Method for producing temperature-sensitive male sterile plants

Abstract

The entire genomes of "PL12" and the original variety thereof were sequenced and compared in order to identify the responsible gene involved in a temperature-sensitive male sterility trait in "PL12" and to provide a method for producing a conditional male sterile plant targeting the gene. As a result, it was found that there was approximately 150 kb of deletion in the seventh chromosome of "PL12". Regarding this region, six types of partial deletion lines were produced by genome editing, and the range in which the responsible mutation was present was successfully narrowed to approximately 10 kb with the presence or absence of a temperature-sensitive male sterility trait in each of the lines as an indicator. Further, as a result of producing a line in which the functions of two genes located in this region were disrupted by genome editing, it was possible to identify the responsible gene involved in the temperature-sensitive male sterility trait. It was also confirmed that the function of the gene was inhibited, and the trait was able to be imparted to Arabidopsis thaliana and tomato as well as rice.

Description

Technical Field

[0001] The present invention relates to a method for producing a temperature-sensitive male-sterile plant, and the plant obtained thereby. The present invention also relates to a method for producing hybrid seeds using the plant, and the seeds obtained thereby.

Background Art

[0002] In hybrids, heterosis such as vigorous growth, high yield, and high quality compared to parental varieties can be obtained. Furthermore, the advantages of hybrid breeding are great, such as the ability to accumulate useful traits due to disease resistance gene groups. Almost all commonly used varieties of vegetables, flowers, corn, etc. are hybrid varieties. Hybrid varieties are also widely popular in rice cultivation in China and the United States.

[0003] However, since hybrid varieties are single-generation varieties, a large amount of crossing work is required each time seed production is carried out. Currently, the labor, time, and cost involved in crossing are bottlenecks. Generally, for crossing between crop varieties, it is necessary to make one variety lack pollen and attach the pollen of the other variety. Currently, in most cases, either manually removing the pistil (emasculation work) or pre-breeding a special male-sterile line is used to make the pollen deficient. For example, in tomatoes, eggplants, etc., since there is no male-sterile line, manual emasculation crossing is carried out in hybrid seed production. If a male-sterile line can be used, a reduction in emasculation work can be expected.

[0004] The main production method of hybrid seeds using male sterile lines is called the three-line method, which requires three lines: a fertility restoration line as the male parent, a male sterile line as the female parent, and its maintenance line (see Figure 1). For the excellent varieties to be used, special breeding is required to endow them with the traits of male sterility and corresponding fertility restoration, which is not only complicated but also time-consuming. Moreover, there are only limited combinations of available cytoplasmic male sterility factors and corresponding fertility restoration factors, and due to the need to introduce these factors through cross-breeding, it is laborious and time-consuming. As a result, it has hindered the utilization of a wide range of genetic resources.

[0005] Therefore, in order to produce hybrid seeds with only two lines (for implementing the two-line method), the use of conditional male sterile lines and transgenic lines as shown in Figure 2, etc. have been mainly considered (Patent Documents 1 to 3, Non-Patent Documents 1 to 3).

[0006] For example, there is temperature-sensitive male sterility, which becomes male sterile only under specific environmental conditions. Depending on the temperature conditions, it can control the state of forming normal pollen and setting seeds and the state of male sterility due to pollen formation failure. From this, it has a great advantage that self-reproduction and cross-reproduction can be easily switched only by temperature conditions, and both the maintenance of the line and the breeding of hybrid varieties can be carried out with one line. Furthermore, if pollen formation can be controlled with high precision, the possibility of mating using flower-visiting insects such as bees will also expand, and further efficiency improvement of seed collection is expected. Therefore, there is a need for a method to establish temperature-sensitive male sterile lines in a wide range of crop species and enable hybrid breeding easily and in a short period.

[0007] As such a temperature-sensitive male sterile line, in rice, "Water Rice Intermediate Female Parent Farm No. 12" (PL12) with "Reimei" as the original variety as shown in Figure 3 has been reported and is also being used (Patent Document 1, Non-Patent Document 1). However, the responsible gene involved in the phenotype of this mutant has not yet been identified. Therefore, it has been difficult to create such temperature-sensitive male sterile lines, and thus to produce hybrid seeds by implementing the two-line method, in varieties other than "Reimei" and in plant species other than rice.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0009]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0010] The present invention has been made in view of the above problems, and an object thereof is to identify a responsible gene involved in the temperature-sensitive male sterility trait in "PL12" and to provide a method for producing conditional male sterile plants targeting the gene.

Means for Solving the Problems

[0011] To achieve the above object, the inventors first performed fine mapping. However, they were unable to narrow down the range from approximately 1.8 Mb near 54 cM on chromosome 7. Therefore, they performed whole-genome sequence analysis of the original varieties "Reimei" and "PL12" and compared the two. As a result, it was found that there is a deletion of approximately 150 kb in the approximately 1.8 Mb region of "PL12".

[0012] Next, as shown in Figure 4, the deletion region of approximately 150 kb was roughly divided into three equal parts, and lines in which these three regions were each deleted were created by genome editing. As a result, it was found that one of these showed the same phenotype as "PL12", narrowing down the candidate region. Similarly, the candidate region was further divided into three parts, and lines in which these three regions were each deleted were created, successfully narrowing down the range where the causal mutation exists to approximately 10 kb.

[0013] Then, based on the information in the database, it was predicted that two genes (ORF1 and ORF2 shown in Figure 4) exist in this 10 kb region. Therefore, the functions of each gene were disrupted by genome editing. As a result, due to the deletion of ORF1, the trait of temperature-sensitive male sterility was exhibited, and it was finally clarified that this gene is the causal gene involved in the temperature-sensitive male sterility trait in "PL12" (hereinafter also referred to as the "temperature-sensitive male sterility gene").

[0014] Furthermore, as a result of suppressing the function of the gene by genome editing, it was found that the same trait of temperature-sensitive male sterility can be obtained in the dicotyledonous plants Arabidopsis thaliana and tomato, leading to the completion of the present invention. Therefore, the present invention provides the following.

[0015] That is, the present invention relates to a method for producing a temperature-sensitive male-sterile plant and the plant obtained thereby. The present invention also relates to a method for producing hybrid seeds using the above plant and the seeds obtained thereby, and more specifically provides the following. <1> A method for producing a temperature-sensitive male-sterile plant, comprising the step of artificially suppressing the function of at least one gene selected from the group consisting of the following (a) to (d) in the plant (a) A gene encoding a protein consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 22 (b) A gene encoding a protein consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 22 (c) A gene encoding an amino acid sequence having 60% or more homology with the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 22 (d) A gene containing DNA that hybridizes under stringent conditions with DNA consisting of the nucleotide sequence encoding the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 22. <2> A temperature-sensitive male-sterile plant in which the function of at least one gene selected from the group consisting of the following (a) to (d) is artificially suppressed (a) A gene encoding a protein consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 22 (b) A gene encoding a protein consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 22 (c) A gene encoding an amino acid sequence having 60% or more homology with the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 22 (d) A gene containing DNA that hybridizes under stringent conditions with DNA consisting of the nucleotide sequence encoding the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 22. <3> A method for producing hybrid seeds, comprising: cultivating the temperature-sensitive male-sterile plant according to <2>, which has been made male-sterile under a restrictive temperature, and cross-pollinating it with any plant, and recovering seeds from the temperature-sensitive male-sterile plant. A method comprising. <4> Hybrid seeds obtained by cultivating the temperature-sensitive male-sterile plant described in <2> under a restrictive temperature and using it as the female parent, and using any plant as the male parent.

Advantages of the Invention

[0016] According to the present invention, it becomes possible to produce temperature-sensitive sterile plants. In particular, except for suppressing the function of the temperature-sensitive male-sterile gene of the present invention, no special breeding is required, and temperature-sensitive sterile plants can be produced.

[0017] Also, as shown in FIGS. 6 and 7, the amino acid sequence of the protein encoded by the temperature-sensitive male-sterile gene of the present invention is highly conserved. Since this gene is also present in Amborella, which is the most primitive angiosperm, it can be seen that this gene is at least common to angiosperms. Therefore, in principle, this gene can be used for all crops. Thus, in the method of the present invention, since the strains used are not limited, it is possible to create male-sterile strains by suppressing the function of this gene in various varieties and strains of various plant species.

[0018] And, by cultivating the obtained temperature-sensitive male-sterile plant as the female parent together with another strain as the male parent under a restrictive temperature, hybrid seeds resulting from the crossing of these two strains can be easily obtained.

Brief Description of the Drawings

[0019]

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Mode for Carrying Out the Invention

[0020] (Method for Producing Temperature-Sensitive Male Sterile Plants) As shown in the examples described below, the present inventors clarified the responsible gene involved in the phenotype of the temperature-sensitive male sterile line "Oryza sativa cv. Nakate Kinmaze 12" (PL12). Furthermore, by suppressing the function of this gene in wild-type rice, Arabidopsis thaliana, and tomatoes using the genome editing method, we also succeeded in conferring the temperature-sensitive male sterile trait on these plants. Therefore, the method for producing a temperature-sensitive male sterile plant of the present invention is characterized by including a step of artificially suppressing the function of the gene (temperature-sensitive male sterility gene), and more specifically provides the following.

[0021] A method for producing a temperature-sensitive male-sterile plant, the method comprising the step of artificially suppressing the function of at least one gene selected from the group consisting of the following (a) to (d) in the plant (a) A gene encoding a protein consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 22 (b) A gene encoding a protein consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 22 (c) A gene encoding an amino acid sequence having 60% or more homology with the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 22 (d) A gene containing DNA that hybridizes under stringent conditions with DNA consisting of the nucleotide sequence encoding the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 22.

[0022] In the present invention, "temperature-sensitive male sterility" means a trait in which, when cultivated under the allowable temperature conditions described below, it normally produces seeds, but when cultivated under the restricted temperature conditions described below, it becomes male-sterile.

[0023] In the present invention, the "plant" to which the temperature-sensitive male sterility trait is to be imparted is not particularly limited as long as it is a plant having the temperature-sensitive male sterility gene described below. For example, monocotyledonous plants (e.g., Gramineae plants such as rice, barley, wheat, sorghum, corn, etc., Amaryllidaceae plants such as onion, leek, etc.) and dicotyledonous plants (e.g., Brassicaceae plants such as Arabidopsis thaliana, Chinese cabbage, rapeseed (oilseed rape), wild cabbage (the original species of cabbage, cauliflower, broccoli, etc.), Solanaceae plants such as tomato, potato, etc., Asteraceae plants such as lettuce, sunflower, etc., Fabaceae plants such as soybean, etc., Cucurbitaceae plants such as cucumber, etc., Apiaceae plants such as carrot, etc., Rosaceae plants such as apple, etc., Musaceae plants such as banana, etc., Amborellaceae plants (Amborella trichopoda)) including angiosperms, gymnosperms, bryophytes, and pteridophytes. Furthermore, gene recombinants and genome editors of these plants (e.g., herbicide-tolerant crops, pest-resistant crops, disease-resistant crops, crops with improved taste, crops with improved storage stability, crops with increased yield) may also be used.

[0024] In the present invention, examples of the "temperature-sensitive male sterility gene" to be targeted for functional suppression, which are genes encoding typical amino acid sequences derived from each plant species, are as shown in Table 1 below.

[0025]

Table 1

[0026] In nature, it is possible for nucleotide sequences to mutate. Accordingly, the encoded amino acids can also change. Therefore, the temperature-sensitive male sterility gene of the present invention includes genes encoding proteins consisting of amino acid sequences in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequences described in any of SEQ ID NOs: 1 to 22, as long as the function thereof can be suppressed to impart the temperature-sensitive male sterility trait.

[0027] Here, "a plurality" usually means within 50 amino acids, preferably within 45 amino acids, more preferably within 40 amino acids, still more preferably within 35 amino acids, yet more preferably within 30 amino acids, still more preferably within 25 amino acids, yet more preferably within 20 amino acids, still more preferably within 15 amino acids, yet more preferably within 10 amino acids (for example, within 9 amino acids, within 8 amino acids, within 7 amino acids, within 6 amino acids), particularly preferably within several amino acids (for example, within 5 amino acids, within 4 amino acids, within 3 amino acids, within 2 amino acids).

[0028] Furthermore, at the current state of the art, those skilled in the art can identify the homologous gene from the same or other plants using the nucleotide sequence information of a specific gene when the gene is obtained. Examples of methods for identifying homologous genes include hybridization techniques (Southern, E.M., J. Mol. Biol., 98:503, 1975) and polymerase chain reaction (PCR) techniques (Saiki, R.K., et al. Science, 230:1350 - 1354, 1985, Saiki, R.K. et al. Science, 239:487 - 491, 1988). To identify homologous genes, a hybridization reaction is usually carried out under stringent conditions. Examples of stringent hybridization conditions include conditions of 6M urea, 0.4% SDS, 0.5x SSC or hybridization conditions of equivalent stringency. By using more stringent conditions, for example, conditions of 6M urea, 0.4% SDS, 0.1x SSC, isolation of genes with higher homology can be expected. The temperature-sensitive male sterile gene of the present invention includes a gene containing DNA that hybridizes under stringent conditions with DNA consisting of a nucleotide sequence encoding the amino acid sequence described in any of SEQ ID NOs: 1 to 22 as long as its function is suppressed to confer the trait of temperature-sensitive male sterility.

[0029] The protein encoded by the identified homologous gene usually has high homology (high similarity), preferably high identity, with that encoded by the specific gene. Here, "high" means at least 40% or more, more preferably 50% or more, still more preferably 60% or more, more preferably 70% or more, still more preferably 80% or more, more preferably 85% or more (for example, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more). The temperature-sensitive male sterile gene of the present invention includes a gene encoding an amino acid sequence having 60% or more homology (similarity) or 40% or more identity with the amino acid sequence described in any of SEQ ID NOs: 1 to 22 as long as its function can be suppressed to impart the trait of temperature-sensitive male sterility. The homology of the amino acid sequence described in SEQ ID NO: 14 with that described in SEQ ID NO: 1 is 65% (the identity is 45%), and the homology of the amino acid sequence described in SEQ ID NO: 14 with that described in SEQ ID NO: 9 or 44 is 73% (the identity is 55%).

[0030] The homology of sequences can be determined using the BLAST program (Altschul et al. J. Mol. Biol., 215: 403-410, 1990). This program is based on the algorithm BLAST by Karlin and Altschul (Proc. Natl. Acad. Sci. USA, 87: 2264-2268, 1990, Proc. Natl. Acad. Sci. USA, 90: 5873-5877, 1993). For example, when analyzing an amino acid sequence by BLAST, the parameters can be, for example, score = 50 and wordlength = 3. Also, when analyzing an amino acid sequence using the Gapped BLAST program, it can be performed as described by Altschul et al. (Nucleic Acids Res. 25: 3389-3402, 1997). When using the BLAST and Gapped BLAST programs, the default parameters of each program are used. The specific methods of these analysis methods are known.

[0031] The "artificial suppression of the function of the temperature-sensitive male sterility gene" of the present invention includes both complete suppression (inhibition) and partial suppression of the function. In addition to the artificial suppression of the expression of the temperature-sensitive male sterility gene, it also includes the artificial suppression of the activity of the protein encoded by the temperature-sensitive male sterility gene. And such artificial suppression can be carried out, for example, by introducing mutations into the coding region, non-coding region, transcriptional control region (promoter region), etc. of the temperature-sensitive male sterility gene.

[0032] In the present invention, the mutation introduced into the temperature-sensitive male sterility gene is not particularly limited as long as it suppresses the function of the gene, and examples include nucleotide substitution, deletion, addition, and / or insertion, but nonsense mutation, frameshift mutation, and null mutation are preferred. Also, the number of mutations introduced into the temperature-sensitive male sterility gene is not particularly limited as long as it suppresses the function of the gene, and it may be 1 or a plurality (for example, 2, 3 or less, 5 or less, 10 or less, 20 or less, 30 or less, 40 or less, 50 or less).

[0033] Such mutations include, for example, as shown in Table 2 described later, nucleotide mutations accompanied by changes or deletions of amino acids at positions 53 and later (about 33% of the whole) of the amino acid sequence set forth in SEQ ID NO: 1. Also, as shown in Table 3 described later, nucleotide mutations accompanied by changes or deletions of amino acids at positions 18 or 19 and later (about 76% of the whole) of the amino acid sequence set forth in SEQ ID NO: 14 are included. Furthermore, nucleotide mutations accompanied by changes or deletions of amino acids at positions 24 and later (about 70% of the whole) of the amino acid sequence set forth in SEQ ID NO: 9 or 44 are included. And due to such deletions, the function of the temperature-sensitive male sterility gene of the present invention is suppressed, and a temperature-sensitive male sterile plant can be obtained.

[0034] Therefore, as the mutation introduced into the temperature-sensitive male sterility gene, it is not necessary to cause the entire amino acid sequence of the protein encoded by the gene to be lost, and it may be introduced into the gene so that a part of it is lost or changed.

[0035] In addition, when a part of the protein expressed due to gene mutation is deleted, usually, 10% or more of the total amino acids may be changed or deleted, preferably 20% or more, more preferably 25% or more, still more preferably 30% or more, further more preferably 35% or more, even more preferably 40% or more, still more preferably 45% or more, even more preferably 50% or more, further more preferably 55% or more, even more preferably 60% or more, still more preferably 65% or more, even more preferably 70% or more, further more preferably 75% or more, even more preferably 80% or more, still more preferably 85% or more, even more preferably 90% or more, further more preferably 95% or more (for example, 96% or more, 97% or more, 98% or more, 99% or more) may be changed or deleted.

[0036] As such a region where the amino acid sequence is changed or deleted, as shown in the examples described later, the C-terminal region is preferable. Also, in FIGS. 6 and 7, a region highly conserved in the amino acid sequence encoded by the temperature-sensitive male sterility gene of the present invention, that is, a region important for exerting its function is suggested. Therefore, such a conserved region is also suitable as a region where the amino acid sequence is changed or deleted. Examples of the conserved region include a region consisting of positions 10 to 75 in the amino acid sequence set forth in SEQ ID NO: 1, or a corresponding region. The corresponding region means, as shown in FIGS. 6, 7, and 14, when using nucleotide and amino acid sequence analysis software (such as GENETYX-MAC, Sequencher, etc.) or BLAST (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi) to align the amino acid sequence set forth in SEQ ID NO: 1 with amino acid sequences derived from other plants (for example, SEQ ID NOs: 2 to 21), it is the region that aligns with the above-mentioned region in the amino acid sequence set forth in SEQ ID NO: 1.

[0037] Introduction of mutations into the temperature-sensitive male sterility gene can be achieved by methods of introducing mutations known to those skilled in the art. Such known methods include genome editing methods, physical mutagenesis methods, methods using chemical mutagens, methods of introducing transposons, etc. into genomic DNA, methods targeting transcripts using siRNA, antisense RNA, RNA having ribozyme activity, etc., but are not limited thereto.

[0038] Among these, from the viewpoint of being able to artificially introduce mutations targeting the temperature-sensitive male sterility gene, genome editing methods, methods targeting transcripts, and the TILLING method described below are preferred.

[0039] The genome editing method is a method of modifying a target gene by using a site-specific nuclease (for example, DNA double-strand cleavage enzymes such as zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), CRISPR-Cas9). For example, methods using fusion proteins such as ZFNs (U.S. Patent Nos. 6265196, 8524500, 7888121, European Patent No. 1720995), TALENs (U.S. Patent Nos. 8470973, 8586363), PPR (pentatricopeptide repeat) fused with a nuclease domain (Nakamura et al., Plant Cell Physiol 53:1171-1179 (2012)), etc., and methods using complexes of guide RNA and proteins such as CRISPR-Cas9 (U.S. Patent No. 8697359, International Publication No. 2013 / 176772), CRISPR-Cpf1 (Zetsche B. et al., Cell, 163(3):759-71, (2015)), Target-AID (K. Nishida et al., Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems, Science, DOI:10.1126 / science.aaf8729, (2016)) can be mentioned.

[0040] Examples of physical mutagenesis methods include heavy ion beam (HIB) irradiation, fast neutron beam irradiation, gamma ray irradiation, and ultraviolet irradiation (see Hayashi et al., Cyclotrons and Their Applications, 2007, 18th International Conference, pp. 237-239, and Kazama et al., Plant Biotechnology, 2008, Vol. 25, pp. 113-117).

[0041] Examples of methods using chemical mutagens include, for example, methods of treating seeds or the like with chemical mutagens (see Zwar and Chandler, Planta, 1995, Vol. 197, pp. 39-48, etc.). There is no particular limitation on the chemical mutagen, but examples include ethyl methanesulfonate (EMS), N-ethyl-N-nitrosourea (ENU), N-methyl-N-nitrosourea (MNU), 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.

[0042] Examples of methods for introducing transposons or the like into genomic DNA include, for example, methods of inserting transposons such as T OS 17 etc. and T-DNA into the genomic DNA of plants (see Kumar et al., Trends Plant Sci., 2001, Vol. 6, No. 3, pp. 127-134, and Tamara et al., Trends in Plant Science, 1999, Vol. 4, No. 3, pp. 90-96).

[0043] For the plants into which mutations have been introduced by the above method, it is possible to confirm, by a known method, that a mutation has been introduced into the temperature-sensitive male sterility gene. Examples of such known methods include the DNA sequencing method (next-generation sequencing method, etc.), the PCR method, the analysis method using a microarray, the Southern blot method, and the Northern blot method. According to such methods, it is possible to determine whether a mutation has been introduced into the temperature-sensitive male sterility gene by comparing the sequence or length of the gene before and after the introduction of the mutation. Further, by using the Northern blot method, the RT-PCR method, the Western blot method, the ELISA method, the analysis method using a microarray, etc., if a decrease in the expression level of the transcription product or translation product of the temperature-sensitive male sterility gene is observed in a plant in which a mutation has been introduced into the transcriptional control region or the like, it is also possible to confirm that the plant is a plant into which a mutation has been introduced into the temperature-sensitive male sterility gene.

[0044] In addition, as another method for confirming that a mutation has been introduced into the temperature-sensitive male sterility gene, TILLING (Targeting Induced Local Lesions IN Genomes) can be mentioned (see Slade et al., Transgenic Res., 2005, Vol. 14, pp. 109-115, and Comai et al., Plant J., 2004, Vol. 37, pp. 778-786). In particular, when non-selective mutations are introduced into the plant genome using the above-mentioned heavy ion beam irradiation, chemical mutagens, etc., after amplifying the temperature-sensitive male sterility gene or a part thereof by PCR, individuals having a mutation in the amplification product can be selected by the above-mentioned TILLING or the like.

[0045] In addition, by crossing the plant into which a mutation has been introduced by the above method with a wild-type plant and performing backcrossing, it is also possible to remove the mutations introduced into the genes other than the target gene.

[0046] Plants in which the function of a temperature-sensitive male sterility gene is suppressed by introducing a mutation into the gene may be heterozygotes of the temperature-sensitive male sterility gene. In such a case, for example, by crossing such heterozygotes to obtain F1 plants, homozygotes having the temperature-sensitive male sterility gene into which the mutation has been introduced are selected from the F1 plants. In this case, the "plant that is a homozygote having the temperature-sensitive male sterility gene into which the mutation has been introduced" includes not only plants having two alleles of the temperature-sensitive male sterility gene having the same mutation, but also a first temperature-sensitive male sterility gene having a first mutation and encoding a protein whose activity is suppressed, and a second temperature-sensitive male sterility gene having a second mutation and encoding a protein whose activity is suppressed.

[0047] In the present invention, as a method for artificially suppressing the function of a temperature-sensitive male sterility gene, in addition to the above-described mutation introduction, a method using DNA encoding dsRNA (double-stranded RNA, such as siRNA) complementary to the transcription product of the temperature-sensitive male sterility gene, a method using DNA (antisense DNA) encoding antisense RNA complementary to the transcription product of the temperature-sensitive male sterility gene, and a method using DNA encoding RNA having ribozyme activity that specifically cleaves the transcription product of the temperature-sensitive male sterility gene (ribozyme method) can be mentioned. Also included are methods targeting the transcription product of the temperature-sensitive male sterility gene.

[0048] In the present invention, the artificial suppression of the temperature-sensitive male sterility gene function can be performed on various plant bodies, seeds, or plant cells according to the above-described methods and the like. Plant cells include not only cultured cells derived from plants but also cells in plant bodies. Furthermore, cells derived from plants in various forms, such as suspension-cultured cells, protoplasts, leaf sections, callus, immature embryos, pollen, etc. are included.

[0049] In the present invention, the DNA encoding the above-described site-specific nuclease, fusion protein or complex of guide RNA and protein, the DNA encoding a transposon, the DNA encoding double-stranded RNA, the DNA encoding antisense RNA, the DNA encoding RNA having ribozyme activity, etc. may be introduced into plant cells in a form inserted into a vector.

[0050] The vector into which the DNA for artificially suppressing the function of the temperature-sensitive male sterility gene is inserted is not particularly limited as long as it can express the inserted gene in plant cells, but it may contain a promoter for constitutively or inducibly expressing the DNA. Examples of the promoter for constitutive expression include the rice ubiquitin promoter, the cauliflower mosaic virus 35S promoter, the rice actin promoter, the maize ubiquitin promoter, etc. Examples of the promoter for inducible expression include promoters known to be expressed by external factors such as infection and invasion of filamentous fungi, bacteria, and viruses, low temperature, high temperature, drying, ultraviolet irradiation, spraying of specific compounds, etc. Furthermore, as the promoter for expressing the DNA encoding short RNAs such as guide RNA and siRNA as the DNA according to the present invention, the U6 promoter and the polIII promoter are preferably used.

[0051] As a method for introducing the DNA or a vector into which the DNA is inserted into plant cells, for example, various methods known to those skilled in the art such as the particle gun method, the particle bombardment method, the method via Agrobacterium (Agrobacterium method), the polyethylene glycol method, the electroporation method (electroporation), etc. can be used.

[0052] Even without taking the form of DNA, the above-mentioned site-specific nuclease, fusion protein, and transposon, as proteins, and the above-mentioned guide RNA, double-stranded RNA, antisense RNA, and RNA having ribozyme activity, as RNA, can introduce mutations even when introduced into plant cells.

[0053] In addition, a temperature-sensitive male sterile plant can be obtained by regenerating a plant from a plant cell in which the function of a gene has been artificially suppressed by the above-mentioned method or the like.

[0054] For example, in rice, several techniques for producing transgenic plants have already been established and widely used in the technical field of the present invention, such as a method of introducing a gene into protoplasts with polyethylene glycol and regenerating a plant (Datta, S.K. In Gene Transfer To Plants (Potrykus I and Spangenberg Eds.) pp66-74, 1995), a method of introducing a gene into protoplasts with an electric pulse and regenerating a plant (Toki et al. Plant Physiol. 100, 1503-1507, 1992), a method of directly introducing a gene into cells by the particle gun method and regenerating a plant (Christou et al. Bio / technology, 9:957-962, 1991), and a method of introducing a gene via Agrobacterium and regenerating a plant (Hiei et al. Plant J. 6:271-282, 1994).

[0055] In the case of Arabidopsis thaliana, the floral dip method (Clough SJ & Bent AF, Plant J 16:735-743, 1998), the method of Akama et al. (Akama et al. Plant Cell Reports 12:7-11, 1992) can be mentioned, and in the present invention, these methods can be preferably used.

[0056] In addition, as methods for producing transformed plants related to wheat, the methods described by Tingay et al. (Tingay S. et al. Plant J. 11: 1369-1376, 1997), Murray et al. (Murray F et al. Plant Cell Report 22: 397-402, 2004), and Travalla et al. (Travalla S et al. Plant Cell Report 23: 780-789, 2005) can be mentioned.

[0057] As a method for regenerating sorghum plants, for example, a method of introducing a gene into immature embryos or callus and regenerating plants by the Agrobacterium method or the particle gun method, or a method of pollinating using pollen introduced with a gene by ultrasonic waves is preferably used (J.A. Able et al., In Vitro Cell. Dev. Biol. 37: 341-348, 2001, A.M. Casas et al., Proc. Natl. Acad. Sci. USA 90: 11212-11216, 1993, V. Girijashankar et al., Plant Cell Rep 24: 513-522, 2005, J.M. JEOUNG et al., Hereditas 137: 20-28, 2002, V Girijashankar et al., Plant Cell Rep 24(9): 513-522, 2005, Zuo-yu Zhao et al., Plant Molecular Biology 44: 789-798, 2000, S. Gurel et al., Plant Cell Rep 28(3): 429-444, 2009, ZY Zhao, Methods Mol Biol, 343: 233-244, 2006, AK Shrawat and H Lorz, Plant Biotechnol J, 4(6): 575-603, 2006, D Syamala and P Devi Indian J Exp Biol, 41(12): 1482-1486, 2003, Z Gao et al., Plant Biotechnol J, 3(6): 591-599, 2005).

[0058] In the case of maize, the methods described by Shillito et al. (Bio / Technology, 7:581, 1989) and Gorden-Kamm et al. (Plant Cell 2:603, 1990) can be mentioned.

[0059] In the case of tomato, the method described by Matsukura et al. (J. Exp. Bot., 44:1837-1845, 1993) can be mentioned.

[0060] In the case of soybean, the method described in the patent gazette (U.S. Patent No. 5,416,011) can be mentioned.

[0061] In the case of potato, the method described by Visser et al. (Theor. Appl. Genet, 78:594, 1989) can be mentioned.

[0062] Also, even in the case of other plants, transformation and regeneration into plants can be carried out using the methods described by Tabei et al. (edited by Yutaka Tabei, "Transformation Protocol [Plant Edition]", Kagaku Dojin Co., Ltd., published on September 20, 2012).

[0063] (Thermosensitive male sterile plants, and their use) By the above methods and the like, a thermosensitive male sterile plant in which the function of the thermosensitive male sterile gene of the present invention is artificially suppressed can be obtained. Therefore, the present invention A thermosensitive male sterile plant in which the function of at least one gene selected from the group consisting of the following (a) to (d) is artificially suppressed (a) A gene encoding a protein consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 22 (b) A gene encoding a protein consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 22 (c) A gene encoding an amino acid sequence having 60% or more homology with the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 22 (d) A gene comprising a DNA that hybridizes under stringent conditions with a DNA consisting of a nucleotide sequence encoding the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 22 is provided.

[0064] Regarding the temperature-sensitive male sterility gene, the artificial suppression of its function, plants to which temperature-sensitive male sterility is imparted by such suppression, etc., are as described above. However, as the temperature-sensitive male sterile plant of the present invention, it is preferably a plant excluding "PL12" (rice intermediate female parent Nong 12), and more preferably a plant excluding "Reimei" in which the function of the temperature-sensitive male sterility gene of the present invention has been artificially suppressed.

[0065] Also, once a plant body in which the function of the temperature-sensitive male sterility gene has been artificially suppressed is obtained, it is possible to obtain progeny from the plant body by sexual reproduction or asexual reproduction. Furthermore, it is also possible to obtain propagation materials (for example, seeds, cuttings, strains, callus, protoplasts, etc.) from the plant body, its progeny, or clones, and mass-produce the plant body based on them. Therefore, the present invention includes progeny and clones of temperature-sensitive male sterile plants, as well as their propagation materials. Note that examples of the propagation materials include seeds, strains, callus, and protoplasts.

[0066] Also, the temperature-sensitive male sterile plant of the present invention can be used in a method for producing hybrid seeds by the two-line method as shown in FIG. 2. Therefore, the present invention cultivating at a restrictive temperature to make it male sterile, the step of cross-pollinating the temperature-sensitive male sterile plant according to claim 2 with any plant, and the step of collecting seeds from the temperature-sensitive male sterile plant, also provides a method for producing hybrid seeds including the above steps.

[0067] In the production of the hybrid seeds of the present invention, the temperature-sensitive male sterile plant of the present invention is cultivated at a restrictive temperature, and a plant in a male sterile state is used.

[0068] "Restriction temperature" means a temperature higher than the cultivation temperature (permissible temperature) at which the temperature-sensitive male-sterile plant of the present invention can form pollen. Such restriction temperature and permissible temperature can be appropriately adjusted by those skilled in the art according to the type of plant, etc.

[0069] Also, cultivation under the restriction temperature may be carried out using a known cultivation method according to the type of plant, etc. Also, the cultivation period is not particularly limited as long as it includes the period from flower bud formation (flowering) to pollen formation. Note that "flower bud" means a growth point that has been growing vegetatively and has differentiated into a growth point that grows reproductively, that is, a flower primordium. The formation period varies depending on the type of plant, its variety / strain, cultivation conditions, etc., but those skilled in the art can judge it by a known method (for example, visual observation). Also, cultivation under the restriction temperature during such a period may be continuous (for example, cultivation under the restriction temperature regardless of day and night), or intermittent (for example, cultivation only during the day under the restriction temperature).

[0070] The "arbitrary plant" to be crossed with the temperature-sensitive male-sterile plant of the present invention is not particularly limited as long as it is a plant maintaining male fertility. For example, it may be of the same species as the temperature-sensitive male-sterile plant of the present invention but of a different strain or variety.

[0071] "Cross-pollination" of the temperature-sensitive male-sterile plant of the present invention with an arbitrary plant, and "recovery of seeds" formed thereby can be appropriately carried out by those skilled in the art using a known method according to the type of plant, etc.

[0072] Also, the seeds thus obtained are seeds of a first-generation hybrid (F1), so-called hybrid seeds, with the temperature-sensitive male-sterile plant of the present invention as the female parent (maternal line) and an arbitrary plant as the male parent (paternal line). Therefore, in the present invention, hybrid seeds with the temperature-sensitive male-sterile plant of the present invention, which was cultivated under the restriction temperature and made male-sterile, as the female parent and an arbitrary plant as the male parent are also provided.

Examples

[0073] Hereinafter, the present invention will be described more specifically based on examples, but the present invention is not limited to the following examples. Also, except for Example 5 regarding tomatoes described later, the following examples were conducted using the materials and methods shown below.

[0074] (Variety, line) For rice, the standard variety "Nipponbare", the temperature-sensitive male sterile line rice "Chukan Mobo 12" (PL12) and its original variety "Reimei" were used. Also, the practical high-yielding rice variety "Hokuriku 193" (Indica rice) was used. For Arabidopsis thaliana, the standard ecotype Columbia was used.

[0075] (Plant cultivation method) Rice was potted under its normal cultivation conditions (28°C during the day / 22°C at night). Flowering was induced under short-day conditions, and cultivation was continued at 28°C during the day / 22°C at night or 33°C during the day / 22°C at night. At the time of heading, pollen formation was observed and seed fertility was confirmed.

[0076] Arabidopsis thaliana was cultivated in a growth chamber set at 21°C continuous light conditions. After bolting, cultivation was continued at 21°C or 27°C, and the pollen formation and fruiting conditions were observed.

[0077] (Genome editing of rice) The CRISPR / Cas9 method commonly used in plants was utilized. More specifically, a vector expressing guide RNA, Cas9, and the hygromycin resistance gene as a selection marker was introduced into calli of the rice cultivar "Nipponbare" or immature embryos of "Hokuriku 193" by the Agrobacterium method. The vector was based on pPZP202 and was capable of expressing guide RNA and Cas9 under the control of the rice U6 promoter and the rice ubiquitin promoter, respectively. Subsequently, regenerated plants were obtained from calli selected for hygromycin resistance, genomic DNA was prepared from their leaves, and the nucleotide sequence of the genome-edited region was confirmed. Those with mutations that shift the reading frame of the codon, such as single-base insertions, were selected. In these mutants, since the gene cannot express the protein that functions, it is in a gene-deficient state.

[0078] Then, the rice cultivars "PL12", "Nipponbare", "Hokuriku 193", and their genome-edited lines were potted and cultivated in a greenhouse under normal cultivation conditions (28°C during the day / 22°C at night). Flowering was induced under short-day conditions, and cultivation was continued at 28°C during the day / 22°C at night or under high-temperature conditions (33°C during the day / 22°C at night for "Nipponbare", 35°C during the day / 25°C at night for "Hokuriku 193"), and pollen formation was observed at the time of heading.

[0079] (Genome editing of Arabidopsis thaliana) Similar to the rice, genome editing was performed using the CRISPR / Cas9 method. The gene introduction method was different from that of the rice. For the transformation of Arabidopsis thaliana, the commonly used floral dip method was used.

[0080] The temperature-sensitive male sterile rice line "PL12" was selected and bred from a mutant population of the cultivar "Reimei" by gamma-ray irradiation. This line forms pollen normally and sets seeds at normal cultivation temperatures (about 28°C), but shows pollen formation failure and no seed setting at high temperatures (about 33°C) (see Figure 3). No abnormalities were observed in vegetative organs such as leaves and pistils, and the sensitive period was found to be several days around 20 days before heading. Genetic analysis has shown that this temperature-sensitive male sterility trait is controlled by a single recessive factor, and it has been reported that the responsible mutation is located on chromosome 7 (see Non-Patent Document 1). Therefore, as described below, an attempt was made to identify the responsible mutation for the temperature-sensitive male sterility trait.

[0081] (Example 1) To identify the responsible mutation for the temperature-sensitive male sterility trait, fine mapping was performed, but the range could not be narrowed down from about 1.8 Mb near 54 cM. Therefore, whole-genome sequence analysis of the original cultivar "Reimei" and "PL12" was performed and compared. As a result, it was found that there is a deletion of about 150 kb in this part of chromosome 7 of "PL12".

[0082] Next, a series of partial deletions was created by introducing double-strand breaks at two positions in the genomic DNA using the CRISPR / Cas9 method with two guide RNAs and deleting the DNA fragment between them. More specifically, as shown in Figure 4, lines with partial deletions in the ranges of (1)-(2), (2)-(3), and (3)-(4) were created so as to approximately trisect the 150-kb deletion region. In order to delete (1)-(2), (2)-(3), and (3)-(4), SEQ ID NOs: 23 and 24, 24 and 25, and 25 and 26 were used as target sequences, and double-strand breaks were generated at two positions by CRISPR / Cas9, respectively.

[0083] As a result, as shown in Figure 5, it was found that one of these (#571) showed the same phenotype as "PL12", and the candidate region was narrowed down to the range of (3)-(4).

[0084] Similarly, three partial deletions were designed inside the deletion region of #571. More specifically, as shown in Fig. 4, strains with partial deletions in the ranges of (3)-(5), (5)-(6), and (6)-(4) were created to approximately trisect the region of (3)-(4). In order to delete (3)-(5), (5)-(6), and (6)-(4), the target sequences of SEQ ID NOs: 25 and 27, 27 and 28, and 28 and 26 were used, and two double-strand breaks were each generated by CRISPR / Cas9.

[0085] As a result, as shown in Fig. 5, since #592 showed the trait of temperature-sensitive male sterility, it was considered that there was a causative mutation in the range of approximately 10 kb from (3) to (5). From the information in the database, it was predicted that two genes (ORF1 and ORF2 shown in Fig. 4) were present in this region.

[0086] Therefore, guide RNAs were designed within each coding region, and attempts were made to disrupt gene functions by introducing frameshift mutations. The amino acid sequence encoded by ORF1 is shown in SEQ ID NO: 1, and the amino acid sequence encoded by ORF2 is shown in SEQ ID NO: 29. The target sequences in the guide RNAs for ORF1 are shown in SEQ ID NO: 30 or 31. The target sequence in the guide RNA for ORF2 is shown in SEQ ID NO: 32. Also, examples of the obtained mutations for ORF1 are shown in Table 2.

[0087]

Table 2

[0088] As a result, as shown in Fig. 5, the strain of #612 showed the trait of temperature-sensitive male sterility. Furthermore, although not shown in the figure, it was confirmed that not only the representative examples of the mutant strains shown in Fig. 5 but also if a frameshift mutation occurred, the trait of temperature-sensitive male sterility would be shown, and it was found that the gene function deletion of ORF1 was the causative mutation of "PL12".

[0089] This gene is predicted to have the ID of LOC_Os07g26794 in the MSU database and Os07g0482700 in the RAP-DB, but it is a novel gene with unknown function. This gene is widely conserved in organisms, especially highly conserved in plant species. The comparison of the predicted amino acid sequences in higher plants is shown in Fig. 6. The sequence conservation is even higher among gramineous crops (see Fig. 7). Also, the temperature-sensitive male sterile gene newly identified in this way was named TMS2.

[0090] (Example 2) The genome-edited lines lacking the said gene were cultivated at the permissive temperature and the restrictive temperature and compared with the standard variety "Nipponbare" before genome editing and the temperature-sensitive male sterile line "PL12", and the appearance of the florets was observed. As a result, as shown in Fig. 8, in the standard variety "Nipponbare", pollen was normally formed regardless of the temperature. On the other hand, in the temperature-sensitive male sterile line "PL12", pollen was normally formed and the anthers were yellow and plump at 28°C, whereas at 33°C, the pollen did not develop and the morphology and color of the anthers became abnormal. And the line in which the said gene was deleted in "Nipponbare" by genome editing showed a temperature-sensitive male sterile phenotype similar to that of "PL12".

[0091] Furthermore, lines in which the gene function was deleted by genome editing of the rice variety "Nipponbare" were cultivated at 28°C and 33°C. As a result, as shown in Fig. 9, at 28°C, they set seeds normally and the panicles drooped, whereas at 33°C, they did not set seeds and the panicles stood upright.

[0092] (Example 3) Using the rice high-yielding variety "Hokuriku 193" (Indica rice), genome editing was performed in the same manner as in Example 2 ("Nipponbare" (Japonica rice)) to delete the temperature-sensitive male sterile gene. The transformation of "Hokuriku 193" was carried out with reference to the 2014 master's thesis of the University of Nagoya by Yuki Higai "Research on the method of rice transformation by Agrobacterium tumefaciens". And the genome-edited line derived from "Hokuriku 193" thus obtained was cultivated in a greenhouse set at normal conditions (28°C) or high-temperature conditions (35°C), and the formed anthers were observed.

[0093] As a result, as shown in Fig. 10, in the genome-edited line derived from "Hokuriku 193", similar to the genome-edited line derived from "Nipponbare", pollen is formed and anthers develop under normal conditions, but it was confirmed that under high-temperature conditions, male sterility occurs due to pollen formation failure.

[0094] Also during the flowering period, pollen of another high-yield variety, "Oonari", was applied to the genome-edited line derived from "Hokuriku 193", and the ripened seeds were germinated and cultivated. Then, genomic DNA was purified from the leaves of each of (1) "Oonari", (2) the genome-edited line derived from "Hokuriku 193", and (3) the individuals obtained by applying pollen of "Oonari" to the genome-edited line derived from "Hokuriku 193" cultivated at high temperature, and the nucleotide sequence of the genome-editing site (mutation insertion site) of the temperature-sensitive male sterility gene was analyzed.

[0095] As a result, as shown in Fig. 11, in the individuals of (3), they were heterozygous for the genomic sequences of (1) and (2), indicating that hybrids were obtained by crossing (1) and (2). Therefore, it was confirmed that the pistil functions normally even when the genome-edited line is cultivated at high temperature, and that the genome-edited line can actually be used for crossing.

[0096] (Example 4) Regarding the dicotyledonous plant Arabidopsis thaliana, an attempt was also made to create a temperature-sensitive male sterile line by deleting the said gene. More specifically, genome editing was performed by the CRISPR / Cas9 method to create a line in which the said gene no longer functions.

[0097] The target sequence in Arabidopsis thaliana is shown in SEQ ID NO: 33. Also, examples of the mutations obtained are shown in Table 3.

[0098] [Table 3]

[0099] Then, strains with gene function deletion due to genome editing of *Arabidopsis thaliana* were cultivated at 21°C and 27°C. As a result, as shown in Fig. 12, at 21°C, they set seeds normally and the pod length was the same as that of the wild type, while at 27°C, they did not set seeds and the pods did not elongate.

[0100] Although not shown in the figure, similar to the above-mentioned rice, it has been confirmed that not only the representative examples of the mutant strains shown in Fig. 12, but also if a frameshift mutation occurs, *Arabidopsis thaliana* also shows the traits related to the temperature-sensitive male sterility.

[0101] Also, similar to Example 3, mating experiments were conducted in *Arabidopsis thaliana*. As a result, although not shown in the figure, seeds were obtained. Therefore, similar to the above-mentioned rice, it is suggested that the suppression of the function of the temperature-sensitive male sterility gene can be used for mating of the genome-edited strain in *Arabidopsis thaliana* without affecting the function of the pistil.

[0102] (Example 5) In tomatoes, genome editing of the temperature-sensitive male sterility gene was performed to establish mutant strains. Specifically, first, the genome sequence information of the temperature-sensitive male sterility gene of tomatoes was obtained from EnsemblePlants. The sequence of the cDNA (wild type) of the gene is shown in SEQ ID NO: 43. Also, the amino acid sequence of the protein encoded by the cDNA is shown in SEQ ID NO: 9 or 44 (SEQ ID NO: 9 and 44 show the same amino acid sequence).

[0103] Then, targeting the obtained genome sequence information, CRISPR / Cas target sequences were searched using CRISPRdirect (http: / / crispr.dbcls.jp / ). As a result, the following two sequences, which are within the exon and have no similar sequences on the tomato genome, were selected as genome editing target sequences. GE51 (within the third exon) 5’-ACCATAGGTGAGAAGTCACGAGG-3’ (SEQ ID NO: 37) GE52 (within the fourth exon) 5’-CCAGGCTGTCTACCAGAGAAATG-3’ (SEQ ID NO: 38).

[0104] Next, a genome editing vector was prepared. As the vector, the genome editing vector for plant genome editing pEgP237-2A-GFP was used. pEgP237-2A-GFP was obtained by license from Professor Gyobu of Tokushima University. For details of the vector, see Ueta et al. (2017) Rapid breeding of parthenocarpic tomato plants using CRISPR / Cas9. Sci Rep. 7:507.

[0105] After digesting pEgP237-2A-GFP with BsaI, it was purified. Next, the following chemically synthesized oligonucleotide DNAs were mixed in equal amounts, then subjected to heat denaturation treatment and annealed. The obtained double-stranded oligonucleotide DNA was mixed with pEgP237-2A-GFP digested with BsaI, inserted into the vector, and ligated. Then, it was introduced into Escherichia coli to amplify the genome editing vector. #51 Sl_tms2_gRNA01F 5’-GATTGACCATAGGTGAGAAGTCACG-3’ (SEQ ID NO: 39) Sl_tms2_gRNA01R 5’-AAACCGTGACTTCTCACGATACCA-3’ (SEQ ID NO: 40) #52 Sl_tms2_gRNA02F 5’-GATTGCATTTCTCTGGTAGACAGCG-3’ (SEQ ID NO: 41) Sl_tms2_gRNA02R 5’-AAACGGCTGTCTACCAGAGAAATG-3’ (SEQ ID NO: 42).

[0106] Then, the genome editing vector constructed in this way was transformed into Rhizobium radiobacter (Agrobacterium tumefaciens) GV2260, and furthermore, the tomato cultivar "Microtom" was transformed using the Agrobacterium method. For the tomato transformation method, refer to Sun et al. (2006) A Highly Efficient Transformation Protocol for Micro-Tom, a Model Cultivar for Tomato Functional Genomics. Plant Cell Physiol. 47:426-431.

[0107] The target sequences of the transformants obtained in this way were confirmed to obtain genome-edited individuals. In the T0 generation, no individuals with homozygous mutations were obtained. Therefore, self-pollination was carried out, and individuals with fixed mutations were selected from the progeny.

[0108] Next, the obtained genome-edited individuals were grown until before flowering at room temperature of 23°C / 18°C (day / night) with a 14-hour day length. Furthermore, they were transferred to a high temperature of 30°C / 28°C (day / night) with a 14-hour day length, and the first flowers were removed. Then, the presence or absence of seeds in the fruits that set was confirmed. The results obtained are shown in Fig. 13.

[0109] As shown in Fig. 13, also in tomatoes, due to the deletion of the temperature-sensitive male sterility gene, seeds are fertile under normal conditions, while no seeds are formed under high temperature conditions, and a temperature-sensitive phenotype was observed as in the case of rice. Tomatoes are self-pollinating plants like rice, and their own pollen adheres to the pistil to form seeds. This indicates the applicability to crops such as vegetables.

[0110] Finally, the amino acid sequences encoded by the temperature-sensitive male sterility genes derived from various plants used in this example and the mutation sites observed in the prepared genome-edited individuals are summarized in Fig. 14.

Industrial Applicability

[0111] As described above, according to the present invention, it is possible to produce temperature-sensitive sterile plants. In particular, it is possible to produce temperature-sensitive sterile plants without requiring special breeding other than suppressing the function of the temperature-sensitive male sterility gene of the present invention.

[0112] Also, as shown in FIGS. 6 and 7, the amino acid sequence of the protein encoded by the temperature-sensitive male sterility gene of the present invention is highly conserved. Therefore, in the method of the present invention, since the strains used are not limited, it is possible to create male sterile strains by suppressing the function of this gene in various varieties and strains of various plant species. For example, by simply suppressing the function of the temperature-sensitive male sterility gene of a useful variety, it can be immediately used for hybrid production.

[0113] Then, by cultivating the obtained temperature-sensitive male sterile plant as the female parent together with another strain as the male parent under the restrictive temperature, hybrid seeds resulting from the cross of these two strains can be easily obtained.

[0114] Thus, the present invention is expected to have a great impact on agricultural production and is an extremely useful technology in the field.

Claims

1. A method for producing a temperature-sensitive male sterile plant, comprising: a step of artificially suppressing the function of at least one gene selected from the group consisting of the following (a) and (c) derived from the plant. (a) A gene encoding a protein consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 22 and having an activity of suppressing the impartation of temperature-sensitive male sterility. (c) A gene encoding a protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 22 and having an activity of suppressing the impartation of temperature-sensitive male sterility.

2. A temperature-sensitive male sterile plant in which the function of at least one gene selected from the group consisting of the following (a) and (c) derived from the plant is artificially suppressed. (a) A gene encoding a protein consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 22 and having an activity of suppressing the impartation of temperature-sensitive male sterility. (c) A gene encoding a protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 22 and having an activity of suppressing the impartation of temperature-sensitive male sterility.

3. A method for producing hybrid seeds, comprising: a step of cultivating at a restrictive temperature to make male sterile, and cross-pollinating the temperature-sensitive male sterile plant according to claim 2 with any plant, and a step of collecting seeds from the temperature-sensitive male sterile plant. The method comprising.

4. Hybrid seeds, wherein the temperature-sensitive male sterile plant according to claim 2, which is cultivated at a restrictive temperature to make male sterile, is used as the female parent, and any plant is used as the male parent.

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