A domestication method of artificial allohexaploid rice aabb based on gene editing

Abstract

This invention discloses a method for domesticating artificial allotetraploid rice AABB based on gene editing, wherein the artificial allotetraploid rice AABB is cultivated rice. O.sativa With spotted wild rice O.punctata This was obtained by hybridization of the parent lines followed by chromosome doubling technology, the method including knocking out or inhibiting chromosomes derived from spotted wild rice O. punctata. SH4 , LABA1 and Bh4 Genes. This invention constructs a dual-target vector that simultaneously targets three genes, and obtains lines with simultaneous mutations in multiple genes through a single genetic transformation. Phenotypic analysis shows that... OsSH4 Knockout reduced the natural grain loss rate from 31% to below 5%; OsLABA1 Knocking out the awn shortened its length from 4.28cm to approximately 1.60cm. OsBh4 Knockout caused the glumes to change from dark brown to yellow. The simultaneous improvement of the above traits significantly enhanced the cultivation level of the artificial allotetraploid AABB while maintaining its original excellent traits such as high biomass and superior grain structure.

Description

Technical Field

[0001] This invention belongs to the fields of plant molecular biology and genetic engineering technology, specifically relating to a method for domesticating artificial allotetraploid rice AABB based on gene editing. Background Technology

[0002] During the long process of artificial domestication and modern breeding, the genetic base of cultivated rice has been continuously narrowed, becoming a key factor restricting its future genetic improvement. Studies have shown that in the process of rice being domesticated from wild species to cultivated species, continuous selection by humans for key traits such as non-shattering grains, compact plant type, and high yield has led to a rapid increase in the frequency of genes controlling these traits, and the genes linked to them have also been fixed. This domestication bottleneck has resulted in the continuous narrowing of the genetic base of cultivated rice.

[0003] Over millions of years of natural evolution, wild rice has accumulated an extremely rich array of superior alleles to adapt to diverse adverse environments. These are precisely the genetic variations that modern cultivated rice has lost or lacks through artificial selection. A high-quality pan-genome map of wild and cultivated rice, created by Academician Han Bin's team, shows that nearly 20% of the genes in common wild rice are unique to cultivated rice. These unique genes are closely related to traits such as disease resistance and environmental adaptability. The abundance and diversity of disease resistance genes in wild rice are significantly higher than in cultivated rice. The research team has precisely located 1184 disease resistance gene loci in wild rice with higher copy numbers than in cultivated rice. Interestingly, the EBT1 gene region has been subject to artificial selection during rice domestication—in the pursuit of high yield and compact plant type in cultivated rice, people may have inadvertently "discarded" the perennial genes of wild rice.

[0004] Wild rice typically exhibits several traits detrimental to agricultural production, including easy grain shattering, long seed dormancy, vining or lodging-prone growth, long awns, small grains, dark husks, and inconsistent maturity. Studies show that while these inherent characteristics—prostrate growth, long awns, black husks, and easy grain shattering—are beneficial for its survival and reproduction in the natural environment, they are not advantageous in modern agricultural production and contradict the requirements of concentrated harvesting, high and stable yields, uniformity, and marketable appearance. Furthermore, the genome structure of wild rice is extremely complex and extensive, with beneficial genes and genes for unfavorable traits often tightly linked. This linkage problem makes effective segregation difficult to achieve using traditional hybridization and backcrossing methods. As a key breeding resource, wild rice's development and utilization efficiency in traditional breeding is severely limited by the linkage of unfavorable traits and reproductive isolation, creating a significant bottleneck in resource utilization.

[0005] The creation of artificial allotetraploid rice AABB originated from cultivated rice ( Oryza sativa AA genome) and spotted wild rice ( Oryza punctataThe artificial allotetraploid rice AABB (a hybrid of the AA and BB genomes) is a distant hybridization. Spotted wild rice, belonging to the non-AA genome wild species of the Oryzae genus, possesses abundant excellent genetic resources for stress resistance and disease resistance. However, due to severe reproductive isolation from cultivated rice, traditional breeding methods struggle to utilize these resources directly. This artificial allotetraploid rice AABB exhibits excellent characteristics such as good self-pollination, strong tillering ability, and plant and leaf morphology resembling cultivated rice. However, it still retains some wild traits, mainly including easy grain shattering, long awns, and black glumes. While these traits represent survival advantages for wild rice in adapting to the natural environment, they contradict the intensive requirements of modern agricultural production. Therefore, how to domesticate this artificial allotetraploid rice AABB to retain the advantages of wild rice while avoiding its disadvantages has become a pressing technical problem for those skilled in the art. Summary of the Invention

[0006] The technical problem to be solved by this invention is to provide a method for the domestication of artificial allotetraploid rice AABB based on gene editing. The above objective of this invention is achieved through the following technical means: This invention provides a method for domesticating artificial allotetraploid rice AABB based on gene editing, wherein the artificial allotetraploid rice AABB is cultivated rice. O.sativa with spotted wild rice O.punctata This is obtained by hybridization of the parent lines followed by chromosome doubling technology, the method including knocking out or inhibiting chromosomes derived from spotted wild rice. O.punctata of SH4 , LABA1 and Bh4 Gene.

[0007] In a preferred embodiment of the present invention, the knockout is performed by transfecting a three-gene dual-target vector.

[0008] In a preferred embodiment of the present invention, the targeting sequence of the three-gene dual-target vector is:

[0009] In a preferred embodiment of the present invention, the three-gene dual-target vector comprises: six sgRNA units (t1-t6) designed for the BB genome; and tRNA sequences inserted between the sgRNA units. hyg Genes; pBR322 ori and pVS1 rep / sta.

[0010] In a preferred embodiment of the present invention, the three-gene dual-target vector is transfected into the callus tissue of artificial allotetraploid rice AABB by Agrobacterium.

[0011] In a preferred embodiment of the present invention, the method further includes a step of identifying gene knockout, wherein the identification step uses the following primers:

[0012] In a preferred embodiment of the present invention, the strains obtained by the domestication method have a natural grain shattering rate of less than 6%, an awn length of less than 2 cm, and a glume color of yellow.

[0013] Compared with the prior art, the present invention has the following beneficial effects: This invention constructs a dual-target vector that simultaneously targets three genes, enabling the generation of strains with simultaneous mutations in multiple genes through a single genetic transformation. More than 50% of these strains involve editing two or more genes. Phenotypic analysis shows that... OsSH4 Knockout reduced the natural grain loss rate from 31% to below 5%; OsLABA1 Knocking out the awn shortened its length from 4.28cm to approximately 1.60cm. OsBh4 Knockout caused the glumes to change from dark brown to yellow. The simultaneous improvement of the above traits significantly enhanced the cultivation level of the artificial allotetraploid AABB while maintaining its original excellent traits such as high biomass and superior grain structure. Attached Figure Description

[0014] Figure 1 Structural map of the three-gene CRISPR / Cas9 editing vector k1-TV-AsarTgk; Figure 2 : SH4 Relative gene expression levels, where data are expressed as mean ± standard deviation. The t-test was used for comparisons between groups. *P < 0.05, **P < 0.01, ***P < 0.001; Figure 3 :LABA1 Relative gene expression levels, where data are expressed as mean ± standard deviation, and t-tests were used for comparisons between groups. * P < 0.05, ** P < 0.01, *** P < 0.001; Figure 4 : Bh4 Relative gene expression levels, where data are expressed as mean ± standard deviation. The t-test was used for comparisons between groups. *P < 0.05, **P < 0.01, ***P < 0.001; Figure 5 Comparison of overall plant morphology (Hainan), scale bar: 15cm; Figure 6 AABB The growth and development process of awn: (A) Overall view of awn development, (BF) Local magnification of A, (B) The differentiation period of spikelet primordia, (C) The differentiation period of secondary branches, (DE) The formation period of pistil and stamen, (F) The formation period of pollen mother cells, scale bar is 100μm; (G) The meiotic division period of pollen mother cells, scale bar is 1cm; Figure 7 AABB CR -31 The growth and development process of awn: (A) Overall view of awn development, (BF) Local magnification of A, (B) The differentiation period of floret primordia, (C) The differentiation period of secondary branches, (DE) The formation period of stamens and pistils, (F) The formation period of pollen mother cells, scale bar is 100μm; (G) The meiotic division period of pollen mother cells, scale bar is 1cm. Figure 8 Comparison of awn morphology, from left to right: cultivated rice, spotted wild rice, AABB, AABB CR -19, AABB CR -20, AABB CR -31; Scale bar is 1cm; Figure 9 : Statistical chart of mango length. Data are expressed as mean ± standard deviation. The t-test was used for comparisons between groups. * P < 0.05, ** P < 0.01, *** P < 0.001, ns is considered not significant. Figure 10 : Abscission layer structure, where AL: abscission layer; RG: rudimentary glume; PE: pedicel; the red box shows a magnified part of the left image; scale bar: 200μm (left image), 100μm (right image). Figure 11 Natural grain fall rate statistics, where data are expressed as mean ± standard deviation, and t-tests were used for comparisons between groups. * P < 0.05, ** P < 0.01, *** P < 0.001, ns is considered not significant; Figure 12 Comparison of grain breaking tensile strength. Data are expressed as mean ± standard deviation. The t-test was used for comparison between groups. *P<0.05, **P<0.01, ***P<0.001, ns is considered not significant. Figure 13 Changes in glume color during seed maturation: (A) Cultivated rice (B) Spotted wild rice (C) AABB (D) AABB CR -19(E)AABB CR -31; from left to right, these represent 3, 6, 9, 12, 15, 18, 21, 24, 27, and 30 days after flowering. The scale bar is 0.3 cm. Figure 14 Temporal changes in chlorophyll content in glumes; Figure 15 Temporal changes in carotenoid content in glumes; Figure 16 Temporal changes in melanin content in glumes; Figure 17The temporal variation of anthocyanin content in glumes was studied. Data are expressed as mean ± standard deviation. The t-test was used for comparisons between groups. * P < 0.05, ** P < 0.01, *** P < 0.001, and ns were considered not significant. Detailed Implementation

[0015] The present invention will be further illustrated below with reference to specific embodiments, but these embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following embodiments are commercially available.

[0016] Example 1: Materials: This invention uses cultivated rice ( O.sativa , AA, 2n = 2 x = 24) and spotted wild rice ( O.punctata , BB, 2n = 2 x =24) was used as a de novo rapid domestication material for artificial allotetraploid rice AABB obtained by chromosome doubling technology after hybridization of the parental lines. This artificial allotetraploid rice AABB has been published in Wang, AY, Zhang, XH, Yang, & CH, et al. (2013). Development and characterization of synthetic amphiploid (aabb) between oryza sativ a and oryza punctata 2013, 189(1), 1-8.

[0017] 1. Assembly of sgRNA expression cassettes and vector construction (1) Synthesize the following target gene amplification primers. Table 1 Target genes and their target sequences

[0018] (2) PCR system Table 2 PCR system

[0019] Table 3 PCR Procedure

[0020] Using 1% agarose gel electrophoresis at 5 V / cm for 20 min, the t1-t6 (1306 bp) electrophoretic fragment was excised under UV light and placed in a system for gel recovery. The recovery procedure is detailed in the manufacturer's kit instructions. The recovered DNA was dissolved and recovered in 30 μL of water (the recovered product was labeled as: rDNAt1). After verification, it was ligated into the vector.

[0021] (3) Enzyme digestion and ligation Table 4 Enzyme digestion and ligation system

[0022] Table 5 Enzyme digestion and ligation reaction conditions

[0023] (4) Transformation Transform 5-10 μL of the ligation product into competent E. coli transformation plates (kanamycin-resistant plates), incubate at 37°C for 12 hours, and then perform plaque PCR identification.

[0024] (5) Plaque PCR identification Ten plaques were selected and simultaneously inoculated into 1.5 mL EP tubes for PCR identification.

[0025] Table 6 Primer list for plaque PCR identification

[0026] (6) PCR system Table 7 Plaque PCR System

[0027] Table 8 Plaque PCR Procedure

[0028] The target band is a fragment of approximately 9277 bp. Take 100 μL of bacterial culture corresponding to 1-3 positive bands for sequencing, and inoculate the remaining 400 μL of bacterial culture into LB broth containing 5-10 mL of kanamycin-resistant culture. Shake the tubes and wait for the sequencing results. Take the tube corresponding to the correct sequencing and extract the plasmid, naming it k1-TV-AsarTgk.

[0029] From this, we can obtain the following... Figure 1 The results of constructing the three-gene dual-target vector are shown below. Figure 1 As shown, the vector is 17840 bp in total length, containing a T-DNA region and a vector backbone region. The positions and functions of each element are clearly defined, providing a reliable tool base for the successful acquisition of multiple mutant lines in this study. The core design feature of this vector is: six sgRNA units (t1-t6) designed specifically for the BB genome, corresponding to… OsSH4, OsLABA1, OsBh4 Each of the three genes has two target sites, enabling simultaneous editing of multiple genes; tRNA sequences are inserted between sgRNA units, and the sgRNA is precisely cleaved by endogenous tRNA processing enzymes to release multiple functional sgRNAs, thereby improving the efficiency of multi-target co-editing. hyg The gene confers resistance to hygromycin, facilitating the selection and regeneration of transformed callus; it also possesses pBR322 ori and pVS1 rep / sta, ensuring stable vector movement between prokaryotic hosts Escherichia coli and Agrobacterium.

[0030] 2. Callus induction and subculture Artificial allotetraploid rice AABB seeds were dehulled, leaving the embryo-bearing half. They were then soaked in mercuric chloride solution, shaken every 5 minutes, for 30 minutes. After soaking, the mercuric chloride solution was removed, and the seeds were rinsed several times with sterile water. Excess water was then blotted dry with filter paper.

[0031] Seed material was inoculated onto induction medium, with 5 seeds evenly distributed on each medium, gently pressed to ensure full contact with nutrients. After inoculation, the material was placed in the dark and cultured at 28°C until sufficient callus tissue grew. The induced callus tissue was carefully detached from the endosperm, and endosperm residue and browned parts were removed. Pale yellow, dense, granular, and dry callus tissue was selected. Approximately 8 callus pieces were inoculated per bottle of medium, with a subculture interval of 14 days.

[0032] During inoculation, avoid contamination of the seeds and culture medium by microorganisms. During callus culture, regularly observe the condition of the material and culture medium. If microbial contamination is found, take immediate action, discarding severely contaminated material or salvaging lightly contaminated material and replacing it with new sterile culture medium.

[0033] Agrobacterium-mediated genetic transformation The recombinant CRISPR / Cas9 vector plasmid was transformed into Agrobacterium tumefaciens competent cells EHA105 via a freeze-thaw method. 1-2 µL of plasmid DNA (approximately 100-200 ng / µL) was added to 100 µL of EHA105 competent cells thawed on ice, gently mixed, and incubated on ice for 30 min. After flash freezing in liquid nitrogen for 1 min, the cells were heat-shocked at 37°C for 5 min. Immediately afterward, the cells were incubated on ice for 2 min, and 800 µL of LLB liquid medium was added. The cells were then incubated at 28°C with shaking at 150 rpm for 2-3 h. The recovered bacterial culture was plated on LB agar plates containing 50 mg / L kanamycin and 20 mg / L rifampin and incubated upside down at 28°C for 36-48 h. Single colonies were picked for colony PCR verification (primers required are shown in Table 9). Positive clones were used for subsequent experiments.

[0034] Table 9. PCR program for recombinant Agrobacterium plaques

[0035] Single colonies of positive recombinant Agrobacterium were picked and inoculated into 5 mL of YEB liquid medium containing the same concentration of kanamycin. The culture was incubated overnight at 28°C with shaking at 200 rpm. The next day, the colonies were transferred to fresh YEB medium at a 1:50 ratio and cultured until OD (outlet count) was reached. 600 =0.8-1.0.

[0036] Transfer the activated bacterial culture to a sterile centrifuge tube and centrifuge at 4,000 rpm for 10 min, discarding the supernatant. Resuspend the bacterial cells in suspension culture medium and adjust the OD. 600 Adjust the concentration to 0.3-0.4. Incubate the adjusted bacterial solution at 28℃ and 100 rpm with shaking for 30-60 min to further activate Agrobacterium.

[0037] Select vigorous, pale yellow, granular embryogenic callus tissue. Under aseptic conditions, pick the callus tissue, removing any browned, water-soaked, or aged portions. Collect the callus in sterile empty bottles for later use.

[0038] Pour the callus tissue into Agrobacterium suspension and soak for 5-10 minutes, gently shaking 1-2 times during this period. Discard the bacterial suspension and transfer the callus to a petri dish lined with sterile filter paper. Dry the callus in a laminar flow hood for 30-60 minutes until there is no obvious bacterial suspension on the surface, but it is still moist. For co-culturing, first place a layer of sterile filter paper on the surface of the culture medium, then evenly place the callus on it, and incubate in the dark at 20°C for 12 hours, then transfer to 25°C for another 2 days of dark incubation.

[0039] After co-culturing, the callus tissue was transferred to a sterile bottle and washed vigorously with sterile water, repeating the washing process 7-8 times. The first 3-4 washes were quickly discarded, and the remaining 3-4 washes were performed by soaking for 3-5 minutes each time, with gentle shaking during the soaking. After discarding the sterile water for the final wash, sterile water containing 500 mg / L carbenicillin or 300 mg / L termethin was added, and the tissue was soaked for 30 minutes, shaking occasionally. The antibiotic solution was then discarded, and the callus was transferred to a culture dish lined with sterile filter paper and allowed to air dry for 1-2 hours until no visible moisture remained on the surface.

[0040] 4. Resistance screening and obtaining regenerated plants Sterilized callus tissue was evenly placed on the surface of the selection medium, with about 10 pieces inoculated per bottle. The culture was carried out at 28°C for 16 hours of light and 8 hours of darkness. After about 14-21 days, non-transformed callus began to turn brown, while new cell clusters appeared on the surface of transformed callus, which were pale yellow and dense. Eventually, non-transformed callus completely turned brown and died, while transformed callus formed obvious resistant clones. The resistant callus that survived the first round of selection was transferred to fresh selection medium and cultured at 28°C for 10-14 days. Callus in good condition was selected for subsequent differentiation culture.

[0041] Differentiation culture of resistant callus: The expanded resistant callus tissue was gently crushed into small pieces of 2-3 mm with sterile forceps, and the browned parts were removed. Then, the callus tissue was evenly placed on the surface of the differentiation medium, about 5-10 pieces per bottle, and cultured at 28℃ under strong light for 16 h and then in the dark for 8 h for 20-30 days.

[0042] Rooting culture of regenerated seedlings: The seedlings obtained from differentiation culture are cut off from the base of the callus tissue. The callus tissue attached to the base of the seedling is removed with a sterile blade to prevent it from continuing to proliferate and consume nutrients. The seedlings are inserted into the rooting medium to a depth of about 0.5-1.0 cm to ensure that the base is in full contact with the medium. The seedlings are cultured at 28℃ for 16 h of light and 8 h of darkness for 10-15 days.

[0043] Transplanting regenerated seedlings to the field: After 10-15 days of rooting culture, select robust plants with well-developed root systems, open the culture container lid, and allow them to acclimatize under natural light in the culture room for 2-3 days. Carefully remove the seedlings, gently rinse the roots with warm water to thoroughly remove any residual culture medium, and transplant them to the field in the evening, continuously observing the seedling growth.

[0044] 5. Molecular identification of gene knockout plants Genomic DNA extraction from regenerated plants: Regenerated plants were transplanted and brought to the 3-5 leaf stage. Approximately 100 mg of young leaves were collected, and DNA was extracted and stored at -20°C. Based on the target gene's location, cross-target primers were designed for target site PCR amplification (as shown in Table 10). The purified PCR products were sent to a sequencing company for bidirectional sequencing.

[0045] Table 10 Primers for Identifying Editing Targets

[0046] 6. Evaluation of the CRISPR / Cas9 system's targeted editing efficiency Use tools such as SnapGene, Chromas, or NCBI Blast to open the sequencing peak diagram and determine the editing type based on the characteristics of the peak diagram.

[0047] The sequencing results were compared with reference sequences of genomes A and B. SNP sites (natural molecular markers) between genomes A and B were used to distinguish alleles from A and B. The genotype of each plant was determined based on the sequencing results.

[0048] Group editing efficiency = × 100% Target success rate = × 100% 7. Gene expression level analysis Total RNA was extracted and cDNA was synthesized. Real-time quantitative PCR was performed using the primers shown in Table 11.

[0049] Table 11 Primer list for real-time quantitative PCR

[0050] 8. Phenotypic Analysis and Statistics 8.1 Morphological comparison of cultivated rice, spotted wild rice, AABB wild type and its mutants Once the materials reached maturity, morphological and agronomic traits were investigated. A completely randomized block design was used, with multiple replicates.

[0051] Plant height: Measured from the ground to the top of the ear excluding the awn (cm); Ear count: The number of tillers that produce ears and seeds per plant; Ear length: The length (cm) from the neck node of the ear to the tip of the ear, excluding the awns. Number of grains per spike: Count the total number of spikelets per spike; Fruit setting rate: Fruit setting rate (%) = (Number of plump kernels ÷ Total number of kernels) × 100%; Grain length and width: Measure the length and width (mm) of 20 plump seeds with vernier calipers; 1000-seed weight: Weigh 1000 seeds randomly (g). awn length: Randomly select 10 spikelets from each plant, measure the length (mm) from the base of the awn to the tip of the awn, and take the average value; Observation of glume color: From the day of flowering to the full maturity of the seed, samples were taken every 3 days, and the color changes were observed and photographed under a stereomicroscope. Shattering rate: Cell sections of the abscission zone were observed and the tensile strength of the grains was measured. The natural shattering rate was also calculated.

[0052] 8.2 Morphological observation during spikelet development Sampling and Fixation: Sampling should begin approximately 25-30 days before heading, with sampling conducted daily or every other day to capture key growth points. When sampling, select representative main stems or large tillers with uniform growth. Carefully remove outer old leaves, preserving the tender leaf sheaths surrounding the young spikelets, avoiding damage to the growth point. For long-term preservation or subsequent sectioning and microscopic observation, immediately place the samples in a fixative solution (FAA:formalin:acetic acid:alcohol = 5:5:90) and refrigerate at 4°C or store at room temperature for later use.

[0053] Microscopic observation: Using a stereomicroscope to observe the overall outline of the young spike, the primordia of the branches and florets, and other external morphological features.

[0054] 8.3 Measurement and Statistical Analysis of Mature Seed Awn Length Sampling: Approximately 25-30 days after heading, when the rice is in the waxy ripening to full ripening stage, the awns have fully elongated and their shape is fixed. 5-10 main panicles are randomly selected from each group of materials and air-dried naturally or at 43℃ until constant weight.

[0055] Manual measurement: Take 5 ears of fruit from each plant, and 5-10 intact awns from each ear. Gently hold the base of the awn with tweezers and measure the straight-line distance from the base to the tip of the awn with a digital vernier caliper. Record the length of each awn and take the average value as the representative value of the ear. The awn may be naturally curved. When measuring, it should be gently straightened but avoid excessive stretching that may cause breakage.

[0056] 8.4 Microscopic observation of abscission layer cells Material collection and fixation: Place fresh rice panicles in FAA fixative, with the fixative volume at least 10 times the material volume. Place the fixative on ice, evacuate for 15 minutes, slowly release the gas, and repeat three times until the material settles to the bottom. Fix the material for 24 hours. For long-term storage, transfer to a 70% ethanol solution. Dehydration: The fixed material is placed in 70% alcohol → 85% alcohol → 95% alcohol → anhydrous ethanol in sequence for dehydration, and each concentration is maintained for 1-2 hours; Transparency: Immerse the dehydrated material in an environmentally friendly dewaxing solution for 4 hours to achieve transparency; Waxing: Keep half of the environmentally friendly dewaxing liquid, add half of the melted paraffin wax and mix, place in a 60℃ oven for 12 hours, replace with a new mixed wax liquid and place for another 12 hours, then pour out the mixed wax liquid, add pure wax liquid and put back in the oven for 24 hours; Embedding: Place the wax-impregnated material into an embedding frame, add molten paraffin, cool and solidify, and freeze at -20°C for 20 minutes before removing for later use; Sectioning: Take the material out of the refrigerator 1 hour in advance and use a manual rotary microtome to cut the embedded material into sections 8-10μm thick; Slide mounting: Place the slide onto the glass slide, drip a few drops of water from the edge of the slide, tilt the slide to allow the water to soak the bottom of the slide and make it fully unfold, and then place the glass slide on a metal constant temperature slide display table at 40°C to dry the surface moisture. Drying the slices: Place the sliced ​​slices in an oven and dry at 60℃ for 24 hours to completely dry them. Dewaxing and rehydration: Place the sections in an environmentally friendly dewaxing solution for 40 min, then transfer them to anhydrous ethanol for 4 min, then place them in ethanol solutions of 95%, 85%, 70%, 50%, 30%, and 15% concentrations for 1 min each, and finally place them in distilled water for 2 min. Staining: Place the baked sections into the staining solution one by one for staining. The 5% toluidine blue solution needs to be stained for 30-60 minutes. Dehydration and clearing: Place the stained sections in distilled water for 2 minutes, then in 15%, 30%, 50%, 70%, 85%, and 95% ethanol solutions for 1 minute each, then in anhydrous ethanol for 2 minutes, and finally in environmentally friendly dewaxing solution for 10 minutes to complete dehydration and clearing. Mounting: Mount the slide with neutral resin, blow it in a fume hood for 15 minutes, lay it flat in a 40℃ oven overnight to dry, and then observe it.

[0057] 8.5 Statistics on natural particle loss rate Ten main stem spikes were selected from each material and labeled with information including material number, heading date, and expected recording date. The number of grains lost was counted at 7, 14, 21, 28, and 35 days after heading to establish a grain loss dynamic curve. The final statistics were based on the maturity date.

[0058] Particle loss rate = × 100%, N drop Number of fallen grains, N remain : The number of grains remaining on the ear.

[0059] 8.6 Measurement of delamination tensile force The abscission pull force is the maximum force required to pull a grain off the spikelet, measured in Newtons (N) or grams per force (gf). This value directly reflects the mechanical strength of the abscission zone—the smaller the pull force, the easier it is for the grain to fall off; the larger the pull force, the more difficult it is for the grain to fall off.

[0060] Sampling: Take 5 main ears of fully mature material, select spikelets from the middle of the ear, cut off the whole spikelets, and retain about 2-3 mm of the spikelet stalk. Measure 10 grains from each ear and take the average value.

[0061] Measurement: After instrument calibration, fix the rachis or twig in the upper clamp and the grains or spikelets in the lower clamp, ensuring the pulling force is perpendicular to the abscission plane, i.e., pulling in the direction of natural grain detachment. Shake the support handle at a uniform speed of 10-20 mm / min to gradually separate the upper and lower clamps, observing the tension gauge reading. Record the maximum tension value when the grain has completely detached from the abscission plane. The measurement time for each grain should be controlled within 5-10 seconds, and obviously abnormal values ​​should be discarded.

[0062] 8.7 Dynamic observation and recording of glume color during seed maturation The experimental procedure for observing and recording the dynamic color of rice glumes during seed maturation is a typical developmental dynamics study. It records the color change of glumes of different materials over developmental time and establishes the correspondence between color and developmental stage.

[0063] Sampling: Select 3 spikelets from each main stem of each material. On the day of heading, attach a tag below the neck node of the spikelet using a thin string or label to avoid damage. Record the material number, heading date, and expected observation date. Designate the heading date as day 0. Subsequent observations should be aligned with the number of days after heading, sampling every 3 days, for approximately 10-12 time points, covering the entire process from heading to full maturity. Immediately photograph and record the glumes color of the harvested kernels to prevent photolysis and oxidation of chlorophyll and carotenoids, which would cause the green color to fade and the yellow color to lighten.

[0064] Photographing: The color of the glumes is recorded by photographing with a stereomicroscope, ensuring that the lens magnification, light source intensity, and background are consistent.

[0065] 8.8 Analysis of the temporal variation of pigment content in glumes The chemical properties of different pigments in glumes vary considerably, thus requiring different extraction and determination methods. Since this is a time-series analysis, samples should be processed immediately after sampling or stored at -80°C. It is recommended to freeze-dry the glumes, grind them into powder, mix thoroughly, and then aliquot and store. The same batch of dry powder should be used for each sample analysis to eliminate moisture interference.

[0066] (1) Chlorophyll content determination: Preparation of Standard Curve: Weigh 5.0 mg each of chlorophyll a and chlorophyll b standards, dissolve them separately in a small amount of 95% ethanol, transfer them to 50 mL brown volumetric flasks, and dilute to volume. The stock solutions of chlorophyll a and chlorophyll b are now 100 mg / L. Take five 10 mL brown volumetric flasks, and transfer chlorophyll a and chlorophyll b stock solutions in gradients, diluting to volume with 95% ethanol to prepare five mixed standard working solutions of different concentrations. Using 95% ethanol as a blank control, measure the absorbance at each concentration point at 665 nm and 649 nm. Plot the chlorophyll a concentration, chlorophyll b concentration, and total chlorophyll concentration (mg / L) on the x-axis, and the corresponding absorbance values ​​(chlorophyll a is represented by OD) on the y-axis. 665 Chlorophyll b uses OD 649 Using y as the ordinate, plot the standard curve to obtain the regression equation. and R 2 value.

[0067] Extraction and determination of chlorophyll content: Weigh 0.2 g of fresh glumes, cut them into small pieces, and place them in a 1.5 mL centrifuge tube. Add 10 steel beads with a diameter of 1 mm, freeze in liquid nitrogen for 1 min, and then grind them into powder using a high-throughput tissue homogenizer. Add 95% ethanol and allow to stand for extraction. Filter the extract into a 25 mL brown volumetric flask, repeatedly wash the residue until colorless, and finally dilute to the mark with 95% ethanol. Shake well and set aside. Using 95% ethanol as a blank control, zero the volume and inject the sample extract into a cuvette. Measure the absorbance at wavelengths of 665 nm and 649 nm. Substitute the measured values ​​into the corresponding standard curve regression equation to calculate the concentration c (mg / L) of chlorophyll a, chlorophyll b, or total chlorophyll in the extract. Convert the chlorophyll content (mg / g) in the sample using the following formula: Lambert-Beer Law: A = κ × c × L A: Absorbance; κ: Absorption coefficient; c: Total chlorophyll concentration; L: Optical path length, which is 1 cm.

[0068] Chlorophyll content (mg / g) =

[0069] (2) Determination of carotenoid content: Preparation of the standard curve: Accurately weigh 5.0 mg of β-carotene standard, dissolve it in a small amount of fat-soluble organic solvent (n-hexane: acetone: anhydrous ethanol = 2:1:1), transfer to a 50 mL brown volumetric flask, and dilute to volume. The stock solution concentration is 100 mg / L. The entire process must be performed in the dark. Take five 10 mL brown volumetric flasks and prepare a series of standard solutions according to the concentration gradient: using the extraction solvent as a blank control, measure the absorbance value of each concentration of standard solution at a wavelength of 450 nm. Repeat the measurement three times for each concentration and take the average value. Perform linear regression with the standard concentration (mg / L) as the x-axis (X) and the absorbance value (A) as the y-axis (Y) to obtain the regression equation. and R 2 value.

[0070] Carotenoid extraction and content determination: Weigh 0.5-1.0 g of fresh glumes, cut them into small pieces, and place them in a 1.5 mL EP tube. Add 10 1 mm steel beads, freeze in liquid nitrogen for 1 min, and then place in a high-throughput tissue homogenizer to quickly grind into powder. Add 10 mL of pre-cooled extraction solvent and vortex thoroughly to form a homogenate. Centrifuge at 5,000 rpm for 10 min at 4℃. Collect the supernatant, transfer it to a separatory funnel, wash with 20 mL of 5% NaCl solution, and discard the aqueous phase. Dehydrate the organic phase using an anhydrous sodium sulfate column and collect it in a 25 mL brown volumetric flask. Dilute to the mark with the extraction solvent. Zero the volume using the extraction solvent as a blank, pour the sample extract into a 1 cm quartz cuvette, and measure the absorbance at 450 nm. If the absorbance exceeds the upper limit of the linear range of the standard curve, dilute appropriately with the extraction solvent and measure again. Substitute the sample absorbance value into the standard curve regression equation to calculate the carotenoid concentration (mg / L) in the extract, and finally convert it into the carotenoid content of the sample: A: absorbance; C: carotenoid concentration in the extract; b is a parameter of the regression equation.

[0071] Carotenoid content (mg / g) =

[0072] (3) Anthocyanin content determination (pH differential method): Reagent preparation: Measure 99 mL of methanol into a reagent bottle, slowly add 1 mL of 37% concentrated hydrochloric acid while stirring, to prepare a 1% hydrochloric acid-methanol solution; weigh 1.86 g of KCl into a beaker, add about 980 mL of distilled water to dissolve, adjust the pH to 1.0 with concentrated hydrochloric acid, transfer the solution to a 1 L volumetric flask, dilute to volume with distilled water, and shake well to prepare a pH 1.0 KCl buffer solution; weigh 54.23 g of sodium acetate into a beaker, add about 950 mL of distilled water to dissolve, adjust the pH to 4.5 with concentrated hydrochloric acid, transfer the solution to a 1 L volumetric flask, dilute to volume with distilled water, and shake well to prepare a pH 4.5 sodium acetate buffer solution.

[0073] Sample pretreatment and anthocyanin extraction: Fresh or -80℃ frozen rice husks were freeze-dried. The dried husks were then ground into a fine powder using a high-throughput tissue grinder, passed through a 60-mesh sieve, and mixed thoroughly. 1.0 g of husk powder was accurately weighed and placed in a 50 mL brown centrifuge tube. 10 mL of pre-cooled 1% hydrochloric acid-methanol extraction solution was added to the centrifuge tube, and the mixture was vortexed to ensure thorough contact between the sample powder and the extraction solution. The centrifuge tube was placed in an ultrasonic cleaner and ultrasonically extracted for 30 min under ice-water bath conditions. After ultrasonication, the centrifuge tube was placed in a centrifuge and centrifuged at 10,000 rpm for 10 min at 4℃. The centrifuge tube was carefully removed, and the supernatant was transferred to a 25 mL brown volumetric flask. 5 mL of extraction solution was added to the precipitate again, and the above extraction, ultrasonication, and centrifugation steps were repeated once. The supernatants from both extractions were combined in the same 25 mL brown volumetric flask. Wash the centrifuge tube and precipitate with the extract in small amounts several times. Add the washings to the volumetric flask. Finally, dilute to the mark with the extract and shake well to obtain the crude anthocyanin extract.

[0074] Sample dilution: Prepare two test tubes: Take two clean 10 mL glass test tubes and label them A (pH 1.0) and B (pH 4.5), respectively. Accurately pipette 1 mL of crude anthocyanin extract into each test tube (A and B). Add 9 mL of pH 1.0 buffer to A and mix well. Add 9 mL of pH 4.5 buffer to B and mix well. Place both test tubes in the dark and allow them to equilibrate at room temperature for 20 min. Take two quartz cuvettes, one as a blank control, and zero the wavelengths at 530 nm and 700 nm using distilled water. Take the diluted solution from test tube A and add it to the cuvette. Read and record the values ​​of A and B. 530nm and A 700nm The absorbance value was measured three times, and the average value was taken. Similarly, the absorbance value of the diluted solution in test tube B was read and recorded in test tube A. 530nm and A 700nm The absorbance value.

[0075] Calculate the absorbance difference (A):

[0076] Calculate the anthocyanin concentration (C) in the extract:

[0077] A: Absorbance; C: Anthocyanin concentration in the extract; M: The molar mass of anthocyanin, expressed as cyanidin-3-O-glucoside, is 449.2 g / mol; : The molar extinction coefficient of the main anthocyanin, cyanidin-3-O-glucoside, at its maximum absorption wavelength near 510 nm, is 26,900 L / (mol·cm); L: the optical path length of the cuvette, which is 1 cm.

[0078] Anthocyanin content (mg / g) =

[0079] (4) Determination of melanin content: Reagent preparation: Weigh 40.0 g NaOH, dilute to 1 L with distilled water, dilute 1 mol / L NaOH 10 times to prepare 0.1 mol / L NaOH solution, accurately weigh 10.0 mg tyrosine, add a small amount of 0.1 mol / L NaOH to dissolve, sonicate for 20 min, and dilute to 10 mL in a brown volumetric flask to a concentration of 1,000 μg / mL.

[0080] Preparation of the standard curve: A full wavelength scan of 200-800 nm was performed on the standard solution. 400 nm was selected as the measurement wavelength. A series of concentration gradient standard solutions were prepared using 0.1 mol / L NaOH. Using 0.1 mol / L NaOH as a blank, the absorbance was measured at 400 nm. Linear regression was performed with the standard concentration (mg / L) as the x-axis and the absorbance value (A) as the y-axis to obtain the regression equation. and R 2 value.

[0081] Melanin Extraction and Content Determination: Weigh 0.5-1.0 g of fresh glumes, cut them into small pieces, and place them in a 1.5 mL EP tube. Add 10 1 mm steel beads, freeze in liquid nitrogen for 1 min, and then place in a high-throughput tissue homogenizer to quickly grind into powder. Add 10 mL of 1 mol / L NaOH, vortex to mix, sonicate in a 60℃ water bath for 60 min, centrifuge at 8,000 rpm for 10 min, collect the supernatant, transfer to a 25 mL brown volumetric flask, and dilute to volume with 0.1 mol / L NaOH (total extraction volume V = 25 mL). Measure the absorbance at 400 nm. Substitute the sample absorbance value into the standard curve regression equation to calculate the melanin concentration (mg / L) in the extract, and finally convert it into the melanin content in the sample. A: absorbance; C: melanin concentration in the extract; a and b are parameters of the regression equation.

[0082] Melanin content (mg / g) =

[0083] 9. Experimental Results 9.1 Genetic transformation and regeneration efficiency In this embodiment, 120 artificial allotetraploid rice AABB seeds were used as starting material, and callus tissue from 99 seeds was induced, with a callus induction rate of 82.5%. After subculture, the growth status of the callus tissue was good or above.

[0084] After genetic transformation, a total of 33 positive regenerated seedlings were obtained, with a transformation efficiency of 39.76%. Gene editing detection was performed on the above positive plants, and 20 mutant plants were detected, with a gene editing efficiency of 60.61% (Table 12).

[0085] The above results indicate that AABB material has strong callus induction ability and is suitable as a genetic transformation recipient. This transformation system shows good applicability in AABB, with high transformation efficiency, and in the successfully transformed plants, more than half achieved effective editing of the target site, indicating a satisfactory editing effect.

[0086] Table 12 Genetic transformation and gene editing efficiency of AABB

[0087] 9.2 Molecular identification of gene knockout plants Target site sequencing was performed on 20 T0 generation mutant lines. The results showed that the editing types of the lines differed. The editing of the A and B genomes in different lines could be divided into three categories: editing of only the B genome, editing of both the A and B genomes, and editing of only a single homologous chromosome.

[0088] sgRNA-1 ( SH4 The editing efficiency of gene knockout target site 1 was low, with editing events detected only on the B genome of three lines: 10, 11, and 29. Among them, 11 was a homozygous mutation (-2 / -2), while the other 17 lines were wild-type at this site.

[0089] sgRNA-2 ( SH4 The editing efficiency of gene knockout target 2 was significantly higher than that of sgRNA-1. A total of 19 lines were edited on the B genome, of which 11 were homozygous mutant lines. There were 9 lines whose A genome was not edited but whose B genome was homozygous mutant, numbered 1, 9, 11, 16, 19, 20, 25, 29 and 31.

[0090] sgRNA-3 ( LABA1 The editing efficiency of gene knockout target 1 is relatively high. Most strains have edits on both genomes. Only strains with edits on the B genome are strain 24 (homozygous mutation, -1 / -1) and strain 29 (heterozygous mutation, -1 / +2).

[0091] sgRNA-4 ( LABA1 The editing efficiency of gene knockout target 2 is similar to that of sgRNA-3, with slightly better editing effect. The strains with only B genome edited are 4 (+1 / +1), 10 (+1 / WT), 11 (+1 / +1) and 24 (-1 / +1).

[0092] sgRNA-5 ( Bh4 Gene knockout target 1) targets Bh4 The design of large-segment deletions in the A genome yielded the best results due to its high editing specificity. The A genome of all strains remained unedited. Eight strains, numbered 1, 3, 9, 11, 19, 20, 29, and 31, exhibited homozygous mutations in the B genome.

[0093] sgRNA-6 ( Bh4 The editing efficiency of gene knockout target 2 is relatively high, but the editing effect is not ideal. The vast majority of strains were edited on both genomes, and no strains with mutations only on the B genome were found.

[0094] When selecting target lines, priority should be given to lines whose genome A has not been edited and whose target gene in genome B has a homozygous mutation, followed by lines whose genome A has not been edited and whose genome B has a heterozygous mutation. In addition to meeting genotypic requirements, a comprehensive evaluation should be conducted based on field growth performance. Mutant regenerated seedlings should exhibit good growth vigor after transplanting, normal tillering, and no obvious developmental defects; lines exhibiting abnormalities such as stunted growth, few tillers, or yellowing leaves should not be selected.

[0095] Based on a combination of genotype and field performance, AABB was ultimately selected. CR-19, AABB CR -20 and AABB CR -31 was used as experimental material for subsequent phenotypic analysis.

[0096] SH4 Gene knockout mutant: AABB CR -19 (WT / WT, WT / WT, WT / WT, -CA / -CA), AABB CR -20 (WT / WT, WT / WT, WT / WT, -C / -C), AABB CR -31 (WT / WT, WT / WT, WT / WT, -C / -C).

[0097] LABA1 Gene knockout mutant: AABB CR -19 (+A / +A, +A / +A, -CT / -CTTG, +A+C / +A+C), AABB CR -20 (+A / +A, -C / -C, -C / -C, -C / -C) and AABB CR -31 (-C / -C, +A+A / +A+A, -C / -C, -C / -C).

[0098] Bh4 Gene knockout mutant: AABB CR -19 (WT / WT, +C / +C, WT / WT, WT / WT), AABB CR -20 (WT / WT, -A / -A, -C / -C, -C / -C) and AABB CR -31 (WT / WT, -A / -A, +C / -C, -C / -C).

[0099] 9.3 Analysis of target gene expression levels in different materials SH4 The expression trends of the gene in spotted wild rice and AABB wild-type were basically the same, with a significant increasing trend in expression levels from flowering to maturity. Figure 2 In cultivated rice, SH4 The expression levels of the three genes were low at all developmental stages. Compared with the wild type, the expression levels of the three genes were lower. SH4 Gene knockout line AABB CR -19, AABB CR -20 and AABB CR -31, the expression level of this gene was extremely low at all stages, almost undetectable, indicating that knocking out this gene has a highly significant impact on its expression level. This phenomenon may stem from the fact that the mutant transcript is recognized and degraded by the nonsense-mediated mRNA degradation pathway in the cell, resulting in a significant decrease in its homeostatic level.

[0100] LABA1 The expression trends of the gene in spotted wild rice and AABB were basically the same, with high expression levels from flowering to maturity and both showing a significant upward trend. Figure 3 In cultivated rice, LABA1 The expression levels of the gene were low at all developmental stages, significantly lower than those of spotted wild rice and AABB. Compared to the AABB wild type, the expression levels of the three genes were... LABA1 Gene knockout line AABB CR -19, AABB CR -20 and AABB CR -31 LABA1 The gene was expressed at certain levels at various stages, but the expression level was much lower than that of its wild type, indicating that knocking out the gene had a significant impact on its expression level.

[0101] Bh4 The expression trends of the gene in spotted wild rice and AABB wild-type were basically the same, with a significant increasing trend in expression levels from flowering to maturity. Figure 4 In cultivated rice, Bh4 Gene expression levels were low at all developmental stages. Compared to the AABB wild-type, expression levels were low in all three developmental stages. Bh4 Gene knockout mutant line AABB CR -19, AABB CR -20 and AABB CR In -31, the expression level of this gene was extremely low at all stages, almost undetectable, indicating that knocking out this gene has a very significant impact on its expression level.

[0102] 9.4 Morphological comparison of cultivated rice, spotted wild rice, AABB wild type and its mutants The results of the agronomic trait survey showed that ( Figure 5 (Table 12) Cultivated rice, spotted wild rice and AABB showed significant differences in several traits, while the three mutant lines of AABB showed consistent performance and were similar to the wild type of AABB.

[0103] Regarding plant height, cultivated rice in Wuhan and Hainan reached 89.26 cm and 63.42 cm, respectively, while spotted wild rice reached 149.65 cm and 70.13 cm, respectively. The AABB wild type reached 76.47 cm and 33.20 cm, respectively. Plant height in both regions fell between the two, but was significantly affected by the environment, with a marked decrease under Hainan cultivation conditions. The three AABB mutant lines in Hainan showed plant heights close to their wild type (33.20 cm), and their behavior was largely consistent.

[0104] Regarding panicle traits, the panicle length of the AABB wild type was 18.88 cm in Wuhan and 7.50 cm in Hainan. The average panicle length of the three mutant lines in Hainan was 7.30 cm, 7.60 cm, and 7.50 cm, respectively, all close to the wild type. In terms of panicle number, the AABB wild type had 17.67 panicles in Wuhan and 24.80 in Hainan. The average panicle number of the three mutant lines in Hainan was 27, 29, and 28, respectively, with a mean of 28, also comparable to the wild type. In comparison, cultivated rice had the longest panicle length (29.30 cm in Wuhan and 26.33 cm in Hainan) and the fewest panicles (8.90 in Wuhan and 9.67 in Hainan); spotted wild rice had the most panicles (42.80 in Wuhan and 33.00 in Hainan). In terms of seed setting rate, the AABB wild type had a seed setting rate of 49.87% in Wuhan and 21.50% in Hainan. The three mutant lines had seed setting rates of 5.40%, 5.68%, and 6.17% in Hainan, all below 7%, significantly lower than the wild type. Cultivated rice had the highest seed setting rate (89.45% in Wuhan and 87.86% in Hainan), followed by spotted wild rice (55.33% in Wuhan and 53.42% in Hainan).

[0105] In terms of grain characteristics, the AABB wild type had the widest grain width (2.98 mm in Wuhan, 2.96 mm in Hainan). The three mutant lines in Hainan had grain widths of 2.92 mm, 3.12 mm, and 2.98 mm, respectively, similar to the wild type. Regarding grain length, the AABB wild type had grains of 8.52 mm in Wuhan and 7.16 mm in Hainan, while the three mutant lines in Hainan had grain lengths of 6.82 mm, 6.92 mm, and 6.90 mm, essentially consistent with the wild type. Cultivated rice had the longest grain length (9.45 mm in Wuhan, 9.33 mm in Hainan) and the narrowest grain width (2.08 mm in Wuhan, 2.06 mm in Hainan). Spotted wild rice had the shortest grain length (5.10 mm in Wuhan, 5.05 mm in Hainan).

[0106] Regarding awn length, cultivated rice is awnless, while the awn length of spotted wild rice is 2.41-3.35 cm, with the AABB wild type having the longest awn (4.28-5.67 cm). The awn length of all three mutant lines was significantly shortened to 1.66-1.90 cm, approximately one-quarter that of the wild type, indicating... LABA1 Gene knockout effectively inhibited the elongation of awns.

[0107] Regarding the color of the hull, cultivated rice is yellow, spotted wild rice and the AABB wild type are black, and the three AABB mutant lines are all yellow, consistent with cultivated rice, confirming... Bh4 Gene knockout causes the glumes to turn from black to yellow.

[0108] Regarding grain shattering, cultivated rice showed no grain shattering, while spotted wild rice and the AABB wild type showed easy grain shattering. The three AABB mutant lines showed moderate grain shattering, with significantly reduced shattering compared to the wild type, indicating... SH4 Gene knockout effectively reduced schistosomes.

[0109] In summary, the simultaneous knockout of the three genes resulted in the AABB mutant lines maintaining a large number of grains and ears, while significantly shortening the awn length, changing the glumes from black to yellow, and significantly reducing grain shattering, thus significantly improving their cultivation level.

[0110] Table 13. Agronomic traits of cultivated rice, spotted wild rice, AABB wild type and its mutants.

[0111] 9.5 Morphological observation during spikelet development AABB wild type ( Figure 6 The development of awns begins in the late stage of spikelet differentiation, and its process is closely related to the developmental stage of the spikelet. During the differentiation of floret primordia, the awn has not yet begun to elongate; during the differentiation of secondary branches, the awn remains dormant and does not elongate significantly; after entering the pistil and stamen formation stage, the awn begins to elongate, and the apical cells of the lemma undergo active division and polar expansion, and the awn morphology begins to appear; by the pollen mother cell formation stage, the awn enters a rapid elongation period, and its length increases significantly; when the pollen mother cell meiosis stage is reached, the elongation of the awn reaches its peak and is basically completed. The LABA1 gene knockout mutant AABBCR-31 ( Figure 7 The awn development process of the mutant was basically consistent with that of the wild type, with no significant delay or advancement in the start time of each stage. During the meiotic division of pollen mother cells, the awn length of the mutant was observed to be slightly shorter than that of the wild type at the same stage, indicating that... LABA1 Gene knockout primarily affects the elongation of the awn, rather than initiating or advancing its developmental process.

[0112] 9.6 Measurement and Statistical Analysis of Mature Seed Awn Length Based on the morphological observation of mature seed awns, it was found that, in addition to cultivated rice, spotted wild rice, AABB wild type, and its three species... LABA1 All gene knockout mutants possessed awn structure. Among them, the AABB wild-type had the longest awn, followed by spotted wild rice. Compared with the AABB wild-type, the three... LABA1 Gene knockout mutant strain AABB CR -19, AABB CR -20 and AABB CR -31, the awn length of different mutant strains was significantly shortened, with the awn length of some mutant strains shortened to about one-quarter of that of the wild type. Figure 8 The above results indicate that... LABA1Gene knockout can effectively inhibit awn elongation, which is an effective strategy to shorten awn length.

[0113] from Figure 8 and Figure 9 It can be seen that cultivated rice exhibits awnless characteristics, while spotted wild rice, AABB, and others show similar characteristics. LABA1 All gene knockout mutants possessed awns, with the AABB mutant having the longest awns, averaging 4.28 cm; followed by spotted wild rice, averaging 2.41 cm. Compared to the AABB wild type, the three mutant types exhibited different characteristics. LABA1 Knockout strain AABB CR -19, AABB CR -20, AABB CR The awn length of all three strains (-31) was significantly shortened, with average values ​​of 1.66 cm, 1.64 cm, and 1.60 cm, respectively. Further statistical analysis showed highly significant differences in awn length among cultivated rice, spotted wild rice, AABB, and their mutant strains (P < 0.001). LABA1 There were no significant differences among gene knockout strains, indicating that awn elongation was significantly inhibited after knocking out the LABA1 gene, and this was consistent across different mutant lines.

[0114] 9.7 Microscopic observation of abscission layer cells The abscission layer is a special layer of cells that forms in the stem tissue during rice seed development. It is located in the stem tissue at the base of the rice grain, close to the junction of the spikelet peduncle and the grain. It begins to form and develop after flowering. The abscission layer cells are usually small in size and tightly packed, forming a clear contrast with the surrounding parenchyma tissue. These cells undergo cell wall degradation when the seed matures, causing the rice grain to separate from the stem. The degree of development of the abscission layer structure directly affects the rice's grain shattering properties.

[0115] In paraffin sections of mature seed abscission tissue, after staining with toluidine blue (e.g.) Figure 10 In the abscission layer, the cell walls and cytoplasm are stained blue-purple to purplish-red. Because the abscission layer cells are rich in pectin and polysaccharides, these acidic polysaccharides, when combined with toluidine blue, often exhibit a characteristic purplish-red color, contrasting sharply with the blue background of the surrounding tissue. In cultivated rice sections, the cells in the abscission layer show no clear boundary with the surrounding tissue, nor do they exhibit continuous fracture surfaces, indicating that the tissue structure remains intact at maturity. In sections of spotted wild rice and AABB, the abscission layer contains 1-2 layers or more of tightly packed small cells, forming a clear boundary with the surrounding parenchyma tissue; fracture surfaces formed by cell wall degradation are even visible, indicating cell separation. Three different mutation types... SH4 Knockout strain AABB CR -19, AABB CR -20, AABB CR-31 shows a blurred abscission structure. Compared to the wild type, the growth state of its abscission structure cells is closer to that of cultivated rice, and no continuous fracture surfaces are observed (e.g. Figure 10 (As shown).

[0116] 9.8 Statistics on natural grain loss rate Natural grain loss rate as follows: Figure 11 As shown, the natural grain shattering rate trends of different materials differed significantly from the day of heading to day 35. Specifically, the grain shattering rate of spotted wild rice and AABB showed a continuous upward trend with increasing days, with a sharp increase between day 14 and day 21; by day 35, the natural grain shattering rates of the two reached 84% and 31%, respectively. In contrast, cultivated rice showed almost no grain shattering throughout the entire statistical period, with a final grain shattering rate of only 1%; while AABB... CR -19, AABB CR -20, AABB CR -31 also exhibited no grain shattering under natural conditions, with a final grain shattering rate of less than 5%, showing a highly significant difference compared to spotted wild rice and the AABB wild type. Among these, AABB... CR The particle loss rate at -31 was even 0. The above results indicate that... SH4 The gene knockout significantly reduced the seed loss rate of the AABB mutant, transforming it from a seed-prone type to a seed-resistant type, thus effectively preventing seed loss before harvest.

[0117] 9.9 Measurement of delamination tensile force Separation tensile strength measurements showed that the spotted wild rice had the lowest grain breaking tensile strength (median 0.90 N < 2.94 N), indicating the weakest connection between the grain and the stem, consistent with its tendency to fall naturally, thus exhibiting the strongest shattering tendency. The AABB wild type had a tensile strength (median 1.07 N < 2.94 N) only slightly higher than the spotted wild rice, also showing a high shattering tendency. In contrast, cultivated rice had the highest separation tensile strength (median 3.46 N), the weakest shattering tendency, and the strongest connection between the grain and the stem. Knockout... SH4 After genes, AABB CR -19, AABB CR -20, AABB CR The median breaking tensile strength values ​​of -31 were 2.75 N, 2.92 N, and 2.95 N, respectively, all between those of cultivated rice and wild-type AABB, and significantly higher than those of AABB. The tensile strength values ​​of the three were very close and around 2.94 N, all showing moderate shattering tendency. Figure 12 The above results indicate that knocking out SH4 Genes can effectively enhance the connection strength between grains and branches, significantly reducing rice grain shattering.

[0118] 9.10 Dynamic changes in glume color during seed maturation During the seed maturation process, cultivated rice, spotted wild rice, AABB and its Bh4 In gene knockout mutants, the glumes are all bright green within 15 days after flowering. Figure 13 Fifteen days after flowering, the color of the glumes of each material began to change systematically. The glumes of cultivated rice gradually changed from bright green to yellow; the glumes of spotted wild rice and AABB rice developed dark brown spots, gradually changing from bright green to brownish-black, with the black color of spotted wild rice being significantly deeper than that of AABB rice; while... Bh4 Gene knockout mutant strain AABB CR -19 and AABB CR The glum color change of -31 differs from that of the AABB wild type. Whether it's a line with only the B genome edited, or a line with both A and B genomes edited and containing heterozygous mutations, the color changes from bright green to yellow. These results indicate that... Bh4 Gene knockout altered the final appearance of the glume color, changing the mutant glume color from the wild-type brownish-black to yellow, confirming that... Bh4 Genes play a key role in the accumulation of pigments in rice husks.

[0119] 9.11 Analysis of the temporal variation of pigment content in glumes Chlorophyll is the core pigment in photosynthesis, responsible for absorbing and converting light energy. In the early heading stage, the glumes are bright green, indicating high photosynthetic activity and supplementing energy supply during the early grain-filling stage. As grain filling progresses, chlorophyll in the glumes gradually degrades, and the nitrogen within is remobilized and transported to the grains for protein synthesis. From the heading stage to full seed maturity, the chlorophyll content in rice glumes generally shows a continuous downward trend, reflecting the natural senescence of the glumes as the grains mature. This applies to cultivated rice, spotted wild rice, AABB rice, and their three... Bh4 Gene knockout mutant strain AABB CR -19, AABB CR -20 and AABB CR -31, all showed the same pattern of change ( Figure 14 This indicates that the B genome or the A and B genomes are in... Bh4 Gene knockout did not have a significant impact on the dynamic changes in chlorophyll content in glumes.

[0120] The changes in carotenoid content in rice husks are closely related to chlorophyll content, and generally show a continuous downward trend. During the heading and flowering stage, carotenoids mainly participate in light energy capture as auxiliary photosynthetic pigments and effectively prevent chlorophyll photo-oxidation through photoprotection. As the grains fill and mature, the synthesis rate of carotenoids in the husks gradually falls below the degradation rate, resulting in a steady downward trend in their content, but the overall change is relatively gradual. Cultivated rice, spotted wild rice, AABB, and other varieties exhibit similar trends. Bh4 Gene knockout mutant strain AABB CR -19, AABB CR -20 and AABB CR -31 showed a consistent pattern in the variation of carotenoid content. Figure 15 ),show Bh4 Gene knockout did not significantly affect the dynamic changes in carotenoid content in glumes.

[0121] Melanin strengthens cell walls, forming a physical barrier to protect cells. Its strong antioxidant activity scavenge free radicals, preventing oxidative damage and helping plants resist abiotic stresses such as ultraviolet radiation, drought, and extreme temperatures. Bh4 The gene functions normally, and the amino acid transporter it encodes can specifically transport tyrosine, thereby driving the synthesis and accumulation of melanin in the glumes, and the melanin content continues to increase. Figure 16 This gives the glumes a dark brown color. In cultivated rice and AABB rice... Bh4 Gene knockout mutant strain AABB CR -19, AABB CR -20 and AABB CR In -31, due to Bh4 The gene function is lost, and the corresponding tyrosine transporter cannot be expressed. The metabolic flow of tyrosine is diverted to other pathways, which eventually causes the color of the husk to change from black to yellow.

[0122] Anthocyanins are a type of flavonoid compound that are mainly produced in rice glumes through flavonoid biosynthesis. Their accumulation mainly occurs during the milk and waxy stages, helping seeds resist ultraviolet damage, protecting photosynthetic pigments such as chlorophyll from photo-oxidation, and enhancing rice's adaptability to abiotic stresses such as high temperature and drought by regulating oxidative stress. Bh4 The gene-mediated melanin synthesis pathway and the anthocyanin synthesis pathway compete for the upstream substrate phenylalanine, which can be converted into tyrosine under the catalysis of phenylalanine hydroxylase. For example... Figure 17 As shown, spotted wild rice and AABB Bh4 The gene function is complete, and tyrosine is efficiently transported to the melanin synthesis pathway, promoting melanin accumulation and giving the glumes a dark brown color. This is observed in cultivated rice and... Bh4 Gene knockout AABB CR -19, AABB CR -20 and AABB CR In -31, due to Bh4 The loss of gene function alters the metabolic pathway of phenylalanine, allowing more substrates to enter the anthocyanin synthesis pathway, ultimately resulting in the yellow color of the glumes due to carotenoids and flavonoids. AABB CR The -19 mutation occurs only in the B genome, preserving the integrity of the A genome. The anthocyanin content and its changing trends in the glumes of this mutant line are more similar to those of cultivated rice than other mutant lines. This phenotypic change is related to the changes in cultivated rice during domestication. Bh4 The phenomenon of natural gene deletion causing the glumes to turn from black to yellow is highly consistent, further revealing... Bh4 Genes are a key factor that influences glume color by regulating the metabolic pathway of phenylalanine, and the loss of their function is an important cause of phenotypic changes.

[0123] In summary, this invention constructs a dual-target vector that simultaneously targets three genes, obtaining lines with simultaneous mutations in multiple genes through a single genetic transformation, with lines having two or more gene edits accounting for more than 50%. Phenotypic analysis shows that... OsSH4 Knockout reduced the natural grain loss rate from 31% to below 5%; OsLABA1 Knocking out the awn shortened its length from 4.28cm to approximately 1.60cm. OsBh4 Knocking out the husks changed their color from dark brown to yellow. Simultaneous improvement of these traits significantly enhanced the cultivability of AABB while maintaining its original high biomass and excellent grain structure.

[0124] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for domesticating artificial allotetraploid rice AABB based on gene editing, wherein the artificial allotetraploid rice AABB is cultivated rice. O.sativa with spotted wild rice O.punctata This is obtained by hybridization of the parent lines followed by chromosome doubling technology, the method including knocking out or inhibiting chromosomes derived from spotted wild rice. O.punctata of SH4 , LABA1 and Bh4 Gene.

2. The domestication method according to claim 1, wherein the knockout is performed by transfecting a three-gene dual-target vector.

3. The domestication method according to claim 2, wherein the target sequence of the three-gene dual-target vector is the nucleotide sequence of SEQ ID NO. 1~6.

4. The domestication method according to claim 3, wherein the three-gene dual-target vector comprises: Six sgRNA units, t1-t6, designed specifically for the BB genome; Insert tRNA sequences between sgRNA units; hyg gene; pBR322 ori and pVS1 rep / sta.

5. The domestication method according to any one of claims 2-4, wherein the three-gene dual-target vector is transfected into the callus of artificial allotetraploid rice AABB by Agrobacterium.

6. The domestication method according to claim 1, the method further includes a step of identifying gene knockout, wherein the identification step uses primers of SEQ ID NO. 7~12.

7. The domestication method according to claim 1, wherein the strain obtained by the domestication method has a natural grain shattering rate of less than 6%, an awn length of less than 2 cm, and a glume color of yellow.

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