Close-linkage molecular marker primer of brown planthopper resistant major gene Bph56 of rice as well as marking method and application of close-linkage molecular marker primer

Abstract

The invention provides a closely linked molecular marker primer of a rice brown planthopper resistant major gene Bph56 as well as a marking method and application of the closely linked molecular marker primer. According to the invention, genotypes of F2 individual plants obtained by hybridizing a rice insect-resistant variety C127 (or) and KW (or) are combined with brown planthopper resistance levels of an F3 family for genetic linkage analysis, a resistance major gene Bph56 carried by the insect-resistant variety C127 is identified, and a region of the resistance major gene Bph56 is reduced to a 77kb fragment limited by markers 6M26.278 and 6M26.355 on two sides and is closely linked with a molecular marker 6M26.355. The molecular marker 6M26.355 disclosed by the invention can be used for effectively detecting whether an insect-resistant variety C127 and derived varieties (lines) of the insect-resistant variety C127 contain the major resistance gene locus or not, so that the selection efficiency of the brown planthopper-resistant character is greatly improved, and the brown planthopper-resistant rice variety (line) containing Bph56 is obtained.

Description

Technical Field

[0001] This invention belongs to the field of molecular genetics and relates to the molecular marker of Bph56, the major gene for resistance to brown planthopper in rice. This invention also relates to the application of this molecular marker in the breeding of brown planthopper-resistant rice varieties. Background Technology

[0002] Rice is one of my country's most important food crops, but in recent decades it has suffered from large-scale pest and disease damage, severely impacting yield and quality. Rice planthoppers are among the most serious field pests in rice-producing areas. The main types of rice planthoppers include the brown planthopper, gray planthopper, and white-backed planthopper, with the brown planthopper causing the most significant damage. The brown planthopper is a monophagous pest, typically laying eggs, reproducing, and feeding only on rice and common wild rice. Adults and nymphs suck sap from the phloem at the base of the rice stem using their stylets, causing yellowing leaves or death of the rice plants. As vectors for rice viral diseases, brown planthoppers can lead to infection, ultimately resulting in reduced yield or crop failure. Since brown planthopper damage primarily occurs during the rice ripening and grain-filling stage, the extensive use of pesticides at this time poses a serious pollution problem to the environment and the rice grains. Developing insect-resistant rice varieties using brown planthopper-resistant genes is the most economical and effective method for the integrated management of brown planthoppers.

[0003] Since the 1970s, over 50 major genes for resistance to brown planthoppers have been reported, of which 17 have been cloned, including Bph1, Bph2, Bph3, Bph6, Bph9, and Bph14. Some of these major genes have been used for varietal genetic improvement in rice breeding programs for resistance to brown planthoppers both domestically and internationally. However, due to the emergence of new biotypes of brown planthoppers, resistant varieties are gradually losing their resistance or facing the risk of losing it altogether. A single resistance gene can gradually lose its effectiveness under long-term cultivation. For example, insect-resistant varieties carrying Bph1 quickly lose their resistance after a few years of widespread cultivation, while varieties carrying both Bph1 and Bph2 exhibit stronger and more durable resistance. Therefore, there is an urgent need in rice production for insect-resistant varieties carrying new and multiple resistance genes.

[0004] Conventional breeding methods often struggle to effectively introduce and aggregate different insect-resistant genes. This invention, based on the identification of molecular markers closely linked to or co-segregating with insect-resistant genes, utilizes marker-assisted selection technology to purposefully introduce and aggregate these genes, thereby breeding durable resistant varieties, delaying the degradation of insect-resistant varieties, and preventing the emergence of new biotypes of brown planthoppers. Summary of the Invention

[0005] The purpose of this invention is to provide a molecular marker for the major gene locus Bph56 for resistance to brown planthopper in rice. By detecting molecular markers closely linked to the major gene locus for resistance to brown planthopper, the resistance of rice plants to brown planthopper can be predicted, thereby accelerating the breeding of brown planthopper-resistant rice varieties.

[0006] The molecular marker for Bph56, the major gene for resistance to brown planthopper in rice, was obtained by PCR amplification using the following primer pairs:

[0007] Labeled primers: 6M26.355

[0008] Upstream primer sequence: GAAACCAAAGAGGCAGTG

[0009] Downstream primer sequence: CTCCATCGACCATACAGG;

[0010] The present invention also provides a molecular marker method for the major gene Bph56 for resistance to brown planthopper in rice. The method involves amplifying the genomic DNA of rice leaves to be tested using the above primer pair. If a 122bp fragment can be amplified using primer 6M26.355, it indicates that the rice to be tested contains the major gene Bph56 for resistance to brown planthopper.

[0011] The process of screening the above-mentioned marker primers is as follows:

[0012] (1) Based on the sequenced genome sequences of rice varieties such as Nipponbare and 9311, and a batch of publicly released SSR or InDel molecular markers, primers were selected and synthesized from the 12 chromosomes of rice according to relatively even genetic distances, as molecular markers for laboratory resistance gene localization and marker-assisted selection. We synthesized approximately 2000 pairs of molecular marker primers, which will be used for subsequent research and technology development.

[0013] (2) A new resistance source, C127, was obtained through previous insect resistance identification, which showed high resistance to brown planthopper. Then, using the brown planthopper-susceptible indica rice variety Kangwen Qingzhan (KW) as the female parent and the brown planthopper-resistant variety C127 as the male parent, hybrid offspring were obtained, and a KW / C127 F2 segregating population was constructed. Each F2 single plant obtained the corresponding F3 family through self-pollination, which was used for seedling insect resistance identification.

[0014] (3) Genomic DNA was extracted from the leaves of individual plants from the parental C127 and KW populations and F2 populations using the CTAB method (Murray & Thompson, 1980 Rapid isolation of high-molecular-weight plant DNA. Nucleic Acids Res 8: 4321-4325). The two parents were screened for polymorphism using the candidate markers developed in method (1). PCR reactions were performed on a Biometra Tone amplification instrument, and the amplification products were analyzed by electrophoresis on a 10% non-denaturing polyacrylamide gel. SSR or InDel markers with polymorphism between the parents were recorded and selected for subsequent genotyping analysis.

[0015] (4) The seedling group method was used to identify plant resistance. The brown planthopper source used in the experiment was a population collected in 2022 from the experimental field of Guangxi University Farm in Nanning City, Guangxi Province, and reproduced on susceptible cultivar 9311. When the rice reached the two-leaf and one-heart stage (about 11 days), 2nd-3rd instar brown planthopper nymphs were inoculated at a ratio of 8 nymphs / seedling. When the mortality rate of the susceptible control cultivar 9311 reached 90%, the survival rate of each family was evaluated according to the method of Huang (Huang et al, 2025 Exploring resistance mechanisms and identifying QTLs for brown planthopper in tropical and subtropical rice (Oryza sativa L.)germplasm. Theoretical and Applied Genetics 138(3): 1-16).

[0016] (5) Based on the average insect resistance level of the F3 family, leaf genomic DNA from 10 extremely insect-resistant individuals and 10 extremely insect-susceptible individuals were selected from the localized population and mixed at the same concentration to construct resistance and susceptibility DNA pools. Simultaneously, primers for molecular markers with polymorphism between parents were used to screen the resistance and susceptibility DNA pools and obtain molecular markers with polymorphism between the two pools. These polymorphic markers indicate a high likelihood of linkage to the resistance trait. Then, based on the chromosome where the linkage marker is located, primers for polymorphism between parents on that chromosome were selected to amplify each individual plant in the F2 segregating population. The PCR procedure was the same as above, and population genotype data was obtained. According to the linkage recombination law, a partial genetic linkage map of rice was constructed using the JoinMap 3.0 software, and the genetic distance between each molecular marker was obtained. Finally, combining the molecular marker genotype data of each individual plant in the F2 population and the corresponding insect resistance level for brown planthopper resistance identification, QTL site scanning of the target chromosome was performed using the QTL IciMapping Version 4.2 software's composite interval mapping method. This method was used to locate the resistance gene locus Bph56 between molecular markers 6M26.211 and 6M27.086 on chromosome 6 using an F3 population comprising 110 families. Figure 1 )

[0017] (6) Based on the preliminary localization results, InDel markers were designed for segments with differences compared to the reference genome sequence, and the polymorphic molecular marker 6M26.355 between the parents was obtained. Simultaneously, genotypes of 600 F3 individuals were screened using molecular markers 6M26.211 and 6M27.231, resulting in 48 recombinant individuals. The newly developed polymorphic markers were used to identify the genotypes of the recombinant individuals, and their insect resistance phenotypes were evaluated. Based on the genotypes and phenotypes of the recombinant individuals, the region containing the resistance gene was narrowed down to between molecular markers 6M26.278 and 6M26.355, corresponding to a physical distance of approximately 80 kb in the Nipponbare genome. No major genes for resistance to brown planthoppers were reported in this region; therefore, the resistance gene locus was named Bph56. Attached Figure Description

[0018] Figure 1 Preliminary mapping of Bph56, the major gene for resistance to brown planthopper in rice germplasm C127. A, Phenotypic distribution of F3 population; B, Preliminary mapping of Bph56.

[0019] Figure 2Genotypes and phenotypes of selected recombinant plants used for fine mapping of the major gene Bph56 for resistance to brown planthopper. Bph56 is located in an approximately 80 kb region between 6M26.278 and 6M26.355, with 6M26.355 tightly linked to the resistance gene. 'n' represents the total number of recombinant plants used for screening. Black, gray, and white represent resistant, neutral, and susceptible genotypes, respectively.

[0020] Figure 3 The band patterns of the amplified products of molecular marker 6M26.355 in different rice materials were detected by 10% PAGE electrophoresis. M represents the marker; 1-4 represent NY50, NY51, ZH11, and 9311, respectively; 5-16 represent the F3 family of KW / C127; 17-18 represent KW and C127, respectively; R and S represent the corresponding resistance and susceptibility band patterns, respectively. Detailed Implementation

[0021] The following embodiments further illustrate the content of the present invention, but should not be construed as limiting the present invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the present invention.

[0022] Example 1: Acquisition of Molecular Markers

[0023] (I) Construction and Phenotypic Identification of KW / C127 F2 Population

[0024] (1) In previous studies, the insect resistance identification of local cultivated rice germplasm resources collected in our laboratory showed that germplasm C127 has high resistance to brown planthopper populations collected from the experimental field of Guangxi University Farm. In order to find simple and effective molecular markers that are closely linked to Bph56, this invention uses the susceptible line KW as the female parent and the brown planthopper resistant rice germplasm C127 as the male parent for hybridization, and then self-pollinates the F1 to construct the F2 segregating population; each F2 single plant obtains the corresponding F3 family through self-pollination.

[0025] (2) The seedling group method was used to identify the insect resistance of the parents and F3 families. To ensure that each family in the parents and F3 population grew uniformly, all test materials were soaked and germinated separately before sowing. Forty seeds of each family (variety) were sown in a 56cm×37cm×8cm plastic tray filled with 5cm of paddy soil. Two replicates were sown for each material in each tray, and two replicates were randomly sown for each of the two parents. Seven days after sowing, the seedlings were thinned and diseased and weak seedlings were removed. When the seedlings reached the two-leaf stage, they were inoculated with 2nd-3rd instar brown planthopper nymphs at a ratio of 8 nymphs per seedling, and finally covered with nylon netting. When more than 90% of the susceptible variety KW died, the survival rate of each family was evaluated according to the method of Huang (Huang et al, 2025 Exploring resistance mechanisms and identifying QTLs for brown planthopper in tropical and subtropical rice (Oryza sativa L.)germplasm. Theoretical and Applied Genetics 138(3): 1-16) (Table 1). The above insect resistance identification was repeated twice, and the average value was taken as the seedling survival rate of each variety or family.

[0026] Table 1: Grading Standards for Rice Resistance to Brown Planthopper

[0027] Survival rate (examined when over 90% of KW die) Resistance level

[0028] 0-30% High Sensitivity (HS)

[0029] 31-50% Sensitivity (S)

[0030] 51-70% moderate antibody (MR)

[0031] 71-90% anti(R)

[0032] 91-100% High Resistance (HR)

[0033] (II) Molecular marker analysis of the KW / C127 F2 population

[0034] (1) Genomic DNA was extracted from the leaves of all individual plants in the parental line and F2 population using the CTAB method (Murray & Thompson, 1980 Rapid isolation of high-molecular-weight plant DNA. Nucleic Acids Res 8: 4321-4325).

[0035] (2) Based on the insect resistance level of the F3 family, leaf genomic DNA from 10 extremely insect-resistant individuals and 10 extremely insect-susceptible individuals were selected from the target population to construct resistance and susceptibility pools. 1786 pairs of molecular markers selected by our laboratory were screened, covering all 12 rice chromosomes. The results showed that the molecular markers 6M23.161, 6M26.211, and 6M27.231 located on the long arm of chromosome 6 exhibited consistent polymorphism between the parents and the resistance and susceptibility DNA pools. No polymorphic markers were found on other chromosomes, thus it was initially determined that the long arm of chromosome 6 may contain resistance gene loci. Subsequently, more polymorphic markers were screened in the target region, and polymorphic marker development was carried out with reference to the resequencing results of the parental C127. A total of 3 polymorphic molecular markers, 6M26.278, 6M27.086, and 6M27.831, were obtained. Furthermore, all individuals in the F2 segregating population were screened using the polymorphic markers to obtain population genotypic data. Based on linkage and exchange patterns, a partial genetic map of rice was constructed using the JoinMap 3.0 software based on population genotype data, and the genetic distances of each molecular marker were obtained. Finally, combining the molecular marker genotypes of each individual plant in the F2 population with the corresponding seedling survival rates for brown planthopper resistance identification, QTL site scanning of the target chromosome was performed using the composite interval mapping method in the IciMapping Version 4.2 software.

[0036] (3) The components of the PCR reaction system are as follows:

[0037] 1 μl of DNA template;

[0038] 0.5 μl of 10 μM primer;

[0039] 2×PCR Mix 5μl

[0040] Add ddH2O to make up to 10 μl.

[0041] The amplification products of the InDel primers used in this experiment are generally between 100-300 bp in length, and the PCR reaction procedure used is as follows:

[0042] 95°C for 5 minutes

[0043] 95°C for 30 seconds

[0044] 55°C 30 sec × 33 cycles

[0045] 72°C 45 sec

[0046] 72°C for 5 minutes

[0047] The conditions used for different primers may vary, mainly in terms of annealing temperature. Sometimes it is necessary to adjust the appropriate annealing temperature for each primer pair. The specific temperature can be set by referring to the values ​​given by the Primer-BLAST program on the NCBI website.

[0048] The amplified products were separated using 10% PAGE gels, and the amplified DNA bands were recorded by silver staining (Zhu et al, 2004 Identification and characterization of a new blast resistance gene located on ricechromosome 1 through linkage and differential analyses. Phytipathology 94:515-519). Polymorphic primers between parents were analyzed in the F2 population to obtain the population genotype.

[0049] The results of group inoculation identification during the seedling stage showed that the survival rates of C127 and KW were 88% and 14%, respectively, indicating that C127 is resistant to brown planthoppers while KW is highly susceptible to them. Figure 1 A). The resistance levels of brown planthoppers in the 110 F3 families showed a continuous distribution, with a minimum value of 0 and a maximum value of 100%. Based on the resistance level, all families could be divided into three phenotypes: resistant, resistant-susceptible segregation, and susceptible. The corresponding genotypes of F2 individuals were denoted as RR (homozygous resistant), Rr (heterozygous resistant), and rr (homozygous susceptible), respectively. The resistance-susceptibility segregation of the F2 population to brown planthoppers conformed to a ratio of 1:2:1 (χc²=0.166<χ²0.05,²=5.99) (Table 2).

[0050] Table 2. Segregation ratio of KW / C127 F2 segregating population to resistance levels of brown planthopper.

[0051] F2 genotype a; number of F2 individuals b; corresponding F3 family survival rate

[0052] RR32RS>70%

[0053] Rr6031%≤RS≤70%

[0054] rr18RS≤30%

[0055] a RR, homozygous insect-resistant; Rr heterozygous insect-resistant; rr, homozygous susceptible; b 1RR: 2Rr: 1rr, fitness test values ​​χ2=0.166, χ20.05=3.84; RS, insect resistance level.

[0056] Genotypes of individual F2 plants in the population were located using polymorphic SSR or InDel molecular markers obtained from screening parents. Simultaneously, QTL scanning was performed based on the resistance values ​​and survival rates of corresponding families. Results showed a significant QTL locus between molecular markers 6M26.211 and 6M27.086 on the long arm of chromosome 6, with a maximum LOD value of 14 and a contribution rate of 35% to the resistance phenotype. Figure 1 B).

[0057] Example 2: Fine mapping of the Bph56 gene

[0058] (1) Screening recombinant single plants and developing new polymorphic molecular markers using molecular markers

[0059] Based on the preliminary mapping results of the resistance gene, the genotypes of 600 F3 single plants were screened using molecular markers 6M26.211 and 6M27.231, resulting in 48 recombinant single plants. Simultaneously, using the resequencing results of variety C127, a new polymorphic molecular marker, 6M26.355, was developed within the preliminarily mapped region. The newly developed polymorphic marker was used to identify the genotypes of the recombinant single plants, and their insect-resistant phenotypes were evaluated.

[0060] (2) Results and Analysis

[0061] The genotypes of 48 recombinant single plants were detected using screened polymorphic markers, and combined with their insect resistance identification results, Bph56 was finally located between 6M26.278 and 6M26.355, and was closely linked to marker 6M26.355. Figure 2 According to the Nipponbare genome sequence, the physical distance between 6M26.278 and 6M26.355 is approximately 80 kb. Therefore, using the molecular marker 6M26.355 to identify the presence of Bph56 is highly efficient, which greatly accelerates the breeding progress of brown planthopper-resistant rice varieties.

[0062] Example 3: Validation of Molecular Markers

[0063] (I) Materials and Methods

[0064] (1) Materials

[0065] Negative varieties: a total of 8 accessions, including susceptible families from the offspring of susceptible strains (species) KW, ZH11, 9311, NY51 and KW×C127 hybrid combinations.

[0066] Positive varieties: a total of 10, insect-resistant families in the offspring of the highly insect-resistant strains (species) NY50, C127 and KW×C127 hybrid combination.

[0067] Molecular marker primer: 6M26.355

[0068] (2) Method

[0069] Genomic DNA was extracted from rice leaf samples using the CTAB extraction method. The sample DNA was amplified using primer 6M26.355. The amplified products were separated using a 10% non-denaturing PAGE gel, and the amplified DNA bands were recorded by silver staining (method as in Example 1).

[0070] (II) Results and Analysis

[0071] Using the above method, PCR amplification was performed on genomic DNA from 18 different samples of rice varieties NY50, NY51, and ZH11. The results showed that a 122bp fragment was amplified in all positive samples, while a 133bp fragment was amplified in the negative samples. This demonstrates that the molecular marker method provided by this invention can accurately screen samples containing the major gene for resistance to brown planthopper, thereby greatly improving the selection efficiency of insect-resistant rice materials.

[0072] like Figure 3 As shown, the non-denaturing PAGE electrophoresis band patterns of the amplified products of molecular marker 6M26.355 in different rice materials are shown. M is a 2000bp marker; the bottom band is 100bp, and the second-to-last band is 250bp. Bands 1-4 represent NY50, NY51, ZH11, and 9311, respectively. Bands 5-16 represent the F3 family of KW / C127; and bands 17-18 represent KW and C127, respectively. S represents KW (susceptible variety, i.e., negative line); R represents C127 (insect-resistant variety, i.e., positive variety); and the arrows indicate the differential bands between resistant and susceptible parents. In positive samples, a 122bp band is present at the same level as R (representing the highly insect-resistant variety C127, a positive variety); however, this band is absent in negative samples.

[0073] In summary, the molecular marker 5M8.54 of this invention can effectively detect whether the insect-resistant variety C127 and its derivative varieties (lines) contain this major resistance gene locus, greatly improving the selection efficiency of brown planthopper-resistant rice plants and obtaining brown planthopper-resistant rice varieties (lines) containing Bph56.

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A tightly linked molecular marker primer for the major gene Bph56, a rice species known for its resistance to brown planthopper, along with its marking method and application, obtained by PCR amplification using the following primer pair: Labeled primers: 6M26.355 Upstream primer sequence: GAAACCAAAGAGGCAGTG Downstream primer sequence: CTCCATCGACCATACAGG.

2. The tightly linked molecular marker primers for the major gene Bph56 of rice resistance to brown planthopper, as described in claim 1, and the marking method and application thereof, characterized in that... Application of molecular markers in breeding rice varieties resistant to brown planthopper.

3. The tightly linked molecular marker primers for the major gene Bph56 of rice resistance to brown planthopper, as described in claim 1, and the marking method and application thereof, characterized in that... A molecular marker method for the major gene Bph56 for resistance to brown planthopper in rice was developed. The above primers were used to amplify the genomic DNA of the rice leaves to be tested, and the amplification products were detected. If primer 6M26.355 could amplify a 122bp fragment, it indicated that the rice to be tested contained the major gene Bph56 for resistance to brown planthopper.

4. A method for screening rice materials resistant to brown planthopper, wherein the primer pair described in claim 1 is used to amplify the genomic DNA of the rice leaves to be tested, and if primer 6M26.355 amplifies a 122bp fragment, the rice to be tested is identified as a resistant rice material carrying the major gene Bph56 for resistance to brown planthopper.

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