Method for evaluating salt tolerance of cotton germplasm at germination stage

Abstract

The application discloses a method for evaluating salt tolerance of cotton germplasm at the germination stage, selects 150mmol / L NaCl as the optimal concentration for evaluating the salt tolerance of cotton, evaluates the salt tolerance of cotton germplasm resources at the concentration, converts six indexes into three new independent indexes through correlation analysis, principal component analysis, comprehensive subordinate function and cluster analysis, and obtains the comprehensive evaluation value of the salt tolerance of the cotton germplasm.

Description

Technical Field

[0001] This invention relates to the field of salt tolerance technology for cotton varieties, specifically a method for evaluating the salt tolerance of cotton germplasm during germination. Background Technology

[0002] Cotton is a major source of natural, high-quality fiber and an important fiber and oilseed crop, playing a vital role in my country's economic development. In recent years, my country's cotton consumption has remained stable at around 8.2 million tons annually, accounting for one-third of global cotton consumption. However, due to rising living standards and increasing demand for food, the competition between cotton and food for land is intensifying. Human activities and global climate change have exacerbated soil salinization, with the global area of ​​saline-alkali land reaching 9.5 × 10⁸ hm². 2 Furthermore, the area affected by soil salinization is increasing by 1 to 1.5 million hectares annually. my country is one of the countries most severely affected by soil salinization, with a total area of ​​approximately 99 million hectares, of which Xinjiang accounts for one-fifth of the national total. Soil salinization is a significant factor affecting cotton yield and quality, severely impacting agricultural production and socio-economic development. Selecting and cultivating salt-tolerant cotton varieties is one of the most economical and effective ways to utilize saline-alkali soils. Expanding cotton planting areas by shifting cotton cultivation to saline-alkali wastelands is of great importance to both the cotton industry and economic development.

[0003] Cotton is a relatively salt-tolerant crop and a pioneer crop in the improvement of saline-alkali land. When the soil salinity is less than 0.2% (34 mmol / L), it is beneficial to cotton emergence and growth, and can even improve yield and quality. However, when the soil salinity is greater than 0.2% (34 mmol / L), it will cause osmotic stress and ion toxicity to cotton. As the soil salinity increases and exceeds a certain critical value, it will significantly affect the growth, development, and physiological metabolism of cotton, leading to a decrease in cotton yield and changes in quality. Identifying and screening the salt tolerance of different cotton germplasm resources, studying the salt tolerance mechanism during cotton germination, and mining salt tolerance-related genes can lay the foundation for the genetic improvement of cotton salt tolerance and the expansion of its planting area. Currently, numerous researchers are evaluating crop resistance to stress during germination, but their methods and screening indicators vary. For example, there is a method for rapidly identifying the salt tolerance of cotton seeds during germination, as described in Chinese Patent Publication No. CN112119857B. This method uses electrophysiological techniques and the rate of chloride ion efflux in the radicle as an indicator of the strength of salt tolerance in cotton seeds. However, analysis of differences between single or multiple indicators cannot provide a comprehensive evaluation of crop resistance to stress. While there have been many previous studies on the identification of cotton salt tolerance, these studies have been limited by the number of tested varieties and the comprehensiveness of the identification methods. Therefore, this study subjected 629 cotton germplasm resources with broad genetic backgrounds to salt stress during germination. Variance analysis, principal component analysis, membership function analysis, cluster analysis, and stepwise regression analysis were performed on the salt tolerance coefficients of various indicators to comprehensively evaluate the salt tolerance of cotton during germination. This aimed to screen for salt-tolerant materials and salt tolerance identification indicators, and establish a rapid and effective evaluation system for germination-stage salt tolerance. This provides material and theoretical basis for the identification and screening of salt tolerance in cotton germplasm resources, the breeding of salt-tolerant varieties, and the discovery of salt-tolerant genes. Summary of the Invention

[0004] The purpose of this invention is to provide a method for evaluating the salt tolerance of cotton germplasm during germination, so as to comprehensively evaluate the salt tolerance of cotton during germination, screen out salt-tolerant materials and salt tolerance identification indicators, and establish a rapid and effective evaluation system for salt tolerance during germination.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for evaluating the salt tolerance of cotton germplasm during germination;

[0006] Choose 150 mmol L -1 NaCl is used as the optimal concentration for evaluating the salt tolerance of cotton. This concentration is employed to evaluate the salt tolerance of cotton germplasm resources. Through correlation analysis, principal component analysis, comprehensive membership function analysis, and cluster analysis, six indicators are transformed into three new independent indicators, yielding a comprehensive evaluation value for the salt tolerance of cotton germplasm. The specific process includes:

[0007] 1) Salt tolerance test

[0008] A. Select cotton seeds that are uniform in size and plump. After disinfecting and soaking them in 10% hydrogen peroxide for 30 minutes, rinse them 4-5 times with sterile water and use filter paper to absorb the moisture from the seed coat surface.

[0009] B. Cut the filter paper into a rectangle 30cm long and 20cm wide. After wetting it in the solution, lay it flat on the test bench. Select 40 seeds and arrange them in two rows (20 seeds per row) 2cm below the top of the filter paper. The two rows are 2cm apart, and the seeds are staggered with the pinholes facing down.

[0010] C. After wetting a filter paper in the solution, align it and lay it flat on the seeds. Fold the filter paper in half from 1.5 to 2 cm from the top (to prevent the solution from evaporating too quickly). Roll the filter paper into a cylindrical shape. Repeat this process 3 times and tie the bottom with a rubber band. Place the filter paper in a germination box with a 3 cm high salt solution. Put 18 filter paper rolls in each germination box.

[0011] D. Place the germination box containing the filter paper roll into the seed germination chamber at a temperature of 30±1℃ and a relative humidity of 60%. Supplement light on the 3rd day, with a light / dark period of 12h / 12h (L / D). Replenish water daily to a uniform liquid level (3cm).

[0012] E. On day 3 and day 7, the number of germinations was counted. On day 7, five seedlings with basically the same growth were randomly selected to measure fresh weight, total seedling length, radicle length and hypocotyl length. Each treatment was repeated 3 times.

[0013] 2) Measurement Indicators

[0014] Germination is indicated when the radicle breaks through the seed coat and shows white markings; use a ruler to measure the total length of the seedling (from the root cap to the point of cotyledon attachment) and the length of the radicle; weigh the seedling after absorbing the surface moisture.

[0015] Germination potential = Number of germinated seeds on day 3 / Number of seeds tested × 100%;

[0016] Germination rate = (Number of germinated seeds on day 7 / Number of seeds tested) × 100%;

[0017] Hypocotyl length = total seedling length - radicle length;

[0018] Relative salt tolerance coefficient of each index = treatment index / control index × 100%;

[0019] 3) Statistical analysis

[0020] Microsoft Excel 2016 was used to organize the experimental data. SPSS Statistics 27 was used to perform statistical description, analysis of variance, principal component analysis and correlation analysis on the experimental data. The R language "cluster, factoextra" software package was used for cluster analysis and visualization. Graphpad Prism was used for plotting.

[0021] The relative values ​​of six traits were converted into three comprehensive indicators by principal component analysis, and the salt tolerance of 629 cotton germplasms was comprehensively evaluated by fuzzy mathematical membership function method.

[0022] Membership function value:

[0023] μ(X i )=(X i -X min ) / (X max -X min i = 1, 2, 3, ..., n

[0024] X i Let X represent the value of the i-th comprehensive index. min Let X represent the minimum value among the i-th comprehensive indicators. max μ(X) represents the maximum value among the i-th comprehensive indicators. i ) represents the membership function value of the i-th comprehensive index;

[0025] Weights of each comprehensive indicator:

[0026]

[0027] W i P represents the weight of the i-th comprehensive indicator among all comprehensive indicators. i This represents the variance contribution rate of the i-th comprehensive index of each cotton germplasm obtained from principal component analysis;

[0028] Salt tolerance comprehensive evaluation value:

[0029]

[0030] The D value represents the comprehensive evaluation value of the salt tolerance of the i-th germplasm under salt stress conditions;

[0031] Cluster analysis: Based on the D value, Euclidean distance, the complete method between groups, and the number of clusters K=5 were used to classify the salt tolerance of cotton germplasm. The salt tolerance of each type of cotton germplasm was defined according to the range of D values.

[0032] Stepwise regression analysis: Stepwise regression analysis was conducted with the comprehensive salt tolerance evaluation value D as the dependent variable and the salt tolerance coefficients of the six indicators as independent variables.

[0033] Preferably, the concentration of the salt solution is selected during treatment at 0, 100, 150, 200, 250, and 300 mmol / L. -1 Six gradient salt stress treatments were applied. On day 7, fresh weight, total seedling length, radicle length, and germination rate were measured and statistically analyzed to determine the appropriate concentration.

[0034] Preferably, the indicators include fresh weight, total seedling length, hypocotyl length, radicle length, germination potential, and germination rate.

[0035] Preferably, under the same conditions, the indicators are measured using four indicators: fresh weight, hypocotyl length, germination potential, and germination rate. The corresponding salt tolerance coefficients are calculated, and the comprehensive evaluation D value calculated by the equation is used to determine the salt tolerance of different cotton germplasm resources.

[0036] Preferably, the correlation analysis shows that different varieties respond differently to salt stress, and a single index cannot comprehensively evaluate the salt tolerance of cotton varieties. The index used should include two or more of the following: fresh weight, total seedling length, hypocotyl length, radicle length, germination potential, and germination rate.

[0037] Preferably, the principal component analysis transforms the salt tolerance coefficients of the original 6 indicators into 3 new independent comprehensive indicators based on the principle that the cumulative contribution rate is greater than 85%. The new comprehensive indicators contain most of the genetic information of the original 6 indicators and can be used to evaluate the salt tolerance of cotton varieties (lines).

[0038] The comprehensive index PC1 was most correlated with relative fresh weight and relative total seedling length, mainly reflecting the total biomass and growth of cotton; the comprehensive index PC2 was most correlated with relative germination potential and relative germination rate, mainly reflecting the germination of cotton seeds under salt stress; the comprehensive index PC3 was most correlated with relative radicle length and relative hypocotyl length (0.634), mainly reflecting the impact of salt stress on the growth of cotton aboveground and underground parts.

[0039] Preferably, the principal component analysis transforms the salt tolerance coefficients of the original 6 indicators into 3 new comprehensive indicators, calculates the membership function values ​​of each of these 3 comprehensive indicators, and calculates the weights of each of the comprehensive indicators based on their variance contribution rates.

[0040] Preferably, after obtaining the membership function value and weight of the comprehensive index, the cluster analysis calculates the comprehensive salt tolerance evaluation value D, evaluates the salt tolerance of cotton germplasm resources based on the value of D, and uses the Euclidean distance "complete" method with a cluster number K=5 to cluster the comprehensive evaluation value D.

[0041] Based on the D-value and clustering results, cotton varieties (lines) are divided into 5 categories: Category 1 (Ⅰ) is of intermediate salt tolerance, with a D-value range of 0.429 to 0.542; Category 2 (Ⅱ) is of salt tolerance, with a D-value range of 0.543 to 0.691; Category 3 (Ⅲ) is of high salt tolerance, with a D-value range of 0.692 to 0.824; Category 4 (Ⅳ) is of salt sensitivity, with a D-value range of 0.248 to 0.428; and Category 5 (Ⅴ) is of high salt sensitivity, with a D-value below 0.248.

[0042] Preferably, when analyzing the salt tolerance of cotton germplasm during germination, a mathematical model for evaluating the salt tolerance of cotton during germination is established. Stepwise regression analysis is performed with the comprehensive salt tolerance evaluation value D as the dependent variable and the salt tolerance coefficients of the six indicators as independent variables to obtain the optimal equation. The relationship between various indicators during cotton germination and salt tolerance is further analyzed to screen out salt tolerance identification indicators.

[0043] Compared with existing technologies, the beneficial effects of this invention are as follows: This method for evaluating the salt tolerance of cotton germplasm during germination involves setting up six gradient salt stress treatments in the early stage, comprehensively analyzing the variance of various indicators among varieties and the salt tolerance coefficient change curves, and selecting 150 mmol / L NaCl as the optimal concentration for evaluating cotton salt tolerance. Using this concentration, the salt tolerance of cotton germplasm resources is evaluated, and the differences in salt tolerance coefficients of six traits, including fresh weight and germination potential, are analyzed. Principal component analysis is used to transform the six indicators into three mutually independent comprehensive indicators, reducing the redundancy of the original indicator information and retaining 91.83% of the original information. Based on this, membership function analysis is used to calculate the comprehensive evaluation value D, and the salt tolerance of cotton during germination is comprehensively evaluated. The maximum distance method is used to classify the D value into five different salt tolerance types. A mathematical model for evaluating the salt tolerance of cotton during germination is established with the comprehensive salt tolerance evaluation value D as the dependent variable and the six salt tolerance coefficients as independent variables, determining that fresh weight, hypocotyl length, germination potential, and germination rate can be used as indicators for identifying cotton salt tolerance. In summary, this study conducted a comprehensive and objective evaluation of the salt tolerance during germination of 629 cotton germplasm resources with rich genetic backgrounds, established an efficient and accurate system for identifying cotton salt tolerance, provided a methodological reference for cotton salt tolerance identification, and provided material and theoretical basis for research on salt tolerance mechanisms, breeding of salt-tolerant varieties, and mining of salt tolerance genes. Attached Figure Description

[0044] Figure 1 This is a schematic diagram of the trait differences and salt tolerance coefficient changes of the first group of cotton varieties under different concentrations of salt stress treatment according to the present invention.

[0045] Figure 2 This is a schematic diagram of the trait differences and salt tolerance coefficient changes of the second group of cotton varieties under different concentrations of salt stress treatment according to the present invention.

[0046] Figure 3This is a schematic diagram of the trait differences and salt tolerance coefficient changes of the third group of cotton varieties under different concentrations of salt stress treatment according to the present invention.

[0047] Figure 4 This is a schematic diagram of the trait differences and salt tolerance coefficient changes of the fourth group of cotton varieties under different concentrations of salt stress treatment according to the present invention.

[0048] Figure 5 This is a schematic diagram of the trait differences and salt tolerance coefficient changes of the fifth group of cotton varieties under different concentrations of salt stress treatment according to the present invention.

[0049] Figure 6 This is a schematic diagram showing the phenotypic characteristics of cotton under salt stress as a control of the present invention.

[0050] Figure 7 This is a schematic diagram of the cluster analysis of salt tolerance of 629 cotton samples in this invention. Detailed Implementation

[0051] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0052] Please see Figure 1-7 The present invention provides a technical solution: a method for evaluating the salt tolerance of cotton germplasm during germination;

[0053] Choose 150 mmol L -1 NaCl is used as the optimal concentration for evaluating the salt tolerance of cotton. This concentration is employed to evaluate the salt tolerance of cotton germplasm resources. Through correlation analysis, principal component analysis, comprehensive membership function analysis, and cluster analysis, six indicators are transformed into three new independent indicators, yielding a comprehensive evaluation value for the salt tolerance of cotton germplasm. The specific process includes:

[0054] 1) Salt tolerance test

[0055] A. Select cotton seeds that are uniform in size and plump. After disinfecting and soaking them in 10% hydrogen peroxide for 30 minutes, rinse them 4-5 times with sterile water and use filter paper to absorb the moisture from the seed coat surface.

[0056] B. Cut the filter paper into a rectangle 30cm long and 20cm wide. After wetting it in the solution, lay it flat on the test bench. Select 40 seeds and arrange them in two rows (20 seeds per row) 2cm below the top of the filter paper. The two rows are 2cm apart, and the seeds are staggered with the pinholes facing down.

[0057] C. After wetting a filter paper in the solution, align it and lay it flat on the seeds. Fold the filter paper in half from 1.5 to 2 cm from the top (to prevent the solution from evaporating too quickly). Roll the filter paper into a cylindrical shape. Repeat this process 3 times and tie the bottom with a rubber band. Place the filter paper in a germination box with a 3 cm high salt solution. Put 18 filter paper rolls in each germination box.

[0058] D. Place the germination box containing the filter paper roll into the seed germination chamber at a temperature of 30±1℃ and a relative humidity of 60%. Supplement light on the 3rd day, with a light / dark period of 12h / 12h (L / D). Replenish water daily to a uniform liquid level (3cm).

[0059] E. On day 3 and day 7, the number of germinations was counted. On day 7, five seedlings with basically the same growth were randomly selected to measure fresh weight, total seedling length, radicle length and hypocotyl length. Each treatment was repeated 3 times.

[0060] 2) Measurement Indicators

[0061] Germination is indicated when the radicle breaks through the seed coat and shows white markings; use a ruler to measure the total length of the seedling (from the root cap to the point of cotyledon attachment) and the length of the radicle; weigh the seedling after absorbing the surface moisture.

[0062] Germination potential = Number of germinated seeds on day 3 / Number of seeds tested × 100%;

[0063] Germination rate = (Number of germinated seeds on day 7 / Number of seeds tested) × 100%;

[0064] Hypocotyl length = total seedling length - radicle length;

[0065] Relative salt tolerance coefficient of each index = treatment index / control index × 100%;

[0066] 3) Statistical analysis

[0067] Microsoft Excel 2016 was used to organize the experimental data. SPSS Statistics 27 was used to perform statistical description, analysis of variance, principal component analysis and correlation analysis on the experimental data. The R language "cluster, factoextra" software package was used for cluster analysis and visualization. Graphpad Prism was used for plotting.

[0068] The relative values ​​of six traits were converted into three comprehensive indicators by principal component analysis, and the salt tolerance of 629 cotton germplasms was comprehensively evaluated by fuzzy mathematical membership function method.

[0069] Membership function value:

[0070] μ(X i )=(X i -X min ) / (X max-X min i = 1, 2, 3, ..., n

[0071] X i Let X represent the value of the i-th comprehensive index. min Let X represent the minimum value among the i-th comprehensive indicators. max μ(X) represents the maximum value among the i-th comprehensive indicators. i ) represents the membership function value of the i-th comprehensive index;

[0072] Weights of each comprehensive indicator:

[0073]

[0074] w i P represents the weight of the i-th comprehensive indicator among all comprehensive indicators. i This represents the variance contribution rate of the i-th comprehensive index of each cotton germplasm obtained from principal component analysis;

[0075] Salt tolerance comprehensive evaluation value:

[0076]

[0077] The D value represents the comprehensive evaluation value of the salt tolerance of the i-th germplasm under salt stress conditions;

[0078] Cluster analysis: Based on the D value, Euclidean distance, the complete method between groups, and the number of clusters K=5 were used to classify the salt tolerance of cotton germplasm. The salt tolerance of each type of cotton germplasm was defined according to the range of D values.

[0079] Stepwise regression analysis: Stepwise regression analysis was conducted with the comprehensive salt tolerance evaluation value D as the dependent variable and the salt tolerance coefficients of the six indicators as independent variables.

[0080] To determine the appropriate concentration for screening salt tolerance during cotton germination, five varieties (lines) were randomly selected for salt tolerance testing at concentrations of 0, 100, 150, 200, 250, and 300 mmol / L. -1 Six gradient salt stress treatments were applied. On day 7, fresh weight, total seedling length, radicle length, and germination rate were measured and recorded. (Refer to...) Figure 1-5 Where FW: fresh weight; SL: total seedling length; RL: radicle length; GR: germination rate; RFW: relative fresh weight; RSL: relative total seedling length; RRL: relative radicle length; RGR: relative germination rate, the phenotypic trends of the five varieties (lines) differed under different salt stress concentrations (a–e). When the treatment concentration was greater than or equal to 200 mmol L... -1At the specified concentrations, fresh weight, seedling length, and root length all showed significant differences compared to the control, and the inhibitory effect on fresh weight, seedling length, and root length became more pronounced with increasing concentration. When the treatment concentrations were 100 and 150 mmol / L... -1 At the same time, the fresh weight, total seedling length, and radicle length of different varieties (lines) differed from the control, indicating that these two concentrations can be used for screening the salt tolerance of different cotton varieties (lines) during seed germination. Analysis of five varieties (lines) at concentrations of 100–300 mmol / L... -1 The relative salt tolerance coefficients (f–j) for different parameters under salt stress show that the relative salt tolerance coefficients for each parameter gradually decrease with increasing concentration. Treatment concentrations are greater than or equal to 200 mmol / L. -1 At a concentration of 100 mmol / L, it severely inhibited cotton growth. -1 At this concentration, the inhibitory effect on cotton growth was least obvious, and it even significantly promoted the growth of variety ST32. Based on Zhang Guowei's research, salt concentrations that are too large or too small in their impact on various indicators are not suitable as a level for salt tolerance assessment. Considering the differences in phenotypic changes and salt tolerance coefficients under different concentrations, 150 mmol / L was selected. -1 NaCl is the optimal concentration for assessing the salt tolerance of cotton.

[0081] Under salt stress and control conditions, the changes in six traits of different varieties (lines) were significantly different, such as Figure 6 As shown in the figure, CK: normal treatment; ST: salt stress; ST-CK: mean difference between salt stress and normal treatment; *** represents P<0.001. In the control group, the coefficients of variation for each trait ranged from 2.79% to 18.01%, with the coefficients of variation decreasing in the following order: hypocotyl length, radicle length, total seedling length, fresh weight, germination potential, and germination rate. In the treatment groups, the coefficients of variation for each trait ranged from 8.96% to 28.12%, with the same arrangement as the control group. Hypocotyl length had the largest coefficient of variation, while germination rate had the smallest. Compared to the control, all six traits were significantly inhibited under salt stress. The mean values ​​of hypocotyl length, radicle length, total seedling length, and fresh weight all decreased by more than 30% compared to the control. Hypocotyl length was the most severely inhibited, with the largest decrease of 42.78%, while germination potential and germination rate were relatively less affected, decreasing by 16.52% and 9.93%, respectively (Table 1). This shows that different varieties respond differently to salt stress, and a single indicator cannot comprehensively evaluate the salt tolerance of cotton varieties.

[0082] Table 1. Phenotypic traits of 629 cotton varieties (lines) under salt stress.

[0083]

[0084]

[0085] To eliminate inherent differences among cotton varieties (lines), the relative salt tolerance coefficients of various indicators were used to comprehensively evaluate the salt tolerance of cotton. Table 2 shows that the relative fresh weight ranged from 0.31 to 1.08, the relative seedling length from 0.24 to 1.05, the relative radicle length from 0.22 to 1.31, the relative hypocotyl length from 0.20 to 1.41, the relative germination potential from 0.19 to 1.13, and the relative germination rate from 0.42 to 1.13. The coefficients of variation for the six relative indicators ranged from 8.57% to 29.12%, with the relative hypocotyl length showing the largest coefficient of variation and the relative germination rate showing the smallest. It can be seen that the salt tolerance coefficients of different indicators vary across different cotton genotypes, indicating that different traits respond differently to salt stress. Therefore, the salt tolerance coefficient of a single indicator cannot accurately evaluate the salt tolerance of cotton varieties (lines).

[0086] Further correlation analysis of the salt tolerance coefficients of various indicators was conducted. Table 3 shows that relative fresh weight, relative total seedling length, and relative germination potential were all significantly positively correlated with other traits. Relative radicle length was significantly positively correlated with relative germination potential. Relative hypocotyl length did not show a significant correlation with relative germination rate. The strongest correlation was found between relative total seedling length and relative radicle length (r = 0.803). ** The correlation between relative hypocotyl length and relative germination rate was the weakest (r = 0.077). The correlations among the salt tolerance coefficients of the six indicators showed high consistency, and the information contained in them partially overlapped.

[0087] Table 2 Salt tolerance coefficients of various cotton traits

[0088]

[0089]

[0090] Table 3 Correlation analysis of salt tolerance coefficients of various cotton traits

[0091]

[0092] * and ** indicate significant correlation at 0.05 and 0.01, respectively.

[0093] Principal component analysis was performed on the salt tolerance coefficients of the six indicators. Based on the principle that the cumulative contribution rate is greater than 85%, the original six salt tolerance coefficients were transformed into three new independent comprehensive indicators, with a cumulative contribution rate of 91.83% (Table 4). This indicates that the new comprehensive indicators contain most of the genetic information of the original six indicators and can be used to evaluate the salt tolerance of cotton varieties (lines). The eigenvalue of the comprehensive index PC1 was 2.919, with a variance contribution rate of 48.65%. It was most correlated with relative fresh weight (0.875) and relative seedling length (0.935), mainly reflecting the total biomass and growth status of cotton. The eigenvalue of the comprehensive index PC2 was 1.649, with a variance contribution rate of 27.48%. It was most correlated with relative germination potential (0.864) and relative germination rate (0.857), mainly reflecting the germination status of cotton seeds under salt stress. The eigenvalue of the comprehensive index PC3 was 0.942, with a variance contribution rate of 15.71%. It was most correlated with relative radicle length (-0.691) and relative hypocotyl length (0.634), mainly reflecting the impact of salt stress on the growth of cotton aboveground and underground parts.

[0094] Table 4 Principal component analysis of salt tolerance coefficients for various cotton traits.

[0095]

[0096]

[0097] Principal component analysis transformed the original six salt tolerance coefficients into three new comprehensive indices. Membership function values ​​were calculated for each of these three comprehensive indices, and their weights were calculated based on their variance contribution rates. The weights for the three comprehensive indices were 0.530, 0.299, and 0.171, respectively. After obtaining the membership function values ​​and weights of the comprehensive indices, the comprehensive salt tolerance evaluation value D (Table 6) was calculated. The salt tolerance of 629 cotton germplasm resources was evaluated based on the magnitude of the D value. The Euclidean distance "complete" method and the number of clusters K=5 were used to cluster the comprehensive evaluation value D. Based on the magnitude of the D value and the clustering results, the 629 varieties (lines) were divided into 5 groups (…). Figure 7The test materials were categorized as follows: Category 1 (Ⅰ) consisted of 188 samples, accounting for 29.89% of the total test materials. These materials were of intermediate salt tolerance, with D values ​​ranging from 0.429 to 0.542. Category 2 (Ⅱ) consisted of 376 samples, accounting for 59.78% of the total test materials. These materials were of salt tolerance, with D values ​​ranging from 0.543 to 0.691. Category 3 (Ⅲ) consisted of 36 samples, accounting for 5.72% of the total test materials. These materials were of high salt tolerance, with D values ​​ranging from 0.698 to 0.824. Category 4 (Ⅳ) consisted of 28 samples, accounting for 4.45% of the total test materials. These materials were of salt sensitivity, with D values ​​ranging from 0.248 to 0.425. Category 5 (Ⅴ) consisted of 1 sample, accounting for 0.16% of the total test materials. This material was of high salt sensitivity, with a D value of 0.063. The high salt tolerance materials are shown in Table 5.

[0098] Table 5. Types, D-values, and rankings of high salt-resistant materials

[0099]

[0100]

[0101] To further analyze the relationship between various indicators during cotton germination and salt tolerance, salt tolerance identification indicators were selected, and a mathematical model for evaluating salt tolerance during cotton germination was established. Stepwise regression analysis was performed with the comprehensive salt tolerance evaluation value D as the dependent variable and the salt tolerance coefficients of the six indicators as independent variables, yielding the optimal equation: D = 0.277RFW + 0.29RGP + 0.189RPL + 0.387RGR0.32, and the coefficient of determination R0.32. 2 =0.992, P<0.001. Analysis of the accuracy of the predicted D value of the equation showed that the prediction accuracy for each variety (line) was above 95.37% (Table 6), proving that the equation can be used to evaluate the salt tolerance of cotton germplasm resources during the germination period. Under the same conditions, only four indicators need to be measured: fresh weight, hypocotyl length, germination potential, and germination rate. The corresponding salt tolerance coefficients can be calculated, and the comprehensive evaluation D value calculated by the equation can be used to determine the salt tolerance of different cotton germplasm resources.

[0102] Salt tolerance in cotton is a complex quantitative trait. Studies have found that there are significant differences in salt tolerance among different cultivars, and the same cultivar also responds differently to salt stress at different growth and development stages and in different organs and tissues.

[0103] In the initial stage of seed germination, salt treatment causes osmotic stress, affecting seed water absorption and swelling, thus delaying germination. Excessively high salt concentrations can also induce dormancy. Simultaneously, salt stress inhibits the decomposition, transformation, and synthesis of stored substances, suppresses the activity of various enzymes, and further inhibits seed germination. After germination, in the seedling stage, osmotic stress reduces the plant's water absorption capacity, decreases the volume of vacuoles in the cytoplasm, affects cell elongation and division, and inhibits the growth and development of the seedling's roots and aboveground parts. + Excessive accumulation disrupts the intracellular ion balance, causing an imbalance between the production and scavenging of reactive oxygen species (ROS), exacerbating membrane lipid peroxidation, damaging cell membrane structure, and leading to cytoplasmic extravasation, resulting in increased conductivity. The activities of ROS-scavenging enzymes such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) are significantly reduced, while malondialdehyde (MDA) content increases markedly. Therefore, previous studies have selected germination potential, germination rate, germination index, vigor index, water absorption rate, conductivity, root length, hypocotyl length, total seedling length, and aboveground / underground dry and fresh weight, as well as SOD, POD, CAT activities, and MDA content as indicators to evaluate the salt tolerance of various crops during germination. However, these indicators exhibit a certain degree of correlation, and using a single indicator or a single evaluation method for multiple indicators cannot accurately and comprehensively evaluate cotton salt tolerance.

[0104] Physiological index measurements are easily affected by individual differences, sampling sites, and sampling times. This study selected six morphological indicators most directly affected by salt stress and combined them with multivariate statistical analysis to conduct a rapid, accurate, and comprehensive evaluation of the salt tolerance of 629 cotton germplasm resources. Correlation analysis showed that, except for germination rate and hypocotyl length, the correlations among the other indicators were significant or highly significant, indicating overlap in the information contained. Principal component analysis was used to transform the six indicators into three independent comprehensive indicators, reducing redundancy of the original indicator information and retaining 91.83% of the original information. Based on this, membership function analysis was used to calculate the comprehensive evaluation value D, which was used to comprehensively evaluate the salt tolerance of cotton during germination. The maximum distance method was used to classify the D value into five different salt tolerance types. A mathematical model for evaluating the salt tolerance of cotton during germination was established with the comprehensive salt tolerance evaluation value D as the dependent variable and the six salt tolerance coefficients as independent variables. Fresh weight, hypocotyl length, germination potential, and germination rate were determined to be indicators for identifying cotton salt tolerance. In summary, this study conducted a comprehensive and objective evaluation of the salt tolerance of 629 cotton germplasm resources with rich genetic backgrounds during the germination period, established an efficient and accurate identification system for cotton salt tolerance, and identified and screened 4 highly salt-tolerant materials and 1 highly salt-sensitive material. This provides a methodological reference for cotton salt tolerance identification and a material basis for the study of salt tolerance mechanisms and the improvement of salt-tolerant varieties.

[0105] Appendix 6: Germplasm number of the tested germplasm, D value, predicted D value and prediction accuracy

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[0124]

[0125] In the preliminary stage of this study, six gradient salt stress treatments were set up. Based on the analysis of variance of various indicators and the salt tolerance coefficient change curves among the five varieties, 150 mmol / L salt was selected. -1 NaCl was used as the optimal concentration for evaluating the salt tolerance of cotton. This concentration was used to evaluate the salt tolerance of 629 cotton germplasm resources. Through correlation analysis, principal component analysis, comprehensive membership function analysis, and cluster analysis, the six indicators were transformed into three new independent indicators, yielding a comprehensive evaluation value for the salt tolerance of the 629 cotton germplasm resources. Based on the D-value, the germplasm was divided into five categories. Finally, the salt-tolerant material ST542 and the salt-sensitive material ST55 were selected. A stepwise regression model was constructed at 150 mmol / L... -1 Under NaCl conditions, fresh weight, hypocotyl length, germination potential, and germination rate can be used as indicators for assessing the salt tolerance of cotton during germination.

[0126] The contents not described in detail in this specification are prior art known to those skilled in the art. Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for evaluating the salt tolerance of cotton germplasm during germination, characterized in that: Choose 150 mmol L -1 NaCl is used as the optimal concentration for evaluating the salt tolerance of cotton. This concentration is employed to evaluate the salt tolerance of cotton germplasm resources. Through correlation analysis, principal component analysis, comprehensive membership function analysis, and cluster analysis, six indicators are transformed into three new independent indicators, yielding a comprehensive evaluation value for the salt tolerance of cotton germplasm. The specific process includes: 1) Salt tolerance test A. Select cotton seeds that are uniform in size and plump. After disinfecting and soaking them in 10% hydrogen peroxide for 30 minutes, rinse them 4-5 times with sterile water and use filter paper to absorb the moisture from the seed coat surface. B. Cut the filter paper into a rectangle 30cm long and 20cm wide. After wetting it in the solution, lay it flat on the test bench. Select 40 seeds and arrange them in two rows (20 seeds per row) 2cm below the top of the filter paper. The two rows are 2cm apart, and the seeds are staggered with the pinholes facing down. C. After wetting a filter paper in the solution, align it and lay it flat on the seeds. Fold the filter paper in half from 1.5 to 2 cm from the top (to prevent the solution from evaporating too quickly). Roll the filter paper into a cylindrical shape. Repeat this process 3 times and tie the bottom with a rubber band. Place the filter paper in a germination box with a 3 cm high salt solution. Put 18 filter paper rolls in each germination box. D. Place the germination box containing the filter paper roll into the seed germination chamber at a temperature of 30±1℃ and a relative humidity of 60%. Supplement light on the 3rd day, with a light / dark period of 12h / 12h (L / D). Replenish water daily to a uniform liquid level (3cm). E. On day 3 and day 7, the number of germinations was counted. On day 7, 5 seedlings with basically the same growth were randomly selected to measure fresh weight, total seedling length, radicle length and hypocotyl length. Each treatment was repeated 3 times. 2) Measurement Indicators Germination is indicated when the radicle breaks through the seed coat and shows white markings; use a ruler to measure the total length of the seedling (from the root cap to the point of cotyledon attachment) and the length of the radicle; weigh the seedling after absorbing the surface moisture. Germination potential = Number of germinated seeds on day 3 / Number of seeds tested × 100%; Germination rate = (Number of germinated seeds on day 7 / Number of seeds tested) × 100%; Hypocotyl length = total seedling length - radicle length; Relative salt tolerance coefficient of each index = treatment index / control index × 100%; 3) Statistical analysis Microsoft Excel 2016 was used to organize the experimental data. SPSS Statistics 27 was used to perform statistical description, analysis of variance, principal component analysis and correlation analysis on the experimental data. The R language "cluster, factoextra" software package was used for cluster analysis and visualization. Graphpad Prism was used for plotting. The relative values ​​of six traits were converted into three comprehensive indicators by principal component analysis, and the salt tolerance of 629 cotton germplasms was comprehensively evaluated by fuzzy mathematical membership function method. Membership function value: μ(X i )=(X i -X min ) / (X max -X min )i=1,2,3,...,n X i Let X represent the value of the i-th comprehensive index. min Let X represent the minimum value among the i-th comprehensive indicators. max μ(X) represents the maximum value among the i-th comprehensive indicators. i ) represents the membership function value of the i-th comprehensive index; Weights of each comprehensive indicator: w i P represents the weight of the i-th comprehensive indicator among all comprehensive indicators. i This represents the variance contribution rate of the i-th comprehensive index of each cotton germplasm obtained from principal component analysis; Salt tolerance comprehensive evaluation value: The D value represents the comprehensive evaluation value of the salt tolerance of the i-th germplasm under salt stress conditions; Cluster analysis: Based on the D value, Euclidean distance, the complete method between groups, and the number of clusters K=5 were used to classify the salt tolerance of cotton germplasm. The salt tolerance of each type of cotton germplasm was defined according to the range of D values. Stepwise regression analysis: Stepwise regression analysis was conducted with the comprehensive salt tolerance evaluation value D as the dependent variable and the salt tolerance coefficients of the six indicators as independent variables.

2. The method for evaluating the salt tolerance of cotton germplasm during germination according to claim 1, characterized in that: The concentration of the salt solution was selected for treatment at 0, 100, 150, 200, 250, and 300 mmol / L. -1 Six gradient salt stress treatments were applied. On day 7, fresh weight, total seedling length, radicle length, and germination rate were measured and statistically analyzed to determine the appropriate concentration.

3. The method for evaluating the salt tolerance of cotton germplasm during germination according to claim 1, characterized in that: The indicators include fresh weight, total seedling length, hypocotyl length, radicle length, germination potential, and germination rate.

4. The method for evaluating the salt tolerance of cotton germplasm during germination according to claim 3, characterized in that: Under the same conditions, the preferred indicators are fresh weight, hypocotyl length, germination potential and germination rate. The corresponding salt tolerance coefficients are calculated, and the comprehensive evaluation D value calculated by the equation is used to determine the salt tolerance of different cotton germplasm resources.

5. The method for evaluating the salt tolerance of cotton germplasm during germination according to claim 3, characterized in that: The correlation analysis shows that different varieties respond differently to salt stress. Using a single index cannot comprehensively evaluate the salt tolerance of cotton varieties. The index used should include two or more of the following: fresh weight, total seedling length, hypocotyl length, radicle length, germination potential, and germination rate.

6. The method for evaluating the salt tolerance of cotton germplasm during germination according to claim 1, characterized in that: Based on the principle that the cumulative contribution rate is greater than 85%, the principal component analysis transforms the salt tolerance coefficients of the original 6 indicators into 3 new independent comprehensive indicators. The new comprehensive indicators contain most of the genetic information of the original 6 indicators and can be used to evaluate the salt tolerance of cotton varieties (lines). The comprehensive index PC1 was most correlated with relative fresh weight and relative total seedling length, mainly reflecting the total biomass and growth of cotton; the comprehensive index PC2 was most correlated with relative germination potential and relative germination rate, mainly reflecting the germination of cotton seeds under salt stress; the comprehensive index PC3 was most correlated with relative radicle length and relative hypocotyl length (0.634), mainly reflecting the impact of salt stress on the growth of cotton aboveground and underground parts.

7. The method for evaluating the salt tolerance of cotton germplasm during germination according to claim 6, characterized in that: The principal component analysis transforms the original six salt tolerance coefficients into three new comprehensive indicators. The membership function values ​​of these three comprehensive indicators are calculated, and their weights are calculated based on the variance contribution rate of each comprehensive indicator.

8. The method for evaluating the salt tolerance of cotton germplasm during germination according to claim 7, characterized in that: After obtaining the membership function values ​​and weights of the comprehensive index, the cluster analysis calculates the comprehensive salt tolerance evaluation value D. The salt tolerance of cotton germplasm resources is evaluated based on the magnitude of the D value. The Euclidean distance "complete" method and the number of clusters K=5 are used to cluster the comprehensive evaluation value D. Based on the D-value and clustering results, cotton varieties (lines) are divided into 5 categories: Category 1 (Ⅰ) is of intermediate salt tolerance, with a D-value range of 0.429 to 0.542; Category 2 (Ⅱ) is of salt tolerance, with a D-value range of 0.543 to 0.691; Category 3 (Ⅲ) is of high salt tolerance, with a D-value range of 0.692 to 0.824; Category 4 (Ⅳ) is of salt sensitivity, with a D-value range of 0.248 to 0.428; and Category 5 (Ⅴ) is of high salt sensitivity, with a D-value below 0.

248.

9. The method for evaluating the salt tolerance of cotton germplasm during germination according to claim 1, characterized in that: When analyzing the salt tolerance of cotton germplasm during germination, a mathematical model for evaluating the salt tolerance of cotton during germination was established. Stepwise regression analysis was performed with the comprehensive salt tolerance evaluation value D as the dependent variable and the salt tolerance coefficients of the six indicators as independent variables to obtain the optimal equation. The relationship between various indicators during the cotton germination period and salt tolerance was further analyzed, and salt tolerance identification indicators were screened.

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