Search

CN-117965788-B - SNP molecular marker related to rice sheath blight resistance, KASP primer group, kit and application

CN117965788BCN 117965788 BCN117965788 BCN 117965788BCN-117965788-B

Abstract

The invention discloses SNP molecular markers related to rice sheath blight resistance, KASP primer sets, kits and applications. The Japanese sunny genome sequence is taken as a reference genome, the SNP molecular marker is positioned at a 33287194bp locus of chromosome 3, the nucleotide sequence at and nearby the SNP locus is shown as SEQ ID No.4, the 24 th nucleotide is the SNP locus, the genotype is TT or CC, and the sheath blight resistance of the rice variety with the genotype of TT is higher than that of the rice variety with the genotype of CC. The invention also provides a KASP primer group for detecting the SNP molecular marker and a detection kit containing the KASP primer group. The KASP primer group or the detection kit can be used for predicting the resistance of rice to banded sclerotial blight, can be used for rice molecular marker assisted breeding, and has important application value in exploring banded sclerotial blight-resistant rice germplasm resources and breeding banded sclerotial blight-resistant rice varieties.

Inventors

  • ZHOU YONGLI
  • CHEN TIANYI
  • WANG DENGJI

Assignees

  • 中国农业科学院作物科学研究所

Dates

Publication Date
20260508
Application Date
20240125

Claims (7)

  1. 1. The application of the SNP molecular marker related to the rice sheath blight resistance in the identification or auxiliary identification of the rice sheath blight resistance or the breeding of rice sheath blight resistance varieties is characterized in that the nucleotide sequence of the SNP molecular marker is shown as SEQ ID No.4, wherein the 24 th nucleotide in the nucleotide sequence shown as SEQ ID No.4 is a SNP locus which is a T/C polymorphism, the genotype of the SNP locus is TT or CC, and the sheath blight resistance of the rice varieties with the genotype of the SNP locus being TT is higher than that of the rice varieties with the genotype being CC.
  2. 2. The method according to claim 1, wherein the rice to be tested with genotype TT of the SNP molecular marker is or is candidate for sheath blight resistance, and the rice to be tested with genotype CC of the SNP molecular marker is or is candidate for sheath blight resistance.
  3. 3. The application of the KASP primer group for detecting SNP molecular markers related to rice sheath blight resistance in identifying or assisting in identifying rice sheath blight resistance or breeding rice sheath blight resistance varieties is characterized in that the KASP primer group consists of two upstream primers A, an upstream primer B and a downstream primer C, wherein the nucleotide sequence of the upstream primer A is the nucleotide sequence shown as SEQ ID No.1 or single-stranded DNA at 22-41 positions in the nucleotide sequence shown as SEQ ID No. 1; The nucleotide sequence of the upstream primer B is shown in SEQ ID No.2 or single-stranded DNA at 22-42 positions in the nucleotide sequence shown in SEQ ID No. 2; the nucleotide sequence of the downstream primer C is shown as SEQ ID No. 3.
  4. 4. The application of a detection kit for identifying or assisting in identifying rice sheath blight resistance to identify or assisting in identifying rice sheath blight resistance or breeding rice sheath blight resistance varieties comprises a KASP primer group, a specific Probe group and KASP HiGeno x Probe Mix, and is characterized in that the KASP primer group consists of two upstream primers A, a primer B and a downstream primer C, wherein the nucleotide sequence of the upstream primer A is the nucleotide sequence shown as SEQ ID No.1 or single-stranded DNA at 22-41 positions in the nucleotide sequence shown as SEQ ID No. 1; The nucleotide sequence of the upstream primer B is shown in SEQ ID No.2 or single-stranded DNA at 22-42 positions in 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 comprises a fluorescent probe A, a quenching probe A, a fluorescent probe B and a quenching probe B, wherein the nucleotide sequence of the fluorescent probe A is shown as SEQ ID No.5, the 5 'end of the fluorescent probe A is connected with a fluorescent group, the nucleotide sequence of the fluorescent probe B is shown as SEQ ID No.6, the 5' end of the fluorescent probe B is connected with a fluorescent group, the nucleotide sequence of the quenching probe A is shown as SEQ ID No.7, the 3 'end of the quenching probe A is connected with a quenching group, the nucleotide sequence of the quenching probe B is shown as SEQ ID No.8, and the 3' end of the quenching probe B is connected with a quenching group.
  5. 5. A method for identifying or aiding in the identification of rice sheath blight resistance, comprising: (1) Extracting genome DNA of a rice sample to be detected; (2) Designing a KASP primer group by taking an SNP molecular marker as a detection target, establishing a PCR amplification system and carrying out PCR amplification, wherein the nucleotide sequence of the SNP molecular marker is shown as SEQ ID No.4, the 24 th nucleotide in the nucleotide sequence shown as SEQ ID No.4 is a SNP locus which is a T/C polymorphism, the genotype of the SNP locus is TT or CC, and the sheath blight resistance of a rice variety with the genotype of the SNP locus being TT is higher than that of a rice variety with the genotype of the SNP locus being CC; the KASP primer group consists of two upstream primers A, an upstream primer B and a downstream primer C, wherein the nucleotide sequence of the upstream primer A is the nucleotide sequence shown in SEQ ID No.1 or single-stranded DNA at 22-41 positions in the nucleotide sequence shown in SEQ ID No. 1; The nucleotide sequence of the upstream primer B is shown in SEQ ID No.2 or single-stranded DNA at 22-42 positions in 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) The method comprises the steps of carrying out fluorescent detection on amplification products, determining genotypes of SNP molecular markers of rice samples to be detected, detecting whether the rice samples are rice with banded sclerotial blight resistance or candidate rice if the genotypes of the SNP molecular markers of the rice samples to be detected are TT, and detecting whether the rice samples are rice with banded sclerotial blight resistance or candidate rice if the genotypes of the SNP molecular markers of the rice samples to be detected are CC.
  6. 6. A method for breeding rice varieties with banded sclerotial blight resistance, comprising: (1) Extracting genome DNA of a rice sample to be detected; (2) Designing a KASP primer group by taking an SNP molecular marker as a detection target, establishing a PCR amplification system and carrying out PCR amplification, wherein the nucleotide sequence of the SNP molecular marker is shown as SEQ ID No.4, the 24 th nucleotide in the nucleotide sequence shown as SEQ ID No.4 is a SNP locus which is a T/C polymorphism, the genotype of the SNP locus is TT or CC, and the sheath blight resistance of a rice variety with the genotype of the SNP locus being TT is higher than that of a rice variety with the genotype of the SNP locus being CC; the KASP primer group consists of two upstream primers A, an upstream primer B and a downstream primer C, wherein the nucleotide sequence of the upstream primer A is the nucleotide sequence shown in SEQ ID No.1 or single-stranded DNA at 22-41 positions in the nucleotide sequence shown in SEQ ID No. 1; The nucleotide sequence of the upstream primer B is shown in SEQ ID No.2 or single-stranded DNA at 22-42 positions in the nucleotide sequence shown in SEQ ID No. 2; The nucleotide sequence of the downstream primer C is shown as SEQ ID No.3, (3) fluorescent detection of amplified products, determination of the genotype of the SNP molecular marker of the rice sample to be detected, and detection of the rice sample as or candidate to be a rice variety with sheath blight resistance if the genotype of the SNP molecular marker of the rice sample to be detected is TT.
  7. 7. The method according to claim 5 or 6, wherein the PCR amplification system established in the step (2) is 2. Mu.L of primer set working solution of primer set 2. Mu. L, KASP, KASP HiGeno x Probe Mix 5. Mu.L and sterile ultra-pure water 2.86. Mu.L of the genomic DNA template solution of the rice sample to be detected; The PCR amplification procedure described in step (2) is: The first step, pre-denaturation at 95 ℃ for 10min; second step ：95℃ 20s、61℃ 40s,95℃ 20s、60.4℃ 40s,95℃ 20s、59.8℃ 40s,95℃ 20s、59.2℃ 40s,95℃ 20s、58.6℃ 40s,95℃ 20s、58℃ 40s,95℃ 20s、57.4℃ 40s,95℃ 20s、56.8℃ 40s,95℃ 20s、56.2℃ 40s,95℃ 20s、61℃ 40s,95℃ 20s、55.6℃ 40s; Third, denaturation at 95 ℃ for 20s, annealing at 55 ℃ for 40s, and 40 cycles; The method for determining the genotype of the SNP molecular marker of the rice sample to be detected in the step (3) comprises the steps of performing fluorescent reading on a QX400 real-time fluorescent quantitative PCR instrument, keeping the temperature at 37 ℃ for 60 seconds, and performing genotyping by adopting a terminal end read fluorescent value.

Description

SNP molecular marker related to rice sheath blight resistance, KASP primer group, kit and application Technical Field The invention relates to SNP molecular markers and detection primers, in particular to SNP molecular markers related to rice sheath blight resistance, KASP detection primer sets and application thereof in identifying rice sheath blight resistance, and belongs to the field of SNP molecular markers related to rice sheath blight resistance and application thereof. Background Sheath blight caused by rhizoctonia solani (Rhizoctonia solani Kuhn) is an important fungal disease in global rice farming, has a great influence on the yield and quality of rice, and generally occurs in rice-planting countries in asia, africa, europe and america. The rice yield can be reduced by 10% -30% in the general year, and the yield can be reduced by 50% in the serious case. For a long time, the prevention and treatment of banded sclerotial blight mainly depend on chemical agents and cultivation measures, and breeding and planting disease-resistant varieties are effective means for preventing and treating banded sclerotial blight. The development of molecular markers for detecting rice sheath blight resistance has important application value in screening rice germplasm resources for sheath blight resistance and rapidly identifying sheath blight resistance plants in the breeding process. KASP (Kompetitive Allele-SPECIFIC PCR, competitive allele-specific PCR) specifically recognizes gene loci through fluorescent probes to achieve genotyping effect, and can be used for detecting SNP loci and InDel loci. Compared with SSR, RFLP, inDel and other molecular markers, the KASP marker has the characteristics of quick detection, low cost, easy large-scale application and the like. KASP marking does not need to be typed according to the size of DNA fragments, can get rid of the defects of relatively complicated steps and low flux of the traditional gel electrophoresis detection method, and is suitable for a high flux molecular detection platform. Therefore, identifying functional SNP loci related to rice sheath blight resistance, developing rice sheath blight resistance KASP molecular markers suitable for a high-throughput molecular detection platform would have important application value for improving rice breeding efficiency and breeding level. Disclosure of Invention One of the purposes of the present invention is to provide SNP molecular markers related to rice sheath blight resistance; the second object of the present invention is to provide a KASP primer set for detecting the SNP molecular marker related to the resistance of rice to banded sclerotial blight; it is a further object of the present invention to provide a detection kit comprising the KASP primer set; The SNP molecular marker or the KASP detection primer group or the detection kit containing the KASP primer group is applied to identification or auxiliary identification or rice sheath blight resistance or rice variety with sheath blight sensitivity. The above object of the present invention is achieved by the following technical solutions: The invention provides an SNP molecular marker (SNP-33287194) related to rice sheath blight resistance, which takes a Japanese genome sequence as a reference genome, wherein the SNP molecular marker is positioned at a 33287194bp locus of chromosome 3, the nucleotide sequence at and nearby the SNP locus is shown as SEQ ID No.4, the 24 th nucleotide in the nucleotide sequence shown as SEQ ID No.4 is a SNP locus and is T/C polymorphism, the genotype is TT or CC, the TT is homozygous type of the SNP locus, the rice to be detected of the genotype of the SNP is TT or candidate of the SNP is sheath blight resistant rice, the CC is homozygous type of the SNP locus is C, the rice to be detected of the genotype of the SNP is CC is or candidate of sheath blight resistant rice, and the sheath blight resistance of the rice variety of which the genotype of the SNP locus is TT is higher than that of the rice variety of which the genotype of the SNP locus is CC. The invention can detect polymorphism or genotype of SNP molecular marker (SNP-33287194) locus related to rice sheath blight resistance by various detection methods, detection instruments or reagents known to the person skilled in the art, including but not limited to DNA sequencing, restriction enzyme fragment length polymorphism, single-stranded conformational polymorphism, denaturing high performance liquid chromatography, SNP chip, various PCR detection kits (including KASP detection kit), wherein the SNP chip comprises a chip based on nucleic acid hybridization reaction, a chip based on single base extension reaction, a chip based on allele specific primer extension reaction, a chip based on "one-step" reaction, a chip based on primer ligation reaction, a chip based on restriction enzyme reaction, a chip based on protein DNA binding reaction and a chip based on fluorescent molec