Molecular marker associated with characters of tomatoes under low phosphorus stress and application of molecular marker

Abstract

The invention relates to the technical field of tomato genetic breeding, in particular to a molecular marker associated with characters of tomatoes under low phosphorus stress and application. The molecular marker comprises a single nucleotide Agt at the 52526904 site of an eleventh chromosome of a tomato genome; g is a nucleotide sequence formed by mutation. By utilizing the molecular marker, not only can the plant height, fresh weight and dry weight in the tomato seedling stage under low-phosphorus stress be predicted and identified, but also the low-phosphorus tolerance degree of the plant can be judged. The molecular marker can provide help for cloning of low-phosphorus tomato seedling stage plant height, fresh weight and dry weight control genes and low-phosphorus-resistant tomato breeding, 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 application. 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 nuclei. It mainly exists in nucleic acids, phospholipids, nucleoproteins and other molecules in the form of phosphate, and participates in plant photosynthesis and respiration. Under low-phosphorus conditions, the main performance characteristics of tomatoes are short plants, small leaf area, and purple-red stems and leaves. Short-term low-phosphorus stress causes purple-red backs of leaves, small leaves and brown spots. Long-term low-phosphorus stress causes dark purple veins, yellowing of old 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, locating tomato phosphorus-efficient genes and developing molecular markers are of great significance for screening and creating phosphorus-efficient materials under low phosphorus and breeding low-phosphorus-tolerant tomato varieties. Summary of the invention

[0003] This application identifies the plant height, fresh weight and dry weight of natural tomato populations under low-phosphorus stress, and through genome-wide association analysis, excavates SNP molecular markers associated with tomato traits under low-phosphorus stress from tomato genome resequencing data. This molecular marker pair can accurately predict and identify the plant height, fresh weight and dry weight of tomato seedlings under low-phosphorus stress, and determine the degree of low-phosphorus tolerance of the plant. This molecular marker can help the cloning of tomato seedling dry weight control genes under low-phosphorus stress and tomato low-phosphorus tolerance breeding, and has important application value.

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

[0005] In the first aspect, the embodiments disclose molecular markers associated with tomato traits under low phosphorus stress. The molecular markers include a nucleotide sequence formed by a single nucleotide A>G mutation at 52526904 of chromosome 11 of the tomato genome SL2.50 ITAG2.4, and the traits include 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.

[0006] In a second aspect, an embodiment discloses a nucleic acid molecule associated with a trait of tomato under low-phosphorus stress. The trait includes at least one of tolerance to low phosphorus in the seedling stage of tomato, 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. The primer pair includes DNA molecules as shown in SEQ ID NOs: 5 and 6. The primer pair performs PCR amplification on a nucleotide sequence at position 52526904 of chromosome 11 of the tomato genome SL2.50 ITAG2.4, and determines the low-phosphorus tolerance of tomato seedlings according to the enzyme digestion results of the PCR amplification product.

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

[0009] In a sixth aspect, the embodiment discloses a method for detecting low-phosphorus tolerance of 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 NO: 5 and 6; performing electrophoresis detection on the enzyme digestion product; judging the low-phosphorus tolerance of the tomato according to 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 non-low-phosphorus tolerant material; if only a band of 710 bp appears, the tomato is a low-phosphorus tolerant material; if 719 bp, 332 bp and 387 bp appear at the same time, the tomato is a material with average low-phosphorus tolerance.

[0010] In the seventh aspect, the embodiment discloses a method for predicting the plant height of tomatoes at the seedling stage under low phosphorus stress. The method comprises: extracting genomic DNA of the tomato material to be tested; performing PCR amplification and enzyme digestion using the primer pair shown in SEQ ID NO: 5 and 6; performing electrophoresis detection on the enzyme digestion product; judging the plant height of the tomato according to the size and number of the 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 band of 710 bp appears, the tomato is a high plant height material; if 719 bp, 332 bp and 387 bp appear at the same time, the tomato is a medium plant height material.

[0011] In an eighth aspect, an 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 and enzyme digestion using a primer pair as shown in SEQ ID NO: 5 and 6; performing electrophoresis detection on the enzyme digestion product; judging the fresh weight of the tomato according to 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 fresh weight material; if only a band of 710 bp appears, the tomato is a high fresh weight material; if 719 bp, 332 bp and 387 bp appear at the same time, the tomato is a medium fresh weight material.

[0012] In the ninth aspect, 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 the tomato material to be tested; performing PCR amplification and enzyme digestion using the primer pair shown in SEQ ID NO: 5 and 6; performing electrophoresis detection on the enzyme digestion product; judging the dry weight of the tomato according to the size and number of the 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 dry weight material; if only a band of 710 bp appears, the tomato is a high dry weight material; if 719 bp, 332 bp and 387 bp appear at the same time, the tomato is a medium dry weight material.

[0013] In a tenth aspect, the embodiments disclose applications 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 applications are selected from at least one of prediction of plant height of tomato seedlings under low phosphorus stress, fresh weight of tomato seedlings under low phosphorus stress, dry weight of tomato seedlings under low phosphorus stress, identification of low phosphorus tolerance, and tomato breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1The electrophoresis results of tomato materials LA0126, LA2413, LA0025, LA0746, LA2640, LA3475, LA3008, LA3856, LA1307, LA4024, LA1544, LA1037, LA3528 and LA0429 detected by using the molecular markers provided in the examples. In the figure, H represents the banding pattern of homozygous materials with high plant height, fresh weight and dry weight (low phosphorus tolerance), and L represents the banding pattern of homozygous materials with low plant height, fresh weight and dry weight (not low phosphorus tolerance).

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

[0016] Figure 3 The electrophoresis results of the tomato material LA0442 detected by the molecular markers provided in the example. 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 scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.

[0018] Example The molecular markers associated with the traits of tomato under low phosphorus stress were screened by genome-wide association analysis. The natural population of tomato materials was treated with low phosphorus stress (41.5 mM K2HPO4·3H2O) for 40 days at the seedling stage, and the plant height, fresh weight and dry weight of each material were measured. The whole genome association analysis was performed on these traits, and the results screened out a SNP molecular marker with A>G at the 52526904th base on chromosome 11 of the tomato genome SL2.50 ITAG2.4, which was associated with the plant height, fresh weight and dry weight of tomato 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 are defined as materials with plant height, fresh weight, and dry weight (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 are defined as materials with medium plant height, fresh weight, and dry weight; materials with plant height less than 38.00 cm, fresh weight less than 5.0000 g and less than 0.5000 g are defined as materials with small plant height, fresh weight, and dry weight (L-type materials). Among them, H-type tomato materials are low-phosphorus tolerant materials, and L-type tomato materials are non-low-phosphorus tolerant materials.

[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] The designed primer pair LP-F (SEQ ID NO: 5) and LP-R (SEQ ID NO: 6) were used to perform PCR amplification on the tomato DNA sample to be tested. The amplified product was digested with TaaI. After the digested product was separated by 3% agarose gel electrophoresis, the tomato's tolerance to low phosphorus was judged according to 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 the three bands of 719bp, 332bp and 387bp appeared at the same time, it was a material with medium tolerance to low phosphorus. Therefore, using this marker, the low-phosphorus tolerance characteristics of the tomato material can be judged at the seedling stage, and its plant height, fresh weight and dry weight at the seedling stage under low-phosphorus stress can be judged to improve breeding efficiency.

[0023] Based on this, the embodiment discloses a primer pair and an endonuclease, including DNA molecules as shown in SEQ ID NO: 5 (LP-F) and SEQ ID NO: 6 (LP-R) and a restriction endonuclease TaaI. The primer pair performs PCR amplification on the nucleotide sequence at position 52526904 of chromosome 11 of the tomato genome SL2.50ITAG2.4. The restriction endonuclease is TaaI, and the PCR product is digested by the endonuclease to judge the low-phosphorus tolerance of tomato seedlings according to 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 of tomatoes. The method comprises: extracting genomic DNA of a tomato material to be tested; performing PCR amplification on the genomic DNA using primers as shown in SEQ ID NOs: 5 and 6; performing enzyme digestion on the PCR amplification product using restriction endonuclease TaaI; performing electrophoresis detection on the enzyme digestion product; judging the low-phosphorus tolerance of tomatoes according to the number and size of 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 band of 710 bp appears, the tomato is a low-phosphorus tolerant material; if three bands of 719 bp, 332 bp, and 387 bp appear at the same time, the tomato is a medium low-phosphorus tolerant material.

[0026] The embodiment discloses a method for predicting the plant height 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 as shown in SEQ ID NOs: 5 and 6; performing enzyme digestion on the PCR amplification product using restriction endonuclease TaaI; performing electrophoresis detection on the enzyme digestion product; judging the height of the tomato plant according to the number and size of the bands detected by electrophoresis; if two bands of 332 bp and 387 bp appear, the tomato is a material with a low plant height; if only a band of 710 bp appears, the tomato is a material with a high plant height; if three bands of 719 bp, 332 bp, and 387 bp appear at the same time, the tomato is a material with a normal 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 as shown in SEQ ID NOs: 5 and 6; performing enzyme digestion on the PCR amplification product using restriction endonuclease TaaI; performing electrophoresis detection on the enzyme digestion product; judging the fresh weight of the tomato according to the number and size of 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 band of 710 bp appears, the tomato is a high fresh weight material; if three bands of 719 bp, 332 bp, and 387 bp appear at the same time, 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 as shown in SEQ ID NOs: 5 and 6; performing enzyme digestion on the PCR amplification product using restriction endonuclease TaaI; performing electrophoresis detection on the enzyme digestion product; judging the dry weight of the tomato according to the number and size of bands detected by electrophoresis; if two bands of 332 bp and 387 bp appear, the tomato is a low dry weight material; if only a band of 710 bp appears, the tomato is a high dry weight material; if three bands of 719 bp, 332 bp, and 387 bp appear at the same time, the tomato is a material of average dry weight.

[0029] The embodiment discloses the application of the molecular marker, the nucleic acid, the primer pair, the endonuclease and the kit. 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, identification of low phosphorus tolerance characteristics of tomato seedlings and tomato breeding.

[0030] In some embodiments, the obtained molecular markers and primers associated with plant height, fresh weight, dry weight and low phosphorus tolerance under low phosphorus stress are used to perform genotype identification on 24 tomato materials, and the plant height, fresh weight and dry weight are predicted based on the identification results, so as to detect and identify the low phosphorus tolerance characteristics, and 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 was 20 μL, including 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 TaqDNA polymerase (5 U / μL), and 15.6 μL ddH2O.

[0033] (3) PCR reaction was carried out on a S1000 PCR instrument produced by Bio-Rad, USA. The PCR amplification program was as follows: 95.0°C pre-denaturation for 3 min; 95.0°C denaturation for 30 s, 55.0°C annealing for 30 s, 72.0°C extension for 60 s, setting the cycle, jump to 95°C, 30 s, cycle 35 times; 72.0°C extension 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 instructions as follows: 8.5 μL of PCR reaction 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 digestion products are detected by 3% agarose gel electrophoresis. If the 710bp band is not digested, the tomato material is judged to have high plant height, fresh weight, and dry weight, high low-phosphorus tolerance, and is a low-phosphorus tolerant material. If the 719bp band is completely digested into two bands of 332bp and 387bp, the material is judged to have low plant height, fresh weight, and dry weight, low low-phosphorus tolerance, and is a non-low-phosphorus tolerant material. If there are three bands of 719bp, 332bp, and 387bp at the same time, the material is judged to have average plant height, fresh weight, and dry weight, and medium low-phosphorus tolerance. Since the phenotype of non-low-phosphorus tolerant materials is a conventional target phenotype in this field, a phenotype that is marked as L but actually has an average phenotype is also judged to be L type.

[0037] like Figure 1 , 2 As shown in Figures 3 and Table 2, the molecular marker can accurately predict plant height, fresh weight and dry weight under low phosphorus stress, with an accuracy rate of 75%, calculated as follows: accuracy rate = number of materials whose marker results are consistent with the actual phenotype in the field / total number of materials. Moreover, the low phosphorus tolerance of the plant can be accurately determined based on the molecular marker. The molecular marker can help clone the control genes of plant height, fresh weight and dry weight of tomato seedlings under low phosphorus stress and provide assistance for tomato low phosphorus tolerance breeding, and has important 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 of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed in the present application should be covered within the protection scope of the present application.

Claims

1. Molecular markers associated with tomato traits under low-phosphorus stress, including a nucleotide sequence formed by a single nucleotide A>G mutation at position 52526904 of chromosome 11 in the tomato genome SL2.50 ITAG2.4, wherein the traits include 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.

2. A nucleic acid molecule associated with traits of tomato under low-phosphorus stress, wherein the traits include 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, and the nucleic acid molecule is shown in SEQ ID NO: 1 or 2.

3. Primer pairs, including: The DNA molecules shown in SEQ ID NO: 5 and 6.

4. A PCR amplification kit, comprising: The primer pair as claimed in claim 3 and other reagents required for amplification.

5. Methods for testing tomato tolerance to low phosphorus 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; Conduct electrophoresis to detect PCR products; The PCR product was digested with TaaI enzyme; 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 not a low-phosphorus tolerant material; If only a band of 710 bp appears, the tomato is a low-phosphorus tolerant material.

6. The method for detecting the plant height of tomato seedlings under low phosphorus stress comprises: 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; Conduct electrophoresis to detect PCR products; The PCR product was digested with TaaI enzyme; The plant height of tomatoes under low phosphorus stress was determined 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 band of 710 bp appears, the tomato is a high plant height material.

7. The method for detecting the fresh weight of tomato seedlings under low phosphorus stress includes: 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; Conduct electrophoresis to detect PCR products; The PCR product was digested with TaaI enzyme; The fresh weight of tomatoes at the 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 band of 710 bp appears, the tomato is a high fresh weight material.

8. 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; Conduct electrophoresis to detect PCR products; 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 a band of 710 bp appears, the tomato is a high dry weight material.

9. The method according to claim 5, 6, 7, 8, wherein the steps of electrophoresis and enzyme digestion detection include: The PCR amplification products were subjected to 1% agarose gel electrophoresis, and the 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.

10. Use of the molecular marker according to claim 1, the nucleic acid molecule according to claim 2, the primer pair according to claim 3 and the kit according to claim 4, wherein the use 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, identification of low-phosphorus tolerance of tomato seedlings and tomato breeding.

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