A molecular marker related to sugar content in tomato fruit and its application

Abstract

This invention discloses a molecular marker related to tomato fruit sugar content and its application, belonging to the field of biotechnology. The invention discloses a molecular marker related to tomato fruit sugar content, ch7:g.49920697T>A, and also develops a genotyping detection method based on this molecular marker. The proposed method can accurately determine the sugar content trait of tomato fruit, providing significant value for tomato fruit sugar content breeding.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a molecular marker related to the sugar content of tomato fruits and an application thereof. Background Art

[0002] As the world's most valuable vegetable crop, tomatoes boast a unique flavor and contribute significantly to human health and nutrition. Sugar content is a key factor influencing tomato taste. Sugars in the fruit primarily exist in the form of fructose and glucose, which together account for approximately 50% of the fruit's total weight. Their content and sugar-to-acid ratio directly influence fruit flavor. However, with the advancement of tomato domestication and improvement, a negative correlation has emerged between fruit size and sugar content, and modern large-fruited tomato varieties generally have low sugar content.

[0003] Research on the sugar content of tomato fruit provides an important theoretical basis for improving fruit quality and economic value. With the continuous development of genomics and molecular biology technologies, it is expected to further reveal the molecular mechanism of sugar accumulation in tomato fruit and provide more powerful tools for precision breeding. Single nucleotide polymorphism (SNP) refers to the change of a single base in the DNA sequence of a population genome, including the insertion, deletion and substitution of a single base. SNP variation sites have the following advantages: large number, wide distribution and representativeness. Molecular markers based on SNPs will not cause separation of markers and target genes, which can greatly improve the accuracy and stability of markers. However, there are currently few reports on SNP molecular markers related to the sugar content of tomato fruits. Summary of the Invention

[0004] The present invention addresses the technical problems existing in the prior art and provides a molecular marker related to the sugar content of tomato fruit and its application. This molecular marker can accurately identify the sugar content of tomato fruit and is of great value for tomato variety breeding.

[0005] One of the objectives of the present invention is to provide a molecular marker related to the sugar content of tomato fruit, characterized in that the sequence of the molecular marker is as shown in SEQ ID NO.1, and there is a T>A mutation at position 152 of SEQ ID NO.1.

[0006] Preferably, the molecular marker is located at position 49920697 bp of chromosome 7 of tomato, and its reference genome is GCA_000188115.3.

[0007] Another object of the present invention is to provide a reagent for identifying the sugar content of tomato fruit, characterized in that the reagent includes primers and / or probes.

[0008] Preferably, the primer combination is SEQ ID NO.2 and SEQ ID NO.3.

[0009] Preferably, the reagents also include DNA extraction reagent, amplification buffer, dNTP, Mg 2+ , DNA polymerase, positive control, negative control, and water.

[0010] Another object of the present invention is to provide a kit for identifying the sugar content of tomato fruits, characterized in that the kit comprises the above-mentioned identification reagent.

[0011] Another object of the present invention is to provide the use of the molecular marker, the identification reagent, or the identification kit in identifying the sugar content of tomato fruits or in tomato variety breeding.

[0012] Preferably, the judgment criteria include that the genotype of the site in high sugar content tomato varieties is TT, the genotype of the site in low sugar content tomato varieties is AA, and the genotype of the site in medium sugar content tomato varieties is TA.

[0013] The present invention has the beneficial effects of disclosing a molecular marker (ch7:g.49920697T>A) related to tomato fruit sugar content and developing a genotyping method based on this molecular marker. This method can accurately determine the sugar content of tomato fruit, providing valuable insights into sugar content-enhancing tomato breeding. DETAILED DESCRIPTION

[0014] The principles and features of the present invention are described below in conjunction with specific embodiments. The examples given are only used to explain the present invention and are not intended to limit the scope of the present invention. Unless otherwise defined, all technical and scientific terms used herein are generally understood by those skilled in the art of the present invention. The terms used in this specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0015] Example 1

[0016] We consulted the literature on the sugar content of tomato fruit and discovered the sugar-related gene Solyc07g040785.1.1 based on the cultivar information included in the Tomato Genome Database (http: / / ted.bti.cornell.edu / ) based on the 150 Tomato Genome ReSequencing Project. We then conducted trait association analysis on the variant sites in this gene and found that ch7:g.49920697T>A could effectively distinguish the sugar content of tomato fruits in the database. We then used the significantly sugar-related variant site ch7:g.49920697T>A to develop molecular markers.

[0017] Example 2

[0018] Detecting molecular markers related to sugar content in tomato fruit comprises the following steps:

[0019] 1) Genomic DNA extraction: Ten tomato samples each of high-sugar content (Japanese Red Ice Cream, Orange Marshmallow, Sweet Wheat), medium-sugar content (Yellow Peach, Millennium, Yellow Saint), and low-sugar content (Duobao, Atomic Grape, Blueberry Tomato) were collected. Genomic DNA was extracted according to the DNA extraction kit and stored at -20°C until use. High sugar content refers to a sugar content greater than or equal to 10%, low sugar content refers to a sugar content less than or equal to 7%, and medium sugar content refers to a sugar content between 7% and 10%.

[0020] 2) High-throughput sequencing analysis: The genomic DNA of the tomato varieties in step 1) was subjected to high-throughput sequencing analysis, and the correlation between the variation of the ch7:g.49920697 site and the soluble sugar content of the fruit of different varieties was analyzed.

[0021] The results are shown in Table 1: The genotype of the g.49920697T>A molecular marker located on chromosome 7 is closely related to the soluble sugar content of the fruit. The genotype of this site is TT in high-sugar content tomato varieties, AA in low-sugar content tomato varieties, and TA in medium-sugar content tomato varieties. The high-throughput sequencing typing results are consistent with the sugar content trait of tomato fruit, confirming that this site has the ability to identify the sugar content of tomato fruit.

[0022] Table 1 Comparison of sugar content and genotypes of different tomato varieties

[0023] Tomato varieties Brix% genotype Japanese red ice cream 10.5±1.3 TT Orange Marshmallows 11.5±1.6 TT sweet wheat 10.1±0.9 TT yellow peach 8.2±0.6 TA Millennium 8.3±0.3 TA Huang Shengnu 8.4±0.2 TA Duobao 5.5±0.8 AA Atomic Grapes 6.2±0.3 AA Blueberry Tomato 5.8±0.6 AA

[0024] Example 3

[0025] A method for identifying the sugar content of tomato fruit comprises the following steps:

[0026] 1) Extract DNA from the sample to be tested: Prepare sample DNA using a DNA extraction kit;

[0027] 2) Prepare the PCR amplification system: 1 μL DNA template, 0.5 μL each of 10 mmol / L forward and reverse primers, 5 μL 2× SYBR GREEN PCR Mastermix, and add ddH2O to a total reaction volume of 10 μL.

[0028] Among them, the forward primer sequence is 5'-GTAGGAATCAATTATATTCC-3' (SEQ ID NO. 2), and the reverse primer sequence is 5'-AGACTAATGCTTATTATTGT-3' (SEQ ID NO. 3);

[0029] PCR reaction conditions: 95°C initial denaturation for 10 min; 20 cycles of 95°C denaturation for 45 sec, 65°C annealing for 50 sec, and 72°C extension for 45 sec; final extension at 72°C for 10 min, and storage at 4°C.

[0030] 4) Sequencing analysis: PCR amplification products were sequenced and analyzed using an ABI3730 sequencer to obtain PCR product sequences and peak plot files. Chromas software was used to view peak plots and perform genotyping.

[0031] 5) Genotyping result judgment criteria: The genotype of this locus in high-sugar tomato varieties is TT, the genotype of this locus in low-sugar tomato varieties is AA, and the genotype of this locus in medium-sugar tomato varieties is TA.

[0032] It is important to note that the above embodiments are intended only to further illustrate and describe the technical solutions of the present invention and are not intended to further limit the technical solutions of the present invention. The methods of the present invention are merely preferred implementations and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A molecular marker related to sugar content in tomato fruit for use in identifying sugar content in tomato fruit or in tomato variety breeding, characterized in that: The sequence of the molecular marker is shown in SEQ ID NO.1, and there is a T>A mutation at position 152 of SEQ ID NO.1; the molecular marker is located at position 49920697bp of tomato chromosome 7, and its reference genome is GCA_000188115.

3. The judgment criteria include that the genotype of this site is TT in high-sugar content tomato varieties, the genotype of this site is AA in low-sugar content tomato varieties, and the genotype of this site is TA in medium-sugar content tomato varieties.

2. A reagent for identifying the sugar content of tomato fruit for use in identifying the sugar content of tomato fruit or in tomato variety breeding, characterized in that: The reagent includes primers and / or probes for identifying the molecular marker according to claim 1, and the judgment criteria for the application include that the genotype of the site in high-sugar content tomato varieties is TT, the genotype of the site in low-sugar content tomato varieties is AA, and the genotype of the site in medium-sugar content tomato varieties is TA.

3. The use according to claim 2, characterized in that The primers are SEQ ID NO.2 and SEQ ID NO.

3.

4. The use according to claim 3, characterized in that The reagents further include at least one or more of a DNA extraction reagent, an amplification buffer, dNTP, Mg2+, a DNA polymerase, a positive control, a negative control, and water.

5. A kit for identifying the sugar content of tomato fruit for use in identifying the sugar content of tomato fruit or in tomato variety breeding, characterized in that: The kit comprises the identification reagent described in any one of claims 2 to 4, and the judgment criteria for application include that the genotype of the site in high-sugar content tomato varieties is TT, the genotype of the site in low-sugar content tomato varieties is AA, and the genotype of the site in medium-sugar content tomato varieties is TA.