Method for rapidly identifying salt tolerance of oat variety resources in large scale and application

Abstract

The invention belongs to the technical field of crop salt tolerance identification, and particularly relates to a method for rapidly identifying the salt tolerance of oat variety resources on a large scale and application. The invention provides a method for rapidly identifying the salt tolerance of oat variety resources on a large scale, which is characterized in that different development stages are combined, multi-index determination and analysis based on morphological and physiological changes are adopted, and quantitative sorting and grading of the salt tolerance of varieties are realized by applying principal component analysis dimensionality reduction and membership function comprehensive evaluation. Moreover, the method has the characteristics of short period, controllable environment, rapidness, high efficiency and the like, and a technical method can be provided for developing the cultivation of salt-tolerant varieties of oats and similar plants and the research on a salt-tolerant mechanism.

Description

Technical Field

[0001] This invention belongs to the field of crop salt tolerance identification technology, specifically relating to a method and application for rapid large-scale identification of salt tolerance in oat varieties. Background Technology

[0002] oat( Avena sativa Oats (L.) are an annual crop belonging to the genus *Oat* of the Poaceae family, used for both food and forage. Their stems and leaves are rich in nutrients and have good palatability, making them a high-quality forage resource. The grains are rich in dietary fiber and β-glucan, offering high nutritional and health benefits. They possess comprehensive stress resistance characteristics, including drought resistance, cold resistance, tolerance to poor soil, and salt tolerance, earning them the reputation of a "pioneer crop for saline-alkali land." Oats have extremely high cultivation value in saline-alkali areas, but the salt tolerance of different varieties varies considerably. Therefore, conducting identification of salt-tolerant oat germplasm and variety breeding is of great significance for the biological management of saline-alkali land, sustainable agricultural and pastoral development, and national food security.

[0003] Currently, crop salt tolerance assessments often rely on single developmental stages (e.g., germination or seedling stages), single stress treatments, or a limited number of morphological indicators. The phenotypic characteristics and physiological responses of plants under salt stress are easily influenced by various factors such as species characteristics and growth status. Therefore, a unified and standardized assessment indicator system for oat salt tolerance has not yet been established. Furthermore, traditional field assessment methods suffer from low efficiency and limited throughput, making it difficult to meet the practical needs of rapid screening and accurate identification of large-scale oat salt-tolerant germplasm resources. Therefore, a systematic, efficient, and accurate comprehensive evaluation method for oat salt tolerance is urgently needed. Summary of the Invention

[0004] The purpose of this invention is to provide a method and application for rapid large-scale identification of salt tolerance of oat varieties. The method can achieve salt tolerance evaluation and screening of a large number of oat varieties in a short period of time, and is simple to operate, low in cost, and accurate in identification results.

[0005] This invention provides a method for rapid, large-scale identification of salt tolerance in oat cultivars, comprising the following steps: Salt stress was applied to oat seeds during their germination period, and the germination characteristics of oat seeds after salt stress treatment were measured. The germination characteristics of oat seeds included relative germination rate, relative plumule length, and relative radicle length. Salt tolerance during the germination period of oat seeds was evaluated based on their germination characteristics, and a comprehensive evaluation value of salt tolerance during the germination period of oats was obtained. Salt stress was applied to oat seedlings during their growth period, and the growth characteristics of the oat seedlings after salt stress treatment were measured. The growth characteristics of the oat seedlings included relative plant height, relative root length, relative root number, and relative SPAD value. Salt tolerance of oat seedlings during their growth period was evaluated based on their growth characteristics, and a comprehensive evaluation value of salt tolerance of oat seedlings during their growth period was obtained. The comprehensive salt tolerance evaluation values ​​during the oat germination period and the comprehensive salt tolerance evaluation values ​​during the oat seedling growth period are weighted and analyzed to obtain a comprehensive weighted value for salt tolerance evaluation. The salt tolerance of oats is evaluated based on the comprehensive weighted value for salt tolerance evaluation. The higher the comprehensive weighted value for salt tolerance evaluation, the stronger the salt tolerance of oats. The salt tolerance evaluation was conducted using principal component analysis and membership function analysis.

[0006] As a preferred embodiment, the salt stress treatment employs a salt concentration gradient treatment.

[0007] As a preferred embodiment, when subjecting oat germination to salt stress, a salt concentration gradient is set up; the salt concentration is 80~150 mM.

[0008] As a preferred option, when subjecting oat seedlings to salt stress treatment, three salt concentration gradients are set for the salt stress treatment; the three salt concentrations are 80~120 mM, 130~170 mM and 180~220 mM.

[0009] As a preferred option, the oat seedlings are cultivated using hydroponics.

[0010] As a preferred embodiment, the oat seedlings are grown at the two-leaf-one-heart stage.

[0011] As a preferred option, when salt stress treatment is carried out during the germination period of oats, the duration of the salt stress treatment is 3 to 7 days.

[0012] As a preferred option, when salt stress treatment is carried out during the oat seedling growth period, the duration of the salt stress treatment is 5 to 10 days.

[0013] As a preferred approach, after obtaining the comprehensive weighted value of salt tolerance evaluation, the method further includes: performing cluster analysis on the comprehensive weighted value of salt tolerance evaluation.

[0014] This invention also provides the application of the method described above in the cultivation of salt-tolerant oat germplasm.

[0015] Beneficial Effects: This invention provides a rapid and large-scale method for identifying the salt tolerance of oat varietal resources. The invention involves salt stress treatment during seed germination, selecting germination rate, plumule length, and radicle length as evaluation indicators, and combining this with membership function analysis to obtain a more objective and accurate assessment of salt tolerance during the oat seed germination stage. Simultaneously, this invention measures multiple indicators of oat seedlings (plant height, root length, root number, and SPAD value), evaluating their salt tolerance using principal component and membership function methods. Then, the salt tolerance of oats is assessed based on the comprehensive salt tolerance evaluation value. Finally, combining the salt tolerance results from the germination and seedling growth stages, a weighted average is performed using the comprehensive D-value to comprehensively consider the salt tolerance of oat germplasm, enabling rapid and accurate identification of oat salt tolerance. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 Figures showing the germination of oat seeds under different concentrations of NaCl. Figure 2 Figures showing the growth of oat seedlings under different concentrations of NaCl treatment; Figure 3 This is a cluster analysis result of salt tolerance of oat germplasm based on the comprehensive weighted value of salt tolerance evaluation during seed germination and seedling growth. Detailed Implementation

[0018] This invention provides a method for rapid, large-scale identification of salt tolerance in oat cultivars, comprising the following steps: Salt stress was applied to oat seeds during their germination period, and the germination characteristics of oat seeds after salt stress treatment were measured. The germination characteristics of oat seeds included relative germination rate, relative plumule length, and relative radicle length. Salt tolerance during the germination period of oat seeds was evaluated based on their germination characteristics, and a comprehensive evaluation value of salt tolerance during the germination period of oats was obtained. Salt stress was applied to oat seedlings during their growth period, and the growth characteristics of the oat seedlings after salt stress treatment were measured. The growth characteristics of the oat seedlings included relative plant height, relative root length, relative root number, and relative SPAD value. Salt tolerance of oat seedlings during their growth period was evaluated based on their growth characteristics, and a comprehensive evaluation value of salt tolerance of oat seedlings during their growth period was obtained. The comprehensive salt tolerance evaluation values ​​during the oat germination period and the comprehensive salt tolerance evaluation values ​​during the oat seedling growth period are weighted and analyzed to obtain a comprehensive weighted value for salt tolerance evaluation. The salt tolerance of each type of oat is evaluated based on the comprehensive weighted value for salt tolerance evaluation. The higher the comprehensive weighted value, the stronger the salt tolerance of the oat. The salt tolerance evaluation was conducted using principal component analysis and membership function analysis.

[0019] Oat germination and seedling growth stages are highly sensitive to environmental stresses (including salt stress), and the early effects of salt stress on seed germination and seedling growth directly influence or determine the later forage (grain) yield of oats. Meanwhile, the plant's response to salt stress is relatively sluggish in later stages of growth and development (e.g., seed setting and maturation). This invention selects two growth stages—the germination and seedling growth stages—for salt tolerance evaluation, which can accurately capture the early stress resistance response characteristics of oats and improve detection efficiency and accuracy.

[0020] The method described in this invention can rapidly and accurately identify the salt tolerance of multiple oat varieties. This invention subjects multiple oat varieties to salt stress treatment during the oat germination period. As one implementation, before salt stress treatment during oat germination, the method further includes pretreatment of the oat seeds. This pretreatment includes seed disinfection. As one implementation, the oat seeds are plump and undamaged. Selecting plump and undamaged oat seeds for the experiment avoids the influence of seed quality on the results. As one implementation, seed disinfection includes soaking the seeds in a 3.5% (w / v) sodium hypochlorite solution for 10 minutes. As another implementation, after seed disinfection, the method further includes seed washing. Washing includes rinsing the disinfected seeds with distilled water until there is no irritating odor; the rinsing is repeated 3-5 times. This pretreatment of oat seeds can kill pathogens and insect eggs on the seed surface, reducing interference.

[0021] After pretreatment of oat seeds, pretreated oat seeds are obtained. This invention then subjectes the pretreated oat seeds to salt stress treatment (denoted as the salt stress treatment group or treatment group). As one embodiment, the salt stress treatment includes treating the pretreated oat seeds with a salt solution. As one embodiment, the salt stress treatment employs a salt concentration gradient. As one embodiment, when conducting salt stress treatment during the oat germination period, one salt concentration gradient is set; the concentration of the salt solution is 80-150 mM. In specific embodiments of this invention, the concentration of the salt solution can be any value within the range of 80-150 mM, for example, 80, 90, 100, 110, 120, 130, 140, or 150 mM. The inventors have found through experiments that when the salt solution concentration reaches 100 mM, the germination rate, radicle length, and plumule length of the oat seeds show significant differences compared to the water control group. Furthermore, this concentration helps reduce experimental costs and is suitable for large-scale operation. As one implementation method, the salt stress treatment also includes a water control group (also referred to as the control group), which uses sterile water to treat the pretreated oat seeds. The oat seeds in the salt stress treatment group and the water treatment group are from the same source, ensuring that the initial state of the seeds is the same; at the same time, the pretreatment conditions for the two groups of oat seeds are identical, avoiding interference from pretreatment differences in subsequent evaluation. The water treatment group in this invention can objectively reflect the true impact of salt stress on oat seed germination, improving the accuracy and reliability of the experimental results.

[0022] In one implementation method, when salt stress treatment is applied during the oat germination period, the duration of the salt stress treatment is 3-7 days. In a specific embodiment of the present invention, the duration of the salt stress treatment can be any value within the range of 3-7 days, such as 3, 4, 5, 6, or 7 days. The timing of salt stress treatment during the oat germination period is beneficial for capturing the differences in the response speed of different oat varieties to salt stress, taking into account both the speed of germination and the uniformity of germination. Applying salt stress to seeds for 3-7 days during the oat germination period allows for a more objective and accurate assessment of the salt tolerance of the corresponding germplasm.

[0023] After salt stress treatment during the germination period of oats, the germination characteristics of oat seeds under salt stress were measured. These characteristics included relative germination rate, relative plumule length, and relative radicle length. Although some oat seeds could germinate after salt stress treatment, the growth of the plumule and radicle was poor, exhibiting typical salt toxicity phenomena under salt stress or adverse conditions (i.e., the lag effect of salt damage). Therefore, using only the germination rate as a single indicator for evaluation during the germination period would lead to inaccurate and unobjective results. This invention selects three indicators—relative germination rate, relative plumule length, and relative radicle length—to evaluate the salt tolerance of oats during the germination period, thereby improving the accuracy and objectivity of the evaluation results.

[0024] As one implementation method, the formula for calculating the relative germination rate is: Relative germination rate = (germination rate of the treatment group / germination rate of the control group) × 100%; where, germination rate = (total number of seeds germinated in 5 days / number of seeds tested) × 100%. In this invention, the radicle breaking through the seed coat by 3 mm is used as the seed germination standard, and the total number of seeds germinating for at least 5 consecutive days starting from the 3rd day of salt stress is counted. As one implementation method, the formula for calculating the relative radicle length is: Relative radicle length = (radicle length of the treatment group / radicle length of the control group) × 100%; as one implementation method, the formula for calculating the relative plumule length is: Relative plumule length = (plump length of the treatment group / plumule length of the control group) × 100%. This invention selects relative germination rate, relative radicle length, and relative plumule length as evaluation indicators, which can intuitively reflect the relative change range of seed germination ability under salt stress, making the evaluation standard uniform and the results more comparable; furthermore, quantifying the degree of salt damage with relative indicators is beneficial for more accurately screening salt-tolerant materials and evaluating the stress effect.

[0025] After obtaining the germination characteristics of oat seeds, this invention analyzes these characteristics using principal component analysis (PCA) to obtain principal components. As one implementation method, PCA is performed using SPSS 27 software, and corresponding principal components are extracted based on the principle that the cumulative contribution rate is greater than 85%. This invention does not have special requirements for the PCA method; any PCA method well-known in the art can be used. In a specific embodiment of this invention, the PCA method includes: Z-score standardization of the original germination characteristic indices of oat seeds (relative germination rate, relative plumule length, and relative radicle length) to eliminate interference from differences in dimensions and magnitudes between different indices; subsequently, the KMO test (KMO value ≥ 0.5) and Bartlett's sphericity test (…) are performed. P <0.05) Verify the applicability of principal component analysis for the data; based on this, calculate the eigenvalues, variance contribution rate (also known as variance percentage), and cumulative variance contribution rate of each indicator, and extract the corresponding principal components according to the principle that the cumulative variance contribution rate is greater than 85%; finally, based on the principal component score coefficient matrix, perform linear combination on the standardized original indicators to calculate the score value of each oat variety on each principal component, which is used for membership function analysis. In the specific implementation of this invention, the obtained germination characteristic values ​​can be input into SPSS 27 software to perform the above principal component analysis process.

[0026] After obtaining the principal components, this invention uses membership function analysis to analyze the principal components and obtains the comprehensive evaluation value of salt tolerance during oat germination, denoted as D1. This invention does not have special requirements for the membership function analysis method; it can be performed according to the membership function analysis method well known in the art. As one implementation method, the membership function analysis method includes: (1) calculating the membership function value μ(Xi), the calculation formula being: μ(Xi) = (i=1,2,3...,n); where Xi is the score of the i-th factor; Xmin is the minimum score of the i-th factor; Xmax is the maximum score of the i-th factor; Xmax and Xmin refer to the maximum and minimum scores of all varieties for this factor; where factor refers to principal component. (2) Calculate the weights of different factors: Wi=Pi / (i=1,2,3......,n); where: Wi is the importance of the i-th factor among all factors; Pi is the contribution rate of the i-th factor; where factor refers to principal component. (3) Comprehensive evaluation of salt tolerance during oat germination, the formula is: D1= (i=1,2,3...,n). This invention evaluates the salt tolerance of each type of oat based on a comprehensive evaluation value D1; the higher the comprehensive evaluation value D1, the stronger the salt tolerance of the oat during germination.

[0027] This invention involves salt stress treatment during the growth period of oat seedlings. As one embodiment, the cultivation of oat seedlings includes germinating oat seeds to obtain oat seedlings. As one embodiment, the pre-germination process includes pre-treatment of the oat seeds; the oat seeds and the pre-treatment method have been discussed above and will not be repeated here. As one embodiment, the germination includes placing the oat seeds in a petri dish pre-lined with a double layer of sterile water-soaked filter paper for germination culture, adding sterile water every 2 days during germination to keep the filter paper moist. As one embodiment, when the radicle of the oat seed reaches 2 cm and the plumule reaches 1 cm, uniformly growing oat seedlings are selected for hydroponics to obtain oat seedlings. As one embodiment, the seedling growth period is the two-leaf-one-heart stage. This invention selects oat seedlings at the two-leaf-one-heart stage for salt stress treatment. At this growth stage, the seedlings have transitioned from heterotrophic to autotrophic, their endosperm nutrients are largely depleted, and they begin to rely entirely on their own photosynthesis, eliminating interference from maternal nutrition and accurately reflecting the salt tolerance of the genotype. The two-leaf-one-heart stage marks the start of vegetative growth, resulting in high population uniformity and effectively reducing errors caused by individual differences. Furthermore, physiological metabolism is active at this stage, with typical stress responses; the leaves at this stage have high photosynthetic efficiency and fully developed stomata. Simultaneously, the root system has strong absorption capacity and is not yet aging, enabling rapid response to salt stress, making it an ideal period for studying early signals of osmotic stress and ion toxicity. Moreover, this stage is convenient for operation, allows for controllable experimental cycles, and the young age of the oat seedlings and the small space required facilitate large-scale identification in incubators or artificial climate chambers. Additionally, this stage is extremely sensitive to salt, allowing for rapid differentiation of salt tolerance differences within a short period (7-14 days), making it suitable for efficient screening.

[0028] As one implementation method, the oat seedlings are cultivated using hydroponics. This invention uses hydroponics to cultivate oat seedlings. When subjecting them to salt stress, the salt solution is directly added to the hydroponic container, ensuring a consistent nutrient environment and salt stress concentration for each root system, avoiding interference from local differences in the results. In conventional crop salt tolerance assessments, seedlings are cultivated using soilless cultivation (vermiculite). During salt stress treatment, the salt solution is directly poured onto the soilless culture medium. However, the formulation and irrigation method of the nutrient solution in soilless cultivation (vermiculite), as well as the implementation of salt treatment, make it difficult to guarantee uniformity and stability. Different root systems cannot be guaranteed to have the same nutrient and salt stress environment, leading to distorted final salt tolerance assessment results. This invention uses hydroponics during the oat seedling stage, ensuring consistency in nutrient composition and salt stress concentration for each oat root system, improving the accuracy of the results.

[0029] As one implementation method, when subjecting oat seedlings to salt stress, three salt concentrations (denoted as salt stress treatments or treatments) are set. Using a single salt concentration can only simulate a specific degree of salt stress environment and cannot cover the true response patterns of seedlings under different salt stress gradients, easily leading to biased and limited evaluation results. Furthermore, different materials have different salt tolerance thresholds, and using only a single concentration makes it difficult to distinguish the differences in salt tolerance between materials, easily resulting in misjudgments or omissions. This invention sets three salt concentrations for salt stress treatment during the oat seedling growth period, comprehensively and systematically reflecting the response patterns of oats under different salt stress intensities, avoiding evaluation biases caused by excessively high or low concentrations, and improving the objectivity and accuracy of the results. As one implementation method, the three salt concentrations are 80-120 mM, 130-170 mM, and 180-220 mM. This invention selects three salt concentration gradients for salt stress treatment, enabling the construction of a dose-response model, thereby more accurately analyzing the salt tolerance of germplasm. The low-concentration (80-120 mM) treatment is mainly used to distinguish salt-sensitive materials; the medium-concentration (130-170 mM) treatment serves as the core evaluation concentration, maximizing the differentiation between materials with different salt tolerance levels, classifying salt tolerance into strong, medium, and weak grades; and the high-concentration (180-220 mM) treatment is mainly used to screen for extremely salt-tolerant germplasm and identify the salt tolerance limit of materials. In specific embodiments of the present invention, the low-concentration salt concentration can be any value within the range of 80-120 mM, such as 80, 90, 100, 110, or 120 mM; the medium-concentration salt concentration can be any value within the range of 130-170 mM, such as 130, 140, 150, 160, or 170 mM; and the high-concentration salt concentration can be any value within the range of 180-220 mM, such as 180, 190, 200, 210, or 220 mM. As one implementation method, the salt stress treatment also includes setting up a water control group (also referred to as the control), in which the oat seedlings are treated with sterile water. The oat seedlings in the salt stress treatment group and the water treatment group of this invention are from the same batch and under the same treatment conditions. The water treatment group in this invention can objectively reflect the true impact of salt stress on the growth of oat seedlings, improving the accuracy and reliability of the experimental results. In a specific embodiment of this invention, when conducting salt tolerance testing on oat seedlings, four treatment groups are set up: a control group, a 100 mM treatment group, a 150 mM treatment group, and a 200 mM treatment group.

[0030] In one implementation method, the salt stress treatment period during the oat seedling growth period is 5-10 days. In specific embodiments of this invention, the salt stress treatment period can be any value within the range of 5-10 days, such as 5, 6, 7, 8, 9, or 10 days. This invention limits the salt stress treatment time during the oat seedling growth period, allowing the seedlings to complete the transition from osmotic stress to ion toxicity, resulting in obvious morphological symptoms that are easy to observe and quantify, while also balancing experimental cycle and efficiency. In this invention, the salt stress treatment time for different salt concentration treatment groups is consistently 5-10 days, allowing for observation of the effects of different salt concentrations on oat seedlings from the same perspective.

[0031] After subjecting oat seedlings to salt stress for 5-10 days during their growth period, the growth characteristics of oat seedlings under different concentrations of salt stress were measured. These growth characteristics included relative plant height, relative root length, relative root number, and relative SPAD value (SPAD, relative chlorophyll content). Existing oat salt tolerance evaluation indicators typically only measure root length and plant height, and determine the salt damage index based on leaf yellowing, then assess the salt tolerance of different varieties based on the salt damage index. These methods are characterized by single indicators, strong subjectivity, and susceptibility to observer judgment and environmental factors, making it difficult to comprehensively and objectively reflect the true salt tolerance level of the plants. This invention employs a comprehensive evaluation system combining relative plant height, relative root length, relative root number (also denoted as relative root count or relative root development), and relative SPAD value. By using relative indicators, systematic errors caused by differences in seed batches, growth environments, and initial growth vigor are eliminated. The indicators are more comprehensive, quantitative, and objective, and can more accurately reflect the true impact of salt stress on oat growth and physiological characteristics, improving the accuracy of salt tolerance evaluation results.

[0032] In one implementation method, the relative plant height = (treatment plant height / control plant height) × 100%; in another implementation method, the relative root length = (treatment root length / control root length) × 100%; in another implementation method, the relative root number = (treatment root number / control root number) × 100%; in another implementation method, the relative SPAD value = (treatment SPAD value / control SPAD value) × 100%. This invention uses relative quantitative data as evaluation indicators, which can intuitively reflect the relative change in salt tolerance of oat seedlings under salt stress, making the evaluation standards unified and the results more comparable; furthermore, quantifying the degree of salt damage with relative indicators is beneficial for more accurately screening salt-tolerant materials and evaluating the effects of stress.

[0033] After obtaining the growth characteristics of oat seedlings after salt stress treatment, principal component analysis (PCA) was used to analyze these characteristics. This invention does not have specific requirements for the PCA method; any well-known PCA method in the art can be used. As one implementation method, SPSS 27 software was used for PCA, and corresponding principal components were extracted based on a cumulative contribution rate greater than 85%. In a specific embodiment of this invention, the PCA method includes: Z-score standardization of the original growth characteristics of oat seedlings (relative plant height, relative root length, relative root number, and relative SPAD value) to eliminate interference from differences in dimensions and magnitudes between different indicators; subsequently, the KMO test (KMO value ≥ 0.5) and Bartlett's sphericity test (…) were performed. P <0.05) Verify the applicability of principal component analysis for the data; on this basis, calculate the eigenvalues, variance contribution rate (also known as variance percentage) and cumulative variance contribution rate of each indicator, and extract the corresponding principal components according to the principle that the cumulative variance contribution rate is greater than 85%; finally, based on the principal component score coefficient matrix, perform linear combination on the standardized original indicators to calculate the score value of each oat variety on each principal component, which is used for membership function analysis.

[0034] After obtaining the principal components, this invention uses membership function analysis to analyze the principal components and obtain the comprehensive evaluation value of salt tolerance during the growth period of oat seedlings. This invention does not have special requirements for the membership function analysis method; it can be performed according to the membership function analysis method well known in the art. As one implementation method, the membership function analysis method includes: (1) calculating the membership function value μ(Xi), the calculation formula is: μ(Xi) = (i=1,2,3...,n); where Xi is the score of the i-th factor; Xmin is the minimum score of the i-th factor; Xmax is the maximum score of the i-th factor; Xmax and Xmin refer to the maximum and minimum scores of all tested varieties for this factor; where factor refers to principal component. (2) Calculate the weights of different factors: Wi=Pi / (i=1,2,3......,n); where: Wi is the importance of the i-th factor among all factors; Pi is the contribution rate of the i-th factor; where factor refers to principal component. (3) Comprehensive evaluation of salt tolerance of oat seedlings during growth period, the formula is: D2= (i=1,2,3...,n). As one implementation method, after obtaining the comprehensive evaluation D2 value after different salt concentration stress treatments, the method also includes calculating the mean of the comprehensive evaluation D2 values ​​after different salt concentration stress treatments (denoted as D). 2均值 This invention assesses the salt tolerance of each type of oat based on the mean of the comprehensive evaluation D2 value; the higher the mean of the comprehensive evaluation D2 value, the stronger the salt tolerance of the oat seedlings during their growth period.

[0035] This invention employs a weighted analysis of the comprehensive salt tolerance evaluation values ​​during the oat germination period and the oat seedling growth period to obtain a weighted comprehensive salt tolerance evaluation value. As one implementation method, the weight ratio for both the oat germination period and the oat seedling growth period is 50%; the weighted comprehensive salt tolerance evaluation value (D...) 加权 The formula for calculating D is: 加权 =50%×D1+50%×D 2均值 This invention utilizes weighted analysis to quantify the contribution of different evaluation indicators, highlighting the dominant role of key indicators in salt tolerance evaluation and avoiding interference caused by differences in the dimensions and magnitudes of various indicators. The response mechanisms and tolerance to salt stress in plants vary significantly at different developmental stages. Observations from a single stage cannot comprehensively reflect the salt tolerance characteristics of oats throughout the entire process from seed germination to seedling growth. Evaluation results are easily distorted due to stage-specific environmental fluctuations, individual differences, or short-term stress responses, making it difficult to objectively and accurately reflect the true salt tolerance level of the material. This invention employs a combined approach of seed germination and seedling growth stages for salt tolerance evaluation, systematically reflecting the response patterns of oats to salt stress at different developmental stages, covering key growth periods, and avoiding the bias and randomness of single-stage evaluations. Furthermore, by comprehensively judging from multiple dimensions such as germination characteristics, growth status, and physiological indicators, the comprehensiveness, stability, and accuracy of the evaluation results can be effectively improved.

[0036] This invention evaluates the salt tolerance of oats based on a comprehensive weighted value of the salt tolerance assessment; it can rank oat varieties according to the comprehensive weighted value of the salt tolerance assessment; the higher the comprehensive weighted value of the salt tolerance assessment, the stronger the salt tolerance of the oats.

[0037] As one implementation method, after obtaining the comprehensive weighted value for salt tolerance evaluation, the method further includes: performing cluster analysis on the comprehensive weighted value. As one implementation method, the cluster analysis is performed using Origin 2024 software with a distance summation method to conduct systematic cluster analysis on all tested variety resources. After obtaining the comprehensive weighted value, this invention uses the default parameters of Origin 2024 software (hierarchical clustering method, Euclidean distance distance type, and a specified K value of 4 for the number of clusters) to perform cluster analysis, which can objectively classify different materials according to their true salt tolerance characteristics, improving the accuracy and rationality of material classification, and providing strong support for the precise screening and grading evaluation of salt-tolerant oat materials. In a specific embodiment of this invention, through systematic cluster analysis, oat germplasm is classified into four types: extremely salt-tolerant, salt-tolerant, salt-sensitive, and extremely salt-sensitive. This invention classifies oat germplasm into four categories, enabling precise differentiation and scientific grading of salt tolerance gradients. This facilitates the rapid screening of highly salt-tolerant germplasm and clearly distinguishes between moderately salt-tolerant and salt-sensitive materials, providing a more reliable classification basis for the systematic classification of oat salt-tolerant germplasm resources.

[0038] This invention also provides the application of the method described above in the cultivation of salt-tolerant oat germplasm. The method of this invention combines different developmental stages, simulates different degrees of salt stress environments by setting multiple salt concentration gradients, analyzes multiple indicators based on morphological and physiological changes, eliminates basic differences between varieties through salt tolerance coefficient standardization, and finally uses principal component analysis for dimensionality reduction and membership function comprehensive evaluation to achieve quantitative ranking and classification of variety salt tolerance. This invention's method is characterized by short cycle time, controllable environment, speed, and high efficiency, and can provide a technical approach for the cultivation of salt-tolerant varieties of oats and similar plants and the study of salt tolerance mechanisms.

[0039] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0040] Example 1 Determination of the optimal salt concentration for treatment: Seeds from two oat varieties, As012 and As018 (variety information is shown in Table 2), were randomly selected for germination experiments under different NaCl salt concentrations. Plump, undamaged oat seeds were selected and disinfected with a 3.5% (w / v) sodium hypochlorite solution for 10 min, then rinsed 3-5 times with distilled water until no irritating odor remained. The disinfected oat seeds were evenly placed in petri dishes (diameter d = 10 cm) with a double layer of sterile, water-soaked filter paper at the bottom, with 50 seeds per dish. The petri dishes containing oat seeds were randomly divided into 5 groups, with one treatment per group and 3 replicates. The specific treatments are as follows: 0 mM group (CK control group): Add 8 mL of sterile distilled water to the petri dish; 50 mM group: Add 8 mL of 50 mmol / L NaCl salt solution to a petri dish; 100 mM group: Add 8 mL of 100 mmol / L NaCl salt solution to a petri dish; 150 mM group: Add 8 mL of 150 mmol / L NaCl salt solution to a petri dish; 200 mM group: Add 8 mL of 200 mmol / L NaCl salt solution to a petri dish.

[0041] The petri dishes containing oat seeds from each group were placed in a light incubator and cultured under dark conditions at 22℃ and 65% relative humidity for 5 consecutive days. During seed germination, 1 mL of sterile water or NaCl solution was added daily to keep the filter paper moist. After 5 days of germination, the seed germination status under different treatment concentrations was observed, and the results are as follows: Figure 1 As shown, the seed germination rate, radicle length, and plumule length under each gradient were measured and statistically analyzed. Further statistical analysis and calculations were performed to obtain the optimal concentration of NaCl salt stress suitable for oat seeds.

[0042] Determination and calculation of germination-related indicators: Seed germination was defined as the radicle breaking through the seed coat by 3 mm. Starting from day 3 of the germination experiment, the number of germinated seeds was counted for at least 5 consecutive days. The length of the radicle and plumule of the germinated seeds were measured. Germination rate, relative germination rate, relative radicle length, and relative plumule length were calculated using the following formulas: Germination rate = (Total number of seeds germinated in 5 days / Number of seeds tested) × 100%; Relative germination rate = (germination rate of treatment group / germination rate of control group) × 100%; Relative radicle length = (radicle length in treatment group / radicle length in control group) × 100%; Relative embryo length = (embryo length of treatment group / embryo length of control group) × 100%. The results are shown in Table 1. The data in the table are the average values ​​of the three repeated experiments.

[0043] By comparing germination rate, radicle length, and plumule length, it was found that when the salt concentration reached 100 mM, the main germination indicators showed significant differences compared with the corresponding indicators in the control group. Therefore, a salt concentration of 100 mM was uniformly used for subsequent large-scale evaluations of salt tolerance in different oat varieties.

[0044] Table 1. Germination of oat seeds under different NaCl concentrations

[0045] Example 2 1. Salt tolerance assessment during seed germination Sixty-five oat varieties from different regions were randomly selected for salt tolerance assessment of oat seed germination. The oat varieties are listed in Table 2.

[0046] Select plump, undamaged oat seeds. Disinfect them with a 3.5% (w / v) sodium hypochlorite solution for 10 minutes, then rinse 3-5 times with distilled water until no irritating odor remains. Arrange the disinfected oat seeds evenly in petri dishes (diameter d=10 cm) with a double layer of sterile, water-moistened filter paper at the bottom, placing 50 seeds in each dish. Randomly divide the petri dishes containing oat seeds into two groups, with one treatment per group and three replicates. The specific treatments are as follows: 0 mM group (CK control group): Add 8 mL of sterile distilled water to the petri dish; 100 mM group: Add 8 mL of 100 mmol / L NaCl salt solution to a petri dish; The petri dishes containing oat seeds in each group were placed in a light incubator and cultured under dark conditions at 22℃ and 65% relative humidity for 5 consecutive days. During seed germination, 1 mL of sterile water or NaCl solution was added daily to keep the filter paper moist. The relative germination rate, relative radicle length, and relative plumule length of seeds under each gradient were measured and statistically analyzed using the same formula as in Example 1. The results are shown in Table 2.

[0047] Principal component analysis was performed on the measured relative germination rate, relative radicle length, and relative plumule length data using SPSS 27 software. Oat variety information, along with the relative germination rate, relative radicle length, and relative plumule length data, were input into SPSS 27. Principal components were extracted based on a cumulative contribution rate greater than 85%, and the results are shown in Table 3. Table 3 shows that the contribution rates of the first two principal components were 61.918% and 23.185%, respectively. These two principal components were selected as the main factors for evaluating the salt tolerance of oat seeds during germination.

[0048] Substitute the selected principal components into the following formula to calculate the membership function value, and then normalize the data as follows: Membership function value: μ(Xi) = (i=1,2,3...,n); In the formula X i For the first i The score values ​​of each factor; X min For the first i Minimum score of each factor; X max For the first i The maximum score of each factor; where factor refers to principal component, and in this experiment there are 2 factors.

[0049] Calculate the weights of different factors: Wi = Pi / (i =1,2,3,......,n); In the formula: Wi For the first i The importance of each factor among all factors; Pi For the first i The contribution rate of each factor; where factor refers to principal component, and in this experiment there are a total of 2 factors.

[0050] The formula for comprehensively evaluating the salt tolerance of oats during germination is: D1= (i=1,2,3...,n); where μ(Xi) For the first i Membership function values ​​of each factor; Wi For the first i The importance of each factor among all factors; where "factor" refers to principal components, and in this experiment, there are a total of 2 factors.

[0051] Based on the comprehensive evaluation D1 value, the salt tolerance of the varietal resources was ranked, and the information of the top ten varieties is shown in Table 4.

[0052] Table 2 Information and Measurement Indicators of 65 Oat Resources (Varieties)

[0053] Table 3 Principal component analysis coefficients of indicators related to seed germination period

[0054] Table 4. Ranking of the top ten oat varieties by salt tolerance based on comprehensive evaluation D1 value.

[0055] In the table, μ1 and μ2 These represent the membership function values ​​of the first and second principal components, respectively. W1 and W2 These represent the weights of the first principal component and the second principal component, respectively. 2. Salt tolerance assessment of oat seedlings during their growth period: Sixty-five oat varieties from different regions were randomly selected for salt tolerance assessment during the seedling growth period. The salt tolerance assessment of oat varieties during the seed germination period was conducted in the same manner as in step 1.

[0056] (1) Seed germination: Select plump and undamaged oat seeds, disinfect them with 3.5% (w / v) sodium hypochlorite solution for 10 min, and rinse them with distilled water 3-5 times until there is no irritating odor. Place the disinfected seeds evenly in petri dishes (diameter d=10 cm) with a double layer of sterile filter paper moistened with sterile water at the bottom, with 50 seeds in each petri dish. Then place the petri dishes containing oat seeds in a light incubator and germinate the seeds under the following conditions: temperature 22℃, relative humidity 65%, no light for the first 5 days, and light intensity of 1000 Lux (photoperiod: 12 h daytime, 2 h nighttime) starting from the 6th day. During the germination period, add sterile water once every 2 days to keep the filter paper moist.

[0057] (2) Seedling transplanting: When the radicle of the oat germination seed reaches 2 cm and the plumule reaches 1 cm (around 3 days), select oat seedlings with uniform growth and transplant them to a hydroponic box with 96 holes (hole diameter d=5 mm). The hydroponic box is filled with 1 / 2 Hoagland nutrient solution, pH 6.5±1, and the nutrient solution is changed every 3 days. The hydroponic box with the transplanted seedlings is placed in a light incubator with a temperature of 22℃, relative humidity of 65%, and light intensity of 3000 Lux (photoperiod: 12 h daytime, 12 h nighttime). When the seedlings reach 10 cm in height, bamboo sticks are inserted into the four corner holes of the hydroponic box and tied with hemp rope as a barrier to prevent the seedlings from falling over.

[0058] (3) Salt stress treatment: When the hydroponic oat seedlings grow to the stage of two leaves and one bud, the hydroponic oat seedlings are randomly divided into 4 groups, with 3 replicates in each group. The specific operation is as follows: Control group: Standard culture, without the addition of NaCl; 100 mM treatment group: Add freshly prepared NaCl solution to the nutrient solution of the hydroponic box planted with seedlings, so that the final NaCl concentration in the nutrient solution is 100 mM. 150 mM treatment group: The operation was the same as the 100 mM group, except that the final NaCl concentration in the nutrient solution was 150 mM. 200 mM treatment group: The operation was the same as the 100 mM group, except that the final NaCl concentration in the nutrient solution was 200 mM. The oat seedlings from each of the above treatment groups were placed back into the same incubator and cultured under the same conditions.

[0059] (4) Determination and analysis of key salt tolerance indicators: After 7 days of cultivation, the growth of oat plants under different salt treatment concentrations was observed. The growth of oat As036 and As043 is shown in the figure below. Figure 2As shown in the figure. Simultaneously, the plant height, root length, and root number of seedlings in each treatment group and the control group were measured sequentially. The obtained data are the average values ​​of the data from each treatment group. A portable chlorophyll meter was used to measure the SPAD value of the second functional leaf. This experiment used oat seedlings at the two-leaf-one-heart stage, with the second functional leaf being the second leaf from the base. The relative plant height, relative root length, relative root number, and relative SPAD value were calculated using the following formulas, and the results are shown in Table 2.

[0060] Relative plant height = (treatment plant height / control plant height) × 100%; Relative root length = (Treatment root length / Control root length) × 100%; Relative root count = (Number of treated roots / Number of control roots) × 100%; Relative SPAD value = (treatment SPAD value / control SPAD value) × 100%; (5) Salt tolerance evaluation and ranking: The salt tolerance of 65 oat germplasm (varieties) under different salt stress treatments was evaluated based on the principal component and membership function method. The salt tolerance of the tested varieties was ranked according to the comprehensive salt tolerance evaluation value (D2 value).

[0061] Principal component analysis was performed on the measured relative plant height, relative root length, relative root number, and relative SPAD value using SPSS 27 software. Principal components were extracted based on a cumulative contribution rate greater than 85%, and the results are shown in Table 5. Table 5 shows that the contribution rates of the first seven principal components were 27.46, 17.702, 13.919, 10.246, 7.243, 6.244, and 5.343, respectively. These seven principal components were selected as the main factors for evaluating the salt tolerance of oat seedlings during their growth period.

[0062] The membership function values ​​of the selected principal components are calculated and then normalized, as shown in the following formula: Membership function value: μ(Xi) = (i=1,2,3...,n); In the formula X i For the first i The score values ​​of each factor; X min For the first i Minimum score of each factor; X max For the first i The maximum score of each factor; where factors refer to principal components, there are a total of 7 factors in this experiment.

[0063] Calculate the weights of different factors: Wi = Pi / (i =1,2,3,......,n); In the formula, Wi represents the importance of the i-th factor among all factors; Pi represents the contribution of the i-th factor; and the factor refers to the principal component. In this experiment, there are a total of 7 factors.

[0064] The formula for the comprehensive evaluation of salt tolerance of oat seedlings during their growth period (D2 value) is as follows: D2= (i=1,2,3...,n); where μ(Xi) For the first i Membership function values ​​of each factor; Wi For the first i The importance of each factor among all factors; where "factor" refers to principal components, and in this experiment, there are a total of 7 factors.

[0065] After obtaining the comprehensive evaluation D2 values ​​after different salt concentration stress treatments, the mean of the comprehensive evaluation D2 values ​​after different salt concentration stress treatments (D 2均值 Based on the mean of the comprehensive evaluation D2 value, the salt tolerance of the varieties was ranked, and the information of the top ten varieties is shown in Table 6.

[0066] Table 5 Principal component analysis coefficients of relevant indicators during the seedling growth period

[0067] Table 6 Seedling stage based on comprehensive evaluation D 2均值 Ranking of the top ten oat varieties by salt tolerance

[0068] In the table, μ1 ~ μ7 These represent the membership function values ​​of the first to seventh principal components, respectively. W1 ~ W2 The values ​​represent the weights of the first to seventh principal components, respectively; the data in the table are the mean values ​​after three salt concentration stress treatments.

[0069] 3. Salt tolerance identification and grading of oat germplasm (varieties) based on comprehensive consideration of salt tolerance during seed germination and seedling growth stages. The comprehensive salt tolerance evaluation D1 value during seed germination and the comprehensive salt tolerance evaluation D2 value during seedling growth were weighted and combined, with each stage having a weight ratio of 50%. The weighted comprehensive salt tolerance evaluation D value (D1) was obtained according to the following formula. 加权 The formula is: D 加权 =50%×D1+50%×D 2均值 .

[0070] Based on the weighted comprehensive evaluation D加权 The salt tolerance of the varieties was ranked by the value, and the information of the top ten varieties is shown in Table 7.

[0071] Table 7. Ranking of the top ten oat varieties by salt tolerance based on D-weighted values ​​at different times.

[0072] Cluster Analysis: Based on the D-weighted values ​​of all varietal resources, a systematic cluster analysis was performed on all varietal resources using the distance summation method with the default parameters of Origin 2024 software. The results are as follows: Figure 3 See Table 7.

[0073] according to Figure 3 It can be seen that 65 oat varieties were classified as extremely salt-tolerant (D). 加权 Values ​​range from 0.8103 to 1.0256, and salt tolerance (D) 加权 Values ​​range from 0.3382 to 0.7639), and sensitive salts (D 加权 Values ​​range from 0.2035 to 0.3013) and extreme sensitive salts (D 加权 The values ​​range from -0.0768 to 0.0433 for four types of germplasm resources, including extremely salt-sensitive germplasm ( Figure 3 Six medium purple varieties and six salt-sensitive germplasm varieties. Figure 3 Nine medium-green varieties and salt-tolerant varieties ( Figure 3 43 medium-blue varieties, and extremely salt-tolerant varieties ( Figure 3 (Seven in red). It can be seen that the method described in this invention can quickly achieve the ranking and classification of salt tolerance among multiple oat varieties.

[0074] As shown in Table 7, oat As061 (Muwang) is a salt-tolerant germplasm, which is consistent with the salt tolerance of Muwang as disclosed in the existing technology ("Study on Salt and Alkali Resistance of Oat Seeds, Hou Xindi et al., Grassland and Grassland Industry, No. 3, 2018").

[0075] Existing techniques (“Response of Germinating Oats to Different Gradient Salt Stress”, Xu Zhenghui, Chinese Journal of Herbivorous Animal Science, Vol. 3, 2020) only assess salt tolerance in oats during the germination stage, indicating that Qingyin 2 is a low germination potential variety with poor salt tolerance (ranked 7th out of 8 varieties). However, this method only reflects the phenotypic characteristics of oats during the germination stage and cannot fully reflect its overall salt tolerance potential. This invention uses a comprehensive salt tolerance evaluation system combining multiple indicators across two key periods—germination and seedling growth—to systematically identify oat variety As056 (Qingyin 2). The results show that this material is an extremely salt-tolerant germplasm.

[0076] This invention combines salt tolerance identification during seed germination with salt tolerance evaluation during seedling growth. Stress treatment with NaCl solutions of varying concentrations is applied, followed by principal component analysis (PCA) for dimensionality reduction and membership function analysis, along with cluster analysis, to quantify, rank, and classify the salt tolerance of varieties. This invention provides a simple, rapid, efficient, and reliable method for evaluating and screening the salt tolerance of large-scale oat varieties. This method is characterized by its short cycle time, controllable environment, speed, and high efficiency, and can provide a technical approach for breeding salt-tolerant varieties of oats and similar plants and for studying salt tolerance mechanisms.

[0077] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for rapid, large-scale identification of salt tolerance in oat varietal resources, characterized in that, Includes the following steps: Salt stress was applied to oat seeds during their germination period, and the germination characteristics of oat seeds after salt stress treatment were measured. The germination characteristics of the oat seeds include relative germination rate, relative plumule length, and relative radicle length; Salt tolerance during the germination period of oat seeds was evaluated based on their germination characteristics, and a comprehensive evaluation value of salt tolerance during the germination period of oats was obtained. Salt stress was applied to oat seedlings during their growth period, and the growth characteristics of the oat seedlings after salt stress treatment were measured. The growth characteristics of the oat seedlings included relative plant height, relative root length, relative root number, and relative SPAD value. Salt tolerance of oat seedlings during their growth period was evaluated based on their growth characteristics, and a comprehensive evaluation value of salt tolerance of oat seedlings during their growth period was obtained. The comprehensive salt tolerance evaluation values ​​during the oat germination period and the comprehensive salt tolerance evaluation values ​​during the oat seedling growth period are weighted and analyzed to obtain a comprehensive weighted value for salt tolerance evaluation. The salt tolerance of oats is evaluated based on the comprehensive weighted value for salt tolerance evaluation. The higher the comprehensive weighted value for salt tolerance evaluation, the stronger the salt tolerance of oats. The salt tolerance evaluation was conducted using principal component analysis and membership function analysis.

2. The method according to claim 1, characterized in that, The salt stress treatment employs a salt concentration gradient treatment.

3. The method according to claim 2, characterized in that, When salt stress is applied during the germination period of oats, a salt concentration gradient is set up for the salt stress treatment; the salt concentration is 80~150 mM.

4. The method according to claim 2, characterized in that, When salt stress treatment is applied to oat seedlings during their growth period, three salt concentration gradients are set up: 80-120 mM, 130-170 mM, and 180-220 mM.

5. The method according to claim 1, characterized in that, The oat seedlings were cultured using hydroponics.

6. The method according to claim 1, characterized in that, The oat seedlings are in the two-leaf-one-heart stage.

7. The method according to claim 1 or 3, characterized in that, When salt stress is applied during the germination period of oats, the duration of the salt stress treatment is 3 to 7 days.

8. The method according to any one of claims 1, 4 to 6, characterized in that, When salt stress is applied to oat seedlings during their growth period, the duration of the salt stress treatment is 5 to 10 days.

9. The method according to claim 1, characterized in that, After obtaining the comprehensive weighted value of salt tolerance evaluation, the method further includes: performing cluster analysis on the comprehensive weighted value of salt tolerance evaluation.

10. The application of the method according to any one of claims 1 to 9 in the cultivation of salt-tolerant oat germplasm.

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