CN113930539A - Development and application of KASP marker of rice high-temperature-resistant gene TT1 - Google Patents

Abstract

The invention discloses KASP marker development and application of a rice high temperature resistant gene TT1, and belongs to the technical field of gene biology. The molecular marker is KASP _ TT1 closely linked with a rice high temperature resistant gene TT1, the KASP _ TT1 is an SNP site in a rice genome, the nucleotide type is A or G, and the nucleotide is the 101 th nucleotide of SEQ ID No.4 in a sequence table. The invention applies KASP technology to carry out genotype identification on the rice high-temperature resistant gene TT1, has the advantages of simple and convenient operation, low cost, short detection period, stable marking, environmental protection and the like, can accurately detect the rice high-temperature resistant gene TT1, and has important significance for promoting high-temperature resistant rice breeding.

Description

Development and application of KASP marker of rice high-temperature-resistant gene TT1

Technical Field

The invention relates to development and application of KASP marker of rice high temperature resistant gene TT1, belonging to the technical field of gene biology.

Background

Rice (Oryza sativa) is an important food crop in China, and the planting area of the rice (Oryza sativa) accounts for 30% of the total planting area of the food crops. The rice is not only staple food of people in China, but also important industrial processing raw materials in China, and is commonly used for preparing starch, brewing wine, vinegar and medicines; meanwhile, the rice straw is good feed, paper making raw material and weaving material (Zhang Zhibo. shallow talk about importance of rice planting [ J ] am technical and scientific ten-year of peasant family, 2014,005: 112-. Therefore, the rice has important strategic significance for national civilian guarantee, and the stable yield and the yield improvement of the rice are important directions for national research.

In recent years, high temperature thermal damage has become an important natural disaster affecting rice production, and the middle and lower reaches of Yangtze river as main rice production areas also become serious disaster areas of high temperature thermal damage. The cultivation of high temperature resistant rice is an important way to solve the problem, the high temperature resistance selection of a target plant is carried out from the DNA level in the rice seedling stage by utilizing the high temperature resistant gene linked molecular marker, the defects in the traditional breeding are overcome, and the method is an effective way for improving the breeding efficiency of the high temperature resistant rice (Tanksley et al, RFLP mapping in plant breeding: New tools for an old science.1989, Biotechnology,7: 257-. At present, part of high temperature resistant genes are excavated, such as OsTT1 gene (huxue, liu, tou cao, etc.. development and application of functional markers of rice high temperature resistant TT1 gene [ J ] molecular plant breeding, 2019, v.17(22): 148-. The genes also develop linked molecular markers, however, although SSR markers, InDel markers or CAPS markers can be used for molecular marker-assisted selection, the detection efficiency is low, aerosol can be generated to pollute the environment, and the method is not suitable for a high-throughput molecular detection platform.

KASP (competitive Allele-Specific PCR) realizes the genotyping effect by specifically identifying gene sites through fluorescent probes, and can be used for detecting SNP sites and InDel sites. Compared with molecular markers such as SSR, RFLP, InDel and the like, the KASP marker has the characteristics of high detection speed, low cost, easiness in large-scale application and the like, does not need to be typed according to the size of a DNA fragment, can get rid of the detection method of the traditional gel electrophoresis, which has relatively complicated steps, low throughput and higher price, and is more suitable for a high-throughput molecular detection platform which is rapidly developed at the present stage. Therefore, the development of the rice grain type KASP molecular marker suitable for the high-throughput molecular detection platform has important significance for popularizing the application of the molecular marker technology and improving the breeding efficiency and the breeding level of rice in China.

Disclosure of Invention

The present invention is implemented by the following technical solutions in view of the technical problems mentioned in the background art:

the KaSP marker of the rice high-temperature resistant gene TT1 is developed, the molecular marker is the KASP marker KASP _ TT1 which is tightly linked with the rice high-temperature resistant gene TT1, the KASP _ TT1 is an SNP site in a rice genome, the nucleotide type of the SNP site is A or G, and the nucleotide type of the SNP site is the 101 th nucleotide of SEQ ID No.4 in a sequence table.

The KASP marker of the rice high temperature resistant gene TT1 is developed in C1, C2, C3 or C4, and the application thereof comprises the following steps:

the C1 is the application of a substance for detecting the polymorphism or genotype of KASP _ TT1 locus in rice genome in identification or auxiliary identification of rice high temperature tolerance;

the C2 is the application of the substance for detecting the polymorphism or genotype of KASP _ TT1 locus in the rice genome in the preparation of identification or auxiliary identification of high-temperature resistant rice products;

the C3 is the application of the substance for detecting the polymorphism or genotype of KASP _ TT1 locus in the rice genome in rice auxiliary breeding or the preparation of rice auxiliary breeding products;

the C4 is the application of the substance for detecting the polymorphism or genotype of KASP _ TT1 locus in rice genome in breeding high-temperature resistant rice resources.

The application of KASP marker development of rice high temperature resistance gene TT1 provides a method for identifying or assisting in identifying rice high temperature resistance, which comprises detecting the genotype of rice to be detected, and identifying or assisting in identifying the rice high temperature resistance according to the genotype of the rice to be detected; the genotype is the genotype of KASP _ TT1 locus in the rice genome; the site KASP _ TT1 is an SNP site in a rice genome, the nucleotide type is A or G, and the site is the 101 th nucleotide of SEQ ID No.4 in a sequence table.

As a preferred example, when the genotype of the KASP _ TT1 site is AA genotype, the rice is high temperature resistant or is selected to be high temperature resistant; when the genotype of the KASP _ TT1 site is AG genotype or GG genotype, the rice is not high-temperature resistant or candidate is not high-temperature resistant; wherein, the AA genotype represents that the nucleotide type of the KASP _ TT1 site in the rice genome is homozygous for A; the GG genotype represents that the nucleotide type of the KASP _ TT1 site in the rice genome is homozygote G; the AG genotype indicates that the nucleotide type of the KASP _ TT1 site in the rice genome is a hybrid type of A and G.

The application of KASP marker development of rice high temperature resistant gene TT1 provides the application of a substance for detecting polymorphism or genotype of KASP _ TT1 locus in rice genome in A1, A2, A3 or A4; the KASP _ TT1 site is an SNP site in a rice genome, the nucleotide type is A or G, and the site is the 101 th nucleotide of SEQ ID No.4 in a sequence table:

the A1 is the application of a substance for detecting the polymorphism or genotype of KASP _ TT1 locus in rice genome in identification or auxiliary identification of rice high temperature tolerance;

the A2 is the application of the substance for detecting the polymorphism or genotype of KASP _ TT1 locus in rice genome in the preparation of identification or auxiliary identification of high temperature resistant rice products;

the A3 is the application of a substance for detecting the polymorphism or genotype of KASP _ TT1 locus in a rice genome in rice auxiliary breeding or preparation of rice auxiliary breeding products;

the A4 is the application of the substance for detecting the polymorphism or genotype of KASP _ TT1 locus in rice genome in breeding high-temperature resistant rice resources.

The application of KASP marker development of rice high temperature resistant gene TT1 provides a method for breeding high temperature resistant rice resources, which comprises the steps of selecting rice with AA as genotype of KASP _ TT1 locus for breeding, wherein the KASP _ TT1 locus is an SNP locus in a rice genome, the nucleotide type of the SNP locus is A or G, and the SNP locus is 101 th nucleotide of SEQ ID No.4 in a sequence table; the AA genotype indicates that the nucleotide type of the KASP _ TT1 site in the rice genome is homozygous for A.

The application of the KASP marker development of the rice high temperature resistant gene TT1 provides a product containing a substance for detecting the polymorphism or genotype of the KASP _ TT1 locus in a rice genome, which is any one of E1-E4:

the E1 is a product for detecting the single nucleotide polymorphism or genotype related to the high temperature tolerance of the rice;

the E2 is a product for identifying or assisting in identifying the high-temperature resistant rice;

the E3 is a product for rice auxiliary breeding;

the E4 is a product for breeding high-temperature resistant rice resources.

As a preferable example, the application of the product containing the substance for detecting the polymorphism or genotype of KASP _ TT1 site in rice genome in B1, B2, B3 or B4;

the B1 is the application of a substance for detecting the polymorphism or genotype of KASP _ TT1 locus in rice genome in identification or auxiliary identification of rice high temperature tolerance;

the B2 is the application of the substance for detecting the polymorphism or genotype of KASP _ TT1 locus in the rice genome in the preparation of identification or auxiliary identification of high-temperature resistant rice products;

the B3 is the application of the substance for detecting the polymorphism or genotype of KASP _ TT1 locus in the rice genome in rice auxiliary breeding or the preparation of rice auxiliary breeding products;

the B4 is the application of the substance for detecting the polymorphism or genotype of KASP _ TT1 locus in rice genome in breeding high-temperature resistant rice resources.

As a preferable example, the substance for detecting the polymorphism or genotype of KASP _ TT1 site in rice genome is D1, D2 or D3 as follows:

the D1 is a PCR primer for amplifying a rice genome DNA fragment including the KASP _ TT1 site, wherein the substance for detecting the polymorphism or genotype of the KASP _ TT1 site in the rice genome contains the PCR primer;

the D2 is the PCR reagent containing the PCR primer, wherein the substance for detecting the polymorphism or genotype of the KASP _ TT1 locus in the rice genome is the PCR reagent;

the D3 is a kit containing the PCR primer D1 or the PCR reagent D2.

As a preferred example, the PCR primers are P1 or P2:

p1, the PCR primer is a primer group consisting of single-stranded DNA with the nucleotide sequence of 22 th to 45 th positions of SEQ ID No.1 in the sequence table, single-stranded DNA with the nucleotide sequence of 22 nd to 45 th positions of SEQ ID No.2 in the sequence table and single-stranded DNA with the nucleotide sequence of SEQ ID No.3 in the sequence table;

p2, the PCR primer is a primer group of single-stranded DNA shown by SEQ ID No.1 in the sequence table, single-stranded DNA shown by SEQ ID No.2 in the sequence table and single-stranded DNA shown by SEQ ID No.3 in the sequence table.

The invention has the beneficial effects that: the invention provides development and application of a KASP molecular marker tightly linked with a rice high-temperature resistant gene TT1, wherein the molecular marker is the KASP marker KASP _ TT1 tightly linked with a rice high-temperature resistant gene TT 1. The marker is developed based on KASP technology, and can detect 15422542 th base of No.3 chromosome of rice genome in high flux. The invention applies KASP technology to carry out genotype identification on the rice high-temperature resistant gene TT1, has the advantages of simple and convenient operation, low cost, short detection period, stable marking, environmental protection and the like, can accurately detect the rice high-temperature resistant gene TT1, and has important significance for promoting high-temperature resistant rice breeding.

Drawings

FIG. 1 is a diagram of a labeling process development;

FIG. 2 is a chart of typing of a variety of material detected using the molecular marker KASP _ TT 1;

FIG. 3 is a graph of typing of F2 population detected using the molecular marker KASP _ TT 1.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in this specification and the appended claims, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It should also be understood that the term "and/or" as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items.

Example 1

Development of KASP marker tightly linked with rice high temperature resistant gene TT1

The development flow of KASP marker closely linked with rice high temperature resistant gene TT1 is shown in figure 1. According to literature reports, a SNP locus which is closely linked with the TT1 gene is obtained.

1. Determination of primers: the closely linked SNP site of the TT1 gene is anchored on the 15422542 th base of the rice chromosome 3 (huxue, hukul, Tuanli, Turongye, etc.. development and application of the functional marker of the rice high temperature resistant TT1 gene [ J ] molecular plant breeding, 2019, v.17(22): 148-. Flanking sequences of closely linked SNP sites of TT1 gene were downloaded from NCBI database and 3 sets of KASP primers were designed altogether using primer5.0 software. After the detection is carried out by utilizing an ArrayTape platform of Douglas Scientific company, 1 set of KASP primers with good polymorphism is selected for subsequent verification, and the primers for detecting the KASP marker tightly linked with the rice high temperature resistant gene TT1 are specifically as follows:

primer X: 5'-gaaggtcggagtcaacggattAAAGCAAGCACAACAGTATTATCA-3' (SEQ ID No.1, lower case letter portion is specific fluorescent tag sequence VIC);

primer Y: 5'-gaaggtgaccaagttcatgctAAAGCAAGCACAACAGTATTATCG-3' (SEQ ID No.2, lower case letter portion is specific fluorescent tag sequence FAM);

Primer R：5’-TTAGGATAGCACTGTAAAACTGCA-3’(SEQ ID No.3)。

the SNP site corresponding to the primer is 15422542 th base of No.3 chromosome of rice genome (corresponding to 101 th nucleotide in sequence 4 in a sequence table).

DNA extraction: extracting genome DNA from rice leaves by a conventional CTAB method.

KASP reaction test

SNP marker amplification and reaction system:

(1) and (3) detecting by using a fluorescent quantitative PCR instrument AB-Q6 Flex:

the detection reaction system of the 5-mu-L PCR fluorescence quantitative instrument comprises: 50ng of genomic DNA, and 0.07. mu.L of a Primer mixture (preferably, the ratio of the Primer mixture: 12. mu.L each of forward Primer X and Primer Y100 pmol. L-1, reverse Primer R100 pmol. L-130. mu.L, ddH2O 46. mu.L, and the same detection object can be achieved by using other reasonable ratios of the Primer mixture), 2.5. mu.L of LGC company 2 XKASP Mix (Low Rox). Editing a sample table, executing a running program and storing data according to an AB-Q6 instrument operation manual of the fluorescent quantitative PCR instrument.

The above reaction system is the preferred reaction system of AB-Q6 Flex, and other reasonable reaction systems can also achieve the same detection purpose.

(2) The selection of the ArrayTape platform from Douglas Scientific

1.6 u L PCR ArrayTape platform detection reaction system includes: genomic DNA 50 ng/. mu.L 0.8. mu.L, Primer Mix 0.03. mu.L (preferred Primer Mix ratios: 12. mu.L for each of forward Primer X and Primer Y100 pmol. L-1, reverse Primer R100 pmol. L-130. mu.L, ddH2O 46. mu.L, the same detection object can be achieved by using other reasonable Primer Mix ratios), LGC 2 XKASP Mix (Std Rox) 0.8. mu.L. According to an ArrayTape platform instrument operation manual, a sample table is compiled, a program is operated, and data are read.

The above reaction system is the preferable reaction system of the ArrayTape platform of Douglas Scientific company, and other reasonable reaction systems can achieve the same detection purpose.

Note: the above is a recommended detection method, and other detection methods capable of achieving the same detection purpose can also be applied to the molecular marker-assisted breeding process of the marker.

Wherein, the 2 XKASP Mix consists of a fluorescent probe A, a fluorescent probe B, a quenching probe A, a quenching probe B, high fidelity Taq enzyme, dNTP, Mg2+, and the like. The nucleotide sequence of the fluorescent probe A is as follows: 5'-GAAGGTCGGAGTCAACGGATT-3', connecting a VIC fluorescent group at the 5 ' end; the nucleotide sequence of the fluorescent probe B is as follows: 5'-GAAGGTGACCAAGTTCATGCT-3', wherein the 5 ' end is connected with a FAM fluorescent group; the nucleotide sequence of the quenching probe A is as follows: 5'-AATCCGTTGACTCCGACCTTC-3', wherein the 3 ' end is connected with a quenching group BHQ; the nucleotide sequence of the quenching probe B is as follows: 5'-AGCATGAACTTGGTCACCTTC-3', wherein the 3 ' end is connected with a quenching group BHQ.

4. And (3) amplification procedure: pre-denaturation at 95 ℃ for 10min for 1 cycle; denaturation at 95 ℃ for 20s, annealing at 55-62 ℃ (preferably 55 ℃) for 60s, setting 40 cycles.

The experiment was carried out while setting a blank control (NTC) without adding template DNA to the reaction system, and 1 or more blank controls were set for each plate.

Analyzing the scanning data, and then determining the genotype of the KASP _ TT1 locus in the rice genome to be detected (namely, detecting whether the 15422542 th base of the No.3 chromosome of the rice genome is A or G), wherein if the fluorescence signal data of the amplification product of the rice to be detected is analyzed to be close to the X axis (VIC signal) by Douglas genotyping software, the genotype of the KASP _ TT1 locus in the rice genome to be detected is AA homozygous (namely, the 15422542 th base of the No.3 chromosome of the rice genome is A homozygous); if the fluorescence signal data of the amplification product of the rice to be detected is analyzed to be close to a Y axis (FAM signal) through Douglas genotyping software, the genotype of the KASP _ TT1 site in the rice genome to be detected is GG homozygous (namely, the 15422542 th base of the No.3 chromosome of the rice genome is G homozygous); if the fluorescence signal data of the amplification product of the rice to be detected is located between the X axis and the Y axis (VIC and FAM signals) through Douglas genotyping software analysis, the genotype of the KASP _ TT1 site in the rice genome to be detected is an AG heterozygote (namely, the 15422542 th base of the No.3 chromosome of the rice genome is an AG heterozygote). The samples shown black in the lower left corner are blank controls.

5. Label typing data analysis

In order to verify the reliability of the KASP _ TT1 site, first, molecular marker detection was performed on 8 rice variety materials using KASP _ TT1 (FIG. 2); then, 8 rice variety materials are subjected to high temperature tolerance test to obtain phenotype data of the materials, and a phenotype identification method and an evaluation standard are shown in the literature (Hushijiao, Lepai, Turongye, etc.. development and application of functional markers of the high temperature resistant TT1 gene of rice [ J ] molecular plant breeding, 2019, v.17(22): 148-. The genotype of the material was detected using the ArrayTape platform from Douglas Scientific, and the experimental design was repeated 2 times to ensure accuracy. The amplification results showed that the KASP _ TT1 site was able to obtain stable PCR products in 8 material samples, and both A and G allelic sites were able to be detected, consistent with the disease resistance of the material (FIG. 2). Therefore, the KASP _ TT1 locus can be used for molecular marker-assisted selective breeding of the rice high-temperature resistant gene TT 1.

TABLE 1.8 phenotypic and genotypic information for test Rice varieties

Serial number

Name of breed

Phenotype

Genotype(s)

Serial number

Name of breed

Phenotype

Genotype(s)

1

CG14

T

AA

5

Yuefeng B

S

GG

2

Minghui 86

S

GG

6

Hengfeng B

S

GG

3

Mian Hui 523

S

GG

7

Ground valley

S

GG

4

Mihui 725

S

GG

8

Wu Yu Jing

S

GG

Note: in the table, 'T' indicates that the phenotype of the material is high temperature resistance; 'S' indicates that the phenotype of the material is not high temperature resistant; 'AA' represents a homozygous high temperature resistant genotype; 'GG' represents a homozygous non-refractory genotype; 'AG' represents a heterozygous non-refractory genotype; ' indicates no detection signal.

Example 2

Application of KASP marker closely linked with rice high-temperature-resistant gene TT1 in molecular marker-assisted selection of rice high-temperature-resistant plants

To examine the utility of the KASP _ TT1 site of the present invention, F1 population was obtained by crossing the refractory rice material CG14 with the non-refractory rice material Michelia angui 725, 48F 2 naturally segregating population was produced by F1 natural selfing, KASP marker detection and phenotype verification were performed on the segregating population (Table 2), and the marker detection and phenotype verification method was referred to in example 1. By performing phenotype and genotype detection and analysis on the segregating population, only 4 genotypes of 48 segregating population single plants are inconsistent with the phenotype result, and the consistency result of the marker KASP _ TT1 and the field resistance is that P is 91.67% (consistency P is the number of plants with the phenotype consistent with the genotype/total number of plants 100%), which indicates that the marker KASP _ TT1 has higher practicability in screening the rice high-temperature resistant plants.

TABLE 2 phenotypic and genotypic information for segregating populations of rice

Material numbering	Resistance to	Genotype(s)	Material numbering	Resistance to	Genotype(s)
						TT1_F2_001	S	GG	TT1_F2_025	T	AA
TT1_F2_002	S	GG	TT1_F2_026	S	AG
						TT1_F2_003	S	GG	TT1_F2_027	T	AA
TT1_F2_004	S	GG	TT1_F2_028	T	AA
						TT1_F2_005	T	AA	TT1_F2_029	S	AA
TT1_F2_006	S	GG	TT1_F2_030	S	AA
						TT1_F2_007	S	AG	TT1_F2_031	S	GG
TT1_F2_008	S	AA	TT1_F2_032	T	AA
						TT1_F2_009	S	AG	TT1_F2_033	S	AG
TT1_F2_010	S	AG	TT1_F2_034	S	AG
						TT1_F2_011	T	AA	TT1_F2_035	S	AG
TT1_F2_012	T	AA	TT1_F2_036	S	GG
						TT1_F2_013	S	AG	TT1_F2_037	S	AG
TT1_F2_014	T	AA	TT1_F2_038	S	AG
						TT1_F2_015	S	AG	TT1_F2_039	T	AA
TT1_F2_016	S	AG	TT1_F2_040	S	GG
						TT1_F2_017	T	AA	TT1_F2_041	T	AA
TT1_F2_018	S	GG	TT1_F2_042	S	AG
						TT1_F2_019	S	AG	TT1_F2_043	T	AA
TT1_F2_020	S	AG	TT1_F2_044	S	GG
						TT1_F2_021	T	AG	TT1_F2_045	S	GG
TT1_F2_022	S	GG	TT1_F2_046	S	AG
						TT1_F2_023	S	AG	TT1_F2_047	S	AG
TT1_F2_024	S	AG	TT1_F2_048	S	GG

Note: in the table, 'T' indicates that the phenotype of the material is high temperature resistance; 'S' indicates that the phenotype of the material is not high temperature resistant; 'AA' represents a homozygous high temperature resistant genotype; 'GG' represents a homozygous non-refractory genotype; 'AG' represents a heterozygous non-refractory genotype; ' indicates no detection signal.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

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<120> development of KASP marker of rice high temperature resistant gene TT1 and application thereof

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Claims (10)

1. The KASP marker development of the rice high temperature resistant gene TT1 is characterized in that: the molecular marker is KASP marker KASP _ TT1 closely linked with rice high temperature resistant gene TT1, the KASP _ TT1 is an SNP site in a rice genome, the nucleotide type is A or G, and the nucleotide is the 101 th nucleotide of SEQ ID No.4 in a sequence table.

2. The application of KASP marker development of rice high temperature resistant gene TT1 according to claim 1, wherein the KASP marker is characterized in that: use in C1, C2, C3 or C4:

the C1 is the application of a substance for detecting the polymorphism or genotype of KASP _ TT1 locus in rice genome in identification or auxiliary identification of rice high temperature tolerance;

the C2 is the application of the substance for detecting the polymorphism or genotype of KASP _ TT1 locus in the rice genome in the preparation of identification or auxiliary identification of high-temperature resistant rice products;

the C3 is the application of the substance for detecting the polymorphism or genotype of KASP _ TT1 locus in the rice genome in rice auxiliary breeding or the preparation of rice auxiliary breeding products;

the C4 is the application of the substance for detecting the polymorphism or genotype of KASP _ TT1 locus in rice genome in breeding high-temperature resistant rice resources.

3. The application of KASP marker development of rice high temperature resistant gene TT1 is characterized in that: the method for identifying or assisting in identifying the high-temperature tolerance of the rice comprises the steps of detecting the genotype of the rice to be detected, and identifying or assisting in identifying the high-temperature tolerance of the rice according to the genotype of the rice to be detected; the genotype is the genotype of KASP _ TT1 locus in the rice genome; the site KASP _ TT1 is an SNP site in a rice genome, the nucleotide type is A or G, and the site is the 101 th nucleotide of SEQ ID No.4 in a sequence table.

4. The application of KASP marker development of rice high temperature resistant gene TT1 according to claim 3, wherein the KASP marker comprises: when the genotype of the KASP _ TT1 locus is an AA genotype, the rice is high-temperature resistant or is selected to be high-temperature resistant; when the genotype of the KASP _ TT1 site is AG genotype or GG genotype, the rice is not high-temperature resistant or candidate is not high-temperature resistant; wherein, the AA genotype represents that the nucleotide type of the KASP _ TT1 site in the rice genome is homozygous for A; the GG genotype represents that the nucleotide type of the KASP _ TT1 site in the rice genome is homozygote G; the AG genotype indicates that the nucleotide type of the KASP _ TT1 site in the rice genome is a hybrid type of A and G.

5. The application of KASP marker development of rice high temperature resistant gene TT1 according to claim 3, wherein the KASP marker comprises: provides the application of a substance for detecting the polymorphism or genotype of KASP _ TT1 locus in rice genome in A1, A2, A3 or A4; the KASP _ TT1 site is an SNP site in a rice genome, the nucleotide type is A or G, and the site is the 101 th nucleotide of SEQ ID No.4 in a sequence table:

the A1 is the application of a substance for detecting the polymorphism or genotype of KASP _ TT1 locus in rice genome in identification or auxiliary identification of rice high temperature tolerance;

the A2 is the application of the substance for detecting the polymorphism or genotype of KASP _ TT1 locus in rice genome in the preparation of identification or auxiliary identification of high temperature resistant rice products;

the A3 is the application of a substance for detecting the polymorphism or genotype of KASP _ TT1 locus in a rice genome in rice auxiliary breeding or preparation of rice auxiliary breeding products;

the A4 is the application of the substance for detecting the polymorphism or genotype of KASP _ TT1 locus in rice genome in breeding high-temperature resistant rice resources.

6. The application of KASP marker development of rice high temperature resistant gene TT1 according to claim 3, wherein the KASP marker comprises: a method for breeding high-temperature-resistant rice resources is provided, comprising the steps of selecting rice with AA as the genotype of KASP _ TT1 site for breeding, wherein the KASP _ TT1 site is an SNP site in a rice genome, the nucleotide type of the SNP site is A or G, and the SNP site is the 101 th nucleotide of SEQ ID No.4 in a sequence table; the AA genotype indicates that the nucleotide type of the KASP _ TT1 site in the rice genome is homozygous for A.

7. The application of KASP marker development of rice high temperature resistant gene TT1 according to claim 3, wherein the KASP marker comprises: provides a product containing a substance for detecting polymorphism or genotype of KASP _ TT1 site in rice genome, which is any one of E1-E4:

the E1 is a product for detecting the single nucleotide polymorphism or genotype related to the high temperature tolerance of the rice;

the E2 is a product for identifying or assisting in identifying the high-temperature resistant rice;

the E3 is a product for rice auxiliary breeding;

the E4 is a product for breeding high-temperature resistant rice resources.

8. The application of KASP marker development of rice high temperature resistant gene TT1 according to claim 7, wherein the KASP marker comprises: the application of the product containing the substance for detecting the polymorphism or genotype of KASP _ TT1 locus in rice genome in B1, B2, B3 or B4;

the B1 is the application of a substance for detecting the polymorphism or genotype of KASP _ TT1 locus in rice genome in identification or auxiliary identification of rice high temperature tolerance;

the B2 is the application of the substance for detecting the polymorphism or genotype of KASP _ TT1 locus in the rice genome in the preparation of identification or auxiliary identification of high-temperature resistant rice products;

the B3 is the application of the substance for detecting the polymorphism or genotype of KASP _ TT1 locus in the rice genome in rice auxiliary breeding or the preparation of rice auxiliary breeding products;

the B4 is the application of the substance for detecting the polymorphism or genotype of KASP _ TT1 locus in rice genome in breeding high-temperature resistant rice resources.

9. The application of the KASP marker of the rice high temperature resistant gene TT1 according to any one of claims 3, 4, 5, 6, 7 or 8, wherein the KASP marker comprises: the substances for detecting the polymorphism or genotype of the KASP _ TT1 site in the rice genome are D1, D2 or D3 as follows:

the D1 is a PCR primer for amplifying a rice genome DNA fragment including the KASP _ TT1 site, wherein the substance for detecting the polymorphism or genotype of the KASP _ TT1 site in the rice genome contains the PCR primer;

the D2 is the PCR reagent containing the PCR primer, wherein the substance for detecting the polymorphism or genotype of the KASP _ TT1 locus in the rice genome is the PCR reagent;

the D3 is a kit containing the PCR primer D1 or the PCR reagent D2.

10. The application of KASP marker development of rice high temperature resistant gene TT1 according to claim 9, wherein the KASP marker comprises: the PCR primer is P1 or P2:

p1, the PCR primer is a primer group consisting of single-stranded DNA with the nucleotide sequence of 22 th to 45 th positions of SEQ ID No.1 in the sequence table, single-stranded DNA with the nucleotide sequence of 22 nd to 45 th positions of SEQ ID No.2 in the sequence table and single-stranded DNA with the nucleotide sequence of SEQ ID No.3 in the sequence table;

p2, the PCR primer is a primer group of single-stranded DNA shown by SEQ ID No.1 in the sequence table, single-stranded DNA shown by SEQ ID No.2 in the sequence table and single-stranded DNA shown by SEQ ID No.3 in the sequence table.

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