Solanaceae male-fertile restorer plant, solanaceae male-fertile restorer method, solanaceae male-fertile restorer plant production method, and solanaceae male-fertile restorer discrimination method

Abstract

At least one of the functional defects or suppressions of the male fertility restorer genes in (a) to (o) of the male fertile restorer plant of the Solanaceae. The restorer genes of (a) to (o) are genes encoding a protein or the like comprising the amino acid sequence shown in any one of SEQ LIDs 1 to 5.

Description

Technical Field

[0001] This invention relates to male fertility restoration plants of the Solanaceae family, methods for restoring male fertility in Solanaceae plants, methods for manufacturing male fertility restoration plants of the Solanaceae family, and methods for determining male fertility restoration in Solanaceae plants.

[0002] This application claims priority based on Japanese Patent Application No. 2024-051561, filed in Japan on March 27, 2024, the contents of which are incorporated herein by reference. Background Technology

[0003] In the hybridization of crops such as vegetables, the process involves collecting F1 seeds from two different parental systems: the seed parent and the pollen parent. To prevent self-pollination from the seed parent system, a process called emasculation is performed, removing the anthers from the seed parent. However, this emasculation process is currently mainly done manually, requiring labor and incurring costs, leading to high seed prices. Furthermore, the involvement of multiple workers raises concerns about the potential leakage of the parental system, a crucial piece of intellectual property. Additionally, manual emasculation has a certain degree of failure rate, potentially resulting in the introduction of pollen from the seed parent and the inclusion of seeds from uncrossed lines.

[0004] As a countermeasure, by making the seed parent male sterile, that is, not producing pollen, or producing sterile, that is, non-hybridizing, inactive pollen, the possibility of pollination of the seed parent can be eliminated even without manual removal of males.

[0005] For example, in rice, hybridization techniques utilizing cytoplasmic male sterility (CMS) are known. As an example of cytoplasmic male sterility, the incompatibility of gene products between the cell nucleus and mitochondria leads to pollen inactivation, preventing hybridization and the production of seeds and fruits. In many plant species, cytoplasm is transferred only from the female side; therefore, cytoplasmic replacement systems, in which different types of cytoplasm are substituted, are used to create CMS systems.

[0006] Patent document 1 discloses a simplified method for preparing male-sterile tomato plants. The method involves fusing protoplasts isolated from tomato plants and treated with cytoplasmic factor desiccation with protoplasts isolated from Solanum plants and treated with nuclear genetic material desiccation, thereby regenerating male-sterile tomatoes from the fused material. This technology aims to create a system with male sterility through protoplast fusion without altering any traits of the target tomato plant other than male sterility, efficiently rendering the target tomato plant male-sterile in a short time.

[0007] Patent document 2 describes a gene containing specific DNA that causes RT-type cytoplasmic male sterility in rice and a method for determining sterility using this gene. In this technology, a mitochondrial gene causing RT-type cytoplasmic male sterility in rice is identified, and this gene can also be used as a DNA marker to identify cytoplasmic male sterile rice systems.

[0008] Existing technical documents Patent documents Patent Document 1: Japanese Patent No. 2824841 Patent Document 2: International Publication No. 2014 / 027502 Summary of the Invention The problem that the invention aims to solve In the technology of Patent Document 1, although a system with male sterility can be obtained from tomatoes (Solanaceae family), the gene for cytoplasmic male sterility is not yet identified. Therefore, although it is possible to use the aforementioned sterility system to produce plants without Solanaceae fruit, propagation is impossible because seeds cannot be produced. To apply the system with male sterility, it is necessary to identify the gene causing cytoplasmic male sterility and to be able to manipulate the on / off state of this gene.

[0009] Patent document 2 discloses the gene for cytoplasmic male sterility in the Oryzae family, but does not disclose the gene causing cytoplasmic male sterility in Solanaceae plants.

[0010] The present invention was made in view of the above circumstances, and its purpose is to provide a new technique for restoring cytoplasmic male sterility in Solanaceae.

[0011] Solution for solving the problem To address the aforementioned issues, the present invention includes the following methods.

[0012] [1] A male fertility-restoring plant of the Solanaceae family, wherein, At least one of the following (a) to (o) fertility restoration genes is functionally impaired or repressed: (a) A fertility restoration gene encoding a protein containing the amino acid sequence shown in sequence number 1; (b) A fertility restoration gene that encodes a protein containing an amino acid sequence in which one or more amino acids are missing, substituted, added or inserted, as shown in sequence number 1, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (c) A fertility restoration gene that encodes a protein containing an amino acid sequence having more than 60% sequence identity with the amino acid sequence shown in sequence number 1, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (d) A fertility restoration gene encoding a protein containing the amino acid sequence shown in sequence number 2; (e) A fertility restoration gene that encodes a protein containing an amino acid sequence in which one or more amino acids are missing, substituted, added or inserted, as shown in sequence number 2, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (f) A fertility restoration gene that encodes a protein containing an amino acid sequence having more than 60% sequence identity with the amino acid sequence shown in sequence number 2, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (g) A fertility restoration gene encoding a protein containing the amino acid sequence shown in sequence number 3; (h) Encodes a protein containing an amino acid sequence in which one or more amino acids are missing, substituted, added or inserted, as shown in sequence number 3, and a fertility restoration gene that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (i) A fertility restoration gene that encodes a protein containing an amino acid sequence having more than 60% sequence identity with the amino acid sequence shown in sequence number 3, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (j) A fertility restoration gene encoding a protein containing the amino acid sequence shown in sequence number 4; (k) Encodes a protein containing an amino acid sequence in which one or more amino acids are missing, substituted, added or inserted, as shown in sequence number 4, and a fertility restoration gene that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (l) A fertility restoration gene that encodes a protein containing an amino acid sequence having more than 60% sequence identity with the amino acid sequence shown in sequence number 4, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (m) A fertility restoration gene encoding a protein containing the amino acid sequence shown in sequence number 5; (n) A fertility restoration gene encoding a protein comprising an amino acid sequence in which one or more amino acids are missing, substituted, added, or inserted, as shown in sequence number 5, and which restores male fertility in Solanaceae plants by causing functional impairment or repression; and (o) A fertility restoration gene that encodes a protein containing an amino acid sequence having more than 60% sequence identity with the amino acid sequence shown in sequence number 5, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition.

[0013] [2] The male fertility restoration plant of Solanaceae as described in [1], wherein the male fertility restoration plant of Solanaceae is Solanum.

[0014] [3] The male fertility restoration plant of Solanaceae as described in [2], wherein the male fertility restoration plant of Solanaceae is tomato.

[0015] [4] The male fertility restoration plant of Solanaceae as described in [2], wherein the male fertility restoration plant of Solanaceae is potato.

[0016] [5] A method for restoring male fertility in Solanaceae plants, wherein, This method includes steps to restore the function of genes that have been impaired or suppressed in Solanaceae plants. The fertility restoration gene is at least one of the following (a) to (o): (a) A fertility restoration gene encoding a protein containing the amino acid sequence shown in sequence number 1; (b) A fertility restoration gene that encodes a protein containing an amino acid sequence in which one or more amino acids are missing, substituted, added or inserted, as shown in sequence number 1, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (c) A fertility restoration gene that encodes a protein containing an amino acid sequence having more than 60% sequence identity with the amino acid sequence shown in sequence number 1, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (d) A fertility restoration gene encoding a protein containing the amino acid sequence shown in sequence number 2; (e) A fertility restoration gene that encodes a protein containing an amino acid sequence in which one or more amino acids are missing, substituted, added or inserted, as shown in sequence number 2, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (f) A fertility restoration gene that encodes a protein containing an amino acid sequence having more than 60% sequence identity with the amino acid sequence shown in sequence number 2, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (g) A fertility restoration gene encoding a protein containing the amino acid sequence shown in sequence number 3; (h) Encodes a protein containing an amino acid sequence in which one or more amino acids are missing, substituted, added or inserted, as shown in sequence number 3, and a fertility restoration gene that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (i) A fertility restoration gene that encodes a protein containing an amino acid sequence having more than 60% sequence identity with the amino acid sequence shown in sequence number 3, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (j) A fertility restoration gene encoding a protein containing the amino acid sequence shown in sequence number 4; (k) Encodes a protein containing an amino acid sequence in which one or more amino acids are missing, substituted, added or inserted, as shown in sequence number 4, and a fertility restoration gene that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (l) A fertility restoration gene that encodes a protein containing an amino acid sequence having more than 60% sequence identity with the amino acid sequence shown in sequence number 4, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (m) A fertility restoration gene encoding a protein containing the amino acid sequence shown in sequence number 5; (n) A fertility restoration gene encoding a protein comprising an amino acid sequence in which one or more amino acids are missing, substituted, added, or inserted, as shown in sequence number 5, and which restores male fertility in Solanaceae plants by causing functional impairment or repression; and (o) A fertility restoration gene that encodes a protein containing an amino acid sequence having more than 60% sequence identity with the amino acid sequence shown in sequence number 5, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition.

[0017] [6] The method for restoring male fertility of Solanaceae plants according to [5], wherein the Solanaceae plant is Solanum.

[0018] [7] The method for restoring male fertility of a Solanaceae plant according to [6], wherein the Solanaceae plant is a tomato.

[0019] [8] The method for restoring male fertility of a Solanaceae plant according to [6], wherein the Solanaceae plant is potato.

[0020] [9] A method for manufacturing a male fertility restored plant of the Solanaceae family, wherein the method comprises: obtaining a Solanaceae plant with restored male fertility by means of the male fertility restoration method of any one of [5] to [8].

[0021]

[10] A method for determining the restoration of male fertility in Solanaceae plants, which is a method for determining whether the fertility of cytoplasmic male sterility in Solanaceae plants has been restored, wherein, The procedure includes the amplification of part or all of the base sequence of at least one of the following (a) to (o): (a) A fertility restoration gene encoding a protein containing the amino acid sequence shown in sequence number 1; (b) A fertility restoration gene that encodes a protein containing an amino acid sequence in which one or more amino acids are missing, substituted, added or inserted, as shown in sequence number 1, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (c) A fertility restoration gene that encodes a protein containing an amino acid sequence having more than 60% sequence identity with the amino acid sequence shown in sequence number 1, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (d) A fertility restoration gene encoding a protein containing the amino acid sequence shown in sequence number 2; (e) A fertility restoration gene that encodes a protein containing an amino acid sequence in which one or more amino acids are missing, substituted, added or inserted, as shown in sequence number 2, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (f) A fertility restoration gene that encodes a protein containing an amino acid sequence having more than 60% sequence identity with the amino acid sequence shown in sequence number 2, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (g) A fertility restoration gene encoding a protein containing the amino acid sequence shown in sequence number 3; (h) Encodes a protein containing an amino acid sequence in which one or more amino acids are missing, substituted, added or inserted, as shown in sequence number 3, and a fertility restoration gene that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (i) A fertility restoration gene that encodes a protein containing an amino acid sequence having more than 60% sequence identity with the amino acid sequence shown in sequence number 3, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (j) A fertility restoration gene encoding a protein containing the amino acid sequence shown in sequence number 4; (k) Encodes a protein containing an amino acid sequence in which one or more amino acids are missing, substituted, added or inserted, as shown in sequence number 4, and a fertility restoration gene that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (l) A fertility restoration gene that encodes a protein containing an amino acid sequence having more than 60% sequence identity with the amino acid sequence shown in sequence number 4, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (m) A fertility restoration gene encoding a protein containing the amino acid sequence shown in sequence number 5; (n) A fertility restoration gene encoding a protein comprising an amino acid sequence in which one or more amino acids are missing, substituted, added, or inserted, as shown in sequence number 5, and which restores male fertility in Solanaceae plants by causing functional impairment or repression; and (o) A fertility restoration gene that encodes a protein containing an amino acid sequence having more than 60% sequence identity with the amino acid sequence shown in sequence number 5, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition.

[0022] Invention Effects According to the present invention, a novel technique for restoring cytoplasmic male sterility in Solanaceae plants can be provided. Attached Figure Description

[0023] Figure 1 This is a graph showing the germination rate of pollen collected from 13 male fertility restoration systems obtained through EMS treatment.

[0024] Figure 2 This is an attached diagram showing the variation content of three systems with variations in the bacteriophage RNA polymerase gene.

[0025] Figure 3 These are images representing pollen germination in three systems where the phage RNA polymerase gene has been knocked out.

[0026] Figure 4 It is an image representing pollen germination in a system where the DNA topoisomerase 1 gene, pentapeptide repeat (PPR) gene, serine / threonine protein kinase gene, or polymerase γ2 (POLYMERASE GAMMA2) gene has been knocked out.

[0027] Figure 5 This is an image of the bands representing the results of restriction enzyme treatment for amplifying the fertility restorer gene in each fertility restorer system of EMS#1, #2, #7, #9, #10, #11, #12, #14, #16, #17, and #20. Detailed Implementation

[0028] The following provides a detailed description of preferred embodiments of the Solanaceae male fertility restoration plant, a method for restoring male fertility in Solanaceae plants, a method for manufacturing the Solanaceae male fertility restoration plant, and a method for determining male fertility restoration in Solanaceae plants. However, the present invention is not limited to the following embodiments.

[0029] [Male fertility-restoring plants in the Solanaceae family] The male fertility-restoring plant of the Solanaceae family in this embodiment is a plant that has restored fertility from cytoplasmic male sterility (CMS), wherein at least one of the following fertility-restoring genes (in other words, at least one fertility-restoring gene selected from the group consisting of (a) to (o)) has a functional deficit or suppression: (a) A fertility restoration gene encoding a protein containing the amino acid sequence shown in sequence number 1; (b) A fertility restoration gene that encodes a protein containing an amino acid sequence in which one or more amino acids are missing, substituted, added or inserted, as shown in sequence number 1, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (c) A fertility restoration gene that encodes a protein containing an amino acid sequence having more than 60% sequence identity with the amino acid sequence shown in sequence number 1, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (d) A fertility restoration gene encoding a protein containing the amino acid sequence shown in sequence number 2; (e) A fertility restoration gene that encodes a protein containing an amino acid sequence in which one or more amino acids are missing, substituted, added or inserted, as shown in sequence number 2, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (f) A fertility restoration gene that encodes a protein containing an amino acid sequence having more than 60% sequence identity with the amino acid sequence shown in sequence number 2, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (g) A fertility restoration gene encoding a protein containing the amino acid sequence shown in sequence number 3; (h) Encodes a protein containing an amino acid sequence in which one or more amino acids are missing, substituted, added or inserted, as shown in sequence number 3, and a fertility restoration gene that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (i) A fertility restoration gene that encodes a protein containing an amino acid sequence having more than 60% sequence identity with the amino acid sequence shown in sequence number 3, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (j) A fertility restoration gene encoding a protein containing the amino acid sequence shown in sequence number 4; (k) Encodes a protein containing an amino acid sequence in which one or more amino acids are missing, substituted, added or inserted, as shown in sequence number 4, and a fertility restoration gene that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (l) A fertility restoration gene that encodes a protein containing an amino acid sequence having more than 60% sequence identity with the amino acid sequence shown in sequence number 4, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (m) A fertility restoration gene encoding a protein containing the amino acid sequence shown in sequence number 5; (n) A fertility restoration gene encoding a protein comprising an amino acid sequence in which one or more amino acids are missing, substituted, added, or inserted, as shown in sequence number 5, and which restores male fertility in Solanaceae plants by causing functional impairment or repression; and (o) A fertility restoration gene that encodes a protein containing an amino acid sequence having more than 60% sequence identity with the amino acid sequence shown in sequence number 5, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition.

[0030] In this specification, "deficiency or suppression of gene function" means that the activity of the gene product is compromised or suppressed, or the expression of the gene is compromised or suppressed, through artificial manipulation of the gene or its expression.

[0031] Gene function loss or suppression can be determined by comparing artificially manipulated fertility-restoring plants with cytoplasmic male-sterile plants as controls. Artificial manipulation can include, but is not limited to, the artificial introduction or expression of nucleic acids, proteins, etc., that cause loss or suppression of gene expression.

[0032] The aforementioned "plants that restore fertility from cytoplasmic male sterility (CMS)" can also be plants possessing orf137 (see Japanese Patent Application Publication No. 2023-32890), which is the causative gene (S factor gene) of cytoplasmic male sterility, and in which at least one of the fertility restoration genes mentioned in (a) to (o) above is functionally deficient or repressed. However, as will be explained in detail below, the present invention is also applicable to Solanaceae plants with cytoplasmic male sterility that possess a gene different from orf137, and therefore the Solanaceae plants of the present invention are not limited to plants possessing the orf137 gene.

[0033] In this specification, "orf137 gene" refers to a gene that encodes a protein containing an amino acid sequence that has more than 80% (preferably more than 95%) sequence identity with the amino acid sequence described in sequence number 6 and causes cytoplasmic male sterility.

[0034] The fertility-restoring genes (a) to (o) mentioned above are genes that restore male fertility in Solanaceae plants with cytoplasmic male sterility through functional loss or suppression, and they are genes located on nuclear DNA. The fertility-restoring genes (a) to (o) can be considered to all play a role in inducing male sterility in Solanaceae plants, and the functional loss or suppression of the fertility-restoring genes (a) to (o) restores cytoplasmic male fertility.

[0035] The fertility restoration gene in (a) above is the phage RNA polymerase gene in tomato (gene ID: Solyc05g010660). The amino acid sequence of the protein encoded by the fertility restoration gene in (c) above has a sequence identity of 60% or more, preferably 75% or more, more preferably 80% or more, further preferably 90% or more, and most preferably 95% or more.

[0036] The fertility restoration gene in (d) above is the DNA topoisomerase 1 gene in tomato (gene ID: Solyc02g081910). The amino acid sequence of the protein encoded by the fertility restoration gene in (f) above has a sequence identity of 60% or more, preferably 75% or more, more preferably 80% or more, further preferably 90% or more, and most preferably 95% or more.

[0037] The fertility restoration gene in (g) above is the pentapeptide repeat (PPR) gene in tomato (gene ID: Solyc07g053060). The amino acid sequence of the protein encoded by the fertility restoration gene in (i) above has a sequence identity of 60% or more, preferably 75% or more, more preferably 80% or more, further preferably 90% or more, and most preferably 95% or more.

[0038] The fertility restoration gene in (j) above is the serine / threonine protein kinase gene in tomato (gene ID: Solyc01g109080). The amino acid sequence of the protein encoded by the fertility restoration gene in (l) above has a sequence identity of 60% or more, preferably 75% or more, more preferably 80% or more, further preferably 90% or more, and most preferably 95% or more.

[0039] The fertility restoration gene mentioned above (m) is the polymerase γ2 gene in tomato (gene ID: Solyc07g064330). The amino acid sequence of the protein encoded by the fertility restoration gene mentioned above (o) has a sequence identity of 60% or more, preferably 75% or more, more preferably 80% or more, further preferably 90% or more, and most preferably 95% or more.

[0040] In the protein encoded by the fertility restoration gene in (b) above, the number of amino acids that can be deleted, substituted, added or inserted in the amino acid sequence shown in sequence number 1 is preferably 1 or more and 30 or less, more preferably 1 or more and 20 or less, more preferably 1 or more and 10 or less, particularly preferably 1 or more and 5 or less, and most preferably 1 or more and 3 or less.

[0041] In the protein encoded by the fertility restoration gene in (e) above, the number of amino acids that can be deleted, substituted, added or inserted in the amino acid sequence shown in sequence number 2 is preferably 1 or more and 30 or less, more preferably 1 or more and 20 or less, more preferably 1 or more and 10 or less, particularly preferably 1 or more and 5 or less, and most preferably 1 or more and 3 or less.

[0042] In the protein encoded by the fertility restoration gene in (h) above, the number of amino acids that can be deleted, substituted, added or inserted in the amino acid sequence shown in sequence number 3 is preferably 1 or more and 30 or less, more preferably 1 or more and 20 or less, more preferably 1 or more and 10 or less, particularly preferably 1 or more and 5 or less, and most preferably 1 or more and 3 or less.

[0043] In the protein encoded by the fertility restoration gene of (k) above, the number of amino acids that can be deleted, substituted, added or inserted in the amino acid sequence shown in sequence number 4 is preferably 1 or more and 30 or less, more preferably 1 or more and 20 or less, more preferably 1 or more and 10 or less, particularly preferably 1 or more and 5 or less, and most preferably 1 or more and 3 or less.

[0044] In the protein encoded by the fertility restoration gene of (n) above, the number of amino acids that can be deleted, substituted, added or inserted in the amino acid sequence shown in sequence number 5 is preferably 1 or more and 30 or less, more preferably 1 or more and 20 or less, more preferably 1 or more and 10 or less, particularly preferably 1 or more and 5 or less, and most preferably 1 or more and 3 or less.

[0045] In this specification, the sequence identity of the target amino acid sequence relative to a reference amino acid sequence (e.g., the amino acid sequence shown in any of sequence numbers 1 to 6) can be determined as follows. First, the reference amino acid sequence and the target amino acid sequence are aligned. In each amino acid sequence, gaps are included in a manner that maximizes sequence identity. Next, the number of identical amino acids in the reference amino acid sequence and the target amino acid sequence is calculated, and the sequence identity can be calculated according to the following formula (1).

[0046] Sequence identity (%) = Number of identical amino acids / Total number of amino acids in the target amino acid sequence × 100… (1) Whether male fertility restoration (cytoplasmic male fertility restoration) in Solanaceae plants occurs by impairing or suppressing the function of the fertility restoration genes (a) to (o) mentioned above can be determined not only by whether the pollen obtained from the Solanaceae plant contains substances that cause pollen tube elongation (pollen germination) (fertility restoration occurs if these substances are present), but also by whether female pollen from normal individuals forms seeds. However, the determination of whether male fertility restoration occurs in Solanaceae plants is not limited to these methods.

[0047] The list of Solanaceae plants is not specifically limited; examples include plants from the genera *Solanum* and *Capsicum*. Examples of plants from the genus *Solanum* include eggplant (*Solanum melongena*), potato (*Solanum tuberosum*), or tomato (*Solanum lycopersicum*). Examples of plants from the genus *Capsicum* include chili pepper or bell pepper (*Capsicum annuum*). Species possessing the *orf137* gene also exist in the cytoplasmic male sterility systems of other Solanaceae plants such as potato.

[0048] It should be noted that by depleting or suppressing the function of any one of the fertility restoration genes in (a) to (c) above, the expression levels of various mRNAs expressed by mitochondrial DNA are inhibited. Furthermore, by depleting or suppressing the function of any one of the fertility restoration genes in (d) to (f) above and / or any one of the fertility restoration genes in (m) to (o) above, the absolute copy number of mitochondrial DNA is reduced. Therefore, even in Solanaceae plants with cytoplasmic male sterility where the causative gene for cytoplasmic male sterility is different from the orf137 gene, fertility restoration can be achieved by depleting or suppressing the function of at least one fertility restoration gene selected from the group consisting of the fertility restoration genes in (a) to (f) and the fertility restoration genes in (m) to (o) above.

[0049] Solanaceous plants include species with edible fruits such as eggplants, tomatoes, chili peppers and green peppers, and species with edible tubers such as potatoes. In addition, they cover a wide range of plants including those cultivated as stimulants such as tobacco, and those used in the horticultural field for ornamental purposes such as physalis and petunias. The techniques of the respective embodiments described in detail in the present specification can be applied to these solanaceous plants for phylogenetic operations such as crossing, and thus have a wide range of utilization.

[0050] The solanaceous plant is particularly preferably a crop, that is, a plant utilized in the agricultural field especially as a food material. In addition to the plant body, the solanaceous plant may also be a plant cultured cell, a plant cell, or a callus.

[0051] [[ID=6)][Method for Restoring Male Fertility of Solanaceous Plants] The method for restoring male fertility of a solanaceous plant according to the present embodiment includes the step of causing the function of at least one fertility restorer gene in the above (a) to (o) of the solanaceous plant to be defective or inhibited. The solanaceous male fertility-restored plant is a solanaceous plant or a progeny thereof obtained by this method for restoring male fertility.

[0052] The method for causing the function of at least one fertility restorer gene in the above (a) to (o) of a solanaceous plant to be defective or inhibited is not particularly limited, and examples thereof include treating a solanaceous plant having cytoplasmic male sterility with a mutagen such as EMS. After treatment with the mutagen, a solanaceous plant with restored male fertility can be obtained by screening for a solanaceous plant in which the function of any one or more of the above fertility restorer genes is defective or inhibited. Alternatively, it is also possible to artificially modify at least one fertility restorer gene in the above (a) to (o) by a genome editing technique, so that the function of the fertility restorer gene is defective or inhibited.

[0053] Examples of the genome editing technique include methods capable of modifying the base sequence of DNA, such as gene targeting methods, CRISPR-Cas systems, zinc finger nucleases, and TALENs, but are not limited thereto. It should be noted that "modification" refers to a change in the base sequence of the target gene.

[0054] The region edited by the genome editing technique may be, for example, at least one fertility restorer gene in the above (a) to (o) and / or its promoter.

[0055] <CRISPR-Cas System> In the CRISPR-Cas system, a guide RNA (gRNA) or a polynucleotide encoding such gRNA and a CRISPR-Cas enzyme or a CRISPR-Cas enzyme are introduced into a Solanaceae plant. The polynucleotides encoding the gRNA and the CRISPR-Cas enzyme are preferably introduced into the Solanaceae plant in a state integrated into the expression vector.

[0056] The aforementioned gRNA contains a base sequence complementary to a portion of the sequence of any of the fertility restoration genes and / or their promoters mentioned in (a) to (o) above. This refers to a substance that constitutes an adaptive immune system in bacteria and archaea, providing acquired resistance to invading foreign nucleic acids, mimicking the hairpin structure of a tracrRNA-crRNA chimera formed by fusing a small RNA fragment (CRISPR-RNA: crRNA) containing a foreign sequence (guide sequence) and a partially complementary RNA (trans-activating crRNA: tracrRNA). The aforementioned gRNA contains, at its 5' end, a polynucleotide sequence consisting of a base sequence upstream of 1 base of the PAM (proto-spacer adjacent motif) sequence contained in any of the fertility restoration genes mentioned in (a) to (o) above, preferably 17 to 24 bases.

[0057] The PAM sequence is present in the fertility restoration genes (a) to (o) mentioned above, which are target genes. It is a sequence that can be recognized by the CRISPR-Cas enzyme, and the sequence varies depending on the bacterial species that is the source of the CRISPR-Cas enzyme.

[0058] CRISPR-Cas enzymes are a family of Cas proteins found in bacteria and archaea that form part of the adaptive immune system that provides resistance to invading foreign nucleic acids. They are endonucleases that recognize the PAM sequence and cleave the double-stranded DNA upstream of it, creating a smooth end. CRISPR-Cas enzymes can be Cas9 or its homologs or variants, or other types.

[0059] Pollen obtained from fertility-restored Solanaceae plants is hemimeric; therefore, by using a CRISPR-Cas system or similar method to delete (knock out) or suppress (knock down) at least one of the fertility restoration genes mentioned in (a) to (o) above, fertility can be restored by deleting or suppressing the fertility restoration gene of at least one allele. However, deleting or suppressing the fertility restoration genes of two alleles can further improve pollen germination rate.

[0060] <Using nucleic acids to suppress the expression of fertility-restoring genes> As a method to suppress the expression of at least one of the fertility restoration genes mentioned in (a) to (o) above, antisense nucleic acids, ribozymes, and RNAi-inducible nucleic acids can be listed.

[0061] As an antisense nucleic acid, for example, it can be listed as a nucleic acid containing a base sequence complementary to the sense sequence (mRNA sequence) of any of the fertility restoration genes mentioned above (a) to (o).

[0062] Those skilled in the art can, based on the base sequence of the cDNA of the fertility restoration gene of (a) above (the base sequence shown in sequence number 7), appropriately set the base sequence of the antisense nucleic acid that inhibits the expression of the fertility restoration gene of (a) above.

[0063] Those skilled in the art can, based on the base sequence of the cDNA of the fertility restoration gene described above (d) (the base sequence shown in sequence number 8), appropriately set the base sequence of the antisense nucleic acid that inhibits the expression of the fertility restoration gene described above (d).

[0064] Those skilled in the art can, based on the base sequence of the cDNA of the fertility restoration gene of (g) above (the base sequence shown in sequence number 9), appropriately set the base sequence of the antisense nucleic acid that inhibits the expression of the fertility restoration gene of (g) above.

[0065] Those skilled in the art can, based on the base sequence of the cDNA of the fertility restoration gene of (j) above (the base sequence shown in sequence number 10), appropriately set the base sequence of the antisense nucleic acid that inhibits the expression of the fertility restoration gene of (j) above.

[0066] Those skilled in the art can, based on the base sequence of the cDNA of the fertility restoration gene of (m) (the base sequence shown in sequence number 11), appropriately set the base sequence of the antisense nucleic acid that inhibits the expression of the fertility restoration gene of (m).

[0067] Ribozymes are catalytically active RNAs that have an active site for reacting with a fertility restoration gene as a target and a substrate-binding site for binding to the fertility restoration gene. Examples of ribozymes, for instance, as substrate-binding sites, include those with sequences complementary to the sequence of the fertility restoration gene mRNA.

[0068] Those skilled in the art can, based on the base sequence of the cDNA of the fertility restoration gene in (a) above (the base sequence shown in sequence number 7), appropriately set the base sequence of the ribozyme that inhibits the expression of the fertility restoration gene in (a) above.

[0069] Those skilled in the art can, based on the base sequence of the cDNA of the fertility restoration gene described above (d) (the base sequence shown in sequence number 8), appropriately set the base sequence of the ribozyme that inhibits the expression of the fertility restoration gene described above (d).

[0070] Those skilled in the art can, based on the base sequence of the cDNA of the fertility restoration gene of (g) above (the base sequence shown in sequence number 9), appropriately set the base sequence of the ribozyme that inhibits the expression of the fertility restoration gene of (g) above.

[0071] Those skilled in the art can, based on the base sequence of the cDNA of the fertility restoration gene of (j) above (the base sequence shown in sequence number 10), appropriately set the base sequence of the ribozyme that inhibits the expression of the fertility restoration gene of (j) above.

[0072] Those skilled in the art can, based on the base sequence of the cDNA of the fertility restoration gene of (m) (the base sequence shown in sequence number 11), appropriately set the base sequence of the ribozyme that inhibits the expression of the fertility restoration gene of (m).

[0073] As RNAi-inducible nucleic acids, examples include siRNA, miRNA, and shRNA. More specifically, examples include double-stranded RNAs containing a portion or all of the base sequence of the mRNA of any of the fertility restoration genes mentioned in (a) to (o) above.

[0074] <Methods for introducing plants into the Solanaceae family> Methods for introducing expression vectors used to alter the sequence of at least one fertility restoration gene and / or its promoter from (a) to (o) above, and methods for introducing the aforementioned nucleic acids used to suppress expression into Solanaceae plants include, for example, the Agrobacterium method, gene gun method, polyethylene glycol (PEG) method, calcium phosphate method, electroporation method, liposome method, and DEAE dextran method. Among these methods, the Agrobacterium method is preferred.

[0075] When using the Agrobacterium method, binary vectors of the pBI or pPZP lineage are preferred. Examples of binary vectors of the pBI lineage include pBIG, pBIN19, pBI101, pBI121, and pBI221. Examples of binary vectors of the pPZP lineage include pPZP100, pPZP200, pPZP500, and pPZP3425.

[0076] [Methods for producing male fertility-restoring plants from the Solanaceae family] The method for manufacturing a male-fertility-restoring plant of the Solanaceae family according to this embodiment includes obtaining a Solanaceae plant with restored male fertility through the "Method for Restoring Male Fertility of Solanaceae Plants" described in this embodiment. The Solanaceae plant obtained by the manufacturing method of this embodiment is a Solanaceae plant that restores fertility from cytoplasmic male sterility (CMS) by causing functional loss or suppression of at least one of the fertility restoration genes mentioned in (a) to (o) above.

[0077] [Methods for determining the restoration of male fertility in Solanaceae plants] The method for determining the restoration of male fertility in Solanaceae plants according to this embodiment is a method for determining whether the fertility of cytoplasmic male sterility in Solanaceae plants has been restored, including the step of amplifying part or all of the regions of at least one of the fertility restoration genes in (a) to (o) above.

[0078] For example, as described in the embodiments below, a Solanaceae plant alters the base sequence of at least one of the fertility restoration genes in (a) to (o) above using genome editing technology, thereby causing the fertility restoration gene to be functionally deficient or suppressed. By amplifying part or all of the fertility restoration gene through PCR or the like, and confirming the base sequence of the amplified product, it is possible to determine whether it is a plant that has restored male fertility.

[0079] As methods for confirming the base sequence of the amplified product, in addition to sequencing the amplified product to confirm the specific base sequence, methods such as confirming the base sequence of the amplified product based on whether the restriction enzyme recognition sequence is included in the amplified product, confirming the length of the amplified product, and confirming whether amplification is possible can be listed, as described in the examples below. Those skilled in the art can appropriately design primer sequences for amplifying the fertility restoration genes (a) to (o) described above.

[0080] The following four groups can be listed as primers used as part of the fertility restoration gene for amplifying (a) above.

[0081] 1. A group of primers containing the base sequence shown in Serial No. 12 on the 3' end side (3' end region) and primers containing the base sequence shown in Serial No. 13 on the 3' end side.

[0082] 2. A group of primers containing the base sequence shown in Serial No. 14 at the 3' end and primers containing the base sequence shown in Serial No. 15 at the 3' end.

[0083] 3. A group of primers containing the base sequence shown in Serial No. 16 at the 3' end and primers containing the base sequence shown in Serial No. 17 at the 3' end.

[0084] 4. A group of primers containing the base sequence shown in Serial No. 18 at the 3' end and primers containing the base sequence shown in Serial No. 19 at the 3' end.

[0085] As a group of primers used to amplify the fertility restoration gene mentioned above (d), one such group can be listed, for example.

[0086] 1. A group of primers containing the base sequence shown in Serial No. 120 at the 3' end and primers containing the base sequence shown in Serial No. 21 at the 3' end.

[0087] For example, the following two groups can be listed as primers used as part of the fertility restoration gene for amplifying the above (g).

[0088] 1. A group of primers containing the base sequence shown in Serial No. 22 at the 3' end and primers containing the base sequence shown in Serial No. 23 at the 3' end.

[0089] 2. A group of primers containing the base sequence shown in Serial No. 24 at the 3' end and primers containing the base sequence shown in Serial No. 25 at the 3' end.

[0090] As a group of primers used to amplify the fertility restoration gene mentioned above (j), one such group can be listed, for example.

[0091] 1. A group of primers containing the base sequence shown in Serial No. 26 at the 3' end and primers containing the base sequence shown in Serial No. 27 at the 3' end.

[0092] The following three groups can be listed as primers used as part of the fertility restoration gene for amplifying the above (m).

[0093] 1. A group of primers containing the base sequence shown in Serial No. 28 at the 3' end and primers containing the base sequence shown in Serial No. 29 at the 3' end.

[0094] 2. A group of primers containing the base sequence shown in Serial No. 30 at the 3' end and primers containing the base sequence shown in Serial No. 31 at the 3' end.

[0095] 3. A group of primers containing the base sequence shown in Serial No. 32 at the 3' end and primers containing the base sequence shown in Serial No. 33 at the 3' end.

[0096] The primers mentioned above can be less than 55 nucleotides in length, less than 45 nucleotides in length, less than 40 nucleotides in length, less than 35 nucleotides in length, or less than 30 nucleotides in length.

[0097] There are no particular restrictions on the parts of the Solanaceae plant from which the fertility restoration gene is amplified; for example, it can be a leaf, a root, a stem, or a pollen.

[0098] This invention is not limited to the embodiments described above, and various modifications can be made within the scope of the invention as described in the claims, which are of course also included within the scope of this invention.

[0099] For example, in the above-described embodiment of "male fertility restoration plant of Solanaceae", the function of at least one fertility restoration gene in (a) to (o) of the male fertility restoration plant of Solanaceae is impaired or suppressed. However, male fertility restoration plants of Solanaceae with impaired or suppressed function of any one of the fertility restoration genes in (a) to (c), (d) to (f), (g) to (i), (j) to (l), and (m) to (o) can also be listed as male fertility restoration plants of Solanaceae in other embodiments of the present invention.

[0100] In addition, in the above-described "Method for Restoring Male Fertility in Solanaceae Plants", the method for restoring male fertility in Solanaceae plants includes a step of causing functional impairment or suppression of at least one of the fertility restoration genes in (a) to (o) above. However, the method for restoring male fertility in Solanaceae plants may also replace this step by including a step of causing functional impairment or suppression of any one of the fertility restoration genes in (a) to (c) above, a step of causing functional impairment or suppression of any one of the fertility restoration genes in (d) to (f) above, a step of causing functional impairment or suppression of any one of the fertility restoration genes in (g) to (i) above, a step of causing functional impairment or suppression of any one of the fertility restoration genes in (j) to (l) above, or a step of causing functional impairment or suppression of any one of the fertility restoration genes in (m) to (o) above. The same applies to the method for manufacturing male-fertility-restoring plants of the Solanaceae family, including those obtained through male fertility restoration methods of Solanaceae plants.

[0101] Example The present invention will be described below through embodiments, but the present invention is not limited to the following embodiments.

[0102] [Solanaceae plants used in experiments] Micro-Tom (TOMJPF0001), a dwarf tomato variety, was backcrossed more than four times in a CMS system of tomatoes known as CMS[P] obtained from NICHIREI, resulting in the replacement of the nuclear genome with Micro-Tom's Dwarf CMS[P] for this experiment. Dwarf CMS[P] was cultivated hydroponically at 23°C, 600 ppm CO2, and 16 hours of light. During cultivation, Otsuka fertilizer (12 g / L for Otsuka No. 1 and 8 g / L for Otsuka No. 2) was used in the nutrient solution, and the EC (electrical conductivity) was adjusted to 1.2 mS.

[0103] [The creation of a new male fertility restoration system] The seeds of Dwarf CMS[P] were soaked in distilled water for 4 hours to allow the water to penetrate to the interior. Next, the seeds were soaked in a 0.5% or 1% EMS (ethyl methanesulfonic acid) aqueous solution for 16 hours using a vortex mixer, followed by rinsing three times with distilled water. Approximately 1800 EMS-treated seeds were then transplanted into a hydroponic culture medium (coconut coir bed, Kaneko Seedling Company) and hydroponically cultivated in a greenhouse.

[0104] M1 generation seeds obtained through the above method were investigated for self-pollination to determine whether they formed self-pollinated seeds. M2 generation seeds (self-pollinated seeds) could be obtained using pollen fertility restoration (RF) systems. These fertility restoration (RF) systems for the M2 generation were named EMS#1, #2, #7, #9, #10, #11, #12, #14, #16, #17, #20, #21, and #22.

[0105] To maintain the new fertility restoration systems, each system was backcrossed into Dwarf CMS[P], and the resulting plant individuals were all cultivated in an indoor environment using rock wool.

[0106] [Germ formation rate of the new male fertility restoration system] Pollen obtained from the aforementioned male fertility restoration systems EMS#1, #2, #7, #9, #10, #11, #12, #14, #16, #17, #20, #21, and #22, the Micro-Tom system as a positive control, and the Dwarf CMS[P] system as a negative control were stained with aniline blue to confirm the germination rate (the proportion of pollen with elongated pollen tubes).

[0107] Figure 1 This is a graph showing the germination rate of pollen collected from 13 male fertility restoration systems obtained through EMS treatment.

[0108] like Figure 1As shown, the germination rate of pollen obtained from the Dwarf CMS[P] system, which served as a negative control, was 0%, with no pollen germinating at all. Conversely, the germination rate of pollen obtained from the Micro-Tom system, which served as a positive control, was 79%.

[0109] In contrast, pollen germination rates ranged from 3% to 36% in male fertility restoration systems EMS#1, #2, #7, #9, #10, #11, #12, #14, #16, #17, #20, #21, and #22. This confirms the male fertility restoration capabilities of male fertility restoration systems EMS#1, #2, #7, #9, #10, #11, #12, #14, #16, #17, #20, #21, and #22.

[0110] [Identification of fertility restoration genes based on BSA-Seq] (Batch DNA production and whole genome decoding) Total DNA was extracted from the leaves of the aforementioned male fertility restoration system using Maxwell 16 Purification Kits (Promega) and Maxwell 16 Instrument (Promega) to create batches of DNA for fertile and sterile groups. Whole-genome sequencing was performed on each batch of DNA using Rhelixa Co., Ltd. The whole-genome sequencing was conducted under Illumina NovaSeq 6000, PE150 (150bp × 2 paired-end), 30G bases per sample, and PCR-free conditions.

[0111] (Data Analysis) Trim Galore (https: / / github.com / FelixKrueger / TrimGalore / blob / master / Docs / Trim_Galore_User_Guide.md) is used to prune the data, removing header sequences, reads with a quality value less than 30, and reads smaller than 20bp.

[0112] Mapping was performed using BWA-MEM (Heng Li. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM.2013), and the reference genome was the nuclear genome SL4.0 of the tomato cultivar Heinz 1706 (https: / / solgenomics.net / organism / Solanum_lycopersicum / genome).

[0113] Variation detection was performed using gatk HaplotypeCaller (McKenna A, et al. 2010. The GenomeAnalysis Toolkit: a MapReduce framework for analyzing next-generation DNAsequencing data. Genome Res. 20(9):1297-1303.), and annotation data were performed using ITAG4.0 (https: / / solgenomics.net / organism / Solanum_lycopersicum / genome) for the tomato cultivar Heinz 1706. SNP-indexes for the fertility and sterility batches were calculated using bcftools (Heng Li. A statistical framework for SNP calling, mutation discovery, association mapping and population genetical parameter estimation from sequencing data. 2011.), and fertility restoration genes were screened by detecting variations with a ΔSNP-index near 0.5. The ΔSNP-index graph was constructed using QTL-seq (Sugihara et al. High-performance pipeline for MutMap and QTL-seq. PeerJ, 2022.). Furthermore, system-specific variants were extracted by comparing them with variant information obtained from variant detection in M2 individuals of each variant.

[0114] (Genetic analysis focusing on genes related to organelle DNA replication) For the M2 generation of each novel fertility restoration system, whole-genome sequencing was performed, and variant detection was conducted for each M2 system. Variants present in genes involved in mitochondrial DNA replication were extracted from the acquired variant information. Gene detection was performed using BLASTP, with an E-value set to 1×10⁻⁶. -10 .

[0115] (Identified fertility restoration gene) Based on the results of the BSA-Seq and organelle DNA replication-related gene analysis described above, the fertility restoration genes for fertility restoration systems EMS#1, #2, #7, #9, #10, #11, #12, #14, #16, #17, #20, #21, and #22 were identified as shown in Table 1 below. Specifically, BSA-Seq identified the phage RNA polymerase gene (gene ID: Solyc05g010660), the pentapeptide repeat (PPR) gene (gene ID: Solyc07g053060), and the serine / threonine protein kinase gene (gene ID: Solyc01g109080) as fertility restoration genes. Organelle DNA replication-related gene analysis identified the DNA topoisomerase 1 gene (gene ID: Solyc02g081910) and the polymerase γ2 gene (gene ID: Solyc07g064330) as fertility restoration genes.

[0116] Table 1 shows the fertility restorer genes identified for each fertility restorer system and the locations of variations generated within each fertility restorer gene. SL4.0 is a widely used reference genome in tomato research; for example, the phage RNA polymerase gene is a gene located in ch05 (chromosome 5).

[0117] [Table 1]

[0118] [Validation of various fertility restoration genes based on CRISPR-Cas9] In this experiment, firstly, in order to confirm that the phage RNA polymerase gene (gene ID: Solyc05g010660) identified as the fertility restoration gene is indeed the fertility restoration gene, the phage RNA polymerase gene of the tomato CMS system Dwarf CMS[P] was knocked out using a CRISPR-Cas9 vector.

[0119] (Construction of CRISPR-Cas9 vector) A CDS for the phage RNA polymerase gene was prepared, and two gRNA sequences (20 bp) were identified on the CDS using CRISPR-P (Yang Lei, et al., A WebTool for Synthetic Single-Guide RNA Design of CRISPR-System in Plants. Molecular Plant, 7(9), 1494-1496, 2014.). The gRNA sequences were 5'-ACTGCGAGAGGCATTCTCAG-3' (sequence number 34) and 5'-CTTGGTTCTGATTTATGCCG-3' (sequence number 35).

[0120] Complementary strand oligonucleotide DNAs of each gRNA sequence were synthesized by Eurofins Genomics Co., Ltd., with ATTG end-5' of one strand and AAAC end-5' of the other strand. 2 μL of each oligonucleotide DNA (50 μM) was added to 46 μL of distilled water, and the mixture was heated at 95°C for 5 minutes, followed by incubation at room temperature for 20 minutes to anneal the oligonucleotide DNAs. These were then mixed with BBSI-treated AtU6 gRNA (pMR217) and AtU6 gRNA (pMR218) and ligated using Ligation high Ver.2 (Toyobo Co., Ltd.). Reaction conditions followed Toyobo's protocol.

[0121] The ligated plasmid was introduced into *E. coli* DH5α (Toyobo Corporation) using a heat shock method. The sequence of the gRNA within the plasmid was confirmed using Sanger sequencing (Eurofins Genomics). AtU6 gRNAs (pMR217, pMR218) containing the target gRNA were mixed with pDe-Cas9-Kan (used as a Cas9 cassette), and the gRNA expression cassette was transferred to pDe-Cas9-Kan using an LR reaction (Thermo Fisher Scientific). The pDe-Cas9-Kan containing the target gRNA sequence was then introduced into *E. coli* DH5α using a heat shock method, and the sequence of the gRNA within the plasmid was confirmed using Sanger sequencing. The prepared plasmid was then introduced into *Agrobacterium* (GV3103) via electroporation.

[0122] (Knockout based on CRISPR-Cas9 vector) The vector prepared as described above was introduced into Dwarf CMS[P] via the Agrobacterium method, resulting in nine transformants. Sanger sequencing was used to investigate variations in the phage RNA polymerase gene, and three independent systems (CR#4, CR#8, and CR#9) with different introduced variations were identified.

[0123] Figure 2 This is an attached diagram showing the variation content of three systems with variations in the bacteriophage RNA polymerase gene.

[0124] like Figure 2 As shown, in the CR#4 system, there is a 4 bp deletion from position 109 to position 112 at the 5' end of exon 9 of the phage RNA polymerase gene.

[0125] In the CR#8 system, there is a 227 bp deletion from the 103rd base from the 5' end of exon 8 of the phage RNA polymerase gene to the 112th base from the 5' end of exon 9. In the CR#9 system, there is a 4 bp deletion in exon 8 of the phage RNA polymerase gene.

[0126] In the CR#9 system, the bases from position 98 to 101 are deleted from the 5' end of exon 8 of the phage RNA polymerase gene.

[0127] These deletions were all detected in a heterozygous state (deletion in only one allele). Furthermore, all were variants accompanied by frameshift mutations, thus suggesting a functional deficit in the phage RNA polymerase gene.

[0128] Figure 3 These are images representing pollen germination in three systems where the phage RNA polymerase gene has been knocked out.

[0129] The investigation into pollen germination on the stigma yielded the following results: Figure 3 As shown, pollen germination was observed in three independent systems: CR#4, CR#8, and CR#9. On the other hand, pollen germination was not confirmed in the unmutated transformant CR#6 (a WT type with the same base sequence as Dwarf CMS[P] for the phage RNA polymerase gene).

[0130] Based on the above results, it was confirmed that male fertility of the cytoplasmic male sterility system can be restored by depriving the phage RNA polymerase gene of function in the tomato CMS system Dwarf CMS[P].

[0131] Figure 4It is an image representing pollen germination in a system where the DNA topoisomerase 1 gene, pentapeptide repeat (PPR) gene, serine / threonine protein kinase gene, or polymerase γ2 gene has been knocked out.

[0132] Similar to the phage RNA polymerase gene, any one of these four genes was knocked out using the CRISPR-Cas9 vector.

[0133] The gRNA used to knock out the DNA topoisomerase 1 gene (gene ID: Solyc02g081910) uses gRNA with the base sequence shown in sequence number 36 as the first site and gRNA with the base sequence shown in sequence number 37 as the second site.

[0134] The gRNA used to knock out the pentapeptide repeat (PPR) gene (gene ID: Solyc07g053060) used gRNA with the base sequence shown in sequence number 38 as the first site and gRNA with the base sequence shown in sequence number 39 as the second site.

[0135] The gRNA used to knock out the serine / threonine protein kinase gene (gene ID: Solyc01g109080) uses gRNA with the base sequence shown in sequence number 40 as the first site and gRNA with the base sequence shown in sequence number 41 as the second site.

[0136] The gRNA used to knock out the polymerase γ2 gene (gene ID: Solyc07g064330) uses gRNA with the base sequence shown in sequence number 42 as the first site and gRNA with the base sequence shown in sequence number 43 as the second site.

[0137] like Figure 4 As shown, even pollen from individuals of any system lacking any of the four genes can be confirmed to germinate.

[0138] Based on these results, it can be confirmed that the DNA topoisomerase 1 gene, pentapeptide repeat (PPR) gene, serine / threonine protein kinase gene, and polymerase γ2 gene are all genes that restore male fertility in cytoplasmic male sterility systems by causing functional deficiencies.

[0139] (DNA marker assay) DNA markers were used to verify the fertility restoration genes for phage RNA polymerase, DNA topoisomerase 1, pentapeptide repeat (PPR), serine / threonine protein kinase, and polymerase γ2. PCR was performed using primers shown in Table 2 for F1 individuals produced by crossing F1 generation of novel fertility restoration systems with pollen from Micro-Tom, and for subsequent generations (BC1F and beyond) obtained by backcrossing each system to Dwarf CMS[P].

[0140] [Table 2]

[0141] In PCR, 10×PCR Buffer (Mg) is mixed. 2+ The PCR reaction solution consisted of 10 μL of the following: 1 μL of dNTP Mixture (each 2.5 mM (mol / L)), 0.8 μL of 10 μM Forward Primer, 0.3 μL of 10 μM Reverse Primer, 0.05 μL of 5 U / μL TaKaRaTaq HS (TAKARA), 0.5 μL of molded DNA, and 7.05 μL of distilled water.

[0142] Using a thermal cycler, a 3-step PCR was performed (initial thermal denaturation at 94°C for 3 minutes, thermal denaturation at 98°C for 10 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 30 seconds (35 cycles of thermal denaturation, annealing, and extension), and final extension at 72°C for 5 minutes).

[0143] After the PCR reaction, add 10 μL of a mixture of 0.1 μL of restriction enzyme, 2 μL of accompanying buffer, and 17.9 μL of distilled water to the PCR product and react overnight at the temperature shown in Table 2.

[0144] Next, a 3w / v% agarose gel containing Midori Green Advance DNA Stain (Genetics, Japan) was prepared, and 6μL of a mixture of 20μL PCR product and 4μL 6×Loading dye was injected.

[0145] Subsequently, electrophoresis was performed at 100V for 45 minutes, and the amplification of the bands was visualized using a UV transilluminator. Molecular weight markers were used using Gene Ladder Wide 1 (Nippon Gene).

[0146] Figure 5This is an image of the bands representing the results of restriction enzyme treatment for amplifying the fertility restorer gene in each fertility restorer system of EMS#1, #2, #7, #9, #10, #11, #12, #14, #16, #17, and #20.

[0147] exist Figure 5 In the images, the left lane contains samples of individuals whose fertility has been restored through mutations in any of the following genes: phage RNA polymerase gene, DNA topoisomerase 1 gene, pentapeptide repeat sequence (PPR) gene, serine / threonine protein kinase gene, and polymerase γ2 gene. The right lane contains samples of Dwarf CMS[P] individuals without mutations in these five genes.

[0148] Under the conditions shown in Table 2 above, the fertility restoration gene was amplified in each fertility restoration system, and the results of restriction enzyme cleavage were as follows: Figure 5 As shown in the left lane of each figure, the systems EMS#1, #2, #7, #9, #10, #11, #12, #14, #16, #17, and #20 are heterozygous combinations of alleles with and without variations. Therefore, bands from one cleaved allele and the other uncleaved allele were identified. For each fertility restoration gene, whether the allele with variation was cleaved, or whether the allele without variation was cleaved, is shown in Table 2.

[0149] On the other hand, in the right lane of the sample of Dwarf CMS[P] with five homozygous fertility restoration genes without variation, a single band from two alleles was identified.

[0150] The results show that by treating the amplification products obtained by PCR amplification using the primer sequences shown in Table 2 with restriction enzymes, it is possible to determine whether the individual Solanaceae plants used as samples contain alleles with variations for each fertility restoration gene (and thus, whether male sterility has been restored).

[0151] Industrial availability According to the present invention, by causing functional impairment or suppression of at least one of the fertility restoration genes in (a) to (o) above, the fertility of Solanaceae plants with cytoplasmic male sterility systems can be restored, and thus can be used industrially.

Claims

1. A male fertility-restoring plant belonging to the Solanaceae family, wherein, At least one of the following (a) to (o) fertility restoration genes is functionally impaired or repressed: (a) A fertility restoration gene encoding a protein containing the amino acid sequence shown in sequence number 1; (b) A fertility restoration gene that encodes a protein containing an amino acid sequence in which one or more amino acids are missing, substituted, added or inserted, as shown in sequence number 1, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (c) A fertility restoration gene that encodes a protein containing an amino acid sequence having more than 60% sequence identity with the amino acid sequence shown in sequence number 1, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (d) A fertility restoration gene encoding a protein containing the amino acid sequence shown in sequence number 2; (e) A fertility restoration gene that encodes a protein containing an amino acid sequence in which one or more amino acids are missing, substituted, added or inserted, as shown in sequence number 2, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (f) A fertility restoration gene that encodes a protein containing an amino acid sequence having more than 60% sequence identity with the amino acid sequence shown in sequence number 2, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (g) A fertility restoration gene encoding a protein containing the amino acid sequence shown in sequence number 3; (h) Encodes a protein containing an amino acid sequence in which one or more amino acids are missing, substituted, added or inserted, as shown in sequence number 3, and a fertility restoration gene that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (i) A fertility restoration gene that encodes a protein containing an amino acid sequence having more than 60% sequence identity with the amino acid sequence shown in sequence number 3, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (j) A fertility restoration gene encoding a protein containing the amino acid sequence shown in sequence number 4; (k) Encodes a protein containing an amino acid sequence in which one or more amino acids are missing, substituted, added or inserted, as shown in sequence number 4, and a fertility restoration gene that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (l) A fertility restoration gene that encodes a protein containing an amino acid sequence having more than 60% sequence identity with the amino acid sequence shown in sequence number 4, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (m) A fertility restoration gene encoding a protein containing the amino acid sequence shown in sequence number 5; (n) A fertility restoration gene that encodes a protein containing an amino acid sequence in which one or more amino acids are missing, substituted, added or inserted, as shown in sequence number 5, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. as well as (o) A fertility restoration gene that encodes a protein containing an amino acid sequence having more than 60% sequence identity with the amino acid sequence shown in sequence number 5, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition.

2. The male fertility-restoring plant of the Solanaceae family according to claim 1, wherein, The male fertility-restoring plant in the Solanaceae family is the genus *Solanum*.

3. The male fertility-restoring plant of the Solanaceae family according to claim 2, wherein, The male fertility-restoring plant of the Solanaceae family mentioned is the tomato.

4. The male fertility-restoring plant of the Solanaceae family according to claim 2, wherein, The male fertility-restoring plant of the Solanaceae family is the potato.

5. A method for restoring male fertility in a Solanaceae plant, wherein, This method includes steps to restore the function of genes that have been impaired or suppressed in Solanaceae plants. The fertility restoration gene is at least one of the following (a) to (o): (a) A fertility restoration gene encoding a protein containing the amino acid sequence shown in sequence number 1; (b) A fertility restoration gene that encodes a protein containing an amino acid sequence in which one or more amino acids are missing, substituted, added or inserted, as shown in sequence number 1, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (c) A fertility restoration gene that encodes a protein containing an amino acid sequence having more than 60% sequence identity with the amino acid sequence shown in sequence number 1, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (d) A fertility restoration gene encoding a protein containing the amino acid sequence shown in sequence number 2; (e) A fertility restoration gene that encodes a protein containing an amino acid sequence in which one or more amino acids are missing, substituted, added or inserted, as shown in sequence number 2, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (f) A fertility restoration gene that encodes a protein containing an amino acid sequence having more than 60% sequence identity with the amino acid sequence shown in sequence number 2, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (g) A fertility restoration gene encoding a protein containing the amino acid sequence shown in sequence number 3; (h) Encodes a protein containing an amino acid sequence in which one or more amino acids are missing, substituted, added or inserted, as shown in sequence number 3, and a fertility restoration gene that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (i) A fertility restoration gene that encodes a protein containing an amino acid sequence having more than 60% sequence identity with the amino acid sequence shown in sequence number 3, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (j) A fertility restoration gene encoding a protein containing the amino acid sequence shown in sequence number 4; (k) Encodes a protein containing an amino acid sequence in which one or more amino acids are missing, substituted, added or inserted, as shown in sequence number 4, and a fertility restoration gene that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (l) A fertility restoration gene that encodes a protein containing an amino acid sequence having more than 60% sequence identity with the amino acid sequence shown in sequence number 4, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (m) A fertility restoration gene encoding a protein containing the amino acid sequence shown in sequence number 5; (n) A fertility restoration gene that encodes a protein containing an amino acid sequence in which one or more amino acids are missing, substituted, added or inserted, as shown in sequence number 5, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. as well as (o) A fertility restoration gene that encodes a protein containing an amino acid sequence having more than 60% sequence identity with the amino acid sequence shown in sequence number 5, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition.

6. The method for restoring male fertility in Solanaceae plants according to claim 5, wherein, The plant in question belongs to the genus *Solanum* of the Solanaceae family.

7. The method for restoring male fertility in Solanaceae plants according to claim 6, wherein, The plant in question is a tomato.

8. The method for restoring male fertility in Solanaceae plants according to claim 6, wherein, The plant in the Solanaceae family is the potato.

9. A method for manufacturing a male fertility-restoring plant of the Solanaceae family, wherein, The method includes: obtaining a Solanaceae plant with restored male fertility by means of the male fertility restoration method of any one of claims 5 to 8.

10. A method for determining the restoration of male fertility in Solanaceae plants, comprising a method for determining whether the fertility of cytoplasmic male sterility in Solanaceae plants has been restored, wherein... The procedure includes the amplification of part or all of the base sequence of at least one of the following (a) to (o): (a) A fertility restoration gene encoding a protein containing the amino acid sequence shown in sequence number 1; (b) A fertility restoration gene that encodes a protein containing an amino acid sequence in which one or more amino acids are missing, substituted, added or inserted, as shown in sequence number 1, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (c) A fertility restoration gene that encodes a protein containing an amino acid sequence having more than 60% sequence identity with the amino acid sequence shown in sequence number 1, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (d) A fertility restoration gene encoding a protein containing the amino acid sequence shown in sequence number 2; (e) A fertility restoration gene that encodes a protein containing an amino acid sequence in which one or more amino acids are missing, substituted, added or inserted, as shown in sequence number 2, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (f) A fertility restoration gene that encodes a protein containing an amino acid sequence having more than 60% sequence identity with the amino acid sequence shown in sequence number 2, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (g) A fertility restoration gene encoding a protein containing the amino acid sequence shown in sequence number 3; (h) Encodes a protein containing an amino acid sequence in which one or more amino acids are missing, substituted, added or inserted, as shown in sequence number 3, and a fertility restoration gene that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (i) A fertility restoration gene that encodes a protein containing an amino acid sequence having more than 60% sequence identity with the amino acid sequence shown in sequence number 3, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (j) A fertility restoration gene encoding a protein containing the amino acid sequence shown in sequence number 4; (k) Encodes a protein containing an amino acid sequence in which one or more amino acids are missing, substituted, added or inserted, as shown in sequence number 4, and a fertility restoration gene that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (l) A fertility restoration gene that encodes a protein containing an amino acid sequence having more than 60% sequence identity with the amino acid sequence shown in sequence number 4, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. (m) A fertility restoration gene encoding a protein containing the amino acid sequence shown in sequence number 5; (n) A fertility restoration gene that encodes a protein containing an amino acid sequence in which one or more amino acids are missing, substituted, added or inserted, as shown in sequence number 5, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition. as well as (o) A fertility restoration gene that encodes a protein containing an amino acid sequence having more than 60% sequence identity with the amino acid sequence shown in sequence number 5, and that restores male fertility in Solanaceae plants by causing functional impairment or inhibition.

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