Cultivation method of nitrogen-efficient rice variety

Abstract

The invention discloses a cultivation method of a nitrogen-efficient rice variety, which comprises the following steps: selecting a breeding material 898 containing an OsTCP19DY gene and a breeding material Ganxiangxi rice No.1 containing an OsCERK1DY gene as donor parents, and respectively hybridizing the donor parents with coma parents as receptor parents; kASP molecular markers of the nitrogen efficient genes OsTCP19DY and OsCERK1DY are developed, designed and identified for molecular marker-assisted selection, and the two nitrogen efficient genes OsTCP19DY and OsCERK1DY are polymerized in a breeding material through continuous backcross, selfing and convergent hybridization; according to the breeding material HZ-OsTCP19DY / OsCERK1DY cultivated by using the cultivation method disclosed by the invention, the utilization efficiency and the yield of nitrogen elements under a low-nitrogen condition are remarkably improved.

Description

Technical Field

[0001] The 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 one of the important factors to promote crop yields. In production, China generally has the problem of high nitrogen fertilizer application and low utilization rate. Excessive application of nitrogen fertilizer may lead to a decrease in crop yield and quality, and the misuse of nitrogen fertilizer will also lead to environmental problems such as eutrophication of water bodies. Although the utilization rate of nitrogen fertilizer in production can be improved through effective field management, the more basic strategy is to improve and cultivate varieties with efficient nitrogen utilization through genetic means. Therefore, cultivating crop varieties that can still maintain growth and ensure yield under low nitrogen levels is of great significance to reducing the use of nitrogen fertilizers in production.

[0003] Dongxiang wild rice (DY) is the northernmost wild rice discovered so far. As a wild relative of cultivated rice, it contains a wealth of favorable genes. With the development of rice functional genomics, some nitrogen-efficient genes have been discovered from Dongxiang wild rice, providing theoretical and technical support for improving rice nitrogen utilization efficiency using genetic means.

[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 29bp of 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 Compared with common cultivated rice, OsTCP19 DY Genes have unique variation sites.

[0005] In 2020, Duanmu Deqiang's group and Cao Yangrong's group jointly published a research paper titled "Natural variation at OsCERK1 regulates arbuscular mycorrhizal symbiosis in rice" in New Phytologist. The members of the research team cloned OsCERK1 from Dongxiang wild rice. DY Gene, which can enhance the symbiosis between rice and arbuscular mycorrhizal fungi (AMF), and improve the nitrogen absorption and utilization rate 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 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, and accelerate the process of rice breeding and green production. DY OsCERK1 DY Rice varieties with both OsTCP19 and OsTCP19 are susceptible to the influence of soil environment in different ecological zones on rice nitrogen absorption and utilization. DY Genes 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 excellent genes. However, at present, there is a lack of simple and effective molecular marker technology to assist the breeding process in the process of cultivating nitrogen-efficient rice. The existing molecular marker technology cannot get rid of the series of lengthy steps such as enzyme cutting, electrophoresis, staining and band detection after completing the polymerase chain reaction (PCR), which increases the operation and labor costs, is inconvenient to operate, 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 need to be analyzed by electrophoresis, staining or adding 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 Higashino-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 DY and OsCERK1 DY By polymerizing in breeding materials, the cultivated breeding materials or lines have broader and more efficient nitrogen absorption and utilization capabilities, 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 was confirmed, and the Dongye-type allele of the gene was a nitrogen-efficient gene; 2) the metabolic pathways of the two Dongye-type nitrogen-efficient genes to be aggregated belonged to different pathways to avoid functional redundancy effects.

[0014] 2. Parent selection

[0015] The following principles are 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 single-gene lines 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 have 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 the breeding objectives, the parent materials are polymerized and bred to obtain rice breeding materials or varieties with wide adaptability and high nitrogen fertilizer absorption and utilization rate; the breeding objectives at least include excellent comprehensive agronomic traits and the aggregation of nitrogen-efficient genes in different metabolic pathways.

[0019] The present invention utilizes a molecular marker-assisted selection method 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, cultivate breeding materials or strains with wide adaptability and nitrogen efficiency, improve rice's absorption and utilization of nitrogen, and meet 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 gene DY The breeding materials with 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 Genes and OsCERK1 DY The genes are all nitrogen efficient genes, and the OsTCP19 DY Genes and OsCERK1 DY Genes belong to different metabolic pathways to avoid redundant effects in function;

[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 used for molecular detection, and the heterozygous plants containing the target gene and with main agronomic traits similar to those of the recurrent parent were selected. The BC2F1 population was obtained by backcrossing with the recurrent parent at the flowering stage. 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 use the above-mentioned KASP molecular marker to perform molecular detection of the target gene at the seedling stage. Among the homozygous plants of the marker genotype, select the plants with main agronomic traits similar to those of the recurrent parent for self-pollination and harvest, and 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 strains, the above KASP molecular markers were still used for molecular detection of target genes at the seedling stage, with OsTCP19 DY BC4F3-OsTCP19, a single plant with homozygous genotype and agronomic traits similar to the recurrent parent DY As the female parent, OsCERK1 DY BC4F3-OsCERK1, a 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 was detected by molecular detection of each plant in the population using the above-mentioned KASP molecular markers at the seedling stage. The plants with double gene homozygous 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 strains, target gene molecular detection was performed at the seedling stage; aboveground plant samples were taken at the mature stage, and nitrogen absorption of the plants was tested by stems, leaves, and ears, and 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 breeding materials F5-OsTCP19 with high nitrogen absorption and utilization efficiency genes DY / OsCERK1 DY ;

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

[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 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 5' end with FAM fluorescent signal tag, forward primer 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 label, forward primer OsCERK1 DY -A HEX fluorescent signal tag was added to the 5' end of FT.

[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 determine the nitrogen accumulation in the roots, stems and leaves of rice, the nitrogen absorption and utilization efficiency of different rice strains is calculated, and the nitrogen absorption of the bred nitrogen-efficient breeding materials is comprehensively evaluated.

[0039] In some embodiments, the OsTCP19 DY The breeding material containing the gene is 898, and the OsCERK1 DY The breeding material of the gene is Ganjundao 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 Humigenes.

[0042] Beneficial effects of the present invention:

[0043] 1. The present invention polymerizes the Higashino-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 promote 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 tolerate low nitrogen in soil. DY The main thing is that it has natural variations that can improve the symbiosis between rice roots and arbuscular mycorrhizae. Arbuscular mycorrhizae colonized on the rice roots can transfer the nitrogen nutrients absorbed by themselves to rice, thereby promoting the absorption of nitrogen by rice.

[0044] 2. The present invention utilizes two Dongye-type nitrogen-efficient genes of different metabolic pathways, and transfers them to the cultivated rice widely used in the same production through molecular marker-assisted selection backcrossing, breeding nitrogen-efficient single-gene lines with the same genetic background and different target genes, and then breeding two-gene lines with different nitrogen-efficient genes through crossbreeding of different single-gene lines. The above breeding procedure has two application advantages: first, the bred nitrogen-efficient single-gene lines can be applied in production as early as possible; second, the flexible combination and crossbreeding of different single-gene lines with the same genetic background can realize the aggregation of different genes and the rapid stabilization of offspring traits, avoid linkage drag, save costs, and shorten the breeding time.

[0045] 3. 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, the present invention expands the planting scale for both backcross and hybrid offspring groups. The advantages are that it can not only screen single plants containing homozygous target genes, but also increase the probability of screening single plants with excellent agronomic traits, reduce costs and workload, and improve 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 rate in different ecological zones. The advantage is that the nitrogen-efficient materials or strains bred can withstand production inspection and reduce the impact of the environment on nitrogen absorption efficiency.

[0047] 5. The present invention develops and designs a method for identifying the target gene OsTCP19 DY and OsCERK1 DY The genotype-based KASP molecular marker can not only quickly and accurately identify OsTCP19 in rice germplasm resources or breeding populations, but also DY and OsCERK1 DY It can detect multiple genes and simultaneously realize high-throughput detection of more sample materials, thus improving selection efficiency and accelerating the breeding process of nitrogen-efficient rice varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 To aggregate the Higashino-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 the OsTCP19 carrying the A allele variant. DY Homozygous for the gene; the pink dots close to the X-axis represent the G allele variant without OsTCP19 DY The red dot between the X and Y axes indicates the homozygous OsTCP19 carrying both the A and G alleles. DY Heterozygotes of the gene; black dots represent negative controls in which double distilled water is used instead of sample template DNA)

[0050] Figure 3 Based on KASP molecular marker b near isogenic line population OsCERK1 DY Genotype detection. (The red dots close to the Y axis represent the OsCERK1 carrying the G allele variant. DY The blue dots near 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 indicates that the gene carries both the G / T allele variant and contains OsCERK1 DY Heterozygotes of the gene; black dots represent negative controls in which double distilled water is used instead of sample template DNA) DETAILED DESCRIPTION

[0051] The present invention will be further described in detail below in conjunction with 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 (a breeding intermediate material, identified as containing OsTCP19 DY gene), mycorrhizal efficient symbiosis gene OsCERK1 DY The donor parent - Ganjundao No. 1 (variety approval number: Ganshendao 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 rice breeding material HZ-OsTCP19 with wide adaptability and high nitrogen fertilizer absorption and utilization rate DY / OsCERK1 DY The process (ref. 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, Ganjundao No. 1, was crossed 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 stages were backcrossed with the recurrent parent Huizhan to obtain the first generation BC1F1 population, which were BC1F1-OsTCP19 DY and BC1F1-OsCERK1 DY express;

[0059] (3) Planting BC1F1-OsTCP19 DY and BC1F1-OsCERK1 DY The planting scale was 48 plants per backcross generation (the same below). The target gene OsTCP19 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 whose main agronomic traits were similar to those of the recurrent parent were selected. The BC2F1 population was obtained by backcrossing with the recurrent parent at the flowering stage. 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 NO.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 rice nitrogen efficient gene OsTCP19 DY and OsCERK1 DY The KASP molecular labeling method: in the synthesis of OsTCP19 DY When using the gene KASP molecular marker primer, the forward primer OsTCP19 DY -FA 5' end with FAM fluorescent signal tag, forward primer OsTCP19 DY -HEX fluorescent signal tag was added to the 5' end of FG; DY Gene KASP molecular marker primers, forward primer OsCERK1 DY -FG 5' end with FAM fluorescent signal label, forward primer OsCERK1 DY-A HEX fluorescent signal tag was added to the 5' end of FT.

[0065] The above 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 to the same PCR reaction system, set up two blank controls with double distilled water instead of sample template DNA, and amplify the DNA of rice germplasm resources or breeding population plants on a PCR instrument;

[0068] 3) The fluorescence signal was read on an Omega fluorescence plate reader and the data were analyzed using Klustercaller data management and analysis software.

[0069] The 10 μL reaction system includes 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 Figure 2-3 , according to the color and position of the data graph, in OsTCP19 DY During genotyping, the green dots close to the Y axis represent the OsTCP19 carrying the A allele variant. DY Homozygous for the gene; the pink dots close to the X-axis represent the homozygous for the G allele without OsTCP19 DY The red dot between the X and Y axes indicates the homozygous OsTCP19 carrying both the A and G alleles. DY Heterozygotes of the gene; black dots represent negative controls in which double-distilled water is used instead of sample template DNA;

[0072] In the presence of OsCERK1 DY During genotyping, the red dots close to the Y axis represent the OsCERK1 carrying G allele. 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 indicates that the gene carries both the G / T allele variant and contains OsCERK1 DYHeterozygotes of the gene; black dots represent negative controls in which double-distilled water was used instead of sample template DNA.

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

[0074] (5) Planting each BC4F1 population, the above-mentioned KASP molecular markers were still used to perform molecular detection of the target gene at the seedling stage, and among the homozygous plants of the marker genotype, the plants with main agronomic traits similar to those of the recurrent parent were selected for self-pollination and harvested to obtain BC4F2 lines, and BC4F2-OsTCP19 was used as the control. DY and BC4F2-OsCERK1 DY express;

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

[0076] (7) Planting BC4F3-OsTCP19 DY and BC4F3-OsCERK1 DY At the seedling stage, the above KASP molecular markers were still used for molecular detection of 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, a homozygous genotype and agronomic traits similar to the recurrent parent, containing 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 was detected by molecular detection of each plant in the population using the above-mentioned KASP molecular markers at the seedling stage. The plants with double gene homozygous 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 The target gene molecular detection was still carried out 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 rate and main traits similar to the recurrent parents were selected to obtain the breeding material HZ-OsTCP19 with high nitrogen efficiency gene polymerization DY / OsCERK1 DY ;

[0081] (12) In 2023 and 2024, weight loss trials and potted plant trials will be 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 mass 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 was 6.83% higher than that of Huizhan, reaching 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 bred 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 may be made without departing from the inventive concept of the present invention, which all fall within the protection scope of the present invention.

Claims

1. A method for breeding nitrogen-efficient rice varieties, characterized in that: The following steps are involved: S1, select OsTCP19 DY Breeding materials containing OsCERK1 gene DY The breeding materials with 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 used for molecular detection, and the heterozygous plants containing the target gene and with main agronomic traits similar to those of the recurrent parent were selected. The BC2F1 population was obtained by backcrossing with the recurrent parent at the flowering stage. 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 use the above-mentioned KASP molecular marker to perform molecular detection of the target gene at the seedling stage. Among the homozygous plants of the marker genotype, select the plants with main agronomic traits similar to those of the recurrent parent for self-pollination and harvest, and 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 strains, the above KASP molecular markers were still used for molecular detection of target genes at the seedling stage, with OsTCP19 DY BC4F3-OsTCP19, a single plant with homozygous genotype and agronomic traits similar to the recurrent parent DY As the female parent, OsCERK1 DY BC4F3-OsCERK1, a 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 was detected by molecular detection of each plant in the population using the above-mentioned KASP molecular markers at the seedling stage. The plants with double gene homozygous 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 strains, target gene molecular detection was performed at the seedling stage; aboveground plant samples were taken at the mature stage, and nitrogen absorption of the plants was tested by stems, leaves, and ears, and 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 breeding materials F5-OsTCP19 with high nitrogen absorption and utilization efficiency genes DY / OsCERK1 DY ; S12. Conduct trials in multiple ecological zones to detect F5-OsTCP19 DY / OsCERK1 DY The nitrogen absorption and utilization efficiency was calculated, the agronomic traits were evaluated, and the applicability of the breeding materials was comprehensively evaluated.

2. The method for breeding nitrogen-efficient rice varieties according to claim 1, characterized in that: 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 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 N0.

6.

3. The method for breeding nitrogen-efficient rice varieties according to claim 2, characterized in that: The forward primer OsTCP19 DY -FA 5' end with FAM fluorescent signal tag, forward primer 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 label, forward primer OsCERK1 DY -A HEX fluorescent signal tag was added to the 5' end of FT.

4. The method for breeding nitrogen-efficient rice varieties according to claim 1, characterized in that: In the steps S3 to S6 and S10 to S11, the planting scale is expanded to more than 48 plants.

5. The method for breeding nitrogen-efficient rice varieties according to claim 1, characterized in that: In the steps S11 to S12, samples are taken at maturity to measure the nitrogen accumulation in the roots, stems and leaves of rice, the nitrogen absorption and utilization efficiency of different rice strains is calculated, and the nitrogen absorption of the bred nitrogen-efficient breeding materials is comprehensively evaluated.

6. The method for breeding nitrogen-efficient rice varieties according to claim 1, characterized in that: The OsTCP19 DY The breeding material containing the gene is 898, and the OsCERK1 DY The breeding material of the gene is Ganjundao No.

1.

7. The method for breeding 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.

8. The method for breeding nitrogen-efficient rice varieties according to claim 7, characterized in that: The recipient parent and the reincarnation parent are Huizhan.

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