A method for cultivating nitrogen-efficient rice varieties

Abstract

The present invention discloses a method for cultivating a nitrogen-efficient rice variety, comprising the following steps: selecting a rice variety containing OsTCP19 DY Breeding material 898 and OsCERK1 DY The breeding material of the gene, Ganjundao No. 1, was used as the donor parent and hybridized with Huizhan as the recipient parent; and the nitrogen efficient gene OsTCP19 was developed, designed and identified. DY and OsCERK1 DY The KASP molecular marker was used for molecular marker-assisted selection, and the two nitrogen-efficient genes OsTCP19 were successfully expressed through continuous backcrossing, self-crossing and polyhybridization. DY and OsCERK1 DY Polymerization in breeding materials; breeding material HZ-OsTCP19 cultivated using the cultivation method of the present invention DY / OsCERK1 DY Nitrogen utilization efficiency and yield under low nitrogen conditions were significantly improved.

Description

Technical Field

[0001] The present invention relates to the technical field of rice molecular breeding, and in particular to a method for cultivating a nitrogen-efficient rice variety. Background Art

[0002] Nitrogen is a key factor in increasing crop yields. Excessive nitrogen fertilizer application can lead to decreased yield and quality, while misuse can also cause environmental problems such as eutrophication. While effective field management can improve nitrogen fertilizer utilization in production, genetically improving varieties with efficient nitrogen utilization is a more fundamental strategy. Therefore, developing crop varieties that can maintain growth and yield under low nitrogen levels is crucial for reducing nitrogen fertilizer use in production.

[0003] Dongxiang wild rice (DY) is the northernmost wild rice discovered to date. As a wild relative of cultivated rice, it contains a rich collection of beneficial genes. Advances in rice functional genomics have uncovered several nitrogen-efficient genes from Dongxiang wild rice, providing theoretical and technical support for genetically improving nitrogen use efficiency in rice.

[0004] In 2021, Chu Chengcai's team published a research paper titled "Genomic basis of geographical adaptation to soil nitrogen in rice" in Nature. The study revealed the molecular basis of nitrogen regulation of rice tillering development. OsTCP19, as an inhibitory factor, can directly bind to the promoter of the dwarf and low-tillering gene (DLT) and negatively regulate its expression, thereby achieving the regulation of rice tillering. The study also found that the OsTCP19 promoter of nitrogen-efficient varieties lacks a 29bp nucleic acid sequence, and the nitrogen-responsive negative regulatory factor - lateral organ boundaries domain (LBD) protein can efficiently bind to this site and inhibit OsTCP19 expression. Experimental verification shows that the allele OsTCP19 exists in Dongxiang wild rice. DY It is a nitrogen efficient gene, and compared with common cultivated rice, OsTCP19 DY Genes have unique mutation sites.

[0005] In 2020, the research groups of Duanmu Deqiang and Cao Yangrong jointly published a research paper titled "Natural variation at OsCERK1 regulates arbuscular mycorrhizal symbiosis in rice" in New Phytologist. The research group members cloned OsCERK1 from Dongxiang wild rice. DY The gene can enhance the symbiosis between rice and arbuscular mycorrhizal fungi (AMF), improving the nitrogen absorption and utilization efficiency of rice by 7.92%.

[0006] After long-term breeding practice, a series of excellent rice varieties and germplasms have been cultivated using Dongxiang wild rice as breeding parent materials, including those containing OsTCP19 DY Rice germplasm 898 and OsCERK1 DY The rice germplasm Ganjundao No. 1 with the gene. Using the above germplasm to improve rice varieties can greatly improve rice's absorption and utilization of nitrogen, accelerate rice breeding and green production. However, it contains a single OsTCP19 DY OsCERK1 DY Rice varieties containing both OsTCP19 and OsTCP19 are susceptible to the influence of soil environment in different ecological zones on rice nitrogen absorption and utilization. DY Gene and OsCERK1 DY Therefore, breeding nitrogen-efficient rice varieties through gene polymerization is a key issue that needs to be solved urgently.

[0007] Molecular marker technology can effectively assist in the selection of superior genes. However, currently, there is a lack of simple and effective molecular marker technology to assist in the breeding process of nitrogen-efficient rice. Existing molecular marker technology cannot get rid of the lengthy steps of polymerase chain reaction (PCR), such as enzyme digestion, electrophoresis, staining, and band detection. This increases operation and labor costs, is inconvenient, and is difficult to use in large-scale breeding applications.

[0008] KASP genotyping technology is a unique competitive allele-specific PCR that can perform high-precision biallelic typing of SNPs and InDels (insertion deletion) on various genomic nucleic acid samples. After the PCR amplification reaction is completed, KASP markers do not require electrophoresis analysis, staining or the addition of fluorescent markers. The genotype analysis can be completed by running the scanning program for 1 minute using a fluorescent quantitative PCR instrument. At the same time, the genotyping results can be easily exported, which saves the time of manual reading of genotypes and avoids the deviation caused by human misreading. In general, KASP technology is simple to operate, stable and accurate in analysis, and low in cost. Using KASP technology to aggregate Higashino-type nitrogen-efficient genes is a fast and effective method for breeding high-yield and high-efficiency rice varieties.

[0009] The present invention utilizes KASP technology to transform the Dongye type nitrogen efficient gene OsTCP19 DY and OsCERK1 DY Aggregation, this new gene combination model, will cultivate more extensive and effective nitrogen-efficient breeding materials to meet the needs of rice production. Summary of the Invention

[0010] In view of the above problems, the present invention provides a method for breeding nitrogen-efficient rice varieties, using KASP technology to transform the Dongye type nitrogen-efficient gene OsTCP19 into DY and OsCERK1 DY By polymerizing in breeding materials, the cultivated breeding materials or varieties have broader and more efficient nitrogen absorption and utilization properties, which is beneficial to the green and efficient production of rice.

[0011] The present invention is achieved in that:

[0012] 1. Gene selection

[0013] The following principles were followed in gene selection: 1) the selected gene had been confirmed, and the Higashino-type allele of the gene was a nitrogen-efficient gene; 2) the metabolic pathways of the two aggregated Higashino-type nitrogen-efficient genes belonged to different pathways to avoid functional redundancy effects.

[0014] 2. Parent selection

[0015] The following principles should be followed in parent selection: 1) Selection of nitrogen-efficient gene donor parents: In principle, breeding intermediate materials with less unfavorable linkage drag should be selected, and it is best to choose a single-gene line containing only the target gene; 2) Selection of nitrogen-efficient gene recipient parents: In principle, varieties or hybrid rice parents that are widely promoted and applied in production, have high fertilizer requirements, and weak tillering ability should be selected, and it is best to choose varieties or hybrid rice parents that still have production potential.

[0016] 3. Breeding methods

[0017] The breeding method adopted is a combination of molecular marker-assisted selection based on KASP technology and conventional breeding (pedigree breeding, comprehensive agronomic trait selection).

[0018] 4. Based on breeding objectives, conduct aggregate breeding of parental materials to obtain rice breeding materials or varieties with broad adaptability and high nitrogen fertilizer absorption and utilization rates; breeding objectives should at least include excellent comprehensive agronomic traits and aggregation of nitrogen-efficient genes from different metabolic pathways.

[0019] The present invention utilizes molecular marker-assisted selection based on KASP technology combined with pedigree breeding to aggregate two Higashino-type nitrogen-efficient genes belonging to different metabolic pathways into a recipient parent with a genetic background, thereby cultivating breeding materials or lines with broad adaptability and high nitrogen efficiency, improving the absorption and utilization of nitrogen by rice, and meeting the needs of green rice production.

[0020] Technical solution: A method for cultivating nitrogen-efficient rice varieties, comprising the following steps:

[0021] S1, select OsTCP19 DY Breeding materials containing OsCERK1 DY The breeding materials of the gene were used as donor parents and hybridized with the recipient parents to obtain F1, and F1-OsTCP19 DY and F1-OsCERK1 DY Said OsTCP19 DY Gene and OsCERK1 DY The genes are all nitrogen efficient genes, the OsTCP19 DY Gene and OsCERK1 DY Genes belong to different metabolic pathways to avoid functional redundancy effects;

[0022] S2. Planting F1-OsTCP19 DY and F1-OsCERK1 DY The seeds and flowering stages were backcrossed with the recurrent parent to obtain the first generation BC1F1 population, which were BC1F1-OsTCP19 DY and BC1F1-OsCERK1 DY express;

[0023] S3. Planting BC1F1-OsTCP19 DY and BC1F1-OsCERK1 DY Population, in the seedling stage using the developed identification target gene OsTCP19 DY and OsCERK1 DYThe KASP molecular markers of the genotype were detected by molecular testing, and the heterozygous plants containing the target gene and with main agronomic traits similar to those of the recurrent parent were selected. The plants were backcrossed with the recurrent parent at the flowering stage to obtain the BC2F1 population. DY and BC2F1-OsCERK1 DY express;

[0024] S4, repeat step S3 until the 4th backcross generation, obtain BC4F1 population, and use BC4F1-OsTCP19 DY and BC4F1-OsCERK1 DY express;

[0025] S5. Plant each BC4F1 population and perform molecular detection of the target gene using the above-mentioned KASP molecular marker at the seedling stage. Among the homozygous plants with the marker genotype, select the plants with main agronomic traits similar to those of the recurrent parent and self-pollinate to obtain BC4F2 lines. DY and BC4F2-OsCERK1 DY express;

[0026] S6, repeat step S5 to obtain BC4F3 strains, BC4F3-OsTCP19 DY and BC4F3-OsCERK1 DY express;

[0027] S7. Planting BC4F3-OsTCP19 DY and BC4F3-OsCERK1 DY At the seedling stage, the KASP molecular markers were still used to detect the target gene, with OsTCP19 DY BC4F3-OsTCP19, a single plant with homozygous genotype and agronomic traits similar to those of the recurrent parent DY As the female parent, OsCERK1 DY BC4F3-OsCERK1 single plant with homozygous genotype and agronomic traits similar to the recurrent parent DY The male parent was pollinated and hybridized to obtain hybrid F1 seeds, and F1-OsTCP19 DY / OsCERK1 DY express;

[0028] S8, planting F1-OsTCP19 DY / OsCERK1 DY Plants were mixed and harvested at maturity to obtain the F2 population, with F2-OsTCP19 DY / OsCERK1 DY express;

[0029] S9, planting F2-OsTCP19 DY / OsCERK1 DY The planting scale was expanded to more than 600 plants. The target gene molecular detection was performed on each plant in the population using the above-mentioned KASP molecular markers at the seedling stage. The plants with double gene homozygosity and agronomic traits similar to those of the recurrent parents were selected for harvesting and F3 seeds were obtained. DY / OsCERK1 DY express;

[0030] S10, planting F3-OsTCP19 DY / OsCERK1 DY Repeat step S9 to obtain F4 seeds, and use F4-OsTCP19 DY / OsCERK1 DY express;

[0031] S11, planting F4-OsTCP19 DY / OsCERK1 DY The target gene was detected at the seedling stage; the aboveground plant samples were taken at the mature stage, and the nitrogen absorption of the plants was tested by stems, leaves, and ears, and the nitrogen absorption and utilization efficiency was calculated; the strains with high nitrogen absorption and utilization rates and main traits similar to the recurrent parents were selected to obtain the breeding material F5-OsTCP19 that aggregated the double nitrogen high-efficiency gene. DY / OsCERK1 DY ;

[0032] S12. Conduct trials in multiple ecological zones to detect F5-OsTCP19 DY / OsCERK1 DY The nitrogen absorption and utilization efficiency of the crops was evaluated, and the agronomic traits were evaluated to comprehensively evaluate the applicability of the breeding materials.

[0033] In some embodiments, in step S3, the target gene OsTCP19 is identified. DY The KASP molecular markers include the forward primer OsTCP19 DY -FA sequence: 5'-GAAGGTGACC AAGTTCATGCTAAAGTTTGTTTAAATTTCAA-3' (SEQ ID NO. 1), forward primer OsTCP19 DY -FG sequence: 5'-GAAGGTCGGAGTCAACGGATTAAAGTTTGTTTAAATTTCAG-3' (SEQ ID NO. 2), and reverse universal primer OsTCP19 DY-R sequence: 5'-TGTGGTGCACATATGAGGAGG-3' (SEQ ID NO.3);

[0034] Used to identify the target gene OsCERK1 DY The KASP molecular markers include the forward primer OsCERK1 DY -FG sequence: 5'-GAAGGTGACCAAGTTCATGCTTTTAAATT CCCTGGTAAAAG-3' (SEQ ID NO. 4), forward primer OsCERK1 DY -FT sequence: 5'-GAAGGTCGGAGTCAACGGATTTTTAAATTCCCTGGTAAAAT-3' (SEQ ID NO. 5), reverse universal primer OsCERK1 DY -R sequence is: 5'-ATCATGGCGCAATT AGGCTT-3' (SEQ ID NO.6).

[0035] In some embodiments, the forward primer OsTCP19 DY -FA's 5' end is labeled with FAM fluorescent signal, and the forward primer is OsTCP19 DY -HEX fluorescent signal tag is added to the 5' end of FG;

[0036] The forward primer OsCERK1 DY -FG 5' end with FAM fluorescent signal tag, forward primer OsCERK1 DY -FT is tagged with a HEX fluorescent signal at the 5' end.

[0037] In some embodiments, in steps S3 to S6 and steps S10 to S11, the planting scale is expanded to more than 48 plants; in step S9, the planting scale is expanded to more than 600 plants, so as to increase the probability of candidate plants having agronomic traits similar to those of the recurrent parent.

[0038] In some embodiments, in steps S11 to S12, samples are taken at maturity to measure the nitrogen accumulation in the roots, stems, and leaves of rice, calculate the nitrogen absorption and utilization efficiency of different rice strains, and comprehensively evaluate the nitrogen absorption of the developed nitrogen-efficient breeding materials.

[0039] In some embodiments, the OsTCP19 DY The breeding material of the gene is 898, which contains OsCERK1 DY The breeding material of the gene is Ganjun Rice No. 1.

[0040] In some embodiments, the recipient parent and the recurrent parent are elite conventional rice varieties or two-line hybrid rice varieties.

[0041] In some embodiments, the recipient parent and the recurrent parent are Humixan.

[0042] Beneficial effects of the present invention:

[0043] 1. The present invention aggregates the Dongye-type nitrogen efficient gene "OsTCP19 DY +OsCERK1 DY ", can better cope with the impact of soil environment in different ecological zones on rice nitrogen absorption and utilization, and avoid the functional defects of a single gene being unable to play its role in promoting efficient nitrogen utilization in rice due to environmental influences. OsTCP19 DY and OsCERK1 DY Genes belong to different metabolic pathways. DY The main reason is that the natural variation plays a key role in regulating tiller number under low nitrogen levels, which improves the adaptability of rice to low nitrogen tolerance in soil. DY The main feature is the natural variation that can enhance the symbiosis between rice roots and arbuscular mycorrhizae. Arbuscular mycorrhizae colonizing on rice roots can transfer the nitrogen nutrients absorbed by themselves to rice, thus promoting the absorption of nitrogen by rice.

[0044] 2. This invention utilizes two Dongye-type nitrogen-efficient genes from different metabolic pathways and transfers them to a widely used cultivated rice cultivar through molecular marker-assisted selection backcrossing. This creates nitrogen-efficient single-gene lines with the same genetic background and different target genes. On this basis, these single-gene lines are then hybridized to create digenic lines that incorporate different nitrogen-efficient genes. This breeding process offers two advantages: first, the resulting nitrogen-efficient single-gene lines can be applied in production as early as possible; second, flexible hybridization of different single-gene lines with the same genetic background allows for the aggregation of different genes and rapid stabilization of offspring traits, avoiding linkage drag, saving costs, and shortening breeding time.

[0045] 3. The present invention expands the planting scale of both backcross and hybrid offspring groups in the process of cultivating rice breeding materials with wide adaptability and high nitrogen fertilizer absorption and utilization rate by aggregating two nitrogen-efficient genes belonging to different metabolic pathways. Its advantages are that it not only screens single plants containing homozygous target genes, but also increases the probability of screening single plants with excellent agronomic traits, reduces costs and workload, and improves test accuracy.

[0046] 4. The present invention identifies the nitrogen utilization performance of rice breeding materials with wide adaptability and high nitrogen fertilizer absorption and utilization efficiency in different ecological zones. The advantage is that the nitrogen-efficient materials or varieties bred can withstand production inspections and reduce the impact of the environment on nitrogen absorption efficiency.

[0047] 5. The present invention is designed to identify the target gene OsTCP19 DY and OsCERK1 DY Genotypic KASP molecular markers can not only quickly and accurately identify OsTCP19 in rice germplasm resources or breeding populations, but also DY and OsCERK1 DY genes, and can simultaneously achieve high-throughput detection of more sample materials, improve selection efficiency, and accelerate the breeding process of nitrogen-efficient rice varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 To aggregate the Dongye-type nitrogen-efficient gene OsTCP19 DY and OsCERK1 DY Cultivation target improved line HZ-OsTCP19 DY / OsCERK1 DY Schematic diagram of the technical route;

[0049] Figure 2 Based on the KASP molecular marker a near-isogenic line population OsTCP19 DY Genotype detection; (The green dots close to the Y axis represent OsTCP19 carrying the A allele variant. DY Homozygous for the gene; the pink dots close to the X-axis represent the homozygous individuals carrying the G allele without OsTCP19. DY The red dot between the X and Y axes represents the homozygote of OsTCP19 carrying both A and G alleles. DY Heterozygotes of the gene; black dots represent negative controls in which double-distilled water was used instead of sample template DNA)

[0050] Figure 3 Based on the KASP molecular marker b near-isogenic population OsCERK1 DY Genotype detection. (The red dots near the Y axis represent OsCERK1 carrying the G allele variant. DY The blue dots close to the X-axis represent homozygotes carrying the T allele without OsCERK1. DY Homozygous for the gene; the green dot in the middle of the X and Y axes represents the OsCERK1 gene carrying both the G / T allele variant. DY Heterozygotes of the gene; black dots represent negative controls in which double-distilled water was used instead of sample template DNA) DETAILED DESCRIPTION

[0051] The present invention will be further described in detail below with reference to the embodiments.

[0052] Example

[0053] 1. Nitrogen-efficient gene donor parent and recipient parent

[0054] (1) Nitrogen-efficient gene OsTCP19 DY The donor parent -889 (breeding intermediate material, identified as containing OsTCP19 DY gene), mycorrhizal efficient symbiosis gene OsCERK1 DY The donor parent is Ganjun Rice No. 1 (variety approval number: Ganshen Rice 20220010, Rice Research Institute, Jiangxi Academy of Agricultural Sciences).

[0055] (2) Recipient parent-Huizhan (variety right number 20191001937, Rice Research Institute, Jiangxi Academy of Agricultural Sciences).

[0056] 2. Aggregation of Dongxiang wild rice nitrogen-efficient gene OsTCP19 DY and OsCERK1 DY Cultivation of the rice breeding material HZ-OsTCP19 with wide adaptability and high nitrogen fertilizer absorption and utilization efficiency DY / OsCERK1 DY The process (refer to Figure 1 ):

[0057] (1) Carrying the nitrogen efficient gene OsTCP19 DY The donor parent -889 and the mycorrhizal efficient symbiotic gene OsCERK1 DY The donor parent Ganjun rice No. 1 was hybridized with the recurrent parent Huizhan to obtain F1, and F1-OsTCP19 DY and F1-OsCERK1 DY express;

[0058] (2) Planting F1-OsTCP19 DY and F1-OsCERK1 DY The seeds and flowering period were backcrossed with the recurrent parent Huizhan to obtain the first generation BC1F1 population, and BC1F1-OsTCP19 DY and BC1F1-OsCERK1 DY express;

[0059] (3) Planting BC1F1-OsTCP19 DY and BC1F1-OsCERK1 DY The planting scale is 48 plants per backcross generation (the same below), and the target gene OsTCP19 developed by the researchers was used in the seedling stage. DY and OsCERK1 DYThe KASP molecular markers a (SEQ ID NO. 1-3) and KASP molecular markers b (SEQ ID NO. 4-6) of different genotypes were molecularly detected, and the heterozygous plants containing the target gene and with main agronomic traits similar to those of the recurrent parent were selected. The plants were backcrossed with the recurrent parent at the flowering stage to obtain BC2F1 populations. The BC2F1-OsTCP19 DY and BC2F1-OsCERK1 DY express.

[0060] Specifically, it was used to identify the target gene OsTCP19 DY The KASP molecular marker a includes the forward primer OsTCP19 DY -FA sequence is shown in SEQ ID NO.1, forward primer OsTCP19 DY -FG sequence is shown in SEQ ID NO.2, and reverse universal primer OsTCP19 DY -R sequence is shown in SEQ ID N0.3; used to identify the target gene OsCERK1 DY The KASP molecular marker b includes the forward primer OsCERK1 DY -FG sequence is shown in SEQ ID NO.4, forward primer OsCERK1 DY -FT sequence is shown in SEQ ID NO.5, reverse universal primer OsCERK1 DY The -R sequence is shown in SEQ ID NO.6 (see Table 1).

[0061] Table 1 Sequence information of markers used in marker-assisted selection

[0062]

[0063]

[0064] Detection of the rice nitrogen-efficient gene OsTCP19 DY and OsCERK1 DY The KASP molecular labeling method: Synthetic OsTCP19 DY When using KASP molecular marker primers, the forward primer OsTCP19 DY -FA's 5' end is labeled with FAM fluorescent signal, and the forward primer is OsTCP19 DY -FG 5' end with HEX fluorescent signal tag; in the synthesis of OsCERK1 DY When using KASP molecular marker primers, the forward primer is OsCERK1 DY -FG 5' end with FAM fluorescent signal tag, forward primer OsCERK1 DY-FT is tagged with a HEX fluorescent signal at the 5' end.

[0065] The above-mentioned molecular marker method comprises the following steps:

[0066] 1) Extraction of genomic DNA from rice plants;

[0067] 2) Add KASP molecular marker a and b primers separately to the same PCR reaction system, and set up two blank controls with double-distilled water instead of sample template DNA. Amplify the DNA of rice germplasm resources or breeding population plants on a PCR instrument;

[0068] 3) Fluorescence signals were read on an Omega fluorescence plate reader and data were analyzed using Klustercaller data management and analysis software.

[0069] The 10 μL reaction system included 2.0 μL of sample template DNA (10 ng / μL), 0.2 μL of fluorescent primer mix (forward primer 1: forward primer 2: reverse primer = 2:2:5), 5.0 μL of 2×KASP reaction mixture, and 2.8 μL of double-distilled water.

[0070] The reaction conditions included pre-denaturation at 94°C for 15 min; denaturation at 94°C for 20 sec, annealing at 61-55°C for 60 sec (0.6°C reduction per cycle), 10 cycles; denaturation at 94°C for 20 sec, annealing at 55°C for 60 sec, 26 cycles.

[0071] refer to Figures 2 and 3 , according to the color and position of the data graph, in the OsTCP19 DY During genotyping, the green dots close to the Y axis represent OsTCP19 carrying the A allele variant. DY Homozygous for the gene; the pink dots close to the X-axis represent the homozygous individuals carrying the G allele without OsTCP19. DY The red dot between the X and Y axes represents the homozygote of OsTCP19 carrying both A and G alleles. DY Heterozygotes of the gene; black dots represent negative controls in which double-distilled water was used instead of sample template DNA;

[0072] In the presence of OsCERK1 DY During genotyping, the red dots close to the Y axis represent OsCERK1 carrying the G allele variant. DY The blue dots close to the X-axis represent homozygotes carrying the T allele without OsCERK1. DY Homozygous for the gene; the green dot in the middle of the X and Y axes represents the OsCERK1 gene carrying both the G / T allele variant. DYBlack circles represent negative controls in which double-distilled water was used instead of sample template DNA.

[0073] (4) Repeat step (3) until the fourth generation of backcrossing to obtain the BC4F1 population, respectively BC4F1-OsTCP19 DY and BC4F1-OsCERK1 DY express;

[0074] (5) Plant each BC4F1 population, and use the above-mentioned KASP molecular marker to perform molecular detection of the target gene at the seedling stage. Among the homozygous single plants of the marker genotype, single plants with main agronomic traits similar to those of the recurrent parent were selected for self-pollination and harvested to obtain BC4F2 lines. DY and BC4F2-OsCERK1 DY express;

[0075] (6) Repeat step (5) until the third generation of self-pollination to obtain BC4F3 strains, respectively BC4F3-OsTCP19 DY and BC4F3-OsCERK1 DY express;

[0076] (7) Planting BC4F3-OsTCP19 DY and BC4F3-OsCERK1 DY At the seedling stage, the KASP molecular markers were still used to detect the target gene. At the flowering stage, the lines with homozygous genotypes and agronomic traits similar to the recurrent parents and containing the target gene OsTCP19 were selected. DY The single strain HZ-OsTCP19 DY As the female parent, the genotype is homozygous and the agronomic traits are similar to the recurrent parent, and it contains the target gene OsCERK1 DY HZ-OsCERK1 DY The male parent was pollinated and hybridized to obtain hybrid F1 seeds, and F1-OsTCP19 DY / OsCERK1 DY express;

[0077] (8) Planting F1-OsTCP19 DY / OsCERK1 DY Plants were mixed and harvested at maturity to obtain the F2 population, with F2-OsTCP19 DY / OsCERK1 DY express;

[0078] (9) Planting F2-OsTCP19 DY / OsCERK1 DYThe planting scale was expanded to more than 600 plants. The target gene molecular detection was performed on each plant in the population using the above-mentioned KASP molecular markers at the seedling stage. The plants with double gene homozygosity and agronomic traits similar to those of the recurrent parents were selected for harvesting and F3 seeds were obtained. DY / OsCERK1 DY express;

[0079] (10) Planting F3-OsTCP19 DY / OsCERK1 DY Repeat step (9) to obtain F4 seeds, and use F4-OsTCP19 DY / OsCERK1 DY express;

[0080] (11) Planting F4-OsTCP19 DY / OsCERK1 DY At the seedling stage, target gene molecular detection was still performed; at the mature stage, aboveground plant samples were taken and the nitrogen absorption of the plants was tested by stems, leaves, and ears, and the nitrogen absorption and utilization efficiency was calculated; strains with high nitrogen absorption and utilization rates and main traits similar to those of the recurrent parents were selected to obtain the breeding material HZ-OsTCP19 that polymerized the double nitrogen high-efficiency gene DY / OsCERK1 DY ;

[0081] (12) In 2023 and 2024, weight loss trials and potted plant trials were conducted in various ecological zones in Jiangxi Province, including Jishui, Gao'an, Ganzhou, Jinxian, Fuzhou, and Xiushui. DY / OsCERK1 DY The results showed that under 75% conventional fertilization, HZ-OsTCP19 DY / OsCERK1 DY The yield of HZ-OsTCP19 in different ecological zones reached the yield level of the control variety Huizhan under 100% conventional fertilization; under the same fertilization conditions, compared with Huizhan, DY / OsCERK1 DY The yield of HZ-OsTCP19 increased by an average of 6.25%. By testing the dry matter weight and nutrient content of rice varieties in pot experiments, under different fertilization conditions (0%, 25%, 50%, 75%, 100%), DY / OsCERK1 DY The nitrogen utilization efficiency of Huizhan increased by 6.83% to 44.91%.

[0082] In the above steps (3) to (6) and steps (10) to (11), the planting scale is expanded to more than 48 plants; in the above step (9), the planting scale is expanded to more than 600 plants, so as to increase the probability of candidate plants having agronomic traits similar to those of the recurrent parent.

[0083] In the above steps (11) to (12), samples are taken at maturity to determine the nitrogen accumulation in the roots, stems and leaves of rice, calculate the nitrogen absorption and utilization efficiency of different rice strains, and comprehensively evaluate the nitrogen absorption of the nitrogen-efficient breeding materials.

[0084] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the creative concept of the present invention, which all fall within the scope of protection of the present invention.

Claims

1. A method for cultivating nitrogen-efficient rice varieties, characterized in that: The following steps are involved: S1, select OsTCP19 DY Breeding materials containing OsCERK1 DY The breeding materials of the gene were used as donor parents and hybridized with the recipient parents to obtain F1, and F1-OsTCP19 DY and F1-OsCERK1 DY express; S2. Planting F1-OsTCP19 DY and F1-OsCERK1 DY The seeds and flowering stages were backcrossed with the recurrent parent to obtain the first generation BC1F1 population, which were BC1F1-OsTCP19 DY and BC1F1-OsCERK1 DY express; S3. Planting BC1F1-OsTCP19 DY and BC1F1-OsCERK1 DY Population, in the seedling stage using the developed identification target gene OsTCP19 DY and OsCERK1 DY The KASP molecular markers of the genotype were detected by molecular testing, and the heterozygous plants containing the target gene and with main agronomic traits similar to those of the recurrent parent were selected. The plants were backcrossed with the recurrent parent at the flowering stage to obtain the BC2F1 population. DY and BC2F1-OsCERK1 DY express; S4, repeat step S3 until the 4th backcross generation, obtain BC4F1 population, and use BC4F1-OsTCP19 DY and BC4F1-OsCERK1 DY express; S5. Plant each BC4F1 population and perform molecular detection of the target gene using the above-mentioned KASP molecular marker at the seedling stage. Among the homozygous plants with the marker genotype, select the plants with main agronomic traits similar to those of the recurrent parent and self-pollinate to obtain BC4F2 lines. DY and BC4F2-OsCERK1 DY express; S6, repeat step S5 to obtain BC4F3 strains, BC4F3-OsTCP19 DY and BC4F3-OsCERK1 DY express; S7. Planting BC4F3-OsTCP19 DY and BC4F3-OsCERK1 DY At the seedling stage, the KASP molecular markers were still used to detect the target gene, with OsTCP19 DY BC4F3-OsTCP19, a single plant with homozygous genotype and agronomic traits similar to those of the recurrent parent DY As the female parent, OsCERK1 DY BC4F3-OsCERK1 single plant with homozygous genotype and agronomic traits similar to the recurrent parent DY The male parent was pollinated and hybridized to obtain hybrid F1 seeds, and F1-OsTCP19 DY / OsCERK1 DY express; S8, planting F1-OsTCP19 DY / OsCERK1 DY Plants were mixed and harvested at maturity to obtain the F2 population, with F2-OsTCP19 DY / OsCERK1 DY express; S9, planting F2-OsTCP19 DY / OsCERK1 DY The planting scale was expanded to more than 600 plants. The target gene molecular detection was performed on each plant in the population using the above-mentioned KASP molecular markers at the seedling stage. The plants with double gene homozygosity and agronomic traits similar to those of the recurrent parents were selected for harvesting and F3 seeds were obtained. DY / OsCERK1 DY express; S10, planting F3-OsTCP19 DY / OsCERK1 DY Repeat step S9 to obtain F4 seeds, and use F4-OsTCP19 DY / OsCERK1 DY express; S11, planting F4-OsTCP19 DY / OsCERK1 DY The target gene was detected at the seedling stage; the aboveground plant samples were taken at the mature stage, and the nitrogen absorption of the plants was tested by stems, leaves, and ears, and the nitrogen absorption and utilization efficiency was calculated; the strains with high nitrogen absorption and utilization rates and main traits similar to the recurrent parents were selected to obtain the breeding material F5-OsTCP19 that aggregated the double nitrogen high-efficiency gene. DY / OsCERK1 DY ; S12. Conduct trials in multiple ecological zones to detect F5-OsTCP19 DY / OsCERK1 DY The nitrogen absorption and utilization efficiency of the crop was evaluated, and the agronomic traits were evaluated, and the applicability of the breeding materials was comprehensively evaluated; Wherein, the OsTCP19 DY The breeding material of the gene is 898, which contains OsCERK1 DY The breeding material of the gene is Ganjundao No. 1, In step S3, the target gene OsTCP19 is identified. DY The KASP molecular markers include the forward primer OsTCP19 DY -FA sequence is shown in SEQ ID NO.1, forward primer OsTCP19 DY -FG sequence is shown in SEQ ID NO.2, and reverse universal primer OsTCP19 DY -R sequence is shown in SEQ ID NO.3; Used to identify the target gene OsCERK1 DY The KASP molecular markers include the forward primer OsCERK1 DY -FG sequence is shown in SEQ ID NO.4, forward primer OsCERK1 DY -FT sequence is shown in SEQ ID NO.5, reverse universal primer OsCERK1 DY -R sequence is shown in SEQ ID NO.

6.

2. The method for cultivating nitrogen-efficient rice varieties according to claim 1, characterized in that: The forward primer OsTCP19 DY -FA's 5' end is labeled with FAM fluorescent signal, and the forward primer is OsTCP19 DY -HEX fluorescent signal tag is added to the 5' end of FG; The forward primer OsCERK1 DY -FG 5' end with FAM fluorescent signal tag, forward primer OsCERK1 DY -FT is tagged with a HEX fluorescent signal at the 5' end.

3. The method for cultivating nitrogen-efficient rice varieties according to claim 1, wherein: In steps S3 to S6 and steps S10 to S11, the planting scale is expanded to more than 48 plants.

4. The method for cultivating nitrogen-efficient rice varieties according to claim 1, characterized in that: In steps S11 to S12, nitrogen accumulation in rice roots, stems, and leaves is measured by sampling at maturity, nitrogen absorption and utilization efficiency of different rice strains is calculated, and nitrogen absorption of the developed nitrogen-efficient breeding materials is comprehensively evaluated.

5. The method for cultivating nitrogen-efficient rice varieties according to claim 1, characterized in that: The recipient parent and the recurrent parent are excellent conventional rice varieties or two-line hybrid rice varieties.

6. The method for cultivating nitrogen-efficient rice varieties according to claim 5, characterized in that: The recipient parent and the reincarnation parent are Huizhan.

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