WO2023033079A1 - Gene involved in regulation of total dry matter production in solanaceous plant and use thereof - Google Patents

Gene involved in regulation of total dry matter production in solanaceous plant and use thereof Download PDF

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WO2023033079A1
WO2023033079A1 PCT/JP2022/032864 JP2022032864W WO2023033079A1 WO 2023033079 A1 WO2023033079 A1 WO 2023033079A1 JP 2022032864 W JP2022032864 W JP 2022032864W WO 2023033079 A1 WO2023033079 A1 WO 2023033079A1
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amino acid
seq
acid sequence
sequence shown
base
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Japanese (ja)
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暁男 大山
哲 松尾
武司 林
宏治 宮武
真咲 岩上
圭太 諏訪部
安章 加賀谷
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国立研究開発法人農業・食品産業技術総合研究機構
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    • 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
    • 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
    • A01H6/82—Solanaceae, e.g. pepper, tobacco, potato, tomato or eggplant
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • 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
    • 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 genes involved in the control of total dry matter production in Solanaceous plants and their use.
  • the yield of agricultural products such as seeds, tubers, and fruits is an important indicator for agricultural management because the increase or decrease is directly linked to profits.
  • the yield of agricultural products is proportional to the amount of dry matter produced and the amount of dry matter distributed to the crop (assimilated product distribution). Therefore, increasing dry matter production or assimilated product distribution can increase the yield of agricultural products.
  • Non-Patent Documents 1 and 2 are listed as candidates for genes used for genetic modification of fruit vegetable plants of the Solanaceae family such as tomatoes.
  • Non-Patent Document 1 describes the fw2.2 gene on chromosome 2, which is a quantitative trait locus (QTL) involved in the difference in fruit size between large and small varieties of tomatoes.
  • QTL quantitative trait locus
  • the fw2.2 gene described in Non-Patent Document 1 is a gene that is ubiquitously present in large variety tomatoes, and is a gene acquired in the process of domestication and large size of small wild tomatoes. It is speculated that That is, the fw2.2 gene is not involved in the control of total dry matter production in solanaceous plants, but is considered to increase cell division and increase the number of ovules. Therefore, the fw2.2 gene only brings about morphological changes in plants, and it is highly likely that it cannot be used to modify yield properties such as total dry matter production.
  • the present invention has been made in view of the above problems, and its object is to identify genes involved in the control of total dry matter production in solanaceous plants including tomatoes, and to provide utilization of these genes. .
  • a method according to an aspect of the present invention is a method for determining the degree of total dry matter production in a solanaceous plant, wherein in the solanaceous plant, the following amino acids (a) to (b): (a) SEQ ID NO: 1 (b) an amino acid corresponding to the 247th amino acid of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1; Alternatively, it includes the step of examining the presence or absence of insertion-causing mutations.
  • a method according to an aspect of the present invention is a method for determining the degree of total dry matter production in a solanaceous plant, wherein in the solanaceous plant, the following amino acids (e) and (f): (e) SEQ ID NO: 2 (f) an amino acid corresponding to the 139th amino acid of the protein consisting of the amino acid sequence shown in SEQ ID NO: 2; The step of testing for the presence or absence of insertion-causing mutations is included.
  • a method according to an aspect of the present invention is a method for determining the degree of total dry matter production in a solanaceous plant, wherein the following amino acids (g) and (h): (g) SEQ ID NO: 3 (h) an amino acid corresponding to the 175th amino acid of the protein consisting of the amino acid sequence shown in SEQ ID NO: 3; The step of testing for the presence or absence of insertion-causing mutations is included.
  • a production method is a method for producing a solanaceous plant with increased total dry matter production, comprising a hybridization step of intraspecifically hybridizing a solanaceous plant, and a solanaceous plant obtained by the hybridization step. and an identification step of identifying a solanaceous plant having an increased total dry matter production by any of the above methods from plants or progeny solanaceous plants.
  • a production method is a method for producing a solanaceous plant having an increased total dry matter production amount, wherein the solanaceae plant having an increased total dry matter production amount from a test solanaceous plant is obtained by any of the above methods. It includes an identification step of identifying a plant and a crossing step of intraspecifically crossing the identified solanaceous plant.
  • a molecular marker according to one aspect of the present invention is a molecular marker related to the control of total dry matter production in a plant of the Solanaceae family, and is a base itself (SNP) corresponding to the bases (a′) to (h′) below.
  • SNP base itself
  • a continuous polynucleotide containing the base (a′) a base corresponding to the 326th base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 4; (b′) a polynucleotide consisting of the base sequence shown in SEQ ID NO: 4 (c′) a base corresponding to the 591st base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 4; (d′) a polynucleotide consisting of the base sequence shown in SEQ ID NO: 4 (e′) a base corresponding to the 397th base of a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 5; (f′) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 5 (g′) a base corresponding to the 401st base of the polynucleotide consisting of the base:
  • a method for producing a solanaceous plant with increased total dry matter production comprising the step of introducing a mutation that deletes or inactivates an amino acid sequence downstream of an amino acid at any position downstream of the corresponding amino acid.
  • the gene according to one aspect of the present invention lacks or lacks an amino acid sequence downstream of the amino acid at any position downstream of the amino acid corresponding to the 73rd amino acid in the amino acid sequence shown in SEQ ID NO: 1 in a solanaceous plant. It consists of a polynucleotide encoding a protein introduced with an inactivating mutation and having the function of increasing the total dry matter production of a plant of the family Solanaceae.
  • the gene according to one aspect of the present invention is a polynucleotide according to any one of the following (1) to (3): (1) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 1; 2) consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and an amino acid corresponding to the 109th amino acid in the amino acid sequence shown in SEQ ID NO: 1 and amino acids downstream thereof; A protein whose sequence is deleted or inactivated, or whose amino acid corresponding to the 247th amino acid in the amino acid sequence shown in SEQ ID NO: 1 is C, and which has the function of regulating the total dry matter production of a solanaceous plant.
  • amino acid corresponding to the 109th amino acid in the amino acid sequence shown in SEQ ID NO: 1 and the amino acid sequence downstream thereof are deleted or inactivated, or the amino acid sequence shown in SEQ ID NO: 1
  • the amino acid corresponding to the 247th amino acid in is C, and 65 or less amino acids are substituted, deleted, added or inserted, and the protein has the function of controlling the total dry matter production of solanaceous plants a polynucleotide encoding a
  • the gene according to one aspect of the present invention is a polynucleotide according to any one of the following (4) to (6): (4) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2; 5) consists of an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2, and the amino acid corresponding to the 133rd amino acid in the amino acid sequence shown in SEQ ID NO: 2 is A, or A polynucleotide encoding a protein in which the amino acid corresponding to the 139th amino acid in the amino acid sequence shown in SEQ ID NO: 2 is N, and which has the function of controlling the total dry matter production of a plant of the family Solanaceae; (6) SEQ ID NO: 2 consisting of an amino acid sequence in which 30 or less amino acids are substituted, deleted, added or inserted into the amino acid sequence shown in SEQ ID NO: 2, and the amino acid corresponding to the 133rd amino acid
  • the gene according to one aspect of the present invention is the polynucleotide according to any one of the following (7) to (9): (7) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 3; 8) consists of an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3, and the amino acid corresponding to the 134th amino acid in the amino acid sequence shown in SEQ ID NO: 3 is P, or A polynucleotide encoding a protein in which the amino acid corresponding to the 175th amino acid in the amino acid sequence shown in SEQ ID NO: 3 is Y, and which has the function of controlling the total dry matter production of a plant of the family Solanaceae; (9) SEQ ID NO: 3, wherein 60 or less amino acids are substituted, deleted, added or inserted into the amino acid sequence shown in SEQ ID NO: 3, and the amino acid corresponding to the 134th amino acid in the amino acid sequence shown in SEQ
  • An expression vector according to one aspect of the present invention comprises any of the above genes.
  • a cell or dicotyledonous plant according to one aspect of the present invention comprises any of the above genes or the above expression vector.
  • a solanaceous plant with an increased total dry matter production is identified, and an eggplant with an increased total dry matter production Can produce family plants.
  • FIG. 1 is a diagram outlining the construction of a recombinant inbred line derived from crossing two F1 breeds and its QTL mapping.
  • Fig. 2 is a diagram explaining the linkage map of linkage group 6 and the position of QTL tfw6.1;
  • FIG. 2 shows the nucleotide and amino acid sequences of Arabidopsis thaliana genome-edited strains used in Examples.
  • FIG. 3 shows the results of comparing the phenotypes of the genome-edited strain of Arabidopsis thaliana used in Examples and the wild type.
  • polynucleotide can also be rephrased as “nucleic acid” or “nucleic acid molecule” and intends a polymer of nucleotides.
  • a “nucleotide sequence” can also be rephrased as a “nucleic acid sequence” or a “nucleotide sequence”, and unless otherwise specified, a sequence of deoxyribonucleotides or a sequence of ribonucleotides is intended.
  • polypeptide can also be rephrased as "protein”.
  • dry matter can be rephrased as “assimilation product” and intends organic compounds such as sugars synthesized by photosynthesis.
  • total dry matter production also referred to as “biomass volume” is intended to be the total amount of dry matter produced.
  • increase in total dry matter production is intended to increase the total amount of dry matter produced. It is intended that the increased leaf area will increase the amount of light received, resulting in an increase in total dry matter production.
  • plants of the Solanaceae family are, for example, tomatoes, potatoes, eggplants, or hot peppers.
  • eggplant broadly includes the cultivated species “Solanum (hereinafter referred to as S) melongena” as well as the wild species “S. incanum” and “S. "S. torvum”, “S. nigrum”, “S. aethiopicum”, “S. macrocarpon”, and “S. Quitoense” )”, and narrowly intended to mean “S. melongena”.
  • tomato is broadly defined as the cultivated species “S. lycopersicum", as well as the wild species “S. cheesmaniae, S. chilense, S. lycopersicum”. S. chmielewskii, S. galapagense, S. habrochaites, S. lycopersicoides, S. neorickii, S. penelli S. pennellii, S. peruvianum, and S. pimpinellifolium, narrowly intended as "S. lycopersicum.”
  • capsicum broadly includes the cultivated species “Capsicum annuum” and the wild species “C. pubescens” and “C. A concept that includes C. baccatum, C. chinense, and C. frutescens, narrowly intended to mean C. anium.
  • pepper is a concept that includes the above-mentioned plants for which terms other than “pepper” are used, such as “green pepper”, “paprika”, and “shishito”, as names of horticultural crops.
  • potato is broadly defined as the cultivated species “S. tuberosum”, as well as the wild species “S. acaule”, “S. sparsipilum” “, “S. leptophyes”, and “S. megistacrobum”, narrowly intended to be “S. tuberosum”.
  • a gene for regulating total dry matter production is a gene that encodes a protein having activity to regulate total dry matter production (total dry matter production regulating activity). If the protein encoded by the gene for controlling total dry matter production has the activity of positively regulating total dry matter production, the presence of the protein will increase total dry matter production. In addition, when the activity of the protein encoded by the gene for controlling total dry matter production is reduced or inhibited, the total dry matter production is reduced more than the plant whose activity is not reduced or inhibited, or not increase.
  • An example of the total dry matter production control gene is a gene involved in the control of the total dry matter production of a solanaceous plant, which consists of the polynucleotide according to any one of the following (1) to (9). : (1) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 1; (2) A polynucleotide consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and encoding a protein having a function of controlling the total dry matter production of Solanaceae plants.
  • an encoding polynucleotide (4) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2; (5) A polynucleotide consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2 and encoding a protein having a function of controlling the total dry matter production of a plant of the family Solanaceae.
  • an encoding polynucleotide (7) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 3;
  • a protein consisting of an amino acid sequence in which 60 or less amino acids are substituted, deleted, added or inserted with respect to the amino acid sequence shown in SEQ ID NO: 3, and having the function of controlling the total dry matter production of a solanaceous plant. Encoding polynucleotide.
  • the polynucleotides (1), (4), and (7) above contain the nucleotide sequence of the tomato-derived tfw6.1 gene or the nucleotide sequence of the coding region (CDS) of the tfw6.1 gene, and measure the total dry matter production Polynucleotides encoding proteins with regulatory functions.
  • the polynucleotides (1), (4), and (7) above include the nucleotide sequence of the gene corresponding to the tfw6.1 gene of the Solanaceae plant or the nucleotide sequence of the CDS of the gene, and the total dry matter production It can be a polynucleotide that encodes a protein that has the function of controlling the amount.
  • sequence identity of the amino acid sequences is preferably 80% or more, or 90% or more, and more preferably 95% or more. , 96% or more, 97% or more, 98% or more, or 99% or more.
  • the number of substituted, deleted, added or inserted amino acids in the amino acid sequence of SEQ ID NO: 1 is preferably 1 to 65, 1 to 60, 1 to 50. , 1 to 40, more preferably 1 to 30, more preferably 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 are particularly preferred.
  • the number of substituted, deleted, added or inserted amino acids in the amino acid sequence of SEQ ID NO: 2 is preferably 1 to 30, 1 to 25, 1 to 20. , more preferably 1 to 15, more preferably 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 are particularly preferred.
  • the number of substituted, deleted, added or inserted amino acids in the amino acid sequence of SEQ ID NO: 3 is preferably 1 to 60, 1 to 50, 1 to 40. , more preferably 1 to 30, more preferably 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3 or 1 to 2 are particularly preferred.
  • a mutated gene derived from tomato or a homologous gene (including an orthologue) derived from a solanaceous plant other than tomato is the above (2), (3), (5), (6), (8) and It is included in the category of polynucleotides of (9).
  • These mutated genes and homologous genes are endogenous genes of Solanaceae plants. [4. Method for determining the degree of total dry matter production in Solanaceous plants] can be molecular markers on these mutated genes or homologous genes.
  • the total dry matter production control gene refers to an artificially mutated gene
  • substitution, deletion, addition or insertion of amino acids is, for example, the Kunkel method (Kunkel, Proc Natl Acad Sci USA, 82: 488-492, 1985), mutagen treatment using drugs, mutagenesis methods using radiation ( ⁇ -rays, heavy ion beams, etc.), genome editing using site-specific nucleases
  • a mutation may be introduced artificially using, for example, a mutation, or it may be derived from a similar mutant polypeptide that exists in nature.
  • the total dry matter production control gene lacks or lacks an amino acid sequence downstream of the amino acid at any position downstream of the amino acid corresponding to the 73rd amino acid in the amino acid sequence shown in SEQ ID NO: 1 in the Solanaceae plant It may be a gene comprising a polynucleotide into which an inactivating mutation has been introduced and which encodes a protein having the function of increasing the total dry matter production of a plant of the family Solanaceae.
  • the polynucleotide is mutated to delete or inactivate the amino acid corresponding to the 109th amino acid of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1 and the amino acid sequence downstream thereof, It may be a gene encoding a protein having a function of increasing the total dry matter production of a solanaceous plant.
  • a gene for controlling total dry matter production can be obtained by artificially introducing a mutation into a gene for controlling total dry matter production of Solanaceae plants using known techniques such as genome editing. The position for introducing mutation into the total dry matter production control gene, the details of the mutation, etc. will be described later [6. Method for Producing Solanaceous Plant with Increased Total Dry Matter Production].
  • the total dry matter production control gene can exist in the form of RNA (eg, mRNA) or in the form of DNA (eg, cDNA or genomic DNA). DNA may be double-stranded or single-stranded.
  • the nucleotide sequence shown in SEQ ID NO: 4, which is an example of the total dry matter production control gene, is the full-length cDNA of the gene encoding the polypeptide shown in SEQ ID NO: 1.
  • the base sequence shown in SEQ ID NO:5 which is another example of the total dry matter production control gene, is the full-length cDNA of the gene encoding the polypeptide shown in SEQ ID NO:2.
  • nucleotide sequence shown in SEQ ID NO: 6 which is another example of the total dry matter production control gene, is the full-length cDNA of the gene encoding the polypeptide shown in SEQ ID NO: 3.
  • the total dry matter yield control gene may contain additional sequences such as the nucleotide sequence of the untranslated region (UTR) in addition to the CDS of the tfw6.1 gene.
  • the method for obtaining (isolating) the gene for controlling total dry matter production is not particularly limited, but for example, a probe that specifically hybridizes with part of the base sequence of the gene for controlling total dry matter production is prepared. and screen a genomic DNA library or a cDNA library.
  • a method for obtaining the total dry matter production control gene a method using an amplification means such as PCR can be mentioned.
  • primers are prepared from the 5′-side and 3′-side sequences (or their complementary sequences) of the cDNA of the total dry matter production control gene, and genomic DNA (or cDNA) is obtained using these primers.
  • genomic DNA or cDNA
  • a large amount of DNA fragments containing the gene for controlling total dry matter production can be obtained by performing PCR or the like using this as a template and amplifying the DNA region sandwiched between the two primers.
  • the origin of the gene for controlling total dry matter production is not particularly limited as long as it is a plant of the Solanaceae family, but it is preferably tomato, potato, eggplant, or hot pepper, more preferably tomato.
  • the total dry matter production control gene can be derived from dicotyledonous plants, including plants of the Solanaceae family. Examples of dicotyledonous plants include Arabidopsis thaliana.
  • Whether or not the isolated candidate gene for controlling the total dry matter production has the activity of controlling the desired total dry matter production is determined by the expression of the candidate gene in a dicotyledonous plant. It can be evaluated by observing whether an increase in total dry matter production is induced compared to non-dicotyledonous plants.
  • Genes controlling total dry matter production can be used to elucidate the mechanism of increasing total dry matter production in dicotyledonous plants.
  • the total dry matter production control gene can be used to prepare a transformant by inserting the sequence into an expression vector or the like and introducing it into a dicotyledonous plant or cell.
  • Dicotyledonous plants with increased total dry matter production can be obtained by cultivating dicotyledonous plants into which a gene for controlling total dry matter production is introduced.
  • the tomato S As a gene for controlling total dry matter production, the tomato S.
  • SEQ ID NO: 4 The first gene
  • SEQ ID NO: 5 the second gene
  • SEQ ID NO: 6 the third gene
  • the third gene which are the CDS nucleotide sequences of the tfw6.1 gene derived from Lycopersicum. Nucleotides.
  • SEQ ID NO: 6 the CDS nucleotide sequences of the tfw6.1 gene derived from Lycopersicum. Nucleotides.
  • the total dry matter production control protein is the protein described in [1.
  • Total dry matter production control gene which is a translation product of the gene described in the column and has at least total dry matter production control activity. If the total dry matter production regulating protein has activity that positively regulates total dry matter production, its presence will increase total dry matter production. In the absence of the total dry matter production control protein, the total dry matter production is reduced or not increased more than in the presence of the total dry matter production control protein.
  • the total dry matter production control protein may be isolated from natural sources or chemically synthesized. More specifically, the proteins are naturally occurring purified products, products of chemical synthetic procedures, and prokaryotic or eukaryotic hosts (e.g., bacterial cells, yeast cells, higher plant cells, insect cells, and mammalian cells). It includes translation products produced by recombinant technology from cells (including animal cells).
  • prokaryotic or eukaryotic hosts e.g., bacterial cells, yeast cells, higher plant cells, insect cells, and mammalian cells. It includes translation products produced by recombinant technology from cells (including animal cells).
  • the total dry matter production control protein is a protein according to any one of (1') to (9') below: (1') a protein consisting of the amino acid sequence shown in SEQ ID NO: 1; (2') a protein consisting of an amino acid sequence having a sequence identity of 80% or more to the amino acid sequence shown in SEQ ID NO: 1 and having the function of controlling the total dry matter production of Solanaceae plants; (3') A protein consisting of an amino acid sequence in which 65 or less amino acids are substituted, deleted, added, or inserted with respect to the amino acid sequence shown in SEQ ID NO: 1, and having the function of controlling the total dry matter production of a solanaceous plant.
  • the proteins (1′), (4′), and (7′) are proteins encoded by the tfw6.1 gene or a gene corresponding to the tfw6.1 gene in Solanaceae plants, and total dry matter It is a protein with production control activity.
  • sequence identity of the amino acid sequences is preferably 80% or more, or 90% or more, preferably 95% or more. More preferably, 96% or more, 97% or more, 98% or more, or 99% or more is particularly preferable.
  • the number of substituted, deleted, added or inserted amino acids in the amino acid sequence of SEQ ID NO: 1 is preferably 1 to 65, 1 to 60, 1 to 50. , 1 to 40, more preferably 1 to 30, more preferably 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 are particularly preferred.
  • the number of substituted, deleted, added or inserted amino acids in the amino acid sequence of SEQ ID NO: 2 is preferably 1 to 30, 1 to 25, 1 to 20. , more preferably 1 to 15, more preferably 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 are particularly preferred.
  • the number of substituted, deleted, added or inserted amino acids in the amino acid sequence of SEQ ID NO: 3 is preferably 1 to 60, 1 to 50, 1 to 40. , more preferably 1 to 30, more preferably 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3 or 1 to 2 are particularly preferred.
  • mutant protein derived from tomato or a homologous protein derived from a solanaceous plant other than tomato is the above (2'), (3'), (5'), (6'), (8') and (9') included in the category of proteins.
  • mutant proteins and homologous proteins are proteins encoded by endogenous genes of Solanaceae plants.
  • the total dry matter production control protein is a polypeptide composed of peptide bonds of amino acids, but it may contain structures other than polypeptides.
  • structures other than polypeptides herein include sugar chains, isoprenoid groups, and the like, but are not limited thereto.
  • the total dry matter production control protein can be a protein from a dicotyledonous plant, including plants of the family Solanaceae, an example of a dicotyledonous plant being Arabidopsis thaliana.
  • Expression vectors, cells, and transformants An expression vector into which the gene for controlling total dry matter production according to one aspect of the present invention has been incorporated, a cell containing the expression vector or the gene for controlling total dry matter production, and the expression vector or the gene for controlling total dry matter production can be expressed.
  • the introduced transformants are also included in the scope of the present invention.
  • the expression vector imparts a trait that controls the total dry matter production to cells or organisms.
  • the type of vector that constitutes the expression vector is not particularly limited, and one that can be expressed in the host cell may be selected as appropriate. That is, an appropriate promoter sequence is selected according to the type of host cell, and the promoter sequence and the total dry matter production control gene are integrated into, for example, a plasmid, phagemid, cosmid, or the like, and used as an expression vector. .
  • Examples of host cells into which expression vectors are introduced include bacterial cells, yeast cells, fungal cells other than yeast cells, and higher eukaryotic cells.
  • Bacterial cells include, for example, E. coli cells.
  • Higher eukaryotic cells include, for example, plant cells and animal cells.
  • Plant cells include, for example, dicotyledonous and monocotyledonous plant cells.
  • Dicotyledonous plant cells include, for example, suspension cultured cells of Solanaceae plants (eg, tobacco BY-2 strain and tomato Sly-1 strain).
  • Monocotyledonous plant cells include, for example, the Oc strain, which is a suspension-cultured cell of rice.
  • Animal cells include insect cells, amphibian cells, reptile cells, avian cells, fish cells, mammalian cells and the like.
  • the total dry matter production control gene is functionally linked to the elements required for transcription (eg, promoter, etc.). Also, if necessary, enhancers, selectable markers, splicing signals, poly-A addition signals, 5'-UTR sequences, etc. may be ligated.
  • a promoter is a DNA sequence that exhibits transcriptional activity in host cells, and can be appropriately selected according to the type of host.
  • Promoter sequences that are operable in host cells include the cauliflower mosaic virus 35S promoter, Agrobacterium nopaline synthase gene promoter and rice ubiquitin gene promoter.
  • the sequence of the promoter region in the total dry matter production control gene may be used.
  • the total dry matter production control gene may be functionally linked to an appropriate terminator (eg, NOS terminator and cauliflower mosaic virus 35S terminator), if necessary.
  • an appropriate terminator eg, NOS terminator and cauliflower mosaic virus 35S terminator
  • the type of appropriate terminator may be appropriately selected according to the type of host cell, as long as it is a sequence capable of terminating the transcription of the gene transcribed by the above promoter.
  • Enhancers are used to increase the efficiency of expression of target genes, and include, for example, the omega sequence of tobacco mosaic virus.
  • the expression vector may further contain a selectable marker.
  • Selectable markers can include, for example, drug resistance genes such as ampicillin, kanamycin, tetracycline, chloramphenicol, neomycin, hygromycin or spectinomycin.
  • the total dry matter production control gene may be linked to a suitable tag sequence for protein purification or a suitable spacer sequence, if necessary.
  • the transformant is meant to include not only cells, tissues, and organs into which the expression vector or the gene for controlling total dry matter production is introduced so as to be expressible, but also individual organisms.
  • Such transformants may be Solanaceae plants.
  • the transformant may be, for example, a microorganism such as E. coli, an animal, or the like.
  • a method for determining the degree of total dry matter production in a solanaceous plant according to one aspect of the present invention is to determine whether the total dry matter production is increasing or not increasing in a solanaceous plant. do.
  • the discrimination method can be used to discriminate individuals with increased total dry matter production in Solanaceous plants.
  • the discrimination method is to determine the genotype of the gene involved in the determination of the total dry matter production, present in the genome of the solanaceous plant, to discriminate the solanaceous plant based on the total dry matter production.
  • the concept of total dry matter production is defined as total dry matter production, which indicates that the total dry matter production of a certain solanaceous plant individual is relatively large or small compared to other solanaceous plant individuals. includes the degree of
  • solanaceous plants such as tomatoes
  • total dry matter production is thought to increase gradually with growth, peak during the continuous production period, and then level off (Reference 1: Saito et al. Hort J 89 : 445-453, 2020). That is, at the early stage of cultivation when the bunches start to bear fruit or when the stems start to grow, the total dry matter production is small. Therefore, the yield can be stably increased by increasing the total dry matter production especially in the early stage of cultivation such as the fruiting period or the stem enlargement period.
  • Solanaceous plants can be candidate plants for breeding material or plants obtained through the process of breeding.
  • Candidate plants for breeding materials include, for example, parent plants used for crossing and plants used for molecular breeding using gene recombination technology.
  • Plants obtained through the process of breeding include, for example, plants obtained by intraspecific crossing of tomato, potato, eggplant, or capsicum belonging to the family Solanaceae, and progeny lines thereof.
  • the solanaceous plant may be a cross-cultivated plant such as a hybrid plant of a tomato belonging to a certain variety and a tomato belonging to another variety, and its progeny.
  • the method for determining the level of total dry matter production in solanaceous plants can be used to determine the level of total dry matter production in dicotyledonous plants, including plants of the Solanaceae family. Examples include Arabidopsis thaliana.
  • solanaceous plant may be a plant obtained by crossing cultivars known to increase total dry matter production, and its progeny.
  • solanaceous plants are plants obtained by crossing a cultivar known to increase total dry matter production with a cultivar whose total dry matter production is unknown, and even if it is a progeny line. good.
  • the solanaceous plant may be a plant obtained by crossing between cultivars whose total dry matter production is unknown or unknown, and its progeny.
  • solanaceous plants are plants obtained by crossing cultivars known to increase total dry matter production with cultivars known to have no increase in total dry matter production, and their progeny. There may be.
  • the solanaceous plant may be a plant obtained by crossing individuals known to increase total dry matter production, and its progeny.
  • plant may refer to part or all of a plant body.
  • the part of the plant body includes, for example, propagation material (eg, leaves, branches, seeds, etc.) and the like.
  • the determination method is the protein according to any one of the following (1′) to (9′) in Solanaceae plants: (1') a protein consisting of the amino acid sequence shown in SEQ ID NO: 1; (2') a protein consisting of an amino acid sequence having a sequence identity of 80% or more to the amino acid sequence shown in SEQ ID NO: 1 and having the function of controlling the total dry matter production of Solanaceae plants; (3') A protein consisting of an amino acid sequence in which 65 or less amino acids are substituted, deleted, added, or inserted with respect to the amino acid sequence shown in SEQ ID NO: 1, and having the function of controlling the total dry matter production of a solanaceous plant.
  • the mutation that affects the function of a protein that has the function of controlling the total dry matter production of a solanaceous plant can be a mutation that causes the protein to express the function of increasing the total dry matter production of the solanaceous plant.
  • the discrimination method by examining the presence or absence of such mutations, it is discriminated whether or not the total dry matter production is increasing in Solanaceous plants.
  • a stop in a polynucleotide encoding any of the proteins (1′) to (9′) in which the mutation that affects the function of the protein that has the function of regulating the total dry matter production of a solanaceous plant It may be a mutation selected from the group consisting of codon mutations, frameshift mutations, and null mutations.
  • the presence or absence of mutations in the total dry matter production control gene can be tested using the molecular markers shown below. Such molecular markers are also included in the scope of the present invention.
  • Molecular markers are the following amino acids (a), (b), (e) to (h) in Solanaceae plants: (a) an amino acid corresponding to the 109th amino acid of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1; (b) an amino acid corresponding to the 247th amino acid of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1; (e) an amino acid corresponding to the 133rd amino acid of the protein consisting of the amino acid sequence shown in SEQ ID NO: 2; (f) an amino acid corresponding to the 139th amino acid of the protein consisting of the amino acid sequence shown in SEQ ID NO: 2; (g) an amino acid corresponding to the 134th amino acid of the protein consisting of the amino acid sequence shown in SEQ ID NO: 3; (h) an amino acid corresponding to the 175th amino acid of the protein consisting of the amino acid sequence shown in SEQ ID NO: 3; is a molecular marker that tests for mutations that cause substitutions, deletions,
  • a protein consisting of the amino acid sequence shown in any one of SEQ ID NOs: 1 to 3 is a protein encoded by a total dry matter production control gene, and for example, a gene consisting of a nucleotide sequence shown in any one of SEQ ID NOs: 4 to 6. is the encoded protein.
  • the protein consisting of the amino acid sequence shown in any one of SEQ ID NOs: 1 to 3 consists of the amino acid sequence of the protein encoded by the standard tomato (S. lycopersicum)-derived tfw6.1 gene.
  • portions corresponding to the above amino acids (a), (b), (e) to (h) for the amino acid sequence of the protein encoded by the reference tomato-derived tfw6.1 gene It may also contain portions with different amino acid sequences.
  • the amino acid positions corresponding to the above amino acids (a), (b), (e) to (h) can be identified by a technique such as homology analysis.
  • amino acid corresponding to the amino acid of refers to an amino acid determined to correspond to the amino acid of (a), (b), (e) to (h) by a method such as homology analysis.
  • Homology analysis methods include, for example, a method by Pairwise Sequence Alignment such as the Needleman-Wunsch method and the Smith-Waterman method, and a method by Multiple Sequence Alignment such as the ClustalW method. Based on this, it is possible to understand the "corresponding amino acid" in the amino acid sequence to be analyzed using the amino acid sequence shown in any of SEQ ID NOs: 1 to 3 as a reference sequence.
  • homologous protein of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1 and the amino acid corresponding to the amino acid (a) or (b) in Solanaceae plants is shown.
  • the amino acid corresponding to (a) is the 351st amino acid
  • the amino acid corresponding to (b) is the 489th amino acid.
  • the amino acid corresponding to the amino acid in (a) is the 333rd amino acid
  • the amino acid corresponding to the amino acid in (b) is It is the 471st amino acid.
  • a protein of Arabidopsis thaliana is shown as an example of a protein homologous to the protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in dicotyledonous plants including solanaceous plants.
  • Arabidopsis thaliana (AT5G48150.1) protein consisting of the amino acid sequence shown in SEQ ID NO: 10, the amino acid corresponding to (a) is the 268th amino acid, and the amino acid corresponding to (b) is the 406th amino acid. is.
  • amino acid corresponding to the amino acid (a) or (b) is, for example, (1) the amino acid (a) or (b) in the protein consisting of the amino acid sequence shown in SEQ ID NO: 1, (2) SEQ ID NO: 1 or (3) the amino acid sequence shown in SEQ ID NO: 1
  • 65 or less amino acids are amino acids corresponding to the amino acids (a) or (b) in a protein consisting of an amino acid sequence having substitutions, deletions, additions or insertions.
  • amino acid corresponding to the amino acid (e) or (f) is, for example, (4) the amino acid (e) or (f) in the protein consisting of the amino acid sequence shown in SEQ ID NO: 2, (5) SEQ ID NO: 2 The amino acid corresponding to the amino acid (e) or (f) in a protein consisting of an amino acid sequence having a sequence identity of 80% or more to the amino acid sequence shown in , or (6) the amino acid sequence shown in SEQ ID NO: 2 On the other hand, 30 or less amino acids are amino acids corresponding to the amino acids (e) or (f) in a protein consisting of an amino acid sequence having substitutions, deletions, additions or insertions.
  • homologous protein of the protein consisting of the amino acid sequence shown in SEQ ID NO: 3 and the amino acid corresponding to the amino acid (g) or (h) in Solanaceae plants is shown.
  • the amino acid corresponding to the amino acid (g) is the 134th amino acid
  • the amino acid corresponding to the amino acid (h) is the 175th amino acid.
  • the amino acid corresponding to the amino acid (g) is the 132nd amino acid
  • the amino acid corresponding to the amino acid (h) is the 173rd amino acid.
  • the amino acid corresponding to the amino acid (g) is the 132nd amino acid
  • the amino acid corresponding to the amino acid (h) is the 173rd amino acid.
  • the amino acid corresponding to the amino acid (g) is the 133rd amino acid
  • the amino acid corresponding to the amino acid (h) is the 174th amino acid.
  • the amino acid corresponding to the amino acid (g) is the 133rd amino acid
  • the amino acid corresponding to the amino acid (h) is the 174th amino acid.
  • a protein of Arabidopsis thaliana is shown as an example of a protein homologous to the protein consisting of the amino acid sequence shown in SEQ ID NO: 3 in dicotyledonous plants including solanaceous plants.
  • Arabidopsis thaliana (AT5G52660.2) protein consisting of the amino acid sequence shown in SEQ ID NO: 16
  • the amino acid corresponding to the amino acid (g) is the 153rd amino acid
  • the amino acid corresponding to the amino acid (h) is the 194th amino acid.
  • amino acid corresponding to the amino acid (g) or (h) is, for example, (7) the amino acid (g) or (h) in the protein consisting of the amino acid sequence shown in SEQ ID NO: 3, (8) SEQ ID NO: 3 or (9) the amino acid sequence shown in SEQ ID NO:3
  • 60 or less amino acids are amino acids corresponding to amino acids (g) or (h) in a protein consisting of an amino acid sequence with substitution, deletion, addition or insertion.
  • An example of a molecular marker is the base itself (SNP) corresponding to the following bases (a') to (h') in Solanaceae plants, or a continuous polynucleotide containing the base: (a') a base corresponding to the 326th base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 4; (b') a base corresponding to the 390th base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 4; (c') a base corresponding to the 591st base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 4; (d') a base corresponding to the 740th base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 4; (e') a base corresponding to the 397th base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 5; (f
  • the bases corresponding to the bases (a') to (h') above are SNPs that cause amino acid substitutions.
  • the above (a') is an SNP that causes a mutation selected from the group consisting of stop codon mutation, frameshift mutation, and null mutation.
  • (b') and (c') above are SNPs that cause synonymous substitutions (silent mutations).
  • the proteins (1) to (9) above express the function of controlling the total dry matter production in Solanaceous plants. can detect the presence or absence of mutations that affect
  • mutations that cause substitution, deletion, addition or insertion of the amino acids corresponding to the above (a) to (h) Presence or absence can be detected.
  • molecular markers examples include SNP markers, AFLP (molecular amplified fragment length polymorphism) markers, RFLP markers, microsatellite markers, SCAR markers, and CAPS markers.
  • the bases themselves corresponding to the bases (a') to (h') above, or the continuous polynucleotides containing the bases, are the SNP markers described in this example or SNP markers that can be identified with them.
  • a SNP marker can be (i) the base corresponding to the SNP itself, (ii) a contiguous polynucleotide containing the SNP, or (iii) a contiguous polynucleotide containing two SNPs.
  • SNP means a DNA polymorphism in which a single base mutation is found in a specific region in the base sequence of DNA.
  • SNP markers SNPs are single nucleotide polymorphisms based on the genome sequence of tomato (S. lycopersicum). The genome sequence of the reference plant tomato (S. lycopersicum) has been published on the solgenomics FTP site (ftp://ftp.solgenomics. net/genomes/Solanum_lycopersicum/annotation/ITAG4.1_release/).
  • (a') to (h') bases are the SNP markers described in this example.
  • the “bases corresponding to the bases (a′) to (h′)” are SNP markers that can be identified with the SNP markers described in this example.
  • “(a') to (h') bases” are mutations on the tfw6.1 gene.
  • the tfw6.1 gene consists of a standard base sequence derived from tomato (S. lycopersicum). Solanaceous plants may contain portions having different nucleotide sequences than SNPs with respect to the reference nucleotide sequence. Since the region on the genome where this SNP marker is located is conserved among many Solanaceae plants, this SNP marker can be identified by techniques such as homology analysis. As a method for homology analysis, the same method as for specifying the "corresponding amino acid" can be used.
  • base corresponding to the base of ⁇ (h') refers to the base on the gene corresponding to the tfw6.1 gene, which was determined to correspond to the base of (a') ⁇ (h') by a method such as homology search. Point.
  • polynucleotides described in (a2) to (h2) and polynucleotides described in (a3) to (h3) below are examples of genes corresponding to the tfw6.1 gene.
  • the base corresponding to (a') is the 1052nd base
  • the base corresponding to (b') is the 1116th base
  • the base corresponding to the base of (c′) is the 1317th base
  • the base corresponding to the base of (d′) is the 1466th base.
  • the base corresponding to the base (a') is the 998th base, and corresponds to the base (b').
  • the base is the 1062nd base
  • the base corresponding to (c') is the 1263rd base
  • the base corresponding to (d') is the 1412nd base.
  • the base corresponding to (a') is the 1010th base
  • the base corresponding to (b') is the 1074th base
  • the base corresponding to the base of (c') is the 1275th base
  • the base corresponding to the base of (d') is the 1424th base.
  • the gene of Arabidopsis thaliana is shown as an example of a gene homologous to the gene consisting of the nucleotide sequence shown in SEQ ID NO: 4 in dicotyledonous plants including solanaceous plants.
  • Arabidopsis thaliana (AT5G52660.2) gene consisting of the nucleotide sequence shown in SEQ ID NO: 20
  • the base corresponding to (a') is the 803rd base
  • the base corresponding to (b') is the 867th base.
  • the base corresponding to the base of (c') is the 1068th base
  • the base corresponding to the base of (d') is the 1217th base.
  • the base corresponding to (g') is the 401st base
  • the base corresponding to (h') is the 523rd base.
  • the gene of Arabidopsis thaliana is shown as an example of a gene homologous to the gene consisting of the nucleotide sequence shown in SEQ ID NO: 6 in dicotyledonous plants including solanaceous plants.
  • Arabidopsis thaliana (AT5G52660.2) gene consisting of the nucleotide sequence shown in SEQ ID NO: 26
  • the base corresponding to (g') is the 458th base
  • the base corresponding to (h') is the 580th base. is the base of
  • the molecular marker is a SNP marker consisting of the SNPs (a') to (h') above.
  • This SNP marker is a SNP marker newly identified by the present inventors, and a person skilled in the art can identify the position of the SNP marker on the genome based on the nucleotide sequence representing each SNP of this SNP marker. can.
  • the SNP of (a') indicates a polymorphism of the 326th base of the base sequence shown in SEQ ID NO: 4 or a base corresponding thereto.
  • the SNP of (b') indicates a polymorphism of the 390th base in the base sequence shown in SEQ ID NO: 4 or a base corresponding thereto.
  • the SNP of (c') indicates a polymorphism of the 591st base in the base sequence shown in SEQ ID NO: 4 or a base corresponding thereto.
  • the SNP of (d') indicates a polymorphism of the 740th base in the base sequence shown in SEQ ID NO: 4 or a base corresponding thereto.
  • the SNP (e') indicates a polymorphism of the 397th base in the base sequence shown in SEQ ID NO: 5 or a base corresponding thereto.
  • the SNP of (f') indicates a polymorphism of the 417th base in the base sequence shown in SEQ ID NO: 5 or a base corresponding thereto.
  • the SNP of (g') indicates a polymorphism of the 401st base in the base sequence shown in SEQ ID NO: 6 or a base corresponding thereto.
  • the SNP of (h') indicates a polymorphism of the 523rd base in the base sequence shown in SEQ ID NO: 6 or a base corresponding thereto.
  • Molecular markers are at least one of the following: SNP (a') is G; SNP (b') is A; SNP (c') is T; SNP (d') is G; SNP (e') is G; SNP (f') is T; SNP (g') is C; SNP (h') is T; When , it can be determined that the solanaceous plant has increased total dry matter production.
  • SNP (a') to SNP (d'), SNP (e') and SNP (f'), or SNP (g') and SNP (h') are analyzed as haplotype blocks. may determine total dry matter production in solanaceous plants.
  • the expression that the solanaceous plant has increased total dry matter production means that the total dry matter production of the individual solanaceous plant having the total dry matter production control protein is higher than the total dry matter production of the individual solanaceous plant having the total dry matter production control protein. It is intended to be relatively large compared to Solanaceae plant individuals.
  • a molecular marker may be a contiguous polynucleotide containing SNP(a') to SNP(h') (hereinafter, polynucleotide (a') to polynucleotide (h')).
  • Polynucleotide (a') is (a1) a polynucleotide consisting of the nucleotide sequence of the region containing SNP (a') in the nucleotide sequence of the tfw6.1 gene, (a2) in the nucleotide sequence of the polynucleotide of (a1),
  • a poly which consists of a nucleotide sequence in which one or several nucleotides are substituted, deleted, added or inserted into a nucleotide sequence other than SNP (a') and has the function of determining the total dry matter production in Solanaceous plants Total dry matter in Solanaceous plants, consisting of a nucleotide sequence having 90% or more identity to a nucleotide sequence other than SNP (a') in the nucleotide sequence of the polynucleotide or (a3) (a1) It is a polynucleotide having a function to determine the amount of production.
  • Polynucleotide (b') is (b1) a polynucleotide consisting of the nucleotide sequence of the region containing SNP (b') in the nucleotide sequence of the tfw6.1 gene, (b2) in the nucleotide sequence of the polynucleotide of (b1),
  • a poly which consists of a nucleotide sequence in which one or several nucleotides are substituted, deleted, added or inserted into a nucleotide sequence other than SNP (b') and has the function of determining the total dry matter production in Solanaceous plants Total dry matter in Solanaceous plants, consisting of a nucleotide sequence having 90% or more identity to a nucleotide sequence other than SNP (b') in the nucleotide sequence of the polynucleotide or (b3) (b1) It is a polynucleotide having a function to determine the amount of production.
  • Polynucleotide (c') is a polynucleotide consisting of the nucleotide sequence of the region containing SNP (c') in the nucleotide sequence of (c1) the tfw6.1 gene, (c2) in the nucleotide sequence of the polynucleotide of (c1),
  • Polynucleotide (d') is (d1) a polynucleotide consisting of the nucleotide sequence of the region containing SNP (d') in the nucleotide sequence of the tfw6.1 gene, (d2) in the nucleotide sequence of the (d1) polynucleotide, A poly which consists of a base sequence in which one or several bases are substituted, deleted, added or inserted into a base sequence other than SNP (d') and has the function of determining the total dry matter production in Solanaceous plants Total dry matter in Solanaceous plants, consisting of a nucleotide sequence having 90% or more identity to a nucleotide sequence other than SNP (d') in the nucleotide sequence of the polynucleotide or (d3) (d1) It is a polynucleotide having a function to determine the amount of production.
  • Polynucleotide (e') is (e1) a polynucleotide consisting of the nucleotide sequence of the region containing SNP (e') in the nucleotide sequence of the tfw6.1 gene, (e2) in the polynucleotide nucleotide sequence of (e1),
  • Polynucleotide (f') is (f1) a polynucleotide consisting of the nucleotide sequence of the region containing SNP (f') in the nucleotide sequence of the tfw6.1 gene, (f2) in the nucleotide sequence of the (f1) polynucleotide, A poly which consists of a nucleotide sequence in which one or several nucleotides are substituted, deleted, added or inserted into a nucleotide sequence other than SNP (f') and has the function of determining the total dry matter production in Solanaceous plants Total dry matter in Solanaceae plants consisting of a nucleotide sequence having 90% or more identity to a nucleotide sequence other than SNP (f') in the nucleotide sequence of the polynucleotide or (f3) (f1) It is a polynucleotide having a function to determine the amount of production.
  • Polynucleotide (g') is (g1) a polynucleotide consisting of the nucleotide sequence of the region containing SNP (g') in the nucleotide sequence of the tfw6.1 gene, (g2) in the nucleotide sequence of the (g1) polynucleotide, A poly which consists of a base sequence in which one or several bases are substituted, deleted, added or inserted into a base sequence other than SNP (g') and has the function of determining the total dry matter production in Solanaceous plants Total dry matter in Solanaceae plants consisting of a nucleotide sequence having 90% or more identity to a nucleotide sequence other than SNP (g') in the nucleotide sequence of the polynucleotide or (g3) (g1) It is a polynucleotide having a function to determine the amount of production.
  • Polynucleotide (h') is (h1) a polynucleotide consisting of the nucleotide sequence of the region containing SNP (h') in the nucleotide sequence of the tfw6.1 gene, (h2) in the nucleotide sequence of the (h1) polynucleotide, A poly which consists of a nucleotide sequence in which one or several nucleotides are substituted, deleted, added or inserted into a nucleotide sequence other than SNP (h') and has the function of determining the total dry matter production in Solanaceous plants Total dry matter in Solanaceae plants, consisting of a nucleotide sequence having 90% or more identity to a nucleotide sequence other than SNP (h') in the nucleotide sequence of the polynucleotide or (h3) (h1) It is a polynucleotide having a function to determine the amount of production.
  • Polynucleotides (a1) to (h1) are, for example, based on the base sequence of the tfw6.1 gene, obtained from solanaceous plants with increased total dry matter production (for example, tomatoes with high total dry matter production (S. lycopersicum Dutch F1 cultivar).
  • the nucleotide sequences of such polynucleotides are obvious to those skilled in the art, and by referring to the genome sequence of tomato (S. lycopersicum) registered in the database mentioned above, or by referring to the total dry matter production It can be determined by decoding the base sequence of the neighboring region of the SNP on the genome of a solanaceous plant with a large number of plants.
  • Polynucleotide (a3) to polynucleotide (h3) in the nucleotide sequence of polynucleotide (a1) to (h1), bases corresponding to SNP (a') to SNP (h') are conserved and have, for example, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to other base sequences.
  • the identity of such nucleotide sequences can be determined, for example, by aligning two nucleotide sequences using analysis software such as BLAST and FASTA.
  • Molecular markers are at least one of the following: the base corresponding to SNP (a') in polynucleotide (a) is G; The base corresponding to SNP (b') in polynucleotide (b) is A; the base corresponding to SNP (c') in polynucleotide (c) is T; the base corresponding to SNP (d') in polynucleotide (d) is G; the base corresponding to SNP (e') in polynucleotide (e) is G; the base corresponding to SNP (f') in polynucleotide (f) is T; the base corresponding to SNP (g') in polynucleotide (g) is C; the base corresponding to SNP (h') in polynucleotide (h) is T; When , it can be determined that the solanaceous plant has increased total dry matter production.
  • Molecular markers are obtained by analyzing polynucleotides (a) to (d), polynucleotides (e) and polynucleotides (f), or polynucleotides (g) and polynucleotides (h) as haplotype blocks.
  • the total dry matter production in the solanaceous plant may be determined.
  • a molecular marker may be a contiguous polynucleotide comprising at least two of SNP(a') through SNP(d').
  • a polynucleotide includes the region between sites SNP(a') to SNP(d') together with sites SNP(a') to SNP(d').
  • Such polynucleotides can be obtained, for example, by referring to the region between the corresponding SNP(a') to SNP(d') sites in Solanaceae plants with increased total dry matter production.
  • the nucleotide sequence of such a polynucleotide at least partially matches the nucleotide sequence of a solanaceous plant with increased total dry matter production.
  • a molecular marker may be a continuous polynucleotide containing SNP (e') and SNP (f').
  • a polynucleotide includes the region between sites of SNP(e') and SNP(f') together with sites of SNP(e') and SNP(f').
  • Such polynucleotides can be obtained, for example, by referring to the region between the corresponding SNP (e') and SNP (f') sites in Solanaceae plants with increased total dry matter production.
  • the nucleotide sequence of such a polynucleotide at least partially matches the nucleotide sequence of a solanaceous plant with increased total dry matter production.
  • a molecular marker may be a continuous polynucleotide containing SNP (g') and SNP (h').
  • a polynucleotide includes the region between sites of SNP(g') and SNP(h') together with sites of SNP(g') and SNP(h').
  • Such polynucleotides can be obtained, for example, by referring to the region between the corresponding SNP (g') and SNP (h') sites in Solanaceae plants with increased total dry matter production.
  • the nucleotide sequence of such a polynucleotide at least partially matches the nucleotide sequence of a solanaceous plant with increased total dry matter production.
  • the method for determining the total dry matter production of Solanaceae plants using the above-described molecular markers is not particularly limited, and for example, a known SNP analysis method for detecting SNPs can be used.
  • a known SNP analysis method for detecting SNPs include a method of SNP analysis by detecting SNPs in PCR-amplified fragments of test samples of Solanaceae plants.
  • a primer set that amplifies a region containing a molecular marker may be used to amplify the region in the DNA of the Solanaceae plant.
  • a primer set is, for example, a primer set that amplifies a region containing at least one of SNP(a') to SNP(h').
  • Amplification of the region in the DNA of the subject of the solanaceous plant is carried out by polymerase chain reaction (PCR) using the DNA extracted from the subject of the solanaceous plant as a template and primers that amplify the region containing the SNP. be able to. Then, the base (genotype) of the SNP in the obtained amplified fragment is determined, and based on the data showing the relationship between the determined base (genotype) and the total dry matter production amount in the solanaceous plant, Determine total dry matter production.
  • PCR polymerase chain reaction
  • the primer set used in PCR is not particularly limited as long as it can amplify the DNA fragment in the region containing the target SNP, and the primer set may be designed to shorten the length of the amplified fragment.
  • the primer set is designed such that the length of the primer-amplified fragment is preferably 700 base pairs (bp) or less, 200 bp or less, 150 bp or less, 120 bp or less, or 100 bp 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 bp or more, 16 bp or more, 17 bp or more, 18 bp or more, or 19 bp or more, or 50 base bp or less, 40 bp or less, or 30 bp or less.
  • a primer set that amplifies a region containing SNP (c′) and SNP (d′) includes, for example, a first primer containing 15 or more consecutive bases in the nucleotide sequence shown in SEQ ID NO: 27, and and a second primer containing 15 or more consecutive bases in the indicated nucleotide sequence.
  • a PCR amplification product consisting of the nucleotide sequence shown in SEQ ID NO: 29 was obtained in Solanaceae plants with increased total dry matter production, corresponding to SNP (c') and SNP (d'). bases can be detected.
  • a primer set that amplifies a region containing SNP (a'), SNP (b') and SNP (c') is, for example, a third primer containing 15 or more consecutive bases in the nucleotide sequence shown in SEQ ID NO: 30 and a fourth primer containing 15 or more consecutive bases in the nucleotide sequence shown in SEQ ID NO:31.
  • PCR amplification products consisting of the nucleotide sequence shown in SEQ ID NO: 32 are obtained in Solanaceae plants with increased total dry matter production, SNP (a'), SNP (b') and SNP A base corresponding to (c') can be detected.
  • a primer set that amplifies a region containing SNP (e′) and SNP (f′) includes, for example, a fifth primer containing 15 or more consecutive bases in the nucleotide sequence shown in SEQ ID NO: 33, and and a sixth primer containing 15 or more consecutive bases in the indicated nucleotide sequence.
  • a PCR amplification product consisting of the nucleotide sequence shown in SEQ ID NO: 35 was obtained in a Solanaceae plant with increased total dry matter production, corresponding to SNP (e') and SNP (f'). bases can be detected.
  • a primer set for amplifying a region containing SNP (g') and SNP (h') includes, for example, a seventh primer containing 15 or more consecutive bases in the nucleotide sequence shown in SEQ ID NO: 36, and and an eighth primer containing 15 or more consecutive bases in the indicated nucleotide sequence.
  • a PCR amplification product consisting of the nucleotide sequence shown in SEQ ID NO: 38 was obtained in a Solanaceae plant with increased total dry matter production, corresponding to SNP (g') and SNP (h'). bases can be detected.
  • PCR in SNP analysis may be either singleplex PCR that amplifies DNA fragments in a reaction system containing a single primer set, or multiplex PCR that amplifies genes in a reaction system containing multiple primer sets.
  • primer sets labeled with fluorescent substances having different wavelengths eg, NED, 6-FAM, VIC, PET
  • the PCR reaction conditions can be appropriately set according to the type of DNA polymerase and PCR equipment used, the length of the amplified fragment, and the like.
  • As the cycle conditions a 3-step PCR method in which 3 steps of denaturation step, annealing step and extension step are used as one cycle, and a 2-step PCR method in which 2 steps of denaturation step, annealing and extension step are used as 1 cycle are applied. can do.
  • An example of PCR reaction conditions is 90-100° C. for 40-60 seconds (eg, 95° C. for 50 seconds), followed by 90-100° C.
  • Annealing temperature includes a condition in which the initial annealing temperature is 60 to 70° C. (eg, 66° C.) and the final annealing temperature is 50 to 60° C. (eg, 56° C.), and the temperature is lowered stepwise in each predetermined cycle.
  • PCR reaction conditions may be adjusted in order to stably detect SNPs according to the condition of the template DNA.
  • Real-time such as TaqMan (registered trademark)-PCR method, Tm-shift genotyping method (Fukuoka et al. Breed Sci 58: 461-464, 2008) for amplification by PCR and identification of SNP markers as PCR in SNP analysis PCR may also be used. That is, TaqMan (registered trademark) probes that detect SNPs contained in amplified fragments amplified using a primer set may be further used.
  • a high-throughput discrimination method can be provided.
  • analysis may be performed by determining the base sequence of amplified fragments using an automatic DNA sequencer or the like.
  • the method for extracting DNA to be amplified by PCR from a specimen of a solanaceous plant is not particularly limited, and a known DNA extraction method can be used.
  • DNA may be extracted using a commercially available DNA extraction kit. Before the DNA extraction step, appropriate pretreatment may be performed depending on the type of specimen, the amount of contaminants, and the like.
  • the DNA extracted from the subject may be washed or purified as necessary for use as a template in PCR reactions.
  • restriction enzyme cleavage fragments obtained by digesting DNA extracted from a subject with two types of restriction enzymes may be amplified by PCR.
  • linkage disequilibrium state is, for example, a linkage disequilibrium state with a linkage disequilibrium coefficient of 0.9 or more.
  • the determination method it is possible to determine whether or not the subject Solanaceae plant has increased total dry matter production using the molecular marker. Therefore, it is possible to select solanaceous plants and their progeny lines with increased total dry matter production based on the determination results.
  • a solanaceous plant with increased total dry matter production is a plant obtained by the production method described below.
  • a solanaceous plant with increased total dry matter production is a plant having the above-described SNP identified by the above-described molecular marker and having increased total dry matter production.
  • a solanaceous plant having an increased total dry matter production amount according to an aspect of the present invention is obtained by intraspecific hybridization of a solanaceous plant and its progeny line, as shown in the production method described later. It is obtained by identifying Solanaceous plants with increased total dry matter production using a marker. A solanaceous plant with an increased total dry matter production that is genetically engineered to have the above SNP is also included in the scope of the present invention. Furthermore, [6. Method for producing a solanaceous plant with increased total dry matter production], in a solanaceous plant, genome editing is performed so as to delete or inactivate the amino acid sequence downstream of the VHIID motif of the GRAS transcription factor.
  • Solanaceous plants with increased total dry matter production are also included in the scope of the present invention.
  • dicotyledonous plants including solanaceous plants, whose total dry matter production is increased by genome editing to delete or inactivate the amino acid sequence downstream of the VHIID motif of the GRAS transcription factor Included in the scope of the present invention.
  • dicotyledonous plants include Arabidopsis thaliana.
  • a production method is a method for producing a solanaceous plant with increased total dry matter production, comprising a hybridization step of intraspecifically hybridizing a solanaceous plant, and a solanaceous plant obtained by the hybridization step. and an identification step of identifying a solanaceous plant having an increased total dry matter production from the plants or progeny of the solanaceous plant by the above-described identification method.
  • solanaceous plants with increased total dry matter production and a description of methods for determining the degree of total dry matter production in solanaceous plants are used to produce solanaceous plants with increased total dry matter production. It is used for the explanation of the method of
  • At least one of the solanaceous plants used in the crossing step may be a solanaceous plant with increased total dry matter production according to one aspect of the present invention.
  • at least one of the solanaceous plants used in the crossing step is a solanaceous plant having an increased total dry matter production that has been selected by a method for determining the degree of total dry matter production of a solanaceous plant according to one embodiment of the present invention.
  • total dry matter is obtained from a test solanaceous plant by a method for determining the degree of total dry matter production in the solanaceous plant according to one aspect of the present invention. It may further comprise an identification step of identifying the solanaceous plant with increased production.
  • a production method includes an identification step of identifying a solanaceous plant having an increased total dry matter production from a test solanaceous plant, and a crossing step of intraspecifically hybridizing the identified solanaceous plant. . That is, the scope of the present invention also includes a production method including an identification step only before the hybridization step and a production method including an identification step before and after the hybridization step.
  • a manufacturing method that includes an identification step only before the hybridization step can be used, for example, as follows. By identifying a solanaceous plant with increased total dry matter production (having a homozygous total dry matter production control gene) before the crossing step, and using such a plant as a parent in F1 breeding, the resulting F1 plant All of them will have the total dry matter production control gene in heterozygotes.
  • a production method that includes an identification step only before the crossing step can also be used for such parental homogenization (selection and fixation) in F1 breeding.
  • the total dry matter production amount is determined from the solanaceous plant obtained by the crossing step or its progeny solanaceous plant by the method for determining the degree of the total dry matter production amount in the solanaceous plant according to one aspect of the present invention. identify solanaceous plants with increased
  • the degree of total dry matter production of a solanaceous plant is determined using a molecular marker, and a solanaceous plant having an increased total dry matter production is selected based on the determination result. can be done.
  • a production method is a method for producing a solanaceous plant with an increased total dry matter production amount, the solanaceous plant corresponding to a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 1.
  • a step of introducing a mutation that deletes or inactivates an amino acid sequence downstream of an amino acid at any position downstream of the amino acid corresponding to the 73rd amino acid in the amino acid sequence shown in SEQ ID NO: 1 in the polynucleotide in the plant contain.
  • methods for producing solanaceous plants with increased total dry matter production by genome editing are also included in the scope of the present invention.
  • a total dry matter production control gene consisting of a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 1 is predicted to encode a GRAS transcription factor.
  • a mutation is artificially introduced by genome editing at any position downstream of the VHIID motif of the GRAS transcription factor, and the amino acid sequence downstream of the VHIID motif is deleted. Solanaceous plants with increased total dry matter production can be produced.
  • the VHIID motif of the GRAS transcription factor is an amino acid sequence corresponding to the 69th to 73rd amino acid sequences in the tomato-derived protein consisting of the amino acid sequence shown in SEQ ID NO: 1 (Reference 2: Li et al. Plant Cell 28: 1025-1034, 2016). Therefore, the production method according to another aspect of the present invention includes the step of introducing a mutation that deletes or inactivates the amino acid sequence downstream of the amino acid at any position downstream of the amino acid corresponding to the 73rd amino acid. contain. Mutations that delete or inactivate amino acid sequences include, for example, stop codon mutations, frameshift mutations, null mutations, and the like.
  • VHIID motifs of GRAS transcription factors in other solanaceous and dicotyledonous plants are as follows.
  • the amino acid sequence corresponding to the 311st to 315th amino acid sequences is the VHIID motif.
  • the amino acid sequence corresponding to the 293rd to 297th amino acid sequences is the VHIID motif.
  • the amino acid sequence corresponding to the 297th to 301st amino acid sequences is the VHIID motif.
  • Arabidopsis thaliana-derived protein consisting of the amino acid sequence shown in SEQ ID NO: 10 the amino acid sequence corresponding to the 228th to 232nd amino acid sequences is the VHIID motif.
  • the amino acid corresponding to the 109th amino acid of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1 and the amino acid sequence downstream thereof may be introduced to eliminate or inactivate the As a result, a stop codon mutation is introduced into the gene for controlling total dry matter production, and the amino acid sequence corresponding to the 109th and subsequent amino acids in the amino acid sequence shown in SEQ ID NO: 1 is deleted, thereby increasing the total dry matter production.
  • a method for producing a solanaceous plant with an increased total dry matter production includes genome editing to delete or inactivate an amino acid sequence downstream of the VHIID motif of a GRAS transcription factor in a dicotyledonous plant including a solanaceous plant. Therefore, it can also be applied to a method for producing dicotyledonous plants with increased total dry matter production. Examples of dicotyledonous plants include Arabidopsis thaliana.
  • FIG. 1 is a diagram illustrating the construction of a recombinant inbred line derived from crossing two F1 cultivars and an overview of its QTL mapping.
  • 'Geronimo'(P1; G1) and 'Momotaro8'(P2; G1) were crossed to generate a quaternary F1 group (G1F1) consisting of 240 strains.
  • Quaternary recombinant inbred lines were then developed by repeated selfing by the single-grain method from each G1F1 plant.
  • GM line seeds were sown in nursery trays containing soil (F7, F9, F10 generation autumn cultivation, Experiments 3-6). In addition, seeds of the GM line were sown on February 1 or 2, 2011 and 2012, and the F8 and F10 generations were cultivated in spring (Experiments 1 and 2). After placing the nursery boxes in the greenhouse for 3 weeks, all plants (one plant per line per experiment) were planted directly onto rockwool slabs (planting density 2.303/m 2 ).
  • Phenotypes of 2-10 traits were collected from 1 plant per line in Mie prefecture or 2 plants per line in Aichi prefecture. Phenotypes of traits related to growth characteristics, yield, and fruit quality were determined in each GM line plant. Sugar content in red ripe marketable fruits was measured using a saccharimeter (PAL-1). A good fruit was defined as a fruit that did not develop any physiological disturbances such as tail rot or cracking. On the other hand, fruits with at least one physiological disorder were defined as defective fruits.
  • Genomic SSR markers derived from non-redundant BAC ends (termed “tma”, “tmb”, “tmc” and “TGS”; total 4047), EST-derived SSR markers (termed “tme” and “TES”; total 2195) , EST-based genomic SSR markers (referred to as “tbm”, total 2510), and cDNA-derived SSR markers published on the website (https://solgenomics.net) (referred to as “tms” in this experiment, total 135). ) was used.
  • SSR allele genotyping Forward primers for SSR alleles were 5′ end-labeled using 6-FAM, NED, PET, or VIC (Applied Biosystems). Genomic DNA of P1P2 and genomic DNA of plants of GM lines of G1F1 and F9 generations were used as templates. PCR was performed in 10 ⁇ L reactions of the Type-it Microsatellite PCR Kit (Qiagen). PCR was started at 95° C. for 5 min, followed by 28 cycles of 95° C. for 30 sec, 60° C. for 90 sec, and 72° C. for 30 sec, and finally 60° C. for 30 min.
  • PCR amplification products were analyzed in an automated sequencer (3730 x1 DNA Analyzer, Applied Biosystems) with a GeneScan-500LIZ Size Standard (Applied Biosystems). Fragment lengths were analyzed using GeneMapper v. 3.7 software (Applied Biosystems).
  • SNP allele genotyping A total of 1536 SNPs were used for genotyping of P1P2 and GM lines. Genotyping was performed using the GoldenGate assay system (Illumina Inc.) according to the manufacturer's protocol. Marker categories were grouped similarly to the SSR markers in Table 1.
  • a linkage map of GM strains was constructed using the Carthagene software (de Givry et al. Bioinformatics 21: 1703-1704, 2005).
  • a linkage map of GM lines was constructed by estimating the frequency of recombination (map distance) between each marker that occurs over the course of generations from each G1F1 plant to progeny plants of each GM line.
  • the SSR markers and SNP markers listed in Table 1 were used.
  • the genotypes of the heterozygous markers in P1P2 (categories 1-7 in Table 1) segregated immediately in the G1F1 generation and recombination frequencies between those marker pairs could be estimated.
  • Recombination frequency estimates were made by focusing on the inheritance of alleles from G1F1 plants to their progeny GM lines, and information on segregation in G1F1 was used only to confirm recombination frequency estimates. Segregation distortions of marker genotypes in the linkage map were identified by chi-square test. The linkage map is MapChart v.1. 2.1 It was drawn using software (Voorrips, J Hered 93: 77-78, 2002).
  • Bayesian QTL mapping using MCMC method Bayesian QTL mapping of quaternary cross herds was performed using genotypes of GM lines and marker genotypes of P1, P2, G1F1 and GM lines (Hayashi et al. Euphytica 183: 277-287, 2012). This mapping method assumed 4 alleles from 4 unknown ancestors (P1 parents and P2 parents) in each QTL. The G1F1 plant marker genotype was used to deduce the P1 and P2 haplotypes. The GM lineage haplotype was inferred from the G1F1 parental haplotype. In addition to the Bayesian method based on the MCMC method, Bayesian analysis using variational approximation for QTL mapping in quaternary crosses (variational approximation) was performed.
  • the tfw6.1 region (defined as sandwiched between two SNP markers, SL2.40ch06_983984R and SL2.40ch06_23559443Y, see FIG. 2) is a heterozygous gene. showed the type.
  • the self-fertilized progeny seeds of this line were collected in seedling trays containing soil on September 8, 2014, and then cultivated in the same manner as in Experiments 7 and 8. After 3 weeks, the plants were potted into rockwool cubes and temporarily planted in a greenhouse (plant factory) of the National Agriculture and Food Research Organization in Tsukuba City, Ibaraki Prefecture.
  • a culture solution manufactured by Otsuka Chemical Co., Ltd.
  • an EC of 1.0 mS/cm was applied to the plants, and the plants were cultivated from September 30 to November 4 of the same year.
  • genomic DNA was isolated according to the above "Isolation of genomic DNA", and this DNA was used as a template, using an SSR marker set located around the tfw6.1 region, and a BS-tag method using a fluorescent dye ( Shimizu and Yano, BMC Research Notes 4:161, 2011; Konishi et al., Vegetable and Tea Industry Research Report 14: 15-22, 2015).
  • Mature fruits were harvested periodically during the cultivation period, divided into good fruits and bad fruits, and the number and fresh weight of the fruits were determined. After cultivation, the plant was cut from the base and divided into immature fruits, good fruits, leaves and stems. For fruits, the number of fruits, fresh weight, and dry weight were determined, and for leaves and stems, fresh weight and dry weight were determined.
  • the dry weight referred to here can be rephrased as the dry matter weight.
  • the dry weight of the fruit harvested during the harvesting period was obtained by converting the fresh weight using the dry weight ratio (dry weight/fresh weight) obtained after the cultivation.
  • Test 2 Analysis of yield components in fixed segregation lines: long-stage cultivation
  • the fixed lines 019G and 019M were sown on February 4, 2019, cultivated in a growth chamber, potted in rockwool cubes on February 28, and rockwool at a planting density of 2.7 (strains/m 2 ). Planted on slabs and cultivated in spring in a greenhouse.
  • Test 3 Immobilization of lines with shortened tfw6.1 regions and region limitation by analysis of yield components
  • the marker genotype of the tfw6.1 region of the truncated line selected above becomes a partially heterozygous allele. Therefore, in order to select lines fixed to high-yield type or Japanese type, self-fertilized seeds of the shortened 3 lines were sown on September 4, 2017, and genotype analysis was performed again. All the cultivation in the growth chamber and the greenhouse was carried out as in Test 1. Genotype analysis was performed using the SSR marker set during growth, and after identifying the fixed lines, the yields (total dry weight) of these lines were compared.
  • RNA-SEQ mutation analysis For the tfw6.1 segregating lines 019G (high-yielding type) and 019M (Japanese type), mRNA mutation analysis was performed using a next-generation sequencer. Total RNA was extracted from immature fruits of each line by the Trizol method (https://ipmb.sinica.edu.tw/microarray/protocol.htm), and a fragmented cDNA library was created using these (TruSeq RNA Sample Prep Kit v2, Illumina). The library was subjected to next-generation sequencer (HiSeq 4000, Illumina) analysis to obtain a lead sequence by paired-end sequencing (approximately 4 Gb/sample).
  • the obtained read sequences were mapped to the reference tomato genome (version SL4.0, ftp://ftp.solgenomics.net/genomes/Solanum_lycopersicum/assembly/build_4.00/) using CLC Genomics Workbench software, and a mutation detection program was used. Detection of non-synonymous substitutions was performed by (Basic Variant Detection, etc.).
  • Genome-edited individuals were selected by RFP fluorescence and base sequence reading, and null segregant individuals in which the mutant allele was homogenized and the vector was removed were selected from their progeny and seeded.
  • the seeds of these genome-edited lines and the seeds of the control wild type (Col-0) were aseptically sown on 1/2 MS medium, cultivated for 4 weeks at 22 ° C. with a light period of 10 hours, and then the phenotypes were compared. .
  • Valid SSR markers were grouped into eight categories according to the allelic combination pattern of each strain (Table 1). Category 7 markers were able to detect four different alleles, indicating that they are useful. However, the marker frequency in the tomato genome was very low. Therefore, in the QTL analysis, different categories of markers were combined and used to compensate for the lack of genetic information in each region of the chromosome. As an example, by category 0 marker A (aa-bb), category 2 marker B (ab-aa), and category 3 marker C (aa-ab), by category 7 marker (ab-cd) Information similar to information can be obtained. A total of 197 SSR markers were selected.
  • Table 2 shows the analysis results of QTL involved in total fruit fresh weight detected by the approximation method.
  • R2 is the estimated proportion of phenotypic variance revealed by QTL.
  • FIG. 2 is a diagram illustrating the linkage map of linkage group 6 and the location of QTL tfw6.1.
  • Table 3 shows the results of component element analysis in pinching cultivation of autumn crops in Test 1.
  • the fixed line 019G with the tfw6.1 allele derived from the high-yielding Dutch tomato has a total fruit weight per plant (total of immature, defective, and good fruit) compared to the fixed line 019M with the allele derived from the Japanese type tomato.
  • Fresh weight fruit dry weight (total fruit weight converted to dry weight), total dry weight, and fruit distribution ratio (fruit dry weight/total dry weight) were all significantly increased.
  • total dry weight refers to the sum of the dry weights of leaves, stems and fruits.
  • the tfw6.1 region was further restricted by comparing the region of the high-yielding marker allele with the region of the Japanese-type marker allele of the line with the truncated tfw6.1 region (Table 4).
  • An increase in total dry matter weight was observed in the line (019_364G) in which the region sandwiched between the markers tbm1344 and tbm0281 had a high-yielding allele.
  • A indicates the genotype of high-yielding tomato
  • B indicates the genotype of Japanese type tomato.
  • RNA-SEQ mutation analysis Within the QTL region limited in Table 4, predicted genes in which non-synonymous substitution occurs between high-yielding type and Japanese type are between marker tbm1344 and marker tbm0265 and outside the long arm side of marker tbm0281. etc. was found. Mutations found in the three predicted genes are shown in SEQ ID NOs:4-6.
  • the present invention can be used in fields such as agriculture and plant breeding.

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Abstract

A method for determining the degree of the total dry matter production in a solanaceous plant, said method comprising, for the following proteins (1) to (3) in the solanaceous plant: (1) a protein consisting of the amino acid sequence represented by SEQ ID NO: 1; (2) a protein consisting of an amino acid sequence having 80% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 1 and having the function of regulating the total dry matter production in the solanaceous plant; and (3) a protein consisting of an amino acid sequence in which not more than 65 amino acids are substituted, deleted, added or inserted into the amino acid sequence represented by SEQ ID NO: 1 and having the function of regulating the total dry matter production in the solanaceous plant, a step for examining the presence or absence of a mutation that affects the regulating function as described above. Thus, provided is a technique for determining the degree of the total dry matter production in a solanaceous plant.

Description

ナス科植物における総乾物生産量の制御に関与する遺伝子及びその利用Genes involved in the control of total dry matter production in Solanaceous plants and their use
　本発明は、ナス科植物における総乾物生産量の制御に関与する遺伝子及びその利用に関する。 The present invention relates to genes involved in the control of total dry matter production in Solanaceous plants and their use.
　種子、塊茎、果実などの農業生産物の収量は、その増減が収益と直結するため、農業経営上の重要な指標となっている。農業生産物の収量は、乾物生産量及び収穫物への乾物分配量（同化産物分配量）に比例する。そのため、乾物生産量又は同化産物分配量を増加させることで、農業生産物の収量を増加させることができる。 The yield of agricultural products such as seeds, tubers, and fruits is an important indicator for agricultural management because the increase or decrease is directly linked to profits. The yield of agricultural products is proportional to the amount of dry matter produced and the amount of dry matter distributed to the crop (assimilated product distribution). Therefore, increasing dry matter production or assimilated product distribution can increase the yield of agricultural products.
　農業生産物の収量を増大させる技術として、補光や資材の選択による光透過率の改善等の環境制御技術があるが、コストがかかる上に安定した収量増加を実現することが困難である。農業生産物の安定した収量増加を実現するために、植物を遺伝的に改変する技術が知られている。 As a technology to increase the yield of agricultural products, there are environmental control technologies such as supplementary lighting and improvement of light transmittance by selecting materials, but it is costly and difficult to achieve a stable increase in yield. Techniques for genetically modifying plants are known in order to achieve stable increases in yields of agricultural products.
　トマトのようなナス科の果菜植物の遺伝的改変に用いる遺伝子の候補として、非特許文献１及び２に記載された遺伝子が挙げられる。非特許文献１には、トマトにおいて、大玉品種と小玉品種との間の果実サイズの違いに関与する量的形質遺伝子座（ＱＴＬ）である、第２染色体上のｆｗ２．２遺伝子について記載されている。 Genes described in Non-Patent Documents 1 and 2 are listed as candidates for genes used for genetic modification of fruit vegetable plants of the Solanaceae family such as tomatoes. Non-Patent Document 1 describes the fw2.2 gene on chromosome 2, which is a quantitative trait locus (QTL) involved in the difference in fruit size between large and small varieties of tomatoes. there is
　非特許文献１に記載されたｆｗ２．２の遺伝子は、大玉品種のトマトに普遍的に存在する遺伝子であり、小玉の野生種のトマトが栽培化・大玉化する過程で獲得された遺伝子であると推測されている。すなわち、ｆｗ２．２の遺伝子は、ナス科植物における総乾物生産量の制御に関与するものではなく、細胞分裂の増大や子室数の増大であると考えられる。したがって、ｆｗ２．２の遺伝子は、植物の形態的変化をもたらすのみであり、総乾物生産量の様な収量性の改変には使用できない可能性が高い。 The fw2.2 gene described in Non-Patent Document 1 is a gene that is ubiquitously present in large variety tomatoes, and is a gene acquired in the process of domestication and large size of small wild tomatoes. It is speculated that That is, the fw2.2 gene is not involved in the control of total dry matter production in solanaceous plants, but is considered to increase cell division and increase the number of ovules. Therefore, the fw2.2 gene only brings about morphological changes in plants, and it is highly likely that it cannot be used to modify yield properties such as total dry matter production.
　イネ等では、総乾物生産量の制御に関与する遺伝子が報告されている一方で、トマトを含むナス科植物では、総乾物生産量の制御に関与する遺伝子は知られていない。 In rice, etc., genes involved in the control of total dry matter production have been reported, but in solanaceous plants, including tomatoes, genes involved in the control of total dry matter production are unknown.
　本発明は、上記課題に鑑みなされたものであり、その目的は、トマトを含むナス科植物において、総乾物生産量の制御に関与する遺伝子を同定し、その遺伝子の利用を提供することにある。 The present invention has been made in view of the above problems, and its object is to identify genes involved in the control of total dry matter production in solanaceous plants including tomatoes, and to provide utilization of these genes. .
　本発明の一態様に係る方法は、ナス科植物における総乾物生産量の程度を判別する方法であって、ナス科植物において、下記（ａ）～（ｂ）のアミノ酸：（ａ）配列番号１に示すアミノ酸配列からなるタンパク質の１０９番目のアミノ酸に相当するアミノ酸；（ｂ）配列番号１に示すアミノ酸配列からなるタンパク質の２４７番目のアミノ酸に相当するアミノ酸；の少なくとも１つに置換、欠損、付加又は挿入を引き起こす変異の有無を検査する工程を含む。 A method according to an aspect of the present invention is a method for determining the degree of total dry matter production in a solanaceous plant, wherein in the solanaceous plant, the following amino acids (a) to (b): (a) SEQ ID NO: 1 (b) an amino acid corresponding to the 247th amino acid of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1; Alternatively, it includes the step of examining the presence or absence of insertion-causing mutations.
　本発明の一態様に係る方法は、ナス科植物における総乾物生産量の程度を判別する方法であって、ナス科植物において、下記（ｅ）及び（ｆ）のアミノ酸：（ｅ）配列番号２に示すアミノ酸配列からなるタンパク質の１３３番目のアミノ酸に相当するアミノ酸；（ｆ）配列番号２に示すアミノ酸配列からなるタンパク質の１３９番目のアミノ酸に相当するアミノ酸；の少なくとも一方に置換、欠損、付加又は挿入を引き起こす変異の有無を検査する工程を含む。 A method according to an aspect of the present invention is a method for determining the degree of total dry matter production in a solanaceous plant, wherein in the solanaceous plant, the following amino acids (e) and (f): (e) SEQ ID NO: 2 (f) an amino acid corresponding to the 139th amino acid of the protein consisting of the amino acid sequence shown in SEQ ID NO: 2; The step of testing for the presence or absence of insertion-causing mutations is included.
　本発明の一態様に係る方法は、ナス科植物における総乾物生産量の程度を判別する方法であって、ナス科植物において、下記（ｇ）及び（ｈ）のアミノ酸：（ｇ）配列番号３に示すアミノ酸配列からなるタンパク質の１３４番目のアミノ酸に相当するアミノ酸；（ｈ）配列番号３に示すアミノ酸配列からなるタンパク質の１７５番目のアミノ酸に相当するアミノ酸；の少なくとも一方に置換、欠損、付加又は挿入を引き起こす変異の有無を検査する工程を含む。 A method according to an aspect of the present invention is a method for determining the degree of total dry matter production in a solanaceous plant, wherein the following amino acids (g) and (h): (g) SEQ ID NO: 3 (h) an amino acid corresponding to the 175th amino acid of the protein consisting of the amino acid sequence shown in SEQ ID NO: 3; The step of testing for the presence or absence of insertion-causing mutations is included.
　本発明の一態様に係る製造方法は、総乾物生産量が増加したナス科植物を製造する方法であって、ナス科植物を種内交雑する交雑工程と、前記交雑工程により得られたナス科植物又はその後代系統のナス科植物から、上記いずれかの方法によって、総乾物生産量が増加したナス科植物を識別する識別工程とを含む。 A production method according to an aspect of the present invention is a method for producing a solanaceous plant with increased total dry matter production, comprising a hybridization step of intraspecifically hybridizing a solanaceous plant, and a solanaceous plant obtained by the hybridization step. and an identification step of identifying a solanaceous plant having an increased total dry matter production by any of the above methods from plants or progeny solanaceous plants.
　本発明の一態様に係る製造方法は、総乾物生産量が増加したナス科植物を製造する方法であって、上記いずれかの方法によって、被験ナス科植物から総乾物生産量が増加したナス科植物を識別する識別工程と、識別したナス科植物を種内交雑する交雑工程とを含む。 A production method according to an aspect of the present invention is a method for producing a solanaceous plant having an increased total dry matter production amount, wherein the solanaceae plant having an increased total dry matter production amount from a test solanaceous plant is obtained by any of the above methods. It includes an identification step of identifying a plant and a crossing step of intraspecifically crossing the identified solanaceous plant.
　本発明の一態様に係る分子マーカーは、ナス科植物における、総乾物生産量の制御に関する分子マーカーであって、下記（ａ’）～（ｈ’）の塩基に相当する塩基自体（ＳＮＰ）か、当該塩基を含む連続したポリヌクレオチド：（ａ’）配列番号４に示す塩基配列からなるポリヌクレオチドの３２６番目の塩基に相当する塩基；（ｂ’）配列番号４に示す塩基配列からなるポリヌクレオチドの３９０番目の塩基に相当する塩基；（ｃ’）配列番号４に示す塩基配列からなるポリヌクレオチドの５９１番目の塩基に相当する塩基；（ｄ’）配列番号４に示す塩基配列からなるポリヌクレオチドの７４０番目の塩基に相当する塩基；（ｅ’）配列番号５に示す塩基配列からなるポリヌクレオチドの３９７番目の塩基に相当する塩基；（ｆ’）配列番号５に示す塩基配列からなるポリヌクレオチドの４１７番目の塩基に相当する塩基；（ｇ’）配列番号６に示す塩基配列からなるポリヌクレオチドの４０１番目の塩基に相当する塩基；（ｈ’）配列番号６に示す塩基配列からなるポリヌクレオチドの５２３番目の塩基に相当する塩基；の少なくとも１つを含む。 A molecular marker according to one aspect of the present invention is a molecular marker related to the control of total dry matter production in a plant of the Solanaceae family, and is a base itself (SNP) corresponding to the bases (a′) to (h′) below. , a continuous polynucleotide containing the base: (a′) a base corresponding to the 326th base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 4; (b′) a polynucleotide consisting of the base sequence shown in SEQ ID NO: 4 (c′) a base corresponding to the 591st base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 4; (d′) a polynucleotide consisting of the base sequence shown in SEQ ID NO: 4 (e′) a base corresponding to the 397th base of a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 5; (f′) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 5 (g′) a base corresponding to the 401st base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 6; (h′) a polynucleotide consisting of the base sequence shown in SEQ ID NO: 6 a base corresponding to the 523rd base of;
　本発明の一態様に係る製造方法は、配列番号１に示すアミノ酸配列からなるタンパク質をコードするポリヌクレオチドに相当するナス科植物におけるポリヌクレオチドにおいて、配列番号１に示すアミノ酸配列の７３番目のアミノ酸に相当するアミノ酸よりも下流のいずれかの位置のアミノ酸よりも下流のアミノ酸配列を欠損又は不活化させる変異を導入する工程を包含する、総乾物生産量が増加したナス科植物を製造する方法である。 In the production method according to one aspect of the present invention, in a polynucleotide in a Solanaceae plant corresponding to a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, at the 73rd amino acid of the amino acid sequence shown in SEQ ID NO: 1 A method for producing a solanaceous plant with increased total dry matter production, comprising the step of introducing a mutation that deletes or inactivates an amino acid sequence downstream of an amino acid at any position downstream of the corresponding amino acid. .
　本発明の一態様に係る遺伝子は、ナス科植物における、配列番号１に示すアミノ酸配列の７３番目のアミノ酸に相当するアミノ酸よりも下流のいずれかの位置のアミノ酸よりも下流のアミノ酸配列を欠損又は不活化させる変異が導入され、ナス科植物の総乾物生産量を増加させる機能を有するタンパク質をコードするポリヌクレオチドからなる。 The gene according to one aspect of the present invention lacks or lacks an amino acid sequence downstream of the amino acid at any position downstream of the amino acid corresponding to the 73rd amino acid in the amino acid sequence shown in SEQ ID NO: 1 in a solanaceous plant. It consists of a polynucleotide encoding a protein introduced with an inactivating mutation and having the function of increasing the total dry matter production of a plant of the family Solanaceae.
　本発明の一態様に係る遺伝子は、以下の（１）～（３）のいずれかに記載のポリヌクレオチド：（１）配列番号１に示すアミノ酸配列からなるタンパク質をコードしているポリヌクレオチド；（２）配列番号１に示すアミノ酸配列に対して、８０％以上の配列同一性を有するアミノ酸配列からなり、かつ、配列番号１に示すアミノ酸配列の１０９番目のアミノ酸に相当するアミノ酸及びその下流のアミノ酸配列が欠損又は不活化されているか、又は、配列番号１に示すアミノ酸配列における２４７番目のアミノ酸に相当するアミノ酸がＣであって、ナス科植物の総乾物生産量を制御する機能を有するタンパク質をコードしているポリヌクレオチド；（３）配列番号１に示すアミノ酸配列の１０９番目のアミノ酸に相当するアミノ酸及びその下流のアミノ酸配列が欠損又は不活化されているか、又は、配列番号１に示すアミノ酸配列における２４７番目のアミノ酸に相当するアミノ酸がＣであって、６５個以下のアミノ酸が置換、欠損、付加又は挿入されたアミノ酸配列からなり、ナス科植物の総乾物生産量を制御する機能を有するタンパク質をコードしているポリヌクレオチド；からなる。 The gene according to one aspect of the present invention is a polynucleotide according to any one of the following (1) to (3): (1) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 1; 2) consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and an amino acid corresponding to the 109th amino acid in the amino acid sequence shown in SEQ ID NO: 1 and amino acids downstream thereof; A protein whose sequence is deleted or inactivated, or whose amino acid corresponding to the 247th amino acid in the amino acid sequence shown in SEQ ID NO: 1 is C, and which has the function of regulating the total dry matter production of a solanaceous plant. (3) the amino acid corresponding to the 109th amino acid in the amino acid sequence shown in SEQ ID NO: 1 and the amino acid sequence downstream thereof are deleted or inactivated, or the amino acid sequence shown in SEQ ID NO: 1 The amino acid corresponding to the 247th amino acid in is C, and 65 or less amino acids are substituted, deleted, added or inserted, and the protein has the function of controlling the total dry matter production of solanaceous plants a polynucleotide encoding a
　本発明の一態様に係る遺伝子は、以下の（４）～（６）のいずれかに記載のポリヌクレオチド：（４）配列番号２に示すアミノ酸配列からなるタンパク質をコードしているポリヌクレオチド；（５）配列番号２に示すアミノ酸配列に対して、８０％以上の配列同一性を有するアミノ酸配列からなり、かつ、配列番号２に示すアミノ酸配列における１３３番目のアミノ酸に相当するアミノ酸がＡ、又は、配列番号２に示すアミノ酸配列における１３９番目のアミノ酸に相当するアミノ酸がＮであって、ナス科植物の総乾物生産量を制御する機能を有するタンパク質をコードしているポリヌクレオチド；（６）配列番号２に示すアミノ酸配列に対して、３０個以下のアミノ酸が置換、欠損、付加又は挿入されたアミノ酸配列からなり、かつ、配列番号２に示すアミノ酸配列における１３３番目のアミノ酸に相当するアミノ酸がＡ、又は、配列番号２に示すアミノ酸配列における１３９番目のアミノ酸に相当するアミノ酸がＮであって、ナス科植物の総乾物生産量を制御する機能を有するタンパク質をコードしているポリヌクレオチド；からなる、遺伝子。 The gene according to one aspect of the present invention is a polynucleotide according to any one of the following (4) to (6): (4) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2; 5) consists of an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2, and the amino acid corresponding to the 133rd amino acid in the amino acid sequence shown in SEQ ID NO: 2 is A, or A polynucleotide encoding a protein in which the amino acid corresponding to the 139th amino acid in the amino acid sequence shown in SEQ ID NO: 2 is N, and which has the function of controlling the total dry matter production of a plant of the family Solanaceae; (6) SEQ ID NO: 2 consisting of an amino acid sequence in which 30 or less amino acids are substituted, deleted, added or inserted into the amino acid sequence shown in SEQ ID NO: 2, and the amino acid corresponding to the 133rd amino acid in the amino acid sequence shown in SEQ ID NO: 2 is A, Alternatively, a polynucleotide encoding a protein in which the amino acid corresponding to the 139th amino acid in the amino acid sequence shown in SEQ ID NO: 2 is N and has the function of controlling the total dry matter production of Solanaceae plants; gene.
　本発明の一態様に係る遺伝子は、以下の（７）～（９）のいずれかに記載のポリヌクレオチド：（７）配列番号３に示すアミノ酸配列からなるタンパク質をコードしているポリヌクレオチド；（８）配列番号３に示すアミノ酸配列に対して、８０％以上の配列同一性を有するアミノ酸配列からなり、かつ、配列番号３に示すアミノ酸配列における１３４番目のアミノ酸に相当するアミノ酸がＰ、又は、配列番号３に示すアミノ酸配列における１７５番目のアミノ酸に相当するアミノ酸がＹであって、ナス科植物の総乾物生産量を制御する機能を有するタンパク質をコードしているポリヌクレオチド；（９）配列番号３に示すアミノ酸配列に対して、６０個以下のアミノ酸が置換、欠損、付加又は挿入されたアミノ酸配列からなり、かつ、配列番号３に示すアミノ酸配列における１３４番目のアミノ酸に相当するアミノ酸がＰ、又は、配列番号３に示すアミノ酸配列における１７５番目のアミノ酸に相当するアミノ酸がＹであって、ナス科植物の総乾物生産量を制御する機能を有するタンパク質をコードしているポリヌクレオチド；からなる。 The gene according to one aspect of the present invention is the polynucleotide according to any one of the following (7) to (9): (7) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 3; 8) consists of an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3, and the amino acid corresponding to the 134th amino acid in the amino acid sequence shown in SEQ ID NO: 3 is P, or A polynucleotide encoding a protein in which the amino acid corresponding to the 175th amino acid in the amino acid sequence shown in SEQ ID NO: 3 is Y, and which has the function of controlling the total dry matter production of a plant of the family Solanaceae; (9) SEQ ID NO: 3, wherein 60 or less amino acids are substituted, deleted, added or inserted into the amino acid sequence shown in SEQ ID NO: 3, and the amino acid corresponding to the 134th amino acid in the amino acid sequence shown in SEQ ID NO: 3 is P, or a polynucleotide encoding a protein having Y as an amino acid corresponding to the 175th amino acid in the amino acid sequence shown in SEQ ID NO: 3 and having a function of controlling the total dry matter production of Solanaceae plants.
　本発明の一態様に係る発現ベクターは、上記いずれかの遺伝子を含む。 An expression vector according to one aspect of the present invention comprises any of the above genes.
　本発明の一態様に係る細胞又は双子葉植物は、上記いずれかの遺伝子又は上記発現ベクターを含む。 A cell or dicotyledonous plant according to one aspect of the present invention comprises any of the above genes or the above expression vector.
　本発明の一態様によれば、ナス科植物における総乾物生産量を制御する遺伝子を用いて、総乾物生産量が増加したナス科植物を識別すること、及び、総乾物生産量が増加したナス科植物を製造することができる。 According to one aspect of the present invention, using a gene that controls the total dry matter production in a solanaceous plant, a solanaceous plant with an increased total dry matter production is identified, and an eggplant with an increased total dry matter production Can produce family plants.
２つのＦ１品種の交配に由来する組換え型自殖系統の構築及びそのＱＴＬマッピングの概要を説明する図である。Brief Description of the Drawings Fig. 1 is a diagram outlining the construction of a recombinant inbred line derived from crossing two F1 breeds and its QTL mapping. 連鎖群６の連鎖地図及びＱＴＬ　ｔｆｗ６．１の位置を説明する図である。Fig. 2 is a diagram explaining the linkage map of linkage group 6 and the position of QTL tfw6.1; 実施例で用いたシロイヌナズナのゲノム編集系統の塩基配列及びアミノ酸配列を示す図である。FIG. 2 shows the nucleotide and amino acid sequences of Arabidopsis thaliana genome-edited strains used in Examples. 実施例で用いたシロイヌナズナのゲノム編集系統と野生型との表現型を比較した結果を示す図である。FIG. 3 shows the results of comparing the phenotypes of the genome-edited strain of Arabidopsis thaliana used in Examples and the wild type.
　本発明の実施の形態について説明すれば、以下の通りである。なお、本発明は、これに限定されるものではない。 A description of the embodiment of the present invention is as follows. However, the present invention is not limited to this.
　本明細書において、「ポリヌクレオチド」は、「核酸」又は「核酸分子」とも換言でき、ヌクレオチドの重合体を意図している。また、「塩基配列」は、「核酸配列」又は「ヌクレオチド配列」とも換言でき、特に言及しない限り、デオキシリボヌクレオチドの配列又はリボヌクレオチドの配列を意図している。本明細書において、「ポリペプチド」は、「タンパク質」とも換言できる。 In the present specification, "polynucleotide" can also be rephrased as "nucleic acid" or "nucleic acid molecule" and intends a polymer of nucleotides. A "nucleotide sequence" can also be rephrased as a "nucleic acid sequence" or a "nucleotide sequence", and unless otherwise specified, a sequence of deoxyribonucleotides or a sequence of ribonucleotides is intended. In the present specification, "polypeptide" can also be rephrased as "protein".
　本明細書において、「乾物」とは、「同化産物」とも換言でき、光合成によって合成される糖などの有機化合物を意図している。本明細書において、「総乾物生産量」とは、「バイオマス量」とも称され、生産される乾物の総量を意図している。本明細書において、「総乾物生産量の増加」とは、生産される乾物の総量が増加することを意図しており、例えば、着果期又は茎部肥大期から収穫期までの間に、葉面積が増大することにより受光量が増大する結果、総乾物生産量が増加することを意図している。 In this specification, "dry matter" can be rephrased as "assimilation product" and intends organic compounds such as sugars synthesized by photosynthesis. As used herein, "total dry matter production", also referred to as "biomass volume", is intended to be the total amount of dry matter produced. As used herein, the term "increase in total dry matter production" is intended to increase the total amount of dry matter produced. It is intended that the increased leaf area will increase the amount of light received, resulting in an increase in total dry matter production.
　本明細書において、「ナス科植物」は、一例として、トマト、ジャガイモ、ナス、又はトウガラシである。 As used herein, "plants of the Solanaceae family" are, for example, tomatoes, potatoes, eggplants, or hot peppers.
　本明細書において、「ナス」は、広義には、栽培種「ソラナム（以下、Ｓと称する）メロンゲナ（Solanum. melongena）」、並びに野生種「Ｓ．インカナム（S. incanum）」、「Ｓ．トルバム（S. torvum）」、「Ｓ．ニグラム（S. nigrum）」、「Ｓ．アエチオピカム（S. aethiopicum）」、「Ｓ．マクロカルポン（S. macrocarpon）」、及び「Ｓ．クイトエンセ（S. quitoense）」を含む概念であり、狭義には「Ｓ．メロンゲナ」を意図している。 As used herein, “eggplant” broadly includes the cultivated species “Solanum (hereinafter referred to as S) melongena” as well as the wild species “S. incanum” and “S. "S. torvum", "S. nigrum", "S. aethiopicum", "S. macrocarpon", and "S. Quitoense" )”, and narrowly intended to mean “S. melongena”.
　本明細書において、「トマト」は、広義には、栽培種「Ｓ．リコペルシクム（S. lycopersicum）」、並びに野生種「Ｓ．ケエスマニ（S. cheesmaniae）、Ｓ．チレンセ（S. chilense）、Ｓ．クミエレウスキイ（S. chmielewskii）、Ｓ．ガラパゲンセ（S. galapagense）、Ｓ．ハブロカイテス（S. habrochaites）、Ｓ．リコペルシコイデス（S. lycopersicoides）、Ｓ．ネオリキイ（S. neorickii）、Ｓ．ペネリ（S. pennellii）、Ｓ．ペルビアナム（S. peruvianum）、及びＳ．ピンピネリフォリウム（S. pimpinellifolium）を含む概念であり、狭義には「Ｓ．リコペルシクム」を意図している。 As used herein, "tomato" is broadly defined as the cultivated species "S. lycopersicum", as well as the wild species "S. cheesmaniae, S. chilense, S. lycopersicum". S. chmielewskii, S. galapagense, S. habrochaites, S. lycopersicoides, S. neorickii, S. penelli S. pennellii, S. peruvianum, and S. pimpinellifolium, narrowly intended as "S. lycopersicum."
　本明細書において、「トウガラシ」は、広義には、栽培種「カプシカム（以下、Ｃと称する）・アニウム（Capsicum annuum）」、並びに野生種「Ｃ．プベッセンス（C. pubescens）」、「Ｃ．バッカータム（C. baccatum）」、「Ｃ．チネンセ（C. chinense）」、及び「Ｃ．フルテッセンス（C. frutescens）」を含む概念であり、狭義には「Ｃ．アニウム」を意図している。また、「トウガラシ」は、園芸作物の呼称として「ピーマン」、「パプリカ」及び「シシトウ」など、「トウガラシ」以外の用語が用いられる上記の植物も含む概念である。 In the present specification, "capsicum" broadly includes the cultivated species "Capsicum annuum" and the wild species "C. pubescens" and "C. A concept that includes C. baccatum, C. chinense, and C. frutescens, narrowly intended to mean C. anium. In addition, "pepper" is a concept that includes the above-mentioned plants for which terms other than "pepper" are used, such as "green pepper", "paprika", and "shishito", as names of horticultural crops.
　本明細書において、「ジャガイモ」は、広義には、栽培種「Ｓ．トゥベローサム（S. tuberosum）」、並びに野生種「Ｓ．アカウレ（S. acaule）」、「Ｓ．スパルシピウム（S. sparsipilum）」、「Ｓ．レプトフィエス（S. leptophyes）」、及び「Ｓ．メジスタクロブム（S. megistacrobum）」を含む概念であり、狭義には「Ｓ．トゥベローサム」を意図している。 As used herein, "potato" is broadly defined as the cultivated species "S. tuberosum", as well as the wild species "S. acaule", "S. sparsipilum" ", "S. leptophyes", and "S. megistacrobum", narrowly intended to be "S. tuberosum".
　〔１．総乾物生産量制御遺伝子〕
　本発明の一態様に係る総乾物生産量制御遺伝子は、総乾物生産量を制御する活性（総乾物生産量制御活性）を有するタンパク質をコードする遺伝子である。総乾物生産量制御遺伝子がコードするタンパク質が総乾物生産量を正に制御する活性を有する場合、当該タンパク質が存在すれば、総乾物生産量が増加する。また、総乾物生産量制御遺伝子がコードするタンパク質の活性が低下しているか又は阻害されると、その活性が低下していないか又は阻害されていない植物よりも総乾物生産量が低減するか又は増加しない。 [1. total dry matter production control gene]
A gene for regulating total dry matter production according to one aspect of the present invention is a gene that encodes a protein having activity to regulate total dry matter production (total dry matter production regulating activity). If the protein encoded by the gene for controlling total dry matter production has the activity of positively regulating total dry matter production, the presence of the protein will increase total dry matter production. In addition, when the activity of the protein encoded by the gene for controlling total dry matter production is reduced or inhibited, the total dry matter production is reduced more than the plant whose activity is not reduced or inhibited, or not increase.
　総乾物生産量制御遺伝子の一例は、ナス科植物の総乾物生産量の制御に関与する遺伝子であって、以下の（１）～（９）のいずれかに記載のポリヌクレオチドからなる遺伝子である：
　（１）配列番号１に示すアミノ酸配列からなるタンパク質をコードしているポリヌクレオチド；
　（２）配列番号１に示すアミノ酸配列に対して、８０％以上の配列同一性を有するアミノ酸配列からなり、ナス科植物の総乾物生産量を制御する機能を有するタンパク質をコードしているポリヌクレオチド；
　（３）配列番号１に示すアミノ酸配列に対して、６５個以下のアミノ酸が置換、欠損、付加又は挿入されたアミノ酸配列からなり、ナス科植物の総乾物生産量を制御する機能を有するタンパク質をコードしているポリヌクレオチド；
　（４）配列番号２に示すアミノ酸配列からなるタンパク質をコードしているポリヌクレオチド；
　（５）配列番号２に示すアミノ酸配列に対して、８０％以上の配列同一性を有するアミノ酸配列からなり、ナス科植物の総乾物生産量を制御する機能を有するタンパク質をコードしているポリヌクレオチド；
　（６）配列番号２に示すアミノ酸配列に対して、３０個以下のアミノ酸が置換、欠損、付加又は挿入されたアミノ酸配列からなり、ナス科植物の総乾物生産量を制御する機能を有するタンパク質をコードしているポリヌクレオチド；
　（７）配列番号３に示すアミノ酸配列からなるタンパク質をコードしているポリヌクレオチド；
　（８）配列番号３に示すアミノ酸配列に対して、８０％以上の配列同一性を有するアミノ酸配列からなり、ナス科植物の総乾物生産量を制御する機能を有するタンパク質をコードしているポリヌクレオチド；
　（９）配列番号３に示すアミノ酸配列に対して、６０個以下のアミノ酸が置換、欠損、付加又は挿入されたアミノ酸配列からなり、ナス科植物の総乾物生産量を制御する機能を有するタンパク質をコードしているポリヌクレオチド。 An example of the total dry matter production control gene is a gene involved in the control of the total dry matter production of a solanaceous plant, which consists of the polynucleotide according to any one of the following (1) to (9). :
(1) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 1;
(2) A polynucleotide consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and encoding a protein having a function of controlling the total dry matter production of Solanaceae plants. ;
(3) A protein consisting of an amino acid sequence in which 65 or less amino acids are substituted, deleted, added or inserted with respect to the amino acid sequence shown in SEQ ID NO: 1, and having the function of controlling the total dry matter production of a solanaceous plant. an encoding polynucleotide;
(4) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2;
(5) A polynucleotide consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2 and encoding a protein having a function of controlling the total dry matter production of a plant of the family Solanaceae. ;
(6) A protein consisting of an amino acid sequence in which 30 or less amino acids are substituted, deleted, added or inserted with respect to the amino acid sequence shown in SEQ ID NO: 2, and having the function of controlling the total dry matter production of a solanaceous plant. an encoding polynucleotide;
(7) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 3;
(8) A polynucleotide consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3 and encoding a protein having a function of controlling the total dry matter production of a plant of the family Solanaceae. ;
(9) A protein consisting of an amino acid sequence in which 60 or less amino acids are substituted, deleted, added or inserted with respect to the amino acid sequence shown in SEQ ID NO: 3, and having the function of controlling the total dry matter production of a solanaceous plant. Encoding polynucleotide.
　上記（１）、（４）、及び（７）のポリヌクレオチドは、トマト由来のｔｆｗ６．１遺伝子の塩基配列又はｔｆｗ６．１遺伝子のコーディング領域（ＣＤＳ）の塩基配列を含み、総乾物生産量を制御する機能を有するタンパク質をコードしているポリヌクレオチドである。また、上記（１）、（４）、及び（７）のポリヌクレオチドは、ナス科植物の上記ｔｆｗ６．１遺伝子に対応する遺伝子の塩基配列又は当該遺伝子のＣＤＳの塩基配列を含み、総乾物生産量を制御する機能を有するタンパク質をコードしているポリヌクレオチドであり得る。 The polynucleotides (1), (4), and (7) above contain the nucleotide sequence of the tomato-derived tfw6.1 gene or the nucleotide sequence of the coding region (CDS) of the tfw6.1 gene, and measure the total dry matter production Polynucleotides encoding proteins with regulatory functions. In addition, the polynucleotides (1), (4), and (7) above include the nucleotide sequence of the gene corresponding to the tfw6.1 gene of the Solanaceae plant or the nucleotide sequence of the CDS of the gene, and the total dry matter production It can be a polynucleotide that encodes a protein that has the function of controlling the amount.
　上記（２）、（５）、及び（８）のポリヌクレオチドに関して、アミノ酸配列の配列同一性は、８０％以上、又は、９０％以上であることが好ましく、９５％以上であることがより好ましく、９６％以上、９７％以上、９８％以上、又は、９９％以上であることが特に好ましい。 Regarding the polynucleotides (2), (5), and (8) above, the sequence identity of the amino acid sequences is preferably 80% or more, or 90% or more, and more preferably 95% or more. , 96% or more, 97% or more, 98% or more, or 99% or more.
　上記（３）のポリヌクレオチドに関して、配列番号１のアミノ酸配列において、置換、欠損、付加又は挿入されたアミノ酸の個数は、１～６５個であることが好ましく、１～６０個、１～５０個、１～４０個、１～３０個であることがより好ましく、１～２５個であることがさらに好ましく、１～２０個、１～１５個、１～１０個、１～５個、１～４個、１～３個、又は１～２個であることが特に好ましい。 Regarding the polynucleotide of (3) above, the number of substituted, deleted, added or inserted amino acids in the amino acid sequence of SEQ ID NO: 1 is preferably 1 to 65, 1 to 60, 1 to 50. , 1 to 40, more preferably 1 to 30, more preferably 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 are particularly preferred.
　上記（６）のポリヌクレオチドに関して、配列番号２のアミノ酸配列において、置換、欠損、付加又は挿入されたアミノ酸の個数は、１～３０個であることが好ましく、１～２５個、１～２０個、１～１５個であることがより好ましく、１～１０個であることがさらに好ましく、１～９個、１～８個、１～７個、１～６個、１～５個、１～４個、１～３個、又は１～２個であることが特に好ましい。 Regarding the polynucleotide of (6) above, the number of substituted, deleted, added or inserted amino acids in the amino acid sequence of SEQ ID NO: 2 is preferably 1 to 30, 1 to 25, 1 to 20. , more preferably 1 to 15, more preferably 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 are particularly preferred.
　上記（９）のポリヌクレオチドに関して、配列番号３のアミノ酸配列において、置換、欠損、付加又は挿入されたアミノ酸の個数は、１～６０個であることが好ましく、１～５０個、１～４０個、１～３０個であることがより好ましく、１～２５個であることがさらに好ましく、１～２０個、１～１５個、１～１０個、１～５個、１～４個、１～３個、又は１～２個であることが特に好ましい。 Regarding the polynucleotide of (9) above, the number of substituted, deleted, added or inserted amino acids in the amino acid sequence of SEQ ID NO: 3 is preferably 1 to 60, 1 to 50, 1 to 40. , more preferably 1 to 30, more preferably 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3 or 1 to 2 are particularly preferred.
　例えば、トマトに由来する変異遺伝子、又は、トマト以外のナス科植物に由来する相同遺伝子（オーソログを含む）が、上記（２）、（３）、（５）、（６）、（８）及び（９）のポリヌクレオチドの範疇に含まれる。これらの変異遺伝子や相同遺伝子は、ナス科植物の内在性（endogenous）の遺伝子である。後述する〔４．ナス科植物における総乾物生産量の程度を判別する方法〕の欄で検査がなされる分子マーカーは、これらの変異遺伝子や相同遺伝子上の分子マーカーでありうる。 For example, a mutated gene derived from tomato, or a homologous gene (including an orthologue) derived from a solanaceous plant other than tomato is the above (2), (3), (5), (6), (8) and It is included in the category of polynucleotides of (9). These mutated genes and homologous genes are endogenous genes of Solanaceae plants. [4. Method for determining the degree of total dry matter production in Solanaceous plants] can be molecular markers on these mutated genes or homologous genes.
　なお、総乾物生産量制御遺伝子が人工的に変異を導入した遺伝子を指す場合、上記「アミノ酸の置換、欠損、付加又は挿入」は、例えば、Ｋｕｎｋｅｌ法（Kunkel, Proc Natl Acad Sci USA, 82: 488-492, 1985）等の部位特異的突然変異誘発法、薬剤を用いた変異原処理、放射線（γ線、重イオンビーム等）の照射による変異誘発手法、部位特異的ヌクレアーゼを利用したゲノム編集等を用いて人工的に変異を導入してもよいし、天然に存在する同様の変異ポリペプチドに由来するものであってもよい。 In addition, when the total dry matter production control gene refers to an artificially mutated gene, the above "substitution, deletion, addition or insertion of amino acids" is, for example, the Kunkel method (Kunkel, Proc Natl Acad Sci USA, 82: 488-492, 1985), mutagen treatment using drugs, mutagenesis methods using radiation (γ-rays, heavy ion beams, etc.), genome editing using site-specific nucleases A mutation may be introduced artificially using, for example, a mutation, or it may be derived from a similar mutant polypeptide that exists in nature.
　また、総乾物生産量制御遺伝子は、ナス科植物における、配列番号１に示すアミノ酸配列の７３番目のアミノ酸に相当するアミノ酸よりも下流のいずれかの位置のアミノ酸よりも下流のアミノ酸配列を欠損又は不活化させる変異が導入され、ナス科植物の総乾物生産量を増加させる機能を有するタンパク質をコードするポリヌクレオチドからなる遺伝子であり得る。さらに、総乾物生産量制御遺伝子において、ポリヌクレオチドは、配列番号１に示すアミノ酸配列からなるタンパク質の１０９番目のアミノ酸に相当するアミノ酸及びその下流のアミノ酸配列を欠損又は不活化させる変異が導入され、ナス科植物の総乾物生産量を増加させる機能を有するタンパク質をコードする遺伝子であり得る。このような総乾物生産量制御遺伝子は、公知のゲノム編集等の技術を利用して、ナス科植物の総乾物生産量制御遺伝子に人工的に変異を導入することによって得ることができる。総乾物生産量制御遺伝子に変異を導入する位置、変異の詳細等については、後述する〔６．総乾物生産量が増加したナス科植物を製造する方法〕の記載を援用する。 In addition, the total dry matter production control gene lacks or lacks an amino acid sequence downstream of the amino acid at any position downstream of the amino acid corresponding to the 73rd amino acid in the amino acid sequence shown in SEQ ID NO: 1 in the Solanaceae plant It may be a gene comprising a polynucleotide into which an inactivating mutation has been introduced and which encodes a protein having the function of increasing the total dry matter production of a plant of the family Solanaceae. Furthermore, in the total dry matter production control gene, the polynucleotide is mutated to delete or inactivate the amino acid corresponding to the 109th amino acid of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1 and the amino acid sequence downstream thereof, It may be a gene encoding a protein having a function of increasing the total dry matter production of a solanaceous plant. Such a gene for controlling total dry matter production can be obtained by artificially introducing a mutation into a gene for controlling total dry matter production of Solanaceae plants using known techniques such as genome editing. The position for introducing mutation into the total dry matter production control gene, the details of the mutation, etc. will be described later [6. Method for Producing Solanaceous Plant with Increased Total Dry Matter Production].
　総乾物生産量制御遺伝子は、ＲＮＡの形態（例えば、ｍＲＮＡ）、又は、ＤＮＡの形態（例えば、ｃＤＮＡ又はゲノムＤＮＡ）で存在し得る。ＤＮＡは、二本鎖であっても、一本鎖であってもよい。総乾物生産量制御遺伝子の一例である、配列番号４に示す塩基配列は、配列番号１に示すポリペプチドをコードする遺伝子の完全長ｃＤＮＡである。また、総乾物生産量制御遺伝子の他の例である、配列番号５に示す塩基配列は、配列番号２に示すポリペプチドをコードする遺伝子の完全長ｃＤＮＡである。さらに、総乾物生産量制御遺伝子の他の例である、配列番号６に示す塩基配列は、配列番号３に示すポリペプチドをコードする遺伝子の完全長ｃＤＮＡである。総乾物生産量制御遺伝子は、ｔｆｗ６．１遺伝子のＣＤＳと共に、非翻訳領域（ＵＴＲ）の塩基配列等の付加的な配列を含むものであってもよい。 The total dry matter production control gene can exist in the form of RNA (eg, mRNA) or in the form of DNA (eg, cDNA or genomic DNA). DNA may be double-stranded or single-stranded. The nucleotide sequence shown in SEQ ID NO: 4, which is an example of the total dry matter production control gene, is the full-length cDNA of the gene encoding the polypeptide shown in SEQ ID NO: 1. In addition, the base sequence shown in SEQ ID NO:5, which is another example of the total dry matter production control gene, is the full-length cDNA of the gene encoding the polypeptide shown in SEQ ID NO:2. Furthermore, the nucleotide sequence shown in SEQ ID NO: 6, which is another example of the total dry matter production control gene, is the full-length cDNA of the gene encoding the polypeptide shown in SEQ ID NO: 3. The total dry matter yield control gene may contain additional sequences such as the nucleotide sequence of the untranslated region (UTR) in addition to the CDS of the tfw6.1 gene.
　総乾物生産量制御遺伝子を取得する（単離する）方法は、特に限定されるものではないが、例えば、総乾物生産量制御遺伝子の塩基配列の一部と特異的にハイブリダイズするプローブを調製し、ゲノムＤＮＡライブラリ又はｃＤＮＡライブラリをスクリーニングすればよい。 The method for obtaining (isolating) the gene for controlling total dry matter production is not particularly limited, but for example, a probe that specifically hybridizes with part of the base sequence of the gene for controlling total dry matter production is prepared. and screen a genomic DNA library or a cDNA library.
　また、総乾物生産量制御遺伝子を取得する方法として、ＰＣＲ等の増幅手段を用いる方法を挙げることができる。例えば、総乾物生産量制御遺伝子のｃＤＮＡのうち、５’側及び３’側の配列（又はその相補配列）の中からそれぞれプライマーを調製し、これらプライマーを用いてゲノムＤＮＡ（又はｃＤＮＡ）等を鋳型にしてＰＣＲ等を行い、両プライマー間に挟まれるＤＮＡ領域を増幅することで、総乾物生産量制御遺伝子を含むＤＮＡ断片を大量に取得できる。 In addition, as a method for obtaining the total dry matter production control gene, a method using an amplification means such as PCR can be mentioned. For example, primers are prepared from the 5′-side and 3′-side sequences (or their complementary sequences) of the cDNA of the total dry matter production control gene, and genomic DNA (or cDNA) is obtained using these primers. A large amount of DNA fragments containing the gene for controlling total dry matter production can be obtained by performing PCR or the like using this as a template and amplifying the DNA region sandwiched between the two primers.
　総乾物生産量制御遺伝子の由来はナス科の植物である限り特に限定されないが、トマト、ジャガイモ、ナス、又はトウガラシの何れかであることが好ましく、トマトであることがより好ましい。また、総乾物生産量制御遺伝子は、ナス科の植物を含む双子葉植物由来であり得る。双子葉植物の例として、シロイヌナズナ（Arabidopsis thaliana）が挙げられる。 The origin of the gene for controlling total dry matter production is not particularly limited as long as it is a plant of the Solanaceae family, but it is preferably tomato, potato, eggplant, or hot pepper, more preferably tomato. Also, the total dry matter production control gene can be derived from dicotyledonous plants, including plants of the Solanaceae family. Examples of dicotyledonous plants include Arabidopsis thaliana.
　なお、単離された総乾物生産量制御遺伝子の候補遺伝子が、所望する総乾物生産量を制御する活性を有するか否かは、双子葉植物において当該候補遺伝子の発現によって、当該候補遺伝子が発現していない双子葉植物と比較して、総乾物生産量の増加が誘導されるかを観察することによって評価することができる。 Whether or not the isolated candidate gene for controlling the total dry matter production has the activity of controlling the desired total dry matter production is determined by the expression of the candidate gene in a dicotyledonous plant. It can be evaluated by observing whether an increase in total dry matter production is induced compared to non-dicotyledonous plants.
　総乾物生産量制御遺伝子は、双子葉植物における、総乾物生産量の増加機構の解明に利用することができる。また、総乾物生産量制御遺伝子は、その配列を発現ベクターに組み込む等して、双子葉植物の植物体又は細胞に導入することによって、形質転換体を作製するために用いることができる。総乾物生産量制御遺伝子が導入された双子葉植物を栽培することで、総乾物生産量が増加した双子葉植物を得ることができる。 　Genes controlling total dry matter production can be used to elucidate the mechanism of increasing total dry matter production in dicotyledonous plants. In addition, the total dry matter production control gene can be used to prepare a transformant by inserting the sequence into an expression vector or the like and introducing it into a dicotyledonous plant or cell. Dicotyledonous plants with increased total dry matter production can be obtained by cultivating dicotyledonous plants into which a gene for controlling total dry matter production is introduced.
　総乾物生産量制御遺伝子として、トマトであるＳ．リコペルシクム由来のｔｆｗ６．１遺伝子のＣＤＳの塩基配列である第１の遺伝子（配列番号４）、第２の遺伝子（配列番号５）、及び第３の遺伝子（配列番号６）の塩基配列からなるポリヌクレオチドが挙げられる。また、総乾物生産量制御遺伝子の範疇には、配列番号４～６に示す塩基配列に対して、８０％以上、８５＆以上、９０％以上、９５％以上、９６％以上、９７％以上、９８％以上、又は、９９％以上の配列同一性を有し、総乾物生産量制御活性を有するポリヌクレオチドからなる遺伝子も含まれる。 As a gene for controlling total dry matter production, the tomato S. The first gene (SEQ ID NO: 4), the second gene (SEQ ID NO: 5), and the third gene (SEQ ID NO: 6), which are the CDS nucleotide sequences of the tfw6.1 gene derived from Lycopersicum. Nucleotides. In addition, in the category of total dry matter production control genes, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% of the base sequences shown in SEQ ID NOS: 4 to 6 Also included are genes consisting of polynucleotides having 99% or more sequence identity and having total dry matter production control activity.
　〔２．総乾物生産量制御タンパク質〕
　本発明の一態様に係る総乾物生産量制御タンパク質は、上記〔１．総乾物生産量制御遺伝子〕欄に記載した遺伝子の翻訳産物であり、少なくとも総乾物生産量制御活性を有する。総乾物生産量制御タンパク質が、総乾物生産量を正に制御する活性を有する場合、当該タンパク質が存在すれば、総乾物生産量が増加する。総乾物生産量制御タンパク質が存在しない場合、総乾物生産量制御タンパク質が存在する場合よりも総乾物生産量が低減するか又は増加しない。 [2. total dry matter production control protein]
The total dry matter production control protein according to one aspect of the present invention is the protein described in [1. Total dry matter production control gene], which is a translation product of the gene described in the column and has at least total dry matter production control activity. If the total dry matter production regulating protein has activity that positively regulates total dry matter production, its presence will increase total dry matter production. In the absence of the total dry matter production control protein, the total dry matter production is reduced or not increased more than in the presence of the total dry matter production control protein.
　総乾物生産量制御タンパク質は、天然の供給源より単離されてもよいし、化学的に合成されてもよい。より具体的には、当該タンパク質は、天然の精製産物、化学的合成手順の産物、及び、原核生物宿主又は真核生物宿主（例えば、細菌細胞、酵母細胞、高等植物細胞、昆虫細胞、及び哺乳動物細胞を含む）から組換え技術によって産生された翻訳産物をその範疇に含む。 The total dry matter production control protein may be isolated from natural sources or chemically synthesized. More specifically, the proteins are naturally occurring purified products, products of chemical synthetic procedures, and prokaryotic or eukaryotic hosts (e.g., bacterial cells, yeast cells, higher plant cells, insect cells, and mammalian cells). It includes translation products produced by recombinant technology from cells (including animal cells).
　総乾物生産量制御タンパク質は、より具体的には、以下の（１’）～（９’）のいずれかに記載のタンパク質である：
　（１’）配列番号１に示すアミノ酸配列からなるタンパク質；
　（２’）配列番号１に示すアミノ酸配列に対して、８０％以上の配列同一性を有するアミノ酸配列からなり、ナス科植物の総乾物生産量を制御する機能を有するタンパク質；
　（３’）配列番号１に示すアミノ酸配列に対して、６５個以下のアミノ酸が置換、欠損、付加又は挿入されたアミノ酸配列からなり、ナス科植物の総乾物生産量を制御する機能を有するタンパク質；
　（４’）配列番号２に示すアミノ酸配列からなるタンパク質；
　（５’）配列番号２に示すアミノ酸配列に対して、８０％以上の配列同一性を有するアミノ酸配列からなり、ナス科植物の総乾物生産量を制御する機能を有するタンパク質；
　（６’）配列番号２に示すアミノ酸配列に対して、３０個以下のアミノ酸が置換、欠損、付加又は挿入されたアミノ酸配列からなり、ナス科植物の総乾物生産量を制御する機能を有するタンパク質；
　（７’）配列番号３に示すアミノ酸配列からなるタンパク質；
　（８’）配列番号３に示すアミノ酸配列に対して、８０％以上の配列同一性を有するアミノ酸配列からなり、ナス科植物の総乾物生産量を制御する機能を有するタンパク質；
　（９’）配列番号３に示すアミノ酸配列に対して、６０個以下のアミノ酸が置換、欠損、付加又は挿入されたアミノ酸配列からなり、ナス科植物の総乾物生産量を制御する機能を有するタンパク質。 More specifically, the total dry matter production control protein is a protein according to any one of (1') to (9') below:
(1') a protein consisting of the amino acid sequence shown in SEQ ID NO: 1;
(2') a protein consisting of an amino acid sequence having a sequence identity of 80% or more to the amino acid sequence shown in SEQ ID NO: 1 and having the function of controlling the total dry matter production of Solanaceae plants;
(3') A protein consisting of an amino acid sequence in which 65 or less amino acids are substituted, deleted, added, or inserted with respect to the amino acid sequence shown in SEQ ID NO: 1, and having the function of controlling the total dry matter production of a solanaceous plant. ;
(4') a protein consisting of the amino acid sequence shown in SEQ ID NO: 2;
(5') a protein consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2, and having the function of controlling the total dry matter production of Solanaceae plants;
(6') A protein consisting of an amino acid sequence in which 30 or less amino acids are substituted, deleted, added or inserted with respect to the amino acid sequence shown in SEQ ID NO: 2, and having the function of regulating the total dry matter production of solanaceous plants. ;
(7') a protein consisting of the amino acid sequence shown in SEQ ID NO: 3;
(8') a protein consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3, and having the function of controlling the total dry matter production of Solanaceae plants;
(9') A protein consisting of an amino acid sequence in which 60 or less amino acids are substituted, deleted, added or inserted with respect to the amino acid sequence shown in SEQ ID NO: 3, and having the function of controlling the total dry matter production of Solanaceous plants .
　上記（１’）、（４’）、及び（７’）のタンパク質は、上記ｔｆｗ６．１遺伝子、又は、ナス科植物における上記ｔｆｗ６．１遺伝子に対応する遺伝子がコードするタンパク質であり、総乾物生産量制御活性を有するタンパク質である。 The proteins (1′), (4′), and (7′) are proteins encoded by the tfw6.1 gene or a gene corresponding to the tfw6.1 gene in Solanaceae plants, and total dry matter It is a protein with production control activity.
　上記（２’）、（５’）、及び（８’）のタンパク質に関して、アミノ酸配列の配列同一性は、８０％以上、又は、９０％以上であることが好ましく、９５％以上であることがより好ましく、９６％以上、９７％以上、９８％以上、又は、９９％以上であることが特に好ましい。 Regarding the proteins (2′), (5′) and (8′) above, the sequence identity of the amino acid sequences is preferably 80% or more, or 90% or more, preferably 95% or more. More preferably, 96% or more, 97% or more, 98% or more, or 99% or more is particularly preferable.
　上記（３’）のタンパク質に関して、配列番号１のアミノ酸配列において、置換、欠損、付加又は挿入されたアミノ酸の個数は、１～６５個であることが好ましく、１～６０個、１～５０個、１～４０個、１～３０個であることがより好ましく、１～２５個であることがさらに好ましく、１～２０個、１～１５個、１～１０個、１～５個、１～４個、１～３個、又は１～２個であることが特に好ましい。 Regarding the protein (3′) above, the number of substituted, deleted, added or inserted amino acids in the amino acid sequence of SEQ ID NO: 1 is preferably 1 to 65, 1 to 60, 1 to 50. , 1 to 40, more preferably 1 to 30, more preferably 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 are particularly preferred.
　上記（６’）のタンパク質に関して、配列番号２のアミノ酸配列において、置換、欠損、付加又は挿入されたアミノ酸の個数は、１～３０個であることが好ましく、１～２５個、１～２０個、１～１５個であることがより好ましく、１～１０個であることがさらに好ましく、１～９個、１～８個、１～７個、１～６個、１～５個、１～４個、１～３個、又は１～２個であることが特に好ましい。 Regarding the protein (6′) above, the number of substituted, deleted, added or inserted amino acids in the amino acid sequence of SEQ ID NO: 2 is preferably 1 to 30, 1 to 25, 1 to 20. , more preferably 1 to 15, more preferably 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 are particularly preferred.
　上記（９’）のタンパク質に関して、配列番号３のアミノ酸配列において、置換、欠損、付加又は挿入されたアミノ酸の個数は、１～６０個であることが好ましく、１～５０個、１～４０個、１～３０個であることがより好ましく、１～２５個であることがさらに好ましく、１～２０個、１～１５個、１～１０個、１～５個、１～４個、１～３個、又は１～２個であることが特に好ましい。 Regarding the protein (9′) above, the number of substituted, deleted, added or inserted amino acids in the amino acid sequence of SEQ ID NO: 3 is preferably 1 to 60, 1 to 50, 1 to 40. , more preferably 1 to 30, more preferably 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3 or 1 to 2 are particularly preferred.
　例えば、トマトに由来する変異タンパク質、又は、トマト以外のナス科植物に由来する相同タンパク質が、上記（２’）、（３’）、（５’）、（６’）、（８’）及び（９’）のタンパク質の範疇に含まれる。これらの変異タンパク質や相同タンパク質は、ナス科植物の内在性の遺伝子にコードされたタンパク質である。 For example, a mutant protein derived from tomato or a homologous protein derived from a solanaceous plant other than tomato is the above (2'), (3'), (5'), (6'), (8') and (9') included in the category of proteins. These mutant proteins and homologous proteins are proteins encoded by endogenous genes of Solanaceae plants.
　総乾物生産量制御タンパク質は、アミノ酸がペプチド結合してなるポリペプチドであるが、ポリペプチド以外の構造を含むものであってもよい。ここでいうポリペプチド以外の構造としては、糖鎖やイソプレノイド基等を挙げることができるが、これに限定されない。 The total dry matter production control protein is a polypeptide composed of peptide bonds of amino acids, but it may contain structures other than polypeptides. Examples of structures other than polypeptides herein include sugar chains, isoprenoid groups, and the like, but are not limited thereto.
　なお、総乾物生産量制御タンパク質として、トマトであるＳ．リコペルシクム由来のタンパク質が挙げられる。また、総乾物生産量制御タンパク質として、ジャガイモ、ナス、又はトウガラシ由来のタンパク質が挙げられる。さらに、総乾物生産量制御タンパク質は、ナス科の植物を含む双子葉植物由来のタンパク質であり得、双子葉植物の例として、シロイヌナズナが挙げられる。 In addition, as a protein for controlling total dry matter production, tomato S. Proteins from Lycopersicum can be mentioned. In addition, proteins derived from potatoes, eggplants, or hot peppers can be mentioned as total dry matter yield control proteins. Furthermore, the total dry matter production control protein can be a protein from a dicotyledonous plant, including plants of the family Solanaceae, an example of a dicotyledonous plant being Arabidopsis thaliana.
　〔３．発現ベクター、細胞、及び形質転換体〕
　本発明の一態様に係る総乾物生産量制御遺伝子が組み込まれた発現ベクター、当該発現ベクター又は総乾物生産量制御遺伝子を含む細胞、並びに、当該発現ベクター又は総乾物生産量制御遺伝子が発現可能に導入された形質転換体についても、本発明の範疇に含まれる。当該発現ベクターは、細胞又は生物個体に、総乾物生産量を制御する形質を付与するものである。 [3. Expression vectors, cells, and transformants]
An expression vector into which the gene for controlling total dry matter production according to one aspect of the present invention has been incorporated, a cell containing the expression vector or the gene for controlling total dry matter production, and the expression vector or the gene for controlling total dry matter production can be expressed. The introduced transformants are also included in the scope of the present invention. The expression vector imparts a trait that controls the total dry matter production to cells or organisms.
　発現ベクターを構成するためのベクターの種類は特に限定されるものではなく、宿主細胞中で発現可能なものを適宜選択すればよい。すなわち、宿主細胞の種類に応じて、適宜プロモータ配列を選択し、当該プロモータ配列と総乾物生産量制御遺伝子とを、例えば、プラスミド、ファージミド、又はコスミド等に組み込んだものを発現ベクターとして用いればよい。 The type of vector that constitutes the expression vector is not particularly limited, and one that can be expressed in the host cell may be selected as appropriate. That is, an appropriate promoter sequence is selected according to the type of host cell, and the promoter sequence and the total dry matter production control gene are integrated into, for example, a plasmid, phagemid, cosmid, or the like, and used as an expression vector. .
　発現ベクターを導入する宿主細胞としては、例えば、細菌細胞、酵母細胞、酵母細胞以外の真菌細胞及び高等真核細胞などが挙げられる。細菌細胞としては、例えば、大腸菌細胞が挙げられる。高等真核細胞としては、例えば、植物細胞及び動物細胞が挙げられる。植物細胞としては、例えば、双子葉植物細胞及び単子葉植物細胞が挙げられる。双子葉植物細胞としては、例えば、ナス科植物の懸濁培養細胞（例えば、タバコＢＹ－２株及びトマトＳｌｙ－１株）が挙げられる。単子葉植物細胞としては、例えば、イネの懸濁培養細胞であるＯｃ株などが挙げられる。動物細胞としては、昆虫細胞、両生類細胞、爬虫類細胞、鳥類細胞、魚類細胞、哺乳動物細胞などが挙げられる。 Examples of host cells into which expression vectors are introduced include bacterial cells, yeast cells, fungal cells other than yeast cells, and higher eukaryotic cells. Bacterial cells include, for example, E. coli cells. Higher eukaryotic cells include, for example, plant cells and animal cells. Plant cells include, for example, dicotyledonous and monocotyledonous plant cells. Dicotyledonous plant cells include, for example, suspension cultured cells of Solanaceae plants (eg, tobacco BY-2 strain and tomato Sly-1 strain). Monocotyledonous plant cells include, for example, the Oc strain, which is a suspension-cultured cell of rice. Animal cells include insect cells, amphibian cells, reptile cells, avian cells, fish cells, mammalian cells and the like.
　発現ベクターにおいて総乾物生産量制御遺伝子は、転写に必要な要素（例えば、プロモータなど）が機能的に連結されている。また、必要に応じて、エンハンサー、選択マーカー、スプライシングシグナル、ポリＡ付加シグナル、及び５’－ＵＴＲ配列などを連結されていてもよい。プロモータは、宿主細胞において転写活性を示すＤＮＡ配列であり、宿主の種類に応じて適宜選択することができる。 In the expression vector, the total dry matter production control gene is functionally linked to the elements required for transcription (eg, promoter, etc.). Also, if necessary, enhancers, selectable markers, splicing signals, poly-A addition signals, 5'-UTR sequences, etc. may be ligated. A promoter is a DNA sequence that exhibits transcriptional activity in host cells, and can be appropriately selected according to the type of host.
　宿主細胞内で作動可能なプロモータ配列としては、カリフラワーモザイクウイルスの３５Ｓプロモータ、アグロバクテリウムのノパリン合成酵素遺伝子プロモータ及びイネユビキチン遺伝子プロモータなどが挙げられる。また、組換え発現プロモータとしては、総乾物生産量制御遺伝子におけるプロモータ領域の配列を用いてもよい。 Promoter sequences that are operable in host cells include the cauliflower mosaic virus 35S promoter, Agrobacterium nopaline synthase gene promoter and rice ubiquitin gene promoter. In addition, as the recombinant expression promoter, the sequence of the promoter region in the total dry matter production control gene may be used.
　発現ベクターにおいて、総乾物生産量制御遺伝子は、必要に応じて、適切なターミネータ（例えば、ＮＯＳターミネータ及びカリフラワーモザイクウイルスの３５Ｓターミネータ）に機能的に結合されてもよい。適切なターミネータの種類は、宿主細胞の種類に応じて適宜選択すればよく、上述のプロモータにより転写された遺伝子の転写を終結できる配列であればよい。エンハンサーは、目的遺伝子の発現効率を高めるために用いられ、例えばタバコモザイクウイルスのオメガ配列が挙げられる。 In the expression vector, the total dry matter production control gene may be functionally linked to an appropriate terminator (eg, NOS terminator and cauliflower mosaic virus 35S terminator), if necessary. The type of appropriate terminator may be appropriately selected according to the type of host cell, as long as it is a sequence capable of terminating the transcription of the gene transcribed by the above promoter. Enhancers are used to increase the efficiency of expression of target genes, and include, for example, the omega sequence of tobacco mosaic virus.
　発現ベクターは、さらに選択マーカーを含有してもよい。選択マーカーとしては、例えば、アンピシリン、カナマイシン、テトラサイクリン、クロラムフェニコール、ネオマイシン、ハイグロマイシン又はスペクチノマイシンのような薬剤耐性遺伝子を挙げることができる。 The expression vector may further contain a selectable marker. Selectable markers can include, for example, drug resistance genes such as ampicillin, kanamycin, tetracycline, chloramphenicol, neomycin, hygromycin or spectinomycin.
　また、発現ベクターにおいて総乾物生産量制御遺伝子は、必要に応じて、好適なタンパク質精製用のタグ配列、または好適なスペーサー配列に結合されていてもよい。 In addition, in the expression vector, the total dry matter production control gene may be linked to a suitable tag sequence for protein purification or a suitable spacer sequence, if necessary.
　また、上記形質転換体とは、上記発現ベクター又は総乾物生産量制御遺伝子が発現可能に導入された細胞、組織及び器官のみならず、生物個体を含む意味である。このような形質転換体は、ナス科植物であり得る。また、形質転換体は、例えば、大腸菌等の微生物、動物等であってもよい。 In addition, the transformant is meant to include not only cells, tissues, and organs into which the expression vector or the gene for controlling total dry matter production is introduced so as to be expressible, but also individual organisms. Such transformants may be Solanaceae plants. Moreover, the transformant may be, for example, a microorganism such as E. coli, an animal, or the like.
　〔４．ナス科植物における総乾物生産量の程度を判別する方法〕
　本発明の一態様に係るナス科植物における総乾物生産量の程度を判別する方法（判別方法）は、ナス科植物において、総乾物生産量が増加していること又は増加していないことを判別する。判別方法は、ナス科植物において、総乾物生産量が増加した個体を判別するために使用することができる。判別方法は、ナス科植物のゲノムに存在する、総乾物生産量の決定に関与する遺伝子の遺伝子型を判定することで、総乾物生産量に基づいてナス科植物を判別する。なお、判別方法において、総乾物生産量の概念には、あるナス科植物個体の総乾物生産量が、他のナス科植物個体と比較して相対的に多い又は少ないことを表す総乾物生産量の程度が含まれる。 [4. Method for Determining Degree of Total Dry Matter Production in Solanaceous Plants]
A method for determining the degree of total dry matter production in a solanaceous plant according to one aspect of the present invention (determination method) is to determine whether the total dry matter production is increasing or not increasing in a solanaceous plant. do. The discrimination method can be used to discriminate individuals with increased total dry matter production in Solanaceous plants. The discrimination method is to determine the genotype of the gene involved in the determination of the total dry matter production, present in the genome of the solanaceous plant, to discriminate the solanaceous plant based on the total dry matter production. In the determination method, the concept of total dry matter production is defined as total dry matter production, which indicates that the total dry matter production of a certain solanaceous plant individual is relatively large or small compared to other solanaceous plant individuals. includes the degree of
　ナス科植物、例えばトマトにおいては、総乾物生産量は、成長に伴って徐々に増加し、連続生産期にピークに達した後に一定になると考えられる（参考文献１：Saito et al. Hort J 89: 445-453, 2020を参照のこと）。すなわち、果房が着果し始めた又は茎部が肥大し始めた栽培初期は、総乾物生産量が少ない。したがって、特に、着果期又は茎部肥大期のような栽培初期における総乾物生産量を増加させることによって、収量を安定的に増加させることができる。 In solanaceous plants, such as tomatoes, total dry matter production is thought to increase gradually with growth, peak during the continuous production period, and then level off (Reference 1: Saito et al. Hort J 89 : 445-453, 2020). That is, at the early stage of cultivation when the bunches start to bear fruit or when the stems start to grow, the total dry matter production is small. Therefore, the yield can be stably increased by increasing the total dry matter production especially in the early stage of cultivation such as the fruiting period or the stem enlargement period.
　ナス科植物は、育種素材の候補植物であるか、育種のプロセスで得られた植物であり得る。育種素材の候補植物としては、例えば、交配に用いる親植物、及び、遺伝子組換え技術を利用した分子育種に用いられる植物が含まれる。また、育種のプロセスで得られた植物としては、例えば、ナス科植物のトマト、ジャガイモ、ナス、又はトウガラシを種内交雑した植物、及びこれらの後代系統である。また、ナス科植物は、ある品種に属するトマトと他の品種に属するトマトとの交雑植物のように品種間交雑した植物、及びその後代系統であってもよい。さらに、ナス科植物における総乾物生産量の程度を判別する方法は、ナス科の植物を含む双子葉植物における総乾物生産量の程度を判別するために用いることが可能であり、双子葉植物の例として、シロイヌナズナが挙げられる。 Solanaceous plants can be candidate plants for breeding material or plants obtained through the process of breeding. Candidate plants for breeding materials include, for example, parent plants used for crossing and plants used for molecular breeding using gene recombination technology. Plants obtained through the process of breeding include, for example, plants obtained by intraspecific crossing of tomato, potato, eggplant, or capsicum belonging to the family Solanaceae, and progeny lines thereof. Also, the solanaceous plant may be a cross-cultivated plant such as a hybrid plant of a tomato belonging to a certain variety and a tomato belonging to another variety, and its progeny. Furthermore, the method for determining the level of total dry matter production in solanaceous plants can be used to determine the level of total dry matter production in dicotyledonous plants, including plants of the Solanaceae family. Examples include Arabidopsis thaliana.
　さらに、ナス科植物は、総乾物生産量が増加していることが分かっている品種同士を交雑した植物、及びその後代系統であってもよい。また、ナス科植物は、総乾物生産量が増加していることが分かっている品種と、総乾物生産量が増加しているか不明な品種とを交雑した植物、及びその後代系統であってもよい。さらに、ナス科植物は、総乾物生産量が増加しているか不明な品種同士を交雑した植物、及びその後代系統であってもよい。また、ナス科植物は、総乾物生産量が増加していることが分かっている品種と、総乾物生産量が増加していないことが分かっている品種とを交雑した植物、及びその後代系統であってもよい。また、ナス科植物は、総乾物生産量が増加していることが分かっている個体同士を交雑した植物、及びその後代系統であってもよい。 In addition, the solanaceous plant may be a plant obtained by crossing cultivars known to increase total dry matter production, and its progeny. In addition, solanaceous plants are plants obtained by crossing a cultivar known to increase total dry matter production with a cultivar whose total dry matter production is unknown, and even if it is a progeny line. good. Furthermore, the solanaceous plant may be a plant obtained by crossing between cultivars whose total dry matter production is unknown or unknown, and its progeny. In addition, solanaceous plants are plants obtained by crossing cultivars known to increase total dry matter production with cultivars known to have no increase in total dry matter production, and their progeny. There may be. Also, the solanaceous plant may be a plant obtained by crossing individuals known to increase total dry matter production, and its progeny.
　本明細書において、植物とは、植物体の一部又は全部であってもよい。植物体の一部としては、例えば、繁殖素材（例えば、葉、枝、種子等）等が挙げられる。 As used herein, the term "plant" may refer to part or all of a plant body. The part of the plant body includes, for example, propagation material (eg, leaves, branches, seeds, etc.) and the like.
　判別方法は、ナス科植物における、下記（１’）～（９’）のいずれかに記載のタンパク質：
　（１’）配列番号１に示すアミノ酸配列からなるタンパク質；
　（２’）配列番号１に示すアミノ酸配列に対して、８０％以上の配列同一性を有するアミノ酸配列からなり、ナス科植物の総乾物生産量を制御する機能を有するタンパク質；
　（３’）配列番号１に示すアミノ酸配列に対して、６５個以下のアミノ酸が置換、欠損、付加又は挿入されたアミノ酸配列からなり、ナス科植物の総乾物生産量を制御する機能を有するタンパク質；
　（４’）配列番号２に示すアミノ酸配列からなるタンパク質；
　（５’）配列番号２に示すアミノ酸配列に対して、８０％以上の配列同一性を有するアミノ酸配列からなり、ナス科植物の総乾物生産量を制御する機能を有するタンパク質；
　（６’）配列番号２に示すアミノ酸配列に対して、３０個以下のアミノ酸が置換、欠損、付加又は挿入されたアミノ酸配列からなり、ナス科植物の総乾物生産量を制御する機能を有するタンパク質；
　（７’）配列番号３に示すアミノ酸配列からなるタンパク質；
　（８’）配列番号３に示すアミノ酸配列に対して、８０％以上の配列同一性を有するアミノ酸配列からなり、ナス科植物の総乾物生産量を制御する機能を有するタンパク質；
　（９’）配列番号３に示すアミノ酸配列に対して、６０個以下のアミノ酸が置換、欠損、付加又は挿入されたアミノ酸配列からなり、ナス科植物の総乾物生産量を制御する機能を有するタンパク質；
の前記制御する機能に影響する変異の有無を検査する工程を含む。 The determination method is the protein according to any one of the following (1′) to (9′) in Solanaceae plants:
(1') a protein consisting of the amino acid sequence shown in SEQ ID NO: 1;
(2') a protein consisting of an amino acid sequence having a sequence identity of 80% or more to the amino acid sequence shown in SEQ ID NO: 1 and having the function of controlling the total dry matter production of Solanaceae plants;
(3') A protein consisting of an amino acid sequence in which 65 or less amino acids are substituted, deleted, added, or inserted with respect to the amino acid sequence shown in SEQ ID NO: 1, and having the function of controlling the total dry matter production of a solanaceous plant. ;
(4') a protein consisting of the amino acid sequence shown in SEQ ID NO: 2;
(5') a protein consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2, and having the function of controlling the total dry matter production of Solanaceae plants;
(6') A protein consisting of an amino acid sequence in which 30 or less amino acids are substituted, deleted, added or inserted with respect to the amino acid sequence shown in SEQ ID NO: 2, and having the function of regulating the total dry matter production of solanaceous plants. ;
(7') a protein consisting of the amino acid sequence shown in SEQ ID NO: 3;
(8') a protein consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3, and having the function of controlling the total dry matter production of Solanaceae plants;
(9') A protein consisting of an amino acid sequence in which 60 or less amino acids are substituted, deleted, added or inserted with respect to the amino acid sequence shown in SEQ ID NO: 3, and having the function of controlling the total dry matter production of Solanaceous plants ;
testing for the presence or absence of mutations that affect said regulating function of .
　ナス科植物の総乾物生産量を制御する機能を有するタンパク質の当該機能に影響する前記変異は、当該タンパク質にナス科植物の総乾物生産量を増加させる機能を発現させる変異であり得る。判別方法は、このような変異の有無を検査することによって、ナス科植物において、総乾物生産量が増加していること又は増加していないことを判別する。 The mutation that affects the function of a protein that has the function of controlling the total dry matter production of a solanaceous plant can be a mutation that causes the protein to express the function of increasing the total dry matter production of the solanaceous plant. In the discrimination method, by examining the presence or absence of such mutations, it is discriminated whether or not the total dry matter production is increasing in Solanaceous plants.
　ナス科植物の総乾物生産量を制御する機能を有するタンパク質の当該機能に影響する前記変異が、前記（１’）～（９’）のタンパク質の何れかをコードしているポリヌクレオチドにおける、ストップコドン変異、フレームシフト変異、及びヌル変異からなる群より選択される変異であり得る。 A stop in a polynucleotide encoding any of the proteins (1′) to (9′) in which the mutation that affects the function of the protein that has the function of regulating the total dry matter production of a solanaceous plant It may be a mutation selected from the group consisting of codon mutations, frameshift mutations, and null mutations.
　判別方法は、以下に示すような分子マーカーを用いて、総乾物生産量制御遺伝子の変異の有無を検査することができる。このような分子マーカーについても、本発明の範疇に含まれる。 For the discrimination method, the presence or absence of mutations in the total dry matter production control gene can be tested using the molecular markers shown below. Such molecular markers are also included in the scope of the present invention.
　分子マーカーは、ナス科植物において、下記（ａ）、（ｂ）、（ｅ）～（ｈ）のアミノ酸：
　（ａ）配列番号１に示すアミノ酸配列からなるタンパク質の１０９番目のアミノ酸に相当するアミノ酸；
　（ｂ）配列番号１に示すアミノ酸配列からなるタンパク質の２４７番目のアミノ酸に相当するアミノ酸；
　（ｅ）配列番号２に示すアミノ酸配列からなるタンパク質の１３３番目のアミノ酸に相当するアミノ酸；
　（ｆ）配列番号２に示すアミノ酸配列からなるタンパク質の１３９番目のアミノ酸に相当するアミノ酸；
　（ｇ）配列番号３に示すアミノ酸配列からなるタンパク質の１３４番目のアミノ酸に相当するアミノ酸；
　（ｈ）配列番号３に示すアミノ酸配列からなるタンパク質の１７５番目のアミノ酸に相当するアミノ酸；
の少なくとも１つに置換、欠損、付加又は挿入を引き起こす変異の有無を検査する分子マーカーである。 Molecular markers are the following amino acids (a), (b), (e) to (h) in Solanaceae plants:
(a) an amino acid corresponding to the 109th amino acid of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1;
(b) an amino acid corresponding to the 247th amino acid of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1;
(e) an amino acid corresponding to the 133rd amino acid of the protein consisting of the amino acid sequence shown in SEQ ID NO: 2;
(f) an amino acid corresponding to the 139th amino acid of the protein consisting of the amino acid sequence shown in SEQ ID NO: 2;
(g) an amino acid corresponding to the 134th amino acid of the protein consisting of the amino acid sequence shown in SEQ ID NO: 3;
(h) an amino acid corresponding to the 175th amino acid of the protein consisting of the amino acid sequence shown in SEQ ID NO: 3;
is a molecular marker that tests for mutations that cause substitutions, deletions, additions or insertions in at least one of
　配列番号１～３のいずれかに示すアミノ酸配列からなるタンパク質は、総乾物生産量制御遺伝子によりコードされたタンパク質であり、一例として、配列番号４～６のいずれかに示す塩基配列からなる遺伝子によりコードされたタンパク質である。 A protein consisting of the amino acid sequence shown in any one of SEQ ID NOs: 1 to 3 is a protein encoded by a total dry matter production control gene, and for example, a gene consisting of a nucleotide sequence shown in any one of SEQ ID NOs: 4 to 6. is the encoded protein.
　配列番号１～３のいずれかに示すアミノ酸配列からなるタンパク質は、基準となるトマト（Ｓ．リコペルシクム）由来のｔｆｗ６．１遺伝子にコードされたタンパク質のアミノ酸配列からなる。ナス科植物においては、基準となるトマト由来のｔｆｗ６．１遺伝子にコードされたタンパク質のアミノ酸配列に対して、上記（ａ）、（ｂ）、（ｅ）～（ｈ）のアミノ酸に相当する部分以外でもアミノ酸配列が異なる部分が含まれ得る。ナス科植物において、上記（ａ）、（ｂ）、（ｅ）～（ｈ）のアミノ酸に相当するアミノ酸の位置は、ホモロジー解析等の手法によって特定することができる。すなわち、ナス科植物の上記ｔｆｗ６．１遺伝子に対応する遺伝子（すなわち植物間で高度に保存されている遺伝子が存在する場合）において、「（ａ）、（ｂ）、（ｅ）～（ｈ）のアミノ酸に相当するアミノ酸」とは、ホモロジー解析等の手法によって、（ａ）、（ｂ）、（ｅ）～（ｈ）のアミノ酸に相当するとされたアミノ酸を指す。 The protein consisting of the amino acid sequence shown in any one of SEQ ID NOs: 1 to 3 consists of the amino acid sequence of the protein encoded by the standard tomato (S. lycopersicum)-derived tfw6.1 gene. In solanaceous plants, portions corresponding to the above amino acids (a), (b), (e) to (h) for the amino acid sequence of the protein encoded by the reference tomato-derived tfw6.1 gene It may also contain portions with different amino acid sequences. In solanaceous plants, the amino acid positions corresponding to the above amino acids (a), (b), (e) to (h) can be identified by a technique such as homology analysis. That is, in the gene corresponding to the tfw6.1 gene of a solanaceous plant (that is, when a highly conserved gene exists among plants), "(a), (b), (e) to (h) The term "amino acid corresponding to the amino acid of" refers to an amino acid determined to correspond to the amino acid of (a), (b), (e) to (h) by a method such as homology analysis.
　なお、ホモロジー解析の方法としては、例えば、Needleman-Wunsch法やSmith-Waterman法等のPairwise Sequence Alignmentによる方法や、ClustalW法等のMultiple Sequence Alignmentによる方法が挙げられ、当業者であれば、これら方法に基づき、配列番号１～３のいずれかに示されるアミノ酸配列を基準配列として用いて、解析対象のアミノ酸配列中における「相当するアミノ酸」を理解することができる。 Homology analysis methods include, for example, a method by Pairwise Sequence Alignment such as the Needleman-Wunsch method and the Smith-Waterman method, and a method by Multiple Sequence Alignment such as the ClustalW method. Based on this, it is possible to understand the "corresponding amino acid" in the amino acid sequence to be analyzed using the amino acid sequence shown in any of SEQ ID NOs: 1 to 3 as a reference sequence.
　ナス科植物における、配列番号１に示すアミノ酸配列からなるタンパク質の相同タンパク質並びに（ａ）又は（ｂ）のアミノ酸に相当するアミノ酸の一例を示す。 An example of the homologous protein of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1 and the amino acid corresponding to the amino acid (a) or (b) in Solanaceae plants is shown.
　配列番号７に示すアミノ酸配列からなるピーマン（CA09g13460）のタンパク質において、（ａ）のアミノ酸に相当するアミノ酸は３５１番目のアミノ酸であり、（ｂ）のアミノ酸に相当するアミノ酸は４８９番目のアミノ酸である。 In the green pepper (CA09g13460) protein consisting of the amino acid sequence shown in SEQ ID NO: 7, the amino acid corresponding to (a) is the 351st amino acid, and the amino acid corresponding to (b) is the 489th amino acid. .
　配列番号８に示すアミノ酸配列からなるナス（Sme2.5_14468.1_g00001.1）のタンパク質において、（ａ）のアミノ酸に相当するアミノ酸は３３３番目のアミノ酸であり、（ｂ）のアミノ酸に相当するアミノ酸は４７１番目のアミノ酸である。 In the eggplant (Sme2.5_14468.1_g00001.1) protein consisting of the amino acid sequence shown in SEQ ID NO: 8, the amino acid corresponding to the amino acid in (a) is the 333rd amino acid, and the amino acid corresponding to the amino acid in (b) is It is the 471st amino acid.
　配列番号９に示すアミノ酸配列からなるジャガイモ（Sotub06g015180.1.1）のタンパク質において、（ａ）のアミノ酸に相当するアミノ酸は３３７番目のアミノ酸であり、（ｂ）のアミノ酸に相当するアミノ酸は４７５番目のアミノ酸である。 In the potato (Sotub06g015180.1.1) protein consisting of the amino acid sequence shown in SEQ ID NO: 9, the amino acid corresponding to (a) is the 337th amino acid, and the amino acid corresponding to (b) is the 475th amino acid. is.
　また、ナス科植物を含む双子葉植物における、配列番号１に示すアミノ酸配列からなるタンパク質の相同タンパク質の一例として、シロイヌナズナのタンパク質を示す。配列番号１０に示すアミノ酸配列からなるシロイヌナズナ（AT5G48150.1）のタンパク質において、（ａ）のアミノ酸に相当するアミノ酸は２６８番目のアミノ酸であり、（ｂ）のアミノ酸に相当するアミノ酸は４０６番目のアミノ酸である。 In addition, a protein of Arabidopsis thaliana is shown as an example of a protein homologous to the protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in dicotyledonous plants including solanaceous plants. In the Arabidopsis thaliana (AT5G48150.1) protein consisting of the amino acid sequence shown in SEQ ID NO: 10, the amino acid corresponding to (a) is the 268th amino acid, and the amino acid corresponding to (b) is the 406th amino acid. is.
　「（ａ）又は（ｂ）のアミノ酸に相当するアミノ酸」は、一例として、（１）配列番号１に示すアミノ酸配列からなるタンパク質における（ａ）又は（ｂ）のアミノ酸、（２）配列番号１に示すアミノ酸配列に対して、８０％以上の配列同一性を有するアミノ酸配列からなるタンパク質における（ａ）又は（ｂ）のアミノ酸に相当するアミノ酸、又は、（３）配列番号１に示すアミノ酸配列に対して、６５個以下のアミノ酸が置換、欠損、付加又は挿入されたアミノ酸配列からなるタンパク質における（ａ）又は（ｂ）のアミノ酸に相当するアミノ酸である。 "An amino acid corresponding to the amino acid (a) or (b)" is, for example, (1) the amino acid (a) or (b) in the protein consisting of the amino acid sequence shown in SEQ ID NO: 1, (2) SEQ ID NO: 1 or (3) the amino acid sequence shown in SEQ ID NO: 1 On the other hand, 65 or less amino acids are amino acids corresponding to the amino acids (a) or (b) in a protein consisting of an amino acid sequence having substitutions, deletions, additions or insertions.
　（ａ）又は（ｂ）のアミノ酸に相当するアミノ酸に置換、欠損、付加又は挿入を引き起こす変異の有無を検出する分子マーカーは、（ａ）及び（ｂ）の両方のアミノ酸に相当するアミノ酸に置換又は欠損を引き起こす変異の有無を検出するものであってもよい。 Molecular markers for detecting the presence or absence of mutations that cause substitution, deletion, addition or insertion in amino acids corresponding to (a) or (b) are substituted with amino acids corresponding to both (a) and (b). Alternatively, the presence or absence of deletion-causing mutations may be detected.
　「（ｅ）又は（ｆ）のアミノ酸に相当するアミノ酸」は、一例として、（４）配列番号２に示すアミノ酸配列からなるタンパク質における（ｅ）又は（ｆ）のアミノ酸、（５）配列番号２に示すアミノ酸配列に対して、８０％以上の配列同一性を有するアミノ酸配列からなるタンパク質における（ｅ）又は（ｆ）のアミノ酸に相当するアミノ酸、又は、（６）配列番号２に示すアミノ酸配列に対して、３０個以下のアミノ酸が置換、欠損、付加又は挿入されたアミノ酸配列からなるタンパク質における（ｅ）又は（ｆ）のアミノ酸に相当するアミノ酸である。 "An amino acid corresponding to the amino acid (e) or (f)" is, for example, (4) the amino acid (e) or (f) in the protein consisting of the amino acid sequence shown in SEQ ID NO: 2, (5) SEQ ID NO: 2 The amino acid corresponding to the amino acid (e) or (f) in a protein consisting of an amino acid sequence having a sequence identity of 80% or more to the amino acid sequence shown in , or (6) the amino acid sequence shown in SEQ ID NO: 2 On the other hand, 30 or less amino acids are amino acids corresponding to the amino acids (e) or (f) in a protein consisting of an amino acid sequence having substitutions, deletions, additions or insertions.
　（ｅ）又は（ｆ）のアミノ酸に相当するアミノ酸に置換、欠損、付加又は挿入を引き起こす変異の有無を検出する分子マーカーは、（ｅ）及び（ｆ）の両方のアミノ酸に相当するアミノ酸に置換又は欠損を引き起こす変異の有無を検出するものであってもよい。 Molecular markers for detecting the presence or absence of mutations that cause substitution, deletion, addition, or insertion in amino acids corresponding to (e) or (f) are substituted with amino acids corresponding to both (e) and (f). Alternatively, the presence or absence of deletion-causing mutations may be detected.
　ナス科植物における、配列番号３に示すアミノ酸配列からなるタンパク質の相同タンパク質並びに（ｇ）又は（ｈ）のアミノ酸に相当するアミノ酸の一例を示す。 An example of the homologous protein of the protein consisting of the amino acid sequence shown in SEQ ID NO: 3 and the amino acid corresponding to the amino acid (g) or (h) in Solanaceae plants is shown.
　配列番号１１に示すアミノ酸配列からなるピーマン（CA06g05790）のタンパク質において、（ｇ）のアミノ酸に相当するアミノ酸は１３４番目のアミノ酸であり、（ｈ）のアミノ酸に相当するアミノ酸は１７５番目のアミノ酸である。 In the green pepper (CA06g05790) protein consisting of the amino acid sequence shown in SEQ ID NO: 11, the amino acid corresponding to the amino acid (g) is the 134th amino acid, and the amino acid corresponding to the amino acid (h) is the 175th amino acid. .
　配列番号１２に示すアミノ酸配列からなるピーマン（Capang06g002314）のタンパク質において、（ｇ）のアミノ酸に相当するアミノ酸は１３２番目のアミノ酸であり、（ｈ）のアミノ酸に相当するアミノ酸は１７３番目のアミノ酸である。 In the green pepper (Capang06g002314) protein consisting of the amino acid sequence shown in SEQ ID NO: 12, the amino acid corresponding to the amino acid (g) is the 132nd amino acid, and the amino acid corresponding to the amino acid (h) is the 173rd amino acid. .
　配列番号１３に示すアミノ酸配列からなるピーマン（Capana06g002501）のタンパク質において、（ｇ）のアミノ酸に相当するアミノ酸は１３２番目のアミノ酸であり、（ｈ）のアミノ酸に相当するアミノ酸は１７３番目のアミノ酸である。 In the green pepper (Capana06g002501) protein consisting of the amino acid sequence shown in SEQ ID NO: 13, the amino acid corresponding to the amino acid (g) is the 132nd amino acid, and the amino acid corresponding to the amino acid (h) is the 173rd amino acid. .
　配列番号１４に示すアミノ酸配列からなるジャガイモ（Sotub06g016400.1.1）のタンパク質において、（ｇ）のアミノ酸に相当するアミノ酸は１３３番目のアミノ酸であり、（ｈ）のアミノ酸に相当するアミノ酸は１７４番目のアミノ酸である。 In the potato (Sotub06g016400.1.1) protein consisting of the amino acid sequence shown in SEQ ID NO: 14, the amino acid corresponding to the amino acid (g) is the 133rd amino acid, and the amino acid corresponding to the amino acid (h) is the 174th amino acid. is.
　配列番号１５に示すアミノ酸配列からなるジャガイモ（PGSC0003DMC400050347）のタンパク質において、（ｇ）のアミノ酸に相当するアミノ酸は１３３番目のアミノ酸であり、（ｈ）のアミノ酸に相当するアミノ酸は１７４番目のアミノ酸である。 In the potato (PGSC0003DMC400050347) protein consisting of the amino acid sequence shown in SEQ ID NO: 15, the amino acid corresponding to the amino acid (g) is the 133rd amino acid, and the amino acid corresponding to the amino acid (h) is the 174th amino acid. .
　また、ナス科植物を含む双子葉植物における、配列番号３に示すアミノ酸配列からなるタンパク質の相同タンパク質の一例として、シロイヌナズナのタンパク質を示す。配列番号１６に示すアミノ酸配列からなるシロイヌナズナ（AT5G52660.2）のタンパク質において、（ｇ）のアミノ酸に相当するアミノ酸は１５３番目のアミノ酸であり、（ｈ）のアミノ酸に相当するアミノ酸は１９４番目のアミノ酸である。 In addition, a protein of Arabidopsis thaliana is shown as an example of a protein homologous to the protein consisting of the amino acid sequence shown in SEQ ID NO: 3 in dicotyledonous plants including solanaceous plants. In the Arabidopsis thaliana (AT5G52660.2) protein consisting of the amino acid sequence shown in SEQ ID NO: 16, the amino acid corresponding to the amino acid (g) is the 153rd amino acid, and the amino acid corresponding to the amino acid (h) is the 194th amino acid. is.
　「（ｇ）又は（ｈ）のアミノ酸に相当するアミノ酸」は、一例として、（７）配列番号３に示すアミノ酸配列からなるタンパク質における（ｇ）又は（ｈ）のアミノ酸、（８）配列番号３に示すアミノ酸配列に対して、８０％以上の配列同一性を有するアミノ酸配列からなるタンパク質における（ｇ）又は（ｈ）のアミノ酸に相当するアミノ酸、又は、（９）配列番号３に示すアミノ酸配列に対して、６０個以下のアミノ酸が置換、欠損、付加又は挿入されたアミノ酸配列からなるタンパク質における（ｇ）又は（ｈ）のアミノ酸に相当するアミノ酸である。 "An amino acid corresponding to the amino acid (g) or (h)" is, for example, (7) the amino acid (g) or (h) in the protein consisting of the amino acid sequence shown in SEQ ID NO: 3, (8) SEQ ID NO: 3 or (9) the amino acid sequence shown in SEQ ID NO:3 On the other hand, 60 or less amino acids are amino acids corresponding to amino acids (g) or (h) in a protein consisting of an amino acid sequence with substitution, deletion, addition or insertion.
　（ｇ）又は（ｈ）のアミノ酸に相当するアミノ酸に置換、欠損、付加又は挿入を引き起こす変異の有無を検出する分子マーカーは、（ｇ）及び（ｈ）の両方のアミノ酸に相当するアミノ酸に置換又は欠損を引き起こす変異の有無を検出するものであってもよい。 Molecular markers for detecting the presence or absence of mutations that cause substitution, deletion, addition, or insertion in amino acids corresponding to (g) or (h) are substituted with amino acids corresponding to both (g) and (h). Alternatively, the presence or absence of deletion-causing mutations may be detected.
　分子マーカーの一例は、ナス科植物における、下記（ａ’）～（ｈ’）の塩基に相当する塩基自体（ＳＮＰ）か、当該塩基を含む連続したポリヌクレオチド：
　（ａ’）配列番号４に示す塩基配列からなるポリヌクレオチドの３２６番目の塩基に相当する塩基；
　（ｂ’）配列番号４に示す塩基配列からなるポリヌクレオチドの３９０番目の塩基に相当する塩基；
　（ｃ’）配列番号４に示す塩基配列からなるポリヌクレオチドの５９１番目の塩基に相当する塩基；
　（ｄ’）配列番号４に示す塩基配列からなるポリヌクレオチドの７４０番目の塩基に相当する塩基；
　（ｅ’）配列番号５に示す塩基配列からなるポリヌクレオチドの３９７番目の塩基に相当する塩基；
　（ｆ’）配列番号５に示す塩基配列からなるポリヌクレオチドの４１７番目の塩基に相当する塩基；
　（ｇ’）配列番号６に示す塩基配列からなるポリヌクレオチドの４０１番目の塩基に相当する塩基；
　（ｈ’）配列番号６に示す塩基配列からなるポリヌクレオチドの５２３番目の塩基に相当する塩基；
の少なくとも１つを含む。すなわち、判別方法は、上記（ａ’）～（ｈ’）の塩基に相当する塩基自体（ＳＮＰ）か、当該塩基を含む連続したポリヌクレオチドを、総乾物生産量の制御に関する分子マーカーとして検査する工程を含む。 An example of a molecular marker is the base itself (SNP) corresponding to the following bases (a') to (h') in Solanaceae plants, or a continuous polynucleotide containing the base:
(a') a base corresponding to the 326th base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 4;
(b') a base corresponding to the 390th base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 4;
(c') a base corresponding to the 591st base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 4;
(d') a base corresponding to the 740th base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 4;
(e') a base corresponding to the 397th base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 5;
(f') a base corresponding to the 417th base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 5;
(g') a base corresponding to the 401st base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 6;
(h') a base corresponding to the 523rd base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 6;
including at least one of That is, in the discrimination method, the base itself (SNP) corresponding to the bases (a') to (h') above, or a continuous polynucleotide containing the base, is examined as a molecular marker related to the control of the total dry matter production. Including process.
　上記（ａ’）～（ｈ’）の塩基に相当する塩基は、アミノ酸置換を引き起こすＳＮＰである。なお、上記（ａ’）は、ストップコドン変異、フレームシフト変異、及びヌル変異からなる群より選択される変異を引き起こすＳＮＰである。また、上記（ｂ’）及び（ｃ’）は、同義置換（サイレント変異）を引き起こすＳＮＰである。 The bases corresponding to the bases (a') to (h') above are SNPs that cause amino acid substitutions. The above (a') is an SNP that causes a mutation selected from the group consisting of stop codon mutation, frameshift mutation, and null mutation. In addition, (b') and (c') above are SNPs that cause synonymous substitutions (silent mutations).
　上記（ａ’）～（ｈ’）の塩基に相当する塩基を分子マーカーとして用いることで、上記（１）～（９）のタンパク質の、ナス科植物における総乾物生産量を制御する機能の発現に影響する変異の有無を検出することができる。上記（ａ’）～（ｈ’）の塩基に相当する塩基を分子マーカーとして用いることで、上記（ａ）～（ｈ）のアミノ酸に相当するアミノ酸に置換、欠損、付加又は挿入を引き起こす変異の有無を検出することができる。 By using the bases corresponding to the bases (a') to (h') above as molecular markers, the proteins (1) to (9) above express the function of controlling the total dry matter production in Solanaceous plants. can detect the presence or absence of mutations that affect By using the bases corresponding to the above (a') to (h') as molecular markers, mutations that cause substitution, deletion, addition or insertion of the amino acids corresponding to the above (a) to (h) Presence or absence can be detected.
　分子マーカーは、一例として、ＳＮＰマーカー、ＡＦＬＰ（分子増幅断片長多型）マーカー、ＲＦＬＰマーカー、マイクロサテライトマーカー、ＳＣＡＲマーカー、ＣＡＰＳマーカーである。 Examples of molecular markers include SNP markers, AFLP (molecular amplified fragment length polymorphism) markers, RFLP markers, microsatellite markers, SCAR markers, and CAPS markers.
　上記（ａ’）～（ｈ’）の塩基に相当する塩基自体、又は当該塩基を含む連続したポリヌクレオチドは、本実施例に記載されたＳＮＰマーカー又はこれと同一視できるＳＮＰマーカーである。ＳＮＰマーカーは、（ｉ）ＳＮＰに相当する塩基自体、（ｉｉ）ＳＮＰを含む連続したポリヌクレオチド、又は、（ｉｉｉ）２つのＳＮＰを含む連続したポリヌクレオチドであり得る。 The bases themselves corresponding to the bases (a') to (h') above, or the continuous polynucleotides containing the bases, are the SNP markers described in this example or SNP markers that can be identified with them. A SNP marker can be (i) the base corresponding to the SNP itself, (ii) a contiguous polynucleotide containing the SNP, or (iii) a contiguous polynucleotide containing two SNPs.
　（ｉ：ＳＮＰマーカー）
　ＳＮＰは、ＤＮＡの塩基配列中のある特定の領域内に一塩基の変異が見られるＤＮＡ多型を意味している。ＳＮＰマーカーにおいて、ＳＮＰは、トマト（Ｓ．リコペルシクム）のゲノム配列を基準とした一塩基多型である。基準植物であるトマト（Ｓ．リコペルシクム）のゲノム配列は、ｓｏｌｇｅｎｏｍｉｃｓ　ＦＴＰサイト（ftp://ftp.solgenomics. net/genomes/Solanum_lycopersicum/annotation/ITAG4.1_release/）に公表されている。 (i: SNP marker)
SNP means a DNA polymorphism in which a single base mutation is found in a specific region in the base sequence of DNA. In SNP markers, SNPs are single nucleotide polymorphisms based on the genome sequence of tomato (S. lycopersicum). The genome sequence of the reference plant tomato (S. lycopersicum) has been published on the solgenomics FTP site (ftp://ftp.solgenomics. net/genomes/Solanum_lycopersicum/annotation/ITAG4.1_release/).
　「（ａ’）～（ｈ’）の塩基」とは、本実施例に記載されたＳＮＰマーカーである。「（ａ’）～（ｈ’）の塩基に相当する塩基」とは、本実施例に記載されたＳＮＰマーカーと同一視できるＳＮＰマーカーである。「（ａ’）～（ｈ’）の塩基」は、ｔｆｗ６．１遺伝子上の変異である。ｔｆｗ６．１遺伝子は、基準となるトマト（Ｓ．リコペルシクム）由来の塩基配列からなる。ナス科植物においては、基準となる塩基配列に対してＳＮＰ以外の部分でも塩基配列が異なる部分が含まれ得る。このＳＮＰマーカーが位置するゲノム上の領域は、多数のナス科植物間で保存されているため、ホモロジー解析等の手法によって、このＳＮＰマーカーを特定することができる。ホモロジー解析の手法としては、「相当するアミノ酸」を特定する場合と同様の手法を用いることができる。 "(a') to (h') bases" are the SNP markers described in this example. The “bases corresponding to the bases (a′) to (h′)” are SNP markers that can be identified with the SNP markers described in this example. "(a') to (h') bases" are mutations on the tfw6.1 gene. The tfw6.1 gene consists of a standard base sequence derived from tomato (S. lycopersicum). Solanaceous plants may contain portions having different nucleotide sequences than SNPs with respect to the reference nucleotide sequence. Since the region on the genome where this SNP marker is located is conserved among many Solanaceae plants, this SNP marker can be identified by techniques such as homology analysis. As a method for homology analysis, the same method as for specifying the "corresponding amino acid" can be used.
　すなわち、他のナス科植物や、ナス科植物を含む双子葉植物において、ｔｆｗ６．１遺伝子に対応する遺伝子（すなわち植物間で高度に保存されている遺伝子）が存在する場合、「（ａ’）～（ｈ’）の塩基に相当する塩基」とは、ホモロジー検索等の手法によって、（ａ’）～（ｈ’）の塩基に相当するとされたｔｆｗ６．１遺伝子に対応する遺伝子上の塩基を指す。例えば、後述する（ａ２）～（ｈ２）に記載のポリヌクレオチドや、（ａ３）～（ｈ３）に記載のポリヌクレオチドが、ｔｆｗ６．１遺伝子に対応する遺伝子の一例である。 That is, when there is a gene corresponding to the tfw6.1 gene (that is, a gene highly conserved among plants) in other solanaceous plants and dicotyledonous plants including solanaceous plants, "(a') The term "base corresponding to the base of ~ (h')" refers to the base on the gene corresponding to the tfw6.1 gene, which was determined to correspond to the base of (a') ~ (h') by a method such as homology search. Point. For example, polynucleotides described in (a2) to (h2) and polynucleotides described in (a3) to (h3) below are examples of genes corresponding to the tfw6.1 gene.
　ナス科植物における、配列番号４に示すｔｆｗ６．１遺伝子のＣＤＳの塩基配列からなる遺伝子の相同遺伝子並びに（ａ’）～（ｄ’）の塩基に相当する塩基の一例を示す。 An example of the homologous gene of the gene consisting of the base sequence of the CDS of the tfw6.1 gene shown in SEQ ID NO: 4 and the bases corresponding to bases (a') to (d') in Solanaceae plants is shown.
　配列番号１７に示す塩基配列からなるピーマン（CA09g13460）の遺伝子において、（ａ’）の塩基に相当する塩基は１０５２番目の塩基であり、（ｂ’）の塩基に相当する塩基は１１１６番目の塩基であり、（ｃ’）の塩基に相当する塩基は１３１７番目の塩基であり、（ｄ’）の塩基に相当する塩基は１４６６番目の塩基である。 In the green pepper (CA09g13460) gene consisting of the nucleotide sequence shown in SEQ ID NO: 17, the base corresponding to (a') is the 1052nd base, and the base corresponding to (b') is the 1116th base. , the base corresponding to the base of (c′) is the 1317th base, and the base corresponding to the base of (d′) is the 1466th base.
　配列番号１８に示す塩基配列からなるナス（Sme2.5_14468.1_g00001.1）の遺伝子において、（ａ’）の塩基に相当する塩基は９９８番目の塩基であり、（ｂ’）の塩基に相当する塩基は１０６２番目の塩基であり、（ｃ’）の塩基に相当する塩基は１２６３番目の塩基であり、（ｄ’）の塩基に相当する塩基は１４１２番目の塩基である。 In the eggplant (Sme2.5_14468.1_g00001.1) gene consisting of the nucleotide sequence shown in SEQ ID NO: 18, the base corresponding to the base (a') is the 998th base, and corresponds to the base (b'). The base is the 1062nd base, the base corresponding to (c') is the 1263rd base, and the base corresponding to (d') is the 1412nd base.
　配列番号１９に示す塩基配列からなるジャガイモ（Sotub06g015180.1.1）の遺伝子において、（ａ’）の塩基に相当する塩基は１０１０番目の塩基であり、（ｂ’）の塩基に相当する塩基は１０７４番目の塩基であり、（ｃ’）の塩基に相当する塩基は１２７５番目の塩基であり、（ｄ’）の塩基に相当する塩基は１４２４番目の塩基である。 In the gene of potato (Sotub06g015180.1.1) consisting of the nucleotide sequence shown in SEQ ID NO: 19, the base corresponding to (a') is the 1010th base, and the base corresponding to (b') is the 1074th base. , the base corresponding to the base of (c') is the 1275th base, and the base corresponding to the base of (d') is the 1424th base.
　また、ナス科植物を含む双子葉植物における、配列番号４に示す塩基配列からなる遺伝子の相同遺伝子の一例として、シロイヌナズナの遺伝子を示す。配列番号２０に示す塩基配列からなるシロイヌナズナ（AT5G52660.2）の遺伝子において、（ａ’）の塩基に相当する塩基は８０３番目の塩基であり、（ｂ’）の塩基に相当する塩基は８６７番目の塩基であり、（ｃ’）の塩基に相当する塩基は１０６８番目の塩基であり、（ｄ’）の塩基に相当する塩基は１２１７番目の塩基である。 In addition, the gene of Arabidopsis thaliana is shown as an example of a gene homologous to the gene consisting of the nucleotide sequence shown in SEQ ID NO: 4 in dicotyledonous plants including solanaceous plants. In the Arabidopsis thaliana (AT5G52660.2) gene consisting of the nucleotide sequence shown in SEQ ID NO: 20, the base corresponding to (a') is the 803rd base, and the base corresponding to (b') is the 867th base. , the base corresponding to the base of (c') is the 1068th base, and the base corresponding to the base of (d') is the 1217th base.
　ナス科植物における、配列番号６に示すｔｆｗ６．１遺伝子のＣＤＳの塩基配列からなる遺伝子の相同遺伝子並びに（ｇ’）～（ｈ’）の塩基に相当する塩基の一例を示す。 An example of the homologous gene of the gene consisting of the nucleotide sequence of the CDS of the tfw6.1 gene shown in SEQ ID NO: 6 and the bases corresponding to bases (g') to (h') in Solanaceae plants is shown.
　配列番号２１に示す塩基配列からなるピーマン（CA06g05790）の遺伝子において、（ｇ’）の塩基に相当する塩基は４０１番目の塩基であり、（ｈ’）の塩基に相当する塩基は５２３番目の塩基である。 In the green pepper (CA06g05790) gene consisting of the nucleotide sequence shown in SEQ ID NO: 21, the base corresponding to (g') is the 401st base, and the base corresponding to (h') is the 523rd base. is.
　配列番号２２に示す塩基配列からなるピーマン（Capang06g002314）の遺伝子において、（ｇ’）の塩基に相当する塩基は３９５番目の塩基であり、（ｈ’）の塩基に相当する塩基は５１７番目の塩基である。 In the green pepper (Capang06g002314) gene consisting of the nucleotide sequence shown in SEQ ID NO: 22, the base corresponding to (g') is the 395th base, and the base corresponding to (h') is the 517th base. is.
　配列番号２３に示す塩基配列からなるピーマン（Capana06g002501）の遺伝子において、（ｇ’）の塩基に相当する塩基は３９５番目の塩基であり、（ｈ’）の塩基に相当する塩基は５１７番目の塩基である。 In the green pepper (Capana06g002501) gene consisting of the nucleotide sequence shown in SEQ ID NO: 23, the base corresponding to (g') is the 395th base, and the base corresponding to (h') is the 517th base. is.
　配列番号２４に示す塩基配列からなるジャガイモ（Sotub06g016400.1.1）の遺伝子において、（ｇ’）の塩基に相当する塩基は３９８番目の塩基であり、（ｈ’）の塩基に相当する塩基は５２０番目の塩基である。 In the gene of potato (Sotub06g016400.1.1) consisting of the nucleotide sequence shown in SEQ ID NO: 24, the base corresponding to (g') is the 398th base, and the base corresponding to (h') is the 520th base. is the base of
　配列番号２５に示す塩基配列からなるジャガイモ（PGSC0003DMC400050347）の遺伝子において、（ｇ’）の塩基に相当する塩基は３９８番目の塩基であり、（ｈ’）の塩基に相当する塩基は５２０番目の塩基である。 In the gene of potato (PGSC0003DMC400050347) consisting of the nucleotide sequence shown in SEQ ID NO: 25, the base corresponding to (g') is the 398th base, and the base corresponding to (h') is the 520th base. is.
　また、ナス科植物を含む双子葉植物における、配列番号６に示す塩基配列からなる遺伝子の相同遺伝子の一例として、シロイヌナズナの遺伝子を示す。配列番号２６に示す塩基配列からなるシロイヌナズナ（AT5G52660.2）の遺伝子において、（ｇ’）の塩基に相当する塩基は４５８番目の塩基であり、（ｈ’）の塩基に相当する塩基は５８０番目の塩基である。 In addition, the gene of Arabidopsis thaliana is shown as an example of a gene homologous to the gene consisting of the nucleotide sequence shown in SEQ ID NO: 6 in dicotyledonous plants including solanaceous plants. In the Arabidopsis thaliana (AT5G52660.2) gene consisting of the nucleotide sequence shown in SEQ ID NO: 26, the base corresponding to (g') is the 458th base, and the base corresponding to (h') is the 580th base. is the base of
　分子マーカーは、上記の（ａ’）～（ｈ’）のＳＮＰからなるＳＮＰマーカーである。このＳＮＰマーカーは、本発明者らが新たに同定したＳＮＰマーカーであり、当業者は、このＳＮＰマーカーのそれぞれのＳＮＰを表す塩基配列に基づき、当該ＳＮＰマーカーのゲノム上の位置を特定することができる。 The molecular marker is a SNP marker consisting of the SNPs (a') to (h') above. This SNP marker is a SNP marker newly identified by the present inventors, and a person skilled in the art can identify the position of the SNP marker on the genome based on the nucleotide sequence representing each SNP of this SNP marker. can.
　（ａ’）のＳＮＰ（以下、ＳＮＰ（ａ’）ともいう）は、配列番号４に示す塩基配列の３２６番目の塩基又はこれに相当する塩基の多型を示している。また、（ｂ’）のＳＮＰ（以下、ＳＮＰ（ｂ’）ともいう）は、配列番号４に示す塩基配列の３９０番目の塩基又はこれに相当する塩基の多型を示している。また、（ｃ’）のＳＮＰ（以下、ＳＮＰ（ｃ’）ともいう）は、配列番号４に示す塩基配列の５９１番目の塩基又はこれに相当する塩基の多型を示している。また、（ｄ’）のＳＮＰ（以下、ＳＮＰ（ｄ’）ともいう）は、配列番号４に示す塩基配列の７４０番目の塩基又はこれに相当する塩基の多型を示している。また、（ｅ’）のＳＮＰ（以下、ＳＮＰ（ｅ’）ともいう）は、配列番号５に示す塩基配列の３９７番目の塩基又はこれに相当する塩基の多型を示している。また、（ｆ’）のＳＮＰ（以下、ＳＮＰ（ｆ’）ともいう）は、配列番号５に示す塩基配列の４１７番目の塩基又はこれに相当する塩基の多型を示している。また、（ｇ’）のＳＮＰ（以下、ＳＮＰ（ｇ’）ともいう）は、配列番号６に示す塩基配列の４０１番目の塩基又はこれに相当する塩基の多型を示している。また、（ｈ’）のＳＮＰ（以下、ＳＮＰ（ｈ’）ともいう）は、配列番号６に示す塩基配列の５２３番目の塩基又はこれに相当する塩基の多型を示している。 The SNP of (a') (hereinafter also referred to as SNP (a')) indicates a polymorphism of the 326th base of the base sequence shown in SEQ ID NO: 4 or a base corresponding thereto. The SNP of (b') (hereinafter also referred to as SNP (b')) indicates a polymorphism of the 390th base in the base sequence shown in SEQ ID NO: 4 or a base corresponding thereto. The SNP of (c') (hereinafter also referred to as SNP (c')) indicates a polymorphism of the 591st base in the base sequence shown in SEQ ID NO: 4 or a base corresponding thereto. The SNP of (d') (hereinafter also referred to as SNP (d')) indicates a polymorphism of the 740th base in the base sequence shown in SEQ ID NO: 4 or a base corresponding thereto. The SNP (e') (hereinafter also referred to as SNP (e')) indicates a polymorphism of the 397th base in the base sequence shown in SEQ ID NO: 5 or a base corresponding thereto. The SNP of (f') (hereinafter also referred to as SNP (f')) indicates a polymorphism of the 417th base in the base sequence shown in SEQ ID NO: 5 or a base corresponding thereto. The SNP of (g') (hereinafter also referred to as SNP (g')) indicates a polymorphism of the 401st base in the base sequence shown in SEQ ID NO: 6 or a base corresponding thereto. The SNP of (h') (hereinafter also referred to as SNP (h')) indicates a polymorphism of the 523rd base in the base sequence shown in SEQ ID NO: 6 or a base corresponding thereto.
　分子マーカーは、以下の少なくとも１つ：
　ＳＮＰ（ａ’）がＧ；
　ＳＮＰ（ｂ’）がＡ；
　ＳＮＰ（ｃ’）がＴ；
　ＳＮＰ（ｄ’）がＧ；
　ＳＮＰ（ｅ’）がＧ；
　ＳＮＰ（ｆ’）がＴ；
　ＳＮＰ（ｇ’）がＣ；
　ＳＮＰ（ｈ’）がＴ；
であるとき、当該ナス科植物は総乾物生産量が増加していると判定することができる。 Molecular markers are at least one of the following:
SNP (a') is G;
SNP (b') is A;
SNP (c') is T;
SNP (d') is G;
SNP (e') is G;
SNP (f') is T;
SNP (g') is C;
SNP (h') is T;
When , it can be determined that the solanaceous plant has increased total dry matter production.
　また、分子マーカーは、ＳＮＰ（ａ’）～ＳＮＰ（ｄ’）、ＳＮＰ（ｅ’）及びＳＮＰ（ｆ’）、又は、ＳＮＰ（ｇ’）及びＳＮＰ（ｈ’）をハプロタイプブロックとして解析することにより、ナス科植物における総乾物生産量を判定してもよい。ここで、ナス科植物は総乾物生産量が増加しているとは、総乾物生産量制御タンパク質を有するナス科植物個体の総乾物生産量が、総乾物生産量制御タンパク質を有さない他のナス科植物個体と比較して相対的に多いことが意図される。 In addition, for molecular markers, SNP (a') to SNP (d'), SNP (e') and SNP (f'), or SNP (g') and SNP (h') are analyzed as haplotype blocks. may determine total dry matter production in solanaceous plants. Here, the expression that the solanaceous plant has increased total dry matter production means that the total dry matter production of the individual solanaceous plant having the total dry matter production control protein is higher than the total dry matter production of the individual solanaceous plant having the total dry matter production control protein. It is intended to be relatively large compared to Solanaceae plant individuals.
　（ｉｉ：ＳＮＰを含むポリヌクレオチド）
　分子マーカーは、ＳＮＰ（ａ’）～ＳＮＰ（ｈ’）を含む連続したポリヌクレオチド（以下、ポリヌクレオチド（ａ’）～ポリヌクレオチド（ｈ’））であってもよい。 (ii: Polynucleotide containing SNP)
A molecular marker may be a contiguous polynucleotide containing SNP(a') to SNP(h') (hereinafter, polynucleotide (a') to polynucleotide (h')).
　ポリヌクレオチド（ａ’）は、（ａ１）ｔｆｗ６．１遺伝子の塩基配列において、ＳＮＰ（ａ’）を含む領域の塩基配列からなるポリヌクレオチド、（ａ２）（ａ１）のポリヌクレオチドの塩基配列において、ＳＮＰ（ａ’）以外の塩基配列に対して、１又は数個の塩基が置換、欠失、付加又は挿入された塩基配列からなり、ナス科植物における総乾物生産量を判定する機能を有するポリヌクレオチド、又は、（ａ３）（ａ１）のポリヌクレオチドの塩基配列において、ＳＮＰ（ａ’）以外の塩基配列に対して、９０％以上の同一性を有する塩基配列からなり、ナス科植物における総乾物生産量を判定する機能を有するポリヌクレオチドである。 Polynucleotide (a') is (a1) a polynucleotide consisting of the nucleotide sequence of the region containing SNP (a') in the nucleotide sequence of the tfw6.1 gene, (a2) in the nucleotide sequence of the polynucleotide of (a1), A poly which consists of a nucleotide sequence in which one or several nucleotides are substituted, deleted, added or inserted into a nucleotide sequence other than SNP (a') and has the function of determining the total dry matter production in Solanaceous plants Total dry matter in Solanaceous plants, consisting of a nucleotide sequence having 90% or more identity to a nucleotide sequence other than SNP (a') in the nucleotide sequence of the polynucleotide or (a3) (a1) It is a polynucleotide having a function to determine the amount of production.
　ポリヌクレオチド（ｂ’）は、（ｂ１）ｔｆｗ６．１遺伝子の塩基配列において、ＳＮＰ（ｂ’）を含む領域の塩基配列からなるポリヌクレオチド、（ｂ２）（ｂ１）のポリヌクレオチドの塩基配列において、ＳＮＰ（ｂ’）以外の塩基配列に対して、１又は数個の塩基が置換、欠失、付加又は挿入された塩基配列からなり、ナス科植物における総乾物生産量を判定する機能を有するポリヌクレオチド、又は、（ｂ３）（ｂ１）のポリヌクレオチドの塩基配列において、ＳＮＰ（ｂ’）以外の塩基配列に対して、９０％以上の同一性を有する塩基配列からなり、ナス科植物における総乾物生産量を判定する機能を有するポリヌクレオチドである。 Polynucleotide (b') is (b1) a polynucleotide consisting of the nucleotide sequence of the region containing SNP (b') in the nucleotide sequence of the tfw6.1 gene, (b2) in the nucleotide sequence of the polynucleotide of (b1), A poly which consists of a nucleotide sequence in which one or several nucleotides are substituted, deleted, added or inserted into a nucleotide sequence other than SNP (b') and has the function of determining the total dry matter production in Solanaceous plants Total dry matter in Solanaceous plants, consisting of a nucleotide sequence having 90% or more identity to a nucleotide sequence other than SNP (b') in the nucleotide sequence of the polynucleotide or (b3) (b1) It is a polynucleotide having a function to determine the amount of production.
　ポリヌクレオチド（ｃ’）は、（ｃ１）ｔｆｗ６．１遺伝子の塩基配列において、ＳＮＰ（ｃ’）を含む領域の塩基配列からなるポリヌクレオチド、（ｃ２）（ｃ１）のポリヌクレオチドの塩基配列において、ＳＮＰ（ｃ’）以外の塩基配列に対して、１又は数個の塩基が置換、欠失、付加又は挿入された塩基配列からなり、ナス科植物における総乾物生産量を判定する機能を有するポリヌクレオチド、又は、（ｃ３）（ｃ１）のポリヌクレオチドの塩基配列において、ＳＮＰ（ｃ’）以外の塩基配列に対して、９０％以上の同一性を有する塩基配列からなり、ナス科植物における総乾物生産量を判定する機能を有するポリヌクレオチドである。 Polynucleotide (c') is a polynucleotide consisting of the nucleotide sequence of the region containing SNP (c') in the nucleotide sequence of (c1) the tfw6.1 gene, (c2) in the nucleotide sequence of the polynucleotide of (c1), A poly which consists of a nucleotide sequence in which one or several nucleotides are substituted, deleted, added or inserted into a nucleotide sequence other than SNP (c′) and has the function of determining the total dry matter production in Solanaceous plants Total dry matter in Solanaceae plants consisting of a nucleotide sequence having 90% or more identity to a nucleotide sequence other than SNP (c') in the nucleotide sequence of the polynucleotide or (c3) (c1) It is a polynucleotide having a function to determine the amount of production.
　ポリヌクレオチド（ｄ’）は、（ｄ１）ｔｆｗ６．１遺伝子の塩基配列において、ＳＮＰ（ｄ’）を含む領域の塩基配列からなるポリヌクレオチド、（ｄ２）（ｄ１）のポリヌクレオチドの塩基配列において、ＳＮＰ（ｄ’）以外の塩基配列に対して、１又は数個の塩基が置換、欠失、付加又は挿入された塩基配列からなり、ナス科植物における総乾物生産量を判定する機能を有するポリヌクレオチド、又は、（ｄ３）（ｄ１）のポリヌクレオチドの塩基配列において、ＳＮＰ（ｄ’）以外の塩基配列に対して、９０％以上の同一性を有する塩基配列からなり、ナス科植物における総乾物生産量を判定する機能を有するポリヌクレオチドである。 Polynucleotide (d') is (d1) a polynucleotide consisting of the nucleotide sequence of the region containing SNP (d') in the nucleotide sequence of the tfw6.1 gene, (d2) in the nucleotide sequence of the (d1) polynucleotide, A poly which consists of a base sequence in which one or several bases are substituted, deleted, added or inserted into a base sequence other than SNP (d') and has the function of determining the total dry matter production in Solanaceous plants Total dry matter in Solanaceous plants, consisting of a nucleotide sequence having 90% or more identity to a nucleotide sequence other than SNP (d') in the nucleotide sequence of the polynucleotide or (d3) (d1) It is a polynucleotide having a function to determine the amount of production.
　ポリヌクレオチド（ｅ’）は、（ｅ１）ｔｆｗ６．１遺伝子の塩基配列において、ＳＮＰ（ｅ’）を含む領域の塩基配列からなるポリヌクレオチド、（ｅ２）（ｅ１）のポリヌクレオチドの塩基配列において、ＳＮＰ（ｅ’）以外の塩基配列に対して、１又は数個の塩基が置換、欠失、付加又は挿入された塩基配列からなり、ナス科植物における総乾物生産量を判定する機能を有するポリヌクレオチド、又は、（ｅ３）（ｅ１）のポリヌクレオチドの塩基配列において、ＳＮＰ（ｅ’）以外の塩基配列に対して、９０％以上の同一性を有する塩基配列からなり、ナス科植物における総乾物生産量を判定する機能を有するポリヌクレオチドである。 Polynucleotide (e') is (e1) a polynucleotide consisting of the nucleotide sequence of the region containing SNP (e') in the nucleotide sequence of the tfw6.1 gene, (e2) in the polynucleotide nucleotide sequence of (e1), A poly which consists of a nucleotide sequence in which one or several nucleotides are substituted, deleted, added or inserted into a nucleotide sequence other than SNP (e′) and has the function of determining the total dry matter production in Solanaceous plants A nucleotide or a nucleotide sequence having 90% or more identity to a nucleotide sequence other than SNP (e') in the polynucleotide nucleotide sequence of (e3) (e1), and total dry matter in Solanaceae plants It is a polynucleotide having a function to determine the amount of production.
　ポリヌクレオチド（ｆ’）は、（ｆ１）ｔｆｗ６．１遺伝子の塩基配列において、ＳＮＰ（ｆ’）を含む領域の塩基配列からなるポリヌクレオチド、（ｆ２）（ｆ１）のポリヌクレオチドの塩基配列において、ＳＮＰ（ｆ’）以外の塩基配列に対して、１又は数個の塩基が置換、欠失、付加又は挿入された塩基配列からなり、ナス科植物における総乾物生産量を判定する機能を有するポリヌクレオチド、又は、（ｆ３）（ｆ１）のポリヌクレオチドの塩基配列において、ＳＮＰ（ｆ’）以外の塩基配列に対して、９０％以上の同一性を有する塩基配列からなり、ナス科植物における総乾物生産量を判定する機能を有するポリヌクレオチドである。 Polynucleotide (f') is (f1) a polynucleotide consisting of the nucleotide sequence of the region containing SNP (f') in the nucleotide sequence of the tfw6.1 gene, (f2) in the nucleotide sequence of the (f1) polynucleotide, A poly which consists of a nucleotide sequence in which one or several nucleotides are substituted, deleted, added or inserted into a nucleotide sequence other than SNP (f') and has the function of determining the total dry matter production in Solanaceous plants Total dry matter in Solanaceae plants consisting of a nucleotide sequence having 90% or more identity to a nucleotide sequence other than SNP (f') in the nucleotide sequence of the polynucleotide or (f3) (f1) It is a polynucleotide having a function to determine the amount of production.
　ポリヌクレオチド（ｇ’）は、（ｇ１）ｔｆｗ６．１遺伝子の塩基配列において、ＳＮＰ（ｇ’）を含む領域の塩基配列からなるポリヌクレオチド、（ｇ２）（ｇ１）のポリヌクレオチドの塩基配列において、ＳＮＰ（ｇ’）以外の塩基配列に対して、１又は数個の塩基が置換、欠失、付加又は挿入された塩基配列からなり、ナス科植物における総乾物生産量を判定する機能を有するポリヌクレオチド、又は、（ｇ３）（ｇ１）のポリヌクレオチドの塩基配列において、ＳＮＰ（ｇ’）以外の塩基配列に対して、９０％以上の同一性を有する塩基配列からなり、ナス科植物における総乾物生産量を判定する機能を有するポリヌクレオチドである。 Polynucleotide (g') is (g1) a polynucleotide consisting of the nucleotide sequence of the region containing SNP (g') in the nucleotide sequence of the tfw6.1 gene, (g2) in the nucleotide sequence of the (g1) polynucleotide, A poly which consists of a base sequence in which one or several bases are substituted, deleted, added or inserted into a base sequence other than SNP (g') and has the function of determining the total dry matter production in Solanaceous plants Total dry matter in Solanaceae plants consisting of a nucleotide sequence having 90% or more identity to a nucleotide sequence other than SNP (g') in the nucleotide sequence of the polynucleotide or (g3) (g1) It is a polynucleotide having a function to determine the amount of production.
　ポリヌクレオチド（ｈ’）は、（ｈ１）ｔｆｗ６．１遺伝子の塩基配列において、ＳＮＰ（ｈ’）を含む領域の塩基配列からなるポリヌクレオチド、（ｈ２）（ｈ１）のポリヌクレオチドの塩基配列において、ＳＮＰ（ｈ’）以外の塩基配列に対して、１又は数個の塩基が置換、欠失、付加又は挿入された塩基配列からなり、ナス科植物における総乾物生産量を判定する機能を有するポリヌクレオチド、又は、（ｈ３）（ｈ１）のポリヌクレオチドの塩基配列において、ＳＮＰ（ｈ’）以外の塩基配列に対して、９０％以上の同一性を有する塩基配列からなり、ナス科植物における総乾物生産量を判定する機能を有するポリヌクレオチドである。 Polynucleotide (h') is (h1) a polynucleotide consisting of the nucleotide sequence of the region containing SNP (h') in the nucleotide sequence of the tfw6.1 gene, (h2) in the nucleotide sequence of the (h1) polynucleotide, A poly which consists of a nucleotide sequence in which one or several nucleotides are substituted, deleted, added or inserted into a nucleotide sequence other than SNP (h') and has the function of determining the total dry matter production in Solanaceous plants Total dry matter in Solanaceae plants, consisting of a nucleotide sequence having 90% or more identity to a nucleotide sequence other than SNP (h') in the nucleotide sequence of the polynucleotide or (h3) (h1) It is a polynucleotide having a function to determine the amount of production.
　（ａ１）のポリヌクレオチド～（ｈ１）のポリヌクレオチドは、例えば、ｔｆｗ６．１遺伝子の塩基配列に基づき、総乾物生産量の増加したナス科植物（例えば、総乾物生産量の多いトマト（Ｓ．リコペルシクムのオランダＦ１品種）から得ることができる。 Polynucleotides (a1) to (h1) are, for example, based on the base sequence of the tfw6.1 gene, obtained from solanaceous plants with increased total dry matter production (for example, tomatoes with high total dry matter production (S. lycopersicum Dutch F1 cultivar).
　（ａ２）のポリヌクレオチド～（ｈ２）のポリヌクレオチドは、（ａ１）のポリヌクレオチド～（ｈ１）のポリヌクレオチドの塩基配列において、ＳＮＰ（ａ’）～ＳＮＰ（ｈ’）に相当する塩基は保存され、それ以外の塩基配列に、数個（例えば１～１０個、好ましくは１～５個、より好ましくは１、２又は３個）の塩基の修飾（置換、欠失、挿入又は付加）を含むものであり得る。このようなポリヌクレオチドの塩基配列は、当該技術分野における当業者にとって明らかであり、上述したデータベースに登録されているトマト（Ｓ．リコペルシクム）のゲノム配列を参照することにより、又は、総乾物生産量の多いナス科植物のゲノム上におけるＳＮＰの近傍領域の塩基配列を解読することにより、決定することができる。 Polynucleotide (a2) to polynucleotide (h2), in the nucleotide sequence of polynucleotide (a1) to (h1), bases corresponding to SNP (a') to SNP (h') are conserved and several (for example, 1 to 10, preferably 1 to 5, more preferably 1, 2 or 3) base modifications (substitutions, deletions, insertions or additions) in other base sequences can contain The nucleotide sequences of such polynucleotides are obvious to those skilled in the art, and by referring to the genome sequence of tomato (S. lycopersicum) registered in the database mentioned above, or by referring to the total dry matter production It can be determined by decoding the base sequence of the neighboring region of the SNP on the genome of a solanaceous plant with a large number of plants.
　（ａ３）のポリヌクレオチド～（ｈ３）のポリヌクレオチドは、（ａ１）のポリヌクレオチド～（ｈ１）のポリヌクレオチドの塩基配列において、ＳＮＰ（ａ’）～ＳＮＰ（ｈ’）に相当する塩基は保存され、それ以外の塩基配列に対して、例えば、９０％以上、９５％以上、９６％以上、９７％以上、９８％以上、又は９９％以上の同一性を有するものであり得る。このような塩基配列の同一性は、例えば、ＢＬＡＳＴ、ＦＡＳＴＡ等の解析ソフトウェアを用いて、２つの塩基配列をアライメントすることによって決定することができる。 Polynucleotide (a3) to polynucleotide (h3), in the nucleotide sequence of polynucleotide (a1) to (h1), bases corresponding to SNP (a') to SNP (h') are conserved and have, for example, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to other base sequences. The identity of such nucleotide sequences can be determined, for example, by aligning two nucleotide sequences using analysis software such as BLAST and FASTA.
　分子マーカーは、以下の少なくとも１つ：
　ポリヌクレオチド（ａ）におけるＳＮＰ（ａ’）に相当する塩基がＧ；
　ポリヌクレオチド（ｂ）におけるＳＮＰ（ｂ’）に相当する塩基がＡ；
　ポリヌクレオチド（ｃ）におけるＳＮＰ（ｃ’）に相当する塩基がＴ；
　ポリヌクレオチド（ｄ）におけるＳＮＰ（ｄ’）に相当する塩基がＧ；
　ポリヌクレオチド（ｅ）におけるＳＮＰ（ｅ’）に相当する塩基がＧ；
　ポリヌクレオチド（ｆ）におけるＳＮＰ（ｆ’）に相当する塩基がＴ；
　ポリヌクレオチド（ｇ）におけるＳＮＰ（ｇ’）に相当する塩基がＣ；
　ポリヌクレオチド（ｈ）におけるＳＮＰ（ｈ’）に相当する塩基がＴ；
であるとき、当該ナス科植物は総乾物生産量が増加していると判定することができる。 Molecular markers are at least one of the following:
the base corresponding to SNP (a') in polynucleotide (a) is G;
The base corresponding to SNP (b') in polynucleotide (b) is A;
the base corresponding to SNP (c') in polynucleotide (c) is T;
the base corresponding to SNP (d') in polynucleotide (d) is G;
the base corresponding to SNP (e') in polynucleotide (e) is G;
the base corresponding to SNP (f') in polynucleotide (f) is T;
the base corresponding to SNP (g') in polynucleotide (g) is C;
the base corresponding to SNP (h') in polynucleotide (h) is T;
When , it can be determined that the solanaceous plant has increased total dry matter production.
　分子マーカーは、ポリヌクレオチド（ａ）～ポリヌクレオチド（ｄ）、ポリヌクレオチド（ｅ）及びポリヌクレオチド（ｆ）、又は、ポリヌクレオチド（ｇ）及びポリヌクレオチド（ｈ）をハプロタイプブロックとして解析することにより、ナス科植物における総乾物生産量を判定してもよい。 Molecular markers are obtained by analyzing polynucleotides (a) to (d), polynucleotides (e) and polynucleotides (f), or polynucleotides (g) and polynucleotides (h) as haplotype blocks. The total dry matter production in the solanaceous plant may be determined.
　（ｉｉｉ：２つ以上のＳＮＰを含むポリヌクレオチド）
　分子マーカーは、ＳＮＰ（ａ’）～ＳＮＰ（ｄ’）の少なくとも２つを含む連続したポリヌクレオチドであってもよい。このようなポリヌクレオチドは、ＳＮＰ（ａ’）～ＳＮＰ（ｄ’）の部位間の領域を、ＳＮＰ（ａ’）～ＳＮＰ（ｄ’）の部位と共に含んでいる。このようなポリヌクレオチドは、例えば、総乾物生産量が増加したナス科植物における対応するＳＮＰ（ａ’）～ＳＮＰ（ｄ’）の部位間の領域を参照することで得られる。このようなポリヌクレオチドの塩基配列は、総乾物生産量が増加したナス科植物の塩基配列と少なくとも部分的に一致する。 (iii: a polynucleotide containing two or more SNPs)
A molecular marker may be a contiguous polynucleotide comprising at least two of SNP(a') through SNP(d'). Such a polynucleotide includes the region between sites SNP(a') to SNP(d') together with sites SNP(a') to SNP(d'). Such polynucleotides can be obtained, for example, by referring to the region between the corresponding SNP(a') to SNP(d') sites in Solanaceae plants with increased total dry matter production. The nucleotide sequence of such a polynucleotide at least partially matches the nucleotide sequence of a solanaceous plant with increased total dry matter production.
　分子マーカーは、ＳＮＰ（ｅ’）及びＳＮＰ（ｆ’）を含む連続したポリヌクレオチドであってもよい。このようなポリヌクレオチドは、ＳＮＰ（ｅ’）及びＳＮＰ（ｆ’）の部位間の領域を、ＳＮＰ（ｅ’）及びＳＮＰ（ｆ’）の部位と共に含んでいる。このようなポリヌクレオチドは、例えば、総乾物生産量が増加したナス科植物における対応するＳＮＰ（ｅ’）及びＳＮＰ（ｆ’）の部位間の領域を参照することで得られる。このようなポリヌクレオチドの塩基配列は、総乾物生産量が増加したナス科植物の塩基配列と少なくとも部分的に一致する。 A molecular marker may be a continuous polynucleotide containing SNP (e') and SNP (f'). Such a polynucleotide includes the region between sites of SNP(e') and SNP(f') together with sites of SNP(e') and SNP(f'). Such polynucleotides can be obtained, for example, by referring to the region between the corresponding SNP (e') and SNP (f') sites in Solanaceae plants with increased total dry matter production. The nucleotide sequence of such a polynucleotide at least partially matches the nucleotide sequence of a solanaceous plant with increased total dry matter production.
　分子マーカーは、ＳＮＰ（ｇ’）及びＳＮＰ（ｈ’）を含む連続したポリヌクレオチドであってもよい。このようなポリヌクレオチドは、ＳＮＰ（ｇ’）及びＳＮＰ（ｈ’）の部位間の領域を、ＳＮＰ（ｇ’）及びＳＮＰ（ｈ’）の部位と共に含んでいる。このようなポリヌクレオチドは、例えば、総乾物生産量が増加したナス科植物における対応するＳＮＰ（ｇ’）及びＳＮＰ（ｈ’）の部位間の領域を参照することで得られる。このようなポリヌクレオチドの塩基配列は、総乾物生産量が増加したナス科植物の塩基配列と少なくとも部分的に一致する。 A molecular marker may be a continuous polynucleotide containing SNP (g') and SNP (h'). Such a polynucleotide includes the region between sites of SNP(g') and SNP(h') together with sites of SNP(g') and SNP(h'). Such polynucleotides can be obtained, for example, by referring to the region between the corresponding SNP (g') and SNP (h') sites in Solanaceae plants with increased total dry matter production. The nucleotide sequence of such a polynucleotide at least partially matches the nucleotide sequence of a solanaceous plant with increased total dry matter production.
　上述した分子マーカーを用いて、ナス科植物における総乾物生産量を判定する方法としては特に限定されず、例えば、ＳＮＰを検出する公知のＳＮＰ分析方法を用いることができる。このような公知のＳＮＰ分析方法には、ナス科植物の被検体のＰＣＲ増幅断片中のＳＮＰを検出することによりＳＮＰ分析する方法が含まれる。 The method for determining the total dry matter production of Solanaceae plants using the above-described molecular markers is not particularly limited, and for example, a known SNP analysis method for detecting SNPs can be used. Such known SNP analysis methods include a method of SNP analysis by detecting SNPs in PCR-amplified fragments of test samples of Solanaceae plants.
　判別方法は、分子マーカーを含む領域を増幅するプライマーセットを用いて、ナス科植物のＤＮＡにおける前記領域を増幅してもよい。このようなプライマーセットは、一例として、ＳＮＰ（ａ’）～ＳＮＰ（ｈ’）の少なくとも１つを含む領域を増幅するプライマーセットである。 In the discrimination method, a primer set that amplifies a region containing a molecular marker may be used to amplify the region in the DNA of the Solanaceae plant. Such a primer set is, for example, a primer set that amplifies a region containing at least one of SNP(a') to SNP(h').
　ナス科植物の被検体のＤＮＡにおける前記領域の増幅は、ナス科植物の被検体から抽出したＤＮＡを鋳型にして、ＳＮＰを含む領域を増幅するプライマーを用いて、ポリメラーゼ連鎖反応（ＰＣＲ）により行うことができる。そして、得られた増幅断片におけるＳＮＰの塩基（遺伝子型）を決定し、決定された塩基（遺伝子型）とナス科植物における総乾物生産量との関係を示すデータに基づいて、ナス科植物における総乾物生産量を判定する。 Amplification of the region in the DNA of the subject of the solanaceous plant is carried out by polymerase chain reaction (PCR) using the DNA extracted from the subject of the solanaceous plant as a template and primers that amplify the region containing the SNP. be able to. Then, the base (genotype) of the SNP in the obtained amplified fragment is determined, and based on the data showing the relationship between the determined base (genotype) and the total dry matter production amount in the solanaceous plant, Determine total dry matter production.
　ＰＣＲにおいて用いるプライマーセットは、標的のＳＮＰを含む領域のＤＮＡ断片を増幅することができるものである限り、特に限定されず、増幅断片の長さが短くなるようにプライマーセットを設計してもよい。例えば、プライマー増幅断片の長さが、好ましくは、７００塩基対（ｂｐ）以下、２００ｂｐ以下、１５０ｂｐ以下、１２０ｂｐ以下、又は１００ｂｐ以下となるようにプライマーセットを設計する。プライマーセットは、フォワードプライマーである第１のプライマーと、リバースプライマーである第２のプライマーとが含まれる。これらのプライマーの長さは、例えば、１５ｂｐ以上、１６ｂｐ以上、１７ｂｐ以上、１８ｂｐ以上、又は１９ｂｐ以上であってもよく、５０塩基ｂｐ以下、４０ｂｐ以下、又は３０ｂｐ以下であってもよい。 The primer set used in PCR is not particularly limited as long as it can amplify the DNA fragment in the region containing the target SNP, and the primer set may be designed to shorten the length of the amplified fragment. . For example, the primer set is designed such that the length of the primer-amplified fragment is preferably 700 base pairs (bp) or less, 200 bp or less, 150 bp or less, 120 bp or less, or 100 bp 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 bp or more, 16 bp or more, 17 bp or more, 18 bp or more, or 19 bp or more, or 50 base bp or less, 40 bp or less, or 30 bp or less.
　プライマーセットの一例を以下に示す。ＳＮＰ（ｃ’）及びＳＮＰ（ｄ’）を含む領域を増幅するプライマーセットは、例えば、配列番号２７に示される塩基配列における１５以上の連続する塩基を含む第１のプライマーと、配列番号２８に示される塩基配列における１５以上の連続する塩基を含む第２のプライマーとからなる、プライマーセットである。このプライマーセットを用いることで、総乾物生産量が増加したナス科植物においては、配列番号２９に示す塩基配列からなるＰＣＲ増幅産物が得られ、ＳＮＰ（ｃ’）及びＳＮＰ（ｄ’）に相当する塩基を検出することができる。 An example of a primer set is shown below. A primer set that amplifies a region containing SNP (c′) and SNP (d′) includes, for example, a first primer containing 15 or more consecutive bases in the nucleotide sequence shown in SEQ ID NO: 27, and and a second primer containing 15 or more consecutive bases in the indicated nucleotide sequence. By using this primer set, a PCR amplification product consisting of the nucleotide sequence shown in SEQ ID NO: 29 was obtained in Solanaceae plants with increased total dry matter production, corresponding to SNP (c') and SNP (d'). bases can be detected.
　ＳＮＰ（ａ’）、ＳＮＰ（ｂ’）及びＳＮＰ（ｃ’）を含む領域を増幅するプライマーセットは、例えば、配列番号３０に示される塩基配列における１５以上の連続する塩基を含む第３のプライマーと、配列番号３１に示される塩基配列における１５以上の連続する塩基を含む第４のプライマーとからなる、プライマーセットである。このプライマーセットを用いることで、総乾物生産量が増加したナス科植物においては、配列番号３２に示す塩基配列からなるＰＣＲ増幅産物が得られ、ＳＮＰ（ａ’）、ＳＮＰ（ｂ’）及びＳＮＰ（ｃ’）に相当する塩基を検出することができる。 A primer set that amplifies a region containing SNP (a'), SNP (b') and SNP (c') is, for example, a third primer containing 15 or more consecutive bases in the nucleotide sequence shown in SEQ ID NO: 30 and a fourth primer containing 15 or more consecutive bases in the nucleotide sequence shown in SEQ ID NO:31. By using this primer set, PCR amplification products consisting of the nucleotide sequence shown in SEQ ID NO: 32 are obtained in Solanaceae plants with increased total dry matter production, SNP (a'), SNP (b') and SNP A base corresponding to (c') can be detected.
　ＳＮＰ（ｅ’）及びＳＮＰ（ｆ’）を含む領域を増幅するプライマーセットは、例えば、配列番号３３に示される塩基配列における１５以上の連続する塩基を含む第５のプライマーと、配列番号３４に示される塩基配列における１５以上の連続する塩基を含む第６のプライマーとからなる、プライマーセットである。このプライマーセットを用いることで、総乾物生産量が増加したナス科植物においては、配列番号３５に示す塩基配列からなるＰＣＲ増幅産物が得られ、ＳＮＰ（ｅ’）及びＳＮＰ（ｆ’）に相当する塩基を検出することができる。 A primer set that amplifies a region containing SNP (e′) and SNP (f′) includes, for example, a fifth primer containing 15 or more consecutive bases in the nucleotide sequence shown in SEQ ID NO: 33, and and a sixth primer containing 15 or more consecutive bases in the indicated nucleotide sequence. By using this primer set, a PCR amplification product consisting of the nucleotide sequence shown in SEQ ID NO: 35 was obtained in a Solanaceae plant with increased total dry matter production, corresponding to SNP (e') and SNP (f'). bases can be detected.
　ＳＮＰ（ｇ’）及びＳＮＰ（ｈ’）を含む領域を増幅するプライマーセットは、例えば、配列番号３６に示される塩基配列における１５以上の連続する塩基を含む第７のプライマーと、配列番号３７に示される塩基配列における１５以上の連続する塩基を含む第８のプライマーとからなる、プライマーセットである。このプライマーセットを用いることで、総乾物生産量が増加したナス科植物においては、配列番号３８に示す塩基配列からなるＰＣＲ増幅産物が得られ、ＳＮＰ（ｇ’）及びＳＮＰ（ｈ’）に相当する塩基を検出することができる。 A primer set for amplifying a region containing SNP (g') and SNP (h') includes, for example, a seventh primer containing 15 or more consecutive bases in the nucleotide sequence shown in SEQ ID NO: 36, and and an eighth primer containing 15 or more consecutive bases in the indicated nucleotide sequence. By using this primer set, a PCR amplification product consisting of the nucleotide sequence shown in SEQ ID NO: 38 was obtained in a Solanaceae plant with increased total dry matter production, corresponding to SNP (g') and SNP (h'). bases can be detected.
　ＳＮＰ分析におけるＰＣＲは、単独のプライマーセットを含む反応系でＤＮＡ断片を増幅するシングルプレックスＰＣＲ、又は、複数のプライマーセットを含む反応系で遺伝子増幅するマルチプレックスＰＣＲ、のいずれであってもよい。マルチプレックスＰＣＲの場合は、異なる波長を有する蛍光物質（例えば、ＮＥＤ、６－ＦＡＭ、ＶＩＣ、ＰＥＴ）により標識したプライマーセットを混合してもよい。 PCR in SNP analysis may be either singleplex PCR that amplifies DNA fragments in a reaction system containing a single primer set, or multiplex PCR that amplifies genes in a reaction system containing multiple primer sets. For multiplex PCR, primer sets labeled with fluorescent substances having different wavelengths (eg, NED, 6-FAM, VIC, PET) may be mixed.
　ＰＣＲの反応条件は、用いるＤＮＡポリメラーゼ及びＰＣＲ装置の種類、増幅断片の長さ等に応じて適宜に設定され得る。サイクル条件としては、変性工程、アニーリング工程及び伸長工程の３工程を１サイクルとする３ステップＰＣＲ法、及び、変性工程とアニーリング及び伸長工程との２工程を１サイクルとする２ステップＰＣＲ法を適用することができる。ＰＣＲ反応条件の一例としては、９０～１００℃で４０～６０秒（例えば、９５℃で５０秒）、次いで、９０～１００℃（例えば、９５℃）で５秒、アニーリング１０～２０秒（例えば、１５秒）、及び６５～８０℃で１０～３０秒（例えば、７２℃で２０秒）の３０～６０サイクル（例えば、４０サイクル）などである。アニーリング温度は、６０～７０℃（例えば、６６℃）の初期アニーリング温度から５０～６０℃（例えば、５６℃）の最終アニーリング温度まで、所定サイクル毎に段階的に低下させる条件が挙げられる。鋳型となるＤＮＡの状態に応じて、ＳＮＰを安定的に検出するために、ＰＣＲ反応条件を調整してもよい。 The PCR reaction conditions can be appropriately set according to the type of DNA polymerase and PCR equipment used, the length of the amplified fragment, and the like. As the cycle conditions, a 3-step PCR method in which 3 steps of denaturation step, annealing step and extension step are used as one cycle, and a 2-step PCR method in which 2 steps of denaturation step, annealing and extension step are used as 1 cycle are applied. can do. An example of PCR reaction conditions is 90-100° C. for 40-60 seconds (eg, 95° C. for 50 seconds), followed by 90-100° C. (eg, 95° C.) for 5 seconds, annealing for 10-20 seconds (eg, , 15 seconds), and 30-60 cycles (eg, 40 cycles) of 65-80° C. for 10-30 seconds (eg, 72° C., 20 seconds). Annealing temperature includes a condition in which the initial annealing temperature is 60 to 70° C. (eg, 66° C.) and the final annealing temperature is 50 to 60° C. (eg, 56° C.), and the temperature is lowered stepwise in each predetermined cycle. PCR reaction conditions may be adjusted in order to stably detect SNPs according to the condition of the template DNA.
　ＳＮＰ分析におけるＰＣＲとして、ＰＣＲによる増幅及びＳＮＰマーカーの特定を行うＴａｑＭａｎ（登録商標）－ＰＣＲ法、Ｔｍ－ｓｈｉｆｔジェノタイピング法（Fukuoka et al. Breed Sci 58: 461-464, 2008）のようなリアルタイムＰＣＲを用いてもよい。すなわち、プライマーセットを用いて増幅した増幅断片に含まれるＳＮＰを検出するＴａｑＭａｎ（登録商標）プローブをさらに用いてもよい。リアルタイムＰＣＲ法を用いることにより、高処理能力の判別方法を提供することができる。なお、ＰＣＲにより増幅した増幅断片においてＳＮＰを特定する方法としては、自動ＤＮＡシークエンサー等を用いて増幅断片の塩基配列を決定することにより、解析してもよい。 Real-time such as TaqMan (registered trademark)-PCR method, Tm-shift genotyping method (Fukuoka et al. Breed Sci 58: 461-464, 2008) for amplification by PCR and identification of SNP markers as PCR in SNP analysis PCR may also be used. That is, TaqMan (registered trademark) probes that detect SNPs contained in amplified fragments amplified using a primer set may be further used. By using the real-time PCR method, a high-throughput discrimination method can be provided. As a method for identifying SNPs in amplified fragments amplified by PCR, analysis may be performed by determining the base sequence of amplified fragments using an automatic DNA sequencer or the like.
　ナス科植物の被検体から、ＰＣＲにより増幅するＤＮＡを抽出する方法としては、特に限定されず、公知のＤＮＡ抽出方法を用いることができる。また、市販のＤＮＡ抽出キットを用いて、ＤＮＡを抽出してもよい。被検体の種類及び夾雑物の量等に応じて、ＤＮＡ抽出工程の前に、適宜に前処理を行ってもよい。また、被検体から抽出されたＤＮＡは、ＰＣＲ反応において鋳型として用いるために、必要に応じて洗浄又は精製してもよい。さらに、被検体から抽出されたＤＮＡを２種類の制限酵素で消化した制限酵素切断断片をＰＣＲにより増幅してもよい。 The method for extracting DNA to be amplified by PCR from a specimen of a solanaceous plant is not particularly limited, and a known DNA extraction method can be used. Alternatively, DNA may be extracted using a commercially available DNA extraction kit. Before the DNA extraction step, appropriate pretreatment may be performed depending on the type of specimen, the amount of contaminants, and the like. In addition, the DNA extracted from the subject may be washed or purified as necessary for use as a template in PCR reactions. Furthermore, restriction enzyme cleavage fragments obtained by digesting DNA extracted from a subject with two types of restriction enzymes may be amplified by PCR.
　また、ナス科植物における総乾物生産量を判定する方法においては、ＳＮＰ（ａ’）～ＳＮＰ（ｈ’）と連鎖不平衡状態にある遺伝子多型を分析し、ＳＮＰ（ａ’）～ＳＮＰ（ｈ’）を同定してもよい。上記連鎖不平衡状態は、一例として、連鎖不平衡係数が０．９以上の連鎖不平衡状態である。 In addition, in the method for determining the total dry matter production of Solanaceae plants, genetic polymorphisms in linkage disequilibrium with SNP (a') to SNP (h') are analyzed, and SNP (a') to SNP ( h') may be identified. The linkage disequilibrium state is, for example, a linkage disequilibrium state with a linkage disequilibrium coefficient of 0.9 or more.
　判別方法によれば、分子マーカーにより、被験ナス科植物が、総乾物生産量が増加しているか否かを判定することができる。したがって、判定結果に基づき総乾物生産量が増加したナス科植物及びその後代系統を選抜することができる。 According to the determination method, it is possible to determine whether or not the subject Solanaceae plant has increased total dry matter production using the molecular marker. Therefore, it is possible to select solanaceous plants and their progeny lines with increased total dry matter production based on the determination results.
　〔５．総乾物生産量が増加したナス科植物〕
　本発明の一態様に係る総乾物生産量が増加したナス科植物は、後述する製造方法によって得られる植物である。総乾物生産量が増加したナス科植物は、上述した分子マーカーで特定される上述したＳＮＰを有し、総乾物生産量が増加した植物である。 [5. Solanaceous plants with increased total dry matter production]
A solanaceous plant with increased total dry matter production according to an aspect of the present invention is a plant obtained by the production method described below. A solanaceous plant with increased total dry matter production is a plant having the above-described SNP identified by the above-described molecular marker and having increased total dry matter production.
　本発明の一態様に係る総乾物生産量が増加したナス科植物は、後述する製造方法に示すように、ナス科植物を種内交雑して得られた植物及びその後代系統から、上述した分子マーカーを用いて総乾物生産量が増加したナス科植物を判別することで得られる。なお、上述したＳＮＰを有するように遺伝子工学的に改変した、総乾物生産量が増加したナス科植物についても、本発明の範疇に含まれる。さらに、後述する〔６．総乾物生産量が増加したナス科植物を製造する方法〕に示すように、ナス科植物において、ＧＲＡＳ転写因子のＶＨＩＩＤモチーフの下流のアミノ酸配列を欠損又は不活化させるようにゲノム編集することにより、総乾物生産量が増加したナス科植物についても、本発明の範疇に含まれる。また、ナス科植物を含む双子葉植物において、ＧＲＡＳ転写因子のＶＨＩＩＤモチーフの下流のアミノ酸配列を欠損又は不活化させるようにゲノム編集することにより、総乾物生産量が増加した双子葉植物についても、本発明の範疇に含まれる。双子葉植物の例として、シロイヌナズナが挙げられる。 A solanaceous plant having an increased total dry matter production amount according to an aspect of the present invention is obtained by intraspecific hybridization of a solanaceous plant and its progeny line, as shown in the production method described later. It is obtained by identifying Solanaceous plants with increased total dry matter production using a marker. A solanaceous plant with an increased total dry matter production that is genetically engineered to have the above SNP is also included in the scope of the present invention. Furthermore, [6. Method for producing a solanaceous plant with increased total dry matter production], in a solanaceous plant, genome editing is performed so as to delete or inactivate the amino acid sequence downstream of the VHIID motif of the GRAS transcription factor. Solanaceous plants with increased total dry matter production are also included in the scope of the present invention. In addition, dicotyledonous plants including solanaceous plants, whose total dry matter production is increased by genome editing to delete or inactivate the amino acid sequence downstream of the VHIID motif of the GRAS transcription factor, Included in the scope of the present invention. Examples of dicotyledonous plants include Arabidopsis thaliana.
　〔６．総乾物生産量が増加したナス科植物を製造する方法〕
　本発明の一態様に係る製造方法は、総乾物生産量が増加したナス科植物を製造する方法であって、ナス科植物を種内交雑する交雑工程と、前記交雑工程により得られたナス科植物又はその後代系統のナス科植物から、上述した判別方法によって、総乾物生産量が増加したナス科植物を識別する識別工程とを含む。 [6. Method for Producing Solanaceous Plant with Increased Total Dry Matter Production]
A production method according to an aspect of the present invention is a method for producing a solanaceous plant with increased total dry matter production, comprising a hybridization step of intraspecifically hybridizing a solanaceous plant, and a solanaceous plant obtained by the hybridization step. and an identification step of identifying a solanaceous plant having an increased total dry matter production from the plants or progeny of the solanaceous plant by the above-described identification method.
　したがって、上述した分子マーカー、総乾物生産量が増加したナス科植物、及び、ナス科植物における総乾物生産量の程度を判別する方法に関する説明を、総乾物生産量が増加したナス科植物を製造する方法の説明に援用する。 Therefore, the above-mentioned molecular markers, solanaceous plants with increased total dry matter production, and a description of methods for determining the degree of total dry matter production in solanaceous plants are used to produce solanaceous plants with increased total dry matter production. It is used for the explanation of the method of
　交雑工程において用いるナス科植物の少なくとも一方は、本発明の一態様に係る総乾物生産量が増加したナス科植物であり得る。また、交雑工程において用いるナス科植物の少なくとも一方は、本発明の一態様に係るナス科植物における総乾物生産量の程度を判別する方法により選抜された総乾物生産量が増加したナス科植物であってもよい。すなわち、本発明の一態様に係る製造方法は、前記交雑工程の前に、本発明の一態様に係るナス科植物における総乾物生産量の程度を判別する方法により、被験ナス科植物から総乾物生産量が増加したナス科植物を識別する識別工程をさらに含み得る。 At least one of the solanaceous plants used in the crossing step may be a solanaceous plant with increased total dry matter production according to one aspect of the present invention. In addition, at least one of the solanaceous plants used in the crossing step is a solanaceous plant having an increased total dry matter production that has been selected by a method for determining the degree of total dry matter production of a solanaceous plant according to one embodiment of the present invention. There may be. That is, in the production method according to one aspect of the present invention, before the hybridization step, total dry matter is obtained from a test solanaceous plant by a method for determining the degree of total dry matter production in the solanaceous plant according to one aspect of the present invention. It may further comprise an identification step of identifying the solanaceous plant with increased production.
　また、本発明の一態様に係る製造方法は、被験ナス科植物から総乾物生産量が増加したナス科植物を識別する識別工程と、識別したナス科植物を種内交雑する交雑工程とを含む。すなわち、本発明は、交雑工程の前にのみ識別工程を含む製造方法や、交雑工程の前後に識別工程を含む製造方法についても、本発明の範疇に含まれる。 In addition, a production method according to an aspect of the present invention includes an identification step of identifying a solanaceous plant having an increased total dry matter production from a test solanaceous plant, and a crossing step of intraspecifically hybridizing the identified solanaceous plant. . That is, the scope of the present invention also includes a production method including an identification step only before the hybridization step and a production method including an identification step before and after the hybridization step.
　交雑工程の前にのみ識別工程を含む製造方法については、例えば、以下のように利用することができる。交雑工程の前に総乾物生産量が増加したナス科植物（総乾物生産量制御遺伝子をホモで有する）を識別し、このような植物をＦ１育種における親とすることで、得られるＦ１植物の全てが総乾物生産量制御遺伝子をヘテロで有することになる。交雑工程の前にのみ識別工程を含む製造方法は、このような、Ｆ１育種における親のホモ化（選抜と固定）に利用することもできる。 A manufacturing method that includes an identification step only before the hybridization step can be used, for example, as follows. By identifying a solanaceous plant with increased total dry matter production (having a homozygous total dry matter production control gene) before the crossing step, and using such a plant as a parent in F1 breeding, the resulting F1 plant All of them will have the total dry matter production control gene in heterozygotes. A production method that includes an identification step only before the crossing step can also be used for such parental homogenization (selection and fixation) in F1 breeding.
　識別工程は、本発明の一態様に係るナス科植物における総乾物生産量の程度を判別する方法により、交雑工程により得られたナス科植物又はその後代系統のナス科植物から、総乾物生産量が増加したナス科植物を識別する。 In the identification step, the total dry matter production amount is determined from the solanaceous plant obtained by the crossing step or its progeny solanaceous plant by the method for determining the degree of the total dry matter production amount in the solanaceous plant according to one aspect of the present invention. identify solanaceous plants with increased
　本発明の一態様に係る製造方法によれば、分子マーカーによりナス科植物において総乾物生産量の程度を判定し、判定結果に基づき選抜した総乾物生産量が増加したナス科植物を製造することができる。 According to the production method according to one aspect of the present invention, the degree of total dry matter production of a solanaceous plant is determined using a molecular marker, and a solanaceous plant having an increased total dry matter production is selected based on the determination result. can be done.
　本発明の他の態様に係る製造方法は、総乾物生産量が増加したナス科植物を製造する方法であって、配列番号１に示すアミノ酸配列からなるタンパク質をコードするポリヌクレオチドに相当するナス科植物におけるポリヌクレオチドにおいて、配列番号１に示すアミノ酸配列の７３番目のアミノ酸に相当するアミノ酸よりも下流のいずれかの位置のアミノ酸よりも下流のアミノ酸配列を欠損又は不活化させる変異を導入する工程を包含する。このように、ゲノム編集により総乾物生産量が増加したナス科植物を製造する方法についても、本発明の範疇に含まれる。 A production method according to another aspect of the present invention is a method for producing a solanaceous plant with an increased total dry matter production amount, the solanaceous plant corresponding to a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 1. A step of introducing a mutation that deletes or inactivates an amino acid sequence downstream of an amino acid at any position downstream of the amino acid corresponding to the 73rd amino acid in the amino acid sequence shown in SEQ ID NO: 1 in the polynucleotide in the plant contain. Thus, methods for producing solanaceous plants with increased total dry matter production by genome editing are also included in the scope of the present invention.
　配列番号１にアミノ酸配列からなるタンパク質をコードしているポリヌクレオチドからなる総乾物生産量制御遺伝子は、ＧＲＡＳ転写因子をコードすると予測される。本発明の他の態様に係る製造方法は、ＧＲＡＳ転写因子のＶＨＩＩＤモチーフの下流の任意の位置に、ゲノム編集により人工的に変異を導入し、ＶＨＩＩＤモチーフの下流のアミノ酸配列を欠損させることによって、総乾物生産量が増加したナス科植物を製造することができる。 A total dry matter production control gene consisting of a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 1 is predicted to encode a GRAS transcription factor. In the production method according to another aspect of the present invention, a mutation is artificially introduced by genome editing at any position downstream of the VHIID motif of the GRAS transcription factor, and the amino acid sequence downstream of the VHIID motif is deleted. Solanaceous plants with increased total dry matter production can be produced.
　ここで、ＧＲＡＳ転写因子のＶＨＩＩＤモチーフは、配列番号１に示すアミノ酸配列からなるトマト由来のタンパク質においては、６９番目～７３番目のアミノ酸配列に相当するアミノ酸の配列である（参考文献２：Li et al. Plant Cell 28: 1025-1034, 2016を参照のこと）。したがって、本発明の他の態様に係る製造方法は、７３番目のアミノ酸に相当するアミノ酸よりも下流のいずれかの位置のアミノ酸よりも下流のアミノ酸配列を欠損又は不活化させる変異を導入する工程を包含する。アミノ酸配列を欠損又は不活化させる変異としては、例えば、ストップコドン変異、フレームシフト変異、ヌル変異等が挙げられる。 Here, the VHIID motif of the GRAS transcription factor is an amino acid sequence corresponding to the 69th to 73rd amino acid sequences in the tomato-derived protein consisting of the amino acid sequence shown in SEQ ID NO: 1 (Reference 2: Li et al. Plant Cell 28: 1025-1034, 2016). Therefore, the production method according to another aspect of the present invention includes the step of introducing a mutation that deletes or inactivates the amino acid sequence downstream of the amino acid at any position downstream of the amino acid corresponding to the 73rd amino acid. contain. Mutations that delete or inactivate amino acid sequences include, for example, stop codon mutations, frameshift mutations, null mutations, and the like.
　他のナス科植物及び双子葉植物におけるＧＲＡＳ転写因子のＶＨＩＩＤモチーフは、以下の通りである。配列番号７に示すアミノ酸配列からなるピーマン由来のタンパク質においては、３１１～３１５番目のアミノ酸配列に相当するアミノ酸配列がＶＨＩＩＤモチーフである。配列番号８に示すアミノ酸配列からなるナス由来のタンパク質においては、２９３～２９７番目のアミノ酸配列に相当するアミノ酸配列がＶＨＩＩＤモチーフである。配列番号９に示すアミノ酸配列からなるジャガイモ由来のタンパク質においては、２９７～３０１番目のアミノ酸配列に相当するアミノ酸配列がＶＨＩＩＤモチーフである。配列番号１０に示すアミノ酸配列からなるシロイヌナズナ由来のタンパク質においては、２２８～２３２番目のアミノ酸配列に相当するアミノ酸配列がＶＨＩＩＤモチーフである。 The VHIID motifs of GRAS transcription factors in other solanaceous and dicotyledonous plants are as follows. In the bell pepper-derived protein consisting of the amino acid sequence shown in SEQ ID NO: 7, the amino acid sequence corresponding to the 311st to 315th amino acid sequences is the VHIID motif. In the eggplant-derived protein consisting of the amino acid sequence shown in SEQ ID NO: 8, the amino acid sequence corresponding to the 293rd to 297th amino acid sequences is the VHIID motif. In the potato-derived protein consisting of the amino acid sequence shown in SEQ ID NO: 9, the amino acid sequence corresponding to the 297th to 301st amino acid sequences is the VHIID motif. In the Arabidopsis thaliana-derived protein consisting of the amino acid sequence shown in SEQ ID NO: 10, the amino acid sequence corresponding to the 228th to 232nd amino acid sequences is the VHIID motif.
　変異を導入する工程においては、配列番号１に示すアミノ酸配列からなるタンパク質をコードするポリヌクレオチドにおいて、配列番号１に示すアミノ酸配列からなるタンパク質の１０９番目のアミノ酸に相当するアミノ酸及びその下流のアミノ酸配列を欠損又は不活化させる変異を導入してもよい。これにより、総乾物生産量制御遺伝子にストップコドン変異が導入され、配列番号１に示すアミノ酸配列の１０９番目以降のアミノ酸配列に相当するアミノ酸配列が欠損することにより、総乾物生産量が増加したナス科植物を製造することができる。 In the step of introducing mutation, in the polynucleotide encoding the protein consisting of the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to the 109th amino acid of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1 and the amino acid sequence downstream thereof may be introduced to eliminate or inactivate the As a result, a stop codon mutation is introduced into the gene for controlling total dry matter production, and the amino acid sequence corresponding to the 109th and subsequent amino acids in the amino acid sequence shown in SEQ ID NO: 1 is deleted, thereby increasing the total dry matter production. Can produce family plants.
　なお、総乾物生産量が増加したナス科植物を製造する方法は、ナス科植物を含む双子葉植物において、ＧＲＡＳ転写因子のＶＨＩＩＤモチーフの下流のアミノ酸配列を欠損又は不活化させるようにゲノム編集することにより、総乾物生産量が増加した双子葉植物を製造する方法に適用することもできる。双子葉植物の例として、シロイヌナズナが挙げられる。 A method for producing a solanaceous plant with an increased total dry matter production includes genome editing to delete or inactivate an amino acid sequence downstream of the VHIID motif of a GRAS transcription factor in a dicotyledonous plant including a solanaceous plant. Therefore, it can also be applied to a method for producing dicotyledonous plants with increased total dry matter production. Examples of dicotyledonous plants include Arabidopsis thaliana.
　本発明は上述した各実施形態に限定されるものではなく、請求項に示した範囲で種々の変更が可能であり、異なる実施形態にそれぞれ開示された技術的手段を適宜組み合わせて得られる実施形態についても本発明の技術的範囲に含まれる。 The present invention is not limited to the above-described embodiments, but can be modified in various ways within the scope of the claims, and can be obtained by appropriately combining technical means disclosed in different embodiments. is also included in the technical scope of the present invention.
　〔材料及び方法〕
　（組換え型自殖系統の構築）
　高収量ビーフステーキ型オランダＦ１品種「Ｇｅｒｏｎｉｍｏ」及び高糖度の大玉日本Ｆ１品種「Ｍｏｍｏｔａｒｏ８」を交雑親として用いた。植物は全て三重県津市の農研機構野菜花き研究部門にある温室内の土が入ったポット中で栽培した。この交雑は四元交雑に相当し、２つのＦ１品種（Ｇ１世代）の４つの親系統を祖先（Ｇ０世代）としている。図１は、２つのＦ１品種の交配に由来する組換え型自殖系統の構築及びそのＱＴＬマッピングの概要を説明する図である。図１に示すように、「Ｇｅｒｏｎｉｍｏ」（Ｐ１；Ｇ１）及び「Ｍｏｍｏｔａｒｏ８」（Ｐ２；Ｇ１）を、２４０株からなる四元Ｆ１群（Ｇ１Ｆ１）を生成するために交配した。ついで、各Ｇ１Ｆ１植物からの単粒系統法による自殖の繰り返しにより、四元組換え型自殖系統を育成した。Ｇ１Ｆ６世代の２０６の組換え型自殖系統を得、これらの農業形質を評価するために、自殖によってそれらを維持した。これら系統をＧＭ系統と称した。 [Materials and methods]
(Construction of recombinant inbred lines)
The high-yield beefsteak-type Dutch F1 cultivar 'Geronimo' and the high-sugar Otama Japanese F1 cultivar 'Momotaro8' were used as crossbred parents. All plants were cultivated in soil-filled pots in a greenhouse at the Vegetable and Flower Research Institute of the National Agriculture and Food Research Organization in Tsu City, Mie Prefecture. This crossing corresponds to a quaternary crossing, with four parental lines of two F1 varieties (G1 generation) as ancestors (G0 generation). FIG. 1 is a diagram illustrating the construction of a recombinant inbred line derived from crossing two F1 cultivars and an overview of its QTL mapping. As shown in FIG. 1, 'Geronimo'(P1; G1) and 'Momotaro8'(P2; G1) were crossed to generate a quaternary F1 group (G1F1) consisting of 240 strains. Quaternary recombinant inbred lines were then developed by repeated selfing by the single-grain method from each G1F1 plant. We obtained 206 recombinant inbred lines of the G1F6 generation and maintained them by selfing to evaluate their agronomic traits. These strains were called GM strains.
　（ＧＭ系統の栽培と２つの場所における表現型の評価）
　三重県において、２０１０年から２０１３年の７月３０日から８月１９日の間に、ＧＭ系統の種子を、土の入った育苗トレーに播種した（Ｆ７、Ｆ９、Ｆ１０世代の秋作栽培、実験３～６）。また、２０１１年及び２０１２年の２月１日又は２月２日にＧＭ系統の種子を播種し、Ｆ８及びＦ１０世代の春作栽培を行った（実験１及び２）。育苗箱を３週間温室内に置いた後、全ての植物（１実験当たり１系統当たり１植物）をロックウールスラブに直接植え付けた（栽植密度２．３０３／ｍ２）。全ての植物を１６度以上の温室内において、電気伝導度（ＥＣ）２．８ｍＳ／ｃｍの培養液（大塚化学製）を施与し、栽培した。１果房当たりの最大の花の数を６に制限し、第３果房（実験３）又は第４果房（実験１、２及び４～６）の上で摘心した。 (GM strain cultivation and phenotypic evaluation in two locations)
In Mie Prefecture, between July 30th and August 19th, 2010, GM line seeds were sown in nursery trays containing soil (F7, F9, F10 generation autumn cultivation, Experiments 3-6). In addition, seeds of the GM line were sown on February 1 or 2, 2011 and 2012, and the F8 and F10 generations were cultivated in spring (Experiments 1 and 2). After placing the nursery boxes in the greenhouse for 3 weeks, all plants (one plant per line per experiment) were planted directly onto rockwool slabs (planting density 2.303/m 2 ). All the plants were cultivated in a greenhouse of 16°C or higher with a culture solution (manufactured by Otsuka Chemical Co., Ltd.) having an electrical conductivity (EC) of 2.8 mS/cm. The maximum number of flowers per cluster was limited to 6 and pinched on the third (experiment 3) or fourth (experiment 1, 2 and 4-6) cluster.
　愛知県において、Ｆ１０世代のＧＭ系統の種子を２０１２年８月２７日（実験７）及び２０１３年８月２８日（実験８）に上記と同様に播種した。実生を、２５℃（昼）／２０℃（夜）、ＣＯ２濃度９００又は１０００ｐｐｍ、日長１６時間の条件で、生育チャンバ内に置いて培養液（ＥＣ＝１．８ｍＳ／ｃｍ、三菱ケミカルアグリドリーム社製）を施与し栽培した。３週間後、全ての植物（１実験当たり１系統当たり２植物）をロックウールスラブ中に直接植え付けた（栽植密度３．０８６／ｍ２）。全ての植物を１３℃以上の温室内において、ＥＣ＝０．８～１．８ｍＳ／ｃｍ（実験７）、又は、ＥＣ＝１．５～２．０ｍＳ／ｃｍ（実験８）の培養液（大塚化学社製）を施与し、栽培した。植物は、第４果房上で摘心した。 In Aichi Prefecture, seeds of the F10 generation GM line were sown on August 27, 2012 (Experiment 7) and August 28, 2013 (Experiment 8) in the same manner as above. Seedlings were placed in a growth chamber under the conditions of 25°C (day)/20°C (night), CO2 concentration of 900 or 1000 ppm, day length of 16 hours and culture medium (EC = 1.8 mS/cm, Mitsubishi Chemical Aguri). Dream Co., Ltd.) was applied and cultivated. After 3 weeks, all plants (2 plants per line per experiment) were planted directly into rockwool slabs (planting density 3.086/m 2 ). All plants in a greenhouse at 13 ° C. or higher, EC = 0.8 to 1.8 mS / cm (experiment 7), or EC = 1.5 to 2.0 mS / cm (experiment 8) culture solution (Otsuka Kagaku Co., Ltd.) and cultivated. Plants were pinched on the 4th bunch.
　２～１０の形質の表現型を、三重県においては１系統当たり１植物、又は、愛知県においては１系統当たり２植物から収集した。生育特性、収量、及び果実の質に関連する形質の表現型を、各ＧＭ系統植物において測定した。赤く熟した市場価値のある果実における糖度を、糖度計（ＰＡＬ－１）を用いて測定した。しり腐れ又はひび割れのようないずれの生理障害も生じていない果実を良果として定義した。一方で、少なくとも１つの生理障害が生じている果実を不良果として定義した。 Phenotypes of 2-10 traits were collected from 1 plant per line in Mie prefecture or 2 plants per line in Aichi prefecture. Phenotypes of traits related to growth characteristics, yield, and fruit quality were determined in each GM line plant. Sugar content in red ripe marketable fruits was measured using a saccharimeter (PAL-1). A good fruit was defined as a fruit that did not develop any physiological disturbances such as tail rot or cracking. On the other hand, fruits with at least one physiological disorder were defined as defective fruits.
　（ＧＭ系統における表現型の相関分析）
　形質間の相関係数を、Ｍｉｃｒｏｓｏｆｔ　Ｅｘｃｅｌ　２０１６により提供される、ｐ値を算出するための相関ツール及びＴＤＩＳＴ機能を用いて算出した。 (Correlation analysis of phenotypes in GM lines)
Correlation coefficients between traits were calculated using the correlation tool for calculating p-values and the TDIST function provided by Microsoft Excel 2016.
　（形質遺伝）
　各実験におけるＧＭ系統の表現型の線形モデルを用いて、遺伝率を算出した。 (trait inheritance)
Heritability was calculated using a linear model of the GM strain phenotype in each experiment.
　（ゲノムＤＮＡの単離）
　ＤＮｅａｓｙ　Ｐｌａｎｔ　Ｍｉｎｉ　Ｋｉｔ（Ｑｉａｇｅｎ）を用いて、Ｐ１及びＰ２の葉のゲノムＤＮＡを単離した。Ｇ１Ｆ１及びＦ９世代のＧＭ系統の植物の葉のゲノムＤＮＡを、ＤＮｅａｓｙ　９６　Ｐｌａｎｔ　Ｋｉｔ（Ｑｉａｇｅｎ）を用いて単離した。Ｑｕａｎｔ－ｉＴ　ＰｉｃｏＧｒｅｅｎ　ｄｓＤＮＡ　ｒｅａｇｅｎｔ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ　Ｋ．Ｋ）及びＡＲＶＯ　ＭＸ（ＰｅｒｋｉｎＥｌｍｅｒ　Ｊａｐａｎ　Ｃｏ．，　Ｌｔｄ．）を用いて、製造者による取扱説明書にしたがって、ゲノムＤＮＡを定量した。 (Isolation of genomic DNA)
P1 and P2 leaf genomic DNA was isolated using the DNeasy Plant Mini Kit (Qiagen). G1F1 and F9 generation plant leaf genomic DNA of the GM line was isolated using the DNeasy 96 Plant Kit (Qiagen). Genomic DNA was quantified using Quant-iT PicoGreen dsDNA reagent (Thermo Fisher Scientific KK) and ARVO MX (PerkinElmer Japan Co., Ltd.) according to the manufacturer's instructions.
　（トマトＳＳＲマーカーのスクリーニング）
　非冗長ＢＡＣ端由来ゲノムＳＳＲマーカー（“ｔｍａ”、“ｔｍｂ”、“ｔｍｃ”、及び“ＴＧＳ”と称する。総数４０４７）、ＥＳＴ由来ＳＳＲマーカー（“ｔｍｅ”及び“ＴＥＳ”と称する。総数２１９５）、ＥＳＴベースゲノムＳＳＲマーカー（“ｔｂｍ”と称する。総数２５１０）、及び、ウェブサイト（https://solgenomics.net）で公開されたｃＤＮＡ由来ＳＳＲマーカー（本実験では“ｔｍｓ”と称する。総数１３５）を用いた。 (Screening for tomato SSR markers)
Genomic SSR markers derived from non-redundant BAC ends (termed “tma”, “tmb”, “tmc” and “TGS”; total 4047), EST-derived SSR markers (termed “tme” and “TES”; total 2195) , EST-based genomic SSR markers (referred to as “tbm”, total 2510), and cDNA-derived SSR markers published on the website (https://solgenomics.net) (referred to as “tms” in this experiment, total 135). ) was used.
　テンプレートとして親（Ｇ１世代）のゲノムＤＮＡを用いて、ポリメラーゼ連鎖反応（ＰＣＲ）後、蛍光標識を行い、ＰＣＲ増幅産物の多型性に基づきマーカーをスクリーニングした。２つの群（Ｇ１Ｆ１及びＦ９世代のＧＭ系統）の遺伝子型を用いて、両親（Ｇ１世代、Ｐ１及びＰ２）アレルの組み合わせパターン（８つのカテゴリー）に、ＳＳＲマーカーを分類した（表１）。 Using parental (G1 generation) genomic DNA as a template, polymerase chain reaction (PCR) was followed by fluorescence labeling, and markers were screened based on the polymorphism of the PCR amplification product. The genotypes of the two groups (G1F1 and F9 generation GM strains) were used to classify the SSR markers into parental (G1 generation, P1 and P2) allele combination patterns (8 categories) (Table 1).
　これらのマーカー（プライマー対）の情報は、ＮＩＶＴＳウェブサイト（http://vegmarks.nivot.affrc.go.jp/）において入手可能である。 Information on these markers (primer pairs) is available on the NIVTS website (http://vegmarks.nivot.affrc.go.jp/).
　（ＳＳＲアレルの遺伝子型解析）
　ＳＳＲアレルのフォワードプライマーを、６－ＦＡＭ、ＮＥＤ、ＰＥＴ、又はＶＩＣ（Ａｐｐｌｉｅｄ　Ｂｉｏｓｙｓｔｅｍｓ）を用いて５’端標識した。Ｐ１Ｐ２のゲノムＤＮＡ、並びにＧ１Ｆ１及びＦ９世代のＧＭ系統の植物のゲノムＤＮＡをテンプレートとして用いた。ＰＣＲを、Ｔｙｐｅ－ｉｔ　Ｍｉｃｒｏｓａｔｅｌｌｉｔｅ　ＰＣＲ　Ｋｉｔ（Ｑｉａｇｅｎ）の反応液１０μＬ中で行った。ＰＣＲは、９５℃で５分からスタートし、９５℃で３０秒、６０℃で９０秒、及び７２℃で３０秒のサイクルを２８サイクル行い、最後に６０℃で３０分とした。ＰＣＲ増幅産物を、ＧｅｎｅＳｃａｎ－５００ＬＩＺ　Ｓｉｚｅ　Ｓｔａｎｄａｒｄ（Ａｐｐｌｉｅｄ　Ｂｉｏｓｙｓｔｅｍｓ）と共に自動シーケンサー（３７３０　ｘ１　ＤＮＡ　Ａｎａｌｙｚｅｒ，　Ａｐｐｌｉｅｄ　Ｂｉｏｓｙｓｔｅｍｓ）において分析した。断片長をＧｅｎｅＭａｐｐｅｒ　ｖ．　３．７　ｓｏｆｔｗａｒｅ（Ａｐｐｌｉｅｄ　Ｂｉｏｓｙｓｔｅｍｓ）において決定した。 (SSR allele genotyping)
Forward primers for SSR alleles were 5′ end-labeled using 6-FAM, NED, PET, or VIC (Applied Biosystems). Genomic DNA of P1P2 and genomic DNA of plants of GM lines of G1F1 and F9 generations were used as templates. PCR was performed in 10 μL reactions of the Type-it Microsatellite PCR Kit (Qiagen). PCR was started at 95° C. for 5 min, followed by 28 cycles of 95° C. for 30 sec, 60° C. for 90 sec, and 72° C. for 30 sec, and finally 60° C. for 30 min. PCR amplification products were analyzed in an automated sequencer (3730 x1 DNA Analyzer, Applied Biosystems) with a GeneScan-500LIZ Size Standard (Applied Biosystems). Fragment lengths were analyzed using GeneMapper v. 3.7 software (Applied Biosystems).
　（ＳＮＰアレルの遺伝子型解析）
　全１５３６のＳＮＰをＰ１Ｐ２及びＧＭ系統の遺伝子型解析に用いた。遺伝子型解析は、ＧｏｌｄｅｎＧａｔｅ　ａｓｓａｙ　ｓｙｓｔｅｍ（Ｉｌｌｕｍｉｎａ　Ｉｎｃ．）を用いて、製造者のプロトコルにしたがって行った。マーカーのカテゴリーは、表１のＳＳＲマーカーと同様に分類した。 (SNP allele genotyping)
A total of 1536 SNPs were used for genotyping of P1P2 and GM lines. Genotyping was performed using the GoldenGate assay system (Illumina Inc.) according to the manufacturer's protocol. Marker categories were grouped similarly to the SSR markers in Table 1.
　（連鎖地図の構築）
　Ｃａｒｔｈａｇｅｎｅ　ｓｏｆｔｗａｒｅ（de Givry et al. Bioinformatics 21: 1703-1704, 2005）を用いて、ＧＭ系統の連鎖地図を構築した。ＧＭ系統の連鎖地図は、各Ｇ１Ｆ１植物から各ＧＭ系統の子孫植物に至る世代の経過に伴って生じる各マーカー間での組換えの頻度（地図距離）を推定することによって、構築した。表１に記載したＳＳＲマーカー及びＳＮＰマーカーを用いた。Ｐ１Ｐ２（表１のカテゴリー１～７）におけるヘテロマーカーの遺伝子型は、Ｇ１Ｆ１世代において即座に分離し、それらのマーカー対間の組換え頻度が推定できた。組換え頻度の推定は、Ｇ１Ｆ１植物からそれらの後代のＧＭ系統へのアレルの遺伝に着目して行い、Ｇ１Ｆ１における分離に関する情報は、組み換え頻度の推定を確認するためにのみ用いた。連鎖地図におけるマーカー遺伝子型の分離歪みをカイ２乗検定により同定した。連鎖地図はＭａｐＣｈａｒｔ　ｖ．　２．１　ｓｏｆｔｗａｒｅ（Voorrips, J Hered 93: 77-78, 2002）を用いて描いた。 (Building a linkage map)
A linkage map of GM strains was constructed using the Carthagene software (de Givry et al. Bioinformatics 21: 1703-1704, 2005). A linkage map of GM lines was constructed by estimating the frequency of recombination (map distance) between each marker that occurs over the course of generations from each G1F1 plant to progeny plants of each GM line. The SSR markers and SNP markers listed in Table 1 were used. The genotypes of the heterozygous markers in P1P2 (categories 1-7 in Table 1) segregated immediately in the G1F1 generation and recombination frequencies between those marker pairs could be estimated. Recombination frequency estimates were made by focusing on the inheritance of alleles from G1F1 plants to their progeny GM lines, and information on segregation in G1F1 was used only to confirm recombination frequency estimates. Segregation distortions of marker genotypes in the linkage map were identified by chi-square test. The linkage map is MapChart v.1. 2.1 It was drawn using software (Voorrips, J Hered 93: 77-78, 2002).
　（ＭＣＭＣ法を用いたベイズＱＴＬマッピング）
　四元交雑群のベイズＱＴＬマッピングを、ＧＭ系統の遺伝子型及びＰ１、Ｐ２、Ｇ１Ｆ１及びＧＭ系統群のマーカー遺伝子型を用いて行った（Hayashi et al. Euphytica 183: 277-287, 2012）。このマッピング法では、各ＱＴＬにおいて４つの未知の祖先（Ｐ１の両親及びＰ２の両親）由来の４つのアレルを仮定した。Ｇ１Ｆ１植物のマーカー遺伝子型は、Ｐ１及びＰ２のハプロタイプを推定するために用いた。ＧＭ系統のハプロタイプは、Ｇ１Ｆ１親のハプロタイプから推測した。ＭＣＭＣ法に基づくベイズ法に加えて、四元交雑におけるＱＴＬマッピングのための変分近似値を用いたベイズ解析（変分近似法）を行った。 (Bayesian QTL mapping using MCMC method)
Bayesian QTL mapping of quaternary cross herds was performed using genotypes of GM lines and marker genotypes of P1, P2, G1F1 and GM lines (Hayashi et al. Euphytica 183: 277-287, 2012). This mapping method assumed 4 alleles from 4 unknown ancestors (P1 parents and P2 parents) in each QTL. The G1F1 plant marker genotype was used to deduce the P1 and P2 haplotypes. The GM lineage haplotype was inferred from the G1F1 parental haplotype. In addition to the Bayesian method based on the MCMC method, Bayesian analysis using variational approximation for QTL mapping in quaternary crosses (variational approximation) was performed.
　（ｔｆｗ６．１分離系統の獲得と採種）
　Ｆ９世代のＧＭ系統のうち、系統０１９については、ｔｆｗ６．１領域（２つのＳNＰマーカー、ＳＬ２．４０ｃｈ０６＿９８３９８４ＲとＳＬ２．４０ｃｈ０６＿２３５５９４４３Ｙに挟まれた間と規定、図２を参照のこと）が、ヘテロの遺伝子型を示していた。この系統の自殖後代種子を、上記と同様、土の入った育苗トレーに２０１４年９月８日に採種後、実験７及び実験８と同様に栽培した。３週間後、植物はロックウールキューブに鉢上げし、茨城県つくば市の農研機構の温室（植物工場）内に仮定植した。ＥＣ＝１．０ｍＳ／ｃｍの培養液（大塚化学社製）を施与し、同年９月３０日～１１月４日まで栽培した。この間に、上記「ゲノムＤＮＡの単離」に従ってゲノムＤＮＡを単離し、このＤＮＡをテンプレートにして、ｔｆｗ６．１領域周辺に座乗するＳＳＲマーカーセットを用い、蛍光色素を利用したＢＳ－ｔａｇ法（Shimizu and Yano, BMC Research Notes 4:161, 2011;小西ら, 野菜茶業研究所研究報告14: 15-22, 2015）によるマルチプレックスＰＣＲを実施した。使用したＳＳＲマーカーセット（セット１：ｔｂｍ０２６０、ｔｂｍ０２６３、ｔｂｍ０２７２、ｔｂｍ０２６５；セット２：ｔｂｍ１３５４、ｔｂｍ２２４１、ｔｂｍ１３４４、ｔｂｍ０２８１）の情報は、公開データベース（VegMarks, https://vegmarks.nivot.affrc.go.jp/VegMarks/app/page/home）から入手可能である。蛍光色素は、６－ＦＡＭ、ＮＥＤ、ＰＥＴ、又はＶＩＣのいずれかを用いた。上記「ＳＳＲアレルの遺伝子型解析」に基づき、多収のオランダ型トマトのアレルをホモ型で有する個体、日本トマト型のアレルをホモ型で持つ個体、ヘテロのアレルを持つ個体を選抜した。多収型アレルを持つ個体を０１９Ｇ、日本型アレルを持つ個体を０１９Ｍ、ヘテロのアレルを持つ個体を０１９Ｈと命名した。１１月４日にこれら植物の入ったロックウールキューブをロックウールスラブ上に定植、さらに栽培し、０１９Ｇ、０１９Ｍ、０１９Ｈの自殖種子を採種した。 (Acquisition and seed collection of tfw6.1 segregating lines)
Among the F9 generation GM lines, for line 019, the tfw6.1 region (defined as sandwiched between two SNP markers, SL2.40ch06_983984R and SL2.40ch06_23559443Y, see FIG. 2) is a heterozygous gene. showed the type. The self-fertilized progeny seeds of this line were collected in seedling trays containing soil on September 8, 2014, and then cultivated in the same manner as in Experiments 7 and 8. After 3 weeks, the plants were potted into rockwool cubes and temporarily planted in a greenhouse (plant factory) of the National Agriculture and Food Research Organization in Tsukuba City, Ibaraki Prefecture. A culture solution (manufactured by Otsuka Chemical Co., Ltd.) with an EC of 1.0 mS/cm was applied to the plants, and the plants were cultivated from September 30 to November 4 of the same year. During this time, genomic DNA was isolated according to the above "Isolation of genomic DNA", and this DNA was used as a template, using an SSR marker set located around the tfw6.1 region, and a BS-tag method using a fluorescent dye ( Shimizu and Yano, BMC Research Notes 4:161, 2011; Konishi et al., Vegetable and Tea Industry Research Report 14: 15-22, 2015). Information on the SSR marker sets used (Set 1: tbm0260, tbm0263, tbm0272, tbm0265; Set 2: tbm1354, tbm2241, tbm1344, tbm0281) can be found in public databases (VegMarks, https://vegmarks.nivot.affrc.go.jp /VegMarks/app/page/home). Either 6-FAM, NED, PET, or VIC was used as the fluorescent dye. Based on the above "genotype analysis of SSR alleles", individuals having a homozygous allele of the high-yielding Dutch tomato, individuals having a homozygous allele of the Japanese tomato, and individuals having a heterozygous allele were selected. An individual with a high yield allele was named 019G, an individual with a Japanese allele was named 019M, and an individual with a heterozygous allele was named 019H. On November 4, rockwool cubes containing these plants were planted on rockwool slabs and further cultivated to collect selfed seeds of 019G, 019M and 019H.
　（試験１：固定した分離系統における収量構成要素の解析：摘心栽培）
　０１９Ｇ及び０１９Ｍは、ｔｆｗ６．１アレルが多収型又は日本型に固定したと考えられる。これらの系統の種子は、２０１７年９月４日に播種後、生育チャンバ内で栽培、９月２８日にロックウールキューブに鉢上げ後、栽植密度３．３（株／ｍ２）でロックウールスラブ上に定植し、温室内で秋作栽培した。定植時の培養液はＥＣ＝２．６ｍＳ／ｃｍとし、栽培終了時までこの濃度で施与した。すべての株は第４果房で摘心し、定植後１２４日目まで栽培した。成熟果実は、栽培期間中定期的に収穫し、良果と不良果とに分け、個数及び新鮮重を求めた。栽培終了後、植物は、株元から切り取り、未熟果、良果、葉、茎、に切り分けた。果実については、果数、新鮮重、及び乾燥重を求め、葉及び茎については、新鮮重及び乾燥重を求めた。ここでいう乾燥重は、乾物重と換言できる。収穫期間中に収穫した果実の乾燥重は、栽培終了後に求めた乾物率（乾燥重／新鮮重）により新鮮重から換算して求めた。 (Test 1: Analysis of yield components in fixed segregation lines: pinching cultivation)
In 019G and 019M, it is considered that the tfw6.1 allele was fixed in the high-yielding type or the Japanese type. Seeds of these lines were cultivated in growth chambers after sowing on Sept. 4, 2017, potted in rockwool cubes on Sept. 28, and rockwool at a planting density of 3.3 (strains/m 2 ). It was planted on a slab and cultivated in autumn in a greenhouse. The culture solution at the time of planting was EC=2.6 mS/cm, and this concentration was applied until the end of cultivation. All strains were pinched at the 4th bunch and cultivated until 124 days after planting. Mature fruits were harvested periodically during the cultivation period, divided into good fruits and bad fruits, and the number and fresh weight of the fruits were determined. After cultivation, the plant was cut from the base and divided into immature fruits, good fruits, leaves and stems. For fruits, the number of fruits, fresh weight, and dry weight were determined, and for leaves and stems, fresh weight and dry weight were determined. The dry weight referred to here can be rephrased as the dry matter weight. The dry weight of the fruit harvested during the harvesting period was obtained by converting the fresh weight using the dry weight ratio (dry weight/fresh weight) obtained after the cultivation.
　（試験２：固定した分離系統における収量構成要素の解析：長段栽培）
　固定系統０１９Ｇ及び０１９Ｍは、２０１９年２月４日に播種し、生育チャンバ内で栽培、２月２８日にロックウールキューブに鉢上げ後、栽植密度２．７（株／ｍ２）でロックウールスラブ上に定植し、温室内で春作栽培した。定植時の培養液はＥＣ＝２．６ｍＳ／ｃｍとし、栽培終了時までこの濃度で施与した。本作では摘心は行わず、定植後１２４日目まで栽培した。成熟果実は、栽培期間中定期的に収穫し、良果と不良果とに分け、個数及び新鮮重を求めた。栽培終了後のデータ収集は、試験１と同様に行った。 (Test 2: Analysis of yield components in fixed segregation lines: long-stage cultivation)
The fixed lines 019G and 019M were sown on February 4, 2019, cultivated in a growth chamber, potted in rockwool cubes on February 28, and rockwool at a planting density of 2.7 (strains/m 2 ). Planted on slabs and cultivated in spring in a greenhouse. The culture solution at the time of planting was EC=2.6 mS/cm, and this concentration was applied until the end of cultivation. In this crop, no pinching was performed, and cultivation was continued until 124 days after planting. Mature fruits were harvested periodically during the cultivation period, divided into good fruits and bad fruits, and the number and fresh weight of the fruits were determined. Data collection after the end of cultivation was performed in the same manner as in Test 1.
　（分離系統のｔｆｗ６．１領域が短縮化した系統の育成）
　ｔｆｗ６．１分離系統のうち、０１９Ｈの自殖種子を、２０１６年８月９日に、試験１と同様に播種後、生育チャンバ内で栽培した。播種後２週間目に、幼苗の葉からＤＮＡを抽出した（ＤＮＡすいすい－Ｐ、株式会社リーゾ）。このＤＮＡをテンプレートにして、上記「ｔｆｗ６．１分離系統の獲得と採種」と同様にマルチプレックスＰＣＲを実施後、「ＳＳＲアレルの遺伝子型解析」に従って遺伝子型解析を行い、ｔｆｗ６．１領域が組換えにより短くなった系統を選抜した。９月８日に、試験１と同様の栽植密度、養液濃度により秋作栽培を実施し、自殖種子を採種した。 (Bringing of strains in which the tfw6.1 region of the segregating strain is shortened)
Of the tfw6.1 segregating lines, selfed seeds of 019H were cultivated in a growth chamber on August 9, 2016 after sowing as in Test 1. Two weeks after seeding, DNA was extracted from the leaves of seedlings (DNA Suisui-P, Rhizo Co., Ltd.). Using this DNA as a template, multiplex PCR was performed in the same manner as in the above "acquisition and seeding of tfw6.1 segregating lines", followed by genotyping according to "SSR allele genotyping", and the tfw6.1 region was grouped. A strain shortened by replacement was selected. On September 8, autumn cultivation was carried out with the same planting density and nutrient solution concentration as in Test 1, and self-grown seeds were harvested.
　（試験３：ｔｆｗ６．１領域が短縮化した系統の固定化と収量構成要素の解析による領域の限定）
　上記で選抜された短縮化された系統のｔｆｗ６．１領域のマーカー遺伝子型は部分的にヘテロのアレルとなる。したがって、これらが多収型か日本型に固定した系統を選抜するため、短縮化した３系統の自殖種子を２０１７年９月４日に播種し、再度遺伝子型解析を行った。生育チャンバ及び温室内の栽培に関しては、全て試験１と同様に行なった。生育途中でＳＳＲマーカーセットを用いた遺伝子型解析を行い、固定系統を判別後、これら系統の収量（総乾物重）を比較した。 (Test 3: Immobilization of lines with shortened tfw6.1 regions and region limitation by analysis of yield components)
The marker genotype of the tfw6.1 region of the truncated line selected above becomes a partially heterozygous allele. Therefore, in order to select lines fixed to high-yield type or Japanese type, self-fertilized seeds of the shortened 3 lines were sown on September 4, 2017, and genotype analysis was performed again. All the cultivation in the growth chamber and the greenhouse was carried out as in Test 1. Genotype analysis was performed using the SSR marker set during growth, and after identifying the fixed lines, the yields (total dry weight) of these lines were compared.
　（ＲＮＡ―ＳＥＱ変異解析）
　ｔｆｗ６．１分離系統０１９Ｇ（多収型）と０１９Ｍ（日本型）について、次世代型シーケンサーを用いたｍＲＮＡの変異解析を行った。それぞれの系統の未熟果実から、トライゾール法（https://ipmb.sinica.edu.tw/microarray/protocol.htm）により総ＲＮＡを抽出し、これらを用いて断片化ｃＤＮＡライブラリを作成した（ＴｒｕＳｅｑ　ＲＮＡ　Ｓａｍｐｌｅ　Ｐｒｅｐ　Ｋｉｔ　ｖ２、イルミナ社）。ライブラリを次世代シーケンサー（ＨｉＳｅｑ　４０００、イルミナ社）解析に供し、ペアエンドシーケンスによるリード配列を取得した（約４Ｇｂ／サンプル）。得られたリード配列をＣＬＣ　Ｇｅｎｏｍｉｃｓ　Ｗｏｒｋｂｅｎｃｈソフトウェアを用い、参照トマトゲノム（バージョンＳＬ４．０、ftp://ftp.solgenomics.net/genomes/Solanum_lycopersicum/assembly/build_4.00/）へマッピングし、変異検出プログラム（Ｂａｓｉｃ　Ｖａｒｉａｎｔ　Ｄｅｔｅｃｔｉｏｎ等）により非同義置換の検出を行った。 (RNA-SEQ mutation analysis)
For the tfw6.1 segregating lines 019G (high-yielding type) and 019M (Japanese type), mRNA mutation analysis was performed using a next-generation sequencer. Total RNA was extracted from immature fruits of each line by the Trizol method (https://ipmb.sinica.edu.tw/microarray/protocol.htm), and a fragmented cDNA library was created using these (TruSeq RNA Sample Prep Kit v2, Illumina). The library was subjected to next-generation sequencer (HiSeq 4000, Illumina) analysis to obtain a lead sequence by paired-end sequencing (approximately 4 Gb/sample). The obtained read sequences were mapped to the reference tomato genome (version SL4.0, ftp://ftp.solgenomics.net/genomes/Solanum_lycopersicum/assembly/build_4.00/) using CLC Genomics Workbench software, and a mutation detection program was used. Detection of non-synonymous substitutions was performed by (Basic Variant Detection, etc.).
　（ゲノム編集による相補性実験）
　シロイヌナズナについて、配列番号１０のＶＨＩＩＤモチーフ（２２８～２３２番目のアミノ酸）の下流、２７１番目のアミノ酸に該当する部分でＮＨＥＪ修復エラーを誘導するようなベクターを構築し（Tsutsui and Higashiyama, Plant Cell Physiol 58(1): 46-56, 2017）、ｆｌｏｒａｌ　ｄｉｐ法（Clough and Bent, Plant J 16(6): 735-743, 1998）によりシロイヌナズナ（Ｃｏｌ－０株）の形質転換を行った。ゲノム編集個体をＲＦＰ蛍光と塩基配列読解とにより選抜し、さらに、それらの後代から変異アレルがホモ化し、かつ、ベクターが除去されたヌルセグリガント個体を選抜し、採種した。これらゲノム編集系統の種子と対照の野生型（Ｃｏｌ－０）の種子とを１／２ＭＳ培地上に無菌播種し、２２℃、明期１０時間の条件で４週間栽培後、表現型を比較した。 (Complementation experiment by genome editing)
For Arabidopsis thaliana, construct a vector that induces an NHEJ repair error at the portion corresponding to the 271st amino acid downstream of the VHIID motif (228th to 232nd amino acids) of SEQ ID NO: 10 (Tsutsui and Higashiyama, Plant Cell Physiol 58 (1): 46-56, 2017), transformation of Arabidopsis thaliana (Col-0 strain) was performed by the floral dip method (Clough and Bent, Plant J 16(6): 735-743, 1998). Genome-edited individuals were selected by RFP fluorescence and base sequence reading, and null segregant individuals in which the mutant allele was homogenized and the vector was removed were selected from their progeny and seeded. The seeds of these genome-edited lines and the seeds of the control wild type (Col-0) were aseptically sown on 1/2 MS medium, cultivated for 4 weeks at 22 ° C. with a light period of 10 hours, and then the phenotypes were compared. .
　〔結果〕
　（ＧＭ系統における表現型の相関分析）
　表現型の相関分析において、総果実新鮮重は果実糖度に対し、負の相関を示した。これらの形質がトレードオフの関係にあることは、他の報告（Bernacchi et al. Theor Appl Genet 97:381-397, 1998; Fulton et al. Theor Appl Genet 95:881-894, 1997; Gur et al. Theor Appl Genet 122:405-420, 2011）においても示されている。 〔result〕
(Correlation analysis of phenotypes in GM lines)
Phenotypic correlation analysis showed a negative correlation between total fruit fresh weight and fruit sugar content. Other reports (Bernacchi et al. Theor Appl Genet 97:381-397, 1998; Fulton et al. Theor Appl Genet 95:881-894, 1997; Gur et al. Theor Appl Genet 122:405-420, 2011).
　三重県で栽培した系統においては、総果実新鮮重と良果新鮮重、総果実新鮮重と良果の数、良果新鮮重と良果の数、及び、平均総果実新鮮重と平均良果新鮮重のそれぞれの間で、有意に高い相関がみられた。作型は表現型に顕著に影響していることが示唆された。 In the lines cultivated in Mie Prefecture, the total fresh fruit weight and the number of good fruits, the total fresh weight of fruits and the number of good fruits, the fresh weight of good fruits and the number of good fruits, and the average total fresh weight of fruits and the average good fruits A significantly high correlation was observed between each of the fresh weights. It was suggested that the planting type had a significant effect on the phenotype.
　（Ｇ１Ｆ１系統及び組換え型自殖系統において有効なＳＳＲマーカー及びＳＮＰマーカーのスクリーニング及び分類）
　有効なＳＳＲマーカーを、それぞれの系統のアレル型の組み合わせパターンにより８つのカテゴリーに分類した（表１）。カテゴリー７のマーカーは、４つの異なるアレルを検出可能であり、当該マーカーが有用であることが示された。しかしながら、トマトゲノムにおけるマーカー頻度は非常に低かった。したがって、ＱＴＬ解析においては、異なるカテゴリーのマーカーを組み合わせ、それらを、染色体の各領域における遺伝情報の不足を補うために用いた。一例として、カテゴリー０のマーカーＡ（ａａ－ｂｂ）、カテゴリー２のマーカーＢ（ａｂ－ａａ）、及びカテゴリー３のマーカーＣ（ａａ－ａｂ）により、カテゴリー７のマーカー（ａｂ－ｃｄ）により得られる情報と同様の情報を得ることができる。合計１９７のＳＳＲマーカーを選択した。 (Screening and classification of effective SSR markers and SNP markers in G1F1 lines and recombinant inbred lines)
Valid SSR markers were grouped into eight categories according to the allelic combination pattern of each strain (Table 1). Category 7 markers were able to detect four different alleles, indicating that they are useful. However, the marker frequency in the tomato genome was very low. Therefore, in the QTL analysis, different categories of markers were combined and used to compensate for the lack of genetic information in each region of the chromosome. As an example, by category 0 marker A (aa-bb), category 2 marker B (ab-aa), and category 3 marker C (aa-ab), by category 7 marker (ab-cd) Information similar to information can be obtained. A total of 197 SSR markers were selected.
　ＳＳＲマーカーの低密度領域をカバーするＳＮＰマーカーのスクリーニング及び遺伝子型の特定を行った。全部で３３８のＳＳＲマーカー及びＳＮＰマーカー（表１）を、連鎖地図構築及びＱＴＬ解析に用いた。 Screening and genotyping of SNP markers covering low-density regions of SSR markers were carried out. A total of 338 SSR and SNP markers (Table 1) were used for linkage map construction and QTL analysis.
Figure JPOXMLDOC01-appb-T000001
 　（連鎖地図の評価）
　ＧＭ系統の連鎖地図は、１２の連鎖群からなり、総遺伝距離１２２１．８ｃＭをカバーした。マーカー間の平均距離は３．７ｃＭであり、最大２８．５ｃＭのギャップがあった。ゲノムカバー率は、トマトゲノムＳＬ３．００(http://solgenomics.net/)の９８．２％であった。連鎖地図の分離歪み率は４％未満（１３／３３８マーカー＝０．０３８５）であった。大きなギャップが存在していたが（２０ｃＭ以上）、ゲノムカバー率及びマーカー間の平均距離（１０ｃＭ未満、Lander and Botstein, Genetics 121: 185-199, 1989）は、連鎖地図が、組換え型自殖系統の全ゲノムの探索に十分であることを示唆していた。
Figure JPOXMLDOC01-appb-T000001
 (Evaluation of linkage map)
The linkage map of the GM lineage consisted of 12 linkage groups, covering a total genetic distance of 1221.8 cM. The average distance between markers was 3.7 cM with a maximum gap of 28.5 cM. The genome coverage was 98.2% of tomato genome SL3.00 (http://solgenomics.net/). The segregation distortion rate of the linkage map was less than 4% (13/338 markers = 0.0385). Although large gaps were present (greater than 20 cM), genomic coverage and average distances between markers (less than 10 cM, Lander and Botstein, Genetics 121: 185-199, 1989) indicated that the linkage map was not consistent with recombinant selfing. It was suggested that it is sufficient for searching the whole genome of the lineage.
　（ＭＣＭＣ法と変分近似法とのマッピング精度の比較）
　全ての形質データを用いてＱＴＬマッピングをする前に、ＱＴＬを検出するための２つのベイズマッピング法であるＭＣＭＣ法と変分近似法とを、総果実新鮮重のデータを用いて比較した。ＱＴＬの検出は、各ＱＴＬ位置がモデルに含まれる事後確率に基づいて行った。各ＱＴＬ位置の事後確率は、ＭＣＭＣ法においては総ＭＣＭＣサンプルに対する当該ＱＴＬを含むモデルが採択されたＭＣＭＣサンプルの比率として算出し、また、変分近似法においてはゲノム中に等間隔で配置された仮想的なＱＴＬのモデルへの包含に関する指示変数γ１の近似事後期待値として得た。２つのベイズ法から得られた結果は概ね等値であったことから、計算時間の短縮のために、計算量の少ない変分近似法のみを残りの形質の解析に適用した。 (Comparison of mapping accuracy between MCMC method and variational approximation method)
Prior to QTL mapping using all trait data, two Bayesian mapping methods for detecting QTLs, the MCMC method and the variational approximation method, were compared using the total fruit fresh weight data. QTL detection was based on the posterior probability that each QTL location was included in the model. The posterior probability of each QTL position was calculated as the ratio of MCMC samples that adopted the model containing that QTL to the total MCMC samples for the MCMC method, and was evenly spaced in the genome for the variational approximation method. was obtained as the approximate posterior expected value of the indicator variable γ 1 for inclusion in the model of the hypothetical QTL. Since the results obtained from the two Bayesian methods were roughly equivalent, only the variational approximation method, which requires less computation, was applied to the analysis of the remaining traits in order to reduce the computational time.
　近似法により検出した総果実新鮮重に関与するＱＴＬの解析結果を表２に示す。表２において、Ｒ２は、ＱＴＬによって明らかにされた表現型分散の推定比率である。図２は、連鎖群６の連鎖地図及びＱＴＬ　ｔｆｗ６．１の位置を説明する図である。 Table 2 shows the analysis results of QTL involved in total fruit fresh weight detected by the approximation method. In Table 2, R2 is the estimated proportion of phenotypic variance revealed by QTL. FIG. 2 is a diagram illustrating the linkage map of linkage group 6 and the location of QTL tfw6.1.
Figure JPOXMLDOC01-appb-T000002
 　（試験１の結果）
　試験１の秋作の摘心栽培における構成要素解析の結果を表３に示す。多収オランダ型トマトに由来するｔｆｗ６．１アレルを持つ固定系統０１９Ｇは、日本型トマト由来のアレルを持つ固定系統０１９Ｍに比べ、株あたりの総果重（未熟果、不良果、良果の合計新鮮重）、果実乾物重（総果重を乾燥重量に換算したもの）、総乾物重、果実分配率（果実乾物重／総乾物重）のいずれも、有意に増大していた。ここで、総乾物重は、葉、茎、果実、の乾燥重量の合計を指す。これらの結果は、地上部バイオマス重の増加により、果実乾物重、さらには総果重が増大したことを示唆している。
Figure JPOXMLDOC01-appb-T000002
 (Results of Test 1)
Table 3 shows the results of component element analysis in pinching cultivation of autumn crops in Test 1. The fixed line 019G with the tfw6.1 allele derived from the high-yielding Dutch tomato has a total fruit weight per plant (total of immature, defective, and good fruit) compared to the fixed line 019M with the allele derived from the Japanese type tomato. Fresh weight), fruit dry weight (total fruit weight converted to dry weight), total dry weight, and fruit distribution ratio (fruit dry weight/total dry weight) were all significantly increased. Here, total dry weight refers to the sum of the dry weights of leaves, stems and fruits. These results suggest that an increase in above-ground biomass weight increased fruit dry weight and also total fruit weight.
　上記解析は、Higashide and Heuvelink (J Amer Soc Hort Sci 134: 460-465, 2009)による構成要素解析を基にした（同論文の図２を参照のこと）。 The above analysis was based on the component analysis by Higashide and Heuvelink (J Amer Soc Hort Sci 134: 460-465, 2009) (see Figure 2 in the same paper).
　（試験２の結果）
　試験２の構成要素解析の結果を表３に示す。春作の長段栽培においても、試験１と同様の結果が得られた。 (Results of test 2)
The results of the component analysis for Test 2 are shown in Table 3. Similar results to those of Test 1 were obtained in long-stage cultivation in spring.
Figure JPOXMLDOC01-appb-T000003
 　（試験３の結果）
　総乾物重について、ｔｆｗ６．１領域が短縮化された系統の、多収型マーカーアレルの領域と、日本型マーカーアレル領域との比較により、ｔｆｗ６．１領域がさらに限定された（表４）。マーカーｔｂｍ１３４４とマーカーｔｂｍ０２８１に挟まれた領域が多収型アレルとなった系統（019_364G）において総乾物重の増大が認められた。なお、表４において、Ａは多収型のトマトの遺伝子型、Ｂは日本型トマトの遺伝子型を示す。
Figure JPOXMLDOC01-appb-T000003
 (Results of Test 3)
For the total dry weight, the tfw6.1 region was further restricted by comparing the region of the high-yielding marker allele with the region of the Japanese-type marker allele of the line with the truncated tfw6.1 region (Table 4). An increase in total dry matter weight was observed in the line (019_364G) in which the region sandwiched between the markers tbm1344 and tbm0281 had a high-yielding allele. In Table 4, A indicates the genotype of high-yielding tomato, and B indicates the genotype of Japanese type tomato.
Figure JPOXMLDOC01-appb-T000004
 　（ＲＮＡ―ＳＥＱ変異解析）
　表４で限定されたQTL領域内で、多収型と日本型の間で非同義置換が生じている予測遺伝子は、マーカーｔｂｍ１３４４とマーカーｔｂｍ０２６５との間、及び、マーカーｔｂｍ０２８１の長腕側の外側などに見出された。３つの予測遺伝子に見出された変異については、配列番号４～６に示す。
Figure JPOXMLDOC01-appb-T000004
 (RNA-SEQ mutation analysis)
Within the QTL region limited in Table 4, predicted genes in which non-synonymous substitution occurs between high-yielding type and Japanese type are between marker tbm1344 and marker tbm0265 and outside the long arm side of marker tbm0281. etc. was found. Mutations found in the three predicted genes are shown in SEQ ID NOs:4-6.
　（ゲノム編集による相補性実験）
　配列番号１０の候補アミノ酸配列についてゲノム編集されたシロイヌナズナ２系統（８１８＃２及び８１８＃９）を実験に供試した。これらのゲノム編集系統の塩基配列及びアミノ酸配列を図３に示す。８１８＃２は、一塩基挿入により、２７１番目のアミノ酸の下流にフレームシフトが生じ、２８２番目以降の配列は欠損していた。また、８１８＃９は、２５塩基の欠失変異により、２６３番目のアミノ酸配列の下流にフレームシフトが生じ、２６８番目以降の配列は欠損していた。 (Complementation experiment by genome editing)
Two Arabidopsis thaliana strains (818#2 and 818#9) genome-edited for the candidate amino acid sequence of SEQ ID NO: 10 were used in the experiment. The base sequences and amino acid sequences of these genome editing strains are shown in FIG. In 818#2, a frameshift occurred downstream of the 271st amino acid due to the insertion of a single nucleotide, and the sequence after the 282nd amino acid was deleted. In addition, 818#9 had a frameshift downstream of the 263rd amino acid sequence due to deletion mutation of 25 bases, and lacked the 268th and subsequent sequences.
　これら変異アレルが固定されたゲノム編集系統と野生型とを培地に播種し、４週間栽培後の表現型を比較した。その結果、ゲノム編集系統では、野生型と比較して植物体が大きくなることが確認された（図４）。この結果は、多収オランダ型トマトに観察される表現型と一致する。 The genome-edited strains in which these mutant alleles were fixed and the wild type were seeded in a medium, and the phenotypes after 4 weeks of cultivation were compared. As a result, it was confirmed that the genome-edited line has a larger plant body than the wild type (Fig. 4). This result is consistent with the phenotype observed in high-yielding Holland type tomatoes.
　本発明は、農業分野、植物育種分野等に利用することができる。 The present invention can be used in fields such as agriculture and plant breeding.

Claims (23)

  1. 　ナス科植物における総乾物生産量の程度を判別する方法であって、
    　ナス科植物において、下記（ａ）～（ｂ）のアミノ酸：
    　（ａ）配列番号１に示すアミノ酸配列からなるタンパク質の１０９番目のアミノ酸に相当するアミノ酸；
    　（ｂ）配列番号１に示すアミノ酸配列からなるタンパク質の２４７番目のアミノ酸に相当するアミノ酸；
    の少なくとも１つに置換、欠損、付加又は挿入を引き起こす変異の有無を検査する工程を含む、方法。     A method for determining the degree of total dry matter production in a solanaceous plant, comprising:
    In solanaceous plants, the following amino acids (a) to (b):
    (a) an amino acid corresponding to the 109th amino acid of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1;
    (b) an amino acid corresponding to the 247th amino acid of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1;
    testing for mutations that cause substitutions, deletions, additions or insertions in at least one of
  2. 　前記検査する工程において、ナス科植物における、下記（ａ’）～（ｄ’）の塩基に相当する塩基自体（ＳＮＰ）か、当該塩基を含む連続したポリヌクレオチド：
    　（ａ’）配列番号４に示す塩基配列からなるポリヌクレオチドの３２６番目の塩基に相当する塩基；
    　（ｂ’）配列番号４に示す塩基配列からなるポリヌクレオチドの３９０番目の塩基に相当する塩基；
    　（ｃ’）配列番号４に示す塩基配列からなるポリヌクレオチドの５９１番目の塩基に相当する塩基；
    　（ｄ’）配列番号４に示す塩基配列からなるポリヌクレオチドの７４０番目の塩基に相当する塩基；
    の少なくとも１つを、総乾物生産量の制御に関する分子マーカーとして検査する、請求項１に記載の方法。     In the step of inspecting, the base itself (SNP) corresponding to the bases (a') to (d') below in the Solanaceae plant, or a continuous polynucleotide containing the base:
    (a') a base corresponding to the 326th base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 4;
    (b') a base corresponding to the 390th base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 4;
    (c') a base corresponding to the 591st base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 4;
    (d') a base corresponding to the 740th base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 4;
    as a molecular marker for control of total dry matter production.
  3. 　前記検査する工程において、
    　前記（ａ）のアミノ酸に相当するアミノ酸及びその下流のアミノ酸配列に置換、欠損、付加又は挿入を引き起こす変異の有無を検査する、請求項１又は２に記載の方法。     In the inspecting step,
    3. The method according to claim 1 or 2, wherein the presence or absence of mutations causing substitution, deletion, addition or insertion in the amino acid sequence corresponding to the amino acid in (a) and its downstream amino acid sequence is examined.
  4. 　前記ナス科植物は、以下の（１）～（３）の何れかに記載のポリヌクレオチド：
    　（１）配列番号１に示すアミノ酸配列からなるタンパク質をコードしているポリヌクレオチド；
    　（２）配列番号１に示すアミノ酸配列に対して、８０％以上の配列同一性を有するアミノ酸配列からなり、ナス科植物の総乾物生産量を制御する機能を有するタンパク質をコードしているポリヌクレオチド；
    　（３）配列番号１に示すアミノ酸配列に対して、６５個以下のアミノ酸が置換、欠損、付加又は挿入されたアミノ酸配列からなり、ナス科植物の総乾物生産量を制御する機能を有するタンパク質をコードしているポリヌクレオチド；
    からなる総乾物生産量制御遺伝子を有するナス科植物である、請求項１～３の何れか１項に記載の方法。     The solanaceous plant is a polynucleotide according to any one of the following (1) to (3):
    (1) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 1;
    (2) A polynucleotide consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and encoding a protein having a function of controlling the total dry matter production of Solanaceae plants. ;
    (3) A protein consisting of an amino acid sequence in which 65 or less amino acids are substituted, deleted, added or inserted with respect to the amino acid sequence shown in SEQ ID NO: 1, and having the function of controlling the total dry matter production of a solanaceous plant. an encoding polynucleotide;
    The method according to any one of claims 1 to 3, which is a solanaceous plant having a gene controlling the total dry matter production amount consisting of.
  5. 　ナス科植物における総乾物生産量の程度を判別する方法であって、
    　ナス科植物において、下記（ｅ）及び（ｆ）のアミノ酸：
    　（ｅ）配列番号２に示すアミノ酸配列からなるタンパク質の１３３番目のアミノ酸に相当するアミノ酸；
    　（ｆ）配列番号２に示すアミノ酸配列からなるタンパク質の１３９番目のアミノ酸に相当するアミノ酸；
    の少なくとも一方に置換、欠損、付加又は挿入を引き起こす変異の有無を検査する工程を含む、方法。     A method for determining the degree of total dry matter production in a solanaceous plant, comprising:
    In solanaceous plants, the following amino acids (e) and (f):
    (e) an amino acid corresponding to the 133rd amino acid of the protein consisting of the amino acid sequence shown in SEQ ID NO: 2;
    (f) an amino acid corresponding to the 139th amino acid of the protein consisting of the amino acid sequence shown in SEQ ID NO: 2;
    A method comprising examining for the presence or absence of a mutation that causes a substitution, deletion, addition or insertion in at least one of
  6. 　前記検査する工程において、
    　ナス科植物における、下記（ｅ’）及び（ｆ’）の塩基に相当する塩基自体（ＳＮＰ）か、当該塩基を含む連続したポリヌクレオチド：
    　（ｅ’）配列番号５に示す塩基配列からなるポリヌクレオチドの３９７番目の塩基に相当する塩基；
    　（ｆ’）配列番号５に示す塩基配列からなるポリヌクレオチドの４１７番目の塩基に相当する塩基；
    の少なくとも一方を、総乾物生産量の制御に関する分子マーカーとして検査する工程を含む、請求項５に記載の方法。     In the inspecting step,
    Bases themselves (SNPs) corresponding to the following bases (e') and (f') in Solanaceae plants, or continuous polynucleotides containing the bases:
    (e') a base corresponding to the 397th base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 5;
    (f') a base corresponding to the 417th base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 5;
    as a molecular marker for control of total dry matter production.
  7. 　前記ナス科植物は、以下の（４）～（６）の何れかに記載のポリヌクレオチド：
    　（４）配列番号２に示すアミノ酸配列からなるタンパク質をコードしているポリヌクレオチド；
    　（５）配列番号２に示すアミノ酸配列に対して、８０％以上の配列同一性を有するアミノ酸配列からなり、ナス科植物の総乾物生産量を制御する機能を有するタンパク質をコードしているポリヌクレオチド；
    　（６）配列番号２に示すアミノ酸配列に対して、３０個以下のアミノ酸が置換、欠損、付加又は挿入されたアミノ酸配列からなり、ナス科植物の総乾物生産量を制御する機能を有するタンパク質をコードしているポリヌクレオチド；
    からなる総乾物生産量制御遺伝子を有するナス科植物である、請求項５又は６に記載の方法。     The solanaceous plant is the polynucleotide according to any one of the following (4) to (6):
    (4) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2;
    (5) A polynucleotide consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2 and encoding a protein having a function of controlling the total dry matter production of a plant of the family Solanaceae. ;
    (6) A protein consisting of an amino acid sequence in which 30 or less amino acids are substituted, deleted, added or inserted with respect to the amino acid sequence shown in SEQ ID NO: 2, and having the function of controlling the total dry matter production of a solanaceous plant. an encoding polynucleotide;
    7. The method according to claim 5 or 6, which is a solanaceous plant having a total dry matter production control gene consisting of.
  8. 　ナス科植物における総乾物生産量の程度を判別する方法であって、
    　ナス科植物において、下記（ｇ）及び（ｈ）のアミノ酸：
    　（ｇ）配列番号３に示すアミノ酸配列からなるタンパク質の１３４番目のアミノ酸に相当するアミノ酸；
    　（ｈ）配列番号３に示すアミノ酸配列からなるタンパク質の１７５番目のアミノ酸に相当するアミノ酸；
    の少なくとも一方に置換、欠損、付加又は挿入を引き起こす変異の有無を検査する工程を含む、方法。     A method for determining the degree of total dry matter production in a solanaceous plant, comprising:
    In solanaceous plants, the following amino acids (g) and (h):
    (g) an amino acid corresponding to the 134th amino acid of the protein consisting of the amino acid sequence shown in SEQ ID NO: 3;
    (h) an amino acid corresponding to the 175th amino acid of the protein consisting of the amino acid sequence shown in SEQ ID NO: 3;
    A method comprising examining for the presence or absence of a mutation that causes a substitution, deletion, addition or insertion in at least one of
  9. 　前記検査する工程において、
    　ナス科植物における、下記（ｇ’）又は（ｈ’）の塩基に相当する塩基自体（ＳＮＰ）か、当該塩基を含む連続したポリヌクレオチド：
    　（ｇ’）配列番号６に示す塩基配列からなるポリヌクレオチドの４０１番目の塩基に相当する塩基；
    　（ｈ’）配列番号６に示す塩基配列からなるポリヌクレオチドの５２３番目の塩基に相当する塩基；
    を、総乾物生産量の制御に関する分子マーカーとして検査する工程を含む、請求項８に記載の方法。     In the inspecting step,
    The base itself (SNP) corresponding to the base of (g') or (h') below in Solanaceae plants, or a continuous polynucleotide containing the base:
    (g') a base corresponding to the 401st base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 6;
    (h') a base corresponding to the 523rd base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 6;
    as a molecular marker for control of total dry matter production.
  10. 　前記ナス科植物は、以下の（７）～（９）の何れかに記載のポリヌクレオチド：
    　（７）配列番号３に示すアミノ酸配列からなるタンパク質をコードしているポリヌクレオチド；
    　（８）配列番号３に示すアミノ酸配列に対して、８０％以上の配列同一性を有するアミノ酸配列からなり、ナス科植物の総乾物生産量を制御する機能を有するタンパク質をコードしているポリヌクレオチド；
    　（９）配列番号３に示すアミノ酸配列に対して、６０個以下のアミノ酸が置換、欠損、付加又は挿入されたアミノ酸配列からなり、ナス科植物の総乾物生産量を制御する機能を有するタンパク質をコードしているポリヌクレオチド；
    からなる総乾物生産量制御遺伝子を有するナス科植物である、請求項８又は９に記載の方法。     The solanaceous plant is a polynucleotide according to any one of the following (7) to (9):
    (7) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 3;
    (8) A polynucleotide consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3 and encoding a protein having a function of controlling the total dry matter production of a plant of the family Solanaceae. ;
    (9) A protein consisting of an amino acid sequence in which 60 or less amino acids are substituted, deleted, added or inserted with respect to the amino acid sequence shown in SEQ ID NO: 3, and having the function of controlling the total dry matter production of a solanaceous plant. an encoding polynucleotide;
    10. The method according to claim 8 or 9, which is a solanaceous plant having a total dry matter production control gene consisting of.
  11. 　前記ナス科植物は、トマト、ジャガイモ、ナス、又はトウガラシである、請求項１～１０のいずれか１項に記載の方法。 The method according to any one of claims 1 to 10, wherein the solanaceous plant is tomato, potato, eggplant, or hot pepper.
  12. 　前記ナス科植物は、育種素材の候補植物であるか、育種のプロセスで得られた植物である、請求項１～１１のいずれか１項に記載の方法。 The method according to any one of claims 1 to 11, wherein the solanaceous plant is a candidate plant for breeding material or a plant obtained through a process of breeding.
  13. 　総乾物生産量が増加したナス科植物を製造する方法であって、
    　ナス科植物を種内交雑する交雑工程と、
    　前記交雑工程により得られたナス科植物又はその後代系統のナス科植物から、請求項１から１２のいずれか１項に記載された方法によって、総乾物生産量が増加したナス科植物を識別する識別工程と
    を含む、製造方法。     A method for producing a solanaceous plant with increased total dry matter production, comprising:
    A crossbreeding step of intraspecifically crossing Solanaceous plants;
    A solanaceous plant having increased total dry matter production is identified from the solanaceous plants obtained by the crossing step or the progeny of the solanaceous plants by the method according to any one of claims 1 to 12. and an identification step.
  14. 　総乾物生産量が増加したナス科植物を製造する方法であって、
    　請求項１から１２のいずれか１項に記載の方法によって、被験ナス科植物から総乾物生産量が増加したナス科植物を識別する識別工程と、
    　識別したナス科植物を種内交雑する交雑工程と
    を含む、製造方法。     A method for producing a solanaceous plant with increased total dry matter production, comprising:
    an identification step of identifying a solanaceous plant with increased total dry matter production from the test solanaceous plant by the method according to any one of claims 1 to 12;
    and a crossing step of intraspecifically crossing the identified solanaceous plants.
  15. 　ナス科植物における、総乾物生産量の制御に関する分子マーカーであって、
    　下記（ａ’）～（ｈ’）の塩基に相当する塩基自体（ＳＮＰ）か、当該塩基を含む連続したポリヌクレオチド：
    　（ａ’）配列番号４に示す塩基配列からなるポリヌクレオチドの３２６番目の塩基に相当する塩基；
    　（ｂ’）配列番号４に示す塩基配列からなるポリヌクレオチドの３９０番目の塩基に相当する塩基；
    　（ｃ’）配列番号４に示す塩基配列からなるポリヌクレオチドの５９１番目の塩基に相当する塩基；
    　（ｄ’）配列番号４に示す塩基配列からなるポリヌクレオチドの７４０番目の塩基に相当する塩基；
    　（ｅ’）配列番号５に示す塩基配列からなるポリヌクレオチドの３９７番目の塩基に相当する塩基；
    　（ｆ’）配列番号５に示す塩基配列からなるポリヌクレオチドの４１７番目の塩基に相当する塩基；
    　（ｇ’）配列番号６に示す塩基配列からなるポリヌクレオチドの４０１番目の塩基に相当する塩基；
    　（ｈ’）配列番号６に示す塩基配列からなるポリヌクレオチドの５２３番目の塩基に相当する塩基；
    の少なくとも１つを含む、分子マーカー。     A molecular marker for the regulation of total dry matter production in solanaceous plants,
    A base itself (SNP) corresponding to the bases (a') to (h') below, or a continuous polynucleotide containing the base:
    (a') a base corresponding to the 326th base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 4;
    (b') a base corresponding to the 390th base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 4;
    (c') a base corresponding to the 591st base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 4;
    (d') a base corresponding to the 740th base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 4;
    (e') a base corresponding to the 397th base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 5;
    (f') a base corresponding to the 417th base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 5;
    (g') a base corresponding to the 401st base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 6;
    (h') a base corresponding to the 523rd base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 6;
    A molecular marker comprising at least one of
  16. 　配列番号１に示すアミノ酸配列からなるタンパク質をコードするポリヌクレオチドに相当するナス科植物におけるポリヌクレオチドにおいて、配列番号１に示すアミノ酸配列の７３番目のアミノ酸に相当するアミノ酸よりも下流のいずれかの位置のアミノ酸よりも下流のアミノ酸配列を欠損又は不活化させる変異を導入する工程を包含する、総乾物生産量が増加したナス科植物を製造する方法。 Any position downstream of the amino acid corresponding to the 73rd amino acid in the amino acid sequence shown in SEQ ID NO: 1 in a polynucleotide in a Solanaceae plant corresponding to a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 A method for producing a solanaceous plant with increased total dry matter production, comprising the step of introducing a mutation that deletes or inactivates an amino acid sequence downstream of the amino acid of .
  17. 　ナス科植物における、配列番号１に示すアミノ酸配列の７３番目のアミノ酸に相当するアミノ酸よりも下流のいずれかの位置のアミノ酸よりも下流のアミノ酸配列を欠損又は不活化させる変異が導入され、ナス科植物の総乾物生産量を増加させる機能を有するタンパク質をコードするポリヌクレオチドからなる、遺伝子。 A mutation that deletes or inactivates the amino acid sequence downstream of the amino acid at any position downstream of the amino acid corresponding to the 73rd amino acid in the amino acid sequence shown in SEQ ID NO: 1 in the Solanaceae plant is introduced. A gene comprising a polynucleotide encoding a protein that has the function of increasing the total dry matter production of a plant.
  18. 　前記ポリヌクレオチドは、配列番号１に示すアミノ酸配列からなるタンパク質の１０９番目のアミノ酸に相当するアミノ酸及びその下流のアミノ酸配列を欠損又は不活化させる変異が導入され、ナス科植物の総乾物生産量を増加させる機能を有するタンパク質をコードする、請求項１７に記載の遺伝子。 The polynucleotide is introduced with a mutation that deletes or inactivates the amino acid corresponding to the 109th amino acid of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1 and the amino acid sequence downstream thereof, and increases the total dry matter production of the Solanaceae plant. 18. The gene of claim 17, which encodes a protein with increasing function.
  19. 　以下の（１）～（３）のいずれかに記載のポリヌクレオチド：
    　（１）配列番号１に示すアミノ酸配列からなるタンパク質をコードしているポリヌクレオチド；
    　（２）配列番号１に示すアミノ酸配列に対して、８０％以上の配列同一性を有するアミノ酸配列からなり、かつ、配列番号１に示すアミノ酸配列の１０９番目のアミノ酸に相当するアミノ酸及びその下流のアミノ酸配列が欠損又は不活化されているか、又は、配列番号１に示すアミノ酸配列における２４７番目のアミノ酸に相当するアミノ酸がＣであって、ナス科植物の総乾物生産量を制御する機能を有するタンパク質をコードしているポリヌクレオチド；
    　（３）配列番号１に示すアミノ酸配列の１０９番目のアミノ酸に相当するアミノ酸及びその下流のアミノ酸配列が欠損又は不活化されているか、又は、配列番号１に示すアミノ酸配列における２４７番目のアミノ酸に相当するアミノ酸がＣであって、６５個以下のアミノ酸が置換、欠損、付加又は挿入されたアミノ酸配列からなり、ナス科植物の総乾物生産量を制御する機能を有するタンパク質をコードしているポリヌクレオチド；
    からなる、遺伝子。     The polynucleotide according to any one of the following (1) to (3):
    (1) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 1;
    (2) consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and an amino acid corresponding to the 109th amino acid in the amino acid sequence shown in SEQ ID NO: 1 and downstream thereof A protein having a deleted or inactivated amino acid sequence, or having C as an amino acid corresponding to the 247th amino acid in the amino acid sequence shown in SEQ ID NO: 1, and having the function of regulating the total dry matter production of a solanaceous plant. a polynucleotide encoding a
    (3) the amino acid corresponding to the 109th amino acid in the amino acid sequence shown in SEQ ID NO: 1 and the amino acid sequence downstream thereof are deleted or inactivated, or correspond to the 247th amino acid in the amino acid sequence shown in SEQ ID NO: 1; Polynucleotide encoding a protein having the function of controlling the total dry matter production of a solanaceous plant, consisting of an amino acid sequence in which the amino acid is C and 65 or less amino acids are substituted, deleted, added or inserted. ;
    A gene consisting of
  20. 　以下の（４）～（６）のいずれかに記載のポリヌクレオチド：
    　（４）配列番号２に示すアミノ酸配列からなるタンパク質をコードしているポリヌクレオチド；
    　（５）配列番号２に示すアミノ酸配列に対して、８０％以上の配列同一性を有するアミノ酸配列からなり、かつ、配列番号２に示すアミノ酸配列における１３３番目のアミノ酸に相当するアミノ酸がＡ、又は、配列番号２に示すアミノ酸配列における１３９番目のアミノ酸に相当するアミノ酸がＮであって、ナス科植物の総乾物生産量を制御する機能を有するタンパク質をコードしているポリヌクレオチド；
    　（６）配列番号２に示すアミノ酸配列に対して、３０個以下のアミノ酸が置換、欠損、付加又は挿入されたアミノ酸配列からなり、かつ、配列番号２に示すアミノ酸配列における１３３番目のアミノ酸に相当するアミノ酸がＡ、又は、配列番号２に示すアミノ酸配列における１３９番目のアミノ酸に相当するアミノ酸がＮであって、ナス科植物の総乾物生産量を制御する機能を有するタンパク質をコードしているポリヌクレオチド；
    からなる、遺伝子。     The polynucleotide according to any one of (4) to (6) below:
    (4) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2;
    (5) consists of an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2, and the amino acid corresponding to the 133rd amino acid in the amino acid sequence shown in SEQ ID NO: 2 is A, or , the amino acid corresponding to the 139th amino acid in the amino acid sequence shown in SEQ ID NO: 2 is N, and a polynucleotide encoding a protein having the function of controlling the total dry matter production of Solanaceae plants;
    (6) Consists of an amino acid sequence in which 30 or less amino acids are substituted, deleted, added or inserted with respect to the amino acid sequence shown in SEQ ID NO: 2, and corresponds to the 133rd amino acid in the amino acid sequence shown in SEQ ID NO: 2 The amino acid corresponding to the 139th amino acid in the amino acid sequence shown in SEQ ID NO: 2 is A, or the amino acid corresponding to the 139th amino acid in the amino acid sequence shown in SEQ ID NO: 2 is N, and encodes a protein having the function of controlling the total dry matter production of solanaceous plants nucleotide;
    A gene consisting of
  21. 　以下の（７）～（９）のいずれかに記載のポリヌクレオチド：
    　（７）配列番号３に示すアミノ酸配列からなるタンパク質をコードしているポリヌクレオチド；
    　（８）配列番号３に示すアミノ酸配列に対して、８０％以上の配列同一性を有するアミノ酸配列からなり、かつ、配列番号３に示すアミノ酸配列における１３４番目のアミノ酸に相当するアミノ酸がＰ、又は、配列番号３に示すアミノ酸配列における１７５番目のアミノ酸に相当するアミノ酸がＹであって、ナス科植物の総乾物生産量を制御する機能を有するタンパク質をコードしているポリヌクレオチド；
    　（９）配列番号３に示すアミノ酸配列に対して、６０個以下のアミノ酸が置換、欠損、付加又は挿入されたアミノ酸配列からなり、かつ、配列番号３に示すアミノ酸配列における１３４番目のアミノ酸に相当するアミノ酸がＰ、又は、配列番号３に示すアミノ酸配列における１７５番目のアミノ酸に相当するアミノ酸がＹであって、ナス科植物の総乾物生産量を制御する機能を有するタンパク質をコードしているポリヌクレオチド；
    からなる、遺伝子。     The polynucleotide according to any one of (7) to (9) below:
    (7) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 3;
    (8) consists of an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3, and the amino acid corresponding to the 134th amino acid in the amino acid sequence shown in SEQ ID NO: 3 is P, or , wherein the amino acid corresponding to the 175th amino acid in the amino acid sequence shown in SEQ ID NO: 3 is Y, and a polynucleotide encoding a protein having the function of controlling the total dry matter production of Solanaceae plants;
    (9) Consists of an amino acid sequence in which 60 or less amino acids are substituted, deleted, added or inserted with respect to the amino acid sequence shown in SEQ ID NO: 3, and corresponds to the 134th amino acid in the amino acid sequence shown in SEQ ID NO: 3 The amino acid corresponding to the 175th amino acid in the amino acid sequence shown in SEQ ID NO: 3 is P, or the amino acid corresponding to the 175th amino acid in the amino acid sequence shown in SEQ ID NO: 3 is Y, and encodes a protein having the function of controlling the total dry matter production of a solanaceous plant nucleotide;
    A gene consisting of
  22. 　請求項１７～２１のいずれか１項に記載の遺伝子を含む、発現ベクター。 An expression vector comprising the gene according to any one of claims 17-21.
  23. 　請求項１７～２１のいずれか１項に記載の遺伝子、又は請求項２２に記載の発現ベクターを含む、細胞又は双子葉植物。 A cell or dicotyledonous plant comprising the gene according to any one of claims 17 to 21 or the expression vector according to claim 22.
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Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
AKIO OYAMA ET AL.: "Breeding technology prospects for high-quality, high-yielding tomato cultivation", NOKO TO ENGEI - AGRICULTURE AND HORTICULTURE, SEIBUNDO SHINKOSHA INC., TOKYO, JP, vol. 93, no. 7, 1 July 2018 (2018-07-01), JP , pages 626 - 631, XP009539939, ISSN: 0369-5247 *
AZUMI ENDO, TOMOHIRO HAYASHI, AKIO OYAMA, MIKIO NAKAZONO, HIROKAZU TAKAHASHI: "P065-A Genetic analysis of dimorphic xylem tissue development in Japanese and Dutch tomato cultivars", IKUSHUGAKU KENKYU - BREEDING RESEARCH, NIHON IKUSHU GAKKAI, TOKYO, JP, vol. 22, no. Suppl. 2, 10 October 2020 (2020-10-10) - 11 October 2020 (2020-10-11), JP , pages 187, XP009539938, ISSN: 1344-7629 *
DATABASE Plant Transcription Factor Database ANONYMOUS : "Solanum lycopersicum", XP093042986, retrieved from PlantTFDB *
DATABASE UNIPROTKB ANONYMOUS : "A0A3Q7GTW2 · A0A3Q7GTW2_SOLLC", XP093042978, retrieved from UNIPROT *
DATABASE UNIPROTKB ANONYMOUS : "A0A3Q7GUK4 · A0A3Q7GUK4_SOLLC", XP093042982, retrieved from UNIPROT *
DATABASE UNIPROTKB ANONYMOUS : "A0A6N2B8S6 · A0A6N2B8S6_SOLCI", XP093042987, retrieved from NCBI *
NIU YILING, ZHAO TINGTING, XU XIANGYANG, LI JINGFU: "Genome-wide identification and characterization of GRAS transcription factors in tomato ( Solanum lycopersicum )", PEERJ, vol. 5, pages e3955, XP093042983, DOI: 10.7717/peerj.3955 *
OHYAMA AKIO; SHIRASAWA KENTA; MATSUNAGA HIROSHI; NEGORO SATOMI; MIYATAKE KOJI; YAMAGUCHI HIROTAKA; NUNOME TSUKASA; IWATA HIROYOSHI: "Bayesian QTL mapping using genome-wide SSR markers and segregating population derived from a cross of two commercial F1hybrids of tomato", THEORETICAL AND APPLIED GENETICS, SPRINGER BERLIN HEIDELBERG, BERLIN/HEIDELBERG, vol. 130, no. 8, 5 May 2017 (2017-05-05), Berlin/Heidelberg, pages 1601 - 1616, XP036277719, ISSN: 0040-5752, DOI: 10.1007/s00122-017-2913-5 *

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