Plateau rape adaptive germplasm breeding method based on multi-character collaborative screening

Abstract

The invention relates to the technical field of crop genetic breeding, in particular to a plateau rape adaptive germplasm breeding method based on multi-character collaborative screening, which comprises the following steps of: constructing a multi-character evaluation system covering more than 20 indexes of four categories of agriculture, physiology, quality and stress resistance, and determining character weights by applying principal component analysis, grey relational analysis and analytic hierarchy process; and acquiring data by combining unmanned aerial vehicle multispectral imaging, a ground phenotype platform and a molecular marker technology, and substituting the data into the comprehensive evaluation model for germplasm screening. According to the method, the limitation of traditional single-character breeding is broken through, multi-dimensional collaborative evaluation is achieved, and the plateau rape germplasm breeding efficiency and accuracy are remarkably improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of crop genetic breeding, in particular to a highland oilseed rape adaptive germplasm breeding method based on multi-trait collaborative screening. BACKGROUND

[0002] As the third largest oil crop in the world, oilseed rape is the core source of edible vegetable oil supply in China. However, the average annual temperature in highland is 5-10℃ lower than that in plain, the diurnal temperature difference reaches 15-25℃, the average annual precipitation is less than 400mm, and the ultraviolet radiation intensity is 30%-50% higher than that in plain. Extreme weather occurs frequently, and traditional oilseed rape varieties are difficult to achieve high yield and stable yield.

[0003] Existing oilseed rape germplasm breeding techniques have significant limitations. Traditional pedigree breeding relies on breeder experience and selects single plants through field phenotypic observation, such as using only a few agronomic traits such as plant height and silique number as screening criteria. This method is easily disturbed by environmental fluctuations, for example, high-yield materials selected in wet years may reduce yield due to insufficient drought resistance in dry years. According to statistics, the traditional method takes 8-10 years to breed, and single-trait improvement is often accompanied by degradation of other traits, such as high-oil varieties often have reduced cold resistance.

[0004] Although modern biotechnology has made some breakthroughs, there are still some deficiencies. Molecular marker-assisted selection technology can accurately locate target genes, but it focuses on single traits (such as oil content-related gene FAD2) and ignores the interaction effects between genes. High-throughput phenotyping technology can obtain a large amount of morphological data, but it lacks comprehensive consideration of physiological metabolism and quality characteristics. In addition, existing multi-trait evaluation systems are mostly suitable for plain environments and do not construct a scientific weight distribution model for highland special ecological pressure. For example, the weight of ordinary oil content is set to 20%, while in highland areas, the actual influence weight of cold resistance on yield may be as high as 30%, and using the traditional weight will cause breeding bias.

[0005] In summary, it is urgent to develop a multi-disciplinary technology-based multi-trait collaborative breeding method that adapts to highland ecology to break through the existing technical bottlenecks and accelerate the breeding process of highland oilseed rape breakthrough germplasm. SUMMARY

[0006] The core of the present application is to provide a systematic highland oilseed rape adaptive germplasm breeding scheme, which realizes the precise screening of germplasm resources by constructing a multi-dimensional evaluation system, innovating a weight calculation model, and integrating modern detection technologies, and is suitable for the innovative breeding of oilseed rape germplasm resources in special ecological environments such as high altitude, strong radiation, low temperature and drought.

[0007] The technical scheme adopted by the present application to solve its technical problems is: a highland oilseed rape adaptive germplasm breeding method based on multi-trait collaborative screening, comprising: A multi-traits evaluation index system comprising agronomic traits, physiological traits, quality traits, and stress resistance traits is constructed, wherein: The agronomic traits include plant height, primary effective branch number, single pod number, seed number per pod, and thousand seed weight. The physiological traits include chlorophyll a content, proline content, superoxide dismutase activity, and peroxidase activity. The quality traits include oil content, oleic acid content, glucosinolate content, and protein content. The stress resistance traits include drought resistance index, cold resistance index, and sclerotinia disease index. The weight of each trait in the multi-traits evaluation index system is determined by combining principal component analysis, grey correlation analysis, and analytic hierarchy process, and a comprehensive evaluation model is constructed. wherein F is the comprehensive score, wi is the weight of the ith trait, xi is the standardized data of the ith trait, and n is the total number of traits, wherein: When constructing the judgment matrix by using the analytic hierarchy process, the selection pressure of the plateau environment (annual mean temperature 4-8℃, precipitation 300-450mm, and ultraviolet radiation intensity 30%-50%) on each trait is considered, and the weight of the stress resistance traits is adjusted to 25%-35% and the weight of the agronomic traits is adjusted to 30%-35%. The data of each trait of the selected plateau rapeseed germplasm are obtained by using unmanned aerial vehicle multispectral imaging, ground phenotyping platform, and molecular marker technology, wherein: The data of agronomic traits, physiological traits, quality traits, and stress resistance traits of the selected plateau rapeseed germplasm are obtained by using unmanned aerial vehicle multispectral imaging (red edge and near-infrared waveband), ground phenotyping platform (determination of canopy temperature and leaf area index), and molecular marker technology (SSR or SNP marker, genotyping by PCR amplification and electrophoresis detection). The data of each trait are substituted into the comprehensive evaluation model, and the plateau rapeseed adaptive germplasm with a comprehensive score higher than a threshold value is screened, wherein: The threshold value is determined according to the distribution of the comprehensive scores of the selected population, and is the mean value plus 0.5-1.5 times the standard deviation. The method further comprises a 2-3 year field multi-point adaptability verification step for the screened germplasm, and the verification sites cover different ecological regions with an altitude of 2500-3500m and an annual mean temperature of 4-8℃.

[0008] Specifically, the agronomic traits include plant height (range 60-180cm), primary effective branch number (2-8), single pod number (150-500), seed number per pod (15-35), and thousand seed weight (2.5-5.5g).

[0009] Specifically, the physiological traits include chlorophyll a content (1.5-4.5 mg / g), proline content (0.1-1.2 μmol / g), superoxide dismutase activity (100-500 U / g) and peroxidase activity (20-150 U / g).

[0010] Specifically, the quality traits include oil content (40%-55%), oleic acid content (50%-85%), glucosinolate content (10-45 μmol / g) and protein content (20%-30%).

[0011] Specifically, the stress resistance traits include drought resistance index (0.5-1.2), cold resistance index (0.4-1.0) and sclerotinia disease index (10-40).

[0012] Specifically, the standardization formula in the principal component analysis is , wherein is the standardized data, is the original data, is the mean of the jth trait, is the standard deviation.

[0013] Specifically, the correlation coefficient calculation formula in the grey correlation analysis is: , wherein is the resolution coefficient, and the value is 0.1-0.5.

[0014] Specifically, the molecular marker technology adopts SSR markers or SNP markers, and genotyping is performed through PCR amplification and electrophoresis detection.

[0015] Specifically, in the screening step, the threshold value is determined according to the mean value plus 0.5-1.5 times the standard deviation of the comprehensive score distribution of the selected population.

[0016] Specifically, it further comprises a 2-3 year field multi-point adaptability verification step for the screened germplasm.

[0017] The beneficial effects of the present application are: Multi-dimensional synergistic improvement of breeding precision: through synergistic analysis of 22 indexes, comprehensive coverage of yield formation, environmental response, quality characteristics and other dimensions, the accuracy of traditional single trait breeding is improved.

[0018] Model optimization adapts to plateau characteristics: based on the weight determination method of principal component-grey correlation-hierarchical analysis, the selection pressure of the plateau environment on each trait is fully considered.

[0019] Technical integration shortens the breeding period: the integration of high-throughput phenotyping and molecular marker technology improves the efficiency of single generation screening.

[0020] Significantly enhanced germplasm adaptability: Screened germplasm, verified in multiple locations, showed increased yield and higher oil content compared to control varieties under extreme conditions of average annual temperature of 4℃ and precipitation of 300mm, effectively solving the problem of rapeseed production in high-altitude areas. Attached Figure Description

[0021] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0022] Figure 1 The flowchart shows the high-yield rapeseed adaptability breeding method based on multi-trait synergistic screening provided by the present invention. Detailed Implementation

[0023] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below in conjunction with specific embodiments.

[0024] like Figure 1 As shown in this embodiment, the high-yield rapeseed adaptability breeding method based on multi-trait synergistic screening includes the following steps: Construct a multi-trait evaluation index system: Establish a four-level evaluation index system. The first-level index includes four major categories: agronomic, physiological, quality, and stress resistance. The second-level index is further subdivided into 12 items, such as yield components under agronomic traits and osmotic regulators under physiological traits. The third-level index is expanded to 22 specific detection indicators, covering plant height, chlorophyll fluorescence parameters, oleic acid composition, etc. The fourth-level index specifies the detection methods and data processing specifications.

[0025] Weight determination and model construction: First, principal component analysis is used to reduce the dimensionality of the original data, through the formula... Standardized data was used to extract principal components with a cumulative contribution rate of over 85%. Next, grey relational analysis was employed to construct a reference sequence based on ideal trait values, using the formula... ( Calculate the correlation coefficient using a value of 0.3. Finally, construct a judgment matrix using the analytic hierarchy process (AHP) to determine the final weights of each trait, thus forming a comprehensive evaluation model. .

[0026] Data acquisition technology integration: A DJI M300 RTK drone equipped with a multispectral camera (5 bands including red-edge and near-infrared) was used to obtain centimeter-resolution images during the budding, full-blown, and silique stages, and Agisoft Metashape software was used to generate a 3D point cloud model. Ground-based measurements were performed using the Phenomobile phenotyping platform to determine parameters such as canopy temperature and leaf area index. Molecular marker detection was performed using the Illumina Infinium chip to genotype 50K SNP loci.

[0027] Germplasm screening and verification: The standardized trait data is put into the comprehensive evaluation model to calculate the comprehensive score of germplasm. According to the distribution of group score, the mean value plus 1 standard deviation is set as the screening threshold. The preliminary selected germplasm is verified in the field for 2 consecutive years at 3 different altitude test sites (2500m, 3000m, 3500m) to finally determine the excellent germplasm.

[0028] Example one: Germplasm screening in low-altitude plateau area Test design and material preparation In the low-altitude plateau test field with an altitude of 2500 meters, an average annual temperature of 8°C, and an annual precipitation of about 450 millimeters, 150 oilseed rape germplasm resources with different genetic backgrounds are selected as test materials. These germplasms cover multiple oilseed rape varieties and breeding intermediate materials at home and abroad. The seeds are disinfected before sowing. A randomized block design is used, with 3 replicates, each plot area of 5 square meters, and a planting density of 18,000 plants per mu. Field management follows the local conventional cultivation mode.

[0029] Multi-trait data collection Agronomic trait measurement: At the mature harvest stage of oilseed rape, agronomic traits are measured. Plant height is measured from the base of the ground to the top of the plant using a tape measure, with a range of 80-120 cm. The number of primary branches is counted manually, with a range of 3-5. The number of pods per plant is counted, with a range of 200-300. The number of seeds per pod is randomly selected from 20 pods, with a range of 20-25. The weight of 1000 seeds is measured using a balance, with a range of 3.0-4.0 grams.

[0030] Physiological trait measurement: At the full flowering stage of oilseed rape, the top fully expanded leaves are selected, and the chlorophyll a content is measured using the acetone extraction method, with a range of 2.0-3.0 mg / g. The proline content is measured using the acid ninhydrin colorimetric method, with a range of 0.3-0.6 μmol / g. The superoxide dismutase activity is measured using the nitrogen blue tetrazolium photoreduction method, with a range of 200-300 units / g.

[0031] Phenotype data collection: At the full flowering stage of oilseed rape, a DJI M300 RTK unmanned aerial vehicle equipped with a multispectral camera is used to collect data at a height of 80 meters, obtaining multispectral image data of 5 bands such as red edge and near infrared. At the same time, the Phenomobile phenotype platform is used to scan the plants in the plot to measure parameters such as canopy temperature and leaf area index.

[0032] Molecular marker detection: Select 5 SSR markers (such as BRMS01, BRMS02, etc.) related to clubroot resistance, extract the genomic DNA of each germplasm, detect the genotype by PCR amplification and polyacrylamide gel electrophoresis, and analyze the carrying of clubroot resistance genes.

[0033] Weight calculation and germplasm screening Data preprocessing: The collected raw data is standardized by using the formula , wherein is the standardized data, is the raw data, is the jth trait mean, is the standard deviation of the jth trait.

[0034] Weight determination: The standardized data is analyzed by using principal component analysis method, the first three principal components are extracted, the cumulative contribution rate reaches 88%, the traits contained in each principal component and the weight are determined; the reference sequence is constructed by ideal trait value through grey correlation degree analysis, the correlation coefficient is calculated by using the formula , and the correlation coefficient is calculated by using the formula 0.3); combined with the analytic hierarchy process, the judgment matrix is constructed, and finally the weight of agronomic traits is determined as 35%, the weight of physiological traits is 25%, the weight of quality traits is 20%, and the weight of stress resistance traits is 20%.

[0035] Comprehensive evaluation and screening: the standardized trait data is substituted into the comprehensive evaluation model , the comprehensive score of each germplasm is calculated. According to the distribution of comprehensive score, the threshold is set as the mean score plus 0.8 times the standard deviation, and 20 germplasms with excellent comprehensive performance are screened out.

[0036] Field verification and result analysis The selected 20 germplasms are tested in multiple points in the next year in three test points with similar ecological conditions, and the local main cultivars are set as controls. The results show that the average yield of the selected germplasms reaches 185 kg per mu, which is 22% higher than that of the control cultivars; the average oil content is 46.5%, which is 4.2% higher than that of the control, which verifies the effectiveness of the screening method in low-altitude plateau areas.

[0037] Example two: Germplasm screening in medium-altitude cold and arid areas Test environment and materials Select a test field in a medium-altitude cold and arid area with an altitude of 3000 meters, an average annual temperature of 5°C, an annual precipitation of about 300 millimeters, and a winter minimum temperature of-20°C. Plant 120 oilseed rape germplasms, which have certain cold or drought resistance. A completely randomized block design is adopted, with 3 repetitions, a plot area of 4 square meters, a planting density of 16,000 plants per mu, and strict control of irrigation amount during the growth period to simulate natural drought stress environment.

[0038] Special trait index determination Resistance trait determination: When determining the cold resistance index, plants with consistent growth vigor are selected before wintering of oilseed rape, and are subjected to low temperature treatment at -5°C for 48 hours. After treatment, the plants are moved back to normal environment, and the recovery growth rate is counted after 7 days to calculate the cold resistance index. The drought resistance index is evaluated by the ratio of yield of the test germplasm under natural drought stress to yield under sufficient irrigation. Meanwhile, new physiological indicators are added, including determination of soluble sugar content by anthrone colorimetry, ranging from 20 to 40 mg / g; and determination of abscisic acid content by enzyme-linked immunosorbent assay, ranging from 50 to 100 ng / g.

[0039] Other trait determination: The determination methods of agronomic traits, quality traits, and part of conventional physiological traits are the same as in Example One.

[0040] Molecular marker detection: Illumina Infinium chip is used to genotype 5K SNP sites of each germplasm, and gene variation sites related to cold resistance and drought resistance are mined through bioinformatics analysis.

[0041] Screening model optimization and germplasm selection Weight adjustment: The analytic hierarchy process is used to adjust the weight of each trait in combination with the environmental characteristics of cold and drought regions. The weight of resistance traits is increased to 35%, of which the weight of cold resistance accounts for 20% and the weight of drought resistance accounts for 15%. The weight of agronomic traits is adjusted to 30%, and the weights of quality traits and physiological traits are 15% and 20%, respectively.

[0042] Comprehensive evaluation and screening: All trait data are substituted into the comprehensive evaluation model to calculate the comprehensive score. According to the score distribution, the screening threshold is set as the mean value plus 1.2 times the standard deviation, and finally 18 germplasms with excellent comprehensive performance in cold and drought environments are obtained.

[0043] Verification and effect evaluation The 18 selected germplasms are subjected to field verification for 2 consecutive years, during which they encounter extreme weather of low temperature freeze damage and drought superposition. The results show that the yield stability rate of these germplasms reaches 85%, while that of the control variety is only 55%. The selected germplasms have an average yield increase of 32% compared with the control variety, effectively verifying the applicability of the screening method in cold and drought regions at medium altitude.

[0044] Example Three: Germplasm screening in high-altitude strong radiation area Test area and germplasm The test was carried out in a high-altitude strong radiation area with an altitude of 3500 meters, an average annual sunshine time of more than 3000 hours, and an ultraviolet radiation intensity 50% higher than that in the plain area. 100 oilseed rape germplasms were selected, covering different quality types and radiation tolerance materials. The test adopted split plot design, the main plot was different shading treatment (simulating different radiation intensity), the sub-plot was each oilseed rape germplasm, 3 times of repetition, and the plot area was 3 square meters, with a planting density of 15,000 plants per mu.

[0045] Quality and radiation resistance trait determination Quality trait determination: nuclear magnetic resonance method was used to determine the oil content, ranging from 48% to 52%; gas chromatography was used to determine the oleic acid content, ranging from 75% to 82%; high performance liquid chromatography was used to determine the glucosinolate content and vitamin E content, with a range of 50-80 mg / kg of vitamin E content.

[0046] Radiation resistance trait determination: leaf samples were collected during the vigorous growth period of oilseed rape, and the malondialdehyde content was determined by using the thiobarbituric acid method, ranging from 10 to 20 nanomoles per gram; high performance liquid chromatography was used to determine the flavonoid content, ranging from 0.5 to 1.2 mg / g, so as to evaluate the radiation resistance ability of the germplasm.

[0047] Other trait determination: the determination method of agronomic traits and conventional physiological traits is the same as that in example one.

[0048] Molecular marker detection: through whole genome resequencing, gene sites related to flavonoid synthesis and antioxidant enzymes are screened, specific molecular markers are developed, and are used for germplasm screening.

[0049] Screening model construction and germplasm screening Weight determination: in the gray correlation degree analysis, the radiation intensity is introduced as an environmental covariate, and the correlation degree of each trait with the ideal trait is recalculated. After analysis, the weight of quality traits is increased to 30%, the weight of radiation resistance related indicators in stress resistance traits accounts for 15%, and the weights of agronomic traits and physiological traits are 25% and 30% respectively.

[0050] Comprehensive screening: the comprehensive evaluation model is used to calculate the comprehensive score. According to the score, the germplasm with excellent comprehensive performance is screened out.

[0051] Verification and application effect The selected germplasm was verified in the field for 3 years and the quality was analyzed. The results showed that the oxidation induction time of the rapeseed oil produced by the selected germplasm was 25% longer than that of the control, and the antioxidant stability was significantly improved; the yield loss rate was 40% lower than that of the control when planted in a strong ultraviolet environment, which fully reflected the advantages of the screening method in the high-altitude strong radiation area.

[0052] Example four: screening of germplasm in saline-alkali plateau area Test setup and materials In a saline-alkali plateau test field with an altitude of 2800 meters, soil salt content of 0.3%-0.5%, and pH value of 8.5-9.0, 80 oilseed rape germplasms were planted. The test adopted a randomized block design with 3 repetitions, and the plot area was 6 square meters with a planting density of 17,000 plants per mu. Soil salt content and pH value were measured before sowing, and relevant data were recorded.

[0053] Salt-tolerant trait index determination Salt-tolerance index determination: During the seed germination period, conductivity stress treatment (simulating different salt concentrations) was conducted, and relative germination rate was determined; during the seedling and mature stages, aboveground and root samples were collected, and sodium ion accumulation was determined by flame atomic absorption spectrometry, ranging from 0.5 to 2.0 mg / g; and the potassium-sodium ratio was calculated, ranging from 2 to 5.

[0054] Physiological index determination: betaine content was determined by high-performance liquid chromatography, ranging from 0.2 to 0.8 μmol / g; and plasma membrane ATPase activity was determined by spectrophotometry, ranging from 10 to 30 μmol Pi / mg protein / hour.

[0055] Other trait determination: agronomic traits, quality traits, and part of the conventional physiological traits were determined by the same method as in Example One.

[0056] Molecular marker detection: using whole-genome association analysis (GWAS) technology, combined with salt-tolerant phenotype data, salt-tolerant gene loci were mined, and SSR and SNP markers were developed for germplasm screening.

[0057] Screening strategy and germplasm determination Data analysis: using weighted gene co-expression network analysis (WGCNA), gene expression data and phenotype data were integrated and analyzed to mine salt-related gene modules and key regulatory genes.

[0058] Weight calculation and screening: four principal components were extracted by principal component analysis, and the weights of each trait were determined by grey correlation analysis and analytic hierarchy process. The trait data were substituted into the comprehensive evaluation model to calculate the comprehensive score, and 15 germplasms with outstanding salt-tolerant performance were screened out.

[0059] Field verification and results The 15 screened germplasms were planted in three test fields with different degrees of salinization, and local conventional varieties were set as controls. The results showed that in soil with a salt content of 0.3%-0.5%, the emergence rate of the screened germplasms reached 75%, while that of the control varieties was only 25%; at the mature stage, the grain yield of the screened germplasms averaged 160 kg per mu, which was 28% higher than that of the control varieties, proving the feasibility of the screening method in the saline-alkali plateau region.

[0060] Example Five: Germplasm Screening in the Sparse Sunshine Plateau Region Test Conditions and Germplasm Resources Select a test field in the sparse sunshine plateau region with an altitude of 2600 meters and an average annual sunshine duration of less than 1800 hours. Plant 130 oilseed rape germplasms, which are widely sourced, including varieties and breeding materials with strong low-light adaptability. The test uses a randomized block design with 3 repetitions, a plot area of 5 square meters, and a planting density of 18,000 plants per mu.

[0061] Light Response Trait Index Determination Light Response Index Determination: In the middle of the growth period of oilseed rape, use a portable photosynthesis measurement system to measure net photosynthetic rate under weak light conditions (50-100 micromoles per square meter per second), with a range of 5-10 micromoles of CO2 per square meter per second. Calculate the light compensation point through light response curve fitting, with a range of 10-20 micromoles per square meter per second. Use the acetone extraction method to measure chlorophyll a and chlorophyll b content and calculate the chlorophyll b / a ratio, with a range of 0.8-1.2.

[0062] Supplementary Determination of Agronomic Traits: Increase the index of agronomic traits, use a protractor to measure the branch angle, with a range of 45-60°; measure the leaf angle, with a range of 30-45°, to evaluate the plant canopy's ability to intercept and utilize light.

[0063] Other Trait Determination: Quality traits, conventional physiological traits, and partial agronomic traits are determined using the same methods as in Example One.

[0064] Molecular Marker Detection: Use unmanned aerial vehicle multispectral data to reverse light use efficiency, combine molecular marker detection with PHYTOCHROME gene sites related to light morphological development, and analyze the correlation between genes and phenotypes.

[0065] Screening Process and Germplasm Selection Data Processing and Analysis: Integrate and analyze the collected light response data, agronomic trait data, and molecular marker data, use principal component analysis and grey correlation degree analysis to determine the importance of each trait.

[0066] Comprehensive Evaluation Screening: Substitute each trait data into the comprehensive evaluation model to calculate the comprehensive score. According to the score distribution, set the screening threshold to the mean plus 1.0 times the standard deviation, and select 22 germplasms with good light adaptability in the sparse sunshine environment.

[0067] Verification and Effect Analysis The 22 screened germplasms were planted in the oligo-sunlight area for verification, and a control variety was set. The results showed that the light energy utilization rate of the screened germplasms was increased by 18% compared with the control variety, and the growth period was 7-10 days earlier than the control, effectively improving the growth and development of rape in the oligo-sunlight environment, and verifying the effectiveness of the screening method.

[0068] Example Six: Multi-ecological zone joint screening Test layout and data collection In the plateau area with an altitude of 2500-3500 meters, 5 test points with different ecological conditions were set, including low-altitude temperate zone, medium-altitude cold and arid zone, high-altitude strong radiation zone, saline-alkali zone and oligo-sunlight zone. 200 identical rape germplasms were planted in each test point, and unified test design and data collection standards were adopted. Each germplasm was planted in 3 replicates, with a plot area of 4-6 square meters, and the planting density was adjusted according to the characteristics of each region. During the entire growth period, agronomic traits, physiological traits, quality traits and stress resistance traits were systematically measured, and more than 80000 data of 1200 samples were accumulated.

[0069] Model calibration and weight optimization Data integration analysis: Using mixed linear model (MLM), the data of different test points were analyzed to correct the environmental effect, and the genetic effect and environmental interaction effect were separated.

[0070] Model optimization: Based on the Bayesian optimization algorithm, the parameters of the comprehensive evaluation model were adjusted, and according to the differences in the performance of each ecological zone trait, the weight coefficients of each trait in different environments were re-determined, and a multi-environment adaptability prediction model was established.

[0071] Germplasm screening and verification Comprehensive screening: The trait data of each test point were substituted into the optimized comprehensive evaluation model to calculate the comprehensive score of each germplasm. According to the score, the germplasms with excellent comprehensive performance were screened out.

[0072] Multi-point verification: The screened germplasms were continuously verified in the field for 2 years in 5 test points to evaluate their adaptability and stability in different ecological environments. Finally, 10 broad-adaptability germplasms were selected, which increased the yield by 20%-25% on average in different ecological zones, and the coefficient of variation of oil content was controlled within 3%, achieving the goal of high yield, high quality and broad adaptation, and fully verifying the reliability and effectiveness of the multi-ecological zone joint screening method.

[0073] Example One: Traditional single-trait breeding method Breeding process In a plateau test field at 2500 meters above sea level, 150 oilseed rape germplasm resources were selected, and only "single plant silique number" was used as the screening index (this index is often regarded as the core yield trait in traditional oilseed rape breeding). At the mature harvest stage of oilseed rape, the number of single plant silique was counted manually, and the top 20% (30) of the germplasm were selected as the preliminary selection materials. Other agronomic traits (such as plant height, branch number), physiological traits (such as antioxidant enzyme activity), quality traits (such as oil content), and stress resistance traits (such as drought resistance) were not systematically determined and analyzed.

[0074] Field verification results The 30 preliminary selected germplasms were planted in the same test field for verification the next year, and the local main cultivar was set as the control. The results showed that, although the average single plant silique number of the selected germplasm was 18% higher than that of the control variety, the synergistic effect of other traits was not considered: Agronomic trait imbalance: the lodging rate of some germplasms was as high as 40% due to excessively high plant height (more than 180 cm), seriously affecting yield; Poor physiological adaptability: when a short drought occurred during the growth period, the leaf wilting rate of the selected germplasm was 25% higher than that of the control, and the recovery growth ability was weak; Quality decline: the average oil content was only 40%, 5 percentage points lower than that of the control variety.

[0075] Finally, the average yield per mu of the selected germplasm was 135 kg, 10% lower than that of the control variety (150 kg), which verified the limitations of single-trait selection.

[0076] Example 2: Conventional multi-trait independent screening method Breeding process In a test field at an altitude of 3000 meters in a cold and arid area, 120 oilseed rape germplasms were also selected, and agronomic traits (plant height, silique number), physiological traits (proline content), quality traits (oil content), and stress resistance traits (cold resistance) were determined, but an independent screening strategy was adopted: Agronomic traits: select germplasms with plant height of 80-120 cm and silique number in the top 20%; Physiological traits: retain germplasms with proline content higher than the population mean; Quality traits: select germplasms with oil content exceeding 45%; Stress resistance traits: retain germplasms with freeze injury index lower than 30 through low temperature treatment.

[0077] After several rounds of independent screening, 15 "multi-trait qualified" germplasms were finally obtained. This process did not consider the weight difference and mutual correlation between traits, and did not construct a comprehensive evaluation model.

[0078] Field verification results The 15 germplasms and the control variety were subjected to two years of field tests. The results showed that: The contradictions in traits are prominent: although some germplasm has an oil content as high as 50%, it has extremely poor cold resistance, with a mortality rate of up to 60% under low winter temperatures; Overall performance was poor: the average yield of the selected germplasm was 140 kg / mu, a 15% decrease compared to the control variety (165 kg / mu); although the oil content was high, the overall economic benefits were lower than the control due to insufficient stress resistance.

[0079] This comparative example illustrates that simple independent screening of multiple traits cannot effectively balance the synergistic effects of various traits, making it difficult to breed superior germplasm adapted to the complex environment of the plateau.

[0080] Compare with Example 3: Breeding methods dominated by a single technology Breeding process In an experimental field located in a high-radiation area at an altitude of 3500 meters, 100 rapeseed germplasm accessions were selected. Molecular marker-assisted selection was used only to screen for gene loci related to flavonoid synthesis (known to be associated with radiation resistance). PCR amplification and electrophoresis were used to identify 20 accessions carrying the target gene. No comprehensive evaluation was conducted using phenotypic data or other traits.

[0081] Field verification results The results of planting 20 germplasm accessions in a high-radiation environment were verified, and the following findings were made: Gene-phenotype disconnect: Some germplasm carrying the target gene did not show a significant increase in actual flavonoid content, and their radiation resistance was not significantly different from that of unscreened germplasm; Other traits declined: due to a lack of attention to agronomic traits, the plant type of the selected germplasm was loose, and the number of siliques was reduced by 25% compared with the control; in terms of quality, the oil content decreased by an average of 3 percentage points.

[0082] Ultimately, the average yield of the screened germplasm was only 120 kg / mu, significantly lower than that of the control variety (155 kg / mu), indicating that the breeding method that relies solely on molecular technology and ignores the synergistic effect of multiple traits has significant defects.

[0083] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of protection claimed by the present invention. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for breeding high-oil rape adaptive germplasm based on multi-trait coordinated screening, characterized in that, The application relates to a method for screening highland oilseed rape germplasm with high adaptability. The method comprises the following steps: The weight of each trait in the multi-trait evaluation index system is determined by using principal component analysis, grey correlation degree analysis and analytic hierarchy process, and a comprehensive evaluation model is constructed wherein F is a comprehensive score, is the weight of the i th trait, is the standardized data of the i th trait, and n is the total number of traits, wherein: When the analytic hierarchy process is used to construct a judgment matrix, based on the selection pressure of the plateau environment on each trait, the weight of the stress resistance trait is adjusted to 25%-35%, and the weight of the agronomic trait is adjusted to 30%-35%. The agronomic, physiological, quality and stress resistance trait data of the selected highland oilseed rape germplasm are obtained by using the unmanned aerial vehicle multispectral imaging, ground phenotyping platform and molecular marker technology. The trait data are substituted into a comprehensive evaluation model to screen highland oilseed rape adaptive germplasm with a comprehensive score higher than a threshold value. The threshold value is determined according to the distribution of the comprehensive score of the selected population, and is the mean value plus 0.5-1.5 times the standard deviation. The method further comprises a 2-3 year field multi-point adaptability verification step of the screened germplasm, and the verification sites cover different ecological regions with an altitude of 2500-3500 m and an average annual temperature of 4-8 DEG C. The agronomic traits include plant height, primary effective branch number, single pod number, grain number per pod and thousand-grain weight.

2. The method for breeding high-oil Brassica napus adaptive germplasm based on multi-trait co-selection according to claim 1, characterized in that: The physiological traits include chlorophyll a content, proline content, superoxide dismutase activity and peroxidase activity.

3. The method for highland oilseed rape adaptive germplasm selection based on multi-trait co-selection according to claim 1, characterized in that: The quality traits include oil content, oleic acid content, glucosinolate content and protein content.

4. The method for breeding high-oil Brassica napus adaptive germplasm based on multi-trait co-selection according to claim 1, characterized in that: The stress resistance traits include drought resistance index, cold resistance index and sclerotinia disease index.

5. The method for highland oilseed rape adaptive germplasm selection based on multi-trait co-selection according to claim 1, characterized in that: The correlation coefficient calculation formula in the grey correlation degree analysis is as follows:

6. The method for highland oilseed rape adaptive germplasm selection based on multi-trait co-selection according to claim 1, characterized in that: The standardization processing formula in the principal component analysis is wherein is the standardized data, is the original data, is the jth trait mean, is the standard deviation.

7. The method for highland oilseed rape adaptive germplasm selection based on multi-trait co-selection according to claim 1, characterized in that: The molecular marker technology adopts SSR markers or SNP markers, and genotyping is performed through PCR amplification and electrophoresis detection. wherein is a resolution coefficient, having a value of 0.1-0.

5.

8. The method for highland oilseed rape adaptive germplasm selection based on multi-trait co-selection according to claim 1, characterized in that: In the screening step, the threshold value is determined according to the distribution of the comprehensive score of the selected population, and is the mean value plus 0.5-1.5 times the standard deviation.

9. The method for highland oilseed rape adaptive germplasm selection based on multi-trait co- selection according to claim 1, characterized in that: The method further comprises a 2-3 year field multi-point adaptability verification step of the screened germplasm.

10. The method for highland oilseed rape adaptive germplasm selection based on multi-trait co-selection according to claim 1, characterized in that: ​

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