Soybean high temperature tolerance evaluation method and application thereof

Abstract

The present application relates to a kind of soybean high temperature tolerance evaluation method and its application, belong to agricultural breeding technical field.The method includes: in summer natural high temperature environment, to the soybean variety to be tested is staged sowing;At full flowering stage, according to the set real-time temperature and effective accumulated temperature threshold, determine normal temperature control and sampling condition of high temperature treatment;Using TTC staining method, pollen is dyed, and according to specific color and transparency standard, pollen vigor is quantitatively divided into four grades;Finally, through the mathematical model established, four normalization indexes of normal temperature control score, high temperature treatment score, heat retention rate and pollen total amount under high temperature are integrated, and comprehensive high temperature tolerance index is obtained by weighted calculation, so that the quantitative evaluation and comparison of soybean germplasm high temperature tolerance ability is realized.The method can be used for quickly and accurately identifying and screening high temperature resistant soybean germplasm resources, and serving soybean high temperature tolerance breeding.

Description

Technical Field

[0001] This invention belongs to the field of agricultural breeding technology, specifically relating to a method for evaluating the high-temperature resistance characteristics of soybeans and its application. Background Technology

[0002] Soybeans are an important dual-purpose crop for grain, oil, and feed, and are the primary source of edible plant protein. High temperatures negatively impact soybean growth, leading to reduced yields. Compared to vegetative growth, reproductive growth is more susceptible to high-temperature stress. Sustained high-temperature stress can cause abortion of seed-setting organs, malformation of male and female gametophytes, fruit deformities, and smaller grain size, severely affecting crop yield and quality. Studies have shown that high-temperature stress during the reproductive growth stage can reduce crop fertility by more than 80%. High-temperature stress also affects anther development and pollen viability. Compared to the control treatment, rice pollen viability decreased by 78.8% and seed setting rate decreased by 48.5% after 10 days of high-temperature treatment. Soybeans are summer dryland crops that prefer warm temperatures but are intolerant of high temperatures. High temperatures during flowering will cause soybeans to drop flowers and reduce pollen viability, thus affecting grain formation. During flowering, high temperatures significantly affect pollen grains and pollen tubes, often leading to abnormal pollen development, reduced pollen viability, pollen abortion, and slowed stigma development, ultimately resulting in decreased soybean fertility and flower and pod drop. High-temperature treatment at 44.1±1.47℃ for 7 days significantly reduced soybean pollen germination rate and the number of pods per plant by 45.49% and 27.10%, respectively. High-temperature treatment at full bloom (45℃) for 8 days reduced the number of pods from flowers by 36.57% and the pod formation rate by 0.90%. Therefore, breeding heat-resistant soybean varieties can help solve the key problem of high-temperature stress affecting soybean production and promote the improvement of soybean production capacity in my country.

[0003] In conclusion, there is an urgent need for a simple, efficient, standardized, and repeatable method for identifying the high-temperature resistance of soybeans. Summary of the Invention

[0004] The purpose of this invention is to address the deficiencies in the prior art by providing a method for evaluating the high-temperature resistance of soybeans and its application.

[0005] To achieve the above objectives, the technical solution of the present invention is: a method for evaluating the high-temperature resistance characteristics of soybeans and its application, comprising:

[0006] The soybean varieties to be evaluated were sown in stages under natural high-temperature conditions.

[0007] During the full bloom period of soybean varieties, sampling conditions for the normal temperature control group and the high temperature treatment group were determined based on the real-time temperature at the time of sampling, the effective accumulated temperature in the 5 days prior to sampling, and the effective accumulated temperature in the 10 days prior to sampling. The conditions for the normal temperature control group were: the actual sampling temperature was below 33℃, the effective accumulated temperature in the 5 days prior to sampling was below 118℃·d, and the effective accumulated temperature in the 10 days prior to sampling was below 226℃·d. The conditions for the high temperature treatment group were: the actual sampling temperature was above 45℃, the effective accumulated temperature in the 5 days prior to sampling was above 120℃·d, and the effective accumulated temperature in the 10 days prior to sampling was above 230℃·d.

[0008] Pollen samples were collected from the room temperature control group and the high temperature treatment group, respectively. After staining with TTC staining method, the pollen viability was divided into four levels according to the degree of staining and transparency: colorless and 100% transparent pollen was non-viable, light purple and 50% transparent pollen was low viability, dark purple and 0% transparent pollen was medium viability, and purplish-black and 0% transparent pollen was high viability.

[0009] The following calculations were performed on the room temperature control group and the high temperature treatment group to obtain the comprehensive high temperature resistance index for each variety to be evaluated:

[0010] (1) Calculate pollen viability score: for varieties In processing The One repetition, The groups were a room-temperature control group and a high-temperature treatment group, with the number of pollen grains at four different viability levels set as follows: ,in These correspond to pollen types with low viability, medium viability, and high viability, respectively; calculate the total number of pollen. and the proportion of pollen at each vitality level ; Calculate the linearly weighted raw vitality score for single-repetition analysis. The weight vector The original scores were linearly mapped to 0-100 to obtain the single-repetition vitality score. The denominator is 4 because ;

[0011] (2) Calculate the weighted mean of variety × treatment: Calculate the variety In processing Weighted mean of pollen viability score ,in and The scores of the room temperature control group and the high temperature treatment group are weighted averages, respectively; the total pollen count under high temperature treatment HT_total is also calculated. ;

[0012] (3) Calculate the heat retention rate: ,in It is a very small constant;

[0013] (4) Calculate the comprehensive heat tolerance index of pollen viability: for , Min-Max normalization was performed separately to obtain HT_score_s and DZ_score_s; First take the logarithm Then, Min-Max normalization is performed to obtain Ret_s; HT_total is first processed... After transformation, Min-Max normalization is performed to obtain HT_total_s; finally, the comprehensive heat tolerance index of pollen viability is calculated: .

[0014] Furthermore, the sowing period is from mid-May to early August, with one sowing period every 10 days, and all varieties are sown separately in the same plot.

[0015] Furthermore, the TTC staining method specifically involves: placing the anthers in TTC staining solution, staining at 35°C in the dark for 2.5 hours, stopping the staining by quick freezing with liquid nitrogen, and then observing under a microscope.

[0016] Furthermore, in (2), -6 .

[0017] Furthermore, in (4), when calculating the comprehensive high-temperature tolerance index of pollen viability, the weighting coefficients of each index are as follows: a is 0.45, b is 0.20, c is 0.25, and d is 0.10.

[0018] Furthermore, in (4), the numerical range of the obtained pollen heat resistance comprehensive value is used to classify the heat resistance level; the method of dividing the numerical range is as follows: several consecutive threshold intervals are set in sequence, and the heat resistance level is mapped to each threshold interval.

[0019] Furthermore, it also includes step S5: calculating the comprehensive heat tolerance index of pollen viability for each variety. The numerical value is used to rank and / or classify the high-temperature resistance of all varieties to be evaluated.

[0020] This invention also provides an application of a method for evaluating the high-temperature resistance of soybeans, applying the above method to soybean breeding.

[0021] Furthermore, soybean breeding focuses on heat-resistant soybeans.

[0022] The present invention also provides a computer-readable storage medium having stored thereon computer program instructions that can be executed by a processor, wherein when the processor executes the computer program instructions, it can implement the steps of the method described above.

[0023] Compared to existing technologies, this invention has the following advantages: This invention evaluates the heat resistance characteristics of soybeans by utilizing the activity of soybean pollen under natural high-temperature conditions. The method is convenient, more in line with actual soybean production, and provides important technical support for developing new high-yield, high-yield, and heat-resistant soybean varieties suitable for high-temperature environments. Attached Figure Description

[0024] Figure 1 Color calipers and transparency for pollen viability at various levels.

[0025] Figure 2 The distribution of pollen comprehensive scores between the control group and the high-temperature treatment group with different materials.

[0026] Figure 3 Comprehensive evaluation and clustering of pollen heat resistance for various materials

[0027] Figure 4 The results of pollen grain staining for heat-resistant and heat-sensitive varieties. Detailed Implementation

[0028] The technical solution of the present invention will now be described in detail with reference to the accompanying drawings.

[0029] It should be noted that the embodiments described below are illustrative, and the methods used, unless otherwise specified, are conventional methods intended to provide further explanation of this application. The materials, reagents, etc., used are commercially available unless otherwise specified. Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0030] It should be noted that the terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the exemplary implementations according to this application; as used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise; furthermore, it should be understood that when the terms “comprising” and / or “including” are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0031] This invention provides a method for evaluating the high-temperature resistance of soybeans and its application, including:

[0032] The soybean varieties to be evaluated were sown in stages under natural high-temperature conditions.

[0033] During the full bloom period of soybean varieties, sampling conditions for the normal temperature control group and the high temperature treatment group were determined based on the real-time temperature at the time of sampling, the effective accumulated temperature in the 5 days prior to sampling, and the effective accumulated temperature in the 10 days prior to sampling. The conditions for the normal temperature control group were: the actual sampling temperature was below 33℃, the effective accumulated temperature in the 5 days prior to sampling was below 118℃·d, and the effective accumulated temperature in the 10 days prior to sampling was below 226℃·d. The conditions for the high temperature treatment group were: the actual sampling temperature was above 45℃, the effective accumulated temperature in the 5 days prior to sampling was above 120℃·d, and the effective accumulated temperature in the 10 days prior to sampling was above 230℃·d.

[0034] Pollen samples were collected from the room temperature control group and the high temperature treatment group, respectively. After staining with TTC staining method, the pollen viability was divided into four levels according to the degree of staining and transparency: colorless and 100% transparent pollen was non-viable, light purple and 50% transparent pollen was low viability, dark purple and 0% transparent pollen was medium viability, and purplish-black and 0% transparent pollen was high viability.

[0035] The following calculations were performed on the room temperature control group and the high temperature treatment group to obtain the comprehensive high temperature resistance index for each variety to be evaluated:

[0036] (1) Calculate pollen viability score: for varieties In processing The One repetition, The groups were a room-temperature control group and a high-temperature treatment group, with the number of pollen grains at four different viability levels set as follows: ,in These correspond to pollen types with low viability, medium viability, and high viability, respectively; calculate the total number of pollen. and the proportion of pollen at each vitality level ; Calculate the linearly weighted raw vitality score for single-repetition analysis. The weight vector The original scores were linearly mapped to 0-100 to obtain the single-repetition vitality score. The denominator is 4 because ;

[0037] (2) Calculate the weighted mean of variety × treatment: Calculate the variety In processing Weighted mean of pollen viability score ,in and The scores of the room temperature control group and the high temperature treatment group are weighted averages, respectively; the total pollen count under high temperature treatment HT_total is also calculated. ;

[0038] (3) Calculate the heat retention rate: ,in It is a very small constant;

[0039] (4) Calculate the comprehensive heat tolerance index of pollen viability: for , Min-Max normalization was performed separately to obtain HT_score_s and DZ_score_s; First take the logarithm Then, Min-Max normalization is performed to obtain Ret_s; HT_total is first processed... After transformation, Min-Max normalization is performed to obtain HT_total_s; finally, the comprehensive heat tolerance index of pollen viability is calculated: .

[0040] The following is a detailed implementation process of the present invention.

[0041] In 2024, 32 soybean varieties from various ecological regions were selected, including Gan Dou 10 (Gan Shen Dou 20170001), Gan Dou 11 (Gan Shen Dou 20190001), Gan Dou 12 (Gan Shen Dou 20190002), Gan Dou 15 (Gan Shen Dou 20210002), Gan Dou 16 (Gan Shen Dou 20220001), Zhong Dou 63 (Guo Shen Dou 20233006), Zhong Dou 41 (Guo Shen Dou 2013014), Zhong Dou 57 (Guo Shen Dou 20210082), and Wan Dou 28 (Guo Shen Dou 28). The following soybean varieties have been approved: Dou 2008004, Xu Dou 16 (National Approved Soybean 2009020), Ji Dou 12 (National Approved Soybean 2001001), Heihe 45 (Heilongjiang Approved Soybean 2007013), Keshan No. 1 (National Approved Soybean 2009002), Zhonghuang 39 (National Approved Soybean 2013016), Youchun 1204 (National Approved Soybean 20170018), Xiangchun Dou 26 (National Approved Soybean 2008024), and Tianlong No. 1 (National Approved Soybean 2008023). Relevant information can be found on the China Seed Industry Big Data Platform.

[0042] Ganxia 2020-1, Jinggan 01-1, and Ganxia 2021-1 were the test materials for the 2022 Jiangxi Province summer soybean variety regional trials. Relevant information can be found in the 2022 Jiangxi Province Major Crop Variety Trial Implementation Plan. Ganxia 2206, Ganxia 2215, and Ganxia 2216 were the test materials for the 2024 Jiangxi Province summer soybean variety regional trials. Relevant information can be found in the 2024 Jiangxi Province Major Crop Variety Trial Implementation Plan. Ganxia 2204, Ganxia 2221, and Ganxia 2213 are new soybean lines created by the Jiangxi Academy of Agricultural Sciences.

[0043] Brazil 11 is a superior soybean germplasm from abroad. Zheng 59 (ZDD24052), Youxian Brown Bean (ZDD14586), Xuzhuang Black Bean (ZDD08257), and Baoshan Soybean (ZDD17593) are soybean resources collected and preserved by the National Crop Germplasm Bank. Relevant information can be found on the China Crop Germplasm Information Network.

[0044] Example 1: Establishment of a comprehensive evaluation method for the high-temperature tolerance of soybean pollen:

[0045] This invention assesses heat tolerance under natural high-temperature conditions. The test site is located in a region with high summer temperatures. Sowing is carried out every 10 days from May to August, with rows 2 meters long, row spacing 0.4 meters, and plant spacing 10 centimeters. Based on the actual field temperature, the highest temperature and effective accumulated temperature of the sampling day, the five days prior, and the ten days prior are monitored / measured. After confirming significant differences in sampling temperatures, further experiments are conducted. Temperature statistics and control / high-temperature confirmation methods are as follows:

[0046] ① Record the real-time field temperature value every hour, and statistically analyze the highest temperature and effective accumulated temperature of the sampling / measurement index on the day, the previous five days and the previous ten days. The effective accumulated temperature is ∑(daily average temperature − reference temperature), and the reference temperature is 10℃.

[0047] ② Select a specific temperature range as the control / high-temperature treatment condition based on the measurement indicators;

[0048] ③ The pollen viability test control / high-temperature treatment temperature ranges are as follows: The actual sampling temperature, accumulated temperature over the 5 days and 10 days prior to sampling were statistically analyzed. When the actual sampling temperature was below 33℃, the accumulated temperature over the 5 days prior to sampling was below 118℃·d, and the accumulated temperature over the 10 days prior to sampling was below 226℃·d, it was considered the normal temperature control group. When the actual sampling temperature was above 45℃, the accumulated temperature over the 5 days prior to sampling was above 120℃·d, and the accumulated temperature over the 10 days prior to sampling was above 230℃·d, it was considered the high-temperature group.

[0049] Pollen viability test: During the peak flowering period, use tweezers to collect 8 flowers of different varieties that are about to open (showing white but not yet open). Carefully remove the anthers from the freshly collected flowers with tweezers, place them in TTC staining solution, and stain at 35°C in the dark for 2.5 hours. Stop the staining by quick freezing with liquid nitrogen.

[0050] ④ Before microscopic examination, thaw the frozen tubes slowly in ice. After complete thawing, gently grind the anthers in the TTC staining solution in the centrifuge tube using a pipette tip. After thorough grinding, gently pipette 10-15 times to mix the solution. Take 10 μL of the supernatant and drop it onto the center of a glass slide. Observe using an optical microscope, using the same magnification and field of view, repeating at least 4 times. Differentiate and count the number of pollen grains in four different viability states (strong viability, moderate viability, weak viability, and no viability) based on color range and transparency, and calculate the percentage of each viability pollen grain in the total number of pollen grains in the field of view.

[0051] ⑤ Using the R packages ggplot2, ape, and data.table, we calculated and analyzed the total pollen count and the percentage of each type of active pollen in the control and high-temperature treatment groups. Further, we calculated the overall high-temperature resistance performance of each material through weighted analysis, Min-Max normalization analysis, and comprehensive high-temperature resistance index analysis. The specific calculation methods are as follows:

[0052] For varieties In processing The There are 1 repetition, and the counts for the four vitality levels (class 0-3) are respectively... The total number of pollen is The corresponding ratio is Further calculate the raw vitality score of single-repetition linear weighted average. The weight vector To facilitate later comparisons, the scores were linearly mapped to 0-100 to obtain the vitality score. (The denominator is 4 because) Subsequently, at the variety × treatment level, the results of multiple replicates were weighted and aggregated by pollen count. ,in and These are the weighted average pollen activity scores under the room temperature control and high temperature treatment, respectively; DZ_total and HT_total were also obtained (respectively...). and the weighted average of the highest vitality level proportion. And record the number of valid replicates. Heat retention rate is defined as... ,in -6 (To avoid abnormal results due to some control groups having a value of 0); to construct a comprehensive heat tolerance index for pollen viability, [the following was observed]: , Min–Max normalization is performed separately to obtain HT_score_s and DZ_score_s, and the logarithm is taken first for each. Then, normalization was performed to obtain Ret_s; the total pollen count under high-temperature treatment was first... After transformation and normalization, HT_total_s is obtained. Finally, HT_score_s (weighted value 0.45), DZ_score_s (weighted value 0.20), Ret_s (weighted value 0.25), and HT_total_s (weighted value 0.10) are weighted, and the final comprehensive high temperature resistance index (T) score for each material is obtained and ranked according to the results. .

[0053] Step 5: Use the Euclidean distance method to perform cluster analysis on the obtained T values, and classify the high temperature resistance into levels based on the cluster analysis results.

[0054] Step 6: Evaluation and application of soybean high-temperature resistance characteristics based on pollen viability. The high-temperature resistance level is divided into levels by the range of the comprehensive value (T) of pollen high-temperature resistance obtained in step 5. The method of dividing the range is as follows: several consecutive threshold intervals are set sequentially, and the level of high-temperature resistance is mapped to each threshold interval.

[0055] Experimental Results and Analysis:

[0056] (1) Statistical analysis of pollen viability in the control group and the high-temperature treatment group

[0057] Pollen viability of 32 varieties under normal temperature control (DZ) and high temperature treatment (HT) conditions was summarized and compared (Table 1). Figure 1 , Figure 2 Overall, the pollen viability score (DZ_score) in the control group ranged from 16.89 to 59.16, with jzs116 having the lowest score of 16.89 and Gan Dou 15 having the highest score of 59.16. The pollen viability score (HT_score) in the high-temperature group ranged from 27.32 to 71.53, with jzs009 having the lowest score of 27.32 and Tianlong 1 having the highest score of 71.53. At the population level, the mean viability score in the control group was 34.84±9.97, and the mean viability score in the high-temperature group was 47.61±9.40, with an average change of 12.76±11.85 between high and normal temperatures. The effect of high-temperature treatment on pollen viability score was significant.

[0058] Of the 32 varieties, 26 showed increased scores under high temperatures (Table 1). Figure 2Among them, the vitality score of Tianlong 1 in the control group was 44.28, and the vitality score of the high-temperature group was 71.53; the vitality score of Zhonggan 601 in the control group was 42.43, and the vitality score of the high-temperature group was 62.80; the vitality score of Ganxia 2215 in the control group was 32.51, and the vitality score of the high-temperature group was 56.94; the vitality score of Ganxia 2216 in the control group was 32.37, and the vitality score of the high-temperature group was 56.43; the vitality score of Ganxia 2213 in the control group was 18.57, and the vitality score of the high-temperature group was 51.77. At the same time, the scores of 6 varieties decreased or remained almost unchanged under high temperature, showing relative sensitivity. Among them, the vitality score of Gandou 15 in the control group was 59.16, and the vitality score of the high-temperature group was 35.82, with a heat resistance retention rate of 0.61; the vitality score of jzs096 in the control group was 39.22, and the vitality score of the high-temperature group was 33.92; and the vitality score of jzs009 in the control group was 29.76, and the vitality score of the high-temperature group was 27.32.

[0059] Regarding the proportion of highly viable pollen, the proportion of highly viable pollen in the control group was 6.00%±7.20%, while that in the high-temperature group was 12.50%±6.90%, an average increase of 6.50%. Among them, the proportion of highly viable pollen in the control group of Zhonggan 601 was 3.96%, while that in the high-temperature group was 33.50%, showing the most significant increase. The proportion of highly viable pollen in the control group of Youchun 1204 was 0%, while that in the high-temperature group was 22.35%. Conversely, the proportion of highly viable pollen in the control group of Gandou 15 was 26.06%, while that in the high-temperature group was 3.61%, indicating that the proportion of highly viable pollen decreased significantly under high temperatures.

[0060] Regarding the total pollen count (Table 1), the total pollen count in the control group was 55,152, while the total pollen count in the high-temperature group was 45,523, approximately 82.5% of the control group. This indicates that the overall amount of statistically significant pollen under high temperatures is lower, but there are significant differences between varieties. Specifically, the total pollen count of Jingyoudou No. 1 in the high-temperature group was 3,899, higher than the 1,289 in its control group. In summary, Tianlong No. 1, Zhonggan 601, Ganxia 2215, and Ganxia 2216 exhibit outstanding high-temperature tolerance, while Gandou No. 15, JZS096, and JZS009 show poor pollen performance in high-temperature environments.

[0061] Table 1. Comprehensive characterization of pollen quantity and activity in the control group and high-temperature treatment group of 32 materials.

[0062]

[0063] (2) Weighted comprehensive evaluation of soybean heat resistance by different indicators

[0064] By normalizing the pollen activity score, total pollen count, and heat resistance retention rate of the control group and the high-temperature group, and further using weighted calculation of each index, the comprehensive value of soybean heat resistance was obtained. Figure 3Based on the comprehensive evaluation value combined with Euclidean distance clustering, the high-temperature resistance level of the pollen in the identified materials was divided into four categories: Category I (extremely sensitive to high temperatures), comprehensive evaluation value < 0.25; Category II (relatively sensitive to high temperatures), 0.25 ≤ comprehensive evaluation value < 0.45; Category III (relatively resistant to high temperatures), 0.45 ≤ comprehensive evaluation value < 0.60; and Category IV (resistant to high temperatures), comprehensive evaluation value ≥ 0.60.

[0065] Example 2: Comprehensive evaluation method for high temperature resistance of soybeans

[0066] Twenty-two varietal resources were selected and sown in stages on May 10th and June 10th, 2025, with sowing requirements and methods consistent with those established in Example 1. Following the temperature range outlined in Example 1, samples were taken from the sown varietal resources, and pollen viability was tested. The selected varietal resources were further identified using the method established in Example 1. Eleven samples were identified as Class IV (heat-tolerant), eight as Class III (relatively heat-tolerant), two as Class II (relatively heat-sensitive), and one sample (Bedford, an overseas resource) as Class I (extremely heat-sensitive). Furthermore, ecological zoning revealed differences in heat tolerance among varietal resources from different ecological regions. Class IV varietal resources were mainly distributed in the Huang-Huai-Hai ecological region (54.55%), Class III resources were mainly from the Northeast ecological region and overseas resources (62.50%), both Class II varietal resources were from the Northeast ecological region, and the Class I Bedford variety was an American variety. Table 2 shows the comprehensive evaluation and grading of the high-temperature resistance of 22 soybean varieties. Figure 4 The pollen staining results were compared between two varieties with extreme differences in high-temperature response: 25GJ095 (high-temperature resistant) and 25GJ107 (high-temperature extremely sensitive).

[0067] Table 2. Comprehensive evaluation and grading of high-temperature resistance of 22 soybean varieties

[0068]

[0069] The above are preferred embodiments of the present invention. Any changes made to the technical solution of the present invention that do not exceed the scope of the technical solution of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for evaluating the high-temperature resistance of soybeans and its application, characterized in that, include: Using pollen viability as an indicator, the soybean varieties to be evaluated were sown in stages under natural high-temperature conditions; During the full bloom period of soybean varieties, a normal temperature control group and a high temperature treatment group were determined based on the real-time temperature at the time of sampling, the effective accumulated temperature in the 5 days prior to sampling, and the effective accumulated temperature in the 10 days prior to sampling. The conditions for the normal temperature control group were: the actual temperature at the time of sampling was below 33℃, the effective accumulated temperature in the 5 days prior to sampling was below 118℃·d, and the effective accumulated temperature in the 10 days prior to sampling was below 226℃·d. The conditions for the high temperature treatment group were: the actual temperature at the time of sampling was above 45℃, the effective accumulated temperature in the 5 days prior to sampling was above 120℃·d, and the effective accumulated temperature in the 10 days prior to sampling was above 230℃·d. Pollen samples were collected from the room temperature control group and the high temperature treatment group, respectively. The TTC staining method was used to stain the pollen. According to the degree of staining and transparency, the pollen viability was divided into four levels: colorless and 100% transparent pollen was inactive, light purple and 50% transparent pollen was low viability pollen, dark purple and 0% transparent pollen was medium viability pollen, and purplish-black and 0% transparent pollen was high viability pollen. The following calculations were performed on the room temperature control group and the high temperature treatment group to obtain the comprehensive high temperature resistance index for each variety to be evaluated: (1) Calculate pollen viability score: for varieties In processing The One repetition, The groups were a room-temperature control group and a high-temperature treatment group, with the number of pollen grains at four different viability levels set as follows: ,in These correspond to pollen types with low viability, medium viability, and high viability, respectively; calculate the total number of pollen. and the proportion of pollen at each vitality level ; Calculate the linearly weighted raw vitality score for single-repetition analysis. The weight vector The original scores were linearly mapped to 0-100 to obtain the single-repetition vitality score. The denominator is 4 because ; (2) Calculate the weighted mean of variety × treatment: Calculate the variety In processing Weighted mean of pollen viability score ,in and The scores of the room temperature control group and the high temperature treatment group are weighted averages, respectively; the total pollen count under high temperature treatment HT_total is also calculated. ; (3) Calculate the heat retention rate: ,in It is a very small constant; (4) Calculate the comprehensive heat tolerance index of pollen viability: for , Min-Max normalization was performed separately to obtain HT_score_s and DZ_score_s; First take the logarithm Then, Min-Max normalization is performed to obtain Ret_s; HT_total is first processed... After transformation, Min-Max normalization is performed to obtain HT_total_s; finally, the comprehensive heat tolerance index of pollen viability is calculated: .

2. The method for evaluating the high-temperature resistance of soybeans according to claim 1, characterized in that, The sowing period is from mid-May to early August.

3. The method for evaluating the high-temperature resistance of soybeans according to claim 1, characterized in that, The TTC staining method specifically involves placing the anthers in TTC staining solution, staining in the dark, stopping the staining process by quick freezing with liquid nitrogen, and then observing under a microscope.

4. The method for evaluating the high-temperature resistance of soybeans according to claim 1, characterized in that, In (2), -6 .

5. The method for evaluating the high-temperature resistance of soybeans according to claim 1, characterized in that, In (4), when calculating the comprehensive heat resistance index of pollen viability, the weighting coefficients of each index are: a = 0.45, b = 0.20, c = 0.25, and d = 0.

10.

6. The method for evaluating the high-temperature resistance of soybeans according to claim 1, characterized in that, (4) The numerical range of the obtained pollen heat resistance comprehensive value is used to classify the heat resistance level; the method of dividing the numerical range is as follows: several consecutive threshold intervals are set in sequence, and the heat resistance level is mapped to each threshold interval.

7. A method for evaluating the high-temperature resistance of soybeans according to any one of claims 1-6, characterized in that, It also includes step S5: calculating the comprehensive heat tolerance index of pollen viability for each variety. The numerical value is used to rank and / or classify the high-temperature resistance of all varieties to be evaluated.

8. An application of a method for evaluating the high-temperature resistance of soybeans, characterized in that, The application of the soybean heat resistance evaluation method as described in any one of claims 1-7 in soybean breeding and germplasm innovation.

9. A computer-readable storage medium having stored thereon computer program instructions executable by a processor, wherein when the processor executes the computer program instructions, it is able to implement the steps of the method as described in any one of claims 1-7.

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