SNP molecular marker related to fruit glossiness in solanum lycopersicum l. and use thereof

Abstract

The present invention belongs to the technical field of vegetable molecular breeding. Disclosed in the present invention are a SNP molecular marker related to the fruit glossiness in Solanum lycopersicum L. and the use thereof. The SNP molecular marker has a nucleotide sequence as shown in SEQ ID NO: 4, and the sequence as shown in SEQ ID NO: 4 has an A / G base mutation at the position of 34 bp. In the present invention, a PARMS SNP molecular marker related to the fruit glossiness in Solanum lycopersicum L. is developed, a specific molecular marker primer for identifying the fruit glossiness in Solanum lycopersicum L. is developed on the basis of positioning results, and the difference in the fruit glossiness in Solanum lycopersicum L. is identified on the basis of detection of the polymorphism at the detection sites. The PARMS SNP molecular marker developed in the present invention co-segregates with the fruit glossiness phenotype in Solanum lycopersicum L., and can realize the determination of fruit glossiness in the seedling stage of Solanum lycopersicum L., so as to reduce the cost of seed production. The molecular marker has important value in assisted breeding and rapid identification of high fruit glossiness varieties of Solanum lycopersicum L.

Description

A SNP molecular marker associated with tomato fruit gloss and its application Technical Field

[0001] The present invention relates to the technical field of vegetable molecular breeding, in particular to a SNP molecular marker related to the glossiness of tomato fruits and an application thereof. Background Art

[0002] Tomatoes (Solanum lycopersicum L.) are one of the most widely cultivated vegetable crops worldwide, rich in nutrients such as folic acid, vitamin C, potassium, and lycopene. With their vibrant color and high nutritional value, tomatoes are a popular fresh-eaten fruit and vegetable, enjoying a unique flavor and rich taste. Their cultivated area is increasing annually. my country leads the world in both tomato production and cultivated area. In recent years, with my country's socioeconomic development and rising living standards, consumers are increasingly choosing fruits and vegetables that prioritize nutrition and flavor. Varieties with bright colors, a clean appearance, and smooth skin are gaining popularity. The appearance of tomatoes encompasses aspects such as color, gloss, and shape. Fruit gloss is an indicator that measures the fruit's ability to reflect light; the greater the light reflection, the higher the light chromaticity. Glossiness is a key appearance trait of cherry tomatoes. Cherry tomatoes with high gloss are popular among consumers for their vibrant and bright appearance, commanding a higher market price than less glossy tomatoes. In the domestic consumer market, as people's living standards rise, their expectations for tomatoes are becoming increasingly sophisticated. Fruits and vegetables with attractive appearance have become the top choice for today's consumers. The sensory quality of tomatoes has become one of the most direct factors influencing consumer purchasing decisions, and tomatoes with good appearance have a strong market. In production, varieties with high gloss are favored by farmers due to their higher prices compared to varieties with less glossiness, leading to the gradual elimination of varieties with less glossiness. Therefore, it is necessary to develop a batch of high-gloss tomato varieties that meet market demand.

[0003] However, in tomato breeding, the evaluation and selection of quality traits is time-consuming and must be performed after the fruit has matured normally (Li Xiaolei et al., 2010). In conventional breeding, the identification of the fruit skin gloss trait must wait until the fruit matures, which is also time-consuming (Zhou Bingyu et al., 2013). Although molecular marker-assisted selection can effectively overcome the shortcomings of traditional breeding, shorten the breeding cycle, and accelerate the breeding process (Zhu Mingtao et al., 2010; Xu Xiangyang et al., 2011; Xu et al., 2015), the current selection of varieties for gloss is relatively blind. There are no trait-linked molecular markers that can be effectively applied in molecular marker-assisted breeding. Therefore, research on the precise mapping of gloss traits is urgently needed.

[0004] Single nucleotide polymorphism (SNP), a third-generation molecular marker technology, has been widely used in germplasm research, variety authenticity verification, and marker-assisted breeding due to its widespread distribution across biological genomes, rich polymorphism, ease of detection and statistics, and high-throughput automated detection. Currently, there are several PCR-based SNP molecular marker detection methods for single-base variations, such as enzyme-amplified polymorphic sequence analysis, high-resolution melting curve analysis, allele-specific PCR, and competitive allele-specific PCR.

[0005] PARMS (Penta-primer amplification refractory mutation system) is a PCR detection technology developed to specifically resolve single-base variants in single-nucleotide polymorphisms (SNPs). This technology allows for rapid and simple SNP allele genotyping. The PARMS SNP PCR reaction uses five primers, two of which are fluorescent universal primers included in the PARMS 2× Master Mix. The remaining three are marker-specific primers and require custom design and synthesis based on the experimental objectives. One of these three primers is locus-specific, and the other two are allele-specific. Each of these two primers has a 21-base universal adapter sequence added to its 5' end to mate with the fluorescent universal primers for amplification. The adapter sequence mates with the FAM fluorophore: 5'-GAAGGTGACCAAGTTCATGCT-3'; the adapter sequence mates with the HEX fluorophore: 5'-GAAGGTCGGAGTCAACGGATT-3'. The basic principles for marker primer design follow universal primer design rules: primer length should be 18–30 bp, and the amplified fragment should be less than 250 bp (including the primer). Shorter is preferred, provided the primer design requirements are met. Allele 1 and Allele 2, specific amplification primers with two different universal adapter primer sequences, bind to the corresponding SNP DNA template after DNA annealing. The PARMS PCR enzyme and buffer system ensure strict allele-specific amplification. Combined with the Locus-specific amplification primers, after the first two rounds of PCR, PCR amplification products with universal adapter sequences are formed. At this point, a universal probe with a reporter fluorescence and a fluorescence quencher group (no fluorescence signal due to the FRET effect in the absence of amplification) can be used for PCR amplification using the PCR amplification product with the universal adapter sequence as a template. Once amplification is successful, the fluorescence quencher and reporter groups on the fluorescent probe dissociate, and the FRET effect disappears. Fluorescence scanning then detects the corresponding fluorescence signal, thereby determining the presence of the corresponding allele.

[0006] PARMS technology offers high accuracy and good stability; its detection cost is low. Detection of different SNP loci only requires the design of three common primers for the corresponding loci, with fluorescently labeled primers serving as universal primers. It is compatible with crude DNA extraction using alkaline boiling, requiring minimal sample preparation time, making it particularly suitable for large-scale, high-throughput detection platforms. In recent years, PARMS SNP detection technology has been increasingly widely used in population genetics research, disease diagnosis, and plant breeding. Therefore, the identification of SNP molecular markers associated with tomato fruit gloss based on PARMS technology, which can be used to determine whether the tomato material being tested is high-gloss, is of great value in assisting the breeding and rapid identification of high-gloss tomato varieties.

[0007] Summary of the Invention

[0008] The purpose of the present invention is to provide a SNP molecular marker related to tomato fruit gloss and its application to solve the problems existing in the above-mentioned prior art. The PARMS SNP molecular marker developed by the present invention co-segregates with the tomato fruit gloss phenotype, which can determine the fruit gloss during the tomato seedling stage, thereby reducing seed production costs. It has important value in assisting the breeding and rapid identification of tomato varieties with high glossiness.

[0009] To achieve the above object, the present invention provides the following solutions:

[0010] The present invention provides a SNP molecular marker related to the glossiness of tomato fruits, the nucleotide sequence of which is shown in SEQ ID NO: 4; there is an A / G base mutation at the 34th bp position of the sequence shown in SEQ ID NO: 4.

[0011] The present invention also provides a primer set for detecting the SNP molecular marker, comprising a forward primer with a nucleotide sequence as shown in SEQ ID NO: 1, a first reverse primer with a nucleotide sequence as shown in SEQ ID NO: 2, and a second reverse primer with a nucleotide sequence as shown in SEQ ID NO: 3.

[0012] Furthermore, the 5' ends of the first reverse primer and the second reverse primer are connected to fluorescent groups of different colors.

[0013] Furthermore, the first reverse primer is connected to a FAM fluorescent group, and the second reverse primer is connected to a HEX fluorescent group.

[0014] The present invention also provides a kit for identifying the glossiness of tomato fruits, comprising the primer set.

[0015] The present invention also provides a method for detecting the glossiness of tomato fruits, comprising the following steps:

[0016] Using the DNA of the tomato sample to be tested as a template, PCR amplification is performed using the primer set to obtain an amplified product;

[0017] The glossiness of the tomato fruit to be tested is determined based on the fluorescence signal of the amplified product.

[0018] Furthermore, if the fluorescence signal is HEX, the tomato to be tested is judged to be genotype A, and the fruit gloss is low; if the fluorescence signal is FAM, the tomato to be tested is judged to be genotype G, and the fruit gloss is high.

[0019] Furthermore, the PCR amplification reaction system includes: 5 μL of 2×PARMS master mix, 0.15 μL of forward primer, 0.15 μL of first reverse primer, 0.4 μL of second reverse primer, 10-100 ng of DNA template, and ddH2O to 10 μL.

[0020] Furthermore, the reaction conditions of the PCR amplification are: 94°C for 15 min; 94°C for 20 s, 65-57°C for 1 min, 10 cycles; 94°C for 20 s, 57°C for 1 min, 32 cycles.

[0021] The present invention also provides an application of the SNP molecular marker, the primer set or the kit in screening high-gloss varieties of tomato fruits.

[0022] The present invention discloses the following technical effects:

[0023] This study developed a PARMS SNP molecular marker associated with tomato fruit gloss. The SNP locus is located at base 63041874 on tomato chromosome 1. Based on the mapping results, specific molecular marker primers were developed to identify tomato fruit gloss. By detecting polymorphisms at the marker locus, differences in tomato fruit gloss were identified. The PARMS SNP molecular marker developed in this study co-segregates with the tomato fruit gloss phenotype, allowing for determination of fruit gloss during the seedling stage, thereby reducing seed production costs. This is of great value in assisting the breeding and rapid identification of high-gloss tomato varieties. It can also accelerate the development of new tomatoes or other fruiting vegetables. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0025] Figure 1 shows a GWAS analysis of tomato fruit gloss; the orange origin in the figure indicates the SNP detected at position 63,041,874 bp on chromosome 1 that is significantly associated with tomato fruit gloss;

[0026] Figure 2 shows the glossiness of the test materials. The glossiness of the fruits of the 31 low-gloss materials ranged from 1.1 to 3.6, and the glossiness of the fruits of the 31 high-gloss materials ranged from 8.9 to 18.7. DETAILED DESCRIPTION

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0030] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0032] Example 1

[0033] Experimental materials: Tomato population materials (from Zhu et al., 2018, Cell 172, 249-261).

[0034] 1. Mining of SNPs associated with tomato fruit gloss: Based on the fruit gloss of 297 germplasm resources (from Guangtao Zhu, et al., Rewiring of the Fruit Metabolome in Tomato Breeding. Cell 172, 2018, Pages 249-261.e12), genome-wide association studies (GWAS) were performed on the genomic data and fruit gloss. A SNP significantly associated with tomato fruit gloss was detected at position 63041874bp on chromosome 1 (NCBI accession number SL2.50 (GCF_000188115.3) of the tomato reference genome) (see Figure 1), and an A / G / - base mutation was found.

[0035] 2. Design of Molecular Marker Primer Combinations for PARMS SNP Detection: Based on the SNP loci identified by GWAS analysis, we searched for position 63041874 on chromosome 1. We designed a locus-specific primer as the forward primer and two allele-specific primers: Allele G primer with a FAM blue fluorescent linker sequence as the first reverse primer; Allele A primer with a HEX green fluorescent linker sequence as the second reverse primer. Primer sequences are shown in Table 1:

[0036] Table 1 Molecular marker primer combinations for SNP detection Note: The underlined sequence in Allele G primer is the FAM fluorescent tag sequence; the underlined sequence in Allele A primer is the HEX fluorescent tag sequence.

[0037] 3. Screening of test germplasm: 31 accessions with high fruit gloss and 31 accessions with low fruit gloss were selected from the GWAS population. The glossiness of the 62 test accessions is shown in Figure 2. The glossiness of the 31 low-gloss accessions ranged from 1.1 to 3.6, while the glossiness of the 31 high-gloss accessions ranged from 8.9 to 18.7.

[0038] 4. Sample material processing: Place a leaf blade approximately 1 cm in length and width into a deep-well plate (96 wells, 1.2 ml); add 100 μL of 0.3 M sodium hydroxide and grind the sample at 50 Hz for 2 min (until the sample is completely ground) using a Shanghai Jingxin tissue grinder; after grinding, centrifuge at 3000 rpm for 1 min and place in a boiling water bath for 2 min; then add 200 μL of 0.2 M Tris-HCl (pH 6.8-7.0) and mix thoroughly, then place in a boiling water bath for another 2 min; after the water bath, centrifuge at 3000 rpm for 1 min, dilute the supernatant 20-fold, and freeze at -20°C to use as a template for subsequent PCR amplification.

[0039] 5. PCR amplification: The PCR amplification reaction system is shown in Table 2 (2×PARMS master mix provided by Wuhan Jingpeptide Biotechnology Co., Ltd.):

[0040] Table 2 PCR amplification reaction system

[0041] PCR amplification reaction conditions are as follows (dual-head 384 PCR instrument ABI Gene Amp 9700):

[0042] Table 3 PCR amplification reaction conditions

[0043] The sequence of the amplified fragment is as follows (SEQ ID NO: 4):

[0044] Note: R represents the A / G base mutation site.

[0045] 6. Genotyping: After PCR, fluorescence signals were read using a TECAN infinite M1000 microplate reader. The online software snpdecoder (http: / / www.snpway.com / snpdecoder / ) was then used to analyze and convert the fluorescence signals, producing clear and intuitive genotyping plots. The genotype results were then output based on color. The results are shown in Table 1. The PARMS genotyping results for 31 low-gloss samples showed that 25 samples had HEX genotypes, consistent with the sample's native genotype A, confirming that the SNP marker site mutated to A, indicating that the tested tomato sample had low fruit gloss. Three samples had a deletion at this site and had genotyping results of FAM, while two samples showed no results. The PARMS genotyping results for 31 high-gloss samples were all FAM, consistent with the sample's genotype G, confirming that the SNP marker site mutated to G, indicating that the tested tomato sample had high fruit gloss. Comprehensive statistics of the results of PARMS testing of 62 test materials showed that the PCR genotyping results of 52 materials were consistent with the material genotype, and the accuracy of this molecular marker reached 90.3%. This shows that using this SNP molecular marker to conduct PARMS experiments on the test samples can effectively detect their genotypes and can be used for the identification of tomato fruit color and auxiliary breeding.

[0046] Table 4 Comparison of genotypes and SNP typing results of PARMS detection in 62 test materials

[0047] Note: Left: 31 parts of low gloss material; Right: 31 parts of high gloss material.

[0048] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A SNP molecular marker associated with tomato fruit gloss, characterized in that: The nucleotide sequence is shown in SEQ ID NO: 4; there is an A / G base mutation at the 34 bp position of the sequence shown in SEQ ID NO:

4.

2. A primer set for detecting the SNP molecular marker according to claim 1, characterized in that: It comprises a forward primer having a nucleotide sequence as shown in SEQ ID NO:1, a first reverse primer having a nucleotide sequence as shown in SEQ ID NO:2 and a second reverse primer having a nucleotide sequence as shown in SEQ ID NO:

3.

3. The primer set according to claim 2, characterized in that The 5' ends of the first reverse primer and the second reverse primer are connected to fluorescent groups of different colors.

4. The primer set according to claim 3, characterized in that: The first reverse primer is connected to a FAM fluorescent group, and the second reverse primer is connected to a HEX fluorescent group.

5. A kit for identifying the glossiness of tomato fruit, characterized in that: The invention comprises the primer set according to any one of claims 2 to 4.

6. A method for detecting the glossiness of tomato fruit, characterized in that: The following steps are involved: Using the DNA of the tomato sample to be tested as a template, performing PCR amplification using the primer set of claim 4 to obtain an amplified product; The glossiness of the tomato fruit to be tested is determined based on the fluorescence signal of the amplified product.

7. The method according to claim 6, characterized in that If the fluorescence signal is HEX, the tomato to be tested is judged to be genotype A, and the fruit has low gloss; if the fluorescence signal is FAM, the tomato to be tested is judged to be genotype G, and the fruit has high gloss.

8. The method according to claim 6, characterized in that The PCR amplification reaction system includes: 2×PARMS master mix 5 μL, forward primer 0.15 μL, first reverse primer 0.15 μL, second reverse primer 0.4 μL, DNA template 10-100 ng, ddH2O supplemented to 10 μL.

9. The method according to claim 6, characterized in that The reaction conditions of the PCR amplification were: 94°C for 15 min; 94°C for 20 s, 65-57°C for 1 min, 10 cycles; 94°C for 20 s, 57°C for 1 min, 32 cycles.

10. Use of the SNP molecular marker according to claim 1, the primer set according to any one of claims 2 to 4, or the kit according to claim 5 in screening tomato fruit varieties with high glossiness.

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