KASP molecular marker for detecting rice blast resistance gene Pi65 and application of KASP molecular marker

Abstract

The invention discloses a KASP molecular marker for detecting a rice blast resistance gene Pi65 and application of the KASP molecular marker. The KASP molecular marker comprises SNP1 and / or SNP2; the SNP1 is located at the 28376936bp position of the 11th chromosome of a Nipponbare MSUv7 reference genome, and the polymorphism of the SNP1 is T or C; the SNP2 is located at the 28377512bp position of the 11th chromosome of a Nipponbare MSUv7 reference genome, and the polymorphism of the SNP2 is A or C. The KASP molecular marker provided by the invention has the characteristics of high typing quality, single copy and high polymorphism, can quickly realize genotype identification of the rice blast resistance gene Pi65 at high throughput and low cost, is used for marker-assisted breeding for rice blast resistance breeding improvement, and has wide application universality.

Description

Technical Field

[0001] This invention belongs to the field of agricultural molecular biology, specifically relating to a KASP molecular marker for detecting the rice blast resistance gene Pi65 and its application. Background Technology

[0002] Rice blast is one of the major diseases affecting rice in all rice-growing regions worldwide, occurring throughout the entire rice growth cycle. Currently, the most economical, effective, and environmentally friendly method for controlling rice blast remains the breeding and planting of rice varieties carrying blast-resistant genes. However, due to the complex and variable nature of the rice blast pathogen, a resistant variety often loses its resistance after a few years of promotion due to mutations in the prevalent strain. Therefore, breeding broad-spectrum and durable resistant varieties is a crucial issue that urgently needs to be addressed in rice breeding and production.

[0003] The disease-resistant gene Pi65, located and cloned by Zheng Wenjing's research team in the northern japonica rice variety Gangyu 129, is a broad-spectrum resistance gene against rice blast, solving the problem of the widespread lack of rice blast resistance genes in northern japonica rice varieties. In rice varieties both domestically and internationally, the disease-resistant gene Pi65 has shown broad-spectrum resistance and stability. Related technologies have revealed that most major varieties cultivated in sub-Saharan Africa carry the disease-resistant gene Pi65, and this gene is significantly associated with reducing the occurrence of rice leaf blast.

[0004] Currently, in the technology of using molecular markers to assist in the breeding of rice varieties resistant to rice blast, the screening of the target gene Pi65 still relies on the InDel-1 molecular marker. The main disadvantages of the InDel-1 molecular marker are: (1) it cannot directly obtain the genotype, and the PCR products of homozygous susceptible genotypes and heterozygous genotypes of Pi65 cannot be distinguished; (2) the detection throughput is low, requiring a large investment of manpower and resources, resulting in high costs. Therefore, the development of KASP markers targeting SNP sites closely linked to the resistance gene Pi65 can accelerate the work of breeders in identifying japonica rice germplasm resources resistant to rice blast and breeding resistant varieties. Summary of the Invention

[0005] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the present invention proposes a KASP molecular marker for detecting the rice blast resistance gene Pi65.

[0006] The present invention also proposes a primer set for detecting the above-mentioned KASP molecular markers.

[0007] The present invention also proposes a reagent kit.

[0008] This invention also proposes a gene chip.

[0009] This invention also proposes applications of the aforementioned KASP molecular markers, primer combinations, kits, and / or gene chips.

[0010] This invention also proposes a method for identifying or assisting in the identification of the rice blast resistance gene Pi65.

[0011] This invention also proposes a method for detecting or assisting in the detection of rice blast resistance.

[0012] This invention also proposes a breeding method for rice resistant to rice blast.

[0013] According to a first aspect of the present invention, a KASP molecular marker for detecting the rice blast resistance gene Pi65 is provided, wherein the KASP molecular marker includes SNP1 and / or SNP2;

[0014] The SNP1 is located at 28,376,936 bp on chromosome 11 of the Nipponbare MSUv7 reference genome, and its polymorphism is either T or C.

[0015] The SNP2 is located at 28377512 bp on chromosome 11 of the Nipponbare MSUv7 reference genome, and its polymorphism is A or C.

[0016] According to a second aspect of the present invention, a primer combination for amplifying the above-mentioned KASP molecular marker is provided.

[0017] In some embodiments of the present invention, the primer combination includes:

[0018] The first primer set used for detecting SNP1 contains a first specific primer sequence as shown in SEQ ID NO.1 and a second specific primer sequence as shown in SEQ ID NO.2; and / or,

[0019] The second primer set used to detect SNP2 contains a third specific primer sequence as shown in SEQ ID NO.4 and a fourth specific primer sequence as shown in SEQ ID NO.5.

[0020] In some embodiments of the present invention, the first specific primer and the second specific primer are respectively connected to different fluorescent groups.

[0021] In some embodiments of the present invention, the third specific primer and the fourth specific primer are respectively linked to different fluorescent groups.

[0022] In some embodiments of the present invention, the fluorescent group includes, but is not limited to, any one of FAM, ROX, CY5, HEX, JOE, CY3, NED, TAMRA, TAXAS RED, VIC, and TET.

[0023] In some embodiments of the present invention, the primer combination further includes universal primers, the universal primers comprising:

[0024] Universal primers for detecting SNP1 have the sequence shown in SEQ ID NO:3; and / or,

[0025] The universal primers used for detecting SNP2 have the sequence shown in SEQ ID NO:6.

[0026] According to a third aspect of the present invention, a kit is provided comprising the primer combination described above.

[0027] According to a fourth aspect of the present invention, a gene chip is provided, the gene chip comprising the above-described primer combination.

[0028] According to a fifth aspect of the present invention, the application of the above-described KASP molecular marker, primer combination, kit, or gene chip in any of the following is proposed:

[0029] (1) Genotyping of the rice blast resistance gene Pi65;

[0030] (2) Detection of the rice blast resistance gene Pi65;

[0031] (3) Identification and screening of rice varieties resistant to rice blast;

[0032] (4) Molecular marker-assisted breeding of rice;

[0033] (5) Rice breeding;

[0034] (6) Prepare products for rice breeding.

[0035] According to a sixth aspect of the present invention, a method for identifying or assisting in the identification of the rice blast resistance gene Pi65 is provided, comprising the following steps:

[0036] S1. Extract genomic DNA from the rice samples;

[0037] S2. Using the above primer combination, kit or liquid phase chip, perform polymorphism detection of the KASP molecular marker on the genomic DNA extracted in step S1, and determine whether the rice to be tested is rice with the rice blast resistance gene Pi65 based on the detection results.

[0038] In some embodiments of the present invention, when the KASP molecular marker is SNP1, if the detected genotype is TT, then the rice to be tested carries a homozygous Pi65 susceptible genotype; if the detected genotype is CC, then the rice to be tested carries a homozygous Pi65 resistant genotype; if the detected genotype is TC, then the rice to be tested has a heterozygous genotype.

[0039] In some embodiments of the present invention, when the KASP molecular marker is SNP2, if the detected genotype is AA, then the rice to be tested carries a homozygous Pi65 susceptible genotype; if the detected genotype is CC, then the rice to be tested carries a homozygous Pi65 resistant genotype; if the detected genotype is AC, then the rice to be tested has a heterozygous genotype.

[0040] In some embodiments of the present invention, the method for extracting genomic DNA from rice samples includes the simplified CTAB method (hexadecyltrimethylammonium bromide method) and the phenol / chloroform extraction method.

[0041] In some embodiments of the present invention, KASP (competitive allele-specific PCR) technology is used to detect polymorphisms of KASP molecular markers.

[0042] According to a seventh aspect of the present invention, a method for detecting or assisting in the detection of rice blast resistance is provided, the method comprising the following steps:

[0043] S1. Extract genomic DNA from the rice samples;

[0044] S2. Using the primer set, kit or liquid phase chip described above, the genomic DNA extracted in step S1 is subjected to polymorphism detection of the KASP molecular marker, and the rice blast resistance of the rice to be tested is determined based on the detection results.

[0045] In some embodiments of the present invention, when the KASP molecular marker is SNP1, if the detected genotype is TT, then the rice to be tested carries a homozygous Pi65 susceptible genotype; if the detected genotype is CC, then the rice to be tested carries a homozygous Pi65 resistant genotype; if the detected genotype is TC, then the rice to be tested has a heterozygous genotype.

[0046] In some embodiments of the present invention, when the KASP molecular marker is SNP2, if the detected genotype is AA, then the rice to be tested carries a homozygous Pi65 susceptible genotype; if the detected genotype is CC, then the rice to be tested carries a homozygous Pi65 resistant genotype; if the detected genotype is AC, then the rice to be tested has a heterozygous genotype.

[0047] In some embodiments of the present invention, the method for extracting genomic DNA from rice samples includes the simplified CTAB method (hexadecyltrimethylammonium bromide method) and the phenol / chloroform extraction method.

[0048] In some embodiments of the present invention, KASP (competitive allele-specific PCR) technology is used to detect polymorphisms of KASP molecular markers.

[0049] According to an eighth aspect of the present invention, a method for breeding rice resistant to rice blast is provided, comprising the following steps: using the above-described method for detecting or assisting in detecting rice blast resistance, selecting rice varieties resistant to rice blast for subsequent breeding.

[0050] According to some embodiments of the present invention, at least the following beneficial effects are achieved: The present invention provides a KASP molecular marker for detecting the rice blast resistance gene Pi65, which has the characteristics of high genotyping quality, single copy, and high polymorphism. It can rapidly, with high throughput and low cost, achieve genotyping of the rice blast resistance gene Pi65, and can be used for marker-assisted breeding for rice blast resistance improvement, with wide applicability.

[0051] This invention provides a KASP molecular marker detection method based on the Douglas Array Tape platform, specifically a KASP (Kompetitive Allele Specific PCR) marker detection method for identifying the rice blast resistance gene Pi65. This method boasts a 90% automation rate, significantly reducing laboratory manpower and human error. It offers high throughput and speed, obtaining 122,880 data points in 8 hours, which is 10 times faster than traditional 96-well plate SNP genotyping methods. Furthermore, it requires minimal reagent consumption (only 0.8 μL / reaction), reducing reagent and consumable costs by 70%-90% compared to traditional 96-well plate SNP genotyping methods.

[0052] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0053] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0054] Figure 1 This is a flowchart of the molecular marker development process in Embodiment 1 of the present invention;

[0055] Figure 2 This is a genotyping result diagram of the molecular marker OS905188_K01 in Example 1 of the present invention;

[0056] Figure 3 This is a genotyping result diagram of the molecular marker OS905189_K01 in Example 1 of the present invention;

[0057] Figure 4 This is an InDel-1 labeled agarose gel electrophoresis image from Example 1 of the present invention, where M is the marker, H2O is the blank control, and 1-13 are sample numbers.

[0058] Figure 5 This is a KASP marker genotyping result detection image of the molecular marker OS905188_K01 for the F1 population in Example 1 of the present invention;

[0059] Figure 6 This is a KASP marker genotyping result detection image of the molecular marker OS905189_K01 for the F1 population in Example 1 of the present invention;

[0060] Figure 7 The molecular marker OS905188_K01 in Example 1 of this invention is for F 2:3 KASP marker typing results of the population;

[0061] Figure 8 The molecular marker OS905189_K01 in Example 1 of this invention is for F 2:3 KASP marker typing results of the population. Detailed Implementation

[0062] The following will describe the concept and technical effects of the present invention clearly and completely with reference to the embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0063] Embodiment of the present invention: A KASP molecular marker for detecting the rice blast resistance gene Pi65.

[0064] The design process of this molecular marker, such as Figure 1As shown, referring to the two papers by Zheng Wenjing's research team on the fine mapping (2016) and cloning (2021) of the disease resistance gene Pi65, the gene sequences of Pi65-R and Pi65-S (InDel-1 30.57Mb~SNP-4 30.63Mb) were obtained, with Nipponbare as the reference genome. SNP polymorphism analysis was performed on the Pi65-R and Pi65-S gene sequences, ultimately obtaining multiple specific SNP sites and flanking sequences approximately 150bp before and after them. Primer sequences for the markers were designed and synthesized, and the markers were then screened to obtain the molecular markers OS905188_K01 and OS905189_K01. OS905188_K01 is located at 28,376,936 bp on chromosome 11 of the Nipponbare MSUv7 reference genome, with polymorphisms of T or C; OS905189_K01 is located at 28,377,512 bp on chromosome 11 of the Nipponbare MSUv7 reference genome, with polymorphisms of A or C. Specifically, the validation of a KASP molecular marker (SNP molecular marker) for detecting the rice blast resistance gene Pi65 is as follows:

[0065] 1. Primer design and synthesis

[0066] For the SNP molecular markers OS905188_K01 and OS905189_K01 obtained from the above screening, KASP primer markers were designed based on the Nipponbare genome, the reference genome of the rice blast resistance gene Pi65, using the online primer design website BatchPrimer3 (http: / / probes.pw.usda.gov / batchprimer3 / ). Each marker set consisted of three primers: two specific primers and one universal primer. The 5' ends of the two specific primers (specific primer PrimerX and specific primer Primer Y) were linked to FAM and HEX fluorescent sequences, respectively. After the design was completed, copy number analysis was performed with reference to the Nipponbare genome. If the marker mismatched with other locations in the genome, further optimization was required; if only a single copy of the polymorphic site at the marker design location existed, it indicated that the marker set was specific. The specific sequence information of the KASP molecular markers is shown in Table 1. The primers were synthesized by Shanghai Sangon Biotech.

[0067] Using the marker OS905188_K01 for detection, if only the fluorescent signal corresponding to Primer X is detected in the PCR product, the detection site base is TT, indicating that the test material carries a homozygous Pi65 susceptible genotype; if only the fluorescent signal corresponding to Primer Y is detected, the detection site base is CC, indicating that the test material carries a homozygous Pi65 resistant genotype; if both Primer X and Primer Y are detected simultaneously, the detection site base is TC, indicating that the test material is a heterozygous genotype.

[0068] Using the marker OS905189_K01 for detection, if only the fluorescent signal corresponding to Primer X is detected in the PCR product, the detection site base is AA, indicating that the test material carries a homozygous Pi65 susceptible genotype; if only the fluorescent signal corresponding to Primer Y is detected, the detection site base is CC, indicating that the test material carries a homozygous Pi65 resistant genotype; if both Primer X and Primer Y are detected simultaneously, the detection site base is AC, indicating that the test material is a heterozygous genotype.

[0069] Table 1. Molecular marker sequence of the disease resistance gene Pi65 KASP

[0070]

[0071] 2 Sample Testing

[0072] (1) DNA sample extraction: After the rice seedlings emerge, leaf samples are taken and genomic DNA is extracted from the samples using the CTAB method.

[0073] Other known methods (such as phenol / chloroform extraction) can also be used for DNA extraction, as long as the genomic DNA of rice can be extracted.

[0074] (2) KASP detection

[0075] KASP test: The KASP reaction test was performed on the Douglas Array Tape genotyping platform.

[0076] Reaction system: The PCR amplification system used was calculated in 0.8 μL volumes as follows: 10-20 ng of dried sample DNA was added to each of two 100 μM specific primers (0.0012 μL each), 0.0030 μL of 100 μM universal primers, 0.4 μL of 2×KASP Master Mix, and 0.3946 μL of ultrapure water. The specific reaction system is shown in Table 2.

[0077] PCR amplification: Performed in a water bath thermal cycler. Touchdown PCR reaction conditions were as follows: pre-denaturation at 94℃ for 15 min; first step amplification reaction: denaturation at 94℃ for 20 s, annealing and extension at 65℃~57℃ for 60 s, 10 cycles, with the annealing and extension temperature decreasing by 0.8℃ per cycle; second step amplification reaction: denaturation at 94℃ for 20 s, annealing and extension at 57℃ for 60 s, 30 cycles.

[0078] Signal scanning and genotyping: After the reaction, the fluorescence data of the KASP reaction products were read using an Arraytape scanning system. The fluorescence scan results were automatically converted into graphs, and then genotyping and data analysis were performed using INTELLICS. In the KASP marker genotyping detection, the sample genotypes were divided into three clusters: the X cluster, the Y cluster, and the heterozygous genotype cluster. The X cluster indicates that the sample contains a homozygous X allele at this KASP marker locus (marked in red on the genotyping graph, located in the upper left corner of the graph), the Y cluster indicates that the sample contains a homozygous Y allele at this KASP marker locus (marked in blue on the genotyping graph, located in the lower right corner of the graph), and the heterozygous genotype cluster indicates that the sample contains heterozygous X and Y alleles at this KASP marker locus (marked in purple on the genotyping graph).

[0079] Table 2 KASP labeling detection reaction system

[0080]

[0081]

[0082] 3. Marker Genotyping Quality Validation

[0083] Based on the above-mentioned KASP detection method, the disease resistance gene Pi65 KASP marker designed above and the known InDel-1 marker were used to detect 13 japonica rice varieties with known rice blast seedling blast resistance types (10 disease-resistant varieties J239, Shennong 014, Longjing 31, Wujing 15, Longjing 26, Suijing 10, Suijing 8, Jijing 111, Longjing 11, and Nanjing 2728, and 3 disease-susceptible varieties Liaoxing 1, Jijing 816, and Liaojing 101). The genotyping quality of the above-mentioned KASP marker was verified by comparing the consistency of the two detection results.

[0084] InDel-1 labeling detection: PCR amplification was performed on a PCR instrument to obtain PCR products. InDel-1 primer 1: ATCTTACCTCAACATTGCC (SEQ ID NO:7), primer 2: AGACATGTTGAAGACGCCT (SEQ ID NO:8). Reaction system: The total volume of the single sample reaction system was 20 μL, including 10 μL of 2×Taq PCR Master Mix, 0.5 μL of 10 μM primers, 1 μL of 100 μg / mL template DNA, and 8 μL of ddH2O. The specific reaction system is shown in Table 3. PCR amplification program for InDel-1 labeling detection: The reaction program was 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 70 s, 35 cycles, 72℃ extension for 5 min, and storage at 4℃. PCR product detection: Take 10 μL of PCR product, electrophoresis it in a 4% agarose gel, stain it with 4S Green Plus nucleic acid dye, and record the experimental results using a gel imaging system. If only a 139 bp fragment is obtained, the sample carries the rice blast resistance gene Pi65; if two fragments of 139 bp and 120 bp are obtained simultaneously, the sample does not contain the rice blast resistance gene Pi65 but contains its susceptible allele.

[0085] Table 3 InDel-1 Labeling Detection Reaction System

[0086]

[0087]

[0088] The detection phenotypic diagrams for OS905188_K01 and OS905189_K01 are as follows: Figure 2-3 As shown in the figure, the KASP marker OS905188_K01 showed that 10 of the 13 japonica rice samples had the CC genotype, including J239, Shennong 014, Longjing 31, Wujing 15, Longjing 26, Suijing 10, Suijing 8, Jijing 111, Longjing 11, and Nanjing 2728, all of which carried the homozygous Pi65 disease-resistant genotype; the other 3 samples had the TT genotype, including Liaoxing 1, Jijing 816, and Liaojing 101, all of which carried the homozygous Pi65 disease-susceptible genotype. The detection results of the KASP marker OS905189_K01 were consistent with those of the marker OS905188_K01. The genotype of the above 10 materials was CC, and all of them carried the homozygous Pi65 disease-resistant genotype; the above 3 materials were AA, and all of them carried the homozygous Pi65 disease-susceptible genotype, which was consistent with the actual results.

[0089] The results of InDel-1 labeling detection are as follows Figure 4As shown in the figure, the 10 materials mentioned above only showed a single fragment of 139bp, indicating that they all carry the rice blast resistance gene Pi65; the 3 materials mentioned above showed two fragments of 139bp and 120bp, indicating that they do not contain the rice blast resistance gene Pi65 but contain its susceptible allele.

[0090] The results of KASP marker detection and InDel-1 marker detection were analyzed, and the results are shown in Table 4. Table 4 shows that the consistency between the two detection results was 100%. Furthermore, materials carrying the Pi65 resistance genotype all exhibited resistance to rice blast, while materials carrying the Pi65 susceptibility genotype all exhibited susceptibility to rice blast. These results indicate that the genotyping results of KASP markers OS905188_K01 and OS905189_K01 are accurate and can be accurately used to detect the Pi65 gene, greatly improving the efficiency and throughput of screening japonica rice resources for the Pi65 resistance gene.

[0091] Table 4. Detection results of KASP and InDel-1 markers

[0092]

[0093]

[0094] 4. Specificity and Practicality Testing

[0095] To test the specificity and applicability of the KASP markers OS905188_K01 and OS905189_K01 in this invention, the corresponding verification was performed using single plants carrying the target genotype in the hybrid offspring, according to the detection method described in section 2 above.

[0096] (1) Screening for true hybrids carrying heterozygous genotypes in the F1 generation of hybrids.

[0097] Using Japonica rice J239 carrying the blast resistance gene Pi65 as the male parent and the susceptible material Jijing 816 as the female parent, 53 F1 generation individual plants were obtained after hybridization. Using Japonica rice J239 carrying the Pi65 blast resistance gene as the female parent and the susceptible material Liaoxing 1 as the male parent, 50 F1 generation individual plants were obtained after hybridization. DNA was extracted from rice leaves of both F1 populations and labeled using the KASP markers OS905188_K01 and OS905189_K01.

[0098] The detection results of KASP tags OS905188_K01 and OS905189_K01 are as follows: Figure 5-6As shown in the figure, the KASP marker OS905188_K01 test results indicate that in the Jijing 816 / J239 F1 population, 46 individual plants tested positive for heterozygous genotypes, making them true hybrids; another 7 individual plants tested positive for homozygous Pi65 susceptible genotypes, making them false hybrids. In the J239 / Lioxing 1 F1 population, only 33 individual plants tested positive for heterozygous genotypes, making them true hybrids; another 17 individual plants tested positive for homozygous Pi65 resistant genotypes, making them false hybrids. The results of the OS905189_K01 marker test are consistent with those of the OS905188_K01 marker test.

[0099] The above results indicate that the Pi65 KASP markers OS905188_K01 and OS905189_K01, which are resistant to rice blast, can accurately and efficiently screen true hybrids in the F1 generation of hybrids, ensuring that the breeding and improvement of japonica rice varieties are advanced based on the correct materials.

[0100] (2) Screening for homozygous individuals carrying the rice blast resistance gene Pi65 in hybrid offspring

[0101] The F1 generation of Jijing 816 / J239 and J239 / Liaoxing 1 were self-pollinated to obtain F2 population seeds. 2：3 Two lines with high seed yields were selected from the population and sown individually in seedling trays in an artificial climate chamber. When the seedlings reached two leaves and one bud, they were sprayed with a suspension of rice blast fungus conidia (2×10⁻⁶). 5 (Inoculation rate: 1 / ml), 6-7 days after inoculation, rice seedling resistance to rice blast was identified and evaluated according to "NY / T 2646-2014 Technical Procedures for Identification and Evaluation of Rice Blast Resistance in Rice Variety Trials". The grading standards for rice seedling blast are shown in Table 5. Simultaneously, F2O3 was extracted. 2：3 The DNA from leaf samples of individual plants in the population was used for genotyping using KASP markers OS905188_K01 and OS905189_K01.

[0102] Table 5 Grading Standards for Rice Seedling Blight Investigation

[0103]

[0104] Table 6. Jijing 816 / J239 and J239 / Liaoxing 1F 2：3 Population resistance to seedling blight and genotyping results

[0105]

[0106]

[0107]

[0108]

[0109]

[0110] After sowing in seedling trays, Jijing 816 / J239 and J239 / Liaoxing 1F 2：3 The groups produced 87 and 88 seedlings respectively. The results of seedling blast identification and KASP marker detection are shown in Table 6. 25CP3 is J239 / Liaoxing 1 F. 2：3 The populations are as follows: 25CP4 is Jijing 816 / J239, 25CP6 is the parent Liaoxing 1, 25CP8 is Jijing 816, and 25CP12 is J239. The marker-based genotyping results are as follows: Figure 7-8 As shown.

[0111] Analysis of the above results shows that single plants with homozygous disease-resistant genotypes all exhibited resistance to rice blast, indicating that the KASP marker in this invention is closely linked to the disease-resistant gene Pi65, and can accurately and quickly screen homozygous individuals carrying the rice blast resistance gene Pi65 in hybrid offspring.

[0112] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A KASP molecular marker for detecting the rice blast resistance gene Pi65, characterized in that, The KASP molecular markers include SNP1 and / or SNP2; The SNP1 is located at 28,376,936 bp on chromosome 11 of the Nipponbare MSUv7 reference genome, and its polymorphism is either T or C. The SNP2 is located at 28377512 bp on chromosome 11 of the Nipponbare MSUv7 reference genome, and its polymorphism is A or C.

2. Primer combinations for amplifying the KASP molecular marker as described in claim 1.

3. The primer combination according to claim 2, characterized in that, The primer combination includes: The first primer set used for detecting SNP1 contains a first specific primer sequence as shown in SEQ ID NO.1 and a second specific primer sequence as shown in SEQ ID NO.2; and / or, The second primer set used to detect SNP2 contains a third specific primer sequence as shown in SEQ ID NO.4 and a fourth specific primer sequence as shown in SEQ ID NO.5; The first and second specific primers are respectively linked to different fluorescent groups; The third and fourth specific primers are each linked to different fluorescent groups.

4. The primer combination according to claim 2, characterized in that, The primer combination further includes universal primers, which include: Universal primers for detecting SNP1 have the sequence shown in SEQ ID NO:3; and / or, The universal primers used for detecting SNP2 have the sequence shown in SEQ ID NO:

6.

5. A reagent kit, characterized in that, The kit comprises the primer combination as described in any one of claims 2-4.

6. A gene chip, characterized in that, The gene chip includes the primer combination as described in any one of claims 2-4.

7. The use of the KASP molecular marker of claim 1, the primer combination of any one of claims 2-4, the kit of claim 5, or the gene chip of claim 6 in any one of the following: (1) Genotyping of the rice blast resistance gene Pi65; (2) Detection of the rice blast resistance gene Pi65; (3) Identification and screening of rice varieties resistant to rice blast; (4) Molecular marker-assisted breeding of rice; (5) Rice breeding; (6) Prepare products for rice breeding.

8. A method for identifying or assisting in the identification of the rice blast resistance gene Pi65, characterized in that, Includes the following steps: S1. Extract genomic DNA from the rice samples; S2. Using the primer combination as described in any one of claims 2-4, the kit as described in claim 5, or the gene chip as described in claim 6, the genomic DNA extracted in step S1 is subjected to polymorphism detection of the KASP molecular marker, and the test rice is determined to be rice with the rice blast resistance gene Pi65 based on the detection results.

9. A method for detecting or assisting in the detection of rice blast resistance, characterized in that, The method includes the following steps: S1. Extract genomic DNA from the rice samples; S2. Using the primer combination as described in any one of claims 2-4, the kit as described in claim 5, or the gene chip as described in claim 6, the genomic DNA extracted in step S1 is subjected to polymorphism detection of the KASP molecular marker, and the rice blast resistance of the rice to be tested is determined based on the detection results.

10. A breeding method for rice resistant to rice blast, characterized in that, The method includes the following steps: using the method described in claim 9, selecting rice varieties resistant to rice blast for subsequent breeding.

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