Long-lastingness regulatory gene of plant belonging to genus dianthus, and use of the same

Abstract

To provide a technique of creating plant belonging to the genus Dianthus having excellent long-lastingness.SOLUTION: A long-lastingness regulatory gene that adjusts long-lastingness of a plant belonging to the genus Dianthus consists of any one of the following polynucleotides (i) to (iii): (i) a polynucleotide encoding protein which consists of a specific amino acid sequence; (ii) a polynucleotide encoding protein which consists of an amino acid sequence having 90% or more of sequence identity with respect to the specific amino acid sequence, and has activity of adjusting long-lastingness of a plant belonging to the genus Dianthus; and (iii) a polynucleotide encoding protein which consists of an amino acid sequence in which 31 or less of amino acids are substituted, deleted, added or inserted with respect to the specific amino acid sequence, and has activity of adjusting long-lastingness of a plant belonging to the genus Dianthus.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a gene for regulating flower retention in Dianthus plants and its use.

Background Art

[0002] For the purpose of improving the quality of carnations, which are Dianthus plants, varieties and lines with good flower retention have been bred. Conventionally, breeding of such carnation varieties with good flower retention has generally been carried out by cross-breeding. However, it is necessary to confirm the quality by subjecting the offspring lines obtained after crossing to a flower retention test for about two years. Therefore, development of a technology that can more efficiently breed carnation varieties with good flower retention is desired.

[0003] As a technology for improving the efficiency of cross-breeding, a method using a DNA marker can be mentioned. Non-Patent Document 1 describes that a comprehensive gene expression analysis was performed on the flower retention of carnations, and a DNA marker related to flower retention specific to a particular variety of carnation was identified. Non-Patent Document 2 describes an aging-related gene related to a low ethylene production amount during aging.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] However, in Non-Patent Documents 1 and 2 as well, the genes involved in the flower retention property of Dianthus plants including carnations have not been identified, and DNA markers for efficiently selecting Dianthus plants have not been found. Therefore, there is a need to identify genes involved in flower retention property and develop DNA markers for efficiently selecting Dianthus plants with good flower retention property.

[0006] One aspect of the present invention aims to realize a technique for creating Dianthus plants with excellent flower retention property.

Means for Solving the Problems

[0007] In order to solve the above problems, a flower retention property regulating gene according to one aspect of the present invention is a flower retention property regulating gene that regulates the flower retention property of Dianthus plants, and is any one of the following polynucleotides (i) to (iii): (i) A polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2; (ii) A polynucleotide encoding a protein consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1 or 2 and having an activity of regulating the flower retention property of Dianthus plants; (iii) A polynucleotide encoding a protein consisting of an amino acid sequence in which 31 or fewer amino acids are substituted, deleted, added or inserted with respect to the amino acid sequence shown in SEQ ID NO: 1 or 2 and having an activity of regulating the flower retention property of Dianthus plants.

[0008] A flower retention property regulating protein according to one aspect of the present invention has an activity of regulating the flower retention property of Dianthus plants, which is encoded by the flower retention property regulating gene according to one aspect of the present invention.

[0009] A determination method for determining the flower retention property of a Dianthus plant according to one aspect of the present invention includes a detection step of detecting the flower retention property regulating gene according to claim 1 or 2 as a molecular marker related to the regulation of the flower retention property of the Dianthus plant in the Dianthus plant.

[0010] In a Dianthus plant with good flower retention property according to one aspect of the present invention, the expression of the flower retention property regulatory gene according to one aspect of the present invention is suppressed.

[0011] A method for producing a Dianthus plant with good flower retention property according to one aspect of the present invention includes a step of suppressing the expression of the flower retention property regulatory gene described in claim 1 or 2 in a Dianthus plant.

Effects of the Invention

[0012] According to one aspect of the present invention, it is possible to realize a technique for creating a Dianthus plant excellent in flower retention property.

Brief Description of the Drawings

[0013]

Figure 1

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Modes for Carrying Out the Invention

[0014] 〔Definitions〕 Hereinafter, the present invention will be described in detail. All the documents described in this specification are incorporated herein by reference. Also, unless otherwise specified in this specification, "A~B" representing a numerical range means "A or more (including A and greater than A), B or less (including B and less than B)".

[0015] As used in this specification, the term "polynucleotide" is used interchangeably with "nucleic acid" or "nucleic acid molecule", and is intended to be a polymer of nucleotides. Here, the nucleic acid can exist in the form of DNA (for example, cDNA or genomic DNA), or in the form of RNA (for example, mRNA). DNA or RNA may be double-stranded or single-stranded. The single-stranded DNA or RNA may be a coding strand (sense strand) or a non-coding strand (antisense strand). When used in this specification, the notation of bases uses the one-letter notation defined by IUPAC and IUB as appropriate.

[0016] In this specification, the term "plant" is intended to be a part or all of a plant body. Examples of a part of a plant body include plant organs (for example, roots, stems, leaves, petals, seeds, fruits, etc.), plant tissues (for example, epidermis, sieve parts, parenchyma, xylem, vascular bundles, etc.), plant cells, callus, etc. In this specification, the term "Dianthus plant" is intended to be plants such as Dianthus japonicus and carnation belonging to the genus Dianthus.

[0017] As used herein, the term "flower retention property" is intended to refer to the degree to which a flower is maintained without wilting, and the term "excellent flower retention property" is intended to mean that, compared to a plant without excellent flower retention property, the flower retention property of a plant with excellent flower retention property is superior and the flower retention period is long. As used herein, the term "having an activity of regulating flower retention property" means that, in a plant in which the polynucleotide encoding the protein having such activity is mutated, or the expression of the polynucleotide is deleted or suppressed, compared to a plant in which the polynucleotide is not mutated or the expression of the polynucleotide is not deleted or suppressed, the flower retention property is excellent. The degree of improvement in the flower retention property of a plant with excellent flower retention property can be, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% with respect to the flower retention period in a plant without excellent flower retention property.

[0018] Also, as used herein, "the expression of a gene (or polynucleotide) is suppressed" is intended to mean that, due to an artificial operation, the endogenous gene (or polynucleotide) originally possessed by a plant itself is mutated (including nucleotide substitution, insertion, addition, or deletion) or deleted, or the expression of the gene is deleted or suppressed. Whether "the expression of a gene (or polynucleotide) is deleted or suppressed" can be determined, for example, based on the amount of polynucleotide extracted from a plant body, which is measured by RT-PCR. The genomic sequence of the carnation cultivar Francesca, which serves as a reference for the base sequence of the flower retention property regulating gene, is published in the carnation database of Kazusa DNA Research Institute (http: / / carnation.kazusa.or.jp / ).

[0019] 〔Flower Retention Property Regulating Gene〕 The flower - holding property - regulating gene according to one aspect of the present invention is a gene encoding a protein having an activity of regulating the flower - holding property of carnation plants. As an example, in carnation plants, when the expression of the protein having an activity of regulating the flower - holding property is lacking or suppressed, the flower - holding property is superior to that of carnation plants in which the expression of the protein is not lacking or suppressed. Also, in carnation plants, when the expression of the protein having an activity of regulating the flower - holding property is not lacking or suppressed, the flower - holding property is lower than that of carnation plants in which the expression of the protein is lacking or suppressed.

[0020] The flower - holding property - regulating gene is a gene involved in regulating the flower - holding property of carnation plants and is any one of the following polynucleotides (i) to (iii): (i) A polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2; (ii) A polynucleotide encoding a protein consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1 or 2 and having an activity of regulating the flower - holding property of carnation plants; (iii) A polynucleotide encoding a protein consisting of an amino acid sequence in which 31 or fewer amino acids are substituted, deleted, added or inserted with respect to the amino acid sequence shown in SEQ ID NO: 1 or 2 and having an activity of regulating the flower - holding property of carnation plants; and is a gene consisting of such polynucleotides.

[0021] The polynucleotide of (i) above contains the nucleotide sequence of the AP2 gene derived from carnation or the nucleotide sequence of the coding region (CDS) of the AP2 gene, and is a polynucleotide encoding a protein having the activity of regulating flower persistence. Further, the polynucleotide of (i) above may contain the nucleotide sequence of the gene corresponding to the above AP2 gene of Dianthus plants or the nucleotide sequence of the CDS of the gene, and is a polynucleotide encoding a protein having the activity of regulating flower persistence. The amino acid sequence shown in SEQ ID NO: 1 is the amino acid sequence encoded by the AP2 gene derived from the reference Francesca, and the amino acid sequence shown in SEQ ID NO: 2 is the amino acid sequence encoded by the AP2 gene derived from Sandroza with excellent flower persistence.

[0022] Regarding the polynucleotide of (ii) above, the sequence identity with the amino acid sequence of SEQ ID NO: 1 or 2 is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. For example, a mutant gene derived from carnation or a homologous gene (including orthologs) derived from a Dianthus plant other than carnation is included in the category of the polynucleotide of (2) above. These mutant genes and homologous genes are endogenous genes of Dianthus plants. The molecular markers examined in the section of [Determination method for determining flower persistence in Dianthus plants] described later may be molecular markers on these mutant genes and homologous genes.

[0023] Regarding the polynucleotide of (iii) above, in the amino acid sequence of SEQ ID NO: 1 or 2, the number of amino acids substituted, deleted, added or inserted may be 1 to 31, 1 to 30, 1 to 28, 1 to 25, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, or 1 to 2.

[0024] When referring to a gene into which a mutation has been artificially introduced into a flower-holding property-regulating gene, the above-mentioned "substitution, deletion, addition, or insertion of an amino acid" may be, for example, a site-directed mutagenesis method such as the Kunkel method (Kunkel et al. (1985): Proc. Natl. Acad. Sci. USA, vol. 82, p488-), mutagenesis treatment using a drug, or a mutagenesis technique by irradiation with radiation (γ-rays, heavy ion beams, etc.). The mutation may be artificially introduced or may be derived from a naturally occurring similar mutant polypeptide.

[0025] The flower-holding property-regulating gene may exist in the form of RNA (e.g., mRNA) or in the form of DNA (e.g., cDNA or genomic DNA). The DNA may be double-stranded or single-stranded. The nucleotide sequence shown in SEQ ID NO: 3, which is an example of the flower-holding property-regulating gene, is a genomic sequence encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, and the nucleotide sequence shown in SEQ ID NO: 4 is a cDNA sequence encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1. Also, the nucleotide sequence shown in SEQ ID NO: 5, which is an example of the flower-holding property-regulating gene, is a genomic sequence encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2. The flower-holding property-regulating gene may include additional sequences such as the nucleotide sequence of the untranslated region (UTR) together with the CDS of the AP2 gene.

[0026] The method for obtaining (isolating) the flower-holding property-regulating gene is not particularly limited. For example, a probe that specifically hybridizes with a part of the nucleotide sequence of the flower-holding property-regulating gene may be prepared and used to screen a genomic DNA library or a cDNA library.

[0027] In addition, as a method for obtaining a flower retention property regulating gene, a method using amplification means such as PCR can be mentioned. For example, among the cDNA of the flower retention property regulating gene, primers are prepared from the sequences on the 5' side and 3' side (or their complementary sequences) respectively, and using these primers, genomic DNA (or cDNA) etc. as a template, PCR etc. is performed, and by amplifying the DNA region sandwiched between both primers, a large amount of DNA fragments containing the flower retention property regulating gene can be obtained.

[0028] The origin of the flower retention property regulating gene is not particularly limited as long as it is a plant of the genus Dianthus, but it is preferably either Dianthus japonicus or carnation, and more preferably carnation.

[0029] Whether or not the isolated candidate gene for the flower retention property regulating gene has the activity to regulate the desired flower retention property can be evaluated by observing whether the improvement of the flower retention property is induced by the deletion or suppression of the expression of the candidate gene in the plant from which it is derived.

[0030] The flower retention property regulating gene can be used to elucidate the mechanism of improving the flower retention property in plants of the genus Dianthus. In addition, the flower retention property regulating gene can be used to produce a transformant by incorporating a sequence that lacks or suppresses its expression into an expression vector and introducing it into a plant body or cell of a plant of the genus Dianthus. By cultivating a plant of the genus Dianthus in which the expression of the flower retention property regulating gene is lacking or suppressed, a plant of the genus Dianthus with improved flower retention property can be obtained.

[0031] Examples of the flower retention property regulating gene include any one of the following polynucleotides (a) to (c): (a) A polynucleotide consisting of the nucleotide sequence shown in any of SEQ ID NOs: 3 to 5; (b) A polynucleotide consisting of a nucleotide sequence having 90% or more sequence identity to the nucleotide sequence shown in any of SEQ ID NOs: 3 to 5, and showing the same function as the polynucleotide in (a) above with respect to the regulation of the flower retention property of plants of the genus Dianthus; (c) A polynucleotide consisting of a nucleotide sequence in which 85 or fewer bases are substituted, deleted, added or inserted with respect to the nucleotide sequence shown in any of SEQ ID NOs: 3 to 5, and having a function equivalent to that of the polynucleotide of (a) above with respect to the regulation of flower-bearing property in Dianthus plants; The gene consisting of is given as an example.

[0032] The polynucleotide of (a) above is a polynucleotide consisting of the nucleotide sequence of the AP2 gene derived from carnation or the nucleotide sequence of the coding region (CDS) of the AP2 gene. Further, the polynucleotide of (a) above consists of the nucleotide sequence of the gene corresponding to the above AP2 gene in Dianthus plants or the nucleotide sequence of the CDS of the gene.

[0033] Regarding the polynucleotide of (b) above, the sequence identity with the nucleotide sequences shown in SEQ ID NOs: 3 to 5 is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. For example, a mutant gene derived from carnation or a homologous gene (including orthologs) derived from a Dianthus plant other than carnation is included in the category of the polynucleotide of (2) above.

[0034] Regarding the polynucleotide of (3) above, in the nucleotide sequences of SEQ ID NOs: 3 to 5, the number of bases substituted, deleted, added or inserted can be 1 to 85, 1 to 80, 1 to 75, 1 to 79, 1 to 65, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, or 1 to 5.

[0035] Examples of the gene for regulating flower retention include polynucleotides consisting of the genomic sequences (SEQ ID NOs: 3 and 5) of the AP2 gene derived from carnation and the cDNA sequence (SEQ ID NO: 4) of the AP2 gene derived from carnation. The nucleotide sequence shown in SEQ ID NO: 3 is the genomic sequence of the AP2 gene derived from Francesco used as a reference, and the nucleotide sequence shown in SEQ ID NO: 4 is the cDNA sequence of the AP2 gene derived from Francesco. The nucleotide sequence shown in SEQ ID NO: 5 is the genomic sequence of the AP2 gene derived from Sandroza, which has excellent flower retention. In addition, within the scope of the gene for regulating flower retention, there are also genes consisting of polynucleotides having a sequence identity of 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more with respect to the nucleotide sequences shown in SEQ ID NOs: 3 to 5 and having the activity of regulating flower retention.

[0036] 〔Protein for regulating flower retention〕 The protein according to one aspect of the present invention is a translation product of the gene described in the above [Gene for regulating flower retention] column and has at least the activity of regulating flower retention. The protein for regulating flower retention may be isolated from a natural source or chemically synthesized. More specifically, the protein includes natural purified products, products of chemical synthesis procedures, and translation products produced by recombinant techniques from prokaryotic hosts or eukaryotic hosts (including, for example, bacterial cells, yeast cells, higher plant cells, insect cells, and mammalian cells).

[0037] The protein for regulating flower retention according to one aspect of the present invention is encoded by the gene for regulating flower retention according to one aspect of the present invention and has the activity of regulating flower retention in Dianthus plants. Typically, the protein for regulating flower retention has the activity of negatively regulating flower retention. Therefore, when the protein for regulating flower retention is not present in Dianthus plants, the flower retention is better than when the protein for regulating flower retention is present, and when the protein for regulating flower retention is present in Dianthus plants, the flower retention is lower than when the protein for regulating flower retention is not present.

[0038] The flower-holding property-regulating protein according to one aspect of the present invention may be any one of the following: (A) A protein consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2; (B) A protein consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1 or 2 and having the activity of regulating the flower-holding property of Dianthus plants; (C) A protein consisting of an amino acid sequence in which 31 or fewer amino acids are substituted, deleted, added or inserted in the amino acid sequence shown in SEQ ID NO: 1 or 2 and having the activity of regulating the flower-holding property of Dianthus plants.

[0039] The protein of (A) above is a protein encoded by the above AP2 gene or a gene corresponding to the above AP2 gene in Dianthus plants, and has the activity of regulating the flower-holding property of Dianthus plants.

[0040] Regarding the protein of (B) above, the sequence identity with the amino acid sequence shown in SEQ ID NO: 1 or 2 is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0041] Regarding the protein of (C) above, in the amino acid sequence of SEQ ID NO: 1 or 2, the number of substituted, deleted, added or inserted amino acids may be 1 to 31, 1 to 30, 1 to 28, 1 to 25, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, or 1 to 2.

[0042] For example, a mutant protein derived from carnation or a homologous protein derived from a Dianthus plant other than carnation is included in the category of the proteins of (B) and (C) above. These mutant proteins and homologous proteins are proteins encoded by endogenous genes of Dianthus plants.

[0043] The flower retention property-regulating protein is a polypeptide formed by peptide bonding of amino acids, but may also include structures other than the polypeptide. Examples of the structures other than the polypeptide herein include, but are not limited to, sugar chains and isoprenoid groups.

[0044] [Expression Vector, Cell, and Transformant] An expression vector that lacks or suppresses the expression of the flower retention property-regulating gene according to one aspect of the present invention, a cell containing the expression vector, a cell in which the expression of the flower retention property-regulating gene is lacking or suppressed, a transformant transformed with the expression vector, and a transformant in which the expression of the flower retention property-regulating gene is lacking or suppressed are also included in the scope of the present invention. The expression vector imparts a trait excellent in flower retention property to a cell or an individual organism.

[0045] Examples of host cells into which the expression vector is introduced include bacterial cells, yeast cells, fungal cells other than yeast cells, and higher eukaryotic cells. Examples of bacterial cells include Escherichia coli cells. Examples of higher eukaryotic cells include plant cells and animal cells. Examples of plant cells include dicotyledonous plant cells and monocotyledonous plant cells. Examples of dicotyledonous plant cells include suspension culture cells of Dianthus plants. Examples of monocotyledonous plant cells include the Oc strain, which is suspension culture cells of rice. Examples of animal cells include insect cells, amphibian cells, reptilian cells, avian cells, fish cells, and mammalian cells.

[0046] The expression vector may contain a polynucleotide that is a gene originally contained in the transformed plant and that mutates, deletes, or suppresses the function of the flowering property regulatory gene. The polynucleotide that mutates, deletes, or suppresses the function of the flowering property regulatory gene may be, for example, a polynucleotide in which the promoter sequence of the flowering property regulatory gene is mutated so as to lack or suppress the expression of the flowering property regulatory gene. The promoter sequence of the flowering property regulatory gene consists of, for example, the nucleotide sequences shown in SEQ ID NOs: 6 and 7. The nucleotide sequence shown in SEQ ID NO: 6 represents the promoter sequence of the AP2 gene derived from Francesca used as a reference, and the nucleotide sequence shown in SEQ ID NO: 7 represents the promoter sequence of the AP2 gene derived from Sandrosa with excellent flowering properties.

[0047] The expression vector may be appropriately designed according to the host cell into which the vector is introduced. That is, according to the type of host cell, the polynucleotide having the above-described mutation may be incorporated into, for example, a plasmid, phagemid, or cosmid, etc., for design. The vector is preferably a vector of the pBI system, pPZP system, or pSMA system that can introduce the polynucleotide of interest into plant cells via Agrobacterium, and a binary vector is particularly preferred.

[0048] In the expression vector, elements necessary for transcription of the polynucleotide that mutates, deletes, or suppresses the function of the gene (for example, a promoter, etc.) are functionally linked to the polynucleotide. Further, the polynucleotide may be linked with a spacer, enhancer, selection marker, splicing signal, polyA addition signal, and 5'-UTR sequence, etc., as necessary. The promoter is a DNA sequence that exhibits transcriptional activity in the host cell and can be appropriately selected according to the type of host, and examples include an actin promoter.

[0049] The expression vector may further contain a selection marker. Examples of the selection marker include drug resistance genes such as ampicillin, kanamycin, tetracycline, chloramphenicol, neomycin, hygromycin, or spectinomycin.

[0050] The expression vector may further contain a selection marker. Examples of the selection marker include drug resistance genes such as ampicillin, kanamycin, tetracycline, chloramphenicol, neomycin, hygromycin, or spectinomycin.

[0051] In addition, the expression vector may be bound to a tag sequence suitable for protein purification or a suitable nadeshiko spacer sequence, if necessary.

[0052] [Determination method for judging the flower retention property of Dianthus plants] The determination method (judgment method) for judging the flower retention property of Dianthus plants according to one aspect of the present invention judges whether the flower retention property is excellent in Dianthus plants. The determination method can be used to judge individuals with excellent flower retention property in Dianthus plants. The determination method judges Dianthus plants based on flower retention property by determining the genotype of the region involved in the expression of the gene involved in the regulation of flower retention property existing in the genome of Dianthus plants. In the determination method, the concept of flower retention property includes that the flower retention property of a certain Dianthus plant individual is relatively high or low compared to other Dianthus plant individuals, and the degree of flower retention property indicating that the flower retention period of a certain Dianthus plant individual is relatively long or short compared to other Dianthus plant individuals.

[0053] The Dianthus plants can be candidate plants for breeding materials or plants obtained in the breeding process. The candidate plants for breeding materials include, for example, parent plants used for crossing and plants used for molecular breeding using genetic recombination techniques. Also, the plants obtained in the breeding process include, for example, plants obtained by intraspecific crossing of carnations or wild carnations of the Dianthus genus and their progeny lines. Further, the Dianthus plants may be plants obtained by intervarietal crossing such as crossing of carnations belonging to one variety and carnations belonging to another variety, and their progeny lines.

[0054] Furthermore, the Dianthus plants may be plants obtained by crossing varieties known to have excellent flower-holding properties and their progeny lines. Also, the Dianthus plants may be plants obtained by crossing a variety known to have excellent flower-holding properties with a variety with unknown or excellent flower-holding properties and their progeny lines. Further, the Dianthus plants may be plants obtained by crossing varieties with unknown or excellent flower-holding properties and their progeny lines. Also, the Dianthus plants may be plants obtained by crossing a variety known to have excellent flower-holding properties with a variety known to have poor flower-holding properties and their progeny lines. Also, the Dianthus plants may be plants obtained by crossing individuals known to have excellent flower-holding properties and their progeny lines.

[0055] Also, the Dianthus plants can be progeny plants of the carnation variety "Sandroza". The carnation variety "Sandroza" can be an example of a variety with excellent flower-holding properties.

[0056] The determination method includes a detection step of detecting, in the Dianthus plants, the flower-holding property-regulating gene according to one aspect of the present invention described above as a molecular marker related to the regulation of the flower-holding property of the Dianthus plants.

[0057] Alternatively, the molecular marker may be a gene polymorphism within a region involved in the expression of the flower-bearing trait regulatory gene. Such a region includes the regions before and after the flower-bearing trait regulatory gene and the region of the flower-bearing trait regulatory gene in the genomic sequence of the Dianthus plant, and is a region involved in the expression of the flower-bearing trait regulatory gene. The region involved in the expression of the flower-bearing trait regulatory gene may be a region between the rear end of the gene located before the flower-bearing trait regulatory gene and the front end of the gene located after the flower-bearing trait regulatory gene in the genomic sequence of the Dianthus plant.

[0058] In the detection step, a genotype that lacks or suppresses the expression of the flower-bearing trait regulatory gene, or a mutation that lacks or suppresses the expression of the flower-bearing trait regulatory gene is detected as a molecular marker. Thus, when it is detected that the expression of the flower-bearing trait regulatory gene is lacking or suppressed in a Dianthus plant, it can be determined that the Dianthus plant is excellent in flower-bearing trait. Also, when it is detected that the expression of the flower-bearing trait regulatory gene is not lacking or suppressed in a Dianthus plant, it can be determined that the Dianthus plant has a low flower-bearing trait.

[0059] In the detection step, it may be performed by quantifying the expression level of the flower-bearing trait regulatory gene contained in the plant. As an example, polynucleotides, such as RNA, are extracted from the plant, and the flower-bearing trait regulatory gene contained in the polynucleotide is quantified by, for example, Real time PCR. The lower the amount of the polynucleotide of the flower-bearing trait regulatory gene, the more excellent the flower-bearing trait can be determined, and the higher the amount of the polynucleotide of the flower-bearing trait regulatory gene, the lower the flower-bearing trait can be determined.

[0060] As an example, the molecular marker used in the detection step is a genotype or mutation that lacks or suppresses the expression of the AP2 gene in the promoter region of the AP2 gene. The molecular marker for detecting such a genotype or mutation is the following polynucleotides (A) to (F): (A); a base corresponding to the 172nd to 173rd bases of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 6 (B); Bases corresponding to the 310th to 327th bases of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 6 (C); Bases corresponding to the 426th to 427th bases of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 6 (D); Bases corresponding to the 984th to 991st bases of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 6 (E); Bases corresponding to the 1063rd to 1072nd bases of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 6 (F); Bases corresponding to the 1120th to 1121st bases of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 6 may include at least one of them.

[0061] In the detection step, among the polynucleotides of (A) to (F) above, at least one may be used as a molecular marker, but it is preferable to use two or more of them as molecular markers.

[0062] That is, the determination method includes a step of examining whether the base itself (SNP) corresponding to the base of the polynucleotide of (A) to (F) above or a continuous polynucleotide containing the base is used as a molecular marker related to the regulation of flower retention. By using the base corresponding to the base of the polynucleotide of (A) to (F) above as a molecular marker, it is possible to detect the presence or absence of mutations that affect the expression of the flower retention regulatory gene. The "base corresponding to the base of the polynucleotide of (A) to (F)" is a SNP marker that can be identified as the same as the SNP marker described in this example. In addition, the "base corresponding to the base of the polynucleotide of (A) to (F)" refers to the base on the gene corresponding to the flower retention regulatory gene that is considered to correspond to the base of the polynucleotide of (A) to (F) by methods such as homology search. For example, the polynucleotide described in (ii) or (iii) above is an example of a gene corresponding to the flower retention regulatory gene.

[0063] The molecular markers include, as an example, SNP markers, AFLP (amplified fragment length polymorphism) markers, RFLP markers, microsatellite markers, SCAR markers, and CAPS markers.

[0064] The method for determining the flower-bearing property in Dianthus plants using the above-described molecular markers is not particularly limited. For example, a known SNP analysis method for detecting SNPs can be used. Such known SNP analysis methods include a method for SNP analysis by detecting SNPs in the PCR amplification fragments of a test sample of Dianthus plants.

[0065] The determination method may amplify the region in the DNA of Dianthus plants using a primer set that amplifies the region containing the molecular marker. Such a primer set is, for example, a primer set that amplifies a region containing at least one of the polynucleotides (A) to (F). Also, an example of such a primer set is primer sets 1 to 6 used in the examples described later, preferably primer sets 1 to 5.

[0066] That is, in the detection step of the determination method, one aspect of the primer set used to amplify the region containing the molecular marker is at least one of the following (d) to (i), preferably at least one of (d) to (h). (d) A combination of an oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 8 and an oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 9; (e) A combination of an oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 10 and an oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 11; (f) A combination of an oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 12 and an oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 13; (g) A combination of an oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 14 and an oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 15; (h) A combination of an oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 16 and an oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 17; and (i) A combination of an oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 18 and an oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 19.

[0067] In addition, with respect to the nucleotide sequence of the oligonucleotide constituting each primer in the above primer set, a modified primer consisting of a nucleotide sequence having 90% or more sequence identity and obtained by modifying some bases in the nucleotide sequence is also included in the scope of the primers according to the present invention. Further, the modified primer may have, for example, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity with the nucleotide sequence shown in any of SEQ ID NOs: 8 to 19 in each of the above primers. In principle, if the modified primer can hybridize with the complementary DNA strand of each of the above primers, it is regarded as equivalent to each primer. Furthermore, this modified primer regarded as equivalent has a function as a primer that hybridizes with the complementary DNA strand of the original primer, and in the nucleotide sequence of the oligonucleotide constituting each of the original primers, for example, 0 to 3, 0 to 2, 1 to 3, or 1 or 2 bases are added, deleted, inserted or substituted. It may be a primer consisting of a modified nucleotide sequence.

[0068] Amplification of the region in the DNA of the subject of Dianthus plants can be performed by polymerase chain reaction (PCR) using DNA extracted from the subject of Dianthus plants as a template and a primer for amplifying the region containing the SNP. Then, the base (genotype) of the SNP in the obtained amplified fragment is determined, and based on the data showing the relationship between the determined base (genotype) and the flower-bearing property in Dianthus plants, the flower-bearing property in Dianthus plants is determined.

[0069] The primer set used in PCR is not particularly limited as long as it can amplify a DNA fragment in the region containing the target SNP, and the primer set may be designed so that the length of the amplified fragment is shortened. For example, the primer set is designed so that the length of the primer amplified fragment is preferably 700 bases or less, 200 bases or less, 150 bases or less, 120 bases or less, or 100 bases or less. The primer set includes a first primer that is a forward primer and a second primer that is a reverse primer. The length of these primers may be, for example, 15 bases or more, 16 bases or more, 17 bases or more, 18 bases or more, or 19 bases or more, and may also be 50 bases or less, 40 bases or less, or 30 bases or less.

[0070] PCR in SNP analysis may be either singleplex PCR that amplifies a DNA fragment in a reaction system containing a single primer set, or multiplex PCR that amplifies a gene in a reaction system containing a plurality of primer sets. In the case of multiplex PCR, primer sets labeled with fluorescent substances having different wavelengths (for example, NED, 6-FAM, VIC, PET) may be mixed.

[0071] The reaction conditions of PCR can be appropriately set according to the type of DNA polymerase and PCR apparatus used, the length of the amplified fragment, etc. As cycle conditions, a three-step PCR method in which three steps of a denaturation step, an annealing step, and an extension step are defined as one cycle, and a two-step PCR method in which two steps of a denaturation step and an annealing and extension step are defined as one cycle can be applied. As an example of PCR reaction conditions, 40 to 60 seconds at 90 to 100°C (for example, 50 seconds at 95°C), then 5 seconds at 90 to 100°C (for example, 95°C), annealing for 10 to 20 seconds (for example, 15 seconds), and 10 to 30 seconds at 65 to 80°C (for example, 20 seconds at 72°C) for 30 to 60 cycles (for example, 40 cycles). Examples of annealing temperature conditions include gradually decreasing the temperature step by step for each predetermined cycle from an initial annealing temperature of 60 to 70°C (for example, 66°C) to a final annealing temperature of 50 to 60°C (for example, 56°C). Depending on the state of the DNA serving as the template, the PCR reaction conditions may be adjusted to stably detect SNPs.

[0072] As PCR in SNP analysis, real-time PCR such as the TaqMan®-PCR method for amplification by PCR and identification of SNP markers, and the Tm-shift genotyping method (Fukuoka et al. Breed Sci 58: 461-464, 2008) may be used. That is, a TaqMan® probe for detecting SNPs contained in the amplified fragment amplified using a primer set may be further used. By using the real-time PCR method, a determination method with high throughput can be provided. In addition, as a method for identifying SNPs in the amplified fragment amplified by PCR, it may be analyzed by determining the nucleotide sequence of the amplified fragment using an automatic DNA sequencer or the like.

[0073] As a method for extracting DNA amplified by PCR from a subject of Dianthus plants, it is not particularly limited, and known DNA extraction methods can be used. Also, DNA may be extracted using a commercially available DNA extraction kit. Depending on the type of subject and the amount of contaminants, etc., pretreatment may be appropriately performed before the DNA extraction step. Further, the DNA extracted from the subject may be washed or purified as necessary for use as a template in the PCR reaction. Furthermore, restriction enzyme cleavage fragments obtained by digesting the DNA extracted from the subject with two types of restriction enzymes may be amplified by PCR.

[0074] In addition, in a method for determining the flower-bearing property in Dianthus plants, gene polymorphisms in linkage disequilibrium with the polynucleotides (A) to (F) above may be analyzed. The linkage disequilibrium state is, for example, a linkage disequilibrium state with a linkage disequilibrium coefficient of 0.9 or more.

[0075] According to the determination method, it is possible to determine whether a test Dianthus plant is excellent in flower-bearing property using a molecular marker. Therefore, based on the determination result, Dianthus plants excellent in flower-bearing property and their progeny lines can be selected.

[0076] [Dianthus plants with good flower-bearing property] The plant according to one aspect of the present invention is a Dianthus plant with good flower-bearing property, in which the expression of the flower-bearing property regulatory gene is deficient or suppressed. The Dianthus plant with good flower-bearing property is excellent in flower-bearing property compared to a Dianthus plant in which the expression of the flower-bearing property regulatory gene is not deficient or suppressed because the function of the flower-bearing property regulatory gene is deficient or suppressed.

[0077] In a Dianthus plant with good flower - retaining property, the deletion or suppression of the expression of the flower - retaining property - regulating gene occurs as a result of known techniques that cause the deletion of the gene itself or the deletion or suppression of the gene expression, such as natural mutation, mutagen treatment, gene recombination, genome editing, or gene knockout. The natural mutation of the above - mentioned gene generally occurs due to replication errors and gene damage. The cause of such damage is exposure to known natural mutagens (such as radiation and ultraviolet rays). The mutagen treatment of the above - mentioned gene can be carried out by artificially applying the above - mentioned mutagen to the plant (and, if necessary, in combination with the suppression of the gene repair function). Examples of the types of mutagens include chemical agents such as ethyl methanesulfonate (EMS), sodium azide, ethidium bromide, and nitrous acid, as well as active rays such as γ - rays, heavy - ion beams, X - rays, neutron beams, and UV. These techniques are preferable from the viewpoint that it is not necessary to add external factors to the plant. The recombination of the above - mentioned gene can be carried out by homologously recombining part or all of the target gene with a recombinant sequence according to known gene recombination techniques.

[0078] The Dianthus plant with good flower - retaining property according to one aspect of the present invention can be obtained, for example, by determining a Dianthus plant with improved flower - retaining property from plants obtained by intraspecific crossing of Dianthus plants and their progeny lines using a molecular marker related to the regulation of flower - retaining property of Dianthus plants. Note that a Dianthus plant with good flower - retaining property that has been genetically engineered to have such a molecular marker is also included in the scope of the present invention. Furthermore, in a Dianthus plant, a Dianthus plant with improved flower - retaining property obtained by genome editing so as to delete or suppress the expression of the flower - retaining property - regulating gene is also included in the scope of the present invention.

[0079] Gypsophila plants can be candidate plants for breeding materials or plants obtained in the breeding process. Candidate plants for breeding materials include, for example, parent plants used for crossing and plants used for molecular breeding using genetic recombination techniques. Plants obtained in the breeding process include, for example, plants obtained by crossing within the genus Gypsophila, plants obtained by crossing within the genus Dianthus, and their progeny lines.

[0080] The Gypsophila plants with good flower-holding property according to one aspect of the present invention include Gypsophila plants determined to be excellent in flower-holding property by the determination method according to one aspect of the present invention, and plants obtained by the method for producing Gypsophila plants with good flower-holding property according to one aspect of the present invention.

[0081] [Method for Producing Gypsophila Plants with Good Flower-Holding Property] The method for producing Gypsophila plants with good flower-holding property of plants according to one aspect of the present invention (hereinafter referred to as the production method) includes a step of deleting or suppressing the expression of the flower-holding property regulatory gene according to one aspect of the present invention in Gypsophila plants. According to the production method according to one aspect of the present invention, Gypsophila plants excellent in flower-holding property can be created.

[0082] The step of deleting or suppressing the gene expression can be carried out by applying known techniques such as natural mutation, mutagen treatment, gene recombination, genome editing, or gene knockout, which either delete the gene itself or delete or suppress the gene expression, to plants having the flower-holding property regulatory gene. As an example, this step may be carried out by transforming a plant with a vector according to one aspect of the present invention using the Agrobacterium method, particle gun method, or electroporation method.

[0083] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope shown in the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention.

Examples

[0084] [AP2 gene flower-holding property regulation function] Genes related to flower-holding were extracted using a microarray constructed from the carnation Expressed Sequence Tag (EST) database (Reference: Tanase et al. (2012) BMC Genomics 13: 292.). The temporal gene expression of these genes during the flower aging process was investigated by real-time PCR. For genes with increased expression during aging, the genomic sequences were investigated, and genes with different genomic sequences (mutations present) were selected in the control Francesca and the good flower-holding variety Sandrosa. The AP2 gene was identified from these results. The results are shown in Fig. 1. Fig. 1 is a diagram showing the results of investigating the expression of the AP2 gene in varieties with different flower-holding properties.

[0085] [Comparison of genomic sequences of carnation AP2 gene] The genomic sequences of the AP2 gene in carnation varieties were compared using the genomic analysis software IGV. Also, the genomic sequence of the AP2 gene in Dianthus was compared together. The results are shown in Fig. 2. Fig. 2 is a diagram schematically showing the results of comparing the genomic sequences of the AP2 gene in carnation varieties. In Fig. 2, the nine rows from the top are the comparison results of carnation varieties, and the bottom row shows the comparison result of Dianthus. In Fig. 2, the light gray lines indicate regions that match the sequence of the control Francesca. The dark gray lines indicate regions with different base sequences from Francesca. The top two varieties in Fig. 2 are varieties with standard flower-holding, and the sequences were almost identical to Francesca. On the other hand, the bottom seven varieties are varieties with good flower-holding, with many regions having different base sequences from Francesca, and the mutations were conserved among the varieties. For the Dianthus sequence, there were many regions that matched Francesca.

[0086] [Comparison of genomic sequences and evaluation of identity] Genomic DNA was extracted from the control variety, Francesca, which has standard flower retention, and Sandro Rosa, which has excellent flower retention, and genomic sequencing was performed using a next-generation sequencer. The genomic sequence and flanking sequences encoding the AP2 gene identified from the results of expression analysis were extracted, and the genomic sequences between the varieties were compared using the analysis software IGV and GENETYX.

[0087] The promoter sequence of the AP2 gene was determined by genomic PCR. First, referring to the genomic sequence of Francesca whose genomic sequence has been published, a Forward primer was designed upstream of the AP2 gene, and the Reverse primer designed for the CDS sequence was used for genomic PCR. Genomic DNA of the control variety Francesca and the good flower retention line Sandro Rosa was extracted and used as a template for genomic PCR. The DNA amplified by PCR was cloned and the sequence was confirmed. The results of comparing the promoter sequences upstream of the AP2 gene are shown in Figure 3. As shown in Figure 3, it was shown that there are regions with different base sequences in the promoter sequences among the varieties. Also, in Figure 4, the results of comparing the amino acid sequences encoded by the AP2 gene in Francesca and Miracle Rouge are shown. As shown in Figure 4, there were regions where the amino acid sequences encoded by the AP2 gene were also different between Francesca and Miracle Rouge.

[0088] 〔Evaluation of the flower retention of carnation varieties〕 The flower retention of each carnation variety was evaluated as follows. The flowers of carnations cultivated in the greenhouse were harvested on the day when they were fully developed and inserted into distilled water. They were transported to a test room with a room temperature of 23°C, a relative humidity of 70%, an illuminance, and a 12-hour day length, and the wilting of the flowers was judged visually every day. The number of days from harvest to wilting was counted as the flower retention days, and it was determined whether the flower retention was excellent (good flower retention) or standard. Wilting was defined as when the petals were rolled inward and lost their ornamental value (during in-rolling), or when the petals turned brown and lost their ornamental value. For cut flowers, a flower retention period of about 7 days was regarded as standard flower retention, and a flower retention period of 10 days or more was determined as a good flower retention line (good flower retention line). The results are shown in Table 1.

[0089]

Table 1

[0090] Next, the determination results of the flower retention property for each variety were associated with the genotypes of the AP2 gene in these varieties. The results are shown in Table 2. The genotype of the AP2 gene was determined by the Direct marker method using primer sets 1 and 2 shown in Table 3 described below.

[0091]

Table 2

[0092] As shown in Tables 1 and 2, the four varieties determined to have standard flower retention properties had the same wild-type genotype of the AP2 gene as that of Francesca, which is the control sequence. Also, the eight varieties determined to have good flower retention properties had mutant genotypes of the AP2 gene that were different from Francesca.

[0093] 〔Discrimination of flower retention property using DNA marker〕 Based on the sequence comparison results as described above and the relationship between the flower retention property and the genotype of the AP2 gene, a DNA marker for discriminating the flower retention property by specifying the genotype of the AP2 gene was designed. Then, using the designed DNA marker, the flower retention property of carnation varieties was discriminated. The flower retention property of carnation varieties was discriminated by comparing the amplification products obtained by amplifying the DNA marker region. The primer sets of primers 1 to 6 shown in Table 3 were used in the reaction system shown in Table 3, respectively.

[0094]

Table 3

[0095] Primer set 1 is a primer set for discriminating the genotype of bases corresponding to the 108th to 593rd bases of the polynucleotide consisting of the base sequence of SEQ ID NO: 7. Primer set 2 is a primer set for discriminating the genotype of bases corresponding to the 15th to 560th bases of the polynucleotide consisting of the base sequence of SEQ ID NO: 6. Primer set 3 is a primer set for discriminating the genotype of bases corresponding to the 8610th to 9318th bases of the polynucleotide consisting of the base sequence of SEQ ID NO: 3. Primer set 4 is a primer set for discriminating the genotype of bases corresponding to the 11281st to 12169th bases of the polynucleotide consisting of the base sequence of SEQ ID NO: 3. Primer set 5 is a primer set for discriminating the genotype of bases corresponding to the 5276th to 5551st bases of the polynucleotide consisting of the base sequences of SEQ ID NO: 3 and 5. Primer set 6 is a primer set for discriminating the genotype of bases corresponding to the 7783rd to 7957th bases of the polynucleotide consisting of the base sequences of SEQ ID NO: 3 and 5.

[0096] Young leaves of each variety of carnation before full expansion were sampled at 50 - 100 mg. After adding liquid nitrogen and grinding, genomic DNA was extracted using the QIAGEN DNeasy Plant Mini kit according to the protocol. The obtained genomic DNA was diluted 2-fold with sterile water and used for the PCR reaction.

[0097] As shown in Table 3, as the PCR reaction system, the following three reaction systems were used respectively to detect the lines shown in Table 1.

[0098] (System using ExTaq) In the system using ExTaq manufactured by Takara Bio Inc., after a reaction at 94°C for 5 minutes using primer set 1 consisting of primer 1F and primer 1R shown in Table 3, or primer set 2 consisting of primer 2F and primer 2R, and the PCR reaction solution shown in Table 4, a denaturation step at 94°C for 30 seconds, an annealing step at 58°C for 20 seconds, and an extension step at 72°C for 30 seconds were performed for 35 - 40 cycles.

[0099]

Table 4

[0100] After the reaction was completed, electrophoresis was performed using a 1% agarose gel to confirm the bands. The results are shown in Figs. 5 and 6. In Figs. 5 and 6, DW represents a negative control without adding genomic DNA. As shown in Fig. 5, when primer set 1 was used, amplification products were obtained only for the good flower-holding Miracle Rouge and Sandroza. Also, as shown in Fig. 6, when primer set 2 was used, amplification products were obtained only for the standard flower-holding Francesca and White Shim. It was shown that good flower-holding plants can be discriminated by the Direct marker method using primer sets of primers 1 and 2.

[0101] (System using KOD-one) In the system using KOD-one manufactured by Toyobo Co., Ltd., a denaturation step at 98°C for 10 seconds, an annealing step at 55°C for 5 seconds, and an extension step at 68°C for 1 second were performed for 35 to 40 cycles using primer set 3 consisting of primer 3F and primer 3R shown in Table 3, primer set 4 consisting of primer 4F and primer 4R, or primer set 6 consisting of primer 6F and primer 6R, and the PCR reaction solution shown in Table 5.

[0102]

Table 5

[0103] After the reaction was completed, electrophoresis was performed using a 2% agarose gel to confirm the bands. The results are shown in Figs. 7 to 9. As shown in Fig. 7, when primer set 3 was used, amplification products were obtained only for Francesca and White Sim with standard flower-holding properties. Also, as shown in Fig. 8, when primer set 4 was used, amplification products were obtained only for Francesca and White Sim with standard flower-holding properties. Furthermore, as shown in Fig. 9, when primer set 6 was used, the positions of the bands where amplification products were detected were different between Francesca and White Sim with standard flower-holding properties, and Miracle Rouge and Sandroza with good flower-holding properties. It was shown that good flower-holding plants can be discriminated by the Direct marker method using primer sets of primers 3, 4, and 6.

[0104] (System using KOD-one and restriction enzymes) In the system using KOD-one and restriction enzymes, after performing a PCR reaction by the system using KOD-one described above using primer set 5 consisting of primer 5F and primer 5R shown in Table 3, restriction enzyme treatment was performed using the enzyme treatment reaction solution shown in Table 6 to digest the amplification product. Thereby, the genomic DNA derived from the good flower-holding line was fragmented.

[0105]

Table 6

[0106] The reaction was carried out at 37 °C for 2 hours. After the reaction was completed, electrophoresis was performed using a 2% agarose gel to confirm the bands. The results are shown in Fig. 10. As shown in Fig. 10, the positions of the bands detected were different between Miracle Rouge and Sandroza with good flower-holding properties and Francesca and White Sim with standard flower-holding properties. By the CAPS method using primer 5, it was possible to discriminate between control plants with standard flower-holding properties and plants with good flower-holding properties.

[0107] 〔Preparation of recombinant carnations〕 (Adjustment of plants) Axillary buds of the carnation cultivar "Ariel" were harvested, sterilized with 70% ethanol, then with hypochlorous acid, and planted on a sterile medium to produce aseptic cultured seedlings. The culture was carried out using Murashige and Skoog (MS) medium or 1 / 2MS medium.

[0108] The MS medium was prepared by mixing one pack of mixed salts for Murashige and Skoog medium (Wako Pure Chemical Industries), 20 g of sucrose, and 1 mL of MS vitamins (×1000). After adjusting the pH to 5.6 - 5.8, it was made up to 1 L. In the case of a solid medium, 2 g / L of gellan gum was added. After mixing all, it was sterilized by autoclaving.

[0109] The 1 / 2MS medium was a medium prepared by halving the salts of the MS medium. One - half pack of mixed salts for Murashige and Skoog medium (Wako Pure Chemical Industries), 20 g of sucrose, and 0.5 mL of MS vitamins (×1000) were mixed. After adjusting the pH to 5.6 - 5.8, it was made up to 1 L. In the case of a solid medium, 2 g / L of gellan gum was added. After mixing all, it was sterilized by autoclaving.

[0110] The MS vitamin stock was prepared by dissolving 10 g of myo - inositol, 0.05 g of nicotinic acid, 0.05 g of pyridoxine hydrochloride, 0.01 g of thiamine hydrochloride, and 0.2 g of glycine in distilled water, adjusting to 100 mL, then dispensing 1 mL each and storing at - 20°C for use.

[0111] (Vector) As the vector, vectors derived from apple latent spherical virus (ALSV) (pEALSR1 and pEALSR2L5R5) were used (Reference 1: Yamagishi & Yoshikawa, Plant Mol Biol 71: 15 - 24, 2009). The ALSV vector incorporating the target gene was introduced into Escherichia coli (DH5α or JM109) and cultured on an LB solid medium containing 50 mg / L of ampicillin. Colonies were picked up, cultured in an LB liquid medium containing 50 mg / L of ampicillin, then the plasmid was extracted and dissolved in TE buffer to a concentration of 2 μg / μL.

[0112] (Recombinant virus) Using the plasmid prepared by the same method as virus sap inoculation (Ooki, 2009), young plants of quinoa or Nicotiana benthamiana, which are host plants for propagation, were mechanically inoculated. Carborundum (600 mesh, Nacalai Tesque) was evenly and thinly sprinkled on the leaves of the propagation host, and 10 μL of a plasmid solution in which vectors pEALSR1 and pEALSR2L5R5 were mixed in equal amounts was rubbed on the entire surface of the leaves. Then, the carborundum on the leaf surface was washed away with sterilized water, and after curing overnight in the dark, it was cultivated in an incubator or a recombinant greenhouse.

[0113] When symptoms were confirmed on the upper leaves 2 - 3 weeks later, new plants of the propagation host were prepared. 2 - 3 volumes of inoculation buffer were added to the infected leaves and ground using a mortar and pestle, and mechanical inoculation was performed again. The infected upper leaves were harvested and stored at -80 °C.

[0114] Recombinant virus RNA was extracted from the infected leaves using TriPure Isolation Reagent (Sigma - Aldrich). The infected leaves were ground using a mortar and pestle, and TriPure Isolation Reagent was added. Then, RNA was extracted according to the protocol of TriPure Isolation Reagent, and the extracted RNA was adjusted to a concentration of 4 μg / μL by ethanol precipitation.

[0115] (Recombinant of Dianthus plants) Inoculation of Dianthus plants such as carnation was performed using the virus RNA inoculation method by particle gun described in Reference 1. To prepare 40 shots of particle gun (RNA: 5 μg / gold particles), 8 mg of gold particles were weighed into a 1.5 ml tube, 50 μl of sterilized water was added and mixed, and ultrasonic treatment was performed for 5 minutes or more.

[0116] Add 50 μl of RNA, 10 μl of 5 M ammonium acetate, and 220 μl of isopropanol gradually with gentle stirring. After adding all and stirring for a while, let it stand at -20°C for 1 hour or more. Centrifuge at 800×g or less to remove the supernatant, and wash the gold particles 4 times with 1 ml of 99.5% ethanol. Finally, suspend the gold particles in 2.4 ml of 99.5% ethanol.

[0117] Set the gold coat tube for the Helios Gene Gun System (Bio-Rad) on the tubing prep station and dry it thoroughly by passing nitrogen gas. Subsequently, pour in the ethanol solution containing the gold particles, fill it evenly, and remove the supernatant ethanol. Dry the gold particles by rotating the tubing prep station and passing nitrogen gas. Pull out the gold coat tube from the tubing prep station and cut it with a tubing cutter to make a cartridge.

[0118] Using the Helios Gene Gun System at a helium pressure of 220 psi, inoculate 2 - 4 shots per individual. As the plant individuals to be tested, similar to the Agrobacterium method, use individuals regenerated from the shoot tip and the tissue immediately below it and cultured aseptically. After spraying water on the plants after firing the particle gun to provide moisture and placing them under dark conditions for 1 day while maintaining the humidity, culture them under normal conditions (25°C, 16-hour day length).

[0119] Fold the tip from the shoot that has grown on the selection medium and transplant it to a 1 / 2MS medium or Hyponex medium without plant hormones. Cut off the tip and perform water cutting for rooting. When new roots are confirmed in water cutting for rooting, plant it in culture soil in an environment with maintained humidity for acclimatization, and transplant it to a pot filled with commercially available horticultural culture soil while observing the plant state. When the plant flowers, harvest the flowers and evaluate the flower retention property. The determination of the flower retention property was performed in the same manner as described above [Experimental methods and results for evaluating flower retention property using markers]. The results are shown in Table 7.

[0120]

Table 7

[0121] As shown in Table 7, the recombinant in which the expression of the wild-type AP2 gene was suppressed had improved flower-holding property.

[0122] 〔Evaluation of flower-holding property in carnation crossing lines using DNA markers〕 Using the methods described in the above 〔Evaluation of flower-holding property of carnation varieties〕 and 〔Discrimination of flower-holding property using DNA markers〕, the flower-holding days and the genotype of the flower-holding property regulatory gene were investigated for carnation crossing lines. The genotype of the flower-holding property regulatory gene was determined by the Direct marker method using primer set 1 (DNA marker 1 (a DNA marker for discriminating good flower-holding lines)), primer sets 2, 3, and 4 (DNA markers 2, 3, and 4 (DNA markers for discriminating standard flower-holding lines)), and primer set 5 (DNA marker 5 (a DNA marker for discriminating standard flower-holding lines and good flower-holding lines)).

[0123] The results of the flower-holding days and DNA marker determination of the carnation crossing lines are shown in Table 8. The flower-holding days of the 66 crossed lines ranged from a minimum of 7 days to a maximum of 30 days. Among the 66 lines, the band of DNA marker 1 (for discriminating good flower-holding lines) was confirmed in 11 lines. The bands of DNA markers 2, 3, and 4 (for discriminating standard flower-holding lines) were confirmed in 63 lines. For DNA marker 5, the band indicating the standard flower-holding line was confirmed in 55 lines, the band indicating the good flower-holding line was confirmed in 3 lines, and both the band indicating the standard flower-holding line and the band indicating the good flower-holding line were confirmed in 8 lines. Based on this, it was determined that 3 lines were homozygous for the mutant gene with good flower-holding property, 8 lines were heterozygous, and 55 lines were homozygous for the wild-type gene with standard flower-holding property.

[0124]

Table 8

[0125] JPEG2025090555000009.jpg232160

[0126] Subsequently, for these 66 lines, the average number of days with flowers was examined for each genotype line discriminated by the markers as described above. The results are shown in Table 9. The average flowering period for all 66 lines was 15.4 days. The lines with mutant gene homozygosity with good flower-holding property had 20.7 days, the heterozygous lines had 17.3 days, and the wild-type gene homozygous lines with standard flower-holding property had 14.8 days. Thus, the lines with the mutant gene marker for good flower-holding property had a significantly longer flowering period than those without it. From the above results, it was shown that the DNA marker related to flower-holding property is useful for the evaluation of flower-holding property.

[0127]

Table 9

Industrial Applicability

[0128] The present invention can be used in the agricultural field, the plant breeding field, etc.

Claims

1. A flower-life regulator gene that regulates the flower-life of plants of the genus Dianthus, Any one of the following polynucleotides (i) to (iii): (i) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2; (ii) a polynucleotide encoding a protein having an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1 or 2 and having an activity of regulating the flower life of plants of the genus Dianthus; (iii) a polynucleotide encoding a protein having an amino acid sequence in which 31 or less amino acids have been substituted, deleted, added or inserted relative to the amino acid sequence shown in SEQ ID NO: 1 or 2 and having an activity of regulating the flower life of plants of the genus Dianthus; This is a gene that regulates flower longevity.

2. Any one of the following polynucleotides (a) to (c): (a) a polynucleotide consisting of a nucleotide sequence shown in any one of SEQ ID NOs: 3 to 5; (b) a polynucleotide having a nucleotide sequence having 90% or more sequence identity to any of the nucleotide sequences shown in SEQ ID NOs: 3 to 5, and exhibiting a function equivalent to that of the polynucleotide of (a) above with respect to regulating the flower life of plants of the genus Dianthus; (c) a polynucleotide consisting of a nucleotide sequence in which 85 or less nucleotides have been substituted, deleted, added or inserted relative to the nucleotide sequence shown in any one of SEQ ID NOs: 3 to 5, and exhibiting a function equivalent to that of the polynucleotide of (a) above with respect to regulating the vase life of plants of the genus Dianthus; The flower life regulator according to claim 1 ,

3. A flower-life regulating protein having an activity of regulating the flower-life of plants of the genus Dianthus, the protein being encoded by the flower-life regulating gene according to claim 1 or 2.

4. A method for determining the flower life of a Dianthus plant, comprising: A method for determining whether a plant is capable of flowering longevity, comprising the step of detecting the gene for regulating flowering longevity according to claim 1 or 2 in a plant of the genus Dianthus as a molecular marker for regulating flowering longevity in plants of the genus Dianthus.

5. The method according to claim 4 , wherein the molecular marker is a gene polymorphism in a region involved in the expression of the flower life regulator gene.

6. The method according to claim 5 , wherein in the detection step, the region in the DNA of the Dianthus plant is amplified using a primer set that amplifies a region including the molecular marker.

7. The method according to claim 6, wherein the primer set is at least one of the following (d) to (h): (d) a combination of an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 8 and an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 9; (e) a combination of an oligonucleotide having the base sequence shown in SEQ ID NO: 10 and an oligonucleotide having the base sequence shown in SEQ ID NO: 11; (f) a combination of an oligonucleotide having the base sequence shown in SEQ ID NO: 12 and an oligonucleotide having the base sequence shown in SEQ ID NO: 13; (g) a combination of an oligonucleotide having the base sequence shown in SEQ ID NO: 14 and an oligonucleotide having the base sequence shown in SEQ ID NO: 15; and (h) A combination of an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 16 and an oligonucleotide consisting of the base sequence shown in SEQ ID NO:

17.

8. The method according to claim 5 , wherein the Dianthus plant is a progeny plant of the carnation cultivar “Sandrosa”.

9. A Dianthus plant having good flower longevity, in which expression of the gene for regulating flower longevity according to claim 1 or 2 is deleted or suppressed.

10. A method for producing a Dianthus plant having long-lasting flowers, comprising the steps of: A method for producing a Dianthus plant having good flower longevity, comprising a step of deleting or suppressing expression of the flower longevity regulating gene according to claim 1 or 2 in a Dianthus plant.

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