SNP (Single Nucleotide Polymorphism) molecular marker of brown planthopper resistant gene Bph33 of rice variety and application of SNP molecular marker

Abstract

The invention relates to an SNP (Single Nucleotide Polymorphism) molecular marker of an anti-brown planthopper major gene locus Bph33 of a rice variety W41123 and application of the SNP molecular marker. The invention discloses an SNP (Single Nucleotide Polymorphism) marker KBPH33 which is closely linked with a rice brown planthopper resistant gene locus Bph33 and has good amplification efficiency. The marker detection is located at the 19571017 site of the No.4 chromosome of Nipponbare rice, the polymorphism is A / G, and a marker primer developed on the basis of a KASP technology is as shown in SEQ ID No: 1-3. The SNP marker disclosed by the invention can be used for detecting a close linkage site of a Bph33 site, can be conveniently and efficiently used for efficiently identifying whether W41123 and derived varieties thereof contain the major gene site or not, can predict the resistance level of brown planthopper in a period, and greatly improves the breeding efficiency.

Description

Technical Field

[0001] This invention belongs to the field of molecular genetics and provides a gene locus for rice resistance to brown planthopper. Bph33 The SNP molecular markers and methods are specifically designed for the breeding of rice varieties resistant to brown planthoppers and the utilization of germplasm resources. Background Technology

[0002] Rice is one of the world's major food crops, and its safe production is crucial for ensuring national food security. 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 number one 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 , Bphi008a ,in Bph3 , Bph14 , Bph6 , Bph15 These methods have been widely applied in actual rice production. Previous researchers constructed an F2 population using the highly resistant brown planthopper indica rice variety W41123 from Vietnam and the susceptible japonica rice variety C418. They identified a major brown planthopper resistance gene, which was located between markers W131 and W115 on the long arm of chromosome 4, with a region size of 49 kb. No brown planthopper resistance genes have been reported within this region; therefore, this resistance site was named […]. Bph33 .

[0004] The phenotypic identification process for brown planthopper resistance is cumbersome and complex, resulting in low breeding efficiency for brown planthopper-resistant rice varieties. Furthermore, it is difficult to combine different brown planthopper resistance genes using conventional breeding methods. Therefore, developing brown planthopper resistance genes is crucial. Bph33Closely linked or co-segregated molecular markers can be used to aggregate one or more insect-resistant target genes or QTLs using marker-assisted selection (MAS) technology. This can accelerate the breeding efficiency of brown planthopper-resistant varieties, select durable resistant varieties, delay the degeneration period of insect-resistant varieties, and prevent the occurrence of new biotypes of brown planthoppers.

[0005] Conventional methods for breeding insect-resistant rice involve insect resistance identification, selecting highly resistant plants based on phenotype, and then hybridizing and backcrossing these plants. However, this method is resource-intensive, time-consuming, and the identification results are easily affected by environmental conditions, leading to errors and low selection efficiency. Using molecular marker-assisted selection (MAG) breeding can effectively reduce breeding costs, shorten the breeding cycle, and allow for targeted multi-gene aggregation, improving breeding efficiency. The marker types mainly used in the literature are SSR and In-Del markers, which have drawbacks such as low polymorphism and small differences in breeding. Furthermore, the EB or polyacrylamide used in the detection process can easily pollute the environment and pose health risks. The development of genes for resistance to brown planthoppers is underway. Bph33 The co-separation of specific molecular markers and the establishment of efficient and environmentally friendly related detection systems are crucial for promoting... Bph33 The application of genes in commercial breeding is of great significance. The KASP-based genotyping method uses computer recording and analysis of fluorescence signals generated during PCR to monitor variant sites, achieving high consistency between the detection results and phenotypes. The detection process eliminates the need for electrophoresis, completely preventing aerosol pollution from PCR products, environmental pollution from Eppendorf products, and the health hazards of formaldehyde. Summary of the Invention

[0006] The purpose of this invention is to provide an SNP molecular marker for detecting genes that resist brown planthopper in rice. Bph33 Does it exist, and how can it improve breeding efficiency?

[0007] To achieve the objectives of this invention, the technical approach is as follows: Based on gene location information, the brown planthopper resistance gene... Bph33 We located the SNP loci within the 19546550-19595500 region of chromosome 4 on the rice reference genome Nipponbare (Oryza sativa Japonica Group IRGSP-1.0) for analysis and mining. For the selected SNP loci, we extracted their flanking sequences and designed primers using the online primer design website SNPWay.

[0008] For these candidate SNP markers, utilize Bph33The designed SNP markers were initially screened using the donor resistant variety W41123, the susceptible control, and common susceptible rice materials. SNP markers that could be specifically distinguished from the resistant donor materials and had good amplification effects were selected.

[0009] The selected resistance gene-linked SNP markers were validated in an F2 population using 86 materials, demonstrating the effectiveness of the method detected by this invention. Bph33 The gene locus is a highly specific resistance locus and can be used for... Bph33 Screening and testing.

[0010] The purpose of this invention is to provide... Bph33 Tightly linked SNP molecular marker K_BPH33 and its applications.

[0011] The technical solution of this invention is as follows: To achieve the objectives of this invention, this invention provides a gene for resistance to brown planthoppers. Bph33 Assisted breeding molecular markers, the molecular markers being related to the rice brown planthopper resistance gene. Bph33 The co-separated SNP marker K_BPH33 was detected at position 19571017 of rice chromosome 4. This SNP marker exhibits an A / G polymorphism. If the site is A, it carries... Bph33 Genes that express resistance to brown planthoppers. For example, rice contains... Bph33 If the gene locus, specifically the base type at that locus, is A, then it is a variety resistant to brown planthoppers.

[0012] Application of SNP molecular marker K_BPH33 in breeding rice with resistance to brown planthopper. The SNP molecular marker is located at base 19571017 on chromosome 4 of the rice reference genome Nipponbare. The SNP molecular marker has a polymorphism of A / G, with the favorable base type being A.

[0013] A specific primer set for detecting SNP molecular markers, the specific primer set comprising the following: (1) Two specific primers: PrimerA:5'-TTCCAGCAAAGGAACCTACCAA-3' (SEQ ID NO.1); PrimerB: 5'-TTCCAGCAAAGGAACCTACCAG-3' (SEQ ID NO. 2); (2) A universal primer PrimerC: 5'-TAAATGTTGCATTGCATAGCGACT-3' (SEQ ID NO. 3).

[0014] Furthermore, the 5' end of Primer A is connected to the FAM fluorescent connector, and the 5' end of Primer B is connected to the HEX fluorescent connector.

[0015] The aforementioned specific primer combination was used in the cultivation of rice resistant to brown planthoppers.

[0016] Furthermore, the application includes the following steps: (1) Extract DNA from the rice sample to be tested; (2) Using the extracted rice sample genomic DNA as a template, PCR amplification was performed using the specific primer combination described in claim 2; (3) The genotype of PCR amplification products was analyzed using a fluorescence detector.

[0017] Furthermore, the PCR reaction conditions in step (2) are as follows: pre-denaturation at 94℃ for 15 minutes; first step amplification reaction: denaturation at 94℃ for 20 seconds, annealing and extension at 61℃~55℃ for 60 seconds, 10 cycles, with the annealing and extension temperature decreasing by 0.6℃ in each cycle; second step amplification reaction: denaturation at 94℃ for 20 seconds, annealing and extension at 55℃ for 60 seconds, 28 cycles.

[0018] Furthermore, in step (3), if only FAM fluorescence is detected in the sample, then the base at the detection site of the sample is determined to be A / A, indicating the presence of homozygous bases. Bph33 Genetically modified rice samples; if only HEX fluorescence is detected in the sample, the base at the detection site is determined to be G / G, indicating the absence of homozygous bases. Bph33 Genetically modified rice samples; if both FAM and HEX fluorescence signals are detected in the sample, the sample is determined to contain heterozygous genes. Bph33 Genetically modified rice samples.

[0019] Furthermore, it contains homozygous... Bph33 Genetically modified rice samples and those containing heterozygotes Bph33 All the genetically modified rice samples were brown planthopper-resistant rice.

[0020] The beneficial effects of the technical solution of this invention are as follows: The SNP molecular markers developed in this invention can efficiently and accurately screen for the desired loci containing resistance to brown planthoppers in different rice varieties. Bph33 resistant rice resources.

[0021] This invention utilizes KASP technology for genotyping of test samples. During the process, DNA is transferred to 384 or 1536 working plates for batch processing based on the number of samples. The operation is largely automated and offers advantages over traditional molecular marker detection methods, including high throughput, rapid operation, short cycle time, accurate sample loading, high sensitivity, and no environmental pollution. It is suitable for large-scale, high-throughput applications. Bph33 Site detection.

[0022] This invention can greatly improve the efficiency of brown planthopper resistance breeding and has high application prospects in molecular marker-assisted breeding for brown planthopper resistance. Attached Figure Description

[0023] Figure 1 This invention relates to a gene for resisting brown planthoppers. Bph33 Flowchart for the development of molecular markers for assisted breeding.

[0024] Figure 2 This is the natural population typing map detected by the SNP marker K_BPH33 in Embodiment 2 of the present invention. Detailed Implementation

[0025] The present invention will be further explained below with reference to the embodiments, but the embodiments do not limit the present invention in any way.

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0027] Unless otherwise specified, all consumables, materials, reagents, etc. used in the following examples are commercially available. Example

[0028] This embodiment illustrates the application of the molecular markers provided by the present invention in detecting brown planthopper resistance genes. Bph33 The specific steps for its application are as follows: 1. Primer design Based on the brown planthopper resistance gene Bph33 The target region was determined by referring to the segment 19546550-19595500 on chromosome 4 of the rice reference genome Nipponbare. SNP sites in and around this segment were analyzed and mined. Flanking sequences were extracted from the selected SNP sites, and primers were designed using the online primer design website SNPWay (http: / / www.snpway.com / ). Each marker set contained three primers, with two specific primers having FAM and HEX fluorescent adapter sequences attached to their 5' ends, respectively. The primers were synthesized by a biotechnology company.

[0029] Table 1 Primer information for molecular marker K_BPH33

[0030] The marker designed based on the KASP reaction principle and the SNP of resistant materials can perform high-throughput sampling on rice materials. Bph33 In the detection of brown planthopper resistance genes, if only FAM fluorescence is detected in the sample, then the base of the sample is A, indicating the presence of homozygous genes. BPH33Genetically modified rice samples; if only HEX fluorescence is detected in the sample, the detection site base is determined to be G, indicating that the sample does not contain homozygous genes. Bph33 Genetically modified rice samples; if both FAM and HEX fluorescence signals are detected in the sample, the sample is determined to contain heterozygous genes. Bph33 Genetically modified rice samples.

[0031] 2. Extraction of genomic DNA from rice leaves using the TPS method (1) Take 50 mg of rice leaves and cut them into 1.2 ml tubes with Eppendorf base. Add one 4 mm steel ball to each well, cover the tube, and freeze it in liquid nitrogen for 5 min. After taking it out, grind it twice in a grinding machine, 1 min each time. (2) Add 600 μl of preheated TPS solution to each well and heat at 65°C with shaking 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 well, mix gently, and freeze at -20℃ for 1-2 h. (4) Centrifuge at 4000 rpm for 10-15 min and discard the supernatant; (5) Add 100 μl of 75% ethanol to each well, gently tap the precipitate, centrifuge at 4000 rpm for 5-10 min, and discard the supernatant; (6) Dry, then add 150 μl to each well. dd Dissolve in H2O overnight and set aside.

[0032] 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.

[0033] Table 2 Reaction system for KASP detection

[0034] The PCR amplification process was carried out in a water bath thermal cycler. The PCR program is shown in Table 3.

[0035] Table 3 PCR amplification reaction conditions

[0036] 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.

[0037] The LGC SNPline genotyping platform and its supporting reagents and consumables used in this invention were all purchased from LGC Ltd. in the UK.

[0038] 3. Labeling and fractal data Use 1 copy Bph33 Donor materials, 8 susceptible control materials, and 8 conventional varieties were labeled and screened initially. Experimental data were read using a Pherastar instrument, and the results are shown in Table 4.

[0039] Table 4. Genotyping results of K_BPH33 molecular markers

[0040] As can be seen from Table 4, containing Bph33 The rice variety tested positive for base A at the K_BPH33 testing site, while the other 16 materials did not. Bph33 The gene was detected with G 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. Bph33 High-throughput detection.

[0041] Example 2: Rice Brown Planthopper Resistance Gene Bph33 Population phenotypic verification of SNP molecular markers in auxiliary breeding.

[0042] Insect resistance was assessed in the parents and F2 generation through seedling inoculation. To ensure uniform growth within each family in the parents and F2 populations, all tested materials were soaked and germinated before sowing. Twenty-five seeds from each family (variety) were sown in a round plastic pot (8.5 cm in diameter, 9.0 cm high) filled with nutrient soil (with a small hole at the bottom for water absorption), with a plant spacing of 2.5 cm. Three pots of each variety were randomly planted. Twenty-eight pots were placed in a 65 cm × 44 cm × 14 cm plastic box (with a water level of approximately 2 cm maintained). Seven days after sowing, thin the seedlings, discarding diseased and weak seedlings, and retaining 20 seedlings per pot. When the seedlings reach the two-leaf-one-heart stage, inoculate them with 2nd-3rd instar brown planthopper nymphs at a ratio of 10 nymphs per seedling. Finally, cover the pot with nylon mesh. When all the susceptible TN1 varieties die, evaluate the resistance of each individual plant on a scale of 0, 1, 3, 5, 7 or 9, referring to the methods of Athwal et al. (1971), IRRI (1988) and Huang et al. (2001) (Table 5). Calculate the resistance level of each family of the parental materials and population by weighted average.

[0043] Table 5. Evaluation criteria for resistance to brown planthopper used in this study.

[0044] Using SNP marker K_BPH33 to target W41123 ( Bph33 Genotyping was performed on 86 individual plants from the donor and C418 F2 population. The specific steps for KASP molecular marker detection were the same as in Example 1. The genotyping results of the markers in the backcross population are as follows: Figure 2 As shown. The markings are represented as Bph33 The genetically homozygous material exhibits a blue fluorescent signal (FAM fluorescence). Bph33 The genetically hybrid material exhibits green fluorescent signals (both FAM and HEX fluorescence signals), while the non-genetically hybrid material... Bph33 The material exhibits a red fluorescent signal (HEX fluorescence).

[0045] 2. Marked phenotypic validation Using the F2 populations of W41123 and C418, the gene in this invention was studied. Bph33 Genetic phenotypic verification was performed using the linkage marker K_BPH33. Bph33 Of the 15 strains, 90% exhibited a phenotype of moderate to high insect resistance, and none contained... Bph33 Of the 12 strains, 90% exhibited the phenotype of insect susceptibility (Table 6), and all 59 strains of the heterozygous material showed a level of insect resistance of at least moderate to high. The phenotypic data corresponded well with the genotypes, further verifying the feasibility and accuracy of the present invention.

[0046] Table 6. K_BPH33 molecular marker typing and phenotypic identification of individuals in the backcross population

[0047] This case verifies the feasibility and accuracy of the SNP marker detection method developed in this invention, and it can be applied to rice brown planthopper resistance genes. Bph33 Screening, identification, and assisted selection breeding.

[0048] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of SNP molecular marker K_BPH33 in breeding of rice with resistance to brown planthopper, characterized in that, The SNP molecular marker is located at base 19571017 of chromosome 4 of rice reference genome Nipponbare, the SNP molecular marker polymorphism is A / G, and the favorable base type is A.

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 A: 5'-TTCCAGCAAAGGAACCTACCAA-3'; Primer B: 5'-TTCCAGCAAAGGAACCTACCAG-3'; (2) one universal primer Primer C: 5'-TAAATGTTGCATTGCATAGCGACT-3'.

3. The specific primer combination according to claim 2, characterized in that, The 5' end of Primer A is connected with a FAM fluorescent linker, and the 5' end of Primer B 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, 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, The PCR reaction condition in step (2) is: 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 A / A, which is a homozygous Bph33 gene rice sample; If the sample only detects HEX fluorescence, it is judged that the sample detection site base is G / G, which does not contain homozygous Bph33 gene rice sample; If both FAM and HEX fluorescent signals are detected in a sample, the sample is judged to contain heterozygote Bph33 Gene rice sample.

8. Use according to claim 7, characterized in that, homozygous for the BPH21 gene Bph33 Gene samples and heterozygous for the BPH21 gene Bph33 Gene samples and heterozygous for the BPH21 gene are brown planthopper resistant rice.

Images