Detection primer combination of SNP (Single Nucleotide Polymorphism) molecular marker of rice heat-resistant gene TT2, kit and application of detection primer combination

Abstract

The invention relates to the technical field of molecular biology and plant molecular breeding, in particular to a detection primer combination of an SNP molecular marker of a rice heat-resistant gene TT2, a kit and application thereof, and the primer combination comprises a primer P1, a primer P2, a primer P3 and a primer P4; the nucleotide sequence of the primer P1 is as shown in SEQ ID NO: 1; the nucleotide sequence of the primer P2 is as shown in SEQ ID NO: 2; the nucleotide sequence of the primer P3 is as shown in SEQ ID NO: 3; the nucleotide sequence of the primer P4 is as shown in SEQ ID NO: 4. The four specific primers of the ARMS-PCR amplification method can efficiently and accurately distinguish the A / A heat-resistant type, the C / C heat-labile type and the A / C heterozygous type of the A / A heat-labile type and the C / C heat-labile type; 28 parts of materials in rice are subjected to TT2 genotype identification, and it is found that excellent heat-resistant haplotype bands can be accurately distinguished.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology and plant molecular breeding, and in particular to a detection primer combination, a kit and an application thereof for a SNP molecular marker of a rice heat-resistant gene TT2. Background Art

[0002] As one of the most important food crops in the world, the yield and quality of rice are of vital significance to ensuring world food security. However, with global warming, high temperature stress has become a key factor affecting rice growth and development, yield formation and quality improvement. In-depth exploration of rice heat resistance mechanisms and seeking effective coping strategies are important topics in the current agricultural research field. In the study of rice heat resistance physiology, scientists have found that high temperature has a significant impact on multiple physiological processes of rice. For example, high temperature can interfere with rice photosynthesis, reduce photosynthetic efficiency, and reduce the accumulation of photosynthetic products. At the same time, high temperature will also affect rice's respiration, water metabolism, nutrient absorption and transport and other physiological processes, destroy the physiological balance in cells, and thus affect the growth and development of plants. Studies have shown that heat-resistant rice varieties can maintain a relatively stable cell membrane structure and function under high temperature stress, reduce electrolyte extravasation, and keep the intracellular environment relatively stable; and can regulate the activity of antioxidant enzyme systems such as superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) to promptly remove excessive accumulated reactive oxygen free radicals in the body and reduce oxidative damage.

[0003] The rice heat tolerance gene TT2 plays a key role in the rice heat tolerance mechanism. The TT2 gene locus encodes a Gγ subunit that controls rice heat tolerance through SCT1-dependent alterations in wax biosynthesis. Under high temperature stress, natural alleles with loss of TT2 function are associated with greater retention of wax and increased heat tolerance. Mechanistically, the transcription factor SCT1 decodes Ca through a calcium-enhanced interaction with calmodulin. 2 + and acts as a negative regulator of its target genes such as wax synthesis regulator 2, OSWR2. Disruption of TT2 leads to heat-triggered Ca 2+ is reduced, thereby weakening the interaction between calmodulin and SCT1, which in turn explains the observed heat-induced changes in wax content. The mutation of the 1637th base of the TT2 gene from C to A makes the material heat-resistant. At present, the development of efficient and universal molecular markers for the TT2 gene is still to be improved, which limits its precise application in heat-resistant rice breeding. The reported molecular markers are linked markers of the TT2 excellent haplotype, which cannot accurately reflect the genotype of the identified material, and there is a risk of genetic separation, or the haplotype is detected by a high-throughput SNP analyzer, which has high experimental costs and instrument matching requirements. Therefore, the development of a simple, efficient, low-cost, green and environmentally friendly detection marker for the TT2 excellent haplotype with low hardware requirements is of great significance for heat-resistant rice breeding. Summary of the invention

[0004] In order to solve the problems raised in the above background technology, the present invention provides a detection primer combination, a kit and application thereof for SNP molecular markers of a heat-resistant gene TT2 of rice.

[0005] A detection primer combination for SNP molecular markers of a rice heat-resistant gene TT2, the primer combination comprising primer P1, primer P2, primer P3 and primer P4;

[0006] The nucleotide sequence of the primer P1 is shown in SEQ ID NO: 1; the nucleotide sequence of the primer P2 is shown in SEQ ID NO: 2; the nucleotide sequence of the primer P3 is shown in SEQ ID NO: 3; and the nucleotide sequence of the primer P4 is shown in SEQ ID NO: 4.

[0007] Furthermore, the primers P1 and P4 are located upstream and downstream of the SNP, respectively. The last base at the 3' end of the primer P2 is the C-type complementary base G at position 1637 of the TT2 gene. The last base at the 3' end of the primer P3 is the A-type base at position 1637 of the TT2 gene.

[0008] Furthermore, the addition ratio of primer P1:primer P2:primer P3:primer P4 in the primer combination is 2:2:1:1.

[0009] A kit for detecting a rice heat-resistant gene TT2, the kit comprising the above primer combination.

[0010] A method for detecting a rice heat-resistant gene TT2 comprises the following steps:

[0011] S1. Extracting genomic DNA of rice to be tested;

[0012] S2. Using the primer combination described in any one of claims 1 to 3, and taking the genomic DNA of the rice to be tested as a template, performing ARMS-PCR amplification on it;

[0013] S3. Detect the ARMS-PCR amplification products in 1% agarose gel, and determine the C / C type, A / A type and A / C type of the TT2 gene based on the band typing of the amplification products.

[0014] Further, the reaction procedure of the ARMS-PCR is as follows:

[0015] Pre-denaturation at 94°C-98°C for 2-10 min; denaturation at 94°C-98°C for 30-60 s, annealing at 42-65°C for 30-60 s, extension at 72°C for 45-60 s, for 28-35 cycles; extension at 72°C for 3-10 min; cooling at 4°C for 5-20 min.

[0016] Furthermore, the ARMS-PCR reaction system is 20 μL in total, and the specific components are as follows:

[0017] DNA polymerase Buffer 4-10μL, MgCl2 solution (25mM) 0.8-3.2μL, dNTP 0.4-2.0μL, DNA polymerase 0.1-1.0μL, P1 primer 0.1-0.5μL, P2 primer 0.1-0.5μL, P3 primer 0.1-0.5μL, P4 primer 0.1-0.5μL, genomic DNA 1.6-4.0μL, and ddH2O to make up the reaction system to 20μL.

[0018] Application of the primer combination or the kit in identifying heat-resistant rice.

[0019] The advantages and beneficial effects of the present invention are as follows: the present invention provides a detection primer and a kit for rapidly identifying excellent haplotypes of TT2; based on the SNP difference at position 1637 of the TT2 gene, four specific primers suitable for the ARMS-PCR amplification method are designed, which can efficiently and accurately distinguish the A / A heat-resistant type, the C / C heat-intolerant type and the A / C heterozygous type thereof; TT2 genotype identification is performed on 28 materials in rice, and it is found that excellent heat-resistant haplotype bands can be accurately distinguished; the detection method of the present invention is low in cost, simple in operation, and green and environmentally friendly; different band types can be clearly distinguished in agarose gel without mixed bands; the detection method of the present invention plays a role in molecular assisted selection in the current heat-resistant breeding process of rice, and can effectively shorten the breeding period. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0021] Figure 1 The results of the detection of different SNP polymorphisms of rice gene TT2 in Example 2 of the present invention;

[0022] Figure 2 This is the detection result of SNP polymorphism of heat-resistant gene TT2 in 28 rice varieties in Example 3 of the present invention. DETAILED DESCRIPTION

[0023] The specific implementation of the present invention is further described below in conjunction with the accompanying drawings and embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and cannot be used to limit the protection scope of the present invention.

[0024] like Figure 1-2 As shown, the present invention provides a technical solution:

[0025] Example 1: Design of excellent haplotype-specific ARMS-PCR detection primers for the heat-resistant gene TT2 in rice

[0026] 1. SNP differential site analysis

[0027] According to the report in Kan Y, Mu XR, Zhang H, Gao J, Shan JX, Ye WW, Lin HX. TT2 controls rice thermotolerance through SCT1-dependent alteration of wax biosynthesis. Nat Plants. 2022 Jan; 8(1): 53-67., the heat resistance of the TT2 gene is caused by the mutation of base 1637 of the genome from C to A, which is a gain-of-function mutation.

[0028] 2. ARMS-PCR Primer Design

[0029] Primer design is based on the C / A polymorphism located at the 1637th base of the TT2 genome, and the homozygous A / A type represents a heat-resistant favorable genotype. The primer combination includes primer P1, primer P2, primer P3 and primer P4, wherein primer P1 and primer P4 are located upstream and downstream of the SNP, the last base at the 3' end of primer P2 is the complementary base G of the C type at the 1637th base of the TT2 gene, and the last base at the 3' end of primer P3 is the A type base at the 1637th base of the TT2 gene;

[0030] The specific nucleotide sequences of each primer combination are as follows:

[0031] Primer P1, SEQ ID NO: 1: 5′-ATTAACCAGTGGCTGCGTCA-3′;

[0032] Primer P2, SEQ ID NO: 2: 5′-CAGGCTGGCTTACTCTCTG-3′;

[0033] Primer P3, SEQ ID NO:3: 5′-CTGCCTCCAGATGCTGA-3′;

[0034] Primer P4, SEQ ID NO:4: 5'-ACACTCAAAAAGCTTGCACGA-3'.

[0035] Example 2: Establishment of an excellent haplotype-specific ARMS-PCR detection method for the heat-resistant gene TT2 in rice

[0036] 1. ARMS-PCR reaction program was: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 40 s, 35 cycles; extension at 72°C for 5 min; cooling at 4°C for 10 min.

[0037] 2. The ARMS-PCR reaction system is 20 μL in total, including 4 μL DNA polymerase buffer, 2 μL MgCl2 solution (25 mM), 0.4 μL dNTP, 0.1 μL DNA polymerase, 0.4 μL P1 primer, 0.4 μL P2 primer, 0.2 μL P3 primer, 0.2 μL P4 primer, 1.6 μL genomic DNA, and finally add ddH2O to make up the ARMS-PCR to 20 μL.

[0038] When the addition ratio of primer P1: primer P2: primer P3: primer P4 is 2:2:1:1, uniform band brightness and no mixed bands can be achieved.

[0039] 3. Band type judgment

[0040] The method for determining the TT2 genotype is as follows: if the size of the amplified product is 570 bp and the band is single, it is determined that the rice sample to be tested contains homozygous C / C bases, representing a non-heat-resistant genotype; if the size of the amplified product is 323 bp and the band is single, it is determined that the rice sample to be tested contains homozygous A / A bases, representing a heat-resistant genotype; if the amplified product contains both 323 bp and 570 bp bands, it is determined that the rice sample to be tested contains heterozygous A / C bases, representing that the TT2 gene is in a heterozygous state. For specific typing, see Figure 1 .

[0041] Case study 3: Screening and validation of detection primers in 28 rice materials

[0042] In order to verify the practicability of the detection primers, 28 rice materials with known TT2 genotypes were used for verification, such as Figure 2 As shown, the detection primer combination can clearly distinguish between excellent haplotypes and non-excellent haplotypes. The results show that only five of the 28 rice samples did not carry the excellent haplotype of TT2, and 23 rice samples carried the excellent haplotype of TT2. The detection results are accurate and reliable, and are completely consistent with the expected results, indicating that the TT2 genotype detection method of the present invention is fully suitable for the molecular assisted selection of heat-resistant rice breeding at this stage.

[0043] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A detection primer combination for SNP molecular markers of rice heat-resistant gene TT2, characterized in that: The primer combination includes primer P1, primer P2, primer P3 and primer P4; The nucleotide sequence of the primer P1 is shown in SEQ ID NO: 1; the nucleotide sequence of the primer P2 is shown in SEQ ID NO: 2; the nucleotide sequence of the primer P3 is shown in SEQ ID NO: 3; and the nucleotide sequence of the primer P4 is shown in SEQ ID NO:

4.

2. The detection primer combination for the SNP molecular marker of the rice heat-resistant gene TT2 according to claim 1, characterized in that: The primers P1 and P4 are located upstream and downstream of the SNP, respectively. The last base at the 3' end of the primer P2 is the C-type complementary base G at position 1637 of the TT2 gene. The last base at the 3' end of the primer P3 is the A-type base at position 1637 of the TT2 gene.

3. The detection primer combination for the SNP molecular marker of the rice heat-resistant gene TT2 according to claim 1, characterized in that: The addition ratio of primer P1:primer P2:primer P3:primer P4 in the primer combination is 2:2:1:

1.

4. A kit for detecting the heat-resistant gene TT2 in rice, characterized in that: The kit comprises the primer combination according to any one of claims 1 to 3.

5. A method for detecting the heat-resistant gene TT2 in rice, characterized in that: The steps include: S1. Extracting genomic DNA of rice to be tested; S2. Using the primer combination described in any one of claims 1 to 3, and taking the genomic DNA of the rice to be tested as a template, performing ARMS-PCR amplification on it; S3. Detect the ARMS-PCR amplification products in 1% agarose gel, and determine the C / C type, A / A type and A / C type of the TT2 gene based on the band typing of the amplification products.

6. The method for detecting the heat-resistant gene TT2 of rice according to claim 5, characterized in that: The reaction procedure of the ARMS-PCR is as follows: Pre-denaturation at 95°C for 2 min; denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 40 s, 35 cycles; extension at 72°C for 5 min; cooling at 4°C for 10 min.

7. The method for detecting the heat-resistant gene TT2 of rice according to claim 5, characterized in that: The ARMS-PCR reaction system is 20 μL in total, and the specific components are as follows: It includes 4μL DNA polymerase Buffer, 2μL 25mM MgCl2 solution, 0.4μL dNTP, 0.1μL DNA polymerase, 0.4μL P1 primer, 0.4μL P2 primer, 0.2μL P3 primer, 0.2μL P4 primer, 1.6μL genomic DNA, and finally add ddH2O to make up the ARMS-PCR to 20μL.

8. Use of the primer combination according to any one of claims 1 to 3 or the kit according to claim 4 in identifying heat-resistant rice.

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