Method for identifying drought resistance of medicago sativa in seedling stage

Abstract

The invention relates to the technical field of agricultural science, in particular to an alfalfa seedling stage drought resistance identification method which comprises the following steps: taking overground part fresh weight, overground part dry weight, root fresh weight and root dry weight of alfalfa seedlings as measurement indexes; respectively measuring the overground part fresh weight, the overground part dry weight, the root fresh weight and the root dry weight of the normal group and the drought stress treatment group; and determining a weight coefficient of each measurement index through a principal component analysis method, calculating a drought resistance measurement value by combining a membership function value, and identifying the drought resistance grade of the alfalfa in the seedling stage according to the D value. Scientific basis is provided for alfalfa drought-resistant variety breeding, germplasm resource screening and cultivation management, and sustainable development of the alfalfa industry in the arid region is promoted.

Description

Technical Field

[0001] The invention relates to the field of agricultural science and technology, and in particular to a method for identifying drought resistance of alfalfa at the seedling stage. Background Art

[0002] Alfalfa is an important forage crop in the world, and its yield and quality are significantly affected by drought stress. As the global climate becomes increasingly dry and warm, drought has become the main abiotic stress factor that restricts the expansion of alfalfa planting areas and the increase in yield. During the seedling stage, alfalfa is highly sensitive to water. Drought causes the seedlings to be short and yellow, grow slowly or even die, which in turn affects the subsequent growth cycle. Therefore, exploring technical indicators and methods for drought resistance identification of alfalfa at the seedling stage and establishing an accurate seedling drought resistance identification technology system are of great significance for screening drought-resistant germplasm resources and accelerating the process of drought-resistant breeding. However, the following problems still exist in the drought resistance identification technology at the seedling stage:

[0003] (1) Lack of unified standardized indicators and methods: A unified system for drought resistance evaluation at the seedling stage of alfalfa has not yet been established. The technical indicators and evaluation methods for drought resistance identification lack consistency, resulting in a lack of a definitive reference standard for the evaluation of resource materials or newly bred varieties. A rapid evaluation system for alfalfa at the seedling stage needs to be established, with clear key evaluation indicators and methods.

[0004] (2) Inconsistent methods for simulating drought: There is no unified standard for the duration and intensity of drought stress at the seedling stage. The drought stress intensities (such as soil moisture control and drought duration) used in different studies vary greatly, resulting in poor comparability of results and affecting the accuracy of identification.

[0005] (3) Evaluation indicators are not representative: Drought resistance identification at the seedling stage involves growth and development, phenotype, and physiological and biochemical indicators. The measurement technology of some indicators is complex and difficult to implement, and the identification cycle is long. It is not suitable for the rapid screening of large groups of materials, resulting in uneven evaluation results.

[0006] In summary, it is necessary to establish a unified technical system for drought resistance identification of alfalfa at the seedling stage to promote the sustainable development of the alfalfa industry in arid areas. Summary of the Invention

[0007] In order to solve the above problems, the present invention provides a method for identifying drought resistance of alfalfa at the seedling stage.

[0008] The present invention is achieved through the following technical solutions: A method for identifying drought resistance of alfalfa at the seedling stage comprises the following steps: The aboveground fresh weight, aboveground dry weight, root fresh weight and root dry weight of alfalfa seedlings were used as the determination indicators.

[0009] The aboveground fresh weight, aboveground dry weight, root fresh weight and root dry weight of alfalfa seedlings in the normal group and drought stress treatment group were measured respectively; the weight coefficient of each measurement index was determined by principal component analysis method, and then the drought resistance measurement value was calculated based on the membership function value, and the drought resistance grade of alfalfa seedlings was identified according to the drought resistance measurement value.

[0010] when D When the value is ≥0.58, it is identified as Class I strong drought-resistant germplasm; when 0.58> D The value is ≥0.42, which is identified as the second category with strong drought resistance. D The value is 0.42> D The value is ≥0.36, and it is identified as the moderate drought-resistant germplasm of category III; D The value is 0.36> D Value ≥ 0.24, identified as Class IV weak drought-resistant germplasm; D The value is 0.24> D The value was ≥0.14, and it was identified as drought-sensitive germplasm of category V.

[0011] Preferably, the calculation formula of the weight coefficient is: .

[0012] The calculation formula of the membership function value is: .

[0013] The calculation formula of the drought resistance metric is: .

[0014] in, j is the weight coefficient, C j It is j The contribution rate of each measurement indicator, μ(X j ) is the membership function value, X j It is j The measured index value, X min All samples in j The minimum value of the measurement index, X max All samples in j The maximum value of the measured index, D is a measure of drought resistance.

[0015] Preferably, the normal group is alfalfa seedlings of the test variety that are watered normally; the relative moisture content of the soil in the normal group is 70% to 80% of the maximum water holding capacity in the field.

[0016] In the drought stress treatment group, the alfalfa seedlings were watered for 9-11 days; the relative soil moisture content in the drought stress treatment group was reduced to 10%-15% of the field water holding capacity.

[0017] Preferably, the alfalfa test variety seedlings are alfalfa test variety seedlings that are 29 to 31 days old.

[0018] Preferably, the maximum field water holding capacity is 35% to 45% of the weight water content of the soil under saturated conditions; the field water holding capacity is 20% to 30% of the weight water content of the soil after drainage is stabilized.

[0019] Preferably, the method for determining the aboveground fresh weight, aboveground dry weight, root fresh weight and root dry weight is to separate the aboveground part and the root with scissors, weigh the aboveground fresh weight and the root fresh weight, then wither, dry to constant weight after withering, and weigh the aboveground dry weight and the root dry weight respectively.

[0020] Preferably, the temperature for fixing is 104°C to 106°C.

[0021] Preferably, the drying temperature is 79°C to 81°C.

[0022] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a method for identifying drought resistance of alfalfa at the seedling stage, comprising the following steps: using the aboveground fresh weight, aboveground dry weight, root fresh weight, and root dry weight of the alfalfa seedlings as measurement indicators; measuring the aboveground fresh weight, aboveground dry weight, root fresh weight, and root dry weight of a normal group and a drought stress treatment group; determining weight coefficients of the measurement indicators using a principal component analysis method, calculating drought resistance metrics based on membership function values, and identifying the drought resistance grade of the alfalfa at the seedling stage based on the drought resistance metrics. When the D value is ≥0.58, it is identified as a strong drought-resistant germplasm of Class I; when it is 0.58>D value ≥0.42, it is identified as a relatively strong drought-resistant germplasm of Class II; when the D value is 0.42>D value ≥0.36, it is identified as a moderate drought-resistant germplasm of Class III; when the D value is 0.36>D value ≥0.24, it is identified as a weak drought-resistant germplasm of Class IV; when the D value is 0.24>D value ≥0.14, it is identified as a drought-sensitive germplasm of Class V.

[0023] Compared with the existing technology, the present invention has achieved significant breakthroughs in scientificity, practicality and operability by establishing a standardized and efficient technical system for drought resistance identification of alfalfa seedlings. This technology focuses precisely on the critical growth stage of alfalfa seedlings and adopts a water control method to simulate field drought conditions (soil moisture content is controlled at 10% to 15% of field water holding capacity). Compared with the traditional repeated drought method, it can reduce more than 50% of the operating steps, greatly improve the identification efficiency, and is particularly suitable for large-scale germplasm resource screening. Based on the growth characteristics of alfalfa seedlings, the present invention innovatively screens out four core evaluation indicators: aboveground fresh weight, aboveground dry weight, root fresh weight and root dry weight, avoiding the errors caused by broad indicators and significantly improving the accuracy of identification. By establishing a standardized process from seedling cultivation, stress treatment to data collection and statistical analysis, it not only fills the gap in the technology for drought resistance identification of alfalfa seedlings, but also ensures the repeatability and comparability of the experimental results. In terms of the evaluation system, a multidimensional analysis method from single drought resistance coefficient (DC) to comprehensive drought resistance coefficient (CDC) was adopted, combined with membership function analysis and D By clustering the values ​​and introducing principal component analysis and linear regression, an objective classification of drought resistance levels (strong drought resistance, relatively strong drought resistance, moderate drought resistance, weak drought resistance, and drought sensitivity) was achieved. This technical system is both scientific and practical. It accurately simulates field drought conditions through water control methods and reduces human error through standardized operations, significantly improving the consistency of results. It provides reliable technical support for alfalfa drought-resistant breeding, cultivation management in arid areas, and scientific research and teaching, and is of great significance to promoting the sustainable development of the alfalfa industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0025] Figure 1 This is a correlation diagram of the six measurement indicators of the present invention.

[0026] Figure 2 This is the cluster analysis of the present invention. DETAILED DESCRIPTION

[0027] To facilitate understanding of the present invention, the present invention will be described more fully below, along with preferred embodiments of the present invention. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the disclosure of the present invention.

[0028] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0029] The beneficial effects of the present invention are described below through specific examples.

[0030] Example 1 1 Materials and Methods 1.1 Materials The experimental materials were 111 core alfalfa germplasm populations, provided by the forage germplasm resource creation, utilization and breeding team of Guyuan Branch of Ningxia Academy of Agricultural and Forestry Sciences. Specific information is shown in Table 1.

[0031] Table 1 Serial number and source of alfalfa materials 1.2 Test methods The experiment was conducted on June 20, 2024, in a drought-resistant shed at the Touying Experimental Base, Guyuan Branch, Ningxia Academy of Agricultural and Forestry Sciences. Drought resistance was evaluated using a seedling potting method. Plastic seedling pots (9 cm high, 8.5 cm bottom diameter, 10 cm mouth diameter) were used. Each pot was filled with 148 g of mixed sieved soil (1:1 substrate: soil ratio), and 30 seeds were sown. From seedling emergence until 30 days later, the seeds were watered regularly (at 8:30 AM) every 1 day to maintain a relative soil moisture content of 70%–80% of the maximum field water holding capacity. After seedlings reached full size, thinning was performed, leaving 10 healthy, evenly growing seedlings per pot. Drought stress treatment was applied after the seedlings reached 30 days old.

[0032] The experiment included two treatments: one with normal watering, the normal group (CK), and a drought stress treatment, in which watering was stopped 30 days after seedling emergence, resulting in drought stress. Each treatment was replicated three times. The normal group was watered daily at 8:30 AM to maintain relative soil moisture at 70%-80% of the maximum field capacity. The drought stress group was not watered. After 10 days of stress, when the soil moisture content remained at 10%-15% of the field capacity, all treatments were measured. Relative soil moisture content was controlled gravimetrically. Maximum field capacity is defined as the weighted soil moisture content of 35%-45% at saturation; field capacity is defined as the weighted soil moisture content of 20%-30% after drainage stabilizes.

[0033] 1.3 Measurement indicators and methods Six phenotypic indices were measured: plant height (PH), shoot fresh weight (SFW), shoot dry weight (SDW), main root length (MRL), root fresh weight (RFW), and root dry weight (RDW). Three seedlings were randomly selected from each pot. Aboveground plant height and root length were measured using a ruler. The shoots and roots were then separated using scissors. The shoot fresh weight and root fresh weight were weighed. Each seedling was then oven-dried at 105°C for sterilization and then dried at 80°C to constant weight. The shoot dry weight and root dry weight were then measured using a balance.

[0034] 1.4 Data Analysis The individual drought resistance coefficient (DC) is the relative value of the measured index. The relative value of the measured index = the drought stress treatment value of the measured index / the normal treatment value. The relative values ​​of the six measured indexes are relative plant height (RPH), relative shoot freshweight (RSFW), relative shoot dry weight (RSDW), relative main root length (RMRL), relative root fresh weight (RRFW), and relative root dry weight (RRDW). The comprehensive drought resistance coefficient (CDC) is the sum of the individual drought resistance coefficients of all measured indexes / the number of measured indexes. The Origin The descriptive statistics and correlation analysis of the individual drought resistance coefficients of the measured indicators were performed using SPSS 2023 software. The principal component analysis and linear stepwise regression analysis were performed using SPSS 25.0. The weight coefficients of the various measured indicators were determined by the principal component analysis method, and the drought resistance measurement value was calculated by combining the membership function value ( D value), and finally the CDC value and D The drought resistance of each material was determined by two methods, CDC value and D The larger the value, the stronger the drought resistance. D The value was considered reliable because a weight coefficient was introduced into the analysis. D The materials were clustered and analyzed based on their values ​​to clarify the drought resistance of each material.

[0035] The specific calculation formula is as follows: (1) (2) (3) (4) (5) (6) Among them, DC is the single drought resistance coefficient, T j are the measured values ​​of each determination index in the drought stress treatment group; CK j is the measured value of each indicator in the normal group, j is the weight coefficient, C j It is j The contribution rate of each measurement indicator, μ(X j ) is the membership function value, X j It is j The measured index value, X min All samples in j The minimum value of the measurement index, X max All samples in j The maximum value of the measured index, D is a measure of drought resistance.

[0036] 2 Results and Analysis 2.1 Effects of drought stress on measured indicators Table 2 shows that the individual drought resistance coefficients for the six parameters measured under water stress at the seedling stage for 111 alfalfa accessions were <1. The data also varied widely, indicating that drought stress at the seedling stage inhibited all alfalfa parameters, but the magnitude of the effects varied. Taproot length was the most tolerant to drought stress, while aboveground fresh weight was the most inhibited. Its individual drought resistance coefficient also had a high coefficient of variation (64.26%), indicating it was the most sensitive to drought stress and could serve as an important screening parameter for drought resistance at the seedling stage. Root fresh weight ranked second in terms of inhibition, and its individual drought resistance coefficient had the highest coefficient of variation (78.60%), indicating that aboveground fresh weight and root fresh weight could also serve as important screening parameters for drought resistance at the seedling stage.

[0037] Table 2 Descriptive statistics of DC values ​​of measurement indicators 2.2 Correlation Analysis There is an extremely significant positive correlation between plant height and taproot length, root fresh weight and root dry weight; there is an extremely significant positive correlation between aboveground fresh weight and aboveground dry weight, root fresh weight and root dry weight; there is an extremely significant positive correlation between aboveground dry weight and root fresh weight and root dry weight; there is an extremely significant positive correlation between taproot length and root fresh weight, and there is an extremely significant positive correlation between root fresh weight and root dry weight. There is a significant positive correlation between plant height and aboveground fresh weight, and between taproot length and root dry weight, as shown in Table 3 and Figure 1 The above correlation analysis of various alfalfa measurement indicators showed that there was an obvious correlation between the various measurement indicators, indicating that alfalfa has a complex drought resistance mechanism in the seedling stage and its drought resistance is polymorphic, which cannot be evaluated by a single measurement indicator. Therefore, it is necessary to analyze and study its comprehensive measurement indicators.

[0038] Table 3 Correlation analysis of various measurement indicators Note: “ / ” indicates that this item does not exist; *: indicates that it is significant at the 0.05 level. p ≤0.05; **: indicates significant at the 0.01 level, p ≤0.01.

[0039] 2.3 Principal Component Analysis By using KMO and Bartlett's sphericity test to perform statistical analysis on the individual drought resistance coefficients of the six determination indices, as shown in Table 4, the metric value of the KMO test is 0.785, and the significance of the Bartlett's sphericity test is 0.000. The above results reflect that the individual drought resistance coefficients of the six determination indices of the 111 alfalfa core germplasm populations are suitable for principal component analysis. The principal component analysis results show that, according to the principle that the contribution rate is greater than 80%, the present invention can extract four principal components, as shown in Table 5, and the contribution rates of these four principal components are 46.374%, 19.449%, 11.901% and 9.161% respectively, and the cumulative contribution rate is 86.885%. That is, these six determination indices are converted into four new comprehensive drought resistance determination indices. According to the differences in loadings, the measurement indicators with higher loadings for the first principal component were root fresh weight, root dry weight, aboveground fresh weight, and aboveground dry weight; the measurement indicators with higher loadings for the second principal component were plant height and taproot length; the measurement indicators with higher loadings for the third principal component were taproot length and aboveground dry weight; and the measurement indicators with higher loadings for the fourth principal component were aboveground fresh weight and taproot length.

[0040] Table 4 KMO and Bartlett's test of sphericity Table 5 Eigenvectors and contribution values ​​of six parameters of alfalfa core germplasm population at seedling stage 2.4 Comprehensive appraisal and evaluation Using CDC value and D The drought resistance of 111 alfalfa core germplasm populations at the seedling stage was evaluated by CDC values. The results showed that the CDC values ​​of the 111 alfalfa core germplasm populations ranged from 0.2301 to 1.7251. D The values ​​range from 0.1424 to 0.6961, with average values ​​of 0.7117 and 0.3694 respectively. D The drought resistance of the core germplasm groups was ranked by the CDC value, as shown in Table 6. It was found that 9 of the core germplasm groups ranked in the top 10 in drought resistance using the two comprehensive evaluation methods appeared in the evaluation of the two methods, which shows that the drought resistance of the core germplasm groups ranked in the top 10 using the CDC value, D The values ​​used to evaluate drought resistance of alfalfa at the seedling stage have certain reliability, but when using D In the drought resistance evaluation, the weight coefficient can scientifically quantify the actual contribution of each measured index of alfalfa, which can improve the objectivity and accuracy of the evaluation. It is recommended that this method can be used to evaluate the drought resistance of alfalfa germplasm resources at the seedling stage.

[0041] Table 6 CDC values ​​and D The top 10 alfalfa core germplasm populations 2.5 Cluster Analysis according to D Cluster analysis was performed on 111 alfalfa germplasm resources, such as Figure 2 As shown, the materials can be divided into 5 categories (Ⅰ-Ⅴ), of which Category Ⅰ has 6 materials. D The values ​​range from 0.5780 to 0.6961, accounting for 5.4% of the total, and are strong drought-resistant materials. There are 25 materials in Class II. D The values ​​range from 0.4212 to 0.5414, accounting for 22.5% of the total, which are strong drought-resistant materials. There are 28 materials in Class III. D The values ​​range from 0.3570 to 0.4124, accounting for 25.2% of the total, which are medium drought-resistant materials. There are 47 materials in Class IV. D The values ​​range from 0.2422 to 0.3444, accounting for 42.3% of the total, which are weak drought-resistant materials. There are 5 materials in Class V. D The values ​​range from 0.1424 to 0.2243, accounting for 4.5% of the total, and are drought-sensitive materials.

[0042] 2.6 Screening of drought resistance identification indicators at the seedling stage The results in Table 7 show that the DC values ​​of the six test parameters of 111 alfalfa core germplasm populations at the seedling stage were D There is a very significant positive correlation between the values, and the DC values ​​of root dry weight and root fresh weight are D The correlation coefficients are 0.8231 and 0.8050 respectively. D The value was the dependent variable, and the drought resistance coefficients of the six determination indicators were the independent variables. A stepwise regression analysis was performed to construct a linear stepwise regression equation for drought resistance evaluation of alfalfa at the seedling stage: y=0.0501+0.0270x1+0.1096x2+0.0264x3+0.1643x4+0.0720x5+0.0352x6, where x1, x2, x3, x4, x5, and x6 represent the six drought resistance determination indicators of plant height, aboveground fresh weight, aboveground dry weight, root length, root fresh weight, and root dry weight, respectively. The determination coefficient of the equation is R 2 The F value is 5.738, and the equation has reached a very significant correlation level. These six independent variables can explain D The total variation of the values ​​reached 100%, indicating that this equation can be used to evaluate the drought resistance of the above 111 alfalfa core germplasm populations at the seedling stage, and the six drought resistance measurement indicators of plant height, aboveground fresh weight, aboveground dry weight, root length, root fresh weight, and root dry weight can be used as the main measurement indicators for the evaluation of alfalfa germplasm resources at the seedling stage.

[0043] Finally, the present invention further conducted a comprehensive analysis by using four methods, namely, single drought resistance coefficient, correlation, principal component and stepwise regression, and explored that the four indicators of aboveground fresh weight, aboveground dry weight, root fresh weight and root dry weight were ideal indicators for drought resistance identification of alfalfa seedlings.

[0044] Table 7 DC values ​​of drought resistance of various indicators and D Correlation analysis of values Note: **: indicates significant at the 0.01 level. p ≤0.01.

[0045] 4 Conclusion Drought stress has an inhibitory effect on all six parameters of alfalfa at the seedling stage. Aboveground fresh weight, aboveground dry weight, root fresh weight, and root dry weight are key parameters for evaluating drought resistance of large alfalfa populations at the seedling stage. D The drought resistance evaluation method is the most ideal method for evaluating the drought resistance of alfalfa germplasm resources at the seedling stage. DThe values ​​were divided into five categories, namely strong drought-resistant, relatively strong drought-resistant, moderate drought-resistant, weak drought-resistant and drought-sensitive. Among them, AG110, AG60, AG51, AG5, AG25 and AG49 germplasms were strong drought-resistant materials, AG87, AG6, AG67, AG89, AG9, AG109, AG95, AG29, AG77, AG21, AG42, AG33, AG36, AG13, AG96, AG90, AG41, AG79, AG92, AG8, AG38, The germplasms AG10, AG108, AG62 and AG11 are highly drought-resistant materials, and the germplasms AG76, AG44, AG85, AG63, AG68, AG81, AG20, AG102, AG26, AG1, AG43, AG84, AG2, AG28, AG75, AG98, AG12, AG107, AG101, AG22, AG71, AG111, AG80, AG78, AG24, AG18, AG103 and AG15 are moderately drought-resistant.

[0046] Example 2 To verify the sensitivity of the D value to extreme drought conditions, a gradient drought stress experiment was designed for a highly drought-resistant accession (AG60) and a drought-sensitive accession (AG15). The experiment used a potting method, setting a gradient of soil moisture content (80%, 50%, 30%, and 15%) to simulate a drought environment ranging from mild to extreme. Each treatment group included three biological replicates, with 10 seedlings of uniform growth retained in each pot. During the drought stress process, soil moisture content was monitored daily, and samples were taken after 7 days of each gradient stress to measure physiological and morphological indicators.

[0047] Physiological indicators were measured spectrophotometrically for leaf proline content (acid ninhydrin method), superoxide dismutase (SOD) activity (nitroblue tetrazolium photoreduction method), and malondialdehyde (MDA) content (thiobarbituric acid method). The results showed that when soil moisture content dropped to 15%, the proline content and SOD activity of the highly drought-resistant material AG60 were significantly higher than those of the sensitive material AG15 (e.g., proline accumulation increased by more than 2-fold and SOD activity increased by 50%). However, the MDA content (a product of membrane lipid peroxidation) was significantly lower than that of AG15 (e.g., a decrease of 30%-50%), indicating that it has stronger antioxidant capacity and cell membrane stability.

[0048] Morphological indicators include wilting severity scores (scaled from 0 to 5, with 0 indicating no wilting and 5 indicating complete death) and regreening rates (the percentage of new leaves emerging is counted after watering is resumed seven days after cessation of stress). AG60 is expected to have a wilting score of ≤2 at 15% water content, with a regreening rate exceeding 80%, while AG15 may reach wilting levels 4-5 and a regreening rate below 20%. Furthermore, dynamic monitoring revealed that drought resistance mechanisms (stomatal closure and root elongation) were activated in AG60 at 30% water content, while AG15 experienced irreversible damage at 50% water content.

[0049] This experiment directly verified the correlation between D value and drought resistance of materials by combining gradient stress with physiological and morphological responses. This demonstrated that D value can sensitively distinguish different drought-resistant materials and provide a reliable basis for selecting alfalfa varieties under extreme drought conditions.

[0050] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0051] The above-described embodiments merely represent several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the patent. A person skilled in the art would be able to make various modifications and improvements without departing from the spirit of the present invention, and these modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be based on the appended claims.

Claims

1. A method for identifying drought resistance of alfalfa at the seedling stage, characterized in that: The following steps are involved: The aboveground fresh weight, aboveground dry weight, root fresh weight and root dry weight of alfalfa seedlings were used as the determination indicators. The aboveground fresh weight, aboveground dry weight, root fresh weight, and root dry weight of alfalfa seedlings in the normal group and the drought stress treatment group were measured respectively. The weight coefficients of each measured index were determined by principal component analysis, and the drought resistance metric values ​​were calculated based on the membership function values. The drought resistance grade of alfalfa seedlings was identified based on the drought resistance metric values. when D When the value is ≥0.58, it is identified as Class I strong drought-resistant germplasm; when 0.58> D The value is ≥0.42, which is identified as the second category with strong drought resistance. D The value is 0.42> D The value is ≥0.36, and it is identified as the moderate drought-resistant germplasm of category III; D The value is 0.36> D Value ≥ 0.24, identified as Class IV weak drought-resistant germplasm; D The value is 0.24> D The value was ≥0.14, and it was identified as drought-sensitive germplasm of category V.

2. The method according to claim 1, characterized in that The calculation formula of the weight coefficient is: ; The calculation formula of the membership function value is: ; ; The calculation formula of the drought resistance metric is: ; in, j is the weight coefficient, C j It is j The contribution rate of each measurement indicator, μ(X j ) is the membership function value, X j It is j The measured index value, X min All samples in j The minimum value of the measurement index, X max All samples in j The maximum value of the measurement index, D is a measure of drought resistance.

3. The method according to claim 1, characterized in that The normal group consisted of alfalfa seedlings that were watered normally; the relative soil moisture content in the normal group was 70% to 80% of the maximum field water holding capacity; In the drought stress treatment group, the alfalfa seedlings were watered for 9-11 days; the relative soil moisture content in the drought stress treatment group was reduced to 10%-15% of the field water holding capacity.

4. The method according to claim 3, characterized in that The alfalfa test variety seedlings refer to alfalfa test variety seedlings that are 29 to 31 days old.

5. The method according to claim 3, characterized in that The maximum field water holding capacity is 35% to 45% of the weight water content of the soil under saturated conditions; the field water holding capacity is 20% to 30% of the weight water content of the soil after drainage is stabilized.

6. The method according to claim 1, wherein The aboveground fresh weight, aboveground dry weight, root fresh weight and root dry weight are determined by separating the aboveground part and the root with scissors, weighing the aboveground fresh weight and the root fresh weight, then withering the green leaves, drying the green leaves to a constant weight, and weighing the aboveground dry weight and the root dry weight respectively.

7. The method according to claim 6, characterized in that The temperature for fixing the greening is 104° C. to 106° C.

8. The method according to claim 6, characterized in that The drying temperature is 79°C to 81°C.

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