CN117837441A - Evaluation and identification method for heat resistance of alfalfa - Google Patents
Evaluation and identification method for heat resistance of alfalfa Download PDFInfo
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Abstract
The invention discloses an evaluation and identification method for heat resistance of alfalfa, and belongs to the technical field of plant science. Evaluating and identifying the heat resistance of the alfalfa by calculating the comprehensive evaluation value D' of the alfalfa; the comprehensive evaluation value D' is calculated by the following mathematical model formula: d' =0.719+0.276×upper fresh weight heat resistance coefficient+0.106×pod activity heat resistance coefficient-1.335×heat damage index; or D' =0.220+0.390×upper fresh weight heat resistance coefficient+0.076×pod activity heat resistance coefficient-0.752×heat damage index+ 0.522 ×recovery index; the heat resistance identification analysis model established by the invention provides an important technical reference for the identification and screening of alfalfa heat-resistant germplasm resources and the breeding of new varieties.
Description
Technical Field
The invention relates to the technical field of plant science, in particular to an evaluation and identification method for heat resistance of alfalfa.
Background
Global warming has become a major problem in crop yield reduction. However, because natural and human factors cause global warming, abnormal high-temperature weather in summer continuously occurs, and high-temperature stress negatively regulates the growth process of plants, including transpiration, photosynthesis, respiration, metabolism and the like of the plants, thereby inhibiting the growth of the plants and even causing death. It is expected that the global average air temperature will rise 7 ℃ at the end of this century. It has been reported that every 3-4 ℃ rise in temperature in africa, asia and the middle east results in 15-35% yield loss of crops, which accelerates grain safety due to population growth. Therefore, the screening of plant germplasm resources with higher heat resistance is of great significance to challenges brought by high-temperature climatic change.
The alfalfa (Medicago sativa L.) belongs to perennial high-quality pasture of alfalfa in the subfamily of Papilionaceae of Leguminosae, is native to Iran, is distributed around the world, is rich in crude protein, vitamins and various mineral elements, is known as 'pasture king', and has the characteristics of strong adaptability and stress resistance, and the like. Meanwhile, the root nodule has the effect of improving soil due to nitrogen fixation and the like. At present, the alfalfa has weak growth vigor, low yield and deteriorated quality in the middle and downstream areas of the Huai river in China due to high temperature and high humidity in summer and the like, so that the development of the alfalfa industry is greatly limited. The research shows that the optimal growth temperature of alfalfa is 20-25 ℃, and death phenomenon occurs in some alfalfa varieties when the summer temperature exceeds 30 ℃. However, different alfalfa varieties have significant differences in tolerance to high temperature stress during different growth periods. The alfalfa thermotolerant germplasm is also an important source of thermotolerant related genes and is also a donor for improving the thermotolerance of thermosensitive alfalfa varieties. At present, a number of thermotolerant genes from alfalfa have been identified. Therefore, it is necessary to establish an effective method for selecting and evaluating heat-resistant alfalfa varieties.
At present, high-temperature identification is carried out by manually simulating natural conditions to exclude interference of other environmental factors in the method for heat-resistant identification of crop germplasm resources. Furthermore, since stress resistance of crops is not determined by a single trait, it is a complex overall trait. Therefore, in recent years, the heat-resistant germplasm resource screening method is mostly based on a multi-index comprehensive evaluation method, and the multi-index comprehensive evaluation method comprises a principal component analysis method, a membership function method, a regression analysis method and other multi-element analysis methods. The alfalfa adopts a membership function method and a grading evaluation method to identify and screen heat-resistant alfalfa germplasm resources. It is difficult to determine the importance of each index in the identification of heat resistance of alfalfa by using the two methods. Therefore, a unified alfalfa heat resistance identification and evaluation standard has not been formed until now due to the numerous identification methods, detection indexes and analysis methods.
Disclosure of Invention
Aiming at the problems, the invention provides an evaluation and identification method for the heat resistance of alfalfa, which is used for evaluating the heat resistance of alfalfa.
The invention aims to provide an evaluation and identification method for heat resistance of alfalfa, which evaluates and identifies the heat resistance of the alfalfa by calculating the comprehensive evaluation value D' of the alfalfa; the comprehensive evaluation value D' is calculated by the following mathematical model formula:
d' =0.719+0.276×upper fresh weight heat resistance coefficient+0.106×pod activity heat resistance coefficient-1.335×heat damage index;
or D' =0.220+0.390×upper fresh weight heat resistance coefficient+0.076×pod activity heat resistance coefficient-0.752×heat damage index+ 0.522 ×recovery index;
the grading criteria for heat resistance are as follows:
| grade of heat resistance | Heat resistance grading standard | Comprehensive evaluation value D' |
| 1 | High sense | 0.0000-0.2000 |
| 2 | Sensitivity to | 0.2001-0.4000 |
| 3 | Zhongnai (Chinese character) | 0.4001-0.6000 |
| 4 | Heat resistant | 0.6001-0.8000 |
| 5 | High tolerance to | 0.8001-1.0000 |
The calculation formula of the heat damage index is as follows: heat damage index = Σ (heat damage number x number of plants of the stage)/(highest number x total number of plants);
the heat injury progression was obtained by the following heat injury symptom evaluation criteria:
the recovery index is calculated by the following formula: recovery index = Σ (recovery series×number of plants at this stage)/(highest series×total number of plants)
The number of recovery stages was obtained by the following recovery symptom evaluation criteria:
preferably, the heat damage progression is obtained according to the following steps:
step 1, dark culturing alfalfa seeds until germination, and transferring the alfalfa seeds into a seedling raising basin for culturing;
and 2, placing the alfalfa seedlings after 4 weeks of culture in an illumination incubator for heat stress treatment, positioning the alfalfa seedlings at the beginning of the 1 st day of heat stress treatment, observing the growth state of the alfalfa seedlings, obtaining the heat damage level according to the heat damage symptom evaluation standard, and carrying out heat damage index evaluation and analysis.
Preferably, in the step 1, alfalfa seeds with consistent sizes are selected, placed in a culture dish paved with two layers of filter paper, after dark culture for 2 days, germinated alfalfa with consistent growth vigor is transferred into a seedling raising square basin, the culture medium is soil, sand and organic fertilizer with the volume ratio of 2:1:1, and placed in a culture chamber, the culture temperature is 25 ℃, the light is 16h, the dark culture is carried out for 8h, each variety is treated with at least 3 biological repetitions, and each basin is 8-10 seedlings.
Preferably, in the step 2, the heat stress treatment condition is that the photoperiod is 16 hours at day and 8 hours at night, the temperature is 39-40 ℃ at day and 30-31 ℃ at night, and the illumination intensity is 4000lx.
Preferably, the recovery stage number is obtained according to the following steps:
and after heat treatment for 7-14 days, the alfalfa seedlings are recovered to grow for 6 days at normal temperature, the positioned alfalfa seedlings are observed every 3 days, the recovery growth state of the alfalfa seedlings is recorded, recovery grades are obtained according to recovery symptom evaluation standards, and recovery index evaluation and analysis are carried out.
Preferably, the above-ground fresh weight heat resistance coefficient and the POD activity heat resistance coefficient are obtained according to the following steps:
s1, after 6 days of growth recovery, determining the fresh weight of the overground part of alfalfa seedlings and POD activity;
s2, measuring the fresh weight of the overground part and the heat resistance coefficient of POD activity.
Preferably, heat resistance = heat stress treatment measurement/control treatment measurement.
Preferably, the caliber of the seedling raising basin is 10cm, the height of the seedling raising basin is 10cm, seedlings are watered every other day during heat stress treatment and recovery treatment, the water adding amount is 50-100ml each time, and heat damage symptom evaluation and recovery symptom evaluation are carried out before watering.
Compared with the prior art, the invention has the following beneficial effects:
(1) According to the invention, different alfalfa varieties are collected widely, high-temperature stress is simulated indoors in a seedling stage, morphological characters such as plant height, fresh weight of overground parts, dry weight of overground parts, relative chlorophyll content, thermal injury index and the like are investigated, physiological indexes such as superoxide dismutase (SOD) activity, peroxidase (POD) activity, catalase (CAT) activity, malondialdehyde (MDA) content and the like are measured, and a multielement analysis method is used for comprehensively evaluating the heat resistance characteristics of alfalfa seedlings, so that indexes suitable for alfalfa seedling stage heat resistance identification are screened, a heat resistance identification analysis model is established, a morphological-based grading evaluation method is established, and important technical references are provided for alfalfa heat-resistant germplasm resource identification and screening and new variety breeding.
(2) According to the invention, the heat damage grade and the recovery grade are evaluated, the heat damage phenomenon and the recovery phenomenon observed in different treatment time are summarized, and the heat stress is finished from light to heavy. In order to classify the heat injury symptoms and recovery symptoms in more detail, the heat injury symptoms and recovery symptoms of each stage comprise various phenomena, and the grading of the invention is more detailed than the prior other grading that each stage comprises only one phenomenon.
(3) Standard for health symptoms of the invention
a. There are definite standards for rating, including the change of leaf color and leaf morphology of different parts of plant;
b. in each grade, the invention not only describes the phenomenon of heat injury symptom position and recovery symptom position, but also specifically positions the plant occurrence position, thereby avoiding coincidence or contradiction with other grades;
c. the criteria for ranking are more detailed, the root neck, middle and upper part are described separately, and in the description of the blade changes, the changes in morphology and color are divided in more detail.
(4) For the health symptom standard, the influence of a single variable on an experiment is reduced, the single variable is changed into a plurality of variables, the error rate of heat damage rating and recovery rating is effectively reduced, and the error of an experiment result is greatly reduced; through multiple angles and overall descriptions, judgment confusion is reduced when judging the heat damage rating and the recovery rating of the plant.
Drawings
FIG. 1 is a graph showing morphological characteristics of 40 alfalfa varieties prior to the 1 st heat treatment;
FIG. 2 is a graph showing morphological characteristics of 40 alfalfa varieties after the 1 st heat treatment;
FIG. 3 is a morphological feature map of 40 alfalfa varieties after a week of heat stress recovery at 1 st;
FIG. 4 is a plot of heat stress morphological characteristics of lot 1, lot 2, of a 30 alfalfa variety;
FIG. 5 is a plot of heat stress morphological characteristics of lot 2, lot 2 of a 30 alfalfa variety;
FIG. 6 is a morphological characterization of 16 alfalfa varieties after a week of heat stress recovery at 3 rd time.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
1. The alfalfa for the parameter is of 50 varieties in total, WL358 (358), WL319 (319), WL343 (343), WL363 (363), WL440 (440), giant 801 (801), giant 995 (995), giant 701 (701), giant 601 (601), giant 201 (201), fingo (TG), wiston (WSD), reindeer (XL), inflite (IST), middi 7 (SD 7), middi 10 (SD 10), baRa420YQ (420 YQ), fuel warrior (RLS), mido 7 (SG 7), super energy (SS), smallpox (TM), queen (HH), gan Nong No. (GN 9), and combinations thereof Gan Nong No. 5 (GN 5), gan Nong No. 6 (GN 6), zhongjia No. 1 (ZM 1), extra-high (TH), zhongjia No. 2 (ZM 2), zhongjia No. 3 (ZM 3), ao Han (AH), polar bear (GIB), victoria (WDLY), tourist (YK), WL525 (525), WL903 (903), WL712 (712), WL656 (656), WL298 (298), WL168 (168), WL 366 (366), WL354 (354), bara310SC (310), algang Gold (AG), alla (AL), king gold (JHH), surprise (JX), alfalfa (MF), paola (PA), trideril (SDL).
The heat resistance screening test was performed three times in 2021, 9 to 11 months (1 st), 2022, 3 to 4 months (2 nd), and 2022, 9 to 11 months (3 rd), respectively.
2. Treatment method
1 st and 2 nd
And (3) material selection: selecting alfalfa seeds with consistent sizes, placing the alfalfa seeds in a culture dish paved with two layers of filter paper, culturing in dark for 2 days, and transferring the germinated alfalfa seeds with consistent growth vigor into a seedling culture square basin with the caliber of 10cm and the height of 10cm, wherein the culture medium is soil: sand: organic fertilizer=2:1:1 (v/v), placed in a culture room at 25 ℃, illuminated for 16h, and dark cultured for 8h. And (3) watering for about 3-4 days according to the dry and wet states of the seedlings, and watering at the morning and afternoon, wherein each hole is watered for 20 mL/time. 5 biological repeats (5 holes) are arranged for each variety of the plug tray, 5 seedlings are planted in each hole, and the plug tray is placed in a glass greenhouse for cultivation. The seedling raising trays are watered regularly and quantitatively every other day, the positions of the seedling raising trays are exchanged every other day to avoid the influence of different external illumination environments on experimental results, and the heat stress treatment is started after the continuous culture is carried out for 30 days. The heat stress treatment is carried out in an illumination intelligent incubator, and the photoperiod of the controllable conditions is set to be 16 hours in daytime and 8 hours at night; temperature: day 40 ℃, night 37 ℃; the illumination intensity is 4000lx. The growth status of alfalfa seedlings was recorded and rated daily from the heat treatment 1d, and the control group was still placed in the greenhouse for cultivation, heat treatment time 6d. The 2 nd time was the 1 st repetition (most varieties were repeated).
3 rd time
On the basis of the problems found in the 1 st and 2 nd experimental processes, a potting test is adopted, seeds with the same size are selected, the seeds are placed in a culture dish paved with two layers of filter paper, the seeds are catalyzed to sprout by using 8% sodium hypochlorite solution and ultrapure water, and dark culture is adopted, after 2d, the germinated alfalfa with the same growth vigor is respectively transferred into a seedling raising square box (3 pots, treatment groups) with the caliber of 10cm and the height of 10cm, wherein the culture medium is soil: sand: organic fertilizer = 2:1:1 (v/v). 6 pots of each variety, 10 plants of each pot, and watering for about 2-3 days according to the dry and wet states of seedlings, wherein each square box of the treatment group is watered for 50 mL/time. The positions of the seedling raising trays are moved and exchanged every 2 days, so that the influence of environmental factors on experiments is avoided. The culture temperature is 25 ℃, the light is irradiated for 16 hours, and the dark culture is carried out for 8 hours. After 4 weeks of sowing, placing the 3-basin seedling trays of the treatment group into an illumination intelligent incubator for heat stress treatment, and setting the photoperiod of the controllable conditions to be 16 hours and 8 hours at night, wherein the temperature is 39-40 ℃ at the daytime and 30-31 ℃ at the night, and the illumination intensity is 4000lx. The soil moisture of the seedling is ensured to be proper, the growth state of alfalfa seedlings is observed and recorded every day from the 1d of heat treatment, and the heat injury index is assessed according to the heat injury symptom assessment standard, and the control group is still placed in a greenhouse for cultivation. After heat treatment for 8d, the plant is placed in a greenhouse, growth is recovered for 6d at normal temperature, recovery indexes are measured, and morphological indexes and physiological indexes of various treatments and controls are calculated statistically. The seedlings are watered every other day during the heat stress treatment and the recovery treatment, the water adding amount is 50-100ml each time,
3. statistics and analysis of heat injury symptoms
(1) 1 st and 2 nd
Rating criteria the alfalfa heat injury symptoms were rated 6 by reference to the rating criteria of Han Ruihong et al (2015), specifically as follows:
level 0: no harm symptom
Stage 1: the plants are slightly wilted, and the edges of the old leaves are yellow or slightly dehydrated;
2 stages: plant wilting, sagging of middle and lower leaves, shrinking, and aging She Bianhuang;
3 stages: the plants seriously wilt, old leaves fall off or dry, upper leaves severely droop, and new leaves lose water and shrink;
4 stages: all plant leaves are dried up or fall off, but the stems remain green;
5 stages: the stems lose green and dry.
(2) 3 rd time
And removing the maximum and minimum seedlings from each variety, randomly selecting 10 seedlings with the same size, and grading the heat injury symptoms of the seedlings. Heat treatment for 14d, the symptoms of heat injury were observed daily on a plant-by-plant basis. The heat damage rating standard is readjusted based on actual conditions in the early alfalfa heat-resistant screening test process and the grading standard studied by the former, and is specifically as follows:
level 0: no heat injury symptoms;
stage 1: the leaves of the root neck and the true leaves are wilted and become yellow after water loss, and the middle and upper parts of the leaves are kept as they are or the leaves are slightly drooped;
2 stages: the root neck cotyledon and true leaf are withered or shed, the middle true leaf is withered and dehydrated to turn yellow, the upper true leaf is dehydrated at the edge, and the upper and middle leaves are drooping;
3 stages: the root neck and the middle leaf are dried up or fall off, the middle leaf has obvious sagging, and the upper true leaf has wilting and water loss yellowing symptoms;
4 stages: the whole plant leaves are dried up or fall off, and the stems have water loss symptoms but are still green;
5 stages: the whole plant leaves are dried or fall off, the stalks lose green, and the plant dies.
After the alfalfa seedlings were subjected to the high temperature treatment for 14d, the temperature of the light incubator was adjusted to 25 ℃/20 ℃ (day/night) for continuous cultivation for 6d, and the recovery stages were counted at 3d and 6d, respectively. The division criteria of the recovery index are adjusted finely with reference to the classification criteria of Han Ruihong et al (2015), the specific recovery index is as follows:
level 0: the whole plant leaves are dried or fall off, the stems lose green, and the plant dies;
stage 1: the stems are still green, but the residual leaves are all dried up and cannot be recovered;
2 stages: the leaf color of the upper residual leaves is recovered to be green, the residual leaves at the root and neck of the middle part are still in a wilting and withering state, and the residual leaves at the middle and upper parts have sagging phenomenon;
3 stages: the leaves of the middle and upper part residual leaves are green, the leaves of the root neck part residual leaves are still in a wilting and withering state, and the leaves of the middle and upper part are straight;
4 stages: the residual leaves of the whole plant are recovered to be green and recovered to be straight.
The heat damage index and the recovery index are calculated as follows:
heat damage index = Σ (heat damage number of stages×number of plants of the stage)/(highest number of stages×total number of plants)
Recovery index = Σ (recovery series×number of plants at this stage)/(highest series×total number of plants)
4. Index measurement
(1) Morphological index determination
After 2 weeks of heat treatment, randomly selecting 3 seedlings with consistent growth vigor from each pot, and measuring the distance from the surface of the substrate to the top of the plant by using a ruler as the Plant Height (PH); manually counting the number of single plant leaves; respectively weighing the fresh weight (AFW) of the overground part and the fresh weight (RFW) of the root; then, the mixture was put in an oven at 105℃for deactivation of enzymes for 1 hour, and then dried at 70℃to constant weight, and the dry weight on the ground (ADW) and the dry weight on the Root (RDW) were weighed, respectively. Other growth index calculation formulas are as follows:
total Fresh Weight (TFW) =stem fresh weight+leaf fresh weight+root fresh weight
Total Dry Weight (TDW) =dry weight of stem+ She Ganchong +dry weight of root
(2) Physiological index measurement
And (3) randomly selecting 3 seedlings from each pot after high-temperature treatment, measuring the relative chlorophyll content SPAD value by using a handheld chlorophyll meter SPAD-502Plus (Konika Co., japan), randomly selecting 3 representative plants from each pot, and measuring the SPAD value by fully expanding the middle leaf of the leaf from the 2 nd leaf position to the 4 th leaf position from the top.
After the high temperature treatment is finished, 3 seedlings are randomly selected from each pot, all the leaves are unfolded from the 2 nd leaf position to the 4 th leaf position from the top end, and the seedlings are quickly frozen by liquid nitrogen and then are preserved in a refrigerator at the temperature of minus 80 ℃ for measuring the content of Malondialdehyde (MDA) and the activities of superoxide dismutase (SOD), peroxidase (POD) and Catalase (CAT). The MDA content, CAT, SOD and POD activity were determined by visible spectrophotometry, and the determination steps were performed according to the corresponding kit specifications (JC 0202-S, JC0103-S, JC0101-S and JC0102-S, kokoku Ming Bio, suzhou). CAT activity was measured by UV spectrophotometry, and the measurement procedure was performed according to the kit instructions (JC 0103-S, koming Bio, suzhou);
POD Activity assay: about 0.1g of leaf tissue was weighed, and 1ml of the extract was added thereto to perform ice bath homogenization. And (5) centrifuging at the temperature of 8000g for 1min at 4 ℃, taking supernatant, and placing the supernatant on ice for testing. The measuring step comprises the following steps: preheating the spectrophotometer for more than 30min, adjusting the wavelength to 470nm, and zeroing the distilled water. And (3) preparing a working solution: mixing the first reagent, the second reagent and the third reagent according to the proportion of 2.6 (ml): 1.5 (ul): 1 (ul); preheating at 25deg.C for more than 10min, and preparing for use. And (3) measuring: 50ul of sample and 950ul of working solution are added into A1 ml glass cuvette, mixed uniformly, and the absorbance A1 at 470nm for 1min and the absorbance A2 after 2min are recorded. Delta a=a2-A1 is calculated.
POD activity calculation: unit definition A470 per minute per gram of tissue in each ml of reaction system varies by 0.01 as an enzyme activity unit. POD (U/g fresh weight) =Δa×v inverse/(w×v sample/V sample total)/0.01/t=2000×Δa/W. V inverse total: 1ml of total volume of the reaction system; v sample: adding a sample volume of 0.05ml; sample V total: adding 1ml of the volume of the extracting solution; t: reaction time, 1min; w: sample weight (g).
Five comprehensive evaluation and heat-resistant evaluation index screening
1. Alfalfa heat resistance grading standard
TABLE 1 alfalfa Heat resistance grading Standard
2. Evaluation according to mathematical model of alfalfa heat resistance evaluation
Raw data processing was performed using microsoft excel 2010. Principal component analysis, correlation analysis, cluster analysis, stepwise regression, etc. were performed using IBM SPSS 25.0 software. The specific index is calculated as follows:
(1) Measurement of Heat resistance coefficient Heatresistance coefficient (HTC)
Htc=heat stress treatment measurement value/control treatment measurement value (1) for each single index
(2) Membership function value of each comprehensive index of different alfalfa varieties (U (X)) i )﹜
U(X i )＝ (X i -X imin )/(X imax -X imin ) ②
Wherein X is i Representing the ith comprehensive index; u (X) i ) Membership function value X representing ith comprehensive index imax And X imin Respectively representing the maximum value and the minimum value of the ith comprehensive index in 20 varieties.
(3) Weights (W) of comprehensive indexes of alfalfa varieties of different genotypes i )
In which W is i Indicating the relative importance of the ith composite index, i.e. the weight of the ith composite index, P i The contribution rate of the i-th composite index is represented.
(4) Comprehensive heat resistance evaluation value of alfalfa varieties with different genotypes
Wherein the D value is a heat resistance comprehensive evaluation value obtained by evaluating and analyzing comprehensive indexes of various alfalfa varieties under the heat stress condition. Based on the comprehensive evaluation D value, the heat resistance of each variety was evaluated according to the classification criteria of table 2. Meanwhile, in order to analyze the relation between each single index and heat resistance, screening accurate heat resistance identification indexes, discussing a mathematical model which can be used for predicting the heat resistance evaluation of the alfalfa, carrying out correlation analysis on stress tolerance coefficients of the indexes and a D value, carrying out stepwise regression analysis by taking the stress tolerance coefficients of the obvious correlation indexes (P <0.05 or P < 0.01) as independent variables and taking the comprehensive evaluation value (D value) as the dependent variable, and establishing an optimal evaluation mathematical model.
6. Analysis of results
1. High temperature stress temperature determination
When the heat-resistant screening experiment was conducted 1 st time, there were fewer seedlings sown, 3 seedlings were left per hole, and there were fewer accidental cases in number (FIGS. 1-3). Experiment 2 based on experiment 1, 30 alfalfa varieties were selected for the experiment (fig. 4-5), and the number of transplanted seedlings was changed from original 3 plants/hole to 6 plants/hole. The quantity is increased, but the culture is carried out under the natural condition of a glass greenhouse, and the plug seedling is found to have side row advantages (good ventilation and light transmission conditions). The growth on both sides is good, the growth in the middle is poor, which is probably the reason for the strong marginal heat resistance and the weak middle, and the influence on the final evaluation result of the experiment is large.
In addition, the 2 nd experiment, since the 1d illumination incubator temperature was set to 40 ℃ at the time of heat treatment, but the actual temperature in the incubator was 43 ℃, resulting in that the set temperature did not coincide with the actual temperature in the incubator, causing serious wilting of all seedlings, the temperature was adjusted back to 40 ℃, the 3 rd treatment was performed, the leaves were basically wilted, and the further progress was not possible, and the 4 th treatment was carried out after placing it in the incubator (fig. 4). Meanwhile, during the experiment, the stress degree of varieties 1-10 and 20-30 is also found to be obviously lower than that of varieties 10-20 (figure 5), which is probably caused by the difference of local environmental temperatures in the incubator. Therefore, the square box nutrition pot replaces the plug in the 3 rd experiment. Batch 2, 1: the number of seedlings to be sown is small, 3 seedlings are reserved in each hole, and the 2 nd batch is changed from the original 3 plants/hole to 6 plants/hole.
The historic highest temperature range of Huaibei and Jianghuai is 38-40 ℃/day, the local area is 40-43 ℃/day, the night temperature is 30-31 ℃, and the high temperature duration is about 7-10d. And (3) carrying out a high-temperature stress treatment test on the control sample on the basis of failure of the 2 nd heat-resistant screening test, wherein the treatment temperature is 39-41 ℃/day to be the optimal temperature for screening the alfalfa heat-resistant varieties. Although the previous two experiments failed, the morphological characteristics of each alfalfa variety in the experimental process provide references for us to reconstruct the evaluation criteria of the heat injury symptoms and recovery symptoms. The 1 st and 2 nd test health symptom rating criteria the alfalfa heat injury symptoms were classified into 6 grades by the grading criteria of Han Ruihong et al (2015), and the specific results are shown in tables 2 and 3. Through apparent property observation (fig. 2-3) and heat injury symptom evaluation results, many varieties such as 701, 601, th and 440 varieties were found to be different, and thus, improvement of heat injury symptom evaluation criteria was required.
Table 240 parts alfalfa variety Heat injury index (1 st time)
Table 330 parts alfalfa variety heat injury index (2 nd time 2 nd batch)
2. 16 alfalfa variety heat resistance analysis (3 rd heat resistance screening test)
2.1 construction of health symptom evaluation criteria of alfalfa
The ranking criteria of the first 2 tests of health symptoms are referred to the ranking criteria of Han Ruihong and the like (2015), and root leaves are found to fall off in the early stage of heat injury treatment, and most of the leaves are not greatly affected at this time and collide with the pre-existing ranking criteria to cause inaccurate recording and error, so that on the basis of the previous study, the health symptom ranking criteria are reconstructed according to the actual condition of the 3 rd test, and specific contents are shown in tables 4-5. The references in tables 4-5 are as follows:
[1] han Ruihong, zhao Dahua, chen Jingjing, lu Shaoyun, guo Zhenfei. Comprehensive evaluation of heat resistance at seedling stage of different alfalfa germplasm resources. Chinese grassland journal, 2015,37 (03): 48-54.
[2] Yang Hui, li Xiaodong, wang Xiaoli, wu Ping, wang Yaxiong, cai Yiming. Evaluation of heat resistance in the seedling stage of alfalfa of different autumn dormancy grades. Modern animal husbandry science, 2017,34 (10): 50.
[3] Gu Kaizhi, chen Guilin seedlings of different eggplant varieties under high temperature stress are studied for heat resistance, J.ecological, 2005, (04): 398-401.
[4] Wu Guosheng, wang Yongjian, cao Wanhong, jiang Yiwei, zhang Lirong. Chinese cabbage Heat pest occurrence rule and heat resistance screening method. North China agricultural journal, 1995, (01): 111-115.
[5] Pang Jiangjiang, sun Xiaodong, cai Xinglai, zhang Wen, zhou Man. Selection of heat resistance index during germination of different quick-service vegetable lines and evaluation of heat resistance. Proc. Of North agriculture and forestry science and technology (Natl.Sci.), 2021,49 (04): 81-92.
[6] Zhang Jingyun, zhao Xiaodong, wanji, xiong Detao, hu Xinlong, miao Na, identification of heat resistance of chinese cabbage and analysis of heat resistance thereof, kenong report, 2014,28 (01): 146-153.
[7] Hu Qiaojiang, chen Longzheng, zhang Yongji, xu Hai, song Bo, su Xiaojun, yuan Xihan. Methods for identifying heat resistance of common cabbage at seedling stage were studied, chinese vegetables, 2011, (02): 56-61.
TABLE 4 comparison of Heat injury symptom evaluation criteria under Heat stress
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2.2 analysis of Heat injury index and recovery index of alfalfa
In the heat treatment process, according to observation statistics (table 6) on the heat-resistant phenotype characters of 16 varieties of alfalfa, 2 varieties with strong heat resistance are provided, and the heat damage indexes of WL656 and WL354 are low and the recovery indexes are high; the varieties with weak heat resistance are WL525 and GIB, and the heat damage index and recovery index are high; during the heat treatment, the temperature is set to 39-40 ℃, so that the death rate is obviously reduced. While at the same time. The heat damage index of each variety is lower than 0.4 before heat treatment, and the heat damage index change is small; from 7d, the heat damage index of each variety was significantly changed. In addition, the heat damage indexes of 701, SD10, WL656 and SDL have smaller change range within 5d, and the reasons are two aspects: on the one hand, it is very heat-resistant; on the other hand stress temperature differences.
Table 616 parts alfalfa variety Heat injury index and recovery index (3 rd time)
2.3 Heat resistance coefficient analysis of individual indices
The heat resistance coefficient can better show the comparison result of each index under the treatment and control conditions. As can be seen from table 7: the heat resistance coefficient of each single index varies significantly between 16 alfalfa varieties. The variation range is 10.46% -96.03%, wherein the variation coefficient of SOD is the largest, and the indexes of CAT, MDA, POD, fresh weight of the overground part, dry weight of the overground part and plant height are the next, which show that the influence of heat stress on the indexes is larger. After heat treatment, 4 morphological index values are obtained for different alfalfa varieties: the plant height, the leaf number, the fresh weight of the overground part and the dry weight of the overground part are all reduced with the comparison ratio (HTC < 1), which shows that the growth of each variety is inhibited to different degrees under the heat stress. The ratio of the physiological index value to the control value after heat treatment is changed in different degrees (STC > 1 or STC < 1) in different varieties. The variation amplitude of each single index under heat stress is different, and the heat resistance of the heat-resistant alloy is difficult to accurately and intuitively evaluate according to any single index, so that the comprehensive evaluation of multiple indexes is required.
TABLE 7 Heat resistance coefficient of 16 alfalfa variety measurement indicators
Note that: m1: plant height; m2: a number of blades; m3: fresh weight of the overground part; m4: dry weight of the aerial parts; m5: survival rate; m6: chlorophyll SPAD value; m7: superoxide dismutase activity; m8: peroxidase activity; m9: catalase activity; m10: malondialdehyde content; m11: a heat damage index; m12: recovery index
Note:M1:Plant height；M2:Leaf number per plant；M3:Aboveground fresh weight；M4:Aboveground dry weight；M5:Survival rate；M6:Chlorophyll SPAD value；M7:Superoxide dismutase activity；M8:Peroxidase activity；M9:Catalase activity；M10:Malondialdehyde content；M11:Heat injury index；M12:Recovery index
2.4 principal component analysis of Heat resistance coefficient
Based on the heat resistance coefficient of each single index of different alfalfa varieties, the feature vector and the contribution rate of each main component are calculated by utilizing SPSS 25.0 software, and different indexes are divided into different main components according to the absolute value of each feature vector. The position of the maximum absolute value of the same index in each factor is the main component to which the index belongs. As can be seen from table 8: the contribution rates of the first 5 converted Composite Indices (CI) were 35.21%, 23.33%, 13.26%, 11.20% and 7.27%, respectively, defined as 1 st (CI) 1 ) To 5 (CI) 5 ) The total contribution rate of the main components reaches 90.27%, which indicates that the 5 comprehensive indexes can represent most of information carried by 12 single indexes, therefore, the 5 main components can be usedThe index is used for analyzing the heat resistance of 16 alfalfa varieties.
Table 8 feature vector and contribution ratio of each comprehensive index
CI: comprehensive evaluation indexes; "x" represents the maximum absolute value of a certain comprehensive evaluation index in each factor
CI：Comprehensiveindex；“*”indicatesthebiggestabsolutevalueofeachindexinallfactors
2.5 comprehensive evaluation of alfalfa seedling Heat resistance
2.5.1 membership function analysis
The membership function values of the various comprehensive indexes of different alfalfa varieties were determined using equation (2) (Table 9). For the same comprehensive index, e.g. CI 1 In other words, U (X 1 ) The maximum value is 1.0000, indicating 701 at CI 1 The heat resistance is the strongest in the comprehensive index, and U (X) 1 ) The minimum value is 0.0000, which indicates that the heat resistance is the weakest on the comprehensive index.
TABLE 9 analysis of membership functions of comprehensive indicators of different alfalfa varieties
2.5.2 comprehensive evaluation and Classification of Heat resistance
Weights for 5 composite metrics were calculated using equation (3), 0.3900,0.2585,0.1469,0.1241 and 0.0805, respectively (Table 9). The comprehensive evaluation value (D value) of the heat resistance of different alfalfa varieties is obtained by utilizing a formula (4) (a table 9), the heat resistance of the different alfalfa varieties is ordered according to the D value, and the highest D value is TG and 701 (0.70), which indicates that the heat resistance is the strongest; the lowest D value was WL525 (0.20), indicating the weakest heat resistance. Systematic clustering analysis (Table 9) was performed on D values according to the grading criteria of Table 2, with 16 alfalfa varieties being classified into 4 categories: heat resistant types (TG, 701, wl656), medium heat resistant types (WL 354, WL319, TH, WL712, SD10, AG, and SDL), heat sensitive types (WL 903, GIB, WL168, WDLY,995, and WL 525).
The apparent property observation (fig. 6) was compared with the comprehensive evaluation results, and the evaluation results were substantially identical to the morphological features.
2.5.3 correlation analysis of the comprehensive evaluation value (D value) with other indices
The correlation between 12 indices of alfalfa under heat treatment was analyzed using the bivariate Pearson simple correlation coefficient method (table 10), and the results showed that the correlation between the majority of indices reached significant and extremely significant levels. Wherein, the plant height is extremely obviously positively correlated with the number of leaves, the fresh weight of the overground part and the dry weight of the overground part, and is obviously negatively correlated with the survival rate; the number of leaves is extremely obviously positively correlated with the fresh weight of the overground part and the dry weight of the overground part, and is obviously correlated with the SPAD value and the POD; the fresh weight of the overground part is extremely obviously and positively correlated with the dry weight of the overground part, and is obviously and positively correlated with the SPAD value and the POD; the dry weight of the overground part is extremely obviously and positively correlated with the SPAD value and the POD; the survival rate is obviously and inversely related to the heat damage index, is extremely obviously and positively related to the recovery index, and is obviously and positively related to the SPAD value; SPAD values are significantly inversely related to CAT and significantly positively related to recovery index; SOD is significantly positively correlated with recovery index. In addition, the D value is extremely significantly inversely correlated with the heat damage index, extremely significantly positively correlated with the number of leaves, the fresh weight of the aerial parts, the dry weight of the aerial parts, the SPAD value, the POD activity and the recovery index, and significantly positively correlated with the survival rate.
TABLE 10 correlation coefficient matrix for individual indices of alfalfa seedlings under heat treatment
Note that: m1: plant height; m2: a number of blades; m3: fresh weight of the overground part; m4: dry weight of the aerial parts; m5: survival rate; m6: chlorophyll SPAD value; m7: superoxide dismutase activity; m8: peroxidase activity; m9: catalase activity; m10: malondialdehyde content; m11: a heat damage index; m12: recovery index
Note:M1:Plant height；M2:Leafnumber per plant；M3:Aboveground fresh weight；M4:Aboveground dry weight；M5:Survival rate；M6:Chlorophyll SPAD value；M7:Superoxide dismutase activity；M8:Peroxidase activity；M9:Catalase activity；M10:Malondialdehyde content；M11:Heat injury index；M12:Recovery index
2.5.4 regression analysis and selection of Heat resistance identification indicators
To analyze the relationship between each single index and heat resistance, a reliable heat resistance identification index is screened out, a mathematical model which can be used for heat resistance evaluation is studied, and a D value of a heat resistance comprehensive evaluation value is taken as a dependent variable, so that the index which is extremely obvious or obviously related to the D value is taken as the dependent variable: the leaf number, the fresh weight of the aerial parts, the dry weight of the aerial parts, the survival rate, the SPAD value, the POD, the heat damage index and the recovery index are taken as independent variables to carry out linear stepwise regression analysis (table 11), and 3 heat resistance evaluation mathematical model formulas are established. Wherein R of the model 2 0.883, 0.931 and 0.964, respectively, means that each key index can explain the cause of 88.3%, 93.1% and 96.4% variation in heat resistance of each kind at present in each model. When F tests are carried out on the models, 3 models are found to pass the F tests, namely that at least one of the fresh weight of the overground part, POD activity, heat damage index and recovery index can influence the heat resistance of each current variety; the Debine-Watson value (2.313) is near the number 2, so that the model has no autocorrelation, the sample data has no association, and the model is good. The mathematical model was used to evaluate the overall heat resistance of 16 alfalfa varieties with average evaluation accuracies of 88.28, 90.86% and 94.84%, respectively (table 12). Furthermore, it was found that in model 1, the lowest estimation accuracy was 60.65%, and the result was poor. Therefore, we consider models 2 and 3 suitable as mathematical models for heat resistance screening evaluation of alfalfa: d' =0.719+0.276×m3+0.106×m8-1.335×m11 (model 2); d' =0.220+0.390×m3+0.076×m8-0.752×m11+0.522 ×m12 (model 3).
TABLE 11 stepwise linear regression analysis results
Note that: ", and", respectively, indicate significant differences at 0.5% and 1% levels; durbin-Watson value: 2.313
Note:“*”and“**”indicates significant difference at 0.5％and 1％level respectively；Durbin-Watson value:2.313
Estimation accuracy analysis of Table 12 regression equation
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (8)
1. A method for evaluating and identifying heat resistance of alfalfa is characterized in that the heat resistance of alfalfa is evaluated and identified by calculating the comprehensive evaluation value D' of alfalfa; the comprehensive evaluation value D' is calculated by the following mathematical model formula:
d' =0.719+0.276×upper fresh weight heat resistance coefficient+0.106×pod activity heat resistance coefficient-1.335×heat damage index;
or D' =0.220+0.390×upper fresh weight heat resistance coefficient+0.076×pod activity heat resistance coefficient-0.752×heat damage index+ 0.522 ×recovery index;
the grading criteria for heat resistance are as follows:
The calculation formula of the heat damage index is as follows: heat damage index = Σ (heat damage number x number of plants of the stage)/(highest number x total number of plants);
the heat injury progression was obtained by the following heat injury symptom evaluation criteria:
the recovery index is calculated by the following formula: recovery index = Σ (recovery number of stages×number of plants of the stage)/(highest number of stages×total number of plants);
the number of recovery stages was obtained by the following recovery symptom evaluation criteria:
2. the method for evaluating and identifying heat resistance of alfalfa according to claim 1, wherein the heat damage progression is obtained according to the following steps:
step 1, dark culturing alfalfa seeds until germination, and transferring the alfalfa seeds into a seedling raising basin for culturing;
and 2, placing the alfalfa seedlings after 4 weeks of culture in an illumination incubator for heat stress treatment, positioning the alfalfa seedlings at the beginning of the 1 st day of heat stress treatment, observing the growth state of the alfalfa seedlings, obtaining the heat damage level according to the heat damage symptom evaluation standard, and carrying out heat damage index evaluation and analysis.
3. The method for evaluating and identifying heat resistance of alfalfa according to claim 2, wherein in step 1, alfalfa seeds with consistent sizes are selected, placed in a culture dish with two layers of filter paper laid, and after dark culture for 2 days, germinated alfalfa is transferred into a seedling raising square basin, wherein the culture medium is soil, sand and organic fertilizer with a volume ratio of 2:1:1; placing in a culture room, culturing at 25deg.C for 16 hr under light and dark for 8 hr, and culturing each variety in dark at least 3 biological replicates per treatment, 8-10 seedlings per pot.
4. The method for evaluating and identifying heat resistance of alfalfa according to claim 2, wherein in step 2, the heat stress treatment condition is photoperiod of 16 hours at night and 8 hours at 39-40 ℃ at 30-31 ℃ at night, and the illumination intensity is 4000lx.
5. The method for evaluating and identifying heat resistance of alfalfa according to claim 2, wherein the recovery stage number is obtained according to the following steps:
and after heat stress treatment for 8 days, the alfalfa seedlings are recovered to grow for 6 days at normal temperature, the positioned alfalfa seedlings are observed every 3 days, the recovery growth state of the alfalfa seedlings is recorded, recovery grades are obtained according to recovery symptom evaluation standards, and recovery index evaluation and analysis are carried out.
6. The method for evaluating and identifying heat resistance of alfalfa according to claim 5, wherein the above-ground fresh weight heat resistance coefficient and the POD activity heat resistance coefficient are obtained by:
s1, after 6 days of growth recovery, determining the fresh weight of the overground part of alfalfa seedlings and POD activity;
s2, measuring the fresh weight of the overground part and the heat resistance coefficient of POD activity.
7. The method for evaluating and identifying heat resistance of alfalfa according to claim 6, wherein the heat resistance coefficient=heat stress treatment measurement value/control treatment measurement value.
8. The method for evaluating and identifying heat resistance of alfalfa according to claim 5, wherein the diameter of the seedling raising basin is 10cm, the height is 10cm, seedlings are watered every other day during heat stress treatment and recovery treatment, the water addition amount is 50-100ml each time, and heat injury symptom evaluation and recovery symptom evaluation are carried out before watering.
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