Method for screening plants of interest gene of japonica rice double parent cross based on high-throughput sequencing

Abstract

The application discloses a japonica rice parent hybrid target gene plant screening method based on high-throughput sequencing and belongs to the field of agricultural biotechnology. The application uses high-throughput sequencing technology to accurately analyze the genetic differences of parent genomes, develops specific molecular markers covering target gene regions, can quickly identify plants containing target genes, greatly improves the screening efficiency and accuracy, shortens the breeding cycle and saves resources, combines molecular marker detection with the phenotypic performance of target traits, fixes target genes in each generation during the construction of hybrid and backcross populations, ensures the stable inheritance of target traits, and verifies the correlation between target genotypes and target traits and their environmental adaptability through joint analysis of genotypes and phenotypes and multi-environment testing, improves the stability and reliability of breeding materials, constructs excellent hybrid combinations and realizes genotype optimization in candidate plant screening and restorer line creation, and improves the quality and yield potential of breeding materials.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of agricultural biotechnology, in particular to a method for screening a japonica rice hybridization target gene plant based on high-throughput sequencing. BACKGROUND

[0002] In recent years, the rapid development of molecular marker assisted breeding and genomics technology provides a new direction for rice breeding.

[0003] However, the traditional breeding method usually relies on phenotypic selection, which is long in cycle, low in efficiency, and easily disturbed by environmental factors, and it is difficult to accurately screen plants with excellent target traits. At present, the specific application of high-throughput sequencing for japonica rice in hybrid breeding is still less, especially how to combine molecular marker development, genotype analysis and hybrid breeding process, which still has certain technical defects. SUMMARY

[0004] The purpose of the present application is to provide a method for screening a japonica rice hybridization target gene plant based on high-throughput sequencing, to solve the problems raised in the background art.

[0005] To achieve the above purpose, the present application provides the following technical scheme: a method for screening a japonica rice hybridization target gene plant based on high-throughput sequencing, comprising the following steps:

[0006] Parent material selection and target gene determination, selecting high-quality japonica rice parent materials, determining genes related to target traits through database analysis, and determining the region and distribution characteristics of target genes according to the breeding requirements of target traits;

[0007] Parent genotype identification and genome comparison, whole genome sequencing of parent materials, obtaining parent genome data, comparing sequencing data to reference genome, identifying genetic differences between parents, screening specific markers in target gene region, and determining the difference fragments of target genes by mining indica-japonica bridge key regions in parent genomes;

[0008] Target region molecular marker development and verification, based on the difference information of parent genomes obtained by high-throughput sequencing, designing molecular markers related to target genes, the molecular markers covering the key sites and upstream and downstream regions of the target gene region, and molecularly verifying the markers of parent materials and hybrid offspring;

[0009] Hybridization and backcross population construction, taking target genes as the core, hybridizing japonica rice parents to obtain F1 generation plants, selecting excellent F1 generation plants for backcross to construct backcross populations, and selecting plants in the population construction process combined with breeding targets;

[0010] High-throughput sequencing and genetic analysis, extracting the genomic DNA of the hybrid offspring plants for PCR amplification, converting the SSR markers or snp markers of the restorer genes and general compatibility genes into Kasp markers, wherein the restorer genes include Rf1 and Rf4, and the general compatibility genes include s5, Sc, pf12 and f5, and constructing an amplicon library of the target region, performing high-throughput sequencing on the amplicon library, performing quality control, alignment and variation analysis on the sequencing data, identifying the genotype of the hybrid offspring plants, and preliminarily screening out plants containing the target genes to provide the recurrent parent of the backcross population;

[0011] Genotype and phenotype combined analysis, combining the genotype analysis results with the population phenotype data, verifying the phenotype correlation of the target genes, and comprehensively analyzing the genotype and phenotype data of the plants to screen out excellent plants with significant genotype and target traits;

[0012] Verification of candidate plants and creation of restorer lines, crossing the selected target plants as candidate materials of restorer lines with sterile lines to construct hybrid combinations, verifying the yield potential and target trait performance of the combinations, and based on the verification results, using high-quality candidate plants as breeding materials to create new restorer lines.

[0013] Further, the parent material selection and target gene determination specifically further include the following steps:

[0014] Selecting parent materials with excellent genetic characteristics from japonica rice excellent varieties or breeding resources, wherein the excellent genetic characteristics include high yield, wide adaptability, salt and alkali resistance, and disease and pest resistance, and materials known to have target traits or possibly related to target genes are preferentially selected;

[0015] Screening target genes significantly associated with target traits, such as restorer ability, seed setting rate or general compatibility, by gene function database analysis;

[0016] Identifying the gene locus, chromosome region and known gene function of the target gene;

[0017] Based on the marker data of the existing genome information of the parent materials and the genetic background of the target traits, inferring the distribution and differential fragment region of the target gene in the parent genome;

[0018] Preliminarily delimiting the target gene region to a specific chromosome range.

[0019] Further, the parent genotype identification and genome comparison specifically further include the following steps:

[0020] Performing whole genome sequencing on the selected japonica rice parent materials, using high-throughput sequencing to generate sequencing data, and the sequencing data depth is greater than or equal to 30x;

[0021] Sequencing data from both parents' genomes were aligned to a reference genome to analyze genetic differences between the two genomes, including single nucleotide polymorphisms and insertions / deletions.

[0022] By combining the gene locus of the target gene with known gene functions, specific markers for the region where the target gene is located are screened.

[0023] Based on the comparative analysis results, combined with specific fragments of the parent genomes, key regions related to heterosis and genetic stability in indica and japonica hybrids were identified, and the distribution and differential information of target gene regions in the parent genomes were determined.

[0024] Furthermore, the development and validation of molecular markers for the target region specifically includes the following steps:

[0025] Based on the differential information of the parent genomes and the localization results of the target gene region, molecular markers covering key sites in the target gene region were designed;

[0026] The molecular markers include single nucleotide polymorphism markers and insertion / deletion markers;

[0027] The molecular markers simultaneously cover key sequences at both ends of the target region and in the middle;

[0028] Molecular markers were used to amplify the biparental materials by PCR, and the accuracy and specificity of the markers were verified by sequencing.

[0029] Based on the marker distribution of parental materials and plants with known phenotypes, high-quality markers that can significantly distinguish the target genotype are screened.

[0030] A molecular detection system was established by combining the selected high-quality markers;

[0031] By comparing the marker detection results with the target phenotype data, it was confirmed that the marker could reflect the target genotype.

[0032] Furthermore, the construction of the hybridization and backcross populations further includes the following steps:

[0033] Select superior single plants from the parent materials as hybridization parents, and carry out artificial hybridization to obtain F1 generation plants. Hybridization is preferably carried out under the condition that the target trait is stable and the flowering period is consistent.

[0034] During the F1 generation plant cultivation process, the growth status and target traits were initially observed, and relevant agronomic traits were recorded.

[0035] F1 generation plants were screened, and individual plants that met the breeding objectives were selected based on the marker detection results of the target gene and the phenotypic expression of the target trait.

[0036] The selected F1 generation plants were used as donor parents and backcrossed with recipient parents to construct a backcross population;

[0037] In the process of constructing backcross populations, the population size is further expanded through multiple generations of backcrossing and self-crossing;

[0038] In each generation of backcrossing and selfcrossing, by combining the results of molecular marker detection and the phenotypic expression of the target trait, plants with high yield, wide adaptability and excellent target traits are selected.

[0039] Furthermore, the high-throughput sequencing and genotyping analysis specifically includes the following steps:

[0040] Genomic DNA was extracted from hybrid offspring plants and amplified by PCR using specific molecular markers targeting the target gene region, so that the amplified fragments covered the key sites and upstream and downstream regions of the target gene.

[0041] After purifying the amplified products, an amplicon library of the target region is constructed by combining the length and coverage of the amplicon sequence.

[0042] High-throughput sequencing platforms were used to perform deep sequencing on amplicon libraries of the target region. Quality control was performed on the sequencing data to remove low-quality reads, adapter sequences, and repetitive sequences.

[0043] Sequencing data is aligned to a reference genome to analyze variation information within the target gene region, including single nucleotide polymorphisms, insertions and deletions, and the distribution of target gene-specific markers.

[0044] By combining the genotype data of the parents, the genotypes of the hybrid offspring are identified and the genotype information is classified and labeled, including homozygous parental type, heterozygous type and non-parental type;

[0045] Based on the variation analysis results of sequencing data, combined with the distribution of target region-specific markers, plants containing the target gene were screened.

[0046] For plants that have undergone preliminary screening, record the variation information and genotype category within the target gene region;

[0047] The screening results were compared and analyzed with the preliminary phenotypic data of the population to identify candidate plants that were significantly associated with the target genotype and the target trait.

[0048] Furthermore, the combined analysis of genotype and phenotype specifically includes the following steps:

[0049] Based on the genotypic data of the selected hybrid offspring plants, key variation information of the target gene region is extracted;

[0050] The correlation analysis between genotype data and population phenotypic data, including the expression of target traits, agronomic traits, and environmental adaptability, was performed.

[0051] Statistical calculations were performed to determine the correlation between the target genotype and the target trait, and the association pattern between genotype and target trait was identified.

[0052] Based on the association analysis results, plants with a significant association between genotype and target trait were initially screened as candidate plants, and the genotype and phenotypic expression of the candidate plants were cross-validated.

[0053] Phenotypic tests of the target traits were conducted on the selected candidate plants under different environmental conditions to evaluate the stability of the target traits and the differences in their performance under multiple environmental conditions.

[0054] Plants that perform well in multiple environments and are consistent with the target genotype are selected as the final high-quality plants.

[0055] Furthermore, the process for setting up different environmental conditions is as follows:

[0056] Extract the environmental parameters corresponding to the candidate plants, including temperature, humidity and light intensity parameters.

[0057] Extract the preset reference parameters corresponding to the condition parameters, wherein the preset reference parameters include temperature reference parameters, humidity reference parameters and light intensity reference parameters;

[0058] Extract the theoretical change in ambient temperature generated by the current light intensity benchmark parameter within a preset light unit time period; wherein the value range of the light unit time period is 30min-60min;

[0059] Extract the actual ambient temperature change generated by the light intensity reference parameter during a preset light unit time period;

[0060] The parameter adjustment coefficient is obtained by using the actual ambient temperature change and the theoretical ambient temperature change of the light intensity reference parameter during a preset illumination unit time period.

[0061] The parameter adjustment coefficient is obtained by the following formula:

[0062]

[0063] Where R represents the parameter adjustment coefficient; T x T represents the theoretical change in ambient temperature caused by the light intensity reference parameter within a preset unit of time during which illumination occurs; sThis represents the actual change in ambient temperature caused by the light intensity reference parameter within a preset light intensity unit time period; L x S represents the distance between the light source and the plant; x This indicates the duration corresponding to a preset unit of illumination time.

[0064] The parameter adjustment coefficient is compared with a preset parameter adjustment coefficient threshold.

[0065] When the parameter adjustment coefficient does not exceed the preset parameter adjustment coefficient threshold, multiple sets of different environmental conditions are set using the preset upper limit values ​​corresponding to the temperature gradient parameter, the upper limit value corresponding to the humidity reference parameter, and the upper limit value corresponding to the light intensity gradient parameter.

[0066] When the parameter adjustment coefficient exceeds the preset parameter adjustment coefficient threshold, the upper limit values ​​corresponding to the temperature gradient parameter, the humidity reference parameter, and the light intensity gradient parameter are adjusted.

[0067] Furthermore, when the parameter adjustment coefficient exceeds a preset parameter adjustment coefficient threshold, the upper limit values ​​corresponding to the temperature gradient parameter, the humidity reference parameter, and the light intensity gradient parameter are adjusted, including:

[0068] When the parameter adjustment coefficient exceeds the preset parameter adjustment coefficient threshold, the upper limit value corresponding to the temperature gradient parameter, the upper limit value corresponding to the humidity reference parameter, and the upper limit value corresponding to the light intensity gradient parameter are retrieved.

[0069] Retrieve parameter adjustment coefficients;

[0070] The upper limits corresponding to the temperature gradient parameter, the humidity reference parameter, and the light intensity gradient parameter are adjusted using the parameter adjustment coefficients to obtain the adjusted upper limits corresponding to the temperature gradient parameter, the humidity reference parameter, and the light intensity gradient parameter.

[0071] The upper limit value of the adjusted temperature gradient parameter is obtained by the following formula:

[0072]

[0073] Among them, T up This indicates the upper limit of the adjusted temperature gradient parameter; T up0 This indicates the upper limit of the temperature gradient parameter before adjustment; R represents the parameter adjustment coefficient.

[0074] Meanwhile, the upper limit value corresponding to the adjusted humidity reference parameter is obtained by the following formula:

[0075]

[0076] Among them, W up This indicates the upper limit value corresponding to the adjusted humidity gradient parameter; W up0 This indicates the upper limit of the humidity gradient parameter before adjustment; T up This indicates the upper limit of the adjusted temperature gradient parameter; T up0 This indicates the upper limit of the temperature gradient parameter before adjustment; R represents the parameter adjustment coefficient.

[0077] Furthermore, the upper limit value corresponding to the adjusted light intensity gradient parameter is obtained by the following formula:

[0078]

[0079] Among them, G up This represents the upper limit of the adjusted illumination intensity gradient parameter; G up0 This represents the upper limit of the light intensity gradient parameter before adjustment; W up This indicates the upper limit value corresponding to the adjusted humidity gradient parameter; W up0 This indicates the upper limit of the humidity gradient parameter before adjustment; T up This indicates the upper limit of the adjusted temperature gradient parameter; T up0 This indicates the upper limit of the temperature gradient parameter before adjustment; R represents the parameter adjustment coefficient.

[0080] Multiple sets of different environmental conditions are set using the adjusted upper limits of the temperature gradient parameter, the humidity baseline parameter, and the light intensity gradient parameter.

[0081] Furthermore, the validation of candidate plants and the creation of restorer lines specifically include the following steps:

[0082] Select the high-quality plants that were finally screened as candidate materials for restorer lines;

[0083] Candidate materials are crossbred with high-quality sterile lines to construct hybrid combinations;

[0084] Hybrid offspring were bred under greenhouse or field conditions, and their target traits and other key agronomic traits were initially observed and the plant performance was recorded.

[0085] Based on the target traits and yield performance, superior hybrid combinations are selected to proceed to the next stage of trials;

[0086] The selected superior hybrid combinations were subjected to field trials. During the trials, data such as the performance of the target traits, yield indicators, and adaptability of the hybrid combinations were recorded.

[0087] By combining the results of multi-environmental tests, we analyzed the stability and yield performance of the target traits of the candidate materials under different environmental conditions, and confirmed the practical application value of the candidate materials based on the test results.

[0088] High-performing candidate materials are used as the base materials for restorer lines. Combined with target traits and genetic background, genotype optimization is carried out, and generation selection and verification are performed on candidate plants.

[0089] To create japonica rice restorer lines that meet the target trait requirements and have a good and stable genetic background.

[0090] Compared with the prior art, the beneficial effects of the present invention are:

[0091] 1. This invention utilizes high-throughput sequencing technology to accurately analyze the whole-genome genetic differences of parental materials, locate specific fragments of target genes, and develop molecular markers covering the target region. These molecular markers are highly sensitive and specific, enabling rapid identification of the target genotype in hybrid offspring. Compared with traditional breeding methods that rely on phenotypic observation, this invention significantly improves the efficiency and accuracy of screening. Combining molecular marker detection with genotypic analysis of high-throughput sequencing data can effectively distinguish between homozygous, heterozygous, and non-target genotype plants, reducing blind spots in screening and thus significantly saving breeding resources.

[0092] 2. This invention combines a multi-generational hybridization and backcross population construction strategy to precisely screen plants in each generation using molecular markers and target phenotypic expression, gradually fixing the inheritance of target genes and achieving stable inheritance of target traits. Simultaneously, during the joint analysis of genotype and phenotype, multi-environmental phenotypic testing of candidate plants verifies the stability and consistency of the target genotype, thereby ensuring that the finally selected plants exhibit excellent target traits under different environmental conditions, laying the foundation for widespread application in breeding practice.

[0093] 3. This invention, by constructing hybrid combinations and comprehensively evaluating their target traits and agronomic traits, can screen out candidate plants with excellent performance and use them to create new restorer lines. Combined with efficient genotypic analysis and phenotypic verification methods, it can achieve genotypic optimization and generation improvement of candidate materials, significantly improving breeding efficiency. At the same time, through a high-throughput, high-precision molecular detection system, it provides an advanced technical platform for the breeding of rice restorer lines and hybrid rice varieties, enhancing the controllability and scientific nature of the breeding process, and ultimately promoting the innovation and development of japonica rice breeding technology. Attached Figure Description

[0094] Figure 1 This is a flowchart illustrating the gene-based plant screening method of the present invention. Detailed Implementation

[0095] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0096] Please see Figure 1 The present invention provides the following technical solutions:

[0097] A method for screening target gene plants in japonica rice through parent-to-parent hybridization based on high-throughput sequencing includes the following steps:

[0098] Parental material selection and target gene determination: Select high-quality japonica rice parental materials, identify genes related to the target trait through database analysis, and clarify the region where the target gene is located and its distribution characteristics according to the breeding requirements of the target trait.

[0099] The process involves identifying the genotypes of both parents and comparing their genomes. Whole-genome sequencing is performed on both parents to obtain their genome data. The sequencing data is then compared to a reference genome to identify genetic differences between the parents, screen for specific markers of the target gene region, and simultaneously identify key bridging regions between the indica and japonica rice genomes of both parents to determine the differentially expressed segments of the target gene.

[0100] The development and validation of molecular markers for target regions involves designing molecular markers related to target genes based on the genomic differences between parents obtained through high-throughput sequencing. These molecular markers cover key sites and upstream and downstream regions of the target gene region, and the markers are validated molecularly on parental materials and hybrid offspring.

[0101] The construction of hybridization and backcross populations involves hybridization of two parents of japonica rice with the target gene as the core, obtaining F1 generation plants, selecting superior F1 generation plants for backcrossing to construct a backcross population, and selecting plants in combination with the breeding objectives during the population construction process.

[0102] High-throughput sequencing and genotyping were performed. Genomic DNA was extracted from the hybrid progeny plants and amplified by PCR. SSR or snp markers of the restorer gene and the wide-compatibility gene were converted into Kasp markers. The restorer gene included Rf1 and Rf4, and the wide-compatibility gene included s5, Sc, pf12 and f5. An amplicon library of the target region was constructed. The amplicon library was sequenced using high-throughput sequencing. The sequencing data were subjected to quality control, alignment and variation analysis to identify the genotype of the hybrid progeny plants. Plants containing the target gene were preliminarily screened to provide recurrent parents for the backcross population.

[0103] Combined genotypic and phenotypic analysis combines the results of genotypic analysis with population phenotypic data to verify the phenotypic association of the target gene. By comprehensively analyzing the genotypic and phenotypic data of the plants, superior plants with significant association between genotype and target trait are screened out.

[0104] The verification of candidate plants and the creation of restorer lines involve using the selected target plants as candidate materials for restorer lines, crossing them with sterile lines to construct hybrid combinations, verifying the yield potential and target trait performance of the combinations, and using the high-quality candidate plants as breeding materials to create new restorer lines.

[0105] In the above embodiments, high-throughput sequencing and molecular marker technology are used to achieve precise localization of target gene regions and rapid screening of target plants, significantly shortening the breeding cycle and reducing screening workload and costs. Whole-genome sequencing combined with molecular marker design comprehensively analyzes the genetic differences between parents and offspring, ensuring the reliability and comprehensiveness of target gene screening. Through joint analysis of genotype and phenotype, the selected candidate plants not only have the target traits but also meet the requirements for environmental adaptability and multi-trait optimization. Through systematic analysis and candidate plant verification, the breeding process is optimized to ensure that the created restorer line material has comprehensive advantages in terms of genetic background, yield, and target traits.

[0106] The selection of parental materials and the determination of target genes also include the following steps:

[0107] Select parental materials with excellent genetic characteristics from superior japonica rice varieties or breeding resource banks. The excellent genetic characteristics include high yield, wide adaptability, salt and alkali resistance, and resistance to diseases and pests. Priority will be given to materials that are known to have the target traits or may be related to the target gene.

[0108] Target genes that are significantly associated with the target trait are screened through gene function database analysis. The target trait includes resilience, seed setting rate, or broad affinity.

[0109] Identify the gene locus, chromosomal region, and known gene function of the target gene;

[0110] Based on existing genomic information of the parents, and combined with the genetic background of the target trait, the distribution and differentially expressed regions of the target gene in the parent genomes are inferred.

[0111] The target gene region is initially delineated to a specific chromosome range.

[0112] In the above embodiments, by screening parental materials with excellent genetic characteristics such as high yield, wide adaptability, and stress resistance from superior japonica rice varieties or breeding resource banks, a foundation can be laid for the inheritance of the target trait, ensuring that the selected parental materials have breeding value. High-quality parental materials can provide a more stable genetic background, reduce interference from non-target traits in hybrid offspring, and thus improve the screening efficiency of target plants. In addition, prioritizing materials that are significantly related to the target trait or whose target genes are known can narrow the screening range and provide a precise direction for subsequent molecular marker development and hybridization experiments.

[0113] In the above embodiments, target genes related to the target trait are screened based on database analysis, clarifying their gene loci, chromosomal regions, and gene functions. This not only provides a theoretical basis for target gene localization but also significantly improves the success rate of subsequent molecular marker design. By combining existing genomic information and marker data from both parents, the distribution and differentially expressed regions of the target gene in the parent genomes can be inferred, enabling precise localization of the target gene region and reducing the risk of blind experiments. In this process, the target gene region is delineated to a specific chromosomal range, providing a clear scope for subsequent molecular marker development while avoiding resource waste caused by an overly broad selection range.

[0114] Parental genotyping and genome comparison specifically include the following steps:

[0115] Whole-genome sequencing was performed on the selected japonica rice parent materials, and high-throughput sequencing was used to generate sequencing data with a sequencing data depth of ≥30×.

[0116] Sequencing data from both parents' genomes were aligned to a reference genome to analyze genetic differences between the two genomes, including single nucleotide polymorphisms and insertions / deletions.

[0117] By combining the gene locus of the target gene with known gene functions, specific markers for the region where the target gene is located are screened.

[0118] Based on the comparative analysis results, combined with specific fragments of the parent genomes, key regions related to heterosis and genetic stability in indica and japonica hybrids were identified, and the distribution and differential information of target gene regions in the parent genomes were determined.

[0119] In the above embodiments, whole-genome sequencing of selected japonica rice parents was performed, and high-throughput sequencing technology was used to generate high-quality sequencing data (depth ≥30×), which comprehensively captured the genetic information of the parents' genomes. High sequencing depth ensured the reliability and accuracy of the data, avoiding the omission of genetic differences due to insufficient sequencing coverage. Furthermore, after aligning the sequencing data to a reference genome, genetic differences between the parents could be clearly analyzed, including single nucleotide polymorphisms (SNPs) and insertion / deletion variations (InDe ls), thereby accurately identifying specific markers in the regions containing target genes.

[0120] In the above embodiments, screening for specific markers for the target region, based on the known gene function of the target gene, facilitates subsequent molecular marker development, improving the markers' specificity and applicability. Simultaneously, comparative analysis of specific fragments from both parent genomes can uncover key genetic regions related to hybrid vigor in indica and japonica varieties, providing important clues for studying hybrid vigor in these varieties. By identifying the differential information of the target gene region in the parent genomes, the genetic background of the target gene can be clarified, ensuring that subsequent experiments are based on explicit genetic information.

[0121] The development and validation of molecular markers for target regions also includes the following steps:

[0122] Based on the differential information of the parent genomes and the localization results of the target gene region, molecular markers covering key sites in the target gene region were designed;

[0123] The molecular markers include single nucleotide polymorphism markers and insertion / deletion markers;

[0124] The molecular markers simultaneously cover key sequences at both ends of the target region and in the middle;

[0125] Molecular markers were used to amplify the biparental materials by PCR, and the accuracy and specificity of the markers were verified by sequencing.

[0126] Based on the marker distribution of parental materials and plants with known phenotypes, high-quality markers that can significantly distinguish the target genotype are screened.

[0127] A molecular detection system was established by combining the selected high-quality markers;

[0128] By comparing the marker detection results with the target phenotype data, it was confirmed that the marker could reflect the target genotype.

[0129] In the above embodiments, molecular markers covering key sites in the target region are designed based on parental genomic differences and the localization results of the target gene region. This accurately reflects the target genotype and significantly improves screening efficiency. The marker design covers both upstream and downstream ends of the target region, as well as key sequences in the middle, improving the comprehensiveness of detection and avoiding the risk of missing important sites. Furthermore, the use of multiple types of molecular markers, such as single nucleotide polymorphism (SNPs) and insertion / deletion (InDeletions) markers, can further improve the sensitivity and reliability of screening.

[0130] In the above embodiments, the accuracy and specificity of the molecular markers were verified by PCR amplification, ensuring that the developed markers could significantly distinguish genotypic differences between parents. Simultaneously, by combining the marker distribution of parental materials and plants with known phenotypes, high-quality markers capable of accurately distinguishing the target genotype were screened. The screening of high-quality markers and the establishment of the molecular detection system provided an effective tool for subsequent high-throughput screening. Furthermore, the comparative analysis of molecular marker detection results with target phenotype data verified the markers' responsiveness to the target genotype, ensuring the accuracy of the screening method.

[0131] The construction of hybridization and backcross populations also includes the following steps:

[0132] Select superior single plants from the parent materials as hybridization parents, and carry out artificial hybridization to obtain F1 generation plants. Hybridization is preferably carried out under the condition that the target trait is stable and the flowering period is consistent.

[0133] During the F1 generation plant cultivation process, the growth status and target traits were initially observed, and relevant agronomic traits were recorded.

[0134] F1 generation plants were screened, and individual plants that met the breeding objectives were selected based on the marker detection results of the target gene and the phenotypic expression of the target trait.

[0135] The selected F1 generation plants were used as donor parents and backcrossed with recipient parents to construct a backcross population;

[0136] In the process of constructing backcross populations, the population size is further expanded through multiple generations of backcrossing and self-crossing;

[0137] In each generation of backcrossing and selfcrossing, by combining the results of molecular marker detection and the phenotypic expression of the target trait, plants with high yield, wide adaptability and excellent target traits are selected.

[0138] In the above embodiments, by selecting superior parental plants for artificial hybridization to obtain F1 generation plants, and completing the hybridization operation under conditions of stable target trait expression and consistent flowering period, the breeding value of F1 generation plants can be improved. During the cultivation of F1 generation plants, plants with excellent target traits are screened based on molecular marker detection results and phenotypic data to ensure that the selected plants have breeding potential. Subsequently, F1 generation plants that meet the breeding objectives are selected as donor parents and backcrossed with recipient parents. A backcross population is constructed through multiple generations of backcrossing and self-crossing. During the backcrossing and self-crossing process, superior individual plants that meet the target traits are selected through molecular marker detection and phenotypic observation, and the population size is expanded to enhance screening stability. By combining the screening strategy of multiple generations of backcrossing and self-crossing, the inheritance of the target gene is gradually fixed, so that the target trait is stably inherited into the offspring plants.

[0139] High-throughput sequencing and genotyping analysis also include the following steps:

[0140] Genomic DNA was extracted from hybrid offspring plants and amplified by PCR using specific molecular markers targeting the target gene region, so that the amplified fragments covered the key sites and upstream and downstream regions of the target gene.

[0141] After purifying the amplified products, an amplicon library of the target region is constructed by combining the length and coverage of the amplicon sequence.

[0142] High-throughput sequencing platforms were used to perform deep sequencing on amplicon libraries of the target region. Quality control was performed on the sequencing data to remove low-quality reads, adapter sequences, and repetitive sequences.

[0143] Sequencing data is aligned to a reference genome to analyze variation information within the target gene region, including single nucleotide polymorphisms, insertions and deletions, and the distribution of target gene-specific markers.

[0144] By combining the genotype data of the parents, the genotypes of the hybrid offspring are identified and the genotype information is classified and labeled, including homozygous parental type, heterozygous type and non-parental type;

[0145] Based on the variation analysis results of sequencing data, combined with the distribution of target region-specific markers, plants containing the target gene were screened.

[0146] For plants that have undergone preliminary screening, record the variation information and genotype category within the target gene region;

[0147] The screening results were compared and analyzed with the preliminary phenotypic data of the population to identify candidate plants that were significantly associated with the target genotype and the target trait.

[0148] In the above embodiments, by extracting genomic DNA from hybrid progeny plants and combining it with PCR amplification using specific molecular markers, an amplicon library of the target region can be accurately constructed. High-throughput sequencing platforms perform deep sequencing on the amplicon library, significantly improving the efficiency and accuracy of detecting variation information in the target gene region. Quality control of the sequencing data (such as removal of low-quality reads, adapter sequences, and repetitive sequences) and alignment analysis can comprehensively capture single nucleotide polymorphisms, insertions / deletions, and specific marker distributions within the target gene region.

[0149] In the above embodiments, by combining the genotype data of both parents and systematically analyzing the genotypic information of the hybrid offspring (including homozygous parental type, heterozygous type, and non-parental type), the genetic background of the hybrid offspring can be clarified, providing a scientific basis for screening plants containing the target gene. Furthermore, combining the genotype of the screened target plant with preliminary phenotypic data can not only verify the correlation between genotype and phenotype but also further screen candidate plants that are significantly associated with the target trait.

[0150] The combined analysis of genotype and phenotype also includes the following steps:

[0151] Based on the genotypic data of the selected hybrid offspring plants, key variation information of the target gene region is extracted;

[0152] The correlation analysis between genotype data and population phenotypic data, including the expression of target traits, agronomic traits, and environmental adaptability, was performed.

[0153] Statistical calculations were performed to determine the correlation between the target genotype and the target trait, and the association pattern between genotype and target trait was identified.

[0154] Based on the association analysis results, plants with a significant association between genotype and target trait were initially screened as candidate plants, and the genotype and phenotypic expression of the candidate plants were cross-validated.

[0155] Phenotypic tests of the target traits were conducted on the selected candidate plants under different environmental conditions to evaluate the stability of the target traits and the differences in their performance under multiple environmental conditions.

[0156] Plants that perform well in multiple environments and are consistent with the target genotype are selected as the final high-quality plants.

[0157] In the above embodiments, by extracting genotypic data from hybrid offspring plants, screening key variation information in the target gene region, and combining this with population phenotypic data (such as target traits, agronomic traits, and environmental adaptability) for association analysis, the association pattern between the target genotype and the target trait can be comprehensively revealed. Statistical calculation of the correlation between the target genotype and the target trait helps to initially screen plants that are significantly associated with the target trait.

[0158] Based on this, candidate plants were subjected to phenotypic testing under multiple environmental conditions to evaluate the stability of their target traits and their adaptability to various environments, ensuring that the final selected plants perform excellently in practical applications. Furthermore, joint analysis of genotype and phenotype can optimize candidate plant selection strategies, achieving precise conversion from genotype to phenotype, and providing a reliable basis for creating high-quality restorer lines with stable inheritance of target traits.

[0159] The validation of candidate plants and the creation of restorer lines also include the following steps:

[0160] Select the high-quality plants that were finally screened as candidate materials for restorer lines;

[0161] Candidate materials are crossbred with high-quality sterile lines to construct hybrid combinations;

[0162] Hybrid offspring were bred under greenhouse or field conditions, and their target traits and other key agronomic traits were initially observed and the plant performance was recorded.

[0163] Based on the target traits and yield performance, superior hybrid combinations are selected to proceed to the next stage of trials;

[0164] The selected superior hybrid combinations were subjected to field trials. During the trials, data such as the performance of the target traits, yield indicators, and adaptability of the hybrid combinations were recorded.

[0165] By combining the results of multi-environmental tests, we analyzed the stability and yield performance of the target traits of the candidate materials under different environmental conditions, and confirmed the practical application value of the candidate materials based on the test results.

[0166] High-performing candidate materials are used as the base materials for restorer lines. Combined with target traits and genetic background, genotype optimization is carried out, and generation selection and verification are performed on candidate plants.

[0167] To create japonica rice restorer lines that meet the target trait requirements and have a good and stable genetic background.

[0168] In the above embodiments, the practical application value of candidate plants is verified, their genetic background and target traits are optimized, high-quality restorer lines are created, and the comprehensive performance of candidate plants is verified through multi-environmental experiments to ensure their wide application and stability. The created restorer lines have significant advantages in terms of yield, adaptability and target traits.

[0169] Meanwhile, the process for setting up different environmental conditions is as follows:

[0170] Extract the environmental parameters corresponding to the candidate plants, including temperature, humidity and light intensity parameters.

[0171] Extract the preset reference parameters corresponding to the condition parameters, wherein the preset reference parameters include temperature reference parameters, humidity reference parameters and light intensity reference parameters;

[0172] Extract the theoretical change in ambient temperature generated by the current light intensity benchmark parameter within a preset light unit time period; wherein the value range of the light unit time period is 30min-60min;

[0173] Extract the actual ambient temperature change generated by the light intensity reference parameter during a preset light unit time period;

[0174] The parameter adjustment coefficient is obtained by using the actual ambient temperature change and the theoretical ambient temperature change of the light intensity reference parameter during a preset illumination unit time period.

[0175] The parameter adjustment coefficient is obtained by the following formula:

[0176]

[0177] Where R represents the parameter adjustment coefficient; T x T represents the theoretical change in ambient temperature caused by the light intensity reference parameter within a preset unit of time during which illumination occurs; s This represents the actual change in ambient temperature caused by the light intensity reference parameter within a preset light intensity unit time period; L x S represents the distance between the light source and the plant; x This indicates the duration corresponding to a preset unit of illumination time.

[0178] The parameter adjustment coefficient is compared with a preset parameter adjustment coefficient threshold.

[0179] When the parameter adjustment coefficient does not exceed the preset parameter adjustment coefficient threshold, multiple sets of different environmental conditions are set using the preset upper limit values ​​corresponding to the temperature gradient parameter, the upper limit value corresponding to the humidity reference parameter, and the upper limit value corresponding to the light intensity gradient parameter.

[0180] When the parameter adjustment coefficient exceeds the preset parameter adjustment coefficient threshold, the upper limit values ​​corresponding to the temperature gradient parameter, the humidity reference parameter, and the light intensity gradient parameter are adjusted.

[0181] The technical effects of the above solution are as follows: By extracting environmental condition parameters (temperature, humidity, light intensity) corresponding to candidate plants and comparing them with preset benchmark parameters, it can be ensured that the environmental conditions are set based on scientific and standardized benchmarks, thereby improving the accuracy of environmental control. By calculating the theoretical and actual changes in ambient temperature generated by the light intensity benchmark parameter within a preset light unit time period, and obtaining the parameter adjustment coefficient accordingly, the specific impact of light intensity on ambient temperature can be quantitatively analyzed. This helps to more accurately predict and adjust environmental conditions to adapt to the growth needs of different plants. Based on the comparison results of the parameter adjustment coefficient and the preset parameter adjustment coefficient threshold, the environmental conditions are dynamically adjusted. When the parameter adjustment coefficient does not exceed the threshold, a more lenient environmental condition setting (upper limit value) is adopted; when it exceeds the threshold, the conditions are adjusted more strictly. This strategy can flexibly respond to different situations, ensuring that plants grow under optimal environmental conditions. By precisely setting and adjusting environmental conditions, the growth environment of plants can be optimized, thereby improving their growth efficiency and quality. This is of great significance for agricultural production, plant research, and other fields. This technical solution not only considers the impact of light intensity on ambient temperature but also enhances the system's adaptability and flexibility through the setting of parameter adjustment coefficients and thresholds. This allows the system to make personalized adjustments based on different plants, different growth stages, and different environmental conditions. This technical solution integrates multiple aspects such as environmental parameter extraction, quantitative analysis, and dynamic adjustment, reflecting the development trend of intelligent agriculture. Optimizing environmental conditions through intelligent means helps improve agricultural production efficiency and quality, and promotes sustainable agricultural development.

[0182] In conclusion, this technical solution improves plant growth efficiency and quality by precisely setting and adjusting environmental conditions, and enhances the system's adaptability and flexibility, which is of great significance for promoting the intelligent development of agriculture.

[0183] Specifically, when the parameter adjustment coefficient exceeds a preset parameter adjustment coefficient threshold, the upper limit values ​​corresponding to the temperature gradient parameter, the humidity reference parameter, and the light intensity gradient parameter are adjusted, including:

[0184] When the parameter adjustment coefficient exceeds the preset parameter adjustment coefficient threshold, the upper limit value corresponding to the temperature gradient parameter, the upper limit value corresponding to the humidity reference parameter, and the upper limit value corresponding to the light intensity gradient parameter are retrieved.

[0185] Retrieve parameter adjustment coefficients;

[0186] The upper limits corresponding to the temperature gradient parameter, the humidity reference parameter, and the light intensity gradient parameter are adjusted using the parameter adjustment coefficients to obtain the adjusted upper limits corresponding to the temperature gradient parameter, the humidity reference parameter, and the light intensity gradient parameter.

[0187] The upper limit value of the adjusted temperature gradient parameter is obtained by the following formula:

[0188]

[0189] Among them, T up This indicates the upper limit of the adjusted temperature gradient parameter; T up0 This indicates the upper limit of the temperature gradient parameter before adjustment; R represents the parameter adjustment coefficient.

[0190] Meanwhile, the upper limit value corresponding to the adjusted humidity reference parameter is obtained by the following formula:

[0191]

[0192] Among them, W up This indicates the upper limit value corresponding to the adjusted humidity gradient parameter; W up0 This indicates the upper limit of the humidity gradient parameter before adjustment; T up This indicates the upper limit of the adjusted temperature gradient parameter; T up0 This indicates the upper limit of the temperature gradient parameter before adjustment; R represents the parameter adjustment coefficient.

[0193] Furthermore, the upper limit value corresponding to the adjusted light intensity gradient parameter is obtained by the following formula:

[0194]

[0195] Among them, G up This represents the upper limit of the adjusted illumination intensity gradient parameter; G up0 This represents the upper limit of the light intensity gradient parameter before adjustment; W up This indicates the upper limit value corresponding to the adjusted humidity gradient parameter; W up0 This indicates the upper limit of the humidity gradient parameter before adjustment; T up This indicates the upper limit of the adjusted temperature gradient parameter; T up0 This indicates the upper limit of the temperature gradient parameter before adjustment; R represents the parameter adjustment coefficient.

[0196] Multiple sets of different environmental conditions are set using the adjusted upper limits of the temperature gradient parameter, the humidity baseline parameter, and the light intensity gradient parameter.

[0197] The technical effect of the above solution is as follows: when the parameter adjustment coefficient exceeds a preset threshold, the solution automatically retrieves the current upper limit values ​​of environmental parameters (temperature, humidity, light intensity) and the parameter adjustment coefficient, and adjusts accordingly. This dynamic response mechanism enhances the system's adaptability to different environmental conditions, ensuring that plants can grow in a more precise and suitable environment. The adjusted upper limits of temperature, humidity, and light intensity are calculated using specific formulas that take into account the influence of the parameter adjustment coefficient on the original upper limits. This calculation method ensures the accuracy and scientific nature of parameter adjustment, avoiding the adverse effects that may result from blind adjustments. By setting multiple sets of different environmental conditions using the adjusted upper limits, the plant's growth environment can be further optimized. This optimization not only helps improve plant growth efficiency and quality but also reduces growth obstacles and disease risks caused by unsuitable environmental conditions. Precise environmental condition settings help reduce unnecessary resource waste, such as energy, water, and fertilizer. By optimizing environmental conditions, plants can be ensured to grow in optimal conditions, thereby improving resource utilization efficiency. This technical solution integrates multiple stages, including parameter extraction, quantitative analysis, dynamic adjustment, and intelligent setting, reflecting the development trend of intelligent and automated agriculture. Optimizing environmental conditions through intelligent means can reduce the frequency and intensity of human intervention, improving agricultural production efficiency and quality. By precisely setting and adjusting environmental conditions, this technical solution contributes to the sustainable development of agricultural production. Optimized environmental conditions not only improve plant growth efficiency and quality but also reduce negative environmental impacts, such as reducing the use of chemical fertilizers and pesticides, and lowering the risk of soil and water pollution.

[0198] In summary, this technical solution, through a dynamic response mechanism, precise and scientific parameter adjustment methods, and optimized environmental conditions, significantly improves plant growth efficiency and quality, enhances the system's adaptability and flexibility, and promotes intelligent and sustainable agricultural development.

[0199] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for screening target gene plants in japonica rice parent-to-parent hybrids based on high-throughput sequencing, characterized in that, Includes the following steps: Parental material selection and target gene determination: Select high-quality japonica rice parental materials, identify genes related to the target trait through database analysis, and clarify the region and distribution characteristics of the target gene according to the breeding requirements of the target trait. The process involves identifying the genotypes of both parents and comparing their genomes. Whole-genome sequencing is performed on both parents to obtain their genome data. The sequencing data is then compared to a reference genome to identify genetic differences between the parents, screen for specific markers of the target gene region, and simultaneously identify key bridging regions between the indica and japonica rice genomes of both parents to determine the differentially expressed segments of the target gene. The development and validation of molecular markers for target regions involves designing molecular markers related to target genes based on the genomic differences between parents obtained through high-throughput sequencing. These molecular markers cover key sites and upstream and downstream regions of the target gene region, and the markers are validated molecularly on parental materials and hybrid offspring. The construction of hybridization and backcross populations involves hybridization of two parents of japonica rice with the target gene as the core, obtaining F1 generation plants, selecting superior F1 generation plants for backcrossing to construct a backcross population, and selecting plants in combination with the breeding objectives during the population construction process. High-throughput sequencing and genotyping were performed. Genomic DNA was extracted from the hybrid progeny plants and amplified by PCR. SSR or snp markers of the restorer gene and the wide-compatibility gene were converted into Kasp markers. The restorer gene included Rf1 and Rf4, and the wide-compatibility gene included s5, Sc, pf12 and f5. An amplicon library of the target region was constructed. The amplicon library was sequenced using high-throughput sequencing. The sequencing data were subjected to quality control, alignment and variation analysis to identify the genotype of the hybrid progeny plants. Plants containing the target gene were preliminarily screened to provide recurrent parents for the backcross population. Combined genotypic and phenotypic analysis combines the results of genotypic analysis with population phenotypic data to verify the phenotypic association of the target gene. By comprehensively analyzing the genotypic and phenotypic data of the plants, superior plants with significant association between genotype and target trait are screened out. Verification of candidate plants and creation of restorer lines: The selected target plants are used as candidate materials for restorer lines and are crossbred with sterile lines to construct hybrid combinations. The yield potential and target trait performance of the combinations are verified. Based on the verification results, high-quality candidate plants are used as breeding materials to create new restorer lines. The combined analysis of genotype and phenotype specifically includes the following steps: Based on the genotypic data of the selected hybrid offspring plants, key variation information of the target gene region is extracted; The correlation analysis between genotype data and population phenotypic data, including the expression of target traits, agronomic traits, and environmental adaptability, was performed. Statistical calculations were performed to determine the correlation between the target genotype and the target trait, and the association pattern between genotype and target trait was identified. Based on the association analysis results, plants with a significant association between genotype and target trait were initially screened as candidate plants, and the genotype and phenotypic expression of the candidate plants were cross-validated. Phenotypic tests of the target traits were conducted on the selected candidate plants under different environmental conditions to evaluate the stability of the target traits and the differences in their performance under multiple environmental conditions. Plants that perform well in multiple environments and are consistent with the target genotype are selected as the final high-quality plants. The process for setting up different environmental conditions is as follows: Extract the environmental parameters corresponding to the candidate plants, including temperature, humidity and light intensity parameters. Extract the preset reference parameters corresponding to the condition parameters, wherein the preset reference parameters include temperature reference parameters, humidity reference parameters and light intensity reference parameters; Extract the theoretical change in ambient temperature generated by the current light intensity benchmark parameter within a preset light unit time period; wherein the value range of the light unit time period is 30min-60min; Extract the actual ambient temperature change generated by the light intensity reference parameter during a preset light unit time period; The parameter adjustment coefficient is obtained by using the actual ambient temperature change and the theoretical ambient temperature change of the light intensity reference parameter during a preset illumination unit time period. The parameter adjustment coefficient is obtained by the following formula: ； Where R represents the parameter adjustment coefficient; T x T represents the theoretical change in ambient temperature caused by the light intensity reference parameter within a preset unit of time during which illumination occurs; s This represents the actual change in ambient temperature caused by the light intensity reference parameter within a preset light intensity unit time period; L x S represents the distance between the light source and the plant; x This indicates the duration corresponding to a preset unit of illumination time. The parameter adjustment coefficient is compared with a preset parameter adjustment coefficient threshold. When the parameter adjustment coefficient does not exceed the preset parameter adjustment coefficient threshold, multiple sets of different environmental conditions are set using the preset upper limit values ​​corresponding to the temperature gradient parameter, the upper limit value corresponding to the humidity reference parameter, and the upper limit value corresponding to the light intensity gradient parameter. When the parameter adjustment coefficient exceeds the preset parameter adjustment coefficient threshold, the upper limit value corresponding to the temperature gradient parameter, the upper limit value corresponding to the humidity reference parameter, and the upper limit value corresponding to the light intensity gradient parameter are adjusted. When the parameter adjustment coefficient exceeds the preset parameter adjustment coefficient threshold, the upper limit value corresponding to the temperature gradient parameter, the upper limit value corresponding to the humidity reference parameter, and the upper limit value corresponding to the light intensity gradient parameter are retrieved. Retrieve parameter adjustment coefficients; The upper limits corresponding to the temperature gradient parameter, the humidity reference parameter, and the light intensity gradient parameter are adjusted using the parameter adjustment coefficients to obtain the adjusted upper limits corresponding to the temperature gradient parameter, the humidity reference parameter, and the light intensity gradient parameter. The upper limit value of the adjusted temperature gradient parameter is obtained by the following formula: ； Among them, T up This indicates the upper limit of the adjusted temperature gradient parameter; T up0 This represents the upper limit of the temperature gradient parameter before adjustment; R represents the parameter adjustment coefficient. Meanwhile, the upper limit value corresponding to the adjusted humidity reference parameter is obtained by the following formula: ； Among them, W up This indicates the upper limit value corresponding to the adjusted humidity gradient parameter; W up0 This indicates the upper limit of the humidity gradient parameter before adjustment; T up This indicates the upper limit of the adjusted temperature gradient parameter; T up0 This represents the upper limit of the temperature gradient parameter before adjustment; R represents the parameter adjustment coefficient. Furthermore, the upper limit value corresponding to the adjusted light intensity gradient parameter is obtained by the following formula: ； Among them, G up This represents the upper limit of the adjusted illumination intensity gradient parameter; G up0 This represents the upper limit of the light intensity gradient parameter before adjustment; W up This indicates the upper limit value corresponding to the adjusted humidity gradient parameter; W up0 This indicates the upper limit of the humidity gradient parameter before adjustment; T up This indicates the upper limit of the adjusted temperature gradient parameter; T up0 This represents the upper limit of the temperature gradient parameter before adjustment; R represents the parameter adjustment coefficient. Multiple sets of different environmental conditions are set using the adjusted upper limits of the temperature gradient parameter, the humidity baseline parameter, and the light intensity gradient parameter.

2. The method for screening target gene plants in japonica rice parent-to-parent hybridization based on high-throughput sequencing as described in claim 1, characterized in that, The selection of parental materials and determination of target genes specifically include the following steps: Select parental materials with excellent genetic characteristics from superior japonica rice varieties or breeding resource banks. The excellent genetic characteristics include high yield, wide adaptability, salt and alkali resistance, and resistance to diseases and pests. Priority will be given to materials that are known to have the target traits or may be related to the target gene. Target genes that are significantly associated with the target trait are screened through gene function database analysis. The target trait includes resilience, seed setting rate, or broad affinity. Identify the gene locus, chromosomal region, and known gene function of the target gene; Based on existing genomic information of the parents, and combined with the genetic background of the target trait, the distribution and differentially expressed regions of the target gene in the parent genomes are inferred. The target gene region is initially delineated to a specific chromosome range.

3. The method for screening target gene plants in japonica rice parent-to-parent hybridization based on high-throughput sequencing as described in claim 1, characterized in that, The parental genotyping and genome comparison also includes the following steps: Whole-genome sequencing was performed on the selected japonica rice parent materials, and high-throughput sequencing was used to generate sequencing data with a sequencing data depth of ≥30×. Sequencing data from both parents' genomes were aligned to a reference genome to analyze genetic differences between the two genomes, including single nucleotide polymorphisms and insertions / deletions. By combining the gene locus of the target gene with known gene functions, specific markers for the region where the target gene is located are screened. Based on the comparative analysis results and combined with specific fragments of the parent genomes, key regions related to heterosis and genetic stability in indica and japonica hybrids were identified, and the distribution and differential information of target gene regions in the parent genomes were determined.

4. The method for screening target gene plants in japonica rice parent-to-parent hybridization based on high-throughput sequencing as described in claim 1, characterized in that, The development and validation of molecular markers for the target region further includes the following steps: Based on the differential information of the parent genomes and the localization results of the target gene region, molecular markers covering key sites in the target gene region were designed; The molecular markers include single nucleotide polymorphism markers and insertion / deletion markers; The molecular markers simultaneously cover key sequences at both ends of the target region and in the middle; Molecular markers were used to amplify the biparental materials by PCR, and the accuracy and specificity of the markers were verified by sequencing. Based on the marker distribution of parental materials and plants with known phenotypes, high-quality markers that can significantly distinguish the target genotype are screened. A molecular detection system was established by combining the selected high-quality markers; By comparing the marker detection results with the target phenotype data, it was confirmed that the marker could reflect the target genotype.

5. The method for screening target gene plants in japonica rice parent-to-parent hybridization based on high-throughput sequencing as described in claim 1, characterized in that, The construction of the hybridization and backcross populations also includes the following steps: Select superior single plants from the parent materials as hybridization parents, and carry out artificial hybridization to obtain F1 generation plants. Hybridization is preferably carried out under the condition that the target trait is stable and the flowering period is consistent. During the F1 generation plant cultivation process, the growth status and target traits were initially observed, and relevant agronomic traits were recorded. F1 generation plants were screened, and individual plants that met the breeding objectives were selected based on the marker detection results of the target gene and the phenotypic expression of the target trait. The selected F1 generation plants were used as donor parents and backcrossed with recipient parents to construct a backcross population; In the process of constructing backcross populations, the population size is further expanded through multiple generations of backcrossing and self-crossing; In each generation of backcrossing and selfcrossing, by combining the results of molecular marker detection and the phenotypic expression of the target trait, plants with high yield, wide adaptability and excellent target traits are selected.

6. The method for screening target gene plants in japonica rice parent-to-parent hybridization based on high-throughput sequencing as described in claim 1, characterized in that, The high-throughput sequencing and genotyping analysis specifically includes the following steps: Genomic DNA was extracted from hybrid offspring plants and amplified by PCR using specific molecular markers targeting the target gene region, so that the amplified fragments covered the key sites and upstream and downstream regions of the target gene. After purifying the amplified products, an amplicon library of the target region is constructed by combining the length and coverage of the amplicon sequence. High-throughput sequencing platforms were used to perform deep sequencing on amplicon libraries of the target region. Quality control was performed on the sequencing data to remove low-quality reads, adapter sequences, and repetitive sequences. Sequencing data is aligned to a reference genome to analyze variation information within the target gene region, including single nucleotide polymorphisms, insertions and deletions, and the distribution of target gene-specific markers. By combining the genotype data of the parents, the genotypes of the hybrid offspring are identified and the genotype information is classified and labeled, including homozygous parental type, heterozygous type and non-parental type; Based on the variation analysis results of sequencing data, combined with the distribution of target region-specific markers, plants containing the target gene were screened. For plants that have undergone preliminary screening, record the variation information and genotype category within the target gene region; The screening results were compared and analyzed with the preliminary phenotypic data of the population to identify candidate plants that were significantly associated with the target genotype and the target trait.

7. The method for screening target gene plants in japonica rice parent-to-parent hybridization based on high-throughput sequencing as described in claim 1, characterized in that, The verification and restoration line creation of the candidate plants specifically includes the following steps: Select the high-quality plants that were finally screened as candidate materials for restorer lines; Candidate materials are crossbred with high-quality sterile lines to construct hybrid combinations; Hybrid offspring were bred under greenhouse or field conditions, and their target traits and other key agronomic traits were initially observed and the plant performance was recorded. Based on the target traits and yield performance, superior hybrid combinations are selected to proceed to the next stage of trials; The selected superior hybrid combinations were subjected to field trials. During the trials, data such as the performance of the target traits, yield indicators, and adaptability of the hybrid combinations were recorded. By combining the results of multi-environmental tests, we analyzed the stability and yield performance of the target traits of the candidate materials under different environmental conditions, and confirmed the practical application value of the candidate materials based on the test results. High-performing candidate materials are used as the base materials for restorer lines. Combined with target traits and genetic background, genotype optimization is carried out, and generation selection and verification are performed on candidate plants. To create japonica rice restorer lines that meet the target trait requirements and have a good and stable genetic background.

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