Rapid breeding method of rice based on explicit very early maturity gene and application

Abstract

This invention discloses a rapid rice breeding method and its application based on a dominant extremely early-maturing gene. Using the dominant extremely early-maturing gene as the core, early-maturing single plants are selected as a backcross breeding bridge tool through maturity trait screening. The method includes: hybridizing rice materials carrying the target beneficial gene and rice materials carrying the extremely early-maturing gene as parents; obtaining an early-maturing bridge plant carrying the target beneficial gene through embryo culture, molecular marker screening, and maturity trait screening; using this early-maturing bridge plant as the male parent and continuously backcrossing it with the rice to be improved, conducting embryo culture, molecular marker screening, and maturity trait screening in each generation, compressing the single-generation backcross cycle to 60-76 days; self-purifying the last backcross generation, and obtaining improved lines through homozygous screening of the target beneficial gene, genetic background verification, and early-maturing trait removal. This invention utilizes the extremely early-maturing gene to accelerate generation iteration, enabling 4-5 generations of rice propagation per year, completing 3-4 backcrosses and 2 selfcrosses within 1.5 years, and can be applied on a large scale to improve multiple traits of rice.

Description

Technical Field

[0001] This invention relates to the field of rice genetics and breeding, specifically to a rapid rice breeding method and its application based on a dominant early-maturing gene. Background Technology

[0002] As a core pillar crop of my country's food security system, rice breeding has always focused on increasing yield, strengthening resistance to pests and diseases, and optimizing rice quality. However, traditional rice breeding methods are inherently time-consuming and inefficient, typically requiring 3-5 years to complete a breeding cycle, making it difficult to meet the market's rapid demand for superior varieties. The emergence of speed breeding technology has revolutionized crop breeding. This technology accelerates the growth and development process by simulating the optimal growth environment for plants and has already been applied to crops such as rapeseed, wheat, and rice. For example, the long-day crop speed breeding system established by Huazhong Agricultural University enables rapeseed to complete a generation in 67 days and wheat in 75 days; the International Rice Research Institute's "Speed ​​Flower" system allows indica and japonica rice to reproduce 4-5 generations per year. However, these technologies generally rely on high-specification artificial climate chambers or intelligent plant factories, resulting in high equipment and maintenance costs. This means that most rice breeding units are still in the exploratory stage and find it difficult to achieve large-scale, routine application.

[0003] Studies have shown that artificially regulating conditions such as high temperature, short day length, and LED light formulations can promote the early expression of rice heading genes (Ehd1, Hd3a, RFT1, etc.), accelerating the transition from vegetative growth to reproductive growth, which is key to shortening the growth cycle. Among these, dominant early-maturing genes can confer early heading characteristics to rice and hybrid offspring and exhibit strong environmental adaptability, making them an important resource for rapid breeding. This provides a new approach for building a rapid breeding system based on dominant early-maturing genes. Summary of the Invention

[0004] This invention aims to provide a rapid rice breeding method and its application based on a dominant early-maturing gene. The core of this invention lies in leveraging the genetic characteristics of the dominant functional gene controlling the early-maturing trait in rice—namely, its "photoperiod insensitivity and ability to stably shorten the growth period"—to construct an integrated and universal rapid breeding system. This system utilizes visible phenotypic screening of early-maturing individual plants as a bridge tool for backcross breeding, encompassing "creation of early-maturing bridge plants—rapid iteration of hybridization and backcrossing—precise screening across multiple generations—efficient finalization of target lines." This method ensures a year-round supply of hybrid materials through staggered sowing, shortens the seed germination cycle through embryo tissue culture, achieves year-round efficient cultivation using a simple temperature-controlled greenhouse, and integrates molecular marker-assisted selection to achieve precise identification of the target gene and genetic background. Ultimately, it yields improved rice lines with stable target traits and a genetic background highly consistent with the recipient within a short period.

[0005] The technical solution adopted in this invention is as follows:

[0006] A rapid rice breeding method based on a dominant early-maturing gene is proposed. This method uses a photoperiod-insensitive, dominant single-gene-controlled early-maturing functional gene as its core, and employs early-maturing individual plants selected based on maturity traits as a bridge tool for backcross breeding to carry out targeted improvement of the rice variety to be improved. The rapid rice breeding method includes the following steps:

[0007] (1) First stage: Cultivation of early-maturing bridge plants:

[0008] Rice materials carrying the target beneficial gene were used as the female parent, and rice materials carrying a photoperiod-insensitive, dominant single-gene-controlled extremely early-maturing functional gene were used as the male parent for cross-pollination. Immature seeds after pollination were cultured into embryos, and after hardening off, seedlings were screened for the target beneficial gene using molecular markers. Seedlings with heterozygous target beneficial gene loci were selected. The seedlings with heterozygous target beneficial gene loci were transplanted for ripening culture, and single plants exhibiting early-maturing characteristics were selected from them as early-maturing bridge plants for backcross breeding.

[0009] (2) Second stage: multi-generation backcross breeding:

[0010] Using the rice variety to be improved as the recurrent parent and the early-maturing bridge plant with heterozygous target beneficial gene loci from the previous generation as the male parent, backcrossing was carried out continuously. Each generation underwent backcross pollination, embryo culture, hardening, dual molecular marker screening, transplanting for ripening, and screening for early-maturing traits in sequence, achieving a reproductive cycle of 60-76 days for each backcross generation. In addition, the first backcross F1 and the last backcross F1 generations were used for preliminary screening of the traits corresponding to the target beneficial genes, and only plants whose phenotypic performance met the breeding target requirements were retained for subsequent breeding stages. Among them, dual molecular marker screening first screened plants with heterozygous target beneficial gene loci, then tested the genetic background similarity between the plants and the recurrent parents, and set gradient thresholds according to the backcross generations for screening.

[0011] (3) Third stage: self-pollination and purification cultivation:

[0012] Qualified F1 plants obtained from the second stage of backcrossing were selected for self-pollination to obtain seeds. Immature seeds after pollination were cultured into embryos and hardened off to obtain a self-pollinated F2 seedling population. Plants homozygous for the target beneficial gene loci were first screened out. Then, the genetic background similarity between the plants and the recurrent parents was verified. Plants with similar genetic backgrounds to the recurrent parents were transplanted for ripening culture. Plants with maturity traits comparable to the recurrent parents were screened out. Subsequently, an agronomic trait survey was conducted to screen out superior plants with core agronomic traits comparable to the recurrent parents.

[0013] If the rice to be improved is a conventional variety, the self-pollinated seeds of superior single plants are harvested directly to obtain rice lines with improved target traits. If the rice to be improved is a sterile line, the fertility of the selected superior sterile plants is induced to restore their fertility, and the self-pollinated seeds are harvested after self-pollination to obtain rice sterile lines with improved target traits.

[0014] In step (1), the target beneficial gene is a functional gene that controls disease resistance, stress resistance, yield traits, quality traits, or other agronomic traits in rice; the photoperiod-insensitive, dominant single-gene-controlled extremely early-maturing functional gene is the SGP(t) gene, or a functional gene that meets the following characteristics: average heading period ≤ 50 days under long-day high-temperature conditions and average heading period ≤ 65 days under short-day low-temperature conditions, and can promote the expression of the flowering glycogen gene Hd3a / RFT1 in the seedling stage; wherein, the long-day high-temperature conditions are: photoperiod 14~16h / d, temperature 28~32℃; the short-day low-temperature conditions are: photoperiod 8~10h / d, temperature 20~24℃.

[0015] In step (2), the rice to be improved is indica rice, japonica rice, or Java rice.

[0016] In steps (1)-(3), the specific process of embryo culture is as follows: take immature seeds 12-14 days after pollination, soak them in 75% ethanol for 30 seconds, rinse them twice with sterile water, soak them in 0.1% mercuric chloride for 10 minutes, and rinse them 4-5 times with sterile water. Then, inoculate them into 1 / 2 MS medium. Cultivate the inoculated seeds until they have 2 true leaves, and then perform hardening treatment to obtain seedlings with 2 leaves and 1 heart.

[0017] Preferably, the inoculated seeds are cultured in an incubator until they reach the two true leaf stage. The environmental parameters of the incubator are: temperature 26~30℃, photoperiod 13h / d, and light intensity 1500μmol·m⁻¹. -2 ·s -1 .

[0018] In steps (1)-(3), the seedling hardening is carried out in a sterile transitional environment in a greenhouse, and the transplanting and ripening culture is carried out in a soilless cultivation environment in a greenhouse; the environmental parameters of the greenhouse are: photoperiod 12~14h / d, corresponding temperature 28~32℃, and light intensity 1500~2000μmol·m -2 ·s -1 The dark period is 10-12 hours per day, corresponding to a temperature of 20-24°C; the relative humidity is 60%-80%.

[0019] In step (2), the genetic background similarity gradient screening threshold for the dual molecular marker screening is: backcross ≥75% for the first generation, ≥87.5% for the second generation, and ≥93% for the third generation and above. The 5 to 10 single plants with the highest similarity are selected in each generation. The backcrossing generations are 3 to 4 times, which are combined with 1 to 2 subsequent self-crosses to achieve stable inheritance of the target trait.

[0020] In step (3), the plant with a genetic background similar to the recurrent parent is a plant with a genetic background similarity of ≥97% to the recurrent parent.

[0021] The above-mentioned rapid rice breeding method is applied to the improvement of rice varieties. The application includes the improvement of rice disease resistance, stress resistance, yield increase or other quality optimization; the rice varieties to be improved include indica rice, japonica rice or Java rice.

[0022] Improved rice varieties were developed using the aforementioned rapid rice breeding methods.

[0023] The significant advantages of this invention are:

[0024] This invention addresses the shortcomings of existing rapid rice breeding technologies, such as high equipment costs, narrow applicability, and reliance on specific genes / varieties. It constructs a universal, low-cost, high-efficiency, and scalable rapid rice breeding system. This system is not limited to specific dominant early-maturing genes, specific target traits, or specific rice varieties. It can achieve a rice reproduction efficiency of 4-5 generations per year and complete a breeding process of 4 hybridizations + 2 self-pollinations within 1.5 years, providing a replicable technical path for the targeted improvement of various rice traits. Attached Figure Description

[0025] Figure 1 Typical agarose gel electrophoresis image of the Xa23 molecular marker for detecting the white leaf blight resistance gene in the BC1F1 hybrid generation.

[0026] Figure 2 Typical agarose gel electrophoresis image of the Pi1 molecular marker for rice blast resistance in backcross BC1F1 generation.

[0027] Figure 3 , Figure 4 , Figure 5 , Figure 6 Typical results of the identification of 48 pairs of SSR molecular markers in the backcross BC3F2 generation. In the figure, the banding patterns of 47 markers in the BC3F2 generation plants are the same as those in Y58S. The detection results of the B11 marker are different from those in Y58S because the Pi1 gene lock marker is the same as the B11 marker (RM224).

[0028] Figure 7 Disease phenotype for identifying rice blast resistance in improved BC3F3 lines.

[0029] Figure 8 Disease phenotype for identifying bacterial blight resistance in improved BC3F3 strains.

[0030] Figure 9 Phenotypic characteristics of improved Y58S and BC3F3 lines and their hybrids with R211.

[0031] Figure 10 Flowchart for rapid breeding and improvement of rice. Detailed Implementation

[0032] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0033] Example 1:

[0034] This embodiment uses the SGP(t) gene-mediated disease resistance improvement of the rice photoperiod-temperature-sensitive male sterile line Y58S as a specific implementation case to verify the effectiveness of the rapid rice breeding method based on the dominant extremely early maturing gene: an early maturing bridge plant was constructed using the SGP(t) gene, and backcrossing was carried out with the rice photoperiod-temperature-sensitive male sterile line Y58S as the variety to be improved and Xa23 / Pi1 as the target beneficial gene (disease resistance). The traditional backcrossing improvement cycle of 3-4 years was compressed to 437 days, and an improved male sterile line with significantly improved resistance and agronomic traits comparable to Y58S was bred, while retaining the yield potential of the original pairing.

[0035] The core breeding strategy of the improved method is as follows: First, the rice extremely early-maturing short-growth-period mutant sgp(t) is crossed with the rice two-line male-sterile line He9S. Combined with molecular marker-assisted selection technology, early-maturing donor materials carrying both Xa23 and Pi1 disease resistance genes are bred. Then, the early-maturing donor material carrying both Xa23 and Pi1 disease resistance genes is used as the male parent and the rice photoperiod-temperature-sensitive male-sterile line Y58S is used as the female parent to carry out backcross breeding. Combined with embryo culture, molecular marker screening and environmental regulation technology, the breeding cycle is shortened, so as to achieve the simultaneous completion of resistance improvement and genetic background restoration.

[0036] In this embodiment, the first hybridization was initiated on March 10, 2024, and the self-pollinated seeds of the BC3F2 generation male-sterile line (BC3F3 generation) were harvested on May 20, 2025. The entire process included one donor creation hybridization (He9S×sgp(t)), three backcrosses (with Y58S), and two self-pollinations. The total cycle for Y58S resistance improvement was 437 days.

[0037] The specific experimental procedure is as follows:

[0038] 1. Pre-experimental preparation

[0039] 1.1 Preparation of Experimental Materials

[0040] (1) Recipient material: Y58S, a photoperiod-thermosensitive male sterile rice line, was used as a recurrent parent for backcross breeding and was propagated in advance. Starting from January 1, 2024, it was sown in greenhouses in stages throughout the year, with one sowing every 15 days and 20-30 plants per sowing period to ensure that each backcross generation had a female parent plant with a suitable flowering period. This material does not carry the resistance genes Xa23 and Pi1, and its breeding method has been published: Deng Qiyun. Breeding of Y58S, a photoperiod-thermosensitive male sterile rice line with wide adaptability [J]. Hybrid Rice, 2005, 20(2):15-18.

[0041] (2) Donor material: rice extremely early maturing short-growth-period mutant sgp(t) (this material only takes about 50 days from sowing to heading), carrying the rice extremely early maturing functional gene SGP(t) controlled by a single gene that is insensitive to photoperiod and incompletely dominant. Its related research has been published: Liu Huaqing, Gu Liqing, Wu Mingji, et al. Genetic analysis and gene mapping of a rice short-growth-period mutant sgp(t) [J]. Chinese Science Bulletin, 2008, 53(13): 1552-1559. Rice two-line male sterile line He9S, this material carries the resistance genes Xa23 and Pi1, and its breeding method has been published: Chen Zhiwei, Guan Huazhong, Mao Damei, et al. Breeding and preliminary application of two-line rice male sterile lines resistant to rice blast and bacterial blight [J]. Journal of Plant Genetic Resources, 2020, 21(5):1078-1088. Starting from January 20, 2024, the early-maturing short-growth mutant rice sgp(t) and the two-line male-sterile rice line He9S will be sown in the greenhouse in stages. One sowing period will be every 15 days, for a total of 3 sowing periods. Each sowing period will have 20-30 plants to ensure the supply of male parent materials required for hybridization and backcrossing generations.

[0042] (3) Control materials: Rice two-line male sterile line He9S was used as a positive control for the identification of resistance genes Xa23 and Pi1. Rice extremely early maturing short growth period mutant sgp(t) was used as a phenotype control for extremely early maturing at the heading stage; rice photoperiod-temperature-sensitive male sterile line Y58S was used as a negative control for the identification of agronomic traits and resistance.

[0043] 1.2 Preparation of Reagents and Instruments

[0044] (1) Molecular marker detection related: Xa23 gene linkage marker M-Xa23 primer, Pi1 gene linkage marker RM224 primer, 48 pairs of rice variety SSR authenticity identification primers (see GB / T 39917-2021), Taq enzyme, dNTPs, agarose, nucleic acid dyes, PCR instrument, electrophoresis instrument, gel imaging system, rice genomic DNA extraction kit.

[0045] Xa23 gene linkage marker M-Xa23 primer sequence:

[0046] Xa23F: 5'-TTGCTCAAGGCTAGGAAAATG-3' (SEQ ID NO.1),

[0047] Xa23R: 5'-CCCCATCAACGAACTACAGG-3' (SEQ ID NO. 2);

[0048] The PCR reaction system and procedure were referenced from: Gao Lijun, Gao Hanliang, Li Rongbai, et al. Optimization and verification of the molecular marker Xa23 gene for resistance to bacterial blight in rice [J]. Molecular Plant Breeding, 2010, 8(04): 660-664.

[0049] Amplifying the genomic DNA of rice varieties reveals the following: if a 200bp characteristic band is amplified, the individual is homozygous for the Xa23 gene; if a 346bp characteristic band is amplified, the individual does not possess the Xa23 gene; and if both 200bp and 346bp characteristic bands are amplified, the individual is heterozygous for the Xa23 gene.

[0050] Pi1 gene linkage marker RM224 primer sequence:

[0051] Pi1F: 5'-ATCGATCGATCTTCACGAGG-3' (SEQ ID NO.3),

[0052] Pi1R: 5'-TGCTATAAAAGGCATTCGGG-3' (SEQ ID NO. 4);

[0053] The PCR reaction system and procedure were referenced from: Chen Zhiwei, Guan Huazhong, Wu Weiren, et al. Screening and application of Pi-1 linked SSR markers for rice blast resistance gene [J]. Journal of Fujian Agriculture and Forestry University (Natural Science Edition), 2005, 34(1): 74-77.

[0054] Amplifying the genomic DNA of rice varieties reveals the following: if a single characteristic band of 157 bp is amplified, the individual is homozygous for the Pi1 gene; if a single characteristic band of 163 bp is amplified, the individual does not possess the Pi1 gene; and if two characteristic bands of 157 bp and 163 bp are amplified, the individual is heterozygous for the Pi1 gene.

[0055] Primer sequences for identifying the authenticity of SSRs of 48 rice varieties:

[0056]

[0057]

[0058]

[0059] The PCR reaction system and procedure refer to GB / T 39917-2021 "Detection of Authenticity and Purity of SSR Molecular Markers in Rice of Major Crop Varieties";

[0060] The percentage of similarity was calculated based on the number of valid detection sites after removing genetically unstable sites from the 48 SSR sites, according to the proportion of sites that matched the genotype of the recurrent parent.

[0061] (2) Related to embryonic tissue culture: 1 / 2 MS medium (i.e., MS basic components halved, with the addition of 30 g / L sucrose and 8 g / L agar, pH adjusted to 5.8), sterile water, 75% (v / v) ethanol, 0.1% (w / v) mercuric chloride, sterile culture dishes, inoculation needles; laminar flow hood, incubator (environmental parameters set as follows: temperature 26~30℃, photoperiod 13h / d, light intensity 1500μmol·m -2 ·s -1 ).

[0062] (3) Cultivation and environmental control: Hoagland's nutrient solution was used for hydroponics. The macronutrients were prepared at the following concentrations: potassium nitrate 0.51 g / L, calcium nitrate tetrahydrate 0.24 g / L, magnesium sulfate heptahydrate 0.49 g / L, and ammonium dihydrogen phosphate 0.136 g / L. Micronutrients were prepared by first preparing a 1000-fold stock solution, which involved weighing out 2.86 g of boric acid, 1.81 g of manganese chloride tetrahydrate, 0.22 g of zinc sulfate heptahydrate, 0.08 g of copper sulfate pentahydrate, and 0.09 g of molybdic acid monohydrate, dissolving them, and then... Prepare a 1L volume of nutrient solution. Add the solution at a ratio of 1mL / L when using. Iron salts must be prepared separately to prevent precipitation: Solution A is 7.45g / L disodium ethylenediaminetetraacetate, and Solution B is 5.57g / L ferrous sulfate heptahydrate. Before use, mix equal volumes of Solutions A and B, then add them to the nutrient solution at a ratio of 1mL / L. To prepare a 1L working solution, take an appropriate amount of distilled water, add 1mL of the macro-element and micro-element stock solutions, and 1mL of the freshly mixed iron salt solution. Make up to 1L and adjust the pH to 5.5-6.5. Hydroponic apparatus. Simple temperature-controlled glass greenhouse: 600m² 2 It is equipped with 400~700nm LED plant growth light source, intelligent temperature control system and mechanical ventilation equipment.

[0063] (4) Related to resistance identification: Rice blast pathogen 501-3, bacterial blight pathogen PXO99, spray inoculator, leaf-cutting inoculator. Rice blast resistance rating refers to the "Technical Specification for Identification and Evaluation of Rice Blast Resistance in Rice Variety Trials" (NY / T2646-2014), and according to Table A.4, the resistance level of improved lines is divided into high resistance, resistance, moderate resistance, moderate susceptibility, susceptibility, and high susceptibility. Bacterial blight resistance rating standard: average lesion length less than 1cm is high resistance, 1.1~3.0cm is resistance, 3.1~5.0cm is moderate resistance, 5.1~12.0cm is moderate susceptibility, 12.1~20.0cm is susceptibility, and greater than 20.1cm is high susceptibility.

[0064] 1.3 Setting of environmental culture parameters

[0065] The environmental parameters for a simple temperature-controlled glass greenhouse are set as follows: light cycle 12~14h / d, corresponding temperature 28~32℃, light intensity 1500~2000μmol·m -2 ·s -1 The dark period is 10-12 hours per day, corresponding to a temperature of 20-24℃; the relative humidity is controlled at 60%-80%, regulated by a combination of natural and mechanical ventilation. Rice transplanting and ripening cultivation uses a hydroponic system, with the Hoagland nutrient solution changed every two weeks to ensure adequate nutrient supply for plant growth.

[0066] 2. Core Experimental Steps

[0067] This embodiment relies on a combination of technologies including short-growth-period gene acceleration for generation, early seedling cultivation through embryo culture, molecular marker-assisted screening, and year-round greenhouse environment control to construct a rice backcrossing improvement process with 4-5 generations per year. The process is carried out in three stages, with each stage operated as follows:

[0068] 2.1 First stage: F1 generation early-maturing bridge strain cultivation (hybridization + embryo culture + molecular marker screening, March 10, 2024 - May 16, 2024)

[0069] (1) Hybrid pollination: On March 10, 2024, rice two-line male sterile line He9S with flowering period was selected as the female parent and rice very early maturing short growth period mutant sgp(t) as the male parent. At the beginning of heading of the female parent plant, fresh pollen of the male parent was collected and manually applied to the stigma of the female parent. After pollination, the plants were bagged and isolated and labeled with information such as hybrid combination and pollination date. Each hybrid combination pollinated 5 to 10 panicles.

[0070] (2) Sampling and disinfection of embryos: 14 days after pollination (March 24, 2024), the hybrid ears were harvested and the immature seeds were removed. The following procedure was followed for disinfection: soaking in 75% (v / v) ethanol for 30 seconds → rinsing twice with sterile water → soaking in 0.1% (w / v) mercuric chloride for 10 minutes (shaking continuously during soaking) → rinsing 4-5 times with sterile water → absorbing the moisture on the seed surface with sterile filter paper.

[0071] (3) Embryo tissue culture and hardening-off: In a clean bench, sterilized immature seeds were inoculated onto sterilized 1 / 2 MS medium, with 10-12 seeds inoculated per petri dish. After covering the petri dish, it was placed in an incubator (with incubator environmental parameters set as described in 1.2) for 10 days (until April 4, 2024), until the seedlings reached the 2-true-leaf stage. Subsequently, the seedlings underwent hardening-off treatment: the petri dishes were transferred from the incubator to a greenhouse (with greenhouse environmental parameters set as described in 1.3). In a clean transition area, the lid was first opened 1 / 3 for 1 day to allow the seedlings to adapt, and then the lid was fully opened for another 2 days to allow the seedlings to gradually adapt to the greenhouse environment for subsequent hydroponics. On April 7, 2024, hardening-off was completed, and the seedlings reached the 2-leaf-1-heart stage.

[0072] (4) Molecular marker screening: At the 2-leaf-1-heart stage of seedlings, 0.1g of fresh leaves from each seedling were taken to extract genomic DNA, and the DNA purity OD was controlled. 260 / OD 280 The effective value was 1.8–2.0. PCR amplification was performed using the Xa23 gene linkage marker M-Xa23 primers and the Pi1 gene linkage marker RM224 primers. The PCR products were separated by agarose gel electrophoresis, and the bands were observed and recorded using a gel imaging system. Seedlings heterozygous for both the Xa23 and Pi1 gene loci were screened. This identification process took 3 days and was carried out simultaneously with the seedling hardening process.

[0073] (5) Transplanting and ripening culture: On April 7, 2024, seedlings that were heterozygous for both Xa23 and Pi1 gene loci after hardening were transplanted to hydroponic ponds in a greenhouse (greenhouse environmental parameters are set as described in 1.3). Simultaneously, seedlings of the rice two-line male sterile line He9S (mother parent) and the rice extremely early-maturing short-growth-period mutant sgp(t) (father parent) were transplanted as controls. The heading date of each individual plant was recorded. From the heterozygous seedlings for both Xa23 and Pi1 gene loci, individual plants with a heading date significantly earlier than the mother parent, and although slightly longer than the father parent, still exhibiting early-maturing characteristics (the cycle from pollination to heading and flowering is 60-76 days) were selected as early-maturing bridge plants for backcross breeding. On May 16, 2024, the early-maturing bridge plant entered the heading stage, while the maternal control plant transplanted at the same time was still in the vegetative growth stage and had not yet headed. Pollen from the early-maturing bridge plant that was flowering at this time was selected to prepare for backcrossing with Y58S.

[0074] 2.2 Second Phase: BC1-BC3 Backcross Generation Breeding (May 16, 2024 - December 8, 2024, total 206 days)

[0075] In this stage, the photoperiod-temperature-sensitive male-sterile rice line Y58S was used as the recurrent parent, and early-maturing bridge plants heterozygous for both Xa23 and Pi1 gene loci obtained from the previous generation were used as donor paternal parents. Three consecutive backcrosses were conducted for improvement. In each backcross generation, early-maturing single plants heterozygous for both Xa23 and Pi1 gene loci (with a cycle of 60-76 days from pollination to heading and flowering) were re-selected from the backcross progeny using molecular markers and heading date surveys to serve as donor paternal parents for the next generation. The key time points for each backcross generation are shown in the table below:

[0076]

[0077] Each backcross generation followed the procedure of hybridization pollination → embryo culture → dual molecular marker screening → transplanting for ripening, with initial resistance screening conducted simultaneously in the BC1F1 and BC3F1 generations. The specific operational steps are as follows:

[0078] (1) Backcrossing: Y58S, the recurrent parent that blooms at the same time, is selected as the female parent, and early-maturing single plants that are heterozygous for both Xa23 and Pi1 gene loci from the previous generation are selected as the male parent. Artificial backcrossing is carried out. After pollination, the plants are bagged and isolated, and the generation, pollination date, single plant number and other information are marked. Five spikes are pollinated per generation.

[0079] (2) Embryo culture and seedling cultivation: Referring to the embryo sampling, disinfection and tissue culture process in 2.1, immature seeds 14 days after backcrossing were treated to obtain robust backcross offspring seedlings, which were hardened off and kept for use until the seedlings reached the 2-leaf-1-heart stage.

[0080] (3) Dual marker screening: When the seedlings reach the 2-leaf-1-heart stage, genomic DNA is extracted from individual plants for dual marker screening. Only plants that pass the dual screening are retained for subsequent culture.

[0081] ① Screening for resistance genes: PCR screening was performed using the Xa23 gene linkage marker M-Xa23 primer and the Pi1 gene linkage marker RM224 primer. The PCR amplification products were separated by agarose gel electrophoresis, and the bands were observed and recorded using a gel imaging system. Only single plants that were heterozygous for both the Xa23 and Pi1 gene loci were retained.

[0082] ② Genetic background similarity screening: 48 pairs of SSR authenticity identification primers were used to detect the genetic background similarity between the single plants that were heterozygous for both the Xa23 and Pi1 gene loci retained in ① and the recurrent parent Y58S. Gradual screening thresholds were set according to backcross generations, namely, genetic background similarity ≥75% for generation BC1, ≥87.5% for generation BC2, and ≥93% for generation BC3. The 5-10 best single plants with the highest similarity in each generation were selected first for subsequent backcross pollination.

[0083] (4) Transplanting and ripening culture: The best positive plants that passed the double screening were transplanted to a greenhouse hydroponic device and cultured according to the environmental parameters in 1.3. The heading date of each plant was investigated and recorded. Plants exhibiting early maturity characteristics (the cycle from pollination to heading and flowering is 60-76 days) were selected for backcrossing in the next generation. When the BC3 backcross was completed, the early-maturing plants obtained by screening that were heterozygous for both Xa23 and Pi1 gene loci had a genetic background similarity of ≥95% with the recurrent parent Y58S, and their agronomic traits (including leaf type and plant type) were similar to those of the recurrent parent Y58S.

[0084] (5) Initial resistance screening: Early-maturing plants that were heterozygous for both the Xa23 and Pi1 gene loci were screened in the BC1F1 and BC3F1 generations, respectively, at the 3-4 leaf stage for initial screening of resistance to rice blast and bacterial blight. For bacterial blight, the leaf-cutting method was used for inoculation (bacterial concentration 1×10⁻⁶). 8 CFU / mL), rice blast disease was treated by spraying (spore concentration 2×10⁻⁶). 5 (Number of plants / mL), and investigate the disease situation 14 days after inoculation. Only the superior single plants with resistance to rice blast and bacterial blight of medium resistance or above are retained for subsequent breeding stages.

[0085] 2.3 Third stage: BC3F2 and BC3F3 generation breeding (self-pollination homozygotes + multi-dimensional precise selection, December 8, 2024 - May 20, 2025, a total of 164 days)

[0086] This stage is the final homozygosity and trait determination stage of breeding. The core objective is to achieve homozygosity of the Xa23 and Pi1 resistance genes and ensure their stable inheritance, effectively eliminate early maturity traits, and restore the genetic background and agronomic traits highly consistent with the recurrent parents, ultimately obtaining a disease-resistant improved rice line that can be directly applied. The specific operations are as follows:

[0087] (1) Self-pollination population preparation: From December 8th to 12th, 2024, select early-maturing single plants with excellent disease resistance from the BC3F1 generation that have passed the second stage of double molecular marker screening and resistance screening, and perform artificial self-pollination. After pollination, the plants are bagged and isolated to prevent contamination by exogenous pollen. The information of the single plants, the self-pollination date and the generation are marked simultaneously. Each plant self-pollinates 10 to 15 ears to obtain BC3F2 generation seeds (i.e., BC3F1 generation self-pollination offspring) and construct a large-scale self-pollination population.

[0088] (2) Embryo culture and seedling cultivation: On December 26, 2024, the embryo culture process (2) and (3) in 2.1 were repeated. The immature seeds of the BC3F2 generation 14 days after self-pollination were treated, and a total of 2510 BC3F2 generation seedlings were obtained.

[0089] (3) Homozygous screening of resistance genes: On January 8, 2025, when the BC3F2 seedlings grew to the 2-leaf-1-heart stage, genomic DNA was extracted from individual plants. PCR amplification was performed using the Xa23 gene linkage marker M-Xa23 primer and the Pi1 gene linkage marker RM224 primer. The PCR amplification products were separated by agarose gel electrophoresis, and the bands were observed and recorded using a gel imaging system. A total of 142 plants were selected that were homozygous for both the Xa23 and Pi1 gene loci.

[0090] (4) Genetic background verification: Using 48 pairs of rice variety SSR authenticity identification primers, 56 superior plants with a genetic background similarity of ≥97% to the recurrent parent Y58S were selected from 142 plants that were homozygous for both Xa23 and Pi1 gene loci obtained in (3).

[0091] (5) Early maturity trait elimination: On March 8, 2025, the photoperiod-sensitive male sterile line Y58S of rice, which was transplanted and cultivated in the same greenhouse at the same time and had the same seedling age, was used as a control. The growth period of 56 excellent plants in (4) was screened, and only plants with the heading period of the recurrent parent Y58S (the cycle from pollination to heading and flowering is 90±2 days) were retained as candidate male sterile line plants.

[0092] (6) Agronomic trait survey: From March 10 to 12, 2025, a core agronomic trait survey was conducted on 18 candidate sterile lines from (5), including plant height, number of tillers, panicle length, and stigma exposure rate. Each plant underwent at least three technical replicate measurements, and the average value was taken as the trait data of that plant. The recurrent parent Y58S was used as a control. Finally, 12 excellent sterile lines with no significant difference in core agronomic traits from the recurrent parent Y58S (P>0.05) were selected.

[0093] (7) Fertility regulation and seed harvesting: On March 12, 2025, 12 superior sterile plants from (6) were divided and propagated, and water and fertilizer management was strengthened to ensure plant growth; on April 1, 2025, the plants entered the fertility sensitive period and were placed in an artificial climate chamber with a photoperiod of 13h / d and a light intensity of 2000μmol·m -2 ·s -1The plants were treated with an average daily temperature of 22℃ for 12 days to induce the restoration of fertility in the photoperiod-sensitive male sterile line. On April 14, 2025, the plants that had restored fertility were transplanted to a greenhouse for further cultivation. On April 25, 2025, the plants entered the flowering period. On May 20, 2025, the mature seeds of the BC3F3 generation produced by self-pollination of the superior male sterile plants of the BC3F2 generation were harvested.

[0094] 3. Resistance identification and verification of agronomic traits and yield

[0095] Resistance identification, agronomic traits, and yield verification were conducted on the BC3F3 generation improved line bred in this embodiment to confirm its resistance improvement effect and production application value. The specific identification results are as follows:

[0096] 3.1 BC3F3 generation resistance identification

[0097] In June 2025, 12 improved BC3F3 generation rice lines and the recurrent parental photoperiod-temperature-sensitive male-sterile line Y58S were planted in a high-incidence area of ​​rice blast in Chadi Township, Shanghang County, Longyan City, Fujian Province, to identify the natural incidence of rice blast. In addition, the resistance of the 12 improved BC3F3 generation rice lines and the recurrent parental photoperiod-temperature-sensitive male-sterile line Y58S to rice blast and bacterial blight was identified at the 3-4 leaf stage. Bacterial blight was detected using the leaf-cutting inoculation method (bacterial concentration 1×10⁻⁶). 8 CFU / mL), rice blast disease was treated by spraying (spore concentration 2×10⁻⁶). 5 (Number of cells / mL), and disease incidence was investigated 14 days after inoculation. Results showed: Rice blast resistance: Natural disease identification showed that all 12 improved lines exhibited moderate resistance, while the recurrent parent Y58S showed high susceptibility; seedling inoculation results were highly consistent with this, with all 12 improved lines showing high resistance, while the recurrent parent Y58S showed high susceptibility, and the lesion area of ​​the improved lines was significantly smaller than that of Y58S. Bacterial blight resistance: Seedling inoculation identification showed that all 12 improved lines exhibited high resistance, while the recurrent parent Y58S showed high susceptibility, and the lesion length of the 12 improved lines was significantly smaller than that of Y58S. These results indicate that the improved lines bred in this example showed significantly enhanced resistance to both rice blast and bacterial blight.

[0098]

[0099] 3.2 Validation of agronomic traits and yield

[0100] In June 2025, hybrid combinations were bred using six improved lines from generation BC3F3 and the photoperiod-temperature-sensitive male-sterile rice line Y58S as the female parent and the indica three-line hybrid rice restorer line R211 as the male parent. The hybrid combinations were planted in the field, and agronomic trait stability and yield were assessed. The results showed that the agronomic traits of the hybrid combinations bred from the improved lines, including plant height, number of effective panicles, panicle length, seed setting rate, number of grains per panicle, and thousand-grain weight, were not significantly different from those of the hybrid combinations bred from Y58S (P>0.05), and the yield levels were comparable (P>0.05). This indicates that the improved lines developed in this example, while achieving enhanced resistance, retained the excellent agronomic traits and yield potential of the original Y58S, and possess practical production application value.

[0101]

[0102] 4. Key Control Points and Precautions for the Experiment

[0103] To ensure the effectiveness of the breeding method of this invention, and to guarantee a shorter breeding cycle and that the improved strain traits meet the standards, the following key control points must be strictly controlled during the experiment:

[0104] (1) Flowering period regulation: Strictly implement the staggered sowing plan to ensure that the flowering periods of the parents of each hybrid and backcross generation are precisely coincided.

[0105] (2) Accuracy of molecular marker screening: During the screening of molecular markers in each generation, the operation procedures of PCR amplification and electrophoresis detection are standardized; SSR marker detection strictly follows the standard requirements of GB / T 39917-2021.

[0106] (3) Aseptic operation of embryo culture: The entire embryo tissue culture is carried out in an aseptic environment. Immature seeds need to be thoroughly disinfected. The culture medium is sterilized at 121℃ and 101kPa for 20 minutes before use. During the culture process, contaminated materials are removed in time to avoid contamination by miscellaneous bacteria and affect the seedling cultivation effect.

[0107] (4) Stability control of cultivation environment: Strictly control greenhouse environmental parameters to avoid large fluctuations in environmental parameters affecting plant growth and development and fertility conversion.

[0108] (5) Experimental record: Record key time nodes such as sowing, pollination, screening, transplanting and harvesting for each generation in detail, and record experimental data such as molecular marker screening, resistance identification results and agronomic trait survey data simultaneously, and retain complete and repeatable experimental records.

Claims

1. A rapid rice breeding method based on a dominant early-maturing gene, characterized in that: Using a photoperiod-insensitive, dominant single-gene-controlled early-maturing functional gene in rice as the core, and screening for early-maturing individual plants based on maturity traits as a bridge tool for backcross breeding, this method facilitates the targeted improvement of rice varieties. The rapid rice breeding method includes the following steps: (1) First stage: Cultivation of early-maturing bridge plants: Rice materials carrying the target beneficial gene were used as the female parent, and rice materials carrying a photoperiod-insensitive, dominant single-gene-controlled extremely early-maturing functional gene were used as the male parent for cross-pollination. Immature seeds after pollination were cultured into embryos, and after hardening off, seedlings were screened for the target beneficial gene using molecular markers. Seedlings with heterozygous target beneficial gene loci were selected. The seedlings with heterozygous target beneficial gene loci were transplanted for ripening culture, and single plants exhibiting early-maturing characteristics were selected from them as early-maturing bridge plants for backcross breeding. (2) Second stage: multi-generation backcross breeding: Using the rice variety to be improved as the recurrent parent and the early-maturing bridge plant with heterozygous target beneficial gene loci from the previous generation as the male parent, backcrossing was carried out continuously. Each generation underwent backcross pollination, embryo culture, hardening, dual molecular marker screening, transplanting for ripening, and screening for early-maturing traits in sequence, achieving a reproductive cycle of 60-76 days for each backcross generation. In addition, the first backcross F1 and the last backcross F1 generations were used for preliminary screening of the traits corresponding to the target beneficial genes, and only plants whose phenotypic performance met the breeding target requirements were retained for subsequent breeding stages. Among them, dual molecular marker screening first screened plants with heterozygous target beneficial gene loci, then tested the genetic background similarity between the plants and the recurrent parents, and set gradient thresholds according to the backcross generations for screening. (3) Third stage: self-pollination and purification cultivation: Qualified F1 plants obtained from the second stage of backcrossing were selected for self-pollination to obtain seeds. Immature seeds after pollination were cultured into embryos and hardened off to obtain a self-pollinated F2 seedling population. Plants homozygous for the target beneficial gene loci were first screened out. Then, the genetic background similarity between the plants and the recurrent parents was verified. Plants with similar genetic backgrounds to the recurrent parents were transplanted for ripening culture. Plants with maturity traits comparable to the recurrent parents were screened out. Subsequently, an agronomic trait survey was conducted to screen out superior plants with core agronomic traits comparable to the recurrent parents. If the rice to be improved is a conventional variety, the self-pollinated seeds of superior single plants are harvested directly to obtain rice lines with improved target traits. If the rice to be improved is a sterile line, the fertility of the selected superior sterile plants is induced to restore their fertility, and the self-pollinated seeds are harvested after self-pollination to obtain rice sterile lines with improved target traits.

2. The rapid rice breeding method according to claim 1, characterized in that: In step (1), the target beneficial gene is a functional gene that controls disease resistance, stress resistance, yield traits, quality traits, or other agronomic traits in rice; the photoperiod-insensitive, dominant single-gene-controlled extremely early-maturing functional gene is... SGP(t) Genes, or functional genes that meet the following characteristics: average heading period ≤ 50 days under long-day, high-temperature conditions, average heading period ≤ 65 days under short-day, low-temperature conditions, and genes that can promote flowering. Hd3a / RFT1 Expression occurred during the seedling stage; the long-day high-temperature conditions were: photoperiod 14~16h / d, temperature 28~32℃; the short-day low-temperature conditions were: photoperiod 8~10h / d, temperature 20~24℃.

3. The rapid rice breeding method according to claim 1, characterized in that: In step (2), the rice to be improved is indica rice, japonica rice, or Java rice.

4. The rapid rice breeding method according to claim 1, characterized in that: In steps (1)-(3), the specific process of embryo culture is as follows: take immature seeds 12-14 days after pollination, soak them in 75% ethanol for 30s, rinse them twice with sterile water, soak them in 0.1% mercuric chloride for 10min, and rinse them 4-5 times with sterile water. Then, inoculate them into 1 / 2MS medium. Cultivate the inoculated seeds until they have 2 true leaves, and then perform hardening treatment to obtain seedlings with 2 leaves and 1 heart.

5. The rapid rice breeding method according to claim 4, characterized in that: The inoculated seeds were cultured in an incubator until they reached the two-true-leaf stage. The incubator environmental parameters were: temperature 26-30℃, photoperiod 13h / d, light intensity 1500μmol·m⁻¹. -2 ·s -1 .

6. The rapid rice breeding method according to claim 1, characterized in that: In steps (1)-(3), the seedling hardening is carried out in a sterile transitional environment in a greenhouse, and the transplanting and ripening culture is carried out in a soilless cultivation environment in a greenhouse; the environmental parameters of the greenhouse are: photoperiod 12~14h / d, corresponding temperature 28~32℃, and light intensity 1500~2000μmol·m -2 ·s -1 The dark period is 10-12 hours per day, corresponding to a temperature of 20-24°C; the relative humidity is 60%-80%.

7. The rapid rice breeding method according to claim 1, characterized in that: In step (2), the genetic background similarity gradient screening threshold for the dual molecular marker screening is: backcross ≥75% for the first generation, ≥87.5% for the second generation, and ≥93% for the third generation and above. The 5 to 10 single plants with the highest similarity are selected in each generation. The backcrossing generations are 3 to 4 times, which are combined with 1 to 2 subsequent self-crosses to achieve stable inheritance of the target trait.

8. The rapid rice breeding method according to claim 1, characterized in that: In step (3), the plant with a genetic background similar to the recurrent parent is a plant with a genetic background similarity of ≥97% to the recurrent parent.

9. The application of the rapid rice breeding method according to any one of claims 1 to 8 in improving rice varieties, characterized in that: The applications include improving rice disease resistance, stress resistance, yield, or other quality optimization; the rice varieties to be improved include indica rice, japonica rice, or Javanese rice.

10. An improved rice strain obtained by the rapid rice breeding method according to any one of claims 1 to 8.

Images