 lines with the genotype "mm" were subjected to the field shedding evaluation test described in (2) above. In addition, the field shedding evaluation test described in (2) above was also performed for "Oonari" and "Oonari Difficult to Shed".

[0092] Figure 3 shows the results of the comparison of the genotypes of "Oonari" and "Oonari shattering difficulty" in the F3 population. W )" and the genotype was "mm(Sh14 mIn Figure 3, the white circles in "Oonari" and "Oonari Difficult to Shatter" indicate the average value per six plants, and the average value per six plants is shown in Figure 3. W and Sh14 m The white circles in the table indicate the average value per 6 plants in each line, and the average value per 32 or 23 lines. When comparing "Oonari" and "Oonari Difficult to Shatter" using Student's test, the p value was less than 0.01, confirming that there was a significant difference in the shedding rate. W The lineage and genotype that was "Sh14 m When a comparison was performed using Welch's test to determine whether the line was "good" or "bad," the p-value was less than 0.01, confirming that there was a significant difference in the rate of grain shedding between the two lines.

[0093] (Test Example 4) Analysis by genome editing Genome editing of the Sh14 gene was performed using "Hokuriku 193" and "Oonari." "Hokuriku 193" is an Indian rice variety, Oryza sativa, known to have a moderate grain shedding tendency.

[0094] (1) Hokuriku Route 193 [CRISPR-Cas9 system expression vector] First, a plasmid vector expressing the CRISPR-Cas9 system containing a sequence encoding the OsCas9 protein (OsCas9) and a sequence encoding a guide RNA (gRNA) (Guide RNA common) was constructed as shown in (b) of FIG. 4, using the sequence shown in the nucleotide sequence of SEQ ID NO: 6 (corresponding to the 150th to 169th sequences of SEQ ID NO: 2) in the gene Os10g0446100 (Sh14 gene) of "Hokuriku 193". In (a) of FIG. 4, the target site of the gRNA (gRNA target sequence recognized by the gRNA) in the map of the genomic region of the gene Os10g0446100 is indicated by an arrow. In addition, the sequence including the nucleotide sequence of the gRNA target sequence and the PAM sequence is shown in SEQ ID NO: 7.

[0095] [Transformation into rice] Using the plasmid vector constructed in (1) above, rice callus was transformed with Agrobacterium. That is, the plasmid vector was first introduced into Agrobacterium (strain: EHA105) and co-cultured with rice seed embryos (rice: Hokuriku 193) for 5 days, and then cultured in a medium containing 50 mg / L hygromycin B and 25 mg / L meropenem (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) for 4 to 5 weeks to allow regeneration.

[0096] [Sequence analysis] Leaves from the regenerated rice seedlings were collected, and genomic DNA was extracted by the SDS method. Using these as a template, PCR was performed using a primer set (Cas_F primer (nucleotide sequence: SEQ ID NO: 8) and Cas_R primer (nucleotide sequence: SEQ ID NO: 9)) capable of amplifying the region containing the target sequence. The nucleotide sequence of the obtained amplified product was decoded by direct sequencing. As a result, two lines were selected: "H-Cas9-1", a line in which a frameshift mutation occurred due to a single base insertion in the target sequence, and "H-Cas9-2", a line in which a frameshift mutation occurred due to a single base deletion. The target sequence and the corresponding sequences of "H-Cas9-1" and "H-Cas9-2" are shown in Table 1 below.

[0097] [Table 1]

[0098] [Evaluation test of shedding habit] "Hokuriku 193" and genome-edited lines "H-Cas9-1" and "H-Cas9-2" were cultivated in deep Wagner pots (diameter 12.7 cm, height 19.8 cm) in a recombinant greenhouse, with one individual per pot and four pots for each variety and line. When the grains in the entire ear turned yellow, the well-grown ears were harvested from each individual, placed in a paper envelope, and dried in a refrigerator at 4°C and 20% humidity for two weeks to prepare ear samples. The bending stress of the obtained ear samples was measured in the same manner as in the bending test in Test Example 1 (2), for a total of 120 grains, with four ears (individuals) per variety and line and 30 grains per ear, and the average value was taken as the bending stress (gf) for that variety and line.

[0099] The results are shown in Figure 5. In Figure 5, the white circles indicate the bending stress (average value of 120 kernels) for each variety and line. Comparisons between varieties and lines were performed using Welch's multiple test, and the p-value was less than 0.01 between "Hokuriku 193" and "H-Cas9-1" and "H-Cas9-2" (between A and B in Figure 5), confirming that there was a significant difference in bending stress between "Hokuriku 193", which shares a common nucleotide sequence of the Sh14 gene with "Oonari", and "H-Cas9-1" and "H-Cas9-2", which had homozygous loss-of-function mutations introduced into the Sh14 gene by genome editing.

[0100] (2) Oonari [Transformation and sequence analysis] First, a plasmid vector expressing the CRISPR-Cas9 system was constructed in the same manner as described above in (1), using the nucleotide sequence shown in SEQ ID NO: 6 (corresponding to positions 150 to 169 of SEQ ID NO: 2) in the "Oonari" gene Os10g0446100 (Sh14 gene) as the target sequence.

[0101] Furthermore, the constructed plasmid vector was used to transform rice (Oonari) with Agrobacterium in the same manner as in (1) above, and the resulting transformants were sequenced. As a result, three lines were selected: "O-Cas9-1", a line in which a frameshift mutation occurred due to a 1-base insertion in the target sequence; "O-Cas9-2", a line in which a frameshift mutation occurred due to a 20-base deletion; and "O-Cas9-3", a line in which a frameshift mutation occurred due to a 1-base deletion. The target sequence and the corresponding sequences of "O-Cas9-1", "O-Cas9-2", and "O-Cas9-3" are shown in Table 2 below.

[0102] [Table 2]

[0103] [Evaluation test of shedding habit] The bending stress was measured for "Oonari" and "Oonari Shatter-resistant" as well as the genome-edited lines "O-Cas9-1", "O-Cas9-2" and "O-Cas9-3" in the same manner as described above in (1). The results are shown in Figure 6. In Figure 6, the white circles indicate the bending stress (average value of 120 kernels) for each variety and line. Comparisons between varieties and lines were performed using Wilcoxon multiple test, and the p-value was less than 0.01 between "Oonari" and "O-Cas9-1," "O-Cas9-2," "O-Cas9-3," and "Oonari Shattering Difficult" (between A and B in Figure 6). This confirmed that there was a significant difference in bending stress between "Oonari" and "O-Cas9-1," "O-Cas9-2," and "O-as9-3," which had homozygous loss-of-function mutations introduced into the Sh14 gene by genome editing, as well as between "Oonari" and "Oonari Shattering Difficult."

[0104] (Test Example 5) Effect of Sh13 gene mutation (1) Creation of the F4 group The population obtained by crossing "Takanari" with "Oonari Difficult to Shatter" was designated as the F4 population. "Oonari" has a tandem duplication mutation in a 6.3 kbp DNA fragment containing the microRNA gene Osa-miR172d, which suppresses grain shedding, resulting in increased expression of this Osa-miR172d, and it is presumed that the locus containing this Osa-miR172d contains the candidate gene Sh13, which causes moderate grain shedding in "Oonari" (Non-Patent Document 7). Furthermore, according to the above Test Example 2(2), the above mutation in the Sh13 gene of "Oonari" is maintained even in "Oonari Difficult to Shatter".

[0105] From the F4 population of "Takanari" and "Oonari Shattering Difficult", 116 lines were sown and 10 individuals per line were transplanted in a row in regular rows (row spacing 30 cm, plant spacing 15 cm). 30 days after transplantation, an equal amount of young leaves was collected from 8 individuals excluding the two individuals at both ends, mixed, and genomic DNA was extracted. Using the obtained genomic DNA as a template, PCR was performed using the primer set designed in Test Example 3 (1) above, and the genotype of the Sh14 gene in each line was determined in the same manner as Test Example 3 (3).

[0106] In addition, PCR was performed using the obtained genomic DNA as a template and the primers DUP_F3 and DUP_R3 described in Non-Patent Document 7, and the presence or absence of an amplification product was confirmed by agarose gel electrophoresis. Lines in which no amplification product was detected were determined to have a wild-type genotype of the Sh13 gene and were classified as "Sh13 W On the other hand, for the lineages for which an amplification product was confirmed, the gene mutation was either homozygous or heterozygous, so for the eight individuals from which genomic DNA was extracted, PCR was performed individually using the primers DUP_F3 and DUP_R3 in the same manner as above, and the presence or absence of an amplification product was confirmed by agarose gel electrophoresis. If an amplification product was confirmed in all eight individuals, the lineage was determined to have a homozygous mutant genotype for the Sh13 gene, and was labeled as "Sh13 m If no amplification product was detected among the eight individuals, the line was determined to be heterozygous for the wild type and mutant type genotype.

[0107] (2) Grain shedding test of F4 population In the above (1), the genotypes of both the Sh13 and Sh14 genes were homozygous for the wild type (Sh13 W Sh14 W ), 17 strains were homozygous for mutant types in both the Sh13 and Sh14 genes (Sh13 m Sh14 m ), 13 strains were homozygous for the wild type of the Sh13 gene and homozygous for the mutant type of the Sh14 gene (Sh13 W Sh14 m ), 14 strains were homozygous for the mutant genotype of the Sh13 gene and homozygous for the wild-type genotype of the Sh14 gene (Sh13 m Sh14 W ) A field grain shedding habit evaluation test was conducted for each of the 21 lines in the same manner as in Test Example 3(2) above.

[0108] Figure 7 shows the percentage of shattered grains in the above-mentioned lines in the F4 population. In Figure 7, the open circles indicate the average value per 6 plants in each line, and the average value per 17, 13, 14, or 21 lines. Comparisons between the lines were performed using the Wilcoxon test, and the percentage of shattered grains in Sh13 was 1.2%. W Sh14 W And Sh13 m Sh14 W and Sh13 W Sh14 m And Sh13 m Sh14 m Between A and C in Figure 7, the p-value was less than 0.01 for all cases, confirming that there was a significant difference in the grain shedding rate.

[0109] This confirmed that the effect of reducing grain shattering caused by homozygous Sh14 mutation is a function of a different mechanism from that of homozygous Sh13 mutation. Furthermore, it was confirmed that the additive effect of homozygous Sh14 and Sh13 mutations further reduces grain shattering.

[0110] (Test Example 6) Expression analysis Among the genes that have been reported to be involved in the control of abscission layer formation, the primer sets shown in Table 3 below that can specifically amplify the SHAT1 gene, SH5 gene, qSH1 gene, and OsSh1 gene were designed. In addition, the primer sets shown in Table 3 below that can specifically amplify the Ubiquitin gene as an internal standard gene were also designed.

[0111] [Table 3]

[0112] Young panicles of "Takanari", "Oonari", and "Oonari shattering difficult" cultivated in the above Test Example 1 (1) were collected, and total RNA was extracted using RNeasy Mini Kit (Qiagen). 1 μg of the obtained total RNA was reverse transcribed using Superscript III First Strand Synthesis Supermix (Invitrogen) to synthesize cDNA. Real-time Quantitative Reverse Transcription PCR (RT-PCR) was performed using each primer set listed in Table 3 with the synthesized cDNA as a template. The expression level of each gene transcript (transcript of SHAT1 gene: SHAT1, transcript of SH5 gene: SH5, transcript of qSH1 gene: qSH1, transcript of OsSh1 gene: OsSh1) in each variety and line was calculated by the ΔΔCt method. The expression level was the relative expression level when the expression level in "Takanari" was set to 1.

[0113] The average relative expression levels of four individuals (lines) per variety and line are shown in Figure 8. In Figure 8, the p-values ​​obtained by Welch's multiple test for comparisons between varieties and lines are shown, and the error bars indicate standard deviations. As shown in Figures 8(a) and 8(b), the expression levels of SHAT1 and SH5 were significantly decreased in Oonari compared to Takanari, and were comparable between Oonari and Oonari Shattering Difficult. As shown in Figures 8(c) and 8(d), the expression levels of qSH1 and OsSh1 were significantly decreased in Oonari compared to Takanari, and even more significantly in Oonari Shattering Difficult. From these results, it was inferred that the step controlled by the Sh14 gene is upstream of the step controlled by the qSH1 gene in the abscission layer formation control system. [Industrial Applicability]

[0114] As described above, according to the present invention, it is possible to provide a novel method for producing rice with low shedding tendency, which can reduce the shedding tendency of rice and obtain rice in which rice seeds (husks) are difficult to detach from the rice ear or rice with moderate shedding tendency, a rice with low shedding tendency, and a method for evaluating rice shedding tendency that can evaluate the shedding tendency of rice. Therefore, according to the present invention, it is also possible to provide a novel method for improving rice shedding tendency, which makes it more difficult for rice seeds to detach from the rice ear. [Sequence List Free Text]

[0115] SEQ ID NO:4 <223> CAPS_F primer SEQ ID NO:5 <223> CAPS_R primer SEQ ID NO:8 <223> Cas_F primer SEQ ID NO:9 <223> Cas_R primer

Claims

1. The method includes a step of artificially deleting a function of a first endogenous gene of rice, A method for producing shatter-resistant rice, characterized in that the first endogenous gene is a gene encoding an amino acid sequence shown in SEQ ID NO: 1 or a gene encoding an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO:

1.

2. A method for producing shatter-resistant rice as described in claim 1, characterized in that it includes a step of artificially introducing a loss-of-function mutation into rice so that the first endogenous gene has the loss-of-function mutation in a homozygous form.

3. The method includes an evaluation step of evaluating the rice grain shattering habit using the loss of the function of the first endogenous gene as an indicator, A method for evaluating rice grain shattering habit, characterized in that the first endogenous gene is a gene encoding an amino acid sequence shown in SEQ ID NO: 1 or a gene encoding an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO:

1.

4. The method for evaluating rice grain shedding habit described in claim 3, characterized in that the evaluation step is a step of evaluating the shedding habit of rice using as an indicator that the first endogenous gene has a homozygous functional loss-type mutated gene.

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