Male fertility-restoring plant of solanaceae, method for restoring male fertility of solanaceous plant, method for producing male fertility-restoring plant of solanaceae, and method for determining male fertility restoration of solanaceous plant

Abstract

To provide a novel cytoplasmic male sterility restoring technology for Solanaceae.SOLUTION: This male fertility-restoring plant of Solanaceae comprises at least one base sequence among (a) to (r), the base sequences (a) to (r) being base sequences having a male fertility-restoring function.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a male fertility restored plant of the Solanaceae family, a method for restoring male fertility to a Solanaceae plant, a method for producing a male fertility restored plant of the Solanaceae family, and a method for distinguishing between male fertility restored plants of the Solanaceae family. [Background technology]

[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 by hand, 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, such as parent lines. 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 cross-breeding.

[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 that utilizes 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 resulting in the failure to produce seeds or fruit. 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 incurable with cytoplasmic factors with protoplasts isolated from Solanum plants and treated to be incurable with 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 affecting any traits of the target tomato plants other than male sterility.

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

[0007] Patent Document 3 discloses a method for producing plants with modified traits, which utilizes a plant expression cassette containing a promoter containing either DNA with a specific sequence number or a partial sequence thereof, and DNA that exhibits specific promoter activity in microspores and, optionally, anther dehiscence tissue, and a heterologous gene operably linked to the promoter. This technology aims to provide a genetic engineering technique that utilizes pollen-specific genes, which is useful for modifying plant traits, such as male sterility, in horticultural plants of the Solanaceae family, such as petunia. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Patent No. 2824841 [Patent Document 2] International Publication No. 2014 / 027502 [Patent Document 3] Japanese Patent Application Laid-Open No. 2003-92937 Summary of the Invention [Problem to be solved by the invention]

[0009] 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.

[0010] Patent Document 2 discloses a gene for cytoplasmic male sterility in the Poaceae family, but does not disclose a gene responsible for cytoplasmic male sterility in plants of the Solanaceae family.

[0011] The technology in Patent Document 3 attempts to genetically manipulate sterility in a type of plant in the Solanaceae family, but the gene manipulated is a gene contained in the nucleus, changing the plant's traits. This technology cannot manipulate sterility on / off by manipulating mitochondrial cytoplasmic genes without changing nuclear genes, as is the case with cytoplasmic male sterility. The technology in Patent Document 3 manipulates plants in the Solanaceae family, such as petunias, in the horticultural field, and is not intended for use in the food field.

[0012] 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. [Means for solving the problem]

[0013] In order to solve the above problems, the present invention includes the following aspects.

[0014] [1] A male fertility restorer plant of the Solanaceae family, which contains at least one of the following nucleotide sequences (a) to (r) in its nuclear genomic DNA: (a) The nucleotide sequence of the region from 83.29 Mbp to 84.65 Mbp from the 5' end of the nuclear genome chromosome 1 of the wild tomato species Solanum pimpinellifolium. (b) a base sequence comprising the base sequence of (a) in which one or more bases are deleted, substituted, added or inserted, and having a male fertility restoration function; (c) a DNA sequence that contains a base sequence having 60% or more sequence identity with the base sequence of (a) and has a male fertility restoration function; (d) The base sequence of the region from 80.03 Mbp to 80.65 Mbp from the 5' end of the nuclear genome chromosome 1 of the cherry tomato species Solanum lycopersicum var. cerasiforme, (e) a base sequence comprising the base sequence of (d) in which one or more bases are deleted, substituted, added or inserted, and having a male fertility restoration function; (f) a base sequence having a sequence identity of 60% or more with the base sequence of (d) and having a male fertility restoration function; (g) a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1; (h) a base sequence encoding 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: 1 and having a male fertility restoration function; (i) a base sequence encoding an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and having a male fertility restoration function; (j) a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2; (k) a base sequence encoding 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: 2 and having a male fertility restoration function; (l) a base sequence encoding an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2 and having a male fertility restoration function; (m) The nucleotide sequence of the region from 38 Mbp to 47 Mbp from the 5' end of the nuclear genome chromosome 2 of Solanum pimpinellifolium, (n) a base sequence comprising the base sequence of (m) in which one or more bases are deleted, substituted, added or inserted, and having a male fertility restoration function; (o) a base sequence having a sequence identity of 60% or more with the base sequence of (m) and having a male fertility restoration function; (p) The nucleotide sequence of the region from 81.89 Mbp to 83.21 Mbp from the 5' end of the nuclear genome chromosome 1 of the wild tomato species Solanum cheesmaniae, (q) a base sequence comprising the base sequence of (p) in which one or more bases have been deleted, substituted, added or inserted, and having a male fertility restoration function; (r) A base sequence that contains a base sequence having 60% or more sequence identity with the base sequence of (p) and has the function of restoring male fertility.

[0015] [2] A male fertility restorer plant of the Solanaceae family described in [1], which is of the genus Solanum.

[0016] [3] The male fertility restorer plant of the Solanaceae family described in [2], which is a tomato.

[0017] [4] The male fertility restorer plant of the Solanaceae family described in [2], which is a potato.

[0018] [5] A method for restoring male fertility to a Solanaceae plant, comprising introducing at least one of the following base sequences (a) to (r) into the nuclear genome of a Solanaceae plant having cytoplasmic male sterility: (a) The nucleotide sequence of the region from 83.29 Mbp to 84.65 Mbp from the 5' end of the nuclear genome chromosome 1 of the wild tomato species Solanum pimpinellifolium. (b) a base sequence comprising the base sequence of (a) in which one or more bases are deleted, substituted, added or inserted, and having a male fertility restoration function; (c) a DNA sequence that contains a base sequence having 60% or more sequence identity with the base sequence of (a) and has a male fertility restoration function; (d) The base sequence of the region from 80.03 Mbp to 80.65 bp from the 5' end of the nuclear genome chromosome 1 of the cherry tomato species Solanum lycopersicum var. cerasiforme, (e) a base sequence comprising the base sequence of (d) in which one or more bases are deleted, substituted, added or inserted, and having a male fertility restoration function; (f) a base sequence having a sequence identity of 60% or more with the base sequence of (d) and having a male fertility restoration function; (g) a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1; (h) a base sequence encoding 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: 1 and having a male fertility restoration function; (i) a base sequence encoding an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and having a male fertility restoration function; (j) a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2; (k) a base sequence encoding 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: 2 and having a male fertility restoration function; (l) a base sequence encoding an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2 and having a male fertility restoration function; (m) The nucleotide sequence of the region from 38 Mbp to 47 Mbp from the 5' end of the nuclear genome chromosome 2 of Solanum pimpinellifolium, (n) a base sequence comprising the base sequence of (m) in which one or more bases are deleted, substituted, added or inserted, and having a male fertility restoration function; (o) a base sequence having a sequence identity of 60% or more with the base sequence of (m) and having a male fertility restoration function; (p) The nucleotide sequence of the region from 81.89 Mbp to 83.21 Mbp from the 5' end of the nuclear genome chromosome 1 of the wild tomato species Solanum cheesmaniae, (q) a base sequence comprising the base sequence of (p) in which one or more bases have been deleted, substituted, added or inserted, and having a male fertility restoration function; (r) A base sequence that contains a base sequence having 60% or more sequence identity with the base sequence of (p) and has the function of restoring male fertility.

[0019] [6] The method for restoring male fertility to a solanaceous plant according to [5], wherein the solanaceous plant is of the genus Solanum.

[0020] [7] The method for restoring male fertility to a solanaceous plant according to [6], wherein the solanaceous plant is a tomato.

[0021] [8] The method for restoring male fertility to a solanaceous plant according to [6], wherein the solanaceous plant is potato.

[0022] [9] A method for producing a male-fertility-restored Solanaceae plant, comprising obtaining a Solanaceae plant whose male fertility has been restored by the method for restoring male fertility to a Solanaceae plant described in any one of [5] to [8].

[0023]

[10] A method for determining whether fertility has been restored in a Solanaceae plant that is cytoplasmic male sterile, comprising a step of confirming whether the Solanaceae plant contains at least one of the following base sequences (a) to (r): (a) The nucleotide sequence of the region from 83.29 Mbp to 84.65 Mbp from the 5' end of the nuclear genome chromosome 1 of the wild tomato species Solanum pimpinellifolium. (b) a base sequence comprising the base sequence of (a) in which one or more bases are deleted, substituted, added or inserted, and having a male fertility restoration function; (c) a DNA sequence that contains a base sequence having 60% or more sequence identity with the base sequence of (a) and has a male fertility restoration function; (d) The base sequence of the region from 80.03 Mbp to 80.65 bp from the 5' end of the nuclear genome chromosome 1 of the cherry tomato species Solanum lycopersicum var. cerasiforme, (e) a base sequence comprising the base sequence of (d) in which one or more bases are deleted, substituted, added or inserted, and having a male fertility restoration function; (f) a base sequence having a sequence identity of 60% or more with the base sequence of (d) and having a male fertility restoration function; (g) a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1; (h) a base sequence encoding 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: 1 and having a male fertility restoration function; (i) a base sequence encoding an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and having a male fertility restoration function; (j) a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2; (k) a base sequence encoding 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: 2 and having a male fertility restoration function; (l) a base sequence encoding an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2 and having a male fertility restoration function; (m) The nucleotide sequence of the region from 38 Mbp to 47 Mbp from the 5' end of the nuclear genome chromosome 2 of Solanum pimpinellifolium, (n) a base sequence comprising the base sequence of (m) in which one or more bases are deleted, substituted, added or inserted, and having a male fertility restoration function; (o) a base sequence having a sequence identity of 60% or more with the base sequence of (m) and having a male fertility restoration function; (p) The nucleotide sequence of the region from 81.89 Mbp to 83.21 Mbp from the 5' end of the nuclear genome chromosome 1 of the wild tomato species Solanum cheesmaniae, (q) a base sequence comprising the base sequence of (p) in which one or more bases have been deleted, substituted, added or inserted, and having a male fertility restoration function; (r) A base sequence that contains a base sequence having 60% or more sequence identity with the base sequence of (p) and has the function of restoring male fertility. [Effects of the Invention]

[0024] According to the present invention, a novel technique for restoring cytoplasmic male sterility in Solanaceae plants can be provided. [Brief explanation of the drawings]

[0025] [Figure 1] 1 is a graph showing the results of linkage analysis of the RF1a1 locus from Solanum pimpinellifolium. [Figure 2] 1 is a diagram showing the results of linkage analysis of the RF1a2 locus derived from Solanum lycopersicum var. cerasiforme. [Figure 3] 1 shows the results of resequencing Solanum lycopersicum var. cerasiforme and the CMS line. [Figure 4] 1 shows the results of a comparison of the genome structures of chromosome 1 among five tomato varieties. [Figure 5] This is an image showing pollen germination of a CMS tomato line into which the pentatricopeptide repeat (PPR) gene (gene ID: SPI01g07345) has been introduced. [Figure 6] This is an image showing pollen germination of a CMS tomato line into which the pentatricopeptide repeat (PPR) gene (gene ID: SPI01g07485) has been introduced. [Figure 7] 1 shows the results of bulk analysis of F1 individuals between a CMS tomato line and Solanum pimpinellifolium. [Figure 8] This is an image showing the results of amplifying a portion of the RF2 locus using DNA obtained from CMS[MSA1], Solanum pimpinellifolium, and their F1 as templates. DETAILED DESCRIPTION OF THE INVENTION

[0026] 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.

[0027] [Male fertility restorer plants of the Solanaceae family] The male fertility restored plant of the Solanaceae family in this embodiment is a plant in which fertility has been restored from cytoplasmic male sterility (CMS), and contains at least one of the following base sequences (a) to (r) (in other words, at least one base sequence selected from the group consisting of (a) to (r), which is a polynucleotide) in its nuclear genomic DNA: (a) The nucleotide sequence of the 83.29-84.65 Mbp region from the 5' end of the nuclear genome chromosome 1 (chromosome 1) of the cultivated tomato species Solanum pimpinellifolium. (b) a base sequence comprising the base sequence of (a) in which one or more bases are deleted, substituted, added or inserted, and having a male fertility restoration function; (c) a DNA sequence that contains a base sequence having 60% or more sequence identity with the base sequence of (a) and has a male fertility restoration function; (d) The base sequence of the region from 80.03 Mbp to 80.65 Mbp from the 5' end of the nuclear genome chromosome 1 (chromosome 1) of the cherry tomato species Solanum lycopersicum var. cerasiforme, (e) a base sequence comprising the base sequence of (d) in which one or more bases are deleted, substituted, added or inserted, and having a male fertility restoration function; (f) a base sequence having a sequence identity of 60% or more with the base sequence of (d) and having a male fertility restoration function; (g) a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1; (h) a base sequence encoding 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: 1 and having a male fertility restoration function; (i) a base sequence encoding an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and having a male fertility restoration function; (j) a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2; (k) a base sequence encoding 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: 2 and having a male fertility restoration function; (l) a base sequence encoding an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2 and having a male fertility restoration function; (m) The nucleotide sequence of the region from 38 Mbp to 47 Mbp from the 5' end of the nuclear genome chromosome 2 (chromosome 2) of Solanum pimpinellifolium, (n) a base sequence comprising the base sequence of (m) in which one or more bases are deleted, substituted, added or inserted, and having a male fertility restoration function; (o) a base sequence having a sequence identity of 60% or more with the base sequence of (m) and having a male fertility restoration function; (p) The base sequence of the region from 81.89 Mbp to 83.21 Mbp from the 5' end of the nuclear genome chromosome 1 (chromosome 1) of the wild tomato species Solanum cheesmaniae, (q) a base sequence comprising the base sequence of (p) in which one or more bases have been deleted, substituted, added or inserted, and having a male fertility restoration function; (r) A base sequence that contains a base sequence having 60% or more sequence identity with the base sequence of (p) and has the function of restoring male fertility.

[0028] The above-mentioned "plant in which fertility has been restored from cytoplasmic male sterility (CMS)" can also be said to be a plant that has orf137 (see Patent Publication No. 2023-32890), the causative gene (S factor gene) of cytoplasmic male sterility, and contains at least one of the above base sequences (a) to (r) in its nuclear genomic DNA.

[0029] As used herein, "orf137 gene" refers to a gene that encodes a protein containing 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: 3 and that causes cytoplasmic male sterility.

[0030] The above base sequences (polynucleotides) (a) to (r) are all base sequences that, when included in the nuclear genomic DNA of a Solanaceae plant that exhibits cytoplasmic male sterility, have the function of restoring male fertility (male fertility restoration function). Male fertility can be restored by introducing a polynucleotide containing at least one of the above base sequences (a) to (r) into the nuclear DNA of a Solanaceae plant that exhibits cytoplasmic male sterility. Each of the genes (a) to (r) may be contained in only one allele (heterozygote) or in both alleles. Fertility restoration occurs when one or more of the genes are contained in one or both alleles.

[0031] The nucleotide sequence of (a) above is a 1.36 Mbp long nucleotide sequence in the region from 83.29 Mbp to 84.65 Mbp from the 5' end of chromosome 1 of the nuclear genome of the fertility restorer line, Solanum pimpinellifolium (LA1670 line). Lines that are naturally fertile, such as Solanum pimpinellifolium, are also generally referred to as "fertility restorer lines." The present inventors have discovered that the nucleotide sequence of this region has male fertility restoration function (activity). Hereinafter, this region will also be referred to as the "RF1a1 locus." The nucleotide sequence of (a) is the nucleotide sequence of the region from the gene shown by gene ID: SPI01g06993 to the gene shown by gene ID: SPI01g07137 on chromosome 1 of the nuclear genome of Solanum pimpinellifolium. Therefore, the nucleotide sequence of (a) includes the gene shown by gene ID: SPI01g06993, the gene shown by gene ID: SPI01g07137, and all genes located between these two genes on chromosome 1.

[0032] The sequence identity of the base sequence of (c) above with the base sequence of (a) above is 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 base sequence of (d) above is a 611 kbp long base sequence located in the region from 80.23 Mbp to 80.65 Mbp from the 5' end of chromosome 1 of the nuclear genome of the fertility restorer line, Solanum lycopersicum var. cerasiforme (LA1673 line). The present inventors have determined that this base sequence has the function of restoring male fertility. Hereinafter, this region will also be referred to as the "RF1a2 locus." The base sequence of (d) is the base sequence of the region from the gene shown by gene ID: SLYcer01g06744 to the gene shown by gene ID: SLYcer01g06812 on chromosome 1 of the nuclear genome of Solanum lycopersicum var. cerasiforme. Therefore, the base sequence of (d) includes the gene shown by gene ID: SLYcer01g06744, the gene shown by gene ID: SLYcer01g06812, and all genes located between these two genes on chromosome 1.

[0034] The sequence identity of the base sequence (f) above with the base sequence (d) above is 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 amino acid sequence shown in SEQ ID NO: 1 is encoded by a gene located between bases 86296512 and 86298197, counting from the 5' end of chromosome 1 of the nuclear genomic DNA of Solanum pimpinellifolium (LA1670 line). This gene is a pentatricopeptide repeat (PPR) gene (gene ID: SPI01g07345). The present inventors have determined that this gene has the function of restoring male fertility. Hereinafter, this gene will also be referred to as the "RF1b gene." An example of the nucleotide sequence (g) above that encodes the amino acid sequence shown in SEQ ID NO: 1 is the nucleotide sequence of the cDNA of the RF1b gene shown in SEQ ID NO: 22.

[0036] The amino acid sequence encoded by the base sequence (i) above has a sequence identity with the base sequence (g) above 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.

[0037] The amino acid sequence shown in SEQ ID NO: 2 is encoded by a gene located between bases 87444727 and 87447736, counting from the 5' end of chromosome 1 of the nuclear genomic DNA of Solanum pimpinellifolium (line LA1670). This gene is a pentatricopeptide repeat (PPR) gene (gene ID: SPI01g07485). The present inventors have determined that this gene has the function of restoring male fertility. Hereinafter, this gene will also be referred to as the "RF1c gene." An example of the nucleotide sequence (j) above that encodes the amino acid sequence shown in SEQ ID NO: 2 is the nucleotide sequence of the cDNA of the RF1c gene shown in SEQ ID NO: 23.

[0038] The sequence identity of the base sequence (l) above with the base sequence (j) above is 60% or more, preferably 75% or more, more preferably 80% or more, even more preferably 90% or more, and most preferably 95% or more.

[0039] The nucleotide sequence of (m) above is a 9 Mbp long nucleotide sequence located in a region of 38 Mbp to 47 Mbp from the 5' end of the nuclear genome chromosome 2 of Solanum pimpinellifolium (strain LA1670). The present inventors have determined that this nucleotide sequence has the function of restoring male fertility. Hereinafter, this region will also be referred to as the "RF2 locus." The base sequence of (m) is the base sequence of the region from the gene shown by gene ID: SPI02g03247 to the gene shown by gene ID: SPI02g04565 on chromosome 2 of the nuclear genome of Solanum pimpinellifolium. Therefore, the base sequence of (m) includes the gene shown by gene ID: SPI02g03247, the gene shown by gene ID: SPI02g04565, and all genes located between these two genes on chromosome 2.

[0040] The sequence identity of the base sequence (o) above with the base sequence (m) above is 60% or more, preferably 75% or more, more preferably 80% or more, even more preferably 90% or more, and most preferably 95% or more.

[0041] The base sequence of (p) above is a 1.32 Mbp long base sequence located in the region from 81.89 Mbp to 83.21 Mbp from the 5' end of chromosome 1 of the nuclear genome of the fertility restorer line Solanum cheesmaniae (LA0166 line). The present inventors have determined that this base sequence has the function of restoring male fertility. Hereinafter, this region will also be referred to as the "RF1d locus." The base sequence of (p) is the base sequence of the region from the gene shown by gene ID: SCH1.0ch01.g39240 to the gene shown by gene ID: SCH1.0ch01.g40750 in the nuclear genome chromosome 1 of Solanum cheesmaniae. Therefore, the base sequence of (p) includes the gene shown by gene ID: SCH1.0ch01.g39240, the gene shown by gene ID: SCH1.0ch01.g40750, and all genes located between these two genes on chromosome 1.

[0042] The sequence identity of the base sequence of (r) above with the base sequence of (p) above is 60% or more, preferably 75% or more, more preferably 80% or more, even more preferably 90% or more, and most preferably 95% or more.

[0043] In the base sequence (b) above, the number of bases that may be deleted, substituted, added, or inserted in the base sequence (a) above 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.

[0044] In the base sequence (e) above, the number of bases that may be deleted, substituted, added, or inserted in the base sequence (d) above 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.

[0045] In the amino acid sequence encoded by the base sequence (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: 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.

[0046] In the amino acid sequence encoded by the base sequence (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: 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.

[0047] In the base sequence (n) above, the number of bases that may be deleted, substituted, added, or inserted in the base sequence (m) above 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.

[0048] In this specification, the sequence identity of a subject base sequence to a reference base sequence (for example, the above-mentioned (a), (d), (m), and (p), or the base sequence shown in SEQ ID NO: 3) can be determined as follows: First, the reference base sequence and the subject base sequence are aligned. Gaps are included in each base sequence to maximize sequence identity. Next, the number of matching bases between the reference base sequence and the subject base sequence is calculated, and the sequence identity can be calculated according to the following formula (1). Sequence identity (%) = number of matched bases / total number of bases in the target sequence × 100 (1)

[0049] Furthermore, herein, the sequence identity of a subject amino acid sequence to a reference amino acid sequence (for example, the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2) 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 amino acids that match the reference amino acid sequence in the amino acid sequence is calculated, and the sequence identity can be calculated according to the following formula (2): Sequence identity (%) = number of matching amino acids / total number of amino acids in the target amino acid sequence × 100 (2)

[0050] Whether or not the above base sequences (b), (c), (e), (f), (h), (i), (k), (l), (n), (o), (q), or (r) have the function of restoring male fertility to cytoplasmic male sterility (male fertility restoration function) can be determined by determining whether or not a Solanaceae plant that has the above-mentioned orf137 gene and the above base sequence (b), (c), (e), (f), (h), (i), (k), (l), (n), (o), (q), or (r) has male fertility.

[0051] Whether or not a Solanaceae plant has male fertility can be determined, for example, by determining whether or not pollen obtained from the plant contains pollen that elongates pollen tubes (pollen germination) (if so, fertility restoration has occurred), or by determining whether or not seeds are formed when the pollen is fertilized with the pollen of a normal plant. However, the determination of whether or not a Solanaceae plant has male fertility, and therefore whether or not the above base sequence (b), (c), (e), (f), (h), (i), (k), (l), (n), (o), (q), or (r) has the function of restoring male fertility to cytoplasmic male sterility (male fertility restoration function), is not limited to these methods.

[0052] 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, potato, and tomato. Examples of plants of the Capsicum include chili peppers and bell peppers. Cytoplasmic male sterile strains of other Solanaceae plants, such as potato, also contain the orf137 gene.

[0053] 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.

[0054] The solanaceous plant is particularly preferably a crop, that is, a plant used in the agricultural field, particularly as a food raw material. The solanaceous plant may be a plant body, a plant cultured cell, a plant cell, or a callus.

[0055] [Method for restoring male fertility to Solanaceae plants] The method for restoring male fertility to a Solanaceae plant of this embodiment includes a step of introducing at least one of the base sequences (a) to (r) above (in other words, at least one base sequence selected from the group consisting of (a) to (r) above) into the nuclear genome of a Solanaceae plant having cytoplasmic male sterility (hereinafter also referred to as the "introduction step"). A "Solanaceae plant having cytoplasmic male sterility" is, in other words, a Solanaceae plant having the above-mentioned orf137 gene. The Solanaceae plant with restored male fertility of the embodiment is a Solanaceae plant obtained by the male fertility restoration method, or a progeny thereof.

[0056] One example of a method for introducing at least one of the above base sequences (a) to (r) into the nuclear genome of a Solanaceae plant having cytoplasmic male sterility is crossbreeding. By crossbreeding a target Solanaceae plant into which at least one of the above base sequences (a) to (r) is to be introduced with a Solanaceae plant having at least one of the above base sequences (a) to (r), it is possible to obtain, in the next generation, an individual Solanaceae plant into which at least one of the above base sequences (a) to (r) has been introduced.

[0057] Examples of Solanaceae plants having the above-mentioned base sequence (a) include Solanum pimpinellifolium (LA1670 line) and its progenies. Examples of Solanaceae plants having the above-mentioned base sequence (b) or (c) include mutants of Solanum pimpinellifolium, closely related species, and progenies thereof.

[0058] An example of a Solanaceae plant having the above-mentioned base sequence (d) is Solanum lycopersicum var. cerasiforme (LA1673 line) or a progeny thereof. Examples of Solanaceae plants having the above-mentioned nucleotide sequence (e) or (f) include mutants of Solanum lycopersicum var. cerasiforme, closely related species, and progenies thereof.

[0059] Examples of Solanaceae plants having the above-mentioned base sequence (g) include Solanum pimpinellifolium (LA1670 line) and its progenies. Examples of Solanaceae plants having the above-mentioned base sequence (h) or (i) include mutants of Solanum pimpinellifolium, closely related species, and progenies thereof.

[0060] Examples of Solanaceae plants having the above-mentioned base sequence (j) include Solanum pimpinellifolium (LA1670 line) and its progenies. Examples of Solanaceae plants having the above-mentioned base sequence (k) or (l) include mutants of Solanum pimpinellifolium, closely related species, and progenies thereof.

[0061] Examples of the Solanaceae plant having the above-mentioned base sequence (m) include Solanum pimpinellifolium (Solanum pimpinellifolium, LA1670 line) and its progeny. Examples of Solanaceae plants having the above-mentioned base sequence (n) or (o) include mutants of Solanum pimpinellifolium, closely related species, and progenies thereof.

[0062] Examples of Solanaceae plants having the above base sequence (p) include Solanum cheesmaniae (LA0166 line) and its progenies. Examples of Solanaceae plants having the above-mentioned base sequence (q) or (r) include mutants of Solanum cheesmaniae, closely related species, and progenies thereof.

[0063] Since the base sequences (g), (h), (i), (j), (k), and (l) above are relatively short in length, known methods such as the Agrobacterium method, particle gun method, polyethylene glycol (PEG) method, calcium phosphate method, electroporation method, liposome method, and DEAE-dextran method can also be used in the introduction step.

[0064] When using the Agrobacterium method, it is preferable to use a pBI or pPZP binary vector. Examples of pBI binary vectors include pBIG, pBIN19, pBI101, pBI121, and pBI221. Examples of pPZP binary vectors include pPZP100, pPZP200, pPZP500, and pPZP3425.

[0065] When a method other than crossbreeding is used to introduce a base sequence, the position for introducing at least one of the base sequences (a) to (r) above is preferably a region that has little impact on the viability of the Solanaceae plant, but is not particularly limited thereto.

[0066] Regardless of the method for introducing a nucleotide sequence, the solanaceous plant into which the nucleotide sequence is to be introduced and the solanaceous plant from which the nucleotide sequence to be introduced is derived may be of the same species or different species.

[0067] The method for restoring male fertility to a solanaceous plant of this embodiment may further include, after the above-mentioned introduction step, a step of backcrossing the solanaceous plant obtained in the introduction step with the target solanaceous plant (hereinafter also referred to as the "backcrossing step").

[0068] By carrying out the backcrossing step, various genes having base sequences other than at least one of the base sequences (a) to (r) above can be made closer to those of the target Solanaceae plant.

[0069] [Method for producing male fertility-restored plants of the Solanaceae family] The method for producing a male fertility-restored plant of the Solanaceae family 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 previous 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 introducing at least one of the base sequences (a) to (r) above into the nuclear genome.

[0070] [Method for determining male fertility restoration in Solanaceae plants] 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 Solanaceae plant that is cytoplasmic male sterility, and includes a step of confirming whether the Solanaceae plant contains at least one of the base sequences (a) to (r) above (hereinafter referred to as the "confirmation step"). In this embodiment, the Solanaceae plant is also a plant that contains the above-mentioned orf137 gene.

[0071] The specific method for the confirmation step is not particularly limited, but examples include amplifying a partial or entire region of at least one of the base sequences (a) to (r) above, or a base sequence located near any of the base sequences (a) to (r). The method for determining whether a Solanaceae plant contains at least one of the base sequences (a) to (r) above from the amplification product is not particularly limited, and for example, the amplification product may be subjected to electrophoresis or the like to determine whether a band is generated, or whether it is cleaved with a restriction enzyme, or the amplification product may be sequenced to confirm a polymorphism (to confirm whether it was amplified from the base sequence (a) to (r) above). For example, if a polymorphism exists in a base sequence located near any of the base sequences (a) to (r) above, the base sequence containing the polymorphism may be sequenced to read the polymorphic base, thereby determining whether or not the plant contains at least one of the base sequences (a) to (r) above.

[0072] The length of each amplification product is not particularly limited, and may be, for example, 50 bp to 2 kbp, or 100 bp to 1 kbp.

[0073] Those skilled in the art can appropriately design primer sequences for determining whether or not a DNA marker contains at least one of the base sequences (a) to (r). Table 1 below shows the sequences of primer pairs for determining whether or not a DNA marker contains the base sequence (a), (d), (g), (j), (m), or (p). The columns "Gene ID," "Gene Name," and "Gene Location" in Table 1 provide information about the gene closest to each DNA marker.

[0074] [Table 1]

[0075] Each primer pair shown in Table 1 is designed so that a portion of a gene or locus (a region having the base sequence (a), (d), (g), (j), (m), or (p) above) is included in the amplification product.

[0076] Table 2 below shows the size (full length) of the amplified product obtained by PCR using each primer set shown in Table 1 when a Solanaceae plant contains the above-mentioned base sequence (a), (d), (g), (j), (m), or (p) in its nuclear genomic DNA (LA-type in Table 2) and when it does not (MT-J-type in Table 2), as well as the difference in size of the amplified product between these two cases. The unit of each size is "bp." [Table 2]

[0077] In the amplification product (gene ID: SPI01g07345, part of the RF1b gene) obtained using the combination of the primer shown in SEQ ID NO: 8 and the primer shown in SEQ ID NO: 9, a polymorphism having different bases can be confirmed in the two cases described above. In detail, when a Solanaceae plant does not contain the RF1b gene, the 410th base counting from the 5' end of the amplification product is C, and when a Solanaceae plant contains the RF1b gene, the 410th base counting from the 5' end of the amplification product is A. To easily distinguish the polymorphism, it is preferable to react the amplified product with EcoN1, a type of restriction enzyme. If the solanaceous plant does not contain the RF1b gene, it will be cleaved by EcoN1, but if the solanaceous plant contains the RF1b gene, it will not be cleaved by EcoN1. In this case, CutSmart Buffer (New England Biolabs) is preferably used as the buffer, and the reaction temperature is preferably 37°C.

[0078] In the amplification product (gene ID: SPI01g07485, part of the RF1c gene) obtained using the pair of primers shown in SEQ ID NO: 10 and SEQ ID NO: 11, a polymorphism having different bases can be confirmed in the two cases described above. Specifically, when a Solanaceae plant does not contain the RF1c gene, the 261st base counting from the 5' end of the amplification product is T, and when a Solanaceae plant contains the RF1c gene, the 261st base counting from the 5' end of the amplification product is G. The polymorphism can be confirmed by sequencing the amplification product.

[0079] In PCR amplification using the four pairs of primers shown in Table 1 to confirm whether a Solanaceae plant contains the RF2 locus, if the size of the amplified product from any one or more of the four pairs is the "LA-type" size shown in Table 2, the Solanaceae plant is recognized as containing the RF2 locus (the base sequence of (m) above). However, the more primer pairs among the four primer pairs that produce amplified products of the "LA-type" size, the higher the probability that the RF2 locus is contained in the nuclear genome. Therefore, the number of primer pairs that produce amplified products of the "LA-type" size is preferably two or more, more preferably three or more, and most preferably four.

[0080] The base sequence of a primer for confirming the presence or absence of the base sequence (a), (d), (g), (j), (m), or (p) is not limited to the base sequence shown in Table 1. For example, a base sequence containing an additional base on the 5'-terminal side of the base sequence shown in Table 1 (a base sequence having each base sequence shown in Table 1 in the 3'-terminal region) can also be suitably used as the base sequence of a primer.

[0081] The primer for confirming the presence or absence of the base sequence (a), (d), (g), (j), (m), or (p) may be 50 nucleotides or less in length, 40 nucleotides or less in length, 35 nucleotides or less in length, 30 nucleotides or less in length, or 25 nucleotides or less in length.

[0082] The part of the Solanaceae plant subjected to the confirmation step is not particularly limited, and may be, for example, a leaf, a root, a stem, or pollen.

[0083] 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.

[0084] [Example of change] In the above-mentioned embodiment of the "Solanaceae male fertility restorer plant," the Solanaceae male fertility restorer plant contains at least one of the above-mentioned base sequences (a) to (r) in its nuclear genomic DNA. However, the male fertility restorer plant of Solanaceae contains at least one of the base sequences (a) to (c) in its nuclear genomic DNA, at least one of the base sequences (d) to (f) in its nuclear genomic DNA, and at least one of the base sequences (g) to (i) in its nuclear genomic DNA may also be used. Other examples of male fertility restorer plants of the Solanaceae family that relate to the present invention include male fertility restorer plants of the Solanaceae family that contain at least one of the base sequences (j) to (l) in their nuclear genomic DNA, male fertility restorer plants of the Solanaceae family that contain at least one of the base sequences (m) to (o) in their nuclear genomic DNA, and male fertility restorer plants of the Solanaceae family that contain at least one of the base sequences (p) to (r) in their nuclear genomic DNA.

[0085] Furthermore, in the embodiment of the "method for restoring male fertility to a solanaceous plant" described above, the method for restoring male fertility to a solanaceous plant includes a step of introducing at least one of the base sequences (a) to (r) into the nuclear genome of a solanaceous plant having cytoplasmic male sterility. However, instead of the above step, the method for restoring male fertility to a solanaceous plant includes a step of introducing at least one of the base sequences (a) to (c) into the nuclear genome of a solanaceous plant having cytoplasmic male sterility, and a step of introducing at least one of the base sequences (d) to (f) into the nuclear genome of a solanaceous plant having cytoplasmic male sterility. The method may include a step of introducing at least one of the base sequences (g) to (i) above into the nuclear genome of a Solanaceae plant having cytoplasmic male sterility, a step of introducing at least one of the base sequences (j) to (l) above into the nuclear genome of a Solanaceae plant having cytoplasmic male sterility, a step of introducing at least one of the base sequences (m) to (o) above into the nuclear genome of a Solanaceae plant having cytoplasmic male sterility, or a step of introducing at least one of the base sequences (p) to (r) above into the nuclear genome of a Solanaceae plant having cytoplasmic male sterility. The same applies to a method for producing a Solanaceae plant with restored male fertility, which includes obtaining a Solanaceae plant with restored male fertility by the method for restoring male fertility to a Solanaceae plant.

[0086] Furthermore, in the above-mentioned embodiment of the "method for determining whether a plant has restored male fertility to a Solanaceae plant," the method for determining whether a plant has restored male fertility to a Solanaceae plant included a step of confirming whether the plant contains at least one of the base sequences (a) to (r) above. However, the method for determining whether a plant has restored male fertility to a Solanaceae plant may instead include a step of confirming whether the plant contains at least one of the base sequences (a) to (c) above, a step of confirming whether the plant contains at least one of the base sequences (d) to (f) above, a step of confirming whether the plant contains at least one of the base sequences (g) to (i) above, a step of confirming whether the plant contains at least one of the base sequences (j) to (l) above, a step of confirming whether the plant contains at least one of the base sequences (m) to (o) above, and / or a step of confirming whether the plant contains at least one of the base sequences (p) to (r) above. [Example]

[0087] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0088] [Identification of gene locus] In this experiment, we first developed a BC1F1 population by crossing 'CMS[MSA1]' with S. pimpinellifolium, and backcrossing the resulting F1 individuals with the maintainer line 'O' (a line that does not have the RF or CMS genes but has a genetic background equivalent to that of 'CMS[MSA1]' in terms of nuclear genes) to create a segregating population that exhibits both fertile and sterile phenotypes. We then searched for RF candidate genes using QTL-seq, which detects causative gene loci from differences in allele frequency.

[0089] (Donor plants and cultivation conditions) The tomato cultivar 'Sekai-ichi' (CMS) and the potato species S. acaule (S. acaule) were used in this experiment. The wild tomato species S. pimpinellifolium (LA1670), a radio-fault (RF) line, and the tomato cultivar 'O' (maintainer) were used. The BC1F1 population, consisting of 244 individuals, was obtained by crossing 'CMS[MSA1]' with S. pimpinellifolium, and then backcrossing 'O' once to the F1 individuals. This population was used to identify RF loci using QTL-seq. Furthermore, the BC3F1 population, consisting of 46 individuals, was used to narrow down RF loci using CAPS markers. The BC1F1 fertile individuals were backcrossed twice with the cultivar 'O'. Cultivation conditions: Seeds were sown in Petri dishes containing filter paper soaked in distilled water. The dishes were sealed with parafilm and left in a dark room at 28°C for approximately one week to germinate. Germinated seeds (approximately 1 cm in height) were transplanted into Jiffy Pots (Sakata Seed) filled with Cocovet (Kaneko Seed) and grown at 25°C and 600 ppm CO2. When the seedlings reached approximately 10 cm in height, they were grown hydroponically in a greenhouse at the Tsukuba Functional Plant Innovation Research Center using a thin film hydroponic system (Nutrient Film Technique, NFT). A concentrated solution was prepared using OAT House Fertilizer Series (OAT Agrio). The concentrated solution was diluted with tap water to adjust the electrical conductivity (EC) to 0.8 mS / cm during seedling growth and 1.2–1.8 mS / cm after seedling growth. The solution was circulated for 15 minutes at 6, 9, 12, 15, and 18 hours.

[0090] (Phenotype survey of the BC1F1 population) For 244 individuals of the BC1F1 population, the inflorescences were bagged before flowering to prevent pollination by pollen from other individuals, and vibration pollination was carried out approximately five times a week. In addition, to evaluate the phenotype of the 244 individuals of the BC1F1 population, the pollen germination ability of each individual was examined using the aniline blue staining method. (Narrowing down RF loci using QTL-seq) First, DNA was extracted from young leaves of 244 individuals of the BC1F1 population, which was the same test sample used for counting the number of seeds and investigating pollen germination ability. Individuals in the BC1F1 population were divided into fertile and sterile individuals. Based on the classification of seed number and the number of repetitions of the aniline blue staining experiment, a total of six bulks were created, three for each fertile and sterile population. The bulk concentration was adjusted to 2 ng / μL by dilution with Tris-EDTA buffer (TE). DNA quality check, sequencing library preparation, and sequencing runs were outsourced to Chemical Dojin Co., Ltd. The next-generation sequencer used was the Illumina HiseqX-ten (Illumina) with a 150 bp paired-end sequence.

[0091] (Calculation of ΔSNP-index and G-value using QTL-seq method) Adapters and reads with a quality score below 30 were removed using TrimGalore (https: / / www.bioinformatics.babraham.ac.uk / projects / trim_galore). Quality-cleared reads were mapped to the S. pimpinellifolium reference genome (SPI_r1.1) and S. lycopersicum reference genome (SL 4.0) using BWA (https: / / github.com / lh3 / bwa). Mutation detection was performed using gatk4 (https: / / github.com / broadinstitute / gatk). ΔSNP-index and G-value calculations were performed using QTLseqr ver. 0.7.5.2. SNPs were filtered for reference allele frequency = 0.2, 100 ≤ TotalSampleDepth ≤ 400, and MinimumSampleDepth = 40. QTL peak regions were extracted with a p-value < 0.01. The reference genome for SPI_r1.1 was prepared by Takei et al., 2021. The reference genome for SL 4.0 was prepared at https: / / solgenomics.net / organism / Solanum_lycopersicum / genome.

[0092] (Narrowing down the RF1a1 locus using CAPS markers) Using SPI_r1.1 as the reference genome, 30 markers were designed based on SNPs present in chromosome 1, 80 Mbp to 90 Mbp, of 'CMS[MSA1]' and used for genotype investigation.

[0093] (Genotyping by PCR and restriction enzyme digestion) After restriction enzyme treatment at 37°C for 2 hours or more, the genotype was investigated by agarose gel electrophoresis. Two grams of agarose was added to 100 ml of Tris-Acetate-EDTA Buffer (TAE), dissolved by boiling in a microwave oven, and then 1 μL of Midori Green "Advance" DNA Stain (Nippon Genetics) was added to prepare a 2% (w / v) agarose gel. 1 μL of 6x Loading dye was added to 5 μL of each PCR product, pipetted, and then 5 μL was applied to each well. 5 μL of Gene Ladder Wide 1 (Nippon Gene) was used as a molecular weight marker. The electrophoresis chamber was filled with TAE buffer, and electrophoresis was performed at 100 V for 20 minutes. A gel imaging system, E-BOX-VX2 / 20M (Bilber-Lumat), was used to detect the amplified products.

[0094] (BC3F1 population creation and phenotyping) To precisely narrow down the RF locus from the homologous recombinant population, BC1F1 fertile individuals were backcrossed twice with the maintainer line 'O' to produce a BC3F1 population of 46 individuals.

[0095] (Comparison of genome structures between S. pimpinellifolium, S. lycopersicum, and S. lycopersicum var. cerasiforme) MCScanX (https: / / github.com / wyp1125 / MCScanX) was used for genome structure comparison. The reference genome (SLYcer_r1.2) of S. lycopersicum var. cerasiforme (LA1673) was prepared by Takei et al., 2021. SPI_r1.1 was used for S. pimpinellifolium, and SL4.0 was used for S. lycopersicum.

[0096] [result] To identify the RF locus in S. pimpinellifolium, we created bulk populations consisting of fertile and sterile populations. The populations were mapped to the S. pimpinellifolium reference genome, SPI_r1.1, and the ΔSNP-index and G-value were calculated using QTL-seq. In all bulks, the G-value and ΔSNP-index showed the highest peaks at physical positions approximately 80 to 90 Mbp on chromosome 1. This suggests that the RF gene is located approximately 80 to 90 Mbp from the 5' end of chromosome 1 in S. pimpinellifolium.

[0097] [Further narrowing down of loci] The RF1 locus, which is primarily responsible for fertility restoration in the RF lines 'LA1670' and 'LA1673', was backcrossed to a CMS line in the genetic background of the cultivar 'OF209' to produce 190 BC5F1 individuals, and linkage analysis using DNA markers was used to narrow down the genetic region. Genes present within the narrowed region were extracted, and genes that were expressed in pollen and whose amino acid sequences differed between the RF lines and the cultivars were selected.

[0098] Figure 1 shows the results of linkage analysis of the RF1a1 locus from Solanum pimpinellifolium. In Figure 1, regions containing heterozygous candidates for the RF1a1 locus are indicated by "H," and regions containing homozygous candidates for CMS (cytoplasmic male sterility) are indicated by "C." Linkage analysis using DNA markers revealed that, as shown in Figure 1, in fertile lines, the region from 83.29 to 84.65 Mbp from the 5' end of chromosome 1 of Solanum pimpinellifolium contained many heterozygous regions for the candidate RF1a1 locus. These results revealed that the base sequence in the region of 83.29 to 84.65 Mbp from the 5' end on chromosome 1 of Solanum pimpinellifolium has the function of restoring male fertility.

[0099] 2 is a diagram showing the results of linkage analysis of the RF1a2 locus derived from Solanum lycopersicum var. cerasiforme. Figure 2 shows the results of linkage analysis using BC3F2 individuals, BC4F1 individuals, and BC3F2 individuals. 3 is a diagram showing the results of resequencing Solanum lycopersicum var. cerasiforme and the CMS line. Figure 3 shows the results of resequencing Solanum lycopersicum var. cerasiforme and the CMS line in BC1F1 individuals. In Figures 2 and 3, heterozygous regions of the RF1a2 locus are shown in white, and CMS-type regions are shown in diagonal lines.

[0100] As shown in Figure 2, in each fertile line, the proportion of heterozygotes was high in the region around 80 Mbp from the 5' end of chromosome 1 of Solanum lycopersicum var. cerasiforme. Furthermore, as shown in Figure 3, resequencing revealed that the base sequence of a 611 kbp region from 80.03 to 80.65 Mbp from the 5' end of chromosome 1 of Solanum lycopersicum var. cerasiforme has the function of restoring male fertility.

[0101] FIG. 4 shows the results of a comparison of the genome structures of chromosome 1 among five tomato varieties. As shown in Figure 4, when we focused on the RF (restorer of fertility) locus on the long arm of chromosome 1, no significant differences in genome structure were observed among the five tomato varieties. Furthermore, it was found that the region from 83.29 to 84.65 Mbp from the 5' end of chromosome 1 of Solanum pimpinellifolium and the region from 81.89 to 83.21 Mbp from the 5' end of chromosome 1 of the nuclear genome of Solanum cheesmaniae share high homology with each other. These results demonstrate that the nucleotide sequence of the 81.89 to 83.21 Mbp region from the 5' end of chromosome 1 of the nuclear genome of Solanum cheesmaniae has the function of restoring male fertility.

[0102] [Identification of RF1b and RF1c genes] In this experiment, we selected a PPR gene present on chromosome 1 of Solanum pimpinellifolium that is expressed in pollen, has amino acid mutations compared to the Heinz line, and is transferred to mitochondria. We then introduced this gene into CMS tomato lines using the Agrobacterium method with T-DNA. Table 3 below shows a list of PPR genes present on chromosome 1 of Solanum pimpinellifolium that are expressed in pollen, have amino acid mutations compared to the Heinz line, and are transferred to mitochondria. [Table 3]

[0103] Figure 5 shows images of pollen germination in a CMS tomato line into which the pentatricopeptide repeat (PPR) gene (gene ID: SPI01g07345) has been introduced. Figure 6 shows images of pollen germination in a CMS tomato line into which the pentatricopeptide repeat (PPR) gene (gene ID: SPI01g07485) has been introduced. In tomatoes into which the pentatricopeptide repeat (PPR) gene (gene ID: SPI01g07345) present on chromosome 1 of Solanum pimpinellifolium had been introduced, pollen germination was observed, and 92 seeds were produced per individual in the T0 generation. In tomato plants into which the pentatricopeptide repeat (PPR) gene (gene ID: SPI01g07485) present on chromosome 1 of Solanum pimpinellifolium was introduced, pollen germination was observed, and one seed was formed per plant in the T0 generation. These results demonstrate that the pentatricopeptide repeat (PPR) gene (gene ID: SPI01g07345) and the pentatricopeptide repeat (PPR) gene (gene ID: SPI01g07485) have the function of restoring male fertility.

[0104] In addition, when we examined the genes in the region where the pentatricopeptide repeat (PPR) gene (gene ID: SPI01g07345) was introduced, we found that they were DNA polymerase III, clamp-loader complex, subunit E, and C-terminal (gene ID: Solyc01g020440), genes that contribute to nuclear DNA replication. When the gene in the region where the pentatricopeptide repeat (PPR) gene (gene ID: SPI01g07485) was introduced was examined, it was found to be a gene called Phototropic-responsive NPH3 family protein (gene ID: Solyc02g092560) that responds to blue light. Thus, since the region into which the gene with male fertility restoration function was introduced was a gene with little relationship to mitochondrial DNA, it was confirmed that fertility restoration was not due to gene disruption, but rather an effect caused by the introduction of the Pentatricopeptide repeat (PPR) gene (Gene ID: SPI01g07345) or the Pentatricopeptide repeat (PPR) gene (Gene ID: SPI01g07485) gene.

[0105] [Identification of the RF2 locus] In this experiment, 36 F1 individuals (F2_#52xMT-J_F1_1 ~ 36) were cultivated by crossing the F2 individuals lacking the RF1a1 locus described above with the tomato cultivar 'Micro-Tom' (male parent). Fertility was assessed by counting the number of seeds and by aniline blue staining. To count the number of seeds, artificially pollinated inflorescences were bagged 0 to 2 days after anthesis, and the number of seeds in the mature fruit was counted. To assess pollen fertility, all individuals were stained with aniline blue to examine the presence or absence of pollen tube elongation. Individuals without confirmed seeds or pollen tube elongation by aniline blue staining were considered sterile. Similarly, 13 BC1F1 individuals obtained by crossing F2 individual #52 (male parent) with a CMS type MT-J line (female parent) were evaluated.

[0106] (Narrowing down RF loci using QTL-seq) DNA was extracted from 36 individuals of the F1 population and 4 individuals of the BC1F1 population with a large number of seeds (approximately 10 seeds / fruit), which were the same test samples used for seed number counting and pollen germination ability investigation.

[0107] (Bulk population sequencing) Individuals in the F1 population were divided into fertile and sterile individuals. Based on the classification of seed number and the number of repetitions of the aniline blue staining experiment, three bulks were created for each of the high fertility population (Bulk1_fer), low fertility population (Bulk3_weak), and sterile population (Bulk2_str). The bulk concentration was adjusted to 2 ng / μL by dilution with Tris-EDTA buffer (TE). DNA quality check, sequencing library preparation, and sequencing runs were outsourced to Relixa Co., Ltd. The next-generation sequencer used was the Illumina Nova-seq (Illumina) with a 150 bp paired-end sequence.

[0108] (Calculation of ΔSNP-index and G-value using QTL-seq method) Adapters and reads with a quality score below 30 were removed using TrimGalore (https: / / www.bioinformatics.babraham.ac.uk / projects / trim_galore). Quality-cleared reads were mapped to the S. pimpinellifolium reference genome (SPI_r1.1) and S. lycopersicum reference genome (SL 4.0) using BWA (https: / / github.com / lh3 / bwa). Mutation detection was performed using gatk4 (https: / / github.com / broadinstitute / gatk). ΔSNP-index and G-value were calculated using QTLseqr ver. 0.7.5.2 based on the results mapped to SL 4.0. SNPs were filtered for reference allele frequency = 0.1, 20 ≤ TotalSampleDepth ≤ 400, and MinimumSampleDepth = 20. QTL peak regions were extracted with a p-value < 0.01. The SL 4.0 reference was created at https: / / solgenomics.net / organism / Solanum_lycopersicum / genome.

[0109] (DNA marker construction) DNA markers were constructed to recognize the insertion or deletion polymorphism specific to the LA1670 line within the QTL region identified by QTLseq (42-51 Mbp on chromosome 2; Table 2). Primer pairs flanking the polymorphisms were designed and used for genotyping. FIG. 7 shows the results of bulk analysis of F1 individuals between a CMS tomato line and Solanum pimpinellifolium. As shown in FIG. 7, a clear peak was confirmed in the region of 42 to 51 Mbp from the 5' end of chromosome 2 of the nuclear genome of Solanum lycopersicum (SL4.0). This revealed that the base sequence of the region from 38 to 47 Mbp from the 5' end of the nuclear genome chromosome 2 of Solanum pimpinellifolium, which corresponds to this region, has the function of restoring male fertility.

[0110] [RF1b and RF1c gene detection] In this experiment, DNA was extracted from the F1 individuals obtained by crossing CMS[MSA1], a CMS line, with Solanum pimpinellifolium (LA1670), a fertility restorer line, and the presence or absence of the RF2 locus was confirmed.

[0111] FIG. 8 is an image showing the results of amplifying a portion of the RF2 locus using DNA obtained from CMS[MSA1], Solanum pimpinellifolium, and their F1 as templates. In Figure 8, "Rf2-1" is a lane into which an amplification product obtained using a set of primers having the base sequences shown in SEQ ID NOs: 12 and 13 was poured. "Rf2-2" is a lane into which an amplification product obtained using a set of primers having the base sequences shown in SEQ ID NOs: 14 and 15 was poured. "Rf2-3" is a lane into which an amplification product obtained using a set of primers having the base sequences shown in SEQ ID NOs: 16 and 17 was poured. "Rf2-4" is a lane into which an amplification product obtained using a set of primers having the base sequences shown in SEQ ID NOs: 18 and 19 was poured.

[0112] As shown in Figure 8, regardless of which primer set was used for PCR amplification, the amplified product from the Solanum pimpinellifolium sample was larger than the amplified product from the CMS[MSA1] sample. This result confirmed that CMS[MSA1] does not have the RF2 locus, while Solanum pimpinellifolium does. For detailed sizes of these amplified products, see Table 2. Furthermore, when the F1 individuals obtained by crossbreeding these lines were used as samples, a relatively small product amplified from the allele derived from CMS[MSA1] and a relatively large product amplified from the allele derived from Solanum pimpinellifolium were confirmed in the lanes using any of the four primer pairs.

[0113] As described above, the four pairs of primers shown in Tables 1 and 2 make it possible to accurately and quickly determine the presence or absence of the RF2 locus and whether it is a heterozygote or a homozygote. [Industrial Applicability]

[0114] According to the present invention, male fertility can be restored by introducing at least one of the above base sequences (a) to (r) into a cytoplasmic male sterile line of a Solanaceae plant, and therefore the present invention is industrially applicable.

Claims

1. A male fertility restorer plant of the Solanaceae family, which contains at least one of the following base sequences (a) to (r) in its nuclear genomic DNA: (a) The base sequence of the region from 83.29 Mbp to 84.65 Mbp from the 5' end of the nuclear genome chromosome 1 of the wild tomato species Solanum pimpinellifolium; (b) a base sequence comprising the base sequence of (a) in which one or more bases are deleted, substituted, added, or inserted, and having a male fertility restoration function; (c) a DNA sequence having a base sequence having 60% or more sequence identity with the base sequence of (a) and having a male fertility restoring function; (d) The base sequence of the region from 80.03 Mbp to 80.65 Mbp from the 5' end of the nuclear genome chromosome 1 of the cherry tomato species Solanum lycopersicum var. cerasiforme; (e) a base sequence comprising the base sequence of (d) in which one or more bases are deleted, substituted, added or inserted, and having a male fertility restoration function; (f) a base sequence having a sequence identity of 60% or more with the base sequence of (d) and having a male fertility restoration function; (g) a base sequence encoding the amino acid sequence shown in SEQ ID NO: 1; (h) a base sequence encoding 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: 1 and having a male fertility restoration function; (i) a base sequence encoding an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and having a male fertility restoration function; (j) a base sequence encoding the amino acid sequence shown in SEQ ID NO: 2; (k) a base sequence encoding 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: 2 and having a male fertility restoration function; (l) a base sequence encoding an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2 and having a male fertility restoration function; (m) the base sequence of the region from 38 Mbp to 47 Mbp from the 5' end of the nuclear genome chromosome 2 of Solanum pimpinellifolium; (n) a base sequence in which one or more bases are deleted, substituted, added, or inserted in the base sequence of (m), and which has a male fertility restoration function; (o) a base sequence having a sequence identity of 60% or more with the base sequence of (m) and having a male fertility restoration function; (p) the base sequence of the region from 81.89 Mbp to 83.21 Mbp from the 5' end of the nuclear genome chromosome 1 of the wild tomato species Solanum cheesemaniae; (q) a base sequence comprising the base sequence of (p) in which one or more bases are deleted, substituted, added or inserted, and having a male fertility restoration function; (r) A base sequence having a sequence identity of 60% or more with the base sequence of (p) and having the function of restoring male fertility.

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

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

4. 3. 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 Solanaceae plant, comprising introducing at least one of the following base sequences (a) to (r) into the nuclear genome of the Solanaceae plant having cytoplasmic male sterility: (a) The base sequence of the region from 83.29 Mbp to 84.65 Mbp from the 5' end of the nuclear genome chromosome 1 of the wild tomato species Solanum pimpinellifolium; (b) a base sequence comprising the base sequence of (a) in which one or more bases are deleted, substituted, added, or inserted, and having a male fertility restoration function; (c) a DNA sequence having a base sequence having 60% or more sequence identity with the base sequence of (a) and having a male fertility restoring function; (d) The base sequence of the region from 80.03 Mbp to 80.65 bp from the 5' end of the nuclear genome chromosome 1 of the cherry tomato species Solanum lycopersicum var. cerasiforme; (e) a base sequence comprising the base sequence of (d) in which one or more bases are deleted, substituted, added or inserted, and having a male fertility restoration function; (f) a base sequence having a sequence identity of 60% or more with the base sequence of (d) and having a male fertility restoration function; (g) a base sequence encoding the amino acid sequence shown in SEQ ID NO: 1; (h) a base sequence encoding 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: 1 and having a male fertility restoration function; (i) a base sequence encoding an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and having a male fertility restoration function; (j) a base sequence encoding the amino acid sequence shown in SEQ ID NO: 2; (k) a base sequence encoding 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: 2 and having a male fertility restoration function; (l) a base sequence encoding an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2 and having a male fertility restoration function; (m) the base sequence of the region from 38 Mbp to 47 Mbp from the 5' end of the nuclear genome chromosome 2 of Solanum pimpinellifolium; (n) a base sequence in which one or more bases are deleted, substituted, added, or inserted in the base sequence of (m), and which has a male fertility restoration function; (o) a base sequence having a sequence identity of 60% or more with the base sequence of (m) and having a male fertility restoration function; (p) the base sequence of the region from 81.89 Mbp to 83.21 Mbp from the 5' end of the nuclear genome chromosome 1 of the wild tomato species Solanum cheesemaniae; (q) a base sequence comprising the base sequence of (p) in which one or more bases are deleted, substituted, added or inserted, and having a male fertility restoration function; (r) A base sequence having a sequence identity of 60% or more with the base sequence of (p) and having the function of restoring male fertility.

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

8. 7. 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 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 claims 5 to 8.

10. A method for determining whether fertility of a cytoplasmic male-sterile plant of the Solanaceae family has been restored, comprising: A method for determining whether a Solanaceae plant has restored male fertility, comprising the step of confirming whether the Solanaceae plant contains at least one of the following base sequences (a) to (r): (a) The base sequence of the region from 83.29 Mbp to 84.65 Mbp from the 5' end of the nuclear genome chromosome 1 of the wild tomato species Solanum pimpinellifolium; (b) a base sequence comprising the base sequence of (a) in which one or more bases are deleted, substituted, added, or inserted, and having a male fertility restoration function; (c) a DNA sequence having a base sequence having 60% or more sequence identity with the base sequence of (a) and having a male fertility restoring function; (d) The base sequence of the region from 80.03 Mbp to 80.65 bp from the 5' end of the nuclear genome chromosome 1 of the cherry tomato species Solanum lycopersicum var. cerasiforme; (e) a base sequence comprising the base sequence of (d) in which one or more bases are deleted, substituted, added or inserted, and having a male fertility restoration function; (f) a base sequence having a sequence identity of 60% or more with the base sequence of (d) and having a male fertility restoration function; (g) a base sequence encoding the amino acid sequence shown in SEQ ID NO: 1; (h) a base sequence encoding 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: 1 and having a male fertility restoration function; (i) a base sequence encoding an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and having a male fertility restoration function; (j) a base sequence encoding the amino acid sequence shown in SEQ ID NO: 2; (k) a base sequence encoding 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: 2 and having a male fertility restoration function; (l) a base sequence encoding an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2 and having a male fertility restoration function; (m) the base sequence of the region from 38 Mbp to 47 Mbp from the 5' end of the nuclear genome chromosome 2 of Solanum pimpinellifolium; (n) a base sequence in which one or more bases are deleted, substituted, added, or inserted in the base sequence of (m), and which has a male fertility restoration function; (o) a base sequence having a sequence identity of 60% or more with the base sequence of (m) and having a male fertility restoration function; (p) the base sequence of the region from 81.89 Mbp to 83.21 Mbp from the 5' end of the nuclear genome chromosome 1 of the wild tomato species Solanum cheesemaniae; (q) a base sequence comprising the base sequence of (p) in which one or more bases are deleted, substituted, added or inserted, and having a male fertility restoration function; (r) A base sequence having a sequence identity of 60% or more with the base sequence of (p) and having the function of restoring male fertility.

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