Molecular marker related to drought resistance of tomatoes and use of molecular marker

Abstract

A molecular marker related to drought resistance of tomatoes and a use of the molecular marker, relating to the technical field of molecular breeding. The nucleotide sequence of the molecular marker is shown as SEQ ID NO. 4, and T / G mutation is present at position 22 bp of the sequence shown in SEQ ID NO. 4. A PARMS primer group for detecting the molecular marker comprises two allele specific primers and one marker site specific primer, the drought resistance of tomatoes can be accurately identified by using the PARMS primer group, and the drought resistance of materials can be determined at the time of young tomato seedling, so that the seed production cost is reduced, and the breeding efficiency is improved. Additionally, the transformation process of new tomatoes or other fruiting vegetables can also be accelerated.

Description

A molecular marker related to tomato drought resistance and its application Technical Field

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

[0002] Tomato (Solanum lycopersicum L.), native to tropical South America, is rich in nutrients such as vitamin C, lycopene, folate, and potassium. It is one of the most widely cultivated vegetable crops worldwide and has high commercial value. With the intensification of global warming, drought stress is becoming an increasingly serious problem for global agriculture. Among the many abiotic stresses, drought and water shortage are the most destructive to crop production and are a major factor limiting crop yield and quality. Tomato is a vegetable crop with high water requirements, and among the environmental stresses, drought stress is the primary limiting factor in tomato yield and quality. China currently has the largest tomato production and cultivated area in the world, but freshwater resources available for agricultural production are extremely scarce. Therefore, exploring tomato drought-resistant genetic resources and breeding drought-resistant tomato varieties are of great scientific and practical significance.

[0003] However, in the breeding of drought-resistant tomato varieties, drought resistance is highly susceptible to environmental influences, and identification of drought resistance in tomatoes requires waiting until the plants reach a certain size. Furthermore, measuring physiological indicators that can measure drought resistance is a cumbersome process. While molecular marker-assisted selection can effectively overcome the shortcomings of traditional breeding, shortening the breeding cycle and accelerating the breeding process, current selection of drought-resistant tomato varieties is relatively unreliable. There are no trait-linked molecular markers that can be effectively applied in molecular marker-assisted breeding. Therefore, research on the precise mapping of drought resistance 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 and rich polymorphism in biological genomes, 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 novel SNP molecular marker detection technology. This technology is based on a PCR-based assay that specifically resolves single-base variants in SNPs. It allows for rapid and simple SNP allele genotyping. PARMS SNP PCR utilizes five primers: two fluorescent universal primers included in the PARMS 2X Master Mix, and the remaining three marker-specific primers are custom-designed and synthesized based on the experimental objectives. One of these three primers is locus-specific, and the other two are allele-specific. A 21-base universal adapter sequence is added to the 5' end of each of the two allele-specific primers to facilitate amplification with the fluorescent universal primers. The basic principles for marker primer design follow general primer design guidelines: primer length should be 18 to 30 bp, and the amplified fragment should be less than 250 bp (including the primer). Shorter primers are preferred, provided they meet the primer design requirements. 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, a PCR amplification product with a universal adapter sequence is formed. At this point, a universal probe with a reporter fluorescence and a fluorescence quencher group (no fluorescence signal due to the FRET effect when no amplification is taking place) 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 group on the fluorescent probe dissociates from the reporter group, and the FRET effect disappears. Fluorescence scanning can then detect the corresponding fluorescence signal, thereby determining the presence of the corresponding allele.

[0006] PARMS technology offers high accuracy and stability, as well as low testing costs. Detection of different SNP loci requires only 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 testing platforms. In recent years, PARMS SNP detection technology has been increasingly applied in population genetics research, disease diagnosis, and plant and animal breeding. Therefore, using PARMS to genotype SNPs for predicting drought resistance in tomatoes is crucial for developing effective molecular markers to assist in the rapid selection and identification of drought-resistant tomato varieties.

[0007] Summary of the Invention

[0008] The present invention aims to provide a molecular marker associated with tomato drought resistance and its application to address the aforementioned problems in the prior art. The molecular marker co-segregates with the tomato drought resistance phenotype, enabling drought resistance determination in tomato seedlings at the seedling stage, thereby reducing seed production costs and improving breeding efficiency.

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

[0010] The present invention provides a molecular marker related to tomato drought resistance. The nucleotide sequence of the molecular marker is shown in SEQ ID NO.4. A T / G mutation exists at the 22nd bp of the sequence shown in SEQ ID NO.4.

[0011] The present invention also provides a PARMS primer set for detecting the molecular marker, comprising two allele-specific primers and one marker site-specific primer; the sequences of the allele-specific primers are shown in SEQ ID NO.1-2; the sequence of the marker site-specific primer is shown in SEQ ID NO.3.

[0012] The present invention also provides a detection kit for the molecular marker, comprising the PARMS primer set.

[0013] The present invention also provides the use of the PARMS primer set or the detection kit in identifying drought resistance of tomatoes.

[0014] The present invention also provides a method for identifying drought resistance of tomatoes, comprising the following steps:

[0015] Using the genomic DNA of the tomato sample to be tested as a template, the template is PCR amplified using the PARMS primer set or the detection kit. After the PCR amplification is completed, the fluorescent signal is read, the fluorescent signal is analyzed and converted, the genotype is identified, and the drought resistance of the tomato is determined based on the genotype;

[0016] If the identified genotype is TT, the drought resistance of the tomato sample to be tested is judged to be strong; if the identified genotype is GG, the drought resistance of the tomato sample to be tested is judged to be weak.

[0017] Furthermore, the PCR amplification program is: pre-denaturation at 94°C for 15 minutes; denaturation at 94°C for 20 seconds, gradient annealing / extension at 65-57°C for 1 minute, 10 cycles; denaturation at 94°C for 20 seconds, annealing / extension at 57°C for 1 minute, 32 cycles.

[0018] Furthermore, the PCR amplification system is: 5 μL of 2×PARMS master mix, 0.15 μL of each 10 μM allele-specific primer, 0.4 μL of 10 μM tag site-specific primer, 10-100 ng of DNA template, and ddH2O to make up to 10 μL.

[0019] The present invention also provides the use of the PARMS primer set or the detection kit in screening tomato varieties or strains with strong drought resistance.

[0020] The present invention also provides the use of the PARMS primer set or the detection kit in assisting the cultivation of drought-resistant tomato varieties.

[0021] Furthermore, the PARMS primer set according to claim 2 or the detection kit according to claim 3 is used to determine the drought resistance of the tomato seedlings during the tomato seedling stage, and tomato seedlings with strong drought resistance are selected for cultivation.

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

[0023] Based on the drought resistance assessment results of 301 tomato germplasm resources, this study conducted a genome-wide association analysis linking genomic data with drought resistance. A single polymorphism (SNP) site significantly associated with tomato drought resistance was detected at position 41,598,797 bp on chromosome 5, which harbors a T / G mutation. Based on the mapping results, specific PARMS primers for tomato drought resistance were developed. The polymorphism at the marker site was then detected to identify differences in tomato drought resistance.

[0024] The molecular marker developed in this invention co-segregates with the tomato drought resistance phenotype, making it possible to determine drought resistance in tomato seedlings, thereby reducing seed production costs and improving breeding efficiency. It can also accelerate the development of new tomatoes and other fruiting vegetables. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] 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.

[0026] Figure 1 shows a genome-wide association analysis of tomato drought resistance. The orange dots in the figure indicate the SNP significantly associated with tomato drought resistance detected at position 41,598,797 bp on chromosome 5.

[0027] Figure 2 shows the representative materials with the highest (i.e., weak drought resistance) and lowest (i.e., strong drought resistance) relative water loss rates after tomato leaf water loss treatment. DETAILED DESCRIPTION

[0028] 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.

[0029] It should be understood that the terms used herein are intended solely to describe particular embodiments and are not intended to limit the present invention. Furthermore, 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, as well as each smaller range between any other stated value or intermediate value within the stated range, is also encompassed by the present invention. The upper and lower limits of these smaller ranges may independently be included or excluded within the range.

[0030] 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.

[0031] 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.

[0032] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0033] Example

[0034] 1. Mining SNPs associated with drought resistance in tomato

[0035] The 301 tomato germplasm accessions used in this study were provided by Huang Sanwen's research group at the Institute of Agricultural Genomics, Chinese Academy of Agricultural Sciences. They were planted in March 2023 at the Jiangsu Academy of Agricultural Sciences' experimental base in Liuhe District, Nanjing. Four plants were planted from each accession, with a spacing of 32 cm between plants. Field management throughout the growing season followed standard greenhouse tomato production practices.

[0036] Thirty days after tomato planting, the fourth leaf was taken for water loss treatment test. The initial fresh weight was FW0. The leaves were placed on filter paper in a light culture room (16 h light / 8 h dark, 25 °C, relative humidity 75%, light intensity 250 μmol·m -2 ·s -1 ), and FW1 was weighed again 24 hours later. Relative leaf water loss was calculated using the formula "(FW0 - FW1) / FW0 × 100% = relative water loss rate" as a measure of drought resistance. Three biological replicates were used for each accession. Based on the drought resistance results of 301 germplasm resources, genome-wide association studies (GWAS) were conducted on the genomic data and drought resistance. A SNP significantly associated with tomato drought resistance was detected at position 41,598,797 bp on chromosome 5 (Figure 1).

[0037] 2. Design of molecular marker primer combinations for PARMS detection of SNPs

[0038] Based on the SNP loci identified in the GWAS analysis, we searched for position 41,598,797 on chromosome 5. We designed one locus-specific primer and two allele-specific primers: an Allele T primer linked to a FAM blue fluorescent linker sequence, and an Allele G primer linked to a HEX green fluorescent linker sequence. The primer sequences are shown in Table 1.

[0039] Table 1 Primers and sequences

[0040] Note: The underlined sequence indicates the specific universal adapter sequence.

[0041] PCR amplified target fragment sequence (SEQ ID NO.4):

[0042] The 22nd bp position in the sequence shown in SEQ ID NO. 4 represents a SNP mutation site, where a T / G mutation exists.

[0043] 3. Screening of test germplasm

[0044] Twelve accessions with high drought resistance and 12 accessions with low drought resistance were selected from the GWAS population. The leaf water loss rates of the 24 accessions after drought treatment are shown in Figure 2. The 12 accessions with high drought resistance had relative leaf water loss rates ranging from 8% to 12%, while the 12 accessions with low drought resistance had relative leaf water loss rates ranging from 23% to 60%.

[0045] 4. Sample Processing

[0046] Take 24 leaves of the test material with a length and width of about 1 cm and place them in 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 (Shanghai Jingxin tissue grinder) for 2 min (until the sample is completely ground); after grinding, centrifuge at 3000 rpm for 1 min and boil in water for 2 min; then add 200 μL of 0.2 M Tris-HCl (pH 6.8-7.0) and mix well, and boil in water again for 2 min; after the water bath is completed, centrifuge at 3000 rpm for 1 min, take the supernatant and dilute it 20 times, freeze it at -20°C, and use it as a template for subsequent PCR amplification.

[0047] 5. PCR Amplification

[0048] The PCR amplification reaction system is shown in Table 2 (PARMS mix Wuhan Jingpeptide):

[0049] Table 2 PCR amplification reaction system

[0050] PCR amplification reaction conditions are shown in Table 3 (dual-head 384 PCR instrument ABI GeneAmp 9700):

[0051] Table 3 PCR amplification reaction conditions

[0052] 6. Genotyping

[0053] After PCR was completed, the fluorescence signal was 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 signal to obtain a clear and intuitive typing diagram. The genotype results were output according to different colors, as shown in Table 4.

[0054] Table 4 Genotyping results

[0055] The genotype of the tomato accessions with strong drought resistance was TT, while the genotype of the tomato accessions with weak drought resistance was GG. As shown in Table 4, the PARMS genotyping results for 11 of the 12 accessions with strong drought resistance were all FAM, consistent with their original genotype of TT, and one accession was HEX. The PARMS genotyping results for the 12 accessions with weak drought resistance were all HEX, consistent with their original genotype of GG, and one accession was FAM. Comprehensive statistical analysis of the PARMS test results for the 24 accessions revealed that the PCR genotyping results for 22 accessions were consistent with the accession genotypes, resulting in an accuracy rate of 91.67% for this molecular marker.

[0056] 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 shall fall within the scope of protection defined by the claims of the present invention.

Claims

1. A molecular marker related to tomato drought resistance, characterized in that, the nucleotide sequence of the molecular marker is shown in SEQ ID NO.4, and there is a T / G mutation at the 22nd bp of the sequence shown in SEQ ID NO.

4.

2. A PARMS primer set for detecting the molecular marker according to claim 1, characterized in that, it includes 2 allele-specific primers and 1 marker-site-specific primer; the sequences of the allele-specific primers are shown in SEQ ID NO.1-2; the sequence of the marker-site-specific primer is shown in SEQ ID NO.

3.

3. A detection kit for the molecular marker according to claim 1, characterized in that, it contains the PARMS primer set according to claim 2.

4. The application of the PARMS primer set according to claim 2 or the detection kit according to claim 3 in identifying tomato drought resistance.

5. A method for identifying tomato drought resistance, characterized in that, it includes the following steps: Using the genomic DNA of the tomato sample to be tested as a template, performing PCR amplification on the template using the PARMS primer set according to claim 2 or the detection kit according to claim 3, reading the fluorescence signal after the PCR amplification is completed, analyzing and converting the fluorescence signal, identifying the genotype, and judging the drought resistance of the tomato according to the genotype; If the identified genotype is TT, it is judged that the drought resistance of the tomato sample to be tested is strong; if the identified genotype is GG, it is judged that the drought resistance of the tomato sample to be tested is weak.

6. According to the method according to claim 5, characterized in that, the program of the PCR amplification is: pre-denaturation at 94°C for 15 min; denaturation at 94°C for 20 s, gradient renaturation / extension at 65 - 57°C for 1 min, 10 cycles; denaturation at 94°C for 20 s, renaturation / extension at 57°C for 1 min, 32 cycles.

7. According to the method according to claim 5, characterized in that, The system for PCR amplification is as follows: 5 μL of 2×PARMS master mix, 0.15 μL each of 10 μM allele-specific primers, 0.4 μL of 10 μM marker-site specific primer, 10 - 100 ng of DNA template, and ddH 2 O is supplemented to 10 μL.

8. The application of the PARMS primer set according to claim 2 or the detection kit according to claim 3 in screening tomato varieties or lines with strong drought resistance.

9. The application of the PARMS primer set according to claim 2 or the detection kit according to claim 3 in assisting the cultivation of tomato drought-resistant varieties.

10. According to the application according to claim 9, characterized in that, judging the drought resistance of the tomato seedlings using the PARMS primer set according to claim 2 or the detection kit according to claim 3 during the tomato seedling stage, and selecting tomato seedlings with strong drought resistance for cultivation.

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