Method for screening japonica rice amphiphilic hybridization target gene plants based on high-throughput sequencing

Abstract

The invention discloses a japonica rice amphiphilic hybridization target gene plant screening method based on high-throughput sequencing, and belongs to the technical field of agricultural biology. A high-throughput sequencing technology is utilized, the genetic difference of parent genomes is accurately analyzed, specific molecular markers covering target gene areas are developed, plants containing target genes can be rapidly recognized, the screening efficiency and accuracy are greatly improved, the breeding period is shortened, resources are saved, and the method is suitable for large-scale popularization and application. Through combination of molecular marker detection and phenotypic expression of target characters, in the construction process of hybridization and backcross populations, target genes are fixed generation by generation, stable inheritance of the target characters, joint analysis of genetic types and phenotypes and multi-environment testing are ensured, correlation between target genotypes and the target characters and environmental adaptability of the target genotypes and the target characters are verified, and the target genotypes and the target characters are identified. The stability and reliability of the breeding material are improved, in candidate plant screening and restorer line creation, an excellent hybrid combination is constructed, genotype optimization is realized, and the quality and yield potential of the breeding material are improved.

Description

Technical Field

[0001] The invention relates to the field of agricultural biotechnology, and in particular to a method for screening target gene plants of japonica rice parental hybridization based on high-throughput sequencing. Background Art

[0002] In recent years, the rapid development of molecular marker-assisted breeding and genomics technology has provided new directions for rice breeding.

[0003] However, traditional breeding methods usually rely on phenotypic selection, which has a long cycle, low efficiency, and is easily affected by environmental factors, making it difficult to accurately screen plants with excellent target traits. Currently, there are still few specific applications of high-throughput sequencing of japonica rice in hybrid breeding, especially how to combine molecular marker development, genotype analysis and hybrid breeding processes, which still has certain technical defects. Summary of the invention

[0004] The purpose of the present invention is to provide a method for screening target gene plants of japonica rice parental hybridization based on high-throughput sequencing to solve the problems raised in the above background technology.

[0005] To achieve the above object, the present invention provides the following technical solution: a method for screening target gene plants of japonica rice parental hybrids based on high-throughput sequencing, comprising the following steps:

[0006] Parent material selection and target gene determination: select high-quality japonica rice parent materials, determine the genes related to the target traits 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 traits;

[0007] Identify the genotypes of the parents and compare their genomes. Perform whole genome sequencing on the parental materials to obtain the parental genome data. Compare the sequencing data to the reference genome, identify the genetic differences between the parents, screen for specific markers in the target gene region, and simultaneously mine the key indica-japonica bridging regions of the parental genomes to determine the differential fragments of the target gene.

[0008] Development and verification of molecular markers in the target region: based on the information of parental genomic differences obtained through high-throughput sequencing, molecular markers related to the target gene are designed. The molecular markers cover the key sites and upstream and downstream regions of the target gene region, and molecular verification is performed on the markers of the parental materials and hybrid offspring;

[0009] Hybridization and backcross population construction: With the target gene as the core, the japonica rice parents are hybridized to obtain F1 generation plants, and excellent F1 generation plants are selected for backcrossing to construct backcross populations. During the population construction process, plants are selected in combination with breeding goals;

[0010] High-throughput sequencing and genotype analysis, extracting genomic DNA from hybrid offspring plants for PCR amplification, converting SSR markers or SNP markers of restoration genes and wide-affinity genes into Kasp markers, wherein the restoration genes include Rf1 and Rf4, and the wide-affinity 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, comparison and variation analysis on the sequencing data, identifying the genotype of the hybrid offspring plants, and preliminarily screening out plants containing the aggregated target genes to provide the recurrent parents of the backcross population;

[0011] Combined genotype and phenotype analysis: combining genotype analysis results with population phenotypic data to verify the phenotypic correlation of the target gene, comprehensively analyzing the genotype and phenotypic data of the plant, and screening out excellent plants with significant correlation between genotype and target traits;

[0012] Verification of candidate plants and creation of restorer lines: The screened target plants are used as restorer line candidate materials, hybridized with sterile lines, and hybrid combinations are constructed to verify the yield potential and target trait performance of the combination. Based on the verification results, high-quality candidate plants are used as breeding materials to create new restorer lines.

[0013] Furthermore, the selection of parent materials and determination of target genes specifically include the following steps:

[0014] Screening parental materials with excellent genetic characteristics from excellent japonica rice varieties or breeding resource banks, wherein the excellent genetic characteristics include high yield, wide adaptability, salt-alkali resistance, and resistance to pests and diseases, and giving priority to materials known to have target traits or that may be related to target genes;

[0015] Screening target genes significantly associated with target traits through gene function database analysis, wherein the target traits include resilience, fruit setting rate or wide affinity;

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

[0017] Based on the marker data of the existing genomic information of the parental materials and the genetic background of the target trait, the distribution of the target gene in the parental genome and the differential fragment regions are inferred;

[0018] The target gene region was initially delineated to a specific chromosome range.

[0019] Furthermore, the parental genotype identification and genome comparison specifically includes the following steps:

[0020] The whole genome of the selected japonica rice parent materials was sequenced, and high-throughput sequencing was used to generate sequencing data with a depth of greater than or equal to 30×;

[0021] Aligning the sequencing data of the parental genomes to the reference genome, and analyzing the genetic differences between the parental genomes, wherein the genetic differences include single nucleotide polymorphisms and insertions and deletions;

[0022] Combine the gene locus of the target gene and the known gene function to screen out specific markers in the region where the target gene is located;

[0023] Based on the comparison analysis results and combined with specific fragments of the parental genomes, the key regions related to indica-japonica hybrid vigor and genetic stability were identified, and the distribution and difference information of the target gene regions in the parental genomes were determined.

[0024] Furthermore, the target region molecular marker development and verification specifically includes the following steps:

[0025] Based on the difference information of the parental genomes and the localization results of the target gene region, molecular markers covering the key sites of the target gene region are designed;

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

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

[0028] PCR amplification of parental materials using molecular markers, combined with sequencing to verify the accuracy and specificity of the markers;

[0029] Based on the distribution of markers of parental materials and plants with known phenotypes, screen for high-quality markers that can significantly distinguish the target genotypes;

[0030] Combine the selected high-quality markers to establish a molecular detection system;

[0031] By comparing the marker test results with the target phenotype data, it is confirmed that the marker can reflect the target genotype.

[0032] Furthermore, the hybridization and backcrossing population construction specifically includes the following steps:

[0033] Select superior individual plants from the parental materials as hybrid parents, perform artificial hybridization to obtain F1 generation plants, preferably perform hybridization under conditions where the target trait is stable and the flowering period is consistent;

[0034] During the cultivation of F1 plants, preliminary observations were made on growth and target traits, and relevant agronomic traits were recorded;

[0035] Screen the F1 generation plants and select the plants that meet the breeding goals based on the marker detection results of the target gene and the phenotypic performance of the target trait;

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

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

[0038] In each generation of backcrossing and selfing, the results of molecular marker detection and the phenotypic expression of the target traits are combined to select plants with high yield, wide adaptability and excellent target traits.

[0039] Furthermore, the high-throughput sequencing and genotype analysis specifically further comprises the following steps:

[0040] Extract genomic DNA from hybrid offspring plants, and perform PCR amplification using specific molecular markers targeting the target gene region, so that the amplified fragment covers the key sites and upstream and downstream regions of the target gene;

[0041] After purifying the amplified product, the amplicon library of the target region is constructed based on the length and coverage of the amplicon sequence;

[0042] A high-throughput sequencing platform was used to perform deep sequencing of the amplicon library of the target region, and the sequencing data was quality controlled to remove low-quality reads, adapter sequences, and repeated sequences;

[0043] Align the sequencing data to the reference genome and analyze the variation information within the target gene region, including single nucleotide polymorphisms, insertions and deletions, and the distribution of target gene-specific markers;

[0044] Combining the genotype data of the parents, identifying the genotype of the hybrid offspring, and classifying and annotating the genotype information, the genotype information includes homozygous parent type, heterozygous type and non-parent type;

[0045] Based on the results of variation analysis of sequencing data and the distribution of specific markers in the target region, plants containing the target gene are screened;

[0046] For the initially screened plants, the variation information and genotype categories within the target gene region were recorded;

[0047] The screening results were compared with the preliminary phenotypic data of the population to screen out candidate plants with significant associations between 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 genotype data of the screened hybrid offspring plants, key variation information of the target gene region is extracted;

[0050] Performing association analysis on genotype data and population phenotypic data, wherein the population phenotypic data includes target trait performance, agronomic traits and environmental adaptability;

[0051] Statistically calculate the correlation between the target genotype and the target trait, and determine the association pattern between the genotype and the target trait;

[0052] According to the results of association analysis, plants with significant association between genotype and target trait were preliminarily selected as candidate plants, and the genotype and phenotypic performance of the candidate plants were cross-validated;

[0053] Conduct phenotypic tests on target traits of the selected candidate plants under different environmental conditions to evaluate the stability of the target traits of the candidate plants and their performance differences under multiple environmental conditions;

[0054] Plants that perform well in multiple environments and are consistent with the target genotype are identified as the high-quality plants that are finally screened out.

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

[0056] Extracting condition parameters contained in the environmental conditions corresponding to the candidate plants, wherein the condition parameters include temperature parameters, humidity parameters and light intensity parameters;

[0057] Extracting 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 ambient temperature change generated by the current illumination intensity reference parameter in a preset illumination unit time period; wherein the illumination unit time period has a value range of 30min-60min;

[0059] Extracting the actual ambient temperature change generated by the illumination intensity reference parameter in a preset illumination unit time period;

[0060] Obtaining a parameter adjustment coefficient using an actual ambient temperature variation amount generated by the illumination intensity reference parameter in a preset illumination unit time period and a theoretical ambient temperature variation amount generated by the illumination intensity reference parameter in 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 Indicates the theoretical ambient temperature change caused by the light intensity reference parameter in the preset light unit time period; T sIndicates the actual ambient temperature change caused by the light intensity reference parameter in the preset light unit time period; L x Indicates the distance between the light source and the plant; S x Indicates the time length corresponding to the preset illumination unit time period;

[0064] Comparing the parameter adjustment coefficient with a preset parameter adjustment coefficient threshold;

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

[0066] When the parameter adjustment coefficient exceeds a 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.

[0067] Further, when the parameter adjustment coefficient exceeds a 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, 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 coefficient;

[0070] The parameter adjustment coefficient is used to adjust 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 to obtain the adjusted 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;

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

[0072]

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

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

[0075]

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

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

[0078]

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

[0080] A plurality of different sets of environmental conditions are set using the adjusted temperature gradient parameter corresponding to the upper limit value, the humidity reference parameter corresponding to the upper limit value, and the light intensity gradient parameter corresponding to the upper limit value.

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

[0082] The high-quality plants finally screened out are selected as candidate materials for the restorer line;

[0083] Cross-breeding candidate materials with high-quality sterile lines to construct hybrid combinations;

[0084] Grow hybrid offspring under greenhouse or field conditions, make preliminary observations of target traits and other key agronomic traits, and record plant performance;

[0085] Combine the target traits and yield performance to select excellent hybrid combinations and enter the next stage of testing;

[0086] Conduct field trials on the selected excellent hybrid combinations, and record the target trait performance, yield index, adaptability and other data of the hybrid combinations during the trials;

[0087] Combine the results of multi-environment tests to analyze the stability of target traits and yield performance of candidate materials under different environmental conditions, and confirm the actual application value of candidate materials based on the test results;

[0088] The candidate materials with excellent performance are used as the basic materials of the restorer line, and the genotype is optimized based on the target traits and genetic background, and the candidate plants are selected and verified for generations;

[0089] Create japonica rice restorer line materials that meet the target trait requirements and have excellent and stable genetic background.

[0090] Compared with the prior art, the present invention has the following beneficial effects:

[0091] 1. Through the application of high-throughput sequencing technology, the present invention can accurately analyze the whole genome genetic differences of parental materials, locate the specific fragments of the target gene, and develop molecular markers covering the target area. These molecular markers have high sensitivity and high specificity, and can quickly identify the target genotype of hybrid offspring plants. Compared with traditional breeding methods that rely on phenotypic observation, the efficiency and accuracy of screening are greatly improved. Combining molecular marker detection with genotype analysis of high-throughput sequencing data can effectively distinguish homozygous, heterozygous and non-target plants, reduce blindness in screening, and thus significantly save breeding resources.

[0092] 2. The present invention combines the construction strategy of multi-generation hybridization and backcrossing populations, uses molecular markers and target phenotypic expressions to perform precise screening in each generation of plants, gradually fixes the inheritance of target genes, and achieves stable inheritance of target traits. At the same time, in the process of joint analysis of genotype and phenotype, multi-environment phenotypic testing of candidate plants is used to verify the stability and consistency of the target genotype, thereby ensuring that the plants finally screened out show excellent target traits under different environmental conditions, laying the foundation for promotion and application in breeding practice.

[0093] 3. The present invention constructs hybrid combinations and conducts comprehensive evaluations of their target traits and agronomic traits, thereby screening out candidate plants with excellent performance and using them to create new restorer line materials. Combined with efficient genotype analysis and phenotypic verification methods, the genotype optimization and generational improvement of candidate materials can be achieved, significantly improving breeding efficiency. At the same time, through a high-throughput, high-precision molecular detection system, an advanced technical platform is provided for the cultivation 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0094] Figure 1 The figure is a schematic diagram of the process of the gene plant screening method of the present invention. DETAILED DESCRIPTION

[0095] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0096] See also Figure 1 , the present invention provides the following technical solutions:

[0097] A method for screening target gene plants of japonica rice parental hybrids based on high-throughput sequencing comprises the following steps:

[0098] Parent material selection and target gene determination: select high-quality japonica rice parent materials, determine the genes related to the target traits 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 traits;

[0099] Identify the genotypes of the parents and compare their genomes. Perform whole genome sequencing on the parental materials to obtain the parental genome data. Compare the sequencing data to the reference genome, identify the genetic differences between the parents, screen for specific markers in the target gene region, and simultaneously mine the key indica-japonica bridging regions of the parental genomes to determine the differential fragments of the target gene.

[0100] Development and verification of molecular markers in the target region: based on the information of parental genomic differences obtained through high-throughput sequencing, molecular markers related to the target gene are designed. The molecular markers cover the key sites and upstream and downstream regions of the target gene region, and molecular verification is performed on the markers of the parental materials and hybrid offspring;

[0101] Hybridization and backcross population construction: With the target gene as the core, the japonica rice parents are hybridized to obtain F1 generation plants, and excellent F1 generation plants are selected for backcrossing to construct backcross populations. During the population construction process, plants are selected in combination with breeding goals;

[0102] High-throughput sequencing and genotype analysis, extracting genomic DNA from hybrid offspring plants for PCR amplification, converting SSR markers or SNP markers of restoration genes and wide-affinity genes into Kasp markers, wherein the restoration genes include Rf1 and Rf4, and the wide-affinity 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, comparison and variation analysis on the sequencing data, identifying the genotype of the hybrid offspring plants, and preliminarily screening out plants containing the aggregated target genes to provide the recurrent parents of the backcross population;

[0103] Combined genotype and phenotype analysis: combining genotype analysis results with population phenotypic data to verify the phenotypic correlation of the target gene, comprehensively analyzing the genotype and phenotypic data of the plant, and screening out excellent plants with significant correlation between genotype and target traits;

[0104] Verification of candidate plants and creation of restorer lines: The screened target plants are used as restorer line candidate materials, hybridized with sterile lines, and hybrid combinations are constructed to verify the yield potential and target trait performance of the combination. Based on the verification results, high-quality candidate plants are used as breeding materials to create new restorer lines.

[0105] In the above embodiments, with the help of high-throughput sequencing and molecular marker technology, accurate positioning of the target gene region and rapid screening of target plants are achieved, which significantly shortens the breeding cycle and reduces the screening workload and cost. Whole genome sequencing is combined with molecular marker design to comprehensively analyze the genetic differences between parents and offspring to ensure the reliability and comprehensiveness of target gene screening. Through the joint analysis of genotype and phenotype, the selected candidate plants not only have the target traits, but also meet the needs of 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 materials have comprehensive advantages in genetic background, yield and target traits.

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

[0107] Screening parental materials with excellent genetic characteristics from excellent japonica rice varieties or breeding resource banks, wherein the excellent genetic characteristics include high yield, wide adaptability, salt-alkali resistance, and resistance to pests and diseases, and giving priority to materials known to have target traits or that may be related to target genes;

[0108] Screening target genes significantly associated with target traits through gene function database analysis, wherein the target traits include resilience, fruit setting rate or wide affinity;

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

[0110] Based on the marker data of the existing genomic information of the parental materials and the genetic background of the target trait, the distribution of the target gene in the parental genome and the differential fragment regions are inferred;

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

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

[0113] In the above-described embodiment, based on the target gene related to the target trait screening database analysis, its gene locus, chromosome region and gene function are clearly defined, not only providing a theoretical basis for the target gene positioning, but also significantly improving the success rate of subsequent molecular marker design. By combining the existing genome information and marker data of the parental material, the distribution of the target gene in the parental genome and the difference fragment region are inferred, the target gene region can be accurately located, reducing the risk of blind experiments. In the process, the target gene region is delineated to the specific chromosome range, providing a clear scope for subsequent molecular marker development, while avoiding the waste of resources caused by the excessive selection range.

[0114] The parental genotype identification and genome comparison specifically includes the following steps:

[0115] The whole genome of the selected japonica rice parent materials was sequenced, and high-throughput sequencing was used to generate sequencing data with a depth of greater than or equal to 30×;

[0116] Aligning the sequencing data of the parental genomes to the reference genome, and analyzing the genetic differences between the parental genomes, wherein the genetic differences include single nucleotide polymorphisms and insertions and deletions;

[0117] Combine the gene locus of the target gene and the known gene function to screen out specific markers in the region where the target gene is located;

[0118] Based on the comparison analysis results and combined with specific fragments of the parental genomes, the key regions related to indica-japonica hybrid vigor and genetic stability were identified, and the distribution and difference information of the target gene regions in the parental genomes were determined.

[0119] In the above embodiment, by performing whole genome sequencing on the selected japonica rice parent materials, high-quality sequencing data (depth ≥ 30×) is generated using high-throughput sequencing technology, which can fully capture the genetic information of the parental genome. High sequencing depth ensures the reliability and accuracy of the data, avoiding the omission of genetic differences caused by insufficient sequencing coverage. In addition, after aligning the sequencing data to the reference genome, the genetic differences between the parents can be clearly analyzed, including single nucleotide polymorphisms (SNPs) and insertion and deletion variations (InDe ls), thereby accurately identifying specific markers in the region where the target gene is located.

[0120] In the above-described embodiment, in conjunction with the known gene function of the target gene, the specific markers of the screening target region contribute to the subsequent molecular marker development, and improve the pertinence and applicability of the marker. Meanwhile, based on the comparison analysis of the parental genome specific fragments, the genetic key region related to the hybrid vigor of indica and japonica rice can also be excavated, providing important clues for studying the hybrid vigor of indica and japonica rice. By identifying the difference information of the target gene region in the parental genome, the genetic background of the target gene can be clearly defined, ensuring that subsequent experiments are carried out based on clear and definite genetic information.

[0121] The development and verification of molecular markers in the target area also includes the following steps:

[0122] Based on the difference information of the parental genomes and the localization results of the target gene region, molecular markers covering the key sites of the target gene region are designed;

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

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

[0125] PCR amplification of parental materials using molecular markers, combined with sequencing to verify the accuracy and specificity of the markers;

[0126] Based on the distribution of markers of parental materials and plants with known phenotypes, screen for high-quality markers that can significantly distinguish the target genotypes;

[0127] Combine the selected high-quality markers to establish a molecular detection system;

[0128] By comparing the marker test results with the target phenotype data, it is confirmed that the marker can reflect the target genotype.

[0129] In the above-mentioned embodiment, molecular markers covering key sites of the target region are designed based on the location results of the parental genome difference information and the target gene region, which can accurately reflect the target genotype and significantly improve the screening efficiency. During the marker design, the upstream and downstream ends of the target region and the intermediate key sequences are covered, which not only improves the comprehensiveness of the detection, but also avoids the risk of missing important sites. In addition, the use of various types of molecular markers such as single nucleotide polymorphism markers (SNPs) and insertion and deletion markers (InDe ls) can further improve the sensitivity and reliability of screening.

[0130] In the above examples, the accuracy and specificity of the molecular markers were verified by PCR amplification to ensure that the developed markers can significantly distinguish the genotypic differences between the parents. At the same time, combined with the distribution of markers of the parental materials and plants with known phenotypes, high-quality markers that can accurately distinguish the target genotype were screened out. The screening of high-quality markers and the establishment of a molecular detection system provide effective tools for subsequent high-throughput screening. In addition, the comparative analysis of the molecular marker detection results and the target phenotypic data verifies the marker's ability to respond to the target genotype, ensuring the accuracy of the screening method.

[0131] The hybridization and backcrossing population construction specifically includes the following steps:

[0132] Select superior individual plants from the parental materials as hybrid parents, perform artificial hybridization to obtain F1 generation plants, preferably perform hybridization under conditions where the target trait is stable and the flowering period is consistent;

[0133] During the cultivation of F1 plants, preliminary observations were made on growth and target traits, and relevant agronomic traits were recorded;

[0134] Screen the F1 generation plants and select the plants that meet the breeding goals based on the marker detection results of the target gene and the phenotypic performance of the target trait;

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

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

[0137] In each generation of backcrossing and selfing, the results of molecular marker detection and the phenotypic expression of the target traits are combined to select plants with high yield, wide adaptability and excellent target traits.

[0138] In the above embodiment, by selecting excellent parental plants for artificial hybridization, F1 generation plants are obtained, and the hybridization operation is completed under the conditions of stable performance of the target trait and consistent flowering period, the breeding value of the F1 generation plants can be improved. In the process of cultivating the F1 generation plants, the plants with excellent target traits are screened in combination with the molecular marker detection results and phenotypic data to ensure that the selected plants have breeding potential. Subsequently, the F1 generation plants that meet the breeding goals are selected as donor parents, backcrossed with the recipient parents, and backcrossing groups are constructed through multi-generation backcrossing and selfing. In the backcrossing and selfing process, excellent plants that meet the target traits are selected through molecular marker detection and phenotypic observation, and the population size is expanded to enhance the screening stability. Combined with the screening strategy of multi-generation backcrossing and selfing, the inheritance of the target gene is gradually fixed, so that the target trait is stably inherited to the offspring plants.

[0139] High-throughput sequencing and genotype analysis specifically include the following steps:

[0140] Extract genomic DNA from hybrid offspring plants, and perform PCR amplification using specific molecular markers targeting the target gene region, so that the amplified fragment covers the key sites and upstream and downstream regions of the target gene;

[0141] After purifying the amplified product, the amplicon library of the target region is constructed based on the length and coverage of the amplicon sequence;

[0142] A high-throughput sequencing platform was used to perform deep sequencing of the amplicon library of the target region, and the sequencing data was quality controlled to remove low-quality reads, adapter sequences, and repeated sequences;

[0143] Align the sequencing data to the reference genome and analyze the variation information within the target gene region, including single nucleotide polymorphisms, insertions and deletions, and the distribution of target gene-specific markers;

[0144] Combining the genotype data of the parents, identifying the genotype of the hybrid offspring, and classifying and annotating the genotype information, the genotype information includes homozygous parent type, heterozygous type and non-parent type;

[0145] Based on the results of variation analysis of sequencing data and the distribution of specific markers in the target region, plants containing the target gene are screened;

[0146] For the initially screened plants, the variation information and genotype categories within the target gene region were recorded;

[0147] The screening results were compared with the preliminary phenotypic data of the population to screen out candidate plants with significant associations between the target genotype and the target trait.

[0148] In the above embodiment, by extracting genomic DNA of hybrid offspring plants and combining PCR amplification of specific molecular markers, the amplicon library of the target region can be accurately constructed. The high-throughput sequencing platform performs deep sequencing of the amplicon library, which significantly improves the efficiency and accuracy of detecting variation information in the target gene region. The quality control of sequencing data (such as removing low-quality reads, linker sequences and repeat sequences) and comparison analysis can comprehensively capture single nucleotide polymorphisms, insertions and deletions, and specific marker distributions in the target gene region.

[0149] In the above embodiment, the genotype information of the hybrid offspring (including homozygous parent type, heterozygous type and non-parent type) is systematically analyzed in combination with the genotype data of the parents, so that the genetic background of the hybrid offspring can be clarified, providing a scientific basis for screening plants containing the target gene. In addition, the genotype of the screened target plant is combined with the preliminary phenotypic data, which can not only verify the correlation between the genotype and the phenotype, but also further screen candidate plants that are significantly associated with the target trait.

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

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

[0152] Performing association analysis on genotype data and population phenotypic data, wherein the population phenotypic data includes target trait performance, agronomic traits and environmental adaptability;

[0153] Statistically calculate the correlation between the target genotype and the target trait, and determine the association pattern between the genotype and the target trait;

[0154] According to the results of association analysis, plants with significant association between genotype and target trait were preliminarily selected as candidate plants, and the genotype and phenotypic performance of the candidate plants were cross-validated;

[0155] Conduct phenotypic tests on target traits of the selected candidate plants under different environmental conditions to evaluate the stability of the target traits of the candidate plants and their performance differences under multiple environmental conditions;

[0156] Plants that perform well in multiple environments and are consistent with the target genotype are identified as the high-quality plants that are finally screened out.

[0157] In the above embodiment, by extracting the genotype data of the hybrid offspring plants, screening the key variation information of the target gene region, and combining the 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 fully revealed. Statistical calculation of the correlation between the target genotype and the target trait helps to preliminarily screen out plants that are significantly associated with the target trait.

[0158] On this basis, the candidate plants are phenotypically tested under multiple environmental conditions to evaluate the stability of their target traits and their adaptability to multiple environments, ensuring that the plants finally selected perform well in practical applications. In addition, the combined analysis of genotype and phenotype can optimize the candidate plant screening strategy, achieve accurate transformation from genotype to phenotype, and provide a reliable basis for creating high-quality restorer line materials with stable inheritance of target traits.

[0159] The verification of candidate plants and creation of restoration lines specifically include the following steps:

[0160] The high-quality plants finally screened out are selected as candidate materials for the restorer line;

[0161] Cross-breeding candidate materials with high-quality sterile lines to construct hybrid combinations;

[0162] Grow hybrid offspring under greenhouse or field conditions, make preliminary observations of target traits and other key agronomic traits, and record plant performance;

[0163] Combine the target traits and yield performance to select excellent hybrid combinations and enter the next stage of testing;

[0164] Conduct field trials on the selected excellent hybrid combinations, and record the target trait performance, yield index, adaptability and other data of the hybrid combinations during the trials;

[0165] Combine the results of multi-environment tests to analyze the stability of target traits and yield performance of candidate materials under different environmental conditions, and confirm the actual application value of candidate materials based on the test results;

[0166] The candidate materials with excellent performance are used as the basic materials of the restorer line, and the genotype is optimized based on the target traits and genetic background, and the candidate plants are selected and verified for generations;

[0167] Create japonica rice restorer line materials that meet the target trait requirements and have excellent and stable genetic background.

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

[0169] At the same time, the setting process of different environmental conditions is as follows:

[0170] Extracting condition parameters contained in the environmental conditions corresponding to the candidate plants, wherein the condition parameters include temperature parameters, humidity parameters and light intensity parameters;

[0171] Extracting 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 ambient temperature change generated by the current illumination intensity reference parameter in a preset illumination unit time period; wherein the illumination unit time period has a value range of 30min-60min;

[0173] Extracting the actual ambient temperature change generated by the illumination intensity reference parameter in a preset illumination unit time period;

[0174] Obtaining a parameter adjustment coefficient using an actual ambient temperature variation amount generated by the illumination intensity reference parameter in a preset illumination unit time period and a theoretical ambient temperature variation amount generated by the illumination intensity reference parameter in 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 Indicates the theoretical ambient temperature change caused by the light intensity reference parameter in the preset light unit time period; T s Indicates the actual ambient temperature change caused by the light intensity reference parameter in the preset light unit time period; L x Indicates the distance between the light source and the plant; S x Indicates the time length corresponding to the preset illumination unit time period;

[0178] Comparing the parameter adjustment coefficient with a preset parameter adjustment coefficient threshold;

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

[0180] When the parameter adjustment coefficient exceeds a 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.

[0181] The technical effect of the above technical solution is: by extracting the environmental condition parameters (temperature, humidity, light intensity) corresponding to the candidate plants and comparing them with the preset benchmark parameters, it can be ensured that the setting of the environmental conditions is based on scientific and standardized benchmarks, thereby improving the accuracy of environmental control. By calculating the theoretical environmental temperature change and the actual environmental temperature change generated by the light intensity benchmark parameter in the preset light unit time period, and obtaining the parameter adjustment coefficient based on this, the specific impact of light intensity on the ambient temperature can be quantitatively analyzed. This helps to more accurately predict and adjust environmental conditions to meet the growth needs of different plants. According to the comparison result between the parameter adjustment coefficient and the preset parameter adjustment coefficient threshold, the setting of environmental conditions is dynamically adjusted. When the parameter adjustment coefficient does not exceed the threshold, a more relaxed environmental condition setting (upper limit value) is adopted; when it exceeds the threshold, the conditions are adjusted more strictly. This strategy can respond flexibly according to different situations to ensure that the plants grow under the best environmental conditions. By accurately setting and adjusting environmental conditions, the growth environment of the plants can be optimized, thereby improving their growth efficiency and quality. This is of great significance to agricultural production, plant research and other fields. This technical solution not only takes into account the impact of light intensity on ambient temperature, but also enhances the adaptability and flexibility of the system by setting parameter adjustment coefficients and thresholds. This enables the system to be personalized according to different plants, different growth stages and different environmental conditions. This technical solution integrates multiple links such as environmental parameter extraction, quantitative analysis, and dynamic adjustment, reflecting the development trend of intelligent agriculture. Optimizing environmental conditions through intelligent means will help improve agricultural production efficiency and quality and promote sustainable agricultural development.

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

[0183] Specifically, 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, 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 coefficient;

[0186] The parameter adjustment coefficient is used to adjust 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 to obtain the adjusted 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;

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

[0188]

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

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

[0191]

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

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

[0194]

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

[0196] A plurality of different sets of environmental conditions are set using the adjusted temperature gradient parameter corresponding to the upper limit value, the humidity reference parameter corresponding to the upper limit value, and the light intensity gradient parameter corresponding to the upper limit value.

[0197] The technical effect of the above technical solution is that when the parameter adjustment coefficient exceeds the preset threshold, the technical solution can automatically retrieve the current environmental parameter upper limit value (temperature, humidity, light intensity) and the parameter adjustment coefficient, and adjust accordingly. This dynamic response mechanism enhances the adaptability of the system to different environmental conditions and ensures that the plants can grow in a more accurate and suitable environment. The adjusted upper limits of temperature, humidity and light intensity are calculated by specific formulas that take into account the influence of the parameter adjustment coefficient on the original upper limit value. This calculation method ensures the accuracy and scientificity of parameter adjustment and avoids the adverse effects that may be caused by blind adjustment. By setting multiple groups of different environmental conditions through the adjusted upper limit value, the growth environment of the plant can be further optimized. This optimization not only helps to improve the growth efficiency and quality of the plant, but also reduces the risk of growth disorders and diseases caused by unsuitable environmental conditions. Accurate environmental condition setting helps to reduce unnecessary waste of resources, such as energy, water resources and fertilizers. By optimizing environmental conditions, it can be ensured that the plants grow in the best state, thereby improving resource utilization efficiency. This technical solution integrates multiple links such as parameter extraction, quantitative analysis, dynamic adjustment and intelligent setting, reflecting the development trend of intelligent and automated agriculture. By optimizing environmental conditions through intelligent means, the frequency and intensity of human intervention can be reduced, and the efficiency and quality of agricultural production can be improved. By accurately setting and adjusting environmental conditions, this technical solution helps to achieve sustainable development of agricultural production. The optimized environmental conditions can not only improve the growth efficiency and quality of plants, but also reduce the negative impact on the environment, such as reducing the use of fertilizers and pesticides, and reducing the risk of pollution of soil and water resources.

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

[0199] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for screening target gene plants of japonica rice parental hybrids based on high-throughput sequencing, characterized in that: The following steps are involved: Parent material selection and target gene determination: select high-quality japonica rice parent materials, determine the genes related to the target traits through database analysis, and clarify the region and distribution characteristics of the target gene according to the breeding requirements of the target traits; Identify the genotypes of the parents and compare their genomes. Perform whole genome sequencing on the parental materials to obtain the parental genome data. Compare the sequencing data to the reference genome, identify the genetic differences between the parents, screen for specific markers in the target gene region, and simultaneously mine the key indica-japonica bridging regions of the parental genomes to determine the differential fragments of the target gene. Development and verification of molecular markers in the target region: based on the information of parental genomic differences obtained through high-throughput sequencing, molecular markers related to the target gene are designed. The molecular markers cover the key sites and upstream and downstream regions of the target gene region, and molecular verification is performed on the markers of the parental materials and hybrid offspring; Hybridization and backcross population construction: With the target gene as the core, the japonica rice parents are hybridized to obtain F1 generation plants, and excellent F1 generation plants are selected for backcrossing to construct backcross populations. During the population construction process, plants are selected in combination with breeding goals; High-throughput sequencing and genotype analysis, extracting genomic DNA from hybrid offspring plants for PCR amplification, converting SSR markers or SNP markers of restoration genes and wide-affinity genes into Kasp markers, wherein the restoration genes include Rf1 and Rf4, and the wide-affinity 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, comparison and variation analysis on the sequencing data, identifying the genotype of the hybrid offspring plants, and preliminarily screening out plants containing the aggregated target genes to provide the recurrent parents of the backcross population; Combined genotype and phenotype analysis: combining genotype analysis results with population phenotypic data to verify the phenotypic correlation of the target gene, comprehensively analyzing the genotype and phenotypic data of the plant, and screening out excellent plants with significant correlation between genotype and target traits; Verification of candidate plants and creation of restorer lines: The screened target plants are used as restorer line candidate materials, hybridized with sterile lines, and hybrid combinations are constructed to verify the yield potential and target trait performance of the combination. Based on the verification results, high-quality candidate plants are used as breeding materials to create new restorer lines.

2. The method for screening target gene plants of japonica rice parental hybridization based on high-throughput sequencing according to claim 1, characterized in that: The parent material selection and target gene determination specifically include the following steps: Screening parental materials with excellent genetic characteristics from excellent japonica rice varieties or breeding resource banks, wherein the excellent genetic characteristics include high yield, wide adaptability, salt-alkali resistance, and resistance to pests and diseases, and giving priority to materials known to have target traits or that may be related to target genes; Screening target genes significantly associated with target traits through gene function database analysis, wherein the target traits include resilience, fruit setting rate or wide affinity; Identify the gene locus, chromosome region and known gene function of the target gene; Based on the marker data of the existing genomic information of the parental materials and the genetic background of the target trait, the distribution of the target gene in the parental genome and the differential fragment regions are inferred; The target gene region was initially delineated to a specific chromosome range.

3. The method for screening target gene plants of japonica rice parental hybridization based on high-throughput sequencing according to claim 1, characterized in that: The parental genotype identification and genome comparison specifically further comprises the following steps: The whole genome of the selected japonica rice parent materials was sequenced, and high-throughput sequencing was used to generate sequencing data with a depth of greater than or equal to 30×; Aligning the sequencing data of the parental genomes to the reference genome, and analyzing the genetic differences between the parental genomes, the genetic differences including single nucleotide polymorphisms and insertions and deletions; Combine the gene locus of the target gene and the known gene function to screen out specific markers in the region where the target gene is located; Based on the comparison analysis results and combined with specific fragments of the parental genomes, the key regions related to indica-japonica hybrid vigor and genetic stability were identified, and the distribution and difference information of the target gene regions in the parental genomes were determined.

4. The method for screening target gene plants of japonica rice parental hybridization based on high-throughput sequencing according to claim 1, characterized in that: The target region molecular marker development and verification specifically includes the following steps: Based on the difference information of the parental genomes and the localization results of the target gene region, molecular markers covering the key sites of the target gene region are designed; The molecular markers include single nucleotide polymorphism markers and insertion and deletion markers; The molecular markers simultaneously cover the key sequences at both ends of the target region, upstream and downstream, and in the middle; PCR amplification of parental materials using molecular markers, combined with sequencing to verify the accuracy and specificity of the markers; Based on the distribution of markers of parental materials and plants with known phenotypes, screen for high-quality markers that can significantly distinguish the target genotypes; Combine the selected high-quality markers to establish a molecular detection system; By comparing the marker test results with the target phenotype data, it is confirmed that the marker can reflect the target genotype.

5. The method for screening target gene plants of japonica rice parental hybridization based on high-throughput sequencing according to claim 1, characterized in that: The hybridization and backcrossing population construction specifically further comprises the following steps: Select superior individual plants from the parental materials as hybrid parents, perform artificial hybridization to obtain F1 generation plants, preferably perform hybridization under conditions where the target trait is stable and the flowering period is consistent; During the cultivation of F1 plants, preliminary observations were made on growth and target traits, and relevant agronomic traits were recorded; Screen the F1 generation plants and select the plants that meet the breeding goals based on the marker detection results of the target gene and the phenotypic performance of the target trait; The selected F1 generation plants are used as donor parents and backcrossed with recipient parents to construct a backcross population; In the process of constructing the backcross population, the population size is further expanded through multiple generations of backcrossing and selfing; In each generation of backcrossing and selfing, the results of molecular marker detection and the phenotypic expression of the target traits are combined to select plants with high yield, wide adaptability and excellent target traits.

6. The method for screening target gene plants of japonica rice parental hybridization based on high-throughput sequencing according to claim 1, characterized in that: The high-throughput sequencing and genotype analysis specifically further comprises the following steps: Extract genomic DNA from hybrid offspring plants, and perform PCR amplification using specific molecular markers targeting the target gene region, so that the amplified fragment covers the key sites and upstream and downstream regions of the target gene; After purifying the amplified product, the amplicon library of the target region is constructed based on the length and coverage of the amplicon sequence; A high-throughput sequencing platform was used to perform deep sequencing of the amplicon library of the target region, and the sequencing data was quality controlled to remove low-quality reads, adapter sequences, and repeated sequences; Align the sequencing data to the reference genome and analyze the variation information within the target gene region, including single nucleotide polymorphisms, insertions and deletions, and the distribution of target gene-specific markers; Combining the genotype data of the parents, identifying the genotype of the hybrid offspring, and classifying and annotating the genotype information, the genotype information includes homozygous parent type, heterozygous type and non-parent type; Based on the results of variation analysis of sequencing data and the distribution of specific markers in the target region, plants containing the target gene are screened; For the initially screened plants, the variation information and genotype categories within the target gene region were recorded; The screening results were compared with the preliminary phenotypic data of the population to screen out candidate plants with significant associations between the target genotype and the target trait.

7. The method for screening target gene plants of japonica rice parental hybridization based on high-throughput sequencing according to claim 1, characterized in that: The combined analysis of genotype and phenotype specifically further comprises the following steps: Based on the genotype data of the screened hybrid offspring plants, key variation information of the target gene region is extracted; Performing association analysis on genotype data and population phenotypic data, wherein the population phenotypic data includes target trait performance, agronomic traits and environmental adaptability; Statistically calculate the correlation between the target genotype and the target trait, and determine the association pattern between the genotype and the target trait; According to the results of association analysis, plants with significant association between genotype and target trait were preliminarily selected as candidate plants, and the genotype and phenotypic performance of the candidate plants were cross-validated; Conduct phenotypic tests on target traits of the selected candidate plants under different environmental conditions to evaluate the stability of the target traits of the candidate plants and their performance differences under multiple environmental conditions; Plants that perform well in multiple environments and are consistent with the target genotype are identified as the high-quality plants that are finally screened out.

8. The method for screening target gene plants of japonica rice parental hybridization based on high-throughput sequencing according to claim 7, characterized in that: The setting process for different environmental conditions is as follows: Extracting condition parameters contained in the environmental conditions corresponding to the candidate plants, wherein the condition parameters include temperature parameters, humidity parameters and light intensity parameters; Extracting 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 ambient temperature change generated by the current illumination intensity reference parameter in a preset illumination unit time period; wherein the illumination unit time period has a value range of 30min-60min; Extracting the actual ambient temperature change generated by the illumination intensity reference parameter in a preset illumination unit time period; Obtaining a parameter adjustment coefficient using the actual ambient temperature variation generated by the illumination intensity reference parameter in a preset illumination unit time period and the theoretical ambient temperature variation generated by the illumination intensity reference parameter in 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 Indicates the theoretical ambient temperature change caused by the light intensity reference parameter in the preset light unit time period; T s Indicates the actual ambient temperature change caused by the light intensity reference parameter in the preset light unit time period; L x Indicates the distance between the light source and the plant; S x Indicates the time length corresponding to the preset illumination unit time period; Comparing the parameter adjustment coefficient with a preset parameter adjustment coefficient threshold; When the parameter adjustment coefficient does not exceed the preset parameter adjustment coefficient threshold, a plurality of different sets of environmental conditions are set using the preset temperature gradient parameter corresponding upper limit value, humidity reference parameter corresponding upper limit value and light intensity gradient parameter corresponding upper limit value; When the parameter adjustment coefficient exceeds a 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.

9. The method for screening target gene plants of japonica rice parental hybridization based on high-throughput sequencing according to claim 8, characterized in that: 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, including: 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 coefficient; The parameter adjustment coefficient is used to adjust 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 to obtain the adjusted 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; The upper limit value of the adjusted temperature gradient parameter is obtained by the following formula: Among them, T up Indicates the upper limit value of the adjusted temperature gradient parameter; T up0 Indicates the upper limit value of the temperature gradient parameter before adjustment; R indicates the parameter adjustment coefficient; At the same time, the upper limit value corresponding to the adjusted humidity reference parameter is obtained by the following formula: Among them, W up Indicates the upper limit value of the adjusted humidity gradient parameter; W up0 Indicates the upper limit value of the humidity gradient parameter before adjustment; T up Indicates the upper limit value of the adjusted temperature gradient parameter; T up0 Indicates the upper limit value of the temperature gradient parameter before adjustment; R indicates the parameter adjustment coefficient; In addition, the upper limit value corresponding to the adjusted light intensity gradient parameter is obtained by the following formula: Among them, G up Indicates the upper limit value of the adjusted light intensity gradient parameter; G up0 Indicates the upper limit value of the light intensity gradient parameter before adjustment; W up Indicates the upper limit value of the adjusted humidity gradient parameter; W up0 Indicates the upper limit value of the humidity gradient parameter before adjustment; T up Indicates the upper limit value of the adjusted temperature gradient parameter; T up0 Indicates the upper limit value of the temperature gradient parameter before adjustment; R indicates the parameter adjustment coefficient; A plurality of different sets of environmental conditions are set using the adjusted temperature gradient parameter corresponding to the upper limit value, the humidity reference parameter corresponding to the upper limit value, and the light intensity gradient parameter corresponding to the upper limit value.

10. The method for screening target gene plants of japonica rice parental hybridization based on high-throughput sequencing according to claim 1, characterized in that: The verification of the candidate plants and the creation of the restorer line specifically include the following steps: The high-quality plants finally screened out are selected as candidate materials for the restoration line; Cross-breeding candidate materials with high-quality sterile lines to construct hybrid combinations; Grow hybrid offspring under greenhouse or field conditions, make preliminary observations of target traits and other key agronomic traits, and record plant performance; Combine the target traits and yield performance to select excellent hybrid combinations and enter the next stage of testing; Conduct field trials on the selected excellent hybrid combinations, and record the target trait performance, yield index, adaptability and other data of the hybrid combinations during the trials; Combine the results of multi-environment tests to analyze the stability of target traits and yield performance of candidate materials under different environmental conditions, and confirm the actual application value of candidate materials based on the test results; The candidate materials with excellent performance are used as the basic materials of the restorer line, and the genotype is optimized based on the target traits and genetic background, and the candidate plants are selected and verified for generations; Create japonica rice restorer line materials that meet the target trait requirements and have excellent and stable genetic background.

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