JP7493802B2 - Genes involved in regulating the amount of assimilates distributed to edible parts in Solanaceae plants and their utilization - Google Patents

Description

The present invention relates to genes involved in regulating the amount of assimilates distributed to edible parts of Solanaceae plants and their use.

The yield of agricultural products such as seeds, tubers, and fruits is an important indicator in agricultural management because increases or decreases in 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 (assimilate allocation). Therefore, the yield of agricultural products can be increased by increasing the amount of dry matter produced or the amount of assimilate allocation.

Technologies for increasing the yield of agricultural products include environmental control techniques such as supplemental lighting and improving light transmittance through the selection of materials, but these are costly and it is difficult to achieve a stable increase in yield. A technique for genetically modifying plants is known to achieve a stable increase in yield of agricultural products.

Candidates for genes to be used in the genetic modification of Solanaceae fruit vegetables such as tomato 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.

Frary et al. Science 289:85-88, 2000 Cong et al. Nat. Genet. 40:800-804, 2008

The fw2.2 gene described in Non-Patent Document 1 is a gene that is universally present in large-fruited tomato varieties, and is presumed to have been acquired during the process of domestication of small-fruited wild tomatoes to produce large-fruited tomatoes. In other words, the fw2.2 gene is not involved in regulating the amount of assimilates distributed to the edible parts, but is thought to increase cell division and the number of locules. Therefore, the fw2.2 gene only brings about morphological changes in the plant, and is unlikely to be used to modify yield, such as the amount of assimilates distributed to the edible parts.

In rice and other plants, genes involved in regulating the amount of assimilates distributed to seeds have been reported, but in tomato, no genes involved in regulating the amount of assimilates distributed to the edible parts are known.

The present invention has been made in consideration of the above problems, and its purpose is to identify a gene involved in regulating the amount of assimilates distributed to the edible parts of fruit vegetables of the Solanaceae family, including tomato, and to provide a use of the gene.

A method according to one embodiment of the present invention is a method for determining the degree of regulation of the amount of assimilate distribution to edible parts in a solanaceous plant, and includes a step of examining the solanaceous plant for the presence or absence of a mutation that causes a substitution or deletion in the following amino acid (a) or (b): (a) the amino acid corresponding to the 870th amino acid in the protein having the amino acid sequence shown in SEQ ID NO:1; (b) the amino acid corresponding to the 402nd amino acid in the protein having the amino acid sequence shown in SEQ ID NO:2.

The production method according to one embodiment of the present invention is a method for producing a solanaceae plant with an increased amount of assimilate distribution to the edible parts, and includes a hybridization step of intraspecifically hybridizing a solanaceae plant with an increased amount of assimilate distribution to the edible parts with another solanaceae plant, and an identification step of identifying a solanaceae plant with an increased amount of assimilate distribution to the edible parts from the solanaceae plant obtained by the hybridization step or a solanaceae plant of a progeny lineage by any of the above methods.

The molecular marker according to one embodiment of the present invention is a molecular marker for regulating the amount of assimilate distribution to edible parts in Solanaceae plants, and is composed of the base itself (SNP) corresponding to the following base (a') or (b'), or a consecutive polynucleotide containing the base: (a') a base corresponding to the 2609th base from the translation start point of the tfw.5.1 gene; (b') a base corresponding to the 1204th base from the translation start point of the tfw.5.1 gene.

The gene according to one embodiment of the present invention is a gene involved in regulating the amount of assimilates distributed to the edible parts of a solanaceae plant, and is a polynucleotide according to any one of (1) to (3) below: (1) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2; (2) a polynucleotide encoding a protein having a sequence identity of 70% or more with the amino acid sequence shown in SEQ ID NO: 1 or 2, in which the amino acid corresponding to the 870th amino acid in the amino acid sequence shown in SEQ ID NO: 1 is E or the amino acid corresponding to the 402nd amino acid in the amino acid sequence shown in SEQ ID NO: 2 is S, and the polynucleotide encoding the protein has a function of regulating the amount of assimilates distributed to the edible parts of a solanaceae plant; (3) a polynucleotide encoding an amino acid sequence in which 300 or less amino acids have been substituted, deleted, added, or inserted with respect to the amino acid sequence shown in SEQ ID NO: 1, in which the amino acid corresponding to the 870th amino acid in the amino acid sequence shown in SEQ ID NO: 1 is E or the amino acid corresponding to the 402nd amino acid in the amino acid sequence shown in SEQ ID NO: 2 is S, and the polynucleotide encoding the protein has a function of regulating the amount of assimilates distributed to the edible parts of a solanaceae plant.

A protein according to one aspect of the present invention is encoded by the above polynucleotide.

An expression vector according to one aspect of the present invention includes the above polynucleotide.

A cell according to one aspect of the present invention contains the above polynucleotide or the above expression vector.

In one embodiment of the present invention, the solanaceae plant incorporates the above polynucleotide or the above expression vector.

According to one aspect of the present invention, by using a gene that regulates the amount of assimilate distribution to the edible parts of a Solanaceae plant, it is possible to identify a Solanaceae plant with an increased amount of assimilate distribution to the edible parts, and to produce a Solanaceae plant with an increased amount of assimilate distribution to the edible parts.

FIG. 1 outlines the construction of recombinant inbred lines derived from a cross of two F1 varieties and their QTL mapping. FIG. 2 is a diagram illustrating a linkage map of linkage group 5 and the position of QTL tfw5.1.

The embodiment of the present invention is described below. However, the present invention is not limited to this embodiment.

In this specification, "polynucleotide" can be alternatively referred to as "nucleic acid" or "nucleic acid molecule" and refers to a polymer of nucleotides. Furthermore, "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. In this specification, "polypeptide" can be alternatively referred to as "protein."

In this specification, "assimilation products" can also be said as "dry matter" and refers to organic compounds such as sugars synthesized by photosynthesis. In this specification, "edible parts" refers to the parts of plants that are primarily edible, and in fruit vegetables, it refers to the edible fruit parts, and in stem vegetables, it refers to the edible tuber parts. In this specification, "increasing the amount of assimilation products distributed to the edible parts" refers to an increase in the amount of assimilation products distributed to the edible parts, and in particular, it refers to an increase in the amount of assimilation products distributed to the edible parts from the fruit-bearing or stem-enlarging stages to the harvesting stage.

In this specification, "Solanaceae plants" are, by way of example, tomatoes, potatoes, eggplants, and chili peppers.

In this specification, "eggplant" is a concept that broadly includes 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," but in the narrow sense it is intended to mean "S. melongena."

In the present specification, the term "tomato" refers broadly to the cultivated species "S. lycopersicum (S. lycopersicum)".
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."

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," and in the narrow sense refers to "C. anium." In addition, "chili pepper" also refers to the above plants for which terms other than "chili pepper" are used, such as "bell pepper," "paprika," and "shishito" as names for horticultural crops.

In this specification, "potato" is a concept that broadly includes 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 is intended to mean "S. tuberosum."

[1. Genes that regulate the amount of assimilates distributed to edible parts]
The gene for regulating the amount of assimilate distribution to edible parts according to one embodiment of the present invention is a gene encoding a protein having an activity for regulating the amount of assimilate distribution to edible parts (activity for regulating the amount of assimilate distribution to edible parts). When a protein has an activity for positively regulating the amount of assimilate distribution to edible parts, the presence of the protein increases the amount of assimilate distribution to edible parts. Furthermore, when the activity of a protein encoded by a gene for regulating the amount of assimilate distribution to edible parts is reduced or inhibited, the amount of assimilate distribution to edible parts is reduced or does not increase compared to a plant in which the activity is not reduced or inhibited.

An example of a gene for regulating the amount of assimilates distributed to edible parts is a gene involved in regulating the amount of assimilates distributed to edible parts of a Solanaceae plant, and is a gene consisting of a polynucleotide described in any one of (1) to (3) below:
(1) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2;
(2) A polynucleotide encoding a protein having a function of regulating the amount of assimilates distributed to edible parts of a Solanaceae plant, the polynucleotide consisting of an amino acid sequence having 70% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1 or 2, in which the amino acid corresponding to the 870th amino acid in the amino acid sequence shown in SEQ ID NO: 1 is E (glutamic acid) or the amino acid corresponding to the 402nd amino acid in the amino acid sequence shown in SEQ ID NO: 2 is S (serine);
(3) A polynucleotide encoding a protein having an amino acid sequence in which 300 or less amino acids have been substituted, deleted, added or inserted relative to the amino acid sequence shown in SEQ ID NO: 1 or 2, and in which the amino acid corresponding to the 870th amino acid in the amino acid sequence shown in SEQ ID NO: 1 is E or the amino acid corresponding to the 402nd amino acid in the amino acid sequence shown in SEQ ID NO: 2 is S, and which has the function of regulating the amount of assimilates distributed to the edible parts of a Solanaceae plant.

The polynucleotide (1) above is a polynucleotide that contains the base sequence of the tfw.5.1 gene derived from tomato or the base sequence of the coding region (CDS) of the tfw.5.1 gene, and encodes a protein that regulates the amount of assimilate distributed to the edible part. The polynucleotide (1) above may also be a polynucleotide that contains the base sequence of a gene corresponding to the tfw.5.1 gene of a solanaceae plant, or the base sequence of the CDS of the gene, and encodes a protein that regulates the amount of assimilate distributed to the edible part.

Regarding the polynucleotide of (2) above, the sequence identity of the amino acid sequence is preferably 70% or more, 75% or more, 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. For example, a mutant gene derived from tomato or a homologous gene (including orthologues) derived from a solanaceae plant other than tomato is included in the category of the polynucleotide of (2) 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 degree of regulation of the amount of assimilate distribution to edible parts in solanaceae plants] described below can be molecular markers on these mutant genes or homologous genes.

With regard to the polynucleotide (3) above, in the amino acid sequence of SEQ ID NO: 1 or 2, the number of substituted, deleted, added or inserted amino acids is preferably 1 to 300, more preferably 1 to 250, 1 to 200, 1 to 150 or 1 to 100, even more preferably 1 to 50, and particularly preferably 1 to 25, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3 or 1 to 2.

When the gene that regulates the distribution of assimilates to the edible part refers to a gene into which a mutation has been artificially introduced, the above-mentioned "substitution, deletion, addition, or insertion of amino acids" may be, for example, a site-specific mutagenesis method such as the Kunkel method (Kunkel et al. (1985): Proc. Natl. Acad. Sci. USA, vol. 82, p. 488-), mutagen treatment using a drug, or a mutagenesis method using radiation (gamma rays, heavy ion beams, etc.), or may be derived from a similar mutant polypeptide that exists in nature.

The gene for regulating the amount of assimilate distribution to the edible part 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 base sequence shown in SEQ ID NO: 3, which is an example of the gene for regulating the amount of assimilate distribution to the edible part, is a full-length cDNA of the gene encoding the polypeptide shown in SEQ ID NO: 1. The base sequence shown in SEQ ID NO: 4, which is another example of the gene for regulating the amount of assimilate distribution to the edible part, is a full-length cDNA of the gene encoding the polypeptide shown in SEQ ID NO: 2. The gene for regulating the amount of assimilate distribution to the edible part may include an additional sequence such as a base sequence of a non-translated region (UTR) in addition to the CDS of the tfw.5.1 gene.

The method for obtaining (isolating) the gene that regulates the amount of assimilate distribution to the edible part is not particularly limited, but for example, a probe that specifically hybridizes with a portion of the base sequence of the gene that regulates the amount of assimilate distribution to the edible part may be prepared and a genomic DNA library or a cDNA library may be screened.

As a method for obtaining the gene that regulates the amount of assimilate distribution to the edible part, an example is a method that uses an amplification method such as PCR. For example, primers are prepared from the 5' and 3' sequences (or their complementary sequences) of the cDNA of the gene that regulates the amount of assimilate distribution to the edible part, and these primers are used to perform PCR or the like using genomic DNA (or cDNA) or the like as a template to amplify the DNA region sandwiched between the two primers, thereby obtaining a large amount of DNA fragments that contain the gene that regulates the amount of assimilate distribution to the edible part.

The origin of the gene that regulates the distribution of assimilates to the edible part is not particularly limited as long as it is a plant of the Solanaceae family, but it is preferably tomato, potato, eggplant, or chili pepper, and more preferably tomato.

Whether or not an isolated candidate gene for a gene regulating the amount of assimilate distributed to edible parts has the activity to regulate the amount of assimilate distributed to the desired edible parts can be evaluated by observing whether expression of the candidate gene in the plant from which it is derived induces an increase in the amount of assimilate distributed to the edible parts.

The gene that regulates the amount of assimilate distribution to edible parts can be used to elucidate the mechanism by which assimilate distribution to edible parts is increased in Solanaceae plants. In addition, the gene that regulates the amount of assimilate distribution to edible parts can be used to create a transformant by introducing the sequence into an expression vector or other means into a plant body or cell of a Solanaceae plant. By cultivating a Solanaceae plant into which the gene that regulates the amount of assimilate distribution to edible parts has been introduced, a Solanaceae plant with an increased amount of assimilate distributed to the edible parts can be obtained.

The gene that regulates the amount of assimilate distribution to the edible part includes a polynucleotide consisting of the base sequence of the first gene (SEQ ID NO: 3) or the second gene (SEQ ID NO: 4), which is the base sequence of the CDS of the tfw.5.1 gene derived from the tomato S. lycopersicum. The category of genes that regulate the amount of assimilate distribution to the edible part also includes genes consisting of polynucleotides that have 70% or more, 75% or more, 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 base sequence shown in SEQ ID NO: 3 or 4 and have activity of regulating the amount of assimilate distribution to the edible part.

[2. Proteins that regulate the distribution of assimilates to edible parts]
The protein regulating the amount of assimilate allocation to the edible part according to one embodiment of the present invention is a translation product of the gene described in the above section [1. Gene regulating the amount of assimilate allocation to the edible part], and has at least activity regulating the amount of assimilate allocation to the edible part. When the protein regulating the amount of assimilate allocation to the edible part has activity to positively regulate the amount of assimilate allocation to the edible part, the presence of the protein increases the amount of assimilate allocated to the edible part. When the protein regulating the amount of assimilate allocation to the edible part is not present, the amount of assimilate allocated to the edible part is reduced or does not increase compared to when the protein regulating the amount of assimilate allocation to the edible part is present.

The protein that regulates assimilate partitioning to edible parts may be isolated from a natural source or may be chemically synthesized. More specifically, the protein includes natural purified products, products of chemical synthesis procedures, and translation products produced by recombinant technology from prokaryotic or eukaryotic hosts, including, for example, bacterial cells, yeast cells, higher plant cells, insect cells, and mammalian cells.

More specifically, the protein that regulates the amount of assimilate distribution to the edible part is a protein described in any one of the following (1') to (3'):
(1') a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2;
(2') a protein consisting of an amino acid sequence having 70% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1 or 2, in which the amino acid corresponding to the 870th amino acid in the amino acid sequence shown in SEQ ID NO: 1 is E, or the amino acid corresponding to the 402nd amino acid in the amino acid sequence shown in SEQ ID NO: 2 is S, and having the function of regulating the amount of assimilate distributed to the edible part of a Solanaceae plant;
(3') A protein consisting of an amino acid sequence in which 300 or less amino acids have been substituted, deleted, added or inserted relative to the amino acid sequence shown in SEQ ID NO: 1 or 2, and in which the amino acid corresponding to the 870th amino acid in the amino acid sequence shown in SEQ ID NO: 1 is E, or the amino acid corresponding to the 402nd amino acid in the amino acid sequence shown in SEQ ID NO: 2 is S, and which has the function of regulating the amount of assimilates distributed to the edible parts of a Solanaceae plant.

The protein (1') is a protein encoded by the tfw.5.1 gene or a gene corresponding to the tfw.5.1 gene in a Solanaceae plant, and has activity of regulating the amount of assimilate distribution to the edible part.

Regarding the above protein (2'), the sequence identity of the amino acid sequence is preferably 70% or more, 75% or more, 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. For example, mutant proteins derived from tomato or homologous proteins derived from solanaceae plants other than tomato are included in the category of the above protein (2'). These mutant proteins and homologous proteins are proteins encoded by endogenous genes of solanaceae plants.

With regard 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 or 2 is preferably 1 to 300, more preferably 1 to 250, 1 to 200, 1 to 150 or 1 to 100, even more preferably 1 to 50, and particularly preferably 1 to 25, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3 or 1 to 2.

The protein that regulates the amount of assimilate distribution to the edible part is a polypeptide formed by peptide bonds between amino acids, but may also contain structures other than polypeptides. Examples of structures other than polypeptides referred to here include, but are not limited to, sugar chains and isoprenoid groups.

As an example of a protein that regulates the amount of assimilates distributed to the edible part, there is a protein derived from S. lycopersicum, which is a tomato. In addition, as an example of a protein that regulates the amount of assimilates distributed to the edible part, there is a protein derived from potato, eggplant, or chili pepper.

3. Expression vectors, cells, and transformants
The present invention also includes within its scope an expression vector incorporating a gene for regulating the amount of assimilate distribution to edible parts according to one embodiment of the present invention, a cell containing the expression vector or the gene for regulating the amount of assimilate distribution to edible parts, and a transformant into which the expression vector or the gene for regulating the amount of assimilate distribution to edible parts has been introduced so as to be expressible. The expression vector confers a trait for regulating the amount of assimilate distribution to edible parts to a cell or an individual organism.

The type of vector used to construct the expression vector is not particularly limited, and one that can be expressed in the host cell may be appropriately selected. In other words, a promoter sequence may be appropriately selected depending on the type of host cell, and the promoter sequence and a gene that regulates the distribution of assimilates to the edible part may be incorporated into, for example, a plasmid, phagemid, or cosmid, and used as the expression vector.

Examples of host cells into which an expression vector is introduced include bacterial cells, yeast cells, fungal cells other than yeast cells, and higher eukaryotic cells. Examples of bacterial cells include Escherichia coli cells. Examples of higher eukaryotic cells include plant cells and animal cells. Examples of plant cells include dicotyledonous plant cells and monocotyledonous plant cells. Examples of dicotyledonous plant cells include suspension culture cells of solanaceae plants (e.g., tobacco strain BY-2 and tomato strain Sly-1). 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.

In the expression vector, the gene for regulating the amount of assimilate distribution to the edible part is functionally linked to elements necessary for transcription (e.g., a promoter, etc.). In addition, an enhancer, a selection marker, a splicing signal, a poly A addition signal, a 5'-UTR sequence, etc. may be linked as necessary. A promoter is a DNA sequence that exhibits transcriptional activity in a host cell, and can be selected appropriately depending on the type of host.

Examples of promoter sequences that can operate in a host cell include the 35S promoter of the cauliflower mosaic virus, the nopaline synthase gene promoter of Agrobacterium, and the rice ubiquitin gene promoter. In addition, the sequence of the promoter region in a gene that regulates the distribution of assimilates to edible parts may be used as a recombinant expression promoter.

In the expression vector, the gene for regulating the amount of assimilate distribution to the edible part may be functionally linked to an appropriate terminator (e.g., the NOS terminator and the 35S terminator of the cauliflower mosaic virus) as necessary. The type of appropriate terminator may be selected appropriately depending on the type of host cell, and may be any sequence that can terminate the 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.

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

In addition, in the expression vector, the gene controlling the amount of assimilate distribution to the edible part may be linked to a suitable tag sequence for protein purification or a suitable spacer sequence, if necessary.

The above-mentioned transformant includes not only cells, tissues, and organs into which the expression vector or the gene for regulating the amount of assimilate distribution to edible parts has been introduced in an expressible manner, but also 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, etc.

4. Method for determining the degree of regulation of the amount of assimilates distributed to edible parts in Solanaceae plants
A method for discriminating the degree of regulation of the amount of assimilation product allocated to the edible part in a solanaceous plant (discrimination method) according to one embodiment of the present invention discriminates whether the amount of assimilation product allocated to the edible part is increased or not increased in the solanaceous plant. The discrimination method can be used to discriminate individuals of solanaceous plants in which the amount of assimilation product allocated to the edible part has increased. The discrimination method discriminates solanaceous plants based on the amount of assimilation product allocated to the edible part by determining the genotype of a gene involved in determining the amount of assimilation product allocated to the edible part, which is present in the genome of the solanaceous plant. Note that in the discrimination method, the concept of the amount of assimilation product allocated to the edible part includes the degree of assimilation product allocation, which indicates whether the amount of assimilation product allocated to the edible part of a certain solanaceous plant individual is relatively large or small compared to other solanaceous plant individuals.

In solanaceae plants, such as tomatoes, the amount of assimilates distributed to the edible parts is thought to gradually increase with growth, peak during the continuous production period, and then plateau (see Reference 1: Saito et al. Hort. J. 89: 445-453, 2020). That is, in the early stages of cultivation when the fruit clusters begin to set or the stems begin to thicken, the amount of assimilates distributed to the edible parts is small. Therefore, by increasing the amount of assimilates distributed to the edible parts, especially in the early stages of cultivation such as the fruit setting or stem thickening periods, it is possible to stably increase the yield.

Solanaceae plants may be candidates for breeding materials or plants obtained by the breeding process. Candidate plants for breeding materials include, for example, parent plants used for crossbreeding and plants used for molecular breeding using recombinant gene technology. Plants obtained by the breeding process include, for example, plants obtained by intraspecific hybridization of tomato, potato, eggplant, or pepper, which are Solanaceae plants, and their progeny lines. Solanaceae plants may also be intervarietal hybridization plants, such as hybridization of a tomato belonging to one variety with a tomato belonging to another variety, and their progeny lines. Solanaceae plants may also be plants obtained by hybridization of varieties known to have an increased amount of assimilate distribution to edible parts, and their progeny lines. Solanaceae plants may also be plants obtained by hybridization of varieties known to have an increased amount of assimilate distribution to edible parts with varieties for which it is unknown whether the amount of assimilate distribution to edible parts has increased, and their progeny lines. Furthermore, the solanaceae plant may be a plant obtained by crossing varieties whose distribution amount of assimilates to edible parts is unknown, and its progeny. The solanaceae plant may also be a plant obtained by crossing varieties whose distribution amount of assimilates to edible parts is known to be increased with varieties whose distribution amount of assimilates to edible parts is known not to be increased, and its progeny. The solanaceae plant may also be a plant obtained by crossing individuals whose distribution amount of assimilates to edible parts is known to be increased, and its progeny.

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

The method of identification can be to test for the presence or absence of a mutation in a gene that regulates the amount of assimilates distributed to the edible part using molecular markers such as those shown below. Such molecular markers are also included in the scope of the present invention.

The molecular marker is an amino acid sequence of the following (a) or (b) in a Solanaceae plant:
(a) an amino acid corresponding to the 870th 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 402nd amino acid of the protein consisting of the amino acid sequence set forth in SEQ ID NO:2;
It is a molecular marker that detects the presence or absence of mutations that cause substitutions or deletions in the chromosome.

The protein consisting of the amino acid sequence shown in SEQ ID NO:1 is a protein encoded by a gene that regulates the amount of assimilates distributed to edible parts, and an example of such a protein is a protein encoded by a gene that consists of the base sequence shown in SEQ ID NO:3. The protein consisting of the amino acid sequence shown in SEQ ID NO:2 is a protein encoded by a gene that regulates the amount of assimilates distributed to edible parts, and an example of such a protein is a protein encoded by a gene that consists of the base sequence shown in SEQ ID NO:4.

The protein consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2 consists of the amino acid sequence of the protein encoded by the reference tfw. 5.1 gene derived from tomato (S. lycopersicum). In a solanaceae plant, the amino acid sequence may include a portion that differs from the amino acid sequence of the protein encoded by the reference tfw. 5.1 gene derived from tomato other than the portion corresponding to the amino acid (a) or (b) above. In a solanaceae plant, the position of the amino acid corresponding to the amino acid (a) or (b) above can be identified by a method such as homology analysis. That is, in a gene corresponding to the tfw. 5.1 gene of a solanaceae plant (i.e., when a gene highly conserved among plants exists), "an amino acid corresponding to the amino acid (a) or (b)" refers to an amino acid that is determined to be equivalent to the amino acid (a) or (b) by a method such as homology analysis.

Methods for homology analysis include, for example, 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. Those skilled in the art can use these methods to understand the "corresponding amino acids" in the amino acid sequence to be analyzed using the amino acid sequence shown in SEQ ID NO: 1 or 2 as a reference sequence.

The following shows an example of a homologous protein of a protein having the amino acid sequence shown in SEQ ID NO:1 in a solanaceae plant, and an amino acid corresponding to the amino acid in (a). In a bell pepper (C. annuum. var. glabriusculum_1) protein having the amino acid sequence shown in SEQ ID NO:5, the amino acid corresponding to the amino acid in (a) is the 850th amino acid. In a bell pepper (C. Annuum_Zunla) protein having the amino acid sequence shown in SEQ ID NO:7, the amino acid corresponding to the amino acid in (a) is the 850th amino acid. In a potato (PGSC0003DMC400027859) protein having the amino acid sequence shown in SEQ ID NO:9, the amino acid corresponding to the amino acid in (a) is the 871st amino acid. In a potato (Sotub05g017810.1.1) protein having the amino acid sequence shown in SEQ ID NO:11, the amino acid corresponding to the amino acid in (a) is the 871st amino acid.

The following shows an example of a homologous protein of a protein consisting of the amino acid sequence shown in SEQ ID NO:2 in a solanaceae plant, and an amino acid corresponding to the amino acid in (b). In a bell pepper (C. annuum. var. glabriusculum_1) protein consisting of the amino acid sequence shown in SEQ ID NO:13, the amino acid corresponding to the amino acid in (b) is the 402nd amino acid. In a bell pepper (C. Annuum_Zunla) protein consisting of the amino acid sequence shown in SEQ ID NO:15, the amino acid corresponding to the amino acid in (b) is the 400th amino acid. In a potato (PGSC0003DMC400056411) protein consisting of the amino acid sequence shown in SEQ ID NO:17, the amino acid corresponding to the amino acid in (b) is the 401st amino acid. In a potato (Sotub05g016440.1.1) protein consisting of the amino acid sequence shown in SEQ ID NO:19, the amino acid corresponding to the amino acid in (b) is the 401st amino acid. In the eggplant (SMEL_003g177800) protein consisting of the amino acid sequence shown in SEQ ID NO:21, the amino acid corresponding to the amino acid in (b) is the 434th amino acid.

"An amino acid corresponding to amino acid (a) or (b)" is, for example, (I) an amino acid (a) or (b) in a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2, (II) an amino acid corresponding to amino acid (a) or (b) in a protein consisting of an amino acid sequence having a sequence identity of 70% or more to the amino acid sequence shown in SEQ ID NO: 1 or 2, or (III) an amino acid corresponding to amino acid (a) or (b) in a protein consisting of an amino acid sequence in which 300 or less amino acids have been substituted, deleted, added, or inserted relative to the amino acid sequence shown in SEQ ID NO: 1 or 2.

A molecular marker that detects the presence or absence of a mutation that causes a substitution or deletion in an amino acid corresponding to amino acid (a) or (b) may be one that detects the presence or absence of a mutation that causes a substitution or deletion in an amino acid corresponding to both amino acids (a) and (b).

An example of a molecular marker is the
(a') a base corresponding to the 2609th base from the translation start site of the tfw.5.1 gene, or
(b') is the base itself (SNP) corresponding to the 1204th base from the translation start point of the tfw.5.1 gene, or a continuous polynucleotide containing the base. That is, the discrimination method includes a step of testing the base itself (SNP) corresponding to the base (a') or (b') above, or a continuous polynucleotide containing the base, as a molecular marker for regulating the amount of assimilate distribution to the edible part.

The base corresponding to the base (a') or (b') above is a SNP that causes an amino acid substitution. By using the base corresponding to the base (a') or (b') above as a molecular marker, the presence or absence of a mutation that causes a substitution or deletion in an amino acid corresponding to the amino acid (a) or (b) above can be detected. As an example, the base corresponding to the 2609th base from the translation start point of the tfw.5.1 gene in (a') above is the 2609th base in the base sequence of SEQ ID NO:3. In addition, the base corresponding to the 1204th base from the translation start point of the tfw.5.1 gene in (b') above is the 1204th base in the base sequence of SEQ ID NO:4.

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

The bases themselves corresponding to the above bases (a') and (b'), or consecutive polynucleotides containing said bases, are the SNP markers described in this embodiment 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.

(i: SNP marker)
SNP means a DNA polymorphism in which a single base mutation is observed in a specific region in the DNA base sequence. In the SNP marker, the SNP is a single nucleotide polymorphism 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/).

"Bases (a') and (b')" are the SNP markers described in this embodiment. "Bases corresponding to bases (a') and (b')" are SNP markers that can be identified with the SNP markers described in this embodiment. "Bases (a') and (b')" are mutations in the tfw. 5.1 gene. The tfw. 5.1 gene consists of a base sequence derived from the reference tomato (S. lycopersicum). In solanaceae plants, the base sequence may include a portion other than the SNP that differs from the reference base sequence. The region on the genome where this SNP marker is located is conserved among many solanaceae plants, so this SNP marker can be identified by a method such as homology analysis. As a method of homology analysis, the same method as that for identifying the "corresponding amino acid" can be used.

That is, when a gene corresponding to the tfw.5.1 gene (i.e., a gene highly conserved among plants) exists in another Solanaceae plant, "bases corresponding to bases (a') and (b')" refers to bases in the gene corresponding to the tfw.5.1 gene that are determined to correspond to bases (a') and (b') by a method such as a homology search. For example, the polynucleotides described in (a2') and (b2') and the polynucleotides described in (a3') and (b3') described below are examples of genes corresponding to the tfw.5.1 gene.

The following shows an example of a homologous gene of a gene consisting of the CDS base sequence of the tfw.5.1 gene shown in SEQ ID NO:3 in a solanaceae plant, and a base corresponding to the base (a'). In the gene of bell pepper (C. annuum. var. glabriusculum_1) consisting of the base sequence shown in SEQ ID NO:6, the base corresponding to the base (a') is the 2549th base. In the gene of bell pepper (C. Annuum_Zunla) consisting of the base sequence shown in SEQ ID NO:8, the base corresponding to the base (a') is the 2549th base. In the gene of potato (PGSC0003DMC400027859) consisting of the base sequence shown in SEQ ID NO:10, the base corresponding to the base (a') is the 2612th base. In the gene of potato (Sotub05g017810.1.1) consisting of the base sequence shown in SEQ ID NO:12, the base corresponding to the base (a') is the 2612th base.

An example of a homologous gene of a gene consisting of the base sequence of the CDS of the tfw.5.1 gene shown in SEQ ID NO: 4 in a solanaceae plant and a base corresponding to the base (b') is shown. In the gene of bell pepper (C. annuum. var. glabriusculum_1) consisting of the base sequence shown in SEQ ID NO: 14, the base corresponding to the base (b') is the 1204th base. In the gene of bell pepper (C. Annuum_Zunla) consisting of the base sequence shown in SEQ ID NO: 16, the base corresponding to the base (b') is the 1198th base. In the gene of potato (PGSC0003DMC400056411) consisting of the base sequence shown in SEQ ID NO: 18, the base corresponding to the base (b') is the 1201st base. In the gene of potato (Sotub05g016440.1.1) consisting of the base sequence shown in SEQ ID NO: 20, the base corresponding to the base (b') is the 1201st base. In the eggplant (SMEL_003g177800) gene consisting of the base sequence shown in SEQ ID NO:22, the base corresponding to the (b') base is the 1300th base.

The molecular marker is a SNP marker consisting of the above SNP (a') or (b'). This SNP marker is a SNP marker newly identified by the present inventors, and a person skilled in the art can identify the position of the SNP marker on the genome based on the base sequence representing each SNP of this SNP marker.

SNP (a') (hereinafter also referred to as SNP(a')) indicates a polymorphism at the 2609th base from the translation start point of the tfw.5.1 gene or a base equivalent thereto. SNP (b') (hereinafter also referred to as SNP(b')) indicates a polymorphism at the 1204th base from the translation start point of the tfw.5.1 gene or a base equivalent thereto.

When (a'') SNP (a') is A or (b'') SNP (b') is T, the molecular marker can determine that the amount of assimilates distributed to the edible parts of the Solanaceae plant is increased. The molecular marker can also determine the amount of assimilates distributed to the edible parts of a Solanaceae plant by analyzing SNP (a') and SNP (b') as a haplotype block. Here, when the amount of assimilates distributed to the edible parts of a Solanaceae plant is increased, it is intended that the amount of assimilates distributed to the edible parts of a certain Solanaceae plant individual is relatively large compared to other Solanaceae plant individuals.

(ii: Polynucleotides containing SNPs)
The molecular marker may be a contiguous polynucleotide containing SNP (a') (hereinafter also referred to as polynucleotide (a')), or a contiguous polynucleotide containing SNP (b') (hereinafter also referred to as polynucleotide (b')).

Polynucleotide (a') is a polynucleotide consisting of the base sequence of a region containing SNP (a') in the base sequence of the tfw.5.1 gene (a1'), a polynucleotide consisting of a base sequence in which one or several bases have been substituted, deleted, added, or inserted in the base sequence of the polynucleotide (a2') (a1') with respect to the base sequence other than SNP (a') and having the function of determining the amount of assimilate distributed to the edible parts of a Solanaceae plant, or a polynucleotide consisting of a base sequence having 90% or more identity with the base sequence other than SNP (a') in the base sequence of the polynucleotide (a3') (a1') and having the function of determining the amount of assimilate distributed to the edible parts of a Solanaceae plant.

Polynucleotide (b') is a polynucleotide consisting of the base sequence of a region containing SNP (b') in the base sequence of the tfw.5.1 gene (b1'); (b2') is a polynucleotide consisting of a base sequence in which one or several bases have been substituted, deleted, added or inserted relative to the base sequence other than SNP (b') in the base sequence of the polynucleotide (b1') and has the function of determining the amount of assimilate distributed to the edible parts of a Solanaceae plant; or (b3') is a polynucleotide consisting of a base sequence having 90% or more identity to the base sequence other than SNP (b') in the base sequence of the polynucleotide (b1') and has the function of determining the amount of assimilate distributed to the edible parts of a Solanaceae plant.

The polynucleotides (a1') and (b1') can be obtained, for example, from a Solanaceae plant with increased assimilate distribution to the edible part based on the base sequence of the tfw.5.1 gene (for example, a tomato (Dutch F1 variety of S. lycopersicum) with increased assimilate distribution to the edible part).

In the polynucleotides (a2') and (b2'), the bases corresponding to SNPs (a') and (b') in the base sequences of the polynucleotides (a1') and (b1') are conserved, and the remaining base sequences may contain modifications (substitutions, deletions, insertions, or additions) of several bases (e.g., 1 to 10, preferably 1 to 5, and more preferably 1, 2, or 3). 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 deciphering the base sequence of the region adjacent to the SNP on the genome of a solanaceae plant that distributes a large amount of assimilates to the edible parts.

In the polynucleotides (a3') and (b3'), the bases corresponding to SNPs (a') and (b') in the base sequences of the polynucleotides (a1') and (b1') are conserved, and the bases may have an identity of, for example, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more to the remaining base sequences. The identity of such base sequences can be determined, for example, by aligning the two base sequences using analysis software such as BLAST or FASTA.

When (a'') the base corresponding to SNP (a') in polynucleotide (a') is A, or (b'') the base corresponding to SNP (b') in polynucleotide (b') is T, it can be determined that the amount of assimilates distributed to the edible parts of the Solanaceae plant is increased. The molecular marker may be used to determine the amount of assimilates distributed to the edible parts of the Solanaceae plant by analyzing polynucleotides (a') and (b') as haplotype blocks.

(iii: polynucleotide containing two SNPs)
The molecular marker may be a continuous polynucleotide (hereinafter also referred to as polynucleotide (c')) containing SNP (a') and SNP (b'). Polynucleotide (c') contains the region between SNP (a') and SNP (b') together with the sites of SNP (a') and SNP (b'). Polynucleotide (c') is obtained, for example, by referring to the region between the corresponding sites of SNP (a') and SNP (b') in a solanaceae plant with increased assimilate distribution to edible parts. The base sequence of polynucleotide (c') at least partially coincides with the base sequence of a solanaceae plant with increased assimilate distribution to edible parts.

The method for determining the amount of assimilates distributed to the edible parts of 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. Such known SNP analysis methods include a method of SNP analysis by detecting SNPs in PCR amplified fragments of a solanaceae plant specimen.

The discrimination method may involve amplifying a region in the DNA of a solanaceae plant using a primer set that amplifies the region including a molecular marker. One example of such a primer set is a primer set that amplifies a region including at least one of SNP (a') and SNP (b').

The region in the DNA of a solanaceae plant specimen can be amplified by polymerase chain reaction (PCR) using primers that amplify the region containing the SNP, with DNA extracted from the solanaceae plant specimen as a template. The base (genotype) of the SNP in the resulting amplified fragment is then determined, and the amount of assimilate distributed to the edible parts of the solanaceae plant is determined based on data showing the relationship between the determined base (genotype) and the amount of assimilate distributed to the edible parts of the solanaceae plant.

The primer set used in PCR is not particularly limited as long as it can amplify a DNA fragment in a region containing the target SNP, and the primer set may be designed so that the length of the amplified fragment is short. 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 50 bp or less, 40 bp or less, or 30 bp or less.

The primer set for amplifying the region containing SNP (a') is, for example, a primer set consisting of a first primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 23 and a second primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 24. By using this primer set, in a Solanaceae plant with increased distribution of assimilates to the edible part, a PCR amplification product consisting of the base sequence shown in SEQ ID NO: 25 can be obtained, and the base corresponding to SNP (a') can be detected.

The primer set that amplifies the region containing SNP (b') is, for example, a primer set consisting of a third primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 26 and a fourth primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 27. By using this primer set, in a Solanaceae plant in which the amount of assimilates distributed to the edible part is increased, a PCR amplification product consisting of the base sequence shown in SEQ ID NO: 28 can be obtained, and the base corresponding to SNP (b') can be detected.

The PCR used 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 having different wavelengths (e.g., NED, 6-FAM, VIC, PET) may be mixed.

The PCR reaction conditions can be appropriately set according to the type of DNA polymerase and PCR device used, the length of the amplified fragment, etc. As cycle conditions, a three-step PCR method in which one cycle is a denaturation step, an annealing step, and an extension step, and a two-step PCR method in which one cycle is a denaturation step, an annealing step, and an extension step, can be applied. An example of the PCR reaction conditions is 90-100°C for 40-60 seconds (e.g., 50 seconds at 95°C), followed by 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., 20 seconds at 72°C) for 30-60 cycles (e.g., 40 cycles). The annealing temperature can be reduced stepwise every predetermined cycle 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). Depending on the state of the template DNA, PCR reaction conditions may be adjusted to stably detect SNPs.

As the PCR in the SNP analysis, real-time PCR such as TaqMan (registered trademark)-PCR method and Tm-shift genotyping method (Fukuoka et al., Breed Sci 58:461-464, 2008) that amplifies by PCR and identifies SNP markers may be used. That is, a TaqMan (registered trademark) probe that detects SNPs contained in the amplified fragment amplified using a primer set may be further used. By using the real-time PCR method, a high-throughput discrimination method can be provided. Note that, as a method for identifying SNPs in the amplified fragment amplified by PCR, analysis may be performed by determining the base sequence of the amplified fragment using an automatic DNA sequencer or the like.

The method for extracting DNA to be amplified by PCR from a solanaceae plant specimen is not particularly limited, and any known DNA extraction method can be used. DNA may also be extracted using a commercially available DNA extraction kit. Depending on the type of specimen and the amount of impurities, etc., pretreatment may be performed appropriately before the DNA extraction step. Furthermore, the DNA extracted from the specimen may be washed or purified as necessary in order to use it as a template in the PCR reaction. Furthermore, the DNA extracted from the specimen may be digested with two types of restriction enzymes, and the restriction enzyme cleavage fragments may be amplified by PCR.

In addition, in a method for determining the amount of assimilates distributed to edible parts of a Solanaceae plant, genetic polymorphisms in linkage disequilibrium with SNP(a') and SNP(b') may be analyzed to identify SNP(a') and SNP(b'). One example of the linkage disequilibrium state is a linkage disequilibrium state with a linkage disequilibrium coefficient of 0.9 or more.

According to the discrimination method, it is possible to determine whether the amount of assimilates distributed to the edible parts of a test Solanaceae plant is increased by using molecular markers. Therefore, based on the determination results, it is possible to select Solanaceae plants and their progeny that have increased amounts of assimilates distributed to the edible parts.

[5. Solanaceae plants with increased assimilate distribution to edible parts]
A solanaceae plant with increased assimilate distribution to edible parts according to one embodiment of the present invention is a plant obtained by the production method described below. The solanaceae plant with increased assimilate distribution to edible parts is a plant having the above-mentioned SNP identified by the above-mentioned molecular marker, and has increased assimilate distribution to edible parts.

As shown in the production method described below, a solanaceae plant having an increased amount of assimilates distributed to edible parts according to one embodiment of the present invention can be obtained by crossbreeding a solanaceae plant having an increased amount of assimilates distributed to edible parts with another solanaceae plant, and identifying the solanaceae plant having an increased amount of assimilates distributed to edible parts from the resulting plant and its progeny using the molecular markers described above. Note that solanaceae plants having an increased amount of assimilates distributed to edible parts that have been genetically engineered to have the SNPs described above are also included in the scope of the present invention.

[6. Method for producing a solanaceous plant having increased amount of assimilates distributed to edible parts]
A production method according to one embodiment of the present invention is a method for producing a Solanaceae plant having an increased amount of assimilate distribution to edible parts, and includes a hybridization step of intraspecifically hybridizing a Solanaceae plant having an increased amount of assimilate distribution to edible parts with another Solanaceae plant, and an identification step of identifying a Solanaceae plant having an increased amount of assimilate distribution to edible parts from the Solanaceae plant obtained by the hybridization step or a Solanaceae plant of a progeny thereof by the discrimination method described above.

Therefore, the above-mentioned explanations regarding the molecular markers, the solanaceae plants with increased distribution of assimilates to the edible parts, and the method for determining the degree of regulation of the distribution of assimilates to the edible parts in solanaceae plants are used to explain the method for producing solanaceae plants with increased distribution of assimilates to the edible parts.

In the hybridization step, the solanaceae plant with increased assimilate distribution to edible parts used as a parent plant may be a solanaceae plant with increased assimilate distribution to edible parts according to one embodiment of the present invention. In addition, the solanaceae plant with increased assimilate distribution to edible parts used in the hybridization step may be a solanaceae plant with increased assimilate distribution to edible parts selected by a method for determining the degree of regulation of the amount of assimilate distribution to edible parts in a solanaceae plant according to one embodiment of the present invention. In other words, 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 solanaceae plant with increased assimilate distribution to edible parts from a test solanaceae plant by a method for determining the degree of regulation of the amount of assimilate distribution to edible parts in a solanaceae plant according to one embodiment of the present invention.

The identification step uses a method for determining the degree of regulation of the amount of assimilate distribution to the edible parts in a solanaceae plant according to one embodiment of the present invention to identify a solanaceae plant with an increased amount of assimilate distribution to the edible parts from among the solanaceae plants obtained by the hybridization step or from the solanaceae plants of the progeny lineage.

According to a production method according to one aspect of the present invention, the degree of regulation of the amount of assimilates distributed to the edible parts of a Solanaceae plant can be determined using molecular markers, and a Solanaceae plant with increased amount of assimilates distributed to the edible parts can be produced based on the determination results.

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

Materials and Methods
(Construction of recombinant inbred lines)
The high-yielding beefsteak-type Dutch F1 variety "Geronimo" and the high-sugar, large-fruited Japanese F1 variety "Momotaro8" were used as cross parents. All plants were grown in pots containing soil in a greenhouse at the Vegetable and Flower Research Division of the National Agriculture and Food Research Organization, Tsu City, Mie Prefecture. This cross corresponds to a four-way cross, with the four parent lines of two F1 varieties (G1 generation) as ancestors (G0 generation). Figure 1 is a diagram outlining the construction of recombinant inbred lines derived from the cross of two F1 varieties and their QTL mapping. As shown in Figure 1, "Geronimo"(P1; G1) and "Momotaro8"(P2; G1) were crossed to generate a four-way F1 group (G1F1) consisting of 240 plants. The four-way recombinant inbred lines were then developed by repeated selfing from each G1F1 plant using the single-grain line method. 206 recombinant inbred lines of the G1F6 generation were obtained and maintained by selfing to evaluate their agronomic traits. These lines were designated GM lines.

Cultivation of GM lines and phenotypic evaluation at two locations
Seeds of GM lines were sown in soil-filled nursery trays between July 30 and August 19 in 2010 and 2013 in Mie Prefecture (F7, F9, and F10 generations, autumn cropping, Experiments 3 to 6). Also, GM lines were sown on February 1 or 2 in 2011 and 2012, and F8 and F10 generations were grown in spring (Experiments 1 and 2). After placing the nursery boxes in a greenhouse for 3 weeks, all plants (one plant per line per experiment) were planted directly on rockwool slabs (planting density 2.303/ ^m2 ). All plants were grown in a greenhouse at 16°C or higher, with a nutrient solution (Otsuka Chemical) of electrical conductivity (EC) 2.8 mS/cm. The maximum number of flowers per truss was limited to 6, and the tops were pinched above the third truss (Experiment 3) or the fourth truss (Experiments 1, 2, and 4 to 6).

In Aichi Prefecture, seeds of the F10 generation GM lines were sown as described above on August 27, 2012 (Experiment 7) and August 28, 2013 (Experiment 8). The seedlings were placed in a growth chamber and cultivated with a culture solution (EC = 1.8 mS/cm, Mitsubishi Chemical Agri Dream Co., Ltd.) at 25°C (day)/20°C (night), _CO2 concentration of 900 or 1000 ppm, and day length of 16 hours. After 3 weeks, all plants (2 plants per line per experiment) were planted directly in rock wool slabs (planting density 3.086/ ^m2 ). All plants were cultivated in a greenhouse at 13°C or higher and cultivated with a culture solution (Otsuka Chemical Co., Ltd.) with EC = 0.8-1.8 mS/cm (Experiment 7) or EC = 1.5-2.0 mS/cm (Experiment 8). Plants were topped above the fourth fruit cluster.

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 in each GM line. Sugar content in red ripe marketable fruits was measured using a saccharometer (PAL-1). Fruits without any physiological disorders such as bottom rot or cracks were defined as good fruits. Meanwhile, fruits with at least one physiological disorder were defined as bad fruits.

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

(Trait Inheritance)
Heritability was calculated using a linear model of the phenotype of the GM lines in each experiment.

(Isolation of Genomic DNA)
Genomic DNA was isolated from leaves of P1 and P2 plants using DNeasy Plant Mini Kit (Qiagen). Genomic DNA was isolated from leaves of plants of GM lines of G1F1 and F9 generations using DNeasy 96 Plant Kit (Qiagen). Genomic DNA was quantified using Quant-iT PicoGreen dsDNA reagent (Thermo Fisher Scientific K.K.) and ARVO MX (PerkinElmer Japan Co., Ltd.) according to the manufacturer's instructions.

(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 experiment; a total of 135) published on the website (https://solgenomics.net).

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 strains of G1F1 and F9 generations), SSR markers were classified into combination patterns (eight categories) of the alleles of both parents (G1 generation, P1, and P2) (Table 1).

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

(Genotype analysis of SSR alleles)
Forward primers for the SSR alleles were 5'-end labeled with 6-FAM, NED, PET, or VIC (Applied Biosystems). Genomic DNA of P1P2 and genomic DNA of plants of GM line of (G1F1 and F9 generations) were used as templates. PCR was performed in 10 μL reaction volume with Type-it Microsatellite PCR Kit (Qiagen). PCR was started at 95°C for 5 min, followed by 28 cycles of 95°C for 30 s, 60°C for 90 s, and 72°C for 30 s, and finally 60°C for 30 min. PCR amplified products were analyzed in an automatic sequencer (3730 x1 DNA Analyzer, Applied Biosystems) together with GeneScan-500LIZ Size Standard (Applied Biosystems). Fragment lengths were analyzed using GeneMapper v. 3.7. The results were determined using Applied Biosystems software.

(SNP allele genotype analysis)
A total of 1536 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. The marker categories were classified in the same way as the SSR markers in Table 1.

(Construction of linkage map)
A linkage map of the GM lines was constructed using Carthagene software (de Givry et al. Bioinformatics 21: 1703-1704, 2005). The linkage map of the GM lines was constructed by estimating the frequency of recombination (map distance) between each marker that occurs with the passage 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 genotypes of heterozygous markers in P1P2 (categories 1 to 7 in Table 1) immediately segregated in the G1F1 generation, allowing the recombination frequency between these marker pairs to be estimated. The recombination frequency was estimated by focusing on the inheritance of alleles from the G1F1 plants to their progeny GM lines, and the information on segregation in G1F1 was used only to confirm the recombination frequency estimate. Segregation distortion of marker genotypes in the linkage map was identified by chi-square test. The linkage map was analyzed using MapChart v. Drawn using 2.1 software.

(Bayesian QTL mapping using MCMC methods)
Bayesian QTL mapping of the four-way cross was performed using the genotypes of the GM line and the marker genotypes of the P1, P2, G1F1 and GM line groups. In this mapping method, four alleles from four unknown ancestors (parents of P1 and parents of P2) were assumed at each QTL. The marker genotypes of the G1F1 plants were used to estimate the haplotypes of P1 and P2. The haplotypes of the GM line were inferred from the haplotypes of the G1F1 parents. In addition to the Bayesian method based on the MCMC method, a Bayesian analysis using variational approximation for QTL mapping in a four-way cross (variational approximation method) was performed.

(Acquisition of tfw5.1 isolates and seed collection)
Among the GM lines of the F9 generation, line 229 showed a heterozygous genotype in the tfw5.1 region (defined here as being between two SSR markers, tbm1306 and tma0146 for convenience, see FIG. 2). Self-pollinated progeny seeds of this line were harvested in a soil-filled seedling tray on September 8, 2014, and then cultivated in the same manner as in Experiments 7 and 8. After 3 weeks, the plants were potted in rock wool cubes and provisionally planted in a greenhouse (plant factory) of the National Agriculture and Food Research Organization in Tsukuba, Ibaraki Prefecture. Culture solution (manufactured by Otsuka Chemical Co., Ltd.) with EC = 1.0 mS / cm was applied, and the plants were cultivated from September 30 to November 4 of the same year. During this time, genomic DNA was isolated according to the above "Isolation of Genomic DNA", and this DNA was used as a template to perform multiplex PCR using a fluorescent dye-based BS-tag method (Shimizu and Yano, BMC Research Notes, 4:161, 2011; Konishi et al., Vegetable and Tea Industry Research Institute Research Report, 14: 15-22, 2015) using an SSR marker set located around the tfw5.1 region. Information on the SSR marker sets used (set 1: tbm1306, TGS0155, TGS0443, tbm1321; set 2: TGS0633, tma0146, TGS0280, tma0252) is available from the public database (VegMarks, https://vegmarks.nivot.affrc.go.jp/VegMarks/app/page/home). The fluorescent dye used was either 6-FAM, NED, PET, or VIC. Based on the above "genotype analysis of SSR alleles," individuals with homozygous alleles of the high-yielding Dutch tomato, individuals with homozygous alleles of the Japanese tomato, and individuals with heterozygous alleles were selected. The individual with the high-yielding allele was named 229G, the individual with the Japanese allele was named 229M, and the individual with the heterozygous allele was named 229H. On November 4, the rockwool cubes containing these plants were planted on rockwool slabs and further cultivated, and self-fertilized seeds of 229G, 229M, and 229H were harvested.

(Test 1: Analysis of yield components in fixed segregating lines)
In 229G and 229M, the tfw5.1 allele is considered to have been fixed to a high-yielding or Japanese type. The seeds of these lines were sown on February 23, 2015, and cultivated in a growth chamber in the same manner as above. On March 19, they were potted in rock wool cubes, planted on rock wool slabs at a planting density of 2.7 (plants/ ^m2 ), and cultivated in a greenhouse for spring cropping. The culture solution at the time of planting was EC = 1.1 mS/cm, and was increased to EC = 1.9 mS/cm on April 30, and was applied at this concentration until the end of cultivation. Cultivation was carried out until the 125th day after planting without pinching. Mature fruits were harvested periodically during the cultivation period, divided into good fruits and bad fruits, and the number and fresh weight were obtained. After the end of cultivation, the plants were cut from the base of the plant and divided into immature fruits, good fruits, 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 said as dry matter weight. The dry weight of the fruits harvested during the harvest period was calculated from the fresh weight using the dry matter ratio (dry weight/fresh weight) measured after the end of cultivation.

(Test 2: Analysis of yield components in fixed segregating lines)
Seeds of the fixed lines 229G and 229M were sown on September 4, 2017, as in Test 1, and cultivated in a growth chamber. On September 28, the plants were potted in rock wool cubes, planted on rock wool slabs at a planting density of 3.3 (plants/ ^m2 ), and cultivated in a greenhouse for autumn cropping. The culture solution at the time of planting was EC = 2.6 mS/cm, and was applied 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, divided into good and bad fruits, and the number and fresh weight were obtained. Data collection after the end of cultivation was performed in the same manner as in Test 1.

(Development of a line in which the tfw5.1 region of the segregating line has been shortened)
Among the tfw5.1 segregation lines, the self-fertilized seeds of 229H were sown on August 9, 2016, in the same manner as in Test 1, and then cultivated in a growth chamber. Two weeks after sowing, DNA was extracted from the leaves of the seedlings (DNA Suisui-P, Rezo Co., Ltd.). Using this DNA as a template, multiplex PCR was performed in the same manner as in the above "Acquisition and seed collection of tfw5.1 segregation lines", and then genotype analysis was performed according to "SSR allele genotype analysis", and lines in which the tfw5.1 region had been shortened by recombination were selected. On September 8, autumn cultivation was performed with the same planting density and nutrient solution concentration as in Test 2, and self-fertilized seeds were collected.

(Test 3: Fixation of lines with truncated tfw5.1 region and restriction of the region by analysis of yield components)
The marker genotype of the tfw5.1 region of the shortened line selected above is a partially hetero allele. Therefore, in order to select lines that are fixed to high-yield or Japanese type, the self-fertilized seeds of the eight shortened lines were sown on September 4, 2017, and genotype analysis was performed again. All cultivation in the growth chamber and greenhouse was performed in the same manner as in Test 2. Genotype analysis was performed using the SSR marker set during growth, and after identifying the fixed line, yield component analysis of these lines was performed in the same manner as in Test 2. In addition, yield comparison was performed between the high-yield type and the Japanese type, but there were two lines in which either type was not sufficiently obtained in this selection. For these lines, yield component analysis was performed again in the spring of 2019 using the fixed seeds obtained here (sown on February 4, 2019, planted on February 28, and cultivated for 109 days after planting). Cultivation management was performed in accordance with Test 1, except that the culture solution concentration was set to EC = 2.6 mS / cm.

〔result〕
(Phenotypic correlation analysis in GM lines)

In phenotypic correlation analysis, total fruit fresh weight was negatively correlated with fruit sugar content. Other reports (Bernacchi et al. 1998; Fulton et al. 1997; Gur et al. 2011) have also shown that these traits have a trade-off relationship.

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

(Screening and typing of effective SSR and SNP markers in G1F1 lines and recombinant inbred lines)
Effective SSR markers were classified into eight categories according to the combination pattern of allele types in each line (Table 1). The marker in category 7 was capable of detecting four different alleles, indicating that the marker was useful. However, the marker frequency in the tomato genome was very low. Therefore, in the QTL analysis, markers in different categories were combined and used to compensate for the lack of genetic information in each region of the chromosome. As an example, marker A (aa-bb) in category 0, marker B (ab-aa) in category 2, and marker C (aa-ab) in category 3 can provide information similar to that obtained by marker (ab-cd) in category 7. A total of 197 SSR markers were selected.

（連鎖地図の評価）
ＧＭ系統の連鎖地図は、１２の連鎖群からなり、総遺伝距離１２２１．８ｃＭをカバーした。マーカー間の平均距離は３．７ｃＭであり、最大２８．５ｃＭのギャップがあった。ゲノムカバー率は、トマトゲノムＳＬ３．００(http://solgenomics.net/)の９８．２％であった。連鎖地図の分離歪み率は４％未満（１３／３３８マーカー＝０．０３８５）であった。大きなギャップが存在していたが（２０ｃＭ以上）、ゲノムカバー率及びマーカー間の平均距離（１０ｃＭ未満、Lander and Botstein 1989）は、連鎖地図が、組換え型自殖系統の全ゲノムの探索に十分であることを示唆していた。 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.

(Evaluation of linkage maps)
The linkage map of 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. The 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 were present (more than 20 cM), the genome coverage and the average distance between markers (less than 10 cM, Lander and Botstein 1989) suggested that the linkage map was sufficient for exploring the entire genome of the recombinant inbred lines.

(Comparison of mapping accuracy between MCMC method and variational approximation method)
Before performing QTL mapping using all trait data, two Bayesian mapping methods for detecting QTLs, the MCMC method and the variational approximation method, were compared using the data of total fruit fresh weight. QTL detection was based on the posterior probability of each QTL location being included in the model. The posterior probability of each QTL location was calculated as the ratio of MCMC samples in which the model including the QTL was adopted to the total MCMC samples in the MCMC method, and was obtained as the approximate posterior expectation value of the indicator variable γ ₁ for the inclusion in the model of hypothetical QTLs equally spaced in the genome in the variational approximation method. Since the results obtained from the two Bayesian methods were roughly equivalent, only the variational approximation method was applied to the analysis of the remaining traits in order to reduce the calculation time.

The analysis results of the QTLs involved in total fruit fresh weight detected by the approximation method are shown in Table 2. In Table 2, ^R2 is the estimated proportion of phenotypic variance explained by the QTL. Figure 2 illustrates the linkage map of linkage group 5 and the location of QTL tfw5.1.

(Results of Test 1)
The results of the component analysis of Test 1 are shown in Table 3. The fixed line 229G with the tfw5.1 allele derived from the high-yielding Dutch tomato had significantly increased total fruit weight (total fresh weight of immature fruit, poor fruit, and good fruit) per plant compared to the fixed line 229M with the allele derived from the Japanese tomato. The average fruit weight (total fruit weight/total fruit number), fruit dry weight (total fruit weight converted to dry weight), and fruit distribution rate (fruit dry weight/total dry weight) were all significantly increased. Here, the total dry weight refers to the total dry weight of the leaves, stems, and all fruits. There was no difference in total dry weight (synonymous with aboveground biomass) or number of fruits between the two lines. These results indicate that the aboveground biomass remained unchanged, while the dry matter distribution to the fruit increased. The distribution of assimilated products (dry matter) to the stems and leaves decreased, the weight per fruit (fresh weight and dry weight) increased, and the yield (fresh fruit weight per individual) increased.

According to a component analysis by Higashide and Heuvelink (J.Amer.Soc.Hort.Sci. 134: 460-465, 2009), the above yield increase effect is thought to be due to an improvement in the dry matter distribution rate to the fruit due to an increase in the fruit sink capacity (see Figure 2 in the same paper).

(Results of Test 2)
The results of the component analysis of Test 2 are shown in Table 3. The results were similar to those of Test 1 in the autumn crop with top pinching.

(Results of Test 3)
The tfw5.1 region was further restricted by comparing the high-yield marker allele region of the line in which the tfw5.1 region was shortened with the Japanese marker allele region for the dry matter partitioning rate to the fruit (Table 4). The partitioning rate is improved in line 1G, in which a marker allele derived from the high-yield type exists in the region between markers tma0252 and tbm1306. There were only two predicted genes with nonsynonymous substitutions in this region: between markers tbm1307 and tbm1306, and between markers tma0146 and TGS0155. In Table 4, A indicates the genotype of the high-yield tomato, and B indicates the genotype of the Japanese tomato.

(RNA-SEQ Mutation Analysis)
Mutation analysis of mRNA was performed using a next-generation sequencer for the tfw5.1 segregation lines 229G (high-yielding type) and 229M (Japanese type). Total RNA was extracted from the unripe fruit of each line by the Trizol method (https://ipmb.sinica.edu.tw/microarray/protocol.htm), and a fragmented cDNA library was created using these (TruSeq RNA Sample Prep Kit v2, Illumina). The library was subjected to next-generation sequencer (HiSeq 4000, Illumina) analysis, and the read sequence was obtained by paired-end sequencing (approximately 4 Gb/sample). The obtained read sequences were mapped to the reference tomato genome (version SL4.0, ftp://ftp.solgenomics.net/genomes/Solanum_lycopersicum/assembly/build_4.00/) using CLC Genomics Workbench software, and nonsynonymous substitutions were detected using a mutation detection program (Basic Variant Detection, etc.). As a result, differences (nonsynonymous substitutions) were found between the high-yielding type and the Japonica type in the amino acid sequences shown in SEQ ID NO: 1 or 2 encoded by two predicted genes in the limited tfw5.1 region (Table 4).

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

Claims (7)

1. A method for determining the degree of regulation of assimilate partitioning to fruit in a tomato plant, comprising:
The method includes a step of testing the plant for the presence or absence of a mutation that causes a substitution or deletion in the following amino acid (a) or (b):
In the inspecting step,
(a) the amino acid corresponding to the 870th amino acid of the protein consisting of the amino acid sequence shown in SEQ ID NO:1 is E (glutamic acid); or (b) the amino acid corresponding to the 402nd amino acid of the protein consisting of the amino acid sequence shown in SEQ ID NO:2 is S (serine);
the plant is determined to be a plant in which the amount of assimilates distributed to the fruit is increased,
The amino acid corresponding to the amino acid (a) or (b) is an amino acid identified as corresponding to the amino acid (a) or (b) based on a homology analysis method using the amino acid sequence shown in SEQ ID NO: 1 or 2 as a reference sequence. In the inspecting step,
In the above-mentioned plant, when the following (a″) or (b″) is satisfied:
(a″) the base corresponding to the 2609th base in the base sequence of SEQ ID NO: 3 is A;
(b'') the base corresponding to the 1204th base in the base sequence of SEQ ID NO: 4 is T;
The plant is determined to have an increased amount of assimilate partitioned to the fruit;
The base corresponding to the base (a'') or (b'') is a base identified as corresponding to the base (a'') or (b'') based on a homology analysis method using the base sequence of SEQ ID NO: 3 or SEQ ID NO: 4 as a reference sequence.
The method of claim 1. The plant contains a polynucleotide according to any one of (1) to (3) below:
(1) a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2;
(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 or 2 and having the function of regulating the amount of assimilate distributed to the fruit of the plant;
(3) A polynucleotide encoding a protein having a function of regulating the amount of assimilate distributed to the fruit of the plant, comprising an amino acid sequence in which 100 or less amino acids have been substituted, deleted, added, or inserted relative to the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence in which 46 or less amino acids have been substituted, deleted, added, or inserted relative to the amino acid sequence shown in SEQ ID NO:2;
The method according to claim 1 or 2, comprising an assimilate distribution control gene comprising: The method according to any one of claims 1 to 3, wherein the plant is a candidate plant for breeding material or a plant obtained by a breeding process. 1. A method for producing a plant that is a tomato with increased assimilate partitioning to the fruit, comprising:
A hybridization step of intraspecifically hybridizing the plant having an increased amount of assimilate distribution to fruits with another plant;
and an identification step of identifying a plant having an increased amount of assimilate distribution to fruit from the plant obtained by the crossbreeding step or a plant of a progeny thereof, by the method described in any one of claims 1 to 4. The method according to claim 5, further comprising, prior to the hybridization step, an identification step of identifying the plant in which the amount of assimilate partitioning from the test plant to the fruit has been increased by the method according to any one of claims 1 to 4. 1. Use of molecular markers for the regulation of assimilate partitioning to fruit in a tomato plant, comprising:
The molecular marker is
A base itself (SNP) corresponding to the following base (a') or (b'), or a consecutive polynucleotide containing the base:
(a') a base corresponding to the 2609th base in the base sequence of SEQ ID NO: 3 ;
(b') a base corresponding to the 1204th base in the base sequence of SEQ ID NO: 4 ;
The base corresponding to the base (a') or (b') is a base that has been identified as corresponding to the base (a') or (b') based on a homology analysis method using the base sequence of SEQ ID NO: 3 or SEQ ID NO: 4 as a reference sequence.

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