Molecular markers associated with tomato traits under low phosphorus stress and their applications

Abstract

The present application relates to the technical field of tomato genetic breeding, and more specifically to molecular markers associated with tomato traits under low-phosphorus stress and their applications. The molecular marker comprises a nucleotide sequence formed by a single nucleotide A>G mutation at position 52526904 of chromosome 11 of the tomato genome. Using this molecular marker, not only can the plant height, fresh weight, and dry weight of tomatoes at the seedling stage under low-phosphorus stress be predicted and identified, but the degree of low-phosphorus tolerance of the plant can also be determined. This molecular marker can assist in cloning genes controlling plant height, fresh weight, and dry weight of tomatoes at the seedling stage under low-phosphorus conditions and in breeding tomatoes for low-phosphorus tolerance, and has important application value.

Description

Technical Field

[0001] The present application relates to the technical field of tomato genetic breeding, and in particular to molecular markers associated with tomato traits under low-phosphorus stress and their applications. Background Art

[0002] Tomato (Solanum lycopersicum) is the world's largest vegetable crop and one of the most widely cultivated vegetables in my country. Phosphorus is a component of cell membranes, cytoplasm, and nucleus, primarily present as phosphate in molecules such as nucleic acids, phospholipids, and nucleoproteins. It participates in plant photosynthesis and respiration. Under low phosphorus conditions, tomatoes exhibit dwarf growth, small leaf area, and purple-red stems and leaves. Short-term low phosphorus stress results in purple-red undersides of leaves, small leaves, and brown spots. Long-term low phosphorus stress causes dark purple veins, yellowing of older leaves, delayed flowering and fruiting, and reduced tomato yield (Zhu et al., 2020; Kalajie et al., 2014). Studying the effects of low phosphorus stress on tomato seedlings and their physiological characteristics, as well as mapping phosphorus-efficient genes and developing molecular markers, is crucial for screening and developing phosphorus-efficient materials and breeding low-phosphorus-tolerant tomato varieties. Summary of the Invention

[0003] This application identified plant height, fresh weight, and dry weight of natural tomato populations under low-phosphorus stress. Using genome-wide association analysis, SNP molecular markers associated with tomato traits under low-phosphorus stress were mined from tomato genome resequencing data. This molecular marker pair can accurately predict and identify plant height, fresh weight, and dry weight of tomato seedlings under low-phosphorus stress, determining the plant's tolerance to low-phosphorus. This molecular marker pair can assist in cloning genes controlling tomato seedling dry weight under low-phosphorus stress and in breeding tomato varieties for low-phosphorus tolerance, thus possessing significant application value.

[0004] To this end, the embodiments of the present application disclose at least the following technical solutions:

[0005] In a first aspect, the embodiments disclose molecular markers associated with traits in tomato plants under low-phosphorus stress. The molecular markers include a nucleotide sequence resulting from a single nucleotide A>G mutation at position 52526904 of chromosome 11 in the tomato genome, SL2.50 ITAG2.4. The traits include at least one of tomato seedling low-phosphorus tolerance, plant height under low-phosphorus stress, fresh weight under low-phosphorus stress, and dry weight under low-phosphorus stress.

[0006] In a second aspect, the embodiments disclose a nucleic acid molecule associated with a trait of tomato under low-phosphorus stress. The trait includes at least one of tomato low-phosphorus tolerance at the seedling stage, plant height under low-phosphorus stress, fresh weight under low-phosphorus stress, and dry weight under low-phosphorus stress. The nucleic acid molecule is as shown in SEQ ID NO: 1 or 2. The nucleic acid molecule is a cleaved 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 cut by TaaI, the material is judged to be a low-phosphorus-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 cut by TaaI into DNA molecules as shown in SEQ ID NO: 3 and SEQ ID NO: 4, the material is judged to be a non-low-phosphorus-tolerant material.

[0007] In a fourth 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 52526904 of chromosome 11 of the tomato genome SL2.50 ITAG2.4, and to determine the tomato seedling stage's tolerance to low phosphorus based on enzyme digestion results of the PCR amplification product.

[0008] In a fifth aspect, the embodiment discloses a PCR amplification kit, comprising the DNA molecule described in the third aspect, and reagents required for PCR amplification and enzyme digestion.

[0009] In a sixth aspect, the embodiments disclose a method for detecting low-phosphorus tolerance in tomatoes. The method comprises: extracting genomic DNA of a tomato material to be tested; performing PCR amplification and enzyme digestion using the primer pair shown in SEQ ID NOs: 5 and 6; performing electrophoresis on the digestion products; and determining the tomato's low-phosphorus tolerance based on the size and number of bands detected by electrophoresis; if two bands of 332 bp (SEQ ID NO: 3) and 387 bp (SEQ ID NO: 4) are present, the tomato is not low-phosphorus tolerant; if only the 710 bp band is present, the tomato is low-phosphorus tolerant; and if all of the 719 bp, 332 bp, and 387 bp bands are present, the tomato has average low-phosphorus tolerance.

[0010] In a seventh aspect, the embodiments disclose a method for predicting tomato plant height at the seedling stage under low phosphorus stress. The method comprises: extracting genomic DNA of a tomato material to be tested; performing PCR amplification and enzyme digestion using the primer pair shown in SEQ ID NOs: 5 and 6; performing electrophoresis on the enzyme digestion products; and determining the tomato plant height based on the size and number of bands detected by electrophoresis; if two bands of 332 bp (SEQ ID NO: 3) and 387 bp (SEQ ID NO: 4) appear, the tomato is a low plant height material; if only the 710 bp band appears, the tomato is a high plant height material; and if all of 719 bp, 332 bp, and 387 bp appear, the tomato is a medium plant height material.

[0011] In an eighth aspect, the embodiments disclose a method for predicting the fresh weight of tomatoes at the seedling stage under low phosphorus stress. The method comprises: extracting genomic DNA of a tomato material to be tested; performing PCR amplification and enzyme digestion using the primer pair shown in SEQ ID NOs: 5 and 6; performing electrophoresis on the digestion products; and determining the fresh weight of the tomato based on the size and number of bands detected by electrophoresis; if two bands of 332 bp (SEQ ID NO: 3) and 387 bp (SEQ ID NO: 4) are present, the tomato is a low fresh weight material; if only the 710 bp band is present, the tomato is a high fresh weight material; and if all of the 719 bp, 332 bp, and 387 bp bands are present, the tomato is a medium fresh weight material.

[0012] In a ninth aspect, embodiments disclose a method for predicting the dry weight of tomatoes at the seedling stage under low phosphorus stress. The method comprises: extracting genomic DNA from a tomato material to be tested; performing PCR amplification and enzyme digestion using the primer pair shown in SEQ ID NOs: 5 and 6; performing electrophoresis on the digestion products; and determining the dry weight of the tomato based on the size and number of bands detected by electrophoresis; if two bands of 332 bp (SEQ ID NO: 3) and 387 bp (SEQ ID NO: 4) are present, the tomato is a low dry weight material; if only the 710 bp band is present, the tomato is a high dry weight material; and if all of the 719 bp, 332 bp, and 387 bp bands are present, the tomato is a medium dry weight material.

[0013] In a tenth aspect, the embodiments disclose uses of the molecular markers described in the first or second aspect, the nucleic acid molecules described in the third aspect, the primer pairs described in the fourth aspect, and the kit described in the fifth aspect. The uses are selected from at least one of the following: prediction of tomato seedling plant height under low-phosphorus stress, prediction of tomato seedling fresh weight under low-phosphorus stress, prediction of tomato seedling dry weight under low-phosphorus stress, identification of low-phosphorus tolerance, and tomato breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1The electrophoresis results for tomato varieties LA0126, LA2413, LA0025, LA0746, LA2640, LA3475, LA3008, LA3856, LA1307, LA4024, LA1544, LA1037, LA3528, and LA0429 using the molecular markers provided in the Examples are shown. In the figure, H represents the banding pattern of homozygous varieties with high plant height, fresh weight, and dry weight (tolerant to low phosphorus levels), and L represents the banding pattern of homozygous varieties with low plant height, fresh weight, and dry weight (intolerant to low phosphorus levels).

[0015] Figure 2 Electrophoresis results for tomato varieties LA2626, LA2093, LA3238, LA2285, LA0404, LA1459, LA1269, LA0113, and LA0466 using the molecular markers provided in the Examples. In the figure, H represents the banding pattern of homozygous varieties with high plant height, fresh weight, and dry weight, and L represents the banding pattern of homozygous varieties with low plant height, fresh weight, and dry weight.

[0016] Figure 3 The electrophoresis results of the tomato material LA0442 using the molecular markers provided in the example are shown. In the figure, H represents the banding pattern of the homozygous material with high plant height, fresh weight, and dry weight, and L represents the banding pattern of the homozygous material with low plant height, fresh weight, and dry weight. DETAILED DESCRIPTION

[0017] In order to make the purpose, technical solutions and advantages of this application more clear, 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] In this example, genome-wide association analysis was used to screen for molecular markers associated with tomato traits under low-phosphorus stress. A natural population of tomato plants was subjected to low-phosphorus stress (41.5 mM K₂HPO₄·3H₂O) for 40 days at the seedling stage. Plant height, fresh weight, and dry weight were measured for each sample. Genome-wide association analysis of these traits was performed. The results identified a single-nucleotide polymorphism (SNP) marker with an A > G residue at position 52526904 on chromosome 11 in the SL2.50 ITAG2.4 region of the tomato genome. This marker was associated with plant height, fresh weight, and dry weight under low-phosphorus stress.

[0019] As shown in Table 1, materials with plant height greater than 58.00 cm, fresh weight greater than 7.0000 g, and dry weight greater than 1.0000 g were defined as H-type materials; materials with plant height less than 58.00 cm and greater than 38.00 cm, fresh weight less than 7.0000 g and greater than 5.0000 g, and dry weight less than 1.0000 g and greater than 0.5000 g were defined as medium plant height, fresh weight, and dry weight materials; and materials with plant height less than 38.00 cm, fresh weight less than 5.0000 g and less than 0.5000 g were defined as small plant height, fresh weight, and dry weight materials (L-type materials). H-type tomato materials are low-phosphorus-tolerant, while L-type tomato materials are not low-phosphorus-tolerant.

[0020] Table 1

[0021] Material name Plant height (cm) Fresh weight (g) Dry weight (g) Material name Plant height (cm) Fresh weight (g) Dry weight (g) LA0025 58.00 12.3000 1.7740 LA1037 42.25 5.0500 0.6068 LA0113 77.83 12.1633 1.5530 LA3856 28.25 4.1950 0.6055 LA0466 70.67 12.4967 1.4503 LA2413 35.67 4.4800 0.5547 LA0746 61.33 6.2567 1.3503 LA0126 37.38 3.8750 0.4515 LA4024 53.67 8.7100 1.2133 LA0442 37.75 2.3575 0.4220 LA0429 59.50 8.9867 1.1470 LA2626 34.00 3.3600 0.3883 LA1307 37.00 6.9000 0.9867 LA2093 43.33 2.3600 0.3690 LA3475 50.67 4.5967 0.7913 LA3238 23.00 3.1267 0.3600 LA3008 32.63 5.4825 0.6558 LA2285 45.17 3.3367 0.3400 LA2640 46.50 5.1650 0.6460 LA0404 33.00 2.7667 0.2753 LA1544 36.83 4.9600 0.6233 LA1459 25.00 2.3500 0.2720 LA3528 41.00 2.9500 0.6230 LA1269 18.33 0.9567 0.1137

[0022] Using the designed primer pair LP-F (SEQ ID NO: 5) and LP-R (SEQ ID NO: 6), PCR amplification was performed on the tomato DNA sample to be tested. The amplified product was digested with TaaI, and the digested product was separated by 3% agarose gel electrophoresis. The tomato's low-phosphorus tolerance was determined based on the number and size of the bands. If two bands of 332bp and 387bp appeared, the tomato was a non-low-phosphorus-tolerant material. If only a band of 710bp appeared, the tomato was a low-phosphorus-tolerant material. If all three bands of 719bp, 332bp, and 387bp appeared at the same time, the tomato was a material with moderate low-phosphorus tolerance. Therefore, using this marker, the low-phosphorus tolerance of the tomato material can be determined at the seedling stage, and its plant height, fresh weight, and dry weight at the seedling stage under low-phosphorus stress can be determined to improve breeding efficiency.

[0023] Based on this, the examples disclose a primer pair and an endonuclease, including the DNA molecules shown in SEQ ID NO: 5 (LP-F) and SEQ ID NO: 6 (LP-R), and the restriction endonuclease TaaI. The primer pair is used to PCR amplify the nucleotide sequence at position 52526904 of chromosome 11 of the tomato genome SL2.50ITAG2.4. The restriction endonuclease is TaaI, which is used to digest the PCR product to determine the low-phosphorus tolerance of tomato seedlings based on the digested amplified product.

[0024] The embodiment discloses a kit, comprising the DNA molecule, restriction endonuclease TaaI, and reagents required for PCR amplification and enzyme digestion.

[0025] The embodiment discloses a method for detecting low-phosphorus tolerance in tomatoes. The method comprises: extracting genomic DNA of a tomato material to be tested; performing PCR amplification on the genomic DNA using primers shown in SEQ ID NOs: 5 and 6; digesting the PCR amplification product using the restriction endonuclease TaaI; performing electrophoresis on the digestion product; and determining the tomato's low-phosphorus tolerance based on the number and size of bands detected by electrophoresis; if two bands of 332 bp and 387 bp appear, the tomato is a material with no low-phosphorus tolerance; if only a band of 710 bp appears, the tomato is a material with low-phosphorus tolerance; and if all three bands of 719 bp, 332 bp, and 387 bp appear, the tomato has moderate low-phosphorus tolerance.

[0026] The embodiment discloses a method for predicting tomato plant height at the seedling stage under low phosphorus stress. The method comprises: extracting genomic DNA from a tomato material to be tested; performing PCR amplification on the genomic DNA using primers shown in SEQ ID NOs: 5 and 6; performing enzyme digestion on the PCR amplification product using the restriction endonuclease TaaI; performing electrophoresis on the digestion product; and determining the tomato plant height based on the number and size of bands detected by electrophoresis; if two bands of 332 bp and 387 bp appear, the tomato is a material with low plant height; if only a band of 710 bp appears, the tomato is a material with high plant height; and if all three bands of 719 bp, 332 bp, and 387 bp appear, the tomato is a material with average plant height.

[0027] The embodiment discloses a method for predicting the fresh weight of tomatoes at the seedling stage under low phosphorus stress. The method comprises: extracting genomic DNA of a tomato material to be tested; performing PCR amplification on the genomic DNA using primers shown in SEQ ID NOs: 5 and 6; digesting the PCR amplification product using the restriction endonuclease TaaI; performing electrophoresis on the digestion product; and determining the fresh weight of the tomato based on the number and size of bands detected by electrophoresis; if two bands of 332 bp and 387 bp are present, the tomato is a low fresh weight material; if only a band of 710 bp is present, the tomato is a high fresh weight material; and if three bands of 719 bp, 332 bp, and 387 bp are present simultaneously, the tomato is a medium fresh weight material.

[0028] The embodiment discloses a method for predicting the dry weight of tomatoes at the seedling stage under low phosphorus stress. The method comprises: extracting genomic DNA of a tomato material to be tested; performing PCR amplification on the genomic DNA using primers shown in SEQ ID NOs: 5 and 6; digesting the PCR amplification product using the restriction endonuclease TaaI; performing electrophoresis on the digestion product; and determining the dry weight of the tomato based on the number and size of bands detected by electrophoresis; if two bands of 332 bp and 387 bp are present, the tomato is a low dry weight material; if only a band of 710 bp is present, the tomato is a high dry weight material; and if all three bands of 719 bp, 332 bp, and 387 bp are present, the tomato is a medium dry weight material.

[0029] The embodiments disclose applications of the molecular marker, the nucleic acid, the primer pair, the endonuclease, and the kit. The applications are selected from at least one of detecting plant height of tomatoes at the seedling stage under low-phosphorus stress, detecting fresh weight of tomatoes at the seedling stage under low-phosphorus stress, detecting dry weight of tomatoes at the seedling stage under low-phosphorus stress, identifying low-phosphorus tolerance characteristics of tomatoes at the seedling stage, and tomato breeding.

[0030] In some embodiments, molecular markers and primers associated with plant height, fresh weight, dry weight, and low-phosphorus tolerance under low-phosphorus stress were used to perform genotyping on 24 tomato materials. The plant height, fresh weight, and dry weight were predicted based on the identification results, and the low-phosphorus tolerance of the tomato materials was then detected and identified. The steps are as follows:

[0031] (1) The genomic DNA of all tomato materials was extracted using the CTAB method.

[0032] (2) PCR amplification of tomato genomic DNA was performed using the pair LP-F (SEQ ID NO: 5) and LP-R (SEQ ID NO: 6). The PCR reaction system (20 μL) contained 0.4 μL LP-F (10 mM), 0.4 μL LP-R (10 mM), 1.0 μL genomic DNA (100 ng / μL), 2.0 μL 10× PCR Buffer, 0.4 μL dNTPs (10 mM), 0.2 μL Taq DNA polymerase (5 U / μL), and 15.6 μL ddH2O.

[0033] (3) PCR reactions were performed on an S1000 PCR instrument manufactured 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 55.0°C for 30 s, extension at 72.0°C for 60 s, cycle setting to 95°C for 30 s, 35 cycles; extension at 72.0°C for 5 min, and storage at 4°C.

[0034] (4) The amplified products were detected by 1% agarose gel electrophoresis, and bands of 710 bp or 719 bp were amplified.

[0035] (5) The amplified 710 bp band was digested with TaaI enzyme (ThermoScientific, 10 U / μL). The digestion system was prepared according to the manufacturer's instructions as follows: 8.5 μL of PCR product, 1 μL of 10× Buffer Tango, and 0.5 μL of TaaI. The digestion was carried out at 37°C for 1.5 h and inactivated at 65°C for 20 min.

[0036] (6) The enzyme-digested products were detected by 3% agarose gel electrophoresis. If the 710bp band was not digested, the tomato material was judged to have high plant height, fresh weight, and dry weight, high low-phosphorus tolerance, and was a low-phosphorus tolerant material. If the 719bp band was completely digested into two bands of 332bp and 387bp, the material was judged to have low plant height, fresh weight, and dry weight, low low-phosphorus tolerance, and was a non-low-phosphorus tolerant material. If there were three bands of 719bp, 332bp, and 387bp at the same time, the material was judged to have average plant height, fresh weight, and dry weight, and average low-phosphorus tolerance. Since the phenotype of the non-low-phosphorus tolerant material is a conventional target phenotype in this field, a phenotype that is marked as L but the actual phenotype is average is also judged to be L type.

[0037] like Figure 1 、 2 As shown in Figures 3 and Table 2, this molecular marker can accurately predict plant height, fresh weight, and dry weight under low-phosphorus stress with an accuracy rate of 75%. This accuracy rate is calculated as follows: Accuracy = Number of accessions whose marker results are consistent with the actual field phenotype / Total accession number. Furthermore, this molecular marker can accurately determine a plant's tolerance to low-phosphorus. This molecular marker can assist in cloning genes controlling plant height, fresh weight, and dry weight in tomato seedlings under low-phosphorus stress and in breeding tomato varieties for low-phosphorus tolerance, thus possessing significant application value.

[0038] Table 2 Results of molecular marker detection and field phenotypic identification of 24 tomato materials

[0039]

[0040] 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 low phosphorus tolerance in tomato seedlings include: Extracting genomic DNA of tomato materials to be tested; PCR amplification was performed using the primer pair shown in SEQ ID NOs: 5 and 6; PCR products were detected by electrophoresis; use TaaI The enzyme digests the PCR product; The low phosphorus tolerance of tomatoes can be judged based on the bands detected by electrophoresis; If two bands of 332 bp and 387 bp appear, the tomato is a non-low-phosphorus-tolerant material; If only a 710 bp band appears, the tomato is a low-phosphorus-tolerant material.

2. A method for detecting plant height of tomatoes at the seedling stage under low phosphorus stress, comprising: Extracting genomic DNA of tomato materials to be tested; PCR amplification was performed using the primer pair shown in SEQ ID NOs: 5 and 6; PCR products were detected by electrophoresis; The PCR product was digested with TaaI enzyme; The plant height of tomatoes under low phosphorus stress was judged based on the bands detected by electrophoresis; If two bands of 332 bp and 387 bp appear, the tomato is a low plant height material; If only a 710 bp band appears, the tomato is a high plant height material.

3. Methods for detecting fresh weight of tomato seedlings under low phosphorus stress, including: Extracting genomic DNA of tomato materials to be tested; PCR amplification was performed using the primer pair shown in SEQ ID NOs: 5 and 6; PCR products were detected by electrophoresis; The PCR product was digested with TaaI enzyme; The fresh weight of tomatoes at seedling stage can be judged based on the bands detected by electrophoresis; If two bands of 332 bp and 387 bp appear, the tomato is a low fresh weight material; If only a 710 bp band appears, the tomato is a high fresh weight material.

4. A method for detecting the dry weight of tomato seedlings under low phosphorus stress, comprising: Extracting genomic DNA of tomato materials to be tested; PCR amplification was performed using the primer pair shown in SEQ ID NOs: 5 and 6; PCR products were detected by electrophoresis; The PCR product was digested with TaaI enzyme; The dry weight of tomato seedlings can be judged based on the bands detected by electrophoresis; If two bands of 332 bp and 387 bp appear, the tomato is a low dry weight material; If only the 710 bp band appears, the tomato is a high dry weight material.

5. The method according to any one of claims 1 to 4, wherein the steps of electrophoresis and enzyme digestion detection comprise: The PCR amplification products were subjected to 1% agarose gel electrophoresis, and a 710 bp or 719 bp band was isolated; The 710 bp or 719 bp band was digested with TaaI enzyme; The enzyme digestion products were subjected to 3% agarose gel electrophoresis.

6. Application of a PCR amplification primer pair, wherein the application is selected from at least one of detecting plant height of tomato seedlings under low phosphorus stress, detecting fresh weight of tomato seedlings under low phosphorus stress, detecting dry weight of tomato seedlings under low phosphorus stress, and identifying low phosphorus tolerance of tomato seedlings; The PCR amplification primer pair includes the DNA molecules shown in SEQ ID NOs: 5 and 6.

7. Application of a PCR amplification kit, wherein the application is selected from at least one of detecting plant height of tomato seedlings under low phosphorus stress, detecting fresh weight of tomato seedlings under low phosphorus stress, detecting dry weight of tomato seedlings under low phosphorus stress, and identifying low phosphorus tolerance of tomato seedlings; The PCR amplification kit includes DNA molecules as shown in SEQ ID NOs: 5 and 6 and other reagents required for amplification.

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