SNP (Single Nucleotide Polymorphism) molecular marker for detecting brown planthopper resistant gene Bph35 (t) of rice and application of SNP molecular marker
By developing the SNP molecular marker K_BPH35(t)-1, which co-isolates with the rice brown planthopper resistance gene Bph35(t), and using KASP technology for efficient and environmentally friendly genotyping, the problem of low rice breeding efficiency was solved, and rapid and accurate screening of brown planthopper resistance genes was achieved, which is suitable for large-scale breeding.
Patent Information
- Application Number
- CN202511635438.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-10
- Publication Date
- 2026-01-09
AI Technical Summary
Existing technologies for breeding rice resistant to brown planthoppers are inefficient. Conventional methods are labor-intensive and resource-intensive and are easily limited by environmental conditions. SSR and In-Del marker polymorphism rates are low, and EB or polyacrylamide pollutes the environment, making it difficult to efficiently screen for brown planthopper-resistant genes.
We developed an SNP molecular marker K_BPH35(t)-1 that co-isolates with the rice brown planthopper resistance gene Bph35(t). We used KASP technology for efficient and environmentally friendly genotyping and achieved rapid screening through specific primer combinations and fluorescence detection.
It improves rice breeding efficiency, shortens the breeding cycle, reduces costs, enables high-throughput and accurate screening of brown planthopper-resistant genes, avoids environmental pollution, and is suitable for large-scale breeding.
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Figure CN121294713A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of molecular biology, specifically to a gene for rice resistance to brown planthopper. Bph35(t) SNP molecular markers and methods for detecting these molecular markers. Background Technology
[0002] Rice is one of the world's three major food crops, and its safe production is of great significance to ensuring national food security and people's survival. During its growth and development, rice is frequently threatened by various pests and diseases, leading to reduced yields or even crop failure. Among these, the brown planthopper is the most serious pest affecting rice production. It not only damages rice plants by sucking sap from the phloem with its stylet, but also transmits grass dwarf virus and toothed leaf dwarf virus, severely threatening rice yield and quality and causing huge economic losses. Control methods for brown planthoppers mainly include biological control and chemical control. Chemical control has advantages such as speed, efficiency, and ease of use; however, the large-scale use of pesticides not only pollutes the ecological environment but also leads to pesticide resistance in brown planthoppers. Therefore, breeding resistant varieties is the most economical and effective means of controlling brown planthoppers.
[0003] At least 46 gene loci for resistance to brown planthoppers have been identified and reported, including 31 dominant genes and 15 recessive genes. Twelve brown planthopper resistance genes have been cloned. Bph3 , Bph6 , Bph9 , Bph14 , Bph15 , Bph18 , Bph26 , Bph29 , Bph30 , Bph32 , Bph40 , Bph1008a ,in Bph3 , Bph14 , Bph6 , Bph15 These methods have been widely applied in actual rice production. Previous researchers constructed an F2 population using the Sri Lankan brown planthopper-resistant indica rice variety Sinna Sivappu and the susceptible japonica rice variety 02428, identifying a major brown planthopper resistance gene. This gene was located on the short arm of chromosome 6. Further fine mapping using SSR markers S6-27 and M-11 pinpointed it to a 125kb region on the short arm of chromosome 6. No brown planthopper resistance genes have been reported within this region; therefore, this resistance site was named […]. BPH35(t) .
[0004] Because the identification process of the brown planthopper resistance is complicated, the breeding efficiency of the rice variety resistant to the brown planthopper is low, and it is difficult to aggregate different brown planthopper resistance genes by using the conventional breeding method. Therefore, the development of the molecular marker closely linked to the brown planthopper resistance gene or the molecular marker co-segregated with the brown planthopper resistance gene, the use of the marker-assisted selection (MAS) technology to aggregate one or more genes or QTLs for the resistance to the brown planthopper, the speeding up of the breeding efficiency of the brown planthopper-resistant variety, the breeding of the durable resistant variety, the delay of the degradation time of the resistant variety, and the prevention of the occurrence of the new biotype of the brown planthopper are achieved.
[0005] The conventional method for the breeding of the rice variety resistant to the brown planthopper is to select the plant with high resistance to the brown planthopper according to the phenotype, to cross and backcross the plant, and to locate the target gene by using the map-based cloning method. However, the method needs to consume a large amount of manpower and material resources, and consumes a long time. The identification result is limited by the environmental conditions, and the result is prone to error, and the selection efficiency is low. The use of the marker-assisted selection breeding can effectively reduce the breeding cost, shorten the breeding cycle, and aggregate the multiple genes with the purpose, so as to improve the breeding efficiency, and add great economic benefits to the actual production of the rice. The marker types mainly used in the literature reports are the SSR and In-Del markers, and the polymorphism rate and the difference are low in the breeding. The use of the EB or polybenzamide in the detection process can pollute the environment and harm the human body.
[0006] Therefore, it is imperative to develop the specific molecular marker co-segregated with the brown planthopper resistance gene, and to establish the efficient and environment-friendly detection method. SUMMARY
[0007] To solve the above technical problems, the purpose of the present application is to provide a SNP molecular marker for detecting the brown planthopper resistance gene of rice Bph35(t) , and improving the breeding efficiency.
[0008] The present application provides the molecular marker for the breeding of the brown planthopper resistance gene Bph35(t) , and the molecular marker is the SNP marker K_BPH35(t)-1 co-segregated with the brown planthopper resistance gene Bph35(t) of the rice. The SNP marker detects the base at the position of 1489635 of the 6th chromosome of the rice, and the polymorphism of the SNP molecular marker is C / A. If the site is C, the plant carrying the Bph35(t) gene shows the resistance to the brown planthopper.
[0009] To achieve the purpose of the present application, the technical solution of the present application is as follows: The first object of the present application is to provide an application of SNP molecular marker K_BPH35(t)-1 in breeding rice with resistance to brown planthopper, wherein the SNP molecular marker is located at base 1489635 of chromosome 6 of rice reference genome shuhui498 (R498) (GCA_026167685.1), the SNP molecular marker polymorphism is C / A, and the beneficial base type is C.
[0010] The second object of the present application is to provide a specific primer combination for detecting the aforementioned SNP molecular marker, which consists of: (1) two specific primers: Primer X: 5'- ATCCTGGGGTGTGTGACGC-3' (SEQ ID NO. 1); Primer Y: 5'- ATCCTGGGGTGTGTGACGA-3' (SEQ ID NO. 2); (2) one universal primer Primer C: 5'- GGGCATTTACATTAGCACAATCCT-3' (SEQ ID NO. 3).
[0011] Further, the 5' end of Primer X is connected with a FAM fluorescent linker, and the 5' end of Primer Y is connected with a HEX fluorescent linker.
[0012] The third object of the present application is to provide a kit comprising the aforementioned specific primer combination.
[0013] The fourth object of the present application is to provide an application of the aforementioned specific primer combination or the aforementioned kit in screening rice with resistance to brown planthopper.
[0014] Further, the application comprises the following steps: (1) extracting DNA of a rice sample to be tested; (2) using the extracted rice sample genomic DNA as a template, performing PCR amplification by using the aforementioned specific primer combination; (3) analyzing the genotype of the PCR amplification product by using a fluorescence detector.
[0015] Further, 94℃ pre-denaturation for 15 minutes; first step amplification reaction: 94℃ denaturation for 20 seconds, 65℃-57℃ annealing and extension for 60 seconds, 10 cycles, and the temperature of annealing and extension decreases by 0.6℃ in each cycle; second step amplification reaction: 94℃ denaturation for 20 seconds, 57℃ annealing and extension for 60 seconds, 26 cycles.
[0016] Further, if only FAM fluorescence is detected in step (3), it is judged that the sample detection site base is C / C, which is a homozygous Bph35(t) gene rice sample; if only HEX fluorescence is detected in the sample, it is judged that the sample detection site base is A / A, which is a non-homozygous Bph35(t) gene rice sample; if both FAM and HEX fluorescence signals are detected in the sample, it is judged that the sample is a heterozygous Bph35(t) gene rice sample.
[0017] Further, the homozygous Bph35(t) gene rice sample and the heterozygous Bph35(t) gene rice sample are both brown planthopper resistant rice.
[0018] The present application is based on the positioning information of the gene, and the anti-brown planthopper gene Bph35(t) is located at the segment of 1489711-1615420 on chromosome 6 of the rice reference genome Shuhui498 (R498), and the SNP sites on both sides of the segment and its vicinity are analyzed and mined. The selected SNP sites are extracted for their flanking sequences, and the online primer design website SNPWay is used for primer design.
[0019] For these candidate SNP markers, the designed SNP markers are initially screened by using Bph35(t) the donor resistant variety Sinna Sivappu, the insect susceptible control and common rice materials, and the SNP markers that can specifically distinguish the resistant donor materials and have good amplification effect are selected.
[0020] About 95 materials are used to verify the selected resistance gene linked SNP markers in the NIL population, which proves that the Bph35(t) gene site detected in the present application is a high-specificity resistance site, which can be used for Bph35(t) screening and detection.
[0021] The present application has the beneficial effects of: The present application develops specific molecular markers that are co-segregated with the anti-brown planthopper gene Bph35(t) and establishes a high-efficiency and environment-friendly detection system, which is of great significance for promoting Bph35(t) the application of the gene in commercial breeding. The KASP-based genotyping method records and analyzes the fluorescence signals generated in the PCR process by computer, realizes the monitoring of the mutation site, and the detection result has high consistency with the phenotype; the detection process does not need electrophoresis, completely eliminates the aerosol pollution of PCR products and the pollution of EB to the environment and the harm of formaldehyde to the human body.
[0022] The SNP molecular marker developed in the application can efficiently and accurately screen the required resistant rice resources containing the resistance to brown planthopper BPH35(t) from different rice varieties.
[0023] The application uses KASP technology to perform genotyping on the sample to be tested, and in the operation process, the DNA is transferred to a 384 or 1536 working plate for batch operation according to the number of the sample to be tested, and the operation process is basically automated, compared with the traditional molecular marker detection method, has the advantages of high throughput, rapid operation, short cycle, accurate sample addition, high sensitivity, environment pollution-free and the like, and is suitable for large-scale, high-throughput BPH35(t) site detection.
[0024] The application can greatly improve the efficiency of brown planthopper resistance breeding, and has high utilization prospect in the aspect of molecular marker assisted breeding of brown planthopper resistance. BRIEF DESCRIPTION OF DRAWINGS
[0025] Figure 1 The flow chart of the development of the molecular marker for the auxiliary breeding of the brown planthopper resistance gene BPH35(t) of the application.
[0026] Figure 2 The figure for detecting natural population genotyping of the SNP marker K_BPH35(t)-1 in the embodiment 2 of the application. DETAILED DESCRIPTION
[0027] The application will be further explained in combination with the embodiments, but the embodiments do not limit the application in any form.
[0028] It should be noted that the following description method is specific and detailed, and different description methods can be used in the application of the application, and the implementation mode of the application will be described in detail in combination with specific implementation cases.
[0029] It should be understood that the following specific implementation cases are only for the purpose of illustration, and are not used to limit the scope of the application. Those skilled in the art can make various modifications and replacements to the application without departing from the purpose and spirit of the application.
[0030] The experimental methods used in the following embodiments are conventional methods unless otherwise specified.
[0031] The consumables, materials and reagents used in the following embodiments can be obtained from commercial channels unless otherwise specified.
[0032] Example 1 Development of SNP molecular marker for auxiliary breeding of rice resistance to brown planthopper Bph35(t) gene This embodiment is used to illustrate the molecular marker provided by the application in the detection of the resistance to brown planthopper geneBph35(t) The application in the above steps is as follows: 1. Primer design According to the positioning interval of the brown planthopper-resistant gene Bph35(t) , the SNP site selected from the segment 1489711-1615420 on chromosome 6 of the rice reference genome Shuhui498 (R498) and the SNP site on both sides of the segment is analyzed and mined, the flanking sequence of the SNP site is extracted, and the online primer design website SNPWay (http: / / www.snpway.com / ) is used for primer design. Each set of markers contains three primers, and two specific primers are connected with FAM and HEX fluorescent linker sequences at the 5' end. The primers are synthesized by Shengong Company.
[0033] Table 1 Primer information of molecular marker K_BPH35(t)-1
[0034] Based on the KASP reaction principle and the SNP design of the resistant and susceptible materials, the Bph35(t) brown planthopper-resistant gene of the rice materials can be detected in a high-throughput manner. If only FAM fluorescence is detected in the sample, the base of the sample is C, which is a pure homozygous BPH35(t) gene rice sample; if only HEX fluorescence is detected in the sample, it is judged that the base of the sample detection site is A, which is a rice sample without pure homozygous gene; if FAM and HEX fluorescence signals are detected in the sample at the same time, it is judged that the sample is a rice sample containing heterozygous BPH35(t) gene. BPH35(t)
[0035] 2. Extracting rice leaf genomic DNA by TPS method (1) Take 50 mg of rice leaves and cut into an Eppendorf base 1.2 ml tube, add 1 4 mm steel ball to each hole, cover the lid and freeze in liquid nitrogen for 5 min, then take out and put into a sample grinder and grind twice, 1 min / time; (2) Add 600 μl of preheated TPS solution to each hole, and heat at 65°C for 45 min; (3) Cool to room temperature, centrifuge at 4000 rpm for 10-15 min, transfer 100 μl of supernatant to a new 96-well PCR plate, add 60-80 μl of pre-cooled isopropanol to each hole, mix gently, and freeze at -20°C for 1-2 h; (4) Centrifuge at 4000 rpm for 10-15 min, remove the supernatant; (5) Add 100 μl of 75% ethanol to each hole, tap the precipitate, centrifuge at 4000 rpm for 5-10 min, and remove the supernatant; (6) Dry, then add 150 μl to each well. dd Dissolve in H2O overnight and set aside.
[0036] 4. Sample loading and KASP reaction testing. The KASP reaction assay was performed on the LGC SNPline genotyping platform. 20 ng of DNA sample was added to each microplate, dried, and then KASP reaction mixture was added. The reaction system is shown in Table 2.
[0037] Table 2 Reaction system for KASP detection
[0038] The PCR amplification process was carried out in a water bath thermal cycler. The PCR program is shown in Table 3.
[0039] Table 3 PCR amplification reaction conditions
[0040] After the reaction is complete, the fluorescence data of the KASP reaction products are read using the Pherastar scanner, and the results of the fluorescence scan are automatically converted into images.
[0041] The LGC SNPline genotyping platform and its supporting reagents and consumables used in this invention were all purchased from LGC Ltd. in the UK.
[0042] 3. Labeling and fractal data Use 1 copy Bph35(t) Donor materials, 5 other insect-resistant and disease-resistant gene donor materials, 8 insect-susceptible control materials, and 8 conventional varieties were initially screened by labeling. Experimental data were read using a Pherastar instrument, and the experimental results are shown in Table 4.
[0043] Table 4. Genotyping results of the K_BPH35(t)-1 molecular marker Variety name Type K_BPH35(t)-1 Sinna Sivappu Bph35(t) / wbph9(t) / wbph10(t) / wbph11(t) / wbph12(t) C Balamawee Bph27(t) A DV85 Xa7 / xa24 / Qbph11 A MR1523 Qsbph / qSBPH5a / qSBPH5b / qSBPH6 A N22 qSBPH4 / qSBPH5 / qSBPH7 A Tetep Pi54 / qSBR11-1 A Ningjing 1 Susceptible material A Ningjing 3 Susceptible material A Ningjing 7 Susceptible material A Nipponbare Susceptible material A Jinnanfeng Susceptible material A USSR5 Susceptible material A TP65 Susceptible material A 02428 Susceptible material A Huaimang 5 Common japonica rice A Changlai japonica Common japonica rice A Jinxiangyu 1 Common japonica rice A Zhennuo 19 Common japonica rice A Yonghui 1540 Indica-japonica three-line hybrid rice A Xiangchang japonica 1 Common japonica rice A Kitaa Ke Common japonica rice A Zhonghua 11 Common japonica rice A As can be seen from Table 4, containing Bph35(t) The rice variety tested positive for base C at the K_BPH35(t)-1 test site, while the other 21 materials did not. Bph35(t) The gene was detected with A at the test site, indicating that the KASP molecular marker described in this invention can rapidly and easily target brown planthopper resistance genes in rice materials. Bph35(t) High-throughput detection.
[0044] Example 2: Rice Brown Planthopper Resistance Gene Bph35(t) Applications of KASP molecular markers 1. Utilize Bph35(t) SNP markers for genotyping The SNP marker K_BPH35(t)-1 was used to detect the genotypes of 95 single plants of the backcross population of Sinna Sivappu and 02428, and the specific steps of KASP molecular marker detection were the same as in Embodiment 1. The results of genotyping of the marker in the backcross population are shown in Table 2. Bph35(t) The marker showed Figure 2 The genetically homozygous material showed a blue fluorescent signal (FAM fluorescence), Bph35(t) the genetically heterozygous material showed a green fluorescent signal (FAM and HEX fluorescent signals), and the material without Bph35(t) showed a red fluorescent signal (HEX fluorescence). Bph35(t)
[0045] 2. Marker phenotype verification The backcross population of Sinna Sivappu and 02428 was used to verify the genetic phenotype of the marker K_BPH35(t)-1 linked to the gene Bph35(t) in the present application. 100% of the 10 lines containing Bph35(t) showed a phenotype of above medium resistance to the insect, and 85.7% of the 14 lines without Bph35(t) showed a phenotype of susceptibility to the insect, and the heterozygous lines showed a phenotype of medium resistance (Table 5). The phenotype data corresponded well to the genotypes, which again verified the feasibility and accuracy of the present application.
[0046] Table 5. K_BPH35(t)-1 molecular marker genotyping and phenotype identification of individuals in the backcross population Material number K_BPH35(t)-1 detection genotype result Resistance gene Comprehensive resistance level Resistance level 16TQ007 A 8.9 Susceptible 16TQ010 A 8.9 Susceptible 16TQ043 A 8.9 Susceptible 16TQ054 A 8.8 Susceptible 16TQ061 A 8.0 Susceptible 16TQ063 A 9.0 Susceptible 16TQ073 A 9.0 Susceptible 16TQ079 A 9.0 Susceptible 16TQ087 A 8.9 Susceptible 16TQ091 A 9.0 Susceptible 16TQ092 A 4.6 Moderately resistant 16TQ093 A 9.0 Susceptible 16TQ138 A 9.0 Susceptible 16TQ171 A 4.8 Moderately resistant 16TQ041 C 1.9 Resistant 16TQ045 C 1.2 Resistant 16TQ052 C 2.2 Resistant 16TQ067 C 2.6 Resistant 16TQ074 C 3.4 Moderately resistant 16TQ076 C 2.4 Resistant 16TQ141 C 2.9 Resistant 16TQ191 C 1.5 Resistant 16TQ128 C 1.7 Resistant 16TQ129 C 2.8 Resistant 16TQ104 C / A 4.5 Moderately resistant 16TQ144 C / A 4.8 Moderately resistant This case verifies the feasibility and accuracy of the SNP marker detection method developed in the present application, which can be applied to the screening and identification of rice brown planthopper resistance genes Bph35(t) and assisted selection breeding.
[0047] The above description is only the preferred embodiments of the present application, and it should be pointed out that for ordinary skilled persons in the technical field, some improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.
Claims
1. Use of a SNP molecular marker K_BPH35(t)-1 in breeding rice having resistance to brown planthopper, characterized in that, The SNP molecular marker is located at base 1489635 of chromosome 6 of rice reference genome shuhui498 (R498), the SNP molecular marker polymorphism is C / A, and the favorable base type is C.
2. A specific primer combination for detecting the SNP molecular marker of claim 1, characterized in that, The specific primer combination consists of: (1) two specific primers: Primer X: 5'- ATCCTGGGGTGTGTGACGC-3'; Primer Y: 5'- ATCCTGGGGTGTGTGACGA-3'; (2) one universal primer Primer C: 5'- GGGCATTTACATTAGCACAATCCT-3'.
3. The specific primer combination according to claim 2, characterized in that, The 5' end of Primer X is connected with a FAM fluorescent linker, and the 5' end of Primer Y is connected with a HEX fluorescent linker.
4. The specific primer combination of claim 2 or 3 is applied in breeding rice with resistance to brown planthopper.
5. Use according to claim 4, characterized in that, The application comprises the following steps: (1) extracting DNA of a rice sample to be tested; (2) using the extracted rice sample genomic DNA as a template, and performing PCR amplification by using the specific primer combination of claim 2; (3) analyzing the genotype of the PCR amplification product by using a fluorescence detector.
6. Use according to claim 5, characterized in that, 94℃ pre-denaturation for 15 minutes; first step amplification reaction: 94℃ denaturation for 20 seconds, 61℃-55℃ annealing and extension for 60 seconds, 10 cycles, and the temperature of annealing and extension decreases by 0.6℃ in each cycle; second step amplification reaction: 94℃ denaturation for 20 seconds, 55℃ annealing and extension for 60 seconds, 28 cycles.
7. Use according to claim 5, characterized in that, Step (3) if the sample only detects FAM fluorescence, it is judged that the sample detection site base is C / C, which is a homozygous Bph35(t) gene rice sample; If the sample only detects HEX fluorescence, it is judged that the sample detection site base is A / A, which does not contain homozygous Bph35(t) Gene rice sample; If both FAM and HEX fluorescent signals are detected in a sample, the sample is judged to contain heterozygous Bph35(t) genetic rice samples.
8. Use according to claim 7, characterized in that, homozygous for the BPH21 gene Bph35(t) Gene rice samples and heterozygous for the BPH21 gene Bph35(t) Both homozygous and heterozygous for the BPH21 gene.