A new material of deep water-tolerant germinated rice and its breeding method and application

Abstract

This invention discloses a new deep-water germination-tolerant rice material, its breeding method, and its application. Utilizing molecular marker-assisted selection (MMR) technology, this invention improves the flood tolerance of the rice variety Yunhangjing 7 by aggregating four beneficial genes: two blast resistance genes (Pi1 and Pi9) and two flood tolerance genes (SUB1 and AG1), resulting in a new deep-water germination-tolerant rice material. This material exhibits rapid deep-water germination, flood tolerance during the seedling stage, suitability for direct seeding, high yield, and good resistance. The breeding method includes hybridization, recrossing, MMR-assisted selection, and backcrossing steps, ultimately selecting the optimal new material lines. This invention successfully creates a new rice material tolerant to deep-water germination and flood tolerance during the seedling stage through gene aggregation using modern MMR-assisted selection technology. This new rice material features high yield and quality, strong stress resistance, and tolerance to deep-water germination.

Description

Technical Field

[0001] This invention relates to the field of rice breeding technology, and in particular to a new rice material resistant to deep water germination, its cultivation method, and its application. Background Technology

[0002] In direct seeding of rice paddies, seeds are often not adequately covered as in traditional transplanting, leaving them vulnerable to pecking by birds and posing a serious threat to the normal growth of rice. To effectively protect the seeds, a water layer is often added to the paddy field. However, most existing rice varieties do not have good flood tolerance and struggle to germinate successfully in such deep water conditions, severely impacting the emergence rate and final yield, thus hindering the further development and widespread application of direct seeding technology.

[0003] Pi1 is a broad-spectrum rice blast resistance gene that has been cloned and widely used in rice blast resistance breeding. Through marker-assisted selection (MAS) technology, the Pi1 gene has been successfully introduced into multiple rice varieties, significantly improving their resistance. For example, studies have shown that introducing the Pi1 gene into maintainer lines such as the recipient parent Zhenshan 97B, and using markers closely linked to the Pi1 gene for MAS selection, resulted in significantly improved resistance in selected plants. Pi9 is an important rice blast resistance gene located at the Pi2 / 9 complex resistance locus on the short arm of chromosome 6 in rice. This gene has been cloned and widely used in rice blast resistance breeding. The Pi9 gene exhibits broad-spectrum and durable resistance in different rice varieties. For example, studies have shown that introducing the Pi9 gene into the rice sterile line Fengyuan A and restorer line Fengyuan B significantly improved their rice blast resistance. In addition, progress has been made in the study of allelic variations of the Pi9 gene, and a number of new allelic variant genes have been discovered. These allelic variant genes have shown different disease resistance frequencies and levels in different rice varieties.

[0004] The SUB1 gene locus contains several ethylene response factor (ERF)-like genes, among which SUB1A is a key gene determining rice flood tolerance. It has been cloned and widely used in rice flood tolerance breeding. Studies have shown that rice varieties possessing the SUB1A-1 allele (such as FR13A) are more resistant to flood damage, surviving up to two weeks even when completely submerged. The SUB1A gene functions by inhibiting ethylene-mediated gibberellin (GA) production, thereby reducing energy consumption and carbohydrate breakdown, and improving rice flood tolerance. The AG1 gene (qAG-9-2, OsTPP7) is a major-effect locus for rice germination flood tolerance. The International Rice Research Institute (IRRI) introduced the AG1 gene into the high-yielding rice variety IR64 through hybridization and backcrossing, obtaining the near-isogenic line IR64-AG1, which significantly improved its germination flood tolerance. Further introduction of the AG1 gene into the superior variety Ciherang-Sub1 also significantly improved its germination flood tolerance.

[0005] Most rice breeding efforts focus on increasing yield, improving quality, and enhancing resistance to diseases and pests. Traditional rice breeding methods mainly rely on phenotypic selection and empirical judgment. Although some results have been achieved to a certain extent, these methods often have limitations such as low efficiency, long cycle, and difficulty in accurately combining multiple superior genes. They are unable to meet the urgent needs of modern agricultural production for rice varieties in terms of comprehensive performance in terms of adaptability, stress resistance, and high yield.

[0006] Existing rice varieties have shortcomings in new cultivation models such as direct seeding in paddy fields. Currently, there is a lack of new deep-water germination-tolerant rice materials that can germinate rapidly in deep-water environments, as well as their cultivation methods and applications. Summary of the Invention

[0007] The purpose of this invention is to provide a new type of rice material that can germinate rapidly in deep water environments, as well as its cultivation method and application.

[0008] In response to the problem that new technologies such as mechanized direct seeding or drone direct seeding in rice paddies make the seeds susceptible to being pecked by birds, requiring water layer protection but most rice varieties are not tolerant of deep water germination, this invention provides the following technical solution: This invention provides the following technical solution: Firstly, this application provides a method for cultivating new rice materials tolerant of deep water germination.

[0009] Secondly, this application provides a new deep-water germination-tolerant rice material obtained by a cultivation method.

[0010] Thirdly, this application provides an application of a polymer gene in the cultivation of new deep-water germination-resistant rice materials.

[0011] The first aspect of this application provides a method for cultivating new rice materials that can germinate in deep water, including the following steps: (1) Parental hybridization: using Yunhangjing 7 as the recipient material and R9001, a rice blast-resistant donor material, as the donor parent, they are hybridized to obtain the Yunhangjing 7 / R9001 hybrid F1 population;

[0012] Using Yunhangjing 7 as the recipient material and CiherangSUB1AG1 as the flood-resistant parent material, they were hybridized to obtain the F1 hybrid population of Yunhangjing 7 / CiherangSUB1AG1.

[0013] The rice blast-resistant donor material R9001 contains resistance genes Pi1 and Pi9, and the flood-resistant material CiherangSUB1AG1 contains the seedling flood-resistant gene SUB1 and the bud flood-resistant gene AG1.

[0014] (2) Recrossing and self-crossing: The F1 population of the hybrid of Yunhangjing 7 / R9001 and the F1 population of the hybrid of Yunhangjing 7 / CiherangSUB1AG1 were recrossed to obtain the F1 seeds of the recrossed population. At the same time, the two single-crossed F1 populations were self-crossed and the self-crossed F2 seeds were collected to form the F2 population of Yunhangjing 7 / R9001 and the F2 population of Yunhangjing 7 / CiherangSUB1AG1, respectively.

[0015] (3) Molecular detection and screening: F1 seeds from recrossing and F2 seeds from self-pollination are planted, and leaves are taken during the tillering stage for molecular detection of target genes. Individual plants containing target genes Pi1, Pi9, SUB1 and AG1 are screened out and recrossing is continued; or F2 self-pollination individual plants are used to continue recrossing to aggregate genes for resistance to rice blast and tolerance to flooding, and recross seeds are obtained.

[0016] (4) Backcrossing: Sow the recrossed seeds, select superior single plants through molecular marker-assisted selection, and backcross them with Yunhang Jing 7 to obtain BC1F1, BC2F1 and BC3F1 seeds in sequence;

[0017] (5) Final screening and obtaining new materials: Sow BC3F1 seeds to obtain BC3F2 seeds;

[0018] Superior strains that integrate genes Pi1, Pi9, SUB1, and AG1 were selected from BC3F2 seeds to obtain new rice materials tolerant to deep-water germination.

[0019] Further, in step (1), Yunhangjing 7, R9001 and CiherangSUB1AG1 are sown and hybridization is carried out during the heading stage to obtain the corresponding hybrid F1 seeds.

[0020] The second aspect of this application provides a new deep-water germination-resistant rice material prepared by a method.

[0021] The third aspect of this application provides the application of the polymerized genes in the cultivation of new deep-water germination-tolerant rice materials. The polymerization of rice blast resistance genes Pi1 and Pi9 and flood tolerance genes SUB1 and AG1 is applied to the cultivation of new deep-water germination-tolerant rice materials. Specifically, the relevant genes in donor material R9001 containing rice blast resistance genes Pi1 and Pi9 and material CiherangSUB1AG1 containing flood tolerance genes SUB1 and AG1 are polymerized into the rice variety Yunhangjing 7 to obtain new deep-water germination-tolerant rice materials.

[0022] Beneficial Effects: This invention utilizes modern molecular marker-assisted selection technology to successfully create a new type of rice material resistant to deep-water germination through gene aggregation. This rice exhibits high yield and quality, strong stress resistance, and the ability to germinate in deep water. The new flood-resistant material obtained by this invention can reduce weed growth in fields during early flooding, which is of great significance for reducing pesticide use and saving costs.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] (1) Enhancing the stress resistance of rice varieties: By combining the genes for resistance to rice blast (Pi1, Pi9) and the genes for tolerance to flooding (SUB1, AG1), the new rice materials cultivated possess both strong deep-water germination ability and good resistance to rice blast, thus improving the growth stability and stress resistance of rice under adverse environments and providing a strong guarantee for high and stable rice yield. This helps to increase the unit yield of paddy fields, achieve increased grain production, and is of great significance to ensuring food security. In 2024, through artificial inoculation to induce disease, the resistance level of the new material 23CA1118-2-1 was level 1, while the resistance level of the control Yunhangjing 7 was level 6, indicating that the stress resistance of the new material was improved. The new material also has good seedling flood resistance. When all 20cm tall seedlings were submerged for 15 days, the seedlings survived and grew normally until maturity after the water was removed, with a survival rate of 95.3%. Through comparative trials of varietal yields, among the 15 new varieties (lines), the yield of the new material 23CA1118-2-1 was 10726.5 kg / ha, an increase of 7.8% compared with the control, ranking third among the 12 varieties.

[0025] (2) Achieving simplified cultivation: This new material is suitable for various simplified cultivation methods such as direct seeding by hand, direct seeding by machine or direct seeding by drone. It can effectively save labor, reduce production costs, and improve the production efficiency of paddy fields. It adapts to the trend of modern agriculture towards simplification and high efficiency, and helps to promote the innovation and promotion of rice planting technology.

[0026] (3) Provide new breeding materials and ideas: It provides a new and excellent breeding material and method for rice breeding work. By carrying out gene aggregation improvement on the existing rice variety Yunhangjing 7, it enriches the germplasm resources of rice, expands the gene pool and selection range of rice breeding, and provides useful reference for the cultivation and improvement of other rice excellent traits. It helps to promote the further development and innovation of rice breeding technology.

[0027] (4) This invention utilizes modern molecular marker-assisted selection technology to conduct in-depth research and precise improvement on the deep-water germination characteristics of rice. By aggregating two key superior genes, namely the rice blast resistance gene (Pi1, Pi9) and the flood tolerance gene (SUB1, AG1), a new deep-water germination-tolerant rice material was successfully created, numbered 23CA1118-2-1 (corresponding field trial number 212). This provides a practical solution to the problem of seed germination in direct seeding of rice fields and opens up a new path for future rice breeding work to develop towards a more mechanized and simplified cultivation direction. It is expected to make an important contribution to ensuring the high efficiency and stability of rice production and promoting the continuous progress of modern agriculture. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 This is a technical roadmap for the breeding of new rice materials according to the present invention.

[0030] Figure 2 This is a comparison diagram of the flood resistance between the parents of the present invention;

[0031] Figure 3 This is a diagram of an experiment on the deep-water germination of the new rice material of this invention, which was submerged for 6 days.

[0032] Figure 4 This is a diagram of an experiment on the deep-water germination of the new rice material of this invention, which was submerged for 12 days.

[0033] Figure 5 This is a diagram of a deep-water germination experiment for the novel material created in this invention.

[0034] Figure 6 This is an experimental diagram showing the survival rate of rice seedlings in the flood-resistant stage of the new rice material of this invention.

[0035] Figure 7 This is an experimental diagram illustrating the disease resistance identification of the new rice material of this invention.

[0036] Figure 8 This is a growth diagram of the new rice material of the present invention. Detailed Implementation

[0037] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the scope of protection of the present invention.

[0038] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0040] In this application, "~ one less" means one or more, and "more than" means two or more. "~ one less item (item) below" or similar expressions refer to any combination of these items, including any combination of single items (items) or multiple items (items). For example, "~ one less item (item) in a, b, or c", or "~ one less item (item) in a, b, and c", can all mean: a, b, c, a~b (i.e., a and b), a~c, b~c, or a~b~c, where a, b, and c can be single or multiple.

[0041] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0042] The first aspect of this application provides a method for cultivating a new rice material that is resistant to deep water germination, including the following steps: (1) Parental hybridization: using Yunhangjing 7 as the recipient material and R9001, a rice blast-resistant donor material, as the donor parent, they are hybridized to obtain the Yunhangjing 7 / R9001 hybrid F1 population;

[0043] Using Yunhangjing 7 as the recipient material and CiherangSUB1AG1 as the flood-resistant parent material, they were hybridized to obtain the F1 hybrid population of Yunhangjing 7 / CiherangSUB1AG1.

[0044] The rice blast-resistant donor material R9001 contains resistance genes Pi1 and Pi9, and the flood-resistant material CiherangSUB1AG1 contains the seedling flood-resistant gene SUB1 and the bud flood-resistant gene AG1.

[0045] (2) Recrossing and self-crossing: The F1 population of the hybrid of Yunhangjing 7 / R9001 and the F1 population of the hybrid of Yunhangjing 7 / CiherangSUB1AG1 were recrossed to obtain the F1 seeds of the recrossed population. At the same time, the two single-crossed F1 populations were self-crossed and the self-crossed F2 seeds were collected to form the F2 population of Yunhangjing 7 / R9001 and the F2 population of Yunhangjing 7 / CiherangSUB1AG1, respectively.

[0046] (3) Molecular detection and screening: F1 seeds from recrossing and F2 seeds from self-pollination are planted, and leaves are taken during the tillering stage for molecular detection of target genes. Individual plants containing target genes Pi1, Pi9, SUB1 and AG1 are screened out and recrossing is continued; or F2 self-pollination individual plants are used to continue recrossing to aggregate genes for resistance to rice blast and tolerance to flooding, and recross seeds are obtained.

[0047] (4) Backcrossing: Sow the recrossed seeds, select superior single plants through molecular marker-assisted selection, and backcross them with Yunhang Jing 7 to obtain BC1F1, BC2F1 and BC3F1 seeds in sequence;

[0048] (5) Final screening and obtaining new materials: Sow BC3F1 seeds to obtain BC3F2 seeds;

[0049] Superior strains that integrate genes Pi1, Pi9, SUB1, and AG1 were selected from BC3F2 seeds to obtain new rice materials tolerant to deep-water germination.

[0050] In some embodiments, in step (3), the F1 population of the planted cross, the F2 population of Yunhangjing 7 / R9001 and the F2 population of Yunhangjing 7 / CiherangSUB1AG1 are selected for molecular detection of target genes to screen out individual plants containing specific genotypes.

[0051] The second aspect of this application provides a new deep-water germination-resistant rice material prepared by a method.

[0052] The third aspect of this application provides the application of the polymerized genes in the cultivation of new deep-water germination-tolerant rice materials. The polymerization of rice blast resistance genes Pi1 and Pi9 and flood tolerance genes SUB1 and AG1 is applied to the cultivation of new deep-water germination-tolerant rice materials. Specifically, the relevant genes in donor material R9001 containing rice blast resistance genes Pi1 and Pi9 and material CiherangSUB1AG1 containing flood tolerance genes SUB1 and AG1 are polymerized into the rice variety Yunhangjing 7 to obtain new deep-water germination-tolerant rice materials.

[0053] Example 1

[0054] The first aspect of this application provides a method for cultivating a new rice material that is resistant to deep water germination, including the following steps: (1) Parental hybridization: using Yunhangjing 7 as the recipient material and R9001, a rice blast-resistant donor material, as the donor parent, they are hybridized to obtain the Yunhangjing 7 / R9001 hybrid F1 population;

[0055] Using Yunhangjing 7 as the recipient material and CiherangSUB1AG1 as the flood-resistant parent material, they were hybridized to obtain the F1 hybrid population of Yunhangjing 7 / CiherangSUB1AG1.

[0056] The blast-resistant donor material R9001 contains the resistance gene Pi1 and the resistance gene Pi9, and the flood-resistant material CiherangSUB1AG1 contains the seedling flood-resistant gene SUB1 and the bud flood-resistant gene AG1. Yunhangjing 7, R9001 and CiherangSUB1AG1 were sown and hybridized at the heading stage to obtain the corresponding hybrid F1 seeds.

[0057] (2) Recrossing and self-crossing: The F1 population of the hybrid of Yunhangjing 7 / R9001 and the F1 population of the hybrid of Yunhangjing 7 / CiherangSUB1AG1 were recrossed to obtain the F1 seeds of the recrossed population. At the same time, the two single-crossed F1 populations were self-crossed and the self-crossed F2 seeds were collected to form the F2 population of Yunhangjing 7 / R9001 and the F2 population of Yunhangjing 7 / CiherangSUB1AG1, respectively.

[0058] (3) Molecular detection and screening: F1 seeds from recross and F2 seeds from selfcross are planted, and leaves are taken during the tillering stage for molecular detection of target genes. Individual plants containing the target genes Pi1, Pi9, SUB1 and AG1 are screened out and recrossing is continued; or F2 plants from selfcross are used to continue recrossing, and genes for resistance to rice blast and tolerance to flooding are aggregated to obtain recross seeds; the F1 population of recross, the F2 population of Yunhangjing 7 / R9001 and the F2 population of Yunhangjing 7 / CiherangSUB1AG1 are selected for molecular detection of target genes, and individual plants containing specific genotypes are screened out.

[0059] (4) Backcrossing: Sow the recrossed seeds, select superior single plants through molecular marker-assisted selection, and backcross them with Yunhang Jing 7 to obtain BC1F1, BC2F1 and BC3F1 seeds in sequence;

[0060] (5) Final screening and obtaining new materials: Sow BC3F1 seeds to obtain BC3F2 seeds;

[0061] Superior rice lines that integrate genes Pi1, Pi9, SUB1, and AG1 were selected from BC3F2 seeds to obtain new deep-water germination tolerant rice materials. The superior line numbered 23CA1118-2-1 (field equivalent number 212) was selected.

[0062] Planting Management:

[0063] 1. Select high-quality seeds and treat them properly before sowing. Sun-dry the seeds for 2 days before soaking to improve the germination rate.

[0064] 2. Prepare the paddy field: Level the paddy field and maintain a water level of 8-10 cm.

[0065] 3. Drone direct seeding: Use drones to sow evenly, with a seeding rate of 3-4 kg per acre; if using large machinery for direct seeding, sow first, then irrigate to maintain a water layer of 8-10 cm.

[0066] 4. Fertilizer and water management: Once the seeds germinate and show green leaves, reduce the water level to 3-4 cm to promote rapid green growth. When the seedlings reach the three-leaf stage, manage fertilizer and water according to the direct seeding method.

[0067] 5. Just like in normal rice paddies, timely and effective prevention and control of pests and diseases are required.

[0068] Example 2

[0069] The difference between Example 2 and Example 1 is as follows:

[0070] The present invention provides a method for cultivating a new type of rice material that is resistant to deep water germination, comprising the following steps: In step (1), in May 2020, Yunhangjing 7, R9001 and CiherangSUB1AG1 were sown at the Changsha base in Hunan Province, and hybridization was carried out during the heading stage to obtain the corresponding hybrid F1 seeds.

[0071] In step (2), in December 2020, at the Lingshui base in Hainan, the F1 populations of Yunhangjing 7 / R9001 and Yunhangjing 7 / CiherangSUB1AG1 were crossbred to obtain crossbred F1 seeds.

[0072] In step (3), the molecular marker-assisted selection step was carried out in May 2021 at the Changsha base in Hunan Province. Target gene molecular detection was performed on the planted F1 cross, the F2 population of Yunhangjing 7 / R9001, and the F2 population of Yunhangjing 7 / CiherangSUB1AG1 to screen out individual plants containing specific genotypes.

[0073] In step (4), the seeding was carried out at the Lingshui base in Hainan and the Changsha base in Hunan in December 2021, May 2022 and December 2022 respectively, and BC1F1, BC2F1 and BC3F1 seeds were obtained in sequence.

[0074] The planting of BC3F1 seeds and the acquisition of BC3F2 seeds were carried out in May 2023 at the Changsha base in Hunan Province. Finally, in March 2024, the superior strain numbered 23CA1118-2-1 (field corresponding number 212) was selected in Yunnan Province.

[0075] Example 3

[0076] This invention relates to a cultivation method for a new type of rice material that can germinate in deep water.

[0077] Example 4

[0078] This invention relates to the application of a gene-polymerization technique in the cultivation of new deep-water germination-tolerant rice materials. Specifically, the gene-polymerization of rice blast resistance genes Pi1 and Pi9 and flood tolerance genes SUB1 and AG1 is applied to the cultivation of new deep-water germination-tolerant rice materials. The method involves polymerizing the relevant genes from donor material R9001 containing rice blast resistance genes Pi1 and Pi9 and material CiherangSUB1AG1 containing flood tolerance genes SUB1 and AG1 into the rice variety Yunhangjing 7 to obtain a new deep-water germination-tolerant rice material.

[0079] Example 5

[0080] This invention relates to a new rice material that integrates four beneficial genes (two blast resistance genes and two flood tolerance genes) and is resistant to germination in deep water. The material is designated as 23CA1118-2-1 (corresponding field trial number 212). This material is characterized by rapid germination in deep water, tolerance to direct seeding, high yield, and good resistance.

[0081] Breeding method: The new line number 23CA1118-2-1 (field corresponding number 212) of this application was obtained through the following breeding method:

[0082] Figure 1 This is a technical roadmap for the creation and breeding of new materials according to the present invention. RP refers to Yunhangjing 7.

[0083] This application also provides applications in rice production. Applying this material to direct seeding of rice by machinery, direct seeding by drones, or in flooded areas of lakes can greatly increase yields, simplify cultivation, and save labor, thus having great application value.

[0084] Implementation: On May 18, 2020, the recipient material Yunhangjing 7, the blast-resistant donor material R90011 (20CA075, R90011 modified to contain resistance genes Pi1 and Pi9), and the flood-tolerant material CiherangSUB1AG1 (containing flood-tolerant genes SUB1 and AG1) were sown at the Changsha base in Hunan Province. At the heading stage, Yunhangjing 7 was crossed with R90011 and CiherangSUB1AG1 respectively to obtain F1 hybrid seeds of Yunhangjing 7 / R90011 and Yunhangjing 7 / CiherangSUB1AG1. Simultaneously, samples of Yunhangjing 7 were taken for blast resistance gene testing, and the results showed that Yunhangjing 7 did not possess the favorable py1 and Pi9 genotypes (Table 1). The flood tolerance of Yunhangjing 7 and the flood-tolerant donor parent were compared, and the differences between the two were significant. Figure 2 The parent plants do not possess the gene for deep-water germination tolerance.

[0085] In December 2020, 16 plants of hybrid F1 (Yunhangjing 7 / R9001) and 29 plants of hybrid F1 (Yunhangjing 7 / CiherangSUB1AG1) were sown at the Lingshui base in Hainan. The two F1 populations were recrossed at the heading stage to obtain F1 seeds from the recross between Yunhangjing 7 / R9001 and Yunhangjing 7 / CiherangSUB1AG1. Simultaneously, the two single-cross F1 plants were self-pollinated and harvested.

[0086] In May 2021, seeds of the recross F1 population of Yunhangjing 7 / R9001 / / Yunhangjing 7 / CiherangSUB1AG1 were sown at the Changsha base in Hunan Province. Five plants were planted in the recross F1 population, along with 160 plants in the F2 population of both Yunhangjing 7 / R9001 and Yunhangjing 7 / CiherangSUB1AG1. Leaf samples were taken at the tillering stage for molecular detection of target genes. Due to the small size of the recross F1 population, no individual plants with the four target genes were identified. Ten homozygous plants for the rice blast resistance genes Pi1 and Pi9 were detected in the F2 population of Yunhangjing 7 / R9001, and 25 heterozygous plants for AG1 and homozygous plants for SUB1 were detected in the F2 population of Yunhangjing 7 / CiherangSUB1AG1. During the heading stage, homozygous single plants of rice blast resistance gene from the F2 population of Yunhangjing 7 / R9001 were crossbred with single plants of flood-tolerant gene from the F2 population of Yunhangjing 7 / CiherangSUB1AG1 to aggregate rice blast resistance and flood-tolerant genes, thus obtaining crossbred seeds.

[0087] In December 2021, seeds of Yunhangjing 7 / R9001 / / Yunhangjing 7 / CiherangSUB1AG1 were sown at the Lingshui base in Hainan. After molecular marker-assisted selection, the best three individual plants were selected and backcrossed with Yunhangjing 7 to obtain BC1F1 seeds.

[0088] In May 2022, BC1F1 seeds were sown at the Changsha base in Hunan Province, and then backcrossed with Yunhang Jing 7 to obtain BC2F1 seeds.

[0089] In December 2022, BC2F1 seeds were sown at the Lingshui base in Hainan and then backcrossed with Yunhang Jing 7 to obtain BC3F1 seeds.

[0090] In May 2023, BC3F1 seeds were sown at the Changsha base in Hunan Province, and BC3F2 seeds were obtained.

[0091] In March 2024, the three best rice lines were selected in Yunnan for deep-water germination and seedling flood tolerance tests. Among them, line number 23CA1118-2-1 (field equivalent number 212) was the best and aggregated four beneficial genes: pi1, pi9, sub1, and AG1. The analysis of blast resistance genes in Yunhang Jing 7 rice is shown in Table 1.

[0092] Table 1

[0093] Gene detection Tag name Gene analysis pikm,pikp,pi1 K_110574 pikm,pikp,pi1 are unfavorable pikm,pikp,pi1 K_110572 pikm,pikp,pi1 are unfavorable pi9 K_060504 pi9 disadvantage

[0094] In April 2024, direct seeding was conducted at an experimental base at an altitude of 1400m, with a seed rate of 37.5kg per hectare. The seed showed vigorous growth, uniform ear layer, moderate growth period, and matured with green stalks. Figure 8 This is a growth diagram of the new rice material of the present invention.

[0095] Example 6

[0096] Take 200 healthy and plump seeds from Example 5, soak them in 75% alcohol for 10 minutes, rinse them three times with distilled water, and then submerge them in a disposable cup at a depth of 10 cm. Repeat this process three times. Place them in a hardening-off room at a temperature of 25 ℃ and a relative humidity of 70% for cultivation. After 6 days, count the number of germinations and the germination rate, and measure the root length and shoot length. Figure 2 This is a comparison diagram of the flood resistance between the parents of the present invention; Figure 3 This is a test diagram of the new rice material of the present invention, which germinates in deep water and is submerged for 6 days. Note: 211: 23CA1118-2-3; 212: 23CA1118-2-1; 213: 23CA1118-3-6; CK: Yunhang Jing 7. Figure 4 This is a test diagram of the new rice material of the present invention, which germinates in deep water and is submerged for 12 days. Note: 212: corresponds to 23CA1118-2-1; CK: Yunhang Jing 7. Figure 5 This is a diagram of a deep-water germination experiment for the novel material created in this invention.

[0097] Example 7

[0098] Seedling flood tolerance assessment

[0099] The normal flood-tolerant seedlings selected from the bud stage in Example 5 were grown to about 20cm. A seedling flooding treatment and a seedling recovery (control) treatment were set up. Each treatment was designed with 3 replicates. 15 seedlings were randomly selected from each replicate and transferred to a culture box with a 3cm thick soil layer. The seedlings were covered with 1cm of sand and continued to be cultured. The seedling flooding treatment was to completely submerge the seedlings for 15 days. The seedling recovery (control) treatment was to maintain a water layer of 2cm. The survival rate was then investigated. Figure 6 This is an experimental diagram showing the survival rate of rice seedlings in the flood-resistant stage of the new rice material of this invention.

[0100] Example 8

[0101] Leaf blight resistance identification

[0102] One hundred seeds of each material from Example 5 were sown in a seedbed. After emergence, when the seedlings reached about 15cm in height, 1050kg of urea per hectare was applied as fertilizer, and Mongolian rice was used as the infection row to induce disease. Leaf blast identification adopted the method of Asaga (1975) of Japan, with a 10-level standard for leaf blast, and a set of disease resistance identification varieties were added: level 0 and level 1 were highly resistant, level 2 and level 3 were resistant, level 4 was moderately resistant, level 5 and level 6 were susceptible, and level 7, level 8, level 9, and level 10 were highly susceptible. Figure 7 This is an experimental diagram illustrating the disease resistance identification of the new rice material of this invention.

[0103] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope. The scope of protection of the present invention is defined by the appended claims, specification, and their equivalents.

Claims

1. A method for cultivating a new rice material tolerant to deep water germination, characterized in that... Includes the following steps: (1) Parental hybridization: Yunhangjing 7 was used as the recipient material and R9001, the blast-resistant donor material, was used as the donor parent. They were hybridized to obtain the F1 hybrid population of Yunhangjing 7 / R9001. Using Yunhangjing 7 as the recipient material and CiherangSUB1AG1 as the flood-resistant parent material, they were hybridized to obtain the F1 hybrid population of Yunhangjing 7 / CiherangSUB1AG1. The rice blast-resistant donor material R9001 contains resistance genes Pi1 and Pi9, and the flood-resistant material CiherangSUB1AG1 contains the seedling flood-resistant gene SUB1 and the bud flood-resistant gene AG1. (2) Recrossing and self-crossing: The F1 population of the hybrid of Yunhangjing 7 / R9001 and the F1 population of the hybrid of Yunhangjing 7 / CiherangSUB1AG1 were recrossed to obtain the F1 seeds of the recrossed population. At the same time, the two F1 populations were self-crossed and the F2 seeds of the self-crossed populations were collected to form the F2 population of Yunhangjing 7 / R9001 and the F2 population of Yunhangjing 7 / CiherangSUB1AG1, respectively. (3) Molecular detection and screening: F1 seeds from recrossing and F2 seeds from self-pollination are planted, and leaves are taken during the tillering stage for molecular detection of target genes. Individual plants containing target genes Pi1, Pi9, SUB1 and AG1 are screened out and recrossing is continued; or F2 self-pollination individual plants are used to continue recrossing to aggregate genes for resistance to rice blast and tolerance to flooding, and recross seeds are obtained. (4) Backcrossing: Sow the recrossed seeds, select superior single plants through molecular marker-assisted selection, and backcross them with Yunhang Jing 7 to obtain BC1F1, BC2F1 and BC3F1 seeds in sequence; (5) Final screening and obtaining new materials: Sow BC3F1 seeds to obtain BC3F2 seeds; Superior strains that integrate genes Pi1, Pi9, SUB1, and AG1 were selected from BC3F2 seeds to obtain new rice materials tolerant to deep-water germination.

2. The method for cultivating new deep-water germination-tolerant rice materials according to claim 1, characterized in that: In step (1), Yunhangjing 7, R9001 and CiherangSUB1AG1 were sown and hybridization was carried out during the heading stage to obtain the corresponding hybrid F1 seeds.

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