A method for screening rapeseed germplasm based on gradient salt-alkali stress
By using a gradient salt-alkali stress method and a comprehensive tolerance index assessment, the problem of identifying pseudo-tolerant materials in rapeseed germplasm screening was solved, enabling refined tolerance classification and cross-generational adaptation potential assessment of rapeseed germplasm, and improving the stability of screening results and breeding application value.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- INNER MONGOLIA AUTONOMOUS REGION ACAD OF AGRI & ANIMAL HUSBANDRY SCI
- Filing Date
- 2026-02-27
- Publication Date
- 2026-06-05
AI Technical Summary
Existing technologies in the screening of salt- and alkali-tolerant rapeseed germplasm do not distinguish the independent effects of salt stress, alkali stress, and combined stress, lack gradient design, and thus cannot identify pseudo-tolerant materials. Tolerance evaluation relies on linear weighting, ignoring the nonlinear nature, making it difficult to maintain stability in complex field environments, and thus limiting its breeding application value.
The gradient salt-alkali stress method was adopted, and rapeseed germplasm was simultaneously treated by setting salt, alkali and salt-alkali combined stress gradient groups. The comprehensive tolerance index was calculated, and the gradient stress-genotype interaction response surface was constructed. The tolerance pattern classification and cross-generational adaptation potential were carried out by objective weighting using the information entropy method and Sigmoid mapping, combined with Gompertz function fitting.
This method enables refined tolerance classification of rapeseed germplasm, improves the stability of screening results and breeding application value, identifies germplasm with both high tolerance stability and cross-generational adaptation potential, avoids subjective bias and linear distortion, and enhances the sensitivity and biological interpretability of tolerance evaluation.
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Figure CN122157793A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of agricultural biotechnology, and in particular to a method for screening salt-tolerant rapeseed germplasm based on gradient salt-alkali stress. Background Technology
[0002] Rapeseed, an important oilseed crop in my country, is often constrained by the increasing salinization of arable land. In recent years, researchers have primarily employed phenotypic evaluation methods based on single stress concentrations, combined with biomass, survival rate, or physiological indicators, to rank rapeseed germplasm resources for salt and alkali tolerance. Some studies have attempted to introduce comprehensive evaluation indices or multi-indicator weighted models to improve screening efficiency, while others have used transcriptomics or QTL mapping to mine stress-resistance-related genes, providing a basis for molecular-assisted selection.
[0003] Current technologies for screening salt- and alkali-tolerant rapeseed germplasm generally employ single stress concentration or static evaluation models. These methods fail to distinguish the independent effects of salt stress, alkali stress, and combined stress, and lack gradient designs to characterize the dynamic response process of tolerance as stress intensity changes. Consequently, they cannot identify pseudo-tolerant materials. Their tolerance evaluation often relies on linear weighting or subjective weighting, ignoring the nonlinear nature of plant stress response. This makes it difficult to effectively distinguish moderately tolerant germplasm. Furthermore, they have not established a functional typing system from phenotypic data to tolerance mechanisms, nor have they considered the cross-generational adaptation potential induced by stress. As a result, the screening results have poor stability in complex saline-alkali environments in the field and limited breeding application value. Summary of the Invention
[0004] In view of the aforementioned existing problems, the present invention is proposed.
[0005] Therefore, this invention provides a method for screening salt-alkali tolerant rapeseed germplasm based on gradient salt-alkali stress. This method addresses the problems of failing to distinguish the independent effects of salt stress, alkali stress, and combined stress, lacking gradient design to characterize the dynamic response process of tolerance as stress intensity changes, resulting in the inability to identify pseudo-tolerant materials. Its tolerance evaluation relies heavily on linear weighting or subjective weighting, ignoring the nonlinear nature of plant stress response, making it difficult to effectively distinguish moderately tolerant germplasm. Furthermore, it fails to establish a functional typing system from phenotypic data to tolerance mechanisms, and does not consider the cross-generational adaptation potential induced by stress. Consequently, the screening results have poor stability in complex saline-alkali environments in the field and limited breeding application value.
[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution:
[0007] This invention provides a method for screening salt-alkali tolerant rapeseed germplasm based on gradient salt-alkali stress, which includes setting up a salt stress gradient group, an alkali stress gradient group and a salt-alkali combined stress gradient group, simultaneously treating multiple rapeseed germplasms, and obtaining phenotypic response data of each rapeseed germplasm under different stress conditions.
[0008] Based on phenotypic response data, the comprehensive tolerance index of each rapeseed germplasm under each stress gradient was calculated;
[0009] Based on the comprehensive tolerance index and salt stress gradient group, alkali stress gradient group and salt-alkali combined stress gradient group, a gradient stress-genotype interaction response surface was constructed for each rapeseed germplasm.
[0010] Geometric feature parameters are extracted from the response surface, and rapeseed germplasm is classified into tolerance patterns based on the geometric feature parameters.
[0011] Candidate rapeseed germplasm was selected based on the tolerance pattern classification results;
[0012] Sublethal salt-alkali stress was applied to the parent plants of candidate rapeseed germplasm, and the offspring seeds were harvested and the offspring plants were cultured under stress-free conditions.
[0013] The progeny plants were placed under high-gradient salt-alkali stress conditions for tolerance testing. Based on the test results and tolerance pattern classification, salt-alkali tolerant rapeseed varieties with both high tolerance stability and cross-generational adaptation potential were identified.
[0014] As a preferred embodiment of the rapeseed salt-alkali tolerant germplasm screening method based on gradient salt-alkali stress described in this invention, the steps of setting up salt stress gradient groups, alkali stress gradient groups, and salt-alkali combined stress gradient groups to simultaneously treat multiple rapeseed germplasms and obtain phenotypic response data of each rapeseed germplasm under different stress conditions are as follows:
[0015] A salt stress gradient set was constructed using sodium chloride solution, with electrical conductivity as the stress intensity index, and at least three gradient levels were set.
[0016] An alkaline stress gradient group was constructed by using a mixed solution of sodium bicarbonate and sodium carbonate, with pH value as the stress intensity index, and at least three gradient levels were set.
[0017] A salt-alkali composite stress gradient group was constructed using sodium chloride and sodium bicarbonate / sodium carbonate combinations to simulate a typical soda-type saline-alkali soil environment, while controlling electrical conductivity and pH value.
[0018] The same group of rapeseed germplasm was planted in the above three stress systems and cultured hydroponically or in a substrate under the same conditions of light, temperature, humidity and nutrient supply.
[0019] After 21 days of stress treatment, five indicators were measured for each germplasm: plant height, aboveground fresh weight, taproot length, chlorophyll SPAD value, and survival status, to obtain a structured phenotypic response dataset.
[0020] As a preferred embodiment of the rapeseed salt-alkali tolerant germplasm screening method based on gradient salt-alkali stress described in this invention, the specific steps of calculating the comprehensive tolerance index of each rapeseed germplasm under each stress gradient based on phenotypic response data are as follows:
[0021] For each indicator in the phenotypic response data, calculate its relative performance value relative to the unstressed control group;
[0022] Based on the degree of variation of all germplasm in the indicators, the information entropy method is used to calculate the objective weight of the indicators to avoid subjective weighting bias.
[0023] The linear weighted score is obtained by multiplying the relative performance values of each indicator by their corresponding weights and then summing the results.
[0024] The linearly weighted score is compressed to the 0-1 interval using a non-linear Sigmoid mapping function.
[0025] As a preferred embodiment of the rapeseed salt-alkali tolerant germplasm screening method based on gradient salt-alkali stress described in this invention, the specific steps for constructing the gradient stress-genotype interaction response surface for each rapeseed germplasm based on the comprehensive tolerance index and salt stress gradient group, alkali stress gradient group, and salt-alkali combined stress gradient group are as follows:
[0026] For each rapeseed germplasm, in the salt stress gradient group, the first response curve was fitted with its electrical conductivity as the independent variable and the corresponding comprehensive tolerance index as the dependent variable;
[0027] In the alkali stress gradient group, with pH value as the independent variable and the corresponding comprehensive tolerance index as the dependent variable, the second response curve was fitted.
[0028] In the salt-alkali combined stress gradient group, a unified combined stress intensity index was constructed based on conductivity and pH value as the independent variable, and its corresponding comprehensive tolerance index was used as the dependent variable to fit the third response curve.
[0029] As a preferred embodiment of the rapeseed salt-alkali tolerant germplasm screening method based on gradient salt-alkali stress described in this invention, the specific steps of extracting geometric feature parameters from the response surface and classifying rapeseed germplasm into tolerance patterns based on the geometric feature parameters are as follows:
[0030] The Gompertz fitting function is mathematically analyzed and expressed as follows:
[0031] ;
[0032] in, For rapeseed germplasm under stress intensity, For the standardized stress intensity variable, The initial decay rate parameter, This represents the stress sensitivity coefficient.
[0033] Setting its second derivative to zero, we can solve for the inflection point, expressed as:
[0034] ;
[0035] in, This is the critical tolerance threshold. Here are the initial decay rate parameters for the Gompertz function. This represents the stress sensitivity coefficient.
[0036] The calculation function is used when the stress intensity ranges from 0 to The average integral over the interval is used as the platform stability index. This reflects the ability of germplasm to maintain high tolerance within commonly used stress ranges;
[0037] The absolute value of the first derivative at the inflection point is calculated as the collapse rate coefficient, which characterizes the severity of performance degradation after exceeding a threshold. The expression is:
[0038] ;
[0039] in, This is the collapse rate coefficient. This represents the theoretical maximum tolerance plateau value of the Gompertz function. The initial decay rate parameter, This is the stress sensitivity coefficient. It is the reciprocal of the natural exponential function at 1.
[0040] Critical tolerance threshold Platform stability index and the collapse rate coefficient Forming a ternary feature vector;
[0041] Cluster analysis or preset threshold discrimination was performed on the ternary feature vectors of all rapeseed germplasms to classify them into four tolerance patterns: ideal tolerance type, widely adaptable intermediate type, sensitive collapse type, and pseudo tolerance type.
[0042] As a preferred embodiment of the rapeseed salt-alkali tolerant germplasm screening method based on gradient salt-alkali stress described in this invention, the specific steps for screening candidate rapeseed germplasm according to the tolerance pattern classification results are as follows:
[0043] Retrieve the classification results of the four tolerance patterns;
[0044] Retaining rapeseed germplasm that was determined to be ideally tolerant indicates robust performance within a wide range of stress and slow decline after exceeding the limit;
[0045] Output the numbers of the ideal tolerant germplasm to obtain the candidate rapeseed germplasm set.
[0046] As a preferred embodiment of the method for screening salt-alkali tolerant rapeseed germplasm based on gradient salt-alkali stress according to the present invention, the specific steps of applying sublethal salt-alkali stress treatment to the parent plants of candidate rapeseed germplasm, harvesting progeny seeds, and cultivating progeny plants under stress-free conditions are as follows:
[0047] The parental seeds of the candidate rapeseed germplasm were planted and cultivated to the four-leaf stage under a controlled greenhouse environment;
[0048] Salt-alkali combined stress was applied, with the stress intensity set below the lethal level, and the treatment was continued for 10 to 14 days to induce physiological adaptation and potential epigenetic memory.
[0049] Resume normal, stress-free culture until flowering and fruiting;
[0050] Harvest F1 generation seeds from individual plants to ensure a clear genetic background;
[0051] F1 generation seeds were sown in sterilized substrate and managed uniformly until the four-leaf stage under conditions free from any salt or alkali stress to obtain offspring plant populations, thus eliminating interference from residual stress from the mother plant.
[0052] As a preferred embodiment of the rapeseed salt-alkali tolerant germplasm screening method based on gradient salt-alkali stress described in this invention, the following steps are taken: The progeny plants are placed under high-gradient salt-alkali stress conditions for tolerance testing. The test results are combined with tolerance pattern classification to determine rapeseed salt-alkali tolerant germplasm that possesses both high tolerance stability and cross-generational adaptation potential.
[0053] High-gradient salt-alkali combined stress treatment was applied to the offspring plants. The stress intensity was higher than that of the parent plants, which was sufficient to distinguish the differences in tolerance.
[0054] A parallel control group was set up simultaneously, consisting of offspring of the same strain that had not experienced parental stress;
[0055] After treatment, the comprehensive tolerance index of the two groups of offspring was calculated and denoted as follows: and ;
[0056] Calculate the stress memory gain value :
[0057] ;
[0058] in, This is the value of the forced memory gain. The overall tolerance index of offspring produced by coerced parents. The overall tolerance index is for control offspring that have not experienced parental stress.
[0059] like If the value is greater than the preset positive threshold, it indicates that the germplasm can transform the parental stress experience into the offspring's representative phenotypic advantage and has a stress memory effect.
[0060] Based on the tolerance pattern classification results, only when the parent belongs to the ideal tolerance type and At that time, it was confirmed that the germplasm not only has strong contemporary tolerance, but also has the potential for cross-generational adaptation.
[0061] The beneficial effects of this invention are as follows: By calculating the comprehensive tolerance index of each germplasm under each gradient, objective weighting is achieved using the information entropy method combined with the Sigmoid nonlinear mapping, transforming the original data of multiple indicators into a unified quantitative scale that conforms to the physiological response law of plants. This helps to avoid subjective bias and linear distortion, improve the sensitivity and biological interpretability of tolerance evaluation, construct a gradient stress-genotype interaction response surface for each germplasm based on the index, and use the Gompertz function to fit and form a continuous dynamic model. This helps to upgrade discrete observation points into individualized tolerance response fingerprints, preserving the evolutionary trajectory of tolerance as stress increases. Attached Figure Description
[0062] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0063] Figure 1 This is a flowchart of a method for screening salt-alkali tolerant rapeseed germplasm based on gradient salt-alkali stress. Detailed Implementation
[0064] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.
[0065] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.
[0066] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.
[0067] Reference Figure 1 This is one embodiment of the present invention, which provides a method for screening salt-alkali tolerant rapeseed germplasm based on gradient salt-alkali stress, comprising the following steps:
[0068] S1. Set up salt stress gradient group, alkali stress gradient group and salt-alkali combined stress gradient group, and simultaneously treat multiple rapeseed germplasms to obtain phenotypic response data of each rapeseed germplasm under different stress conditions.
[0069] Furthermore, a salt stress gradient group was constructed using sodium chloride solution, with electrical conductivity as the stress intensity index, and at least three gradient levels were set.
[0070] An alkaline stress gradient group was constructed by using a mixed solution of sodium bicarbonate and sodium carbonate, with pH value as the stress intensity index, and at least three gradient levels were set.
[0071] A salt-alkali composite stress gradient group was constructed using sodium chloride and sodium bicarbonate / sodium carbonate combinations to simulate a typical soda-type saline-alkali soil environment, while controlling electrical conductivity and pH value.
[0072] The same group of rapeseed germplasm was planted in the above three stress systems and cultured hydroponically or in a substrate under the same conditions of light, temperature, humidity and nutrient supply.
[0073] After 21 days of stress treatment, five indicators were measured for each germplasm: plant height, aboveground fresh weight, taproot length, chlorophyll SPAD value, and survival status, to obtain a structured phenotypic response dataset.
[0074] It should be noted that by constructing three independent gradient systems for salt stress, alkali stress, and salt-alkali combined stress, and simultaneously treating the same group of rapeseed germplasm, the physiological effects of salt damage and alkali damage can be effectively decoupled, avoiding the mechanism confusion caused by traditional mixed stress. At the same time, by using five multi-dimensional phenotypic indicators and unified environmental control, the comparability and biological representativeness of phenotypic data are improved, providing a high signal-to-noise ratio basic dataset for subsequent tolerance quantification.
[0075] S2. Based on the phenotypic response data, calculate the comprehensive tolerance index of each rapeseed germplasm under each stress gradient.
[0076] Furthermore, for each indicator in the phenotypic response data, its relative performance value relative to the unstressed control group was calculated.
[0077] Based on the degree of variation of all germplasm in the indicators, the information entropy method is used to calculate the objective weight of the indicators to avoid subjective weighting bias.
[0078] The linear weighted score is obtained by multiplying the relative performance values of each indicator by their corresponding weights and then summing the results.
[0079] The linearly weighted score is compressed to the 0-1 interval using a non-linear Sigmoid mapping function.
[0080] It should be noted that by introducing the information entropy method to achieve objective allocation of index weights, the subjective bias caused by human weighting is eliminated. Furthermore, the Sigmoid nonlinear mapping function is used to compress the linear weighted score, so that the comprehensive tolerance index changes slowly in the low tolerance zone, has high discrimination in the medium tolerance zone, and tends to saturate in the high tolerance zone. This is more in line with the real physiological response law of plants under stress, thereby improving the accuracy and biological interpretability of tolerance evaluation.
[0081] S3. Based on the comprehensive tolerance index and salt stress gradient group, alkali stress gradient group and salt-alkali combined stress gradient group, the gradient stress-genotype interaction response surface of each rapeseed germplasm was constructed.
[0082] Furthermore, for each rapeseed germplasm, in the salt stress gradient group, the first response curve was fitted with its electrical conductivity as the independent variable and the corresponding comprehensive tolerance index as the dependent variable;
[0083] In the alkali stress gradient group, with pH value as the independent variable and the corresponding comprehensive tolerance index as the dependent variable, the second response curve was fitted.
[0084] In the salt-alkali combined stress gradient group, a unified combined stress intensity index was constructed based on conductivity and pH value as the independent variable, and its corresponding comprehensive tolerance index was used as the dependent variable to fit the third response curve.
[0085] It should be noted that by integrating electrical conductivity and pH value into a unified composite stress intensity index, a standardized expression of different types of salt and alkali stress in mathematical space was achieved. By combining the Gompertz function to fit the three types of response curves respectively, a gradient stress-genotype interaction response surface exclusive to each rapeseed germplasm was successfully constructed. This laid a functional foundation for extracting geometric features from dynamic response patterns and broke through the static limitations of traditional single-point tolerance evaluation.
[0086] S4. Extract geometric feature parameters from the response surface and classify rapeseed germplasm into tolerance patterns based on the geometric feature parameters.
[0087] Furthermore, the Gompertz fitting function is mathematically analyzed, and its expression is:
[0088] ;
[0089] in, For rapeseed germplasm under stress intensity, For the standardized stress intensity variable, The initial decay rate parameter, This represents the stress sensitivity coefficient.
[0090] Setting its second derivative to zero, we can solve for the inflection point, expressed as:
[0091] ;
[0092] in, This is the critical tolerance threshold. Here are the initial decay rate parameters for the Gompertz function. This represents the stress sensitivity coefficient.
[0093] The calculation function is used when the stress intensity ranges from 0 to The average integral over the interval is used as the platform stability index. This reflects the ability of germplasm to maintain high tolerance within commonly used stress ranges;
[0094] The absolute value of the first derivative at the inflection point is calculated as the collapse rate coefficient, which characterizes the severity of performance degradation after exceeding a threshold. The expression is:
[0095] ;
[0096] in, This is the collapse rate coefficient. This represents the theoretical maximum tolerance plateau value of the Gompertz function. The initial decay rate parameter, This is the stress sensitivity coefficient. It is the reciprocal of the natural exponential function at 1.
[0097] Critical tolerance threshold Platform stability index and the collapse rate coefficient Forming a ternary feature vector;
[0098] Cluster analysis or preset threshold discrimination was performed on the ternary feature vectors of all rapeseed germplasms to classify them into four tolerance patterns: ideal tolerance type, widely adaptable intermediate type, sensitive collapse type, and pseudo tolerance type.
[0099] It should be noted that by performing differential geometric analysis on the response surface, morphological characteristics such as inflection point location, platform stability, and collapse rate were transformed into quantifiable parameters for the first time, realizing a refined classification of rapeseed germplasm tolerance mechanisms; in particular, the identification of pseudo-tolerant types effectively avoids the risk of sudden inactivation due to environmental fluctuations in field applications, improving the reliability and breeding value of screening results.
[0100] S5. Based on the tolerance pattern classification results, candidate rapeseed germplasm are selected.
[0101] Furthermore, the classification results of the four tolerance patterns are invoked;
[0102] Retaining rapeseed germplasm that was determined to be ideally tolerant indicates robust performance within a wide range of stress and slow decline after exceeding the limit;
[0103] Output the numbers of the ideal tolerant germplasm to obtain the candidate rapeseed germplasm set.
[0104] It should be noted that only ideal tolerance-type germplasm was retained as candidates to ensure that the selected materials simultaneously possess the three core advantages of high critical tolerance threshold, strong platform stability, and low collapse rate. These represent the stress-resistant types with stable yield potential among current rapeseed germplasm. The screening strategy abandons the traditional method of relying solely on a single stress point or total biomass, and starts from the dynamic nature of the response, thereby improving the hit rate of excellent germplasm and the success rate of subsequent verification.
[0105] S6. Apply sublethal salt-alkali stress to the parent plants of the candidate rapeseed germplasm, harvest the offspring seeds, and cultivate the offspring plants under stress-free conditions.
[0106] Furthermore, the parent seeds of the candidate rapeseed germplasm are planted and cultivated to the four-leaf stage under a controlled greenhouse environment;
[0107] Salt-alkali combined stress was applied, with the stress intensity set below the lethal level, and the treatment was continued for 10 to 14 days to induce physiological adaptation and potential epigenetic memory.
[0108] Resume normal, stress-free culture until flowering and fruiting;
[0109] Harvest F1 generation seeds from individual plants to ensure a clear genetic background;
[0110] F1 generation seeds were sown in sterilized substrate and managed uniformly until the four-leaf stage under conditions free from any salt or alkali stress to obtain offspring plant populations, thus eliminating interference from residual stress from the mother plant.
[0111] It should be noted that by inducing potential epigenetic memory by applying sublethal stress to the parents and raising offspring under stress-free conditions, the maternal effect and intergenerational adaptation contribution were effectively separated. The design ensures that the phenotypic differences of the offspring originate from the heritable regulatory mechanisms triggered by the parental stress experience, rather than residual ions or physiological damage. This provides a clean and reliable experimental system for verifying whether tolerance has the potential for intergenerational transmission.
[0112] S7. Place the offspring plants under high-gradient salt-alkali stress conditions for tolerance testing. Combine the test results with tolerance pattern classification to determine the salt-alkali tolerant rapeseed germplasm that has both high tolerance stability and cross-generational adaptation potential.
[0113] Furthermore, the offspring plants were subjected to a high-gradient salt-alkali combined stress treatment, with the stress intensity being higher than that of the parent plants, which was sufficient to distinguish the differences in tolerance.
[0114] A parallel control group was set up simultaneously, consisting of offspring of the same strain that had not experienced parental stress;
[0115] After treatment, the comprehensive tolerance index of the two groups of offspring was calculated and denoted as follows: and ;
[0116] Calculate the stress memory gain value :
[0117] ;
[0118] in, This is the value of the forced memory gain. The overall tolerance index of offspring produced by coerced parents. The overall tolerance index is for control offspring that have not experienced parental stress.
[0119] like If the value is greater than the preset positive threshold, it indicates that the germplasm can transform the parental stress experience into the offspring's representative phenotypic advantage and has a stress memory effect.
[0120] Based on the tolerance pattern classification results, only when the parent belongs to the ideal tolerance type and At that time, it was confirmed that the germplasm not only has strong contemporary tolerance, but also has the potential for cross-generational adaptation.
[0121] It should be noted that by introducing the innovative index of stress memory gain, a quantitative assessment of the intergenerational adaptability of rapeseed was achieved for the first time. Combined with the results of contemporary tolerance model classification, the selected germplasm not only showed robust performance under contemporary stress, but also passed on its stress resistance advantage to its offspring. It has both high tolerance stability and evolutionary adaptation potential, providing a breakthrough germplasm resource for breeding new rapeseed varieties that are truly suitable for long-term planting in saline-alkali land.
[0122] In summary, this invention transforms multi-index raw data into a unified quantitative scale that conforms to the physiological response laws of plants by calculating the comprehensive tolerance index of each germplasm under various gradients, objectively weighting it using the information entropy method and combining it with the Sigmoid nonlinear mapping. This avoids subjective bias and linear distortion, improves the sensitivity and biological interpretability of tolerance evaluation, and constructs a gradient stress-genotype interaction response surface for each germplasm based on the index. A continuous dynamic model is formed by fitting the model using the Gompertz function. This transforms discrete observation points into individualized tolerance response fingerprints, preserving the evolutionary trajectory of tolerance as stress increases.
[0123] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.
Claims
1. A method for screening oilseed rape germplasm for salt tolerance based on gradient salt-alkali stress, characterized in that: include: Salt stress gradient group, alkali stress gradient group and salt-alkali combined stress gradient group were set up to simultaneously treat multiple rapeseed germplasms and obtain phenotypic response data of each rapeseed germplasm under different stress conditions. Based on phenotypic response data, the comprehensive tolerance index of each rapeseed germplasm under each stress gradient was calculated; Based on the comprehensive tolerance index and salt stress gradient group, alkali stress gradient group and salt-alkali combined stress gradient group, a gradient stress-genotype interaction response surface was constructed for each rapeseed germplasm. Geometric feature parameters are extracted from the response surface, and rapeseed germplasm is classified into tolerance patterns based on the geometric feature parameters. Candidate rapeseed germplasm was selected based on the tolerance pattern classification results; Sublethal salt-alkali stress was applied to the parent plants of candidate rapeseed germplasm, and the offspring seeds were harvested and the offspring plants were cultured under stress-free conditions. The progeny plants were placed under high-gradient salt-alkali stress conditions for tolerance testing. Based on the test results and tolerance pattern classification, salt-alkali tolerant rapeseed varieties with both high tolerance stability and cross-generational adaptation potential were identified.
2. The method for screening salt-alkali tolerant rapeseed germplasm based on gradient salt-alkali stress as described in claim 1, characterized in that: The steps for setting up salt stress gradient groups, alkali stress gradient groups, and salt-alkali combined stress gradient groups to simultaneously treat multiple rapeseed germplasms and obtain phenotypic response data of each rapeseed germplasm under different stress conditions are as follows: A salt stress gradient set was constructed using sodium chloride solution, with electrical conductivity as the stress intensity index, and at least three gradient levels were set. An alkaline stress gradient group was constructed by using a mixed solution of sodium bicarbonate and sodium carbonate, with pH value as the stress intensity index, and at least three gradient levels were set. A salt-alkali composite stress gradient group was constructed using sodium chloride and sodium bicarbonate / sodium carbonate combinations to simulate a typical soda-type saline-alkali soil environment, while controlling electrical conductivity and pH value. The same group of rapeseed germplasm was planted in the above three stress systems and cultured hydroponically or in a substrate under the same conditions of light, temperature, humidity and nutrient supply. After 21 days of stress treatment, five indicators were measured for each germplasm: plant height, aboveground fresh weight, taproot length, chlorophyll SPAD value, and survival status, to obtain a structured phenotypic response dataset.
3. The method for screening salt-alkali tolerant rapeseed germplasm based on gradient salt-alkali stress as described in claim 2, characterized in that: The specific steps for calculating the comprehensive tolerance index of each rapeseed germplasm under various stress gradients based on phenotypic response data are as follows: For each indicator in the phenotypic response data, calculate its relative performance value relative to the unstressed control group; Based on the degree of variation of all germplasm in the indicators, the information entropy method is used to calculate the objective weight of the indicators to avoid subjective weighting bias. The linear weighted score is obtained by multiplying the relative performance values of each indicator by their corresponding weights and then summing the results. The linearly weighted score is compressed to the 0-1 interval using a non-linear Sigmoid mapping function.
4. The method for screening salt-alkali tolerant rapeseed germplasm based on gradient salt-alkali stress as described in claim 3, characterized in that: The gradient stress-genotype interaction response surface for each rapeseed germplasm is constructed based on the comprehensive tolerance index and salt stress gradient group, alkali stress gradient group, and salt-alkali combined stress gradient group. The specific steps are as follows: For each rapeseed germplasm, in the salt stress gradient group, the first response curve was fitted with its electrical conductivity as the independent variable and the corresponding comprehensive tolerance index as the dependent variable; In the alkali stress gradient group, with pH value as the independent variable and the corresponding comprehensive tolerance index as the dependent variable, the second response curve was fitted. In the salt-alkali combined stress gradient group, a unified combined stress intensity index was constructed based on conductivity and pH value as the independent variable, and its corresponding comprehensive tolerance index was used as the dependent variable to fit the third response curve.
5. The method for screening salt-alkali tolerant rapeseed germplasm based on gradient salt-alkali stress as described in claim 4, characterized in that: The specific steps for extracting geometric feature parameters from the response surface and classifying rapeseed germplasm tolerance patterns based on these geometric feature parameters are as follows: The Gompertz fitting function is mathematically analyzed and expressed as follows: ； in, For rapeseed germplasm under stress intensity, For the standardized stress intensity variable, The initial decay rate parameter, This represents the stress sensitivity coefficient. Setting its second derivative to zero, we can solve for the inflection point, expressed as: ; in, This is the critical tolerance threshold. Here are the initial decay rate parameters for the Gompertz function. This represents the stress sensitivity coefficient. The calculation function is used when the stress intensity ranges from 0 to The average integral over the interval is used as the platform stability index. This reflects the ability of germplasm to maintain high tolerance within commonly used stress ranges; The absolute value of the first derivative at the inflection point is calculated as the collapse rate coefficient, which characterizes the severity of performance degradation after exceeding a threshold. The expression is: ; in, This is the collapse rate coefficient. This represents the theoretical maximum tolerance plateau value of the Gompertz function. The initial decay rate parameter, This is the stress sensitivity coefficient. It is the reciprocal of the natural exponential function at 1. Critical tolerance threshold Platform stability index and the collapse rate coefficient Forming a ternary feature vector; Cluster analysis or preset threshold discrimination was performed on the ternary feature vectors of all rapeseed germplasms to classify them into four tolerance patterns: ideal tolerance type, widely adaptable intermediate type, sensitive collapse type, and pseudo tolerance type.
6. The method for screening salt-alkali tolerant rapeseed germplasm based on gradient salt-alkali stress as described in claim 5, characterized in that: The specific steps for selecting candidate rapeseed germplasm based on the tolerance pattern classification results are as follows: Retrieve the classification results of the four tolerance patterns; Retaining rapeseed germplasm that was determined to be ideally tolerant indicates robust performance within a wide range of stress and slow decline after exceeding the limit; Output the numbers of the ideal tolerant germplasm to obtain the candidate rapeseed germplasm set.
7. The method for screening salt-alkali tolerant rapeseed germplasm based on gradient salt-alkali stress as described in claim 6, characterized in that: The specific steps for subjecting parental plants of candidate rapeseed germplasm to sublethal salt-alkali stress, harvesting progeny seeds, and cultivating progeny plants under stress-free conditions are as follows: The parental seeds of the candidate rapeseed germplasm were planted and cultivated to the four-leaf stage under a controlled greenhouse environment; Salt-alkali combined stress was applied, with the stress intensity set below the lethal level, and the treatment was continued for 10 to 14 days to induce physiological adaptation and potential epigenetic memory. Resume normal, stress-free culture until flowering and fruiting; Harvest F1 generation seeds from individual plants to ensure a clear genetic background; F1 generation seeds were sown in sterilized substrate and managed uniformly until the four-leaf stage under conditions free from any salt or alkali stress to obtain offspring plant populations, thus eliminating interference from residual stress from the mother plant.
8. The method for screening salt-alkali tolerant rapeseed germplasm based on gradient salt-alkali stress as described in claim 7, characterized in that: The process involves placing progeny plants under high-gradient salt-alkali stress conditions for tolerance testing. Combining the test results with tolerance pattern classification, salt-alkali tolerant rapeseed germplasm exhibiting both high tolerance stability and cross-generational adaptability potential is determined. The specific steps are as follows: High-gradient salt-alkali combined stress treatment was applied to the offspring plants. The stress intensity was higher than that of the parent plants, which was sufficient to distinguish the differences in tolerance. A parallel control group was set up simultaneously, consisting of offspring of the same strain that had not experienced parental stress; After treatment, the comprehensive tolerance index of the two groups of offspring was calculated and denoted as follows: and ; Calculate the stress memory gain value : ; in, This is the value of the forced memory gain. The overall tolerance index of offspring produced by coerced parents. The overall tolerance index is for control offspring that have not experienced parental stress. like If the value is greater than the preset positive threshold, it indicates that the germplasm can transform the parental stress experience into the offspring's representative phenotypic advantage and has a stress memory effect. A rapeseed germplasm with high tolerance and stability as well as cross-generational adaptability was tested for adaptability in multiple saline-alkali land locations in the field. Based on the tolerance pattern classification results, only when the parent belongs to the ideal tolerance type and At that time, it was confirmed that the germplasm not only has strong contemporary tolerance, but also has the potential for cross-generational adaptation.
9. The method for screening salt-alkali tolerant rapeseed germplasm based on gradient salt-alkali stress as described in claim 8, characterized in that: The salt-alkali tolerant rapeseed variety, which possesses both high tolerance and stability as well as cross-generational adaptability potential, underwent field adaptation verification at multiple saline-alkali land locations. The specific steps were as follows: Select three or more test sites with different salinity and alkalinity types; Uniform field plots were set up at each test site, and the germplasm and the locally cultivated sensitive control varieties were planted simultaneously. Survival rate, biomass and photosynthetic parameters were measured during key growth stages, and yield per plant and seed oil content were measured at maturity. If the field comprehensive adaptability index is not lower than 0.80 at all test sites, then the germplasm is confirmed to have cross-regional promotion and application value.
10. The method for screening salt-alkali tolerant rapeseed germplasm based on gradient salt-alkali stress as described in claim 9, characterized in that: The specific steps for selecting three or more test sites with different salinity types are as follows: A uniform field plot design was set up at each experimental site, using a randomized block design, with no fewer than 3 replicate plots for each germplasm species. Before sowing, the content of soil in the 0–20 cm topsoil layer at each test site was measured to determine the salinity type and intensity level. During the critical growth stages of rapeseed, plant survival rate, leaf area index and photosynthetic rate were recorded simultaneously. Harvest and thresh individually at maturity, and measure the number of siliques per plant, the number of seeds per silique, the weight of 1000 seeds, and the oil content of the seeds.