Genes involved in the regulation of total dry matter production in Solanaceae plants and their utilization

Abstract

To provide technologies for determining the degree of total dry matter production in Solanaceae plants.SOLUTION: Provided is a method for determining the degree of total dry matter production in a Solanaceae plant, the method comprising examining the presence or absence of mutations that affect control functions of the following proteins (1) to (3) in a Solanaceae plant: (1) a protein consisting of a specific amino acid sequence; (2) a protein consisting of an amino acid sequence having a sequence identity of 80% or more to a specific amino acid sequence and having the function of controlling the total dry matter production of the Solanaceae plant; and (3) a protein consisting of an amino acid sequence in which 65 or less amino acids are substituted, deleted, added, or inserted into a specific amino acid sequence, and having the function of controlling the total dry matter production of the Solanaceae plant.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a gene involved in the control of total dry matter production in Solanaceae plants and the use thereof. [Background technology]

[0002] The yield of agricultural products such as seeds, tubers, and fruits is an important indicator in agricultural management because increases or decreases in the yield are directly linked to profits. The yield of agricultural products is proportional to the amount of dry matter produced and the amount of dry matter allocated to the harvested product (the amount of assimilate allocated). Therefore, the yield of agricultural products can be increased by increasing the amount of dry matter produced or the amount of assimilate allocated.

[0003] While environmental control techniques such as supplemental lighting and improved light transmittance through the selection of materials are available to increase the yield of agricultural products, these are costly and difficult to achieve a stable increase in yield. A known technique for achieving a stable increase in yield of agricultural products is the genetic modification of plants.

[0004] Candidate genes for use in genetic modification of Solanaceae fruit vegetables such as tomatoes include the genes described in Non-Patent Documents 1 and 2. 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-fruited and small-fruited varieties in tomatoes. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Frary et al. Science 289:85-88, 2000 [Non-patent document 2] Cong et al. Nat Genet 40:800-804, 2008 Summary of the Invention [Problem to be solved by the invention]

[0006] The fw2.2 gene described in Non-Patent Document 1 is a gene universally present in large-fruited tomato varieties, and is presumed to have been acquired during the domestication process of small-fruited wild tomatoes to produce larger fruit. In other words, the fw2.2 gene is not thought to be involved in controlling total dry matter production in Solanaceae plants, but rather in increasing cell division and locule number. Therefore, the fw2.2 gene only brings about morphological changes in plants, and is unlikely to be used to modify yield, such as total dry matter production.

[0007] While genes involved in the control of total dry matter production have been reported in rice and other plants, no genes are known to be involved in the control of total dry matter production in Solanaceae plants, including tomato.

[0008] The present invention has been made in view of the above-mentioned problems, and an object of the present invention is to identify a gene involved in the control of total dry matter production in Solanaceae plants including tomato, and to provide a use of the gene. [Means for solving the problem]

[0009] A method according to one embodiment of the present invention is a method for determining the level of total dry matter production in a Solanaceae plant, and includes the step of examining the Solanaceae plant for the presence or absence of a mutation that causes a substitution, deletion, addition, or insertion in at least one of the following amino acids (a) to (b): (a) the amino acid corresponding to the 109th amino acid in the protein consisting of the amino acid sequence shown in SEQ ID NO: 1; and (b) the amino acid corresponding to the 247th amino acid in the protein consisting of the amino acid sequence shown in SEQ ID NO: 1.

[0010] A method according to one embodiment of the present invention is a method for determining the level of total dry matter production in a Solanaceae plant, and includes a step of examining the Solanaceae plant for the presence or absence of a mutation that causes a substitution, deletion, addition, or insertion in at least one of the following amino acids (e) and (f): (e) the amino acid corresponding to the 133rd amino acid in the protein consisting of the amino acid sequence set forth in SEQ ID NO: 2; and (f) the amino acid corresponding to the 139th amino acid in the protein consisting of the amino acid sequence set forth in SEQ ID NO: 2.

[0011] A method according to one embodiment of the present invention is a method for determining the level of total dry matter production in a Solanaceae plant, and includes a step of examining the Solanaceae plant for the presence or absence of a mutation that causes a substitution, deletion, addition, or insertion in at least one of the following amino acids (g) and (h): (g) the amino acid corresponding to the 134th amino acid in the protein consisting of the amino acid sequence set forth in SEQ ID NO: 3; and (h) the amino acid corresponding to the 175th amino acid in the protein consisting of the amino acid sequence set forth in SEQ ID NO: 3.

[0012] A production method according to one embodiment of the present invention is a method for producing a Solanaceae plant with increased total dry matter production, and includes a hybridization step of intraspecifically hybridizing Solanaceae plants, and an identification step of identifying a Solanaceae plant with increased total dry matter production from the Solanaceae plant obtained by the hybridization step or a Solanaceae plant of a progeny lineage, using any of the above methods.

[0013] A production method according to one embodiment of the present invention is a method for producing a solanaceous plant with increased total dry matter production, and includes an identification step of identifying a solanaceous plant with increased total dry matter production from a test solanaceous plant by any of the above-described methods, and a hybridization step of intraspecifically hybridizing the identified solanaceous plant.

[0014] A molecular marker according to one embodiment of the present invention is a molecular marker for controlling total dry matter production in Solanaceae plants, and includes at least one of the bases (SNP) corresponding to the bases (a') to (h') below, or consecutive polynucleotides containing such bases: (a') a base corresponding to the 326th base of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 4; (b') a base corresponding to the 390th base of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 4; (c') a base corresponding to the 591st base of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 4; (d') a base corresponding to the 740th base of 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 base sequence shown in SEQ ID NO: 5; (f') a base corresponding to the 417th base of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 5; (g') a base corresponding to the 401st base of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 6; or (h') a base corresponding to the 523rd base of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 6.

[0015] A production method according to one embodiment of the present invention is a method for producing a solanaceous plant with increased total dry matter production, comprising the step of introducing a mutation into a polynucleotide encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 in a solanaceous plant, such that the mutation 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 set forth in SEQ ID NO: 1.

[0016] A gene according to one embodiment of the present invention comprises a polynucleotide encoding a protein having the function of increasing the total dry matter production of a Solanaceae plant, into which a mutation has been introduced 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 a Solanaceae plant.

[0017] A gene according to one embodiment of the present invention comprises a polynucleotide selected from the group consisting of the following polynucleotides (1) to (3): (1) a polynucleotide encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1; (2) a polynucleotide encoding a protein having an amino acid sequence that is 80% or more identical to the amino acid sequence set forth in SEQ ID NO: 1, in which the amino acid corresponding to the 109th amino acid in the amino acid sequence set forth in SEQ ID NO: 1 and the amino acid sequence downstream thereof are deleted or inactivated, or the amino acid corresponding to the 247th amino acid in the amino acid sequence set forth in SEQ ID NO: 1 is C, and which has the function of regulating the total dry matter production in Solanaceae plants; (3) a polynucleotide encoding a protein having an amino acid sequence in which the amino acid corresponding to the 109th amino acid in the amino acid sequence set forth in SEQ ID NO: 1 and the amino acid sequence downstream thereof are deleted or inactivated, or the amino acid corresponding to the 247th amino acid in the amino acid sequence set forth in SEQ ID NO: 1 is C, and in which 65 or fewer amino acids have been substituted, deleted, added, or inserted, and which has the function of regulating the total dry matter production in Solanaceae plants.

[0018] A gene according to one embodiment of the present invention is a polynucleotide selected from the group consisting of any of the following polynucleotides (4) to (6): (4) a polynucleotide encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2; (5) a polynucleotide encoding a protein having an amino acid sequence with 80% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, wherein the amino acid corresponding to the 133rd amino acid in the amino acid sequence set forth in SEQ ID NO: 2 is A or the amino acid corresponding to the 139th amino acid in the amino acid sequence set forth in SEQ ID NO: 2 is N, and wherein the protein has the function of regulating the total dry matter production in Solanaceae plants; or (6) a polynucleotide having an amino acid sequence with 30 or fewer amino acids substituted, deleted, added, or inserted relative to the amino acid sequence set forth in SEQ ID NO: 2, wherein the amino acid corresponding to the 133rd amino acid in the amino acid sequence set forth in SEQ ID NO: 2 is A or the amino acid corresponding to the 139th amino acid in the amino acid sequence set forth in SEQ ID NO: 2 is N, and wherein the polynucleotide encodes a protein having the function of regulating the total dry matter production in Solanaceae plants.

[0019] A gene according to one embodiment of the present invention comprises a polynucleotide selected from the group consisting of any of the following polynucleotides (7) to (9): (7) a polynucleotide encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 3; (8) a polynucleotide encoding a protein having an amino acid sequence with 80% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 3, wherein the amino acid corresponding to the 134th amino acid in the amino acid sequence set forth in SEQ ID NO: 3 is P or the amino acid corresponding to the 175th amino acid in the amino acid sequence set forth in SEQ ID NO: 3 is Y, and wherein the protein has the function of regulating the total dry matter production in Solanaceae plants; or (9) a polynucleotide having an amino acid sequence in which 60 or fewer amino acids have been substituted, deleted, added, or inserted relative to the amino acid sequence set forth in SEQ ID NO: 3, wherein the amino acid corresponding to the 134th amino acid in the amino acid sequence set forth in SEQ ID NO: 3 is P or the amino acid corresponding to the 175th amino acid in the amino acid sequence set forth in SEQ ID NO: 3 is Y, and wherein the polynucleotide encodes a protein having the function of regulating the total dry matter production in Solanaceae plants.

[0020] An expression vector according to one aspect of the present invention comprises any one of the above genes.

[0021] A cell or dicotyledonous plant according to one aspect of the present invention comprises any one of the above genes or the above expression vector. [Effects of the Invention]

[0022] According to one aspect of the present invention, by using a gene that controls total dry matter production in a solanaceae plant, it is possible to identify a solanaceae plant with increased total dry matter production and to produce a solanaceae plant with increased total dry matter production. [Brief explanation of the drawings]

[0023] [Figure 1] FIG. 1 outlines the construction of recombinant inbred lines derived from the cross of two F1 varieties and their QTL mapping. [Figure 2]FIG. 1 is a diagram illustrating a linkage map of linkage group 6 and the location of QTL tfw6.1. [Figure 3] FIG. 1 shows the nucleotide sequence and amino acid sequence of the genome-edited Arabidopsis line used in the Examples. [Figure 4] FIG. 1 shows the results of comparing the phenotypes of the genome-edited Arabidopsis lines used in the Examples with the wild type. DETAILED DESCRIPTION OF THE INVENTION

[0024] The following describes an embodiment of the present invention, but the present invention is not limited to this.

[0025] As used herein, the term "polynucleotide" can be alternatively referred to as "nucleic acid" or "nucleic acid molecule," and refers to a polymer of nucleotides. Furthermore, the term "base sequence" can be alternatively referred to as "nucleic acid sequence" or "nucleotide sequence," and refers to a sequence of deoxyribonucleotides or a sequence of ribonucleotides, unless otherwise specified. As used herein, the term "polypeptide" can be alternatively referred to as "protein."

[0026] As used herein, "dry matter" can also be referred to as "assimilates," and refers to organic compounds such as sugars synthesized by photosynthesis. As used herein, "total dry matter production" is also referred to as "biomass amount," and refers to the total amount of dry matter produced. As used herein, "increase in total dry matter production" refers to an increase in the total amount of dry matter produced, for example, an increase in total dry matter production as a result of increased light reception due to an increase in leaf area from the fruit-bearing or stem-thickening stage to the harvest stage.

[0027] As used herein, "solanaceous plants" are, by way of example, tomatoes, potatoes, eggplants, or peppers.

[0028] In this specification, the term "eggplant" is broadly defined to include the cultivated species "Solanum melongena (hereinafter referred to as S)," as well as the wild species "S. incanum," "S. torvum," "S. nigrum," "S. aethiopicum," "S. macrocarpon," and "S. quitoense," and in the narrow sense, it refers to "S. melongena."

[0029] In this specification, "tomato" refers to the cultivated species "S. lycopersicum (S. lycopersicum)" in a broad sense. This concept includes "S. ersicum" as well as the wild species "S. cheesmaniae, S. chilense, S. chmielewskii, S. galapagense, S. habrochaites, S. lycopersicoides, S. neorickii, S. pennellii, S. peruvianum, and S. pimpinellifolium," and in the narrow sense it is intended to mean "S. lycopersicum."

[0030] In this specification, the term "chili pepper" broadly refers to the cultivated species "Capsicum annuum" and the wild species "C. pubescens," "C. baccatum," "C. chinense," and "C. frutescens," but in the narrow sense refers to "C. anium." Furthermore, the term "chili pepper" also refers to the above-mentioned plants for which terms other than "chili pepper" are used, such as "bell pepper," "paprika," and "shishito pepper," as horticultural crop names.

[0031] In this specification, the term "potato" is used in a broad sense to include the cultivated species "S. tuberosum" as well as the wild species "S. acaule," "S. sparsipilum," "S. leptophyes," and "S. megistacrobum," and in the narrow sense, it refers to "S. tuberosum."

[0032] [1. Genes controlling total dry matter production] A total dry matter production control gene according to one embodiment of the present invention is a gene encoding a protein having activity that controls total dry matter production (total dry matter production control activity). When a protein encoded by a total dry matter production control gene has activity that positively controls total dry matter production, the presence of the protein increases total dry matter production. Furthermore, when the activity of a protein encoded by a total dry matter production control gene is reduced or inhibited, total dry matter production is reduced or does not increase compared to a plant in which the activity is not reduced or inhibited.

[0033] An example of a total dry matter production control gene is a gene involved in the control of total dry matter production in a Solanaceae plant, and is a gene consisting of any one of the following polynucleotides (1) to (9): (1) a polynucleotide encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1; (2) a polynucleotide encoding a protein having an amino acid sequence that has 80% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1 and that has the function of regulating the total dry matter production of a Solanaceae plant; (3) a polynucleotide encoding a protein having an amino acid sequence in which 65 or fewer amino acids have been substituted, deleted, added, or inserted relative to the amino acid sequence set forth in SEQ ID NO: 1, and having the function of regulating the total dry matter production of a Solanaceae plant; (4) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2; (5) a polynucleotide encoding a protein having an amino acid sequence having 80% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 2 and having the function of regulating the total dry matter production of a Solanaceae plant; (6) a polynucleotide encoding a protein having an amino acid sequence in which 30 or fewer amino acids have been substituted, deleted, added, or inserted relative to the amino acid sequence set forth in SEQ ID NO: 2, and having the function of regulating the total dry matter production of a Solanaceae plant; (7) A polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 3; (8) a polynucleotide encoding a protein having an amino acid sequence having 80% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 3 and having the function of regulating the total dry matter production of a Solanaceae plant; (9) A polynucleotide encoding a protein having an amino acid sequence in which 60 or fewer amino acids have been substituted, deleted, added, or inserted relative to the amino acid sequence shown in SEQ ID NO: 3, and having the function of controlling the total dry matter production of Solanaceae plants.

[0034] The polynucleotides (1), (4), and (7) above are polynucleotides that contain the nucleotide sequence of the tfw6.1 gene derived from tomato or the nucleotide sequence of the coding region (CDS) of the tfw6.1 gene and encode a protein that functions to control total dry matter production. The polynucleotides (1), (4), and (7) above may also be polynucleotides that contain the nucleotide sequence of a gene corresponding to the tfw6.1 gene of a Solanaceae plant or the nucleotide sequence of the CDS of the gene and encode a protein that functions to control total dry matter production.

[0035] With respect to the polynucleotides (2), (5), and (8) above, the sequence identity of the amino acid sequences is preferably 80% or more, or 90% or more, more preferably 95% or more, and particularly preferably 96% or more, 97% or more, 98% or more, or 99% or more.

[0036] With regard to the polynucleotide (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, more preferably 1 to 60, 1 to 50, 1 to 40, or 1 to 30, even more preferably 1 to 25, and particularly preferably 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, or 1 to 2.

[0037] With regard to the polynucleotide (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, more preferably 1 to 25, 1 to 20, or 1 to 15, even more preferably 1 to 10, and particularly preferably 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.

[0038] With regard to the polynucleotide (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, more preferably 1 to 50, 1 to 40, or 1 to 30, even more preferably 1 to 25, and particularly preferably 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, or 1 to 2.

[0039] For example, mutant genes derived from tomato or homologous genes (including orthologs) derived from Solanaceae plants other than tomato are included in the categories of polynucleotides (2), (3), (5), (6), (8), and (9) above. These mutant genes and homologous genes are endogenous genes of Solanaceae plants. The molecular markers tested in the section "4. Method for determining the level of total dry matter production in Solanaceae plants" described below may be molecular markers on these mutant genes or homologous genes.

[0040] When the total dry matter production control gene refers to a gene into which a mutation has been artificially introduced, the above-mentioned "amino acid substitution, deletion, addition, or insertion" may be an artificial mutation introduced using, for example, a site-specific mutagenesis method such as the Kunkel method (Kunkel, Proc Natl Acad Sci USA, 82: 488-492, 1985), mutagen treatment using a drug, a mutagenesis method using radiation (gamma rays, heavy ion beams, etc.), genome editing using site-specific nucleases, or may be derived from a similar mutant polypeptide that exists in nature.

[0041] The total dry matter production control gene may be a gene consisting of a polynucleotide into which a mutation has been introduced that deletes or inactivates an amino acid sequence downstream of any amino acid corresponding to the 73rd amino acid in the amino acid sequence set forth in SEQ ID NO: 1 in a solanaceous plant, thereby encoding a protein that functions to increase total dry matter production in a solanaceous plant. Furthermore, the polynucleotide in the total dry matter production control gene may be a gene into which a mutation has been introduced that deletes or inactivates an amino acid corresponding to the 109th amino acid in the protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 and the amino acid sequence downstream thereof, thereby encoding a protein that functions to increase total dry matter production in a solanaceous plant. Such a total dry matter production control gene can be obtained by artificially introducing a mutation into the total dry matter production control gene of a solanaceous plant using known techniques such as genome editing. The location of the mutation to be introduced into the total dry matter production control gene, details of the mutation, and other details are incorporated herein by reference in Section 6. "Method for Producing a Solanaceous Plant with Increased Total Dry Matter Production," described below.

[0042] The total dry matter production control gene may exist in the form of RNA (e.g., mRNA) or DNA (e.g., cDNA or genomic DNA). The DNA may be double-stranded or single-stranded. The nucleotide sequence shown in SEQ ID NO: 4, which is an example of a total dry matter production control gene, is a full-length cDNA of the gene encoding the polypeptide shown in SEQ ID NO: 1. The nucleotide sequence shown in SEQ ID NO: 5, which is another example of a total dry matter production control gene, is a full-length cDNA of the gene encoding the polypeptide shown in SEQ ID NO: 2. The nucleotide sequence shown in SEQ ID NO: 6, which is another example of a total dry matter production control gene, is a full-length cDNA of the gene encoding the polypeptide shown in SEQ ID NO: 3. The total dry matter production control gene may contain an additional sequence, such as a nucleotide sequence of an untranslated region (UTR), in addition to the CDS of the tfw6.1 gene.

[0043] The method for obtaining (isolating) the total dry matter production control gene is not particularly limited, but for example, a probe that specifically hybridizes with a portion of the base sequence of the total dry matter production control gene may be prepared and a genomic DNA library or a cDNA library may be screened.

[0044] Another method for obtaining the total dry matter production control gene is to use an amplification method such as PCR. For example, primers are prepared from the 5' and 3' sequences (or their complementary sequences) of the cDNA of the total dry matter production control gene, and PCR or the like is performed using these primers and genomic DNA (or cDNA) as a template to amplify the DNA region sandwiched between the two primers, thereby obtaining a large amount of DNA fragments containing the total dry matter production control gene.

[0045] The origin of the total dry matter production control gene is not particularly limited as long as it is a plant of the Solanaceae family, but is preferably tomato, potato, eggplant, or chili pepper, and more preferably tomato. The total dry matter production control gene may also be derived from a dicotyledonous plant, including a plant of the Solanaceae family. An example of a dicotyledonous plant is Arabidopsis thaliana.

[0046] Whether or not an isolated candidate gene for a total dry matter production control gene has the activity to control the desired total dry matter production can be evaluated by observing whether expression of the candidate gene in a dicotyledonous plant induces an increase in total dry matter production compared to a dicotyledonous plant in which the candidate gene is not expressed.

[0047] The total dry matter production control gene can be used to elucidate the mechanism of increased total dry matter production in dicotyledonous plants. Furthermore, the total dry matter production control gene can be used to produce a transformant by incorporating its sequence into an expression vector and introducing it into a dicotyledonous plant or cell. By cultivating a dicotyledonous plant into which the total dry matter production control gene has been introduced, a dicotyledonous plant with increased total dry matter production can be obtained.

[0048] Examples of total dry matter production control genes include polynucleotides consisting of the nucleotide sequences of 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 nucleotide sequences of the CDS of the tfw6.1 gene derived from S. lycopersicum (tomato). The total dry matter production control gene also includes genes consisting of polynucleotides that have 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to the nucleotide sequences set forth in SEQ ID NOs: 4 to 6 and have total dry matter production control activity.

[0049] [2. Total dry matter production control protein] A total dry matter production control protein according to one embodiment of the present invention is a translation product of a gene described in the above section [1. Total dry matter production control gene] and has at least total dry matter production control activity. When a total dry matter production control protein has activity that positively controls total dry matter production, the presence of the protein increases total dry matter production. In the absence of the total dry matter production control protein, total dry matter production decreases or does not increase compared to the presence of the total dry matter production control protein.

[0050] The total dry matter production control protein may be isolated from a natural source or chemically synthesized. More specifically, the protein includes purified natural products, products of chemical synthesis procedures, and translation products produced by recombinant technology from prokaryotic or eukaryotic hosts (e.g., bacterial cells, yeast cells, higher plant cells, insect cells, and mammalian cells).

[0051] More specifically, the total dry matter production control protein is a protein described in any one of the following (1') to (9'): (1') a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1; (2') a protein having an amino acid sequence having 80% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1 and having the function of regulating the total dry matter production of a Solanaceae plant; (3') a protein consisting of an amino acid sequence in which 65 or fewer amino acids have been substituted, deleted, added, or inserted relative to the amino acid sequence set forth in SEQ ID NO: 1, and having the function of regulating the total dry matter production of a Solanaceae plant; (4') a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2; (5') a protein consisting of an amino acid sequence having 80% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, and having the function of regulating the total dry matter production of a Solanaceae plant; (6') a protein consisting of an amino acid sequence in which 30 or fewer amino acids have been substituted, deleted, added, or inserted relative to the amino acid sequence set forth in SEQ ID NO: 2, and having the function of regulating the total dry matter production of a Solanaceae plant; (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 to the amino acid sequence shown in SEQ ID NO: 3, and having the function of regulating the total dry matter production of a Solanaceae plant; (9') A protein consisting of an amino acid sequence in which 60 or fewer amino acids have been substituted, deleted, added, or inserted relative to the amino acid sequence shown in SEQ ID NO: 3, and having the function of regulating the total dry matter production of Solanaceae plants.

[0052] The above proteins (1'), (4'), and (7') are proteins encoded by the above tfw6.1 gene or a gene corresponding to the above tfw6.1 gene in a Solanaceae plant, and are proteins having total dry matter production control activity.

[0053] With respect to the proteins (2'), (5'), and (8') above, the sequence identity of the amino acid sequence is preferably 80% or more, or 90% or more, more preferably 95% or more, and particularly preferably 96% or more, 97% or more, 98% or more, or 99% or more.

[0054] With respect to 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, more preferably 1 to 60, 1 to 50, 1 to 40, or 1 to 30, even more preferably 1 to 25, and particularly preferably 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, or 1 to 2.

[0055] With respect to 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, more preferably 1 to 25, 1 to 20, or 1 to 15, even more preferably 1 to 10, and particularly preferably 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.

[0056] With respect to 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, more preferably 1 to 50, 1 to 40, or 1 to 30, even more preferably 1 to 25, and particularly preferably 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, or 1 to 2.

[0057] For example, mutant proteins derived from tomato or homologous proteins derived from Solanaceae plants other than tomato are included in the categories of proteins (2'), (3'), (5'), (6'), (8'), and (9') above. These mutant proteins and homologous proteins are proteins encoded by endogenous genes of Solanaceae plants.

[0058] The total dry matter production control protein is a polypeptide formed by peptide bonds between amino acids, but may also contain structures other than polypeptides, such as, but not limited to, sugar chains and isoprenoid groups.

[0059] Examples of total dry matter production control proteins include proteins derived from S. lycopersicum, which is a tomato. Other examples of total dry matter production control proteins include proteins derived from potato, eggplant, or chili pepper. Furthermore, total dry matter production control proteins may be proteins derived from dicotyledonous plants, including plants of the Solanaceae family, such as Arabidopsis thaliana.

[0060] 3. Expression Vectors, Cells, and Transformants The present invention also encompasses an expression vector incorporating a total dry matter production control gene according to one embodiment of the present invention, a cell containing the expression vector or the total dry matter production control gene, and a transformant into which the expression vector or the total dry matter production control gene has been introduced in an expressible manner. The expression vector confers a trait for controlling total dry matter production to a cell or an individual organism.

[0061] The type of vector used to construct the expression vector is not particularly limited, and may be appropriately selected from those capable of expression in host cells. That is, a promoter sequence may be appropriately selected depending on the type of host cell, and the promoter sequence and the total dry matter production control gene may be incorporated into, for example, a plasmid, a phagemid, or a cosmid, and used as the expression vector.

[0062] Host cells into which expression vectors can be introduced include, for example, bacterial cells, yeast cells, fungal cells other than yeast cells, and higher eukaryotic cells. Examples of bacterial cells include Escherichia coli cells. Examples of higher eukaryotic cells include plant cells and animal cells. Examples of plant cells include dicotyledonous plant cells and monocotyledonous plant cells. Examples of dicotyledonous plant cells include suspension culture cells of Solanaceae plants (e.g., tobacco BY-2 strain and tomato Sly-1 strain). Examples of monocotyledonous plant cells include the Oc strain, which is a suspension culture cell of rice. Examples of animal cells include insect cells, amphibian cells, reptile cells, avian cells, fish cells, and mammalian cells.

[0063] In the expression vector, the total dry matter production control gene is functionally linked to elements necessary for transcription (e.g., a promoter, etc.). Furthermore, an enhancer, a selection marker, a splicing signal, a poly(A) addition signal, a 5'-UTR sequence, etc. may also be linked as necessary. A promoter is a DNA sequence that exhibits transcriptional activity in host cells and can be appropriately selected depending on the type of host.

[0064] Examples of promoter sequences operable in host cells include the 35S promoter of cauliflower mosaic virus, the nopaline synthase gene promoter of Agrobacterium, and the rice ubiquitin gene promoter. Furthermore, the promoter sequence of the promoter region in the total dry matter production control gene may also be used as a recombinant expression promoter.

[0065] In the expression vector, the total dry matter production control gene may be functionally linked to an appropriate terminator (e.g., the NOS terminator and the 35S terminator of the cauliflower mosaic virus) as needed. The type of appropriate terminator may be selected appropriately depending on the type of host cell, and may be any sequence that can terminate transcription of the gene transcribed by the above-mentioned promoter. Enhancers are used to increase the expression efficiency of the target gene, and examples of such enhancers include the omega sequence of the tobacco mosaic virus.

[0066] The expression vector may further contain a selection marker, such as a drug resistance gene for ampicillin, kanamycin, tetracycline, chloramphenicol, neomycin, hygromycin, or spectinomycin.

[0067] Furthermore, 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.

[0068] The term "transformant" refers not only to cells, tissues, and organs into which the expression vector or the total dry matter production control gene has been introduced in an expressible manner, but also to individual organisms. Such a transformant may be a plant of the Solanaceae family. The transformant may also be, for example, a microorganism such as Escherichia coli, an animal, or the like.

[0069] 4. Method for determining the degree of total dry matter production in Solanaceae plants A method for determining the level of total dry matter production in a solanaceous plant (discrimination method) according to one embodiment of the present invention determines whether or not total dry matter production is increased in the solanaceous plant. The discrimination method can be used to identify solanaceous plants with increased total dry matter production. The discrimination method discriminates solanaceous plants based on total dry matter production by determining the genotype of genes involved in determining total dry matter production, which are present in the genome of the solanaceous plant. Note that in the discrimination method, the concept of total dry matter production includes the degree of total dry matter production, which indicates whether the total dry matter production of a certain solanaceous plant individual is relatively high or low compared to other solanaceous plant individuals.

[0070] In solanaceous plants, such as tomatoes, total dry matter production gradually increases with growth, peaks during the continuous production period, and then plateaus (see Reference 1: Saito et al. Hort J 89: 445-453, 2020). That is, total dry matter production is low in the early stages of cultivation when fruit clusters begin to set or stems begin to thicken. Therefore, by increasing total dry matter production, especially in the early stages of cultivation, such as during fruit set or stem thickening, yield can be steadily increased.

[0071] Solanaceae plants may be candidate plants for breeding material or plants obtained through a breeding process. Candidate plants for breeding material include, for example, parent plants used in crossbreeding and plants used in molecular breeding using genetic engineering. Plants obtained through a breeding process include, for example, plants obtained by intraspecific hybridization of Solanaceae plants such as tomato, potato, eggplant, or chili pepper, and their progeny. Solanaceae plants may also be intervarietal hybrids, such as hybrids between one tomato variety and another tomato variety, and their progeny. Furthermore, the method for determining the level of total dry matter production in Solanaceae plants can be used to determine the level of total dry matter production in dicotyledonous plants, including Solanaceae plants. An example of a dicotyledonous plant is Arabidopsis thaliana.

[0072] Furthermore, the solanaceae plant may be a plant obtained by crossing varieties known to have increased total dry matter production with each other, and its progeny line. Furthermore, the solanaceae plant may be a plant obtained by crossing varieties known to have increased total dry matter production with varieties for which it is unknown whether the total dry matter production has increased, and its progeny line. Furthermore, the solanaceae plant may be a plant obtained by crossing varieties for which it is unknown whether the total dry matter production has increased, and its progeny line. Furthermore, the solanaceae plant may be a plant obtained by crossing varieties known to have increased total dry matter production with varieties for which it is not known whether the total dry matter production has increased, and its progeny line. Furthermore, the solanaceae plant may be a plant obtained by crossing individuals known to have increased total dry matter production with each other, and its progeny line.

[0073] In this specification, the term "plant" may refer to a part or the whole of a plant body. Examples of a part of a plant body include propagation materials (e.g., leaves, branches, seeds, etc.).

[0074] The method for distinguishing includes detecting a protein in a Solanaceae plant, the protein being any one of the following (1') to (9'): (1') a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1; (2') a protein having an amino acid sequence having 80% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1 and having the function of regulating the total dry matter production of a Solanaceae plant; (3') a protein consisting of an amino acid sequence in which 65 or fewer amino acids have been substituted, deleted, added, or inserted relative to the amino acid sequence set forth in SEQ ID NO: 1, and having the function of regulating the total dry matter production of a Solanaceae plant; (4') a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2; (5') a protein consisting of an amino acid sequence having 80% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, and having the function of regulating the total dry matter production of a Solanaceae plant; (6') a protein consisting of an amino acid sequence in which 30 or fewer amino acids have been substituted, deleted, added, or inserted relative to the amino acid sequence set forth in SEQ ID NO: 2, and having the function of regulating the total dry matter production of a Solanaceae plant; (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 to the amino acid sequence shown in SEQ ID NO: 3, and having the function of regulating the total dry matter production of a Solanaceae plant; (9') a protein consisting of an amino acid sequence in which 60 or fewer amino acids have been substituted, deleted, added, or inserted relative to the amino acid sequence set forth in SEQ ID NO: 3, and having the function of regulating the total dry matter production of a Solanaceae plant; The method includes a step of examining the presence or absence of a mutation that affects the regulatory function of the gene.

[0075] The mutation affecting the function of a protein that controls total dry matter production in Solanaceae plants may be a mutation that causes the protein to express a function of increasing total dry matter production in Solanaceae plants. The discrimination method determines whether or not total dry matter production is increased in Solanaceae plants by examining the presence or absence of such a mutation.

[0076] The mutation affecting the function of a protein having the function of controlling the total dry matter production of a Solanaceae plant may be a mutation selected from the group consisting of a stop codon mutation, a frameshift mutation, and a null mutation in a polynucleotide encoding any of the proteins (1') to (9').

[0077] The discrimination method can examine the presence or absence of a mutation in the total dry matter production control gene using the molecular markers shown below. Such molecular markers are also included in the scope of the present invention.

[0078] The 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 set forth in SEQ ID NO: 1; (b) an amino acid corresponding to the 247th amino acid of the protein consisting of the amino acid sequence set forth in SEQ ID NO: 1; (e) an amino acid corresponding to the 133rd amino acid of the protein consisting of the amino acid sequence set forth in SEQ ID NO: 2; (f) an amino acid corresponding to the 139th amino acid of the protein consisting of the amino acid sequence set forth in SEQ ID NO: 2; (g) an amino acid corresponding to the 134th amino acid of the protein consisting of the amino acid sequence set forth in SEQ ID NO: 3; (h) an amino acid corresponding to the 175th amino acid of the protein consisting of the amino acid sequence set forth in SEQ ID NO: 3; It is a molecular marker that tests for the presence or absence of mutations that cause substitutions, deletions, additions, or insertions in at least one of the above.

[0079] A protein consisting of an 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 an example is a protein encoded by a gene consisting of a base sequence shown in any one of SEQ ID NOs: 4 to 6.

[0080] A protein consisting of an amino acid sequence set forth in any one of SEQ ID NOS: 1 to 3 has the amino acid sequence of a protein encoded by the reference tfw6.1 gene derived from tomato (S. lycopersicum). In Solanaceae plants, the amino acid sequence may contain portions that differ from the amino acid sequence of a protein encoded by the reference tfw6.1 gene derived from tomato, in addition to the portions corresponding to the amino acids (a), (b), and (e) to (h) above. In Solanaceae plants, the positions of the amino acids corresponding to the amino acids (a), (b), and (e) to (h) above can be identified by techniques such as homology analysis. That is, in a gene corresponding to the tfw6.1 gene of a Solanaceae plant (i.e., in the case where a gene highly conserved among plants exists), "amino acids corresponding to amino acids (a), (b), and (e) to (h)" refer to amino acids that have been determined to correspond to amino acids (a), (b), and (e) to (h) by techniques such as homology analysis.

[0081] Examples of homology analysis methods include pairwise sequence alignment methods such as the Needleman-Wunsch method and the Smith-Waterman method, and multiple sequence alignment methods such as the ClustalW method. Based on these methods, a person skilled in the art can determine the "corresponding amino acids" in the amino acid sequence to be analyzed by using the amino acid sequence shown in any of SEQ ID NOs: 1 to 3 as a reference sequence.

[0082] The following shows examples of proteins in Solanaceae plants that are homologous to the protein consisting of the amino acid sequence shown in SEQ ID NO: 1, and amino acids corresponding to amino acids (a) or (b).

[0083] In the protein of bell pepper (CA09g13460) consisting of the amino acid sequence shown in SEQ ID NO: 7, the amino acid corresponding to amino acid (a) is the 351st amino acid, and the amino acid corresponding to amino acid (b) is the 489th amino acid.

[0084] 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 (a) is the 333rd amino acid, and the amino acid corresponding to (b) is the 471st amino acid.

[0085] In the potato protein (Sotub06g015180.1.1) consisting of the amino acid sequence shown in SEQ ID NO: 9, the amino acid corresponding to amino acid (a) is the 337th amino acid, and the amino acid corresponding to amino acid (b) is the 475th amino acid.

[0086] Furthermore, an Arabidopsis protein is shown as an example of a homologous protein of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in dicotyledonous plants including Solanaceae plants. In the Arabidopsis protein (AT5G48150.1) consisting of the amino acid sequence shown in SEQ ID NO: 10, the amino acid corresponding to amino acid (a) is the 268th amino acid, and the amino acid corresponding to amino acid (b) is the 406th amino acid.

[0087] Examples of "amino acids corresponding to amino acids (a) or (b)" include (1) amino acids (a) or (b) in a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, (2) amino acids corresponding to amino acids (a) or (b) in a protein consisting of an amino acid sequence that has 80% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1, or (3) amino acids corresponding to amino acids (a) or (b) in a protein consisting of an amino acid sequence in which 65 or fewer amino acids have been substituted, deleted, added, or inserted relative to the amino acid sequence shown in SEQ ID NO: 1.

[0088] A molecular marker that detects the presence or absence of a mutation that causes a substitution, deletion, addition, or insertion of an amino acid corresponding to amino acid (a) or (b) may be a molecular marker that detects the presence or absence of a mutation that causes a substitution or deletion of an amino acid corresponding to both amino acids (a) and (b).

[0089] Examples of "amino acids corresponding to amino acids (e) or (f)" include (4) amino acids (e) or (f) in a protein consisting of the amino acid sequence shown in SEQ ID NO: 2, (5) amino acids corresponding to amino acids (e) or (f) in a protein consisting of an amino acid sequence having 80% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 2, or (6) amino acids corresponding to amino acids (e) or (f) in a protein consisting of an amino acid sequence in which 30 or fewer amino acids have been substituted, deleted, added, or inserted relative to the amino acid sequence shown in SEQ ID NO: 2.

[0090] A molecular marker that detects the presence or absence of a mutation that causes a substitution, deletion, addition, or insertion of an amino acid corresponding to amino acid (e) or (h) may be one that detects the presence or absence of a mutation that causes a substitution or deletion of an amino acid corresponding to both amino acids (e) and (h).

[0091] The following shows examples of proteins in Solanaceae plants that are homologous to the protein consisting of the amino acid sequence set forth in SEQ ID NO: 3, as well as amino acids corresponding to amino acids (g) or (h).

[0092] In the protein of bell pepper (CA06g05790) consisting of the amino acid sequence shown in SEQ ID NO: 11, the amino acid corresponding to amino acid (g) is the 134th amino acid, and the amino acid corresponding to amino acid (h) is the 175th amino acid.

[0093] In the protein of bell pepper (Capang06g002314) consisting of the amino acid sequence shown in SEQ ID NO: 12, the amino acid corresponding to amino acid (g) is the 132nd amino acid, and the amino acid corresponding to amino acid (h) is the 173rd amino acid.

[0094] In the protein of bell pepper (Capana06g002501) consisting of the amino acid sequence shown in SEQ ID NO: 13, the amino acid corresponding to amino acid (g) is the 132nd amino acid, and the amino acid corresponding to amino acid (h) is the 173rd amino acid.

[0095] In the potato protein (Sotub06g016400.1.1) consisting of the amino acid sequence shown in SEQ ID NO: 14, the amino acid corresponding to amino acid (g) is the 133rd amino acid, and the amino acid corresponding to amino acid (h) is the 174th amino acid.

[0096] In the potato protein (PGSC0003DMC400050347) consisting of the amino acid sequence shown in SEQ ID NO: 15, the amino acid corresponding to amino acid (g) is the 133rd amino acid, and the amino acid corresponding to amino acid (h) is the 174th amino acid.

[0097] Furthermore, an Arabidopsis protein is shown as an example of a homologous protein of the protein consisting of the amino acid sequence shown in SEQ ID NO: 3 in dicotyledonous plants including Solanaceae 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 amino acid (g) is the 153rd amino acid, and the amino acid corresponding to amino acid (h) is the 194th amino acid.

[0098] Examples of "amino acids corresponding to amino acids (g) or (h)" include (7) amino acids (g) or (h) in a protein consisting of the amino acid sequence shown in SEQ ID NO: 3, (8) amino acids corresponding to amino acids (g) or (h) in a protein consisting of an amino acid sequence having 80% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 3, or (9) amino acids corresponding to amino acids (g) or (h) in a protein consisting of an amino acid sequence in which 60 or fewer amino acids have been substituted, deleted, added, or inserted relative to the amino acid sequence shown in SEQ ID NO: 3.

[0099] A molecular marker that detects the presence or absence of a mutation that causes a substitution, deletion, addition, or insertion of an amino acid corresponding to amino acid (g) or (h) may be one that detects the presence or absence of a mutation that causes a substitution or deletion of an amino acid corresponding to both amino acids (g) and (h).

[0100] An example of a molecular marker is a single nucleotide polynucleotide (SNP) corresponding to the following bases (a') to (h') in a 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 set forth in SEQ ID NO: 4; (b') a base corresponding to the 390th base of the polynucleotide consisting of the base sequence set forth in SEQ ID NO: 4; (c') a base corresponding to the 591st base of the polynucleotide consisting of the base sequence set forth in SEQ ID NO: 4; (d') a base corresponding to the 740th base of the polynucleotide consisting of the base sequence set forth in SEQ ID NO: 4; (e') a base corresponding to the 397th base of the polynucleotide consisting of the base sequence set forth in SEQ ID NO: 5; (f') a base corresponding to the 417th base of the polynucleotide consisting of the base sequence set forth 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 set forth in SEQ ID NO: 6; That is, the discrimination method includes a step of testing the bases themselves (SNPs) corresponding to the bases (a') to (h') above, or a consecutive polynucleotide containing the bases, as molecular markers related to the control of total dry matter production.

[0101] The bases corresponding to the bases (a') to (h') above are SNPs that cause amino acid substitutions. Note that (a') above is a SNP that causes a mutation selected from the group consisting of a stop codon mutation, a frameshift mutation, and a null mutation. Furthermore, (b') and (c') above are SNPs that cause synonymous substitutions (silent mutations).

[0102] By using the bases corresponding to the above bases (a') to (h') as molecular markers, it is possible to detect the presence or absence of a mutation that affects the expression of the function of the above proteins (1) to (9) to control total dry matter production in Solanaceae plants.By using the bases corresponding to the above bases (a') to (h') as molecular markers, it is possible to detect the presence or absence of a mutation that causes a substitution, deletion, addition, or insertion of an amino acid corresponding to the above amino acid (a) to (h).

[0103] Examples of molecular markers include SNP markers, AFLP (amplified fragment length polymorphism) markers, RFLP markers, microsatellite markers, SCAR markers, and CAPS markers.

[0104] The bases themselves corresponding to the above bases (a') to (h') or consecutive polynucleotides containing such bases are the SNP markers described in this Example or SNP markers that can be identified therewith. The SNP markers can be (i) the bases themselves corresponding to the SNP, (ii) consecutive polynucleotides containing the SNP, or (iii) consecutive polynucleotides containing two SNPs.

[0105] (i: SNP marker) SNP refers to a DNA polymorphism in which a single base mutation occurs within a specific region of the DNA base sequence. 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), is published on the solgenomics FTP site (ftp: / / ftp.solgenomics.net / genomes / Solanum_lycopersicum / annotation / ITAG4.1_release / ).

[0106] The "bases (a') to (h')" refer to the SNP markers described in this example. The "bases corresponding to the bases (a') to (h')" refer to SNP markers that can be identified as the SNP markers described in this example. The "bases (a') to (h')" refer to mutations in the tfw6.1 gene. The tfw6.1 gene consists of a base sequence derived from the reference tomato (S. lycopersicum). Solanaceae plants may contain base sequence differences other than SNPs relative to the reference base sequence. Because the region of the genome where this SNP marker is located is conserved among many Solanaceae plants, this SNP marker can be identified by a method such as homology analysis. The homology analysis method can be the same as that used to identify the "corresponding amino acid."

[0107] That is, when a gene corresponding to the tfw6.1 gene (i.e., a gene highly conserved among plants) exists in other solanaceae plants or dicotyledonous plants including solanaceae plants, "bases corresponding to bases (a') to (h')" refers to bases in the gene corresponding to the tfw6.1 gene that are determined to correspond to bases (a') to (h') by a method such as homology search. For example, the polynucleotides described in (a2) to (h2) and the polynucleotides described in (a3) ​​to (h3) below are examples of genes corresponding to the tfw6.1 gene.

[0108] 1 shows an example of a homologous gene of a gene consisting of the CDS base sequence of the tfw6.1 gene shown in SEQ ID NO: 4 in a Solanaceae plant, and bases corresponding to bases (a') to (d').

[0109] In the gene of bell pepper (CA09g13460) consisting of the base sequence shown in SEQ ID NO: 17, the base corresponding to base (a') is the 1052nd base, the base corresponding to base (b') is the 1116th base, the base corresponding to base (c') is the 1317th base, and the base corresponding to base (d') is the 1466th base.

[0110] In the eggplant (Sme2.5_14468.1_g00001.1) gene consisting of the base sequence shown in SEQ ID NO: 18, the base corresponding to base (a') is the 998th base, the base corresponding to base (b') is the 1062nd base, the base corresponding to base (c') is the 1263rd base, and the base corresponding to base (d') is the 1412th base.

[0111] In the potato (Sotub06g015180.1.1) gene consisting of the base sequence shown in SEQ ID NO: 19, the base corresponding to base (a') is the 1010th base, the base corresponding to base (b') is the 1074th base, the base corresponding to base (c') is the 1275th base, and the base corresponding to base (d') is the 1424th base.

[0112] Furthermore, the Arabidopsis gene is shown as an example of a homologous gene of the gene consisting of the nucleotide sequence shown in SEQ ID NO: 4 in dicotyledonous plants including Solanaceae plants. In the Arabidopsis thaliana (AT5G52660.2) gene consisting of the nucleotide sequence shown in SEQ ID NO: 20, the base corresponding to base (a') is the 803rd base, the base corresponding to base (b') is the 867th base, the base corresponding to base (c') is the 1068th base, and the base corresponding to base (d') is the 1217th base.

[0113] 1 shows an example of a homologous gene of a gene consisting of the CDS base sequence of the tfw6.1 gene shown in SEQ ID NO: 6 in a Solanaceae plant, and bases corresponding to bases (g') to (h').

[0114] In the gene of bell pepper (CA06g05790) consisting of the base sequence shown in SEQ ID NO: 21, the base corresponding to base (g') is the 401st base, and the base corresponding to base (h') is the 523rd base.

[0115] In the gene of bell pepper (Capang06g002314) consisting of the base sequence shown in SEQ ID NO: 22, the base corresponding to base (g') is the 395th base, and the base corresponding to base (h') is the 517th base.

[0116] In the gene of bell pepper (Capana06g002501) consisting of the base sequence shown in SEQ ID NO: 23, the base corresponding to base (g') is the 395th base, and the base corresponding to base (h') is the 517th base.

[0117] In the potato (Sotub06g016400.1.1) gene consisting of the base sequence shown in SEQ ID NO: 24, the base corresponding to base (g') is the 398th base, and the base corresponding to base (h') is the 520th base.

[0118] In the potato gene (PGSC0003DMC400050347) consisting of the base sequence shown in SEQ ID NO: 25, the base corresponding to base (g') is the 398th base, and the base corresponding to base (h') is the 520th base.

[0119] Furthermore, the Arabidopsis gene is shown as an example of a homologous gene of the gene consisting of the nucleotide sequence shown in SEQ ID NO: 6 in dicotyledonous plants including Solanaceae plants. In the Arabidopsis thaliana (AT5G52660.2) gene consisting of the nucleotide sequence shown in SEQ ID NO: 26, the base corresponding to base (g') is the 458th base, and the base corresponding to base (h') is the 580th base. The molecular markers are SNP markers consisting of the above SNPs (a') to (h'). These SNP markers have been newly identified by the present inventors, and those skilled in the art can identify the genomic locations of these SNP markers based on the nucleotide sequences representing the respective SNPs of the SNP markers.

[0120] The SNP (a') (hereinafter also referred to as SNP(a')) indicates a polymorphism of the 326th base in the base sequence shown in SEQ ID NO: 4 or a base corresponding thereto. The SNP (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 (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 (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. Furthermore, SNP (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 equivalent thereto. SNP (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 equivalent thereto. SNP (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 equivalent thereto.

[0121] Molecular markers include 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 the above expression is satisfied, it can be determined that the total dry matter production of the Solanaceae plant has increased.

[0122] Alternatively, the total dry matter production in a Solanaceae plant may be determined by analyzing molecular markers consisting of SNPs (a') to (d'), SNPs (e') and (f'), or SNPs (g') and (h') as haplotype blocks. Here, "the total dry matter production of a Solanaceae plant is increased" means that the total dry matter production of an individual Solanaceae plant having a total dry matter production control protein is relatively higher than that of another individual Solanaceae plant not having a total dry matter production control protein.

[0123] (ii: Polynucleotide containing SNP) The molecular marker may be a continuous polynucleotide containing SNP(a') to SNP(h') (hereinafter referred to as polynucleotide(a') to polynucleotide(h')).

[0124] Polynucleotide (a') is (a1) a polynucleotide consisting of the base sequence of a region containing SNP (a') in the base sequence of the tfw6.1 gene; (a2) a polynucleotide consisting of a base sequence in which one or more bases have been substituted, deleted, added, or inserted relative to the base sequence of the polynucleotide of (a1) other than SNP (a'), and having the function of determining the total dry matter production in a Solanaceae plant; or (a3) ​​a polynucleotide consisting of a base sequence that is 90% or more identical to the base sequence of the polynucleotide of (a1) other than SNP (a'), and having the function of determining the total dry matter production in a Solanaceae plant.

[0125] Polynucleotide (b') is (b1) a polynucleotide consisting of the base sequence of a region containing SNP (b') in the base sequence of the tfw6.1 gene; (b2) a polynucleotide consisting of a base sequence in which one or more bases have been substituted, deleted, added, or inserted relative to the base sequence of the polynucleotide of (b1) other than SNP (b'), and having the function of determining the total dry matter production in a Solanaceae plant; or (b3) a polynucleotide consisting of a base sequence that is 90% or more identical to the base sequence of the polynucleotide of (b1) other than SNP (b'), and having the function of determining the total dry matter production in a Solanaceae plant.

[0126] Polynucleotide (c') is (c1) a polynucleotide consisting of the base sequence of a region containing SNP (c') in the base sequence of the tfw6.1 gene; (c2) a polynucleotide consisting of a base sequence in which one or more bases have been substituted, deleted, added, or inserted relative to the base sequence other than SNP (c') in the base sequence of the polynucleotide (c1), and has the function of determining the total dry matter production in a Solanaceae plant; or (c3) a polynucleotide consisting of a base sequence that is 90% or more identical to the base sequence other than SNP (c') in the base sequence of the polynucleotide (c1), and has the function of determining the total dry matter production in a Solanaceae plant.

[0127] Polynucleotide (d') is (d1) a polynucleotide consisting of the base sequence of a region containing SNP (d') in the base sequence of the tfw6.1 gene; (d2) a polynucleotide consisting of a base sequence in which one or more bases have been substituted, deleted, added, or inserted relative to the base sequence other than SNP (d') in the base sequence of the polynucleotide (d1), and has the function of determining the total dry matter production in a Solanaceae plant; or (d3) a polynucleotide consisting of a base sequence that is 90% or more identical to the base sequence other than SNP (d') in the base sequence of the polynucleotide (d1), and has the function of determining the total dry matter production in a Solanaceae plant.

[0128] Polynucleotide (e') is (e1) a polynucleotide consisting of the base sequence of a region containing SNP (e') in the base sequence of the tfw6.1 gene; (e2) a polynucleotide consisting of a base sequence in which one or more bases have been substituted, deleted, added, or inserted relative to the base sequence other than SNP (e') in the base sequence of the polynucleotide (e1), and has the function of determining the total dry matter production in a Solanaceae plant; or (e3) a polynucleotide consisting of a base sequence that is 90% or more identical to the base sequence other than SNP (e') in the base sequence of the polynucleotide (e1), and has the function of determining the total dry matter production in a Solanaceae plant.

[0129] Polynucleotide (f') is (f1) a polynucleotide consisting of the base sequence of a region containing SNP (f') in the base sequence of the tfw6.1 gene; (f2) a polynucleotide consisting of a base sequence in which one or more bases have been substituted, deleted, added, or inserted relative to the base sequence other than SNP (f') in the base sequence of the polynucleotide (f1), and has the function of determining the total dry matter production in a Solanaceae plant; or (f3) a polynucleotide consisting of a base sequence that is 90% or more identical to the base sequence other than SNP (f') in the base sequence of the polynucleotide (f1), and has the function of determining the total dry matter production in a Solanaceae plant.

[0130] Polynucleotide (g') is (g1) a polynucleotide consisting of the base sequence of a region containing SNP (g') in the base sequence of the tfw6.1 gene; (g2) a polynucleotide consisting of a base sequence in which one or more bases have been substituted, deleted, added, or inserted relative to the base sequence other than SNP (g') in the base sequence of the polynucleotide (g1), and has the function of determining the total dry matter production in a Solanaceae plant; or (g3) a polynucleotide consisting of a base sequence that is 90% or more identical to the base sequence other than SNP (g') in the base sequence of the polynucleotide (g1), and has the function of determining the total dry matter production in a Solanaceae plant.

[0131] Polynucleotide (h') is (h1) a polynucleotide consisting of the base sequence of a region containing SNP (h') in the base sequence of the tfw6.1 gene; (h2) a polynucleotide consisting of a base sequence in which one or more bases have been substituted, deleted, added, or inserted relative to the base sequence other than SNP (h') in the base sequence of the polynucleotide (h1), and has the function of determining the total dry matter production in a Solanaceae plant; or (h3) a polynucleotide consisting of a base sequence that is 90% or more identical to the base sequence other than SNP (h') in the base sequence of the polynucleotide (h1), and has the function of determining the total dry matter production in a Solanaceae plant.

[0132] The polynucleotides (a1) to (h1) can be obtained, for example, from a Solanaceae plant with increased total dry matter production (e.g., a tomato (S. lycopersicum Dutch F1 variety) with high total dry matter production) based on the nucleotide sequence of the tfw6.1 gene.

[0133] Polynucleotides (a2) to (h2) may have the bases corresponding to SNPs (a') to (h') conserved in the base sequences of polynucleotides (a1) to (h1), but may contain modifications (substitutions, deletions, insertions, or additions) of several (e.g., 1 to 10, preferably 1 to 5, and more preferably 1, 2, or 3) bases in the remaining base sequences. The base sequences of such polynucleotides are clear to those skilled in the art and can be determined by referring to the genome sequence of tomato (S. lycopersicum) registered in the above-mentioned database, or by decoding the base sequence of the region adjacent to the SNP in the genome of a Solanaceae plant with high total dry matter production.

[0134] Polynucleotides (a3) ​​to (h3) may have, in the base sequences of polynucleotides (a1) to (h1), bases corresponding to SNPs (a') to (h'), conserved, and may have, for example, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the remaining base sequences. Such base sequence identity can be determined by aligning the two base sequences using analysis software such as BLAST or FASTA.

[0135] Molecular markers include 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 the above expression is satisfied, it can be determined that the total dry matter production of the Solanaceae plant has increased.

[0136] The molecular markers may be analyzed using polynucleotides (a) to (d), polynucleotides (e) and (f), or polynucleotides (g) and (h) as haplotype blocks to determine the total dry matter production in a Solanaceae plant.

[0137] (iii: Polynucleotide containing two or more SNPs) The molecular marker may be a continuous polynucleotide containing at least two of SNPs (a') to (d'). Such a polynucleotide contains the region between SNPs (a') to (d') along with the sites of SNPs (a') to (d'). Such a polynucleotide can be obtained, for example, by referencing the region between the corresponding sites of SNPs (a') to (d') in a Solanaceae plant with increased total dry matter production. The nucleotide sequence of such a polynucleotide at least partially matches the nucleotide sequence of a Solanaceae plant with increased total dry matter production.

[0138] The molecular marker may be a contiguous polynucleotide containing SNP(e') and SNP(f'). Such a polynucleotide contains the region between SNP(e') and SNP(f') along with the sites of SNP(e') and SNP(f'). Such a polynucleotide can be obtained, for example, by referencing the region between the corresponding sites of SNP(e') and SNP(f') in a Solanaceae plant with increased total dry matter production. The nucleotide sequence of such a polynucleotide at least partially matches the nucleotide sequence of a Solanaceae plant with increased total dry matter production.

[0139] The molecular marker may be a contiguous polynucleotide containing SNP(g') and SNP(h'). Such a polynucleotide contains the region between SNP(g') and SNP(h') along with the sites of SNP(g') and SNP(h'). Such a polynucleotide can be obtained, for example, by referencing the region between corresponding SNP(g') and SNP(h') sites in a Solanaceae plant with increased total dry matter production. The nucleotide sequence of such a polynucleotide at least partially matches the nucleotide sequence of a Solanaceae plant with increased total dry matter production.

[0140] The method for determining the total dry matter production in a Solanaceae plant using the above-mentioned molecular markers is not particularly limited, and for example, a known SNP analysis method for detecting SNPs can be used, including a method for SNP analysis by detecting SNPs in PCR-amplified fragments of a test Solanaceae plant.

[0141] The discrimination method may involve amplifying a region in the DNA of a Solanaceae plant using a primer set that amplifies the region containing a molecular marker. One example of such a primer set is a primer set that amplifies a region containing at least one of SNPs (a') to (h').

[0142] The region in the DNA of a solanaceous plant specimen can be amplified by polymerase chain reaction (PCR) using DNA extracted from the solanaceous plant specimen as a template and primers that amplify the region containing the SNP. The base (genotype) of the SNP in the resulting amplified fragment is then determined, and the total dry matter production of the solanaceous plant is estimated based on data showing the relationship between the determined base (genotype) and the total dry matter production of the solanaceous plant.

[0143] The primer set used in PCR is not particularly limited as long as it can amplify a DNA fragment 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 so 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 may be 50 bp or less, 40 bp or less, or 30 bp or less.

[0144] An example of a primer set is shown below. A primer set that amplifies a region containing SNP(c') and SNP(d') is, for example, a primer set consisting of a first primer containing 15 or more consecutive bases in the nucleotide sequence shown in SEQ ID NO: 27 and a second primer containing 15 or more consecutive bases in the nucleotide sequence shown in SEQ ID NO: 28. By using this primer set, a PCR amplification product consisting of the nucleotide sequence shown in SEQ ID NO: 29 can be obtained in Solanaceae plants with increased total dry matter production, allowing the detection of bases corresponding to SNP(c') and SNP(d').

[0145] A primer set that amplifies a region containing SNP(a'), SNP(b'), and SNP(c') is, for example, a primer set consisting of 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, a PCR amplification product consisting of the nucleotide sequence shown in SEQ ID NO: 32 can be obtained in Solanaceae plants with increased total dry matter production, and the bases corresponding to SNP(a'), SNP(b'), and SNP(c') can be detected.

[0146] A primer set that amplifies a region containing SNP(e') and SNP(f') is, for example, a primer set consisting of a fifth primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 33 and a sixth primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 34. By using this primer set, a PCR amplification product consisting of the base sequence shown in SEQ ID NO: 35 can be obtained in Solanaceae plants with increased total dry matter production, and the bases corresponding to SNP(e') and SNP(f') can be detected.

[0147] A primer set that amplifies a region containing SNP(g') and SNP(h') is, for example, a primer set consisting of a seventh primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 36 and an eighth primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 37. By using this primer set, a PCR amplification product consisting of the base sequence shown in SEQ ID NO: 38 can be obtained in Solanaceae plants with increased total dry matter production, allowing the detection of bases corresponding to SNP(g') and SNP(h').

[0148] PCR in SNP analysis may be either singleplex PCR, which amplifies DNA fragments in a reaction system containing a single primer set, or multiplex PCR, which amplifies genes in a reaction system containing multiple primer sets. In the case of multiplex PCR, primer sets labeled with fluorescent substances with different wavelengths (e.g., NED, 6-FAM, VIC, PET) may be mixed.

[0149] PCR reaction conditions can be appropriately set depending on the type of DNA polymerase and PCR instrument used, the length of the amplified fragment, and other factors. Cycling conditions include a three-step PCR method, in which one cycle consists of three steps: denaturation, annealing, and extension; and a two-step PCR method, in which one cycle consists of two steps: denaturation, annealing, and extension. An example of PCR reaction conditions is 90-100°C for 40-60 seconds (e.g., 95°C for 50 seconds), followed by 30-60 cycles (e.g., 40 cycles) of 90-100°C (e.g., 95°C) for 5 seconds, annealing for 10-20 seconds (e.g., 15 seconds), and 65-80°C for 10-30 seconds (e.g., 72°C for 20 seconds). The annealing temperature can be gradually decreased from an initial annealing temperature of 60-70°C (e.g., 66°C) to a final annealing temperature of 50-60°C (e.g., 56°C) every predetermined number of cycles. Depending on the state of the template DNA, PCR reaction conditions may be adjusted to stably detect SNPs.

[0150] As the PCR in SNP analysis, real-time PCR such as TaqMan®-PCR or Tm-shift genotyping (Fukuoka et al., Breed Sci 58: 461-464, 2008), which amplifies and identifies SNP markers by PCR, may be used. That is, a TaqMan® probe may be further used to detect SNPs contained in the amplified fragments amplified using a primer set. The use of real-time PCR can provide a high-throughput discrimination method. SNPs in the amplified fragments amplified by PCR may be identified by analyzing the nucleotide sequence of the amplified fragments using an automated DNA sequencer or the like.

[0151] The method for extracting DNA to be amplified by PCR from a solanaceous plant specimen is not particularly limited, and known DNA extraction methods can be used. Alternatively, DNA may be extracted using a commercially available DNA extraction kit. Depending on the type of specimen and the amount of contaminants, appropriate pretreatment may be performed before the DNA extraction step. Furthermore, the DNA extracted from the specimen may be washed or purified as necessary for use as a template in the PCR reaction. Furthermore, the DNA extracted from the specimen may be digested with two restriction enzymes, and the resulting restriction enzyme fragments may be amplified by PCR.

[0152] In addition, in the method for determining the total dry matter production in a Solanaceae plant, genetic polymorphisms in linkage disequilibrium with SNP(a') to SNP(h') may be analyzed to identify SNP(a') to SNP(h'). The linkage disequilibrium state is, for example, a linkage disequilibrium state with a linkage disequilibrium coefficient of 0.9 or more.

[0153] According to the discrimination method, it is possible to determine whether a test Solanaceae plant has increased total dry matter production using molecular markers, and therefore, based on the determination results, it is possible to select Solanaceae plants with increased total dry matter production and their progeny lines.

[0154] [5. Solanaceae plants with increased total dry matter production] A solanaceous plant with increased total dry matter production according to one embodiment of the present invention is a plant obtained by the production method described below. The solanaceous plant with increased total dry matter production has the SNP identified by the molecular marker described above.

[0155] According to one embodiment of the present invention, a solanaceous plant with increased total dry matter production can be obtained by identifying a solanaceous plant with increased total dry matter production from plants obtained by intraspecific hybridization of solanaceous plants and their progeny lines using the molecular markers described above, as described in the production method described below. Note that solanaceous plants with increased total dry matter production that have been genetically engineered to contain the SNPs described above are also included within the scope of the present invention. Furthermore, as described in [6. Method for Producing a Solanaceous Plant with Increased Total Dry Matter Production] below, a solanaceous plant with increased total dry matter production obtained by genome editing to delete or inactivate the amino acid sequence downstream of the VHIID motif of a GRAS transcription factor is also included within the scope of the present invention. Furthermore, a dicotyledonous plant, including a solanaceous plant, with increased total dry matter production obtained by genome editing to delete or inactivate the amino acid sequence downstream of the VHIID motif of a GRAS transcription factor is also included within the scope of the present invention. An example of a dicotyledonous plant is Arabidopsis thaliana.

[0156] 6. Method for producing a solanaceous plant with increased total dry matter production One embodiment of the production method of the present invention is a method for producing a solanaceous plant with increased total dry matter production, and includes a hybridization step of intraspecifically hybridizing solanaceous plants, and an identification step of identifying a solanaceous plant with increased total dry matter production from the solanaceous plant obtained by the hybridization step or a solanaceous plant of a progeny lineage using the above-mentioned discrimination method.

[0157] Therefore, the above-mentioned descriptions of molecular markers, solanaceous plants with increased total dry matter production, and methods for determining the degree of total dry matter production in solanaceous plants are incorporated herein by reference to describe methods for producing solanaceous plants with increased total dry matter production.

[0158] At least one of the solanaceous plants used in the hybridization step may be a solanaceous plant with increased total dry matter production according to one embodiment of the present invention. Furthermore, at least one of the solanaceous plants used in the hybridization step may be a solanaceous plant with increased total dry matter production selected by the method for determining the level of total dry matter production in solanaceous plants according to one embodiment of the present invention. That is, the production method according to one embodiment of the present invention may further include, prior to the hybridization step, an identification step of identifying a solanaceous plant with increased total dry matter production from test solanaceous plants by the method for determining the level of total dry matter production in solanaceous plants according to one embodiment of the present invention.

[0159] Furthermore, a production method according to one embodiment of the present invention includes an identification step of identifying a Solanaceae plant with increased total dry matter production from test Solanaceae plants, and a hybridization step of intraspecifically hybridizing the identified Solanaceae plants. That is, the scope of the present invention also includes production methods that include an identification step only before the hybridization step, and production methods that include an identification step before or after the hybridization step.

[0160] A production method that includes an identification step only before the crossing step can be used, for example, as follows: By identifying a Solanaceae 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, all of the resulting F1 plants will have a heterozygous total dry matter production control gene. A production method that includes an identification step only before the crossing step can also be used for homozygous parental selection (selection and fixation) in F1 breeding.

[0161] The identification step uses a method for determining the degree of total dry matter production in a Solanaceae plant according to one embodiment of the present invention to identify a Solanaceae plant with increased total dry matter production from the Solanaceae plants obtained by the crossbreeding step or from Solanaceae plants of its progeny lineage.

[0162] According to a production method of one embodiment of the present invention, the level of total dry matter production in a Solanaceae plant can be determined using molecular markers, and a Solanaceae plant with increased total dry matter production can be produced by selecting based on the determination results.

[0163] A production method according to another embodiment of the present invention is a method for producing a solanaceae plant with increased total dry matter production, and includes the step of introducing a mutation into a polynucleotide encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 in a solanaceae plant, such that an amino acid sequence downstream of an amino acid at any position downstream of the amino acid corresponding to amino acid 73 in the amino acid sequence set forth in SEQ ID NO: 1 is deleted or inactivated. Such methods for producing solanaceae plants with increased total dry matter production by genome editing are also included in the scope of the present invention.

[0164] The total dry matter production control gene, which is composed of a polynucleotide encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, is predicted to encode a GRAS transcription factor. Another embodiment of the production method of the present invention involves artificially introducing a mutation by genome editing into any position downstream of the VHIID motif of the GRAS transcription factor, thereby deleting the amino acid sequence downstream of the VHIID motif, thereby producing a Solanaceae plant with increased total dry matter production.

[0165] Here, the VHIID motif of the GRAS transcription factor is a sequence of amino acids corresponding to the 69th to 73rd amino acids in a tomato-derived protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 (see Reference 2: Li et al. Plant Cell 28: 1025-1034, 2016). Therefore, a production method according to another embodiment of the present invention includes 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. Examples of mutations that delete or inactivate an amino acid sequence include a stop codon mutation, a frameshift mutation, and a null mutation.

[0166] The VHIID motifs of GRAS transcription factors in other solanaceous plants and dicotyledonous plants are as follows: In the bell pepper-derived protein consisting of the amino acid sequence set forth in SEQ ID NO: 7, the amino acid sequence corresponding to the amino acid sequence at positions 311 to 315 is the VHIID motif; In the eggplant-derived protein consisting of the amino acid sequence set forth in SEQ ID NO: 8, the amino acid sequence corresponding to the amino acid sequence at positions 293 to 297 is the VHIID motif; In the potato-derived protein consisting of the amino acid sequence set forth in SEQ ID NO: 9, the amino acid sequence corresponding to the amino acid sequence at positions 297 to 301 is the VHIID motif; and In the Arabidopsis-derived protein consisting of the amino acid sequence set forth in SEQ ID NO: 10, the amino acid sequence corresponding to the amino acid sequence at positions 228 to 232 is the VHIID motif.

[0167] In the step of introducing a mutation, a mutation may be introduced into a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 to delete or inactivate the amino acid corresponding to the 109th amino acid in the protein consisting of the amino acid sequence shown in SEQ ID NO: 1 and the amino acid sequence downstream thereof. This introduces a stop codon mutation into the total dry matter production control gene, deleting the amino acid sequence corresponding to the amino acid sequence from position 109 onwards in the amino acid sequence shown in SEQ ID NO: 1, thereby enabling the production of a Solanaceae plant with increased total dry matter production.

[0168] The method for producing a Solanaceae plant with increased total dry matter production can also be applied to a method for producing a dicotyledonous plant, including a Solanaceae plant, by genome editing to delete or inactivate the amino acid sequence downstream of the VHIID motif of the GRAS transcription factor. An example of a dicotyledonous plant is Arabidopsis thaliana.

[0169] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention. [Example]

[0170] Materials and Methods (Construction of recombinant inbred lines) The high-yielding, beefsteak-type Dutch F1 cultivar "Geronimo" and the large-fruited, high-sugar-content Japanese F1 cultivar "Momotaro8" were used as hybrid parents. All plants were grown in pots filled with soil in a greenhouse at the National Agriculture and Food Research Organization's Vegetable and Floriculture Research Division in Tsu City, Mie Prefecture. This cross corresponds to a four-way cross, with the four parental lines of the two F1 cultivars (G1 generation) as the ancestors (G0 generation). Figure 1 outlines the construction of recombinant inbred lines derived from the cross of the two F1 cultivars and their QTL mapping. As shown in Figure 1, "Geronimo" (P1;G1) and "Momotaro8" (P2;G1) were crossed to generate a four-way F1 population (G1F1) consisting of 240 plants. Four-way recombinant inbred lines were then developed by repeated self-pollination from each G1F1 plant using the single-seed method. We obtained 206 recombinant inbred lines of the G1F6 generation and maintained them by selfing to evaluate their agronomic traits. These lines were designated GM lines.

[0171] (Growth of GM lines and evaluation of phenotypes at two locations) In Mie Prefecture, seeds of GM lines were sown in seedling trays containing soil between July 30 and August 19, 2010 and 2013 (autumn cultivation of F7, F9, and F10 generations, Experiments 3 to 6). Also, seeds of GM lines were sown on February 1 or 2 in 2011 and 2012 for spring cultivation of F8 and F10 generations (Experiments 1 and 2). After placing the seedling trays in a greenhouse for 3 weeks, all plants (one plant per line per experiment) were directly planted onto rockwool slabs (planting density 2.303 / m). 2 All plants were grown in a greenhouse at temperatures above 16°C, using a nutrient solution (Otsuka Chemical) with an electrical conductivity (EC) of 2.8 mS / cm. The maximum number of flowers per truss was limited to six, and the tops were pinched above the third truss (Experiment 3) or the fourth truss (Experiments 1, 2, and 4–6).

[0172] Seeds of the F10 generation GM lines were sown in Aichi Prefecture on August 27, 2012 (Experiment 7) and August 28, 2013 (Experiment 8) in the same manner as above. Seedlings were grown in a growth chamber at 25°C (day) / 20°C (night), CO2 concentration of 900 or 1000 ppm, and a 16-hour photoperiod, and were supplied with nutrient solution (EC = 1.8 mS / cm, Mitsubishi Chemical Agri Dream Co., Ltd.). After 3 weeks, all plants (two plants per line per experiment) were directly planted in rockwool slabs (planting density 3.086 m / m). 2 All plants were grown in a greenhouse at a temperature of 13°C or higher, and were supplied with a nutrient solution (Otsuka Chemical Co., Ltd.) with an EC of 0.8-1.8 mS / cm (Experiment 7) or an EC of 1.5-2.0 mS / cm (Experiment 8). The plants were pinched above the fourth fruit cluster.

[0173] Phenotypes of 2 to 10 traits were collected from one plant per line in Mie Prefecture or two plants per line in Aichi Prefecture. Phenotypes of traits related to growth characteristics, yield, and fruit quality were measured for each GM line. The sugar content of red, ripe, marketable fruit was measured using a saccharimeter (PAL-1). Fruits free of any physiological disorders such as bottom rot or cracking were defined as good fruits. Fruits with at least one physiological disorder were defined as poor fruits.

[0174] (Phenotypic correlation analysis in GM lines) Correlation coefficients between traits were calculated using the correlation tool and TDIST function provided by Microsoft Excel 2016 to calculate p-values.

[0175] (trait inheritance) Heritability was calculated using a linear model of the phenotype of the GM lines in each experiment.

[0176] (Genomic DNA isolation) Genomic DNA was isolated from P1 and P2 leaves using the DNeasy Plant Mini Kit (Qiagen). Genomic DNA was isolated from leaves of G1F1 and F9 GM line plants 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.

[0177] (Tomato SSR marker screening) We used non-redundant BAC end-derived genomic SSR markers (referred to as "tma," "tmb," "tmc," and "TGS"; a total of 4047), EST-derived SSR markers (referred to as "tme" and "TES"; a total of 2195), EST-based genomic SSR markers (referred to as "tbm"; a total of 2510), and cDNA-derived SSR markers (referred to as "tms" in this study; a total of 135) published on the website (https: / / solgenomics.net).

[0178] Using parental (G1 generation) genomic DNA as a template, polymerase chain reaction (PCR) was performed, followed by fluorescent labeling, and markers were screened based on the polymorphism of the PCR amplified products. Using the genotypes of the two groups (GM lines of the G1F1 and F9 generations), SSR markers were classified into eight categories of allele combination patterns of the parents (G1 generation, P1, and P2) (Table 1).

[0179] Information on these markers (primer pairs) is available on the NIVTS website (http: / / vegmarks.nivot.affrc.go.jp / ).

[0180] (Genotyping of SSR alleles) The forward primers for the SSR alleles were 5'-labeled using 6-FAM, NED, PET, or VIC (Applied Biosystems). Genomic DNA from P1P2 and GM plants from the G1F1 and F9 generations were used as templates. PCR was performed in 10 μL reactions using the Type-it Microsatellite PCR Kit (Qiagen). PCR started at 95°C for 5 minutes, followed by 28 cycles of 95°C for 30 seconds, 60°C for 90 seconds, and 72°C for 30 seconds, and then a final cycle of 60°C for 30 minutes. PCR amplification products were analyzed using an automated sequencer (3730 x1 DNA Analyzer, Applied Biosystems) with a GeneScan-500LIZ Size Standard (Applied Biosystems). Fragment lengths were determined using GeneMapper v. 3.7 software (Applied Biosystems).

[0181] (SNP allele genotyping) A total of 1,536 SNPs were used for genotyping of the P1P2 and GM lines. Genotyping was performed using the GoldenGate assay system (Illumina Inc.) according to the manufacturer's protocol. Marker categories were classified in the same way as SSR markers in Table 1.

[0182] (Construction of linkage maps) A linkage map of the GM lines was constructed using Carthagene software (de Givry et al. Bioinformatics 21: 1703-1704, 2005). The linkage map was constructed by estimating the recombination frequency (map distance) between each marker over the course of generations from each G1F1 plant to the progeny plants of each GM line. The SSR and SNP markers listed in Table 1 were used. The heterozygous marker genotypes in P1P2 (categories 1–7 in Table 1) segregated immediately in the G1F1 generation, allowing the recombination frequency between these marker pairs to be estimated. The recombination frequency estimates were based on the inheritance of alleles from the G1F1 plants to their progeny GM lines; information on segregation in G1F1 was used only to confirm the recombination frequency estimates. Segregation distortion of marker genotypes in the linkage map was identified using a chi-square test. Linkage maps were drawn using MapChart v. 2.1 software (Voorrips, J Hered 93: 77-78, 2002).

[0183] (Bayesian QTL mapping using MCMC methods) Bayesian QTL mapping of a four-way cross was performed using the genotypes of the GM lines and the marker genotypes of P1, P2, G1F1, and the GM lines (Hayashi et al. Euphytica 183: 277-287, 2012). This mapping method assumed four alleles at each QTL derived from four unknown ancestors (the parents of P1 and the parents of P2). Marker genotypes of G1F1 plants were used to estimate the haplotypes of P1 and P2. The haplotypes of the GM lines were inferred from the haplotypes of the G1F1 parents. In addition to Bayesian methods based on MCMC methods, a Bayesian analysis using variational approximation (variational approximation method) was also performed for QTL mapping in a four-way cross.

[0184] (tfw6.1 isolates and seed collection) Among the F9 generation GM lines, line 019 showed a heterozygous genotype for the tfw6.1 region (defined as the region between two SNP markers, SL2.40ch06_983984R and SL2.40ch06_23559443Y; see Figure 2). Self-pollinated progeny seeds of this line were collected in soil-filled seedling trays on September 8, 2014, and then cultivated as in Experiments 7 and 8. After 3 weeks, the plants were potted in rockwool cubes and transplanted into a greenhouse (plant factory) at the National Agriculture and Food Research Organization (NARO) in Tsukuba, Ibaraki Prefecture. The plants were cultivated in a nutrient solution (Otsuka Chemical Co., Ltd.) with an EC of 1.0 mS / cm from September 30 to November 4 of the same year. During this time, genomic DNA was isolated as described above in the "Genomic DNA Isolation" section. Using this DNA as a template, multiplex PCR was performed using a set of SSR markers located around the tfw6.1 region using the fluorescent dye-based BS-tag method (Shimizu and Yano, BMC Research Notes 4:161, 2011; Konishi et al., Vegetable and Tea Science Research Institute Research Bulletin 14:15-22, 2015). Information on the SSR marker sets used (Set 1: tbm0260, tbm0263, tbm0272, tbm0265; Set 2: tbm1354, tbm2241, tbm1344, tbm0281) is available from the public database (VegMarks, https: / / vegmarks.nivot.affrc.go.jp / VegMarks / app / page / home). The fluorescent dyes used were 6-FAM, NED, PET, or VIC. Based on the above-mentioned "SSR allele genotype analysis," we selected individuals with homozygous high-yielding Dutch-type tomato alleles, homozygous Japanese-type tomato alleles, and heterozygous Japanese-type tomato alleles. The individual with the high-yielding allele was named 019G, the individual with the Japanese-type allele 019M, and the individual with the heterozygous Japanese allele 019H. On November 4, the rockwool cubes containing these plants were planted on rockwool slabs and further cultivated. Self-pollinated seeds of 019G, 019M, and 019H were collected.

[0185] (Test 1: Analysis of yield components in fixed segregating lines: pinching cultivation) In 019G and 019M, the tfw6.1 allele is thought to have been fixed for high-yielding or Japanese varieties. Seeds of these lines were sown on September 4, 2017, cultivated in a growth chamber, potted in rockwool cubes on September 28, and planted at a density of 3.3 plants / m. 2 ) on rockwool slabs and cultivated in a greenhouse for autumn cultivation. The nutrient solution at planting time was adjusted to EC = 2.6 mS / cm and maintained at this concentration until the end of cultivation. All plants were pinched at the fourth fruit cluster and cultivated until 124 days after planting. Mature fruits were harvested periodically during the cultivation period, separated into good and bad fruits, and the number and fresh weight were measured. After cultivation was completed, the plants were cut at the base and separated into immature fruit, good fruit, leaves, and stems. The number of fruits, fresh weight, and dry weight were measured for the fruits, and the fresh weight and dry weight were measured for the leaves and stems. Dry weight here can be interpreted as dry matter weight. The dry weight of fruits harvested during the harvest period was calculated by converting the fresh weight using the dry matter ratio (dry weight / fresh weight) determined after cultivation was completed.

[0186] (Test 2: Analysis of yield components in fixed segregating lines: Long-row cultivation) The fixed lines 019G and 019M were sown on February 4, 2019, cultivated in a growth chamber, and then potted in rockwool cubes on February 28 at a planting density of 2.7 plants / m. 2 ) on rock wool slabs and cultivated in a greenhouse for spring cultivation. The nutrient solution at the time of planting was set to EC = 2.6 mS / cm, and this concentration was applied until the end of cultivation. No top pinching was performed on this plant, and it was cultivated until the 124th day after planting. Mature fruits were harvested periodically during the cultivation period, and separated into good and bad fruits, and the number and fresh weight were measured. Data collection after the end of cultivation was performed in the same manner as in Experiment 1.

[0187] (Creation of a line with a shortened tfw6.1 region from a segregating line) Self-pollinated seeds of 019H, one of the tfw6.1 segregants, were sown on August 9, 2016, as in Experiment 1, and then cultivated in a growth chamber. Two weeks after sowing, DNA was extracted from seedling leaves (DNA Suisui-P, Reezo Co., Ltd.). Using this DNA as a template, multiplex PCR was performed as described above in "Obtaining tfw6.1 segregants and seed collection." Genotyping was then performed according to the "SSR allele genotyping analysis" section, and lines with shortened tfw6.1 regions due to recombination were selected. On September 8, autumn cultivation was carried out using the same planting density and nutrient solution concentration as in Experiment 1, and self-pollinated seeds were harvested.

[0188] (Test 3: Isolation of lines with truncated tfw6.1 region and analysis of yield components to narrow down the region) The marker genotypes in the tfw6.1 region of the truncated lines selected above are partially heterozygous. Therefore, to select lines that are fixed to high-yielding or Japanese types, self-pollinated seeds of the three truncated lines were sown on September 4, 2017, and genotype analysis was performed again. Cultivation in the growth chamber and greenhouse was performed in the same manner as in Experiment 1. Genotype analysis was performed using an SSR marker set during growth, and after identifying the fixed lines, the yield (total dry weight) of these lines was compared. (RNA-SEQ mutation analysis) Next-generation sequencing was used to analyze mRNA mutations in the tfw6.1 isolates 019G (high-yielding) and 019M (Japanese-type). Total RNA was extracted from unripe fruit of each isolate using the Trizol method (https: / / ipmb.sinica.edu.tw / microarray / protocol.htm), and fragmented cDNA libraries were prepared using TruSeq RNA Sample Prep Kit v2 (Illumina). The libraries were then analyzed using a next-generation sequencer (HiSeq 4000, Illumina) to obtain paired-end sequence reads (approximately 4 Gb per sample). The resulting reads 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 nonsynonymous substitutions were detected using a mutation detection program (e.g., Basic Variant Detection).

[0189] (Genome editing complementation experiments) A vector was constructed to induce NHEJ repair errors at the 271st amino acid downstream of the VHIID motif (amino acids 228–232) of SEQ ID NO: 10 in Arabidopsis (Tsutsui and Higashiyama, Plant Cell Physiol 58(1): 46–56, 2017). Arabidopsis (Col-0 strain) was transformed using the floral dip method (Clough and Bent, Plant J 16(6): 735–743, 1998). Genome-edited individuals were selected using RFP fluorescence and nucleotide sequencing. Null segregants, in which the mutant allele was homozygous and the vector was eliminated, were selected from their progeny and harvested. Seeds from these genome-edited lines and wild-type (Col-0) control lines were aseptically sown on 1 / 2 MS medium and grown for 4 weeks at 22°C with a 10-hour photoperiod. Phenotypes were then compared.

[0190] 〔result〕 (Phenotypic correlation analysis in GM lines)

[0191] In phenotypic correlation analysis, total fruit fresh weight was negatively correlated with fruit soluble solids (SOx). The trade-off between these traits has also been reported in other studies (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).

[0192] In the lines cultivated in Mie Prefecture, significantly high correlations were observed between total fresh fruit weight and fresh weight of good fruits, total fresh fruit weight and number of good fruits, fresh weight of good fruits and number of good fruits, and average total fresh fruit weight and average fresh weight of good fruits. This suggests that cropping type has a significant influence on the phenotype.

[0193] (Screening and classification of effective SSR and SNP markers in G1F1 lines and recombinant inbred lines) The effective SSR markers were classified into eight categories based on the allele combination patterns of each line (Table 1). The marker in category 7 was able to detect four different alleles, demonstrating its usefulness. However, its frequency in the tomato genome was very low. Therefore, in QTL analysis, markers from different categories were combined and used to compensate for the lack of genetic information in each chromosomal region. For example, marker A (aa-bb) in category 0, marker B (ab-aa) in category 2, and marker C (aa-ab) in category 3 could provide information similar to that obtained by markers in category 7 (ab-cd). A total of 197 SSR markers were selected.

[0194] SNP markers covering the low density region of SSR markers were screened and genotyped. A total of 338 SSR and SNP markers (Table 1) were used for linkage map construction and QTL analysis. [Table 1] (Evaluation of linkage maps) The linkage map for the GM lines consisted of 12 linkage groups and covered 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. Genome coverage was 98.2% of the 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 existed (more than 20 cM), the genome coverage and average distance between markers (less than 10 cM; Lander and Botstein, Genetics 121: 185-199, 1989) suggested that the linkage map was sufficient for exploring the entire genome of the recombinant inbred lines.

[0195] (Comparison of mapping accuracy between MCMC method and variational approximation method) Before performing QTL mapping using all trait data, two Bayesian mapping methods for QTL detection, 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 of each QTL location being included in the model. For the MCMC method, the posterior probability of each QTL location was calculated as the proportion of MCMC samples in which a model including that QTL was adopted relative to the total MCMC samples. For the variational approximation method, the posterior probability was calculated as the approximate posterior expected value of the indicator variable γ1 for the inclusion of hypothetical QTLs evenly spaced throughout the genome. Since the results obtained from the two Bayesian methods were generally equivalent, to reduce computational time, only the less computationally intensive variational approximation method was applied to the remaining trait analyses.

[0196] The results of the analysis of QTLs related to total fruit fresh weight detected by the approximation method are shown in Table 2. In Table 2, R 2is the estimated proportion of phenotypic variance explained by the QTL. Figure 2 illustrates the linkage map of linkage group 6 and the location of QTL tfw6.1. [Table 2]

[0197] (Test 1 results) Table 3 shows the results of component analysis for the autumn-cropped pinching cultivation in Experiment 1. Compared with the fixed line 019M, which has an allele derived from a high-yielding Dutch tomato, the fixed line 019G, which has a tfw6.1 allele derived from a high-yielding Dutch tomato, had significantly higher total fruit weight per plant (total fresh weight of immature, poor, and good fruit), fruit dry matter weight (total fruit weight converted to dry weight), total dry matter weight, and fruit partitioning ratio (fruit dry matter weight / total dry matter weight). Here, total dry matter weight refers to the sum of the dry weights of leaves, stems, and fruit. These results suggest that increased aboveground biomass weight led to increased fruit dry matter weight and, in turn, total fruit weight.

[0198] 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).

[0199] (Test 2 results) The results of the component analysis for Test 2 are shown in Table 3. Similar results to Test 1 were obtained for the long-row cultivation in spring. [Table 3]

[0200] (Test 3 results) Comparison of the high-yielding marker allele region in the truncated tfw6.1 region with the Japonica marker allele region further narrowed down the tfw6.1 region for total dry matter weight (Table 4). Increased total dry matter weight was observed in the line (019_364G) in which the region between markers tbm1344 and tbm0281 contained a high-yielding allele. In Table 4, A indicates the high-yielding tomato genotype, and B indicates the Japonica tomato genotype. [Table 4]

[0201] (RNA-SEQ mutation analysis) Within the QTL region defined in Table 4, predicted genes where nonsynonymous substitutions have occurred between the high-yielding type and the Japonica type were found between markers tbm1344 and tbm0265 and on the outside of the long arm of marker tbm0281. The mutations found in the three predicted genes are shown in SEQ ID NOs: 4 to 6.

[0202] (Genome editing complementation experiments) Two Arabidopsis lines (818#2 and 818#9) that were genome-edited for the candidate amino acid sequence of SEQ ID NO: 10 were used in the experiment. The nucleotide and amino acid sequences of these genome-edited lines are shown in Figure 3. In 818#2, a single-base insertion caused a frameshift downstream of the 271st amino acid, resulting in a deletion of the sequence from 282 onwards. In addition, in 818#9, a 25-base deletion mutation caused a frameshift downstream of the 263rd amino acid sequence, resulting in a deletion of the sequence from 268 onwards.

[0203] The genome-edited lines with fixed mutant alleles were sown on culture medium and compared with wild-type plants for 4 weeks. The results confirmed that the genome-edited lines exhibited larger plant bodies than the wild-type (Figure 4). This result is consistent with the phenotype observed in high-yielding Dutch tomatoes. [Industrial Applicability]

[0204] The present invention can be used in the fields of agriculture, plant breeding, etc.

Claims

1. 1. A method for determining the degree of total dry matter production in a tomato plant, comprising: The method includes a step of examining the presence or absence of a mutation that causes an amino acid deficiency in the plant, which is described in (a) below: In the inspecting step, (a) determining that a tomato plant has an increased total dry matter production when the tomato plant is deficient in an amino acid sequence corresponding to the 109th amino acid of the protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 and the amino acid sequence downstream thereof; The mutation is associated with a QTL on chromosome 6 for increased total dry matter production.

2. In the step of inspecting, at least one of the following (a') to (d') is detected in the tomato plant: (a') the base corresponding to the 326th base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 4 is G; (b') the base corresponding to the 390th base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 4 is A; (c') the base corresponding to the 591st base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 4 is T; (d') the base corresponding to the 740th base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 4 is G; The method according to claim 1, wherein the plant is determined to be a plant with increased total dry matter production when

3. The tomato plant contains a polynucleotide selected from 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 encoding a protein having an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1 and having the function of regulating the total dry matter production of the tomato plant; (3) a polynucleotide encoding a protein having an amino acid sequence in which 30 or fewer amino acids have been substituted, deleted, added, or inserted relative to the amino acid sequence set forth in SEQ ID NO: 1, and having the function of regulating the total dry matter production of the tomato plant; The method according to claim 1 or 2, wherein the plant is a tomato having a total dry matter production control gene comprising:

4. The method according to any one of claims 1 to 3, wherein the tomato plant is a candidate plant for breeding material or a plant obtained in a breeding process.

5. 1. A method for producing a plant that is a tomato with increased total dry matter production, comprising: A hybridization step of intraspecifically hybridizing the tomato plant; an identifying step of identifying the tomato plant with increased total dry matter production by the method according to any one of claims 1 to 4 from the tomato plant obtained by the crossbreeding step or a tomato plant of a progeny line thereof; A manufacturing method comprising:

6. 1. A method for producing a plant that is a tomato with increased total dry matter production, comprising: an identifying step of identifying a test tomato plant having increased total dry matter production by the method of any one of claims 1 to 4; a hybridization step of intraspecifically hybridizing the identified tomato plants; A manufacturing method comprising:

7. 1. Use of molecular markers for increased total dry matter production in a tomato plant, comprising: The molecular marker is at least one of the following bases (a') to (d') themselves (SNP) or a consecutive 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; It consists of The molecular marker detects a QTL on chromosome 6 for increased total dry matter production.

8. a step of introducing a mutation into a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in a tomato plant, in which 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; A method for producing a plant that is a tomato with increased total dry matter production.

9. A gene in a tomato plant, in which a mutation has been introduced that causes a deletion of the amino acid corresponding to the 109th amino acid and the amino acid sequence downstream thereof in a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, and which consists of a polynucleotide that encodes a protein having the function of increasing the total dry matter production of said tomato plant.

10. A polynucleotide according to any one of the following (2) to (3): (2) A polynucleotide encoding a protein having a function of regulating the total dry matter production of a tomato plant, the polynucleotide consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1, and lacking the amino acid corresponding to the 109th amino acid of the amino acid sequence shown in SEQ ID NO: 1 and the amino acid sequence downstream thereof; (3) A polynucleotide encoding a protein having the function of regulating the total dry matter production of a tomato plant, the polynucleotide consisting of an amino acid sequence in which 30 or fewer amino acids have been substituted, deleted, added, or inserted in the amino acid sequence shown in SEQ ID NO: 1, and in which 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; A gene consisting of A gene, the expression of which increases total dry matter production compared to a tomato plant in which the gene is not expressed.

11. An expression vector comprising the gene according to claim 9 or 10.

12. A cell or a dicotyledonous plant comprising the gene according to claim 9 or 10, or the expression vector according to claim 11.

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