SNP molecular marker, KASP primer set, kit and application related to rice sheath blight resistance

Abstract

The application discloses a SNP molecular marker related to rice sheath blight resistance, a KASP primer set, a kit and application. With Nipponbare genome sequence as a reference genome, the SNP molecular marker is located at a 33287194bp site on a 3rd chromosome, a nucleotide sequence at the SNP site and nearby is shown as SEQ ID No. 4, a 24th nucleotide is a SNP site, and a rice variety with a genotype of TT has higher sheath blight resistance than a rice variety with a genotype of CC. The application further provides a KASP primer set for detecting the SNP molecular marker and a detection kit containing the KASP primer set. The KASP primer set or the detection kit can be used for predicting the sheath blight resistance of rice, and can be used for rice molecular marker assisted breeding, and has important application value in exploring rice germplasm resources resistant to sheath blight and breeding rice varieties resistant to sheath blight.

Description

Technical Field

[0001] This invention relates to SNP molecular markers and detection primers, particularly to SNP molecular markers related to rice sheath blight resistance, KASP detection primer sets, and their application in identifying rice resistance to sheath blight, belonging to the field of SNP molecular markers related to rice sheath blight resistance and their applications. Background Technology

[0002] Rice sheath blight, caused by *Rhizoctonia solani* Kuhn, is a significant fungal disease in global rice cultivation, severely impacting rice yield and quality. It is prevalent in rice-growing countries across Asia, Africa, Europe, and the Americas. In normal years, it can lead to yield reductions of 10%-30%, and in severe cases, up to 50%. For a long time, control of rice sheath blight has relied primarily on chemical agents and cultivation practices. Breeding and planting resistant varieties are effective means of controlling the disease. Developing molecular markers for detecting rice resistance to rice sheath blight has important applications in screening resistant rice germplasm resources and rapidly identifying resistant plants during the breeding process.

[0003] KASP (Kompetitive Allele-Specific PCR) achieves genotyping by specifically recognizing gene loci using fluorescent probes, and can be used to detect SNP and InDel loci. Compared with molecular markers such as SSR, RFLP, and InDel, KASP markers offer advantages such as rapid detection, low cost, and ease of large-scale application. KASP markers do not require genotyping based on DNA fragment size, overcoming the relatively cumbersome and low-throughput drawbacks of traditional gel electrophoresis methods, making them suitable for high-throughput molecular detection platforms. Therefore, identifying functional SNP loci associated with rice sheath blight resistance and developing KASP molecular markers suitable for high-throughput molecular detection platforms will have significant application value in improving rice breeding efficiency and quality. Summary of the Invention

[0004] One objective of this invention is to provide SNP molecular markers associated with rice resistance to sheath blight;

[0005] The second objective of this invention is to provide a KASP primer set for detecting the SNP molecular markers associated with rice resistance to sheath blight;

[0006] A third objective of this invention is to provide a detection kit containing the aforementioned KASP primer set;

[0007] The fourth objective of this invention is to apply the SNP molecular marker or KASP detection primer set or the detection kit containing the KASP primer set to the identification or auxiliary identification of rice resistance to sheath blight, or to the breeding of rice varieties resistant to sheath blight or susceptible to sheath blight.

[0008] The above-mentioned objective of this invention is achieved through the following technical solution:

[0009] One aspect of this invention provides a SNP molecular marker (SNP-33287194) associated with rice resistance to sheath blight. Using the Nipponbare genome sequence as a reference genome, this SNP molecular marker is located at 33287194 bp on chromosome 3. The nucleotide sequence at and near this SNP site is shown in SEQ ID No. 4. Specifically, the 24th nucleotide in the nucleotide sequence shown in SEQ ID No. 4 is the SNP site, exhibiting a T / C polymorphism. The genotype is TT or CC. TT is a homozygous form of the SNP site with the genotype T. Rice varieties with the SNP genotype TT are, or are candidates, rice varieties resistant to sheath blight. CC is a homozygous form of the SNP site with the genotype C. Rice varieties with the SNP genotype CC are, or are candidates, rice varieties susceptible to sheath blight. Rice varieties with the SNP genotype TT exhibit higher resistance to sheath blight than rice varieties with the SNP genotype CC.

[0010] This invention can detect the polymorphism or genotype of the SNP molecular marker (SNP-33287194) site associated with resistance to rice sheath blight using various detection methods, instruments, or reagents known to those skilled in the art, including but not limited to: DNA sequencing, restriction fragment length polymorphism, single-strand conformation polymorphism, denaturing high-performance liquid chromatography, SNP chips, and various PCR detection kits (including KASP detection kits); wherein, the SNP chip includes chips based on nucleic acid hybridization reaction, chips based on single-base extension reaction, chips based on allele-specific primer extension reaction, chips based on "one-step" reaction, chips based on primer ligation reaction, chips based on restriction endonuclease reaction, chips based on protein DNA binding reaction, and chips based on fluorescent molecule DNA binding reaction.

[0011] The present invention can also combine the substance for detecting the SNP site polymorphism and genotype with other substances (such as substances for detecting single nucleotide polymorphisms or genotypes of other molecular markers related to rice resistance to sheath blight) to prepare products for identifying rice varieties resistant to sheath blight.

[0012] Another aspect of the present invention provides a KASP primer set for detecting SNP molecular markers associated with rice resistance to sheath blight, the KASP primer set being tagged or untagged with a marker. The marker refers to any atom or molecule that can be used to provide a detectable effect and can be linked to a nucleic acid. Markers include, but are not limited to, dyes; radioactive markers, such as 32P; binding moieties, such as biotin; haptens, such as digoxigenin (DIG); luminescent, phosphorescent, or fluorescent moieties; and fluorescent dyes alone or in combination with moieties whose emission spectra can be inhibited or shifted by fluorescence resonance energy transfer (FRET). The marker can provide a signal detectable by fluorescence, radioactivity, colorimetry, gravimetric determination, X-ray diffraction or absorption, magnetism, enzyme activity, etc. The marker can be a charged moiety (positive or negative charge) or, optionally, charge-neutral. The marker can include nucleic acid or protein sequences or combinations thereof, provided that the sequence containing the marker is detectable. In some embodiments, nucleic acids are detected directly without a marker (e.g., direct sequence reading).

[0013] As a preferred embodiment of the present invention, the present invention provides a set of KASP primers for detecting SNP molecular markers related to rice resistance to sheath blight. The KASP primer set consists of two upstream primers A and B and one downstream primer C. The upstream primer A has the nucleotide sequence shown in SEQ ID No. 1 or the single-stranded DNA at positions 22-41 of the nucleotide sequence shown in SEQ ID No. 1. The nucleotide sequence shown in SEQ ID No. 1 consists of 44 nucleotides, with nucleotides 1-21 being the FAM sequence (as a marker) and nucleotides 22-41 being a specific sequence.

[0014] The nucleotide sequence of the upstream primer B is the single-stranded DNA at positions 22-42 of the nucleotide sequence shown in SEQ ID No. 2. The nucleotide sequence shown in SEQ ID No. 2 consists of 42 nucleotides, with nucleotides 1-21 being the HEX sequence (as a marker) and nucleotides 22-42 being the specific sequence.

[0015] The nucleotide sequence of the downstream primer C is shown in SEQ ID No. 3; the upstream primer A has a FAM fluorescent tag sequence at the 5' end, and the fluorescent signal of the FAM group can be read by an enzyme-linked immunosorbent assay (ELISA) reader or a real-time PCR instrument.

[0016] Another aspect of the present invention provides a detection kit for identifying or assisting in the identification of rice sheath blight resistance, comprising: a KASP primer set, a specific probe set, and a KASP HiGeno 2x Probe Mix; wherein the KASP primer set consists of two upstream primers A and B and one downstream primer C; wherein the nucleotide sequence of the upstream primer A is the nucleotide sequence shown in SEQ ID No. 1 or is single-stranded DNA at positions 22-41 of the nucleotide sequence shown in SEQ ID No. 1, the nucleotide sequence of the upstream primer B is the nucleotide sequence shown in SEQ ID No. 2 or is single-stranded DNA at positions 22-42 of the nucleotide sequence shown in SEQ ID No. 2, and the nucleotide sequence of the downstream primer C is shown in SEQ ID No. 3.

[0017] The specific probe set includes fluorescent probe A, quencher probe A, fluorescent probe B, and quencher probe B; the nucleotide sequence of fluorescent probe A is shown in SEQ ID No. 5, with a fluorescent group attached to the 5' end, preferably a FAM fluorescent group; the nucleotide sequence of fluorescent probe B is shown in SEQ ID No. 6, with a fluorescent group attached to the 5' end, preferably a HEX fluorescent group; the nucleotide sequence of quencher probe A is shown in SEQ ID No. 7, with a quencher group attached to the 3' end, preferably a BHQ gene; the nucleotide sequence of quencher probe B is shown in SEQ ID No. 8, with a quencher group attached to the 3' end, preferably a BHQ group.

[0018] The KASP HiGeno 2x Probe Mix contains high-fidelity Taq enzyme, dNTPs, and Mg. 2+ These can be purchased commercially (product of Beijing Jiacheng Biotechnology Co., Ltd. (item number AQP-001S)).

[0019] Another aspect of the present invention is to provide a method for identifying or assisting in the identification of rice sheath blight resistance, comprising:

[0020] (1) Extract genomic DNA from the rice sample to be tested;

[0021] (2) Using the SNP molecular marker (SNP-33287194) associated with rice resistance to sheath blight as the detection target, a KASP primer set was designed to establish a PCR amplification system and PCR amplification was performed.

[0022] (3) Detect the amplification products with fluorescence to determine the genotype of the SNP molecular marker (SNP-33287194) in the rice sample to be tested; if the genotype of the SNP molecular marker in the rice sample to be tested is TT, then the rice sample to be tested is or is a candidate rice resistant to sheath blight; if the genotype of the SNP molecular marker in the rice sample to be tested is CC, then the rice sample to be tested is or is a candidate rice susceptible to sheath blight.

[0023] This invention further provides a method for breeding rice varieties resistant to sheath blight, comprising:

[0024] (1) Extract genomic DNA from the rice sample to be tested;

[0025] (2) Using the SNP molecular marker (SNP-33287194) associated with rice resistance to sheath blight as the detection target, a KASP primer set was designed to establish a PCR amplification system and PCR amplification was performed.

[0026] (3) Detect the amplification product with fluorescence to determine the genotype of the SNP molecular marker (SNP-33287194) in the rice sample to be tested; if the genotype of the SNP molecular marker in the rice sample to be tested is TT, then the rice sample to be tested is or is a candidate rice variety with resistance to sheath blight.

[0027] The present invention further provides a method for breeding rice varieties susceptible to sheath blight, comprising:

[0028] (1) Extract genomic DNA from the rice sample to be tested;

[0029] (2) Using the SNP molecular marker (SNP-33287194) associated with rice resistance to sheath blight as the detection target, a KASP primer set was designed to establish a PCR amplification system and PCR amplification was performed.

[0030] (3) Detect the amplification product with fluorescence to determine the genotype of the SNP molecular marker (SNP-33287194) in the rice sample to be tested; if the genotype of the SNP molecular marker in the rice sample to be tested is CC, then the rice sample to be tested is or is a candidate rice variety susceptible to sheath blight.

[0031] As a preferred embodiment of the present invention, the PCR amplification system established in step (2) is as follows: 2 μL of genomic DNA template solution of the rice sample to be tested, 0.14 μL of KASP primer working solution, 5 μL of KASP HiGeno 2x ProbeMix, and 2.86 μL of sterile ultrapure water.

[0032] As a preferred embodiment of the present invention, the PCR amplification procedure in step (2) is as follows:

[0033] Step 1: Pre-denaturation at 95℃ for 10 min;

[0034] Step 2: 95℃ for 20s, 61℃ for 40s, 95℃ for 20s, 60.4℃ for 40s, 95℃ for 20s, 59.8℃ for 40s, 95℃ for 20s, 59.2℃ for 40s, 95℃ for 20s, 58.6℃ for 40s, 95℃ for 20s, 58℃ for 40s, 95℃ for 20s, 57.4℃ for 40s, 95℃ for 20s, 56.8℃ for 40s, 95℃ for 20s, 56.2℃ for 40s, 95℃ for 20s, 61℃ for 40s, 95℃ for 20s, 55.6℃ for 40s;

[0035] Step 3: Denaturation at 95℃ for 20 seconds, annealing at 55℃ for 40 seconds, 40 cycles.

[0036] As a preferred embodiment of the present invention, the method for determining the genotype of the SNP molecular marker (SNP-33287194) in step (3) of the rice sample to be tested is as follows: the fluorescence plate is read in a QX400 real-time fluorescence quantitative PCR instrument at a temperature of 37°C for 60s, and the fluorescence value is read from the terminal end for genotyping.

[0037] The rice sample to be tested in this invention can be any one of the following rice materials: AUSPADDY(RED)::IRGC 44978-1, LARABORO::IRGC 79342-1, AUS 295::IRGC 29083-1, AUS299::IRGC 29087-1, ARC 12067::IRGC 21881-1, AUS 329::IRGC 29116-1, LAL MOTI::IRGC 82168-1, DEVARASI::IRGC 16173-1, AUS171::IRGC 29004-1, AUS 308::IRGC 29096-1, MANSARA DHAN::IRGC 86940-1, KURULU WEE(WHITE)::IRGC 66518-1, ZARBASAIL::IRGC37346-1, ARC 12101::IRGC 21907-1, BEGMI 135::IRGC 27845-1, MADISA::IRGC 27568-1, M 142::IRGC 35054-1, ARC 14901::IRGC 41811-1, BARI SUTAR::IRGC 52410-1, KOYRA::IRGC 77267-1, LAL TAURA::IRGC 35017-1, JHUL DIGA::IRGC 26478-1, UPRH 58::IRGC61525-1, AUS 439::IRGC 29221-1, DANGAR::IRGC 76296-1, MOTIA::IRGC 28036-1, SHITTAAMAN::IRGC 37596-1, CT 58::GERVEX 1491-C1, POLIZESTI 28::GERVEX 1170-C1, JUBILIENI::GERVEX 992-C1, SANT ANDREA::IRGC 65732-1, RPC 12::GERVEX 1505-C1, SELN 244A6-20::GERVEX 1273-C1, Gongchengxiang, THAIPERLA::GERVEX 696-C1, SANGHAI::GERVEX 1264-C1, CT 23::GERVEX 1488-C1, S102::GERVEX 1671-C1, SR 113::GERVEX 553-C1, KULON::GERVEX 1473-C1, SETTANTUNO::GERVEX1279-C1、CIGALON::GERVEX 1514-C1、DELTA::GERVEX 1519-C1、68-2::IRGC 14546-1、ESCARLATE::GERVEX887-C1、Qiutianxiaoting、PLOVDIV 22::GERVEX 1167-C1、T 757::GERVEX1316-C1、AUGUSTO::GERVEX 1643-C1、MELAS::GERVEX 1684-C1、ROCCA::IRGC 50351-1、CAPATAZ::GERVEX 521-C1、M 203::IRGC 76309-1、MEJANES2::GERVEX 1560-C1。

[0038] Furthermore, the rice samples to be tested mentioned above are offspring obtained from any one or two of the following rice materials as parents: AUS PADDY(RED)::IRGC 44978-1, LARABORO::IRGC 79342-1, AUS295::IRGC29083-1, AUS299::IRGC 29087-1, ARC 12067::IRGC 21881-1, AUS 329::IRGC 29116-1, LAL MOTI::IRGC 82168-1, DEVARASI::IRGC 16173-1, AUS171::IRGC 29004-1, AUS 308::IRGC 29096-1, MANSARADHAN::IRGC 86940-1, KURULU WEE(WHITE)::IRGC 66518-1, ZARBASAIL::IRGC 37346-1, ARC 12101::IRGC 21907-1, BEGMI 135::IRGC 27845-1, MADISA::IRGC 27568-1, M 142::IRGC 35054-1, ARC 14901::IRGC 41811-1, BARI SUTAR::IRGC 52410-1, KOYRA::IRGC 77267-1, LAL TAURA::IRGC 35017-1, JHUL DIGA::IRGC26478-1, UPRH 58::IRGC 61525-1, AUS 439::IRGC 29221-1, DANGAR::IRGC 76296-1, MOTIA::IRGC 28036-1, SHITTAAMAN::IRGC 37596-1, CT 58::GERVEX 1491-C1, POLIZESTI28::GERVEX 1170-C1, JUBILIENI::GERVEX 992-C1, SANT ANDREA::IRGC 65732-1, RPC12::GERVEX 1505-C1, SELN 244A6-20::GERVEX 1273-C1, Gongchengxiang, THAIPERLA::GERVEX 696-C1, SANGHAI::GERVEX 1264-C1, CT 23::GERVEX 1488-C1, S102::GERVEX1671-C1, SR 113::GERVEX 553-C1, KULON::GERVEX 1473-C1, SETTANTUNO::GERVEX1279-C1, CIGALON::GERVEX 1514-C1, DELTA::GERVEX 1519-C1, 68-2::IRGC 14546-1, ESCARLATE::GERVEX 887-C1, Qiutianxiaoting, PLOVDIV 22::GERVEX 1167-C1, T 757::GERVEX 1316-C1, AUGUSTO::GERVEX 1643-C1, MELAS::GERVEX 1684-C1, ROCCA::IRGC50351-1, CAPATAZ::GERVEX 521-C1, M 203::IRGC 76309-1, MEJANES2::GERVEX 1560-C1.

[0039] This invention provides a SNP molecular marker (SNP-33287194) associated with rice resistance to rice sheath blight and designs a KASP primer set for detecting this SNP molecular marker, as well as a detection kit containing this KASP primer set for identifying or assisting in the identification of rice resistance to rice sheath blight. Using the KASP primer set provided by this invention or the detection kit containing this KASP primer set, it can be used to predict rice resistance to rice sheath blight, for early screening of rice varieties, and for marker-assisted breeding of rice. It has significant application value in the research of discovering rice germplasm resources resistant to rice sheath blight and breeding rice varieties resistant to rice sheath blight.

[0040] This invention relates to the definition of abbreviations and key terms.

[0041] The sheath blight described in this invention may be sheath blight caused by RH9 or other strains.

[0042] In this invention, the rice resistant to sheath blight can be rice with a relative lesion height of less than or equal to 40% in the disease resistance test.

[0043] In this invention, the rice susceptible to sheath blight can be rice with a relative lesion height of ≥75% in the disease resistance test.

[0044] SNP: Single nucleotide polymorphism.

[0045] KASP: Competitive allele-specific PCR. Attached Figure Description

[0046] Figure 1 The image shows the genotype detection results of the SNP molecular marker (SNP-33287194) associated with rice resistance to sheath blight. Detailed Implementation

[0047] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, it should be understood that the embodiments described are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but such modifications or substitutions all fall within the protection scope of the present invention.

[0048] Unless otherwise specified, the experimental methods used in the following examples or test cases are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0049] The rice materials used in the following examples or test cases have been disclosed in the reference: Wang W, Mauleon R, Hu Z, et al. Genomic variation in 3010 diverse accessions of Asian cultivated rice, Nature, 2018, 557(7703):43-49. The public can obtain the biological materials from the applicant. The biological materials are only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0050] The strain RH9 of the herbarium blight pathogen is described in the article “Pan Xuebiao, Zou Junhuang, Chen Zongxiang, et al. Molecular marker localization of major QTLs for resistance to herbarium blight in rice variety Jasmine85 [J]. Science Bulletin, 1999(15):1629-1635”, which can be obtained by the public from the Institute of Crop Science, Chinese Academy of Agricultural Sciences.

[0051] Example 1: Identification of SNP molecular markers associated with rice resistance to sheath blight

[0052] Fifty-three rice varieties were selected from the global core rice germplasm resources (Wang W, Mauleon R, Hu Z, et al. Genomic variation in 3010 diverse accessions of Asian cultivated rice, Nature, 2018, 557(7703):43-49.) that have been sequenced (average depth 14×). These varieties were inoculated with rice sheath blight strain RH9. Based on the high density of SNP genotypes, genome-wide association analysis was performed on the resistance and susceptibility phenotypes of rice sheath blight in the 563 rice varieties. Combined with linkage disequilibrium (LD) analysis, a region on chromosome 3 associated with rice sheath blight resistance was found. The SNP loci within this region that were most significantly associated with rice sheath blight resistance were selected for further analysis. Using the Nipponbare genome sequence as a reference, the SNP locus is located at 33287194 bp on chromosome 3. The nucleotide sequence of this SNP locus (abbreviated as SNP-33287194) and its vicinity is shown in SEQ ID No. 4. The 24th nucleotide is the SNP locus, exhibiting a T / C polymorphism. Rice varieties with the TT genotype at this SNP locus showed significantly higher resistance to sheath blight than rice varieties with the CC genotype at the same SNP locus.

[0053] Example 1: Establishment and Detection Results of a Method for Identifying Rice Sheath Blight Resistance Using the SNP Molecular Marker (SNP-33287194)

[0054] 1. Convert SNP markers to KASP markers and design a primer set for detecting these markers.

[0055] SNP markers were converted to KASP markers for use in marker-assisted selection breeding.

[0056] A primer set for detecting KASP markers based on KASP technology was designed, referred to as the KASP primer set. The KASP primer set consists of two upstream primers (primer A and primer B) and one downstream primer (primer C).

[0057] The nucleotide sequence of primer A is shown in Sequence 1, Sequence 1 (SEQ ID No. 1): 5'- GAAGGTGACCAAGTTCA TGCT TCGCCGATATACCTTCGGAC-3'.

[0058] The nucleotide sequence of primer B is shown in Sequence 2, Sequence 2 (SEQ ID No. 2): 5'- GAAGGTCGGAGTCAACG GATTG TCGCCGATATACCTTCGGAT-3'.

[0059] The nucleotide sequence of primer C is shown in Sequence 3.

[0060] Sequence 3 (SEQ ID No. 3): 5'-GTGAGATTTACTACCTTGGATGAGG-3'.

[0061] The specific SNP is located at nucleotide 24 of the DNA molecule shown in SEQ ID No. 4 in the rice genome.

[0062] Primer A is a primer with a FAM fluorescent tag sequence (underlined bases) at the 5' end, and primer C amplifies the fragment at the C site of the SNP. The fluorescent signal of the FAM group can be read using an ELISA reader or a real-time PCR instrument.

[0063] Primer B is a primer with a HEX fluorescent tag sequence (underlined bases) at the 5' end, and primer C amplifies the fragment at the SNP site T. The fluorescent signal of the HEX group can be read using an ELISA reader or a real-time PCR instrument.

[0064] 2. Establishment of a detection method for rice sheath blight resistance using KASP primer sets.

[0065] 2.1 Preparation of template DNA

[0066] Genomic DNA was extracted from the leaves of the rice plants to be tested and diluted to obtain a template solution. The DNA concentration in the template solution was 30-40 ng / μL.

[0067] 2.2 Perform KASP

[0068] Primer working solution: First, dilute the three primers to 100mM with ddH2O, and then prepare the primer working solution according to the following formula: Primer A 12μL, Primer B 12μL, Primer C 30μL, ddH2O 46μL.

[0069] KASP HiGeno 2x Probe Mix is ​​a product of Beijing Jiacheng Biotechnology Co., Ltd. (Catalog No. AQP-001S). KASP HiGeno 2x Probe Mix contains fluorescent probe A, fluorescent probe B, quencher probe A, quencher probe B, high-fidelity Taq enzyme, dNTPs, and Mg. 2+ wait.

[0070] The fluorescent probe A has the sequence 5'-GAAGGTGACCAAGTTCATGCT-3' (SEQ ID No. 5), with a FAM fluorescent group attached to its 5' end. The fluorescent probe B has the sequence 5'-GAAGGTCGGAGTCAACGGATT-3' (SEQ ID No. 6), with a HEX fluorescent group attached to its 5' end. The quencher probe A has the sequence 5'-AGCATGAACTTGGTCACCTTC-3' (SEQ ID No. 7), with a BHQ quencher group attached to its 3' end. The quencher probe B has the sequence 5'-AATCCGTTGACTCCGACCTTC-3' (SEQ ID No. 8), with a BHQ quencher group attached to its 3' end.

[0071] The KASP reaction system consisted of 1 μL template solution, 0.14 μL primer working solution, 5 μL KASP HiGeno 2xProbe Mix, and 3.86 μL sterile ultrapure water.

[0072] KASP was performed on a QX400 qPCR instrument using a touch-down amplification program.

[0073] KASP's response procedure:

[0074] Step 1: Pre-denaturation at 95℃ for 10 minutes;

[0075] Step 2: 95℃ for 20s, 61℃ for 40s, 95℃ for 20s, 60.4℃ for 40s, 95℃ for 20s, 59.8℃ for 40s, 95℃ for 20s, 59.2℃ for 40s, 95℃ for 20s, 58.6℃ for 40s, 95℃ for 20s, 58℃ for 40s, 95℃ for 20s, 57.4℃ for 40s, 95℃ for 20s, 56.8℃ for 40s, 95℃ for 20s, 56.2℃ for 40s, 95℃ for 20s, 61℃ for 40s, 95℃ for 20s, 55.6℃ for 40s;

[0076] Step 3: 95℃ denaturation for 20s, 55℃ annealing for 40s, 44 cycles.

[0077] The experiment also included a blank control (NTC) in the reaction system without template DNA, with one or more blank controls in each plate.

[0078] 2.3 Perform fluorescence scanning.

[0079] After completing step 2.2, the fluorescence plate was immediately read at 37°C for 60 seconds, and the fluorescence value was read from the terminal end for genotyping.

[0080] The FAM excitation wavelength is 485 nm, and the emission wavelength is 520 nm. The HEX excitation wavelength is 535 nm, and the emission wavelength is 556 nm. The system reference fluorescence ROX excitation wavelength is 575 nm, and the emission wavelength is 610 nm.

[0081] If only the HEX group shows a fluorescent signal, then the genotype of SNP-33287194 in the rice being tested is TT (i.e., SNP-33287194 in the genome is homozygous for T); if only the FAM group shows a fluorescent signal, then the genotype of SNP-33287194 in the rice being tested is CC (i.e., SNP-33287194 in the genome is homozygous for C).

[0082] If the genotype of the SNP locus in the rice being tested is TT, then the rice being tested is or is a candidate for rice resistant to sheath blight. If the genotype of the SNP locus in the rice being tested is CC, then the rice being tested is or is a candidate for rice susceptible to sheath blight. The sheath blight resistance of the rice resistant to sheath blight is higher than that of the rice susceptible to sheath blight. The rice with the genotype TT at the SNP locus has higher or is a candidate for higher resistance to sheath blight than the rice with the genotype CC at the SNP locus.

[0083] 2.4 Identification of rice sheath blight resistance

[0084] 2.4.1 Inoculate strain RH9 onto PDA solid medium and incubate at 28°C for 72 hours. Once the medium is covered with mycelia, use a 5mm punch to cut out mycelial blocks. Place the mycelial blocks into PDA medium containing 0.8–1.0 cm sterile wooden short toothpicks and incubate for 3 days until the toothpick inoculum is covered with mycelia.

[0085] 2.4.2 Fifty-four rice materials from Table 1 were selected as experimental materials. The seeds of the rice to be tested were sown in seedling trays containing nutrient soil that had been sprayed with soil fungicide. After being cultivated in a greenhouse for about 25 days, the rice was transplanted to a net house for planting. Each variety was set up with 3 replicates, with 2 rows planted in each replicate and 6 seedlings transplanted in each row.

[0086] 2.4.3 When the rice plants are in the peak tillering stage in step 2.4.2, take the toothpicks with mycelium obtained in step 2.4.1 and use the toothpick embedding method (refer to the literature: Pan Xuebiao, Zou Junhuang, Chen Zongxiang, et al. Molecular marker localization of major QTLs for resistance to sheath blight in rice variety Jasmine85 [J]. Science Bulletin, 1999(15):1629-1635) to artificially inoculate the rice plants, inoculating 5-6 tillers per plant.

[0087] 2.4.4 After completing step 2.4.3, continue cultivation for 30 days. After 30 days, measure the relative lesion height of each plant (relative lesion height (%) = lesion height / plant height * 100). For each plant, measure the lesion height of 3 inoculated tillers and the plant height. Repeat the survey for 6 plants. Take the average value of the relative lesion height.

[0088] According to the IRRI (2002) standard evaluation system for rice, rice varieties are classified into disease susceptibility levels based on their resistance to rice sheath blight: no infection observed (level 0), relative lesion height below 20% (level 1), relative lesion height 20%–30% (level 3), relative lesion height 31%–45% (level 5), relative lesion height 46%–65% (level 7), and relative lesion height greater than 65% (level 9) (IRRI (2002) Standard evaluation system for rice. International Rice Research Institute, Manila).

[0089] 2. Experimental Results

[0090] The genotype of SNP-33287194 in the rice sample was detected according to the methods described in 2.1-2.3 above. The resistance of the rice to sheath blight was determined based on the genotype results: if the genotype of SNP-33287194 was TT, the rice was resistant to sheath blight; if the genotype was CC, the rice was susceptible to sheath blight. The sheath blight resistance of the resistant rice was higher than that of the susceptible rice.

[0091] The test results are shown in Tables 1 and 2. Figure 1 Of the 54 rice materials in Table 1, 27 were resistant to sheath blight and 27 were susceptible to sheath blight.

[0092] Table 1. Relative lesion height and SNP locus genotypes after rice germplasm resources were inoculated with Rhizoctonia solani RH9.

[0093]

[0094]

[0095] Figure 1In the study, TT represented the genotype of SNP-33287194 in rice, CC represented the genotype of SNP-33287194 in rice, and NTC represented the blank control without template DNA. These results indicated that all 27 rice accessions resistant to rice sheath blight (SNP-33287194) had the genotype TT, while all 27 accessions susceptible to rice sheath blight (SNP-33287194) had the genotype CC. Rice with the genotype TT showed significantly higher resistance to rice sheath blight than rice with the genotype CC. Based on the IRRI (International Rice Research Institute, Manila) standard evaluation system for rice (2002), rice varieties with the genotype TT in SNP-33287194 exhibited resistance to rice sheath blight.

[0096] Therefore, the results of this invention in identifying rice sheath blight resistance using SNP-33287194 are consistent with the actual results of rice sheath blight resistance testing. Thus, by detecting polymorphisms or genotypes of SNP-33287194 in the rice genome, rice sheath blight resistance can be identified rapidly and accurately.

[0097] In breeding rice resistant to sheath blight, it is best to select rice with the genotype TT at the aforementioned SNP locus as the parent for breeding; in breeding rice susceptible to sheath blight, it is best to select rice with the genotype CC at the aforementioned SNP locus as the parent for breeding.

[0098] Table 2. Analysis of the relationship between SNP-33287194 genotype and rice sheath blight resistance.

[0099] genotype Relative lesion height Resistance to sheath blight TT <![CDATA[0.31±0.07 a ]]> Disease resistance CC <![CDATA[0.86±0.05 b ]]> Highly susceptible to disease

[0100] Note: Different superscript letters in the same column indicate significant differences (P<0.05).

[0101] As shown in Table 2, the average relative lesion height of homozygous TT rice varieties was 0.31, indicating that the TT genotype showed resistance to sheath blight. The average relative lesion height of homozygous CC rice varieties was 0.86, indicating that all of them were susceptible to the disease. This shows that there is a significant difference in the relative lesion height between the two genotypes.

Claims

1. The application of SNP molecular markers related to rice resistance to rice sheath blight in identifying or assisting in the identification of rice resistance to rice sheath blight or in breeding rice varieties resistant to rice sheath blight, characterized in that, The nucleotide sequence of the SNP molecular marker is shown in SEQ ID No. 4; wherein, the 24th nucleotide in the nucleotide sequence shown in SEQ ID No. 4 is the SNP site, which is a T / C polymorphism, and the genotype of the SNP site is TT or CC. Rice varieties with the genotype of the SNP site TT have higher resistance to sheath blight than rice varieties with the genotype of the SNP site CC.

2. The application according to claim 1, characterized in that, The rice varieties tested with the SNP molecular marker genotype TT are or candidates for rice resistant to sheath blight; the rice varieties tested with the SNP molecular marker genotype CC are or candidates for rice susceptible to sheath blight.

3. The application of KASP primer sets for detecting SNP molecular markers related to rice resistance to rice sheath blight in identifying or assisting in the identification of rice resistance to rice sheath blight or in breeding rice varieties resistant to rice sheath blight, characterized in that, The KASP primer set consists of two upstream primers A and B, and one downstream primer C; wherein the nucleotide sequence of the upstream primer A is the nucleotide sequence shown in SEQ ID No. 1 or the single-stranded DNA at positions 22-41 of the nucleotide sequence shown in SEQ ID No. 1; The nucleotide sequence of the upstream primer B is as shown in SEQ ID No. 2 or is the single-stranded DNA at positions 22-42 of the nucleotide sequence shown in SEQ ID No. 2; The nucleotide sequence of the downstream primer C is shown in SEQ ID No.

3.

4. The application of a detection kit for identifying or assisting in the identification of rice sheath blight resistance in the identification or breeding of rice sheath blight resistant varieties, the detection kit comprising: KASP primer set, specific probe set and KASP HiGeno 2x Probe Mix; characterized in that the KASP primer set consists of two upstream primers A and B and one downstream primer C; wherein the nucleotide sequence of the upstream primer A is the nucleotide sequence shown in SEQ ID No. 1 or the single-stranded DNA at positions 22-41 of the nucleotide sequence shown in SEQ ID No. 1; The nucleotide sequence of the upstream primer B is as shown in SEQ ID No. 2 or is the single-stranded DNA at positions 22-42 of the nucleotide sequence shown in SEQ ID No. 2; The nucleotide sequence of the downstream primer C is shown in SEQ ID No. 3; The specific probe set includes fluorescent probe A, quencher probe A, fluorescent probe B, and quencher probe B; the nucleotide sequence of fluorescent probe A is shown in SEQ ID No. 5, with a fluorescent group attached to its 5' end; the nucleotide sequence of fluorescent probe B is shown in SEQ ID No. 6, with a fluorescent group attached to its 5' end; the nucleotide sequence of quencher probe A is shown in SEQ ID No. 7, with a quencher group attached to its 3' end; and the nucleotide sequence of quencher probe B is shown in SEQ ID No. 8, with a quencher group attached to its 3' end.

5. A method for identifying or assisting in the identification of rice sheath blight resistance, characterized in that, include: (1) Extract genomic DNA from the rice sample to be tested; (2) A KASP primer set was designed with SNP molecular markers as detection targets to establish a PCR amplification system and PCR amplification was performed; the nucleotide sequence of the SNP molecular markers is shown in SEQ ID No. 4; wherein, the 24th nucleotide in the nucleotide sequence shown in SEQ ID No. 4 is an SNP site, which is a T / C polymorphism, and the genotype of the SNP site is TT or CC. Rice varieties with the genotype of the SNP site TT have higher resistance to sheath blight than rice varieties with the genotype of the SNP site CC. The KASP primer set consists of two upstream primers A and B, and one downstream primer C; wherein the nucleotide sequence of the upstream primer A is the nucleotide sequence shown in SEQ ID No. 1 or the single-stranded DNA at positions 22-41 of the nucleotide sequence shown in SEQ ID No. 1; The nucleotide sequence of the upstream primer B is as shown in SEQ ID No. 2 or is the single-stranded DNA at positions 22-42 of the nucleotide sequence shown in SEQ ID No. 2; The nucleotide sequence of the downstream primer C is shown in SEQ ID No. 3; (3) Detect the amplification products with fluorescence to determine the genotype of the SNP molecular marker in the rice sample to be tested; if the genotype of the SNP molecular marker in the rice sample to be tested is TT, then the rice sample to be tested is or is a candidate rice resistant to sheath blight; if the genotype of the SNP molecular marker in the rice sample to be tested is CC, then the rice sample to be tested is or is a candidate rice susceptible to sheath blight.

6. A method for breeding rice varieties resistant to sheath blight, characterized in that, include: (1) Extract genomic DNA from the rice sample to be tested; (2) A KASP primer set was designed with SNP molecular markers as detection targets to establish a PCR amplification system and PCR amplification was performed; the nucleotide sequence of the SNP molecular markers is shown in SEQ ID No. 4; wherein, the 24th nucleotide in the nucleotide sequence shown in SEQ ID No. 4 is an SNP site, which is a T / C polymorphism, and the genotype of the SNP site is TT or CC. Rice varieties with the genotype of the SNP site TT have higher resistance to sheath blight than rice varieties with the genotype of the SNP site CC. The KASP primer set consists of two upstream primers A and B, and one downstream primer C; wherein the nucleotide sequence of the upstream primer A is the nucleotide sequence shown in SEQ ID No. 1 or the single-stranded DNA at positions 22-41 of the nucleotide sequence shown in SEQ ID No. 1; The nucleotide sequence of the upstream primer B is as shown in SEQ ID No. 2 or is the single-stranded DNA at positions 22-42 of the nucleotide sequence shown in SEQ ID No. 2; The nucleotide sequence of the downstream primer C is shown in SEQ ID No. 3; (3) Detect the amplification product with fluorescence to determine the genotype of the SNP molecular marker of the rice sample to be tested; if the genotype of the SNP molecular marker of the rice sample to be tested is TT, then the rice sample to be tested is or is a candidate rice variety with resistance to sheath blight.

7. The method according to claim 5 or 6, characterized in that, The PCR amplification system established in step (2) is as follows: 2 μL of genomic DNA template solution of the rice sample to be tested, 0.14 μL of KASP primer working solution, 5 μL of KASP HiGeno 2xProbe Mix, and 2.86 μL of sterile ultrapure water; The PCR amplification procedure described in step (2) is as follows: Step 1: Pre-denaturation at 95℃ for 10 minutes; Step 2: 95℃ for 20s, 61℃ for 40s, 95℃ for 20s, 60.4℃ for 40s, 95℃ for 20s, 59.8℃ for 40s, 95℃ for 20s, 59.2℃ for 40s, 95℃ for 20s, 58.6℃ for 40s, 95℃ for 20s, 58℃ for 40s, 95℃ for 20s, 57.4℃ for 40s, 95℃ for 20s, 56.8℃ for 40s, 95℃ for 20s, 56.2℃ for 40s, 95℃ for 20s, 61℃ for 40s, 95℃ for 20s, 55.6℃ for 40s; Step 3: Denaturation at 95℃ for 20 seconds, annealing at 55℃ for 40 seconds, 40 cycles; The method for determining the genotype of the SNP molecular marker in the rice sample to be tested in step (3) is as follows: the fluorescence plate is read in a QX400 real-time fluorescence quantitative PCR instrument at a temperature of 37℃ for 60s, and the fluorescence value is read from the terminal end for genotyping.

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