Method for creating brown planthopper and southern black-streaked dwarf virus resistant rice material through polygene polymerization

Abstract

The invention discloses a method for creating a brown planthopper and southern black-streaked dwarf virus resistant rice material through polygene polymerization, and belongs to the technical field of rice molecular breeding. A restoring line 9311 which is large in popularization area in production and excellent in comprehensive character is selected as a recurrent parent, and near-isogenic line materials of seven resistance genes are constructed respectively. And hybridizing near-isogenic lines of different genes in pairs, and combining molecular marker-assisted selection and a conventional breeding technology to obtain 81 homozygous materials respectively carrying double genes, three genes, four genes, five genes and six genes. 9 rice materials resistant to brown planthopper and southern black-streaked dwarf virus are finally screened through field morbidity investigation and artificial insect inoculation identification. The strain bred by the method disclosed by the invention is well coordinated in yield and resistance, and the disease and pest resistance is remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of crop molecular breeding, and relates to a method for creating a rice material with multiple genes pyramided to be resistant to both brown planthopper and southern rice black-streaked dwarf virus and the method thereof. Background Art

[0002] Rice (Oryza sativa L.) is a very important food crop. The brown planthopper (BPH, Nilaparvata lugens) is the most important pest in rice planting areas, causing huge losses to rice production. According to the records of the China Agricultural Yearbook, the annual occurrence area of brown planthopper in China exceeds 20 million hectares, and the rice loss caused by it is about 3 million tons every year. At present, chemical agents are mainly used to control brown planthopper in production. However, the abuse of pesticides not only increases the production and labor costs of farmers, but also causes irreversible harm to the environment. At the same time, pesticides can also kill the natural enemies of brown planthopper, enhance the drug resistance of pests, and instead induce the resurgence of brown planthopper. Therefore, cultivating insect-resistant varieties by using rice resistance genes is the most economical and effective prevention and control strategy at present. It is found that planting rice varieties with different resistances can effectively control the growth of brown planthopper populations and, to a certain extent, slow down the mutation rate of their biotypes or virulent types, so as to achieve the purpose of durable resistance. Due to the co-evolution of brown planthopper and rice, varieties with resistance conferred by a single resistance gene are easily eliminated, while pyramiding multiple resistance genes can make rice varieties have a broader spectrum of resistance to brown planthopper and a longer duration. However, when multiple genes are simultaneously pyramided into the same rice variety, the interaction effects between resistance genes are difficult to predict. Therefore, it is particularly important to accurately evaluate the interaction effects between resistance genes, and screening out resistance gene combinations with strong positive interaction effects is of great significance for the genetic improvement of rice against brown planthopper.

[0003] Southern rice black-streaked dwarf virus (SRBSDV) is a newly discovered rice virus species in China and is the most important and harmful rice virus disease in southern China and countries in the Indochina Peninsula at present. The annual incidence area of this virus is between 4 million and 6 million mu. In 2020 and 2023, southern rice black-streaked dwarf virus was included in the list of the first-class crop pests and diseases by the Ministry of Agriculture and Rural Affairs. It was statistically found in 2022 that the currently cultivated rice varieties do not have broad-spectrum resistance to this disease. At present, the prevention and control of this disease mainly rely on physical and chemical methods to control white-backed planthopper to reduce the spread of the virus among rice plants, or use chemical agents to inhibit the reproduction and spread of the virus in the host. Based on this, screening rice variety resources resistant to virus, identifying rice antiviral genes, carrying out rice antiviral breeding, improving existing high-quality varieties, establishing a perfect identification spectrum of antiviral germplasm resources, and exploring and utilizing antiviral germplasm resources have important theoretical and practical guiding significance.

[0004] At present, more than 40 brown planthopper (BPH) resistance genes and 12 quantitative trait loci (QTLs) for resistance to southern rice black-streaked dwarf virus (SRBSDV) have been mapped in rice. The mapping and cloning of these genes provide the possibility for molecular marker-assisted selection (MAS) breeding to improve the disease and insect resistance characteristics of rice. By combining MAS with conventional breeding techniques, multiple genes can be efficiently pyramided in the same material. The resistance spectra of individual resistance genes may vary. After gene pyramiding, the resistance spectra of the genes can be effectively broadened and the resistance level can be further enhanced, thus achieving the goal of durable broad-spectrum resistance. Previously, we successfully constructed near-isogenic line (NIL) materials carrying multiple BPH resistance genes (BPH3, BPH6, BPH9, BPH14, BPH31, BPH32, and BPH33) based on the indica rice 9311 background. On this basis, using MAS and conventional breeding methods, through field disease incidence surveys and artificial insect inoculation identification, 9 rice materials with dual resistance to BPH and SRBSDV were finally screened out. These improved lines can be used either as intermediate materials for disease and insect resistance improvement or directly as new restorer line male parents to hybridize with excellent two-line sterile lines currently used in production to select new hybrid rice combinations with high resistance, high quality, and high yield, providing an effective solution to the problem of poor disease and insect resistance of current rice varieties in China. Summary of the Invention

[0005] The object of the present invention is to provide a method for pyramiding multiple genes to create rice materials with dual resistance to brown planthopper and southern rice black-streaked dwarf virus. To achieve the technical object of the present invention, the technical scheme adopted by the present invention is as follows: The first aspect of the present invention provides a method for pyramiding multiple genes to create rice materials with dual resistance to brown planthopper and southern rice black-streaked dwarf virus, comprising the following steps: (1) Using the homozygous resistance gene near-isogenic lines carrying a single resistance gene under the rice 9311 background as the basic materials, cross them in pairs, that is, cross BPH3-NIL with BPH14-NIL, BPH9-NIL with BPH33-NIL, and BPH6-NIL with BPH31-NIL to obtain the F1 generations of 3 kinds of hybrid combinations; (2) After planting the F1 generation resulting from the cross between BPH3-NIL and BPH14-NIL by tiller lines, PCR detection was carried out using MAS molecular marker-assisted breeding technology to screen out F1 single plants with heterozygous resistant genotypes for the corresponding target resistance genes, denoted as 3 / 14; after planting the F1 generation resulting from the cross between BPH6-NIL and BPH31-NIL by tiller lines, PCR detection was carried out using MAS molecular marker-assisted breeding technology to screen out F1 single plants with heterozygous resistant genotypes for the corresponding target resistance genes, denoted as 6 / 31; after planting the F1 generation resulting from the cross between BPH31-NIL and BPH33-NIL by tiller lines, PCR detection was carried out using MAS molecular marker-assisted breeding technology to screen out F1 single plants with heterozygous resistant genotypes for the corresponding target resistance genes, denoted as 9 / 33; (3) Cross 3 / 14 with 6 / 31 to obtain the F1 generation carrying four resistance genes BPH3, BPH6, BPH14, and BPH31; cross 9 / 33 with the homozygous resistant gene near-isogenic line BPH32-NIL carrying a single resistance gene under the background of rice 9311 to obtain the F1 generation carrying three resistance genes BPH9, BPH32, and BPH33; (4) After planting the F1 generation carrying four resistance genes BPH3, BPH6, BPH14, and BPH31 by tiller lines, PCR detection was carried out using MAS molecular marker-assisted breeding technology to screen out F1 single plants with heterozygous resistant genotypes for the corresponding target resistance genes, denoted as 3 / 6 / 14 / 31; after planting the F1 generation carrying three resistance genes BPH9, BPH32, and BPH33 by tiller lines, PCR detection was carried out using MAS molecular marker-assisted breeding technology to screen out F1 single plants with heterozygous resistant genotypes for the corresponding target resistance genes, denoted as 9 / 32 / 33; (5) Cross 3 / 6 / 14 / 31 with 9 / 32 / 33 to obtain the F1 generation carrying seven resistance genes BPH3, BPH6, BPH9, BPH14, BPH31, BPH32, and BPH33; (6) After planting the F1 generation carrying seven resistance genes BPH3, BPH6, BPH9, BPH14, BPH31, BPH32, and BPH33 by tiller lines, PCR detection was carried out using MAS molecular marker-assisted breeding technology to screen out F1 single plants with heterozygous resistant genotypes for the corresponding target resistance genes, and bagged to harvest selfed seeds F2; After planting the F2 generation of the ramet lines, PCR detection was carried out using the MAS molecular marker-assisted breeding technology to screen out the F2 single plants with homozygous resistant genotypes for the corresponding target resistance genes. Then, they were continuously self-crossed for three generations to obtain the F5 generation, which is the pyramiding family with homozygous multiple genes. Among the F5 generation, the rice materials with dual resistance to brown planthopper and southern black-streaked dwarf virus are: the pyramiding families with homozygous triple genes of BPH14, BPH3, and BPH32; the pyramiding families with homozygous triple genes of BPH14, BPH32, and BPH9; the pyramiding families with homozygous triple genes of BPH33, BPH3, and BPH32; the pyramiding families with homozygous triple genes of BPH33, BPH32, and BPH9; the pyramiding families with homozygous triple genes of BPH33, BPH32, and BPH31; the pyramiding families with homozygous quadruple genes of BPH14, BPH6, BPH32, and BPH31; the pyramiding families with homozygous quadruple genes of BPH14, BPH33, BPH3, and BPH32; the pyramiding families with homozygous quadruple genes of BPH14, BPH33, BPH6, and BPH9; the pyramiding families with homozygous quintuple genes of BPH14, BPH33, BPH6, BPH32, and BPH9. Among them, BPH3 is a gene cluster composed of three genes with gene numbers LOC_Os04g12540, LOC_Os04g12560, and LOC_Os04g12580; the gene number of BPH6 is LOC_Os04g35210; BPH9 is a gene cluster composed of two genes with gene numbers LOC_Os12g37280 and LOC_Os12g37290; the gene number of BPH14 is LOC_Os03g63150; the gene number of BPH31 is LOC_Os11g29150; the gene number of BPH32 is LOC_Os06g03240; the gene number of BPH33 is LOC_Os04g02520. The molecular marker of the BPH3 gene is 939, and the primers of the molecular marker 939 are SEQ ID NO.1 - 2; the molecular marker of the BPH6 gene is 39 - 1, and the primers of the molecular marker 39 - 1 are SEQ ID NO.3 - 4; the molecular marker of the BPH9 gene is HJ12, and the primers of the molecular marker HJ12 are SEQ ID NO.5 - 6; the molecular marker of the BPH14 gene is Z14, and the primers of the molecular marker Z14 are SEQ ID NO.7 - 8; the molecular marker of the BPH31 gene is 31 - 1, and the primers of the molecular marker 31 - 1 are SEQ ID NO.9 - 10; the molecular marker of the BPH32 gene is 32 - 5, and the primers of the molecular marker 32 - 5 are SEQ ID NO.11 - 12; the molecular marker of the BPH33 gene is H99, and the primers of the molecular marker H99 are SEQ ID NO.13 - 14.

[0006] The second aspect of the present invention provides a molecular marker primer composition, which includes: primers for molecular marker 939, SEQ ID NO.1-2; primers for molecular marker 39-1, SEQ ID NO.3-4; primers for molecular marker HJ12, SEQ ID NO.5-6; primers for molecular marker Z14, SEQ ID NO.7-8; primers for molecular marker 31-1, SEQ ID NO.9-10; primers for molecular marker 32-5, SEQ ID NO.11-12; primers for molecular marker H99, SEQ ID NO.13-14.

[0007] The third aspect of the present invention provides the application of the above-mentioned molecular marker primer composition in creating rice materials resistant to brown planthopper, rice materials resistant to southern rice black-streaked dwarf virus, and rice materials resistant to both brown planthopper and southern rice black-streaked dwarf virus.

[0008] The fourth aspect of the present invention provides a kit for creating rice materials resistant to brown planthopper, rice materials resistant to southern rice black-streaked dwarf virus, and rice materials resistant to both brown planthopper and southern rice black-streaked dwarf virus, and the kit contains the above-mentioned molecular marker primer composition.

[0009] The fifth aspect of the present invention provides the application of the above-mentioned kit in creating rice materials resistant to brown planthopper, rice materials resistant to southern rice black-streaked dwarf virus, and rice materials resistant to both brown planthopper and southern rice black-streaked dwarf virus.

[0010] The advantages of the present invention are as follows: (1) The nine kinds of rice materials obtained in the present invention, which aggregate different resistance genes, show resistant and highly resistant levels to brown planthopper, and can significantly reduce the incidence of southern rice black-streaked dwarf virus. Since the nine gene aggregation materials carry different genes from each other, it effectively ensures gene diversity and avoids the problem that a single gene is prone to failure, providing the possibility for achieving durable and broad-spectrum resistance to brown planthopper and virus diseases.

[0011] (2) The present invention can improve the efficiency of assistant selection in conventional breeding and save costs. By detecting the brown planthopper resistance gene locus in rice plants through molecular markers, resistant single plants can be quickly identified at the seedling stage, and susceptible single plants can be eliminated in time without insect inoculation identification, which not only saves production costs, but also improves the selection efficiency of resistant materials and shortens the breeding cycle of rice varieties.

[0012] (3) The present invention adopts the method of intercrossing single-gene near-isogenic lines, which can achieve the rapid aggregation of multiple resistance genes under the same genetic background, reduce the detection steps of linkage drag and genetic background recovery rate, and improve the selection efficiency of aggregation breeding. Description of the Drawings

[0013] Figure 1: The SRBSDV incidence rate of the F5 generation carrying the BPH32 gene was significantly reduced. A and B respectively represent the field SRBSDV incidence rates of the F5 generation with and without the BPH32 gene; C is the significant analysis of the incidence rate difference between the F5 generation with and without the BPH32 gene, and **** indicates a highly significant difference between the two; D is the field disease incidence situation of the F5 generation with and without the BPH32 gene.

[0014] Figure 2 : Phenotypes of healthy and diseased plants of some single-gene near-isogenic lines and the F5 generation. 9311 is the control, and the percentage numbers in parentheses represent the incidence rate. Mock is the healthy plant, SRBSDV is the diseased plant, 3, 6, 9... respectively represent the near-isogenic lines homozygous for the resistance gene carrying BPH3, the near-isogenic lines homozygous for the resistance gene carrying BPH6, the near-isogenic lines homozygous for the resistance gene carrying BPH9..., and the others are polygene homozygous pyramiding families. For example, 14 / 33 represents the pyramiding family homozygous for the BPH14 and BPH33 genes, and 14 / 3 / 32 represents the pyramiding family homozygous for the BPH14, BPH3, and BPH32 genes. The materials with relatively low incidence rates are marked in red.

[0015] Figure 3 : The brown planthopper resistance phenotypes of the pyramiding families homozygous for five genes in the F5 generation. A and B respectively represent the phenotypes of the control 9311 and the pyramiding family before and after insect inoculation. 14 / 33 / 6 / 32 / 9 represents the pyramiding family homozygous for the BPH14, BPH33, BPH6, BPH32, and BPH9 genes. Scale: 10 cm.

[0016] Figure 4: Molecular marker gel images of 9 polygene homozygous pyramiding families carrying resistance genes in the F5 generation. Z14, H99, 939, 39-1, 32-5, 31-1, and HJ12 are molecular markers, and the resistance genes linked or co-segregated with these markers, such as BPH14 and BPH33 in parentheses; except for Figure b, lanes 1 to 9 are 9 polygene homozygous pyramiding families 14 / 3 / 32, 14 / 32 / 9, 33 / 3 / 32, 33 / 32 / 9, 33 / 32 / 31, 14 / 6 / 32 / 31, 14 / 33 / 3 / 32, 14 / 33 / 6 / 9, 14 / 33 / 6 / 32 / 9. For example, 14 / 3 / 32 represents a pyramiding family homozygous for three genes BPH14, BPH3, and BPH32, and 14 / 33 / 6 / 32 / 9 represents a pyramiding family homozygous for five genes BPH14, BPH33, BPH6, BPH32, and BPH9. Lanes 10 and 11 represent the insect-resistant parent (donor) and the insect-susceptible parent 9311 (recipient), respectively. Lane 12 is the marker, and the marker is a DNA size gradient indicator band. The bands indicate 100bp, 200bp, 300bp, and 400bp from small to large. Lane 1 in Figure b is the marker, and lanes 2 to 10 are 9 polygene homozygous pyramiding families 14 / 3 / 32, 14 / 32 / 9, 33 / 3 / 32, 33 / 32 / 9, 33 / 32 / 31, 14 / 6 / 32 / 31, 14 / 33 / 3 / 32, 14 / 33 / 6 / 9, 14 / 33 / 6 / 32 / 9, respectively. Lanes 11 and 12 are the donor and recipient, respectively. Detailed implementation manners

[0017] To make the content of the present invention easier to understand, the technical solutions of the present invention will be further described below in conjunction with the detailed implementation manners, but the present invention is not limited thereto.

[0018] Seven homozygous resistance gene near-isogenic lines in the background of indica rice 9311 used in the following examples, namely BPH3-NIL, BPH6-NIL, BPH9-NIL, BPH14-NIL, BPH31-NIL, BPH32-NIL, and BPH33-NIL, were obtained by using indica rice 9311 as the recurrent parent and introgressing the single resistance genes BPH3, BPH6, BPH9, BPH14, BPH31, BPH32, and BPH33 into 9311 through hybridization and backcrossing, and their construction methods have all been disclosed in the prior art. Among them, BPH3 is a gene cluster composed of three genes with gene numbers LOC_Os04g12540, LOC_Os04g12560, and LOC_Os04g12580; the gene number of BPH6 is LOC_Os04g35210; BPH9 is a gene cluster composed of two genes with gene numbers LOC_Os12g37280 and LOC_Os12g37290; the gene number of BPH14 is LOC_Os03g63150; the gene number of BPH31 is LOC_Os11g29150; the gene number of BPH32 is LOC_Os06g03240; the gene number of BPH33 is LOC_Os04g02520. For the construction methods of BPH3-NIL and BPH32-NIL, see: He et al. 2020. Molecular Breeding 40(10):95. DOI:10.1007 / s11032-020-01175-z; for the construction method of BPH6-NIL, see: Xiao et al. 2016. Scientific Reports 6:38159. DOI:10.1038 / srep38159; for the construction method of BPH9-NIL, see: Wang et al. 2017. Rice 10:51. DOI:10.1186 / s12284-017-0194-x; for the construction method of BPH14-NIL, see: Hu et al. 2013. Pest Management Science 69:802-808. DOI:10.1002 / ps.3437; for the construction method of BPH31-NIL, see: Zhou Mingsong. Construction and Resistance Evaluation of Pyramiding Lines of Brown Planthopper Resistance Genes in Rice and Fine Mapping of Bph31 [D]. Huazhong Agricultural University; for the construction method of BPH33-NIL, see: Hu et al. Rice 11:55. DOI:10.1186 / s12284-018-0249-7.The donor of BPH3 is rice Rathu Heenati (RH), the donor of BPH6 is rice Swarnalata, the donor of BPH9 is rice Pokkali, the donor of BPH14 is rice B5, the donor of BPH31 is rice AUS69, the donor of BPH32 is rice Ptb33, and the donor of BPH33 is rice KOLAYAL.

[0019] The molecular marker information used in the MAS molecular marker-assisted breeding technology in the following examples is shown in Table 1.

[0020] Table 1 Molecular marker sequence information used in MAS The band size of the PCR amplification product of the primer of molecular marker 939 is as follows: If a single band of 132 bp appears, it indicates that the tested rice is a homozygous genotype of the BPH3 resistance allele (i.e., homozygous resistant genotype); if a single band of 142 bp appears, it indicates that the tested rice does not carry the BPH3 resistance allele (i.e., homozygous sensitive genotype); if there are two bands of 132 bp and 142 bp, it indicates that the tested rice is a heterozygous genotype of the BPH3 allele (i.e., heterozygous resistant genotype).

[0021] The band size of the PCR amplification product of the primer of molecular marker 39-1 is as follows: If a single band of 136 bp appears, it indicates that the tested rice is a homozygous genotype of the BPH6 resistance allele (i.e., homozygous resistant genotype); if a single band of 125 bp appears, it indicates that the tested rice does not carry the BPH6 resistance allele (i.e., homozygous sensitive genotype); if there are two bands of 136 bp and 125 bp, it indicates that the tested rice is a heterozygous genotype of the Bph6 allele (i.e., heterozygous resistant genotype).

[0022] The band size of the PCR amplification product of the primer of molecular marker HJ12 is as follows: If a single band of 129 bp appears, it indicates that the tested rice is a homozygous genotype of the BPH9 resistance allele (i.e., homozygous resistant genotype); if a single band of 123 bp appears, it indicates that the tested rice does not carry the BPH9 resistance allele (i.e., homozygous sensitive genotype); if there are two bands of 129 bp and 123 bp, it indicates that the tested rice is a heterozygous genotype of the Bph9 allele (i.e., heterozygous resistant genotype).

[0023] The band sizes of the PCR amplification products of the primers of molecular marker Z14 are as follows: If a single band of 90 bp appears, it indicates that the rice to be tested is of the homozygous genotype of the BPH14 resistance allele (i.e., the homozygous resistant genotype); if a single band of 99 bp appears, it indicates that the rice to be tested does not carry the BPH14 resistance allele (i.e., the homozygous sensitive genotype); if two bands of 90 bp and 99 bp appear, it indicates that the rice to be tested is of the Bph14 allelic heterozygous genotype (i.e., the heterozygous resistant genotype).

[0024] The band sizes of the PCR amplification products of the primers of molecular marker 31-1 are as follows: If a single band of 198 bp appears, it indicates that the rice to be tested is of the homozygous genotype of the BPH31 resistance allele (i.e., the homozygous resistant genotype); if a single band of 163 bp appears, it indicates that the rice to be tested does not carry the BPH31 resistance allele (i.e., the homozygous sensitive genotype); if two bands of 198 bp and 163 bp appear, it indicates that the rice to be tested is of the BPH31 allelic heterozygous genotype (i.e., the heterozygous resistant genotype).

[0025] The band sizes of the PCR amplification products of the primers of molecular marker 32-5 are as follows: If a single band of 176 bp appears, it indicates that the rice to be tested is of the homozygous genotype of the BPH32 resistance allele (i.e., the homozygous resistant genotype); if a single band of 168 bp appears, it indicates that the rice to be tested does not carry the BPH32 resistance allele (i.e., the homozygous sensitive genotype); if two bands of 176 bp and 168 bp appear, it indicates that the rice to be tested is of the BPH32 allelic heterozygous genotype (i.e., the heterozygous resistant genotype).

[0026] The band sizes of the PCR amplification products of the primers of molecular marker H99 are as follows: If a single band of 96 bp appears, it indicates that the rice to be tested is of the homozygous genotype of the BPH33 resistance allele (i.e., the homozygous resistant genotype); if a single band of 102 bp appears, it indicates that the rice to be tested does not carry the BPH33 resistance allele (i.e., the homozygous sensitive genotype); if two bands of 96 bp and 102 bp appear, it indicates that the rice to be tested is of the BPH33 allelic heterozygous genotype (i.e., the heterozygous resistant genotype).

[0027] Example 1: (1) Using the homozygous resistant gene near-isogenic lines BPH3-NIL, BPH6-NIL, BPH9-NIL, BPH14-NIL, BPH31-NIL, BPH33-NIL containing a single resistant gene as the basic materials, cross them in pairs (i.e., cross BPH3-NIL with BPH14-NIL, cross BPH6-NIL with BPH31-NIL, cross BPH31-NIL with BPH33-NIL) to obtain the F1 generations of 3 hybrid combinations, and ensure that at least 20 hybrid seeds are obtained for each hybrid combination.

[0028] (2) After planting the F1 generation obtained by crossing BPH3-NIL and BPH14-NIL by tiller line, PCR detection was carried out using MAS molecular marker-assisted breeding technology to screen out F1 single plants with heterozygous resistance genotypes for the corresponding target resistance genes, denoted as 3 / 14; after planting the F1 generation obtained by crossing BPH6-NIL and BPH31-NIL by tiller line, PCR detection was carried out using MAS molecular marker-assisted breeding technology to screen out F1 single plants with heterozygous resistance genotypes for the corresponding target resistance genes, denoted as 6 / 31; after planting the F1 generation obtained by crossing BPH31-NIL and BPH33-NIL by tiller line, PCR detection was carried out using MAS molecular marker-assisted breeding technology to screen out F1 single plants with heterozygous resistance genotypes for the corresponding target resistance genes, denoted as 9 / 33.

[0029] (3) Cross 3 / 14 with 6 / 31 to obtain the F1 generation carrying four resistance gene combinations (BPH3 + BPH6 + BPH14 + BPH31), ensuring at least 100 seeds are obtained; cross 9 / 33 with the homozygous resistance gene near-isogenic line BPH32-NIL carrying a single resistance gene to obtain the F1 generation carrying three resistance gene combinations (BPH9 + BPH32 + BPH33), ensuring at least 100 seeds are obtained.

[0030] (4) After planting the F1 generation carrying four resistance gene combinations (BPH3 + BPH6 + BPH14 + BPH31) by tiller line, PCR detection was carried out using MAS molecular marker-assisted breeding technology to screen out F1 single plants with heterozygous resistance genotypes for the corresponding target resistance genes, denoted as 3 / 6 / 14 / 31; after planting the F1 generation carrying three resistance gene combinations (BPH9 + BPH32 + BPH33) by tiller line, PCR detection was carried out using MAS molecular marker-assisted breeding technology to screen out F1 single plants with heterozygous resistance genotypes for the corresponding target resistance genes, denoted as 9 / 32 / 33.

[0031] (5) Cross 3 / 6 / 14 / 31 with 9 / 32 / 33 to obtain the F1 generation carrying seven resistance gene combinations (BPH3 + BPH6 + BPH9 + BPH14 + BPH31 + BPH32 + BPH33), ensuring at least 500 seeds are obtained.

[0032] (6) After planting the F1 generation carrying seven resistance gene combinations (BPH3 + BPH6 + BPH9 + BPH14 + BPH31 + BPH32 + BPH33) by tiller line, PCR detection was carried out using MAS molecular marker-assisted breeding technology to screen out F1 single plants with heterozygous resistance genotypes for the corresponding target resistance genes, and bag them to collect selfed seeds F2, which is the seven-gene segregation population.

[0033] (7) After planting the seven-gene segregation population of the ramets, PCR detection was carried out using the MAS molecular marker-assisted breeding technology to screen out the F2 single plants with homozygous resistant genotypes for the corresponding target resistance genes. After selfing for three consecutive generations, the F5 generation was obtained, which is the pyramiding family with multiple genes homozygous (Table 2).

[0034] (8) The F5 generation was planted in the field for natural incidence identification of Southern rice black-streaked dwarf virus (SRBSDV). The disease nursery is located in Tuanshu Village, Yuanjiang County, Yuxi City, Yunnan Province (101°06′E, 23°30′N). It is located on the low-latitude plateau, belonging to the monsoon climate, warm in winter and hot in summer, surrounded by mountains, which is conducive to the perennial occurrence and prevalence of white-backed planthoppers and southern black virus. The F5 generation and the control indica rice 9311 were sown in the seedling nursery at the same time. After 35 days, 4 rows were transplanted for each material, with 10 plants in each row, and the plant spacing was 15 cm × 18 cm, and transplanted into the large field. Normal fertilizer management was carried out, and no pesticides and fungicides were applied throughout the growth period. The experiment was set with 3 replicates and arranged in a randomized block design. After 60 days, the number of dwarfed plants of the tested rice materials was investigated one by one, and the average incidence rate (%) = 100 × the number of dwarfed plants of the tested variety / the total number of plants of the tested variety was calculated. And the disease resistance levels were divided according to the relevant standards formulated by the National Rice Virus Disease Cooperative Group: Immune: 0; High resistance: 0.1% - 5.0%; Resistance: 5.1% - 15.0%; Moderate resistance: 15.1% - 30.0%; Moderate susceptibility: 30.1% - 50.0%; High susceptibility: 50.1% - 100.0%. The results showed (Table 2) that the incidence rate of the pyramiding family carrying BPH32 was significantly lower than that of the pyramiding family not carrying BPH32, and the two-tailed value of the two-sample heteroscedastic t-test between the two groups was 3.11×10 -15 , reaching a highly significant level of difference ( Figure 1 ). In addition, the incidence rate of the pyramiding family carrying both BPH33 and BPH3 was also significantly lower than that of the pyramiding family not carrying these two genes, and the two-tailed value of the t-test was 3.73×10 -4 , reaching a highly significant level of difference. Finally, we screened out a variety of disease-resistant pyramiding families with an incidence rate not exceeding 30% in the F5 generation. Among them, the incidence rate of the pyramiding family carrying 5 resistance gene combinations (BPH6 + BPH9 + BPH14 + BPH32 + BPH33) was only 10%, while the incidence rate of the control indica rice 9311 was higher than 70% (Table 2, Figure 2 ).

[0035] Table 2 Natural field incidence rate of SRBSDV of 81 multi-gene homozygous pyramiding families (9) Identify the resistance of the disease-resistant pyramiding lines with an incidence rate not exceeding 30% screened in (8) and the control indica rice to brown planthopper at the seedling stage in the greenhouse. The identification method refers to: Huang et al. 2001. Theoretical and Applied Genetics 102: 929-934. https: / / doi.org / 10.1007 / s001220000455. Sow the susceptible control 9311 and the materials to be identified at the same time. Sow 12 plump seeds of each material in the same black plastic seedling box with an open bottom (diameter 7.2 cm, bottom diameter 5 cm, height 8 cm), and fill the seedling box with soil suitable for cultivating rice. When the plants grow to the three-leaf stage, retain 9 seedlings with consistent growth in each row and place the seedling tray box inside a 200-mesh screen. Introduce 2nd-instar brown planthopper nymphs at a rate of 8-10 per seedling. When more than 90% of the plants in the susceptible control die, the resistance level can be evaluated according to the damage degree of each seedling, and the resistance score value of each material is obtained through weighted average calculation. When the dead seedling rate of the control 9311 reaches 90%, 9 pyramiding lines are at the resistant or highly resistant level, that is, 9 new materials resistant to both brown planthopper and southern black-streaked dwarf virus are screened from the F5 generation (Table 3, Figure 3 , Figure 4 ): Pyramiding lines homozygous for three genes (BPH14 + BPH3 + BPH32), pyramiding lines homozygous for three genes (BPH14 + BPH32 + BPH9), pyramiding lines homozygous for three genes (BPH33 + BPH3 + BPH32), pyramiding lines homozygous for three genes (BPH33 + BPH32 + BPH9), pyramiding lines homozygous for three genes (BPH33 + BPH32 + BPH31), pyramiding lines homozygous for four genes (BPH14 + BPH6 + BPH32 + BPH31), pyramiding lines homozygous for four genes (BPH14 + BPH33 + BPH3 + BPH32), pyramiding lines homozygous for four genes (BPH14 + BPH33 + BPH6 + BPH9), pyramiding lines homozygous for five genes (BPH14 + BPH33 + BPH6 + BPH32 + BPH9).

[0036] Table 3 Disease and insect resistance information of pyramiding lines homozygous for multiple genes The above are only the preferred embodiments of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope covered by the present invention.

Claims

1. A method for creating rice materials resistant to brown planthopper and southern black-streaked dwarf disease by multi-gene polymerization, characterized in that: The following steps are involved: (1) Using homozygous near-isogenic lines carrying a single resistance gene in the rice 9311 background as basic materials for pairwise hybridization, BPH3 -NIL and BPH14 -NIL hybridization, BPH9 -NIL and BPH33 -NIL hybridization, BPH6 -NIL and BPH31 -NIL hybridization, to obtain the F1 generation of three hybrid combinations; (2) Planting of ramet lines BPH3 -NIL and BPH14 -NIL hybrid F1 generation, PCR detection was performed using MAS molecular marker-assisted breeding technology to screen out F1 plants with heterozygous resistance genotypes corresponding to the target resistance genes, recorded as 3 / 14; the ramet line was planted BPH6 -NIL and BPH31 -NIL hybrid F1 generation, PCR detection was performed using MAS molecular marker-assisted breeding technology to screen out F1 plants with heterozygous resistance genotypes corresponding to the target resistance genes, recorded as 6 / 31; the ramet line was planted BPH31 -NIL and BPH33 -NIL hybrid F1 generation, PCR detection was performed using MAS molecular marker-assisted breeding technology to screen out F1 plants with heterozygous resistance genotypes for the corresponding target resistance genes, recorded as 9 / 33; (3) Hybridizing 3 / 14 with 6 / 31 yields four resistance genes BPH3, BPH6, BPH14 and BPH31 F1 generation; 9 / 33 and the homozygous resistance gene near isogenic line carrying a single resistance gene in the rice 9311 background BPH32 -NIL hybridization, carrying three resistance genes BPH9, BPH32 and BPH33 F1 generation; (4) Planting of ramet lines carrying four resistance genes BPH3, BPH6, BPH14 and BPH31 After the F1 generation, PCR detection was performed using MAS molecular marker-assisted breeding technology to screen out F1 plants with heterozygous resistance genotypes corresponding to the target resistance genes, recorded as 3 / 6 / 14 / 31; the ramet line was planted with three resistance genes BPH9, BPH32 and BPH33 After the F1 generation was generated, PCR detection was performed using MAS molecular marker-assisted breeding technology to screen out the F1 plants with heterozygous resistance genotypes for the corresponding target resistance genes, which were recorded as 9 / 32 / 33; (5) Hybridizing 3 / 6 / 14 / 31 with 9 / 32 / 33 yields seven resistance genes BPH3, BPH6, BPH9, BPH14, BPH31, BPH32 and BPH33 F1 generation; (6) Planting of ramet lines carrying seven resistance genes BPH3, BPH6, BPH9, BPH14, BPH31, BPH32 and BPH33 After the F1 generation, PCR detection was performed using MAS molecular marker-assisted breeding technology to screen out F1 plants with heterozygous resistance genotypes corresponding to the target resistance genes, and the self-cross F2 was collected by bagging; (7) After the F2 plants were planted, PCR detection was performed using MAS molecular marker-assisted breeding technology to screen out F2 plants with homozygous resistance genotypes for the corresponding target resistance genes. The plants were then self-pollinated for three consecutive generations to obtain the F5 generation, which was a polygenetic homozygous aggregate family. Among the F5 generation, the rice materials that were resistant to both brown planthopper and southern black-streaked dwarf disease included: BPH14, BPH3 and BPH32 Three-gene homozygous aggregate family, BPH14, BPH32 and BPH9 Three-gene homozygous aggregate family, BPH33, BPH3 and BPH32 Three-gene homozygous aggregate family, BPH33, BPH32 and BPH9 Three-gene homozygous aggregate family, BPH33, BPH32 and BPH31 Three-gene homozygous aggregate family, BPH14, BPH6, BPH32 and BPH31 Four-gene homozygous aggregate family, BPH14, BPH33, BPH3 and BPH32 Four-gene homozygous aggregate family, BPH14, BPH33, BPH6 and BPH9 Four-gene homozygous aggregate family, BPH14, BPH33, BPH6, BPH32 and BPH9 A clustered family homozygous for the five genes.

2. The method according to claim 1, characterized in that: BPH3 It is from gene number LOC_Os04g12540 、 LOC_Os04g12560 、 A gene cluster consisting of three genes in LOC_Os04g12580; BPH6 The gene number is LOC_Os04g35210; BPH9 It is caused by gene number LOC_Os12g37280 、 A gene cluster consisting of 2 genes in LOC_Os12g37290; BPH14 The gene number is LOC_Os03g63150; BPH31 The gene number is LOC_Os11g29150; BPH32 The gene number is LOC_Os06g03240; BPH33 The gene number is LOC_Os04g02520.

3. The method according to claim 1, characterized in that: BPH3 The molecular marker of the gene is 939, and the primers of the molecular marker 939 are SEQ ID NO.1~2; BPH6 The molecular marker of the gene is 39-1, and the primer of the molecular marker 39-1 is SEQ ID NO.3~4; BPH9 The molecular marker of the gene is HJ12, and the primers of the molecular marker HJ12 are SEQ ID NO.5~6; BPH14 The molecular marker of the gene is Z14, and the primers of the molecular marker Z14 are SEQ ID NO.7-8; BPH31 The molecular marker of the gene is 31-1, and the primers of the molecular marker 31-1 are SEQ ID NO.9~10; BPH32 The molecular marker of the gene is 32-5, and the primers of the molecular marker 32-5 are SEQ ID NO.11~12; BPH33 The molecular marker of the gene is H99, and the primers of the molecular marker H99 are SEQ ID NO.13~14.

4. A molecular marker primer composition, characterized in that: The primer composition includes: primers for molecular marker 939, SEQ ID NOs. 1 to 2; primers for molecular marker 39-1, SEQ ID NOs. 3 to 4; primers for molecular marker HJ12, SEQ ID NOs. 5 to 6; primers for molecular marker Z14, SEQ ID NOs. 7 to 8; primers for molecular marker 31-1, SEQ ID NOs. 9 to 10; primers for molecular marker 32-5, SEQ ID NOs. 11 to 12; and primers for molecular marker H99, SEQ ID NOs. 13 to 14.

5. Use of the molecular marker primer composition according to claim 4 in creating rice materials resistant to brown planthopper, rice materials resistant to southern black-streaked dwarf disease, and rice materials resistant to both brown planthopper and southern black-streaked dwarf disease.

6. A kit for creating rice materials resistant to brown planthopper, rice materials resistant to southern black-streaked dwarf disease, and rice materials resistant to both brown planthopper and southern black-streaked dwarf disease, characterized in that: The kit comprises the molecular marker primer composition according to claim 5.

7. Use of the kit according to claim 6 in creating rice materials resistant to brown planthopper, rice materials resistant to southern black-streaked dwarf disease, and rice materials resistant to both brown planthopper and southern black-streaked dwarf disease.

Images