JP7602907B2 - Solanum lycopersicum and its production method - Google Patents

Description

The present invention relates to a novel Solanum lycopersicum with unprecedented characteristics and a method for producing the same.

γ-Aminobutyric acid (sometimes called GABA or GABA) is produced from glutamic acid through a decarboxylation reaction by 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 to treat symptoms associated with the aftereffects of head trauma, and as a functional health ingredient in foods.

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 (scientific name: Solanum lycopersicum L.) have been improved to increase their γ-aminobutyric acid content.

For example, Patent Document 1 discloses a gene encoding plant-derived glutamic acid decarboxylase (GAD) that synthesizes γ-aminobutyric acid from glutamic acid (Glu). Patent Document 1 also discloses that a polynucleotide encoding a functional plant GAD enzyme has been incorporated 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.

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

Non-Patent Document 2 discloses that, in order to increase the GABA content in tomatoes, genome editing technology was used to delete the autoinhibitory domains of two endogenous GAD genes, resulting in the accumulation of 7-15 times the amount of GABA. Furthermore, Non-Patent Document 3 discloses that a tomato that accumulates high amounts of GABA through genome editing was crossed with a pure line 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 accumulating high amounts of GABA in the fruit.

Special Publication No. 2005-506034 JP 2009-027987 A

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 Efficient increase of γ-aminobutyric acid (GABA) content in tomato fruits by targeted mutagenesis, SCIENTIFIC Reports, 2017, volume 7, page 7057 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

As mentioned above, although there are examples of producing tomatoes with high GABA accumulation using genetic engineering and genome editing techniques, tomatoes with the characteristic of accumulating high levels of GABA through cross breeding without using these techniques were not 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 or genome editing techniques, and a method for producing the same.

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

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, and 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 according to (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.

(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 pennellii that 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 and does not have the low fruit weight-related gene located on chromosome 3.
(5) The method for producing Solanum lycopersicum according to (4), wherein 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 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 line and the cultivated species Solanum lycopersicum M82.

(8) Use of 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, wherein 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 a homozygous or heterozygous type for the wild type, in Solanum pennellii that accumulates γ-aminobutyric acid, as a DNA marker that can be determined to have a γ-aminobutyric acid metabolism-related gene located on chromosome 3.
(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 multiple mutation sites contained in the base sequence of SEQ ID NO: 1 and the base sequence of SEQ ID NO: 2.

(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 a homozygous type 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.

(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 described in (12) is 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.

The Solanum lycopersicum of the present invention has the characteristics that it accumulates an extremely high amount of γ-aminobutyric acid per unit fruit weight, and the decrease in fruit weight due to high accumulation of γ-aminobutyric acid is extremely small, resulting in an extremely high content of γ-aminobutyric acid.

In addition, the method for producing Solanum lycopersicum according to the present invention utilizes a first Solanum lycopersicum having a γ-aminobutyric acid metabolism-related gene located on chromosome 3 in Solanum pennellii, which accumulates γ-aminobutyric acid, to produce a Solanum lycopersicum hybrid line that has the γ-aminobutyric acid metabolism-related gene and does not have the low fruit weight-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.

Alignment of DNA regions amplified with marker No. 2 Alignment of DNA regions amplified by marker No. 32

The Solanum lycopersicum of 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-mentioned chromosome 3. We have focused on the fact that, among the introgression lines between Solanum pennelli and Solanum lycopersicum, those containing a specific region of chromosome 3 of Solanum pennelli have relatively small fruit weight but accumulate high amounts of γ-aminobutyric acid. We have then assumed that the γ-aminobutyric acid metabolism-related gene and the low fruit weight-related gene are present in the specific region.

The Solanum lycopersicum of the present invention has the novel characteristics of having extremely high levels of accumulated γ-aminobutyric acid per unit fruit weight and extremely little loss in fruit weight due to high accumulation of γ-aminobutyric acid, by isolating these γ-aminobutyric acid metabolism-related genes and low fruit weight-related genes.

Here, 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 that suppress the metabolism of γ-aminobutyric acid, genes involved in the biosynthesis of glutamic acid, which is a substrate for γ-aminobutyric synthesis, and genes that suppress the metabolism of glutamic acid. 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 more γ-aminobutyric acid than the homologous genes in Solanum lycopersicum.

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 pennellii are thought to function to reduce fruit weight more than their homologous genes in Solanum lycopersicum.

In addition, 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. UC DAVIS C.M. 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 part of the third chromosome has been replaced, has relatively small fruit weight but exhibits traits such as high accumulation of gamma-aminobutyric acid.

Therefore, the Solanum lycopersicum of 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. From the progeny lines obtained, a genotype that has a γ-aminobutyric acid metabolism-related gene and does not have the low fruit weight-related gene located on the third chromosome is selected.

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 over several generations, backcrossing is performed over several generations, or self-crossing and backcrossing are appropriately repeated.

The cultivated species of Solanum lycopersicum is not particularly limited, and generally available species 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.

Specifically, whether the obtained progeny has the above-mentioned γ-aminobutyric acid metabolism-related gene and does not have the above-mentioned low fruit weight-related gene can be determined using the first DNA marker and the second DNA marker. In other words, the first DNA marker and the second DNA marker are DNA markers that can distinguish the genotype that has the γ-aminobutyric acid metabolism-related gene and does not have the above-mentioned 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.

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 described later, the results of DNA marker analysis show that the base sequences of the first DNA marker shown in FIG. 1 are the cultivated type, with "PK993" and "Heinz1706" on the third and fourth lines from the top, and the wild type, with "TK8728" and "19F273" on the first and second lines from the top. The results of comparison of the base sequences of SEQ ID NO: 1 and SEQ ID NO: 2 are shown in Table 1.

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 base sequences different from SEQ ID NO: 1 contained in the first DNA marker include base sequences that preserve 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 have 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 of the above mutation sites 1-1 to 1-10, preferably three or more, more preferably five or more, even more preferably seven or more, and most preferably ten mutation sites, 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.

In addition, the second DNA marker may be, for example, a wild type nucleotide sequence of SEQ ID NO: 3 and a cultivated type nucleotide sequence of SEQ ID NO: 4. That is, the nucleotide sequence of SEQ ID NO: 3 is derived from the wild species Solanum pennellii LA0716, and the nucleotide sequence of SEQ ID NO: 4 is derived from the cultivated species Solanum lycopersicum M82. The results of pairwise alignment and comparison of the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 4 are shown in FIG. 2. As shown in the examples described later, the results of DNA marker analysis show that the nucleotide sequences of the second DNA markers shown in FIG. 2 are cultivated type nucleotide sequences of "PK993", "19F273", and "Heinz1706" in the first to third lines from the top, and the wild type nucleotide sequence of "TK8728" in the fourth line from the top. The results of comparison of the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 4 are shown in Table 2.

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 itself, and may be a base sequence different from SEQ ID NOs: 3 and 4, so long as it preserves at least one, preferably two or more, more preferably three or more, and even more preferably four types of mutation sites among the above mutation sites 2-1 to 2-4. Examples of base sequences different from SEQ ID NO: 3 contained in the second DNA marker include base sequences that preserve at least one, preferably two or more, more preferably three or more, and even more preferably four types of mutation sites among the above mutation sites 2-1 to 2-4, and have 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.

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

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 gamma-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.

On the other hand, the mating type of each of the first and second DNA markers can be determined appropriately 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 that detects at least one of the multiple mutation sites contained in the first DNA marker and the multiple mutation sites contained in the second DNA marker.

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 type of each 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 or the LAMP (loop-mediated isothermal amplification) method.

In addition to this method, the mating 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 or the like. Furthermore, the mating types of the first and second DNA markers can be determined using the so-called SSCP (Single Strand Conformation Polymorphism) method.

In addition, as a different method, when the sequence pattern of the mutation site for each of the first and second DNA markers is a recognition sequence for a restriction enzyme, a DNA fragment containing the mutation site can be amplified, and the mating type 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.

Furthermore, for each of the first and second DNA markers, a method of designing a probe that hybridizes specifically 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.

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 pattern of each mutation site. In this case, the first and second DNA markers are amplified using the designed primers, and the junction type of each of the first and second DNA markers can be determined based on the presence or absence of amplification.

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

However, when determining the mating type of the first DNA marker, it is not necessary to identify all of the sequence patterns of the 10 mutation sites described above; it is sufficient to identify the sequence patterns of at least one, or two to nine, of the 10 types of mutation sites. These 10 mutation sites are in linkage disequilibrium, so by identifying the sequence patterns of a small number of mutation sites (for example, one), the sequence patterns of the other mutation sites can be predicted with high accuracy.

Furthermore, the first DNA marker being heterozygous means that it has both a wild-type sequence pattern and a cultivated-type sequence pattern for the mutation site shown in Table 1.

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 sequence patterns of the four mutation sites shown in Table 2 are revealed, at least two, preferably three, and most preferably four, sites have a wild type sequence pattern. The second DNA marker being of the cultivated type means that when all sequence patterns of the four mutation sites shown in Table 2 are revealed, at least two, preferably three, and most preferably four, sites have a cultivated type sequence pattern.

However, when determining the mating type of the second DNA marker, it is not necessary to clarify all four of the above-mentioned mutation sites; it is sufficient to clarify the sequence pattern of at least one, or two or three, of the four types of mutation sites. These four mutation sites are in linkage disequilibrium, so 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.

Furthermore, the second DNA marker being homozygous for the cultivated species type means that it has the cultivated species type sequence pattern for the mutation site shown in Table 2.

By analyzing the first and second DNA markers for the progeny obtained by crossbreeding in the above manner, it is possible to determine whether or not Solanum pennellii has the characteristic of having a γ-aminobutyric acid metabolism-related gene located on chromosome 3, but not having a low fruit weight-related gene located on said 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 an extremely high accumulation amount of γ-aminobutyric acid per unit fruit weight, and the decrease in fruit weight associated with high accumulation of γ-aminobutyric acid is extremely small, and therefore has the characteristic of an extremely high γ-aminobutyric acid content.

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 of fruit (e.g., 1 mg). The Solanum lycopersicum of 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. Significant means statistically significant, and can be determined by methods such as Student's t-test and Welch's t-test. For example, the Solanum lycopersicum according to the present invention accumulates 105% of the amount of gamma-aminobutyric acid, preferably 110% of the amount of gamma-aminobutyric acid, preferably 130% of the amount of gamma-aminobutyric acid, preferably 150% of the amount of gamma-aminobutyric acid, preferably 170% of the amount of gamma-aminobutyric acid, preferably 200% of the amount of gamma-aminobutyric acid, preferably 230% of the amount of gamma-aminobutyric acid, preferably 250% of the amount of gamma-aminobutyric acid, preferably 300% of the amount of gamma-aminobutyric acid, preferably 350% of the amount of gamma-aminobutyric acid.

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 particular limitation. As shown in the examples described later, the progeny with high accumulation of γ-aminobutyric acid obtained by crossing Solanum pennellii, which has high accumulation 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 the characteristic that, despite high accumulation of γ-aminobutyric acid, it has a higher fruit weight than the cultivated species Solanum lycopersicum used as one of the parents, or, even if the fruit weight is reduced, the degree of 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 55 to 100%, more preferably 60 to 100%, even more preferably 70 to 100%, even more preferably 80 to 100%, and most preferably 85 to 100%.

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.

[Example 1]
In Example 1, a novel hybrid line that accumulates a large amount of γ-aminobutyric acid was developed using processing tomatoes.
<Ingredients>
The strains of Solanum lycopersicum used in this example are shown in Table 3. TK8728 was obtained from UC Davis C.M. 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 strain and the cultivated species Solanum lycopersicum M82.

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

Methods
<Creation of DNA markers>
Based on the base sequence information of wild species Solanum pennellii LA0716 line and Solanum lycopersicum published in a public database (Sol Genomics Network https://solgenomics.net/), a DNA marker capable of detecting polymorphism between wild species Solanum pennellii LA0716 line and Solanum lycopersicum Heinz1706 was prepared. The position and base sequence of the primer on chromosome 3, annealing temperature and band pattern of the prepared DNA marker are shown in Table 4. The position of the primer on chromosome 3 was specified using version SL3.0 of the map published in a public database.

なお、表４に示した各プライマーの塩基配列を上から順に配列番号５～１８とした。

The base sequences of the primers shown in Table 4 are SEQ ID NOs: 5 to 18, in order from top to bottom.

<DNA marker testing>
PCR was performed using DNA extracted from each Solanum lycopersicum lineage as a template. The PCR products were electrophoresed using a capillary electrophoresis device (Microchip Electrophoresis Device for DNA/RNA Analysis MCE-202, manufactured by Shimadzu Corporation). The genotype was identified from the band pattern obtained as a result of the electrophoresis. The PCR reaction conditions are shown in Table 5. Capillary electrophoresis was performed according to the specified protocol of the device.

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

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

The results of GABA content, fruit weight, and DNA marker analysis for the progeny produced in this example are summarized in Table 7.

<Analysis of TK8728>
As shown in Table 6, the GABA content of the introgression TK8728 was increased by about three times compared to the background variety Solanum lycopersicum M82, but the fruit weight was reduced to about 50%. In TK8728, a part of the third chromosome is replaced with the genotype of the wild species Solanum pennellii LA0716 line. This suggests that a gene that increases the GABA content (hereinafter referred to as "γ-aminobutyric acid metabolism related gene") and a gene that reduces the fruit weight (hereinafter referred to as "low fruit weight related gene") may be located in the region.

<Analysis of F2 generation>
In the F2 generation, 16F281 had a higher GABA content and a lower fruit weight than 16F280. As a result of the Student's t-test, the GABA content of 16F281 was significantly higher (p=0.000051<0.05) and the fruit weight was significantly lower (p=0.020<0.05) than 16F280. From this result and the results of the DNA marker analysis, it is inferred that the gamma-aminobutyric acid metabolism-related genes and low fruit weight-related genes are present between No. 1 and No. 3. In the F2 generation, the individual in which No. 2 was a heterozygote was selfed to obtain the F3 generation. Furthermore, in the F3 generation, the individual in which No. 2 was a heterozygote was selfed to obtain the F4 generation.

<Analysis of the F4 generation>
In the F4 generation, 18F308 had a higher GABA content and lower fruit weight than 18F307. Based on this result and the results of DNA marker analysis, it is inferred that the γ-aminobutyric acid metabolism-related genes and low fruit weight-related genes are located near No. 2. In the F4 generation, individuals with high GABA content and fruit weight were selected and self-fertilized to obtain the F5 generation. Furthermore, in the F5 generation, individuals with high GABA content and fruit weight were selected and self-fertilized to obtain the F6 generation.

<Analysis of the F6 generation>
In the F6 generation, a line (19F273) was obtained that had about twice the GABA content compared to the control variety PK993, while retaining 85% or more of the fruit weight. Looking at the results of DNA marker analysis, 19F273 No. 2 and No. 31 were wild types, and No. 32, No. 8, No. 6, and No. 3 were cultivated types. As a result of Student's t-test, the GABA content of 19F273 was significantly higher than that of PK993 (p = 0.01 < 0.05). On the other hand, the fruit weight of 19F273 was significantly lower than that of PK993 (p = 0.047 < 0.05). However, the fruit weight of 19F273 was significantly higher than that of 16F281, a line with a high GABA content (p = 0.0008 < 0.05), but no significant difference was observed when compared to 16F280, a line with a normal GABA content (p = 0.27 > 0.05).

From these results, it was determined that in 19F273, the γ-aminobutyric acid metabolism-related genes and the low fruit weight-related genes could be separated. From the above, it became clear that by selecting Solanum lycopersicum in which the gene regions No. 2 and No. 31 are of the wild type and the gene region No. 32 is of the cultivated type, it is possible to develop a new Solanum lycopersicum that is high in GABA but has reduced loss in fruit weight.

<Sequence analysis of the region amplified by DNA markers>
For TK8728, 19F273, PK993, and Heinz1706, sequence analysis was performed on the region amplified by marker No. 2. Sequencing was outsourced to Eurofins Genomics, Inc. The sequence alignment results are shown in FIG. 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).

For TK8728, 19F273, PK993, and Heinz1706, sequence analysis of the region amplified by marker No. 32 was performed 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).

[Example 2]
In Example 2, a novel hybrid line that accumulates a large amount of γ-aminobutyric acid was developed using fresh tomatoes.
<Ingredients>
The Solanum lycopersicum used in this example is 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.
Methods
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.
<Results>
The results of measuring GABA content and fruit weight are shown in Table 8.

The results of GABA content, fruit weight, and DNA marker analysis for the progeny produced in this example are summarized in Table 9.

<Analysis of BC4F2 generation>
As shown in Table 8, in the BC4F2 generation, 20K101-1 had the same GABA content and fruit weight as PK941. On the other hand, 20K101-3 had a higher GABA content than PK941, but the fruit weight was lower. 20K101-2 had the same fruit weight as PK941, but the GABA content was also higher. In addition, from the results of DNA marker analysis shown in Table 9, in 20K101-2, only No. 2 was a heterozygote, and No. 31 and No. 32 were cultivated types. Therefore, it was revealed that by selecting Solanum lycopersicum in which the gene region of No. 2 is a wild type or heterozygote and the genotype of No. 32 is a cultivated type, it is possible to cultivate Solanum lycopersicum that has a high GABA content while suppressing the decrease in fruit weight. In addition, No. Based on the results of Examples 1 and 2, it was considered that No. 31 may be either a wild type or a cultivated type.

The present invention can be used, for example, to breed new varieties of Solanum lycopersicum that accumulate high amounts of GABA.

Claims (7)

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 . 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. 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. 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. 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. A DNA marker detection kit including 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. 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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