Male fertility restoration plant of solanaceae family, method for restoring male fertility in solanaceae plant, method for producing male fertility restoration plant of solanaceae family, and method for determining male fertility restoration in solanaceae plant

Abstract

The male fertility restoration plant of the Solanaceae family is deficient or suppressed in the function of at least one of the fertility restoring genes (a)-(o). The fertility restoring genes (a)-(o) are genes coding for a protein or the like containing an amino acid sequence represented by any one of SEQ ID NOs: 1-5.

Description

Solanaceae male fertility restored plants, method for restoring male fertility to solanaceae plants, method for producing solanaceae male fertility restored plants, and method for determining male fertility restored plants

[0001] The present invention relates to a male fertility-restoring plant of the Solanaceae family, a method for restoring male fertility to a Solanaceae plant, a method for producing a male fertility-restoring plant of the Solanaceae family, and a method for distinguishing between male fertility-restoring plants of the Solanaceae family.This application claims priority to Japanese Patent Application No. 2024-051561, filed on March 27, 2024, the contents of which are incorporated herein by reference.

[0002] In the breeding of vegetables and other crops, F1 seed production involves crossing two different parent lines, a seed parent and a pollen parent. To prevent self-pollination from the seed parent line, a process called emasculation is performed, in which the anthers are removed from the seed parent. However, this emasculation process is currently primarily performed manually, which requires labor and is costly, resulting in a rise in the price of seeds obtained through seed production. Furthermore, the need for a large number of workers poses a risk of the leakage of valuable intellectual property, namely the parent line. Manual emasculation also has a certain risk of failure, which can lead to contamination with pollen from the seed parent, resulting in seeds that do not represent the desired lineage.

[0003] As a countermeasure to these problems, the seed parent can be made male sterile, i.e., to stop producing pollen or to produce sterile, i.e., inactive, non-cross-fertilizing pollen, thereby eliminating the possibility of pollen from the seed parent being pollinated without the need for manual male sterilization.

[0004] For example, in rice, a breeding technique utilizing cytoplasmic male sterility (CMS) is known. One example of cytoplasmic male sterility is the phenomenon in which incompatibility between the cell nucleus and mitochondrial gene products causes pollen to become inactive, preventing mating and preventing the production of seeds and fruits. In many plant species, cytoplasm is transmitted only from the female side, so CMS lines are produced using cytoplasm replacement lines in which heterologous cytoplasm has been replaced.

[0005] Patent Document 1 discloses a simple method for producing male-sterile tomato plants, which involves fusing protoplasts isolated from tomato plants and treated to be inactivated by cytoplasmic factors with protoplasts isolated from Solanum plants and treated to be inactivated by nuclear genetic material, and then regenerating male-sterile tomatoes from the fusion product. This technique aims to create a line with male sterility by protoplast fusion, and to efficiently render the target tomato plants male-sterile in a short period of time without causing any changes in the target tomato plants' traits other than male sterility.

[0006] Patent Document 2 describes a gene containing a specific DNA that causes rice RT-type cytoplasmic male sterility, and a method for identifying sterility using the gene. This technology identifies a mitochondrial gene that causes rice RT-type cytoplasmic male sterility, and by using this gene as a DNA marker, it can also be used to identify rice lines that are cytoplasmic male sterile.

[0007] Japanese Patent No. 2824841 International Publication No. 2014 / 027502

[0008] Although the technology of Patent Document 1 can obtain male-sterile lines in tomatoes of the Solanaceae family, the gene responsible for the cytoplasmic male sterility has not been identified. Therefore, although it is possible to produce fruitless plants of the Solanaceae family using the aforementioned sterile lines, they cannot produce seeds and therefore cannot be propagated. To apply male-sterile lines, it is desirable to identify the gene responsible for cytoplasmic male sterility and make it possible to control its on / off state.

[0009] Patent Document 2 discloses a gene for cytoplasmic male sterility in grasses, but does not disclose a gene responsible for cytoplasmic male sterility in plants of the Solanaceae family.

[0010] The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a novel technique for restoring cytoplasmic male sterility in Solanaceae plants.

[0011] In order to solve the above problems, the present invention includes the following aspects.

[0012] [1] A male fertility restorer plant of the Solanaceae family, in which the function of at least one fertility restorer gene selected from the following (a) to (o) is deleted or suppressed: (a) a fertility restorer gene encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 1; (b) a fertility restorer gene encoding a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence set forth in SEQ ID NO: 1, and which causes male fertility restoration in Solanaceae plants when the function is lost or suppressed; (c) a fertility restorer gene encoding a protein comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1, and which causes male fertility restoration in Solanaceae plants when the function is lost or suppressed; (d) a fertility restorer gene encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2; (e) a fertility restorer gene encoding a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence set forth in SEQ ID NO: 2, and which causes male fertility restoration in Solanaceae plants when the function is lost or suppressed; (f) a fertility restorer gene that encodes a protein comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2, and whose function is lost or suppressed to restore male fertility to Solanaceae plants; (g) a fertility restorer gene that encodes a protein comprising the amino acid sequence shown in SEQ ID NO: 3; (h) a fertility restorer gene that encodes a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 3, and whose function is lost or suppressed to restore male fertility to Solanaceae plants; (i) a fertility restorer gene that encodes a protein comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3, and whose function is lost or suppressed to restore male fertility to Solanaceae plants; (j) a fertility restorer gene that encodes a protein comprising the amino acid sequence shown in SEQ ID NO: 4; (k) a fertility restorer gene that encodes a protein containing an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted in the amino acid sequence shown in SEQ ID NO: 4, and whose function is lost or suppressed to restore male fertility to a Solanaceae plant;(l) A fertility restorer gene that encodes a protein comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and whose function is lost or suppressed to restore male fertility to Solanaceae plants; (m) A fertility restorer gene that encodes a protein comprising the amino acid sequence shown in SEQ ID NO: 5; (n) A fertility restorer gene that encodes a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 5, and whose function is lost or suppressed to restore male fertility to Solanaceae plants; (o) A fertility restorer gene that encodes a protein comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 5, and whose function is lost or suppressed to restore male fertility to Solanaceae plants.

[0013] [2] The male fertility restorer plant of the Solanaceae family according to [1], which is of the genus Solanum.

[0014] [3] The male fertility restorer plant of the Solanaceae family according to [2], which is a tomato.

[0015] [4] The male fertility restorer plant of the Solanaceae family according to [2], which is a potato.

[0016] [5] A method for restoring male fertility to a solanaceous plant, comprising a step of deleting or suppressing the function of a fertility restorer gene of the solanaceous plant, wherein the fertility restorer gene is at least one of the following (a) to (o): (a) a fertility restorer gene encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 1; (b) a fertility restorer gene encoding a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence set forth in SEQ ID NO: 1, and which causes male fertility restoration in Solanaceae plants when the function is lost or suppressed; (c) a fertility restorer gene encoding a protein comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1, and which causes male fertility restoration in Solanaceae plants when the function is lost or suppressed; (d) a fertility restorer gene encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2; (e) a fertility restorer gene encoding a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence set forth in SEQ ID NO: 2, and which causes male fertility restoration in Solanaceae plants when the function is lost or suppressed; (f) a fertility restorer gene that encodes a protein comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2, and whose function is lost or suppressed to restore male fertility to Solanaceae plants; (g) a fertility restorer gene that encodes a protein comprising the amino acid sequence shown in SEQ ID NO: 3; (h) a fertility restorer gene that encodes a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 3, and whose function is lost or suppressed to restore male fertility to Solanaceae plants; (i) a fertility restorer gene that encodes a protein comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3, and whose function is lost or suppressed to restore male fertility to Solanaceae plants; (j) a fertility restorer gene that encodes a protein comprising the amino acid sequence shown in SEQ ID NO: 4; (k) a fertility restorer gene that encodes a protein containing an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted in the amino acid sequence shown in SEQ ID NO: 4, and whose function is lost or suppressed to restore male fertility to a Solanaceae plant;(l) A fertility restorer gene that encodes a protein comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and whose function is lost or suppressed to restore male fertility to Solanaceae plants; (m) A fertility restorer gene that encodes a protein comprising the amino acid sequence shown in SEQ ID NO: 5; (n) A fertility restorer gene that encodes a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 5, and whose function is lost or suppressed to restore male fertility to Solanaceae plants; (o) A fertility restorer gene that encodes a protein comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 5, and whose function is lost or suppressed to restore male fertility to Solanaceae plants.

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

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

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

[0020] [9] A method for producing a male-fertility-restored plant of the Solanaceae family, comprising obtaining a Solanaceae plant whose male fertility has been restored by the method for restoring male fertility to a Solanaceae plant according to any one of [5] to [8].

[0021]

[10] A method for determining whether fertility has been restored in a cytoplasmic male sterility plant of the Solanaceae family, comprising the step of amplifying part or all of the base sequence of at least one fertility restorer gene selected from the following (a) to (o): (a) a fertility restorer gene encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 1; (b) a fertility restorer gene encoding a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence set forth in SEQ ID NO: 1, and which causes male fertility restoration in Solanaceae plants when the function is lost or suppressed; (c) a fertility restorer gene encoding a protein comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1, and which causes male fertility restoration in Solanaceae plants when the function is lost or suppressed; (d) a fertility restorer gene encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2; (e) a fertility restorer gene encoding a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence set forth in SEQ ID NO: 2, and which causes male fertility restoration in Solanaceae plants when the function is lost or suppressed; (f) a fertility restorer gene that encodes a protein comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2, and whose function is lost or suppressed to restore male fertility to Solanaceae plants; (g) a fertility restorer gene that encodes a protein comprising the amino acid sequence shown in SEQ ID NO: 3; (h) a fertility restorer gene that encodes a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 3, and whose function is lost or suppressed to restore male fertility to Solanaceae plants; (i) a fertility restorer gene that encodes a protein comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3, and whose function is lost or suppressed to restore male fertility to Solanaceae plants; (j) a fertility restorer gene that encodes a protein comprising the amino acid sequence shown in SEQ ID NO: 4;(k) a fertility restorer gene that encodes a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 4, and whose function is lost or suppressed to restore male fertility to Solanaceae plants; (l) a fertility restorer gene that encodes a protein comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and whose function is lost or suppressed to restore male fertility to Solanaceae plants; (m) a fertility restorer gene that encodes a protein comprising the amino acid sequence shown in SEQ ID NO: 5; (n) a fertility restorer gene that encodes a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 5, and whose function is lost or suppressed to restore male fertility to Solanaceae plants; (o) a fertility restorer gene that encodes a protein comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 5, and whose function is lost or suppressed to restore male fertility to Solanaceae plants.

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

[0023] Graph showing the germination rate of pollen collected from 13 male fertility restorer lines obtained by EMS treatment. Diagram showing the mutation details of three lines having mutations in the phage-type RNA polymerase gene. Images showing pollen germination of three lines in which the phage-type RNA polymerase gene has been knocked out. Images showing pollen germination of lines in which the DNA topoisomerase 1 gene, the Pentatricopeptide repeat (PPR) gene, the Serine / Threonine-protein kinase gene, or the POLYMERASE GAMMA2 gene has been knocked out. This is an image of bands showing the results of amplifying the fertility restorer gene for each of the fertility restorer lines EMS#1, #2, #7, #9, #10, #11, #12, #14, #16, #17, and #20 and treating them with restriction enzymes.

[0024] Preferred embodiments of the present invention will be described below in detail with respect to a male fertility-restoring plant of the Solanaceae family, a method for restoring male fertility to a Solanaceae plant, a method for producing a male fertility-restoring plant of the Solanaceae family, and a method for determining whether a male fertility has been restored to a Solanaceae plant, although the present invention is not limited to the following embodiments.

[0025] [Solanaceae male fertility restorer plant] The Solanaceae male fertility restorer plant of this embodiment is a plant in which fertility has been restored from cytoplasmic male sterility (CMS), and in which the function of at least one fertility restorer gene selected from the group consisting of (a) to (o) below (in other words, at least one fertility restorer gene selected from the group consisting of (a) to (o)) is deleted or suppressed. (a) a fertility restorer gene encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 1; (b) a fertility restorer gene encoding a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence set forth in SEQ ID NO: 1, and which causes male fertility restoration in Solanaceae plants when the function is lost or suppressed; (c) a fertility restorer gene encoding a protein comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1, and which causes male fertility restoration in Solanaceae plants when the function is lost or suppressed; (d) a fertility restorer gene encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2; (e) a fertility restorer gene encoding a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence set forth in SEQ ID NO: 2, and which causes male fertility restoration in Solanaceae plants when the function is lost or suppressed; (f) a fertility restorer gene that encodes a protein comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2, and whose function is lost or suppressed to restore male fertility to Solanaceae plants; (g) a fertility restorer gene that encodes a protein comprising the amino acid sequence shown in SEQ ID NO: 3; (h) a fertility restorer gene that encodes a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 3, and whose function is lost or suppressed to restore male fertility to Solanaceae plants; (i) a fertility restorer gene that encodes a protein comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3, and whose function is lost or suppressed to restore male fertility to Solanaceae plants; (j) a fertility restorer gene that encodes a protein comprising the amino acid sequence shown in SEQ ID NO: 4;(k) a fertility restorer gene that encodes a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 4, and whose function is lost or suppressed to restore male fertility to Solanaceae plants; (l) a fertility restorer gene that encodes a protein comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and whose function is lost or suppressed to restore male fertility to Solanaceae plants; (m) a fertility restorer gene that encodes a protein comprising the amino acid sequence shown in SEQ ID NO: 5; (n) a fertility restorer gene that encodes a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 5, and whose function is lost or suppressed to restore male fertility to Solanaceae plants; (o) a fertility restorer gene that encodes a protein comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 5, and whose function is lost or suppressed to restore male fertility to Solanaceae plants.

[0026] As used herein, "the function of a gene is deleted or suppressed" means that the activity of the gene product is deleted or suppressed, or the expression of the gene is deleted or suppressed, by artificial manipulation to control the gene or its expression.

[0027] Whether a gene function is lost or suppressed can be determined, for example, by comparing a fertility-restored plant produced by artificial manipulation with a cytoplasmic male sterility-inhibited control plant. Artificial manipulation includes, but is not limited to, the artificial introduction or expression of a nucleic acid or protein that causes the loss or suppression of the expression of the target gene.

[0028] The above-mentioned "plant in which fertility has been restored from cytoplasmic male sterility (CMS)" may be a plant that has orf137 (see JP 2023-32890 A), the gene (S factor gene) responsible for cytoplasmic male sterility, and in which the function of at least one of the fertility restorer genes (a) to (o) above is deleted or suppressed. However, as will be described in detail later, the present invention can also be applied to cytoplasmic male sterile Solanaceae plants that have a gene other than the orf137 gene as the causative gene for cytoplasmic male sterility, and therefore the Solanaceae plants of the present invention are not limited to plants that have the orf137 gene.

[0029] As used herein, "orf137 gene" refers to a gene that encodes a protein comprising an amino acid sequence that has 80% or more (preferably 95% or more) sequence identity with the amino acid sequence set forth in SEQ ID NO: 6 and that causes cytoplasmic male sterility.

[0030] The fertility restorer genes (a) to (o) are all genes present in nuclear DNA that restore male fertility when their functions are lost or suppressed in Solanaceae plants that have cytoplasmic male sterility. The fertility restorer genes (a) to (o) all function to assist the male sterility-causing function of orf137 in Solanaceae plants, and it is believed that cytoplasmic male fertility is restored when the functions of the fertility restorer genes (a) to (o) are lost or suppressed.

[0031] The fertility restorer gene (a) above is a phage-type RNA polymerase gene in tomato (gene ID: Solyc05g010660). The amino acid sequence of the protein encoded by the fertility restorer gene (c) above has sequence identity with the amino acid sequence shown in SEQ ID NO: 1 of 60% or more, preferably 75% or more, more preferably 80% or more, even more preferably 90% or more, and most preferably 95% or more.

[0032] The fertility restorer gene (d) above is the DNA topoisomerase 1 gene in tomato (gene ID: Solyc02g081910). The amino acid sequence of the protein encoded by the fertility restorer gene (f) above has sequence identity with the amino acid sequence shown in SEQ ID NO: 2 of 60% or more, preferably 75% or more, more preferably 80% or more, even more preferably 90% or more, and most preferably 95% or more.

[0033] The fertility restorer gene (g) above is the Pentatricopeptide repeat (PPR) gene in tomato (gene ID: Solyc07g053060). The amino acid sequence of the protein encoded by the fertility restorer gene (i) above has sequence identity with the amino acid sequence shown in SEQ ID NO: 3 of 60% or more, preferably 75% or more, more preferably 80% or more, even more preferably 90% or more, and most preferably 95% or more.

[0034] The fertility restorer gene (j) above is a serine / threonine-protein kinase gene in tomato (gene ID: Solyc01g109080). The amino acid sequence of the protein encoded by the fertility restorer gene (l) above has sequence identity with the amino acid sequence shown in SEQ ID NO: 4 of 60% or more, preferably 75% or more, more preferably 80% or more, even more preferably 90% or more, and most preferably 95% or more.

[0035] The fertility restorer gene (m) is the POLYMERASE GAMMA2 gene in tomato (gene ID: Solyc07g064330). The amino acid sequence of the protein encoded by the fertility restorer gene (o) has sequence identity with the amino acid sequence shown in SEQ ID NO: 5 of 60% or more, preferably 75% or more, more preferably 80% or more, even more preferably 90% or more, and most preferably 95% or more.

[0036] In the protein encoded by the fertility restorer gene (b) above, the number of amino acids that may be deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 1 is preferably 1 to 30, more preferably 1 to 20, more preferably 1 to 10, particularly preferably 1 to 5, and most preferably 1 to 3.

[0037] In the protein encoded by the fertility restorer gene (e) above, the number of amino acids that may be deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 2 is preferably 1 to 30, more preferably 1 to 20, more preferably 1 to 10, particularly preferably 1 to 5, and most preferably 1 to 3.

[0038] In the protein encoded by the fertility restorer gene (h) above, the number of amino acids that may be deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 3 is preferably 1 to 30, more preferably 1 to 20, more preferably 1 to 10, particularly preferably 1 to 5, and most preferably 1 to 3.

[0039] In the protein encoded by the fertility restorer gene (k) above, the number of amino acids that may be deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 4 is preferably 1 to 30, more preferably 1 to 20, more preferably 1 to 10, particularly preferably 1 to 5, and most preferably 1 to 3.

[0040] In the protein encoded by the fertility restorer gene (n) above, the number of amino acids that may be deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 5 is preferably 1 to 30, more preferably 1 to 20, more preferably 1 to 10, particularly preferably 1 to 5, and most preferably 1 to 3.

[0041] Herein, the sequence identity of a subject amino acid sequence to a reference amino acid sequence (for example, an amino acid sequence shown in any of SEQ ID NOs: 1 to 6) can be determined as follows. First, the reference amino acid sequence and the subject amino acid sequence are aligned. Gaps are included in each amino acid sequence to maximize sequence identity. Next, the number of identical amino acids in the reference amino acid sequence and the subject amino acid sequence is calculated, and the sequence identity can be calculated according to the following formula (1): Sequence identity (%) = number of identical amino acids / total number of amino acids in the subject amino acid sequence × 100 (1)

[0042] Whether male fertility restoration (cytoplasmic male fertility restoration) has occurred in a Solanaceae plant due to the loss or suppression of the function of the fertility restorer genes (a) to (o) above can be determined by whether or not the pollen obtained from the Solanaceae plant contains pollen tubes that elongate (pollen germination) (if so, fertility restoration has occurred), or by pollinating the pistil of a normal plant with the pollen and determining whether or not seeds are formed. However, methods for determining whether male fertility restoration has occurred in a Solanaceae plant are not limited to these.

[0043] The type of the Solanaceae plant is not particularly limited, and examples thereof include plants of the genus Solanum and plants of the genus Capsicum. Examples of plants of the Solanaceae include eggplant (Solanum melongena), potato (Solanum tuberosum), and tomato (Solanum lycopersicum). Examples of plants of the Capsicum include chili peppers and bell peppers (Capsicum annuum). Cytoplasmic male sterile strains of other Solanaceae plants, such as potato, also contain the orf137 gene.

[0044] Furthermore, by deleting or suppressing the function of any of the fertility restorer genes (a) to (c) above, the expression levels of various mRNAs expressed from mitochondrial DNA are suppressed. Furthermore, by deleting or suppressing the function of any of the fertility restorer genes (d) to (f) above and / or any of the fertility restorer genes (m) to (o) above, the absolute copy number of mitochondrial DNA is reduced. Therefore, even in cytoplasmic male sterile Solanaceae plants that have a gene other than the orf137 gene as the causative gene for cytoplasmic male sterility, fertility can be restored by deleting or suppressing the function of at least one or more fertility restorer genes selected from the group consisting of the fertility restorer genes (a) to (f) above and the fertility restorer genes (m) to (o) above.

[0045] Solanaceae plants include species whose fruits are edible, such as eggplant, tomato, chili pepper, and bell pepper, as well as species whose tubers are edible, such as potatoes. They also include a wide variety of plants, such as tobacco, which is cultivated as a luxury item, and plants used in horticulture for ornamental purposes, such as physalis or petunia. The techniques according to the embodiments detailed in this specification can be applied to the genetic manipulation, such as crossbreeding, of these Solanaceae plants, and therefore have a wide range of applications.

[0046] The solanaceous plant is particularly preferably a crop, that is, a plant used in the agricultural field, particularly as a food raw material. Furthermore, the solanaceous plant may be a plant body, a plant cultured cell, a plant cell, or a callus.

[0047] [Method for restoring male fertility to a Solanaceae plant] The method for restoring male fertility to a Solanaceae plant of this embodiment comprises a step of deleting or suppressing the function of at least one of the fertility restorer genes (a) to (o) in the Solanaceae plant. The above-mentioned male fertility-restored Solanaceae plant is a Solanaceae plant obtained by the male fertility restoration method or a progeny thereof.

[0048] The means for deleting or suppressing the function of at least one of the fertility restorer genes (a) to (o) in a Solanaceae plant is not particularly limited, but examples include treating a Solanaceae plant with cytoplasmic male sterility with a mutagen such as EMS. After treatment with the mutagen, Solanaceae plants in which the function of one or more of the above fertility restorer genes is deleted or suppressed can be screened to obtain Solanaceae plants with restored male fertility. Alternatively, genome editing technology can be used to artificially modify at least one of the fertility restorer genes (a) to (o) to delete or suppress the function of the fertility restorer gene.

[0049] Genome editing techniques include, but are not limited to, methods that can modify DNA base sequences, such as gene targeting, CRISPR-Cas systems, zinc finger nucleases, and TALENs. Note that "modification" refers to a change in the base sequence of a target gene.

[0050] The region to be edited by genome editing technology may be, for example, at least one of the fertility restorer genes (a) to (o) above and / or its promoter.

[0051] <CRISPR-Cas System> In the CRISPR-Cas system, a guide RNA (gRNA) or a polynucleotide encoding the gRNA, and a CRISPR-Cas enzyme or a polynucleotide encoding the CRISPR-Cas enzyme are introduced into a Solanaceae plant. The polynucleotide encoding the gRNA and the polynucleotide encoding the CRISPR-Cas enzyme are preferably introduced into the Solanaceae plant in a state where they are incorporated into an expression vector.

[0052] The gRNA comprises a base sequence complementary to a portion of the sequence of any one of the fertility restorer genes and / or their promoters described above (a) to (o), and constitutes an adaptive immune system that provides acquired resistance to invading foreign nucleic acids in bacteria and archaea. It means a mimic of the hairpin structure of a tracrRNA-crRNA chimera formed by fusing a small RNA fragment (CRISPR-RNA: crRNA) containing a foreign sequence (guide sequence) with an RNA (trans-activating crRNA: tracrRNA) partially complementary to the crRNA. The gRNA comprises a polynucleotide in the 5'-terminal region consisting of a base sequence complementary to the base sequence of preferably 17 to 24 bases, from one base upstream of the PAM (proto-spacer adjacent motif) sequence contained in any one of the fertility restorer genes described above (a) to (o).

[0053] The PAM sequence is present in the fertility restorer genes (a) to (o) above, which are the target genes, and is a sequence that can be recognized by the CRISPR-Cas enzyme, and the sequence differs depending on the bacterial species from which the CRISPR-Cas enzyme is derived.

[0054] CRISPR-Cas enzymes are a family of Cas proteins that constitute an adaptive immune system in bacteria and archaea that provides acquired resistance to invading foreign nucleic acids, and are endonucleases that recognize the above-mentioned PAM sequence and cleave double-stranded DNA upstream of it so that the ends become blunt. The CRISPR-Cas enzyme may be Cas9, a homolog or variant thereof, or another type.

[0055] Since pollen obtained from a fertility-restored Solanaceae plant is haploid, when at least one of the fertility restorer genes (a) to (o) above is deleted (knocked out) or suppressed (knocked down) using a CRISPR-Cas system or the like, fertility can be restored by deleting or suppressing the fertility restorer gene of at least one allele. However, deleting or suppressing the fertility restorer genes of both alleles can further increase the pollen germination rate.

[0056] <Suppression of expression of fertility restorer gene using nucleic acid> Examples of means for suppressing the expression of at least one of the fertility restorer genes (a) to (o) above include antisense nucleic acids, ribozymes, and RNAi-inducing nucleic acids.

[0057] Examples of antisense nucleic acids include nucleic acids containing a base sequence complementary to the sense sequence (mRNA sequence) of any of the fertility restorer genes (a) to (o) above.

[0058] Those skilled in the art can appropriately design the base sequence of the antisense nucleic acid that suppresses the expression of the fertility restorer gene (a) based on the base sequence of the cDNA of the fertility restorer gene (a) (base sequence shown in SEQ ID NO: 7). Those skilled in the art can appropriately design the base sequence of the antisense nucleic acid that suppresses the expression of the fertility restorer gene (d) based on the base sequence of the cDNA of the fertility restorer gene (d) (base sequence shown in SEQ ID NO: 8). Those skilled in the art can appropriately design the base sequence of the antisense nucleic acid that suppresses the expression of the fertility restorer gene (g) based on the base sequence of the cDNA of the fertility restorer gene (g) (base sequence shown in SEQ ID NO: 9). Those skilled in the art can appropriately design the base sequence of the antisense nucleic acid that suppresses the expression of the fertility restorer gene (j) based on the base sequence of the cDNA of the fertility restorer gene (j) (base sequence shown in SEQ ID NO: 10). A person skilled in the art can appropriately design the base sequence of the antisense nucleic acid that suppresses the expression of the fertility restorer gene (m) above based on the base sequence of the cDNA of the fertility restorer gene (m) above (the base sequence shown in SEQ ID NO: 11).

[0059] A ribozyme is a catalytic RNA that has an active site that reacts with a target fertility restorer gene and a substrate-binding site that binds to the fertility restorer gene. Examples of ribozymes include ribozymes whose substrate-binding site has a sequence complementary to the sequence of the mRNA of the fertility restorer gene.

[0060] Those skilled in the art can appropriately design the base sequence of the ribozyme that suppresses the expression of the fertility restorer gene (a) based on the base sequence of the cDNA of the fertility restorer gene (a) (base sequence shown in SEQ ID NO: 7). Those skilled in the art can appropriately design the base sequence of the ribozyme that suppresses the expression of the fertility restorer gene (d) based on the base sequence of the cDNA of the fertility restorer gene (d) (base sequence shown in SEQ ID NO: 8). Those skilled in the art can appropriately design the base sequence of the ribozyme that suppresses the expression of the fertility restorer gene (g) based on the base sequence of the cDNA of the fertility restorer gene (g) (base sequence shown in SEQ ID NO: 9). Those skilled in the art can appropriately design the base sequence of the ribozyme that suppresses the expression of the fertility restorer gene (j) based on the base sequence of the cDNA of the fertility restorer gene (j) (base sequence shown in SEQ ID NO: 10). A person skilled in the art can appropriately design the base sequence of the ribozyme that suppresses the expression of the fertility restorer gene (m) above based on the base sequence of the cDNA of the fertility restorer gene (m) above (the base sequence shown in SEQ ID NO: 11).

[0061] Examples of RNAi-inducing nucleic acids include siRNA, miRNA, and shRNA. More specifically, examples include double-stranded RNAs containing a base sequence identical to part or all of the sequence of the mRNA of any of the fertility restorer genes (a) to (o) above.

[0062] <Method of Introduction into Solanaceae Plants> Methods for introducing into solanaceae plants an expression vector for modifying the sequence of at least one of the fertility restorer genes (a) to (o) and / or its promoter, or the above-mentioned nucleic acid for suppressing expression, can be exemplified by the Agrobacterium method, particle gun method, polyethylene glycol (PEG) method, calcium phosphate method, electroporation method, liposome method, and DEAE-dextran method. Among these introduction methods, the Agrobacterium method can be preferably used.

[0063] When using the Agrobacterium method, it is preferable to use a pBI-based or pPZP-based binary vector. Examples of pBI-based binary vectors include pBIG, pBIN19, pBI101, pBI121, and pBI221. Examples of pPZP-based binary vectors include pPZP100, pPZP200, pPZP500, and pPZP3425.

[0064] [Method for producing a male fertility-restored plant of Solanaceae] The method for producing a male fertility-restored plant of Solanaceae of this embodiment includes obtaining a Solanaceae plant with restored male fertility by the "method for restoring male fertility to a Solanaceae plant" of the above embodiment. The Solanaceae plant obtained by the production method of this embodiment is a Solanaceae plant in which fertility has been restored from cytoplasmic male sterility (CMS) by deleting or suppressing the function of at least one of the fertility restorer genes (a) to (o) above.

[0065] [Method for determining whether male fertility has been restored in a Solanaceae plant] The method for determining whether male fertility has been restored in a Solanaceae plant of this embodiment is a method for determining whether fertility has been restored to a cytoplasmic male sterility plant in the Solanaceae family, and includes a step of amplifying a partial or entire region of at least one of the fertility restorer genes (a) to (o) above.

[0066] For example, as described in the Examples below, whether a Solanaceae plant is a plant in which the base sequence of at least one of the fertility restorer genes (a) to (o) above has been modified using genome editing technology to eliminate or suppress the function of the fertility restorer gene, thereby restoring male fertility, can be determined by amplifying part or all of the fertility restorer gene by PCR or the like and confirming the base sequence of the amplified product.

[0067] Methods for confirming the base sequence of the amplified product include sequencing the amplified product to confirm the specific base sequence, as well as methods for confirming the base sequence of the amplified product based on whether or not a restriction enzyme recognition sequence is contained in the amplified product, as will be described in the Examples below, methods for confirming the length of the amplified product, methods for confirming whether or not it is amplifiable, etc. Primer sequences for amplifying the fertility restorer genes (a) to (o) above can be designed appropriately by those skilled in the art.

[0068] Examples of primer pairs for amplifying a portion of the fertility restorer gene (a) above include the following four pairs: 1. A pair of a primer containing the nucleotide sequence shown in SEQ ID NO: 12 at its 3'-end (3'-terminal region) and a primer containing the nucleotide sequence shown in SEQ ID NO: 13 at its 3'-end. 2. A pair of a primer containing the nucleotide sequence shown in SEQ ID NO: 14 at its 3'-end and a primer containing the nucleotide sequence shown in SEQ ID NO: 15 at its 3'-end. 3. A pair of a primer containing the nucleotide sequence shown in SEQ ID NO: 16 at its 3'-end and a primer containing the nucleotide sequence shown in SEQ ID NO: 17 at its 3'-end. 4. A pair of a primer containing the nucleotide sequence shown in SEQ ID NO: 18 at its 3'-end and a primer containing the nucleotide sequence shown in SEQ ID NO: 19 at its 3'-end.

[0069] An example of the pair of primers for amplifying a portion of the fertility restorer gene (d) above is one of the following pairs: 1. A pair of a primer containing the nucleotide sequence shown in SEQ ID NO: 120 at its 3' end and a primer containing the nucleotide sequence shown in SEQ ID NO: 21 at its 3' end.

[0070] Examples of primer pairs for amplifying a portion of the fertility restorer gene (g) above include the following two pairs: 1. A pair of a primer containing the nucleotide sequence shown in SEQ ID NO: 22 at its 3' end and a primer containing the nucleotide sequence shown in SEQ ID NO: 23 at its 3' end. 2. A pair of a primer containing the nucleotide sequence shown in SEQ ID NO: 24 at its 3' end and a primer containing the nucleotide sequence shown in SEQ ID NO: 25 at its 3' end.

[0071] An example of a primer pair for amplifying a portion of the fertility restorer gene (j) above is the following pair: 1. A primer pair comprising a primer containing the nucleotide sequence shown in SEQ ID NO: 26 at its 3' end and a primer containing the nucleotide sequence shown in SEQ ID NO: 27 at its 3' end.

[0072] Examples of primer pairs for amplifying a portion of the fertility restorer gene (m) above include the following three pairs: 1. A pair of a primer containing the nucleotide sequence shown in SEQ ID NO: 28 at its 3' end and a primer containing the nucleotide sequence shown in SEQ ID NO: 29 at its 3' end. 2. A pair of a primer containing the nucleotide sequence shown in SEQ ID NO: 30 at its 3' end and a primer containing the nucleotide sequence shown in SEQ ID NO: 31 at its 3' end. 3. A pair of a primer containing the nucleotide sequence shown in SEQ ID NO: 32 at its 3' end and a primer containing the nucleotide sequence shown in SEQ ID NO: 33 at its 3' end.

[0073] Each of the above primers may be 55 nucleotides or less in length, 45 nucleotides or less in length, 40 nucleotides or less in length, 35 nucleotides or less in length, or 30 nucleotides or less in length.

[0074] The part of the Solanaceae plant to be subjected to amplification of the fertility restorer gene is not particularly limited, and may be, for example, a leaf, a root, a stem, or pollen.

[0075] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the invention described in the claims, and it goes without saying that these modifications are also included within the scope of the present invention.

[0076] For example, in the embodiment of the "Solanaceae male fertility restorer plant" described above, the male fertility restorer plant of Solanaceae has a deletion or suppression of the function of at least one of the fertility restorer genes (a) to (o) above. However, other examples of the male fertility restorer plant of Solanaceae according to the present invention include a male fertility restorer plant of Solanaceae in which the function of any of the fertility restorer genes (a) to (c) has been deleted or suppressed, a male fertility restorer plant of Solanaceae in which the function of any of the fertility restorer genes (d) to (f) has been deleted or suppressed, a male fertility restorer plant of Solanaceae in which the function of any of the fertility restorer genes (g) to (i), a male fertility restorer plant of Solanaceae in which the function of any of the fertility restorer genes (j) to (l), and a male fertility restorer plant of Solanaceae in which the function of any of the fertility restorer genes (m) to (o) has been deleted or suppressed.

[0077] Furthermore, in the above-described embodiment of the "method for restoring male fertility to a solanaceous plant," the method for restoring male fertility to a solanaceous plant includes a step of deleting or suppressing the function of at least one of the fertility restorer genes (a) to (o) above. However, the method for restoring male fertility to a solanaceous plant may include, instead of the above step, a step of deleting or suppressing the function of any of the fertility restorer genes (a) to (c) above, a step of deleting or suppressing the function of any of the fertility restorer genes (d) to (f) above, a step of deleting or suppressing the function of any of the fertility restorer genes (g) to (i) above, a step of deleting or suppressing the function of any of the fertility restorer genes (j) to (l) above, or a step of deleting or suppressing the function of any of the fertility restorer genes (m) to (o) above. The same applies to a method for producing a male-fertility-restored Solanaceae plant, which comprises obtaining a Solanaceae plant whose male fertility has been restored by the method for restoring male fertility to a Solanaceae plant.

[0078] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0079] [Solanaceae Plants Used in the Experiment] The tomato CMS line known as CMS[P], obtained from Nichirei Corporation, was backcrossed with the dwarf tomato variety Micro-Tom (TOMJPF0001) four or more times to replace the nuclear genome with Micro-Tom, resulting in Dwarf CMS[P], used in this experiment. Dwarf CMS[P] was grown at a temperature of 23°C and CO ２ The plants were grown hydroponically in a 600 ppm solution with a 16-hour photoperiod. During the cultivation period, Otsuka fertilizer (Otsuka No. 1 12 g / L, Otsuka No. 2 8 g / L) was used in the nutrient solution, and the EC (electrical conductivity) was adjusted to 1.2 mS.

[0080] [Creation of a new male fertility restored line] Dwarf CMS[P] seeds were soaked in distilled water for 4 hours to allow the water to penetrate the inside. Next, the seeds were soaked in a 0.5% or 1% aqueous solution of EMS (ethyl methane sulfonate), soaked for 16 hours using a rotator, and then washed three times with distilled water. Approximately 1,800 EMS-treated seeds were transplanted into a medium specifically for hydroponics (Cocobed, Kaneko Seeds) and hydroponically grown in a greenhouse. The M1 generation seeds thus obtained were cultivated and self-fertilized to investigate the formation of self-fertilized seeds. In the lines in which pollen fertility was restored, M ２ We were able to obtain the seeds of the next generation (self-fertilized seeds). ２ These fertility restorer (RF) lines of the generations were designated EMS#1, #2, #7, #9, #10, #11, #12, #14, #16, #17, #20, #21, and #22.

[0081] To maintain the newly obtained fertility restorer lines, each line was backcrossed to Dwarf CMS[P], and the resulting plants were cultivated indoors using rock wool.

[0082] [Germination rate of new male fertility restorer lines] The germination rate (the percentage of pollen with elongated pollen tubes) of pollen obtained from the above-mentioned male fertility restorer lines EMS#1, #2, #7, #9, #10, #11, #12, #14, #16, #17, #20, #21, and #22, the Micro-Tom line as a positive control, and the Dwarf CMS[P] line as a negative control was confirmed by staining with aniline blue.

[0083] FIG. 1 is a graph showing the germination rate of pollen collected from 13 male fertility restorer lines obtained by EMS treatment.

[0084] As shown in Figure 1, the germination rate of pollen obtained from the negative control Dwarf CMS[P] line was 0%, meaning that none of the pollen germinated, while the germination rate of pollen obtained from the positive control Micro-Tom line was 79%.

[0085] In contrast, 3 to 36% of the pollen germinated in the male fertility restorer lines EMS#1, #2, #7, #9, #10, #11, #12, #14, #16, #17, #20, #21, and #22, confirming that male fertility was restored in the male fertility restorer lines EMS#1, #2, #7, #9, #10, #11, #12, #14, #16, #17, #20, #21, and #22.

[0086] [Identification of fertility restorer genes using BSA-Seq] (Preparation of bulk DNA and whole genome sequencing) Using Maxwell 16 Purification Kits (Promega) and Maxwell 16 Instrument (Promega), total DNA was extracted from the leaves of the male fertility restorer lines described above, and bulk DNA of fertile and sterile populations was prepared. Whole genome sequencing was performed on each bulk DNA at Rhelixa Co., Ltd. Whole genome sequencing was performed using Illumina NovaSeq 6000, PE150 (150 bp x 2 paired-end), 30 G bases per sample, and PCR-free conditions.

[0087] (Data Analysis) Data were trimmed using Trim Galore (https: / / github.com / FelixKrueger / TrimGalore / blob / master / Docs / Trim_Galore_User_Guide.md), which removed adapters and reads with a quality score of less than 30 and reads shorter than 20 bp. Mapping was performed using BWA-MEM (Heng Li. Aligning sequence reads, clone sequences, and assembly contigs with BWA-MEM.2013), with the tomato cultivar Heinz 1706 nuclear genome SL4.0 (https: / / solgenomics.net / organism / Solanum_lycopersicum / genome) as the reference genome. Mutation detection was performed using gatk HaplotypeCaller (McKenna A, et al. 2010. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 20(9):1297-1303.), and annotation data from ITAG 4.0 (https: / / solgenomics.net / organism / Solanum_lycopersicum / genome) for the tomato cultivar Heinz 1706 was used. SNP-indexes for fertile and sterile bulks were calculated using bcftools (Heng Li. A statistical framework for SNP calling, mutation discovery, association mapping, and population genetic parameter estimation from sequencing data. 2011.), and fertility restorer genes were narrowed down by detecting mutations with a ΔSNP-index of approximately 0.5.The ΔSNP-index graph was created using QTL-seq (Sugihara et al. High-performance pipeline for MutMap and QTL-seq. PeerJ, 2022). Mutations specific to each line are shown as M of each mutant. ２ The mutations were extracted by comparing them with the mutation information obtained by individual variant calling.

[0088] (Gene analysis focusing on organelle DNA replication-related genes) M ２ For each generation, whole genome sequencing was performed and ２ Variant calling of the strains was performed. From the obtained mutation information, mutations present in genes involved in mitochondrial DNA replication were extracted. BLASTP was used to detect genes, and the E-value was set to 1 x 10 －１０ was set to.

[0089] (Identified Fertility Restorer Genes) As a result of the above-mentioned BSA-Seq and organelle DNA replication-related gene analysis, the fertility restorer genes shown in Table 1 below were identified for the fertility restorer lines EMS#1, #2, #7, #9, #10, #11, #12, #14, #16, #17, #20, #21, and #22. Specifically, BSA-Seq identified the phage-type RNA polymerase gene (gene ID: Solyc05g010660), the pentatricopeptide repeat (PPR) gene (gene ID: Solyc07g053060), and the serine / threonine-protein kinase gene (gene ID: Solyc01g109080) as fertility restorer genes, and organelle DNA replication-related gene analysis identified the DNA topoisomerase 1 gene (gene ID: Solyc02g081910) and the POLYMERASE GAMMA2 gene (gene ID: Solyc07g064330) as fertility restorer genes. Table 1 shows the fertility restorer genes identified for each fertility restorer line and the location of the mutations in each fertility restorer gene. SL4.0 is a reference genome widely used in tomato research, and for example, the phage-type RNA polymerase gene is located on ch05 (chromosome 5).

[0090]

[0091] [Verification of each fertility restorer gene using CRISPR-Cas9] In this experiment, first, to confirm that the phage-type RNA polymerase gene (gene ID: Solyc05g010660), which had been identified as a fertility restorer gene, was truly a fertility restorer gene, the phage-type RNA polymerase gene of the tomato CMS line Dwarf CMS[P] was knocked out using a CRISPR-Cas9 vector.

[0092] (Construction of CRISPR-Cas9 Vector) The CDS of the phage-type RNA polymerase gene was prepared, and two gRNA sequences (20 bp) were determined on the CDS using CRISPR-P (Yang Lei, et al., A Web Tool 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' (SEQ ID NO: 34) and 5'-CTTGGTTCTGATTTATGCCG-3' (SEQ ID NO: 35), respectively.

[0093] Complementary oligo DNA strands for each gRNA sequence were synthesized by Eurofins Genomics, Inc., with ATTG at one 5' end and AAAC at the other. Two microliters of each oligo DNA (50 μM) was added to 46 μL of distilled water, heated at 95°C for 5 minutes, and then left at room temperature for 20 minutes to anneal each oligo DNA. BbsI-treated AtU6gRNA (pMR217) and AtU6gRNA (pMR218) were mixed and ligated using Ligation High Ver. 2 (Toyobo). Reaction conditions followed Toyobo's protocol. The ligated plasmid was introduced into Escherichia coli DH5α (Toyobo) by heat shock. The gRNA sequence within the plasmid was confirmed by Sanger sequencing (Eurofins Genomics). AtU6gRNA (pMR217, pMR218) containing the desired gRNA was mixed with the Cas9 cassette pDe-Cas9-Kan, and the gRNA expression cassette was transferred to pDe-Cas9-Kan using an LR reaction (Thermo Fisher Scientific). pDe-Cas9-Kan containing the desired gRNA sequence was introduced into Escherichia coli DH5α by heat shock, and the gRNA sequence within the plasmid was confirmed by Sanger sequencing. The constructed plasmid was then introduced into Agrobacterium (GV3103) by electroporation.

[0094] (Knockout using CRISPR-Cas9 vector) The vector prepared as described above was introduced into Dwarf CMS[P] using the Agrobacterium method, and nine transformants were obtained. The presence or absence of mutations in the phage-type RNA polymerase gene was investigated using Sanger sequencing, and three independent lines (CR#4, CR#8, and CR#9) with different introduced mutations were confirmed.

[0095] FIG. 2 shows the mutation details of three strains with mutations in the phage-type RNA polymerase gene.

[0096] As shown in Figure 2, in the CR#4 line, a 4-bp deletion of bases 109 to 112 from the 5' end of exon 9 of the Phage-type RNA polymerase gene was present. In the CR#8 line, a 227-bp deletion of bases was present, from the 103rd base counting from the 5' end of exon 8 of the Phage-type RNA polymerase gene to the 112th base from the 5' end of exon 9. In the CR#9 line, a 4-bp deletion was present in exon 8 of the Phage-type RNA polymerase gene. In the CR#9 line, a deletion of bases 98-101 from the 5' end of exon 8 of the Phage-type RNA polymerase gene was present. These deletions were detected in a heterozygous state (deletion in only one allele), and because they were all frameshift mutations, they suggested that the function of the phage-type RNA polymerase gene was impaired.

[0097] FIG. 3 shows images showing pollen germination of three lines in which the phage-type RNA polymerase gene has been knocked out.

[0098] Pollen germination on the stigma was examined, and pollen germination was observed in each of the three independent lines, CR#4, CR#8, and CR#9, as shown in Figure 3. On the other hand, no pollen germination was observed in the transformant CR#6 (WT type with the same base sequence as Dwarf CMS[P] for the phage-type RNA polymerase gene) into which no mutation had been introduced.

[0099] From the above results, it was confirmed that male fertility can be restored in a cytoplasmic male sterile line by deleting the function of the phage-type RNA polymerase gene in the tomato CMS line Dwarf CMS[P].

[0100] 4 shows images of pollen germination in lines in which the DNA topoisomerase 1 gene, the Pentatricopeptide repeat (PPR) gene, the Serine / Threonine-protein kinase gene, or the POLYMERASE GAMMA2 gene were knocked out. Any of these four genes, as well as the phage-type RNA polymerase gene, were knocked out using the CRISPR-Cas9 vector.

[0101] For knocking out the DNA topoisomerase1 gene (gene ID: Solyc02g081910), a gRNA having the base sequence shown in SEQ ID NO: 36 was used as the first position, and a gRNA having the base sequence shown in SEQ ID NO: 37 was used as the second position. For knocking out the Pentatricopeptide repeat (PPR) gene (gene ID: Solyc07g053060), a gRNA having the base sequence shown in SEQ ID NO: 38 was used as the first position, and a gRNA having the base sequence shown in SEQ ID NO: 39 was used as the second position. For knocking out the serine / threonine-protein kinase gene (gene ID: Solyc01g109080), a gRNA having the base sequence shown in SEQ ID NO: 40 was used as the first position, and a gRNA having the base sequence shown in SEQ ID NO: 41 was used as the second position. The gRNA for knocking out the POLYMERASE GAMMA2 gene (gene ID: Solyc07g064330) uses a gRNA having the base sequence shown in SEQ ID NO: 42 as the first position, and a gRNA having the base sequence shown in SEQ ID NO: 43 as the second position. As shown in Figure 4, germination was confirmed in pollen from individuals of any lineage lacking any of the four genes. From these results, it was confirmed that the DNA topoisomerase1 gene, Pentatricopeptide repeat (PPR) gene, Serine / threonine-protein kinase gene, and POLYMERASE GAMMA2 gene are all genes that restore male fertility in cytoplasmic male sterile lines by causing a loss of function.

[0102] (DNA Marker Test) The fertility restorer genes, i.e., the phage-type RNA polymerase gene, the DNA topoisomerase 1 gene, the pentacopeptide repeat (PPR) gene, the serine / threonine-protein kinase gene, and the POLYMERASE GAMMA2 gene, were verified using DNA markers. PCR was performed using the primers shown in Table 2 below on F1 individuals produced by crossing Micro-Tom pollen with the F1 generation of the new fertility restorer line, and on populations of BC1F1 and subsequent generations obtained by backcrossing each line to Dwarf CMS[P].

[0103]

[0104] In PCR, 10x PCR Buffer (Mg ２＋ A PCR reaction solution was prepared in a total volume of 10 μL by mixing 1 μL of dNTP mixture (2.5 mM (mol / L) each), 0.8 μL of dNTP mixture (2.5 mM (mol / L) each), 0.3 μL of 10 μM forward primer, 0.3 μL of 10 μM reverse primer, 0.05 μL of 5 U / μL TaKaRa Taq HS (TAKARA Corporation), 0.5 μL of template DNA, and 7.05 μL of distilled water.

[0105] Using a thermal cycler, a 3-step PCR (initial heat denaturation at 94 ° C for 3 minutes, heat denaturation at 98 ° C for 10 seconds, annealing at 55 ° C for 30 seconds, extension at 72 ° C for 30 seconds (35 cycles of heat denaturation, annealing, and extension), final extension at 72 ° C for 5 minutes) was performed. After the PCR reaction, 0.1 μL of the restriction enzyme shown in Table 2, 2 μL of the accompanying buffer, and 17.9 μL of distilled water were mixed to a total volume of 10 μL and added to the PCR product, and the reaction was allowed to proceed overnight at the temperature shown in Table 2. Next, a 3 w / v% agarose gel containing Midori Green Advance DNA Stain (Nippon Genetics Co., Ltd.) was prepared, and 6 μL of a mixture of 20 μL of PCR product and 4 μL of 6x Loading dye was poured onto the gel. Then, electrophoresis was performed at 100 V for 45 minutes, and the band amplification was visualized using a UV transilluminator. Gene Ladder Wide 1 (Nippon Gene Co., Ltd.) was used as a molecular weight marker.

[0106] Figure 5 shows band images showing the results of amplifying the fertility restorer gene for each of the fertility restorer lines EMS#1, #2, #7, #9, #10, #11, #12, #14, #16, #17, and #20 and treating them with restriction enzymes.

[0107] In each image in Figure 5, the left lane is a sample of an individual whose fertility was restored by mutation of any of the following genes: phage-type RNA polymerase gene, DNA topoisomerase 1 gene, pentacopeptide repeat (PPR) gene, serine / threonine-protein kinase gene, and POLYMERASE GAMMA2 gene; and the right lane is a sample of a Dwarf CMS[P] individual that does not have mutations in these five genes.

[0108] The fertility restorer genes for each fertility restorer line were amplified under the conditions shown in Table 2 above and digested with restriction enzymes. As shown in the left lane of each figure in Figure 5, each of the EMS#1, #2, #7, #9, #10, #11, #12, #14, #16, #17, and #20 lines is heterozygous, having both a mutated allele and a non-mutated allele, and therefore a band derived from one cleaved allele and a band derived from the other non-cleaved allele were observed. For each fertility restorer gene, see Table 2 to see whether the mutated allele or the non-mutated allele was cleaved. On the other hand, in the right lane, which uses Dwarf CMS[P] as a sample, which has five non-mutated fertility restorer genes in a homozygous state, a single band derived from both alleles was observed. These results revealed that by treating the amplified products obtained by PCR amplification using the primer sequences shown in Table 2 with restriction enzymes, it is possible to determine whether or not a sample Solanaceae plant contains a mutated allele for each fertility restoration gene (and thus whether or not male sterility has been restored).

[0109] According to the present invention, by deleting or suppressing the function of at least one of the fertility restorer genes (a) to (o) above, the fertility of a cytoplasmic male sterile line of a Solanaceae plant can be restored, making the invention industrially applicable.

Claims

1. A male fertility restorer plant of the Solanaceae family, in which the function of at least one of the following fertility restorer genes (a) to (o) is deleted or suppressed: (a) a fertility restorer gene encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 1; (b) a fertility restorer gene encoding a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence set forth in SEQ ID NO: 1, and which causes male fertility restoration in Solanaceae plants when the function is lost or suppressed; (c) a fertility restorer gene encoding a protein comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1, and which causes male fertility restoration in Solanaceae plants when the function is lost or suppressed; (d) a fertility restorer gene encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2; (e) a fertility restorer gene encoding a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence set forth in SEQ ID NO: 2, and which causes male fertility restoration in Solanaceae plants when the function is lost or suppressed; (f) a fertility restorer gene that encodes a protein comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2, and whose function is lost or suppressed to restore male fertility to Solanaceae plants; (g) a fertility restorer gene that encodes a protein comprising the amino acid sequence shown in SEQ ID NO: 3; (h) a fertility restorer gene that encodes a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 3, and whose function is lost or suppressed to restore male fertility to Solanaceae plants; (i) a fertility restorer gene that encodes a protein comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3, and whose function is lost or suppressed to restore male fertility to Solanaceae plants; (j) a fertility restorer gene that encodes a protein comprising the amino acid sequence shown in SEQ ID NO: 4; (k) a fertility restorer gene that encodes a protein containing an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted in the amino acid sequence shown in SEQ ID NO: 4, and whose function is lost or suppressed to restore male fertility to a Solanaceae plant;(l) A fertility restorer gene that encodes a protein comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and whose function is lost or suppressed to restore male fertility to Solanaceae plants; (m) A fertility restorer gene that encodes a protein comprising the amino acid sequence shown in SEQ ID NO: 5; (n) A fertility restorer gene that encodes a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 5, and whose function is lost or suppressed to restore male fertility to Solanaceae plants; (o) A fertility restorer gene that encodes a protein comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 5, and whose function is lost or suppressed to restore male fertility to Solanaceae plants.

2. The male fertility restorer plant of the Solanaceae family according to claim 1, which is of the genus Solanum.

3. The male fertility restorer plant of the Solanaceae family according to claim 2, which is a tomato.

4. The male fertility restorer plant of the Solanaceae family according to claim 2, which is a potato.

5. A method for restoring male fertility to a solanaceous plant, comprising a step of deleting or suppressing the function of a fertility restorer gene of the solanaceous plant, wherein the fertility restorer gene is at least one of the following (a) to (o): (a) a fertility restorer gene encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 1; (b) a fertility restorer gene encoding a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence set forth in SEQ ID NO: 1, and which causes male fertility restoration in Solanaceae plants when the function is lost or suppressed; (c) a fertility restorer gene encoding a protein comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1, and which causes male fertility restoration in Solanaceae plants when the function is lost or suppressed; (d) a fertility restorer gene encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2; (e) a fertility restorer gene encoding a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence set forth in SEQ ID NO: 2, and which causes male fertility restoration in Solanaceae plants when the function is lost or suppressed; (f) a fertility restorer gene that encodes a protein comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2, and whose function is lost or suppressed to restore male fertility to Solanaceae plants; (g) a fertility restorer gene that encodes a protein comprising the amino acid sequence shown in SEQ ID NO: 3; (h) a fertility restorer gene that encodes a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 3, and whose function is lost or suppressed to restore male fertility to Solanaceae plants; (i) a fertility restorer gene that encodes a protein comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3, and whose function is lost or suppressed to restore male fertility to Solanaceae plants; (j) a fertility restorer gene that encodes a protein comprising the amino acid sequence shown in SEQ ID NO: 4; (k) a fertility restorer gene that encodes a protein containing an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted in the amino acid sequence shown in SEQ ID NO: 4, and whose function is lost or suppressed to restore male fertility to a Solanaceae plant;(l) A fertility restorer gene that encodes a protein comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and whose function is lost or suppressed to restore male fertility to Solanaceae plants; (m) A fertility restorer gene that encodes a protein comprising the amino acid sequence shown in SEQ ID NO: 5; (n) A fertility restorer gene that encodes a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 5, and whose function is lost or suppressed to restore male fertility to Solanaceae plants; (o) A fertility restorer gene that encodes a protein comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 5, and whose function is lost or suppressed to restore male fertility to Solanaceae plants.

6. The method for restoring male fertility to a solanaceous plant according to claim 5, wherein the solanaceous plant is of the genus Solanum.

7. The method for restoring male fertility to a solanaceous plant according to claim 6, wherein the solanaceous plant is a tomato.

8. The method for restoring male fertility to a solanaceous plant according to claim 6, wherein the solanaceous plant is potato.

9. A method for producing a male-fertility-restored Solanaceae plant, comprising obtaining a Solanaceae plant having restored male fertility by the method for restoring male fertility to a Solanaceae plant according to any one of claims 5 to 8.

10. A method for determining whether or not fertility has been restored in a cytoplasmic male sterility plant of the Solanaceae family, comprising the step of amplifying part or all of the base sequence of at least one of the following fertility restorer genes (a) to (o): (a) a fertility restorer gene encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 1; (b) a fertility restorer gene encoding a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence set forth in SEQ ID NO: 1, and which causes male fertility restoration in Solanaceae plants when the function is lost or suppressed; (c) a fertility restorer gene encoding a protein comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1, and which causes male fertility restoration in Solanaceae plants when the function is lost or suppressed; (d) a fertility restorer gene encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2; (e) a fertility restorer gene encoding a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence set forth in SEQ ID NO: 2, and which causes male fertility restoration in Solanaceae plants when the function is lost or suppressed; (f) a fertility restorer gene that encodes a protein comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2, and whose function is lost or suppressed to restore male fertility to Solanaceae plants; (g) a fertility restorer gene that encodes a protein comprising the amino acid sequence shown in SEQ ID NO: 3; (h) a fertility restorer gene that encodes a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 3, and whose function is lost or suppressed to restore male fertility to Solanaceae plants; (i) a fertility restorer gene that encodes a protein comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3, and whose function is lost or suppressed to restore male fertility to Solanaceae plants; (j) a fertility restorer gene that encodes a protein comprising the amino acid sequence shown in SEQ ID NO: 4; (k) a fertility restorer gene that encodes a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 4, and that restores male fertility to a Solanaceae plant when its function is lost or suppressed; (l) a fertility restorer gene that encodes a protein comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and that restores male fertility to a Solanaceae plant when its function is lost or suppressed;(m) a fertility restorer gene that encodes a protein comprising the amino acid sequence shown in SEQ ID NO: 5; (n) a fertility restorer gene that encodes a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 5, and whose function is lost or suppressed to restore male fertility to Solanaceae plants; (o) a fertility restorer gene that encodes a protein comprising an amino acid sequence that has 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 5, and whose function is lost or suppressed to restore male fertility to Solanaceae plants.

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