Genes controlling flower life in Dianthus plants and their uses

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 the flower life of plants of the genus Dianthus and uses thereof. [Background technology]

[0002] With the aim of improving the quality of carnations, a plant in the Dianthus genus, efforts are being made to develop cultivars and lines with long-lasting flowers. Until now, cultivating carnation cultivars with long-lasting flowers has typically been done through crossbreeding, but the progeny lines obtained after crossbreeding must be subjected to a vase life test over a period of approximately two years to confirm their quality. Therefore, there is a need for the development of technology that can more efficiently breed carnation cultivars with long-lasting flowers.

[0003] Techniques for improving the efficiency of crossbreeding include methods using DNA markers. Non-Patent Document 1 describes the identification of DNA markers related to the flower life of specific carnation varieties through comprehensive gene expression analysis of carnation flower life. Non-Patent Document 2 describes senescence-related genes associated with low ethylene production during senescence. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Boxriker MR et al. (2017), Scientia Horticulturae. 217: 61-72 [Non-patent document 2] Tanase K et al. (2013), J Japan Soc Hort Sci 82 (2) 179-187 Summary of the Invention [Problem to be solved by the invention]

[0005] However, even in Non-Patent Documents 1 and 2, the gene involved in the vase life of plants of the genus Dianthus, including carnation, has not been identified, and no DNA marker for efficiently selecting plants of the genus Dianthus has been found. Therefore, there is a need to identify the gene involved in vase life and develop a DNA marker for efficiently selecting Dianthus plants with long vase life.

[0006] An object of one aspect of the present invention is to realize a technique for producing a Dianthus plant having excellent vase life. [Means for solving the problem]

[0007] In order to achieve the above object, a flower-life regulating gene according to one aspect of the present invention is a gene for regulating the flower-life of plants of the genus Dianthus, and is a gene for regulating the flower-life of plants of the genus Dianthus, the gene comprising 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 fewer 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 the activity of regulating the flower life of plants of the genus Dianthus.

[0008] A flower life regulatory protein according to one embodiment of the present invention is encoded by a flower life regulatory gene according to one embodiment of the present invention and has an activity of regulating the flower life of plants of the genus Dianthus.

[0009] A method for determining the flower longevity of Dianthus plants according to one embodiment of the present invention includes a detection step of detecting the flower longevity regulatory gene described in claim 1 or 2 in Dianthus plants as a molecular marker for regulating the flower longevity of Dianthus plants.

[0010] In a Dianthus plant having good vase life according to one aspect of the present invention, the expression of a gene that regulates vase life according to one aspect of the present invention is suppressed.

[0011] A method for producing a Dianthus plant with long flower life according to one aspect of the present invention includes a step of suppressing the expression of the gene for regulating flower life according to claim 1 or 2 in a Dianthus plant. [Effects of the Invention]

[0012] According to one aspect of the present invention, a technique for producing a Dianthus plant having excellent vase life can be realized. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 shows the results of investigating the expression of the AP2 gene in varieties with different vase life, as conducted in the Examples. [Figure 2] FIG. 1 is a diagram schematically showing the results of a comparison of the genomic sequences of the AP2 gene in carnation cultivars in an example. [Figure 3] FIG. 1 shows the results of a comparison of promoter sequences upstream of the AP2 gene in an example. [Figure 4] FIG. 1 shows the results of a comparison of amino acid sequences encoded by AP2 genes in an example. [Figure 5] FIG. 1 shows the results of determining vase life using DNA markers in an example. [Figure 6] FIG. 1 shows the results of determining vase life using DNA markers in an example. [Figure 7] FIG. 1 shows the results of determining vase life using DNA markers in an example. [Figure 8] FIG. 1 shows the results of determining vase life using DNA markers in an example. [Figure 9] FIG. 1 shows the results of determining vase life using DNA markers in an example. [Figure 10] FIG. 1 shows the results of determining vase life using DNA markers in an example. DETAILED DESCRIPTION OF THE INVENTION

[0014] [Definition] The present invention will be described in detail below. All of the documents described in this specification are incorporated herein by reference. Furthermore, unless otherwise specified in this specification, "A to B" representing a numerical range means "A or more (including A and greater than A) and B or less (including B and less than B)."

[0015] As used herein, the term "polynucleotide" is used interchangeably with "nucleic acid" or "nucleic acid molecule," and refers to a polymer of nucleotides. Here, nucleic acids can exist in the form of DNA (e.g., cDNA or genomic DNA) or RNA (e.g., mRNA). DNA or RNA may be double-stranded or single-stranded. Single-stranded DNA or RNA may be a coding strand (sense strand) or a non-coding strand (antisense strand). As used herein, bases are represented by the single-letter symbols defined by IUPAC and IUB, as appropriate.

[0016] As used herein, the term "plant" refers to a part or the whole of a plant. Examples of parts of a plant include plant organs (e.g., roots, stems, leaves, petals, seeds, fruits, etc.), plant tissues (e.g., epidermis, phloem, parenchyma, xylem, vascular bundles, etc.), plant cells, callus, etc. As used herein, the term "Dianthus plants" refers to plants belonging to the genus Dianthus, such as Dianthus quinqueradiata and carnation.

[0017] As used herein, the term "flower life" refers to the degree to which flowers remain intact without wilting, and the term "long-lasting flower life" refers to the superior flower life or longer flower life of a plant with long-lasting flower life compared to a plant without long-lasting flower life. As used herein, the term "having an activity to regulate flower life" refers to the superior flower life of a plant in which a polynucleotide encoding a protein with the activity is mutated or in which expression of the polynucleotide is deleted or suppressed, compared to a plant in which the polynucleotide is not mutated or in which expression of the polynucleotide is not deleted or suppressed. The degree of improvement in flower life of a plant with long-lasting flower life may 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% relative to the flower life of a plant without long-lasting flower life.

[0018] Furthermore, as used herein, "the expression of a gene (or polynucleotide) is suppressed" means that the endogenous gene (or polynucleotide) inherent in the plant has been mutated (including nucleotide substitution, insertion, addition, or deletion) or deleted, or that the expression of the gene has been deleted or suppressed, due to artificial manipulation. Whether or not "the expression of a gene (or polynucleotide) is deleted or suppressed" can be determined based on the amount of polynucleotide extracted from the plant, as measured by RT-PCR, for example. The genome sequence of the carnation cultivar Francesco, which serves as the basis for the base sequence of the flower-vase regulatory gene, has been published in the carnation database of the Kazusa DNA Research Institute (http: / / carnation.kazusa.or.jp / ).

[0019] [Vase life control gene] A flower-longevity regulating gene according to one embodiment of the present invention is a gene encoding a protein having an activity for regulating the flower longevity of plants of the genus Dianthus. For example, when the expression of a protein having an activity for regulating the flower longevity is absent or suppressed in a Dianthus plant, the flower longevity is longer than that of a Dianthus plant in which the expression of the protein is absent or suppressed. Furthermore, when the expression of a protein having an activity for regulating the flower longevity is absent or suppressed in a Dianthus plant, the flower longevity is shorter than that of a Dianthus plant in which the expression of the protein is absent or suppressed.

[0020] The flower-life regulating gene is a gene involved in regulating the flower-life of plants of the genus Dianthus, 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 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 vase life of plants of the genus Dianthus; (iii) A gene consisting of a polynucleotide encoding a protein having an amino acid sequence in which 31 or fewer 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 the activity of regulating the flower life of plants of the genus Dianthus.

[0021] The polynucleotide (i) above is a polynucleotide that 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 encodes a protein having activity to regulate vase life. The polynucleotide (i) above may also be a polynucleotide that contains the nucleotide sequence of a gene corresponding to the AP2 gene of a plant of the genus Dianthus or the nucleotide sequence of the CDS of that gene and encodes a protein having activity to regulate vase life. The amino acid sequence shown in SEQ ID NO: 1 is the amino acid sequence encoded by the AP2 gene derived from the reference plant Francesco, and the amino acid sequence shown in SEQ ID NO: 2 is the amino acid sequence encoded by the AP2 gene derived from Sandrosa, which has excellent vase life.

[0022] With regard to the polynucleotides 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, mutant genes derived from carnations or homologous genes (including orthologs) derived from plants of the genus Dianthus other than carnations are included in the category of polynucleotides of (2) above. These mutant genes and homologous genes are endogenous genes of plants of the genus Dianthus. The molecular markers tested in the section "Method for determining flower longevity in plants of the genus Dianthus" described below may be molecular markers on these mutant genes or homologous genes.

[0023] With respect to the polynucleotide (iii) above, the number of substituted, deleted, added or inserted amino acids in the amino acid sequence of SEQ ID NO: 1 or 2 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] In addition, when the flower-life regulatory gene refers to a gene into which a mutation has been artificially introduced, the above-mentioned "amino acid substitution, deletion, addition, or insertion" may be an artificial mutation introduced using, for example, site-directed mutagenesis such as the Kunkel method (Kunkel et al. (1985): Proc. Natl. Acad. Sci. USA, vol. 82, p. 488-), mutagen treatment using a drug, or mutagenesis by irradiation with radiation (gamma rays, heavy ion beams, etc.), or may be derived from a similar mutant polypeptide that exists in nature.

[0025] The flower longevity regulatory gene may exist in the form of RNA (e.g., mRNA) or 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 a flower longevity regulatory 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. Furthermore, the nucleotide sequence shown in SEQ ID NO: 5, which is an example of a flower longevity regulatory gene, is a genomic sequence encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2. The flower longevity regulatory gene may contain an additional sequence, such as a nucleotide sequence of an untranslated region (UTR), in addition to the CDS of the AP2 gene.

[0026] The method for obtaining (isolating) the flower life regulating gene is not particularly limited, but for example, a probe that specifically hybridizes with a portion of the base sequence of the flower life regulating gene may be prepared and a genomic DNA library or a cDNA library may be screened.

[0027] Another method for obtaining a gene that regulates flower longevity is to use an amplification method such as PCR. For example, primers are prepared from the 5' and 3' sequences (or their complementary sequences) of the cDNA of the gene that regulates flower longevity, and these primers are used to perform PCR or the like using genomic DNA (or cDNA) as a template, thereby amplifying the DNA region between the two primers, thereby obtaining a large amount of a DNA fragment containing the gene that regulates flower longevity.

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

[0029] Furthermore, whether or not an isolated candidate gene for a flower-life regulating gene has the activity to regulate the desired flower-life can be evaluated by observing whether an improvement in flower-life is induced by deleting or suppressing the expression of the candidate gene in the plant from which it is derived.

[0030] The flower-life regulator gene can be used to elucidate the mechanism for improving flower life in Dianthus plants. Furthermore, the flower-life regulator gene can be used to produce a transformant by introducing a sequence that deletes or suppresses its expression into an expression vector and then introducing the vector into a plant or cell of a Dianthus plant. By cultivating a Dianthus plant in which the expression of the flower-life regulator gene is deleted or suppressed, a Dianthus plant with improved flower life can be obtained.

[0031] The flower life regulatory gene is any one of the following polynucleotides (a) to (c): (a) a polynucleotide consisting of a nucleotide sequence set forth 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 in regulating the vase life of plants of the genus Dianthus; (c) a polynucleotide consisting of a nucleotide sequence in which 85 or fewer nucleotides have been substituted, deleted, added, or inserted relative to the nucleotide sequence set forth in any one of SEQ ID NOs: 3 to 5, and which exhibits a function equivalent to that of the polynucleotide of (a) above in regulating the vase life of plants of the genus Dianthus; An example is a gene consisting of:

[0032] The polynucleotide (a) above is a polynucleotide comprising the nucleotide sequence of the AP2 gene derived from carnation or the nucleotide sequence of the coding region (CDS) of the AP2 gene. The polynucleotide (a) above also comprises the nucleotide sequence of a gene corresponding to the AP2 gene of a plant of the genus Dianthus or the nucleotide sequence of the CDS of the gene.

[0033] The polynucleotides of (b) above have sequence identities 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 the base sequences of SEQ ID NOs: 3 to 5. For example, mutant genes derived from carnation or homologous genes (including orthologs) derived from plants of the genus Dianthus other than carnation are included in the category of polynucleotides of (2) above.

[0034] With respect to the polynucleotides (3) above, the number of substituted, deleted, added or inserted bases in the base sequences of SEQ ID NOs: 3 to 5 may 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 flower-vase life regulators include polynucleotides consisting of the genomic sequence of the AP2 gene derived from carnation (SEQ ID NOS: 3 and 5) and the cDNA sequence of the AP2 gene derived from carnation (SEQ ID NOS: 4). The nucleotide sequence shown in SEQ ID NOS: 3 is the genomic sequence of the AP2 gene derived from Francesco, which serves as a reference, and the nucleotide sequence shown in SEQ ID NOS: 4 is the cDNA sequence of the AP2 gene derived from Francesco. The nucleotide sequence shown in SEQ ID NOS: 5 is the genomic sequence of the AP2 gene derived from Sandrosa, which has excellent flower-vase life. The category of flower-vase life regulators also includes genes consisting of polynucleotides that have 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 sequence identity to the nucleotide sequences shown in SEQ ID NOS: 3 to 5 and have flower-vase life regulator activity.

[0036] [Flower life regulation protein] A protein according to one embodiment of the present invention is a translation product of a gene described in the above section entitled "Vase Life Regulatory Gene" and has at least the activity of regulating the vase life of flowers. The vase life regulator protein may be isolated from a natural source or chemically synthesized. More specifically, the protein includes purified natural products, products of chemical synthesis procedures, and translation products produced by recombinant technology from prokaryotic or eukaryotic hosts (e.g., bacterial cells, yeast cells, higher plant cells, insect cells, and mammalian cells).

[0037] A flower life regulatory protein according to one embodiment of the present invention is encoded by a flower life regulatory gene according to one embodiment of the present invention and has an activity of regulating flower life in Dianthus plants. Typically, the flower life regulatory protein has an activity of negatively controlling flower life. Therefore, when the flower life regulatory protein is not present in a Dianthus plant, the flower life is longer than when the flower life regulatory protein is present, and when the flower life regulatory protein is present in a Dianthus plant, the flower life is shorter than when the flower life regulatory protein is not present.

[0038] The flower life regulating protein according to one embodiment 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 an activity to regulate the vase life of plants of the genus Dianthus; (C) A protein consisting of an amino acid sequence in which 31 or fewer 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 the activity of regulating the flower life of plants of the genus Dianthus.

[0039] The protein (A) is a protein encoded by the AP2 gene or a gene corresponding to the AP2 gene in plants of the genus Dianthus, and has the activity of regulating the vase life of plants of the genus Dianthus.

[0040] With respect to the protein (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] With respect to the protein (C) above, the number of substituted, deleted, added or inserted amino acids in the amino acid sequence of SEQ ID NO: 1 or 2 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, mutant proteins derived from carnation or homologous proteins derived from plants of the genus Dianthus other than carnation are included in the above categories of proteins (B) and (C). These mutant proteins and homologous proteins are proteins encoded by endogenous genes of plants of the genus Dianthus.

[0043] The vase life regulatory protein is a polypeptide formed by peptide bonds between amino acids, but may also contain structures other than polypeptides, such as, but not limited to, sugar chains and isoprenoid groups.

[0044] [Expression vectors, cells, and transformants] The present invention also includes, within its scope, an expression vector that deletes or suppresses the expression of a gene that regulates flower longevity according to one embodiment of the present invention, cells containing the expression vector, cells in which expression of a gene that regulates flower longevity has been deleted or suppressed, a transformant transformed with the expression vector, and a transformant in which expression of a gene that regulates flower longevity has been deleted or suppressed. The expression vector confers a trait of excellent flower longevity to a cell or an individual organism.

[0045] Host cells into which expression vectors can be introduced include, for example, 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 plants of the genus Dianthus. Examples of monocotyledonous plant cells include the Oc strain, which is a suspension culture cell of rice. Examples of animal cells include insect cells, amphibian cells, reptile cells, avian cells, fish cells, and mammalian cells.

[0046] The expression vector may contain a polynucleotide that mutates, deletes, or suppresses the function of a flower longevity regulatory gene, which is a gene originally contained in the transformed plant. An example of a polynucleotide that mutates, deletes, or suppresses the function of a flower longevity regulatory gene may be a polynucleotide in which the promoter sequence of the flower longevity regulatory gene has been mutated so as to delete or suppress expression of the flower longevity regulatory gene. Examples of promoter sequences for flower longevity regulatory genes include 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 Francesco, which serves as a reference, and the nucleotide sequence shown in SEQ ID NO: 7 represents the promoter sequence of the AP2 gene derived from Sandrosa, which has excellent flower longevity.

[0047] An expression vector may be designed appropriately depending on the host cell into which the vector will be introduced. That is, depending on the type of host cell, the vector may be designed by incorporating a polynucleotide having the above-described mutation into, for example, a plasmid, a phagemid, or a cosmid. The vector is preferably a pBI-, pPZP-, or pSMA-based vector, which can introduce a polynucleotide of interest into plant cells via Agrobacterium, and a binary vector is particularly preferred.

[0048] In the expression vector, elements (e.g., promoter) necessary for transcription of a polynucleotide that mutates, deletes, or suppresses the function of a gene are operably linked to the polynucleotide. Furthermore, the polynucleotide may be linked, as necessary, to a spacer, enhancer, selection marker, splicing signal, poly(A) addition signal, 5'-UTR sequence, and the like. The promoter is a DNA sequence that exhibits transcriptional activity in host cells and can be appropriately selected depending on the type of host; for example, an actin promoter is used.

[0049] The expression vector may further contain a selection marker, such as a drug resistance gene for ampicillin, kanamycin, tetracycline, chloramphenicol, neomycin, hygromycin, or spectinomycin.

[0050] The expression vector may further contain a selection marker, such as a drug resistance gene for ampicillin, kanamycin, tetracycline, chloramphenicol, neomycin, hygromycin, or spectinomycin.

[0051] Furthermore, the expression vector may be linked to a suitable tag sequence for protein purification or a suitable caryophyllus pacer sequence, if necessary.

[0052] [Method for determining the flower life of Dianthus plants] A method for determining the flower longevity of Dianthus plants (determination method) according to one embodiment of the present invention determines whether a Dianthus plant has excellent flower longevity. The determination method can be used to identify Dianthus plants with excellent flower longevity. The determination method determines the genotype of a region involved in the expression of a gene involved in regulating flower longevity, present in the genome of the Dianthus plant, thereby determining Dianthus plants based on their flower longevity. Note that in the determination method, the concept of flower longevity includes whether the flower longevity of a certain Dianthus plant individual is relatively long or short compared to other Dianthus plant individuals, as well as the degree of flower longevity, which indicates whether the flower longevity of a certain Dianthus plant individual is relatively long or short compared to other Dianthus plant individuals.

[0053] Dianthus plants can be candidate plants for breeding material or plants obtained through a breeding process. Candidate plants for breeding material include, for example, parent plants used in crossbreeding and plants used in molecular breeding using genetic engineering techniques. Furthermore, plants obtained through a breeding process include, for example, plants obtained by intraspecific hybridization of Dianthus plants, such as carnations or Dianthus japonica, and their progeny lines. Dianthus plants can also be intervarietal hybrids, such as hybrids between one carnation variety and another carnation variety, and their progeny lines.

[0054] Furthermore, the Dianthus plant may be a plant obtained by crossbreeding varieties known to have excellent flower longevity, and its progeny. Furthermore, the Dianthus plant may be a plant obtained by crossbreeding a variety known to have excellent flower longevity with a variety whose excellent flower longevity is unknown, and its progeny. Furthermore, the Dianthus plant may be a plant obtained by crossbreeding varieties whose excellent flower longevity is unknown, and its progeny. Furthermore, the Dianthus plant may be a plant obtained by crossbreeding a variety known to have excellent flower longevity with a variety whose excellent flower longevity is unknown, and its progeny. Furthermore, the Dianthus plant may be a plant obtained by crossbreeding individuals known to have excellent flower longevity with individuals whose excellent flower longevity is unknown, and its progeny.

[0055] Furthermore, the Dianthus plant may be a progeny plant of the carnation cultivar "Sandrosa." The carnation cultivar "Sandrosa" is an example of a cultivar with excellent vase life.

[0056] The determination method includes a detection step of detecting, in a Dianthus plant, the flower life regulatory gene according to one aspect of the present invention as a molecular marker for regulation of flower life in Dianthus plants.

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

[0058] In the detection step, a genotype that lacks or suppresses the expression of a gene that regulates flower longevity, or a mutation that lacks or suppresses the expression of a gene that regulates flower longevity, is detected as a molecular marker. Thus, if it is detected that the expression of a gene that regulates flower longevity is lacking or suppressed in a Dianthus plant, the Dianthus plant can be determined to have excellent flower longevity. Furthermore, if it is detected that the expression of a gene that regulates flower longevity is not lacking or suppressed in a Dianthus plant, the Dianthus plant can be determined to have short flower longevity.

[0059] The detection step may be performed by quantifying the expression level of a flower-life regulatory gene contained in the plant. For example, polynucleotides, such as RNA, are extracted from the plant, and the flower-life regulatory gene contained in the polynucleotides is quantified, for example, by real-time PCR. The lower the amount of polynucleotides of the flower-life regulatory gene, the better the flower-life can be determined to be, and the higher the amount of polynucleotides of the flower-life regulatory gene, the shorter the flower-life can be determined to be.

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

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

[0062] That is, the determination method includes a step of testing the bases (SNPs) themselves corresponding to the bases of the polynucleotides (A) to (F) above, or a consecutive polynucleotide containing such bases, as molecular markers related to the regulation of flower longevity. By using the bases corresponding to the bases of the polynucleotides (A) to (F) above as molecular markers, the presence or absence of mutations affecting the expression of the flower longevity regulatory gene can be detected. The "bases corresponding to the bases of the polynucleotides (A) to (F)" refer to SNP markers that can be identified as SNP markers described in this example. Furthermore, the "bases corresponding to the bases of the polynucleotides (A) to (F)" refer to bases in the gene corresponding to the flower longevity regulatory gene that have been identified as corresponding to the bases of the polynucleotides (A) to (F) by a homology search or other method. For example, the polynucleotides described in (ii) or (iii) above are examples of genes corresponding to the flower longevity regulatory gene.

[0063] Examples of molecular markers include SNP markers, AFLP (amplified fragment length polymorphism) markers, RFLP markers, microsatellite markers, SCAR markers, and CAPS markers.

[0064] The method for determining the vase life of a Dianthus plant using the above-mentioned molecular markers is not particularly limited, and for example, a known SNP analysis method for detecting SNPs can be used, including a method for SNP analysis by detecting SNPs in PCR-amplified fragments of a Dianthus plant specimen.

[0065] The determination method may involve amplifying a region in the DNA of a Dianthus plant using a primer set that amplifies a region containing a molecular marker. One example of such a primer set is a primer set that amplifies a region containing at least one of the polynucleotides (A) to (F). Examples of such primer sets include primer sets 1 to 6, preferably primer sets 1 to 5, used in the Examples described below.

[0066] That is, one embodiment of the primer set used to amplify the region containing the molecular marker in the detection step of the determination method 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 base sequence shown in SEQ ID NO: 18 and an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 19.

[0067] In addition, modified primers comprising a base sequence having 90% or more sequence identity to the base sequences of the oligonucleotides constituting each primer in the primer set, and having modified base sequences obtained by modifying some bases in the base sequence, are also included within the scope of the primers of the present invention. Furthermore, 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 to the base sequence shown in any of SEQ ID NOS: 8 to 19 in each of the above primers. In principle, a modified primer is considered equivalent to each of the above primers if it can hybridize with the complementary DNA strand of each of the above primers. Furthermore, this modified primer considered to be equivalent may be a primer that functions as a primer that hybridizes with the complementary DNA strand of the original primer, and is composed of an oligonucleotide having a modified base sequence in which, for example, 0 to 3, 0 to 2, 1 to 3, or 1 or 2 bases have been added, deleted, inserted, or substituted in the base sequence of the oligonucleotides constituting each original primer.

[0068] The region in the DNA of a Dianthus plant specimen can be amplified by polymerase chain reaction (PCR) using DNA extracted from the Dianthus plant specimen as a template and primers that amplify the region containing the SNP. The base (genotype) of the SNP in the resulting amplified fragment is then determined, and the vase life of the Dianthus plant is judged based on data showing the relationship between the determined base (genotype) and the vase life of the Dianthus plant.

[0069] The primer set used in PCR is not particularly limited as long as it can amplify a DNA fragment containing the target SNP. The primer set may be designed to shorten the length of the amplified fragment. For example, the primer set is designed so that the length of the primer-amplified fragment is preferably 700 bases (bases) or less, 200 b or less, 150 b or less, 120 b or less, or 100 b 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 b or more, 16 b or more, 17 b or more, 18 b or more, or 19 b or more, or 50 b or less, 40 b or less, or 30 b or less.

[0070] PCR in SNP analysis may be either singleplex PCR, which amplifies DNA fragments in a reaction system containing a single primer set, or multiplex PCR, which amplifies genes in a reaction system containing multiple primer sets. In the case of multiplex PCR, primer sets labeled with fluorescent substances with different wavelengths (e.g., NED, 6-FAM, VIC, PET) may be mixed.

[0071] PCR reaction conditions can be appropriately set depending on the type of DNA polymerase and PCR instrument used, the length of the amplified fragment, and other factors. Cycling conditions include a three-step PCR method, in which one cycle consists of three steps: denaturation, annealing, and extension; and a two-step PCR method, in which one cycle consists of two steps: denaturation, annealing, and extension. An example of PCR reaction conditions is 90-100°C for 40-60 seconds (e.g., 95°C for 50 seconds), followed by 30-60 cycles (e.g., 40 cycles) of 90-100°C (e.g., 95°C) for 5 seconds, annealing for 10-20 seconds (e.g., 15 seconds), and 65-80°C for 10-30 seconds (e.g., 72°C for 20 seconds). The annealing temperature can be gradually decreased from an initial annealing temperature of 60-70°C (e.g., 66°C) to a final annealing temperature of 50-60°C (e.g., 56°C) every predetermined number of cycles. Depending on the state of the template DNA, PCR reaction conditions may be adjusted to stably detect SNPs.

[0072] As PCR for SNP analysis, real-time PCR such as TaqMan®-PCR or Tm-shift genotyping (Fukuoka et al., Breed Sci 58: 461-464, 2008), which amplifies and identifies SNP markers by PCR, may be used. That is, a TaqMan® probe may be further used to detect SNPs contained in the amplified fragments amplified using a primer set. The use of real-time PCR can provide a high-throughput determination method. SNPs in the amplified fragments amplified by PCR may be identified by analyzing the nucleotide sequence of the amplified fragments using an automated DNA sequencer or the like.

[0073] The method for extracting DNA to be amplified by PCR from a Dianthus plant specimen is not particularly limited, and known DNA extraction methods can be used. Alternatively, DNA may be extracted using a commercially available DNA extraction kit. Depending on the type of specimen and the amount of contaminants, appropriate pretreatment may be performed before the DNA extraction step. Furthermore, the DNA extracted from the specimen may be washed or purified as necessary for use as a template in the PCR reaction. Furthermore, the DNA extracted from the specimen may be digested with two restriction enzymes, and the resulting restriction enzyme fragments may be amplified by PCR.

[0074] Furthermore, in the method for determining the vase life of a Dianthus plant, genetic polymorphisms that are 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 has excellent vase life using molecular markers, and therefore, Dianthus plants with excellent vase life and their progeny lines can be selected based on the determination results.

[0076] [Dianthus plants with long-lasting flowers] A plant according to one aspect of the present invention is a Dianthus plant with long flower life, in which the expression of a gene regulating flower life is deleted or suppressed. Due to the deletion or suppression of the function of the gene regulating flower life, the Dianthus plant with long flower life has superior flower life compared to Dianthus plants in which the expression of the gene regulating flower life is not deleted or suppressed.

[0077] In Dianthus plants with long flower life, the loss or suppression of expression of a gene that regulates flower life has occurred as a result of known methods for deleting the gene itself or for deleting or suppressing gene expression, such as spontaneous mutation, mutagen treatment, gene recombination, genome editing, or gene knockout. Spontaneous mutation of the gene generally occurs due to replication errors and genetic damage. Such damage can be caused by exposure to known naturally occurring mutagens (e.g., radiation or ultraviolet light). Mutagen treatment of the gene can be carried out by artificially applying the mutagen to the plant (and, if necessary, in combination with suppression of gene repair function). Examples of mutagens include chemical agents such as ethyl methanesulfonate (EMS), sodium azide, ethidium bromide, and nitrous acid, as well as actinic rays such as gamma rays, heavy ion beams, X-rays, neutron rays, and UV rays. These methods are preferred because they do not require the addition of exogenous factors to the plant. The gene recombination can be carried out by homologously recombining part or all of the target gene with a recombination sequence according to known gene recombination techniques.

[0078] One embodiment of the present invention provides a Dianthus plant with improved flower longevity, for example, by identifying Dianthus plants with improved flower longevity from plants obtained by intraspecific hybridization of Dianthus plants and their progeny using molecular markers related to the regulation of flower longevity in Dianthus plants. Dianthus plants with improved flower longevity that have been genetically engineered to contain such molecular markers are also included in the scope of the present invention. Furthermore, Dianthus plants with improved flower longevity that have been obtained by genome editing to eliminate or suppress the expression of genes that regulate flower longevity are also included in the scope of the present invention.

[0079] Dianthus plants can be candidate plants for breeding material or plants obtained through a breeding process. Candidate plants for breeding material include, for example, parent plants used in crossbreeding and plants used in molecular breeding using genetic engineering techniques. Furthermore, plants obtained through a breeding process include, for example, plants obtained by intrageneric hybridization of Dianthus plants, plants obtained by intraspecific hybridization of carnations, and their progeny lines.

[0080] A Dianthus plant having good flower-life properties according to one embodiment of the present invention includes a Dianthus plant determined to have excellent flower-life properties by a determination method according to one embodiment of the present invention, and a plant obtained by a Dianthus plant having good flower-life properties according to one embodiment of the present invention.

[0081] [Method for producing Dianthus plants with long-lasting flowers] A method for producing a Dianthus plant with long flowering time 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 a Dianthus plant's flowering time regulatory gene according to one aspect of the present invention. According to the production method according to one aspect of the present invention, a Dianthus plant with excellent flowering time can be produced.

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

[0083] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of 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. [Example]

[0084] [The AP2 gene's function in controlling flower longevity] Genes related to flower longevity were extracted using a microarray constructed from a 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 senescence process was investigated using real-time PCR. The genomic sequences of genes whose expression increases during senescence were examined, and genes with different genomic sequences (mutations) between the control Francesco and the long-lasting flower cultivar Sandrosa were selected. From these results, the AP2 gene was identified. The results are shown in Figure 1. Figure 1 shows the results of investigating AP2 gene expression in cultivars with different flower longevity.

[0085] [Comparison of the genomic sequence of the carnation AP2 gene] The genome analysis software IGV was used to compare the AP2 gene genomic sequences of carnation cultivars. The AP2 gene genomic sequence of Dianthus spp. was also compared. The results are shown in Figure 2. Figure 2 is a schematic diagram showing the comparison of the AP2 gene genomic sequences of carnation cultivars. The top nine rows in Figure 2 show the comparison results for carnation cultivars, and the bottom row shows the comparison results for Dianthus spp. In Figure 2, light gray lines indicate regions that match the sequence of the control cultivar Francesco. Dark gray lines indicate regions where the nucleotide sequence differs from Francesco. The top two cultivars in Figure 2 are cultivars with standard vase life and their sequences are nearly identical to Francesco. On the other hand, the bottom seven cultivars are cultivars with long vase life and have many regions where the nucleotide sequence differs from Francesco, and these variations are conserved among cultivars. The Dianthus sequence had many regions that matched Francesco.

[0086] [Comparison of genome sequences and evaluation of identity] Genomic DNA was extracted from the control cultivars, Francesco, which has a standard flowering period, and Sand Rosa, which has an excellent flowering period, and genome sequencing was performed using a next-generation sequencer. The genome sequence encoding the AP2 gene identified from the results of expression analysis and its surrounding sequences were extracted, and the genome sequences between the cultivars were compared using the analysis software IGV and GENETYX.

[0087] The promoter sequence of the AP2 gene was determined by genomic PCR. First, a forward primer was designed upstream of the AP2 gene based on the published genome sequence of Francesco. A reverse primer designed to the CDS sequence was used in genomic PCR. The genomes of the control cultivar Francesco and the long-lasting cultivar Sand Rosa were extracted and used as templates for genomic PCR. The PCR-amplified DNA was cloned and the sequence confirmed. Figure 3 shows the results of a comparison of the promoter sequence upstream of the AP2 gene. As shown in Figure 3, regions of the promoter sequence with different nucleotide sequences existed between the cultivars. Figure 4 also shows the results of a comparison of the amino acid sequences encoded by the AP2 gene in Francesco and Miracle Rouge. As shown in Figure 4, there were also regions of amino acid sequences encoded by the AP2 gene that differed between Francesco and Miracle Rouge.

[0088] [Evaluation of flower life of carnation varieties] The vase life of each carnation cultivar was evaluated as follows. Carnations grown in a greenhouse were harvested on the day their flowers fully expanded and placed in distilled water. They were then brought into a testing room with a room temperature of 23°C, relative humidity of 70%, light intensity, and a 12-hour day length. The flowers were visually inspected daily for wilting. The number of days from harvest to wilting was counted as vase life, and the cultivars were judged as having excellent vase life (good vase life) or standard vase life. Wilting was defined as when the petals curled inward and lost their ornamental value (in-rolling), or when the petals browned and lost their ornamental value. For cut flowers, a vase life of approximately 7 days was considered standard, and a vase life of 10 days or more was considered good vase life. The results are shown in Table 1.

[0089] [Table 1]

[0090] Next, the results of the flower-life assessment for each variety were linked to the genotype 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 below.

[0091] [Table 2]

[0092] As shown in Tables 1 and 2, the four cultivars judged to have standard vase life were wild-type cultivars with the same AP2 gene genotype as the control cultivar Francesco. The eight cultivars judged to have good vase life were mutant cultivars with AP2 gene genotypes different from Francesco.

[0093] [Determining flower life using DNA markers] Based on the sequence comparison results and the relationship between vase life and AP2 gene genotype, DNA markers were designed to identify the AP2 gene genotype and determine vase life. The designed DNA markers were then used to determine the vase life of carnation cultivars. The vase life of carnation cultivars was determined by comparing the amplification products obtained by amplifying the DNA marker region. Primer sets 1 to 6 shown in Table 3 were used in the reaction systems shown in Table 3.

[0094] [Table 3]

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

[0096] 50-100 mg of young leaves from each carnation cultivar were sampled before they fully expanded. After crushing them with liquid nitrogen, genomic DNA was extracted using the QIAGEN DNeasy Plant Mini kit according to the protocol. The resulting genomic DNA was diluted 2-fold with sterile water and used in PCR reactions.

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

[0098] (ExTaq-based system) In a system using Takara Bio's ExTaq, 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 were used, and after a reaction at 94°C for 5 minutes, 35 to 40 cycles were performed, including 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.

[0099] [Table 4]

[0100] After the reaction was completed, the bands were confirmed by electrophoresis on a 1% agarose gel. The results are shown in Figures 5 and 6. In Figures 5 and 6, DW represents the negative control to which no genomic DNA was added. As shown in Figure 5, when primer set 1 was used, amplification products were obtained only from Miracle Rouge and Sand Rosa, which have long-lasting flower life. Furthermore, as shown in Figure 6, when primer set 2 was used, amplification products were obtained only from Francesco and White Sim, which have standard flower life. It was demonstrated that the Direct Marker method using the primer set of primers 1 and 2 can distinguish plants with long-lasting flower life.

[0101] (System using KOD-one) In a system using Toyobo KOD-one, 35 to 40 cycles of 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 using primer set 3 consisting of primers 3F and 3R, primer set 4 consisting of primers 4F and 4R, or primer set 6 consisting of primers 6F and 6R shown in Table 3, and the PCR reaction solution shown in Table 5.

[0102] [Table 5]

[0103] After the reaction was completed, the bands were confirmed by electrophoresis on a 2% agarose gel. The results are shown in Figures 7 to 9. As shown in Figure 7, when primer set 3 was used, amplification products were obtained only for Francesco and White Sim, which have standard vase life. Similarly, as shown in Figure 8, when primer set 4 was used, amplification products were obtained only for Francesco and White Sim, which have standard vase life. Furthermore, as shown in Figure 9, when primer set 6 was used, the bands at which amplification products were detected differed between Francesco and White Sim, which have standard vase life, and Miracle Rouge and Sand Rosa, which have long vase life. This demonstrates that the Direct Marker method using the primer set of primers 3, 4, and 6 can distinguish plants with long vase life.

[0104] (KOD-one and restriction enzyme system) In the system using KOD-one and restriction enzymes, PCR was performed using primer set 5 consisting of primer 5F and primer 5R shown in Table 3 in the system using KOD-one described above, followed by digestion of the amplified product by restriction enzyme treatment using the enzyme treatment reaction solution shown in Table 6. This resulted in fragmentation of the genomic DNA from the line with good flower life.

[0105] [Table 6]

[0106] The reaction was carried out at 37°C for 2 hours. After the reaction was completed, the bands were confirmed by electrophoresis on a 2% agarose gel. The results are shown in Figure 10. As shown in Figure 10, the positions of the bands detected for Miracle Rouge and Sand Rosa, which have long-lasting flower life, were different from those for Francesco and White Sim, which have standard flower life. The CAPS method using primer 5 made it possible to distinguish between control plants with standard flower life and plants with long-lasting flower life.

[0107] [Production of recombinant carnations] (Plant Adjustment) Axillary buds of the carnation cultivar "Ariel" were harvested, sterilized in 70% ethanol, then sterilized in hypochlorite, and planted on sterile medium to produce axenic seedlings. Murashige-Skoog (MS) medium or 1 / 2 MS medium was used for the culture.

[0108] MS medium was prepared by mixing one packet of Murashige-Skoog medium mixed salts (Wako Pure Chemical Industries), 20 g of sucrose, and 1 mL of MS vitamins (×1000). The pH was adjusted to 5.6–5.8, and the mixture was diluted to 1 L. For solid medium, 2 g / L of gellan gum was added. After mixing, the mixture was sterilized in an autoclave.

[0109] 1 / 2 MS medium was prepared by reducing the salt content of MS medium by half. It was prepared by mixing 1 / 2 a packet of Murashige-Skoog medium mixed salts (Wako Pure Chemical Industries), 20 g of sucrose, and 0.5 mL of MS vitamins (×1,000). The pH was adjusted to 5.6-5.8, and the volume was increased to 1 L. For solid medium, 2 g / L of gellan gum was added. After mixing, the mixture was sterilized in an autoclave.

[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 to make 100 mL, which was then dispensed in 1 mL aliquots and stored at -20°C.

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

[0112] (recombinant virus) Using the plasmid prepared using a method similar to that used for virus sap inoculation (Ohki, 2009), we mechanically inoculated seedlings of quinoa or Nicotiana benthamiana, the propagation hosts. Carborundum (600 mesh, Nacalai Tesque) was sprinkled evenly and thinly on the leaves of the propagation hosts, and 10 μL of a plasmid solution containing equal amounts of the vectors pEALSR1 and pEALSR2L5R5 was rubbed onto the entire surface of the leaves. The carborundum on the leaf surface was then washed off with sterilized water, and the plants were left to rest overnight in the dark before being grown in an incubator or a recombinant greenhouse.

[0113] After 2–3 weeks, when symptoms were observed on the upper leaves, new propagation hosts were prepared, and the infected leaves were crushed in a mortar and pestle with 2–3 volumes of inoculation buffer, followed by mechanical inoculation again. The infected upper leaves were harvested and stored at −80°C.

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

[0115] (Modification of Dianthus plants) Carnations and other plants of the Dianthus genus were inoculated using the viral RNA inoculation method using a particle gun as described in Reference 1. To prepare enough for 40 particle gun shots (RNA: 5 μg / gold particle), 8 mg of gold particles was weighed out and placed in a 1.5 ml tube, and 50 μl of sterilized water was added, mixed, and sonicated for at least 5 minutes.

[0116] 50 μl of RNA, 10 μl of 5M ammonium acetate, and 220 μl of isopropanol were added in small portions with gentle stirring. After all the components were added and stirred for a while, the mixture was left to stand at -20°C for at least 1 hour. The mixture was centrifuged at 800 × g or less to remove the supernatant, and the gold particles were washed four times with 1 ml of 99.5% ethanol. Finally, the gold particles were suspended in 2.4 ml of 99.5% ethanol.

[0117] A gold-coated tube for the Helios Gene Gun System (Bio-Rad) was placed in a tubing prep station and thoroughly dried by passing nitrogen gas through it. An ethanol solution containing gold particles was then poured into the tube, filling it uniformly, and the supernatant ethanol was removed. The gold particles were dried by rotating the tubing prep station and passing nitrogen gas through it. The gold-coated tube was removed from the tubing prep station and cut into cartridges using a tubing cutter.

[0118] Two to four shots per plant were inoculated using a Helios Gene Gun System at a helium pressure of 220 psi. As with the Agrobacterium method, the plants used for the experiments were regenerated from the shoot apex and tissue immediately below it, and then axenically cultured. After firing the particle gun, the plants were moistened by spraying with water using a spray bottle. The plants were then placed in a shaded area for one day while maintaining the humidity, and then cultured under standard conditions (25°C, 16-hour photoperiod).

[0119] The tips of shoots growing on the selective medium were broken off and transplanted into 1 / 2 MS medium or Hyponex medium containing no plant hormones. The tips were then cut off and placed in water for rooting. Once new roots were confirmed in the water-based rooting, the plants were planted in culture soil in a humid environment for acclimatization, and then transplanted into pots filled with commercially available horticultural soil while observing the plant's condition. Once the plants flowered, the flowers were harvested and evaluated for flower longevity. Flower longevity was assessed in the same manner as described above (Experimental method and results for evaluating flower longevity using markers). The results are shown in Table 7.

[0120] [Table 7]

[0121] As shown in Table 7, the recombinant plants in which the expression of the wild-type AP2 gene was suppressed showed improved vase life.

[0122] [Evaluation of vase life in carnation hybrids using DNA markers] Using the methods described above in "Evaluating the flower life of carnation cultivars" and "Distinguishing flower life using DNA markers," carnation hybrid lines were investigated for flower life duration and the genotypes of genes that regulate flower life. The genotypes of genes that regulate flower life were determined by the Direct Marker method using primer set 1 (DNA marker 1 (DNA marker that distinguishes lines with long flower life)), primer sets 2, 3, and 4 (DNA markers 2, 3, and 4 (DNA markers that distinguish lines with standard flower life)), and primer set 5 (DNA marker 5 (DNA marker that distinguishes lines with standard flower life from lines with long flower life)).

[0123] Table 8 shows the results of the vase life and DNA marker identification of the carnation hybrid lines. The minimum vase life of the 66 hybrid lines was 7 days and the maximum was 30 days. Of the 66 lines, 11 lines showed a band for DNA marker 1 (which distinguishes lines with long vase life). 63 lines showed bands for DNA markers 2, 3, and 4 (which distinguish lines with standard vase life). Furthermore, for DNA marker 5, only the band indicating a line with standard vase life was confirmed in 55 lines, only the band indicating a line with good vase life was confirmed in 3 lines, and both the band indicating a line with standard vase life and the band indicating a line with good vase life were confirmed in 8 lines. Based on this, it was determined that 3 lines were homozygous for the mutant gene for long vase life, 8 lines were heterozygous, and 55 lines were homozygous for the wild-type gene for standard vase life.

[0124] [Table 8]

[0125] JPEG0007779575000009.jpg232160

[0126] Next, the average vase life of these 66 lines was examined for each genotype identified by the markers described above. The results are shown in Table 9. The average vase life of all 66 lines was 15.4 days. The average vase life of the mutant gene homozygous lines, which exhibit good vase life, was 20.7 days, the average vase life of the heterozygous lines was 17.3 days, and the average vase life of the wild-type gene homozygous lines, which exhibited standard vase life, was 14.8 days. Thus, lines carrying the mutant gene marker for good vase life had significantly longer vase life than lines without the marker. These results demonstrate that DNA markers for vase life are useful for evaluating vase life.

[0127] [Table 9] [Industrial Applicability]

[0128] The present invention can be used in the fields of agriculture, plant breeding, etc.

Claims

1. A flower-life regulatory 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 vase 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 the nucleotide sequence shown in any one of SEQ ID NOs: 3 to 5; (b) a polynucleotide consisting of 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 vase life of plants of the genus Dianthus; (c) a polynucleotide consisting of a nucleotide sequence in which 85 or fewer 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 which exhibits a function equivalent to that of the polynucleotide of (a) above in regulating the vase life of plants of the genus Dianthus; The flower life regulatory gene according to claim 1, comprising:

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

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

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

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 containing 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 consisting of the base sequence shown in SEQ ID NO: 10 and an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 11; (f) a combination of an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 12 and an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 13; (g) a combination of an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 14 and an oligonucleotide consisting of 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 with long-lasting flowering, in which expression of the gene for controlling flowering time according to claim 1 or 2 is deleted or suppressed.

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

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