[go: up one dir, main page]

WO2025115990A1 - Plante de stévia ayant une teneur élevée en rébaudioside m - Google Patents

Plante de stévia ayant une teneur élevée en rébaudioside m Download PDF

Info

Publication number
WO2025115990A1
WO2025115990A1 PCT/JP2024/042255 JP2024042255W WO2025115990A1 WO 2025115990 A1 WO2025115990 A1 WO 2025115990A1 JP 2024042255 W JP2024042255 W JP 2024042255W WO 2025115990 A1 WO2025115990 A1 WO 2025115990A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
plant
content
present
rebm
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/JP2024/042255
Other languages
English (en)
Japanese (ja)
Inventor
正良 平井
一考 岩城
兼太郎 落合
克郎 宮川
絵美 岡田
真樹 小牧
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Suntory Holdings Ltd
Original Assignee
Suntory Holdings Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Suntory Holdings Ltd filed Critical Suntory Holdings Ltd
Publication of WO2025115990A1 publication Critical patent/WO2025115990A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/14—Asteraceae or Compositae, e.g. safflower, sunflower, artichoke or lettuce
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/20—Carbocyclic rings
    • C07H15/24—Condensed ring systems having three or more rings
    • C07H15/256—Polyterpene radicals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/04—Plant cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/56—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical directly bound to a condensed ring system having three or more carbocyclic rings, e.g. daunomycin, adriamycin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae

Definitions

  • the present invention relates to stevia plants that have a high content of useful steviol glycosides such as rebaudioside M, and screening methods for the same.
  • Patent Document 1 discloses a functional sweetener composition that contains vitamins, a high-intensity sweetener, and a sweetness-improving composition.
  • Steviol glycosides are known as the sweet components contained in stevia extract.
  • Stevia extract is mainly extracted and refined from stevia leaves.
  • Stevia is a perennial plant of the Asteraceae family native to Paraguay in South America, and its scientific name is Stevia Rebaudiana Bertoni.
  • Stevia contains components that are about 300 times sweeter than sugar, so it is cultivated to extract these sweet components and use them as a natural sweetener.
  • Various glycosides have been reported as steviol glycosides, including rebaudioside A (hereinafter, "rebaudioside” may be abbreviated as "Reb”), RebB, RebC, RebD, RebE, RebM, and RebN (Patent Document 2).
  • RebM cannot be obtained in large quantities from natural stevia plants, so there is a demand for efficient acquisition of it.
  • the present invention provides a stevia plant that contains useful steviol glycosides such as RebM at a higher content than wild-type stevia species, as well as a method for producing and screening the plant.
  • the present invention provides the following: [1] A stevia plant having at least one of the following genetic characteristics (1) to (5): (1) Homozygosity or heterozygosity for an allele in which the sequence corresponding to positions 150 to 151 of SEQ ID NO:1 is T. (2) Homozygosity or heterozygosity for an allele in which the base at the position corresponding to position 151 of SEQ ID NO:2 is TAGGTGGTGACACTGTAGC. (3) Homozygous or heterozygous for an allele in which the base at the position corresponding to position 151 of SEQ ID NO:3 is A.
  • a stevia plant having at least one of the following chemical characteristics: (i) to (v) (i) The rebaudioside (Reb) M content is 1.8% or more per unit mass of dried leaves. (ii) The difference between the RebM content and the stevioside (ST) content per unit mass of dried leaves is 0.9% or more. (iii) The difference between the RebM content and the RebE content per unit mass of dried leaves is 1.3% or more.
  • the ratio of the RebM content to the RebN content per unit mass of dry leaves, RebM/RebN, is 1.52 or more.
  • the ratio of the RebM content to the stevioside content per unit mass of dried leaves, RebM/stevioside, is 1.83 or more.
  • tissue, tissue culture or cell according to [5] above which is selected from an embryo, a meristematic cell, a pollen, a leaf, a root, a root tip, a petal, a protoplast, a leaf slice and a callus.
  • a method for producing a steviol glycoside-containing extract comprising a step of obtaining an extract from a plant body described in any one of [1] to [3] above, a dead tissue or dead cell described in [4] above, a seed, tissue, dried leaf, tissue culture or cell described in [5] above, or a tissue, tissue culture or cell described in [6] above.
  • a method for producing steviol glycoside comprising a step of purifying steviol glycoside from the extract described in [9] above.
  • a method for producing a food or beverage, a sweetener composition, a flavoring, or a pharmaceutical comprising the steps of providing an extract of a plant body described in any one of [1] to [3] above, an extract of a dead tissue or dead cell described in [4] above, an extract of a seed, tissue, dried leaf, tissue culture, or cell described in [5] above, an extract of a tissue, tissue culture, or cell described in [6] above, or an extract described in [9] above, and adding the extract to a raw material for the food or beverage, a sweetener composition, a flavoring, or a pharmaceutical.
  • a method for producing a food or beverage, a sweetener composition, a flavoring, or a pharmaceutical comprising the steps of purifying steviol glycoside from the extract described in [9] above, and adding the purified steviol glycoside to a raw material for the food or beverage, a sweetener composition, a flavoring, or a pharmaceutical.
  • the present invention makes it possible to obtain stevia plants that contain more useful steviol glycosides, to provide a means for producing such plants, to provide leaves from such plants, and to provide foods, beverages, etc. that contain useful steviol glycosides obtained from the leaves.
  • 1 is a diagram showing the positions of mutations related to genetic feature (1) in the base sequence of SEQ ID NO: 1.
  • the left side of the slash in parentheses is the wild-type sequence, and the right side is the mutant-type sequence.
  • 2 is a diagram showing the positions of mutations related to genetic feature (2) in the base sequence of SEQ ID NO: 2.
  • the left side of the slash in parentheses is the wild-type sequence, and the right side is the mutant-type sequence.
  • 3 is a diagram showing the positions of mutations related to genetic feature (3) in the base sequence of SEQ ID NO: 3.
  • the left side of the slash in parentheses is the wild-type sequence, and the right side is the mutant-type sequence.
  • FIG. 4 is a diagram showing the positions of mutations related to genetic feature (4) in the base sequence of SEQ ID NO: 4.
  • the left side of the slash in parentheses is the wild-type sequence, and the right side is the mutant-type sequence.
  • 5 is a diagram showing the positions of mutations related to genetic feature (5) in the base sequence of SEQ ID NO: 5.
  • the left side of the slash in parentheses is the wild-type sequence, and the right side is the mutant-type sequence.
  • FIG. 6 shows the results of an analysis of whether or not a Stevia plant has the genetic characteristic (2′), (4′), or (5).
  • Plant of the Present Invention 1-1 Stevia plant having at least one of the genetic characteristics (1) to (5)
  • the present invention provides a stevia plant having at least one of the genetic characteristics (1) to (5) below (hereinafter, sometimes referred to as "plant A of the present invention” or “stevia plant A of the present invention”).
  • plant A of the present invention or “stevia plant A of the present invention”
  • Homozygosity or heterozygosity for an allele in which the base at the position corresponding to position 151 of SEQ ID NO:2 is TAGGTGGTGACACTGTAGC.
  • genetic characteristics (1)-(4) are heterozygous as follows: (1') Heterozygosity for an allele in which the sequence corresponding to positions 150 to 151 of SEQ ID NO:1 is T. (2') Heterozygosity for an allele in which the base at the position corresponding to position 151 of SEQ ID NO:2 is TAGGTGGTGACACTGTAGC. (3') Heterozygosity for an allele in which the base at the position corresponding to position 151 of SEQ ID NO:3 is A. (4') Heterozygosity for an allele in which the base at the position corresponding to position 151 of SEQ ID NO:4 is A.
  • a position/portion corresponding to means, if a sequence identical to a reference sequence (e.g., SEQ ID NOs: 1 to 5, etc.) exists in the genome, a position/portion in that sequence present in the genome (e.g., positions 150 to 151, position 151, etc.), and, if a sequence identical to the reference sequence does not exist in the genome, means a position/portion in a sequence in the genome that corresponds to the position/portion in the reference sequence.
  • a reference sequence e.g., SEQ ID NOs: 1 to 5, etc.
  • Whether a sequence identical to or corresponding to a reference sequence exists in the genome can be determined, for example, by amplifying the genomic DNA of the target stevia plant with primers that can amplify the reference sequence by PCR, sequencing the amplified product, and performing alignment analysis of the obtained sequence with the reference sequence.
  • Non-limiting examples of sequences corresponding to the reference sequence include base sequences having sequence identity of 60% or more, 70% or more, 75% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 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, 98.1% or more, 98.4% or more, 98.7% or more, 99% or more, 99.2% or more, 99.5% or more, or 99.8% or more.
  • the position/portion corresponding to the position/portion in the reference sequence in the genome can be determined in consideration of the base sequence before and after the position/portion in the reference sequence. For example, by performing alignment analysis between a reference sequence and a sequence in the genome that corresponds to the reference sequence, it is possible to determine the position/portion in the sequence in the genome that corresponds to the position/portion in the reference sequence.
  • the "portion corresponding to positions 150-151 of SEQ ID NO:1" of genetic characteristic (1) of the present invention is the 150-151 position from the 5' side of the portion in the genome consisting of the same nucleotide sequence as SEQ ID NO:1.
  • the genome of a stevia plant has a portion consisting of a nucleotide sequence that is not identical to SEQ ID NO:1 but corresponds to it, the genome does not have a portion consisting of the same nucleotide sequence as SEQ ID NO:1, so the "portion corresponding to positions 150-151 of SEQ ID NO:1" does not necessarily correspond to positions 150-151 from the 5' side of the portion corresponding to SEQ ID NO:1.
  • the "portion corresponding to positions 150-151 of SEQ ID NO:1" in the genome of such a stevia plant can be specified.
  • positions selected from the group consisting of (1) the portion corresponding to positions 150 to 151 of SEQ ID NO:1, (2) the position corresponding to position 151 of SEQ ID NO:2, (3) the position corresponding to position 151 of SEQ ID NO:3, (4) the position corresponding to position 151 of SEQ ID NO:4, and (5) the position corresponding to position 151 of SEQ ID NO:5 may be collectively referred to as the "polymorphic site of the present invention” or the "mutation site of the present invention.”
  • each of the positions/portions (1) to (5) above may be referred to as the "polymorphic site (1),” the "polymorphic site (2),” or the “mutation site (1),” “mutation site (2),” etc.
  • a sequence corresponding to SEQ ID NO:1 refers to a portion of a base sequence that has sequence identity of 60% or more, 70% or more, 75% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 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, 98.1% or more, 98.4% or more, 98.7% or more, 99% or more, 99.2% or more, 99.5% or more, or 99.8% or more to the base sequence of SEQ ID NO:1.
  • the "portion consisting of a base sequence corresponding to SEQ ID NO:1" includes a portion of the genome of a Stevia plant that can be amplified by PCR using a forward primer that hybridizes to a complementary sequence of a portion from positions 1 to 149 from the 5' end of SEQ ID NO:1 (i.e., from the 5' end of SEQ ID NO:1 to the base one base 5' end of the mutation site (1)) and a reverse primer that hybridizes to a portion from positions 1 to 150 from the 3' end of SEQ ID NO:1 (i.e., from the 3' end of SEQ ID NO:1 to the base one base 3' end of the mutation site (1)).
  • the genetic characteristic (1) of the present invention has been described as an example here, but the same applies to the genetic characteristics (2) to (5) of the present invention.
  • the "portion consisting of a nucleotide sequence corresponding to SEQ ID NO:1" includes, for example, a portion of the genome of a Stevia plant that can be amplified by PCR using a forward primer containing the nucleotide sequence of SEQ ID NO:6 and a reverse primer containing the nucleotide sequence of SEQ ID NO:7.
  • the "portion consisting of a nucleotide sequence corresponding to SEQ ID NO:2" includes, for example, a portion of the genome of a stevia plant that can be amplified by PCR using a forward primer containing the nucleotide sequence of SEQ ID NO:8 and a reverse primer containing the nucleotide sequence of SEQ ID NO:9.
  • the "portion consisting of a nucleotide sequence corresponding to SEQ ID NO: 4" includes, for example, a portion of the genome of a Stevia plant that can be amplified by PCR using a forward primer containing the nucleotide sequence of SEQ ID NO: 10 and a reverse primer containing the nucleotide sequence of SEQ ID NO: 11.
  • the "portion consisting of a nucleotide sequence corresponding to SEQ ID NO:5" includes, for example, a portion of the genome of a stevia plant that can be amplified by PCR using a forward primer containing the nucleotide sequence of SEQ ID NO:12 and a reverse primer containing the nucleotide sequence of SEQ ID NO:13.
  • the "allele in which the sequence corresponding to positions 150 to 151 of SEQ ID NO: 1 is T” comprises the base sequence of SEQ ID NO: 14, 15, 16, or 17.
  • the "allele in which the base at the position corresponding to position 151 of SEQ ID NO: 2 is TAGGTGGTGACACTGTAGC” comprises the base sequence of SEQ ID NO: 18, 19, 20, or 21.
  • the "allele in which the base at the position corresponding to position 151 of SEQ ID NO: 3 is A" includes the base sequence of SEQ ID NO: 22, 23, 24, or 25.
  • the "allele in which the base at the position corresponding to position 151 of SEQ ID NO:4 is A” includes the base sequence of SEQ ID NO:26, 27, 28, or 29.
  • the "allele in which the base at the position corresponding to position 151 of SEQ ID NO:5 is A” includes the base sequence of SEQ ID NO:30, 31, 32, or 33.
  • mutations selected from the group consisting of (1) a TG to T mutation at a portion corresponding to positions 150 to 151 of SEQ ID NO:1, (2) a T to TAGGTGGTGACACTGTAGC mutation at a position corresponding to position 151 of SEQ ID NO:2, (3) a G to A mutation at a position corresponding to position 151 of SEQ ID NO:3, (4) a T to A mutation at a position corresponding to position 151 of SEQ ID NO:4, and (5) a T to A mutation at a position corresponding to position 151 of SEQ ID NO:5 may be collectively referred to as "polymorphisms of the present invention” or “mutations of the present invention.”
  • each of the above mutations (1) to (5) may be referred to as "polymorphism (1) of the present invention,” “polymorphism (2) of the present invention,” “mutation (1) of the present invention,” “mutation (2) of the present invention,” etc.
  • the above genetic characteristics can be determined using PCR, TaqMan PCR, sequencing, microarray, Invader, TILLING, RAD (random amplified polymorphic DNA), restriction fragment length polymorphism (RFLP), PCR-SSCP, AFLP (amplified fragment length polymorphism), SSLP (simple sequence length polymorphism), CAPS (cleaved amplified polymorphic sequence), dCAPS (derived cleaved amplified polymorphic sequence), allele-specific oligonucleotide (ASO), ARMS, and denaturing gradient gel electrophoresis.
  • RAD random amplified polymorphic DNA
  • RFLP restriction fragment length polymorphism
  • PCR-SSCP PCR-SSCP
  • AFLP amplified fragment length polymorphism
  • SSLP simple sequence length polymorphism
  • CAPS cleaved amplified polymorphic sequence
  • dCAPS derived cleaved amplified polymorphic sequence
  • Detection can be performed by, but is not limited to, the following methods: dynamic (DGGE) method, CCM (chemical cleavage of mismatch) method, DOL method, MALDI-TOF/MS method, TDI method, padlock probe method, molecular beacon method, DASH (dynamic allele specific hybridization) method, UCAN method, ECA method, PINPOINT method, PROBE (primer oligo base extension) method, VSET (very short extension) method, Survivor assay, Sniper assay, Luminex assay, GOOD method, LCx method, SNaPshot method, Mass ARRAY method, pyrosequencing method, SNP-IT method, melting curve analysis method, etc.
  • the genetic characteristic of the present invention can be detected by the dCAPS method using the following combination of primer sets and restriction enzymes.
  • a candidate plant has a mutation (1')
  • PCR amplification is performed on the genomic DNA of the candidate plant using a forward primer having the nucleotide sequence shown in SEQ ID NO: 34 and a reverse primer having the nucleotide sequence shown in SEQ ID NO: 35, and the resulting PCR product (about 144 bp long, for example, SEQ ID NO: 36) is treated with the restriction enzyme Hyp188I, but only a band about 144 bp long (for example, SEQ ID NO: 36) is obtained.
  • PCR amplification is performed in the same manner as above, and the resulting PCR product (about 145 bp long, for example, SEQ ID NO: 37) is treated with the restriction enzyme Hyp188I, a band about 117 bp long (for example, SEQ ID NO: 38) and a band about 28 bp long (for example, SEQ ID NO: 39) are obtained. Therefore, when a band about 144 bp long is observed by the above dCAPS method, it can be determined that the plant has the genetic characteristic (1') of the present invention.
  • PCR amplification is performed on the genomic DNA of the candidate plant using a forward primer having the base sequence shown in SEQ ID NO: 40 and a reverse primer having the base sequence shown in SEQ ID NO: 41, and the resulting PCR product (about 103 bp long, for example, SEQ ID NO: 42) is treated with the restriction enzyme NdeI, resulting in an about 78 bp long band (for example, SEQ ID NO: 43) and an about 25 bp long band (for example, SEQ ID NO: 44).
  • PCR amplification is performed in the same manner as above, and the resulting PCR product (about 103 bp long, for example, SEQ ID NO: 45) is treated with the restriction enzyme NdeI, only an about 103 bp long band (for example, SEQ ID NO: 45) is obtained. Therefore, when the above dCAPS method is used to confirm an about 78 bp long band and/or an about 25 bp long band derived from the decomposition product and an about 103 bp long band derived from the non-decomposition product, it can be determined that the plant has the genetic characteristic (4') of the present invention. An example of genetic feature (4') detected using the above method is shown in Figure 6.
  • PCR amplification is performed on the genomic DNA of the candidate plant using a forward primer having the base sequence shown in SEQ ID NO: 46 and a reverse primer having the base sequence shown in SEQ ID NO: 47, and the resulting PCR product (about 161 bp long, for example, SEQ ID NO: 48) is treated with the restriction enzyme MfeI, resulting in an about 130 bp long band (for example, SEQ ID NO: 49) and an about 31 bp long band (for example, SEQ ID NO: 50).
  • PCR amplification is performed on the genomic DNA of the candidate plant using a forward primer having the base sequence shown in SEQ ID NO: 52 and a reverse primer having the base sequence shown in SEQ ID NO: 53, a PCR product (about 206 bp long, for example, SEQ ID NO: 54) is obtained.
  • PCR amplification is performed in the same manner as above, a PCR product (about 188 bp long, for example, SEQ ID NO: 55) is obtained.
  • plant A of the present invention has at least one characteristic selected from RebM content, the ratio of RebM content to RebN content (RebM/RebN), the ratio of RebM content to ST content (RebM/ST), RebM-ST (the difference between RebM content and ST content; the same applies below), and RebM-RebE, higher than a stevia plant not having genetic characteristics (1'), (2'), (4'), and (5) (hereinafter sometimes referred to as a control plant).
  • “Higher than a control plant” means, for example, that when grown under the same cultivation conditions, the content (e.g., content in dried leaves), content ratio or content difference of the above-mentioned component is higher, preferably statistically significantly higher, than that of a control plant.
  • the content, content ratio, or content difference of the above components in the dried leaves is more than 1-fold, about 1.1-fold or more, about 1.2-fold or more, about 1.3-fold or more, about 1.4-fold or more, about 1.5-fold or more, about 1.6-fold or more, about 1.7-fold or more, about 1.8-fold or more, about 1.9-fold or more, about 2-fold or more, about 2.1-fold or more, about 2.2-fold or more, about 2.3-fold or more, about 2.4-fold or more, about 2.5-fold or more, about 2.6-fold or more, about 2.7-fold or more, about 2.8-fold or more, about 2.9-fold or more, about 3-fold or more, about 3.1-fold or more, about 3.2-fold or more, about 3.3-fold or more, about 3.4-fold or more, about 3.5-fold or more, about 3.6-fold or more of the content, content ratio, or content difference in the control plant.
  • the average value of the former is greater than the average value of the latter
  • the P value of the two-sided test is less than 0.05, preferably less than 0.01, and particularly preferably less than 0.005.
  • the contents, etc. to be compared may be the average values of multiple individuals.
  • Total Steviol Glycoside is a collective term for measurable steviol glycosides, not including unknown steviol glycosides or steviol glycosides present in amounts below the detection limit.
  • TSG is any combination of two or more selected from the group consisting of RebA, RebB, RebC, RebD, RebE, RebF, RebG, RebI, RebJ, RebK, RebM, RebN, RebO, RebQ, RebR, dulcoside A, rubusoside, steviol monoside, steviolbioside, and stevioside.
  • TSG consists of RebA, RebB, RebC, RebD, RebE, RebF, RebI, RebJ, RebM, RebN, and stevioside.
  • plant A of the present invention may have a plurality of the above genetic characteristics (1) to (5).
  • the number of genetic characteristics may be any of 2 to 5 types, i.e., 2, 3, 4, or 5 types.
  • plant A of the present invention has genetic characteristics (1'), (2'), (4'), and (5).
  • the present invention provides a stevia plant having at least one of chemical characteristics (i) to (v) (hereinafter, sometimes collectively referred to as "plant B of the present invention” or “Stevia plant B of the present invention”).
  • plant B of the present invention or “Stevia plant B of the present invention”
  • the RebM content is 1.8% or more per unit mass of dry leaves.
  • the difference between the RebM content and the stevioside content per unit mass of dried leaves is 0.9% or more.
  • the difference between the RebM content and the RebE content per unit mass of dried leaves is 1.3% or more.
  • the ratio of the RebM content to the RebN content per unit mass of dry leaves, RebM/RebN, is 1.52 or more.
  • the ratio of the RebM content to the stevioside content per unit mass of dried leaves, RebM/steviol, is 1.83 or more.
  • the content of RebM is 1.8% or more per unit mass of the dried leaves means, for example, that a given mass of dried leaves (e.g., 50 mg) contains RebM at a ratio of 1.8% by mass or more (e.g., 0.9 mg or more).
  • the ratio of RebM per unit mass of the dried leaves in this embodiment is not limited and may be, for example, 1.8% or more, 1.9% or more, 1.95% or more, 2.0% or more, 2.1% or more, 2.2% or more, 2.3% or more, 2.4% or more, 2.5% or more, etc.
  • the upper limit of the ratio of RebM per unit mass of the dried leaves is not particularly limited and may be, for example, 10%, 8%, 7%, 6%, 5%, 4%, 3%, 2.5%, etc.
  • dried leaves refers to fresh leaves of the Stevia plant of the present invention that have been dried to reduce the moisture content to 3 to 4% by weight.
  • the difference between the RebM content and the stevioside content is 0.9% or more per unit mass of dried leaves means, for example, that the difference between the amount of RebM and the amount of stevioside contained in a given mass of dried leaves (e.g., 50 mg) is 0.9% or more by mass.
  • the difference between the RebM content and the stevioside content per unit mass of dried leaves is not limited, and may be, for example, 0.9% or more, 1.0% or more, 1.1% or more, 1.2% or more, 1.3% or more, 1.4% or more, 1.5% or more, 1.6% or more, 1.7% or more, 1.8% or more, 1.9% or more, 2.0% or more, etc.
  • the upper limit of the difference between the RebM content and the stevioside content per unit mass of dried leaves is not particularly limited, and may be, for example, 5%, 4%, 3%, 2.5%, 2%, etc.
  • the difference between the RebM content and the RebE content is 1.3% or more per unit mass of the dried leaves means, for example, that the difference between the amount of RebM and the amount of RebE contained in a given mass of dried leaves (e.g., 50 mg) is 1.3% or more by mass.
  • the difference between the amount of RebM and the amount of RebE per unit mass of the dried leaves is not limited and may be, for example, 1.3% or more, 1.4% or more, 1.5% or more, 1.6% or more, 1.7% or more, 1.8% or more, 1.9% or more, 2.0% or more, 2.1% or more, 2.2% or more, 2.3% or more, 2.4% or more, etc.
  • the upper limit of the difference between the RebM content and the stevioside content per unit mass of the dried leaves is not particularly limited and may be, for example, 5%, 4%, 3%, 2.5%, 2%, etc.
  • the ratio of RebM content to RebN content, RebM/RebN is 1.52 or more per unit mass of dried leaves means, for example, that the ratio of RebM to RebN contained in a given mass of dried leaves (e.g., 50 mg) is 1.52 or more.
  • RebM/RebN per unit mass of dried leaves is not limited and may be, for example, 1.52 or more, 1.6 or more, 1.7 or more, 1.75 or more, 1.8 or more, 1.83 or more, 1.85 or more, 1.90 or more, 1.95 or more, 2.0 or more, 2.1 or more, etc.
  • the upper limit of the ratio of RebM to RebN per unit mass of dried leaves is not particularly limited, and may be, for example, 20.0, 10.0, 9.0, 8.0, 7.0, 6.0, 5.0, etc.
  • the ratio of the RebM content to the stevioside content, RebM/ST, of 1.83 or more per unit mass of dried leaves means, for example, that the ratio of RebM to stevioside contained in a given mass of dried leaves (e.g., 50 mg) is 1.83 or more.
  • RebM/ST per unit mass of dried leaves is not limited and may be, for example, 1.83 or more, 1.9 or more, 2.0 or more, 2.1 or more, 2.4 or more, 2.5 or more, 2.8 or more, 3.0 or more, 3.1 or more, 3.3 or more, 3.5 or more, 3.7 or more, 3.9 or more, 4.0 or more, 4.5 or more, 5.0 or more, 5.5 or more, 6.0 or more, etc.
  • the upper limit of the ratio of RebM to stevioside per unit mass of dried leaves is not particularly limited, but may be, for example, 20.0, 10.0, 9.0, 8.0, 7.0, 6.0, 5.0, etc.
  • plant B of the present invention may have a plurality of the above chemical characteristics.
  • the number of chemical characteristics may be any of 2 to 5 types, i.e., 2, 3, 4, or 5 types.
  • the present invention provides a stevia plant having at least one of the genetic characteristics (1) to (5) and at least one of the chemical characteristics (i) to (v) (hereinafter, sometimes collectively referred to as "plant C of the present invention” or “Stevia plant C of the present invention”).
  • plant C of the present invention or “Stevia plant C of the present invention”
  • the plants A to C of the present invention may be collectively referred to as “the plants of the present invention.”
  • Steviol glycosides such as RebD, RebE, RebM, and RebN can be extracted in the form of an extract by reacting fresh or dried leaves of the plant of the present invention with an appropriate solvent (aqueous solvent such as water, or organic solvent such as alcohol, ether, and acetone).
  • aqueous solvent such as water, or organic solvent such as alcohol, ether, and acetone.
  • the extraction conditions can be referenced to the methods described in Ohta et al., J. Appl. Glycosci., Vol. 57, No. 3, 199-209 (2010) or WO2010/038911, or the methods described in the Examples below.
  • the extract thus obtained can be purified into individual steviol glycosides, such as RebD, RebE, RebM, and RebN, by using known methods such as a gradient of ethyl acetate or other organic solvent:water, high performance liquid chromatography (HPLC), gas chromatography, time-of-flight mass spectrometry (TOF-MS), and ultra (High) Performance Liquid chromatography (UPLC).
  • HPLC high performance liquid chromatography
  • TOF-MS time-of-flight mass spectrometry
  • UPLC ultra (High) Performance Liquid chromatography
  • the content of steviol glycosides such as RebD, RebE, RebM, and RebN can be measured by the method described in Ohta et al. or WO2010/038911, or the method described in the Examples below. Specifically, for example, the content can be measured by sampling fresh leaves from the stevia plants A to C of the present invention and performing LC-MS/MS.
  • the plant body of the present invention may be one obtained by a recombinant gene technique or its progeny (hereinafter, sometimes referred to as a "recombinant plant body”), or one obtained by a non-recombinant technique or its progeny (hereinafter, sometimes referred to as a "non-recombinant plant body”).
  • non-recombinant techniques include hybridization, self-pollination, and other methods of inducing mutations in genes of a host cell (or a host plant body) without introducing a foreign gene.
  • Such methods include a method of using a mutagen on plant cells. Examples of such mutagen are ethyl methanesulfonate (EMS) and sodium azide.
  • EMS can be used to treat plant cells at concentrations of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, and the like.
  • the treatment time is 1 to 48 hours, 2 to 36 hours, 3 to 30 hours, 4 to 28 hours, 5 to 26 hours, or 6 to 24 hours.
  • the treatment procedure itself is publicly known, but for example, it can be carried out by immersing the water-absorbed seeds that have undergone the water absorption process in a treatment solution containing the mutagen at the above concentration for the treatment time described above.
  • plant cells can be irradiated with radiation or light such as X-rays, gamma rays, ion beams (e.g., heavy ion beams), or ultraviolet rays.
  • radiation or light such as X-rays, gamma rays, ion beams (e.g., heavy ion beams), or ultraviolet rays.
  • cells irradiated with an appropriate amount of ultraviolet light can be cultured in a selective medium, and then cells, calli, or plants having the desired traits can be selected.
  • the irradiation intensity is 0.01-100Gr, 0.03-75Gr, 0.05-50Gr, 0.07-25Gr, 0.09-20Gr, 0.1-15Gr, 0.1-10Gr, 0.5-10Gr, 1-10Gr, the irradiation distance is 1cm-200m, 5cm-100m, 7cm-75m, 9cm-50m, 10cm-30m, 10cm-20m, 10cm-10m, and the irradiation time is 1min-2years, 2min-1year, 3min-0.5years, 4min-1month, 5min-2weeks, 10min-1week.
  • the irradiation intensity, distance, and time vary depending on the type of radiation and the state of the target of irradiation (cells, callus, plant body), but can be adjusted appropriately by a person skilled in the art.
  • plant cells may undergo mutations during cultivation, and therefore it is preferable to reinstate them into an individual plant for more stable maintenance of traits.
  • Plants obtained by subsequently subjecting the plant body of the present invention to genetic modification e.g., by genome editing, etc.
  • a host e.g., a plant body obtained by subjecting the plant body of the present invention as a host to genetic modification to further impart another trait
  • the plant body of the present invention includes not only the entire plant body, but also parts or derivatives thereof.
  • the plant body of the present invention may include plant organs (e.g., leaves, petals, stems, roots, seeds, etc.), plant tissues (e.g., epidermis, phloem, parenchyma, xylem, vascular bundles, palisade tissue, spongy tissue, etc.), or various forms of plant cells (e.g., suspension culture cells), protoplasts, leaf slices, callus, etc.
  • plant organs e.g., leaves, petals, stems, roots, seeds, etc.
  • plant tissues e.g., epidermis, phloem, parenchyma, xylem, vascular bundles, palisade tissue, spongy tissue, etc.
  • various forms of plant cells e.g., suspension culture cells
  • protoplasts e.g., leaf slices, callus, etc.
  • the plant body of the present invention may also include tissue cultures or plant cultured cells. This is because the plant body can be regenerated by culturing such tissue cultures or plant cultured cells. Examples of regenerative forms of the plant body of the present invention include, but are not limited to, embryos, meristematic cells, pollen, leaves, roots, root tips, petals, protoplasts, leaf slices, and callus.
  • the present invention provides a method for producing the Stevia plant of the present invention (hereinafter, sometimes referred to as "Production Method A of the present invention"), which comprises a step of crossing the Stevia plant of the present invention with a second Stevia plant.
  • the Stevia plant produced by this method can have the same phenotype and genetic characteristics as the plant of the present invention.
  • the ranges of the contents and mass ratios of each component, the combinations of each characteristic (chemical characteristic and/or genetic characteristic) in the plant obtained by production method A of the present invention are as described above for the plant of the present invention.
  • crossing means crossing any one of the plants of the present invention (first generation (S1)) with a second plant (S1) to obtain a child plant (a plant produced by the production method of the present invention (second generation (S2)).
  • Backcrossing is a preferred crossing method.
  • “Backcrossing” is a technique in which, for example, a child plant (S2) born between a plant of the present invention and a second plant is further crossed with a plant of the present invention (i.e., a plant having the genetic characteristic of the present invention) (S1) to produce a plant having the genetic characteristic of the present invention.
  • the second plant (S1) used in the production method of the present invention has the same phenotype and genetic characteristics as the plant of the present invention, this is essentially backcrossing. It is preferable to perform crossing over two or more generations, but when the genetic characteristics are heterozygosity, etc., a plant having a desired combination of genetic characteristics may be obtained in one generation.
  • the plant of the present invention can be produced by self-pollination, which can be carried out by self-pollinating the pistil of the plant of the present invention with pollen from the stamen of the plant of the present invention.
  • the plant produced by the production method of the present invention has the same phenotype and genetic characteristics as the plant of the present invention, so it is possible to produce a stevia plant having a phenotype equivalent to that of the plant of the present invention by further crossing the plant produced by the production method of the present invention with a third stevia plant.
  • the plant body of the present invention can be produced by regenerating the plant body by culturing the tissue culture or plant cell culture described above.
  • the culture conditions are the same as those for culturing tissue culture or plant cell culture of wild-type stevia plants and are well known (Protocols for In Vitro cultures and secondary metabolite analysis of aromatic and medicinal plants, Method in molecular biology, vol. 1391, pp113-123).
  • the plant of the present invention can be produced by introducing the mutation of the present invention into the genome of a stevia plant.
  • the present invention provides a method for producing the stevia plant of the present invention (hereinafter, sometimes referred to as "production method B of the present invention"), which comprises the step of introducing at least one of the following mutations into the genome of a stevia plant: (1) A mutation that changes the sequence corresponding to positions 150 to 151 of SEQ ID NO:1 to T. (2) A mutation that changes the base at the position corresponding to position 151 of SEQ ID NO:2 to TAGGTGGTGACACTGTAGC. (3) A mutation that changes the base at the position corresponding to position 151 of SEQ ID NO:3 to A.
  • the introduction of the mutation may be carried out by a recombinant gene technique or a non-recombinant gene technique.
  • An example of a recombinant gene technique is a method of introducing a desired mutation into the genome of a host cell (or a host plant) by genome editing or the like.
  • the non-recombinant gene technique is as described above for the plant of the present invention.
  • the present invention provides a method for screening a Stevia plant having at least one of the chemical characteristics (i) to (v) from a test Stevia plant, the method comprising the step of detecting the presence and/or absence of at least one of the genetic characteristics (1) to (5) (hereinafter, sometimes referred to as the "screening method of the present invention").
  • the screening method of the present invention may further comprise a step of selecting, from among the test plants, a plant body in which the presence of at least one of the above-mentioned genetic characteristics has been detected.
  • the presence of the genetic features of the present invention may be determined, for example, by: (I) the presence of an allele in which the sequence corresponding to positions 150 to 151 of SEQ ID NO: 1 is T (for example, an allele containing the nucleotide sequence of SEQ ID NO: 14, 15, 16, or 17; hereinafter, sometimes referred to as "allele i"); (II) the presence of an allele in which the base at the position corresponding to position 151 of SEQ ID NO: 2 is TAGGTGGTGACACTGTAGC (for example, an allele containing the base sequence of SEQ ID NO: 18, 19, 20, or 21; hereinafter, sometimes referred to as "allele ii”); (III) the presence of an allele in which the base at the position corresponding to position 151 of SEQ ID NO: 3 is A (for example, an allele containing the base sequence of SEQ ID NO: 22, 23, 24, or 25; hereinafter, sometimes referred to as "allele iii”); (IV) the presence of
  • the absence of a genetic feature of the present invention may be, for example, (i) the absence of allele i; (ii) absence of allele ii; (iii) absence of allele iii; (iv) the absence of allele iv, and/or (v) can be determined by detecting the presence of an allele other than allele v.
  • methods for detecting genetic characteristics of the present invention include, but are not limited to, PCR, TaqMan PCR, sequencing, microarray, Invader, TILLING, RAD, RFLP, PCR-SSCP, AFLP, SSLP, CAPS, dCAPS, ASO, ARMS, DGGE, CCM, DOL, MALDI-TOF/MS, TDI, padlock probe, molecular beacon, DASH, UCAN, ECA, PINPOINT, PROBE, VSET, Survivor assay, Sniper assay, Luminex assay, GOOD, LCx, SNaPshot, Mass ARRAY, pyrosequencing, SNP-IT, and melting curve analysis.
  • the genetic characteristic (1) of the present invention can be detected, for example, by the dCAPS method using the following primer set and restriction enzymes.
  • Primer set A primer set comprising a forward primer comprising a contiguous sequence of 15 to 25 bases from the 3' end of SEQ ID NO: 34, and a reverse primer comprising a sequence complementary to any contiguous sequence of 15 or more bases located on the 3' side of position 26 of SEQ ID NO: 36 or 37 (e.g., SEQ ID NO: 35).
  • Restriction enzyme Hyp188I or its isoschizomer
  • the genetic characteristic (4) of the present invention can be detected, for example, by the dCAPS method using the following primer set and restriction enzymes.
  • Primer set A primer set comprising a forward primer containing any sequence of 15 or more consecutive bases located on the 5' side of position 78 of SEQ ID NO: 42 or 45 (e.g., SEQ ID NO: 40), and a reverse primer containing a consecutive sequence 15 to 25 bases long from the 3' end of SEQ ID NO: 41.
  • Restriction enzyme NdeI or its isoschizomer
  • the genetic characteristic (5) of the present invention can be detected, for example, by the dCAPS method using the following primer set and restriction enzymes.
  • Primer set A primer set comprising a forward primer comprising a contiguous sequence of 15 to 28 bases from the 3' end of SEQ ID NO: 46, and a reverse primer comprising a sequence (e.g., SEQ ID NO: 47) complementary to any contiguous sequence of 15 or more bases located on the 3' side of position 29 of SEQ ID NO: 48 or 51.
  • Restriction enzyme MfeI or its isoschizomer
  • the genetic characteristic (2) of the present invention can be detected, for example, using the following primer set.
  • Primer set A primer set comprising a forward primer comprising a contiguous sequence of 15 to 25 bases from the 3' end of SEQ ID NO:52, and a reverse primer comprising a sequence complementary to any contiguous sequence of 15 or more bases located on the 3' side of position 26 of SEQ ID NO:54 or 55 (e.g., SEQ ID NO:53).
  • the primer sequences can be optimized within the range that satisfies the above conditions.
  • optimizing primer design see, for example, Sambrook and Russell, "Molecular Cloning: A Laboratory Manual” 3rd Edition (2001), Cold Spring Harbor Laboratory Press.
  • the above primers may be 15-50 bases long, 18-48 bases long, 20-45 bases long, 30-40 bases long, etc. It is also possible to design primer sets other than those mentioned above and select the corresponding restriction enzymes based on the genetic characteristics of the present invention.
  • the genetic characteristic (1), (4), or (5) of the present invention can be detected by, for example, the dCAPS method using a primer set having the following sequences and a restriction enzyme:
  • a primer set having the following sequences and a restriction enzyme The above-mentioned combinations of primer sets and restriction enzymes are merely examples, and a person skilled in the art can find other combinations of primer sets and restriction enzymes that can detect the genetic features of the present invention.
  • the screening method of the present invention may further include a step of measuring the content of RebM, stevioside, RebE and/or RebN in tissues (e.g., leaves) of a test stevia plant in which at least one of the genetic features (1) to (5) of the present invention is detected.
  • the measurement of the content of RebM, stevioside, RebE and/or RebN is as described in the section on the plant of the present invention.
  • an individual having a high RebM content, a difference between the RebM content and the stevioside content, a difference between the RebM content and the RebE content, a ratio of the RebM content to the RebN content, and/or a ratio of the RebM content to the stevioside content may be selected, and this may be crossed with another stevia plant, and the screening method of the present invention may be applied to the resulting offspring plant.
  • the screening method of the present invention may include one or more of the following steps.
  • the selected individuals having a high RebM content, the difference between RebM content and stevioside content, the difference between RebM content and RebE content, the ratio of RebM content to RebN content, and/or the ratio of RebM content to stevioside content may be, for example, the top 50%, top 40%, top 30%, top 20%, top 10%, top 5%, top 4%, top 3%, top 2%, or top 1% of individuals in terms of height having a high RebM content, the difference between RebM content and stevioside content, the difference between RebM content and RebE content, the ratio of RebM content to RebN content, and/or the ratio of RebM content to stevioside content, among the test stevia plants in which at least one of the genetic characteristics (1) to (5) of the present invention has been detected.
  • the other stevia plant to be crossed may or may not contain the genetic features (1) to (5) of the present invention.
  • steps (iv) to (vii) can be repeated multiple times. In this manner, stevia plants having higher RebM content, difference between RebM content and stevioside content, difference between RebM content and RebE content, ratio of RebM content to RebN content, and/or ratio of RebM content to stevioside content can be screened.
  • the test stevia plant may be a natural plant or a non-genetically modified plant.
  • the non-genetically modified plant is as described in the section on the plant of the present invention.
  • the test stevia plant may include a stevia plant that has been subjected to a mutagenesis treatment and its progeny.
  • the mutagenesis is as described in the section on the plant of the present invention, and includes treatment with a mutagen, treatment with radiation or light, etc.
  • the present invention also provides the primer sets and combinations thereof described above, for example, the primer sets described in Table 1 above, and combinations of these primer sets.
  • the present invention further provides primer sets capable of amplifying by PCR a region having a nucleotide sequence selected from SEQ ID NOs: 14 to 33 and 56 to 70, for example, a primer set of a forward primer containing the nucleotide sequence of SEQ ID NO: 6 and a reverse primer containing the nucleotide sequence of SEQ ID NO: 7, a primer set of a forward primer containing the nucleotide sequence of SEQ ID NO: 8 and a reverse primer containing the nucleotide sequence of SEQ ID NO: 9, a primer set of a forward primer containing the nucleotide sequence of SEQ ID NO: 10 and a reverse primer containing the nucleotide sequence of SEQ ID NO: 11, a primer set of a forward primer containing the nucleotide sequence of SEQ ID NO: 12 and a reverse primer containing the nucle
  • the present invention provides a probe capable of detecting the presence and/or absence of the genetic feature of the present invention (hereinafter, sometimes referred to as the "probe of the present invention").
  • the probe of the present invention may have a structure suitable for various detection methods for the presence and/or absence of the genetic feature of the present invention (e.g., real-time PCR such as TaqMan PCR).
  • the probe of the present invention may contain a base sequence that is complementary to a portion of the genome containing the mutation site of the present invention.
  • Non-limiting examples of such probes include those that contain a sequence complementary to a base sequence selected from SEQ ID NOs: 14-16, 18-20, 22-24, 26-28, 30-32, and 56-70.
  • SEQ ID NOs: 14-16, 18-20, 22-24, 26-28, and 30-32 are specific to alleles containing the mutation of the present invention
  • SEQ ID NOs: 56-70 are specific to alleles not containing the mutation of the present invention.
  • the presence of the genetic feature of the present invention can be detected by detecting both an allele containing a mutation of the present invention and an allele not containing a mutation of the present invention
  • the absence of the genetic feature of the present invention can be detected by detecting only an allele containing a mutation of the present invention or only an allele not containing a mutation of the present invention.
  • the probe of the present invention preferably has a label.
  • the probe of the present invention has a label and a polynucleotide containing a base sequence complementary to a base sequence selected from SEQ ID NOs: 14-16, 18-20, 22-24, 26-28, 30-32, and 56-70.
  • the present invention further provides a kit comprising the above primer set and the corresponding restriction enzyme.
  • the kit of the present invention comprises the primer set shown in Table 1 and the corresponding restriction enzyme.
  • the present invention also provides a kit comprising a primer set capable of amplifying by PCR a region having a base sequence selected from the group consisting of SEQ ID NOs: 14 to 33 and 56 to 70, and the corresponding probe of the present invention.
  • primer sets, probes, and kits can be used to detect the genetic characteristics (1) to (5) of the present invention, and can be used in the screening method of the present invention. Furthermore, these primer sets and kits may include instructions including explanations regarding the detection of the genetic characteristics (1) to (5) of the present invention and the screening method of the present invention, such as instruction manuals, site information including information on the method of use (e.g., URL, two-dimensional code), and media on which information on the method of use is recorded (e.g., flexible disk, CD, DVD, Blu-ray disk, memory card, USB memory), etc.
  • site information including information on the method of use
  • media on which information on the method of use is recorded e.g., flexible disk, CD, DVD, Blu-ray disk, memory card, USB memory
  • the present invention provides a screening kit for the Stevia plant of the present invention, comprising a reagent for detecting the presence and/or absence of at least one of the genetic features (1) to (5).
  • the reagent may comprise a primer and/or a probe for use in the CAPS method, the dCAPS method, or the TaqMan PCR method.
  • the reagent for detecting the presence and/or absence of at least one of the genetic features (1) to (5) comprises a combination of a primer set and a restriction enzyme for detecting at least one of the above genetic features (1) to (5) by the dCAPS method, for example, a combination of a primer set and a restriction enzyme as set forth in Table 1, or a combination of a primer set for amplifying a mutation site of the present invention (e.g., a site containing a sequence selected from SEQ ID NOs: 14 to 33) and a probe having a base sequence complementary to a site related to at least one of the genetic features (1) to (5) (e.g., a site containing a sequence selected from SEQ ID NOs: 14 to 16, 18 to 20, 22 to 24, 26 to 28, 30 to 32), which can be used in the TaqMan PCR method, etc.
  • a primer set and a restriction enzyme for detecting at least one of the above genetic features (1) to (5) by the dCAPS method
  • dead tissue or dead cell of the present invention In a further embodiment of the present invention, dead tissue or cells of the stevia plant of the present invention, the stevia plant selected by the screening method of the present invention, or the stevia plant produced by the production method of the present invention (hereinafter, sometimes referred to as "dead tissue or dead cell of the present invention").
  • “Dead” means a state in which the plant has no ability to reproduce, grow, reproduce, or grow, and includes natural death without artificial manipulation, as well as death caused by artificial manipulation such as cutting, crushing, heating (including heating through a gas such as air, heating through a liquid such as water, and heating through steam such as water vapor), freezing, drying, and freeze-drying.
  • Dead tissue means tissue in which all of the cells contained therein are dead, and includes dead plant bodies (plant bodies in which all of the cells contained therein are dead).
  • specific examples of dead tissue or dead cell of the present invention include dead tissue or cells other than seeds. More specifically, dead embryos, meristematic cells, pollen, leaves, roots, root tips, petals, protoplasts, leaf slices, and calluses are included.
  • the dead tissue or cells of the present invention can be used as a raw material for the below-described extracts, steviol glycosides, medicines, flavorings, foods and beverages, and the like.
  • a method for producing an extract containing steviol glycosides (hereinafter, sometimes referred to as the "method for producing the extract of the present invention"), which comprises a step of obtaining an extract from a plant of the present invention, a stevia plant selected by the screening method of the present invention, or a stevia plant produced by the production method of the present invention, or dead tissue or dead cells of the plant, or seeds, leaves (e.g., dried leaves or fresh leaves), tissues, tissue cultures, or cells of the plant.
  • an extract containing steviol glycosides from the plant of the present invention a stevia plant selected by the screening method of the present invention, or a stevia plant produced by the production method of the present invention, seeds, leaves (e.g., dried leaves or fresh leaves), tissues, tissue cultures, or cells of said plant, or dead tissues or dead cells of the present invention (hereinafter, sometimes referred to as "the extract of the present invention").
  • the extract of the present invention is preferably produced by the production method of the extract of the present invention.
  • the production method of the steviol glycoside of the present invention comprising a step of purifying steviol glycosides from the extract of the present invention.
  • the production method of steviol glycosides of the present invention may further comprise a step of obtaining an extract containing steviol glycosides from the plant of the present invention, a stevia plant selected by the screening method of the present invention, or a stevia plant produced by the production method of the present invention, or dead tissues or dead cells of the present invention.
  • An extract containing steviol glycosides can be obtained, for example, by reacting fresh or dried leaves of the plant of the present invention with an appropriate solvent (aqueous solvent such as water, or organic solvent such as alcohol, ether, and acetone).
  • an appropriate solvent aqueous solvent such as water, or organic solvent such as alcohol, ether, and acetone.
  • the extraction conditions can be determined by referring to the methods described in Ohta et al. or WO2010/038911 or the methods described in the Examples below.
  • Steviol glycosides can also be purified from extracts containing steviol glycosides using known methods such as ethyl acetate or other organic solvent:water gradients, chromatography (e.g., High Performance Liquid Chromatography (HPLC), Ultra (High) Performance Liquid Chromatography (UPLC), gas chromatography, etc.), and Time-of-Flight mass spectrometry (TOF-MS).
  • HPLC High Performance Liquid Chromatography
  • UPLC Ultra (High) Performance Liquid Chromatography
  • TOF-MS Time-of-Flight mass spectrometry
  • the type of steviol glycoside is not particularly limited and includes RebA, RebB, RebC, RebD, RebE, RebF, RebG, RebI, RebJ, RebK, RebM, RebN, RebO, RebQ, RebR, dulcoside A, rubusoside, steviolmonoside, steviolbioside, stevioside, etc.
  • the steviol glycoside includes RebD, RebE, RebM, RebN, stevioside, or a combination thereof.
  • the extract of the present invention has a higher RebM content, a higher difference between the RebM content and the stevioside content, a higher difference between the RebM content and the RebE content, a higher ratio of the RebM content to the RebN content, and/or a higher ratio of the RebM content to the stevioside content, compared to a Stevia species not having any of the genetic characteristics (2'), (4'), and (5) of the present invention.
  • the extract of the present invention has a RebM content, a difference between the RebM content and the stevioside content, a difference between the RebM content and the RebE content, a ratio of the RebM content to the RebN content, and/or a ratio of the RebM content to the stevioside content of about 50% or more, about 100% or more, about 150% or more, about 200% or more, about 250% or more, about 300% or more, about 350% or more, about 400% or more, about 450% or more, about 500% or more, about 550% or more, about 600% or more, about 650% or more, compared to an extract obtained from a Stevia plant body not having any of the genetic characteristics (2'), (4'), and (5) of the present invention.
  • the concentration may be about 700% or more, about 750% or more, about 800% or more, about 850% or more, about 900% or more, about 950% or more, about 1000% or more, about 1050% or more, about 1100% or more, about 1150% or more, about 1200% or more, about 1250% or more, about 1300% or more, about 1350% or more, about 1400% or more, about 1450% or more, about 1500% or more, about 1550% or more, about 1600% or more, about 1650% or more, about 1700% or more, about 1750% or more, about 1800% or more, about 1850% or more, about 1900% or more, about 1950% or more, about 2000% or more, about 2050% or more, or about 2100% or more.
  • the extract of the present invention and the extract obtained from a Stevia plant having none of the genetic characteristics (2'), (4'), or (5) of the present invention may be obtained by the same method.
  • the present invention provides a method for producing a food, drink, sweetener composition, flavor, or pharmaceutical product, which includes a step of mixing the extract of the present invention and/or the steviol glycoside obtained by the method for producing a steviol glycoside of the present invention with other ingredients, such as raw materials for a food, drink, sweetener, flavor, or pharmaceutical product.
  • the present invention provides a novel food, drink, sweetener composition, flavor, or pharmaceutical product having an increased steviol glycoside content obtained by the above-mentioned production method.
  • the food and drink mentioned here includes beverages and foods.
  • the present invention provides a novel beverage, food, sweetener composition, flavor, or pharmaceutical product, and also provides a method for producing the beverage, food, sweetener composition, flavor, or pharmaceutical product.
  • the present invention provides a method for producing a steviol glycoside-containing composition, comprising a step of mixing the extract of the present invention and/or the steviol glycoside obtained by the method for producing a steviol glycoside of the present invention with other ingredients.
  • the present invention provides a steviol glycoside-containing composition obtained by the production method, which contains the steviol glycoside and other ingredients.
  • the above-mentioned "other ingredients” may include ingredients that are not present in natural stevia plants or non-natural ingredients. Therefore, the composition may be a non-natural composition.
  • the composition may be, for example, a pharmaceutical composition, a flavor composition, a beverage composition, or a food composition.
  • the present invention provides a nucleotide sequence related to the plant of the present invention.
  • the nucleotide sequence of the stevia plant having the genetic characteristic (1) comprises or consists of a nucleotide sequence selected from SEQ ID NOs: 14 to 17.
  • the nucleotide sequence of the stevia plant having the genetic characteristic (2) comprises or consists of a nucleotide sequence selected from SEQ ID NOs: 18 to 21.
  • the nucleotide sequence of the stevia plant having the genetic characteristic (3) comprises or consists of a nucleotide sequence selected from SEQ ID NOs: 22 to 25.
  • the nucleotide sequence of the stevia plant having the genetic characteristic (4) comprises or consists of a nucleotide sequence selected from SEQ ID NOs: 26 to 29.
  • the nucleotide sequence of the stevia plant having the genetic characteristic (5) comprises or consists of a nucleotide sequence selected from SEQ ID NOs: 30 to 33.
  • Example 1 Identification of mutations related to high RebM phenotype The steviol glycoside content of stevia lines cultivated at Suntory World Research Center was measured, and genome sequencing was performed. Specifically, 0.25 g of fresh leaves were sampled from each line, dried by freeze-drying, and 0.05 g of the crushed dried material was added to 100 times the amount (5 mL) of pure water. Extraction was performed by ultrasonic treatment for 20 minutes, followed by centrifugation and filtration, and the resulting solution was diluted 60 times with 32% acetonitrile to prepare a sample solution.
  • Tables 2-3 show the steviol glycoside contents of representative strains of the high RebM group, the low RebM group with a relatively low RebM content, and the medium RebM group with a lower RebM content than the high RebM group but a higher RebM content than the low RebM group.
  • Table 4 also shows the differences and ratios of the contents of various steviol glycosides.
  • TSG indicates total steviol glycosides.
  • the value of each steviol glycoside is the weight percentage in the dried leaves, and the value of TSG is the sum of the values of all measured steviol glycosides (i.e., RebA, RebB, RebC, RebD, RebE, RebF, RebI, RebJ, RebM, RebN, and ST).
  • the numbers indicate average values, "+” indicates P ⁇ 0.05, "++” indicates P ⁇ 0.01, meaning that the high RebM group has a higher content than the mid RebM and low RebM groups, "-" indicates P ⁇ 0.05, “--” indicates P ⁇ 0.01, meaning that the high RebM group has a lower content than the mid RebM and low RebM groups, and " ⁇ " indicates that no significant difference was observed between the contents of the two groups at the significance level of 0.05.
  • genomic DNA was extracted from fresh leaves of each individual tested in Example 1, and PCR was performed using the above dCAPS primers corresponding to each genetic characteristic.
  • restriction enzymes corresponding to each genetic feature (4') and (5) were added to the PCR products, and the enzyme reaction was carried out at 37°C.
  • the restriction enzyme-treated PCR products were electrophoresed using a microchip electrophoresis device, LabChip GX Touch HT (PerkinElmer), and the presence or absence of the genetic feature was determined based on the resulting band pattern.
  • Genetic feature (2') is heterozygous, so individuals in which both a PCR product of approximately 206 bp and a PCR product of approximately 188 bp were observed were determined to have the genetic feature.
  • Genetic feature (4') is heterozygous, so individuals in which both decomposition product and non-decomposition product bands were observed were determined to have the genetic feature.
  • Genetic feature (5) is homozygous, so individuals in which a decomposition product band was observed were determined to have the genetic feature.
  • genetic features (2'), (4'), and (5) tended to be detected in high RebM strains.
  • genetic features (2'), (4'), and (5) tended not to be detected in medium RebM and low RebM strains.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Botany (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Mycology (AREA)
  • Environmental Sciences (AREA)
  • Developmental Biology & Embryology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Cell Biology (AREA)
  • Nutrition Science (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Physiology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne une plante de stévia ayant au moins l'une des caractéristiques génétiques (1) à (5).
PCT/JP2024/042255 2023-12-01 2024-11-29 Plante de stévia ayant une teneur élevée en rébaudioside m Pending WO2025115990A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2023204266 2023-12-01
JP2023-204266 2023-12-01

Publications (1)

Publication Number Publication Date
WO2025115990A1 true WO2025115990A1 (fr) 2025-06-05

Family

ID=95897032

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2024/042255 Pending WO2025115990A1 (fr) 2023-12-01 2024-11-29 Plante de stévia ayant une teneur élevée en rébaudioside m

Country Status (1)

Country Link
WO (1) WO2025115990A1 (fr)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016049531A1 (fr) * 2014-09-26 2016-03-31 Purecircle Usa Inc. Marqueurs de polymorphisme mononucléotidique (snp) pour le stévia
WO2019074089A1 (fr) * 2017-10-12 2019-04-18 サントリーホールディングス株式会社 Plante stévia riche en rébaudioside m
WO2020061456A1 (fr) * 2018-09-22 2020-03-26 Purecircle Usa Inc Cultivar de stévia '16265046'
WO2020106716A1 (fr) * 2018-11-20 2020-05-28 Purecircle Usa, Inc. Cultivar de stévia aneuploïde 'ap-1'
WO2021084325A1 (fr) * 2019-11-01 2021-05-06 Purecircle Usa, Inc. Cultivar de stévia '18136109'
WO2021230257A1 (fr) * 2020-05-12 2021-11-18 サントリーホールディングス株式会社 Plante de stévia a forte teneur en rébaudoside m et son procédé de criblage

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016049531A1 (fr) * 2014-09-26 2016-03-31 Purecircle Usa Inc. Marqueurs de polymorphisme mononucléotidique (snp) pour le stévia
WO2019074089A1 (fr) * 2017-10-12 2019-04-18 サントリーホールディングス株式会社 Plante stévia riche en rébaudioside m
WO2020061456A1 (fr) * 2018-09-22 2020-03-26 Purecircle Usa Inc Cultivar de stévia '16265046'
WO2020106716A1 (fr) * 2018-11-20 2020-05-28 Purecircle Usa, Inc. Cultivar de stévia aneuploïde 'ap-1'
WO2021084325A1 (fr) * 2019-11-01 2021-05-06 Purecircle Usa, Inc. Cultivar de stévia '18136109'
WO2021230257A1 (fr) * 2020-05-12 2021-11-18 サントリーホールディングス株式会社 Plante de stévia a forte teneur en rébaudoside m et son procédé de criblage

Similar Documents

Publication Publication Date Title
JP7756123B2 (ja) 高レバウジオシドｍ含有ステビア植物
JP7520722B2 (ja) 高レバウジオシドｄ含有ステビア植物
JP7337068B2 (ja) 甘味成分高含有型ステビア植物及びそのスクリーニング方法
US12389848B2 (en) Stevia plant having high rebaudioside M content ratio and screening method for same
WO2021230256A1 (fr) Plante stevia riche en rébaudioside d
JP7739174B2 (ja) 花芽形成能の低いステビア植物
JP7564803B2 (ja) 花粉形成能の低いステビア植物
JP2024012453A (ja) チラミン低含有ステビア植物
WO2025115990A1 (fr) Plante de stévia ayant une teneur élevée en rébaudioside m
WO2025005216A1 (fr) Plante de stévia ayant une teneur élevée en rébaudioside n
JP7737366B2 (ja) 高ステビオール配糖体含有ステビア植物及びそのスクリーニング方法
WO2024005142A1 (fr) Procédé de criblage de plantes de stévia
EP4151083A1 (fr) Plante stévia à haute teneur en rébaudioside e
WO2024248125A1 (fr) Plante de stévia riche en rebe et son procédé de criblage
WO2022220152A1 (fr) Plante de stevia riche en composants nutritionnels

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24897674

Country of ref document: EP

Kind code of ref document: A1