High Aspartic Acid Tomato

Abstract

To provide a tomato (Solanum lycopersicum) with an increased aspartic acid content of its fruit and a method for producing the same.SOLUTION: The present invention discloses Solanum lycopersicum, including a gene responsible for the trait of high aspartic acid content on the chromosome 4, the responsible gene being included in the gene region of chromosome 4 of Solanum chmielewskii LA1318 strain corresponding to the gene region of 60,001,206 bp to 61,179,940 bp of chromosome 4 of Solanum lycopersicum (SL2.50).SELECTED DRAWING: None

Description

Technical Field

[0006] , , ,

[0001] The present invention relates to, for example, tomatoes (Solanum lycopersicum) with an increased aspartic acid content in fruits.

Background Art

[0002] Aspartic acid is generally known to have functions such as promoting the excretion of ammonia, which is a toxin in the human body, and promoting fatigue recovery by using amino acids for gluconeogenesis. Therefore, there is a demand for tomato fruits containing a high content of aspartic acid.

[0003] On the other hand, the aspartic acid content of tomatoes is about 71 mg / 100 g FW (Exhibition: Food Composition Database (Ministry of Education, Culture, Sports, Science and Technology)). Although the wild tomato Solanum chmielewskii LA1318 line has a significantly higher aspartic acid content compared to cultivated varieties and other wild species, its fruit weight and fruit set are poor, and its taste is extremely bad. In addition, although there is a report (Non-Patent Document 1) on producing tomatoes with a high aspartic acid content by genetic recombination, there is no report on tomatoes with increased aspartic acid using the wild Solanum chmielewskii LA1318 line as a material.

[0004] In addition, Patent Documents 1 and 2 disclose a plant in which amino acids contained in the edible part of tomatoes are enhanced and a method for producing the same by contacting a salt tolerance-imparting agent during hydroponic cultivation.

[0005] Patent Documents 3 to 5 disclose transgenic plants that highly accumulate free amino acids in the edible parts of plants and methods for producing the same. Patent Documents 3 to 5 are related to the glutamate dehydrogenase (GDH) gene or the glutamate decarboxylase (GAD) gene.

Prior Art Documents

Patent Documents

[0006] [Patent Document 1] Japanese Patent Publication No. 2019-162141 [Patent Document 2] WO 2018 / 061870 [Patent Document 3] WO 2003 / 000041 [Patent Document 4] Japanese Patent Publication No. 2001-238556 [Patent Document 5] Japanese Patent Publication No. 2001-238555 [Non-patent literature]

[0007] [Non-Patent Document 1] Snowden CJ et al., A tonoplast Glu / Asp / GABA exchanger that affects tomato fruit amino acid composition., Plant J, 2015, 81(5), pp. 651-660 [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] In view of the above circumstances, the present invention aims to provide tomatoes (Solanum lycopersicum) with increased aspartic acid content in the fruit, and a method for producing the same. [Means for solving the problem]

[0009] In order to solve the above problems, we conducted intensive research and discovered that a gene exists in a specific gene region on chromosome 4 of the Solanum kimielskii LA1318 line that increases the aspartic acid content in tomato fruits. Furthermore, we found that the aspartic acid content in fruits of Solanum lycopersicum containing this gene in a homozygous or heterozygous state on chromosome 4 was significantly higher than that of fruits of Solanum lycopersicum (cultivated type) that did not contain this gene, thus completing the present invention.

[0010] In other words, the present invention encompasses the following: [1] Solanum lycopersicum, The gene causing the high aspartic acid content trait is located on chromosome 4. The gene responsible for the high aspartic acid content trait is located in the gene region of chromosome 4 of the Solanum kimielskii LA1318 lineage, which corresponds to the gene region of 60,001,206 bp to 61,179,940 bp on chromosome 4 of Solanum lycopersicum (SL2.50). The aforementioned Solanum lycopersicum. [2] The solanum lycopersicum described in [1], wherein the gene causing the high aspartic acid content trait is included in the gene region of chromosome 4 of the solanum kimielskii LA1318 line, which corresponds to the gene region of 60,483,830 bp to 60,557,813 bp of chromosome 4 of solanum lycopersicum (SL2.50). [3] Solanum lycopersicum, PCR using primer sets containing DNA with the nucleotide sequence of SEQ ID NO: 5 and DNA with the nucleotide sequence of SEQ ID NO: 6, primer sets containing DNA with the nucleotide sequence of SEQ ID NO: 7 and DNA with the nucleotide sequence of SEQ ID NO: 8, primer sets containing DNA with the nucleotide sequence of SEQ ID NO: 11 and DNA with the nucleotide sequence of SEQ ID NO: 12, or primer sets containing DNA with the nucleotide sequence of SEQ ID NO: 15 and DNA with the nucleotide sequence of SEQ ID NO: 16 allows for the detection of aspartic acid-high-content genes present on chromosome 4 in a homozygous or heterozygous state. The aforementioned Solanum lycopersicum. [4] The Solanum lycopersicum described in any one of [1] to [3], wherein the aspartic acid content of its fruit is higher than that of the cultivated Solanum lycopersicum. [5] The solanum lycopersicum described in [4] has a lower glutamic acid content in its fruit compared to the cultivated solanum lycopersicum.

[0011] [6] A method for producing Solanum lycopersicum, The process of obtaining the F1 generation by crossing a first parent variety containing the gene for high aspartic acid content on chromosome 4 with any second parent variety, and A step of selecting Solanum lycopersicum in the obtained F1 generation or subsequent generations that contain the gene causing the high aspartic acid content trait on chromosome 4 in a homozygous or heterozygous state, Includes, The gene responsible for the high aspartic acid content trait is located in the gene region of chromosome 4 of the Solanum kimielskii LA1318 lineage, which corresponds to the gene region of 60,001,206 bp to 61,179,940 bp on chromosome 4 of Solanum lycopersicum (SL2.50). The aforementioned method. [7] The method according to [6], wherein the gene causing the high aspartic acid content trait is included in the gene region of chromosome 4 of the Solanum kimielskii LA1318 line, which corresponds to the gene region of 60,483,830 bp to 60,557,813 bp of chromosome 4 of Solanum lycopersicum (SL2.50). [8] The selection step is performed by PCR using a primer set containing DNA having the base sequence of SEQ ID NO: 5 and DNA having the base sequence of SEQ ID NO: 6, a primer set containing DNA having the base sequence of SEQ ID NO: 7 and DNA having the base sequence of SEQ ID NO: 8, a primer set containing DNA having the base sequence of SEQ ID NO: 11 and DNA having the base sequence of SEQ ID NO: 12, or a primer set containing DNA having the base sequence of SEQ ID NO: 15 and DNA having the base sequence of SEQ ID NO: 16, to detect aspartic acid high-content trait genes present on chromosome 4 in a homozygous or heterozygous state, according to the method of [6] or [7]. [9] The method according to any one of [6] to [8], wherein the aspartic acid content of the fruit of the produced Solanum lycopersicum is higher than that of the cultivated Solanum lycopersicum.

[10] The glutamic acid content of the fruits of the Solanum lycopersicum produced is lower compared to the cultivated Solanum lycopersicum, [9] the method described.

[11] A method for identifying Solanum lycopersicum, The process includes detecting a gene region containing the gene responsible for the high aspartic acid content trait on chromosome 4 of Solanum lycopersicum, The detection step is performed by PCR using a primer set containing DNA having the nucleotide sequence of SEQ ID NO: 5 and DNA having the nucleotide sequence of SEQ ID NO: 6, a primer set containing DNA having the nucleotide sequence of SEQ ID NO: 7 and DNA having the nucleotide sequence of SEQ ID NO: 8, a primer set containing DNA having the nucleotide sequence of SEQ ID NO: 11 and DNA having the nucleotide sequence of SEQ ID NO: 12, or a primer set containing DNA having the nucleotide sequence of SEQ ID NO: 15 and DNA having the nucleotide sequence of SEQ ID NO: 16, and detecting the aspartic acid high-content trait-causing gene present in homozygous or heterozygous form on chromosome 4 by PCR. The method.

Effects of the Invention

[0012] According to the present invention, there are provided Solanum lycopersicum having an increased aspartic acid content in its fruit and a method for producing the same. In ordinary tomatoes, the fruit contains 71 mg / 100 g FW of aspartic acid, whereas in Solanum lycopersicum according to the present invention, the aspartic acid content in the fruit is 190 to 380 mg / 100 g FW.

[0013] In addition, by using a tomato variety grown using Solanum lycopersicum according to the present invention, the amount of aspartic acid in fresh tomatoes, tomato juice, and tomato processed products can be increased.

[0014] Furthermore, according to Solanum lycopersicum according to the present invention, an effect of promoting ammonia excretion and promoting fatigue recovery can be expected in fresh tomatoes and tomato processed products due to the increase in the amount of aspartic acid.

[0015] Also, in the high aspartic acid genetic resource Solanum chmielewskii LA1318 line, the fruit weight is significantly reduced compared to fresh tomato varieties, but when Solanum lycopersicum according to the present invention is used, the reduction in fruit weight can be suppressed, and since the fruit also undergoes red ripening, it is also suitable as a fresh tomato.

[0016] Furthermore, according to the Solanum lycopersicum of the present invention, the increase in aspartic acid content and the resulting decrease in glutamic acid content reduce the umami flavor that remains as an aftertaste, while also improving sweetness and reducing acidity. Therefore, the Solanum lycopersicum of the present invention is suitable for tomato processed foods such as beverages and desserts, or as a tomato for fresh consumption. [Brief explanation of the drawing]

[0017] [Figure 1] This graph shows the polymorphic sites and number of polymorphisms specific to individuals with high aspartic acid content on chromosome 4 in Example 1. [Figure 2] The table and graph show the results of comparing the genotype and aspartic acid content of Kc15-148-1 in Example 2. [Figure 3] The table and graph show the results of comparing the genotypes and aspartic acid content of Kc14-1026-5-2 and Kc15-148-1-1 in Example 3. [Figure 4] This graph shows the results of comparing the genotype and aspartic acid content of primer No. 4 in Example 4. [Figure 5] This figure shows the results of the polymorphism investigation using each primer set in Example 4. [Modes for carrying out the invention]

[0018] The present invention will be described in detail below. The Solanum lycopersicum according to the present invention contains, in a homozygous or heterozygous form, the gene for high aspartic acid content that is included in the gene region of chromosome 4 of the Solanum kimielskii LA1318 line, which corresponds to the gene region of chromosome 4 of Solanum lycopersicum (SL2.50) from 60,001,206 bp to 61,179,940 bp. The gene region on chromosome 4 of the Solanum kimielskii LA1318 strain that corresponds to the gene region of 60,001,206 bp to 61,179,940 bp on chromosome 4 of Solanum lycopersicum (SL2.50), which is the gene causing the high aspartic acid content trait, can be determined by aligning and comparing the base sequence of the above gene region on chromosome 4 of Solanum lycopersicum (SL2.50) with the base sequence of chromosome 4 of the Solanum kimielskii LA1318 strain. Preferably, the gene causing the high aspartic acid content trait is included in the gene region on chromosome 4 of the Solanum kimielskii LA1318 strain that corresponds to the gene region of 60,483,830 bp to 60,557,813 bp on chromosome 4 of Solanum lycopersicum (SL2.50).

[0019] The inventors focused on the fact that the Solanum kimielskii LA1318 strain has the trait of having a high aspartic acid content in its fruit, and conceived that one or more genes causing the high aspartic acid content trait may exist in the aforementioned gene region of chromosome 4. The Solanum kimielskii LA1318 strain is available from the TGRC (Tomato Genetics Resource Center) in the United States (also referred to herein as "TK3273"). Furthermore, the genetic information for the above-mentioned Solanum lycopersicum (SL2.50) is available from the Sol Genomics Network of the Solanaceae Global Network.

[0020] Here, "genes causing high aspartic acid content" refer to genes that directly or indirectly cause high aspartic acid content in tomato fruits. Since the Solanum kimielskii LA1318 line exhibits the above trait, it is presumed that the genes causing high aspartic acid content in the above region on chromosome 4 of this line have mutations that enhance the activity of genes involved in promoting aspartic acid biosynthesis, mutations that delete or weaken the activity of genes involved in suppressing aspartic acid biosynthesis, and mutations that delete or weaken the activity of genes involved in promoting aspartic acid degradation, compared to the cultivated variety genes of Solanum lycopersicum.

[0021] Hereinafter, the gene causing the high aspartic acid content trait, which is located in the above gene region on chromosome 4 of the Solanum kimielskii LA1318 line and increases the aspartic acid content in the fruit, may be referred to as the "causative gene derived from Solanum kimielskii LA1318."

[0022] Furthermore, the inventors have confirmed that the known glutamine metabolism-related enzymes, glutamate decarboxylase (GAD) and glutamate dehydrogenase (GDH), are not located in the aforementioned gene region on chromosome 4 of the Solanum kimielskii LA1318 lineage.

[0023] In the Solanum lycopersicum according to the present invention, the causative gene derived from Solanum kimielskii LA1318 is located on chromosome 4 in either homozygous or heterozygous forms. Since the above trait of the Solanum kimielskii LA1318 line is considered to be a codominant trait, fruits with increased aspartic acid content can be obtained even in heterozygous forms.

[0024] Furthermore, in the Solanum lycopersicum according to the present invention, the causative gene derived from Solanum kimielskii LA1318 may have 90% or more identity with the base sequence of the causative gene derived from Solanum kimielskii LA1318, as long as the aforementioned trait is not impaired, more preferably 95% or more, and even more preferably 98% or more identity.

[0025] Furthermore, the Solanum lycopersicum according to the present invention contains a causative gene derived from Solanum kimielskii LA1318 on chromosome 4, and as long as the aforementioned trait is not impaired, chromosome 4 may contain part or all of the gene region on chromosome 4 of the Solanum kimielskii LA1318 strain corresponding to the gene region of 60,001,206 bp to 61,179,940 bp of chromosome 4 of Solanum lycopersicum (SL2.50), or the gene region of 60,483,830 bp to 60,557,813 bp of chromosome 4 of the Solanum kimielskii LA1318 strain.

[0026] The aspartic acid content of the fruit of Solanum lycopersicum according to the present invention is characterized by being higher than that of cultivated Solanum lycopersicum, preferably 20% or more, more preferably 30% or more, and even more preferably 50% or more of the aspartic acid content of the fruit of cultivated Solanum lycopersicum. "Cultivated type" refers to Solanum lycopersicum that has not undergone modification to place the causative gene derived from Solanum kimielskii LA1318 on chromosome 4 in a homozygous or heterozygous locus. Furthermore, the fruit referred to here is a red, ripe fruit.

[0027] The aspartic acid content in the fruit can be measured by conventional methods. For example, it can be measured using a biochemistry analyzer (Model 2900, YSI Corporation) on the juice obtained by crushing. When measured by this method, for example, the aspartic acid concentration in the fruit of Solanum lycopersicum that does not have the causative gene derived from Solanum kimielskii LA1318 on chromosome 4 in a homozygous or heterozygous state is about 30 mg%, while the aspartic acid concentration in the fruit of Solanum lycopersicum according to the present invention is preferably 60 mg% or more, more preferably 100 mg% or more, and even more preferably 200 mg% or more.

[0028] Furthermore, the glutamic acid content of the fruit of Solanum lycopersicum according to the present invention is lower than that of cultivated Solanum lycopersicum, preferably 50% or less, more preferably 30% or less, and even more preferably 20% or less, compared to the glutamic acid content of the fruit of cultivated Solanum lycopersicum.

[0029] The glutamic acid content in the fruit can be measured by the same method as described above for measuring the aspartic acid content in the fruit. For example, the glutamic acid concentration in the fruit of Solanum lycopersicum that does not have the causative gene derived from Solanum kimielskii LA1318 on chromosome 4 in a homozygous or heterozygous state is about 200 mg%, while the glutamic acid concentration in the fruit of Solanum lycopersicum according to the present invention is preferably 150 mg% or less, more preferably 100 mg% or less, and even more preferably 50 mg% or less.

[0030] Furthermore, the ratio of aspartic acid concentration (content) to glutamic acid concentration (content) in the fruit of Solanum lycopersicum according to the present invention (Asp / Glu ratio) is, for example, 1 to 2, preferably 2 to 5, and particularly preferably 5 to 10.

[0031] Furthermore, in the fruit of Solanum lycopersicum according to the present invention, the expression of traits that increase aspartic acid content and / or decrease glutamic acid content does not affect the sugar content.

[0032] The Solanum lycopersicum according to the present invention can be produced by genetic engineering techniques. Examples of genetic engineering techniques include known genome editing technologies, specifically, methods such as CRISPR, ZFN, and TALEN, which are used to manipulate the genome of Solanum lycopersicum to include the causative gene derived from Solanum kimielskii LA1318. Furthermore, it can also be obtained by the following manufacturing methods.

[0033] Specifically, first, as the first parent variety, a first parent variety is prepared that contains the gene for the high aspartic acid content trait, which is located on chromosome 4 of the Solanum kimielskii LA1318 line, corresponding to the gene region of chromosome 4 of Solanum lycopersicum (SL2.50) from 60,001,206 bp to 61,179,940 bp, preferably the gene for the high aspartic acid content trait, which is located on chromosome 4 of the Solanum kimielskii LA1318 line, corresponding to the gene region of chromosome 4 from 60,483,830 bp to 60,557,813 bp. The first parent variety can preferably be Solanum lycopersicum, or it may be the Solanum kimielskii LA1318 line, or a hybrid of the Solanum kimielskii LA1318 line and another cultivated variety.

[0034] Any second parent variety can be used, preferably Solanum lycopersicum. Furthermore, the second parent variety may not contain the causative gene derived from Solanum kimielskii LA1318 or the aforementioned gene region containing said causative gene.

[0035] Then, by performing the steps of crossing the first parent variety with the second parent variety to obtain the F1 generation, and selecting Solanum lycopersicum in the obtained F1 generation or subsequent generations that contain, in a homozygous or heterozygous state, the aspartic acid high-content trait gene contained in the gene region of the Solanum kimielskii LA1318 line corresponding to Solanum kimielskii LA1318 line, in a homozygous or heterozygous state, the aspartic acid high-content trait gene in the fruit can be produced. Furthermore, the manufactured Solanum lycopersicum exhibits a reduced glutamic acid content in its fruits. The resulting F1 generation progeny can be obtained by processes such as self-pollinating the F1 generation to obtain the F2 generation, backcrossing the F1 generation with the first or second parent variety, or crossing the F1 generation with any Solanum lycopersicum. In addition, in this manufacturing method, the processes of obtaining and selecting these progeny may be repeated multiple times.

[0036] The process of selecting a desired Solanum lycopersicum can be carried out by detecting the gene region containing the causative gene derived from Solanum kimielskii LA1318 on chromosome 4 using any marker.

[0037] For example, PCR using a primer set containing DNA with the base sequence of SEQ ID NO: 5 and DNA with the base sequence of SEQ ID NO: 6 (corresponding to the forward primer and reverse primer of primer name: 04g074490 in the following example, respectively) detects the chromosome 4 marker of the Solanum kimielskii LA1318 strain corresponding to 60,483,986 bp of chromosome 4 of Solanum lycopersicum (SL2.50). PCR using a primer set containing DNA with the base sequence of SEQ ID NO: 7 and DNA with the base sequence of SEQ ID NO: 8 (corresponding to the forward primer and reverse primer of primer name: 04g074530 in the following example, respectively) detects the chromosome 4 marker of the Solanum kimielskii LA1318 strain corresponding to 60,520,092 bp of chromosome 4 of Solanum lycopersicum (SL2.50). PCR using a primer set containing DNA with the base sequence of SEQ ID NO: 11 and DNA with the base sequence of SEQ ID NO: 12 (corresponding to the forward primer and reverse primer of primer name: 04g074610 in the following examples, respectively) detects the chromosome 4 marker of the Solanum kimielskii LA1318 strain corresponding to 60,557,703 bp of chromosome 4 of Solanum lycopersicum (SL2.50). PCR using a primer set containing DNA with the base sequence of SEQ ID NO: 15 and DNA with the base sequence of SEQ ID NO: 16 (corresponding to the forward primer and reverse primer of primer name: 04g074900 in the following examples, respectively) detects the chromosome 4 marker of the Solanum kimielskii LA1318 strain corresponding to 60,818,232 bp of chromosome 4 of Solanum lycopersicum (SL2.50).

[0038] In a preferred embodiment, the selection step is performed by PCR using a primer set containing DNA having the base sequence of SEQ ID NO: 5 and DNA having the base sequence of SEQ ID NO: 6, a primer set containing DNA having the base sequence of SEQ ID NO: 7 and DNA having the base sequence of SEQ ID NO: 8, a primer set containing DNA having the base sequence of SEQ ID NO: 11 and DNA having the base sequence of SEQ ID NO: 12, or a primer set containing DNA having the base sequence of SEQ ID NO: 15 and DNA having the base sequence of SEQ ID NO: 16, to detect the aspartic acid high-content trait gene present on chromosome 4 in a homozygous or heterozygous state. Then, the marker detected by the primer set is selected to indicate the presence of a Solanum lycopersicum gene and / or a gene region containing it, which originates from Solanum kimielskii LA1318, in a homozygous or heterozygous state on chromosome 4.

[0039] In addition, the following primers may be included as alternatives: primers having a base sequence in which one or more bases (for example, 1 to 10, 1 to 5, 1 to 3, preferably 1 or 2) are deleted, substituted, inserted, or added in the base sequence indicated by the sequence number of each primer described above, and which have the respective primer function (primers that anneal (hybridize) to a predetermined position in the causative gene and / or gene region containing it derived from Solanum kimielski LA1318, and act as a starting point to induce the extension reaction).

[0040] In the selection process described above, one or more markers may be detected in the selected Solanum lycopersicum that indicate the presence of a causative gene derived from Solanum kimielskii LA1318 on chromosome 4 in a homozygous or heterozygous state.

[0041] Preferably, one or more markers selected from the group consisting of a marker for chromosome 4 of Solanum lycopersicum (SL2.50) corresponding to 60,483,986 bp, a marker for chromosome 4 of Solanum lycopersicum (SL2.50) corresponding to 60,520,092 bp, a marker for chromosome 4 of Solanum lycopersicum (SL2.50) corresponding to 60,557,703 bp, and a marker for chromosome 4 of Solanum lycopersicum (SL2.50) corresponding to 60,818,232 bp are detected.

[0042] If the detection of the marker indicates that the causative gene and / or gene region containing it, derived from Solanum kimielskii LA1318, is present on chromosome 4 in a homozygous or heterozygous state, then the Solanum lycopersicum strain is determined to be a variety that produces fruits with increased aspartic acid content.

[0043] From another perspective, the Solanum lycopersicum according to the present invention can also be described as a Solanum lycopersicum in which the causative gene and / or gene region containing the Solanum kimielskii LA1318 is detected on chromosome 4 in a homozygous or heterozygous state by PCR using any of the above primer sets.

[0044] Another aspect of the present invention is a method for identifying Solanum lycopersicum, comprising the step of detecting the causative gene and / or gene region containing it on chromosome 4 of Solanum lycopersicum by performing PCR using any of the above primer sets. In this identification method, if the causative gene and / or gene region containing it, derived from Solanum lycopersicum LA1318, is shown to be present on chromosome 4 in a homozygous or heterozygous state, the Solanum lycopersicum is identified as a strain that produces fruits with increased aspartic acid content. [Examples]

[0045] The present invention will be described in more detail below using examples, but the technical scope of the present invention is not limited to these examples.

[0046] [Example 1] Narrowing down the locus region of the gene causing the high aspartic acid content trait in Solanum lycopersicum 1. Materials and Methods In the first half of 2017, 25 plants each of Kc15-165-3 (17J11) and Kc15-165-4 (17J12), which exhibit a high aspartic acid content trait that is isolated between individuals in Solanum lycopersicum, were cultivated in Kagome's fields. Kc15-165-3 and Kc15-165-4 are backcross lines (BC4F3 generation) created by crossing TK3273 with a processing variety (TK5352). Leaves and ripe fruits were sampled from each individual. The sampled ripe fruits were crushed in a mixer for 1 minute, packed into sample bottles, boiled for 30 minutes, and subjected to aspartic acid analysis.

[0047] From a total of 50 plants (25 plants each from two lines), 10 individuals with high aspartic acid content (high-aspartic acid bulk), 10 individuals with low aspartic acid content (low-aspartic acid bulk), and 10 individuals with moderate aspartic acid content (medium-aspartic acid bulk) were selected. Leaves sampled from each bulk were mixed and bulk sequencing (whole-genome sequencing analysis) was performed. The genome sequence data was used to search for mutations related to the high-aspartic acid content trait.

[0048] 2. Results and Discussion Table 1 shows the aspartic acid analysis results for each individual of Kc15-165-3 (17J11) and Kc15-165-4 (17J12). Based on these results, 10 individuals with an Asp / Glu ratio of 3 or higher (high Asp-containing bulk) were selected as high-aspartic acid bulk, 10 individuals with an Asp / Glu ratio of approximately 1 (medium Asp-containing bulk) were selected as medium-aspartic acid bulk, and 10 individuals with an Asp / Glu ratio of approximately 0.5 (low Asp-containing bulk) were selected as low-aspartic acid bulk.

[0049] [Table 1]

[0050] Whole-genome sequencing was performed on each bulk sample, and differences in nucleotide sequences from the reference sequence (hereinafter referred to as "polymorphisms") were extracted. From the extracted polymorphisms, polymorphisms present in the gene sequence were selected, and further polymorphisms accompanied by changes in the amino acid sequence were selected. Since the high aspartic acid content trait is codominant, it was assumed that individuals with high aspartic acid content bulks would have the causative polymorphism homozygous, individuals with medium content bulks would have the polymorphism heterozygous, and individuals with low content bulks would have the same nucleotide sequence as normal (no polymorphism). Therefore, the selected polymorphisms were compared among the bulk samples, and only those that were homozygous in high aspartic acid content bulks, heterozygous in medium content bulks, and polymorphic-free (same sequence as the reference sequence) in low content bulks were selected. As a result, the selected polymorphisms were concentrated in the 59-63 Mb region of chromosome 4 (Figure 1). From this, it was considered that the causative gene for the high aspartic acid content trait is located in this region, and further fine-tuning was attempted.

[0051] [Example 2] Fine Mapping 1. Materials and Methods Kc15-148-1 was used for evaluation. Kc15-148 is an F2 line produced by crossing a TK3273 backcross line (14Z125-2) with a fresh mini-pair variety (PK868), and it was expected that aspartic acid content would be segregated among individuals. Therefore, from Kc15-148-1, individuals with wild-type (homozygous), heterozygous, and cultivated-type characteristics in the 59-63Mb region of chromosome 4, as well as individuals with segregated genotypes within this region, were selected for evaluation.

[0052] Table 2 shows the primer sets used for fine mapping. DNA was extracted from 39 Kc15-148-1 individuals. PCR was performed using the extracted DNA and the primer sets shown in Table 2, and the amplified DNA fragments were detected using a capillary electrophoresis apparatus (MCE-202 / maltiNA, Shimadzu Corporation). The PCR conditions are shown in Table 3. Selected individuals were cultivated, and the locus region of the gene causing the high aspartic acid content was narrowed down by comparing the genotype results with aspartic acid and glutamic acid content.

[0053] [Table 2]

[0054] [Table 3]

[0055] Simple DNA extraction method and marker testing using the KOD FX system The DNA extraction and marker testing described above were performed as follows: 1) Items to prepare Scissors, bottled 70% ethanol, 1.5ml tubes or 96-well deep-well plate, crushing alumina beads (φ3mm or 4mm), tube holder (when using 1.5ml tubes), deep-well plate mat (when using 96-well deep-well plate), marker pen, extraction buffer (100mM Tris-HCl (pH9.5), 1M KCl, 10mM EDTA), and test seedlings. 2) Extraction method (1) Half of the cotyledon (slightly smaller than a little fingernail) was cut off with scissors and placed in a 1.5 ml tube or a 96-well deep-well plate; (2) Add 100 μL of extraction buffer and the crushing beads, then close the lid. When using a 96-well deep-well plate, securely cover with the deep-well plate mat. When using a 1.5 ml tube: use φ3 mm crushing beads. When using a 96-well deep-well plate: use φ4 mm crushing beads. (3) A 1.5 ml tube or a 96-well deep-well plate was placed in the Geno Grinder and crushed at a rotation speed of 1000 rpm for 1 minute; (4) 0.5 μL was used as the PCR template (it is better to add the premix first, as it will dry out if added to the tube first). 3) Marker testing (1) A premix solution was prepared with the following composition: (The following instructions are for 50 samples; for 96 samples, prepare two copies of the following instructions.) 2X PCR Buffer for KOD FX 625μL dNTP (2mM) 250μL F Primer 50μL R Primer 50μL (If using multiple primers, reduce the amount of sterile water by the amount of liquid in each primer.) Sterile water 237.5μL KOD FX 12.5μL; (2) Dispense 24.5 μl of the premix solution into PCR tubes (96-well plates); (3) The PCR template was dispensed in 0.5 μL portions into PCR tubes (96-well plates); (4) PCR was performed using the following program: 94℃ 2 min 1 time ·98℃ 10sec, Tm℃ 30sec, 68℃ 1min 40 times; (5) The results were confirmed using an agarose gel or capillary electrophoresis apparatus.

[0056] 2. Results and Discussion Figure 2 shows the results of comparing the genotype and aspartic acid content of Kc15-148-1. Individuals that showed the wild type with both the 04g072670 primer and the 04g079160 primer (wild type) had high aspartic acid content and low glutamic acid content (Asp / Glu = 7.22). Individuals that showed the heterozygous type with both primers (heterozygous) had relatively high aspartic acid content, but also higher glutamic acid content compared to wild-type homozygous individuals (Asp / Glu = 1.23). Furthermore, individuals that showed the cultivated type with both primers (cultivated type) had low aspartic acid content and high glutamic acid content (Asp / Glu = 0.15). From this, it was determined that the high aspartic acid content trait lies in the region between the 04g072670 primer and the 04g079160 primer investigated in this example. Furthermore, in these individuals, where the test results for both primers were wild-type, heterozygous, and cultivated-type, it was inferred that recombination did not occur within this region (meaning this region was entirely wild-type, heterozygous, and cultivated-type).

[0057] Comparing individuals with segregated genotypes within this region, individuals homozygous for wild-type (segregates A, B, C) in the region from primer 04g074490 to primer 04g076240 had high aspartic acid content (Asp / Glu > 4.8), while individuals homozygous for cultivated-type (segregate I) had low aspartic acid content (Asp / Glu ≈ 0.1). Heterozygous individuals (segregates D, E, F, G, H) had an Asp / Glu ratio of approximately 1.0. Since genotype and aspartic acid content were linked, the locus region of the gene responsible for the high aspartic acid content trait was narrowed down to the region indicated by the black box: 60,001,206 ~ 61,863,326 bp (SL2.50). Therefore, we decided to confirm that the genotype in this region is linked to the high aspartic acid content trait in other lines as well, and to attempt to further narrow down the locus region.

[0058] [Example 3] Fine Mapping 2 1. Materials and Methods Kc14-1026-5-2 (15Z155-2) and Kc15-148-1-1 were used for evaluation. Kc14-1026-5-2 is a backcross line (BC3F2) created by crossing TK3273 with a fresh round variety (PK515), and it was expected that aspartic acid content would vary among individuals. Therefore, from Kc14-1026-5-2, individuals with wild-type (homozygous), heterozygous, and cultivated-type characteristics within a narrowed region of chromosome 4 were selected and evaluated.

[0059] Furthermore, in order to further narrow down the region, we used the offspring (Kc15-148-1-1) obtained from individuals of Kc15-148-1 isolate G (see Figure 2) whose genotype in the region narrowed down in the evaluation of Example 2 was heterozygous. We selected individuals with wild-type, heterozygous, and cultivated-type genotypes within the narrowed-down region of chromosome 4, as well as individuals with segregated genotypes within the region, and performed evaluations. The primer set, DNA extraction method, PCR conditions, and detection method used for marker selection were the same as in Example 2.

[0060] 2. Results and Discussion Figure 3 shows the results of comparing the genotypes and aspartic acid content of Kc14-1026-5-2 and Kc15-148-1-1. For Kc14-1026-5-2, the Asp-F3 / R3 primer and the 04g074610 primer, both located within the narrowed region, were used for genotype analysis. With both primers, individuals homozygous for the wild type (wild type) had high Asp content (Asp / Glu = 7.03), while individuals homozygous for the cultivated type (cultivated type) had low content (Asp / Glu = 0.16). Heterozygous individuals had an Asp / Glu ratio of 1.10, and the high aspartic acid content trait was consistent with the polymorphism detection results for both primer sets.

[0061] In Kc15-148-1-1, the 04g074740 primer, 04g074900 primer, and 04g076240 primer, all located within the narrowed region, were used for genotyping. Using both the 04g074740 and 04g074900 primers, individuals homozygous for wild-type had an Asp / Glu ratio of 6.61, heterozygous individuals had an Asp / Glu ratio of 1.03, and cultivated-type individuals had an Asp / Glu ratio of 0.13, consistent with the polymorphism detection results for the high aspartic acid content trait using this primer set. On the other hand, in these individuals, the genotype tested with the 04g076240 primer was consistently wild-type. Based on these results and the results in Figure 2, it was determined that the causative gene for the high aspartic acid content trait is located in the region indicated by the black box, from 60,001,206 to 61,179,940 bp (SL2.50).

[0062] [Example 4] Narrowing down the locus region of the gene causing the high aspartic acid content trait in Solanum lycopersicum 1. Materials and Methods For fine mapping, we used Kc18-121-1, the F2 generation obtained by crossing and self-pollinating a variety with a high aspartic acid content trait (possessive variety), Kc15-165-8 (17J14-5), with a variety without the high aspartic acid content trait (non-possessive variety), Kc17-332-1 (18F250-B). As controls, Kc15-165-8-5 (17J14-5-1) and PK993 were used as possessive and non-possessive varieties, respectively.

[0063] Fifty seeds of Kc18-121-1 were sown, and leaves were sampled from each plant. DNA was extracted according to the simplified DNA extraction method using the KOD FX system and marker testing described in Example 2. PCR was performed using the extracted DNA and primers No. 3, 4, and 6 shown in Table 2 of Example 2, according to the conditions in Table 3 of Example 2. Based on the PCR results, transplanting and cultivation were carried out according to the genotype (Table 4).

[0064] [Table 4]

[0065] 2. Evaluation Method For varieties other than PK993, fully ripened fruit was sampled from each individual plant, and for PK993, bulk samples were taken from five plants. Crushed samples were then prepared. The washed fruit was crushed in a mixer for 1 minute, packed into sample bottles, and boiled for 30 minutes to prepare the crushed samples. Using the prepared samples, high content analysis of radioisotopes, lycopene, glutamic acid, and aspartic acid was performed according to the analytical procedure.

[0066] To reconfirm the genotype, leaves were sampled from each individual plant, and DNA was extracted again according to the simplified DNA extraction method using the KOD FX system and marker testing described in Example 2. PCR was performed using the extracted DNA and primers No. 3, 4, 6, and 8 shown in Table 2 of Example 2, according to the conditions in Table 3 of Example 2.

[0067] 3. Results and Discussion Table 5 shows the results of the investigations into aspartic acid and glutamic acid content and genotype. Individuals F270, F271_A, and F271_B all had higher aspartic acid content compared to individuals F270_C and F272. From this, we considered that the gene responsible for the high aspartic acid content trait is located near primer No. 4 (the region from primer No. 3 to primer No. 6, chromosome 4, 60,483,830 bp to 60,557,813 bp (SL2.50)), where the genotypes of individuals F270, F271_A, and F271_B are common (Figure 4). In the tests conducted in this example, the high aspartic acid content trait and other cultivation characteristics of Kc15-165-8-5(F270) were confirmed to be fixed. Therefore, the next generation seeds collected (Kc15-165-8-5-1, 19F270-1) were designated as PK1521.

[0068] [Table 5]

[0069] Thirteen genes were found in the narrowed-down region (Table 6). Therefore, polymorphisms on these 13 genes were investigated using the bulk sequencing data obtained when the locus of the causative gene was narrowed down in Example 3. As a result, one gene lacked polymorphism specific to individuals exhibiting this trait, and two genes exhibited polymorphism but without amino acid substitutions. It was hypothesized that the causative gene was among the remaining 10 genes. Since the association between these 10 genes and aspartic acid has not been previously reported, it was highly likely that the causative gene was a novel gene involved in aspartic acid biosynthesis or metabolism.

[0070] [Table 6]

[0071] When PCR was performed with primer No. 4, a 240 bp band was detected in PK993 (F272), a non-possessing variety, but no band was detected in the non-possessing individual of Kc18-121-1 (F271_C) (Figure 5, center). Therefore, among Kc18-121-1 individuals, both homozygous and heterozygous types with primer No. 4 showed the same band pattern, making it impossible to identify the genotype (F271_A, F271_B). F270 and F271_A had high aspartic acid content, and based on the preceding and succeeding genotypes, the No. 4 genotype was presumed to be homozygous (Table 5). On the other hand, although it was difficult to determine the genotype of F271_B because the preceding and succeeding genotypes were different, its aspartic acid content was as high as that of F270 and F271_A, suggesting that primer No. 4, where the causative gene is presumed to reside, was likely homozygous.

[0072] Furthermore, the band size differed between non-possessing varieties when tested with primer No. 3 and primer No. 6 (Figure 5). From this, we hypothesized that multiple alleles exist at the locations of primers No. 3, No. 4, and No. 6 in cultivated varieties (non-possessing varieties).

[0073] Since it was possible to select individuals with a high aspartic acid content trait derived from Solanum kimielskii using the prepared No. 4 primer, we believe it can be used as a DNA marker.

[0074] summary We performed fine mapping of the high aspartic acid content trait and attempted to develop a DNA marker usable in processing tomato varieties. The results showed that the causative gene was located in the region from 60,483,830 bp to 60,557,813 bp (SL2.50) on chromosome 4. Using the primer set 04g074530-F / R, which detects polymorphism in this region, it was possible to select individuals with the high aspartic acid content trait derived from Solanum kimielskii, even in processing varieties. Based on these results, we will utilize 04g074530-F / R as a breeding marker for high aspartic acid content.

Claims

1. Solanum lycopersicum, It contains the gene responsible for the high aspartic acid content trait on chromosome 4, The gene responsible for the high aspartic acid content trait is contained in a gene region of chromosome 4 of the Solanum lycopersicum LA1318 line, which corresponds to a gene region of 60,001,206 bp to 61,179,940 bp on chromosome 4 of Solanum lycopersicum (SL2.50). The above Solanum lycopersicum.

2. The Solanum lycopersicum according to claim 1, wherein the gene responsible for the high aspartic acid content trait is contained in the gene region of chromosome 4 of the Solanum lycopersicum LA1318 strain, which corresponds to the gene region of 60,483,830 bp to 60,557,813 bp of chromosome 4 of Solanum lycopersicum (SL2.50).

3. Solanum lycopersicum, A gene responsible for the trait of high aspartic acid content, which is present in a homozygous or heterozygous form on chromosome 4, is detected by PCR using a primer set containing a DNA having the nucleotide sequence of SEQ ID NO: 5 and a DNA having the nucleotide sequence of SEQ ID NO: 6, a primer set containing a DNA having the nucleotide sequence of SEQ ID NO: 7 and a DNA having the nucleotide sequence of SEQ ID NO: 8, a primer set containing a DNA having the nucleotide sequence of SEQ ID NO: 11 and a DNA having the nucleotide sequence of SEQ ID NO: 12, or a primer set containing a DNA having the nucleotide sequence of SEQ ID NO: 15 and a DNA having the nucleotide sequence of SEQ ID NO:

16. The above Solanum lycopersicum.

4. 4. Solanum lycopersicum according to any one of claims 1 to 3, wherein the aspartic acid content of the fruit is higher than that of cultivated Solanum lycopersicum.

5. 5. Solanum lycopersicum according to claim 4, the glutamic acid content of whose fruits is lower compared to that of cultivated Solanum lycopersicum.

6. A method for producing Solanum lycopersicum, comprising: A step of crossing a first parent variety containing a gene responsible for the trait of high aspartic acid content on chromosome 4 with an arbitrary second parent variety to obtain an F1 generation; and selecting Solanum lycopersicum that contains the gene responsible for the trait of high aspartic acid content on chromosome 4 in a homozygous or heterozygous form from the obtained F1 generation or its progeny; Including, The gene responsible for the high aspartic acid content trait is contained in a gene region of chromosome 4 of the Solanum lycopersicum LA1318 line, which corresponds to a gene region of 60,001,206 bp to 61,179,940 bp on chromosome 4 of Solanum lycopersicum (SL2.50). The method.

7. The method according to claim 6, wherein the gene responsible for the high aspartic acid content trait is contained in a gene region of chromosome 4 of the Solanum chmierzkii LA1318 strain, which corresponds to the gene region of 60,483,830 bp to 60,557,813 bp of chromosome 4 of Solanum lycopersicum (SL2.50).

8. 8. The method according to claim 6 or 7, wherein the selection step is carried out by PCR using a primer set comprising DNA having the nucleotide sequence of SEQ ID NO: 5 and DNA having the nucleotide sequence of SEQ ID NO: 6, a primer set comprising DNA having the nucleotide sequence of SEQ ID NO: 7 and DNA having the nucleotide sequence of SEQ ID NO: 8, a primer set comprising DNA having the nucleotide sequence of SEQ ID NO: 11 and DNA having the nucleotide sequence of SEQ ID NO: 12, or a primer set comprising DNA having the nucleotide sequence of SEQ ID NO: 15 and DNA having the nucleotide sequence of SEQ ID NO: 16, to detect the gene responsible for the high aspartic acid content trait, which is present in a homozygous or heterozygous form on chromosome 4.

9. 9. The method according to claim 6, wherein the aspartic acid content of the produced fruit of Solanum lycopersicum is higher than that of cultivated Solanum lycopersicum.

10. 10. The method according to claim 9, wherein the glutamic acid content of the produced fruit of Solanum lycopersicum is lower compared to that of cultivated Solanum lycopersicum.

11. A method for identifying Solanum lycopersicum, comprising: detecting a gene region containing a gene responsible for the high aspartic acid content trait on chromosome 4 of Solanum lycopersicum, The detection step is carried out by PCR using a primer set comprising DNA having the nucleotide sequence of SEQ ID NO: 5 and DNA having the nucleotide sequence of SEQ ID NO: 6, a primer set comprising DNA having the nucleotide sequence of SEQ ID NO: 7 and DNA having the nucleotide sequence of SEQ ID NO: 8, a primer set comprising DNA having the nucleotide sequence of SEQ ID NO: 11 and DNA having the nucleotide sequence of SEQ ID NO: 12, or a primer set comprising DNA having the nucleotide sequence of SEQ ID NO: 15 and DNA having the nucleotide sequence of SEQ ID NO: 16, thereby detecting the gene responsible for the high aspartic acid content trait present in a homozygous or heterozygous form on chromosome 4. The method.

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