Breeding method and application of aerospace carrying rice mutant strain Diantun 502 aviation M6-8

Abstract

The invention discloses a breeding method and application of a space-carrying rice mutant strain Diantun 502 aviation M6-8, and relates to the technical field of rice breeding, and the technical key points are as follows: the method comprises the following steps: by taking conventional fragrant soft rice 'Diantun 502' breeder's seeds with the amylose content of 8.2% as materials, carrying out space-carrying space mutagenesis to obtain SP0-generation seeds; then through ground multi-generation system breeding, phenotypic screening and STR molecular marker identification are combined, and excellent mutant strains with soft rice characters, fragrance, precocity, strong tillering and rice blast resistance are screened out; and finally, selecting the fragrant and soft type rice mutant strain 'Diantun 502 aviation M6-8' with excellent comprehensive characters through a multi-point product comparison test. The mutant strain keeps the taste quality of fragrant and soft rice, meanwhile, the yield is increased by 8.1% or above, the rice blast resistance is remarkably enhanced, the growth period is shortened, synergistic improvement of fragrance, softness, morning, abundance and resistance is achieved, and the mutant strain can serve as a new variety to be directly popularized or serve as a high-quality parent to be used for cross breeding.

Description

Technical Field

[0001] This invention relates to the field of rice breeding technology, specifically to a method and application for breeding a space-borne rice mutant strain, Diantun 502 Hang M6-8. Background Technology

[0002] Fragrant soft rice, an important category of high-end staple food in my country, is highly favored by the market for its fragrant, soft, chewy, and smooth taste. Yunnan, as the world's origin of soft rice and a center of rice genetic diversity, possesses abundant local soft rice resources. Its low amylose content and resistance to retrogradation in cold weather make it a valuable gene pool for high-quality rice breeding. However, traditional fragrant soft rice has long faced the industry dilemma of "high quality but low yield, high yield but low quality," specifically manifested in yields more than 20% lower than hybrid rice, poor disease resistance, susceptibility to lodging, and narrow adaptability. Taking the main Yunnan variety "Diantun 502" as an example, after nearly thirty years of promotion, it has suffered severe degeneration, with rice blast incidence reaching 100%, yield decline exceeding 40%, and significant reduction in quality and resistance. The industry urgently needs to cultivate breakthrough new varieties that combine high quality and high yield.

[0003] To address the aforementioned problems, space-induced mutation breeding technology has become a crucial breakthrough. This technology utilizes the unique microgravity and high-energy particle radiation of the space environment to induce heritable variations in the crop genome, offering advantages such as high mutation frequency, wide variety of mutation types, and short breeding cycles, making it suitable for rapidly creating new germplasm. Based on this technological approach, this invention uses Diantun 502 seeds carried aboard the Shenzhou-13 manned spacecraft for space-induced mutation, aiming to create new rice germplasm that significantly improves yield, resistance, and adaptability while maintaining the excellent quality of fragrant and soft rice through a combination of space-induced mutation and multi-generational selection on the ground.

[0004] Through systematic breeding, this project successfully obtained the mutant strain "Dian Tun 502 Hang M6-8". This strain retains the turbidity of the soft rice endosperm and its aroma, while exhibiting early maturity, strong tillering, large panicles with many grains, high seed setting rate, and an 8.1% increase in yield compared to the original strain, along with significantly improved resistance. Genetic analysis confirmed a difference between this strain and the original strain at the STR marker RM85 locus, classifying it as a new material with a clearly defined genetic background. This mutant strain not only provides core germplasm for the renewal and rejuvenation of soft rice varieties, but its high thousand-grain weight characteristic can also serve as a key parent for breeding high-quality and high-yield fragrant soft rice. Summary of the Invention

[0005] The purpose of this invention is to solve the above-mentioned problems by using spacecraft to select and breed superior mutant new materials, thereby addressing the long-standing issues in the existing fragrant rice industry such as the mismatch between quality and yield, insufficient stress resistance, variety degradation, and low breeding efficiency.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: A method for breeding and applying a space-borne rice mutant strain, Dian Tun 502 Hang M6-8, comprising the following steps:

[0007] Step 1: Select the original seeds of "Dian Tun 502" conventional fragrant soft rice with an amylose content of 8.2%, package them, and send them into space with the manned spacecraft. They will stay in orbit for 6 months and return to Earth to obtain SP0 generation seeds.

[0008] Step 2: SP1 generation is harvested by single transplanting and equal mixing of single main panicles; SP2 generation is used to construct a mutant population and screen for superior single plants that are early-maturing, have strong tillering, are resistant to rice blast, and have both soft rice traits and aroma; SP3-SP5 generation is propagated through low-heat valleys to screen for lines with stable agronomic traits and consistent aroma.

[0009] Step 3: Genomic DNA was extracted from young leaves of 12 superior SP5 lines and PCR amplified using FAM fluorescently modified RM85 primers. After capillary electrophoresis and software analysis, genetically differential lines showing a bimodal characteristic at the RM85 locus of 78bp and 93bp were screened.

[0010] Step 4: Set up test sites in different low, medium and high altitude areas of indica rice region, conduct variety comparison test through random block design, and select the best-performing fragrant and soft rice mutant strain Diantun 502HangM6-8 by combining yield, resistance and rice quality indicators.

[0011] Furthermore, the original seeds of “Diantun 502” mentioned in step 1) must meet the degradation characteristics of loss of resistance to rice blast and decline in yield potential. During the entire process of carrying the seeds, they are sealed and preserved in a special biological sample bag.

[0012] Furthermore, the specific criteria for identifying the traits of SP2 generation soft rice in step 2) are as follows: the brown rice endosperm is milky white, cloudy and turbid, and non-transparent or semi-transparent. This trait does not segregate in the offspring after screening.

[0013] Furthermore, the specific method of propagation of SP3-SP5 generations in step 2) is as follows: SP3 generation is sown in Yuanjiang, SP4 generation is compared in Kunming, and SP5 generation is expanded to Yuanjiang in the winter of 2024. Each generation adopts the single-planting method and conventional fertilizer and water management.

[0014] Further, the PCR amplification system in step 3) is as follows: 17 μl of Qingke Gold Mix, 1 μl of 10 μM PrimerF, 1 μl of 10 μM PrimerR, 1 μl of gDNA template, and a total system of 20 μl; the amplification program is as follows: 98℃ pre-denaturation for 2 min, 35 cycles, 72℃ extension for 5 min, and storage at 4℃.

[0015] Further, the pretreatment for capillary electrophoresis in step 3) includes: mixing ABI HiDi Formamide and GeneScan 500LIZ internal standard at a ratio of 130:1 to prepare a mix; adding 10 μl of mix and 0.5 μl of sample template to each 96-well reaction well; centrifuging at 4000 rpm; pre-denaturing at 95°C for 5 min; immediately cooling at -20°C; thawing and mixing; and then detecting on an ABI 3730xl genetic analyzer.

[0016] Furthermore, the superior mutant strains selected in step 4) must meet the following criteria: amylose content 8.5%, rice blast resistance level ≤ 3, growth period 139 days, and thousand-grain weight 33.9g.

[0017] Another objective of this invention is to provide a high-yield cultivation method for the fragrant and soft rice mutant obtained by the above method, comprising the following steps:

[0018] Seed treatment: Select "Dian Tun 502 Hang M6-8" seeds, sun-dry the seeds for 2 days before soaking, and soak the seeds in "Shibaoke" for 36 hours;

[0019] Seedling raising and transplanting: The seedling age should be controlled at 30-35 days. Cultivate strong seedlings and transplant them with tillers and pesticides. Apply 5-8 kg of urea per mu 5 days before transplanting and carry out integrated pest and disease control at the same time.

[0020] Density control: Use a transplanting density of 4 inches × 8 inches to ensure a basic seedling density of about 40,000 plants per acre;

[0021] Water and fertilizer management: Fertilization follows the principle of "promoting tillering in the early stage, strengthening seedlings in the middle stage, and promoting grain production in the later stage", with heavy base fertilizer, early topdressing, increased potassium fertilizer, and appropriate application of ear and grain fertilizer; water management adopts the model of "shallow water for transplanting, 1 inch of water for greening, thin water for tillering, sufficient seedlings for drying the field, 1 inch of water for promoting ear production, and moist water for strong grains".

[0022] Pest and disease control: Implement integrated pest and disease control measures for rice blast and other diseases to ensure healthy plant growth.

[0023] Another objective of this invention is to provide an application of a fragrant and soft rice mutant obtained through the above-mentioned method. The mutant strain “Dian Tun 502 Hang M6-8” with the preservation number CCTCC No. P202602 is used as a new fragrant and soft rice variety and is promoted for planting in similar ecological zones such as the cool and temperate japonica rice area, the high temperature area of ​​indica rice area, and the tropical rice area in Yunnan. This mutant strain has both the turbidity of the soft rice endosperm and the natural aroma, and its high yield, disease resistance and adaptability are significantly better than the original strain “Dian Tun 502”.

[0024] Another objective of this invention is to provide an application of the fragrant and soft rice mutant obtained by the above method, using the "Dian Tun 502 Hang M6-8" mutant as the core parent for breeding, and hybridizing or backcrossing it with rice varieties that have resistance to bacterial blight, rice planthopper resistance or high yield potential, thereby aggregating superior genes to cultivate new varieties.

[0025] Compared with existing technologies, the beneficial effects of this solution are as follows: This invention, through a technical approach combining aerospace mutagenesis and ground-based multi-generational systematic breeding, successfully cultivated the "Dian Tun 502 Hang M6-8" mutant strain. While fully preserving the core quality characteristics of fragrant and soft rice, it achieved a comprehensive breakthrough in yield, resistance, and adaptability. This mutant strain has an amylose content of 8.5%, and its endosperm is milky white and cloudy, with a natural aroma reminiscent of popcorn or almonds. Its eating quality, which does not become stale when cold, is consistent with the original variety. At the same time, the yield is increased by 8.1% compared to the original variety, and the total number of grains per panicle, seed setting rate, and thousand-grain weight are increased by 11.1%, 16.0%, and 14.5%, respectively. The rice blast resistance has been improved from level 7 susceptibility to level 3 moderate resistance, and the growth period has been shortened by 12.7 days. This effectively solves the pain points of traditional fragrant and soft rice, which are characterized by "high quality but low yield, and high yield but poor quality" and insufficient resistance, achieving a synergistic aggregation of five excellent traits: fragrance, softness, early maturity, high yield, and resistance.

[0026] This invention constructs an integrated breeding system of "space mutagenesis-ground multi-generation directional screening-STR molecular marker identification-multi-point ecological verification", which significantly improves breeding efficiency and germplasm reliability. Space-induced mutagenesis utilizes the combined environment of microgravity and cosmic rays in space to significantly increase the frequency and diversity of gene mutations, making it easier to obtain breakthrough mutations compared to traditional mutagenesis techniques. During ground-based breeding, rich variations are preserved through mixed harvesting of the SP1 generation, early maturity and stress resistance phenotypes are precisely screened in the SP2 generation, and low-heat valley generation (SP3-SP5) accelerates trait homozygosity. Combined with visual identification of soft rice traits and dual verification by the aroma chewing / cooking method, the targeted enrichment of target traits is achieved. The key innovation lies in the introduction of STR molecular marker RM85 site detection, which precisely screens genetically differentiated lines at the molecular level, avoiding the subjectivity and misjudgment risk of traditional phenotypic screening. It clarifies the genetic background differences between mutants and the original species, ensuring the uniqueness and stability of the new germplasm. The entire breeding cycle is significantly shortened compared to traditional methods, and the obtained mutants have uniform agronomic traits and clear genetic backgrounds, solving the core problems of "low efficiency, mixed germplasm, and poor stability" in traditional breeding. Attached Figure Description

[0027] Figure 1 This is a capillary electrophoresis peak diagram of the mutant strain XH8 (i.e., Dian Tun 502 Hang M6-8) at the STR marker RM85 site in an embodiment of the present invention;

[0028] Figure 2This is a capillary electrophoresis peak diagram of the control variety Diantun 502 (CK) at the STR marker RM85 site in an embodiment of the present invention;

[0029] Figure 3 This is a DNA sequence alignment diagram based on the mutation site of STR marker RM85 in an embodiment of the present invention;

[0030] Figure 4 This is a schematic diagram of mutant strain selection in an embodiment of the present invention;

[0031] Figure 5 This is a comparison of the yield traits of the mutant strain and the control variety (CK) in the embodiments of the present invention;

[0032] Figure 6 This is a photograph of the grain appearance of the mutant strain "Diantun 502 Hang M6-8" in an embodiment of the present invention;

[0033] Figure 7 These are photographs of the appearance of rice (brown rice / polished rice) from the mutant strain "Diantun 502 Hang M6-8" in this embodiment of the invention.

[0034] Figure 8 This is a general flowchart of the space-borne mutation and ground-based multi-generation system selection method in this embodiment of the invention. Detailed Implementation

[0035] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be described in further detail below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort should fall within the scope of protection of the present invention.

[0036] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0037] Example 1: Spaceborne Mutation and Ground-based Multi-Generation System Selection Method

[0038] 1. Mutagenic materials and spaceborne applications

[0039] Selected from the original seeds of "Dian Tun 502," a variety that has undergone years of purification and rejuvenation, this conventional fragrant and soft rice variety has an amylose content of 8.2% and a light aroma. However, it has lost its resistance to rice blast, resulting in a decline in yield potential. One thousand plump seeds were packaged into special biological sample bags and sent into space aboard the Shenzhou-13 manned spacecraft on October 16, 2021. The seeds remained in orbit for approximately six months (until April 16, 2022), exposed to a comprehensive mutagenic environment including microgravity, cosmic rays, and high vacuum. The seed generation that returned to Earth was designated SP0.

[0040] 2. Ground-based first-generation (SP1) planting and mixed harvesting

[0041] In the spring of 2022, the seedlings were planted at the Yunnan Academy of Agricultural Sciences research base (Lufeng City). All SP0 generation seeds were germinated, then raised in a moist film-covered nursery, and transplanted individually to the field at a planting size of 15cm × 25cm. Conventional fertilization and watering were applied. After maturity, to avoid losing beneficial mutations, a strategy of single-plant harvesting and equal-volume mixing was adopted. Specifically, 20 grains were randomly harvested from the main spike of each surviving plant in the field, and the grains from all plants were thoroughly mixed to form a mixed SP1 generation seed population. This method aimed to preserve as many variant types as possible. The original seed "Diantun 502" was also planted as a control (CK).

[0042] 3. Second-generation (SP2) population construction and initial phenotypic screening

[0043] In 2023, the Yunnan Academy of Agricultural Sciences research base sowed SP1 generation mixed seeds, cultivating approximately 12,000 seedlings, which were then transplanted individually to construct the SP2 generation mutant population. Systematic observation was conducted throughout the entire growth period. The key selection criteria were: ① Growth period: Individual plants maturing more than 5 days earlier than the control (CK); ② Plant type and tillering: Individual plants with strong tillering ability (more than 30% more tillers per plant than CK), moderate plant height, and sturdy stems; ③ Disease resistance: Individual plants exhibiting significantly fewer leaf disease (especially rice blast) lesions than CK plants under naturally induced conditions. Approximately 500 individual plants initially selected in the field were further evaluated as follows:

[0044] 3.1 Identification of soft rice trait: Based on the single recessive inheritance pattern of the soft rice trait and the cloudy (cloudy) appearance of the endosperm of the soft rice trait, after the seeds of the SP2 generation are milled into brown rice, the soft rice trait and transparent rice can be distinguished by the naked eye. After the seed identification of the SP2 generation, only the seeds with the soft rice trait are selected for planting, and their offspring will all have the soft rice trait. The soft rice trait will no longer segregate in the offspring.

[0045] Specifically, 20 mature grains were taken from each plant, hulled using a brown rice machine, and the endosperm appearance was observed under bright light. Soft rice exhibited an endosperm that was milky white and cloudy (as shown in the image). Figure 6(As shown); non-soft rice (ordinary rice) has a transparent or translucent endosperm. Only single plants with typical soft rice characteristics are retained.

[0046] 3.2 Initial Aroma Screening: Aroma identification primarily employs the traditional grain chewing method. Personnel sensitive to aroma characteristics are trained for the assessment. Specifically, the previously identified soft rice plants are ground into brown rice and chewed for 1-2 minutes. Three trained tasters independently evaluate the rice. A popcorn-like aroma is used as the standard; plants that unanimously pass the evaluation are considered aromatic.

[0047] The identification process began with the selection of individual plants from the SP2 generation population. In the field, plants were first selected based on yield potential. From each selected plant, 20-30 grains were randomly taken and milled into brown rice for evaluation of appearance quality. Plants with poor appearance quality were discarded. Then, aroma traits were assessed on a plant-by-plant basis using a chewing method. Plants lacking aroma traits were discarded, while those exhibiting aroma traits were retained for further generations. Simultaneously, molecular markers were used to determine the aroma traits of some higher-generation materials, and cooking methods were also used to confirm the aroma traits of the materials.

[0048] After dual screening in the field and indoors, 15 superior individual plants that simultaneously possessed the four traits of early maturity, strong tillering, soft rice, and aroma were selected and numbered H1 to H15. The remaining individual plants were eliminated.

[0049] 4. Generation extension and trait stabilization from the third to the fifth generation (SP3-SP5)

[0050] SP3 generation (winter 2023) utilized a winter generation facility in Yuanjiang, Yunnan (a low-heat river valley region) to sow and transplant seeds of 15 individual plants (H1-H15) separately. The aim was to accelerate the generation process and rapidly achieve homozygous stability of variant traits.

[0051] In spring 2024, SP4 generation seeds of each SP3 line were planted in 12 rows (approximately 500 plants per line) in Lufeng City for a comparative experiment.

[0052] Trait identification: Record the growth period, plant height, effective panicles, and number of grains per panicle for each line. Continue to use the chewing method to identify aroma, and for the first time use the steaming method for verification: take 20 grams of rice from each line and steam it uniformly. The evaluation team smells the aroma of the cooked rice, focusing on the consistency within the line. Eliminate 3 lines with severe segregation or unstable aroma, and retain 12 superior lines with uniform agronomic traits and stable aroma (numbered XH1-XH12).

[0053] SP5 generation (winter 2024) was propagated again in Yuanjiang to expand the breeding of 12 superior lines. The harvested seeds were used for two purposes: ① a portion for molecular marker identification; ② a portion for multi-site comparison experiments.

[0054] 5. Sixth Generation (SP6) Multi-point Comparison and Final Selection

[0055] In 2025, experimental sites were established in three ecological zones within Yunnan Province: Site I (Lufeng City, 1360 meters above sea level), Site II (Yuanjiang, 370 meters above sea level), and Site III (Menghai County, 1200 meters above sea level). A randomized block design was adopted with three replicates and a plot area of ​​15 square meters. The test materials consisted of 12 mutant lines (XH1-XH12) and the control "Diantun 502" (CK).

[0056] This invention measures the yield of plots, investigates the effective panicles, number of grains per panicle, seed setting rate, and thousand-grain weight, and sets up a disease nursery in Menghai County to conduct artificial inoculation and identification, and investigates the incidence of leaf blast and neck blast according to the 0-10 standard.

[0057] The XH8 strain demonstrated the best overall performance in the three-point trials, with an average yield increase of 8.1% compared to the control (CK), an earlier maturity of 12.7 days, and a blast resistance level of 3 (moderately resistant), while the CK was 7 (susceptible). Rice quality testing revealed a mesochain starch content of 8.5%. Therefore, XH8 was selected as the optimal mutant strain and named "Dian Tun 502 Hang M6-8". The preservation number for this superior mutant strain is CCTCC No. P202602, with a preservation date of February 2026, and the preservation institution is the China Center for Type Culture Collection.

[0058] The general flowchart of the spaceborne mutation and ground-based multi-generation system selection method provided by this invention is attached. Figure 8 As shown, the agronomic traits of the mutant strain and the original species of Diantun 502 were identified as follows. Figure 4-7 As shown, the mutant strain "Diantun 502 Hang M6-8" has fuller grains compared to the original strain, and the rice retains the characteristic cloudy appearance of soft rice. The main agronomic traits are compared in Table 1 below:

[0059] Table 1 Comparison of agronomic traits

[0060]

[0061] Conclusion: This mutant strain showed significantly higher yield than the control in multiple demonstration sites, with good overall growth, clear leaf color, uniform plant growth in the field, full grains, no premature aging, good color change in the later stage, good resistance, good uniformity, high yield, and wide adaptability. The entire growth period was 137.6 days, plant height was 115.2 cm, panicle length was 27.9 cm, effective panicles were 215,000 / mu, total grains per panicle were 181.0, filled grains were 158.6, seed setting rate was 87.6%, 1000-grain weight was 33.9 g, and the rice was soft (endosperm turbidity was observed). Figure 6 It has a fragrance (as determined by chewing and steaming methods).

[0062] Example 2: Molecular genetic identification method for mutant strains

[0063] 1. Experimental Materials

[0064] Plant materials: young leaves of the mutant strain “Diantun 502 Hang M6-8” (i.e. XH8) and the original species “Diantun 502” (CK).

[0065] Main reagents and instruments: As listed in Table 2 below, including TSINGKE plant genomic DNA extraction kit, TSINGKE Gold Mix, ABI HiDi Formamide, GeneScan 500LIZ internal standard, and rice STR fluorescent primers (RM series). The main instruments were a Langji A300 PCR instrument and an ABI 3730xl genetic analyzer.

[0066] Table 2 Experimental Instruments and Reagents

[0067]

[0068] 2. Experimental Procedure

[0069] The specific steps for DNA extraction are as follows:

[0070] The specific steps for using the TSINGKE Plant Genomic DNA Extraction Kit (General Type) are as follows:

[0071] 2.1 Place the Spin Column in the Collection Tube, add 250 μl of Buffer BL, and centrifuge at 12000 rpm / min for 1 min to activate the silica membrane;

[0072] 2.2 Take 50 mg of sample tissue and grind it thoroughly in liquid nitrogen. After grinding, place it in a 1.5 ml centrifuge tube, add 400 μl of Buffer gP1, vortex for 1 min, and incubate in a 65℃ water bath for 10 ~ 30 min. During this time, you can take it out and invert it to mix well to ensure complete lysis.

[0073] 2.3 Add 150 μl of Buffer gP2, vortex for 1 min, and incubate on ice for 5 min;

[0074] 2.4 Centrifuge at 12000 rpm / min for 5 min, and transfer the supernatant to a new centrifuge tube;

[0075] 2.5 Add an equal volume of anhydrous ethanol to the supernatant, immediately shake thoroughly to mix, transfer all liquid to a Spin Column, centrifuge at 12,000 rpm / min for 30 s, and discard the waste liquid;

[0076] 2.6 Add 500 μl of Buffer Pw (with anhydrous ethanol added before use) to the Spin Column, centrifuge at 12000 rpm / min for 30 s, and discard the waste liquid;

[0077] 2.7 Add 500 μl of Wash Buffer (with anhydrous ethanol added before use) to the Spin Column, centrifuge at 12000 rpm / min for 30 s, and discard the waste liquid;

[0078] 2.8 Repeat step 7;

[0079] 2.9 Place the Spin Column back into the Collection Tube, centrifuge at 12,000 rpm / min for 2 min, and then air dry for 1 min after opening the lid;

[0080] 2.10 Remove the Spin Column and place it in a clean centrifuge tube. Add 50-100 μl of TE Buffer (preheated at 65°C) to the center of the adsorption membrane, incubate at 20-25°C for 2 min, and centrifuge at 12,000 rpm / min for 2 min.

[0081] 3. Synthesis of modified primers: Fluorescent modified primers were synthesized by adding FAM fluorescent groups to the 5' end of the differentially labeled RM85 F primers, and PAGE primers were synthesized from the R primers. The primer sequences are shown in Table 3 below:

[0082] Table 3 Primer Sequences

[0083]

[0084] 4. PCR amplification

[0085] DNA samples were extracted and used as PCR templates. Amplification was performed using fluorescently modified primers with Qingke Gold Mix (green). The components of the amplification system are shown in Table 4 below:

[0086] Table 4 Components of the amplification system

[0087]

[0088] The above amplification systems were amplified according to the amplification procedures in Table 5 below:

[0089] Table 5 Amplification Procedure

[0090]

[0091] 5. Electrophoresis detection: Take 2 μL of the amplified PCR product and perform agarose gel electrophoresis at 300V for 12 minutes to obtain the identification gel image. Determine the template concentration from the gel image and dilute with water to the concentration required for capillary electrophoresis.

[0092] 6. Capillary detection

[0093] 6.1 Mix HiDi and GS500 internal standards at a ratio of 130:1 to prepare a mix.

[0094] 6.2 Dispense the mix using a domestic 96-well reaction plate, adding 10 μL of mix to each well.

[0095] 6.3 corresponds to adding 0.5 μL of sample template to a 96-well plate and centrifuging to 4000 rpm.

[0096] 6.4 Heat the mixing plate at 95°C for 5 minutes using a metal bath heater to pre-deform it, then immediately place it at -20°C after removing it from the heat.

[0097] 6.5 After cooling, remove the sample, centrifuge at 4000 rpm, thaw, mix well, and then perform capillary electrophoresis on a 3730 sequencer.

[0098] 7. Data Analysis and Results

[0099] The raw data of this invention were analyzed using GeneMapper® Software v6.0. The software automatically corrected the fragment size based on the internal standard and output the peak diagram and fragment size (bp) of the sample at the RM85 marker site. The data are shown in Table 6 below.

[0100] Table 6 Genotyping results of mutant strains and original strains at the RM85 marker site.

[0101]

[0102] like Figure 1 and Figure 2 As shown in the peak diagram, the original species “Dian Tun 502” exhibits a single peak (93 bp fragment size) at the RM85 site, while the mutant strain “Dian Tun 502 Hang M6-8” shows a double peak (78 bp and 93 bp fragment sizes). This indicates that space-induced mutagenesis led to variations in the DNA sequence at this site (such as insertions / deletions or changes in the number of repeat units), confirming at the molecular level that “Dian Tun 502 Hang M6-8” is a new germplasm with a different genetic background from the original species. Further sequence comparison of the mutation sites confirmed single-base mutations and deletions, as shown in the attached diagram. Figure 3 As shown.

[0103] Example 3: High-yield cultivation methods for mutant strains

[0104] Sowing and seedling raising: Sun-dry the seeds for 2 days before soaking, and then disinfect them by soaking them in "Shibaike" pesticide for 36 hours. Cultivate strong seedlings of appropriate age, controlling the seedling age to 30-35 days, and transplant them with pesticide and tillers.

[0105] Reasonable planting density: The recommended planting density is 4 inches × 9 inches (approximately 13.3cm × 30cm), ensuring that there are about 40,000 basic seedlings per acre.

[0106] Fertilizer and water management: Fertilization should follow the principle of "promoting tillering in the early stage, strengthening seedlings in the middle stage, and promoting grain production in the later stage". Apply heavy base fertilizer, apply tillering fertilizer early, and increase potassium fertilizer. Water management should follow the principle of "shallow water for transplanting, small amount of water for greening, thin water for tillering, sufficient seedlings for drying the field, and alternating between dry and wet conditions in the later stage".

[0107] Pest and disease control: Focus on the prevention and control of rice blast, sheath blight and stem borers, and adhere to the principle of prevention first and unified prevention and control.

[0108] Pest and disease control: Timely implementation of integrated pest management to ensure healthy plant growth.

[0109] Example 4: Application of mutant strains

[0110] 1. Direct promotion as a breakthrough new variety of fragrant soft rice: This mutant strain has completed multiple generations of breeding and multi-location trials, exhibiting stable traits, excellent quality, and significantly higher yield than the original variety and similar soft rice varieties. It can apply for variety approval in accordance with the "Major Crop Variety Approval Methods," and upon approval, it can be directly promoted and planted as a main high-yield and high-quality fragrant soft rice variety in Yunnan and similar ecological zones, rapidly enhancing the market supply capacity of high-end soft rice.

[0111] 2. As a core parent for high-quality breeding: This mutant strain is a rare and precious germplasm resource that integrates "fragrant, soft, early-maturing, high-yielding, and resistant" traits. With a high thousand-grain weight, it can be used as a male or female parent to hybridize with other varieties possessing special resistance (such as bacterial blight and rice planthopper) or higher yield potential. This will aggregate more superior genes in the offspring, breeding "super-parent" new varieties. Using it as a donor parent and high-yielding but inferior-quality varieties as recurrent parents, backcrossing can introduce superior traits such as "fragrant" and "soft" into a high-yielding background, resolving the contradiction between high yield and high quality. Its specific STR marker (RM85) and aroma and soft rice traits can serve as targets for marker-assisted selection or for map-based cloning of key genes controlling these traits, deepening the genetic research on fragrant and soft rice traits.

[0112] 3. For developing high-end rice brands: Utilizing its superior taste and unique geographical origin (space-induced mutation, rare Yunnan soft rice), combined with green organic cultivation techniques, a high-end rice brand can be created. Through a marketing model of "variety characteristics + geographical indication + green certification + brand story," the market value of its products can be increased by 2-3 yuan / kg compared to ordinary rice, resulting in significant economic benefits and powerfully promoting rural industrial revitalization.

[0113] The above specific embodiments are merely explanations of the present invention and are not intended to limit the present invention. After reading this specification, those skilled in the art can make modifications to these embodiments without contributing any inventive step, but as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. A method and application for breeding a space-borne rice mutant strain, Dian Tun 502 Hang M6-8, characterized in that, Includes the following steps: Step 1: Select the original seeds of "Dian Tun 502" conventional fragrant soft rice with an amylose content of 8.2%, package them, and send them into space with the manned spacecraft. They will stay in orbit for 6 months and return to Earth to obtain SP0 generation seeds. Step 2: SP1 generation is harvested by single transplanting and equal mixing of single main panicles; SP2 generation is used to construct a mutant population and screen for superior single plants that are early-maturing, have strong tillering, are resistant to rice blast, and have both soft rice traits and aroma; SP3-SP5 generation is propagated through low-heat valleys to screen for lines with stable agronomic traits and consistent aroma. Step 3: Genomic DNA was extracted from young leaves of 12 superior SP5 lines and PCR amplified using FAM fluorescently modified RM85 primers. After capillary electrophoresis and software analysis, genetically differential lines showing a bimodal characteristic at the RM85 locus of 78bp and 93bp were screened. Step 4: Set up test sites in different low, medium and high altitude areas of indica rice region, conduct variety comparison test through random block design, and select the best-performing fragrant and soft rice mutant strain Diantun 502HangM6-8 by combining yield, resistance and rice quality indicators.

2. The breeding method according to claim 1, characterized in that, The original seeds of "Diantun 502" mentioned in step 1) must meet the degradation characteristics of loss of resistance to rice blast and decline in yield potential. During the entire process, the seeds are sealed and stored in a special biological sample bag.

3. The breeding method according to claim 1, characterized in that, The specific criteria for identifying the traits of SP2 generation soft rice in step 2) are as follows: the brown rice endosperm is milky white, cloudy and turbid, and non-transparent or semi-transparent. This trait does not separate in the offspring after screening.

4. The breeding method according to claim 1, characterized in that, The specific method of propagation of SP3-SP5 generations in step 2) is as follows: SP3 generation is sown in Yuanjiang, SP4 generation is compared with Lufeng City, and SP5 generation is expanded to Yuanjiang in the winter of 2024. Each generation adopts the single-planting mode and conventional fertilizer and water management.

5. The breeding method according to claim 1, characterized in that, The PCR amplification system in step 3) is as follows: 17 μl of Qingke Gold Mix, 1 μl of 10 μM PrimerF, 1 μl of 10 μM PrimerR, 1 μl of gDNA template, and a total system of 20 μl; the amplification program is as follows: 98℃ pre-denaturation for 2 min, 35 cycles, 72℃ extension for 5 min, and storage at 4℃.

6. The breeding method according to claim 1, characterized in that, Step 3) pretreatment for capillary electrophoresis includes: mixing ABI HiDi Formamide and GeneScan 500LIZ internal standard at a ratio of 130:1 to prepare a mix; adding 10 μl of mix and 0.5 μl of sample template to each 96-well reaction well; centrifuging at 4000 rpm; pre-denaturing at 95℃ for 5 min; immediately cooling at -20℃; thawing and mixing; and then detecting on an ABI 3730xl genetic analyzer.

7. The breeding method according to claim 1, characterized in that, The superior mutant strains selected in step 4) must meet the following criteria: amylose content 8.5%, rice blast resistance level ≤ 3, growth period 139 days, and thousand-grain weight 33.9g.

8. A high-yield cultivation method for a fragrant and soft-type rice mutant obtained by the method described in any one of claims 1-7, characterized in that, Includes the following steps: Seed treatment: Select "Dian Tun 502 Hang M6-8" seeds, sun-dry them for 2 days before soaking, and soak them in "Shi Bao Ke" for 36 hours; Seedling raising and transplanting: The seedling age should be controlled at 30-35 days. Cultivate strong seedlings and transplant them with tillers and pesticides. Apply 5-8 kg of urea per mu 5 days before transplanting and carry out integrated pest and disease control at the same time. Density control: Use a transplanting density of 4 inches × 8 inches to ensure a basic seedling density of about 40,000 plants per acre; Water and fertilizer management: Fertilization follows the principle of "promoting tillering in the early stage, strengthening seedlings in the middle stage, and promoting grain production in the later stage", with heavy base fertilizer, early topdressing, increased potassium fertilizer, and appropriate application of ear and grain fertilizer; water management adopts the model of "shallow water for transplanting, small amount of water for greening, thin water for tillering, sufficient seedlings for drying the field, small amount of water for promoting ear production, and moist water for strong grains". Pest and disease control: Implement integrated pest and disease control measures for rice blast and other diseases to ensure healthy plant growth.

9. The application of a fragrant and soft-type rice mutant obtained by the method described in any one of claims 1-7, characterized in that, The mutant strain "Dian Tun 502 Hang M6-8" with the preservation number CCTCC No. P202602 was promoted and planted in the indica rice area of ​​Yunnan as a new fragrant soft rice variety. This mutant strain has both the endosperm turbidity characteristic of soft rice and natural aroma, and its yield, disease resistance and adaptability are significantly better than the original strain "Dian Tun 502".

10. The application of a fragrant and soft-type rice mutant obtained by the method described in any one of claims 1-7, characterized in that, The mutant strain "Diantun 502 Hang M6-8" was used as the core parent for breeding. It was hybridized or backcrossed with rice varieties that had resistance to bacterial blight, rice planthopper resistance, or high yield potential to combine superior genes and breed new varieties.

Images