Regulation of Heat Tolerance of Rice Heading Period and Application of Molecular Marker

Abstract

The application discloses a main-effect QTL for regulating heat-resistance of rice heading stage, molecular markers and application, belongs to the field of rice breeding and molecular biology technology. qHTC-5.1 The main-effect QTL is named as qHTC- 5.1 The main-effect QTL site is located on the 5th chromosome, the genetic distance is 72.24-82.66 cM, the physical distance is 16852357-19308227 bp, and the LOD value is as high as 3.74. The molecular markers Indel htc-1 and the molecular markers Indel htc-2 are set on the upstream and downstream of the QTL site respectively. The molecular markers are applied to breeding rice with strong heat-resistance to high temperature, and the breeding efficiency is greatly improved.

Description

Technical Field

[0001] This invention relates to the fields of rice breeding and molecular biology, and more specifically to the major QTLs and molecular markers and their applications for regulating heat tolerance during the heading stage of rice. Background Technology

[0002] Rice ( Oryza sativa L. (a type of corn) is a major grain crop in my country, serving as the primary food source for approximately 65% ​​of the country's population, and its planting area accounts for 35% of the total grain planting area nationwide. [1] As global warming intensifies, the frequency, duration, and severity of extreme heat events are likely to increase significantly, having a sustained negative impact on rice cultivation and thus seriously threatening sustainable agricultural development and global food security. [2] High temperatures and heat stress affect rice throughout its entire growth cycle, with the heading stage being the most sensitive period. High temperatures during panicle differentiation can lead to floret degeneration, poor panicle development, and pollen sterility. Heat stress during flowering can result in poor grain filling, impaired grain setting, reduced grain weight accumulation, and increased grain chalkiness. [3-5] Therefore, in-depth analysis of the genetic mechanism of heat tolerance during the heading stage of rice and the discovery of related regulatory genes are of great significance for breeding high-yield, heat-tolerant new rice varieties, thereby ensuring rice production under high-temperature stress.

[0003] To date, previous studies have made some progress in understanding the genetic mechanisms of heat tolerance during the heading stage of rice. (Chen et al.) [6] Using heat-tolerant rice interfering genes (RILs) constructed from a cross between the heat-tolerant variety 'T226' and the susceptible variety 'T219', QTL loci for heat tolerance during the rice flowering period were mapped, identifying a total of 7 related QTLs. Among them, the gene for strong heat tolerance originated from a major QTL. qHt3 Its LOD value is as high as 13.6. (Zhang et al.) [7] QTLs discovered using NIL populations TT3 Further research revealed that under heat stress, the E3 ubiquitin ligase, which is regulated and located in the cytoplasmic membrane, translocates to the endosome. It then mediates vacuolar degradation by ubiquitination of chloroplast precursor proteins, protecting thylakoids from heat stress and reducing the damage to chloroplasts caused by heat stress.

[0004] With the development of molecular biology techniques and map-based cloning, the functions and mechanisms of action of genes regulating heat tolerance in rice have gradually become clear. (Li et al.) [8] A major gene encoding a 26S proteasome α2 subunit was successfully isolated and cloned using a backcross population. OgTT1 This can promote the degradation rate of ubiquitinated substrates by the proteasome in cells at high temperatures, thereby reducing the types and quantities of toxic denatured proteins accumulated in cells under high-temperature stress and alleviating cell death caused by high temperatures. (Liu et al.)[9] Discover OsNTL3 Overexpression of an NAC transcription factor that encodes a C-terminal transmembrane domain OsNTL3 In response to heat stress and endoplasmic reticulum stress inducers, migration from the plasma membrane to the nucleus significantly improved the heat tolerance of rice seedlings. Mechanistic studies have found that... OsNTL3 direct combination OsbZIP74 The promoter regulates its expression under thermal stress, and OsNTL3 Upregulation under heat stress depends on OsbZIP74 This reveals the origin of OsNTL 3 and OsbZIP74 Mediated regulatory circuits and their signal responses between the endoplasmic reticulum, plasma membrane, and nucleus under thermal stress.

[0005] Previous researchers have made some progress in identifying QTLs related to heat tolerance in rice, but related research results are rarely reported in marker-assisted breeding for heat tolerance. Furthermore, the consistency of QTLs identified in different studies is insufficient, and environmental interactions are significant, limiting their breeding application value. Therefore, integrating multi-dimensional omics data to accurately locate stable major-effect QTLs and efficiently screen and validate key candidate genes has become a core task in overcoming the bottlenecks in molecular breeding for heat tolerance.

[0006] Therefore, how to provide a major QTL, molecular marker and application for regulating heat resistance during the heading stage of rice is a technical problem that urgently needs to be solved by those skilled in the art.

[0007] The references mentioned above are as follows: 1 Han Q, Chen YF, Liu XY, et al. Quality attributes of paddy rice during storage as affected by accumulated temperature[J]. Frontiers inNutrition, 2024, 10(3): 1337110. 2 Fang Wenying, Chen Jiaqi, Chu Daiwei, et al. Characteristics and control measures of high temperature heat damage during flowering and grain filling period of single-season rice [J]. Chinese Rice, 2024, 30(04): 98-100. 3 Huang D, Zhang Z, Fan Y, et al. Detection of QTL for High-Temperature Tolerance in Rice Using a High-Density Bin Map [J]. Agronomy, 2023, 13(6): 1582. 4 Nakamura S, Satoh A, Aizawa M, et al. Characteristics ofPhysicochemical Properties of Chalky Grains of Japonica Rice Generated byHigh Temperature during Ripening [J]. Foods, 2021, 11(1): 97. 5 Zhang C X, Fu G F, Yang X Q, et al. Heat Stress Effects areStronger on Spikelets Than on Flag Leaves in Rice Due to Differences inDissipation Capacity [J]. Journal of Agronomy and Crop Science, 2015, 202(5):394-408. 6 CHEN Q Q, YU S B, LI C H. Identification of QTLs for heat toleranceat flowering stage in rice[J]. Scientia Agricultura Sinica, 2008, 41(2): 315-321. 7 Zhang H, Zhou J F, Kan Y, et al. A genetic module at one locus inrice protects chloroplasts to enhance thermotolerance[J].Science, 2022, 376(6599):1293-1300. 8 Li X M, Chao D Y, Wu Y, et al. Natural alleles of a proteasome α2subunit gene contribute to thermotolerance and adaptation of African rice[J].Nature Genetics, 2015, 47(7): 827-833. 9 Liu X, Lyu Y, Yang W, et al. A membrane-associated NACtranscription factor OsNTL3 is involved in thermotolerance in Rice[J]. PlantBiotechnology Journal, 2019, 18(5): 1317-1329. Summary of the Invention

[0008] In view of this, the present invention provides major QTLs and molecular markers for regulating heat tolerance during the heading stage of rice, and their applications.

[0009] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: The major-effect QTL regulating heat tolerance at the heading stage of rice is located on chromosome 5 of rice and is named... qHTC- 5.1 The genetic distance is 72.24~82.66 cM, and the physical distance is 16852357~19308227 bp.

[0010] When 50% of the rice plants in the experimental field begin to head (the middle of the young panicle emerges from the flag leaf sheath), the rice is considered to have entered the heading stage. This invention defines heat resistance by a heat resistance coefficient, i.e., heat resistance coefficient = seed setting rate of the heat stress group / seed setting rate of the control group (natural high-temperature treatment or artificial heat treatment can be performed). The higher the heat resistance coefficient, the stronger the heat resistance, and vice versa.

[0011] The molecular markers for the major QTLs regulating heat tolerance at the heading stage of rice mentioned above include two closely linked pairs of molecular markers, Indel htc-1 and Indel htc-2; wherein, The primer pair for the molecular marker Indel htc-1 is: Upstream primer htc-1-F: 5'-ATGGCGCGGGCTAACTAAG-3', SEQ ID NO.1; Downstream primer htc-1-R: 5'-TGCGTTGGTACAGAAGGGAC-3', SEQ ID NO.2; The primer pair for the molecular marker Indel htc-2 is: Upstream primer htc-2-F: 5'-CGCTTTATGGATGGGAACGC-3', SEQ ID NO.3; Downstream primer htc-2-R: 5'-TTCTGCATGAGCCATGTGGA-3', SEQ ID NO.4.

[0012] The above-mentioned major QTLs for regulating heat tolerance during the heading stage of rice are applied in the breeding of new heat-tolerant rice varieties.

[0013] The application of the above-mentioned molecular markers in the breeding of new heat-tolerant rice varieties.

[0014] A method for breeding heat-resistant rice, the process of which is as follows: Rice DNA was extracted, and PCR amplification was performed on the DNA using the primer pairs of the molecular markers Indel htc-1 and Indel htc-2 mentioned above. The amplification products were detected by electrophoresis, and the heat resistance of rice was analyzed by banding.

[0015] Furthermore, The PCR amplification reaction system consisted of: 2 μL of 10 μmol / L upstream primer, 2 μL of 10 μmol / L downstream primer, 1 μL of DNA template, and 6 μL of Mixase.

[0016] Furthermore, The PCR amplification reaction program was as follows: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 8 s, 32 cycles of amplification, and a final extension at 72℃ for 5 min.

[0017] A breeding kit for rice with strong heat resistance at the heading stage includes primer pairs of the aforementioned molecular markers Indel htc-1 and Indel htc-2.

[0018] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects: This invention uses 120 recombinant inbred lines obtained by continuous self-pollination of the F1 generation of a hybrid rice variety, Huazhan (male parent), and Reyan 2 (female parent). A high-density genetic map of this population was used for QTL mapping analysis, detecting a QTL with a high LOD value of 3.74. Molecular marker-assisted breeding technology can effectively address the problem of incomplete understanding of related genes. By constructing genetic linkage maps and analyzing quantitative trait loci (QTLs), molecular markers tightly linked to major QTLs related to heat tolerance at the heading stage of rice can be effectively identified. These molecular markers can be used to screen rice offspring, significantly improving breeding efficiency while saving costs. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0020] Figure 1 This is a flowchart of the genetic material construction process used in the major-effect QTL mapping of rice heading stage heat resistance in Example 1 of the present invention.

[0021] Figure 2 The frequency distribution of heat resistance characteristics of the RILs population in Example 1 of this invention is shown, where A is the frequency distribution of fruit set rate in 2023, B is the frequency distribution of fruit set rate in 2024, C is the frequency distribution of fruit set rate in 2025, D is the frequency distribution of heat resistance coefficient I, and E is the frequency distribution of heat resistance coefficient II.

[0022] Figure 3 The main QTL for regulating heat tolerance during the heading stage of rice in Example 1 of this invention is... qHTC-5.1 Its location on chromosome 5.

[0023] Figure 4 This is an electrophoresis diagram of the primer pair of molecular marker Indel htc-1 amplified in the parents, their F1 generation, and the RILs population in Example 2 of the present invention. In the diagram, 1 is Reyan 2, 2 is Huazhan, 3 is the F1 generation of the Huazhan / Reyan 2 hybrid, 4-9 are heat-resistant lines in the RILs population of the Huazhan / Reyan hybrid combination, and 10-11 are heat-sensitive lines in the RILs population of the Huazhan / Reyan hybrid combination.

[0024] Figure 5 This is an electrophoresis diagram of the primer pair of molecular marker Indel htc-2 in Example 2 of the present invention, amplified in the parents and their F1 generation and RILs population. In the diagram, 1 is Reyan 2, 2 is Huazhan, 3 is the F1 generation of the Reyan 2 / Huazhan hybrid, 4-9 are heat-resistant lines in the RILs population of the Reyan 2 / Huazhan hybrid combination, and 10-11 are heat-sensitive lines in the RILs population of the Huazhan / Reyan hybrid combination.

[0025] Figure 6 This is an electrophoresis diagram of the primer pair of molecular marker Indel htc-1 in Example 3 of the present invention, amplified in the parents and their BC3F1 generation. In the diagram, 1 is 9311, 2 is Huazhan, 3 is the F1 generation of the 9311 / Huazhan hybrid, 4-9 are heat-resistant lines in the BC3F1 population of the 9311 / Huazhan hybrid, and 10-11 are heat-sensitive lines in the BC3F1 population of the 9311 / Huazhan hybrid.

[0026] Figure 7 This is an electrophoresis diagram of the primer pair of molecular marker Indel htc-2 in Example 3 of the present invention, amplified in the parents and their BC3F1 generation. In this diagram, 1 is 9311, 2 is Huazhan, 3 is the F1 generation of the 9311 / Huazhan hybrid, 4-9 are heat-resistant lines in the BC3F1 population of the 9311 / Huazhan hybrid, and 10-11 are heat-sensitive lines in the BC3F1 population of the 9311 / Huazhan hybrid. Detailed Implementation

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Example 1

[0029] QTL mapping of the main effect of regulating heat tolerance during heading stage in rice

[0030] 1. Acquisition of experimental materials Using Huazhan as the donor parent and the local rice variety Reyan 2 as the recipient parent, hybridization was carried out to obtain the F1 generation. The F1 generation was then subjected to 12 consecutive generations of bagged self-pollination using the single-seed method (the offspring lines did not exhibit phenotypic segregation), ultimately yielding 120 stable genetic lines (F1). 12 All strains are phenotypically stable, such as Figure 1 As shown.

[0031] Select parent lines and seeds of each strain (F) 12 Sixty seeds of each parent were selected and disinfected (first treated with 70% ethanol for 1 minute, then with 10% NaClO for 30 minutes, and finally rinsed several times with deionized water). The seeds were then wrapped in a damp towel and placed in a 37℃ constant temperature incubator for 2 days to germinate (the water was changed once a day during this period). Seeds with uniform white sprouts were selected and sown in the seedbed. After 30 days, 24 seedlings of each parent line and each strain with good growth were selected and transplanted. All rice materials were planted in the experimental field of the College of Biochemistry, Zhejiang Normal University, Jinhua City, Zhejiang Province (119.5666°E, 29.0896°N), with a plant spacing of 20cm×20cm and 4 rows and 6 columns per strain. Conventional water and fertilizer management was carried out, including regular weeding, pest control and fertilization, until the seedlings reached the heading stage.

[0032] 2. Determination of heat resistance during the heading stage of rice Based on years of meteorological data, Jinhua typically experiences high temperatures from late July to early August each year. When the average daily temperature reaches or exceeds 32°C, it meets the requirements for high-temperature stress in rice. By adjusting the transplanting time, the heading period of the experimental materials was concentrated within the high-temperature period from July 20th to August 20th each year. Taking 2023-2025 as an example, the rice transplanting dates for 2023, 2024, and 2025 were June 12th, June 17th, and June 24th, respectively. Meteorological data shows that 2024 was an extreme high-temperature year, with 21 days of average daily temperature exceeding 32°C and a monthly average temperature of 32.51°C, thus classifying it as a high-temperature year. 2023 and 2025 had 0 and 12 days exceeding 32°C, respectively, and can be classified as relatively low-temperature years. Each year, three typical plants with no obvious marginal effect are selected from each line, and their main panicle seed setting rate is statistically analyzed. The heat resistance coefficient of each rice line under high temperature is also calculated.

[0033] Seed setting rate = number of filled grains per ear / total number of grains per ear × 100%.

[0034] Heat resistance coefficient = annual fruit setting rate at high temperature / annual fruit setting rate at low temperature, i.e.: Heat resistance coefficient I = 2024 fruit set rate / 2023 fruit set rate.

[0035] Heat resistance coefficient II = 2024 fruit set rate / 2025 fruit set rate.

[0036] The results are as follows Figure 2 As shown, the data on heat tolerance during the heading stage of rice exhibit a normal distribution and a wide range, demonstrating the genetic characteristics of quantitative traits.

[0037] 3. QTL Positioning Analysis Genetic map constructed using high-density SNPs and Indel markers previously developed in the laboratory

[10] Quantitative trait loci (QTL) mapping was performed on heat tolerance at the heading stage of rice. The relationship between markers and quantitative trait phenotypic values ​​across the entire chromosome set was analyzed using R-QTL software. Each QTL was located at its corresponding linkage group, and its genetic effect was estimated. If a molecular marker with a LOD > 2.0 was detected, it was considered that one QTL existed between the two markers corresponding to the highest LOD value.

[0038] Ultimately, we identified a major-effect QTL located on chromosome 5 between the Indel htc-1 and Indel htc-2 markers. This QTL was mapped across multiple years of the heat tolerance trait, with a high LOD value of 3.74. Its genetic distance ranged from 72.24 to 82.66 cM, and its physical distance from 16,852,357 to 19,308,227 bp. We named it... qHTC-5.1 ,like Figure 3As shown.

[0039] The references mentioned above are as follows: 10 Chen Jun, Xu Jiangmin, Zhou Yinan, et al. Discovery of QTLs and candidate gene analysis for resistance to bacterial blight in rice [J]. Acta Botanica Sinica, 2025, 60(5): 831-845.

[0040] Example 2

[0041] Molecular marker-assisted selection

[0042] At QTL sites qHTC-5.1 Molecular markers Indel htc-1 and Indel htc-2 were set upstream and downstream, respectively, and primers were designed.

[0043] The primer pair for the molecular marker Indel htc-1 is: Upstream primer: 5'-ATGGCGCGGGCTAACTAAG-3', SEQ ID NO.1; Downstream primer: 5'-TGCGTTGGTACAGAAGGGAC-3', SEQ ID NO.2.

[0044] The primer pair for the molecular marker Indel htc-2 is: Upstream primer: 5'-CGCTTTATGGATGGGAACGC-3', SEQ ID NO.3; Downstream primer: 5'-TTCTGCATGAGCCATGTGGA-3', SEQ ID NO.4.

[0045] The detection method is as follows: Rice leaves from the parental varieties Huazhan and Reyan 2, as well as the test lines, were collected, and genomic DNA was extracted. The genomic DNA was then amplified by PCR using the aforementioned molecular markers.

[0046] PCR reaction system: 2 μL upstream primer (10 μmol / L), 2 μL downstream primer (10 μmol / L), 1 μL DNA template, 6 μL Mixase.

[0047] The reaction procedure was as follows: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 8 s, amplification for 32 cycles, and finally 72℃ final extension for 5 min.

[0048] PCR amplification products were detected by 4% agarose gel electrophoresis. Analysis of the electrophoretic bands showed that both Indel HTC-1 and Indel HTC-2 bands tended towards the parental line Huazhan (HTC-1: approximately 245 bp; HTC-2: approximately 225 bp), indicating strong heat tolerance at the heading stage of this rice line. Conversely, if both bands tended towards Reyan 2 (HTC-1: approximately 240 bp; HTC-2: approximately 230 bp), it indicated poor heat tolerance at the heading stage of this rice line.

[0049] Specifically: Six phenotypically stable and heat-tolerant lines (natural high-temperature stress or artificial heat stress of 38℃ during the heading stage) and two phenotypically stable and heat-sensitive lines (heat stress ≤0.40, determined by natural high-temperature stress during the heading stage and artificial heat stress at 38℃ during the heading stage, and the seed setting rate of the heat-stressed group and the control group were calculated after maturity; if the heat stress coefficient was ≥0.80, the line was rated as heat-tolerant) and two heat-sensitive lines were selected from the RILs population of the parental lines Reyan 2, Huazhan and their F1 generation, and the Reyan 2 / Huazhan hybrid combination were rated as heat-tolerant lines) and the control group were selected as heat-sensitive lines. DNA was extracted from these lines and then PCR amplification was performed using primer pairs Indel htc-1 and Indel htc-2.

[0050] pass Figure 4 and Figure 5 It can be seen that in the RILs population of the Reyan 2 / Huazhan hybrid, the bands of 6 lines are biased towards Huazhan, indicating the retention of the excellent trait of strong heat resistance, while the bands of 2 lines are biased towards Reyan 2, indicating the retention of the heat-sensitive trait. Comparing the heat resistance of the tested rice lines with the results predicted by the above band analysis, the bands of the heat-resistant lines in the population all tend towards Huazhan, while the bands of the heat-sensitive lines all tend towards Reyan 2, showing that the predicted results are consistent with the actual test results.

[0051] Example 3

[0052] Application of QTLs related to heat tolerance at rice heading stage in rice breeding

[0053] The molecular markers set in Example 2 can be applied to molecular-assisted breeding of rice. The specific implementation method is as follows: Other rice varieties with poor heat tolerance, such as 9311, are crossed with Huazhan to obtain the corresponding F1 generation. Backcrossing is then performed using 9311 as the recurrent parent until the BC3F1 generation. DNA from some individual plants in the BC3F1 generation is extracted, and then PCR amplification is performed using primer pairs Indel htc-1 and Indel htc-2. The PCR amplification products are detected by 4% agarose gel electrophoresis. Analysis of the band patterns shows that both Indel htc-1 and Indel htc-2 bands tend towards the parent Huazhan, indicating that this rice line has strong heat tolerance at the heading stage. Using this method for screening and targeted selection, rice varieties with strong heat tolerance at the heading stage and retaining the excellent traits of 9311 can be obtained, greatly improving breeding efficiency.

[0054] In this laboratory, the heat-resistant rice variety 9311 was backcrossed with Huazhan using this molecular marker. Using the aforementioned method, targeted selection was performed, resulting in six different rice progeny lines with strong heat resistance at the heading stage and retaining the superior traits of 9311, as well as two rice progeny lines with weaker heat resistance. Figure 6 and Figure 7 It can be seen that the bands of the tested lines 4-9 are biased towards Huazhan, indicating that they retain the excellent trait of strong heat resistance; the bands of the tested lines 10-11 are biased towards Reyan 2, indicating that their genetic background is closer to Reyan 2 and their heat resistance is weaker.

[0055] Eight different rice progeny lines selected above were subjected to 38℃ heat stress in a constant temperature incubator during the heading stage, with a 28℃ control group. After maturity, the seed setting rate of the heat stress group and the control group were counted, and the heat tolerance coefficient was calculated. The seed setting rate of rice lines with stripes 4-9 under heat stress was 74.55-96.21%, and the heat tolerance coefficient was 0.81-0.95. The seed setting rate of rice lines with stripes 10-11 under heat stress was 12.83% and 29.17%, respectively, and the heat tolerance coefficient was 0.16 and 0.37, respectively.

[0056] In summary, the major-effect QTL for regulating heat tolerance at the heading stage of rice in this invention can effectively increase the heat tolerance of rice. During the breeding process, it can effectively enhance the heat tolerance of rice and accelerate the optimization of rice varieties. Furthermore, in the process of molecular-assisted breeding of rice, it can cultivate rice varieties with stronger heat tolerance, assist in the early screening of heat-tolerant rice, and optimize rice quality and yield. This method is simple, safe, and effective, beneficial to improving the economic value of rice varieties, balancing economic and ecological benefits, and suitable for large-scale promotion and application.

[0057] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0058] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A major-effect QTL for regulating heat tolerance during the heading stage of rice, characterized in that, The major-effect QTL is located on chromosome 5 of rice and is named qHTC-5.1 The genetic distance is 72.24~82.66 cM, and the physical distance is 16852357~19308227 bp.

2. The molecular marker for the major-effect QTL regulating heat tolerance at the heading stage of rice as described in claim 1, characterized in that, The molecular markers include two closely linked pairs of molecular markers, Indel htc-1 and Indel htc-2; wherein... The primer pair for the molecular marker Indel htc-1 is: Upstream primer htc-1-F: 5'-ATGGCGCGGGCTAACTAAG-3', SEQ ID NO.1; Downstream primer htc-1-R: 5'-TGCGTTGGTACAGAAGGGAC-3', SEQ ID NO.2; The primer pair for the molecular marker Indel htc-2 is: Upstream primer htc-2-F: 5'-CGCTTTATGGATGGGAACGC-3', SEQ ID NO.3; Downstream primer htc-2-R: 5'-TTCTGCATGAGCCATGTGGA-3', SEQ ID NO.

4.

3. The application of the major-effect QTL for regulating heat tolerance during the heading stage of rice as described in claim 1 in the breeding of new heat-tolerant rice varieties.

4. The application of the molecular marker described in claim 2 in the breeding of new heat-resistant rice varieties.

5. A method for breeding heat-resistant rice, characterized in that, The process is as follows: Rice DNA was extracted, and the DNA was amplified by PCR using the primer pair of molecular markers Indel htc-1 and Indel htc-2 as described in claim 2. The amplification products were detected by electrophoresis, and the heat resistance of rice was analyzed by banding.

6. The method as described in claim 5, characterized in that, The PCR amplification reaction system consisted of: 2 μL of 10 μmol / L upstream primer, 2 μL of 10 μmol / L downstream primer, 1 μL of DNA template, and 6 μL of Mixase.

7. The method as described in claim 5, characterized in that, The PCR amplification reaction program was as follows: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 8 s, 32 cycles of amplification, and a final extension at 72℃ for 5 min.

8. A breeding kit for rice with strong heat resistance at the heading stage, characterized in that, The primer pair includes the molecular markers Indel htc-1 and Indel htc-2 as described in claim 2.

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