InDel molecular marker FM1 of rice anti-chilo suppressalis gene SBR11 and application of InDel molecular marker FM1

Abstract

The invention discloses an InDel molecular marker FM1 of a rice anti-chilo suppressalis gene SBR11 and application of the InDel molecular marker FM1. The InDel molecular marker FM1 is closely linked with a rice striped rice borer resistant gene SBR11. Experiments prove that the InDel molecular marker FM1 is adopted to carry out PCR amplification on rice genome DNA, and the chilo suppressalis resistance of a rice material (carrying the chilo suppressalis resistant gene SBR11) with a strip with the size of 120 bp in a PCR product is higher than that of a rice material (not carrying the chilo suppressalis resistant gene SBR11) without the strip with the size of 120 bp in the PCR product. The InDel molecular marker FM1 provided by the invention can be used for identifying whether rice carries the chilo suppressalis resistant gene SBR11 or not, chilo suppressalis resistance and molecular marker-assisted selective breeding, especially early identification and screening of the chilo suppressalis resistance of rice, and the breeding efficiency of chilo suppressalis resistant rice breeding is greatly improved.

Description

Technical Field

[0001] This invention relates to the field of rice molecular breeding technology, specifically to the InDel molecular marker FM1 of the rice rice stem borer resistance gene SBR11 and its application. Background Technology

[0002] Research reports indicate that crop diseases and pests cause 20%-40% of global crop yield losses, with the majority of these losses caused directly by pests or through pest-borne diseases. Rice (Oryza sativa L.) yield has a significant impact on food security and social stability. Among the more than 200 pests that harm rice (Oryza sativa L.), the rice stem borer (Chilosuppressalis Walker) is extremely widespread, causing substantial economic losses due to yield reductions. Therefore, effective control of the rice stem borer is crucial for maintaining stable rice yields.

[0003] Chemical control has long been the primary means of mitigating losses caused by these pests. However, the overuse of chemical pesticides over the past 50 years has led to insect resistance, and the environmental pollution and food safety problems caused by their long-term use are becoming increasingly widespread. The increasing difficulty and cost of developing new pesticides are also negatively impacting economic development. How to effectively control pests while ensuring food and environmental safety is a major challenge. Therefore, there is an urgent need to find alternative pest control methods to replace chemical pesticides, enabling sustainable crop production.

[0004] Cloning insect-resistant genes and developing new insect-resistant varieties have provided new ideas for crop pest control because they are economically and environmentally friendly, making them particularly important for ensuring rice yield and quality. Although research on rice insect-resistant genes has been conducted for many years and a large number of insect-resistant genes have been screened, the focus has been mainly on resistance to brown planthoppers. Research progress on rice stem borers, another major pest of rice, has been relatively slow, and the available gene resources are very limited.

[0005] The qGRB1 or SBR11 (LOC_Os11g37700) gene is an endogenous rice stem borer resistance gene extracted from rice germplasm resources, located on chromosome 11 of the rice genome. To date, there have been no reports on the development of molecular markers targeting rice stem borer resistance genes or on marker-assisted breeding of rice varieties resistant to rice stem borers. Therefore, developing functional molecular markers closely linked to the SBR11 gene and applying them to the breeding of new rice varieties resistant to rice stem borers can significantly improve the breeding efficiency of rice varieties resistant to rice stem borers, and is an important aspect of marker-assisted selection breeding for rice varieties resistant to rice stem borers. Summary of the Invention

[0006] The technical problem this invention aims to solve is how to quickly and accurately detect the rice stem borer resistance gene SBR11, thereby enabling the identification of rice stem borer resistance and improving the efficiency of rice breeding for rice resistant to rice stem borers.

[0007] To address the aforementioned technical problems, this invention first provides a primer pair for identifying or assisting in the identification of rice stem borer resistance.

[0008] The primer pair provided by this invention for identifying or assisting in the identification of rice stem borer resistance consists of primer F and primer R;

[0009] The primer F is either a1) or a2):

[0010] a1) The single-stranded DNA molecule shown in SEQ ID NO.1;

[0011] a2) A single-stranded DNA molecule of SEQ ID NO.1 with one or more nucleotide substitutions and / or deletions and / or additions that have the same function as SEQ ID NO.1;

[0012] The primer R is either a3) or a4) as follows:

[0013] a3) The single-stranded DNA molecule shown in SEQ ID NO.2;

[0014] a4) A single-stranded DNA molecule that has undergone substitution and / or deletion and / or addition of one or more nucleotides of SEQ ID NO.2 and has the same function as SEQ ID NO.2.

[0015] In the primer pair described above, the molar ratio of primer F to primer R is 1:1.

[0016] To address the aforementioned technical problems, the present invention further provides a PCR reagent or kit containing the aforementioned primer pairs; the PCR reagent or kit functions as any one of the following b1)-b5):

[0017] b1) Detection or auxiliary detection of the rice stem borer resistance gene SBR11;

[0018] b2) Screening or assisted screening of rice varieties carrying the rice stem borer resistance gene SBR11;

[0019] b3) Identify or assist in the identification of rice stem borer resistance;

[0020] b4) Screening or assisted screening of rice resistant to rice stem borer;

[0021] b5) Rice breeding or variety improvement.

[0022] In some implementations, the PCR reagent or kit may also include other reagents for PCR amplification, such as 2×TSINGKE Master Mix.

[0023] In some implementations, the kit may also include a negative control, such as sterile water.

[0024] In some implementations, the kit may also include a positive control, such as genomic DNA from IC 25690.

[0025] To address the aforementioned technical problems, this invention also provides new uses for the primer pairs or the PCR reagents or kits described above.

[0026] This invention provides the use of the above primer pairs or the above PCR reagents or kits in any one of the following c1)-c10):

[0027] c1) Detection or auxiliary detection of the rice stem borer resistance gene SBR11;

[0028] c2) Prepare products for detecting or assisting in the detection of the rice stem borer resistance gene SBR11;

[0029] c3) Screening or auxiliary screening of rice carrying the rice stem borer resistance gene SBR11;

[0030] c4) Prepare rice products for screening or auxiliary screening of rice carrying the rice stem borer resistance gene SBR11;

[0031] c5) Identification or auxiliary identification of rice stem borer resistance;

[0032] c6) Prepare products for identifying or assisting in the development of rice stem borer resistance;

[0033] c7) Screening or assisted screening of rice resistant to rice stem borer;

[0034] c8) Prepare products for screening or assisting in screening rice resistant to rice stem borer;

[0035] c9) Rice breeding or variety improvement;

[0036] c10) is used to prepare products for rice breeding or variety improvement.

[0037] To address the aforementioned technical problems, this invention also provides new uses for InDel molecular labeling or for detecting InDel molecular labeling substances.

[0038] This invention provides the application of InDel molecular markers or substances for detecting InDel molecular markers in any of the following c1)-c10):

[0039] c1) Detection or auxiliary detection of the rice stem borer resistance gene SBR11;

[0040] c2) Prepare products for detecting or assisting in the detection of the rice stem borer resistance gene SBR11;

[0041] c3) Screening or auxiliary screening of rice carrying the rice stem borer resistance gene SBR11;

[0042] c4) Prepare rice products for screening or auxiliary screening of rice carrying the rice stem borer resistance gene SBR11;

[0043] c5) Identification or auxiliary identification of rice stem borer resistance;

[0044] c6) Prepare products for identifying or assisting in the development of rice stem borer resistance;

[0045] c7) Screening or assisted screening of rice resistant to rice stem borer;

[0046] c8) Prepare products for screening or assisting in screening rice resistant to rice stem borer;

[0047] c9) Rice breeding or variety improvement;

[0048] c10) Prepare products for rice breeding or variety improvement;

[0049] The InDel molecular marker is located at positions 98-109 of SEQ ID NO.4 and is a double-stranded DNA molecule with a single-stranded nucleotide sequence at positions 98-109 of SEQ ID NO.4.

[0050] Furthermore, the substance used to detect the InDel molecular marker is a substance used to detect whether the rice to be tested contains the InDel molecular marker.

[0051] Furthermore, the substance for detecting whether the rice sample contains the InDel molecular marker includes a primer pair for amplifying a DNA fragment containing the InDel molecular marker. The primer pair consists of a forward primer and a reverse primer, wherein the forward primer is a single-stranded DNA that specifically binds upstream of the InDel molecular marker, and the reverse primer is a single-stranded DNA that specifically binds downstream of the InDel molecular marker.

[0052] In some implementations, the forward primer is primer F as described above; and the reverse primer is primer R as described above.

[0053] To address the aforementioned technical problems, this invention also provides a method for detecting or assisting in the detection of the rice stem borer resistance gene SBR11.

[0054] The method for detecting or assisting in the detection of the rice stem borer resistance gene SBR11 provided by the present invention includes the following steps: detecting whether the rice genome contains the InDel molecular marker, and detecting or assisting in the detection of the rice stem borer resistance gene SBR11 based on whether the rice genome contains the InDel molecular marker; wherein the InDel molecular marker is located at positions 98-109 of SEQ ID NO.4.

[0055] In the above-mentioned method for detecting or assisting in the detection of the rice stem borer resistance gene SBR11, the method for detecting or assisting in the detection of the rice stem borer resistance gene SBR11 based on whether the rice genome contains the InDel molecular marker is as follows: if the rice genome to be tested contains the InDel molecular marker, then the rice to be tested carries the rice stem borer resistance gene SBR11; otherwise, the rice to be tested does not carry the rice stem borer resistance gene SBR11.

[0056] To address the aforementioned technical problems, this invention also provides a method for identifying or assisting in the identification of rice stem borer resistance.

[0057] The method for identifying or assisting in the identification of rice stem borer resistance provided by the present invention includes the following steps: detecting whether the rice genome contains an InDel molecular marker, and identifying or assisting in the identification of rice stem borer resistance based on whether the rice genome contains an InDel molecular marker; wherein the InDel molecular marker is located at positions 98-109 of SEQ ID NO.4.

[0058] The method for identifying or assisting in the identification of rice stem borer resistance, as described above, involves identifying or assisting in the identification of rice stem borer resistance based on whether the rice genome contains the InDel molecular marker. Rice containing the InDel molecular marker exhibits higher resistance to the rice stem borer than rice without the InDel molecular marker. This higher resistance is manifested in the lower rice stem borer damage rate of rice containing the InDel molecular marker compared to rice without the InDel molecular marker.

[0059] To address the aforementioned technical problems, this invention also provides a method for screening or assisting in the screening of rice carrying the rice stem borer resistance gene SBR11.

[0060] The present invention provides a screening or assisted screening method for rice carrying the rice stem borer resistance gene SBR11, comprising the following steps: detecting whether the rice genome contains the InDel molecular marker, and selecting rice whose genome contains the InDel molecular marker; wherein the InDel molecular marker is located at positions 98-109 of SEQ ID NO.4. Rice whose genome contains the InDel molecular marker is rice carrying the rice stem borer resistance gene SBR11.

[0061] To address the aforementioned technical problems, the present invention also provides a method for screening or assisting in the screening of rice resistant to rice stem borer.

[0062] The method for screening or assisting in screening rice resistant to rice stem borer provided by this invention includes the following steps: detecting whether the rice genome contains the InDel molecular marker, and selecting rice whose genome contains the InDel molecular marker; the InDel molecular marker is located at positions 98-109 of SEQ ID NO.4. Rice whose genome contains the InDel molecular marker is rice resistant to rice stem borer.

[0063] To address the aforementioned technical problems, this invention ultimately provides a method for rice breeding or variety improvement.

[0064] The method for rice breeding or variety improvement provided by the present invention includes the following steps: detecting whether the rice genome contains an InDel molecular marker, and selecting rice with an InDel molecular marker in its genome as a parent for breeding; wherein the InDel molecular marker is located at positions 98-109 of SEQ ID NO.4.

[0065] In the above-mentioned methods for rice breeding or variety improvement, the indicators for breeding or variety improvement include resistance to rice stem borer.

[0066] In the above-mentioned methods for rice breeding or variety improvement, the purpose of the breeding or variety improvement includes selecting rice varieties carrying the rice stem borer resistance gene SBR11 or rice varieties resistant to rice stem borers.

[0067] In any of the methods described above, the primer pairs described above can be used to detect whether the rice genome contains the InDel molecular marker.

[0068] In some embodiments, the method for detecting or assisting in the detection of the rice stem borer resistance gene SBR11 includes the following steps: extracting genomic DNA from the rice to be tested, performing PCR amplification using the genomic DNA as a template to obtain PCR products; detecting the PCR products by electrophoresis, and detecting or assisting in the detection of the rice stem borer resistance gene SBR11 based on the size of the PCR products: if a band of 120 bp is present in the PCR product, then the rice to be tested carries the rice stem borer resistance gene SBR11; otherwise, the rice to be tested does not carry the rice stem borer resistance gene SBR11.

[0069] In some specific implementations, the method for detecting or assisting in the detection of the rice stem borer resistance gene SBR11 in rice is as follows, based on the size of the PCR product: if the PCR product contains only a 120bp band or both 120bp and 132bp bands, then the rice being tested carries the rice stem borer resistance gene SBR11; if the PCR product contains only a 132bp band, then the rice being tested does not carry the rice stem borer resistance gene SBR11.

[0070] In some embodiments, the method for identifying or assisting in the identification of rice stem borer resistance includes the following steps: extracting genomic DNA from the rice to be tested, performing PCR amplification using the genomic DNA as a template to obtain PCR products; detecting the PCR products by electrophoresis, and identifying or assisting in the identification of rice stem borer resistance based on the size of the PCR products: rice with a 120 bp band in the PCR product has higher resistance to rice stem borer than rice without a 120 bp band in the PCR product. This higher resistance to rice stem borer is reflected in the fact that rice with a 120 bp band in the PCR product has a lower rate of rice stem borer damage than rice without a 120 bp band in the PCR product.

[0071] In some specific implementations, the method for identifying or assisting in the identification of rice stem borer resistance, based on the size of the PCR product, is as follows: rice with only a 120bp band in the PCR product has higher resistance to rice stem borer than rice with both 120bp and 132bp bands in the PCR product; rice with both 120bp and 132bp bands in the PCR product has higher resistance to rice stem borer than rice with only a 132bp band in the PCR product. Rice with only a 120bp band in its PCR product exhibits higher resistance to the rice stem borer than rice with both 120bp and 132bp bands in its PCR product. This is reflected in the lower rice stem borer infestation rate of rice with only a 120bp band in its PCR product compared to rice with both 120bp and 132bp bands in its PCR product.

[0072] In some embodiments, the method for screening or assisting in screening rice carrying the rice stem borer resistance gene SBR11 includes the following steps: extracting genomic DNA from the rice to be tested, performing PCR amplification using the genomic DNA as a template to obtain PCR products; detecting the PCR products by electrophoresis, and selecting rice varieties containing a 120bp band in the PCR products.

[0073] In some specific implementations, the method for screening or assisting in screening rice carrying the rice stem borer resistance gene SBR11 selects rice varieties that have only a 120bp band in their PCR products and rice varieties that have bands of 120bp and 132bp in their PCR products.

[0074] In some implementations, the method for screening or assisting in screening rice resistant to rice stem borer includes the following steps: extracting genomic DNA from the rice to be tested, performing PCR amplification using the genomic DNA as a template to obtain PCR products; detecting the PCR products by electrophoresis, and selecting rice varieties containing a band of 120 bp in the PCR products.

[0075] In some specific implementations, the method for screening or assisting in screening rice resistant to rice stem borer selects rice varieties that have only a 120bp band in their PCR product and rice varieties that have bands of 120bp and 132bp in their PCR product.

[0076] In some preferred embodiments, the method for screening or assisting in screening rice resistant to rice stem borer selects rice varieties whose PCR products contain only a 120bp band.

[0077] In some embodiments, the method for rice breeding or variety improvement includes the following steps: extracting genomic DNA from the rice to be tested, performing PCR amplification using the genomic DNA as a template to obtain PCR products; detecting the PCR products by electrophoresis, and selecting rice varieties with a 120bp band in the PCR products as parents for breeding.

[0078] In some specific implementations, in the rice breeding or variety improvement method, rice varieties with only a 120bp band in the PCR product and rice varieties with bands of 120bp and 132bp in the PCR product are selected as parents for breeding.

[0079] In some preferred embodiments, in the method of rice breeding or variety improvement, rice varieties in which only a 120bp band exists in the PCR product are selected as parents for breeding.

[0080] The reaction system for any of the above-mentioned PCR amplifications is as follows: 1.5 μL genomic DNA, 7.5 μL 2×TSINGKE MasterMix, 4.5 μL ddH2O, 0.75 μL primer F, and 0.75 μL primer R. The final concentration of primer F and primer R in the reaction system is 0.5 μM.

[0081] The reaction procedure for PCR amplification of any of the above is as follows: 95℃ pre-denaturation for 3 min; 95℃ for 15 s, 56℃ for 30 s, 72℃ for 15 s, 35 cycles; 72℃ extension for 5 min.

[0082] The nucleotide sequence of the PCR product corresponding to any of the above-mentioned bands of 120 bp in size is shown in SEQ ID NO.3.

[0083] The nucleotide sequence of the PCR product corresponding to any of the above-mentioned bands of 132 bp in size is shown in SEQ ID NO.4.

[0084] The nucleotide sequence of any of the above-mentioned rice stem borer resistance genes SBR11 (the gene name may also be qGRB1) is shown in SEQ ID NO.5.

[0085] The damage rate of the rice stem borer mentioned above can be obtained as follows: Individual rice plants are planted in a netted greenhouse. When the rice reaches the tillering stage, rice stem borer larvae are introduced (one young larva per tiller). After the damage symptoms stabilize, the damage rate of the rice stem borer is investigated. The damage rate (%) is calculated as follows: (Number of damaged tillers / Total number of tillers per plant) × 100%.

[0086] The rice mentioned above can be any rice germplasm resource, variety, strain or single plant commonly found in this technical field.

[0087] In some embodiments, the rice is at least one of the following materials: ARC 13156, RL ​​GOPHER, DILBAKSH, DO LEUANG, GINMASARI, IAS22-8PALMAR, IC 25690, K 113, MONMINEUPLEU, TAIPEI167, TAM, CT 45, NIA TACUARI, EA HOUM, LITCHIKIANG, Huangpinuo, Zimangfeie2, and OS 4, as well as Wushan Simiao as the recipient parent, IC 25690 as the recurrent parent, and the BC4F2 population of individual plants is obtained by backcrossing 4 times and selfcrossing 1 time after hybridization.

[0088] This invention provides an InDel molecular marker FM1, which is tightly linked to the rice stem borer resistance gene SBR11. Its primer sequences are shown in SEQ ID NO.1 and SEQ ID NO.2. Experiments have demonstrated that PCR amplification of rice genomic DNA using the InDel molecular marker FM1 yields PCR products. Rice materials containing a 120 bp band in the PCR product (carrying the SBR11 resistance gene) exhibit higher resistance to the rice stem borer than rice materials without a 120 bp band in the PCR product (not carrying the SBR11 resistance gene). The InDel molecular marker FM1 provided by this invention can be used to identify whether rice carries the SBR11 resistance gene, assess rice stem borer resistance, and for marker-assisted selection breeding, especially for the early identification and screening of rice stem borer resistance, significantly improving the breeding efficiency of rice resistant to the rice stem borer. Attached Figure Description

[0089] Figure 1 The results of identification of 18 rice varieties using the InDel molecular marker FM1 are presented.

[0090] Figure 2 Application of InDel molecular marker FM1 in marker-assisted selection breeding. Detailed Implementation

[0091] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0092] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0093] The rice insect-resistant material IC25690 described in the following examples is described in the literature “Wang, W., Mauleon, R., Hu, Z., Chebotarov, D., Tai, S., Wu, Z., Li, M., Zheng, T., Fuentes, RR, Zhang, F., Mansueto, L., Copetti, D., Sanciangco, M., Palis, KC, Xu, J., Sun, C., Fu, B., Zhang, H., Gao, Y., Zhao, X., … Leung, H. (2018). Genomic variation in 3,010 diverse accessions of Asian cultivated rice. Nature, 557(7703), 43-49. https: / / doi.org / 10.1038 / s41586-018-0063-9”.

[0094] The rice insect-sensitive material Changhui 70 in the following examples is a new plant variety that Yangtze University applied for protection registration with the state on December 29, 2018 and was authorized for publication. The application number is 20184678.9, the publication number is CNA025018E, and the variety right number is CNA20184678.9.

[0095] The rice germplasm resources in the following examples are ARC 13156, RL ​​GOPHER, DILBAKSH, DO LEUANG, GINMASARI, IAS22-8PALMAR, IC 25690, K 113, MONMINEUPLEU, TAIPEI 167, TAM, CT 45, NIATACUARI, EA HOUM, LITCHIKIANG, Huangpinuo, Zimangfeie2, and OS. 4 are recorded in the literature "Wang, W., Mauleon, R., Hu, Z., Chebotarov, D., Tai, S., Wu, Z., Li, M., Zheng, T., Fuentes, RR, Zhang, F., Mansueto ,L.,Copetti,D.,Sanciangco,M.,Palis,KC,Xu,J.,Sun,C.,Fu,B.,Zhang,H.,Gao,Y.,Zhao,X.,…Leung,H.(2018).Genomic variation in 3,010diverseaccessions of Asian cultivated rice.Nature,557(7703),43-49.https: / / doi.org / 10.1038 / s41586-018-0063-9".

[0096] Example 1: Development and validation of the InDel molecular marker FM1 for the rice stem borer resistance gene SBR11.

[0097] I. Development of the InDel molecular marker FM1 for the rice rice stem borer resistance gene SBR11

[0098] 1. Referring to the published Nipponbare reference genome sequence and its physical location (https: / / rice.uga.edu / cgi-bin / sequence_display.cgi?orf=LOC_Os11g37700.1), sequence alignment analysis was performed on the SBR11 gene of the re-sequencing insect-resistant material IC25690 and the insect-susceptible material Changhui 70. The gene sequences of the insect-resistant material IC25690 and the insect-susceptible material Changhui 70 are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively. The sequence alignment analysis results showed that, compared with the insect-susceptible material Changhui 70, the SBR11 gene sequence of the insect-resistant material IC25690 had a 12bp deletion, which was located at positions 98-109 of SEQ ID NO.4.

[0099] 2. For the 12bp insertion / deletion sequence discovered in step 1, the InDel molecular marker FM1 was designed, and its primer sequence is as follows:

[0100] FM1-F: 5'-TGATTGATTGATTGAACTCTTCGAT-3' (SEQ ID NO. 1);

[0101] FM1-R: 5'-GGGAGATGCCTTGCAAAAT-3' (SEQ ID NO. 2).

[0102] In practical applications, the InDel molecular marker FM1 can be used to amplify the genomic DNA of rice materials by PCR. Then, denaturing polyacrylamide gel electrophoresis can be used to detect the size of the PCR product to determine whether the rice material carries the rice stem borer resistance gene SBR11 and its resistance to rice stem borer: if a 120bp band is present in the PCR product, the rice material carries the rice stem borer resistance gene SBR11 and is a rice stem borer resistant material; otherwise, the rice material does not carry the rice stem borer resistance gene SBR11 and is a rice stem borer susceptible material.

[0103] II. Validation of the InDel molecular marker FM1 for the rice stem borer resistance gene SBR11

[0104] Materials tested: ARC 13156, RL ​​GOPHER, DILBAKSH, DO LEUANG, GINMASARI, IAS 22-8PALMAR, IC 25690, K 113, MONMINUPLEU, TAIPEI 167, TAM, CT 45, NIA TACUARI, EA HOUM, LITCHIKIANG, Huangpinuo, Zimangfeie and OS 4.

[0105] 1. Genotyping

[0106] Genomic DNA was extracted from each test material using the CTAB method. Then, using the genomic DNA as a template, PCR amplification was performed using the InDel molecular marker FM1 from step one to obtain PCR products. The PCR products were separated by electrophoresis on an 8% polyacrylamide gel and then silver stained for color development.

[0107] The PCR amplification system (15 μL) consisted of: 1.5 μL template DNA, 0.75 μL primer FM1-F, 0.75 μL primer FM1-R, 7.5 μL 2×TSINGKE Master Mix (Beijing Qingke Biotechnology Co., Ltd., catalog number TSE003), and 4.5 μL ddH2O. The final concentration of primers FM1-F and FM1-R in the PCR amplification system was 0.5 μM.

[0108] The PCR amplification reaction program is as follows: 95℃ pre-denaturation for 3 min; 95℃ for 15 s, 56℃ for 30 s, 72℃ for 15 s, 35 cycles; 72℃ extension for 5 min.

[0109] The results are as follows Figure 1 As shown in the figure. The results showed that materials ARC 13156 and IC 25690 were both materials carrying the rice stem borer resistance gene SBR11, and their PCR amplification bands were both 120bp in size; materials RL GOPHER, DILBAKSH, DO LEUANG, GINMASARI, IAS22-8PALMAR, K 113, MONMINEUPLEU, TAIPEI 167, TAM, CT 45, NIA TACUARI, EA HOUM, LITCHIKIANG, Huangpinuo, Zimangfeie, and OS 4 were materials that did not carry the rice stem borer resistance gene SBR11, and their PCR amplification bands were all 132bp in size.

[0110] 2. Phenotypic identification of rice stem borer resistance

[0111] The resistance of each tested material to the rice stem borer was identified. The specific steps are as follows: Individual plants of different tested materials were planted in a net house. When the rice reached the tillering stage, rice stem borer larvae were introduced. One young rice stem borer larva was introduced into each tiller. After the damage symptoms stabilized, the damage rate (DR) of each tested material was investigated. The damage rate (%) was calculated as follows: {Number of damaged tillers (including dead sheaths, dead hearts, and whiteheads) / Total number of tillers per plant} × 100%. A higher damage rate indicates lower resistance to the rice stem borer, and a lower damage rate indicates higher resistance to the rice stem borer.

[0112] The results are shown in Table 1. The results showed that the rice stem borer infestation rates of materials ARC 13156 and IC 25690 carrying the rice stem borer resistance gene SBR11 were 34.55% and 10.14%, respectively, both exhibiting strong resistance to the rice stem borer. In contrast, the rice stem borer infestation rates of materials without the rice stem borer resistance gene SBR11 (RL GOPHER, DILBAKSH, DO LEUANG, GINMASARI, IAS22-8PALMAR, K113, MONMINEUPLEU, TAIPEI 167, TAM, CT 45, NIA TACUARI, EAHOUM, LITCHIKIANG, Huangpinuo, Zimangfeie, and OS 4) ranged from 40.74% to 100%, with an average of 68.12%, all exhibiting poor resistance to the rice stem borer.

[0113] The above results show that the InDel molecular marker FM1 of the present invention can effectively identify the rice stem borer resistance of the tested rice. Applying the InDel molecular marker FM1 to rice molecular marker-assisted selection breeding can greatly improve the breeding efficiency of rice resistant to rice stem borer.

[0114] Table 1. Phenotypic identification of rice germplasm resources resistant to rice stem borer.

[0115]

[0116]

[0117] Example 2: Application of the InDel molecular marker FM1 of the rice stem borer resistance gene SBR11 in marker-assisted selection breeding.

[0118] I. Test Materials

[0119] 1. Using Wushan Simiao (a rice variety susceptible to rice stem borer) as the female parent and IC 25690 (a rice variety resistant to rice stem borer) as the male parent, hybridization was carried out to obtain the F1 generation.

[0120] 2. Backcross the F1 generation obtained in step 1 with Wushan Simiao to obtain the BC1F1 generation. Using the InDel molecular marker FM1, select individual plants carrying the SBR11 gene from the BC1F1 population that have comprehensive traits (including agronomic traits such as leaf type, plant height, ear type, and heading date as observed in the field) that are close to those of Wushan Simiao. Backcross these plants with Wushan Simiao to obtain the BC2F1 generation. Repeat this process until the BC4F1 generation is obtained.

[0121] 3. Using the InDel molecular marker FM1, single plants carrying the SBR11 gene and whose comprehensive traits (including agronomic traits such as plant leaf type, plant height, ear type, and heading date as observed in the field) are very similar to Wushan Simiao in the BC4F1 generation population were selected for self-pollination to obtain the BC4F2 generation segregating population. From the BC4F2 segregating population, 30 single plants were randomly selected as test materials and named as number 1-30.

[0122] II. Genotype Testing

[0123] Genomic DNA was extracted from the test materials (numbered 1-30), IC 25690, and Wushan Simiao. Then, using the genomic DNA as a template, PCR amplification was performed using the codominant molecular marker FM1 from Example 1 to obtain PCR products. The PCR products were separated by electrophoresis on an 8% polyacrylamide gel and then silver stained for color development.

[0124] The results are as follows Figure 2 As shown in the figure. The results showed that materials numbered 3, 4, 5, 12, 16, 19, 24, and 28 were all homozygous bands (PCR products showed only a 120bp band), and their amplification bands were the same size as the IC 25690 amplification band, indicating that materials numbered 3, 4, 5, 12, 16, 19, 24, and 28 carried homozygous IC 25690 alleles on the SBR11 gene; materials numbered 1, 2, 6, 8, 9, 11, 13, 14, 17, 18, 20, 21, 23, 26, and 30 were all heterozygous bands (PCR products showed both 120bp and 132bp bands), and one of their amplification bands was the same size as the IC 25690 amplification band. The 25690 amplified band was the same size as the other amplified band, which was the same size as the Wushan Simiao amplified band. This indicates that materials numbered 1, 2, 6, 8, 9, 11, 13, 14, 17, 18, 20, 21, 23, 26, and 30 carry heterozygous IC 25690 alleles in the SBR11 gene. Materials numbered 7, 10, 15, 22, 25, 27, and 29 were all homozygous (the PCR product only showed a 132bp band) and were the same size as the Wushan Simiao amplified band. This indicates that materials numbered 7, 10, 15, 22, 25, 27, and 29 carry homozygous Wushan Simiao alleles in the SBR11 gene and do not carry the IC 25690 allele.

[0125] III. Phenotypic Identification of Rice Stem Borer Resistance

[0126] The rice stem borer resistance phenotype of each test material was identified according to the method in step 2 of Example 1. The results are shown in Table 2.

[0127] Table 2. Results of rice stem borer resistance identification of 30 individual plants from the BC4F2 self-pollinated population.

[0128]

[0129] Based on the electrophoresis results of the PCR products of each material, seven rice materials carrying the rice stem borer resistance gene SBR11 (numbered 3, 4, 5, 12, 16, 19, 24, and 28) were selected for subsequent breeding of rice varieties resistant to rice stem borers. In practical applications, the InDel molecular marker FM1 can be used to determine early and accurately whether the rice germplasm materials carry the rice stem borer resistance gene SBR11 through PCR detection and electrophoresis, thereby achieving rapid, efficient, and accurate identification of rice stem borer resistance and accelerating the breeding process.

[0130] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. Primer pair, wherein the primer pair consists of primer F and primer R; The primer F is either a1) or a2): a1) The single-stranded DNA molecule shown in SEQ ID NO.1; a2) A single-stranded DNA molecule of SEQ ID NO.1 that has undergone substitution and / or deletion and / or addition of one or more nucleotides and has the same function as SEQ ID NO.1; The primer R is either a3) or a4) as follows: a3) The single-stranded DNA molecule shown in SEQ ID NO.2; a4) A single-stranded DNA molecule of SEQ ID NO.2 with one or more nucleotide substitutions and / or deletions and / or additions that have the same function as SEQ ID NO.

2.

2. A PCR reagent or kit containing the primer pair of claim 1; the PCR reagent or kit functions as any one of the following b1)-b5): b1) Detection or auxiliary detection of the rice stem borer resistance gene SBR11; b2) Screening or assisted screening of rice varieties carrying the rice stem borer resistance gene SBR11; b3) Identify or assist in the identification of rice stem borer resistance; b4) Screening or assisted screening of rice resistant to rice stem borer; b5) Rice breeding or variety improvement.

3. The use of the primer pair of claim 1 or the PCR reagent or kit of claim 2 in any one of the following c1)-c10): c1) Detection or auxiliary detection of the rice stem borer resistance gene SBR11; c2) Prepare products for detecting or assisting in the detection of the rice stem borer resistance gene SBR11; c3) Screening or auxiliary screening of rice carrying the rice stem borer resistance gene SBR11; c4) Prepare rice products for screening or auxiliary screening of rice carrying the rice stem borer resistance gene SBR11; c5) Identification or auxiliary identification of rice stem borer resistance; c6) Prepare products for identifying or assisting in the development of rice stem borer resistance; c7) Screening or assisted screening of rice resistant to rice stem borer; c8) Prepare products for screening or assisting in screening rice resistant to rice stem borer; c9) Rice breeding or variety improvement; c10) is used to prepare products for rice breeding or variety improvement.

4. Application of InDel molecular labeling or substances for detecting InDel molecular labeling in any of the following c1)-c10): c1) Detection or auxiliary detection of the rice stem borer resistance gene SBR11; c2) Prepare products for detecting or assisting in the detection of the rice stem borer resistance gene SBR11; c3) Screening or auxiliary screening of rice carrying the rice stem borer resistance gene SBR11; c4) Prepare rice products for screening or auxiliary screening of rice carrying the rice stem borer resistance gene SBR11; c5) Identification or auxiliary identification of rice stem borer resistance; c6) Prepare products for identifying or assisting in the development of rice stem borer resistance; c7) Screening or assisted screening of rice resistant to rice stem borer; c8) Prepare products for screening or assisting in screening rice resistant to rice stem borer; c9) Rice breeding or variety improvement; c10) Prepare products for rice breeding or variety improvement; The InDel molecular marker is located at positions 98-109 of SEQ ID NO.

4.

5. The application according to claim 4, characterized in that: The substance used to detect the InDel molecular marker is the substance used to detect whether the rice to be tested contains the InDel molecular marker; Alternatively, the substance used to detect whether the rice to be tested contains the InDel molecular marker includes the primer pair described in claim 1.

6. A method for detecting or assisting in the detection of the rice stem borer resistance gene SBR11, comprising the following steps: detecting whether the rice genome contains an InDel molecular marker, and detecting or assisting in the detection of the rice stem borer resistance gene SBR11 based on whether the rice genome contains an InDel molecular marker; wherein the InDel molecular marker is located at positions 98-109 of SEQ ID NO.

4.

7. A method for identifying or assisting in the identification of rice stem borer resistance, comprising the following steps: detecting whether the rice genome contains an InDel molecular marker, and identifying or assisting in the identification of rice stem borer resistance based on whether the rice genome contains an InDel molecular marker; wherein the InDel molecular marker is located at positions 98-109 of SEQ ID NO.

4.

8. A method for screening or assisting in screening rice carrying the rice stem borer resistance gene SBR11, comprising the following steps: detecting whether the rice genome contains an InDel molecular marker, and selecting rice whose genome contains an InDel molecular marker; wherein the InDel molecular marker is located at positions 98-109 of SEQ ID NO.

4.

9. A method for screening or assisting in the selection of rice resistant to rice stem borer, comprising the following steps: detecting whether the rice genome contains an InDel molecular marker, and selecting rice whose genome contains an InDel molecular marker; wherein the InDel molecular marker is located at positions 98-109 of SEQ ID NO.

4.

10. A method for rice breeding or variety improvement, comprising the following steps: detecting whether the rice genome contains an InDel molecular marker, and selecting rice varieties containing an InDel molecular marker in their genome as parents for breeding; wherein the InDel molecular marker is located at positions 98-109 of SEQ ID NO.4.

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