Breeding method of high-yield hybrid rice capable of resisting rice leaf smut disease

Abstract

The invention provides a method for breeding high-yield hybrid rice capable of resisting rice leaf smut disease, which comprises the following steps of: combining a plurality of technical means such as parent material selection, molecular marker-assisted selection, cross breeding, gene editing, transgenosis, tissue culture, multi-environment field test, genetic diversity protection and utilization, comprehensive performance evaluation and the like; the hybrid rice variety with high disease resistance and high yield potential is finally obtained. Screening parent materials with high disease resistance and high yield potential through field evaluation and laboratory detection; the genotype is rapidly identified by using a molecular marker technology to ensure that the filial generation carries disease-resistant and high-yield genes; performing cross breeding and gene editing; introducing an exogenous disease-resistant gene through an agrobacterium tumefaciens-mediated transgenic technology; using a tissue culture technology to rapidly breed excellent individuals; field trials are carried out in multiple environments, and stability and adaptability are evaluated; a germplasm resource library is established, and genetic diversity is protected; comprehensively evaluating the disease resistance, the yield, the plant character and the rice quality of the variety.

Description

Technical Field

[0001] The invention relates to the technical field of rice breeding, and in particular to a breeding method for high-yield hybrid rice resistant to rice leaf smut. Background Art

[0002] Traditional rice breeding methods have shortcomings in improving crop yield and disease resistance, such as long breeding cycle, narrow genetic basis, low selection efficiency, difficulty in taking into account multiple traits, environmental dependence and lack of precision. The present invention aims to effectively solve these problems and improve breeding efficiency and comprehensive performance of varieties through the application of multiple technologies. Summary of the invention

[0003] The present invention provides a breeding method for high-yield hybrid rice resistant to rice leaf smut, which rapidly obtains hybrid rice varieties with high disease resistance and high yield by screening parent materials of disease-resistant genes and high-yield genes, utilizing molecular marker-assisted selection, gene editing and transgenic technologies, and combining tissue culture technology and multi-environment field tests, and ensures the genetic diversity of breeding materials and continuous improvement of varieties through the protection and utilization of genetic diversity.

[0004] In order to solve the above technical problems, the present invention provides the following technical solution: a breeding method for high-yield hybrid rice resistant to rice leaf smut, comprising the following steps:

[0005] S1. Parent material selection:

[0006] From existing rice varieties, parent materials with high resistance to rice leaf smut and parent materials with high yield potential were selected through field evaluation and laboratory testing;

[0007] S2, Molecular marker-assisted selection:

[0008] Using single nucleotide polymorphism markers, simple repeat sequence markers and sequence-specific amplified region markers, the parental materials screened in step S1 are subjected to genotyping, and parental materials carrying multiple disease-resistant genes and high-yield genes are selected;

[0009] S3. Hybrid breeding:

[0010] Hybridizing the parental materials selected in step S2 to obtain the first generation hybrid (F1);

[0011] S4. Gene Editing:

[0012] The hybrid first generation obtained in step S3 is subjected to gene editing using the CRISPR / Cas9 system to knock out or enhance key genes related to disease resistance and yield. Specific editing sites include but are not limited to:

[0013] Rice leaf smut resistance genes: such as Pi-ta, Pi-k, Pi-21, etc.;

[0014] High-yield genes: such as OsSPL14, OsGS1, OsMADS2, etc.;

[0015] S5. Transgenic:

[0016] Using Agrobacterium-mediated gene transformation, exogenous rice leaf smut resistance genes (such as Pi-z, Pi-kh, etc.) are introduced into the first generation of hybrids after gene editing in step S4 to further improve their disease resistance;

[0017] S6. Tissue culture:

[0018] Rapidly propagate the transgenic hybrid rice obtained in step S5 through callus culture and endosperm culture techniques to select individuals with excellent disease resistance and yield;

[0019] S7. Multi-environment field trial:

[0020] Conduct field trials in different geographical regions and disease pressure conditions, record and analyze trial data, and evaluate disease resistance and yield performance of rice varieties;

[0021] S8. Conservation and utilization of genetic diversity:

[0022] Establish a germplasm resource bank to collect and preserve a variety of rice gene resources with disease resistance and high yield potential for subsequent selection and improvement of breeding materials;

[0023] S9. Comprehensive performance evaluation:

[0024] Through multi-year and multi-point field trials, the disease resistance, yield, plant traits and rice quality of rice varieties are evaluated to ensure that the final hybrid rice varieties have excellent overall performance.

[0025] Preferably: the field evaluation and laboratory testing in step S1 include:

[0026] Field evaluation: conduct disease resistance evaluation under different disease pressures, record and analyze the disease incidence and disease index of rice;

[0027] Laboratory testing: Detect the presence and expression of disease-resistant genes and high-yield genes in rice through PCR amplification and sequencing technology.

[0028] Preferably: the molecular marker-assisted selection technology in step S2 includes but is not limited to single nucleotide polymorphism (SNP) markers, simple sequence repeat (SSR) markers, and sequence-specific amplified region (SSAR) markers.

[0029] Preferably: the CRISPR / Cas9 system in step S4 comprises:

[0030] Design sgRNA sequences targeting target genes; use vector systems to introduce sgRNA and Cas9 enzymes into hybrid first-generation rice cells; obtain gene-edited rice plants through plant tissue culture technology; test gene-edited rice plants to confirm the knockout or enhancement effects of target genes.

[0031] Preferably, the Agrobacterium-mediated gene transformation method in step S5 comprises:

[0032] a. Select exogenous rice leaf smut resistance genes (such as Pi-z, Pi-kh, etc.);

[0033] b. Using Agrobacterium to introduce foreign genes into the gene-edited hybrid rice cells;

[0034] c. Obtaining transgenic rice plants through endosperm culture technology;

[0035] d. Test transgenic rice plants to confirm the insertion and expression of exogenous genes.

[0036] Preferably: the tissue culture technology in step S6 includes:

[0037] a. Callus culture: Induce and culture the callus of rice seeds or seedlings to obtain callus;

[0038] b. Endosperm culture: The endosperm of rice seeds is cultured in vitro to obtain regenerated plants;

[0039] c. Through screening and identification, select individuals with excellent disease resistance and yield.

[0040] Preferably, the multi-environment field test in step S7 includes:

[0041] a. Conduct trials in different geographical areas, including but not limited to low altitude, high altitude, coastal and inland areas;

[0042] b. Conduct the test under different disease pressure conditions, including but not limited to light disease area, moderate disease area and severe disease area;

[0043] c. Record and analyze test data to evaluate the disease resistance and yield performance of rice varieties.

[0044] Preferably, the establishment of the germplasm resource bank in step S8 includes:

[0045] a. Collect and preserve rice varieties from different sources, including wild species, local varieties and improved varieties;

[0046] b. Identify and evaluate the genetic diversity of germplasm resources through genome sequencing and molecular marker technology;

[0047] c. Use bioinformatics technology to establish a germplasm resource database to facilitate the selection and improvement of subsequent breeding materials.

[0048] Preferably: the comprehensive performance evaluation in step S9 includes:

[0049] a. Disease resistance evaluation: The disease resistance of rice varieties is evaluated through artificial inoculation of rice leaf smut fungus;

[0050] b. Yield evaluation: Evaluate the yield performance of rice varieties through multi-year and multi-site field trials;

[0051] c. Plant trait evaluation: measuring and analyzing rice plant height, tillering number, root development and other trait indicators;

[0052] d.Quality assessment: Evaluate the rice quality of rice varieties through rice quality testing.

[0053] One or more technical solutions provided in the embodiments of the present application have at least the following technical effects or advantages:

[0054] The present invention improves the selection efficiency and accuracy through molecular marker-assisted selection; realizes the precise improvement of target genes through gene editing and transgenic technology; accelerates the breeding process through tissue culture technology; verifies the stability and adaptability of varieties through multi-environment field trials; and ensures the genetic diversity of breeding materials and the continuous improvement of varieties through the protection and utilization of genetic diversity.

[0055] Other advantages, objectives and features of the present invention will be set forth in part in the following description and, in part, will be apparent to those skilled in the art based on an examination of the following or may be taught from the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 A flow chart of parent material selection and molecular marker-assisted selection in a breeding method for high-yield hybrid rice resistant to rice leaf smut of the present invention;

[0057] Figure 2 A flow chart of hybrid breeding, gene editing and transgenic of a breeding method for high-yield hybrid rice resistant to rice leaf smut of the present invention;

[0058] Figure 3 The present invention is a flow chart of tissue culture, multi-environment field test and comprehensive performance evaluation of a breeding method for high-yield hybrid rice resistant to rice leaf smut. DETAILED DESCRIPTION

[0059] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0060] It should be noted that the terms “vertical”, “horizontal”, “up”, “down”, “left”, “right” and similar expressions used in this document are only for illustrative purposes and do not represent the only implementation method.

[0061] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by technicians in the technical field to which the present invention belongs; the terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention; the term "and / or" used herein includes any and all combinations of one or more related listed items.

[0062] like Figure 1 , 2 As shown in 3, a breeding method for high-yield hybrid rice resistant to rice leaf smut comprises the following steps:

[0063] S1. Parent material selection: From existing rice varieties, parent materials with high resistance to rice leaf smut and parent materials with high yield potential are screened out through field evaluation and laboratory testing; the field evaluation includes disease resistance evaluation under different disease pressures, and recording and analyzing the disease incidence and disease index of rice; the laboratory testing includes detecting the presence and expression of disease resistance genes and high-yield genes of rice through PCR amplification and sequencing technology;

[0064] S2. Molecular marker-assisted selection: using single nucleotide polymorphism (SNP) markers, simple sequence repeat (SSR) markers and sequence-specific amplified region (SSAR) markers, the parental materials screened in step S1 are genotyped and parental materials carrying multiple disease resistance genes and high-yield genes are selected;

[0065] S3, hybrid breeding: hybridizing the parental materials selected in step S2 to obtain the first generation of hybrids (F1);

[0066] S4. Gene editing: Using the CRISPR / Cas9 system to perform gene editing on the hybrid first generation obtained in step S3, knocking out or enhancing key genes related to disease resistance and yield, and specific editing sites include but are not limited to rice leaf smut resistance genes (such as Pi-ta, Pi-k, Pi-21, etc.) and high-yield genes (such as OsSPL14, OsGS1, OsMADS2, etc.); the CRISPR / Cas9 system includes designing sgRNA sequences for target genes, using a vector system to introduce sgRNA and Cas9 enzymes into hybrid first generation rice cells, and obtaining gene-edited rice plants through plant tissue culture technology; detecting the gene-edited rice plants to confirm the knockout or enhancement effect of the target gene;

[0067] S5. Genetic modification: using Agrobacterium-mediated gene transformation method, introducing exogenous rice leaf smut resistance genes (such as Pi-z, Pi-kh, etc.) into the hybrid first generation after gene editing in step S4, to further improve its disease resistance; selecting exogenous rice leaf smut resistance genes (such as Pi-z, Pi-kh, etc.), using Agrobacterium to introduce the exogenous genes into the rice cells of the hybrid first generation after gene editing, and obtaining transgenic rice plants through endosperm culture technology; detecting the transgenic rice plants to confirm the insertion and expression of the exogenous genes;

[0068] S6. Tissue culture: Rapidly propagate the transgenic hybrid rice obtained in step S5 through callus culture and endosperm culture techniques, and screen out individuals with excellent disease resistance and yield; callus culture includes inducing callus culture of rice seeds or seedlings to obtain callus; endosperm culture includes in vitro culturing the endosperm of rice seeds to obtain regenerated plants; through screening and identification, select individuals with excellent disease resistance and yield;

[0069] S7. Multi-environment field trials: Conduct field trials in different geographical regions and disease pressure conditions, record and analyze test data, and evaluate the disease resistance and yield performance of rice varieties; the different geographical regions include but are not limited to low altitude, high altitude, coastal and inland areas; the different disease pressure conditions include but are not limited to light disease areas, moderate disease areas and severe disease areas; record and analyze test data, and evaluate the disease resistance and yield performance of rice varieties;

[0070] S8. Conservation and utilization of genetic diversity: Establish a germplasm resource bank to collect and preserve a variety of rice gene resources with disease resistance and high yield potential for the selection and improvement of subsequent breeding materials; the establishment of the germplasm resource bank includes collecting and preserving rice varieties from different sources, including wild species, local varieties and improved varieties; identify and evaluate the genetic diversity of germplasm resources through genome sequencing and molecular marker technology; use bioinformatics technology to establish a germplasm resource database to facilitate the selection and improvement of subsequent breeding materials;

[0071] S9. Comprehensive performance evaluation: Through multi-year and multi-point field trials, the disease resistance, yield, plant traits and rice quality of rice varieties are evaluated to ensure that the hybrid rice varieties finally obtained have excellent comprehensive performance; the comprehensive performance evaluation includes disease resistance evaluation, through artificial inoculation of rice leaf smut fungus experiments, to evaluate the disease resistance of rice varieties; yield evaluation, through multi-year and multi-point field trials, to evaluate the yield performance of rice varieties; plant trait evaluation, measuring and analyzing rice plant height, tiller number, root development and other trait indicators; quality evaluation, through rice quality testing, to evaluate the rice quality of rice varieties.

[0072] In this embodiment, the specific examples of each step are as follows:

[0073] Parent material selection: The selection of parent materials is the basis of the entire breeding process. Through field evaluation and laboratory testing, rice varieties with high disease resistance and high yield potential can be effectively screened, providing high-quality materials for subsequent hybridization and genetic improvement. By combining comprehensive field and laboratory evaluation, it is ensured that the selected parent materials have high disease resistance and high yield potential, laying a solid foundation for the subsequent breeding process.

[0074] Molecular marker-assisted selection: Molecular marker technology can quickly and accurately identify the genotype of parental materials, improve the efficiency and accuracy of selection, and ensure that hybrid offspring carry multiple disease-resistant genes and high-yield genes. Molecular marker-assisted selection technology can improve the accuracy and efficiency of breeding, ensure that the selected parental materials carry multiple disease-resistant and high-yield genes, and thus significantly improve the comprehensive performance of hybrid offspring.

[0075] Hybrid breeding: Hybrid breeding is an important means to achieve genetic recombination. By hybridizing parental materials carrying multiple excellent genes, a hybrid generation with excellent comprehensive performance can be effectively obtained. Hybrid breeding can effectively integrate multiple excellent genes to produce a hybrid generation with high disease resistance and high yield potential, providing a high-quality material foundation for subsequent gene editing and transgenics.

[0076] Gene editing: Using the CRISPR / Cas9 system for gene editing can accurately knock out or enhance target genes, improving rice disease resistance and yield. CRISPR / Cas9 technology has a high degree of precision and flexibility, and can specifically modify target genes, effectively improving rice disease resistance and yield, providing strong technical support for breeding.

[0077] Genetic modification: Through the Agrobacterium-mediated gene transformation method, exogenous disease-resistant genes are introduced into the gene-edited hybrid generation to further improve its disease resistance. The Agrobacterium-mediated gene transformation method has the characteristics of high transformation efficiency and low cost, and can effectively introduce exogenous disease-resistant genes into rice, significantly improving its disease resistance.

[0078] Tissue culture: Tissue culture technology can quickly reproduce genetically modified hybrid rice, ensuring a large number of high-quality individuals and improving breeding efficiency. Tissue culture technology can quickly reproduce a large number of high-quality individuals, improving breeding efficiency, ensuring the smooth progress of the breeding process and ultimately obtaining high-quality varieties.

[0079] Multi-environment field trials: Multi-environment field trials can verify the stability and adaptability of rice varieties in different geographical regions and disease pressure conditions, ensuring their reliability and practicality in actual production. Multi-environment field trials can comprehensively evaluate the stability and adaptability of rice varieties, ensuring their performance under different environmental conditions, and have important practical production value.

[0080] Protection and utilization of genetic diversity: Establishing a germplasm resource bank to collect and preserve a variety of rice gene resources will not only help protect genetic diversity, but also provide a rich material basis for subsequent breeding. Establishing a germplasm resource bank will help protect and utilize rich genetic diversity, provide a diverse material basis for subsequent breeding, and promote innovation and development in breeding work.

[0081] Comprehensive performance evaluation: Through multi-year and multi-point field trials, the final hybrid rice varieties are comprehensively evaluated to ensure that they have excellent comprehensive performance in terms of disease resistance, yield, plant traits and rice quality. Comprehensive performance evaluation can comprehensively evaluate various performance indicators of rice varieties to ensure that the final rice varieties have excellent comprehensive performance and meet actual production needs.

[0082] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for breeding high-yield hybrid rice resistant to rice leaf smut, characterized in that: The following steps are involved: S1. Parent material selection: From existing rice varieties, parent materials with high resistance to rice leaf smut and parent materials with high yield potential were selected through field evaluation and laboratory testing; S2, Molecular marker-assisted selection: Using single nucleotide polymorphism markers, simple repeat sequence markers and sequence-specific amplified region markers, the parental materials screened in step S1 are subjected to genotyping, and parental materials carrying multiple disease-resistant genes and high-yield genes are selected; S3. Hybrid breeding: Hybridizing the parental materials selected in step S2 to obtain the first generation hybrid (F1); S4. Gene Editing: The hybrid first generation obtained in step S3 is subjected to gene editing using the CRISPR / Cas9 system to knock out or enhance key genes related to disease resistance and yield. Specific editing sites include but are not limited to: Rice leaf smut resistance genes: such as Pi-ta, Pi-k, Pi-21, etc.; High-yield genes: such as OsSPL14, OsGS1, OsMADS2, etc.; S5. Transgenic: Using Agrobacterium-mediated gene transformation, exogenous rice leaf smut resistance genes (such as Pi-z, Pi-kh, etc.) are introduced into the first generation of hybrids after gene editing in step S4 to further improve their disease resistance; S6. Tissue culture: Rapidly propagate the transgenic hybrid rice obtained in step S5 through callus culture and endosperm culture techniques to select individuals with excellent disease resistance and yield; S7. Multi-environment field trial: Conduct field trials in different geographical regions and disease pressure conditions, record and analyze trial data, and evaluate disease resistance and yield performance of rice varieties; S8. Conservation and utilization of genetic diversity: Establish a germplasm resource bank to collect and preserve a variety of rice gene resources with disease resistance and high yield potential for subsequent selection and improvement of breeding materials; S9. Comprehensive performance evaluation: Through multi-year and multi-point field trials, the disease resistance, yield, plant traits and rice quality of rice varieties are evaluated to ensure that the final hybrid rice varieties have excellent overall performance.

2. The breeding method according to claim 1, characterized in that: The field evaluation and laboratory testing in step S1 include: Field evaluation: conduct disease resistance evaluation under different disease pressures, record and analyze the disease incidence and disease index of rice; Laboratory testing: Detect the presence and expression of disease-resistant genes and high-yield genes in rice through PCR amplification and sequencing technology.

3. The breeding method according to claim 1, characterized in that: The molecular marker-assisted selection technology in step S2 includes but is not limited to single nucleotide polymorphism (SNP) markers, simple sequence repeat (SSR) markers, and sequence-specific amplified region (SSAR) markers.

4. The breeding method according to claim 1, characterized in that: The CRISPR / Cas9 system in step S4 includes: designing a sgRNA sequence targeting a target gene; introducing sgRNA and Cas9 enzyme into hybrid first-generation rice cells using a vector system; obtaining gene-edited rice plants through plant tissue culture technology; and detecting the gene-edited rice plants to confirm the knockout or enhancement effect of the target gene.

5. The breeding method according to claim 1, characterized in that: The Agrobacterium-mediated gene transformation method in step S5 comprises: a. Select exogenous rice leaf smut resistance genes (such as Pi-z, Pi-kh, etc.); b. Using Agrobacterium to introduce foreign genes into the gene-edited hybrid rice cells; c. Obtaining transgenic rice plants through endosperm culture technology; d. Test transgenic rice plants to confirm the insertion and expression of exogenous genes.

6. The breeding method according to claim 1, characterized in that: The tissue culture technique in step S6 includes: a. Callus culture: Induce and culture the callus of rice seeds or seedlings to obtain callus; b. Endosperm culture: The endosperm of rice seeds is cultured in vitro to obtain regenerated plants; c. Through screening and identification, select individuals with excellent disease resistance and yield.

7. The breeding method according to claim 1, characterized in that: The multi-environment field test in step S7 includes: a. Conduct trials in different geographical areas, including but not limited to low altitude, high altitude, coastal and inland areas; b. Conduct the test under different disease pressure conditions, including but not limited to light disease area, moderate disease area and severe disease area; c. Record and analyze test data to evaluate the disease resistance and yield performance of rice varieties.

8. The breeding method according to claim 1, characterized in that: The establishment of the germplasm resource bank in step S8 includes: a. Collect and preserve rice varieties from different sources, including wild species, local varieties and improved varieties; b. Identify and evaluate the genetic diversity of germplasm resources through genome sequencing and molecular marker technology; c. Use bioinformatics technology to establish a germplasm resource database to facilitate the selection and improvement of subsequent breeding materials.

9. The breeding method according to claim 1, characterized in that: The comprehensive performance evaluation in step S9 includes: a. Disease resistance evaluation: The disease resistance of rice varieties is evaluated through artificial inoculation of rice leaf smut fungus; b. Yield evaluation: Evaluate the yield performance of rice varieties through multi-year and multi-site field trials; c. Plant trait evaluation: measuring and analyzing rice plant height, tillering number, root development and other trait indicators; d.Quality assessment: Evaluate the rice quality of rice varieties through rice quality testing.

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