Molecular markers associated with ammonium tolerance in tomato seedlings and their application

Abstract

The present application relates to the technical field of tomato genetic breeding, and specifically to molecular markers associated with ammonium tolerance in tomato seedlings and their applications. The molecular marker comprises a nucleotide sequence formed by a single nucleotide A>G mutation at position 62760934 of chromosome 8 of the tomato genome SL2.50ITAG2.4. The molecular markers, nucleic acid molecules, primer pairs, kits, methods and applications provided in the present application can accurately and efficiently determine the ammonium tolerance of tomatoes at the seedling stage, effectively reducing the planting scale of breeding materials, alleviating the workload of later field phenotypic identification, accelerating the breeding process and improving breeding efficiency. The molecular marker is a universal molecular marker with little restriction on genetic materials and a wide range of applications.

Description

Technical Field

[0001] The present application relates to the technical field of tomato genetic breeding, and in particular to molecular markers associated with ammonium tolerance in tomato seedlings and their applications. Background Art

[0002] Tomato (Solanum lycopersicum) is an annual herb of the Solanaceae family, Solanum genus. It is rich in lycopene and vitamin C. It also contains vitamins and mineral elements that have a protective effect on the cardiovascular system, can reduce the incidence of heart disease, and help gastric juice digest fat and protein.

[0003] Nitrogen plays a key role in the formation of crop yield and quality. Nitrogen deficiency in plants leads to smaller branches and leaves, dull color, and low yield. Nitrogen fertilizers widely used in agricultural production include nitrate nitrogen (NO3 - ) and ammonium nitrogen (NH4 + ). After applying nitrate nitrogen fertilizer, more than half is not used by plants, and it leads to a series of environmental problems. Compared with nitrate nitrogen fertilizer, the nitrogen fertilizer utilization rate is higher when ammonium nitrogen fertilizer is applied. However, high concentrations and single ammonium nitrogen usually cause severe growth retardation of plants, manifested as chlorosis of leaves, inhibition of root growth, stunted stem growth and development, etc., which is called ammonium toxicity. Different tomato varieties have different sensitivities to ammonium nitrogen. Cultivating tomato ammonium nitrogen tolerant materials can improve the flexibility of nitrogen fertilizer application, improve the nitrogen utilization efficiency of tomatoes, and protect the ecological environment.

[0004] Currently, tomato breeding primarily relies on conventional breeding methods, which are time-consuming and labor-intensive, with poor phenotypic accuracy and susceptibility to environmental factors. Molecular marker-assisted breeding (MAS) utilizes the close linkage between molecular markers and target trait genes. By detecting molecular markers, the presence of target genes can be detected, enabling selection for target traits. MAS offers the advantages of early, rapid, accurate, and environmentally friendly selection. Summary of the Invention

[0005] The present application discovered a site related to the tolerance of tomatoes to high concentrations of ammonium nitrogen in the roots during the seedling stage, which determines the tolerance of tomatoes to high concentrations of ammonium nitrogen in the roots during the seedling stage. Sensitivity to ammonium nitrogen is determined by the dry weight ratio, that is, the ratio of the dry weight of tomato seedlings grown under pure ammonium nitrogen to the dry weight of tomato seedlings grown under pure nitrate nitrogen. The dry weight ratio of ammonium-sensitive tomatoes is relatively small, while the dry weight ratio of high-ammonium-tolerant varieties is relatively large. Molecular markers were developed based on the sequence differences of this site, and the tolerance of tomatoes to ammonium nitrogen was identified during the seedling stage. This molecular marker can effectively distinguish the sensitivity of tomato germplasm resources to high concentrations of ammonium nitrogen, and can be combined with conventional breeding methods to cultivate new varieties of tomatoes that are resistant to high ammonium. At the same time, the present application also lays an important foundation for analyzing the artificial domestication process of tomatoes, cloning tomato ammonium-tolerant genes, and then analyzing the molecular mechanism of tomato ammonium tolerance.

[0006] To this end, the embodiments of the present application disclose at least the following technical solutions:

[0007] In a first aspect, the embodiments disclose molecular markers associated with ammonium tolerance in tomato seedlings, including a nucleotide sequence formed by a single nucleotide A>G mutation at position 62760934 of chromosome 8 of the tomato genome (database number, Tomato SL2.50 ITAG2.4, https: / / solgenomics.sgn.cornell.edu / ).

[0008] In a third aspect, an embodiment discloses a nucleic acid molecule. The nucleic acid molecule is associated with ammonium tolerance in tomato seedlings, and the nucleic acid molecule includes a nucleotide sequence as shown in any one of SEQ ID NOs: 1 to 4. The nucleic acid molecule is a cleavage amplified polymorphic sequence (CAPS). For example, when it is detected that the tomato material to be tested contains a DNA molecule as shown in SEQ ID NO: 1 and cannot be cleaved by SalI, the material is judged to be an ammonium-tolerant material. When it is detected that the tomato material to be tested contains a DNA molecule as shown in SEQ ID NO: 2 and can be cleaved by SalI into DNA molecules as shown in SEQ ID NO: 3 and SEQ ID NO: 4, the material is judged to be a non-ammonium-tolerant material.

[0009] In a third aspect, the embodiments disclose a primer pair comprising DNA molecules as shown in SEQ ID NOs: 5 and 6. The primer pair is used to perform PCR amplification on a nucleotide sequence comprising position 62760934 of chromosome 8 of the tomato genome SL2.50 ITAG2.4, and the ammonium tolerance of tomato seedlings is determined based on the enzyme digestion results of the PCR amplification product.

[0010] In a fourth aspect, the embodiment discloses a kit comprising the DNA molecule described in the third aspect, reagents required for PCR amplification, and reagents required for enzyme cleavage.

[0011] In a fifth aspect, embodiments disclose a method for detecting ammonium tolerance in tomato seedlings. The method comprises: extracting genomic DNA from a tomato material to be tested; performing PCR amplification on the genomic DNA using the primer pair shown in SEQ ID NOs: 5 and 6; performing enzyme digestion and electrophoresis on the PCR product; and determining the ammonium tolerance of the tomato based on the bands detected by electrophoresis. If only the 991 bp band (SEQ ID NO: 1) appears, the tomato is an ammonium-tolerant material; if two bands, 621 bp (SEQ ID NO: 3) and 370 bp (SEQ ID NO: 4), appear, the tomato is not ammonium-tolerant.

[0012] In a sixth aspect, embodiments disclose uses of the molecular marker described in the first aspect, the nucleic acid molecule described in the second aspect, the primer pair described in the third aspect, or the kit described in the fourth aspect. These uses include at least one of detecting ammonium tolerance in tomato seedlings, detecting dry weight ratio in tomato seedlings, and tomato breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a GWAS result diagram of the aboveground dry weight ratio of tomatoes provided in the examples.

[0014] Figure 2 These are the electrophoresis results of tomato materials LA3846, LA0113, LA2413, LA0533, and LA1478 detected using the molecular markers provided in the examples. In the figure, H represents the banding pattern of homozygous materials with a high dry weight ratio (ammonium resistance), and L represents the banding pattern of homozygous materials with a low dry weight ratio (non-ammonium resistance).

[0015] Figure 3 The electrophoresis results of tomato materials LA1162, LA1457, LA2283, LA3214, LA0126, LA1286, LA1461, and LA2184 detected using the molecular markers provided in the examples. In the figure, H represents the band pattern of homozygous materials with high dry weight ratios, and L represents the band pattern of homozygous materials with low dry weight ratios.

[0016] Figure 4 The electrophoresis results of tomato materials LA1244, LA1589, LA1543, LA2133, LA1218, LA2009, LA1673, LA1482, LA4133, LA0395, LA0089, LA4024, and LA2184 detected using the molecular markers provided in the examples. In the figure, H represents the banding pattern of the homozygous material with a high dry weight ratio, and L represents the banding pattern of the homozygous material with a low dry weight ratio. DETAILED DESCRIPTION

[0017] In order to make the purpose, technical solutions and advantages of this application clearer, the present application is further described in detail below with reference to the examples. It should be understood that the specific examples described herein are only used to explain this application and are not intended to limit this application. Reagents not described in detail in this application are all conventional reagents and can be obtained from commercial channels; methods not specifically described in detail are all conventional experimental methods and can be obtained from the prior art.

[0018] Conventional tomato breeding methods cannot determine the plant dry weight ratio at the seedling stage, cannot determine the ammonium tolerance of tomatoes at the seedling stage, cannot effectively distinguish between heterozygous and homozygous materials, and are significantly affected by the environment. The molecular markers, nucleic acid molecules, primer pairs, kits, methods, and applications provided in this application can accurately and efficiently determine the ammonium tolerance of tomatoes at the seedling stage, effectively reducing the planting scale of breeding materials, alleviating the workload of later field phenotyping, accelerating the breeding process, and improving breeding efficiency. This molecular marker is universal, is less restricted by genetic material, and has a wide range of applications.

[0019] Mining molecular markers associated with ammonium tolerance in tomato seedlings

[0020] The present invention uses genome-wide association analysis to identify molecular markers associated with ammonium tolerance in tomato seedlings. The specific process includes:

[0021] 1) Plant and manage a natural population of 26 tomato materials and preserve aboveground samples of each plant.

[0022] 2) High ammonium stress (ammonium nitrogen source (AA): 6 mM (NH)2SO4) was applied to the plants at the seedling stage. The fresh weight and dry weight of each material were obtained after 40 days. At least 5 individual plants were surveyed for each material.

[0023] 3) The plants were treated with nitrate nitrogen (nitrate nitrogen source (NN): 12 mM NaNO3) at the seedling stage. The fresh weight and dry weight of each material were obtained 40 days later. At least 5 individual plants were surveyed for each material.

[0024] 4) Arrange the aboveground dry weight data for each material and calculate the ratio of the dry weight of the material after ammonium nitrogen treatment to the dry weight of the material after nitrate nitrogen treatment, which is the aboveground dry weight ratio. Materials with an aboveground dry weight ratio greater than 1 are defined as ammonium-tolerant materials (marked "H"). Materials with an aboveground dry weight ratio greater than 0.75 and less than 1 are generally ammonium-tolerant materials. Materials with an aboveground dry weight ratio less than 0.75 are defined as non-ammonium-tolerant materials (marked "L") (as shown in Table 1).

[0025] 5) DNA of all tomato materials was extracted using the CTAB method, high-throughput sequencing libraries were constructed, and sequencing analysis was performed using HiSeq4000 (Illumina), with a data volume of 35G for each material.

[0026] 6) Bioinformatics and Genome-Wide Association Analysis: The sequenced fragments of each sample were aligned to the tomato reference genome (database ID: Tomato SL2.50 ITAG2.4, https: / / solgenomics.sgn.cornell.edu / ) using bwa software. After alignment, variants were identified using samtools and bcftools. After quality filtering of variant sites, a genome-wide SNP matrix was generated and converted to a bed file. Genome-wide association analysis was performed in tomato using EMMAX.

[0027] like Figure 1 The results showed that there was an A>G SNP molecular marker at base 62760934 of chromosome 8 in the tomato genome SL2.50 ITAG2.4.

[0028] Table 1 Phenotypic identification results of 26 tomato materials

[0029] Material name Ammonium / nitrate-dry weight Ammonium resistance Material name Ammonium / nitrate-dry weight Ammonium resistance LA3846 1.36601 H LA0113 0.37886 L LA2413 1.1643 H LA1162 0.26257 L LA0533 1.99189 H LA2133 0.88272 generally LA2181 1.46541 H LA1218 0.79612 generally LA1478 1.29287 H LA1457 0.59193 L LA0126 1.98058 H LA2283 0.50399 L LA1286 1.4127 H LA3214 0.4793 L LA1244 1.3913 H LA1543 0.27392 L LA1589 1.46437 H LA2184 0.41637 L LA1461 1.49485 H LA0395 0.41096 L LA2009 1.23063 H LA0089 0.84818 generally LA1673 1.62857 H LA4133 0.35722 L LA1482 1.03023 H LA4024 0.34091 L

[0030] Application of molecular markers

[0031] Using the designed primer pair ggz-F and ggz-R, PCR amplification was performed on a tomato DNA sample to be tested. The amplified product was digested with SalI, and the digested products were separated by 2% agarose gel electrophoresis. The ammonium tolerance of the tomato was determined based on the bands. If two bands of 621 bp and 370 bp were present, the tomato was non-ammonium-tolerant. If only the 991 bp band was present, the tomato was ammonium-tolerant. Therefore, using this molecular marker, the aboveground dry weight ratio of tomatoes can be determined at the seedling stage, thereby determining the ammonium tolerance of tomatoes and improving breeding efficiency.

[0032] Based on this, the examples disclose a primer pair comprising the DNA molecules shown in SEQ ID NO: 5 (ggz-F) and SEQ ID NO: 6 (ggz-R). The primer pair is used to PCR amplify the nucleotide sequence encompassing position 62760934 of chromosome 8 in the tomato genome, SL2.50 ITAG2.4. The PCR product amplified by the primer pair is digested with the endonuclease, and the digested product can be used to determine the ammonium tolerance of tomato seedlings.

[0033] The embodiment discloses a kit including the above primer pairs, reagents required for PCR amplification, and reagents required for enzyme digestion. The reagents required for PCR amplification include 2X M5 HiPerplus Taq HiFi PCR mix and nuclease-free ddH2O. The reagents required for enzyme digestion include SalI enzyme and buffer.

[0034] The embodiment discloses a method for detecting ammonium tolerance in tomato seedlings. The method comprises: extracting tomato genomic SL2.50 ITAG2.4 DNA; performing PCR amplification on the genomic DNA using the primer pair shown in SEQ ID NOs: 5 and 6; digesting the PCR amplification product using the restriction endonuclease SalI; performing electrophoresis on the digestion product; and determining the ammonium tolerance of the tomato based on the bands detected by electrophoresis; if two bands of 621 bp and 370 bp are present, the tomato is non-ammonium tolerant; if only a band of 991 bp is present, the tomato is ammonium tolerant.

[0035] The embodiments disclose applications of the molecular marker, the nucleic acid molecule, the primer pair, or the kit, including at least one of detecting ammonium tolerance of tomatoes at the seedling stage, detecting dry weight ratio of tomatoes at the seedling stage, and tomato breeding.

[0036] In some embodiments, genotyping and dry weight ratio prediction are performed on 26 tomato materials, and then whether the 26 tomato materials have ammonium tolerance is predicted based on the dry weight ratio. The steps are as follows:

[0037] (1) PCR amplification of tomato genomic DNA was performed using primer pairs ggz-F and ggz-R. The total volume of the PCR reaction system was 20 μL, containing: 500 ng tomato genomic GCA_000188115.4 DNA (80-150 ng / μL), 0.5 μL ggz-F (10 mM), 0.5 μL ggz-R (10 mM), 10 μL 2X M5 HiPerplus Taq HiFi PCR mix (Beijing Polymer Biotechnology Co., Ltd.), and the remainder of nuclease-free ddH2O. The PCR reaction was performed on an S1000 PCR instrument produced by Bio-Rad, USA. The PCR amplification program was as follows: pre-denaturation at 95.0°C for 3 min; denaturation at 95.0°C for 30 s, annealing at 50.0°C for 30 s, extension at 72.0°C for 60 s, cycle jump to 95°C for 30 s, cycle 35 times; extension at 72.0°C for 5 min, and storage at 4°C.

[0038] (2) The amplified products were detected by 1% agarose gel electrophoresis, and a 991 bp band was amplified.

[0039] (3) The amplified 991bp band was digested with SalI enzyme (Thermo Scientific, 10U / μL). The enzyme digestion system was prepared according to the instructions as follows: 1μL SalI (10U / μL, ER0641, Thermo Scientific TM ), 5 μL PCR reaction product, 2 μL ddH2O and 2 μL 10× Buffer O (Thermo ScientificTM The enzyme was digested at 37°C for 3 h and inactivated at 65°C for 20 min.

[0040] (4) The enzyme-digested products are subjected to 2% agarose gel electrophoresis. If the 991bp band is not cleaved by the enzyme, the tomato material is judged to have a high dry weight ratio and high ammonium resistance, and is an ammonium-resistant material. If the 991bp band is cleaved by the enzyme into two bands of 621bp and 370bp, the material is judged to have a low dry weight ratio and low ammonium resistance, and is a non-ammonium-resistant material. If there are three bands of 991bp, 621bp and 370bp at the same time, the material is judged to have an average dry weight ratio and average ammonium resistance. Since the phenotype of the non-ammonium-resistant material is a conventional target phenotype in this field, a phenotype that is judged to be L by the marker but is actually an average phenotype is also judged to be L type.

[0041] like Figures 2-4 As shown in Table 2, the accuracy of the marker was 73.08% based on the aboveground dry weight ratio phenotype of this batch of tomatoes (Table 2).

[0042] The above identification results demonstrate that the molecular markers, nucleic acid molecules, primer pair kits, methods, and applications provided herein can accurately and efficiently determine ammonium tolerance in tomato seedlings, effectively reducing the planting scale of breeding materials, alleviating the workload of later field phenotypic identification, accelerating the breeding process, and improving breeding efficiency. This molecular marker is universal, with minimal genetic material limitations and wide application potential.

[0043] Table 2 Results of molecular marker detection and field phenotypic identification of 26 tomato materials

[0044]

[0045]

[0046] The above is only a preferred specific implementation method of the present application, but the scope of protection of the present application is not limited thereto. Any changes or replacements that can be easily thought of by any technician familiar with this technical field within the technical scope disclosed in this application should be covered by the scope of protection of the present application.

Claims

1. Methods for detecting ammonium tolerance of tomato seedlings, including: Extracting genomic DNA of tomato materials to be tested; PCR amplification of the genomic DNA using the primer pair shown in SEQ ID NOs: 5 and 6; PCR products were detected by electrophoresis; The PCR product was digested with SalI enzyme; The ammonium tolerance of tomatoes can be judged based on the bands detected by electrophoresis; If two bands of 621 bp and 370 bp appear, the tomato is a non-ammonium-tolerant material; If only a 991 bp band appears, the tomato is an ammonium-tolerant material.

2. The method according to claim 1, wherein the step of performing electrophoresis detection on the PCR product comprises performing 1% agarose gel electrophoresis on the PCR amplification product to separate a 991 bp band.

3. The method according to claim 2, wherein the enzymatic cleavage step comprises: The 991 bp band was digested with SalI enzyme; and The enzyme digestion products were subjected to 2% agarose gel electrophoresis.

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