Evaluation method for identifying preliminary salt tolerance of millet in germination period and application of evaluation method

Abstract

The invention belongs to the technical field of agricultural production, and discloses an evaluation method for identifying the preliminary salt tolerance of foxtail millet in a germination period, which comprises the following steps: determining five key indexes of relative germination potential, relative germination rate, relative root length, relative bud length and relative germination index in the germination period of foxtail millet; the salt tolerance of the millet germplasm resources in the germination period can be preliminarily evaluated by establishing a regression equation to estimate the D value. The simple, convenient and scientific method for identifying the salt tolerance of the millet germplasm resources in the germination period is established, convenience is provided for screening salt-tolerant germplasm of millet, and the method is a premise for carrying out research on a salt-tolerant mechanism of millet.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural production, and in particular to an evaluation method and application of preliminary salt tolerance identification of millet during germination period. Background Art

[0002] Soil salinization is a recognized global environmental problem that has a significant impact on the sustainable development of agriculture worldwide. Soil salinization is the main abiotic stress that affects the growth, development and productivity of crops worldwide. Salt stress has adverse effects on various phenotypic traits, physiological functions and biochemical indices of plants, especially during the reproductive period, such as seed germination and seedling growth. Millet (Setarial italica (L.) P. Beauv.) is an important food crop grown in border areas. Compared with large crops such as corn and wheat, it has more drought and salt resistance. As one of the oldest domesticated crops, millet (Setaria italica (L.) P. Beauv.) is a typical environmentally friendly crop with characteristics such as drought resistance, salt tolerance, high water use efficiency, wide adaptability, tolerance to adverse soil conditions, and strong yield stability. Screening millet varieties with good salt tolerance lays the foundation for developing salt-tolerant germplasm resources suitable for saline-alkali land cultivation, breeding salt-tolerant varieties, improving saline-alkali land and promoting sustainable agricultural development.

[0003] Salt tolerance is a complex quantitative trait, and the determination of a single trait cannot well reflect the salt tolerance of plants. There are many methods to evaluate plant salt tolerance, among which the membership function method is the most commonly used identification method, that is, calculating the membership function value and D value based on weights. Membership function analysis can integrate more traits to screen and evaluate salt-tolerant germplasm of different plant varieties. The relative values ​​of germination potential, germination rate, bud length and root length under salt stress and control conditions during the germination period of millet can better distinguish the differences in salt tolerance of different germplasms. For a long time, many researchers have identified the salt tolerance of crops such as millet and sorghum during the germination period, but there is a lack of unified identification standards to standardize and unify them, which makes the identification of millet salt tolerance difficult and inaccurate. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art and provide an evaluation method and application for preliminary salt tolerance identification of millet during germination period.

[0005] The technical solution adopted by the present invention to solve its technical problem is:

[0006] A method for evaluating the preliminary salt tolerance of millet during the germination period, wherein the method is to determine five key indicators, namely, relative germination potential, relative germination rate, relative root length, relative bud length, and relative germination index, during the germination period of millet, and to establish a regression equation to estimate the D value, so as to preliminarily evaluate the salt tolerance of millet germplasm resources during the germination period;

[0007] Wherein, the regression equation is:

[0008] y=0.204X1+0.205X2+1.329X3+0.473X4+0.283X5-0.222(R 2 =1, P<0.01), where X1, X2,

[0009] X3, X4, and X5 represent relative germination potential, relative germination rate, relative root length, relative shoot length, and relative germination index, respectively, and -0.222 is the constant of the random error term;

[0010] The dependent variable y is the D value.

[0011] Furthermore, the estimation accuracy of the regression equation is greater than 99%.

[0012] Furthermore, according to the D value, the average value of D The salt tolerance of millet germplasm was classified by the standard deviation SD, and its salt tolerance was divided into 5 levels:

[0013] (1) That is, D ≥ 0.7324 is a highly salt-tolerant variety HST;

[0014] (2) That is, 0.7324>D≥0.6351 is the salt-tolerant variety ST;

[0015] (3) That is, 0.6351>D≥0.3309 is the medium salt-tolerant variety MST;

[0016] (4) That is, 0.3309>D≥0.2336, which is a salt-sensitive variety SS;

[0017] (5) That is, D < 0.2336 is a highly salt-sensitive variety HSS.

[0018] Furthermore, the specific steps are as follows:

[0019] (1) Determination of salt concentration

[0020] The concentration of 170mmol / L was selected as the treatment concentration for the salt tolerance identification of millet during the germination period;

[0021] Select salt-tolerant millet materials in the germination period, select 50 seeds of each variety with full grains and uniform size and place them in a disposable sterile culture dish covered with double-layer sterilized filter paper; conduct preliminary experiments to screen the optimal salt stress concentration, and first set the NaCl solution concentration to 0, 50, 100, 150, 200, 250, 300, and 350mmol / L, a total of 8 treatment concentrations, and then set the concentration between 150-200mmol / L and set a 10mmol / L interval to re-screen to determine the final salt stress concentration, and repeat each treatment three times; add 7mL of NaCl solution of different concentrations to each variety, and add an equal amount of distilled water to the control and place them evenly in the culture dish. Cultivate at 25±2℃, 70% humidity, and 16 / 8h day / night. Add sterile water on the 6th day to ensure that the roots are completely immersed in the salt solution. No additional salt solution is added during the cultivation process. Cultivate for 8 days in total, and measure the germination rate on the 8th day of cultivation;

[0022] (2) Screening and identification of salt-tolerant millets

[0023] Select salt-tolerant millet materials in the germination period, select each variety with full grains and uniform size, select a concentration of 170mmol / L as the stress concentration, add 7mL of NaCl solution of different concentrations to each variety, add an equal amount of distilled water to the control and place them evenly in a culture dish; culture at 25±2℃, 70% humidity, day / night 16 / 8h conditions, supplement with sterile water on the 6th day to ensure that the roots are completely immersed in the salt solution, no additional salt solution is added during the culture process, culture for 8 days in total, and determine the germination rate on the 8th day of culture; put the same variety of millet, i.e. red millet, in different batches as a reference, and repeat each treatment three times to check whether the experimental conditions are consistent between different batches;

[0024] (3) Measurement items

[0025] The germination standard was that the root length was at least equal to the seed length, and the sprout length was at least 1 / 2 of the seed length. During this period, the number of germinated seeds was recorded at the same time every 2, 4, 6, and 8 days. The germination potential was counted on the 4th day of culture, and the germination rate was counted on the 8th day. The phenotypic traits were measured after 8 days of treatment. The root length and sprout length were measured with a ruler, and 10 samples were randomly selected from each culture dish to calculate the average value. The root length was measured by measuring the length from the junction of the hypocotyl and the radicle to the root tip with a ruler. The sprout length was measured by measuring the length from the cotyledon to the root tip of the plant with a ruler.

[0026] The calculation formulas for germination potential, germination rate, relative germination rate, germination index, relative germination index, vitality index and relative vitality index are as follows:

[0027] The following formulas are used to calculate the various trait indicators:

[0028] 1) Germination potential (GP, %) = G4 / 50×100, where G4 is the number of germinated seeds on the fourth day;

[0029] 2) germination rate (GR, %) = G8 / 50 × 100, G8 is the number of germinated seeds on the eighth day;

[0030] 3)

[0031] In the formula, Gt is the number of germinated seeds on the 2nd, 4th, 6th, and 8th days, and Dt is the corresponding germination days;

[0032] 4) Relative germination potential (RGP, %) = treatment germination potential / control germination potential × 100;

[0033] 5) Relative germination rate (RGR, %) = treatment germination rate / control germination rate × 100;

[0034] 6) Relative germination index (RGI, %) = treatment germination index / control germination index × 100;

[0035] 7) Relative root length (RRL, %) = treatment root length / control root length × 100;

[0036] 8) Relative shoot length (RBL, %) = treatment shoot length / control shoot length × 100;

[0037] 9) Coefficient of variation CV = (standard deviation SD / mean value) × 100;

[0038] (4) Data statistics and analysis

[0039] Microsoft Excel 2019 was used for basic data processing, and SPSS26.0 and Origin 2021 software were used to perform correlation analysis, principal component analysis, membership function analysis, cluster analysis, and normal distribution diagrams of related indicators;

[0040] The membership function method was used to conduct a comprehensive evaluation of the salt tolerance of millet germplasm resources in Shanxi Province, and the membership function value was calculated. The calculation formula is as follows:

[0041]

[0042] In the formula, μ(X j ) represents the membership function value of the jth comprehensive index in a specific variety, X j represents the jth comprehensive index value in a specific germplasm; X max and X min Respectively represent the maximum and minimum values ​​of the jth comprehensive index of all varieties; each comprehensive index has a membership function value, and μ(X j ) is between 0 and 1;

[0043] Then, according to the relative importance of each trait, a certain weight is assigned. The weight formula is as follows:

[0044]

[0045] Where W j Indicates the importance of the jth comprehensive index among all the measured indicators; P j is the correlation coefficient between the jth comprehensive index and the salt tolerance characteristics of each variety, representing the contribution rate of the jth comprehensive index of each variety;

[0046] Finally, the membership function values ​​of each index are added together to obtain the comprehensive evaluation value of each variety, namely, the D value;

[0047]

[0048] In the formula, D represents the comprehensive evaluation value of salt tolerance of each variety under NaCl stress, X j represents the jth comprehensive index value in a specific germplasm, W j Indicates the importance of the jth comprehensive indicator among all the measured indicators;

[0049] The salt tolerance of millet varieties can be determined according to the size of the D value; the larger the D value, the stronger the salt tolerance of the variety, and vice versa, the weaker the salt tolerance of the variety;

[0050] (5) Salt tolerance evaluation

[0051] The standard for millet salt tolerance was established based on previous studies (2024 Singh, 2012 Chen, 2020 Li). The D value distribution was comprehensively evaluated based on five salt tolerance-related traits. The average value of D The salt tolerance of millet germplasm was classified into 5 levels based on the salt tolerance and standard deviation SD, namely It is a highly salt-tolerant variety, HST; It is a salt-tolerant variety, ST; It is a medium salt-tolerant variety, MST; It is a salt-sensitive variety SS; It is a highly salt-sensitive variety, HSS;

[0052] (6) Evaluation and prediction of salt tolerance during millet germination

[0053] In order to predict the salt tolerance of millet during the germination period, SPSS26.0 was used to perform multivariate stepwise regression analysis on the D value (dependent variable y) and five salt tolerance related indicators (independent variable X). According to the scores of the five indicators, four indicators (relative germination potential, relative root length, relative shoot length, and relative germination index) were screened out, and a mathematical evaluation model for salt tolerance was established: y = 0.204X1 + 0.205X2 + 1.329X3 + 0.473X4 + 0.283X5 - 0.222 (R2 =1, P<0.01), where X1, X2, X3, X4 and X5 represent relative germination potential, relative germination rate, relative root length, relative shoot length and relative germination index, respectively, and -0.222 is the constant of the random error term; the comprehensive score and regression value were tested and found that their estimation accuracy was greater than 99%, indicating that the equation was accurate and effective in predicting the salt tolerance of millet germplasm. Therefore, the relative germination potential, relative germination rate, relative root length, relative shoot length and relative germination index of millet were measured during the germination period, and the D value was estimated through the regression equation, which can preliminarily judge the salt tolerance of the germplasm.

[0054] The evaluation method described above is used for the identification of salt tolerance of millet during the germination period.

[0055] The advantages and positive effects achieved by the present invention are:

[0056] 1. The identification of millet salt-tolerant germplasm resources is the primary problem that needs to be solved in millet salt-tolerant breeding. The present invention establishes a simple and scientific method for identifying the salt tolerance of millet germplasm resources during the germination period, which provides convenience for the screening of millet salt-tolerant germplasm and is also a prerequisite for conducting research on the salt tolerance mechanism of millet.

[0057] 2. The present invention uses NaCl salt solutions of different concentrations to treat 10 millet germplasms, and by analyzing the relative germination potential, relative germination rate, relative bud length and relative root length during the germination period, it is determined that the NaCl concentration suitable for the identification of salt tolerance during the germination period of millet is 170mmol / L. At this concentration, the salt tolerance of 333 germplasm resources was comprehensively evaluated using principal component analysis and cluster analysis, and the millet salt tolerance grade was classified using the membership function value method. The millet salt tolerance during the germination period was evaluated using the comprehensive evaluation D value of the five related traits of relative germination potential, relative germination rate, relative root length, relative bud length and relative germination index. The results showed that there was a very significant correlation between the five salt tolerance indices. Two representative principal components with a cumulative contribution rate of 85.911% were extracted from the five individual trait indices through principal component analysis. The scores of the two principal components were analyzed by the membership function value method to obtain the comprehensive score value (D value) for the salt tolerance evaluation of different varieties. Based on the D value, 333 local millet varieties in Shanxi were divided into five salt tolerance types, including 17 highly salt-tolerant millet materials, 37 salt-tolerant materials, 223 moderately salt-tolerant materials, 36 salt-sensitive materials, and 20 highly salt-sensitive materials. At the same time, regression analysis was used to establish a regression equation that can be used to evaluate the salt tolerance of millet during the germination period.

[0058] y=0.204X1+0.205X2+1.329X3+0.473X4+0.283X5-0.222. It shows that the relative germination potential, relative germination rate, relative root length, relative bud length and relative germination index measured during the germination period of millet can be used as evaluation indicators for the identification of millet salt tolerance. This study established an efficient and reliable evaluation method for the preliminary identification of millet salt tolerance during the germination period. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 The figure is a diagram of the optimal NaCl solution concentration for evaluating the salt tolerance of millet determined by the two indicators of relative germination rate and germination phenotype in the present invention; wherein (A) the change of the relative germination rate of millet under the stress of different concentrations of NaCl solution, (B) the change of the variance of the relative germination rate of millet under the stress of different concentrations of NaCl solution, and (C) the seed germination phenotype;

[0060] Figure 2 The optimal NaCl concentration diagram for the salt tolerance evaluation of millet according to the two indicators of relative germination rate and germination phenotype in the present invention; (A) the change of relative germination rate of millet under the stress of different concentrations of NaCl solution, (B) the change of variance of relative germination rate of millet under the stress of different concentrations of NaCl solution, (C) germination phenotype;

[0061] Figure 3 The present invention shows the classification of 333 Shanxi millet germplasms based on D value (A), the distribution of D value (B), and the salt tolerance classification diagram of 333 Shanxi millet germplasms based on D value; among them, HST, highly salt-tolerant; ST, salt-tolerant; MST, moderately salt-tolerant; SS, salt-sensitive; HSS, highly salt-sensitive. DETAILED DESCRIPTION

[0062] The present invention will be further described below in conjunction with the embodiments. The following embodiments are descriptive rather than restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0063] The various experimental operations involved in the specific embodiments are all routine techniques in the art. For parts not specially annotated in this document, ordinary technicians in the art can implement them by referring to various commonly used reference books, scientific and technological literature or related instructions, manuals, etc. before the filing date of this invention.

[0064] A method for evaluating the preliminary salt tolerance of millet during the germination period, wherein the method is to determine five key indicators, namely, relative germination potential, relative germination rate, relative root length, relative bud length, and relative germination index, during the germination period of millet, and to establish a regression equation to estimate the D value, so as to preliminarily evaluate the salt tolerance of millet germplasm resources during the germination period;

[0065] Wherein, the regression equation is:

[0066] y=0.204X1+0.205X2+1.329X3+0.473X4+0.283X5-0.222(R 2 =1, P<0.01), where X1, X2,

[0067] X3, X4, and X5 represent relative germination potential, relative germination rate, relative root length, relative shoot length, and relative germination index, respectively, and -0.222 is the constant of the random error term;

[0068] The dependent variable y is the D value.

[0069] Preferably, the estimation accuracy of the regression equation is greater than 99%.

[0070] Preferably, according to the D value, the average value of D The salt tolerance of millet germplasm was classified by the standard deviation SD, and its salt tolerance was divided into 5 levels:

[0071] (1) That is, D ≥ 0.7324 is a highly salt-tolerant variety HST;

[0072] (2) That is, 0.7324>D≥0.6351 is the salt-tolerant variety ST;

[0073] (3) That is, 0.6351>D≥0.3309 is the medium salt-tolerant variety MST;

[0074] (4) That is, 0.3309>D≥0.2336, which is a salt-sensitive variety SS;

[0075] (5) That is, D < 0.2336 is a highly salt-sensitive variety HSS.

[0076] Preferably, the specific steps are as follows:

[0077] (1) Determination of salt concentration

[0078] The concentration of 170mmol / L was selected as the treatment concentration for the salt tolerance identification of millet during the germination period;

[0079] Select salt-tolerant millet materials in the germination period, select 50 seeds of each variety with full grains and uniform size and place them in a disposable sterile culture dish covered with double-layer sterilized filter paper; conduct preliminary experiments to screen the optimal salt stress concentration, and first set the NaCl solution concentration to 0, 50, 100, 150, 200, 250, 300, and 350mmol / L, a total of 8 treatment concentrations, and then set the concentration between 150-200mmol / L and set a 10mmol / L interval to re-screen to determine the final salt stress concentration, and repeat each treatment three times; add 7mL of NaCl solution of different concentrations to each variety, and add an equal amount of distilled water to the control and place them evenly in the culture dish. Cultivate at 25±2℃, 70% humidity, and 16 / 8h day / night. Add sterile water on the 6th day to ensure that the roots are completely immersed in the salt solution. No additional salt solution is added during the cultivation process. Cultivate for 8 days in total, and measure the germination rate on the 8th day of cultivation;

[0080] (2) Screening and identification of salt-tolerant millets

[0081] Select salt-tolerant millet materials in the germination period, select each variety with full grains and uniform size, select a concentration of 170mmol / L as the stress concentration, add 7mL of NaCl solution of different concentrations to each variety, add an equal amount of distilled water to the control and place them evenly in a culture dish; culture at 25±2℃, 70% humidity, day / night 16 / 8h conditions, supplement with sterile water on the 6th day to ensure that the roots are completely immersed in the salt solution, no additional salt solution is added during the culture process, culture for 8 days in total, and determine the germination rate on the 8th day of culture; put the same variety of millet, i.e. red millet, in different batches as a reference, and repeat each treatment three times to check whether the experimental conditions are consistent between different batches;

[0082] (3) Measurement items

[0083] The germination standard was that the root length was at least equal to the seed length, and the sprout length was at least 1 / 2 of the seed length. During this period, the number of germinated seeds was recorded at the same time every 2, 4, 6, and 8 days. The germination potential was counted on the 4th day of culture, and the germination rate was counted on the 8th day. The phenotypic traits were measured after 8 days of treatment. The root length and sprout length were measured with a ruler, and 10 samples were randomly selected from each culture dish to calculate the average value. The root length was measured by measuring the length from the junction of the hypocotyl and the radicle to the root tip with a ruler. The sprout length was measured by measuring the length from the cotyledon to the root tip of the plant with a ruler.

[0084] The calculation formulas for germination potential, germination rate, relative germination rate, germination index, relative germination index, vitality index and relative vitality index are as follows:

[0085] The following formulas are used to calculate the various trait indicators:

[0086] 1) Germination potential (GP, %) = G4 / 50×100, where G4 is the number of germinated seeds on the fourth day;

[0087] 2) germination rate (GR, %) = G8 / 50 × 100, G8 is the number of germinated seeds on the eighth day;

[0088] 3)

[0089] In the formula, Gt is the number of germinated seeds on the 2nd, 4th, 6th, and 8th days, and Dt is the corresponding germination days;

[0090] 4) Relative germination potential (RGP, %) = treatment germination potential / control germination potential × 100;

[0091] 5) Relative germination rate (RGR, %) = treatment germination rate / control germination rate × 100;

[0092] 6) Relative germination index (RGI, %) = treatment germination index / control germination index × 100;

[0093] 7) Relative root length (RRL, %) = treatment root length / control root length × 100;

[0094] 8) Relative shoot length (RBL, %) = treatment shoot length / control shoot length × 100;

[0095] 9) Coefficient of variation CV = (standard deviation SD / mean value) × 100;

[0096] (4) Data statistics and analysis

[0097] Microsoft Excel 2019 was used for basic data processing, and SPSS26.0 and Origin 2021 software were used to perform correlation analysis, principal component analysis, membership function analysis, cluster analysis, and normal distribution diagrams of related indicators;

[0098] The membership function method was used to conduct a comprehensive evaluation of the salt tolerance of millet germplasm resources in Shanxi Province, and the membership function value was calculated. The calculation formula is as follows:

[0099]

[0100] In the formula, μ(X j ) represents the membership function value of the jth comprehensive index in a specific variety, X j represents the jth comprehensive index value in a specific germplasm; X max and X min Respectively represent the maximum and minimum values ​​of the jth comprehensive index of all varieties; each comprehensive index has a membership function value, and μ(X j ) is between 0 and 1;

[0101] Then, according to the relative importance of each trait, a certain weight is assigned. The weight formula is as follows:

[0102]

[0103] Where W j Indicates the importance of the jth comprehensive index among all the measured indicators; P j is the correlation coefficient between the jth comprehensive index and the salt tolerance characteristics of each variety, representing the contribution rate of the jth comprehensive index of each variety;

[0104] Finally, the membership function values ​​of each index are added together to obtain the comprehensive evaluation value of each variety, namely, the D value;

[0105]

[0106] In the formula, D represents the comprehensive evaluation value of salt tolerance of each variety under NaCl stress, X j represents the jth comprehensive index value in a specific germplasm, W j Indicates the importance of the jth comprehensive indicator among all the measured indicators;

[0107] The salt tolerance of millet varieties can be determined according to the size of the D value; the larger the D value, the stronger the salt tolerance of the variety, and vice versa, the weaker the salt tolerance of the variety;

[0108] (5) Salt tolerance evaluation

[0109] The standard for millet salt tolerance was established based on previous studies (2024 Singh, 2012 Chen, 2020 Li). The D value distribution was comprehensively evaluated based on five salt tolerance-related traits. The average value of D The salt tolerance of millet germplasm was classified into 5 levels based on the salt tolerance and standard deviation SD, namely It is a highly salt-tolerant variety, HST; It is a salt-tolerant variety, ST; It is a medium salt-tolerant variety, MST; It is a salt-sensitive variety SS; It is a highly salt-sensitive variety, HSS;

[0110] (6) Evaluation and prediction of salt tolerance during millet germination

[0111] In order to predict the salt tolerance of millet during the germination period, SPSS26.0 was used to perform multivariate stepwise regression analysis on the D value (dependent variable y) and five salt tolerance related indicators (independent variable X). According to the scores of the five indicators, four indicators (relative germination potential, relative root length, relative shoot length, and relative germination index) were screened out, and a mathematical evaluation model for salt tolerance was established: y = 0.204X1 + 0.205X2 + 1.329X3 + 0.473X4 + 0.283X5 - 0.222 (R2 =1, P<0.01), where X1, X2, X3, X4 and X5 represent relative germination potential, relative germination rate, relative root length, relative shoot length and relative germination index, respectively, and -0.222 is the constant of the random error term; the comprehensive score and regression value were tested and found that their estimation accuracy was greater than 99%, indicating that the equation was accurate and effective in predicting the salt tolerance of millet germplasm. Therefore, the relative germination potential, relative germination rate, relative root length, relative shoot length and relative germination index of millet were measured during the germination period, and the D value was estimated through the regression equation, which can preliminarily judge the salt tolerance of the germplasm.

[0112] The evaluation method described above is used for the identification of salt tolerance of millet during the germination period.

[0113] Specifically, the relevant preparation and detection are as follows:

[0114] 2. Materials and Methods

[0115] 2.1 Test materials

[0116] 333 Shanxi millet local varieties preserved in the National Germplasm Resources Medium-term Bank (Taiyuan) were selected as experimental materials (Table 1), which covered the basic genetic diversity of Shanxi millet and could better represent the genetic variation of Shanxi millet local varieties.

[0117] Table 1333 Sources of Shanxi local varieties

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130] 2.2 Experimental methods

[0131] 2.2.1 Determination of salt concentration

[0132] The screening of salt-tolerant millet materials during the germination period was carried out in the constant temperature germination room of the Agricultural Gene Resources Research Center of Shanxi Agricultural University. For each variety, 50 seeds with full grains and uniform size were selected and placed in a disposable sterile culture dish with a diameter of 9 cm and covered with double-layer sterilized filter paper. Seven varieties from 7 prefecture-level cities in Shanxi from north to south were randomly selected, including CC013 (Shuozhou Shuocheng), CC037 (Datong Lingqiu), CC099 (Yangquan Yuxian), CC192 (Lvliang Xingxian), CC225 (Changzhi Xiangyuan), CC503 (Linfen Anze), and CC622 (Xinzhou Jingle) for preliminary experimental screening of the optimal salt stress concentration. The NaCl solution concentration was first set to 0, 50, 100, 150, 200, 250, 300, and 350 mmol / L for a total of 8 treatment concentrations, and then the concentration was re-screened again at a spacing of 10 mmol / L between 150-200 mmol / L to determine the final salt stress concentration. Each treatment was repeated three times. 7 mL of NaCl solution of different concentrations was added to each variety, and the same amount of distilled water was added to the control and evenly placed in the culture dish. The culture was carried out at 25±2℃, 70% humidity, and 16 / 8h day / night. On the 6th day, 1 mL of sterile water was added to ensure that the roots were completely immersed in the salt solution. No additional salt solution was added during the culture process. The culture was carried out for 8 days in total, and the germination rate was measured on the 8th day of culture.

[0133] 2.2.2 Screening and identification of salt-tolerant millets

[0134] 333 millet local varieties were treated with the concentration selected in the pre-experiment as the stress concentration. 7 mL of NaCl solution of different concentrations was added to each variety. The control was added with an equal amount of distilled water and evenly placed in a culture dish. The culture was carried out at 25 ± 2 ° C, 70% humidity, and 16 / 8 h day / night. Sterile water was added on the 6th day to ensure that the roots were completely immersed in the salt solution. No additional salt solution was added during the culture process. The total culture was 8 days, and the germination rate was measured on the 8th day of culture. The same variety of millet (red millet) was placed in different batches as a reference, and each treatment was repeated three times to check whether the experimental conditions were consistent between different batches.

[0135] 2.3 Measurement items

[0136] The germination standard is that the root length is at least as long as the seed and the sprout length is at least 1 / 2 of the seed length. The number of germinated seeds is recorded at the same time every 2, 4, 6, and 8 days. The germination potential is counted on the 4th day of culture, and the germination rate is counted on the 8th day. The phenotypic traits are measured after 8 days of treatment. The root length and sprout length are measured with a ruler. 10 samples are randomly selected from each culture dish to calculate the average value. The root length is measured by measuring the length from the junction of the hypocotyl and the radicle to the root tip with a ruler; the sprout length is measured by measuring the length from the cotyledon to the root tip of the plant with a ruler.

[0137] The calculation formulas for germination potential, germination rate, relative germination rate, germination index, relative germination index, vitality index and relative vitality index are as follows:

[0138] The following formulas are used to calculate the various trait indicators:

[0139] (1) Germination potential (GP, %) = G4 / 50 × 100, where G4 is the number of germinated seeds on the fourth day;

[0140] (2) germination rate (GR, %) = G8 / 50 × 100, where G8 is the number of germinated seeds on the eighth day;

[0141] (3)

[0142] In the formula, Gt is the number of germinated seeds on the 2nd, 4th, 6th, and 8th days, and Dt is the corresponding germination days;

[0143] (4) Relative germination potential (RGP, %) = treatment germination potential / control germination potential × 100;

[0144] (5) Relative germination rate (RGR, %) = treatment germination rate / control germination rate × 100;

[0145] (6) Relative germination index (RGI, %) = treatment germination index / control germination index × 100;

[0146] (7) Relative root length (RRL, %) = treatment root length / control root length × 100;

[0147] (8) Relative shoot length (RBL, %) = treatment shoot length / control shoot length × 100;

[0148] (9) Coefficient of variation CV = (standard deviation SD / mean value) × 100.

[0149] 2.4 Data Statistics and Analysis

[0150] Microsoft Excel 2019 was used for basic data processing, and SPSS26.0 and Origin 2021 software were used for correlation analysis, principal component analysis, membership function analysis, cluster analysis, and normal distribution diagrams of related indicators. The membership function method was used to conduct a comprehensive evaluation of salt tolerance of 333 Shanxi millet germplasm resources. The membership function values ​​were referenced in the literature (Zhang H, Yuan C, Mao G, et al. Evaluation of saline-alkali and drought tolerance in maize varieties [J]. Bangladesh Journal of Botany, 2019, 48 (4): 1047-1063.

[0151] Chen Erying, Qin Ling, Yang Yanbing, et al. Differences and comprehensive evaluation of salt-alkali tolerance of millet varieties under production conditions [J]. Chinese Agricultural Science, 2019, 52(22): 4050-4065.) The calculation formula is as follows:

[0152]

[0153] In the formula, μ(X j ) represents the membership function value of the jth comprehensive index in a specific variety, X j represents the jth comprehensive index value in a specific germplasm; X max and X min They represent the maximum and minimum values ​​of the jth comprehensive index of all varieties. Each comprehensive index has a membership function value, and μ(X j ) is between 0 and 1.

[0154] Then, according to the relative importance of each trait, a certain weight is assigned. The weight formula is as follows:

[0155]

[0156] Where W j Indicates the importance of the jth comprehensive index among all the measured indicators; P j is the correlation coefficient between the j-th comprehensive index and the salt tolerance characteristics of each variety, representing the contribution rate of the j-th comprehensive index of each variety.

[0157] Finally, the membership function values ​​of each indicator are added together as the comprehensive evaluation value (D value) for evaluating each variety.

[0158]

[0159] Where D represents the comprehensive evaluation value of salt tolerance of each variety under NaCl stress.

[0160] The salt tolerance of millet varieties can be determined based on the size of the D value. The larger the D value, the stronger the salt tolerance of the variety, and vice versa.

[0161] 2.5 Salt tolerance rating evaluation

[0162] The salt tolerance grade standard of millet was established based on previous studies (Singh 2024, Chen 2012, Li 2020). The D value distribution was comprehensively evaluated based on the five salt tolerance-related traits. The average value of D The salt tolerance of 333 Shanxi millet germplasms was classified into 5 levels based on the salt tolerance index and standard deviation (SD). It is a highly salt-tolerant variety (HST); It is a salt-tolerant variety (ST); It is a medium salt-tolerant variety (MST); It is a salt-sensitive variety (SS); It is a highly salt-sensitive variety (HSS).

[0163] 3. Results and Analysis

[0164] 3.1 Determination of germination salt concentration

[0165] The results of this experiment show that there are differences in the relative germination rate of millet under different salt concentration treatments. The relative germination rate is one of the important indicators to measure whether the seeds are salt tolerant. Figure 1 It can be seen that low-concentration salt stress (150mmol / LNaCl solution and below) during seed germination prolongs the seed swelling process, but the final germination rate is relatively less affected. High-concentration salt stress (250mmol / LNaCl solution and above) will cause the seeds to be unable to fully absorb water, resulting in the seeds being unable to germinate within a certain period of time. The variance analysis of the relative germination rate of millet showed that when the concentration of NaCl solution was 150-200mmol / L, the variance of the relative germination rate of millet was the largest; then, in the range of 150-200mmol / L, the NaCl solution concentration was screened again at intervals of 10mmo / L ( Figure 2 ), when the salt concentration reached 170 and 200mmol / L, the variance was large; but when the NaCl solution concentration was 200mmol / L, the sprout and root length of millet were strongly inhibited, and the degree of inhibition on the root was greater than that on the sprout. In summary, when the NaCl solution concentration was 170mmol / L, the relative germination rate of each variety was significantly different compared with the control, and in order to ensure that indicators such as sprout length and root length could be measured, we selected a concentration of 170mmol / L as the treatment concentration for the salt tolerance identification of millet during the germination period.

[0166] 3.2 Quantitative traits during germination

[0167] Under 170mmol / LNaCl stress, 5 salt tolerance related test traits, including germination potential, germination rate, germination index, root length, and bud length, were obtained from 333 millet materials, and the relative values ​​(treatment value / control value) were calculated. As shown in Table 2, the coefficient of variation of the five salt tolerance related indicators is in the following order from large to small: relative germination potential > relative germination index > relative bud length > relative germination rate > relative root length. The relative bud length of most varieties is greater than the relative root length, indicating that the inhibition of the root system under salt stress is greater than the inhibition of the bud, and the root of millet during the germination period is more sensitive to salt stress than the bud.

[0168] Table 2 Descriptive statistics of salt tolerance-related traits of millet during germination

[0169]

[0170]

[0171] 3.3 Correlation analysis of salt tolerance indexes during germination period

[0172] The correlation analysis of the five indices of relative germination potential, relative germination rate, relative root length, relative bud length, and relative germination index (Table 3) showed that the relative germination rate was extremely significantly positively correlated with the relative values ​​of the other four traits. The correlation coefficients of relative germination rate with relative germination potential, relative root length, relative bud length, and relative germination index were 0.78, 0.18, 0.47, and 0.83, respectively; relative germination potential, relative germination rate, relative root length, relative bud length, and relative germination index were all extremely significantly positively correlated; among them, the correlation coefficient between relative germination index and relative germination potential was the highest, which was 0.93 (Table 3). The results show that the higher the vitality of the variety itself, the better the quality, the longer the root buds, the less inhibition, the stronger the ability to resist salt stress, and the higher the relative germination rate, which is the basis for ensuring germination in the later stage. The above results show that the five traits can be used for principal component analysis (PCA).

[0173] Table 3 Correlation of various indicators during the germination period of millet varieties (lines)

[0174]

[0175] 3.4 Principal component analysis of salt tolerance indexes during germination period

[0176] After data standardization, principal component analysis was performed on the various indicators of 333 millet local varieties (lines) under salt stress (Table 4), and two representative principal components with a cumulative contribution rate of 85.911% were extracted, which represented most of the information of the five original indicators and could be used as comprehensive indicators for the evaluation of millet salt tolerance. The contribution rate of the first principal component was 62.918%, and the eigenvalue was 3.146, which indicated that the first principal component played a major role in the salt tolerance of millet. Among them, the relative germination index, relative germination potential, and relative germination rate had higher loads, which meant that these three indicators had a larger weight in the first principal component, had a more significant impact on it, and were directly related to the germination of seeds. Therefore, it can be inferred that the first principal component mainly reflects the germination ability of millet under salt stress. The contribution rate of the second principal component was 22.993%, and the eigenvalue was 1.150. The relative root length and relative shoot length had higher loadings, indicating that these two indicators had a significant impact on the second principal component. The relative root length and relative shoot length were directly related to the germination process of seeds. Therefore, the second principal component mainly described the germination of millet under salt stress.

[0177] Table 4 Principal component analysis of various indicators of millet varieties (lines) during germination period

[0178]

[0179] 3.5 Comprehensive evaluation and analysis of salt tolerance of millet during germination

[0180] The data was standardized according to formula (1), and the weights of the two comprehensive indicators were calculated to be 0.7324 and 0.2676 respectively. According to the contribution rate of each comprehensive indicator, the D value of the comprehensive salt tolerance evaluation of millet during the germination period was calculated using formula (3). The distribution of D values ​​of the comprehensive salt tolerance evaluation of 333 Shanxi millets is as follows: Figure 3 As shown. The range of D value is 0.1238~0.8632. The largest D value is CC183 Qingruan Gu (D=0.8632) from Yu County, Yangquan City, indicating that it has the strongest salt tolerance; the smallest D value is CC582 Zhua Zhua Gu (D=0.1238) from Xia County, Yuncheng City, indicating that it has the weakest salt tolerance.

[0181] Based on the comprehensive evaluation of the D value distribution of the five salt tolerance related traits, the average value of D The salt tolerance of 333 Shanxi millet germplasms was classified into 5 levels based on the salt tolerance index and standard deviation (SD) (Table 5).

[0182] (1) That is, D ≥ 0.7324 was highly salt-tolerant varieties (HST), including 17 varieties;

[0183] (2) That is, 0.7324>D≥0.6351 is salt-tolerant varieties (ST), including 37 varieties;

[0184] (3) That is, 0.6351>D≥0.3309 is medium salt tolerance (MST), including 223 varieties;

[0185] (4) That is, 0.3309>D≥0.2336, which is salt-sensitive variety (SS), including 36 varieties;

[0186] (5) That is, D < 0.2336 is a highly salt-sensitive variety (HSS), including 20 varieties.

[0187] Table 5 Extreme salt resistant materials

[0188]

[0189] 3.6 Evaluation and prediction of salt tolerance of millet during germination

[0190] In order to predict the salt tolerance of millet during the germination period, SPSS26.0 was used to perform multivariate stepwise regression analysis on the D value (dependent variable y) and five salt tolerance related indicators (independent variable X). According to the scores of the five indicators, four indicators (relative germination potential, relative root length, relative shoot length, and relative germination index) were screened out, and a mathematical evaluation model for salt tolerance was established: y=0.204X1+0.205X2+1.329X3+0.473X4+0.283X5-0.222(R 2 =1, P<0.01), where X1, X2, X3, X4 and X5 represent relative germination potential, relative germination rate, relative root length, relative shoot length and relative germination index, respectively, and -0.222 is the constant of the random error term. The comprehensive score and regression value were tested and found to have an estimation accuracy of more than 99%, indicating that the equation has high accuracy and good effect in predicting the salt tolerance of millet germplasm. Therefore, the relative germination potential, relative germination rate, relative root length, relative shoot length and relative germination index of millet were measured during the germination period, and the D value was estimated by regression equation, which can preliminarily judge the salt tolerance of the germplasm.

[0191] Table 6 Precision analysis of regression equation

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200] 4. Conclusion and discussion

[0201] The problem of saline-alkali land in the world is indeed very serious. According to statistics, about 1 billion hectares of land in the world are affected by salinization, which is a shocking figure. This means that more than 20% of the world's irrigated land and more than 6% of the world's total agricultural area. To hold the rice bowl in our own hands, we must not only increase crop yields, but also increase agricultural yields in saline-alkali soils. Salinization refers to the accumulation of too many soluble salts in the soil. These salts gradually accumulate in the soil, making the salt concentration in the soil exceed the range that crops can tolerate for normal growth. These high concentrations of soluble salts limit the absorption of water by the roots through their high osmotic pressure, thereby reducing plant growth. Germination is the starting point of the growth and development process of all crops. Salinity may lead to a significant reduction in germination rate. When planted in saline-alkali land, the germination of salt-sensitive crops is significantly inhibited, resulting in reduced yields. Millet is one of the oldest domesticated food crops in northern China. As a functional food source and model crop, most millets have a certain tolerance to salt stress, and phenotypic identification is an important means to study salt tolerance traits. Therefore, it is of great significance to the breeding and application of millet to identify and screen the salt tolerance of millet during the germination period through a rapid and effective method.

[0202] The present invention randomly selected 7 varieties from north to south in Shanxi for concentration screening under different salt concentrations, and finally determined 170mmol / L as the stress concentration of NaCl solution. 333 Shanxi millet germplasms were subjected to stress treatment with 170mmol / L NaCl solution during the germination period, and their salt tolerance was comprehensively evaluated by measuring salt tolerance-related traits and calculating 5 salt tolerance-related indices (RGP, RGR, RRL, RBL, RGI) using principal component analysis and membership function method. Among them, there are 17 highly salt-tolerant millet materials (CC199, CC059, CC050, CC383, CC577, CC063, CC136, CC139, CC197, CC182, CC225, CC354, CC554, CC151, CC168, CC106, and CC183), 37 salt-tolerant materials, 223 moderately salt-tolerant materials, 36 salt-sensitive materials, and 20 highly salt-sensitive materials (CC582, CC563, CC580, CC146, CC124, CC001, CC326, CC153, CC007, CC238, CC540, CC537, CC535, CC568, CC376, CC154, CC005, CC583, CC571, and CC161). The results of stepwise regression analysis showed that by measuring the five key indicators of relative germination potential, relative germination rate, relative root length, relative bud length and relative germination index during the germination period of millet, the regression equation was established to estimate the D value, which can preliminarily evaluate the salt tolerance of millet germplasm resources during the germination period. An efficient and reliable method for evaluating the salt tolerance of millet during the germination period was established. This method is not only simple and easy to implement, but also has high accuracy and reliability, which provides strong support for the study of the salt tolerance mechanism of millet and the breeding of high salt tolerance varieties.

[0203] These millet germplasms are important resources and are of great significance to salt-tolerant breeding and the study of salt-tolerant mechanisms. Millet salt tolerance is a complex quantitative trait that is controlled by multiple genes and requires comprehensive consideration of the influence of genetic and environmental factors. In current research, in order to overcome the difficulty of salt-tolerant environments, it is still necessary to screen and collect salt-tolerant germplasm resources and explore salt-tolerant genes to achieve the purpose of molecular breeding. Therefore, screening millet salt-tolerant varieties is of great significance to the development and utilization of saline-alkali land and the identification of key genes related to salt stress, thereby promoting agricultural production and environmental improvement.

[0204] In the existing technology, the salt tolerance of millet during the germination period is evaluated with relative germination potential, relative germination rate, relative root length and relative bud length under salt stress as indicators, but the contribution values ​​of the four indicators are not clear. The present invention measures 333 millet samples for five indicators, namely, relative germination potential, relative germination rate, relative bud length, relative root length and relative germination index during the germination of millet, performs correlation analysis and principal component analysis on the five indicators, determines the contribution of each indicator to the salt tolerance of millet during the germination period, establishes a regression equation, determines the contribution value of each indicator, and provides a clear evaluation model for the identification of salt tolerance during the germination period of millet. The relative germination potential, relative germination rate, relative root length, relative bud length and relative germination index of millet are measured during the germination period, and the D value is estimated by the regression equation, so that the salt tolerance of germplasm during the germination period can be more accurately judged.

[0205] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. A method for evaluating the preliminary salt tolerance of millet during the germination period, characterized in that: The method is to determine five key indicators, namely, relative germination potential, relative germination rate, relative root length, relative bud length, and relative germination index, during the germination period of millet, and establish a regression equation to estimate the D value, so as to preliminarily evaluate the salt tolerance of millet germplasm resources during the germination period; Wherein, the regression equation is: y=0.204X1+0.205X2+1.329X3+0.473X4+0.283X5-0.222(R 2 =1, P<0.01), where X1, X2, X3, X4, and X5 represent relative germination potential, relative germination rate, relative root length, relative shoot length, and relative germination index, respectively, and -0.222 is the constant of the random error term; The dependent variable y is the D value.

2. The evaluation method according to claim 1, characterized in that: The estimated accuracy of the regression equation is greater than 99%.

3. The evaluation method according to claim 1, characterized in that: According to the D value, the average value of D The salt tolerance of millet germplasm was classified by the standard deviation SD, and its salt tolerance was divided into 5 levels: (1) That is, D ≥ 0.7324 is a highly salt-tolerant variety HST; (2) That is, 0.7324>D≥0.6351 is the salt-tolerant variety ST; (3) That is, 0.6351>D≥0.3309 is the medium salt-tolerant variety MST; (4) That is, 0.3309>D≥0.2336, which is a salt-sensitive variety SS; (5) That is, D < 0.2336 is a highly salt-sensitive variety HSS.

4. The evaluation method according to any one of claims 1 to 3, characterized in that: The specific steps are as follows: (1) Determination of salt concentration The concentration of 170mmol / L was selected as the treatment concentration for the salt tolerance identification of millet during the germination period; Select salt-tolerant millet materials in the germination period, select 50 seeds of each variety with full grains and uniform size and place them in a disposable sterile culture dish covered with double-layer sterilized filter paper; conduct preliminary experiments to screen the optimal salt stress concentration, and first set the NaCl solution concentration to 0, 50, 100, 150, 200, 250, 300, and 350mmol / L, a total of 8 treatment concentrations, and then set the concentration between 150-200mmol / L and set a 10mmol / L interval to re-screen to determine the final salt stress concentration, and repeat each treatment three times; add 7mL of NaCl solution of different concentrations to each variety, and add an equal amount of distilled water to the control and place them evenly in the culture dish. Cultivate at 25±2℃, 70% humidity, and 16 / 8h day / night. Add sterile water on the 6th day to ensure that the roots are completely immersed in the salt solution. No additional salt solution is added during the cultivation process. Cultivate for 8 days in total, and measure the germination rate on the 8th day of cultivation; (2) Screening and identification of salt-tolerant millets Select salt-tolerant millet materials in the germination period, select each variety with full grains and uniform size, select a concentration of 170mmol / L as the stress concentration, add 7mL of NaCl solution of different concentrations to each variety, add an equal amount of distilled water to the control and place them evenly in a culture dish; culture at 25±2℃, 70% humidity, day / night 16 / 8h conditions, supplement with sterile water on the 6th day to ensure that the roots are completely immersed in the salt solution, no additional salt solution is added during the culture process, culture for 8 days in total, and determine the germination rate on the 8th day of culture; put the same variety of millet, i.e. red millet, in different batches as a reference, and repeat each treatment three times to check whether the experimental conditions are consistent between different batches; (3) Measurement items The germination standard is that the root length is at least as long as the seed and the sprout length is at least 1 / 2 of the seed length. The number of germinated seeds is recorded at the same time every 2, 4, 6, and 8 days. The germination potential was counted on the 4th day of culture, and the germination rate was counted on the 8th day. The phenotypic traits were measured 8 days after treatment. The root length and shoot length were measured with a ruler, and 10 samples were randomly selected from each culture dish to calculate the average value. The root length was measured by measuring the length from the junction of the hypocotyl and the radicle to the root tip with a ruler. The shoot length was measured by measuring the length from the cotyledon to the root tip of the plant with a ruler. The calculation formulas for germination potential, germination rate, relative germination rate, germination index, relative germination index, vitality index and relative vitality index are as follows: The following formulas are used to calculate the various trait indicators: 1) Germination potential (GP, %) = G4 / 50×100, where G4 is the number of germinated seeds on the fourth day; 2) germination rate (GR, %) = G8 / 50 × 100, G8 is the number of germinated seeds on the eighth day; 3) In the formula, Gt is the number of germinated seeds on the 2nd, 4th, 6th, and 8th days, and Dt is the corresponding germination days; 4) Relative germination potential (RGP, %) = treatment germination potential / control germination potential × 100; 5) Relative germination rate (RGR, %) = treatment germination rate / control germination rate × 100; 6) Relative germination index (RGI, %) = treatment germination index / control germination index × 100; 7) Relative root length (RRL, %) = treatment root length / control root length × 100; 8) Relative shoot length (RBL, %) = treatment shoot length / control shoot length × 100; 9) Coefficient of variation CV = (standard deviation SD / mean value) × 100; (4) Data statistics and analysis Microsoft Excel 2019 was used for basic data processing, and SPSS26.0 and Origin 2021 software were used to perform correlation analysis, principal component analysis, membership function analysis, cluster analysis, and normal distribution diagrams of related indicators; The membership function method was used to conduct a comprehensive evaluation of the salt tolerance of germplasm resources and calculate the membership function value. The calculation formula is as follows: In the formula, μ(X j ) represents the membership function value of the jth comprehensive index in a specific variety, X j represents the jth comprehensive index value in a specific germplasm; X max and X min Respectively represent the maximum and minimum values ​​of the jth comprehensive index of all varieties; each comprehensive index has a membership function value, and μ(X j ) is between 0 and 1; Then, according to the relative importance of each trait, a certain weight is assigned. The weight formula is as follows: Where W j Indicates the importance of the jth comprehensive index among all the measured indicators; P j is the correlation coefficient between the jth comprehensive index and the salt tolerance characteristics of each variety, representing the contribution rate of the jth comprehensive index of each variety; Finally, the membership function values ​​of each index are added together to obtain the comprehensive evaluation value of each variety, namely, the D value; In the formula, D represents the comprehensive evaluation value of salt tolerance of each variety under NaCl stress, X j represents the jth comprehensive index value in a specific germplasm, W j Indicates the importance of the jth comprehensive indicator among all the measured indicators; The salt tolerance of millet varieties can be determined according to the size of the D value; the larger the D value, the stronger the salt tolerance of the variety, and vice versa, the weaker the salt tolerance of the variety; (5) Salt tolerance evaluation The standard for millet salt tolerance was established based on previous studies (2024 Singh, 2012 Chen, 2020 Li). The D value distribution was comprehensively evaluated based on five salt tolerance-related traits. The average value of D The salt tolerance of millet germplasm was classified into 5 levels based on the salt tolerance and standard deviation SD, namely It is a highly salt-tolerant variety, HST; It is a salt-tolerant variety, ST; It is a medium salt-tolerant variety, MST; It is a salt-sensitive variety SS; It is a highly salt-sensitive variety, HSS; (6) Evaluation and prediction of salt tolerance during millet germination In order to predict the salt tolerance of millet during the germination period, SPSS26.0 was used to conduct multivariate stepwise regression analysis on the D value (dependent variable y) and five salt tolerance related indicators (independent variable X). According to the scores of the five indicators, four indicators (relative germination potential, relative root length, relative shoot length, and relative germination index) were selected, and a mathematical evaluation model for salt tolerance was established: y=0.204X1+0.205X2+1.329X3+0.473X4+0.283X5-0.222(R 2 =1, P<0.01), where X1, X2, X3, X4 and X5 represent relative germination potential, relative germination rate, relative root length, relative shoot length and relative germination index, respectively, and -0.222 is the constant of the random error term; the comprehensive score and regression value were tested and found that their estimation accuracy was greater than 99%, indicating that the equation was accurate and effective in predicting the salt tolerance of millet germplasm. Therefore, the relative germination potential, relative germination rate, relative root length, relative shoot length and relative germination index of millet were measured during the germination period, and the D value was estimated through the regression equation, which can preliminarily judge the salt tolerance of the germplasm.

5. Use of the evaluation method according to any one of claims 1 to 4 in the identification of salt tolerance of millet during the germination period.

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