Solanum lycopersicum and its production method

Abstract

To make a novel Solanum lycopersicum that accumulates a high content of GABA by cross breeding.SOLUTION: The novel Solanum lycopersicum has a γ-aminobutyric acid metabolism-related gene located at chromosome 3 of Solanum pennellii, which accumulates γ-aminobutyric acid, and does not have a low fruit weight-related gene located at the chromosome 3.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a novel Solanum lycopersicum having unprecedented characteristics and a method for producing the same. [Background technology]

[0002] γ-Aminobutyric acid (GABA) is produced from glutamic acid by decarboxylation with glutamic acid decarboxylase. γ-Aminobutyric acid functions as an inhibitory transmitter in the central nervous system of humans and mammals. γ-Aminobutyric acid can be used as a medical drug for the treatment of symptoms associated with the sequelae of head trauma, and as a functional health ingredient in foods.

[0003] γ-Aminobutyric acid is found in many foods, including vegetables (tomatoes, kale, peppers, etc.), fruits (melons, grapes, bananas, etc.), and lactic acid fermentation products (pickles, yogurt, etc.). In particular, tomatoes (Solanum lycopersicum L.) have been improved to increase their γ-aminobutyric acid content.

[0004] For example, Patent Document 1 discloses a gene encoding glutamic acid decarboxylase (GAD) derived from a plant, which synthesizes γ-aminobutyric acid from glutamic acid (Glu). Patent Document 1 also discloses that a polynucleotide encoding a functional plant GAD enzyme is integrated into the plant genome to produce a transformed plant in order to accumulate high levels of GABA in the plant. Patent Document 1 also discloses, as publicly known information, sequence information for GAD in Arabidopsis thaliana, GAD in tobacco, GAD in petunia, and GAD in tomato.

[0005] Patent Document 2 discloses a composition containing γ-aminobutyric acid at a high concentration, using tomato fruits containing γ-aminobutyric acid at a high concentration, specifically, tomato fruits containing 800 to 3000 mg of γ-aminobutyric acid per kg of raw fruit. Non-Patent Document 1 discloses that the amount of GABA contained in tomato fruits increases due to salt stress. However, Non-Patent Document 1 also discloses that salt stress causes a decrease in fruit weight and the occurrence of fruit end rot.

[0006] In addition, Non-Patent Document 2 discloses that in order to increase the GABA content of tomatoes, the autoinhibitory domains of two endogenous GAD genes were deleted using genome editing technology, resulting in accumulation of 7 to 15 times the amount of GABA. Furthermore, Non-Patent Document 3 discloses that a tomato that highly accumulates GABA by genome editing was crossed with a pure lineage tomato variety "Aichi First" to produce a hybrid line, and the fruit traits such as GABA accumulation were evaluated. The hybrid line produced in Non-Patent Document 3 has the characteristic of highly accumulating GABA in the fruit. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Special Publication No. 2005-506034 [Patent Document 2] JP 2009-027987 A [Non-patent literature]

[0008] [Non-Patent Document 1] Metabolic Alterations in Organic Acids and γ-Aminobutyric Acid in Developing Tomato (Solanum lycopersicum L.) Fruits, Plant and Cell Physiology, 2010, Aug; 51(8): 1300-1314 [Non-Patent Document 2] Efficient increase of γ-aminobutyric acid (GABA) content in tomato fruits by targeted mutagenesis, SCIENTIFIC Reports, 2017, volume 7, page 7057 [Non-Patent Document 3] Utilization of a Genome-Edited Tomato (Solanum lycopersicum) with High Gamma Aminobutyric Acid Content in Hybrid Breeding, J Agric Food Chem, 2018, 66, 4, 963-971 Summary of the Invention [Problem to be solved by the invention]

[0009] As mentioned above, although there are examples of producing tomatoes with high GABA accumulation using genetic engineering and genome editing techniques, tomatoes with the property of accumulating high levels of GABA through cross breeding without using these techniques have not been known. In view of the above-mentioned circumstances, the present invention aims to provide a novel tomato (Solanum lycopersicum) that accumulates high levels of GABA through cross breeding without using genetic engineering and genome editing techniques, and a method for producing the same. [Means for solving the problem]

[0010] In order to achieve the above-mentioned objective, the inventors conducted intensive research and discovered that by using Solanum lycopersicum having a γ-aminobutyric acid metabolism-related gene located on chromosome 3 in Solanum pennellii, which accumulates γ-aminobutyric acid, it is possible to produce Solanum lycopersicum that has the γ-aminobutyric acid metabolism-related gene but does not have the low fruit weight-related gene located on chromosome 3, thereby completing the present invention.

[0011] The present invention encompasses the following: (1) Solanum lycopersicum, which has a γ-aminobutyric acid metabolism-related gene located on chromosome 3 in Solanum pennellii that accumulates γ-aminobutyric acid, but does not have a low fruit weight-related gene located on the same chromosome 3. (2) Solanum lycopersicum described in (1), in which a first DNA marker having the base sequence of SEQ ID NO: 1 as the wild type and the base sequence of SEQ ID NO: 2 as the cultivated type is a homozygous or heterozygous type of the wild type, and a second DNA marker having the base sequence of SEQ ID NO: 3 as the wild type and the base sequence of SEQ ID NO: 4 as the cultivated type is a homozygous type of the cultivated type. (3) Solanum lycopersicum described in (1) above, which is a progeny line of an introgression line IL3-3 (accession No. LA3488) between the wild species Solanum pennellii LA0716 and the cultivated species Solanum lycopersicum M82.

[0012] (4) A method for producing Solanum lycopersicum, comprising the steps of: creating a hybrid line whose ancestors are a first Solanum lycopersicum having a γ-aminobutyric acid metabolism-related gene located on chromosome 3 in Solanum pennelli, which accumulates γ-aminobutyric acid, and an arbitrary second Solanum lycopersicum; and selecting a Solanum lycopersicum hybrid line that has the γ-aminobutyric acid metabolism-related gene but does not have the low fruit weight-related gene located on chromosome 3. (5) A method for producing Solanum lycopersicum according to (4), characterized in that the step of producing the hybrid line comprises producing an inbred hybrid line and / or a backcross line. (6) A method for producing Solanum lycopersicum described in (4), characterized in that in the above-mentioned selection step, a hybrid line is selected in which a first DNA marker having the base sequence of SEQ ID NO: 1 as the wild type and the base sequence of SEQ ID NO: 2 as the cultivated type is a homozygous or heterozygous type of the wild type, and a second DNA marker having the base sequence of SEQ ID NO: 3 as the wild type and the base sequence of SEQ ID NO: 4 as the cultivated type is a homozygous type of the cultivated type. (7) The method for producing Solanum lycopersicum according to (4), wherein the first Solanum lycopersicum is an introgression line IL3-3 (Accession No. LA3488) between the wild species Solanum pennellii LA0716 strain and the cultivated species Solanum lycopersicum M82.

[0013] (8) Use of a pair of polynucleotides consisting of a polynucleotide identified by the base sequence of SEQ ID NO: 1 and a polynucleotide identified by the base sequence of SEQ ID NO: 2, which can be determined to have a γ-aminobutyric acid metabolism-related gene located on chromosome 3 in Solanum pennellii that accumulates γ-aminobutyric acid when the base sequence of SEQ ID NO: 1 is the wild type and the base sequence of SEQ ID NO: 2 is the cultivated type, and the hybrid line is homozygous or heterozygous for the wild type, as a DNA marker. (9) The use according to (8), characterized in that it is determined whether a hybrid line is of a wild type homozygous or heterozygous type based on at least one of the multiple mutation sites contained in the base sequence of SEQ ID NO: 1 and the base sequence of SEQ ID NO: 2.

[0014] (10) Use of a pair of polynucleotides consisting of a polynucleotide identified by the base sequence of SEQ ID NO: 3 and a polynucleotide identified by the base sequence of SEQ ID NO: 4, wherein the base sequence of SEQ ID NO: 3 is the wild type and the base sequence of SEQ ID NO: 4 is the cultivated type, and when a hybrid line is homozygous for the cultivated type, the pair of polynucleotides can be determined to not have a low fruit weight-related gene located on chromosome 3 in Solanum pennellii as a DNA marker. (11) The use according to (10), characterized in that it is determined whether a hybrid line is homozygous for a cultivated species type based on at least one of multiple mutation sites contained in the base sequence of SEQ ID NO: 3 and the base sequence of SEQ ID NO: 4.

[0015] (12) A DNA marker detection kit comprising a detection means for detecting at least one of a plurality of mutation sites contained in a pair of polynucleotides consisting of a polynucleotide specified by the base sequence of SEQ ID NO:1 and a polynucleotide specified by the base sequence of SEQ ID NO:2, and a plurality of mutation sites contained in a pair of polynucleotides consisting of a polynucleotide specified by the base sequence of SEQ ID NO:3 and a polynucleotide specified by the base sequence of SEQ ID NO:4. (13) The DNA marker detection kit according to (12), characterized in that the detection means includes a pair of primers for amplifying a region including the mutation site to be detected, and / or a pair of probes consisting of a wild-type probe that hybridizes to the wild-type at the mutation site to be detected and a cultivated-type probe that hybridizes to the cultivated-type. Effect of the Invention

[0016] The Solanum lycopersicum of the present invention has the characteristics of having an extremely high accumulation amount of γ-aminobutyric acid per unit fruit weight, and extremely little reduction in fruit weight due to high accumulation of γ-aminobutyric acid, resulting in an extremely high γ-aminobutyric acid content.

[0017] In addition, the method for producing Solanum lycopersicum according to the present invention can produce a Solanum lycopersicum hybrid line that has a γ-aminobutyric acid metabolism-related gene located on chromosome 3 in Solanum pennelli, which accumulates γ-aminobutyric acid, by utilizing the first Solanum lycopersicum, which has the γ-aminobutyric acid metabolism-related gene located on chromosome 3. Therefore, according to the method for producing Solanum lycopersicum according to the present invention, it is possible to produce Solanum lycopersicum with an extremely high γ-aminobutyric acid content. [Brief description of the drawings]

[0018] [Figure 1] Alignment of DNA region amplified by marker No. 2 [Diagram 2] Alignment of DNA regions amplified by marker No. 32 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The Solanum lycopersicum according to the present invention is characterized in that it has a γ-aminobutyric acid metabolism-related gene located on chromosome 3 in Solanum pennelli, which accumulates γ-aminobutyric acid, and does not have a low fruit weight-related gene located on the above chromosome 3. Among the introgression lines between Solanum pennelli and Solanum lycopersicum, we focused on the fact that one containing a specific region on chromosome 3 of Solanum pennelli has relatively small fruit weight but accumulates a large amount of γ-aminobutyric acid. We then assumed that the γ-aminobutyric acid metabolism-related gene and the low fruit weight-related gene are present in the specific region.

[0020] The Solanum lycopersicum of the present invention has the novel characteristics of having extremely high accumulation of γ-aminobutyric acid per unit fruit weight and extremely little reduction in fruit weight due to high accumulation of γ-aminobutyric acid, by separating these γ-aminobutyric acid metabolism-related genes and low fruit weight-related genes.

[0021] Here, the γ-aminobutyric acid metabolism-related genes refer to genes related to the trait of increasing the amount of γ-aminobutyric acid in fruits, and are thought to include, for example, genes involved in γ-aminobutyric acid synthesis (e.g., glutamic acid decarboxylase gene), genes suppressing the metabolism of γ-aminobutyric acid, genes involved in the biosynthesis of glutamic acid, which is a substrate for γ-aminobutyric synthesis, genes suppressing the metabolism of glutamic acid, etc. These genes involved in γ-aminobutyric acid synthesis (e.g., glutamic acid decarboxylase gene), etc. located on the third chromosome of Solanum pennellii are thought to function to accumulate γ-aminobutyric acid at a higher level than the homologous genes in Solanum lycopersicum.

[0022] In addition, low fruit weight-related genes refer to genes related to traits that reduce fruit weight, and are thought to include, for example, genes related to fruit growth. These fruit growth-related genes located on chromosome 3 of Solanum pennelli are thought to function to reduce fruit weight more than their homologous genes in Solanum lycopersicum.

[0023] The introgression line between Solanum pennellii and Solanum lycopersicum is not particularly limited, but for example, a line in which a part of the chromosome of the cultivated species Solanum lycopersicum M82 is replaced with a chromosome of the wild species Solanum pennellii LA0716 can be used. The UC Davis CM Rick Tomato Genetics Resource Center (TGRC http: / / tgrc.ucdavis.edu) provides various introgression lines in which a part of the chromosome of the cultivated species Solanum lycopersicum M82 is replaced with a chromosome of the wild species Solanum pennellii LA0716. Among these introgressions, the introgression line IL3-3 (accession No. LA3488), in which a part of the 3rd chromosome is replaced, has a relatively small fruit weight but shows a trait of high accumulation of γ-aminobutyric acid.

[0024] Therefore, the Solanum lycopersicum according to the present invention can be produced, for example, from the introgression line IL3-3 (Accession No. LA3488). That is, a large number of progeny lines are produced by crossing the introgression line IL3-3 with another cultivated species, Solanum lycopersicum, as parents. Among the progeny lines obtained, genotypes having a γ-aminobutyric acid metabolism-related gene and not having the low fruit weight-related gene located on the above-mentioned chromosome 3 are selected.

[0025] Here, the crossbreeding is not particularly limited, and any crossbreeding technique commonly used in breeding and the like can be used. For example, crossbreeding techniques include self-crossing and backcrossing. In producing the Solanum lycopersicum of the present invention, a method can be adopted in which the above-mentioned introgression line IL3-3 and another cultivated species of Solanum lycopersicum are used as parents, and self-crossing is performed for several generations, backcrossing is performed for several generations, or self-crossing and backcrossing are appropriately repeated.

[0026] The cultivated species of Solanum lycopersicum is not particularly limited, and generally available ones can be used. Examples of the cultivated species of Solanum lycopersicum that can be used include M82, Furikoma, Tomato Intermediate Mother No. 3, NT604, Natsunokoma, Yellow Carol, Del Harvest No. 2, Takii Otama 193, Olmeca, Lycoball, TY First, KRN-2011, Sweet Memory, TTM056, Aitaki No. 1, SAKTOM12, DG07-1, TONTELLE, NINIVE, PRS, etc.

[0027] Specifically, whether the obtained progeny has the above-mentioned γ-aminobutyric acid metabolism-related gene and does not have the low fruit weight-related gene can be determined using the first DNA marker and the second DNA marker. That is, the first DNA marker and the second DNA marker are DNA markers that can distinguish the genotype having the γ-aminobutyric acid metabolism-related gene and not having the low fruit weight-related gene. More specifically, genomic DNA is extracted from the obtained progeny according to a standard method, and the first DNA marker and the second DNA marker in the extracted genomic DNA are analyzed.

[0028] The first DNA marker may have, for example, the base sequence of SEQ ID NO: 1 as the wild type and the base sequence of SEQ ID NO: 2 as the cultivated type. That is, the base sequence of SEQ ID NO: 1 is derived from the wild species Solanum pennellii LA0716, and the base sequence of SEQ ID NO: 2 is derived from the cultivated species Solanum lycopersicum M82. The results of pairwise alignment and comparison of the base sequences of SEQ ID NO: 1 and SEQ ID NO: 2 are shown in FIG. 1. As shown in the Examples below, the first DNA marker shown in FIG. 1 has, as a result of DNA marker analysis, the base sequences of "PK993" and "Heinz1706" on the third and fourth lines from the top are the cultivated type, and the base sequences of "TK8728" and "19F273" on the first and second lines from the top are the wild type. The results of comparison of the base sequences of SEQ ID NO: 1 and SEQ ID NO: 2 are shown in Table 1.

[0029] [Table 1]

[0030] The first DNA marker is specified by the base sequences of SEQ ID NOs: 1 and 2, but is not limited to SEQ ID NOs: 1 and 2 themselves, and may be a base sequence different from SEQ ID NOs: 1 and 2, as long as it preserves at least one, preferably three or more, more preferably five or more, even more preferably seven or more, and most preferably ten types of mutation sites among the above mutation sites 1-1 to 1-10. Examples of the base sequence different from SEQ ID NO: 1 contained in the first DNA marker include a base sequence that preserves at least one, preferably three or more, more preferably five or more, even more preferably seven or more, and most preferably ten types of mutation sites among the above mutation sites 1-1 to 1-10, and has a degree of identity of 90% or more with SEQ ID NO: 1, preferably a degree of identity of 95% or more with SEQ ID NO: 1, more preferably a degree of identity of 97% or more with SEQ ID NO: 1, and most preferably a degree of identity of 99% or more with SEQ ID NO: 1. Similarly, examples of the base sequence different from SEQ ID NO:2 contained in the first DNA marker include a base sequence that conserves at least one, preferably three or more, more preferably five or more, even more preferably seven or more, and most preferably ten mutation sites among the above mutation sites 1-1 to 1-10, and has a degree of identity of 90% or more with SEQ ID NO:2, preferably a degree of identity of 95% or more with SEQ ID NO:1, more preferably a degree of identity of 97% or more with SEQ ID NO:1, and most preferably a degree of identity of 99% or more with SEQ ID NO:1.

[0031] In addition, the second DNA marker may have, for example, the base sequence of SEQ ID NO: 3 as the wild type and the base sequence of SEQ ID NO: 4 as the cultivated type. That is, the base sequence of SEQ ID NO: 3 is derived from the wild species Solanum pennellii LA0716, and the base sequence of SEQ ID NO: 4 is derived from the cultivated species Solanum lycopersicum M82. The results of pairwise alignment and comparison of the base sequence of SEQ ID NO: 3 and the base sequence of SEQ ID NO: 4 are shown in FIG. 2. As shown in the Examples described later, the second DNA marker shown in FIG. 2 has the base sequences of the cultivated type, “PK993”, “19F273”, and “Heinz1706” in the first to third lines from the top, and the base sequence of “TK8728” in the fourth line from the top, as a result of DNA marker analysis. The results of comparison of the base sequence of SEQ ID NO: 3 and the base sequence of SEQ ID NO: 4 are shown in Table 2.

[0032] [Table 2]

[0033] The second DNA marker is specified by the base sequences of SEQ ID NOs: 3 and 4, but is not limited to SEQ ID NOs: 3 and 4 themselves, and may be a base sequence different from SEQ ID NOs: 3 and 4 as long as it preserves at least one, preferably two or more, more preferably three or more, and even more preferably four of the above mutation sites 2-1 to 2-4. The base sequence different from SEQ ID NO: 3 contained in the second DNA marker may be a base sequence that preserves at least one, preferably two or more, more preferably three or more, and even more preferably four of the above mutation sites 2-1 to 2-4, and has a degree of identity of 90% or more with SEQ ID NO: 3, preferably a degree of identity of 95% or more with SEQ ID NO: 3, more preferably a degree of identity of 97% or more with SEQ ID NO: 3, and most preferably a degree of identity of 99% or more with SEQ ID NO: 3. Similarly, examples of the base sequence different from SEQ ID NO:4 contained in the second DNA marker include a base sequence that conserves at least one, preferably two or more, more preferably three or more, and even more preferably four of the above mutation sites 2-1 to 2-4, and has a degree of identity of 90% or more with SEQ ID NO:4, preferably a degree of identity of 95% or more with SEQ ID NO:4, more preferably a degree of identity of 97% or more with SEQ ID NO:4, and most preferably a degree of identity of 99% or more with SEQ ID NO:4.

[0034] The first DNA marker and / or the second DNA marker described above are each polynucleotides in a specific region present in genomic DNA, and are polynucleotides that can distinguish whether a species is of the wild type or the cultivated type.

[0035] That is, the polynucleotide specified by the base sequence of SEQ ID NO: 1 and the polynucleotide specified by the base sequence of SEQ ID NO: 2 can be used to determine whether a hybrid line has the characteristic of having the above-mentioned γ-aminobutyric acid metabolism-related gene. Also, the polynucleotide specified by the base sequence of SEQ ID NO: 3 and the polynucleotide specified by the base sequence of SEQ ID NO: 4 can be used to determine whether a hybrid line has the characteristic of not having the above-mentioned low fruit weight-related gene.

[0036] On the other hand, the mating type of each of the first and second DNA markers can be determined based on a conventional method. Specifically, the mating type of each of the first and second DNA markers can be determined by using a detection means for detecting at least one of the above-mentioned multiple mutation sites contained in the first DNA marker and the above-mentioned multiple mutation sites contained in the second DNA marker.

[0037] For example, the first DNA marker and the second DNA marker have a 56-base deletion mutation (mutation site 1-3) and a 10-base deletion mutation (mutation site 2-3), respectively, as differences between the wild type and the cultivated type. Therefore, as the detection means, a pair of primers is designed to sandwich the mutation site, and the mating types of the first DNA marker and the second DNA marker can be determined based on the base length of the DNA fragment amplified by a nucleic acid amplification reaction using the primers. Nucleic acid amplification can be performed, for example, by the PCR method, but it may also be performed by other known amplification methods, such as the LCR (ligase chain reaction) method and the LAMP (loop-mediated isothermal amplification) method.

[0038] In addition to this method, the junction types of the first and second DNA markers can be determined by directly reading the base sequences of the first and second DNA markers using a sequencer, etc. Furthermore, the junction types of the first and second DNA markers can be determined by using the so-called SSCP (Single Strand Conformation Polymorphism) method.

[0039] Furthermore, as a different method, when the sequence pattern of the mutation site for each of the first and second DNA markers serves as a restriction enzyme recognition sequence, a DNA fragment containing the mutation site can be amplified, and the mating types of each of the first and second DNA markers can be determined based on the presence or absence of cleavage of the obtained DNA fragment by the restriction enzyme. In addition, the dCAPS (derived CAPS) method, in which a restriction enzyme recognition site is created by a primer set including an intentionally designed mismatch primer, may also be used.

[0040] Furthermore, for each of the first and second DNA markers, a method of designing a probe that specifically hybridizes for each sequence pattern for each mutation site can be applied as the detection means. Specifically, a pair of probes is designed, consisting of a wild-type probe that hybridizes to the wild-type at the mutation site to be detected and a cultivated-type probe that hybridizes to the cultivated-type. Then, for each of the first and second DNA markers, a DNA fragment containing the mutation site to be detected is amplified, and the amplified DNA fragment is hybridized with the pair of probes, thereby determining the mating type of each of the first and second DNA markers.

[0041] Furthermore, a method can be applied in which primers are designed for each of the first and second DNA markers by partially including the sequence patterns of the respective mutation sites. In this case, the first and second DNA markers are amplified using the designed primers, and the zygosity of each of the first and second DNA markers can be determined based on the presence or absence of amplification.

[0042] The first DNA marker described above contains one deletion mutation and nine single nucleotide polymorphisms. The first DNA marker is of the wild type, which means that when all the sequence patterns of the 10 types of mutation sites shown in Table 1 are revealed, at least 5 sites, preferably 6 sites, more preferably 7 sites, more preferably 8 sites, even more preferably 9 sites, and most preferably 10 sites are of the wild type sequence pattern. The first DNA marker is of the cultivated type, which means that when all the sequence patterns of the 10 types of mutation sites shown in Table 1 are revealed, at least 5 sites, preferably 6 sites, more preferably 7 sites, more preferably 8 sites, even more preferably 9 sites, and most preferably 10 sites are of the cultivated type sequence pattern.

[0043] However, when determining the mating type of the first DNA marker, it is not necessary to identify all of the sequence patterns of the above-mentioned 10 mutation sites, and it is sufficient to identify the sequence patterns of at least one, or 2 to 9, of the 10 types of mutation sites. Since these 10 mutation sites are in linkage disequilibrium, identifying the sequence patterns of a small number of mutation sites (for example, one) allows highly accurate prediction of sequence patterns of other mutation sites.

[0044] Furthermore, the first DNA marker being heterozygous means that it has both the wild type sequence pattern and the cultivated type sequence pattern with respect to the mutation site shown in Table 1.

[0045] On the other hand, the second DNA marker described above contains one deletion mutation and three single nucleotide polymorphisms. The second DNA marker being of the wild type means that when all the sequence patterns of the four mutation sites shown in Table 2 are revealed, at least two, preferably three, and most preferably four, are of the wild type. The second DNA marker being of the cultivated type means that when all the sequence patterns of the four mutation sites shown in Table 2 are revealed, at least two, preferably three, and most preferably four, are of the cultivated type.

[0046] However, when determining the mating type of the second DNA marker, it is not necessary to clarify all of the above-mentioned four mutation sites, and it is sufficient to clarify the sequence pattern of at least one, or two or three, of the four types of mutation sites. Since these four mutation sites are in linkage disequilibrium, by identifying the sequence pattern of a small number of mutation sites (for example, one), the sequence patterns of the other mutation sites can be predicted with high accuracy.

[0047] Furthermore, the second DNA marker being homozygous for the cultivar type means that it has a cultivar-type sequence pattern with respect to the mutation site shown in Table 2.

[0048] By analyzing the first and second DNA markers for the progeny obtained by the crossing as described above, it can be determined whether or not the progeny has the characteristic of having a γ-aminobutyric acid metabolism-related gene located on chromosome 3 in Solanum pennelli and not having a low fruit weight-related gene located on chromosome 3. As a result of the determination, Solanum lycopersicum having the characteristic of having a γ-aminobutyric acid metabolism-related gene and not having a low fruit weight-related gene located on chromosome 3 has a very high accumulation amount of γ-aminobutyric acid per unit fruit weight and a very small decrease in fruit weight due to high accumulation of γ-aminobutyric acid, and therefore has a very high γ-aminobutyric acid content.

[0049] Here, the amount of γ-aminobutyric acid accumulated per unit fruit weight is not particularly limited, but can be evaluated, for example, as the weight of γ-aminobutyric acid contained per unit weight (e.g., 1 mg) of fruit. The Solanum lycopersicum according to the present invention shows a significantly higher amount of γ-aminobutyric acid accumulated compared to the amount of γ-aminobutyric acid accumulated in the cultivated species Solanum lycopersicum used as one of the parents. Significance means statistical significance, and can be determined by methods such as Student's t-test and Welch's t-test. For example, the Solanum lycopersicum of the present invention accumulates 105% of the γ-aminobutyric acid amount, preferably 110% of the γ-aminobutyric acid amount, preferably 130% of the γ-aminobutyric acid amount, preferably 150% of the γ-aminobutyric acid amount, preferably 170% of the γ-aminobutyric acid amount, preferably 200% of the γ-aminobutyric acid amount, preferably 230% of the γ-aminobutyric acid amount, preferably 250% of the γ-aminobutyric acid amount, preferably 300% of the γ-aminobutyric acid amount, preferably 350% of the γ-aminobutyric acid amount, compared to the cultivated Solanum lycopersicum used as one of the parents.

[0050] In addition, the reduction in fruit weight due to high accumulation of γ-aminobutyric acid can be evaluated, for example, by the weight of the fruit or the diameter of the fruit, without being particularly limited. As shown in the examples described later, the progeny of the high accumulation of γ-aminobutyric acid obtained by crossing Solanum pennelli, which has a high accumulation amount of γ-aminobutyric acid, with the cultivated species Solanum lycopersicum tends to have a significantly reduced fruit weight compared to the cultivated species Solanum lycopersicum. However, the Solanum lycopersicum according to the present invention has a characteristic that, despite the high accumulation of γ-aminobutyric acid, the fruit weight is higher than that of the cultivated species Solanum lycopersicum used as one of the parents, or even if the fruit weight is reduced, the degree of the reduction is extremely small. For example, the Solanum lycopersicum of the present invention preferably has a fruit weight of 100 to 110% of the fruit weight of the cultivated species Solanum lycopersicum used as one of the parents, preferably has a fruit weight of 55 to 100%, more preferably has a fruit weight of 60 to 100%, even more preferably has a fruit weight of 70 to 100%, even more preferably has a fruit weight of 80 to 100%, and most preferably has a fruit weight of 85 to 100%. EXAMPLES

[0051] The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to the following examples.

[0052] [Example 1] In Example 1, a novel hybrid line that accumulates high amounts of γ-aminobutyric acid was developed using processing tomatoes. <Material> The strains of Solanum lycopersicum used in this example are shown in Table 3. TK8728 was obtained from the UC Davis CM Rick Tomato Genetics Resource Center (TGRC http: / / tgrc.ucdavis.edu). TK8728 is an introgression line IL3-3 (accession No. LA3488) between the wild species Solanum pennellii LA0716 and the cultivated species Solanum lycopersicum M82.

[0053] This was crossed with the processing tomato variety PK993 cultivated by Kagome Co., Ltd. to obtain the F1 generation. The F1 generation was self-propagated to obtain the F2 generation (16F280, 16F281, 16F282). The self-propagation of the F2 generation was repeated twice to obtain the F4 generation (18F308, 18F307). The self-propagation of the F4 generation was repeated twice to obtain the F6 generation (19F273). These plants were cultivated in a field owned by Kagome Co., Ltd.

[0054]

Table 3

[0055] <Method> <Creation of DNA Marker> Based on the nucleotide sequence information of the wild species Solanum pennellii LA0716 line and Solanum lycopersicum published in a publicly known database (Sol Genomics Network https: / / solgenomics.net / ), a DNA marker capable of detecting polymorphisms between the wild species Solanum pennellii LA0716 line and Solanum lycopersicum Heinz1706 was created. Table 4 shows the positions and nucleotide sequences of the primers on chromosome 3, the annealing temperature, and the band pattern in the created DNA marker. To identify the positions of the primers on chromosome 3, the map version SL3.0 published in a publicly known database was used.

[0056]

Table 4

[0057] <DNA Marker Assay> PCR was performed using DNA extracted from each Solanum lycopersicum line as a template. The PCR product was electrophoresed using a capillary electrophoresis apparatus (microchip electrophoresis apparatus for DNA / RNA analysis MCE-202, manufactured by Shimadzu Corporation). The genotype was identified from the band pattern obtained as a result of electrophoresis. The reaction conditions for PCR are shown in Table 5. Capillary electrophoresis was performed according to a predetermined protocol of the apparatus.

[0058]

Table 5

[0059] <Measurement of GABA content and fruit weight> Ripe red fruits were harvested from each Solanum lycopersicum line. After harvesting, the fruit weight was measured. The fruit weight was measured using an electronic balance (ME1002E, manufactured by Mettler). The fruits for which the fruit weight measurement was completed were crushed in a mixer for 1 minute. After crushing, they were filled into a bottle container and heated in a boiling water bath for 30 minutes. After heating, they were filtered through filter paper (quantitative filter paper No. 5A, manufactured by ADVANTEC). The filtrate was used as a sample for measuring the GABA content. The GABA was measured by the following method. The filtrate was diluted 10-fold (by volume) with 3% sulfosalicylic acid, then filtered through a membrane filter (DISMIC-25CS0.20μm 25CS020AN, manufactured by ADVANTEC), and measured using an amino acid automatic analyzer (L-8900, manufactured by Hitachi, Ltd.). The amount of GABA was calculated from a calibration curve prepared using a commercially available standard.

[0060] <Results> The results of measuring the GABA content and fruit weight are shown in Table 6.

[0061]

Table 6

[0062] In addition, the results of GABA content, fruit weight, and DNA marker analysis for the progeny produced in this example are summarized in Table 7.

[0063]

Table 7

[0064] <Analysis of TK8728> As shown in Table 6, in the introgression line TK8728, compared with the background variety Solanum lycopersicum M82, the GABA content increased up to about 3-fold, but the fruit weight decreased up to about 50%. In TK8728, a part of chromosome 3 has been replaced by the genotype of the wild species Solanum pennellii LA0716 line. From this, it was suggested that in this region, there may be a gene that highly contains GABA (hereinafter referred to as "γ-aminobutyric acid metabolism-related gene") and a gene that reduces fruit weight (hereinafter referred to as "low fruit weight-related gene").

[0065] <Analysis of F2 generation> In the F2 generation, compared with 16F280, 16F281 had a higher GABA content and a lower fruit weight. As a result of the Student's t-test, the GABA content of 16F281 was significantly higher (p = 0.000051 < 0.05) compared with 16F280, and the fruit weight was significantly lower (p = 0.020 < 0.05). From this result and the result of DNA marker analysis, it was inferred that the γ-aminobutyric acid metabolism-related gene and the low fruit weight-related gene exist between No. 1 and No. 3. In the F2 generation, individuals with No. 2 being heterozygous were self-propagated to obtain the F3 generation. Furthermore, in the F3 generation, individuals with No. 2 being heterozygous were self-propagated to obtain the F4 generation.

[0066] <Analysis of F4 generation> In the F4 generation, compared with 18F307, 18F308 had a higher GABA content and a lower fruit weight. From these results and those of DNA marker analysis, it was inferred that the genes related to γ-aminobutyric acid metabolism and those related to low fruit weight are present near No. 2. In the F4 generation, individuals with high GABA content and fruit weight were selected and self-propagated to obtain the F5 generation. Furthermore, in the F5 generation, individuals with high GABA content and fruit weight were selected and self-propagated to obtain the F6 generation.

[0067] <Analysis of the F6 generation> In the F6 generation, a line (19F273) was obtained in which the GABA content increased by about twofold compared with the control variety PK993, while the fruit weight remained at 85% or more. Looking at the results of DNA marker analysis, in 19F273, No. 2 and No. 31 are wild-type, and No. 32, No. 8, No. 6, and No. 3 are cultivated-type. As a result of Student's t-test, the GABA content of 19F273 was significantly higher compared with PK993 (p = 0.01 < 0.05). On the other hand, the fruit weight of 19F273 was significantly lower compared with PK993 (p = 0.047 < 0.05). However, the fruit weight of 19F273 was significantly higher compared with 16F281, which is a line with a high GABA content (p = 0.0008 < 0.05), and there was no significant difference compared with 16F280, which is a line with a normal GABA content (p = 0.27 > 0.05).

[0068] From these results, it was determined that in 19F273, the genes related to γ-aminobutyric acid metabolism and those related to low fruit weight could be separated. From the above, by selecting Solanum lycopersicum in which the gene regions of No. 2 and No. 31 are wild-type and the gene region of No. 32 is cultivated-type, it became clear that a new Solanum lycopersicum with a high GABA content and a suppressed decrease in fruit weight could be cultivated.

[0069] <Sequence analysis of the region amplified by the DNA marker> Sequence analysis of the region amplified by marker No. 2 was performed for TK8728, 19F273, PK993, and Heinz1706. Sequencing was outsourced to Eurofins Genomics, Inc. The sequence alignment results are shown in Figure 1. For marker No. 2, the wild-type base sequence is shown in SEQ ID NO: 1, and the cultivated-type base sequence is shown in SEQ ID NO: 2. In the region amplified by marker No. 2, 10 mutations linked to the trait of high GABA content were confirmed (first DNA marker).

[0070] Sequence analysis of the region amplified by marker No. 32 was performed for TK8728, 19F273, PK993, and Heinz1706 in the same manner as described above. The sequence alignment results are shown in Figure 2. For marker No. 32, the wild-type base sequence is shown in SEQ ID NO: 3, and the cultivated-type base sequence is shown in SEQ ID NO: 4. In the region amplified by marker No. 32, four mutations linked to the trait of reduced fruit weight were confirmed (second DNA marker).

[0071] [Example 2] In Example 2, a novel hybrid line that accumulates high amounts of γ-aminobutyric acid was developed using fresh tomatoes. <Material> The Solanum lycopersicum used in this example are as follows. TK8728 is the same as that used in Example 1. This was crossed with PK941, a fresh tomato variety developed by Kagome Co., Ltd., to obtain the F1 generation. PK941 was backcrossed four times to the F1 generation to obtain the BC4F1 generation. The BC4F1 generation was further self-pollinated to obtain the BC4F2 generation (20K101-1, 20K101-2, 20K101-3). These plants were cultivated in a Venlo-type greenhouse owned by Kagome Co., Ltd. <Method> The DNA markers used were the same as those produced in Example 1. DNA marker assay, measurement of GABA content, and measurement of fruit weight were performed in the same manner as in Example 1. <Result> The results of measuring GABA content and fruit weight are shown in Table 8.

[0072]

Table 8

[0073] In addition, for the progeny produced in this example, the results of GABA content, fruit weight, and DNA marker analysis were summarized and shown in Table 9.

[0074]

Table 9

[0075] <Analysis of BC4F2 generation> As shown in Table 8, in the BC4F2 generation, 20K101-1 had a GABA content and fruit weight similar to those of PK941. On the other hand, 20K101-3 had a higher GABA content than PK941, but the fruit weight decreased. 20K101-2 had a fruit weight equivalent to that of PK941 while also having a higher GABA content. Also, from the results of the DNA marker analysis shown in Table 9, since 20K101-2 had only No. 2 as heterozygous and No. 31 and No. 32 as cultivated species types, it became clear that by selecting Solanum lycopersicum with a wild species type or heterozygous type in the gene region of No. 2 and a cultivated species type in the genotype of No. 32, a Solanum lycopersicum with high GABA content and suppressed reduction in fruit weight can be bred. Note that No. 31 was considered to be either a wild species type or a cultivated species type based on the results of Examples 1 and 2.

Industrial Applicability

[0076] The present invention can be used, for example, in breeding new varieties of Solanum lycopersicum that highly accumulate GABA.

Claims

1. Solanum lycopersicum having a γ-aminobutyric acid metabolism-related gene located on chromosome 3 in Solanum pennellii that accumulates γ-aminobutyric acid, and not having a low fruit weight-related gene located on chromosome 3; a first DNA marker having the base sequence of SEQ ID NO: 1 as a wild type and the base sequence of SEQ ID NO: 2 as a cultivated type, which is a homozygous or heterozygous type of the wild type; and a second DNA marker having the base sequence of SEQ ID NO: 3 as a wild type and the base sequence of SEQ ID NO: 4 as a cultivated type, which is a homozygous type of the cultivated type. Solanum lycopersicum.

2. The Solanum lycopersicum according to claim 1, which is a progeny line of an introgression line IL3-3 (Accession No. LA3488) between the wild species Solanum pennellii LA0716 line and the cultivated species Solanum lycopersicum M82.

3. A step of preparing a hybrid line having as its ancestors a first Solanum lycopersicum having a γ-aminobutyric acid metabolism-related gene located on chromosome 3 in Solanum pennellii that accumulates γ-aminobutyric acid and an arbitrary second Solanum lycopersicum; and selecting a Solanum lycopersicum hybrid line having the γ-aminobutyric acid metabolism-related gene and not having the low fruit weight-related gene located on the third chromosome. The step of selecting comprises selecting a hybrid line in which a first DNA marker having the base sequence of SEQ ID NO: 1 as a wild type and the base sequence of SEQ ID NO: 2 as a cultivated type is a homozygous or heterozygous type of the wild type, and a second DNA marker having the base sequence of SEQ ID NO: 3 as a wild type and the base sequence of SEQ ID NO: 4 as a cultivated type is a homozygous type of the cultivated type. How to grow Solanum lycopersicum.

4. The method for producing Solanum lycopersicum according to claim 3, characterized in that the step of producing the hybrid line comprises producing an inbred hybrid line and / or a backcross line.

5. The method for producing Solanum lycopersicum according to claim 3, characterized in that the first Solanum lycopersicum is an introgression line IL3-3 (Accession No. LA3488) between the wild species Solanum pennellii LA0716 line and the cultivated species Solanum lycopersicum M82.

6. A DNA marker detection kit comprising a detection means for detecting at least one of a plurality of mutation sites contained in a pair of polynucleotides consisting of a polynucleotide specified by the base sequence of SEQ ID NO:1 and a polynucleotide specified by the base sequence of SEQ ID NO:2, and a plurality of mutation sites contained in a pair of polynucleotides consisting of a polynucleotide specified by the base sequence of SEQ ID NO:3 and a polynucleotide specified by the base sequence of SEQ ID NO:

4.

7. The DNA marker detection kit described in claim 6, characterized in that the detection means includes a pair of primers that amplify a region including the mutation site to be detected, and / or a pair of probes consisting of a wild-type probe that hybridizes to the wild-type at the mutation site to be detected and a cultivated-type probe that hybridizes to the cultivated-type.

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