SNP (Single Nucleotide Polymorphism) molecular marker related to tomato peel thickness character and application of SNP molecular marker

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) molecular marker related to tomato peel thickness character and application thereof, and belongs to the technical field of nucleic acid-containing determination methods. A representative and universal SNP site related to the tomato peel thickness is obtained through identification of a whole genome association analysis method and is at least one of the 57968691 site, the 58439462 site, the 60203161 site and the 60338017 site of the full-length sequence of the third chromosome of a tomato genome, a primer group is further developed, detection is conducted through a KASP method, wide verification is achieved in different materials, and the SNP site can be used for identifying the tomato peel thickness. And a group typing effect which is highly consistent and has clear boundaries is obtained. Based on the detection method provided by the invention, rapid, accurate and high-throughput detection of the tomato peel thickness can be realized, and great help is provided for improving the breeding efficiency, reducing the breeding cost, accelerating the breeding process and researching genetic improvement of varieties.

Description

Technical Field

[0001] The present invention belongs to the technical field of nucleic acid determination methods, and particularly relates to a SNP molecular marker associated with tomato peel thickness traits and an application thereof. Background Art

[0002] tomato( Solanum lycopersicum Solanum lycopersicum L.) is an annual herbaceous plant of the Solanaceae family. Its fruit can be eaten fresh or processed into by-products such as juice and jam. It is an important part of people's balanced diet and is deeply loved by consumers for its unique flavor and rich nutrients such as lycopene, ascorbic acid, and flavonoids.

[0003] Tomato fruit development is a complex biological process regulated by multiple genetic and environmental factors. Pericarp formation is a crucial step in tomato fruit development. Pericarp typically refers to the outer skin of the fruit. Pericarp thickness is associated with numerous traits, including yield, appearance quality, flavor quality, cracking resistance, and storage and transportation resistance. For example, thin-skinned tomatoes offer a superior taste and are a breeding goal for fresh-eating tomatoes. Thick-skinned tomatoes, on the other hand, are less prone to cracking and are more resistant to storage and transportation. Therefore, cultivating and producing tomato varieties with varying pericarp thicknesses based on specific production needs can meet diverse and specialized consumer demands and achieve efficient, high-quality production.

[0004] Genome-wide association study (GWAS) is an important method for studying the relationship between genetic variation and complex traits. Through genome-wide association analysis of large-scale samples, genes and mutation sites associated with specific phenotypic characteristics can be identified. Minkyung Kim et al. used GWAS technology to analyze and explore quantitative trait loci (QTL) related to eight traits of tomato fruit (including skin thickness). The results showed that single nucleotide polymorphism (SNP) sites SLA805176 and SLA775228 on chromosome 2, SNP site SLA769530 on chromosome 9, and SNP site SLA790046 on chromosome 12 were correlated with tomato skin thickness (Kim et al. et al . Genome-wide association study identifies QTL for eight fruittraits in cultivated tomato ( Solanum lycopersicum L.) . Horticulture Research ,2021,8:203.).

[0005] Competitive allele-specific PCR (KASP) uses allele-specific primers for PCR amplification and fluorescent dye-based detection of the amplified products. It can simultaneously detect multiple loci in a single reaction and boasts advantages such as high efficiency, accuracy, reliability, low cost, and ease of automation and high-throughput operation. It is a leading genotyping method worldwide and has been widely used in fields such as biomedicine and plant and animal breeding. By combining KASP genotyping with mutation sites identified through GWAS analysis, molecular markers associated with specific traits can be developed. This allows for rapid identification of target traits in the laboratory, saving costs and significantly improving breeding efficiency. For example, patent documents CN 117683938 A and CN 118406794 A disclose KASP molecular markers tightly linked to tomato fruit width and length, respectively. These markers can be rapidly identified using PCR amplification.

[0006] Tomato peel thickness is a quantitative trait controlled by multiple genes. Discovering more KASP molecular markers related to it is of great significance for a comprehensive understanding of the genetic basis of peel thickness trait and molecular breeding research of tomatoes. Summary of the Invention

[0007] The purpose of the present invention is to provide a molecular marker related to the tomato peel thickness trait, which is used to quickly identify the tomato peel thickness trait, improve the accuracy and efficiency of breeding screening, and provide tomato breeders with a convenient and accurate screening method.

[0008] To achieve the above object, the present invention adopts the following technical solutions: The present invention obtains multiple single nucleotide polymorphism sites related to the phenotypic characteristics of tomato peel thickness through genome-wide association analysis of large-scale samples, further designs KASP primers, performs typing and identification in tomato populations, and develops molecular markers with good typing effects.

[0009] The present invention uses the screened single nucleotide polymorphism site as a detection target for screening or identifying tomato peel thickness. The single nucleotide polymorphism site is at least one of positions 57968691, 58439462, 60203161, and 60338017 of the full-length sequence of chromosome 3 of the tomato genome, wherein the 57968691 position has a T>C polymorphism, the 58439462 position has a G>A polymorphism, the 60203161 position has a T>C polymorphism, and the 60338017 position has a G>A polymorphism; the tomato genome sequence number is GCF_000188115.5.

[0010] Specifically, the single nucleotide polymorphism sites are as follows: The SL_57968691 site is located at position 57968691 of the full-length sequence of chromosome 3 in the tomato genome version SL3.1 (sequence number GCF_000188115.5 in the NCBI database). The base is T or C. When the genotype is TT, the tomato peel thickness is thinner, and when the genotype is CC, the tomato peel thickness is thicker. The SL_58439462 site is located at position 58439462 of the full-length sequence of chromosome 3 in the SL3.1 version of the tomato genome. The base is G or A. When the genotype is GG, the tomato peel is thinner, and when the genotype is AA, the tomato peel is thicker. The SL_60203161 site is located at position 60203161 of the full-length sequence of chromosome 3 in the SL3.1 version of the tomato genome. The base is T or C. When the genotype is TT, the tomato peel thickness is thinner, and when the genotype is CC, the tomato peel thickness is thicker. The SL_60338017 site is located at position 60338017 of the full-length sequence of chromosome 3 of the tomato genome version SL3.1. The base is G or A. When the genotype is GG, the tomato skin thickness is thinner, and when the genotype is AA, the tomato skin thickness is thicker.

[0011] The research of the present invention shows that using the above-mentioned SNP sites as detection targets in the tomato population has obtained highly consistent and clearly defined population typing effects, which can clearly distinguish tomato materials with different genotypes, and the difference in peel thickness of materials with different genotypes reaches an extremely significant difference level.

[0012] Furthermore, the application includes: using genomic DNA from a tomato plant to be tested as a template, performing a PCR reaction using a primer set specifically detecting the single nucleotide polymorphism site, determining the genotype of the tomato to be tested based on the PCR product amplified by the corresponding primer pair, and predicting tomato peel thickness. This method can be used to screen tomato germplasm at the seedling stage or even earlier.

[0013] The present invention can also develop a corresponding detection kit using the above-mentioned SNP site as a detection target, wherein the detection kit includes a primer set for specifically detecting the SNP site in the tomato genome, and the primer set includes two specific forward primers and a universal reverse primer.

[0014] Another object of the present invention is to provide a method for detecting the thickness of tomato peel, the method comprising the following steps: (1) Extracting genomic DNA from the tomato plants to be tested; (2) Using genomic DNA from tomato plants as a template, PCR amplification was performed using a primer set for amplifying a KASP molecular marker associated with tomato peel thickness. The primer set was used to specifically detect at least one single nucleotide polymorphism site at positions 57968691, 58439462, 60203161, and 60338017 of the full-length sequence of chromosome 3 of the tomato genome, wherein a T>C polymorphism existed at position 57968691, a G>A polymorphism existed at position 58439462, a T>C polymorphism existed at position 60203161, and a G>A polymorphism existed at position 60338017; the tomato genome sequence number was GCF_000188115.5; (3) The PCR products were tested and the tomato was genotyped. For the polymorphism at position 57968691, when the genotype was TT, the tomato peel was judged to be thin, and when the genotype was CC, the tomato peel was judged to be thick. For the polymorphism at position 58439462, when the genotype was GG, the tomato peel was judged to be thin, and when the genotype was AA, the tomato peel was judged to be thick. For the polymorphism at position 60203161, when the genotype was TT, the tomato peel was judged to be thin, and when the genotype was CC, the tomato peel was judged to be thick. For the polymorphism at position 60338017, when the genotype was GG, the tomato peel was judged to be thin, and when the genotype was AA, the tomato peel was judged to be thick.

[0015] Preferably, in step (1), genomic DNA is extracted from leaves of seedlings of the tomato variety to be tested.

[0016] The present invention develops a KASP molecular marker associated with the tomato peel thickness trait based on the aforementioned SNP locus. Specifically, using the SNP locus as coordinates, nucleotide fragments upstream and downstream of the genome are intercepted as KASP molecular markers. Primers are designed based on the SNP locus in the KASP molecular marker to specifically detect the SNP locus in the tomato genome.

[0017] Preferably, the KASP molecular marker is a nucleotide sequence of 40 to 100 bp upstream and downstream, taking the SNP site as the coordinate.

[0018] More preferably, the nucleotide sequence of the KASP molecular marker is shown in any one of SEQ ID NOs. 1 to 4.

[0019] The present invention designs a primer set according to the SNP site in the KASP molecular marker, and the primer set includes a specific forward primer 1, a specific forward primer 2 and a universal reverse primer.

[0020] Specifically, the nucleotide sequences of the primer set for detecting the polymorphism at position 57968691 are shown as SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7; the nucleotide sequences of the primer set for detecting the polymorphism at position 58439462 are shown as SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10; the nucleotide sequences of the primer set for detecting the polymorphism at position 60203161 are shown as SEQ ID NO.11, SEQ ID NO.12, and SEQ ID NO.13; and the nucleotide sequences of the primer set for detecting the polymorphism at position 60338017 are shown as SEQ ID NO.14, SEQ ID NO.15, and SEQ ID NO.16.

[0021] The present invention shows that the use of the primer set for KASP amplification has verified that a highly consistent and clearly defined population typing effect is obtained in a tomato population.

[0022] Preferably, the 5' ends of the specific forward primer 1 and the specific forward primer 2 are connected to two different fluorescent tag sequences, respectively. Different genotyping can be achieved by identifying the fluorescent tags.

[0023] More preferably, the 5' ends of the two specific forward primers are labeled with a FAM group and a HEX group, respectively.

[0024] Preferably, in step (2), the PCR reaction system is: 0.8 μL of 20 ng / μL template DNA, 0.75 μL of 2× KASP premix, and 0.05 μL of primer mixture, wherein the primer mixture is prepared by mixing forward primer 1, forward primer 2, and reverse primer at a concentration of 100 μM with ddH2O in a volume ratio of 12:12:30:46.

[0025] Preferably, in step (2), the PCR reaction procedure is as follows: step 1: pre-denaturation at 94°C for 15 minutes; step 2: 94°C for 20 seconds, annealing at 61-55°C for 60 seconds, for a total of 10 cycles, with the temperature decreasing by 0.6°C each cycle; step 3: denaturation at 94°C for 20 seconds, annealing at 55°C for 60 seconds, for a total of 26 cycles.

[0026] The present invention has the following beneficial effects: (1) The present invention identified representative and universal SNP sites associated with tomato peel thickness through genome-wide association analysis. The primer set further developed was tested using the KASP method and was widely verified in different materials, achieving highly consistent and well-defined population typing results.

[0027] (2) The detection method developed based on the SNP loci of the present invention can realize rapid, accurate and high-throughput detection of tomato peel thickness. It can perform rapid, accurate and high-throughput identification of tomato peel thickness at the tomato seedling stage, realize early selection of breeding targets, and is of great help in improving breeding efficiency, reducing breeding costs, accelerating breeding progress and genetic improvement research of varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the Manhattan plot of the whole gene association analysis of peel thickness in the present invention. The dotted line in the figure represents P = 1×10 -8.514 The threshold level for significant correlation.

[0029] Figure 2 This is the KASP marker typing diagram of the SL_57968691 marker site in 37 materials.

[0030] Figure 3 This is the KASP marker typing diagram of the SL_58439462 marker site in 37 materials.

[0031] Figure 4 This is the KASP marker typing diagram of the SL_60203161 marker site in 37 materials.

[0032] Figure 5 This is the KASP marker typing diagram of the SL_60338017 marker site in 37 materials.

[0033] Figure 6 This is the KASP marker typing diagram of the SL_57968691 marker site in 262 materials.

[0034] Figure 7 This is the KASP marker typing diagram of the SL_58439462 marker site in 262 materials.

[0035] Figure 8 This is the KASP marker typing diagram of the SL_60203161 marker site in 262 materials.

[0036] Figure 9 This is the KASP marker typing diagram of the SL_60338017 marker site in 262 materials.

[0037] Figure 10 This is a phenotypic difference diagram of the allelic variation of pericarp thickness at the SL_57968691 marker locus in 262 materials.

[0038] Figure 11 This is a phenotypic difference map of the allelic variation of pericarp thickness at the SL_58439462 marker locus in 262 materials.

[0039] Figure 12 This is a phenotypic difference map of the allelic variation of pericarp thickness at the SL_60203161 marker locus in 262 materials.

[0040] Figure 13 This is a phenotypic difference map of the allelic variation of pericarp thickness at the SL_60338017 marker locus in 262 materials. DETAILED DESCRIPTION

[0041] The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the following embodiments are provided for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0042] Unless otherwise specified, the experimental methods used in the following examples were generally carried out under conventional conditions or conditions recommended by the manufacturers.

[0043] Unless otherwise specified, the materials and reagents used in the following examples can be purchased from commercial sources.

[0044] Example 1: Mining SNP sites associated with tomato peel thickness 1. Materials 241 natural tomato populations originated from the germplasm resources preserved by the Tomato and Pepper Group of the Vegetable Research Institute of Zhejiang Academy of Agricultural Sciences. The names of the 241 natural tomato population materials are: T16-134A, T16-065AA, T16-031E, T16-040A, T16-025B, T15-205-0-0, T15-207-0-0, T15-211-0-4-4-1-0, T15-196, T15-202-0, KP-13, T15-042-0-3-1-2, T17-117CD1, T17-103A, T17-113CA, T17-097AB, T18-222AA, S11-30A, T17-115A-1, T17-132, T17-107CA, T18-235 A1, T18-206A1, T18-216A, T17-006AB, T11-002B, F820AA, T15-210-0-5- 2-11, T09-901A-0, 7818D-0, T15-184-2-1-0-2-2-1, T15-089, T18-201, T18-203, T18-185, T18-198, T18-164A1, T06-104A, T15-113, T17-119A, T17-107CB, T18-241AA, T18-205AA, T18-158A, T18-200A, T06-002-0, PI 634844, ZJU-035 (TC1320), ZJU-033 (H18103), ZJU-031 (HZZ8), ZJU-030 (TH107), ZJ U-029 (T16), ZJU-026 (HYZ3), ZJU-023 (RD17-301), ZJU-018 (ZRS_7), ZJU-015 (Gls-I P2), ZJU-013 (LA3320), ZJU-010 (Ty-5+P2), ZJU-008 (CLN2413), ZJU-007 (PSL-5), ZJU-006 (LPI-1), ZJU-005 (T18431), ZJU-003 (171011), ZJU-002 (Microtom), T07-021, T09-078A, T09-089-2-0, T09-101-7-1, T09-004A, T09-011A-0, T09-111A-0, T09-110A-0, T09-915F2-1-0, PI 127806, PI 127805, PI 126952, PI 126947, PI 126934, PI 126932, PI 126931, PI 126925, PI 126924, PI 126432, PI<h2 style=";text-align:left;direction:ltr">118407、PI112215、PI 110596、T09-921A-0、T09-910A-0、PI 406976、PI 406970、PI 406930、PI406925、PI 406907、PI 379021、PI 379006、PI 379003、PI 379001、PI 378996、PI 375937、PI 370093、PI 365966、PI 365964、PI 365930、PI 365927、PI 365923、PI 365922、PI390649、PI 390509、PI 390689、PI 406770、PI 390706、PI 390727、PI 390724、PI 390749、PI 190256、PI 204981、PI 205009、PI 211840、PI 230327、PI 303662、PI 279734、PI279372、PI 279371、PI 279370、PI 279368、T17-087A、T16-131C、T11-024P、T14-044C、T17-090A、T13-024A、T12-040A、T12 -031A、T9179、T18-131A、T14-011A、T13-004D、T13-013C、T12-032RA、T12-023-0、T20-01 6A, T01-199A, T9248-2, T11-023A, T11-066, T17-086A, 27A27A, CRA66, 271807BA, 93170-3-1-2-0, 17LD-5A, T04-101, T03-14-0, T17-204, T17-205, T17-017AD, T11-083, T17-043 A1、T18-017AA、T18-048CA、T18-021A1、T20-003-0、T17-139、T17-195、T17-202、T18-073 BA、T18-077AA、T18-086A、T18-074A、T18-042ED、T18-053、T9022、T18-079CA、T18-009A、 T19-137A、T15-040-2-1-4-3-0、T15-015-3-4-4-1-0、T18-088、T19-042-0-1、T19-029、T 19-104A、T19-092-0-0、T19-083A、T19-250D、T19-215A、T19-148A、T19-194、T19-197、PI407548, PI 407544, PI 407430, PI 95588, PI 435238, PI414174, PI 407552, PI 407555, PI 407556, PI 407545, PI 406797, PI 438556, PI 438591, PI 422397, PI 406832, PI 406819, PI 406824, PI 406777, PI 406884, PI 406877, PI406879, PI 406836, PI 406894, PI 406895, PI 406888, PI 406889, PI 438896,PI 560323, PI 487625, PI 452284, PI 647555, PI 560324, PI 379061, PI 379055, PI 379037, PI390500, PI 390502, PI 390406, PI 390410, PI 321749, PI 340905, PI 344102, PI 365913, PI 365915, PI 390507, T19-172A, T18-083, T07-043B, T18-087, T19-209, T18-089, 11051, 11052.

[0045] 2. Data Collection The natural population was planted in the Yangdu Scientific Research and Innovation Base of Zhejiang Academy of Agricultural Sciences. Three holes were planted in the seedling tray for each tomato material, and six plants were retained in each hole. After germination, the strongest plants were selected for transplantation, and five plants were planted for each material. After the fruits matured, three complete and disease-free fruits in the commercial maturity stage were randomly selected. The tomato fruit composite phenotype extraction and evaluation technology based on image recognition technology developed by the Tomato and Pepper Group of the Vegetable Research Institute of Zhejiang Academy of Agricultural Sciences was used to obtain the peel thickness data.

[0046] 3. Screening sites The SNPs obtained from resequencing 241 tomato materials were screened using the criteria of minimum allele frequency (MAF) > 0.05 and missingness rate less than 20%. Only sites with biallelic genes were retained, resulting in 8,668,967 SNPs. GEMMA was used to perform association analysis on related traits in different populations, with a P = 1 × 10 -8.514 The significant correlation threshold level was used to screen out potential candidate SNPs.

[0047] 4. Filter results Four regions significantly associated with pericarp thickness were identified genome-wide ( Figure 1 ), of which a region on chromosome 3 of the tomato genome had the greatest genetic contribution to pericarp thickness. Thirteen SNPs were selected for initial screening.

[0048] The tomato genome version is SL3.1, with the specific sequence number GCF_000188115.5. The 13 SNP sites are: 1. SL_57968691, located at position 57968691 in the full-length sequence of chromosome 3 of the tomato genome version SL3.1, with a base of T or C; 2. SL_58439462, located at position 58439462 in the full-length sequence of chromosome 3 of the tomato genome version SL3.1, with a base of G or A; 3. SL_60203161 site, located at position 60203161 in the full-length sequence of chromosome 3 of the tomato genome version SL3.1, with a base of T or C; 4. SL_60338017, located at position 60338017 of the full-length sequence of chromosome 3 of the tomato genome version SL3.1, with a base of G or A; 5. SL_57623177, located at position 57623177 of the full-length sequence of chromosome 3 of the tomato genome version SL3.1, with a base of G or C; 6. SL_58057668, located at position 58057668 of the full-length sequence of chromosome 3 of the tomato genome version SL3.1, with a base of C or T; 7. SL_58363546, located at position 58363546 of the full-length sequence of chromosome 3 of the tomato genome version SL3.1, with bases A or G; 8. SL_58432413, located at position 58432413 of the full-length sequence of chromosome 3 of the tomato genome version SL3.1, with a base of G or A; 9. SL_60176091, located at position 60176091 of the full-length sequence of chromosome 3 of the tomato genome version SL3.1, with a base of C or A; 10. SL_65935114, located at position 65935114 of the full-length sequence of chromosome 3 of the tomato genome version SL3.1, with a base of C or A; 11. SL_66009308, located at position 66009308 of the full-length sequence of chromosome 3 of the tomato genome version SL3.1, with bases A or C; 12. SL_66030262, located at position 66030262 of the full-length sequence of chromosome 3 of the tomato genome version SL3.1, with bases A or G; 13. SL_66043774 site is located at position 66043774 of the full-length sequence of chromosome 3 of the tomato genome version SL3.1, and the base is T or A.

[0049] Example 2: Design and selection of SNP primers related to tomato peel thickness 1. Based on the 13 sites provided in Example 1, 100 bp of sequences upstream and downstream of the relevant SNP sites were downloaded from NCBI to design KASP primer sets. The primer set for each site included two specific forward primers F1 and F2 and a universal reverse primer R. The 5' ends of the two specific forward primers were connected to a fluorescent tag sequence carrying a FAM group and a fluorescent tag sequence carrying a HEX group, respectively, as shown in Table 1.

[0050] Table 1. Nucleotide and primer sequence of 13 SNP sites

[0051] 2. Thirteen loci on chromosome 3 of the tomato genome were screened based on whether they could produce highly consistent and clearly defined population typing results.

[0052] 37 samples and 1 ddH2O were randomly selected from the 241 tomato materials in Example 1 as NTC blank controls. The names of the 37 sample materials were: PI 390706, PI 390727, PI 406894, PI 438896, PI560323, PI 487625, PI 452284, PI 647555, PI 560324, PI 379061, PI 379055, PI 379037, PI 390500, PI 390502, PI 390406, PI 390410, PI 321749, PI 340905, PI 344102, PI 365915, PI 390507, T19-172A, T18-083, T07-043B, 11052, T17-132, PI 126947, 93170-3-1-2-0, PI 127805, PI 126952, PI 126934, PI 126931, PI 126925, PI 126932, PI 110596, PI406888, PI 406889.

[0053] Tomato genomic DNA was extracted by magnetic bead method. The tomato genomic DNA to be tested was used as template. The genomic DNA samples of the 37 tomato materials were amplified and fluorescently detected using 13 pairs of KASP primer sets on the IntelliQube genotyping platform.

[0054] PCR reaction system: DNA 0.8 μL, 2× KASP Master Mix 0.75 μL, KASP Assay Mix 0.05 μL. KASP Assay Mix was prepared by mixing 100 μM specific forward primer F1, specific forward primer F2, universal reverse primer R, and ddH2O in a volume ratio of 12:12:30:46. PCR reaction program: pre-denaturation at 94°C for 15 min, denaturation at 94°C for 20 s, annealing at 61-55°C (temperature decreased by 0.6°C each cycle) for 60 s, for a total of 10 cycles; denaturation at 94°C for 20 s, annealing at 55°C for 60 s, for a total of 26 cycles.

[0055] Fluorescence data was read and analyzed using the IntelliQube fully automated PCR instrument. Four significant signal clusters were observed. The red dots near the horizontal axis were the same color as the fluorescent linker of forward primer F1, indicating that the sample carried a FAM fluorescent signal. The blue dots near the vertical axis were the same color as the fluorescent linker of forward primer F2, indicating that the sample carried a HEX fluorescent signal. The dots clustered in the middle carried both signals. The black hollow dots represented the amplification signal of the negative control (NTC), and the gray hollow dots indicated that no amplification signal was detected in the sample.

[0056] like Figure 2-Figure 5 Finally, four primer sets with significant fluorescence signal aggregation typing trends and more sample signal amplification data were selected, namely SL_57968691 primer set, SL_58439462 primer set, SL_60203161 primer set, and SL_60338017 primer set.

[0057] Example 3: Application of KASP molecular markers for selecting tomato peel thickness 1. Data Collection A total of 262 tomato accessions were collected, as shown in Table 2. Accessions numbered 1 to 113 were sourced from the germplasm bank maintained by the Tomato and Pepper Group of the Vegetable Research Institute of the Zhejiang Academy of Agricultural Sciences, while accessions numbered 114 to 262 were sourced from a tomato farmer's germplasm procurement website. All 262 accessions were planted in the mulberry garden base of the Zhejiang Academy of Agricultural Sciences. Twelve seeds per accession were sown in seedling trays. After germination, four plants were selected and transplanted to a hydroponic greenhouse in the mulberry garden. After fruit ripening, three intact, disease-free fruits at commercial maturity were randomly selected. Peel thickness data were obtained using a tomato fruit composite phenotypic extraction and evaluation technology developed by the Tomato and Pepper Group of the Vegetable Research Institute of the Zhejiang Academy of Agricultural Sciences, based on image recognition technology.

[0058] 2. Selection of tomato peel thickness using KASP molecular markers (1) Extract genomic DNA of the tomato variety to be tested.

[0059] (2) The four primer pairs in Example 2 were used to perform KASP amplification on the genomic DNA of the tomato variety to be tested.

[0060] PCR reaction system: contains 0.8 μL 20 ng / μL genomic DNA, 0.75 μL 2× KASP MasterMix (from LGC), and 0.05 μL KASP Assay Mix (100 μM specific forward primer F1, specific forward primer F2, universal reverse primer R, and ddH2O in a volume ratio of 12:12:30:46); PCR reaction program: 94°C for 15 min, 94°C for 20 s, annealing at 61-55°C for 60 s, for a total of 10 cycles, with the temperature decreasing by 0.6°C each cycle; denaturation at 94°C for 20 s, annealing at 55°C for 60 s, for a total of 26 cycles.

[0061] (3) Use a fluorescent quantitative PCR instrument to detect the PCR products and determine the genotype of the tomato variety at the SNP site corresponding to each primer set.

[0062] Fluorescence data was read and analyzed using the IntelliQube fully automated PCR instrument. Three significant signal clusters were observed. The red dots near the horizontal axis matched the color of the fluorescent linker of forward primer F1, indicating that the sample carried a FAM fluorescent signal. The blue dots near the vertical axis matched the color of the fluorescent linker of forward primer F2, indicating that the sample carried a HEX fluorescent signal. The purple dots clustered in the middle represented samples carrying both signals.

[0063] like Figures 6 to 9As shown, the four groups of KASP markers provided in Example 2 achieved highly consistent and clearly defined population typing results in 262 different types of tomato populations, confirming that the SNP site combination provided in Example 1 has good versatility and stability in tomato variety resources with different genetic backgrounds.

[0064] The data of tomato fruit genotype detected by KASP molecular markers and the fruit skin thickness phenotype collected in the field are shown in Table 2.

[0065] Table 2

[0066] Note: / in the table indicates no fluorescent signal was detected.

[0067] like Figure 10-13 Combined with the peel thickness phenotype in Table 2, the detection results of the SL_57968691 primer set showed that among the 262 materials, the average peel width of the materials with the genotype TT was 5.319 mm, and the average peel width of the materials with the genotype CC was 7.997 mm. The difference in peel width between the two genotypes reached an extremely significant level ( p <0.0001), the CC genotype material has a wider pericarp thickness than the TT genotype material; The results of the SL_58439462 primer set showed that among the 262 materials, the average pericarp width of the materials with the GG genotype was 5.426 mm, and the average pericarp width of the materials with the AA genotype was 7.864 mm. The difference in pericarp width between the two genotypes reached an extremely significant level ( p <0.0001), the AA genotype material has a wider pericarp thickness than the GG genotype material; The results of the SL_60203161 primer set showed that among the 262 materials, the average pericarp width of the materials with the genotype TT was 5.439 mm, and the average pericarp width of the materials with the genotype CC was 7.626 mm. The difference in pericarp width between the two genotypes reached an extremely significant level ( p <0.0001), the CC genotype material has a wider pericarp thickness than the TT genotype material; The results of the SL_60338017 primer set showed that among the 262 materials, the average pericarp width of the materials with the GG genotype was 5.319 mm, and the average pericarp width of the materials with the AA genotype was 7.997 mm. The difference in pericarp width between the two genotypes reached an extremely significant level ( p <0.0001), the AA genotype material had a thicker and wider pericarp than the GG genotype material.

[0068] The four markers described above produced highly consistent and well-defined population typing results across different tomato varieties, with highly significant differences in the population typing results, indicating that these four markers can be used to guide selection for peel thickness. Applying these four markers to molecular marker-assisted breeding for genetic improvement of tomato quality, combined with conventional breeding, could accelerate the breeding process for varieties with varying peel thicknesses, such as processing tomatoes and tomato varieties with different taste characteristics, by selecting for different peel thicknesses.

Claims

1. Application of single nucleotide polymorphism sites as detection targets in screening or identifying tomato peel thickness, characterized in that: The single nucleotide polymorphism site is at least one of positions 57968691, 58439462, 60203161, and 60338017 of the full-length sequence of chromosome 3 of the tomato genome, wherein there is a T>C polymorphism at position 57968691, a G>A polymorphism at position 58439462, a T>C polymorphism at position 60203161, and a G>A polymorphism at position 60338017; the tomato genome sequence number is GCF_000188115.

5.

2. The use according to claim 1, characterized in that The application includes: using the genomic DNA of the tomato plant to be tested as a template, performing a PCR reaction using a primer set that specifically detects the single nucleotide polymorphism site, determining the genotype of the tomato to be tested, and predicting the tomato peel thickness.

3. A method for detecting the thickness of tomato peel, characterized in that: The following steps are involved: (1) Extracting genomic DNA from the tomato plants to be tested; (2) Using genomic DNA from tomato plants as a template, PCR amplification was performed using a primer set for amplifying a KASP molecular marker associated with tomato peel thickness. The primer set was used to specifically detect at least one single nucleotide polymorphism site at positions 57968691, 58439462, 60203161, and 60338017 of the full-length sequence of chromosome 3 of the tomato genome, wherein a T>C polymorphism existed at position 57968691, a G>A polymorphism existed at position 58439462, a T>C polymorphism existed at position 60203161, and a G>A polymorphism existed at position 60338017; the tomato genome sequence number was GCF_000188115.5; (3) The PCR products were tested and the tomato was genotyped. For the polymorphism at position 57968691, when the genotype was TT, the tomato peel was judged to be thin, and when the genotype was CC, the tomato peel was judged to be thick. For the polymorphism at position 58439462, when the genotype was GG, the tomato peel was judged to be thin, and when the genotype was AA, the tomato peel was judged to be thick. For the polymorphism at position 60203161, when the genotype was TT, the tomato peel was judged to be thin, and when the genotype was CC, the tomato peel was judged to be thick. For the polymorphism at position 60338017, when the genotype was GG, the tomato peel was judged to be thin, and when the genotype was AA, the tomato peel was judged to be thick.

4. The method for detecting the thickness of tomato peel according to claim 3, wherein: The KASP molecular marker is a nucleotide sequence of 40 to 100 bp upstream and downstream, with the single nucleotide polymorphism site as the coordinate.

5. The method for detecting the thickness of tomato peel according to claim 4, wherein: The nucleotide sequence of the KASP molecular marker is shown in any one of SEQ ID NOs. 1 to 4.

6. The method for detecting the thickness of tomato peel according to any one of claims 3 to 5, characterized in that: The primer set includes a specific forward primer 1, a specific forward primer 2 and a universal reverse primer. The nucleotide sequences of the primer set for detecting the polymorphism at position 57968691 are shown as SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7; the nucleotide sequences of the primer set for detecting the polymorphism at position 58439462 are shown as SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10; the nucleotide sequences of the primer set for detecting the polymorphism at position 60203161 are shown as SEQ ID NO.11, SEQ ID NO.12 and SEQ ID NO.13; and the nucleotide sequences of the primer set for detecting the polymorphism at position 60338017 are shown as SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO.

16.

7. The method for detecting the thickness of tomato peel according to claim 6, wherein: The 5' ends of the specific forward primer 1 and the specific forward primer 2 are respectively connected to two different fluorescent tag sequences.

8. The method for detecting the thickness of tomato peel according to claim 7, wherein: The 5' ends of the two specific forward primers were labeled with a FAM group and a HEX group, respectively.

9. The method for detecting the thickness of tomato peel according to claim 3, wherein: In step (2), the PCR reaction system is: 0.8 μL of 20 ng / μL template DNA, 0.75 μL of 2× KASP premix, and 0.05 μL of primer mixture, wherein the primer mixture is prepared by mixing forward primer 1, forward primer 2, and reverse primer at a concentration of 100 μM with ddH2O in a volume ratio of 12:12:30:

46.

10. The method for detecting the thickness of tomato peel according to claim 3, wherein: In step (2), the PCR reaction procedure is as follows: Step 1: pre-denaturation at 94°C for 15 minutes; Step 2: 94°C for 20 seconds, annealing at 61-55°C for 60 seconds, for a total of 10 cycles, with the temperature decreasing by 0.6°C each cycle; Step 3: denaturation at 94°C for 20 seconds, annealing at 55°C for 60 seconds, for a total of 26 cycles.

Images