Rice Brown Planthopper Resistance Gene BPH33.2 and Its Molecular Marker and Application

Abstract

The invention provides a rice brown planthopper resistance gene BPH33.2 The present invention uses a genetic population obtained by hybridization, backcrossing and selfing of the susceptible variety 9311 (♀) with the Sri Lankan highly resistant varieties KOLAYAL and POLIYAL (♂), and locates a new brown planthopper resistance gene on the short arm of rice chromosome 4 by pooled sequencing (BSA‑seq). BPH33.2 , through genetic transformation BPH33.2 Gene, so that susceptible rice appears resistant to brown planthopper phenotype; at the same time, gene knockout BPH33.2 Gene, causing insect-resistant rice to lose the brown planthopper resistance phenotype, thus confirming BPH33.2 The present invention also provides a rice brown planthopper resistance gene BPH33.2 Tightly linked molecular markers, through experimental detection of molecular markers linked or co-segregated with these resistance gene loci, can accurately predict the brown planthopper resistance of rice plants at the seedling stage and accelerate the breeding of brown planthopper-resistant rice varieties.

Description

Technical Field

[0001] The invention belongs to the field of plant genetic engineering, and specifically relates to a rice brown planthopper resistance gene BPH33.2 and a tightly linked molecular marker thereof, and application of the molecular marker in rice brown planthopper resistance breeding and variety improvement. Background Art

[0002] Rice (Oryza sativa) is one of the most important food crops in the world, and the stability of its yield is crucial to safeguarding the national economy and people's lives. As a model research crop of the Gramineae family, rice has a relatively small genome, very mature transgenic technology, and increasingly in-depth functional genomic research. Therefore, rice has shown unparalleled superiority and importance in social and economic development, national security, and scientific research.

[0003] Brown planthopper (BPH, Nilaparvata lugens) is the most important pest in rice-growing areas. Brown planthopper has strong explosive power and migratory ability, and its damage area is large and the degree of damage is serious. Brown planthopper has become a serious threat to the safe production of rice in my country.

[0004] At present, chemical agents are mainly used to control brown planthoppers in production. However, the abuse of pesticides not only increases farmers' production and labor costs, but also causes irreversible harm to the environment. At the same time, pesticides can kill the natural enemies of brown planthoppers, enhance the resistance of pests, and induce the resurgence of brown planthoppers. Using rice resistance genes to breed insect-resistant varieties is currently the most economical and effective prevention and control strategy. The International Rice Research Institute (IRRI) has proved in the practice of controlling brown planthoppers that planting rice varieties with different resistances can effectively control the crazy growth of brown planthopper populations and slow down the mutation rate of their biotypes or virulence to a certain extent, thereby achieving the purpose of lasting resistance. Therefore, the continuous discovery and cloning of new resistance genes and their application in breeding are key research topics for solving the comprehensive prevention and control of brown planthoppers. In addition, the cloning of resistance genes and the elucidation of insect resistance mechanisms can enrich the research results of rice functional genomics and ensure that my country is at the world's leading level in related fields.

[0005] There are abundant rice germplasm resources resistant to brown planthoppers in nature, and identifying and cloning insect-resistant genes in these resistant rice has always been the focus of scientists' research. As of 2022, researchers have identified at least 40 brown planthopper resistance genes or QTLs, all of which come from wild rice and indica rice resources. With the continuous deepening of rice functional genomics research, some brown planthopper resistance genes have also been cloned one after another. So far, 15 resistance genes have been cloned in rice. Among them, domestic research teams have made major contributions and cloned 13 of them. Professor He Guangcun's team at Wuhan University cloned the first brown planthopper resistance gene in rice, Bph14, which encodes a helix-helix-nucleotide binding site leucine repeat motif (CC-NB-LRR). Its unique LRR region may play an important role in signal recognition response and activation of defense response after brown planthopper invasion (Duetal.2009PNAS 106:22163-22168).

[0006] Previously, our laboratory introduced more than 300 varieties or strains that may be resistant to various BPH biotypes from IRRI. Through seedling resistance evaluation, we screened more than 20 highly resistant varieties (Hu et al. 2016 Rice 11:55). Among them, two local indica rice varieties from Sri Lanka, KOLAYAL and POLIYAL, showed high resistance to brown planthoppers in Fuzhou and Wuhan, China. In order to locate the resistance gene, we constructed BC1F sequences of susceptible varieties 9311 and KOLAYAL, 9311 and POLIYAL 2:3 The present invention successfully cloned a new brown planthopper resistance gene through the forward genetics map-based cloning method, and developed a series of molecular markers that are closely linked or co-segregated with the resistance gene. With the help of molecular marker-assisted selection (MAS) technology, insect-resistant genes can be precisely introduced or aggregated in a targeted manner, thereby selecting durable resistant varieties, effectively suppressing the brown planthopper population, saving labor and pesticide costs, and increasing rice yields. Summary of the invention

[0007] The purpose of the present invention is to provide a rice brown planthopper resistance gene BPH33.2 and its molecular markers and applications. By experimentally detecting molecular markers linked or co-segregated with these resistance gene loci, the brown planthopper resistance of rice plants can be accurately predicted at the seedling stage, thereby accelerating the selection of brown planthopper-resistant rice varieties.

[0008] The present invention adopts the forward genetics method to locate the resistance gene BPH33.2 by constructing a genetic population of resistant and susceptible materials. By genetically transforming the BPH33.2 gene, the susceptible rice shows a brown planthopper resistance phenotype; at the same time, the BPH33.2 gene is knocked out by gene to make the insect-resistant rice lose the brown planthopper resistance phenotype, thereby confirming the function of BPH33.2.

[0009] The present invention provides a rice brown planthopper resistance gene BPH33.2, the nucleotide sequence of which is shown in SEQ ID NO.1, the gene is 8,674 bp in length, has 4 exons and 3 introns, the first exon segment is 2514-2778 bp, the second exon segment is 3456-3724 bp, the third exon segment is 4535-7732 bp, and the fourth exon segment is 7940-8674 bp. Further, the cDNA of the rice brown planthopper resistance gene BPH33.2 is 4470 bp in length, the sequence is shown in SEQ ID NO.2, and it encodes 1072 amino acids. The present invention also provides a protein encoded by the rice brown planthopper resistance gene BPH33.2, the amino acid sequence of which is shown in SEQ ID NO.3.

[0010] Because the codons in the gene coding region are degenerate, the present invention also includes replacing, adding and / or deleting one or more nucleotides in the polynucleotide sequence encoding the above protein, and having a nucleotide sequence encoding a protein with the same function as the above.

[0011] It should be understood that, without affecting the activity of the BPH33.2 protein, those skilled in the art can make various substitutions, additions and / or deletions of one or several amino acids to the amino acid sequence shown in SEQ ID NO.3 to obtain an amino acid sequence with equivalent functions, thereby applying it to the genetic improvement of rice resistance to brown planthoppers.

[0012] The present invention also provides a molecular marker closely linked to the rice brown planthopper resistance gene BPH33.2, which is obtained by conventional PCR amplification using one of the following primer pairs:

[0013] 1) Labeling primer H99,

[0014] Left primer sequence, 5'-CACTGTGGTTACAACAGAGGT-3'

[0015] Right primer sequence, 5'-TCTCTTCTCGTTGCTGCTCA-3'

[0016] 2) Labeling primer H79,

[0017] Left primer sequence, 5'-CCGTGAGTTCACTTGTAA-3'

[0018] Right primer sequence, 5'-GTACGATTTGACCAGCGAG-3'

[0019] 3) Labeled primer 33-3,

[0020] Left primer sequence, 5'-CCTCGCTTACGAAATTAGTG-3'

[0021] Right primer sequence, 5'-TGTGGCAGAGCAAGAGGG-3'

[0022] 4) Labeling primer 33-4,

[0023] Left primer sequence, 5'-GCTTTCGGATTATCTTCACTA-3'

[0024] Right primer sequence, 5'-AGCTCCCTAAGCTCAACCA-3'

[0025] The present invention also provides a molecular marker method for identifying whether a rice variety contains the BPH33.2 major effect gene, that is, using one of the above primer pairs to amplify the rice genome DNA to be tested, if the primer H99 can amplify a 96bp DNA fragment, or H79 can amplify a 140bp DNA fragment, or 33-3 can amplify a 330bp DNA fragment, or 33-4 can amplify an 868bp DNA fragment, it indicates that the brown planthopper resistance gene site BPH33.2 of the insect-resistant rice variety exists; if the primer H99 can only amplify a 112bp DNA fragment, or the primer H79 can only amplify a 148bp DNA fragment, or the primer 33-3 can not amplify any DNA fragment, or the primer 33-4 can not amplify any DNA fragment, it indicates that the brown planthopper resistance gene site BPH33.2 does not exist in the insect-resistant rice variety. Among them, markers H99 and H79 are tightly linked markers on both sides of the physical position of the BPH33.2 gene. They are co-dominant markers, that is, they can simultaneously distinguish between homozygous and heterozygous resistant genotypes; markers 33-3 and 33-4 are specific markers designed in the coding region of the BPH33.2 gene. They are dominant markers, that is, they cannot distinguish between homozygous and heterozygous resistant genotypes, but can distinguish between homozygous susceptible genotypes.

[0026] The present invention provides a method for cultivating plants with brown planthopper resistance by using transgenic technology, comprising:

[0027] 1) transforming plant callus cells with a polynucleotide containing the BPH33.2 gene; the polynucleotide sequence is shown in SEQ ID NO.1 or SEQ ID NO.2;

[0028] 2) Regenerating transformed plant cells into plants;

[0029] 3) cultivating the regenerated plants and expressing the polynucleotide, and harvesting T1 generation seeds;

[0030] 4) sowing T1 generation seeds, detecting the BPH33.2 gene using the above-mentioned molecular markers 33-3 and 33-4, and harvesting T2 generation seeds that are homozygous for the gene.

[0031] The present invention also provides a method for producing plants with brown planthopper resistance by conventional breeding (non-transgenic), the method comprising hybridizing plants with brown planthopper resistance gene BPH33.2 with other plants to produce offspring plants with brown planthopper resistance. The plants are monocotyledonous plants. Preferably, the plants are rice.

[0032] The advantages of the present invention are:

[0033] (1) The present invention successfully cloned a rice brown planthopper resistance gene BPH33.2 using the map-based cloning method in forward genetics and verified its function in rice brown planthopper resistance.

[0034] (2) The BPH33.2 gene of the present invention is a completely dominant insect-resistant gene. There is no significant difference in resistance to brown planthoppers between homozygous and heterozygous genes, and the gene can be better applied to insect-resistant breeding of hybrid rice.

[0035] (3) The BPH33.2 gene confers strong resistance to brown planthoppers at the seedling, tillering and adult stages of rice, and has good resistance to multiple biotypes and pathogenic types of brown planthoppers, as well as white-backed planthoppers, which is of great significance for insect-resistant breeding.

[0036] (4) The molecular markers that are closely linked or co-segregated with the resistance gene in the present invention are highly reliable and easy to identify. By simply detecting these molecular markers through simple experiments, the presence or absence of brown planthopper resistance in rice plants can be accurately predicted, and then the brown planthopper resistance can be predicted. The detection is convenient, fast and efficient without being affected by the environment.

[0037] (5) The present invention can improve the auxiliary selection efficiency of conventional breeding and save costs. By detecting the brown planthopper resistance gene loci in rice plants through molecular markers, highly resistant plants can be quickly identified only in the seedling stage, and susceptible plants can be eliminated in time, which not only saves production costs, but also improves the selection efficiency of resistant materials and shortens the breeding cycle of rice varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1:Nilophaga resistance phenotype of the BPH33 near-isogenic line. A, B, seedling resistance of the near-isogenic line; C, D, honeydew secreted by the brown planthopper on the near-isogenic line, the area after ninhydrin staining (purple part). 9311 and NIL-BPH33 represent near-isogenic lines that do not carry and carry the BPH33 gene homozygous, respectively; scale: 5 cm.

[0039] Figure 2 :Precise localization of BPH33. The arrows above indicate molecular markers, the numbers below indicate the physical positions of the corresponding molecular markers on chromosomes, the numbers in brackets indicate the number of plants whose genotype and phenotype of the recombinant plants are inconsistent on the corresponding molecular markers; the white square columns represent the homozygous 9311 genotype, the black columns represent the homozygous KOLAYAL (KOL) genotype, and the gray columns represent the recombination breakpoints; n is the number of plants contained in the segregation population used to screen the recombinant plants, 16 to 100 are the numbers of the recombinant plants, and the right side indicates the phenotype of the recombinant plants' resistance to brown planthoppers at the seedling stage, S and R represent susceptible and resistant, and the smaller the resistance score, the more resistant it is.

[0040] Figure 3 : Schematic diagram of the BPH33 candidate gene segment.

[0041] Figure 4 : Phenotypes of knockout families of the BPH33 candidate gene. A, seedling resistance of the bph33.1 / bph33.3 ​​double knockout mutant; B, seedling resistance of the bph33.2 / bph33.3 ​​double knockout mutant; C, seedling resistance of the bph33.2 single knockout mutant.

[0042] Figure 5 :The phenotypes of the transgenic T2 family with background expression of the BPH33 candidate gene. A, seedling resistance of the transgenic T2 family with background expression of BPH33.1; B, seedling resistance of the transgenic T2 family with background expression of BPH33.3; C, seedling resistance of the transgenic T2 family with background expression of BPH33.2.

[0043] Figure 6 : Gel images of BPH33.2 molecular markers. M, DNA ladder marker; Lanes 1-8 are 8 samples in the F2 population, Lanes 9 and 10 are the resistant donor KOLAYAL and the susceptible control 9311, respectively. A, B, C and D are gel images of markers H99, H79, 33-3 and 33-4, respectively. S, susceptible; R, resistant.

[0044] Figure 7 :Nilophaga resistance of BPH33.2 improved lines and their hybrid rice. Figure A shows the seedling resistance of conventional improved lines carrying homozygous BPH33.2 to Nilophaga resistance; Figure B shows the seedling resistance of hybrid rice improved lines carrying heterozygous BPH33.2 to Nilophaga resistance. DETAILED DESCRIPTION

[0045] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0046] Example 1: Fine localization and cloning verification of BPH33.2

[0047] Construction of near-isogenic lines of 1BPH33 and evaluation of their effects

[0048] In order to precisely locate the BPH33 gene and to construct a molecular marker-assisted selection system for the BPH33 gene for application in insect-resistant breeding, the applicant constructed a near-isogenic line of BPH33 in the background of the 9311 rice variety. The specific steps are: first, using molecular markers H99 and H79 on both sides of BPH33, a single plant with a homozygous insect-resistant parent genotype at BPH33 locus is selected from a BC1F3 family (the insect-resistant parent KOLAYAL is hybridized with the insect-susceptible parent 9311, the obtained F1 is backcrossed with 9311 once to obtain BC1F1, and then self-pollinated twice to obtain BC1F3), and backcrossed with 9311 to obtain BC2F1, and then the BC2F1 heterozygous for 9311 and BPH33 locus is further backcrossed to obtain BC3F1, and a single plant with a heterozygous genotype at BPH33 locus is self-pollinated to obtain BC3F2, and then a single plant with a homozygous resistant parent genotype at BPH33 locus is selected, and then self-pollinated to obtain BC3F3, i.e., a near-isogenic line (NIL-BPH33) of BPH33. The seedling resistance and the honeydew area of ​​brown planthoppers are measured for the near-isogenic line and the parents, respectively. The improved seedling group identification method is used as the main method for identifying brown planthopper resistance in this example. The insect-sensitive control, insect-resistant control and materials to be identified are sown at the same time. 20 full seeds are sown for each material in a separate black plastic seedling box (caliber 7.2cm, bottom diameter 5cm, height 8cm). When the plant grows to the three-leaf stage, 12 seedlings with consistent growth are retained in each row, and the seedling tray is placed in a 200-mesh gauze. 2-year-old brown planthopper nymphs are inoculated according to the number of 8-10 heads per seedling. When more than 90% of the plants in the insect-sensitive control die, the resistance grade can be evaluated according to the degree of damage to each seedling, and the resistance score value of each material is obtained after weighted average calculation. Honeydew area test: Select rice plants in the four-leaf stage with consistent growth, transplant them into plastic cups (caliber 9cm, 500ml), put one seedling in each plastic cup, and cultivate them with the prepared nutrient solution. The mouth of the cup is sealed with a round filter paper, and then a small hole is drilled in the center of the filter paper. The rice stalks are inserted into the filter paper with a hole in the center, and the cup containing the brown planthopper nymphs covers the stalks. Then, a similar plastic cup is placed upside down on it. Five nymphs are placed in each cup. After feeding for 48 hours, the filter paper is removed, and the number is written on the edge of the filter paper with a pencil. It is placed in an oven (constant temperature of 50°C) to dry for 20 minutes. After drying, it is taken out and evenly sprayed with 0.25% ninhydrin / ethanol solution, and placed in a 60°C oven for color development for 30 minutes. The purple part displayed on the filter paper is the area of ​​nectar secreted by the brown planthopper. After taking a picture and scanning it, the stained area is calculated on the imageJ software. The results are as follows: Figure 1 As shown in the figure, 9311 was susceptible to brown planthoppers, while NIL-BPH33 was resistant. The honeydew area secreted by brown planthoppers on 9311 was significantly larger than that on NIL-BPH33.

[0049] Fine positioning of 2BPH33

[0050] In order to precisely locate the BPH33 gene, the applicant selected a BC3F1 family with heterozygous genotypes at molecular markers H14 and H84 (Hu et al. 2018 Rice 11: 55), and obtained a BC3F2 segregation population containing 6600 individual plants after self-pollination. According to the results of the preliminary positioning, all individual plants were genotyped using tightly linked markers on both sides of the target gene fragment, and individual plants that underwent recombination exchange between the molecular markers on both sides were selected (i.e., the H14 site was homozygous 9311 genotype B, the H84 site was heterozygous genotype H; or the H14 site was heterozygous genotype H, the H84 site was homozygous 9311 genotype B, etc.), and the individual plants that underwent recombination exchange were transplanted to the field for planting. After maturity, the individual plants were collected as self-pollination seeds for subsequent genotype and phenotypic analysis. Develop molecular markers that are evenly distributed and polymorphic between the two parents in the target gene segment, continue to perform genotyping analysis on these recombinant strains, and construct a physical genetic map of the target segment based on the physical position of the molecular markers on the chromosome. Figure 2 ). Select 1 to 2 plants of the same exchange type and conduct progeny testing. BPH33 in this example is a dominant gene. For the HA and AH exchange types, we cannot accurately determine which genotype provides the phenotypic effect. Therefore, for the identification of the brown planthopper seedling resistance of the recombinant plants, we adopt the method of progeny testing. That is, after the recombinant plant plants are harvested and numbered, 1 to 2 plants of the same exchange type are selected and sown in a 96-well PCR plate, and about 50 seeds are sown for each number. Then, the molecular markers in the target segment are used for genotyping, and AB and BA type plants are selected. About 8 to 12 plants can be selected for each strain and planted in rows. After that, seedling resistance identification is carried out, and then the phenotype of the recombinant plant is determined based on the average resistance score of the selected progeny. Combining the genotype and phenotype of the recombinant plant, BPH33 was finally precisely located between markers H99 and H101, and the corresponding reference sequence Nipponbare has an interval of 60kb ( Figure 2 ).

[0051] Candidate gene analysis of 3BPH33

[0052] In order to find out which candidate genes are in the BPH33 fine-mapping interval, the applicant performed third-generation genome sequencing on the parent KOLAYAL carrying BPH33. The results showed that the 60kb interval of the reference sequence Nipponbare was as long as 160kb in KOLAYAL, 100kb more. In these 160kb, FGENESH was used to predict genes, and a total of 4 genes were found. The first gene is very similar to the sequence of Nipponbare and encodes a C2H2 zinc finger transcription factor. The next three genes all encode a class of atypical LRR proteins, and there are multiple copies, which are significantly different in structure from the reference genome. Therefore, they were identified as reliable candidate genes for BPH33 and named BPH33.1, BPH33.2 and BPH33.3, respectively, with 6, 2 and 4 copies, respectively ( Figure 3 ).

[0053] Functional validation of candidate genes of 4BPH33

[0054] In order to find out which one of BPH33.1, BPH33.2 and BPH33.3 is BPH33, or whether they are all BPH33, the applicant conducted transgenic verification using gene knockout and genetic complementation experiments.

[0055] First, CRISPR-Cas9 technology was used to simultaneously knock out BPH33.1 and BPH33.3, as well as BPH33.2 and BPH33.3 in the insect-resistant BPH33 near-isogenic line. The results showed that most bph33.1 / bph33.3 ​​homozygous mutants were insect-resistant, with no difference in resistance from the near-isogenic line; while all bph33.2 / bph33.3 ​​homozygous mutants were susceptible to insects, with significant differences in resistance from the near-isogenic line ( Figure 4 ). Further analysis revealed that all bph33.1 / bph33.3 ​​double mutants with susceptible phenotypes had a large deletion between the BPH33.1 and BPH33.3 genes, that is, the BPH33.2 gene was also missing, and they were actually bph33.1 / bph33.2 / bph33.3 ​​triple mutants ( Figure 3 ). These results indirectly indicate that neither BPH33.1 nor BPH33.3 is BPH33, and BPH33.2 is most likely BPH33. The applicant further designed a specific target site on the second exon of BPH33.2 and obtained a homozygous single knockout mutant of BPH33.2. The results of resistance identification found that all BPH33.2 mutants were susceptible to insects ( Figure 4 ), thus confirming that BPH33.2 is BPH33.

[0056] On the other hand, the applicant cloned the genomic sequences (promoter + 5'UTR + CDS + 3'UTR) of BPH33.1, BPH33.2 and BPH33.3 in the insect-resistant near-isogenic line of BPH33, and connected them into the PCAMBIA1300 vector, which is the recombinant expression vector of the candidate gene. Then, these three vectors were transformed into the susceptible rice varieties Kasalash and Zhonghua 11 through Agrobacterium-mediated genetic transformation. The homozygous T2 generation of transgenic families were identified for resistance to brown planthoppers at the seedling stage. The results showed that the transgenic families of BPH33.1 and BPH33.3 were susceptible to insects, while the transgenic family of BPH33.2 was resistant to insects ( Figure 5 ), these results further proved that BPH33.2 is BPH33. The nucleotide sequence of BPH33.2 is shown in SEQ ID NO.1, and its cDNA sequence is shown in SEQ ID NO.2.

[0057] Example 2: Development and validation of BPH33.2 molecular markers

[0058] 1BPH33.2 molecular marker development and identification

[0059] The molecular markers linked to the BPH33.2 gene were developed with reference to the RiceVarMap database of Huazhong Agricultural University ( http: / / ricevarmap.ncpgr.cn / ). First, search for chromosome 4 0-1 in the database The mutation information of all InDels (insertion and deletion) in the M interval is obtained, and then the mutation sites with InDels of 3 to 30 bp are screened out. Then, specific primers are designed at a distance of 50 to 60 bp on both sides of this segment, so that the size of the amplified target fragment containing this deletion is about 100 bp. The primer length is preferably 18 to 20 bp, and the GC content is generally 40 to 60%. The selected primers are blasted on the database to eliminate primers with low specificity. Then, primers uniformly distributed in the 0-1M interval are selected for DNA fragment amplification between the resistant and sensitive parents. The PCR products are separated on 4% polyacrylamide gel and silver stained. The color is developed in 0.5 mol / L NaOH-formaldehyde solution, and the band type is photographed and preserved on the gel imaging instrument. Primers with obvious differences in the band type of the resistant and sensitive parents (upper and lower differences, no common bands) are candidate molecular markers. H99 and H79 are the molecular markers that are closely linked on both sides of BPH33.2 that were screened out.

[0060] In addition, based on the CDS sequence of the cloned BPH33.2, it was compared with the sequences of two other homologous genes BPH33.1 and BPH33.3, as well as the sequences of multiple third-generation sequencing varieties (R498, 9311, Zhenshan 97 (ZS97), Minghui 63 (MH63), and Nipponbare (NPB)), and the sequence of another cloned brown planthopper resistance gene BPH30 in this section. Then, primers were designed in the BPH33.2-specific section, and two pairs of primers, 33-3 and 33-4, were obtained. Whenever the target fragment can be amplified using these two pairs of primers, it proves that the functional allele of BPH33.2 exists, and if the target fragment cannot be amplified, it indicates that the allele is not carried.

[0061] H99 labeled primer:

[0062] Left primer sequence, 5'-CACTGTGGTTACAACAGAGGT-3'

[0063] Right primer sequence, 5'-TCTCTTCTCGTTGCTGCTCA-3'

[0064] H79 marker primer:

[0065] Left primer sequence, 5'-CCGTGAGTTCACTTGTAA-3'

[0066] Right primer sequence, 5'-GTACGATTTGACCAGCGAG-3'

[0067] 33-3 marker primer:

[0068] Left primer sequence, 5'-CCTCGCTTACGAAATTAGTG-3'

[0069] Right primer sequence, 5'-TGTGGCAGAGCAAGAGGG-3'

[0070] 33-4 marker primer:

[0071] Left primer sequence, 5'-GCTTTCGGATTATCTTCACTA-3'

[0072] Right primer sequence, 5'-AGCTCCCTAAGCTCAACCA-3'

[0073] Validation of 2BPH33.2 molecular marker

[0074] To verify the efficiency of BPH33.2 molecular marker in screening BPH33.2 functional alleles, we conducted a 9311 / KOLIYAL BC3F 2:3Molecular marker genotyping of 92 families in the segregating population was performed. Combined with the seedling resistance phenotype of each family, t-test and association analysis confirmed that markers H99, H79, 33-3 and 33-4 co-segregated with the resistance-susceptibility phenotype of brown planthopper ( Figure 6 ).

[0075] Example 3: Molecular marker-assisted selection of improved rice lines resistant to brown planthoppers carrying the BPH33.2 functional allele. Using molecular marker-assisted selection (MAS), combined with conventional backcross breeding technology, the BPH33.2 functional allele was introduced into excellent insect-susceptible rice varieties (9311, Huanghuazhan, Huazhan, Wushansimiao, 19xiang, Guang 8B), and a series of rice lines with significantly improved brown planthopper resistance and excellent other agronomic traits were obtained, which can be used as intermediate materials for the cultivation of new brown planthopper-resistant rice varieties. The specific operation method is: the variety to be improved is used as the recurrent parent, and it is hybridized with the parent KOLAYAL carrying BPH33.2 to obtain F1 hybrids. Then the recurrent parent is hybridized with F1 to obtain BC1F1, and the BPH33.2 heterozygous single plants are selected by MAS and hybridized with the recurrent parent to obtain BC2F1. Then, MAS was used to select BC2F1 heterozygous for BPH33.2 and hybridized with the recurrent parent to obtain BC3F1. BC3F1 plants heterozygous for BPH33.2 and with agronomic traits closest to those of the recurrent parent were selected for self-pollination to obtain BC3F2. MAS was used to select plants with homozygous KOLAYAL genotype for BPH33.2 and excellent agronomic traits for self-pollination to obtain BC3F3. BC3F3 plants with excellent agronomic traits were further selected for self-pollination to obtain BC3F4. After four consecutive generations of self-pollination, BC3F8 was obtained, which is an improved strain that is homozygous for BPH33.2 and has excellent and stable agronomic traits. The results of the identification of brown planthopper resistance at the seedling stage showed that the improved strain had significantly improved insect resistance compared with the control that did not carry the BPH33.2 gene ( Figure 7 A).

[0076] In addition, the improved line of 9311 (carrying BPH33.2) was hybridized with the two-line sterile lines Guangzhan 63S and Y58S to obtain BPH33.2 heterozygous two-line hybrid rice. The results of insect resistance identification showed that the improved two-line hybrid rice had significantly improved brown planthopper resistance compared with the control ( Figure 7 B) These results indicate that the function of BPH33.2 is not restricted by the genetic background of rice and is a completely dominant gene, which has a good prospect in breeding insect-resistant hybrid rice.

[0077] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A rice gene for resistance to brown planthoppers BPH33.2 , characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

1.

2. The rice brown planthopper resistance gene according to claim 1 BPH33.2 , characterized in that: Its cDNA sequence is shown as SEQ ID NO.

2.

3. The rice brown planthopper resistance gene according to claim 1 BPH33.2 , characterized in that: The amino acid sequence of the protein encoded by it is shown in SEQ ID NO.

3.

4. A rice plant containing the brown planthopper resistance gene according to claim 1 BPH33.2 recombinant vector, recombinant bacteria, expression cassette or transgenic cell line.

5. The rice brown planthopper resistance gene according to claim 1 BPH33.2 Application in improving rice resistance to brown planthopper.

6. A method for preparing transgenic rice with brown planthopper resistance, characterized in that: The expression box containing the polynucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.2 is transferred into the susceptible rice variety to obtain transgenic rice with brown planthopper resistance.

7. A rice brown planthopper resistance gene as claimed in claim 1 BPH33.2 Tightly linked molecular marker primers, characterized in that: The sequences of the molecular marker primers are as follows: Left primer 5'-GCTTTCGGATTATCTTCACTA-3', Right primer 5′-AGCTCCCTAAGCTCAACCA-3′.

8. Use of the molecular marker primer according to claim 7 in breeding rice resistant to brown planthopper.

9. A method for screening rice for resistance to brown planthoppers, characterized in that: Amplify the rice genomic DNA to be tested by the molecular marker primers described in claim 7, and detect the amplified product; if the molecular marker primers can amplify a DNA fragment of 868 bp, it indicates that the rice brown planthopper resistance gene described in any one of claims 1 to 3 exists in the rice. BPH33.2 If no DNA fragment can be amplified using the molecular marker primers, it indicates that the rice brown planthopper resistance gene described in any one of claims 1 to 3 does not exist in the rice. BPH33.2 .

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