A molecular marker related to tomato drought resistance and its application

Abstract

The present invention discloses a molecular marker related to drought resistance of tomatoes and its application, belonging to the technical field of molecular breeding. The nucleotide sequence of the molecular marker is shown in SEQ ID NO.4, and there is a T / G mutation at the 22 bp of the sequence shown in SEQ ID NO.4. The PARMS primer set for detecting the molecular marker includes 2 allele-specific primers and 1 marker site-specific primer. The PARMS primer set can accurately identify the drought resistance of tomatoes, and can determine the drought resistance of materials in the tomato seedling stage, thereby reducing the cost of seed production and improving breeding efficiency. At the same time, it can also accelerate the breeding process of new tomatoes or other fruit and vegetable vegetables.

Description

Technical Field

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

[0002] Tomato (Solanum lycopersicum L.) is native to tropical South America. It is rich in vitamin C, lycopene, folic acid, potassium and other nutrients. It is one of the most widely cultivated vegetable crops in the world and has high commercial value. As global warming intensifies, drought stress has caused more and more serious problems for global agriculture. Among many abiotic stresses, drought and water shortage are the most destructive to crop production and are an important factor limiting crop yield and quality. Tomato is a vegetable crop with a high water requirement. Among environmental stresses, drought stress is the main limiting factor limiting tomato yield and quality. China is currently the country with the largest tomato production and planting area in the world, but freshwater resources that can be used for agricultural production are very scarce. Therefore, it is of great scientific and practical significance to explore tomato drought-resistant genetic resources and breed drought-resistant tomato varieties.

[0003] However, in the breeding of drought-resistant tomato varieties, drought resistance is easily affected by the environment, and the identification of drought resistance of tomatoes needs to wait until the plants grow to a certain size, and the steps to measure the physiological indicators that can measure drought resistance are relatively cumbersome. Although molecular marker-assisted selection breeding can effectively overcome the defects of traditional breeding, shorten the breeding cycle, and accelerate the breeding process, the current selection of drought-resistant tomatoes is relatively blind, and there are no trait-linked molecular markers that can be effectively applied to the molecular marker-assisted breeding process. Therefore, it is urgent to carry out research on the precise positioning of drought resistance.

[0004] As the third generation of molecular marker technology, single nucleotide polymorphism (SNP) has been widely used in germplasm resource research, variety authenticity identification, and molecular marker-assisted breeding due to its wide distribution and rich polymorphism in biological genomes, easy detection and statistics, and high-throughput automated detection. There are currently a variety of PCR-based SNP molecular marker detection methods for single-base variations, such as enzyme-amplified polymorphic sequence method, high-resolution melting curve, allele-specific PCR, and competitive allele-specific PCR (KompetitiveAllele Specific PCR).

[0005] PARMS (Penta-primer amplification refractory mutation system) is a new type of SNP molecular marker detection technology. This technology is a PCR detection technology developed based on the specific resolution of SNP single-base variant sites. This technology can quickly and easily detect SNP allele genotypes. PARMSSNP uses 5 primers for PCR reaction, of which 2 fluorescent universal primers are included in PARMS2X Master Mix, and the remaining 3 are marker-specific primers, which need to be custom-designed and synthesized according to the purpose of the experiment. Among these 3 primers, one is a marker locus-specific primer (Locus specific primer) and the other two are SNP allele-specific primers (Allele specific primer). The 5' of the two Allele specific primers is added with a specific universal adapter sequence of 21 bases for matching amplification with the fluorescent universal primers. The basic principle of marker primer design can adopt the general primer design rules, the primer length is 18 to 30 bp, and the amplified fragment is less than 250 bp (including primers). Under the premise of meeting the primer design conditions, the shorter the better. Allele1 and Allele 2 specific amplification primers with two different universal adapter primer sequences bind to the corresponding SNP DNA template after DNA renaturation. The PARMS PCR enzyme and buffer system can ensure strict allele-specific amplification. With the Locus specific amplification primer, after the first two rounds of PCR, a PCR amplification product with a universal adapter sequence is formed. At this time, the universal probe with a reporter fluorescence and a fluorescence quenching group (no fluorescence signal due to the FRET effect when there is no amplification) can be used as a template for PCR amplification using the PCR amplification product with a universal adapter sequence. Once the amplification is successful, the fluorescence quenching group on the fluorescent probe dissociates from the reporter group, and the FRET effect disappears. At this time, a fluorescence scan can be performed to detect the corresponding fluorescence signal, so it can be known whether the corresponding allele exists.

[0006] PARMS technology has high accuracy and good stability; the detection cost is low. For the detection of different SNP sites, only three common primers for the corresponding sites need to be designed, and the primers with fluorescent labels are universal primers; it is compatible with crude DNA alkaline boiling extraction, and the sample preparation time is extremely short, which is 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 animal and plant breeding. Therefore, the use of PARMS technology to type SNP sites for predicting tomato drought resistance is of great significance for the development of effective molecular markers for auxiliary breeding and rapid identification of drought-resistant tomato varieties. Summary of the invention

[0007] The purpose of the present invention is to provide a molecular marker related to tomato drought resistance and its application to solve the problems existing in the above-mentioned prior art. The molecular marker of the present invention is co-segregated with the tomato drought resistance phenotype, which can determine the drought resistance of the material in the tomato seedling stage, thereby reducing the seed production cost and improving the breeding efficiency.

[0008] To achieve the above object, the present invention provides the following solutions:

[0009] The invention provides a molecular marker related to drought resistance of tomatoes. The nucleotide sequence of the molecular marker is shown in SEQ ID NO.4. A T / G mutation exists at the 22 bp position of the sequence shown in SEQ ID NO.4.

[0010] The present invention also provides a PARMS primer set for detecting the molecular marker, comprising 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.

[0011] The present invention also provides a detection kit for the molecular marker, comprising the PARMS primer set.

[0012] The present invention also provides the use of the PARMS primer set or the detection kit in identifying drought resistance of tomatoes.

[0013] The present invention also provides a method for identifying drought resistance of tomatoes, comprising the following steps:

[0014] Using the genomic DNA of the tomato sample to be tested as a template, using the PARMS primer set or the detection kit to perform PCR amplification on the template, reading the fluorescent signal after the PCR amplification is completed, analyzing and converting the fluorescent signal, identifying the genotype, and judging the drought resistance of the tomato according to the genotype;

[0015] 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.

[0016] Furthermore, the PCR amplification program is: pre-denaturation at 94°C for 15 min; denaturation at 94°C for 20 s, gradient annealing / extension at 65-57°C for 1 min, 10 cycles; denaturation at 94°C for 20 s, annealing / extension at 57°C for 1 min, 32 cycles.

[0017] Furthermore, the PCR amplification system is: 2×PARMS master mix 5 μL, 10 μM allele-specific primers 0.15 μL each, 10 μM marker site-specific primers 0.4 μL, DNA template 10-100 ng, ddH2O to 10 μL.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] The present invention discloses the following technical effects:

[0022] Based on the identification results of drought resistance of 301 tomato germplasm resources, the present invention conducts genome-wide association analysis with drought resistance, and detects a SNP site significantly associated with tomato drought resistance at the 41598797bp position of chromosome 5, which has a T / G mutation. Based on the positioning results, specific PARMS primers for identifying drought resistance of tomato are developed, and the polymorphism at the marker site is detected to identify the difference in drought resistance of tomato.

[0023] The molecular marker developed by the present invention is co-segregated with the tomato drought resistance phenotype, which can determine the drought resistance of materials in the tomato seedling stage, thereby reducing seed production costs and improving breeding efficiency. At the same time, it can also accelerate the breeding process of new tomatoes or other fruit and vegetable 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 drawings required for use in the embodiments will be briefly introduced below. 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 creative work.

[0025] Figure 1 This is a genome-wide association analysis of tomato drought resistance. The orange dots in the figure show the SNPs significantly associated with tomato drought resistance detected at position 41598797bp on chromosome 5;

[0026] Figure 2They are 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

[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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

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

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 a conflict with any incorporated document, the content 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 of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary 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

[0033] 1. Mining SNPs associated with drought resistance in tomato

[0034] The 301 tomato germplasm materials for the test were provided by Huang Sanwen's research group at the Institute of Agricultural Genomics, Chinese Academy of Agricultural Sciences. They were planted in the experimental base of Jiangsu Academy of Agricultural Sciences in Liuhe District, Nanjing in March 2023. Four plants were planted for each germplasm material, with a spacing of 32 cm between plants. Field management during the entire growth period was carried out in accordance with general tomato greenhouse cultivation production management measures.

[0035] After 30 days of 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 ), FW1 was weighed again after 24 hours, and the relative water loss rate of leaves was calculated by the formula "(FW0-FW1) / FW0×100%=relative water loss rate" as an indicator of drought resistance. Three biological replicates were performed for each material. Based on the identification results of drought resistance of 301 germplasm resources, genome data was analyzed for drought resistance by genome-wide association study (GWAS). A SNP significantly associated with tomato drought resistance was detected at the 41598797bp position of chromosome 5 ( Figure 1 ).

[0036] 2. Design of Molecular Marker Primer Combinations for PARMS Detection of SNPs

[0037] According to the SNP sites obtained from the GWAS analysis results, the 41598797bp position of chromosome 5 was searched, and a locus specific primer and two SNP allele specific primers were designed. Allele T primer was connected to the FAM blue fluorescent linker sequence, and Allele G primer was connected to the HEX green fluorescent linker sequence. The primer sequences are shown in Table 1.

[0038] Table 1 Primers and sequences

[0039]

[0040] Note: Specific universal adapter sequences are underlined.

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

[0042] CGTAGGTGGCATCGTAGTCAAKTGACGGTCTGTCCTGCTAGTTC.

[0043] The 22nd bp in the sequence shown in SEQ ID NO.4 represents a SNP mutation site, where a T / G mutation exists.

[0044] 3. Screening of test germplasm

[0045] From the GWAS population, 12 materials with strong drought resistance and 12 materials with weak drought resistance were selected. The leaf water loss rate of the 24 test materials after drought treatment was as follows: Figure 2 As shown, the relative water loss rate of leaves of 12 materials with strong drought resistance after drought treatment was between 8% and 12%, and the relative water loss rate of leaves of 12 materials with weak drought resistance after drought treatment was between 23% and 60%.

[0046] 4. Sample Processing

[0047] Take 24 leaves of the test material with a length and width of about 1 cm and put them 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 (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 for 2 min again; after the water bath, 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.

[0048] 5. PCR Amplification

[0049] The PCR amplification reaction system is shown in Table 2 (PARMS mix Wuhan Jingpeptide):

[0050] Table 2 PCR amplification reaction system

[0051]

[0052] PCR amplification reaction conditions are shown in Table 3 (dual-head 384PCR instrument ABI Gene Amp 9700):

[0053] Table 3 PCR amplification reaction conditions

[0054]

[0055]

[0056] 6. Genotyping

[0057] After PCR was completed, the fluorescence signal was read using a TECAN infinite M1000 microplate reader, and then the online software snpdecoder (http: / / www.snpway.com / snpdecoder / ) was used to analyze and convert the fluorescence signal to obtain a clear and intuitive typing diagram, and the genotype results were output according to different colors, as shown in Table 4.

[0058] Table 4 Genotyping results

[0059]

[0060] The genotype of the tomato material with strong drought resistance is TT, and the genotype of the tomato material with weak drought resistance is GG. As shown in Table 4, the genotyping results of PARMS detection of 12 materials with strong drought resistance showed that 11 materials had genotyping results of FAM, which was consistent with the genotype TT of the material itself, and another material had genotyping result of HEX; the genotyping results of PARMS detection of 12 materials with weak drought resistance showed that 11 materials had genotyping results of HEX, which was consistent with the genotype GG of the material itself, and another material had genotyping result of FAM. Comprehensive statistics of the results of PARMS detection of 24 test materials showed that the PCR genotyping results of 22 materials were consistent with the material genotype, and the accuracy of the molecular marker reached 91.67%.

[0061] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A molecular marker related to drought resistance of tomato, 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 comprises 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 molecular marker detection kit according to claim 1, characterized in that: Comprising the PARMS primer set according to claim 2.

4. Use of the PARMS primer set according to claim 2 or the detection kit according to claim 3 in identifying drought resistance of tomatoes.

5. A method for identifying drought resistance of tomatoes, characterized in that: The steps include: Using the genomic DNA of the tomato sample to be tested as a template, using the PARMS primer set described in claim 2 or the detection kit described in claim 3 to perform PCR amplification on the template, reading the fluorescent signal after the PCR amplification is completed, analyzing and converting the fluorescent 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. The method according to claim 5, characterized in that The PCR amplification program is: pre-denaturation at 94°C for 15 min; denaturation at 94°C for 20 s, gradient annealing / extension at 65-57°C for 1 min, 10 cycles; denaturation at 94°C for 20 s, annealing / extension at 57°C for 1 min, 32 cycles.

7. The method according to claim 5, characterized in that The PCR amplification system is: 2×PARMS mastermix 5 μL, 10 μM allele-specific primers 0.15 μL each, 10 μM tag site-specific primers 0.4 μL, DNA template 10-100 ng, ddH2O to 10 μL.

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

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

10. The use according to claim 9, characterized in that: During the tomato seedling stage, the drought resistance of the tomato seedlings is determined using the PARMS primer set described in claim 2 or the detection kit described in claim 3, and tomato seedlings with strong drought resistance are selected for cultivation.

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