SNP molecular marker associated with stress-induced leaf abscission in tomato and uses thereof

Abstract

This application discloses a SNP molecular marker associated with stress-induced leaf drop in tomato and its application. The SNP molecular marker comprises the nucleotide sequence formed by a G>T single nucleotide mutation at position 57663293 of chromosome 3 in the tomato genome. This SNP molecular marker can effectively address the shortcomings of conventional breeding methods, including the inability to determine leaf drop under no-stress conditions and the influence of environmental factors on leaf drop phenotype assessment. Using this SNP molecular marker, the leaf drop phenotype under stress can be accurately and efficiently identified during the tomato seedling stage, thereby effectively reducing the planting scale of breeding materials, alleviating the workload of later phenotypic identification, rapidly screening for superior target plants, accelerating the breeding process, and improving breeding efficiency.

Description

Technical Field

[0001] This application relates to the field of tomato genetic breeding marker screening technology, specifically to SNP molecular markers associated with stress-induced tomato leaf drop and their applications. Background Technology

[0002] Tomato (Solanum lycopersicum) is an important horticultural crop widely cultivated globally. Due to its vibrant color, unique flavor, rich nutrition, and wide range of uses, tomatoes are widely accepted and loved by people around the world. Tomatoes were one of the first crops to have their genetic linkage maps constructed and were among the first higher plants used in gene transformation research.

[0003] Currently, tomato breeding mainly uses conventional methods. Traditional breeding methods have drawbacks such as being time-consuming, labor-intensive, inaccurate, and susceptible to environmental factors. However, molecular marker-assisted breeding based on molecular marker-assisted selection (MAS) technology utilizes the high correlation between molecular markers and target trait genes. By detecting molecular markers, the presence of the target gene can be detected, thus achieving the purpose of selecting the target trait. It has the advantages of being early, rapid, accurate, and unaffected by environmental conditions.

[0004] Plant organ abscission refers to the process by which parts of a plant (such as flowers, fruits, and leaves) detach from the parent plant in response to developmental and environmental signals. In tomato cultivation, plants often experience leaf drop due to biotic and abiotic stresses, and this drop is controlled by a combination of factors. There are three main causes of leaf drop: normal physiological abscission, abscission caused by an imbalance between source and sink supply and demand, and abscission caused by abiotic stress. Abiotic stress-induced abscission may be due to changes in the plant's internal hormone levels. For example, ethylene and abscisic acid can induce dehydration in drought or salt stress, thereby promoting leaf or fruit abscission and protecting the plant from adverse damage. The areas of the plant most prone to organ abscission are often the abscission zone, the formation of which is generally controlled by the plant's genetic makeup. Reasonable regulation of tomato leaf abscission can not only reduce production costs and improve land use efficiency but also effectively increase the yield and quality of agricultural products.

[0005] However, few universal molecular markers have been found in tomatoes, and no molecular markers associated with stress-induced tomato leaf drop have been reported or published. Therefore, developing molecular markers associated with stress-induced tomato leaf drop is of great significance for the breeding of superior tomato varieties. Summary of the Invention

[0006] This application has discovered a SNP molecular marker associated with stress-induced tomato leaf drop. This SNP molecular marker provides technical support for the cloning of genes controlling stress-induced tomato leaf drop and for tomato breeding, and has important application value.

[0007] Therefore, the embodiments of this application provide at least the following technical solutions:

[0008] In a first aspect, embodiments of this application provide SNP molecular markers associated with stress-induced leaf drop in tomatoes. The SNP molecular markers include nucleotide sequences formed by a G>T single nucleotide mutation at position 57663293 of chromosome 3 of the tomato genome.

[0009] In some embodiments, the genotype at position 57663293 on chromosome 3 of the tomato genome is TT, which is associated with a non-deciduous tomato phenotype, and GG, which is associated with a deciduous tomato phenotype.

[0010] In some embodiments, the SNP molecular marker has a nucleotide sequence as shown in SEQ ID NO:1 and / or SEQ ID NO:2.

[0011] Secondly, embodiments of this application provide a primer pair. The primer pair includes SID-F as shown in SEQ ID NO:3 and SID-R as shown in SEQ ID NO:4.

[0012] Thirdly, embodiments of this application provide a nucleic acid molecule associated with stress-induced tomato leaf drop, the nucleic acid molecule including the aforementioned SNP molecular marker.

[0013] Fourthly, embodiments of this application provide a kit for identifying the deciduous trait of tomatoes, which includes the aforementioned primer pair.

[0014] Fifthly, embodiments of this application provide a method for identifying the leaf-deciduous trait of tomatoes. The method includes: performing PCR amplification using the DNA of the tomato to be tested as a template; purifying and sequencing the amplification product; determining the genotype at locus 57663293 on chromosome 3 of the tomato genome based on the sequencing results; and identifying the leaf-deciduous trait of the tomato based on the genotype.

[0015] Sixthly, embodiments of this application provide another method for identifying the leaf-fall trait of tomatoes. The method includes: using the DNA of the tomato to be tested as a template, performing PCR amplification on it using the primers described in the first aspect to obtain an amplification product; digesting the amplification product with the SacI restriction endonuclease to obtain a digested product; and analyzing the digested product to determine the leaf-fall trait of the tomato.

[0016] Seventhly, embodiments of this application provide the application of the aforementioned SNP molecular markers, primer pairs, kits, nucleic acid molecules, or methods in tomato breeding. Attached Figure Description

[0017] Figure 1 The results of a genome-wide association analysis (GWAS) of tomato leaf fall provided for this example.

[0018] Figure 2 The agarose gel electrophoresis results of different types of tomato materials amplified using SID labeling are provided in the example; lane 1 is M, and the others are electrophoresis bands of amplification products from 10 materials. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Reagents not described in detail herein are all conventional reagents and are commercially available.

[0020] In the following experimental procedures, unless otherwise specified, all operations shall be performed in accordance with the methods provided in Molecular Cloning: A Laboratory Manual (3rd Edition, translated by Huang Peitang et al., Beijing: Science Press, 2002).

[0021] This example demonstrates the identification of stress-induced leaf drop phenotypes in tomato materials. Using genome resequencing data and genome-wide association analysis (GWAS), single nucleotide polymorphism (SNP) sites highly associated with stress-induced leaf drop were identified, revealing a strong correlation between SNPs and stress-induced leaf drop traits in tomatoes. This example further utilizes molecular markers obtained through two strategies for fine-tuning the leaf drop trait, ultimately developing a single enzyme-amplified polymorphic sequence (CAPS) marker, SID, highly associated with stress-induced leaf drop. This example demonstrates the identification of tomato germplasm resources using the SID marker, achieving 100% accuracy.

[0022] The technical solutions provided in this embodiment effectively address the shortcomings of conventional breeding methods, including the inability to determine leaf drop under stress-free conditions and the susceptibility of leaf drop trait determination to environmental influences. These solutions allow for accurate and efficient identification of leaf drop phenotypes under stress conditions during the tomato seedling stage, thereby effectively reducing the planting scale of breeding materials, alleviating the workload of later phenotypic identification, rapidly screening for superior target plants, accelerating the breeding process, and improving breeding efficiency. More importantly, the molecular markers described are universal, less limited by genetic materials, and have a wide range of applications. This embodiment provides closely related molecular markers for cloning stress-induced tomato leaf drop control genes and for tomato breeding, possessing significant application value.

[0023] Based on this, this embodiment provides an SNP molecular marker associated with stress-induced tomato leaf drop, wherein the SNP molecular marker includes a nucleotide sequence formed by a G>T single nucleotide mutation at position 57663293 of chromosome 3 of the tomato genome. In some embodiments, the tomato phenotype associated with TT at position 57663293 of chromosome 3 of the tomato genome is a non-leaf-forming phenotype, and the tomato phenotype associated with GG is a leaf-forming phenotype.

[0024] In some embodiments, the SNP molecular marker has a nucleotide sequence as shown in SEQ ID NO:1 or SEQ ID NO:2.

[0025] Based on this, an embodiment provides a primer pair comprising SID-F as shown in SEQ ID NO:3 and SID-R as shown in SEQ ID NO:4. This primer pair can be used for PCR amplification to obtain a nucleic acid fragment containing the 57663293rd site of chromosome 3.

[0026] The embodiments provide a nucleic acid molecule associated with stress-induced tomato leaf drop, the nucleic acid molecule comprising the aforementioned SNP molecular marker. In some embodiments, the nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO:1 or SEQ ID NO:2.

[0027] The example provides a kit for identifying the deciduous trait of tomatoes, which includes the aforementioned primer pair.

[0028] The example provides a method for identifying the leaf-shedding trait of tomatoes, comprising: performing PCR using the DNA of the tomato as a template.

[0029] Amplification; purification and sequencing of the amplified product; determination of the genotype at locus 57663293 on chromosome 3 of the tomato genome based on the sequencing results; analysis of the correlation between the genotype and the tomato leaf-fall trait to identify tomato varieties with stress-induced leaf fall. Further, the genotype includes the GG genotype and the TT genotype, wherein the TT genotype represents tomato varieties with stress-induced leaf fall. In some embodiments, the nucleotide sequences of the primers used for PCR amplification are shown in SEQ ID NO:3 and SEQ ID NO:4.

[0030] The embodiment also provides another method for identifying the deciduous trait of tomatoes, including: using the DNA of the tomato to be tested as a template, performing PCR amplification on it using the primers to obtain an amplification product; digesting the amplification product with the SacI restriction endonuclease to obtain a digested product; and analyzing the digested product to determine the deciduous trait of the tomato. In some embodiments, if the digested product shows a band of 187 bp in electrophoresis, the material is determined to be non-deciduous; if the digested product shows a band of 161 bp in electrophoresis, the material is determined to be deciduous.

[0031] The examples provide the application of the aforementioned SNP molecular markers, primer pairs, nucleic acid molecules, kits, or methods in tomato breeding.

[0032] The present application will be further described below with reference to more specific embodiments. Of course, the following embodiments should not be construed as limiting the present application.

[0033] 1. Identification of SNP sites closely associated with stress-induced tomato leaf drop

[0034] This application utilizes genome-wide association analysis to identify SNPs closely associated with stress-induced tomato leaf drop. The specific method is as follows:

[0035] Tomato seedlings (CM Rick Tomato Genetics Resource Center, https: / / tgrc.ucdavis.edu) at the 5-leaf-1-heart stage were treated with 300 mM sodium chloride. The leaf drop and number of fallen leaves were recorded for each individual plant, and 5 plants were planted per material. Bioinformatics and genome-wide association analysis were performed using tomato resequencing data: the sequencing fragments of each sample were aligned to the tomato reference genome (Heinz) using bwa software. After alignment, variant identification was performed using samtools and bcftools software. After filtering the quality of variant sites, genome-wide association analysis was performed. Figure 1 The results of the genome-wide association analysis of stress-induced leaf drop in tomatoes are shown in the figure. As can be seen from the figure, the peak on chromosome 3 is the associated position of the leaf drop trait.

[0036] The example compared the regions associated with GWAS and linkage localization, and found that the SNP site located at 57663293 (SL2.50 Heinz 1706) on chromosome 3 of the tomato genome was screened and found that the SNP site was highly associated with stress-induced leaf drop in tomatoes.

[0037] 2. Application of SID molecular markers in tomato breeding

[0038] In this embodiment, primers were designed to amplify the target fragment containing the SNP locus at position 57663293 on chromosome 3 for PCR amplification of the molecular markers obtained above that are associated with stress-induced tomato leaf drop. The leaf drop morphology of tomato materials was determined or identified based on the PCR amplification product and / or the genotype of the SNP locus.

[0039] In some embodiments, the genotype of this SNP locus was identified in 10 tomato materials (CM Rick Tomato Genetics Resource Center, https: / / tgrc.ucdavis.edu), and the leaf drop phenotype of the materials was predicted based on the identification results. The steps included: using extracted tomato genomic DNA at a concentration of 80-150 ng / μL as a template, and performing PCR amplification using SID-F as described in SEQ ID NO.3 and SID-R as described in SEQ ID NO.4 as primers. The PCR reaction was performed on a Bio-Rad S1000 PCR instrument, and the PCR reaction system is shown in Table 1. The specific PCR amplification program was as follows: 95.0 ℃ pre-denaturation for 3 min; 95.0 ℃ denaturation for 30 s, 55.0 ℃ annealing for 30 s, 72.0 ℃ extension for 45 s, cycled, then skipped to 95 ℃ for 30 s, cycled 34 times; 72.0 ℃ extension for 5 min, and stored at 4 ℃.

[0040] Table 1

[0041]

[0042] like Figure 2 As shown, the amplified products were analyzed by 1% agarose gel electrophoresis, and all amplified bands containing the mutation site 57663293 on chromosome 3 (SEQ ID NO:1) were 187 bp in length. Sequencing revealed that the mutation site 57663293 on chromosome 3 was located at position 162 bp of this 187 bp. Sequencing results indicate that if the base at position 162 bp shown in SEQ ID NO:1 is G, meaning the mutation site 57663293 on chromosome 3 of the tomato material genome is G, then the genotype of this mutation site is GG, and the tomato material exhibits a deciduous phenotype. If the base at position 162 bp shown in SEQ ID NO:1 is T, then the tomato material exhibits a non-deciduous phenotype. Since tomatoes are natural genetic material, for the leaf-falling trait, the genotypes at mutation site 57663293 on chromosome 3 of the tomato genome are all homozygous, namely the GG genotype (D type) and the TT genotype (N type). Therefore, the leaf-falling phenotype of tomatoes can be determined by determining the specific genotype of the tomato.

[0043] In some embodiments, the same primers as described above are used to amplify the extracted tomato genome by PCR, producing a 187 bp product. This 187 bp product is then digested with enzymes, and the leaf drop morphology of the tomato is determined based on the number and length of the digested bands. Specific steps include: digesting the amplified 187 bp band with SacI (Thermo Scientific, 10 U / μl). The enzyme digestion system is prepared according to the instructions as follows: PCR reaction product, 5 μl; ddH2O, 8.5 μl; 10×Fast Digest Buffer, 1 μl; Fast Digest XhoI, 0.5 μl; the reaction system is incubated at 37 ℃ for 2 h; the digested product is then obtained.

[0044] like Figure 2 The image shows the enzyme digestion products of 10 tomato samples after 2% agarose gel electrophoresis. Five samples showed only a 187 bp band (indicating they could not be digested), indicating they were non-deciduous varieties. The other five samples showed a 161 bp band, indicating they were deciduous varieties. Further analysis based on the deciduous phenotype of these tomato samples showed that the accuracy of using this molecular marker to detect deciduous phenotype was 100%.

[0045] The above analysis results indicate that molecular marker identification and screening in breeding, based on specific bands or genotypes, allows for the selection of non-deciduous materials without stress treatment during the seedling stage, improving selection efficiency, reducing the workload of later plant transplanting and screening, and accelerating the breeding process. Furthermore, this marker possesses excellent versatility and can be accurately used to predict the stress-induced deciduous phenotype of various tomato materials.

Claims

1. A method for identifying the leaf-falling trait induced by sodium chloride stress in tomatoes, the method comprising: PCR amplification was performed using the DNA of the tomato to be tested as a template; The amplification product was purified and sequenced, and the genotype at locus 57663293 on chromosome 3 of the tomato genome was determined based on the sequencing results. The deciduous phenotype of the tomato was identified based on the genotype. The genotype at position 57663293 on chromosome 3 of the tomato genome is TT, which is associated with a non-deciduous phenotype, while the genotype is GG, which is associated with a deciduous phenotype. The tomato genome version is SL2.50 Heinz 1706.

2. A method for identifying the leaf-fall trait induced by sodium chloride stress in tomatoes, the method comprising: DNA from the tomato to be tested was amplified by PCR using primer pairs, including SID-F as shown in SEQ ID NO:3 and SID-R as shown in SEQ ID NO:4, to obtain amplification products; The amplification product was digested with SacI restriction endonuclease to obtain the digested product; The enzyme digestion products were analyzed to determine the leaf drop trait of tomatoes; If the enzyme digestion product shows a band of 187 bp in electrophoresis, the material is determined to be non-deciduous; if the enzyme digestion product shows a band of 161 bp in electrophoresis, the material is determined to be deciduous.

3. The application of the primer pair of claim 2, or a kit including the primer pair of claim 2, or the method of any one of claims 1-2, in the breeding of tomato deciduous trait induced by sodium chloride stress.

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