Drought-tolerant rice breeding and cultivation method for strengthening root development

Abstract

The invention discloses a drought-enduring rice breeding and cultivation method for strengthening root development, relates to the technical field of rice breeding and cultivation, and aims to solve the problem of poor cultivation effect of drought-enduring rice. According to the method, target genes are introduced through molecular marker-assisted backcross by utilizing gene polymerization, key genes are confirmed through instantaneous silencing before gene editing, target specificity is verified, gradient stress and single plant collaborative scoring are synchronously identified, characters are accurately polymerized, the off-target risk is reduced, stable collaboration of plant root systems and drought tolerance is ensured, and the drought tolerance is improved. The dynamic microenvironment adapts to different growth stages of plants, gradient drought stress training is carried out in stages, drought tolerance, yield, agronomic traits and quality are evaluated and considered, cooperation of cultivation and breeding is achieved, drought tolerance is enhanced, comprehensive performance of varieties is guaranteed, and actual production requirements are met.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of rice breeding and cultivation, in particular to a drought-tolerant rice breeding and cultivation method for strengthening root system development. BACKGROUND

[0002] In the existing drought-tolerant breeding of rice, germplasm screening mainly relies on a single index, and no clear correlation between root system development and drought tolerance is established, resulting in insufficient targeting of the screened materials. The dimension of root system phenotype analysis is single, and it is difficult to accurately quantify morphological and functional characteristics. Moreover, there is a lack of molecular markers closely linked to deep root and drought tolerance traits, and the breeding efficiency is low. At the same time, the genetic background is not accurately introduced during gene aggregation, the combination of microenvironment regulation and drought stress training during the cultivation stage is loose, and the evaluation of breeding does not take into account drought tolerance, yield and quality, so the practical performance of the final variety is limited. SUMMARY

[0003] The present application relates to the field of rice breeding and cultivation, in particular to a drought-tolerant rice breeding and cultivation method for strengthening root system development.

[0004] To achieve the above-mentioned purpose, the present application provides the following technical scheme: A drought-tolerant rice breeding and cultivation method for strengthening root system development, comprising the following steps: S1: Basic germplasm material screening: screening basic germplasm materials with correlated traits of strengthening root system development and drought tolerance from existing gene banks; Among them, the core indicators of root system development and drought tolerance are first confirmed; S2: Root system phenotype analysis: image analysis of the morphological traits of the root system of the basic germplasm material; S3: Development of gene molecular markers: according to the image analysis results of the basic germplasm material, new molecular markers are developed for the close linkage of drought tolerance and deep root traits of the basic germplasm material; Among them, according to the core indicators of root system development and drought tolerance, the corresponding indicator data in the image analysis results of the basic germplasm material are confirmed, and the phenotype data of the basic germplasm material are obtained after confirmation; The core indicators of root system development and drought tolerance in the phenotype data are subjected to genomic DNA extraction, and a DNA mixed pool is constructed by equal mixing method; After the DNA mixed pool is constructed, the polymorphic marker sites are screened, and the screening process is as follows: S301: DNA sequencing and analysis: whole genome resequencing or simplified genome sequencing is performed on the two gene pools in the DNA mixed pool to obtain single nucleotide polymorphism and insertion and deletion variation information at the genome level; S302: Polymorphic marker screening: through bioinformatics analysis, the sequence differences between the two gene pools in the DNA mixed pool are compared, and the variation sites that appear frequently in the deep root drought tolerance gene pool and rarely or not in the shallow root sensitive gene pool are screened out, and the candidate marker region is determined according to the variation site; S303: Marker site annotation: the candidate marker site is located on the rice reference genome, the rice reference genome is extracted from the gene database, the gene function of the site region is analyzed through the gene annotation database, and the region located in the internal or upstream and downstream regulation region of the root development and water stress response related gene is marked; S4: Gene aggregation breeding: according to the new type of molecular marker result, the genes controlling drought tolerance and deep root traits are aggregated in the same genetic background, and the target strain is obtained after the aggregation is completed; Among them, the donor parent is screened from the new type of molecular marker result, and the donor parent includes a deep root trait donor parent and a drought tolerance donor parent; The recipient parent is then confirmed, and the recipient parent is a rice variety with excellent agronomic traits but weak root development and poor drought tolerance.

[0005] Preferably, the basic germplasm material with enhanced root development and drought tolerance related traits is screened from the existing gene library, including: The core indicators of root development include deep root proportion, root-shoot ratio, total root length, root surface area, root hair density and root vessel diameter; the core indicators of drought tolerance include leaf water retention rate, drought recovery rate and seed setting rate; Based on the confirmed core indicators, targeted retrieval is performed from the germplasm material information of the existing rice gene library, and the germplasm material that does not meet the indicators is excluded after retrieval; The germplasm material that meets the indicators is cultured at the germination stage, and the germination stage culture process includes culture condition setting, root trait observation and pre-screening elimination; The germplasm material screened after germination stage culture is transplanted to a greenhouse pot, simulating field drought conditions, while observing root development traits and drought tolerance phenotypes, and establishing the correlation between the two; After the correlation between root development traits and drought tolerance phenotypes is established, the final basic germplasm material is obtained.

[0006] Preferably, the image analysis of the morphological traits of the root system of the basic germplasm material includes: Before the morphological traits of the root system of the basic germplasm material are analyzed by image analysis, the root system is subjected to standardization treatment, which comprises the following steps: first, selecting the basic germplasm material in the growth period, using the stratified root extraction method to excavate the selected basic germplasm material, then immersing the excavated root system in a phosphoric acid buffer solution containing 2% formaldehyde for 24 hours, and finally grouping the root system according to the germplasm material number, growth period and treatment group; The root system of the basic germplasm material subjected to the standardization treatment is subjected to multi-dimensional image automatic acquisition, wherein the acquisition equipment comprises a visible light camera, a near-infrared camera and a laser scanning device. The visible light camera captures the morphological contour and branching structure of the root system by shooting two-dimensional images of the root system from the top, front, back, left and right five fixed angles. The near-infrared camera synchronously acquires near-infrared images, and distinguishes between live roots and dead roots by using the difference in near-infrared absorption between the root system and water. The laser scanning device scans the point cloud of the root system, and records the three-dimensional coordinate information of the root system surface. After the multi-dimensional image acquisition is completed, image preprocessing is performed, which comprises denoising processing, contrast enhancement and image segmentation of the multi-dimensional images; After the image preprocessing, three-dimensional model reconstruction is performed, and the three-dimensional model reconstruction process is as follows: S201: Feature point matching: identifying the key feature points of the root system in the multi-dimensional images subjected to the image preprocessing, and calculating the spatial coordinates of the points by using a stereo matching algorithm; S202: Point cloud fusion: fusing the point cloud data acquired by the laser scanning with the coordinates of the identified key feature points, to generate a three-dimensional point cloud model containing the details of the root system surface; S203: Grid construction: performing grid processing on the generated three-dimensional point cloud model, to generate a continuous root system surface model; Extracting the root system trait parameters from the reconstructed three-dimensional model, wherein the root system trait parameters comprise morphological parameters and functional parameters, the morphological parameters comprise length parameters, area and volume parameters and depth parameters, and the functional parameters comprise topological structure, root hair characteristics and spatial distribution; Finally, the image analysis of the morphological traits is completed.

[0007] Preferably, the new molecular marker for the close linkage between the drought tolerance and deep root traits of the basic germplasm material of rice is obtained according to the image analysis results of the basic germplasm material, and the new molecular marker comprises the following steps: The DNA mixed pool comprises a deep root drought tolerance gene pool and a shallow root sensitive gene pool; The marker region is verified by population association analysis, and the marker verification is as follows: 1-2 pairs of hybrid combinations are selected in the marker region, and a separation population or a recombinant inbred line population is constructed according to the hybrid combinations, the separation population is subjected to root image analysis and drought tolerance identification, and the single plant is subjected to genotype detection by using the marker region, finally, interval mapping method is used to calculate the linkage exchange rate and correlation coefficient of the candidate marker and the target trait, and the marker site with a logarithmic odds ratio ≥ 3.0 and a phenotypic explanation rate ≥ 10% is screened out; Finally, the screened marker site is subjected to specific primer design and standardization treatment, and a new type of molecular marker is obtained after specific primer design and standardization treatment.

[0008] Preferably, according to the results of the new type of molecular marker, the genes controlling drought tolerance and deep root traits are aggregated into the same genetic background, comprising: The two parents in the donor parent are crossed, and the F1 hybrid is obtained after crossing, and the F1 hybrid is used as the father or mother to cross with the recipient parent, and the backcross generation is obtained after backcrossing, and the backcross generation is introduced into the genetic background of the recipient parent; The backcross generation is subjected to new type of molecular marker detection, and the backcross generation with completed new type of molecular marker detection is subjected to second backcrossing with the recipient parent to obtain backcross generation two, and the backcross generation two is subjected to new type of molecular marker detection; The backcross generation two is subjected to third backcrossing with the recipient parent to obtain backcross generation three; The backcross generation three is selfed to obtain selfed generation two, and the selfed generation two is planted into strains, and 10-15 strains are randomly selected from each strain for new type of molecular marker detection; The unstable strains with a trait variation coefficient > 10% in the selfed generation two are removed, and the stable strains with a variation coefficient ≤ 5% are retained after removal, and the stable strains are labeled as selfed generation three; Finally, the selfed generation three is selfed, and the selfed generation four is obtained after selfing, and the selfed generation four is subjected to molecular marker rechecking, phenotype rechecking and comprehensive evaluation of agronomic traits; According to the results of molecular marker rechecking, phenotype rechecking and comprehensive evaluation of agronomic traits, the final strain is screened out, and the final strain is the target strain.

[0009] Preferably, it further comprises: According to the root development core index and the drought tolerance core index, the core evaluation index of the root trait and the drought tolerance is confirmed; The target strains are grouped according to the number, each group containing 30-50 rice seedlings, and the 30-50 rice seedlings are set as control group and positive control group; The rice seedlings with control group and positive control group are planted by greenhouse potting, and the rice seedlings after planting are subjected to stress treatment and trait synchronous identification; The stress treatment and trait synchronous identification process is: S501: mild drought stress identification: this stage is stress 1 period, lasting for 15 days, the soil water content is reduced to 40%-50% of the maximum field water holding capacity, and the root system traits are measured every 5 days, and the drought tolerance index is measured every 7 days during the period; S502: moderate drought stress identification: this stage is stress 2 period, lasting for 20 days, for the mild drought stress identification qualified strain, the soil water content is further reduced to 25%-35% of the maximum field water holding capacity, and the root system surface area, root hair density and root vessel diameter are measured every 7 days during the period; drought recovery rate and seed setting rate are measured every 10 days; S503: recovery growth verification: this stage is recovery period, lasting for 15 days, for the moderate drought stress identification qualified strain, the normal irrigation is restored, and the root regeneration capacity and leaf recovery rate are measured during the period, and the root system core index and drought tolerance core index are measured again; The rice seedlings qualified by the recovery growth verification are subjected to single plant level correlation detection, the single plant level correlation detection is that the root system traits and drought tolerance index of the rice seedlings qualified by the recovery growth verification are measured, and the synergistic comprehensive score of each rice seedling is calculated; Finally, the excellent strain is screened according to the calculation result.

[0010] Preferably, it also includes: Identifying key genes related to root system development and drought tolerance from the excellent strain; Using virus-induced gene silencing technology, the key genes are subjected to transient silencing, and the transient silencing process is: constructing a gene silencing vector, transforming the excellent strain seedlings by agrobacterium mediation, measuring the root system traits and drought tolerance index of the plants after silencing; if the target traits decrease after silencing, it is confirmed that the gene is a key gene, and is included in the genome editing range; The key genes subjected to transient silencing are subjected to editing target point design, and the editing target point design includes target point position selection and target point specificity verification, wherein the target point position selection is: selecting a key functional domain of a gene coding region, or a core regulatory element of a gene promoter region; the target point specificity verification is: through local alignment search tool, the whole genome of rice is aligned to ensure that the target point sequence is uniquely matched in the rice genome; at the same time, a target point specificity detection primer is designed; After the editing target point design is completed, the editing vector construction is carried out, and the editing vector construction process is: S601: vector skeleton selection: using a binary vector for rice transformation, the binary vector includes a Cas9 protein coding gene, a target sgRNA expression box and a selection marker gene; S602: sgRNA expression cassette construction: according to the target sequence, synthesize sgRNA oligonucleotide primers, connect with the gene promoter in the vector skeleton, form sgRNA expression unit; S603: vector assembly and verification: insert sgRNA expression unit, Cas9 protein coding gene and screening marker gene into vector skeleton in turn, verify the correctness of vector construction by restriction endonuclease; According to the completed editing vector, the cells of the excellent strain are introduced into the cells of the excellent strain, and the introduction method includes Agrobacterium-mediated transformation or gene gun method; After the introduction is completed, the cells are transferred to the selection medium, and the successfully transformed cell groups are screened out; The screened transformed cells are transferred to the differentiation medium to induce the formation of regenerated seedlings, and after the regenerated seedlings are transplanted to the soil, genomic DNA is extracted, and editing events are detected by polymerase chain reaction and sequencing technology, and it is confirmed whether the target gene site is modified as expected, and individuals that are not edited or off-target are excluded; Finally, the edited rice seedlings are verified at the phenotype and molecular level, and the modification and regulation of the key genes in the excellent strain are completed after verification.

[0011] Preferably, it also includes: According to the root characteristics and drought tolerance requirements of the modified and regulated excellent strain, the screening index of the microbial inoculant is confirmed, and the screening index includes functional index, synergistic index and safety index; According to the screening index, the microbial inoculants in the inoculant library are screened, and the screened inoculants are purified and expanded, and the final microbial inoculants are obtained after purification and expansion; The microenvironment construction process is: first, the soil texture of the planting area is improved, then the improved soil is pasteurized, the pasteurized soil is inoculated with microbial inoculants, and the soil is incubated at room temperature for 7 days for inoculant activation, so that the concentration of the inoculant in the soil reaches 10 6 -10 7 CFU / g; The rice seedlings of the excellent strain are subjected to root soaking treatment, and after the root soaking treatment is completed, the rice seedlings are planted in the constructed microenvironment, and the microenvironment during planting is dynamically regulated; The dynamic regulation time includes: microenvironment regulation during planting period: 1-15 days after planting; microenvironment regulation during growth period: 16-60 days after planting; microenvironment strengthening during adversity period: drought stress training period; After dynamic regulation, the construction effect of the microenvironment is evaluated, including short-term effect evaluation and long-term effect evaluation, the short-term effect evaluation is 30 days after construction, and the long-term effect evaluation is 60-90 days after construction; Finally, the microenvironment is optimized according to the evaluation results.

[0012] Preferably, it also includes: Before the drought stress training, the training object is confirmed, and the training object is selected as the excellent strain plant which grows well in the micro environment and is in the tillering peak period to the initial stage of the embryo; After the training object is confirmed, the drought stress training in the early cultivation period is carried out, and the drought stress training process in the early cultivation period is as follows: S8011: Mild stress adaptation: stress intensity regulation and micro environment coordination management are carried out on the training object on the 1st-7th day; S8012: Moderate stress induction: stress intensity regulation and physiological regulation assistance are carried out on the training object on the 8th-14th day; S8013: Stress recovery and consolidation: recovery irrigation and response effect evaluation are carried out on the training object on the 15th-21st day; The drought stress training in the breeding middle period is carried out on the training object, and the drought stress training process in the breeding middle period is as follows: S8021: Progressive drought initiation: stress intensity regulation and functional strengthening of the agent are carried out on the training object on the 1st-10th day; S8022: Continuous drought strengthening: stress intensity regulation and soil environment management are carried out on the training object on the 11th-21st day; S8023: Drought and rehydration cycle training: cycle regulation and root protection are carried out on the training object on the 22nd-28th day; The index data in the drought stress training process are monitored in real time, and the index data include soil index, drought stress training and agent index; Finally, the drought stress training in the breeding middle period and the early cultivation period of the excellent strain is completed.

[0013] Preferably, it also includes: First, the selection and breeding standard is formulated, and the selection and breeding standard includes survival bottom line index, physiological drought tolerance index, growth maintenance index and recovery capacity index; According to the selection and breeding standard, the drought-tolerant rice which meets the drought tolerance standard is repeatedly verified, and the drought-tolerant rice which passes the repeated verification of drought tolerance is subjected to yield experiment, and the evaluation standard of the yield experiment is the potential yield, yield stability and drought resistance index of the rice; The agronomic characters of the rice in the whole growth period are evaluated, and the evaluation standard is plant type structure, lodging resistance, growth period and ear character; Then, the quality of the rice is analyzed, and the quality analysis standard is processing quality, appearance quality and taste quality; Finally, according to the results of repeated verification of drought tolerance, yield experiment, evaluation of agronomic characters and quality analysis, the comprehensive performance evaluation results of the selected candidate varieties are obtained.

[0014] Compared with the prior art, the application has the following beneficial effects: 1. The application provides a drought-tolerant rice breeding and cultivation method for strengthening root system development, which comprises the following steps:

[0015] 2. The application provides a drought-tolerant rice breeding and cultivation method for strengthening root system development, which comprises the following steps:

[0016] 3. The application provides a drought-tolerant rice breeding and cultivation method for strengthening root system development, which comprises the following steps: BRIEF DESCRIPTION OF DRAWINGS

[0017] Fig. 1 Figure 1 is a schematic diagram of the drought-tolerant rice breeding and cultivation steps of the application; Fig. 2 Figure 2 is a schematic diagram of the drought stress training steps in the early cultivation stage of the application; Fig. 3 Figure 3 is a schematic diagram of the drought stress training steps in the middle breeding stage of the application. DETAILED DESCRIPTION

[0018] The technical solutions in the embodiments of the application will be clearly and completely described below with reference to the drawings in the embodiments of the application. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments of the application. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the application.

[0019] Please refer to Figs. 1-3 The technical solutions provided in the embodiments are as follows: The application provides a drought-tolerant rice breeding and cultivation method for strengthening root system development, which comprises the following steps: S1: basic germplasm material screening: screening basic germplasm materials with drought tolerance and root system development related traits from existing gene banks; S2: root phenotype analysis: image analysis of the morphological traits of the root system of the basic germplasm materials; S3: Gene molecular marker development: According to the image analysis results of the basic germplasm materials, new molecular markers are developed for the close linkage of drought tolerance and deep root traits of rice in the basic germplasm materials; S4: Gene pyramiding breeding: According to the results of new molecular markers, genes controlling drought tolerance and deep root traits are pyramided into the same genetic background, and the target line is obtained after pyramiding; S5: Synchronous identification: The line with root traits and drought tolerance in the target line is identified, and the excellent line is obtained after identification; S6: Gene regulation and editing: Through genome editing technology, the key genes of the excellent line are modified and regulated; S7: Microenvironment construction: Microbial inoculants are screened, and the excellent line with modification and regulation is constructed after screening; S8: Cultivation dynamic control: According to the constructed microenvironment, drought stress training is carried out on the excellent line in the breeding and early cultivation stages; S9: Rice breeding evaluation: According to the results of drought stress training, rice that meets the drought tolerance standard is selected, and the comprehensive performance of the selected candidate varieties is evaluated.

[0020] Screening of basic germplasm materials related to root development and drought tolerance from existing gene banks, including: First, the core indicators of root development and drought tolerance are confirmed; The core indicators of root development include deep root ratio, root-shoot ratio, total root length, root surface area, root hair density and root vessel diameter; the core indicators of drought tolerance include leaf water retention rate, drought recovery rate and seed setting rate; Based on the confirmed core indicators, targeted retrieval is carried out from the germplasm material information of the existing rice gene bank, and the germplasm materials that do not meet the indicators are excluded after retrieval; The germplasm materials that meet the indicators are cultured in the germination stage, and the germination stage culture process includes culture condition setting, root trait observation and pre-screening elimination; The germplasm materials screened in the germination stage are transplanted to the greenhouse potting, simulating the field drought conditions, and observing the root development traits and drought phenotype, establishing their correlation; After the correlation between root development traits and drought phenotype is established, the final basic germplasm material is obtained.

[0021] Specifically, the root development index covers depth, volume, absorption efficiency and transport capacity, and completely covers the key characteristics of root water absorption and water transport; the drought tolerance index combines physiology, recovery ability and yield, reflects not only the drought tolerance mechanism, but also the practical application value of the material, avoids the screening deviation caused by a single index, excludes materials that do not meet the index through gene library information retrieval, reduces the sample size of subsequent cultivation from the source; and then eliminates materials with poor early-stage traits through pre-screening in the germination stage, avoids invalid investment in greenhouse potting, greatly saves manpower, material resources and time cost, improves screening efficiency, and simulates field drought conditions in the greenhouse potting, rather than relying only on the controllable environment in the laboratory, which can more truly reflect the performance of germplasm materials under natural drought stress, reduce the problem of laboratory compliance but field failure, make the screened materials more meet the actual production needs, and through the correlation between root development traits and drought tolerance phenotype, not only basic germplasm materials are screened, but also which root traits play a key role in drought tolerance are determined, providing a theoretical basis for subsequent gene positioning and molecular breeding, and extending the application scenarios of screening results.

[0022] The morphological traits of the root system of the basic germplasm material are analyzed by image analysis, including: Before the image analysis of the morphological traits of the root system of the basic germplasm material, the root system is standardized, and the standardization processing is as follows: first, select the basic germplasm material in the growth period, use the layered root extraction method to excavate the selected basic germplasm material, then immerse the excavated root system in a phosphate buffer solution containing 2% formaldehyde for 24 hours, and finally group the root system according to the germplasm material number, growth period and treatment group; The root system of the basic germplasm material after standardization processing is collected by multi-dimensional image automatic collection, wherein the collection equipment includes a visible light camera, a near-infrared camera and a laser scanning device, the visible light camera captures the morphological contour and branch structure of the root system by shooting two-dimensional images from the top, front, back, left and right five fixed angles, the near-infrared camera synchronously collects near-infrared images, and the laser scanning device records the three-dimensional coordinate information of the root surface by point cloud scanning; After the multi-dimensional image collection is completed, image preprocessing is performed, and the image preprocessing includes denoising processing, contrast enhancement and image segmentation of the multi-dimensional image; After the image preprocessing, three-dimensional model reconstruction is performed, and the three-dimensional model reconstruction process is as follows: S201: Feature point matching: identifying key feature points of the root system in the multi-dimensional image after image preprocessing, and calculating the spatial coordinates of the points by a stereo matching algorithm; S202: Point cloud fusion: fusing the point cloud data obtained by laser scanning and the coordinates of the identified key feature points to generate a three-dimensional point cloud model containing the details of the root surface; S203: mesh construction: meshing the generated three-dimensional point cloud model to generate a continuous root system surface model; extracting root system trait parameters from the reconstructed three-dimensional model, the root system trait parameters including morphological parameters and functional parameters, the morphological parameters including length parameters, area and volume parameters, and depth parameters; the functional parameters including topological structure, root hair characteristics, and spatial distribution; Finally, the image analysis of morphological traits is completed.

[0023] Specifically, the layered root extraction method is used to reduce root excavation damage, 2% formaldehyde phosphate buffer is used for fixation to avoid sample degradation, grouping according to number, growth period and treatment group ensures traceability, effectively eliminates the interference of sample preparation differences on the analysis results, and improves the data comparability. The visible light camera captures two-dimensional morphology and branching structure, the near-infrared camera distinguishes between live roots and dead roots by using water absorption difference, and the laser scanning obtains three-dimensional coordinates, breaking through the limitation of single imaging, covering not only morphological contour but also physiological activity and spatial position information, providing rich data sources for subsequent analysis. Image preprocessing eliminates environmental interference and enhances root feature signals through denoising, contrast enhancement and segmentation; in three-dimensional reconstruction, feature point matching accurately calculates spatial coordinates, point cloud fusion integrates laser data and image features, mesh construction generates a continuous surface model, and layer-by-layer progression realizes accurate conversion of two-dimensional information to three-dimensional structure, avoiding parameter extraction bias. The extracted morphological parameters such as length and area reflect the basic characteristics of root system, and the functional parameters such as topological structure and root hair characteristics are related to the water and nutrient absorption capacity of root system. Not only the morphological analysis is completed, but also the data support for exploring the correlation between root morphology and physiological function is provided, meeting the research needs of multiple scenes such as breeding and cultivation.

[0024] According to the image analysis results of the basic germplasm material, new type molecular markers for the close linkage of drought tolerance and deep root traits of rice in the basic germplasm material are obtained, including: Among them, according to the core indicators of root development and the core indicators of drought tolerance, the corresponding index data in the image analysis results of the basic germplasm material are confirmed, and the phenotype data of the basic germplasm material are obtained after confirmation; The core indicators of root development and the core indicators of drought tolerance in the phenotype data are subjected to genome DNA extraction, and the DNA mixed pool is constructed by using the equal mixing method. The DNA mixed pool includes a deep root drought tolerance gene pool and a shallow root sensitive gene pool; After the DNA mixed pool is constructed, polymorphic marker sites are screened, and the screening process is as follows: S301: DNA sequencing and analysis: whole genome resequencing or simplified genome sequencing is performed on the two gene pools in the DNA mixed pool to obtain variation information of single nucleotide polymorphism and insertion and deletion at the genome level; S302: Polymorphic marker screening: by bioinformatics analysis, comparing the sequence differences of the two gene pools in the DNA mixed pool, and screening the variation sites that appear frequently in the deep-rooted drought-resistant gene pool and rarely or not in the shallow-rooted sensitive gene pool, and determining the candidate marker region according to the variation sites; S303: Marker site annotation: locating the candidate marker site to the rice reference genome, extracting the rice reference genome from the gene database, analyzing the gene function of the site region through the gene annotation database, and marking the region located in the internal or upstream and downstream regulation region of the root development and water stress response related gene; The marker region is verified by marker point using population association analysis. The marker point verification is: first, select 1-2 pairs of hybrid combinations in the marker region, construct a segregation population or a recombinant inbred line population according to the hybrid combination, analyze the root image and identify the drought resistance of the constructed segregation population, and at the same time, detect the genotype of a single plant using the marker region. Finally, the interval mapping method is used to calculate the linkage exchange rate and correlation coefficient of the candidate marker and the target trait, and the marker site with a logarithmic odds ratio value ≥ 3.0 and a phenotype explanation rate ≥ 10% is screened out. Finally, the screened marker point site is designed and standardized by specific primers. The specific primer design and standardization process obtains a new type of molecular marker.

[0025] Specifically, the phenotype data directly connects the core indicators of root development and drought resistance, and is derived from the previous standardized image analysis to avoid phenotype recording bias. Based on this, a gene pool is constructed to ensure that the subsequent molecular marker development always focuses on the target traits of deep root and drought resistance, eliminates the misplacement of phenotype and genotype, improves the accuracy of association analysis, enriches the target trait related variations by constructing two extreme trait gene pools of deep root drought resistance and shallow root sensitivity, reduces the redundant data and cost of single sequencing; compared with whole population sequencing, the sequencing amount is greatly reduced while the target variation signal is strengthened, the efficiency of subsequent polymorphism screening is improved, the comprehensive variation is obtained by combining whole genome and reduced genome sequencing, the specific sites are screened by comparing the sequence differences of the two pools, and then anchored to the rice reference genome, focusing on the root development and water stress response related gene region by combining functional annotation, avoiding invalid markers caused by random screening, improving the association of candidate markers and target traits, verifying by F2 segregation population or recombinant inbred lines, simultaneously carrying out phenotype identification and genotype detection, calculating the linkage exchange rate and phenotype explanation rate by interval mapping, and setting a clear threshold to effectively exclude false positive markers, ensuring the stability and effectiveness of the final marker. The final marker is designed and standardized by specific primers, which can be directly used for rapid detection of target materials in subsequent breeding without complex pretreatment, reducing the application threshold and providing an efficient tool for large-scale breeding.

[0026] In order to solve the problems of low gene aggregation efficiency, inaccurate genetic background introduction, lack of root system and drought tolerance collaborative identification, low gene editing target specificity and high off-target risk, and difficulty in efficient breeding of excellent strains with target traits in the prior art, please refer to Figs. 1-3 The embodiment provides the following technical solutions: According to the new molecular marker results, the genes controlling the drought tolerance and deep root traits are aggregated in the same genetic background, including: The donor parents are screened from the new molecular marker results, and the donor parents include deep root trait donor parents and drought tolerance donor parents; The recipient parent is confirmed, and the recipient parent is a rice variety with excellent agronomic traits but weak root development and poor drought tolerance; The two parents in the donor parent are crossed, and the F1 hybrid is obtained after crossing, and the F1 hybrid is used as a male parent or a female parent to backcross with the recipient parent, and the backcross generation is obtained after backcrossing, and the backcross generation is introduced into the genetic background of the recipient parent; The backcross generation is detected by the new molecular marker, the backcross generation with the new molecular marker detection is backcrossed with the recipient parent for the second time, and the backcross generation is obtained, and the backcross generation is detected by the new molecular marker; The backcross generation is backcrossed with the recipient parent for the third time, and the backcross generation is obtained; The backcross generation is self-crossed, and the self-crossed generation is obtained, and the self-crossed generation is planted into strains, and 10-15 strains are randomly selected for new molecular marker detection; The unstable strains with a trait variation coefficient of more than 10% in the self-crossed generation are removed, and the stable strains with a variation coefficient of less than or equal to 5% are reserved after removal, and the stable strains are marked as the self-crossed generation; Finally, the self-crossed generation is self-crossed, and the self-crossed generation is obtained after self-crossing, and the self-crossed generation is detected by the molecular marker, the phenotype is retested, and the agronomic traits are comprehensively evaluated; According to the results of molecular marker rechecking, phenotype retesting and comprehensive evaluation of agronomic traits, the final strain is screened out, and the final strain is the target strain.

[0027] Specifically, the donor parents correspond to deep root and drought tolerance key traits, respectively, and the recipient parents lock varieties with good agronomic traits but weak target traits. This not only clearly targets gene aggregation, but also ensures that the final lines retain high yield, stress resistance and other practical agronomic traits, avoids the loss of excellent traits due to blind aggregation, and achieves the improvement goal of making up for short boards and preserving advantages. Through three rounds of backcross, the residual non-target genes in the donor parent are greatly reduced, and the linkage of undesirable traits is reduced; subsequent multiple generations of selfing promote genotype homozygosity, avoid trait separation caused by heterozygosity, and ensure stable inheritance of target traits, while considering the controllability and purity of the genetic background. From the first backcross generation to the second selfing generation, new molecular markers are used to track target genes throughout the process, replacing the blindness of traditional phenotype screening, and quickly excluding individuals that do not carry target genes; especially in the second selfing generation, through marker detection combined with coefficient of variation screening, the line with genotype homozygosity and consistent traits is accurately locked, greatly shortening the screening period. Finally, through molecular marker rechecking, phenotype rechecking, and agronomic trait evaluation, a triple verification system of genotype, phenotype, and agronomic traits is formed to ensure that the selected target lines not only meet the deep root and drought tolerance requirements, but also meet the requirements of excellent agronomic traits in actual agricultural production, avoiding the problem of meeting the laboratory standards but not being suitable for field use. Among them, the gene aggregation table is shown in the following figure:

[0028] S5: simultaneous identification: the line with coordinated root traits and drought tolerance in the target line is identified, and the excellent line is obtained after the identification is completed; According to the core indicators of root development and drought tolerance, the core evaluation indicators of the line with coordinated root traits and drought tolerance are confirmed; Group the target lines according to the number, each group contains 30-50 rice seedlings, and set up 30-50 rice seedlings for control group and positive control; The rice seedlings with control group and positive control set up are planted in a greenhouse, and the planted rice seedlings are subjected to stress treatment and trait simultaneous identification; The stress treatment and trait simultaneous identification process is as follows: S501: mild drought stress identification: this stage is stress 1 period, lasting for 15 days, the soil water content is reduced to 40%-50% of the maximum field water holding capacity, and the root traits are measured every 5 days during the period, and the drought tolerance index is measured every 7 days; S502: moderate drought stress identification: this stage is stress 2 period, lasting for 20 days, for the lines that pass the mild drought stress identification, the soil water content is further reduced to 25%-35% of the maximum field water holding capacity, and the root surface area, root hair density and root vessel diameter are measured every 7 days during the period; drought recovery rate and seed setting rate are measured every 10 days; S503: recovery growth verification: this stage is the recovery period, lasting for 15 days, the medium drought stress qualified line is resumed normal irrigation, the root regeneration capacity and the leaf recovery rate are determined during the period, the root core index and the drought resistance core index are determined again; The rice seedlings qualified in the recovery growth verification are subjected to single plant level correlation detection, the single plant level correlation detection is that the root traits and drought resistance indexes of the rice seedlings qualified in the recovery growth verification are determined respectively, and the synergistic comprehensive score of each rice seedling is calculated; Finally, the excellent lines are screened according to the calculation results.

[0029] Specifically, the root development and drought resistance core indexes confirmed in the early stage are directly used to avoid evaluation deviation caused by index switching, ensure that the synergistic judgment always focuses on key traits, provide a unified standard for subsequent screening, reduce subjective errors, the sample size of 30-50 plants in each group meets the statistical requirements, and a control group and a positive control are set to effectively distinguish the influence of environmental factors and genotypes on traits, avoid result misjudgment caused by single treatment, and improve data reliability. The gradient design from light drought to medium drought simulates the process of gradually increasing drought in the field; the determination frequency and indexes are adjusted at different stages, and recovery growth verification is increased to comprehensively evaluate the comprehensive performance of the lines under drought stress and after disaster, avoid ignoring the recovery capacity in actual production by only looking at static drought resistance, and finally determine the traits and calculate the synergistic comprehensive score at the single plant level to break through the limitation of group average data masking individual differences, accurately locate the excellent single plants with real synergistic root and drought resistance, and ensure that the screened lines have stable synergistic phenotypes to meet the subsequent breeding or production application requirements.

[0030] S6: gene regulation and editing: the key genes of the excellent lines are modified and regulated through genome editing technology; The key genes related to root development and drought resistance are identified from the excellent lines; The virus-induced gene silencing technology is used to transiently silence the key genes, and the transient silencing process is as follows: a gene silencing vector is constructed, the excellent line seedlings are transformed by Agrobacterium mediation, and the root traits and drought resistance indexes of the plants after silencing are determined; if the target traits decrease after silencing, the gene is confirmed as a key gene and included in the genome editing range; The key genes subjected to transient silencing are designed for editing target points, including target point position selection and target point specificity verification, wherein the target point position selection is to select the key functional domain of the gene coding region or the core regulatory element of the gene promoter region; the target point specificity verification is to align the rice whole genome by using a local alignment search tool to ensure that the target point sequence is uniquely matched in the rice genome; at the same time, a target point specificity detection primer is designed. After the design of the editing target is completed, the editing vector construction is carried out, and the editing vector construction process is as follows: S601: Vector skeleton selection: a binary vector for rice transformation is used, and the binary vector comprises a Cas9 protein coding gene, a target sgRNA expression cassette and a screening marker gene; S602: sgRNA expression cassette construction: according to the target sequence, an sgRNA oligonucleotide primer is synthesized and connected with a gene promoter in the vector skeleton to form an sgRNA expression unit; S603: Vector assembly and verification: the sgRNA expression unit, the Cas9 protein coding gene and the screening marker gene are sequentially inserted into the vector skeleton, and the correctness of the vector construction is verified by restriction endonuclease; According to the constructed editing vector, cells of the excellent strain are introduced, and the introduction method comprises Agrobacterium-mediated transformation or a gene gun method; After the introduction is completed, the cells are transferred to a selection medium, and successfully transformed cell groups are screened out; The screened transformed cells are transferred to a differentiation medium to induce the formation of regenerated seedlings, the regenerated seedlings are transplanted to soil, genomic DNA is extracted, and the editing event is detected by polymerase chain reaction and sequencing technology, and it is confirmed whether the target gene site is modified as expected and whether the individual is not edited or off-target; Finally, the edited rice seedlings are verified at the phenotype and molecular levels, and the modification and regulation of the key gene in the excellent strain are completed after the verification is qualified.

[0031] Specifically, only the gene with the target trait decreased after silencing is included in the editing range through virus-induced gene silencing transient verification, non-key genes are excluded, the editing object is directly associated with root development and drought tolerance, the editing pertinence is improved from the source, the target gene function domain or promoter core element is selected to ensure that the gene function can be effectively regulated after editing; meanwhile, the specificity is verified through whole genome comparison to ensure that the target sequence is unique, specific detection primers are matched to greatly reduce the off-target probability and improve the editing safety, a mature binary vector is used to integrate Cas9, sgRNA expression cassette and screening marker, the elements are complete and suitable for the rice transformation system; the correctness of the vector is verified by restriction endonuclease to avoid experimental failure caused by vector construction failure, to ensure the subsequent transformation efficiency, the target site modification is confirmed by PCR and sequencing after transformation, the off-target individuals are excluded, and the phenotype and molecular levels are double-verified to ensure that the editing is successful and the trait meets the standard, to avoid the problem that the editing is successful but the trait is not improved, and finally to ensure the practical value of the modification and regulation.

[0032] In order to solve the problems of insufficient combination of microbial inoculants and breeding in the prior art, unstable application effect, lack of phased system design of drought stress training, non-uniform evaluation standard of breeding, and not considering comprehensive performance such as drought resistance, yield and quality, resulting in low breeding efficiency, please refer to Figs. 1-3 The embodiment provides the following technical solutions: S7: Microenvironment construction: screening of microbial inoculants, and microenvironment construction of excellent strains after modification and control are completed; According to the root characteristics and drought resistance demand of the excellent strains after modification and control, the screening indexes of the microbial inoculants are confirmed, and the screening indexes include functional indexes, synergistic indexes and safety indexes; According to the screening indexes, the microbial inoculants in the inoculant library are screened, the qualified inoculants are purified and expanded, and the final microbial inoculants are obtained after purification and expansion are completed; The microenvironment construction process is: first, the soil texture of the planting area is improved, then the improved soil is pasteurized, the pasteurized soil is inoculated with microbial inoculants, and the soil is placed at room temperature for 7 days for inoculant activation, so that the concentration of the inoculants in the soil reaches 10 6 -10 7 CFU / g; The rice seedlings of the excellent strains are subjected to root soaking treatment, and after the root soaking treatment is completed, the rice seedlings are planted in the constructed microenvironment, and the microenvironment during planting is dynamically regulated; The dynamic regulation time includes: microenvironment regulation during planting: 1-15 days after planting; microenvironment regulation during growth: 16-60 days after planting; microenvironment intensification during adversity: during drought stress training; After dynamic regulation, the construction effect of the microenvironment is evaluated, including short-term effect evaluation and long-term effect evaluation, the short-term effect evaluation is 30 days after construction, and the long-term effect evaluation is 60-90 days after construction; Finally, the microenvironment is optimized according to the evaluation results.

[0033] Specifically, taking function, synergy and safety as core indexes, the inoculants can assist root development and improve drought resistance, and antagonism with plants or introduction of safety hazards is avoided, so as to realize accurate matching of inoculants and strains from the source, improve soil texture to adapt to root growth, and then remove interference of miscellaneous bacteria through pasteurization, and then activate to 10 6 -10 7The specific concentration of CFU / g not only guarantees the dominant colonization of the target microbial agent, but also avoids the fluctuation of effect caused by insufficient concentration or competition of miscellaneous bacteria, provides a stable microbial environment for plant growth, regulates in stages according to the planting period, growth period and adversity period, accurately matches the microenvironment needs of rice at different growth stages, especially strengthens the regulation in the adversity period, forms synergy with the drought tolerance characteristics of the excellent strain, further improves the stress resistance, measures the short-term effect and stable performance in the long term, optimizes the microenvironment combined with the evaluation results to avoid the problem of short-term effectiveness but long-term failure, forms a closed loop of construction, evaluation and optimization, ensures that the microenvironment continuously supports the excellent strain, and improves the practical application value.

[0034] S8: Cultivation dynamic control: drought stress training is carried out on the excellent strain in the breeding middle period and the pre-cultivation period according to the constructed microenvironment; Before the drought stress training, the training object is first confirmed, and the training object is selected as the excellent strain plant growing well in the microenvironment and in the tillering peak period to the initial booting period; After the training object is confirmed, drought stress training is carried out in the pre-cultivation period, and the drought stress training process in the pre-cultivation period is as follows: S8011: Mild stress adaptation: stress intensity regulation and microenvironment synergistic management are carried out on the training object on the 1st-7th day; S8012: Moderate stress induction: stress intensity regulation and physiological regulation assistance are carried out on the training object on the 8th-14th day; S8013: Stress recovery and consolidation: recovery irrigation and response effect evaluation are carried out on the training object on the 15th-21st day; The table of the pre-cultivation period drought stress training is as follows:

[0035]

[0036] Then, the training object is subjected to drought stress training in the breeding middle period, and the drought stress training process in the breeding middle period is as follows: S8021: Progressive drought initiation: stress intensity regulation and microbial agent function strengthening are carried out on the training object on the 1st-10th day; S8022: Continuous drought strengthening: stress intensity regulation and soil environment management are carried out on the training object on the 11th-21st day; S8023: Drought and rehydration cycle training: cycle regulation and root protection are carried out on the training object on the 22nd-28th day; The table of the breeding middle period drought stress training is as follows:

[0037]

[0038] Real-time monitoring of index data during drought stress training, including soil indicators, drought stress training, and fungicide indicators; Finally, the drought stress training of the excellent strain breeding in the middle and early stages of cultivation is completed.

[0039] Specifically, plants growing well in the microenvironment and in the tillering peak to the initial booting stage are selected. This period is the key stage for rice to be sensitive to drought and for the stress resistance mechanism to be easily induced. Avoiding weak seedlings or non-critical period plants that result in ineffective training ensures that the stress training can precisely act on the core period of resistance capacity construction. In the early stage of cultivation, from mild adaptation to moderate induction to recovery and consolidation, and in the middle stage of breeding, from gradual start-up, continuous strengthening to drought and recovery cycle, the physiological logic of low-intensity adaptation to high-intensity induction to recovery and consolidation is followed. Avoiding sudden strong stress that causes plant damage, gradually activating root regulation, osmotic regulation and other drought tolerance mechanisms, the plant forms stable and sustainable stress resistance. In the training, the microenvironment management, fungicide function strengthening and physiological regulation are combined to deeply integrate the stress training and the microenvironment constructed in the early stage. The advantages of fungicide root promotion and soil environment optimization are used to assist plants in coping with stress, forming a synergistic effect of stress induction and microenvironment support, improving the efficiency of drought tolerance capacity construction, and monitoring soil, stress, and fungicide indicators in real time. The stress intensity and microenvironment parameters can be adjusted in time to avoid excessive stress that causes plant death or insufficient stress that affects training effectiveness, ensuring that the training is within a controllable range and improving the stability and reliability of the results.

[0040] S9: Rice breeding evaluation: breeding rice that meets the drought tolerance standard according to the results of drought stress training, and evaluating the comprehensive performance of the selected candidate varieties; First, the breeding standards are established, including survival bottom line indicators, physiological drought tolerance indicators, growth maintenance indicators, and recovery capacity indicators; According to the breeding standards, the rice that meets the drought tolerance standard is repeatedly verified for drought tolerance, and the rice that passes the repeated verification of drought tolerance is subjected to yield experiments. The evaluation criteria for yield experiments are the potential yield, yield stability, and drought resistance index of rice. The agronomic traits of rice during the entire growth period are evaluated, and the evaluation criteria are plant structure, lodging resistance, growth period, and ear traits; Then, the quality of rice is analyzed, and the quality analysis criteria are processing quality, appearance quality, and taste quality; Finally, according to the results of repeated verification of drought tolerance, yield experiments, agronomic trait evaluation, and quality analysis, the comprehensive performance evaluation results of the selected candidate varieties are obtained.

[0041] Specifically, the four indexes of survival bottom line, physiological drought tolerance, growth maintenance and recovery ability avoid the problems of drought tolerance but difficult to survive or survival but growth stagnation caused by single standard, define the core characteristics of drought-tolerant varieties comprehensively, exclude accidental drought-tolerant individuals through repeated verification first, ensure the stability of drought tolerance; then evaluate yield from potential yield, yield stability and drought resistance index, avoid ineffective varieties with drought tolerance but low yield, and ensure stable yield performance in drought environment, meet the core needs of agricultural production of resistance and high yield, evaluate plant type, lodging resistance, growth period and ear traits, which are all key indicators of field planting and management, ensure that the varieties not only tolerate drought, but also adapt to actual cultivation scenarios, reduce planting difficulty and risk, analyze processing quality, appearance quality and taste quality, break through the limitation of heavy resistance and high yield, light quality, ensure that the varieties meet the production needs and meet the market requirements for rice quality, and improve the comprehensive application value.

[0042] It should be noted that, in this document, the terms such as first and second are used merely to distinguish one entity or action from another entity or action, and do not necessarily require or imply these entities or actions to be in any actual relationship or order. Moreover, the terms "comprises", "comprising" or any other variations thereof are intended to cover non-exclusive inclusions, so that a process, method, article or apparatus including a series of elements includes not only those elements, but also other elements not explicitly listed or inherent to such a process, method, article or apparatus.

[0043] Although the embodiments of the present application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present application.

Claims

1. A method for breeding and cultivating drought-resistant rice with enhanced root development, characterized in that, Includes the following steps: S1: Screening of basic germplasm materials: Screening basic germplasm materials from existing gene banks to enhance traits associated with root development and drought resistance; First, the core indicators of root development and drought resistance will be confirmed. S2: Root phenotypic analysis: Image analysis of the morphological characteristics of the root system of basic germplasm materials; S3: Development of gene molecular markers: Based on the image analysis results of basic germplasm materials, novel molecular markers were developed to closely link the rice drought resistance and deep root traits of basic germplasm materials. Among them, based on the core indicators of root development and drought resistance, the corresponding indicator data in the image analysis results of the basic germplasm materials were confirmed, and the phenotypic data of the basic germplasm materials were obtained after confirmation. Genomic DNA was extracted from the core indicators of root development and drought resistance in the phenotypic data, and a DNA pool was constructed using the equal-volume mixing method. After the DNA mixing pool is constructed, polymorphic marker sites are screened. The screening process is as follows: S301: DNA Sequencing and Analysis: Perform whole-genome resequencing or simplified genome sequencing on two gene pools in the DNA pool to obtain genome-level information on single nucleotide polymorphisms and insertion / deletion variations; S302: Polymorphic marker screening: Through bioinformatics analysis, the sequence differences between the two gene pools in the DNA mixing pool are compared, and variant sites that appear frequently in the deep-rooted drought-resistant gene pool but appear infrequently or not at all in the shallow-rooted sensitive gene pool are screened out. Candidate marker regions are determined based on the variant sites. S303: Marker site annotation: Candidate marker sites are located on the rice reference genome, which is extracted from the gene database. The gene function of the region where the site is located is analyzed through the gene annotation database, and the regions located inside or in the upstream and downstream regulatory regions of genes related to root development and water stress response are marked. S4: Gene aggregation breeding: Based on the results of novel molecular markers, genes controlling drought resistance and deep root traits are aggregated into the same genetic background, and the target line is obtained after aggregation. Among them, donor parents were screened from the results of novel molecular markers, including donor parents with deep roots and donor parents with drought resistance; The recipient parent was then identified as a rice variety with excellent agronomic traits but weak root development and poor drought resistance.

2. The method for breeding and cultivating drought-resistant rice with enhanced root development according to claim 1, characterized in that, The screening of basic germplasm materials from existing gene banks to enhance traits associated with root development and drought resistance includes: Core indicators of root development include deep root ratio, root-to-shoot ratio, total root length, root surface area, root hair density, and root xylem diameter; core indicators of drought resistance include leaf water retention rate, drought recovery rate, and fruit setting rate. Based on the confirmed core indicators, a targeted search was conducted on the germplasm material information in the existing rice gene bank, and germplasm materials that did not meet the indicators were excluded after the search. Germplasm materials that meet the criteria are cultured during the germination period. The germination period culture process includes: setting culture conditions, observing root traits, and pre-screening and elimination. Germplasm materials screened during germination were transplanted to greenhouse pots to simulate field drought conditions. Root development traits and drought tolerance phenotypes were observed to establish the correlation between the two. The final basic germplasm material was obtained by establishing the association between root development traits and drought resistance phenotype.

3. The method for breeding and cultivating drought-resistant rice with enhanced root development according to claim 2, characterized in that, The image analysis of the morphological characteristics of the root system of the basic germplasm materials includes: Before performing image analysis on the morphological characteristics of the root system of basic germplasm materials, the root system was first standardized. The standardization process was as follows: First, basic germplasm materials in the growth period were selected, and the roots of the selected basic germplasm materials were dug up using the stratified root digging method. Then, the dug roots were immersed in phosphate buffer containing 2% formaldehyde for 24 hours for fixation. Finally, the roots were grouped according to the germplasm material number, growth period and treatment group. The root systems of the standardized basic germplasm materials were subjected to automated multi-dimensional image acquisition. The acquisition equipment included a visible light camera, a near-infrared camera, and a laser scanning device. The visible light camera captured two-dimensional images of the root system from five fixed angles (top, front, back, left, and right) to capture the morphological outline and branching structure of the root system. The near-infrared camera simultaneously acquired near-infrared images and used the difference in near-infrared absorption between the root system and water to distinguish between living and dead roots. The laser scanning device performed point cloud scanning on the root system to record the three-dimensional coordinate information of the root surface. After the multi-dimensional image acquisition is completed, image preprocessing is performed, which includes denoising, contrast enhancement and image segmentation of the multi-dimensional image. After image preprocessing, a 3D model is reconstructed. The 3D model reconstruction process is as follows: S201: Feature point matching: Identify key feature points of the root system in a multi-dimensional image after image preprocessing, and calculate the spatial coordinates of the points using a stereo matching algorithm; S202: Point cloud fusion: The point cloud data obtained by laser scanning is fused with the coordinates of key feature points identified to generate a three-dimensional point cloud model containing details of the root system surface. S203: Mesh Construction: Mesh the generated 3D point cloud model to generate a continuous root surface model; Root system trait parameters are extracted from the reconstructed 3D model. These parameters include morphological and functional parameters. Morphological parameters include length, area and volume, and depth. Functional parameters include topology, root hair characteristics, and spatial distribution. Finally, the image analysis of morphological characteristics was completed.

4. The method for breeding and cultivating drought-resistant rice with enhanced root development according to claim 3, characterized in that, Based on the image analysis results of the basic germplasm materials, novel molecular markers were used to identify the close linkage between the rice drought resistance and deep root traits of the basic germplasm materials, including: The DNA mixing pool includes a deep-rooted drought-resistant gene pool and a shallow-rooted sensitive gene pool; The marker regions were validated using population association analysis. The validation process involved selecting 1-2 pairs of hybridization combinations within the marker regions, constructing segregating populations or recombinant inbred line populations based on the hybridization combinations, performing root image analysis and drought resistance identification on the constructed segregating populations, and simultaneously using the marker regions to detect the genotypes of individual plants. Finally, the linkage exchange rate and correlation coefficient between candidate markers and target traits were calculated using interval mapping, and marker loci with a log-dominance ratio ≥3.0 and a phenotypic explanation rate ≥10% were selected. Finally, the selected marker sites were subjected to specific primer design and standardization, resulting in novel molecular markers.

5. The method for breeding and cultivating drought-resistant rice with enhanced root development according to claim 4, characterized in that, The aggregation of genes controlling drought resistance and deep root traits into the same genetic background based on novel molecular marker results includes: Two parents from the donor parent are crossed to obtain the F1 generation hybrid. The F1 generation hybrid is then used as the male or female parent to backcross with the recipient parent to obtain the backcross generation. The backcross generation is then introduced into the genetic background of the recipient parent. The first generation of backcrosses was subjected to novel molecular marker detection. The first generation of backcrosses with the recipient parent after novel molecular marker detection was performed a second backcross to obtain the second generation of backcrosses. The second generation of backcrosses was then subjected to novel molecular marker detection. Then, the second generation backcross is backcrossed a third time with the recipient parent to obtain the third generation backcross; The third generation of backcrosses was self-crossed to obtain the second generation of self-crosses. The second generation of self-crosses were planted into lines, and 10-15 plants were randomly selected from each line for novel molecular marker detection. Unstable lines with a coefficient of variation >10% in the second generation of self-pollination were removed, and stable lines with a coefficient of variation ≤5% were retained. The stable lines were then labeled as the third generation of self-pollination. Finally, the third generation of self-pollination was self-pollinated to obtain the fourth generation of self-pollination. The fourth generation of self-pollination was then subjected to molecular marker retesting, phenotypic retesting, and comprehensive evaluation of agronomic traits. The final strains were selected based on the results of molecular marker retesting, phenotypic retesting, and comprehensive evaluation of agronomic traits. These final strains were then designated as the target strains.

6. The method for breeding and cultivating drought-resistant rice with enhanced root development according to claim 5, characterized in that, Also includes: S5: Simultaneous identification: Identify the target lines in which root traits and drought resistance are synergistic, and obtain superior lines after the identification is completed; Based on the core indicators of root development and drought resistance, the core evaluation indicators for the synergy between root traits and drought resistance were identified. The target lines were grouped according to their numbers, with each group containing 30-50 rice seedlings. The 30-50 rice seedlings were used as a control group and a positive control group. The rice seedlings that were set up as control and positive control groups were grown in greenhouse pots. The planted rice seedlings were subjected to stress treatment and their traits were identified simultaneously. The procedure for simultaneous identification of stress treatment and traits is as follows: S501: Mild drought stress identification: This stage is the first stage of stress, lasting 15 days, reducing the soil moisture content to 40%-50% of the field maximum water holding capacity. During this period, root traits are measured every 5 days and drought resistance indicators are measured every 7 days. S502: Moderate drought stress identification: This stage is the second stage of stress, lasting 20 days. For the line that passed the mild drought stress identification, the soil moisture content was further reduced to 25%-35% of the field maximum water holding capacity. During this period, the root surface area, root hair density and root xylem diameter were measured every 7 days; the drought recovery rate and fruit setting rate were measured every 10 days. S503: Growth Recovery Verification: This stage is the recovery period, lasting 15 days. Normal irrigation is restored to the lines that have passed the moderate drought stress assessment. During this period, the root regeneration capacity and leaf recovery rate are measured, and the core indicators of the root system and the core indicators of drought resistance are measured again. The rice seedlings that have passed the growth recovery verification were subjected to single-plant-level correlation testing. The single-plant-level correlation testing was conducted by measuring the root characteristics and drought resistance indicators of the rice seedlings that have passed the growth recovery verification, and calculating the synergy comprehensive score of each rice seedling. Finally, superior strains were selected based on the calculation results.

7. The method for breeding and cultivating drought-resistant rice with enhanced root development according to claim 6, characterized in that, Also includes: S6: Gene regulation editing: Modifying and regulating key genes in superior strains through genome editing technology; Identify key genes related to root development and drought resistance in superior strains; Virus-induced gene silencing technology was used to transiently silence key genes. The transient silencing process was as follows: a gene silencing vector was constructed, and seedlings of superior strains were transformed through Agrobacterium-mediated transformation. The root traits and drought resistance indicators of the silenced plants were measured. If the target trait decreased after silencing, the gene was confirmed as a key gene and included in the genome editing scope. The key genes that are transiently silenced are designed as editing targets. The design of editing targets includes target location selection and target specificity verification. Target location selection involves selecting key functional domains in the gene coding region or core regulatory elements in the gene promoter region. Target specificity verification involves comparing the entire rice genome using local alignment search tools to ensure that the target sequence uniquely matches in the rice genome. At the same time, primers for target specificity detection are designed. After the editing target design is completed, the editing vector is constructed. The editing vector construction process is as follows: S601: Vector backbone selection: A binary vector for rice transformation was used, which contains the Cas9 protein-coding gene, the target sgRNA expression cassette, and the selection marker gene; S602: Construction of sgRNA expression cassette: Based on the target sequence, sgRNA oligonucleotide primers are synthesized and linked to the gene promoter in the vector backbone to form sgRNA expression units; S603: Vector assembly and verification: The sgRNA expression unit, Cas9 protein coding gene and selection marker gene were sequentially inserted into the vector backbone, and the correctness of the vector construction was verified by restriction endonuclease. The constructed editing vector is introduced into cells of superior strains, and the introduction methods include Agrobacterium-mediated transformation or gene gun method. After the cells were introduced, they were transferred to a selective culture medium, and successfully transformed cell clusters were screened out. The selected transformed cells were transferred to differentiation medium to induce the formation of regenerated seedlings. After the regenerated seedlings were transplanted into the soil, genomic DNA was extracted. Editing events were detected by polymerase chain reaction and sequencing technology. At the same time, it was confirmed whether the target gene site was modified as expected, and individuals that were not edited or off-target were excluded. Finally, the successfully edited rice seedlings were validated at the phenotypic and molecular levels. After successful validation, the modification and regulation of key genes in the superior lines were completed.

8. The method for breeding and cultivating drought-resistant rice with enhanced root development according to claim 7, characterized in that, Also includes: S7: Microenvironment construction: Screening of microbial agents, followed by microenvironment construction of superior strains after modification and regulation; Based on the root characteristics and drought resistance requirements of the modified and regulated superior strains, the screening indicators for microbial agents were confirmed. The screening indicators included functional indicators, synergistic indicators, and safety indicators. The microbial agents in the microbial agent library are screened according to the screening criteria. The qualified agents are purified and propagated. After purification and propagation, the final microbial agents are obtained. The microenvironment construction process is as follows: First, the soil texture of the planting area is improved; then, the improved soil is pasteurized; the pasteurized soil is inoculated with microbial agents; and the soil is left to stand at room temperature for 7 days to activate the agents, so that the concentration of the agents in the soil reaches 10. 6 -10 7 CFU / g; Superior rice seedlings were subjected to root soaking treatment. After the root soaking treatment, the rice seedlings were planted in a constructed microenvironment, and the microenvironment was dynamically regulated during the planting process. Dynamic regulation time includes microenvironment regulation during the transplanting period: 1-15 days after planting; microenvironment regulation during the growth period: 16-60 days after planting; and microenvironment enhancement during the adversity period: during drought stress training. After dynamic regulation is completed, the construction effect of the microenvironment is evaluated, including short-term effect evaluation and long-term effect evaluation. The short-term effect evaluation is conducted on the 30th day after construction, and the long-term effect evaluation is conducted on the 60th-90th day after construction. Finally, the microenvironment is optimized based on the evaluation results.

9. A method for breeding and cultivating drought-resistant rice with enhanced root development according to claim 8, characterized in that, Also includes: S8: Cultivation dynamic control: Drought stress training of superior strains during the mid-breeding and early cultivation stages based on the constructed microenvironment; Before conducting drought stress training, the training subjects should be identified. The training subjects should be selected from superior plant lines that grow well in the microenvironment and are in the peak tillering stage to the early heading stage. After the training subjects are identified, drought stress training in the early stage of cultivation will be conducted. The training process for drought stress in the early stage of cultivation is as follows: S8011: Mild stress adaptation: Stress intensity regulation and microenvironment co-management are carried out on trainees from day 1 to day 7; S8012: Moderate stress induction: Stress intensity and physiological regulation were assisted in training subjects from day 8 to day 14; S8013: Stress Recovery and Consolidation: On days 15-21, assess the recovery and response effectiveness of the trainees; Then, the training subjects were subjected to mid-breeding drought stress training. The mid-breeding drought stress training process is as follows: S8021: Gradual drought initiation: Stress intensity regulation and microbial agent function enhancement are performed on the trainees from day 1 to day 10; S8022: Sustained drought intensification: Stress intensity regulation and soil environment management were carried out on the training subjects from day 11 to day 21; S8023: Drought and Re-watering Cycle Training: Cycle regulation and root protection are carried out on the trainees from day 22 to day 28; Real-time monitoring of indicator data during drought stress training, including soil indicators, drought stress training data, and microbial agent indicators; Finally, drought stress training was completed during the mid-term of breeding of superior strains and the early stage of cultivation.

10. A method for breeding and cultivating drought-resistant rice with enhanced root development according to claim 9, characterized in that, Also includes: S9: Rice Breeding Evaluation: Based on the results of drought stress training, rice varieties that meet drought resistance standards are selected and bred, and the comprehensive performance of the selected candidate varieties is evaluated. First, breeding standards are formulated, including survival baseline indicators, physiological drought resistance indicators, growth maintenance indicators, and recovery capacity indicators. According to the breeding standards, the drought resistance of rice that meets the drought resistance standards is repeatedly verified. Rice that passes the repeated verification of drought resistance is then subjected to yield experiments. The evaluation criteria for the yield experiments are the potential yield, yield stability and drought resistance index of rice. The agronomic traits of rice throughout its entire growth period were evaluated, with the evaluation criteria including plant structure, lodging resistance, growth period, and panicle traits. Next, the quality of the rice was analyzed, with the quality analysis standards being processing quality, appearance quality, and taste quality. Finally, based on the results of repeated verification of drought resistance, yield experiments, agronomic trait evaluation, and quality analysis, the comprehensive performance evaluation results of the selected candidate varieties were obtained.

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