Maize inbred line heat resistance dual-verification screening method based on flowering phase multi-index joint evaluation

Abstract

The invention discloses a maize inbred line heat resistance dual-verification screening method based on flowering phase multi-index joint evaluation. The method comprises the following steps: step 1, material pretreatment and high-temperature stress; step 2, measuring a flowering phase specific index of the material to be tested; 3, weighting the indexes by using an entropy weight-TOPSIS model, constructing a heat resistance comprehensive index HTI, and screening a heat-resistant inbred line; 4, field verification: carrying out field verification on the heat-resistant corn inbred line obtained in the step 3, and if the field verification reaches the standard, judging that the heat-resistant corn inbred line is a real heat-resistant corn inbred line; and if the field verification does not reach the standard, returning to the step 1 to carry out indoor verification again. The screening accuracy and reliability of the heat-resistant maize inbred line are enhanced; in addition, the accuracy and the efficiency are high, and support is provided for mining of corn heat-resistant materials and breeding of heat-resistant varieties.

Description

Technical Field

[0001] The present invention belongs to the technical field of agricultural breeding, and specifically relates to a double-verification screening method for heat tolerance of maize inbred lines based on multi-index joint evaluation during the flowering period. Background Art

[0002] The flowering period of maize (tasseling - silking stage) is the most sensitive growth stage to high temperature. Short-term extreme high temperature is very likely to cause pollen inactivation and silking delay, resulting in pollination failure. Therefore, how to screen out maize species that are less sensitive to high temperature during the flowering period of maize has become a technical problem to be urgently solved.

[0003] An inbred line is a line obtained by self-pollination under artificial control for several generations, continuously eliminating poor ear rows, and selecting individual plants with better agronomic traits for self-pollination, so as to obtain a line with relatively uniform agronomic traits and a relatively simple genetic basis, which is called an inbred line. The hybrid offspring with strong vitality and high yield produced by crossing between excellent inbred lines is called an inter-inbred hybrid, and its agronomic traits are superior to those of varieties or inter-varietal hybrids. Generally, the yield increases by 20 - 30%. It is widely used in maize.

[0004] As the core material for the breeding of hybrids, the heat tolerance of inbred lines directly affects the stability of hybrid combinations. The existing heat tolerance screening methods for maize inbred lines have two major deficiencies:

[0005] (1) Most heat tolerance indicators are relatively single, and the indicators have not been standardized and weighted, so it is impossible to accurately capture the key thresholds of high-temperature response during the flowering period;

[0006] (2) Ignoring the specificity of inbred lines: There are differences in the heat tolerance mechanisms between inbred lines and hybrids (such as the loss of heterosis), and the existing hybrid screening models are not applicable to the evaluation of inbred lines. Summary of the Invention

[0007] Object of the Invention: The present invention makes improvements in view of the problems existing in the above-mentioned prior art, that is, the present invention discloses a double-verification screening method for heat tolerance of maize inbred lines based on multi-index joint evaluation during the flowering period. The present invention integrates indicators such as morphological development, reproductive organ function, and hormone dynamics during the flowering period, conducts a systematic evaluation of core indicators such as reproductive development stability, pollen viability, and hormone regulation network under high-temperature stress, and establishes an accurate identification system for heat tolerance of inbred lines to solve the problem of poor accuracy of heat tolerance of inbred lines.

[0008] Technical Solution: A double-verification screening method for heat tolerance of maize inbred lines based on multi-index joint evaluation during the flowering period, the steps are as follows:

[0009] Step 1: Material pretreatment and high-temperature stress:

[0010] (11) Select maize inbred line varieties that have been self-purified for more than 6 generations as the test materials, and conduct phenotypic consistency screening in an artificial climate chamber before sowing;

[0011] (12) Set a control group and a treatment group for the test materials, and then use the artificial climate chamber to conduct high-temperature treatment during the flowering period of the test materials, where:

[0012] - Control group: Day temperature 28±2°C / night temperature 22±1°C, photoperiod 14h / 10h, lasting for 7-10 days;

[0013] - Treatment group: Day temperature 40±1°C / night temperature 28±1°C, lasting for 7-10 days; On the 2nd - 3rd day after the start of natural pollination in the field, from 10:00 am to 1:00 pm every day, use a movable infrared radiator to hang 15-25 cm above the tassel, and use a thermal imager to monitor the surface temperature of the tassel in real time, so that the surface temperature of the tassel is maintained at 41±0.5°C for 3 hours;

[0014] Step two: Determination of the flowering period specific indicators of the test materials

[0015] (21) Determine the reproductive development indicators, which consist of:

[0016] Duration of tassel shedding, in hours, used to characterize pollen viability;

[0017] Silking synchrony, referring to the number of days between tasseling and silking, in days, abbreviated as ASI;

[0018] Silk browning index, with the grading standard of 0 level no browning → 4 level full browning;

[0019] (22) Determination of the hormones of the test materials:

[0020] (221) Determination of the contents of jasmonic acid (JA) and ethylene (ETH) in the tassel;

[0021] (222) Determination of the polar transport rate of auxin in the ear;

[0022] (23) Determination of the antioxidant enzyme activities of the test materials:

[0023] (231) Determination of the SOD (superoxide dismutase) activity of the ear leaf;

[0024] (232) Determination of the POD (peroxidase) activity of the ear leaf;

[0025] Step three: Use the entropy weight-TOPSIS model to assign weights to the indicators, construct the heat tolerance comprehensive index HTI, and then screen heat-tolerant inbred lines:

[0026] (31) Calculate the relative values of the indicators measured in step two

[0027] If the indexes measured in Step 2 are the duration of tassel shedding, the browning index of silk, the jasmonic acid content in the tassel, the ethylene content in the tassel, the polar transport rate of auxin in the ear, the SOD activity of the ear leaf, and the POD activity of the ear leaf, their relative value RV i is calculated by the following formula:

[0028] where i is the sample number;

[0029] If the index measured in Step 2 is the silking synchrony, its relative value RV i is calculated by the following formula:

[0030] where: i is the sample number;

[0031] (32) Perform extreme value standardization on the relative value RV i obtained in step (31):

[0032] Use the range method to standardize the relative value RV i obtained in step (31), and the standardization calculation formula is as follows:

[0033] where:

[0034] Z ij is the standardized value of the i-th sample on the j-th index; RV ij is the relative value of the i-th sample on the j-th index; max(RV j ) is the maximum relative value among all samples on the j-th index; min(RV j ) is the minimum relative value among all samples on the j-th index; i is the sample number, i is a positive integer, i ≤ 500; j is the number of indexes, j is a positive integer, 1 ≤ j ≤ 8;

[0035] (33) Using the weight assignment method, calculate the weights of the data standardized in step (32) respectively through formulas (4) to (6), and the calculation formula is as follows:

[0036]

[0037] In the formula: p ij is the index probability matrix; n is the number of indexes, 200 ≤ n ≤ 500; Z ij is the standardized value of the i-th sample on the j-th index; e j is the information entropy of the j-th index; W j is the weight of the j-th index;

[0038] (34) Calculation of the heat tolerance comprehensive index HTI, and its calculation formula is as follows:

[0039]

[0040] In the formula, HTI i is the heat tolerance comprehensive index of the i-th sample; n is the number of indicators, where 200 ≤ n ≤ 500;

[0041] (35) Grading:

[0042] If the heat tolerance comprehensive index HTI ≥ 75%, it is identified as a heat-tolerant maize inbred line indoors and proceeds to Step Four;

[0043] Otherwise, it is identified as a heat-intolerant maize inbred line indoors and this operation ends;

[0044] Step Four: Field verification:

[0045] Field verification is carried out on the heat-tolerant maize inbred lines identified indoors in Step Three. If the field verification meets the standards, it is determined as a truly heat-tolerant maize inbred line; if the field verification does not meet the standards, it is necessary to return to Step One for indoor verification again.

[0046] Background: The sister line of the heat-tolerant maize inbred line A (such as A') is introduced and crossed with the same conventional line (such as D) as a control group. Essentially, this is to exclude the interference of the genetic background and accurately lock in the genetic contribution of heat tolerance.

[0047] The sister line A' of the heat-tolerant maize inbred line A has a similar genetic background to the heat-tolerant maize inbred line A (>95% genomic identity), but fails the heat tolerance screening (low HTI); by comparing the HTI differences between A×D and A'×D, it can be directly attributed to the heat tolerance gene of A, excluding the interference of other genetic or environmental factors.

[0048] Furthermore, the specific steps of Step Four are as follows:

[0049] (41) The heat-tolerant maize inbred lines screened indoors are verified through sister line hybridization. The HTI of the hybrids assembled by the heat-tolerant maize inbred lines needs to be more than 10% higher than the average value of the heat-tolerant maize inbred line parents;

[0050] (42) Hybridizations are carried out by setting treatment groups and control groups respectively, where:

[0051] Treatment group: Heat-tolerant maize inbred line × Conventional maize inbred line;

[0052] Control group: Sister line of the heat-tolerant maize inbred line × Conventional maize inbred line, where:

[0053] The conventional maize inbred line is Zheng 58, and its indoor HTI is 65%;

[0054] (43) Hybrid heat tolerance evaluation:

[0055] Perform the same high-temperature stress treatment on the hybrids in the treatment group and the control group as in step one, and calculate their HTI;

[0056] If the HTI of the hybrids assembled with heat-tolerant inbred lines ≥ (HTI of heat-tolerant maize inbred lines + HTI of conventional maize inbred lines) × 0.5 × 110%, then the field verification is up to standard, and it is judged as a truly heat-tolerant maize inbred line; otherwise, if the field verification is not up to standard, it is necessary to return to step one to re-perform the indoor verification.

[0057] Furthermore, the specific steps of the pre-sowing phenotypic uniformity screening in step (11) are as follows:

[0058] (111) Number the i test materials respectively, sow each test material evenly in flower pots, sow 3 seeds in each flower pot, with a sowing depth of 5 cm, then gently cover a thin layer of soil, water thoroughly, and then place them in an artificial climate chamber for cultivation, watering, fertilizing, and pest control regularly, where: i is a positive integer, i ≤ 500;

[0059] The nutrient soil in the flower pots is evenly mixed by leaf mold soil, peat soil, and perlite in a ratio of 3:2:1;

[0060] The temperature of the artificial climate chamber is controlled at 25 - 30 °C, the light intensity is 3000 - 5000 lux, the light time is 12 - 14 hours per day, and the air humidity is maintained at 60% - 70%;

[0061] (112) According to the phenotypic characteristics of the test materials, determine the observation indicators, organize the data of the observed and recorded indicators, calculate the coefficient of variation of each indicator, and take the coefficient of variation ≤ 5% as the standard for good phenotypic uniformity, where:

[0062] The indicators include plant height, ear height, number of leaves, leaf color, leaf shape, stem diameter, number of male inflorescence branches, color of female inflorescence filaments, and ear shape.

[0063] Furthermore, for the qualitative traits of leaf color, leaf shape, color of female inflorescence filaments, and ear shape, they need to be converted into quantifiable variation indicators, where:

[0064] Leaf color: light green = 1; green = 2; dark green = 3; purple = 4; Leaf shape: lanceolate = 1, broad lanceolate = 2, oval = 3; Color of female inflorescence filaments: yellowish green = 1; light red = 2; dark red = 3; variegated = 4; Ear shape: cylindrical = 1; conical = 2

[0065] Furthermore, in step (12), the daytime temperature of the treatment group reaches the peak high temperature from 10:00 to 16:00 every day;

[0066] In step (12), the wavelength of the movable infrared radiator is 800 - 1200 nm, and the power density is 2.5 kW / m 2 .

[0067] During the pulsed high-temperature treatment in the treatment group in step (12), the female ear is covered with a reflective aluminum foil sunshade to maintain the temperature around it ≤ 32°C.

[0068] Furthermore, in step (12), the humidity of the control group and the treatment group is uniformly controlled at 60 - 70%.

[0069] Furthermore, the specific steps for the determination of the jasmonic acid content in the male ear in step (221) are as follows:

[0070] (2211) Sampling: At the tasseling stage, collect fresh male ear tissues, quickly freeze them in liquid nitrogen, and store them at -80°C for later use;

[0071] (2212) Extraction: Take 0.5 g of the frozen sample, place it in a pre-cooled mortar, add liquid nitrogen and grind it into a powder, add 3 - 5 mL of 80% methanol extraction solution, extract at 4°C for 4 h, centrifuge at 1000 g for 15 min, take the supernatant, and filter it through a 0.22 μm filter membrane to obtain the sample to be measured;

[0072] (2213) Place the sample to be measured obtained in step (2212) into a high-performance liquid chromatograph for determination, where:

[0073] The chromatographic conditions are as follows:

[0074] Chromatographic column: C18 reversed-phase column (such as Waters Atlantis T3, 2.1×100 mm, 3 μm); Mobile phase: Phase A is an aqueous solution of formic acid with a mass concentration of 0.1%, and phase B is an acetonitrile solution of formic acid with a mass concentration of 0.1%; Gradient elution: 0 - 5 min, 10% B → 90% B; 5 - 7 min, 90% B is maintained; 7 - 7.1 min, 90% B → 10% B; 7.1 - 10 min, 10% B is balanced; Flow rate: 0.3 mL / min, Column temperature: 40°C;

[0075] Mass spectrometry conditions: Ion source: Electrospray ionization (ESI-); Monitoring mode: Multiple reaction monitoring (MRM); Quantitative ion pairs: JA (m / z 209.1 → 59.0), internal standard D2-JA (m / z 211.1 → 61.0);

[0076] (2214) Data processing: Calculate the jasmonic acid content in the male ear by the external standard method or the internal standard method, and the result is expressed as ng / g fresh weight.

[0077] Further, the specific steps for measuring the ethylene content in the tassel in step (221) are as follows:

[0078] (s2211) Sample treatment: Sampling: Take 1 g of fresh tassel tissue and immediately put it into a 10 mL airtight syringe, then seal it.

[0079] Ethylene release: Place the syringe in a constant temperature incubator at 25 °C and incubate it in the dark for 1 hour. During this period, gently shake it once every 10 minutes.

[0080] (s2212) ETH gas collection

[0081] Use a 1 mL airtight needle to extract 1 mL of the gas at the top of the syringe and inject it into the injection port of a gas chromatograph (GC). Among them:

[0082] The chromatographic conditions are as follows: Chromatographic column: Porapak Q packed column (2 m × 3 mm); Carrier gas: High-purity nitrogen, flow rate 30 mL / min;

[0083] Detector: Hydrogen flame ionization detector (FID), temperature 250 °C; Column temperature: 80 °C, injection port temperature 150 °C;

[0084] Standard curve: Use ethylene standard gas (10 - 100 μL / L) to draw a standard curve;

[0085] (s2213) Data calculation

[0086] Calculate the ethylene content in the tassel according to the standard curve, and the result is expressed in μL / kg·h.

[0087] Further, the specific steps for measuring the polar transport rate of auxin in the ear (fluorescence method) in step (222) are as follows:

[0088] (2221) Preparation of ear segments:

[0089] Select ears with consistent growth, and use a blade to cut them into segments with a length of 2 - 5 cm, retaining the morphological upper and lower ends. Immediately immerse the tissue segments in the transport buffer and pre-equilibrate them in the dark at 25 °C for 30 minutes to restore tissue activity. Among them:

[0090] The pH value of the transport buffer is 5.5, and its composition is as follows: 1 mM KCl, 1 mM CaCl2, 1 mM MES, 1% sucrose, and the balance is water;

[0091] (2222) Application of the marker:

[0092] Dissolve the fluorescently labeled IAA (Alexa Fluor 488-IAA) in the transport buffer and add 10 μL of the fluorescent IAA solution dropwise to the cut surface at the base of the ear; Blank control: Only add the transport buffer (without fluorescent IAA);

[0093] Inhibitor control: Add a polar transport inhibitor to the buffer. Lay the treated ears flat in a petri dish on wet filter paper and incubate at 25 °C for 2 - 4 hours in the dark. Among them: The polar transport inhibitor is NPA with a concentration of 10 μM;

[0094] (2223) Transport detection:

[0095] Quickly freeze the samples with liquid nitrogen to terminate the transport, immediately perform cryosectioning, control the section thickness at 20 - 50 μm, fix the sections on glass slides, observe the distribution of the labeled substances with a fluorescence microscope, and use the image analysis software ImageJ to measure the transport distance of the fluorescence signal;

[0096] (2224) Data calculation: Calculate the polar transport rate of auxin in the ear through the following formula:

[0097] Transport rate (mm / h) = Transport distance (mm) / Incubation time (h).

[0098] Furthermore, the specific steps for the determination of the SOD (superoxide dismutase) activity of the ear leaf in step (231) are as follows:

[0099] (2311) Enzyme solution extraction: Weigh 0.5 g of fresh ear leaf of the test material, add liquid nitrogen and grind it into a powder. Add 5 mL of pre-cooled SOD extraction buffer to it, homogenize in an ice bath, centrifuge at 12,000 × g for 20 minutes at 4 °C, take the supernatant and divide it into aliquots to obtain the enzyme solution, and store it at -20 °C for later use. Among them: The composition of the SOD extraction buffer is as follows:

[0100] 50 mM phosphate buffer (pH value is 7.8), and contains 1 wt% PVP (polyvinylpyrrolidone, to prevent phenolic interference);

[0101] 0.1 mM EDTA;

[0102] (2312) SOD activity determination: Establish a reaction system control group and a reaction system determination group. Among them:

[0103] Reaction system control group: 0.1 mL of SOD extraction buffer + 2.9 mL of SOD reaction solution;

[0104] Reaction system determination group: 0.1 mL of SOD extraction buffer + 0.1 mL of the enzyme solution obtained in step (2311) + 2.8 mL of SOD reaction solution;

[0105] Light reaction: After mixing the control group and the measurement group of the reaction system respectively, react them under 4000 lux light for 15 minutes;

[0106] Terminate the reaction: Let it stand still in the dark for 5 minutes, and immediately measure the absorbance at 560 nm. Among them:

[0107] The SOD reaction solution is fixed volume to 3.0 mL / tube, and its components are as follows: 1.5 mL of 50 mM phosphate buffer (pH value is 7.8);

[0108] 0.3 mL of 130 mM methionine (final concentration 13 mM); 0.3 mL of 750 μM NBT (final concentration 75 μM); 0.3 mL of 100 μM EDTA (final concentration 10 μM); 0.3 mL of 100 μM riboflavin (final concentration 10 μM);

[0109] (2313) Calculate the SOD activity of the ear leaf, and its calculation formula is as follows:

[0110]

[0111] Furthermore, the specific steps for measuring the POD (peroxidase) activity of the ear leaf in step (232) are as follows:

[0112] (2321) Enzyme solution extraction: Weigh 0.5 g of fresh ear leaf of the test material, add liquid nitrogen and grind it into powder, add 5 mL of pre-cooled POD extraction buffer to it, homogenize in an ice bath, centrifuge at 12,000×g at 4 °C for 20 minutes, take the supernatant and divide it into aliquots to obtain the enzyme solution, and store it at -20 °C for later use. Among them: The composition of the POD extraction buffer is as follows:

[0113] 50 mM phosphate buffer (pH value is 6.0) and contains 1 wt% PVP (polyvinylpyrrolidone, to prevent phenolic interference);

[0114] 0.1 mM EDTA;

[0115] (2322) POD activity measurement:

[0116] Control group of the reaction system: 2 mL of phosphate buffer + 0.9 mL of o-methoxyphenol + 0.1 mL of hydrogen peroxide + 0.1 mL of ultrapure water;

[0117] Measurement group of the reaction system: 2 mL of phosphate buffer + 0.9 mL of o-methoxyphenol + 0.1 mL of hydrogen peroxide + 0.1 mL of the enzyme solution obtained in step (2321);

[0118] Reaction start and detection: 30 °C constant temperature water bath → immediately mix well → measure the absorbance at 470 nm (OD 470 ) every 30 seconds for a total of 3 minutes;

[0119] (2323) Calculate the POD activity of the ear leaf, and the calculation formula is as follows:

[0120]

[0121] The main innovation of the present invention lies in:

[0122] 1. Initiate the directional stress technology during the high-temperature sensitive period of flowering. Through the temperature control of the male ear / female ear in different time periods, that is, apply a pulsed high temperature of 41 °C / 3 h alone during the pollen shedding period of the male ear to simulate the field heat stress fluctuation mode;

[0123] 2. Develop a "double verification" system for the heat tolerance of inbred lines: The results of indoor screening need to be verified through the hybridization combination of sister lines. The hybrid HTI assembled by heat-tolerant inbred lines needs to be more than 10% higher than the average value of the parents. Ensure that the selected inbred lines are not only heat-tolerant themselves, but also can stably inherit the heat-tolerant characteristics to the hybrids, avoiding the disconnection between the laboratory results and the actual field application.

[0124] Beneficial effects: The beneficial effects of the double-verification screening method for the heat tolerance of maize inbred lines based on multi-index joint evaluation during the flowering period disclosed by the present invention are as follows:

[0125] 1. According to the actual needs of the breeding goal, the present invention determines the comprehensive heat tolerance index through multi-index joint evaluation, further determines the reliable heat tolerance screening standard, and further strengthens the accuracy and reliability of the screening of heat-tolerant maize inbred lines through the double-verification means of combining indoor experiments and field experiments;

[0126] 2. The method provided by the present invention is used to identify and screen heat-tolerant maize inbred lines, which solves the problem that the heat tolerance results identified in the climate chamber environment do not match the field. Through the field identification after hybridization with sister inbred lines, the gene interference problem is excluded. This method has high precision and efficiency, solves the problems of difficult screening and identification, low efficiency, and poor repeatability of heat-tolerant maize materials, and provides support for the excavation of heat-tolerant maize materials and the breeding of heat-tolerant varieties. Specific implementation manners

[0127] The specific implementation manners of the present invention will be described in detail below.

[0128] Example 1

[0129] The double-verification screening method for the heat tolerance of maize inbred lines based on multi-index joint evaluation during the flowering period is as follows:

[0130] Step 1: Material pretreatment and high-temperature stress:

[0131] (11) Select 200 maize inbred line varieties that have been self-purified for more than 6 generations as the test materials, and conduct phenotypic consistency screening in an artificial climate chamber before sowing;

[0132] (12) Set up a control group and a treatment group for the test materials, and then use an artificial climate chamber to conduct high-temperature treatment on the test materials during the flowering period, where:

[0133] - Control group: Day temperature 28±2°C / night temperature 22±1°C, photoperiod 14h / 10h, lasting for 8 days;

[0134] - Treatment group: Day temperature 40±1°C / night temperature 28±1°C, lasting for 8 days; From the 2nd to the 3rd day after the start of natural pollen scattering in the field, from 10:00 am to 1:00 pm every day, use a movable infrared radiator to hang 20 cm above the tassel, and use a thermal imager to monitor the surface temperature of the tassel in real time, so that the surface temperature of the tassel is maintained at 41±0.5°C for 3 hours;

[0135] Step two: Determination of the flowering period specific indicators of the test materials

[0136] (21) Determine the reproductive development indicators, which consist of:

[0137] Duration of tassel pollen scattering, in hours, used to characterize pollen viability;

[0138] Silking synchrony, referring to the number of days between tasseling and silking, in days, abbreviated as ASI;

[0139] Silk browning index, the grading standard is 0 level no browning → 4 level full browning;

[0140] (22) Determination of the hormones of the test materials:

[0141] (221) Determination of the contents of jasmonic acid (JA) and ethylene (ETH) in the tassel;

[0142] (222) Determination of the polar transport rate of auxin in the ear;

[0143] (23) Determination of the antioxidant enzyme activities of the test materials:

[0144] (231) Determination of the activity of superoxide dismutase (SOD) in the ear leaf;

[0145] (232) Determination of the activity of peroxidase (POD) in the ear leaf;

[0146] Step three: Use the entropy weight-TOPSIS model to assign weights to the indicators, construct a heat tolerance comprehensive index HTI, and then screen heat-tolerant inbred lines:

[0147] (31) Calculate the relative values of the indicators measured in step two

[0148] If the indexes measured in Step 2 are the duration of tassel anthesis, the browning index of silk, the jasmonic acid content in the tassel, the ethylene content in the tassel, the polar transport rate of auxin in the ear, the SOD activity of the ear leaf, and the POD activity of the ear leaf, their relative value RV i is calculated as follows:

[0149] where i is the sample number;

[0150] If the index measured in Step 2 is the silk emergence synchrony, its relative value RV i is calculated as follows:

[0151] where: i is the sample number;

[0152] (32) Perform extreme value standardization on the relative value RV i obtained in Step (31):

[0153] Use the range method to standardize the relative value RV i obtained in Step (31), and the standardization calculation formula is as follows:

[0154] where:

[0155] Z ij is the standardized value of the i-th sample on the j-th index; RV ij is the relative value of the i-th sample on the j-th index; max(RV j ) is the maximum relative value among all samples on the j-th index; min(RV j ) is the minimum relative value among all samples on the j-th index; i is the sample number, i is a positive integer, i ≤ 200; j is the number of indexes, j is a positive integer, 1 ≤ j ≤ 8;

[0156] (33) Using the weight assignment method, calculate the weights of the data standardized in Step (32) through Formulas (4) to (6) respectively, and the calculation formula is as follows:

[0157]

[0158] In the formula: p ij is the index probability matrix; n is the number of indexes, n = 200; Z ij is the standardized value of the i-th sample on the j-th index; e j is the information entropy of the j-th index; W j is the weight of the j-th index;

[0159] (34) Calculate the heat tolerance comprehensive index HTI, and the calculation formula is as follows:

[0160]

[0161] In the formula, HTI i is the comprehensive heat tolerance index of the i-th sample; n is the number of indicators, n = 200;

[0162] (35) Grading:

[0163] If the comprehensive heat tolerance index HTI ≥ 75%, it is identified as a heat-tolerant maize inbred line indoors and enters Step 4;

[0164] Otherwise, it is identified as a heat-intolerant maize inbred line indoors and this operation ends;

[0165] Step 4: Field verification:

[0166] The heat-tolerant maize inbred lines identified indoors obtained in Step 3 are verified in the field. If the field verification meets the standard, it is judged as a truly heat-tolerant maize inbred line; if the field verification does not meet the standard, it is necessary to return to Step 1 to re-conduct the indoor verification.

[0167] Furthermore, the specific steps of Step 4 are as follows:

[0168] (41) The heat-tolerant maize inbred lines screened indoors are verified through sib-cross combination. The HTI of the hybrid seeds obtained by combining heat-tolerant maize inbred lines needs to be more than 10% higher than the average value of the heat-tolerant maize inbred line parents;

[0169] (42) Set treatment groups and control groups for hybridization respectively, where:

[0170] Treatment group: Heat-tolerant maize inbred line × Conventional maize inbred line;

[0171] Control group: Sib line of heat-tolerant maize inbred line × Conventional maize inbred line, where:

[0172] The conventional maize inbred line is Zheng 58, and its indoor HTI is 65%;

[0173] (43) Evaluation of the heat tolerance of hybrid seeds:

[0174] Perform the same high-temperature stress treatment on the hybrid seeds of the treatment group and the control group as in Step 1, and calculate their HTI,

[0175] If the HTI of the hybrid seeds obtained by combining heat-tolerant inbred lines ≥ (HTI of heat-tolerant maize inbred line + HTI of conventional maize inbred line) × 0.5 × 110%, the field verification meets the standard and it is judged as a truly heat-tolerant maize inbred line; otherwise, the field verification does not meet the standard and it is necessary to return to Step 1 to re-conduct the indoor verification.

[0176] Furthermore, the specific steps of the phenotypic consistency screening in the artificial climate chamber before sowing in Step (11) are as follows:

[0177] (111) Number i portions of the test materials respectively, sow each portion of the test materials evenly in flower pots, sow 3 seeds in each flower pot, with a sowing depth of 5 cm, then gently cover with a thin layer of soil, water thoroughly, and then place them in an artificial climate chamber for cultivation, watering, fertilizing, and pest and disease control regularly, where: i is a positive integer, i ≤ 200;

[0178] The nutrient soil in the flower pots is evenly mixed by leaf mold soil, peat soil, and perlite in a ratio of 3:2:1;

[0179] The temperature of the artificial climate chamber is controlled at 28 °C, the light intensity is 4000 lux, the light time is 13 hours per day, and the air humidity is maintained at 65%;

[0180] (112) According to the phenotypic characteristics of the test materials, determine the observation indexes, organize the data of the indexes recorded in the observation, calculate the coefficient of variation of each index, and take the coefficient of variation ≤ 5% as the standard of better phenotypic consistency, where:

[0181] The indexes include plant height, ear height, number of leaves, leaf color, leaf shape, stem diameter, number of male spike branches, color of female spike filaments, and ear shape.

[0182] Furthermore, for the qualitative traits of leaf color, leaf shape, color of female spike filaments, and ear shape, they need to be converted into quantifiable variation indexes, where:

[0183] Leaf color: light green = 1; green = 2; dark green = 3; purple = 4; Leaf shape: lanceolate = 1, broad lanceolate = 2, oval = 3; Color of female spike filaments: yellowish green = 1; light red = 2; dark red = 3; variegated = 4; Ear shape: cylindrical = 1; conical = 2

[0184] Furthermore, in step (12), the daytime temperature of the treatment group reaches the peak high temperature from 10:00 to 16:00 every day;

[0185] In step (12), the wavelength of the movable infrared radiator is 1000 nm, and the power density is 2.5 kW / m 2 .

[0186] In step (12), during the pulsed high-temperature treatment of the treatment group, the female spike is covered with a reflective aluminum foil sunshade to maintain the temperature around it ≤ 32 °C.

[0187] Furthermore, in step (12), the humidity of the control group and the treatment group is uniformly controlled at 65%.

[0188] Furthermore, the specific steps for measuring the jasmonic acid content in the male spike in step (221) are as follows:

[0189] (2211) Sampling: At the tasseling stage, collect fresh tassel tissues, quickly put them into liquid nitrogen for quick freezing, and store them at -80 °C for later use;

[0190] (2212) Extraction: Take 0.5 g of the frozen sample, place it in a pre-cooled mortar, add liquid nitrogen and grind it into powder, add 4 mL of 80% methanol extraction solution, extract at 4 °C for 4 h, centrifuge at 1000 g for 15 min, take the supernatant, and filter it through a 0.22 μm filter membrane to obtain the sample to be measured;

[0191] (2213) Place the sample to be measured obtained in step (2212) into a high performance liquid chromatograph for determination, where:

[0192] The chromatographic conditions are as follows:

[0193] Chromatographic column: C18 reverse phase column (such as Waters Atlantis T3, 2.1×100 mm, 3 μm); Mobile phase: Phase A is an aqueous solution of formic acid with a mass concentration of 0.1%, and phase B is an acetonitrile solution of formic acid with a mass concentration of 0.1%; Gradient elution: 0 - 5 min, 10% B → 90% B; 5 - 7 min, maintain 90% B; 7 - 7.1 min, 90% B → 10% B; 7.1 - 10 min, balance with 10% B; Flow rate: 0.3 mL / min, Column temperature: 40 °C;

[0194] Mass spectrometry conditions:

[0195] Ion source: Electrospray ionization (ESI-); Monitoring mode: Multiple reaction monitoring (MRM); Quantitative ion pair: JA (m / z209.1 → 59.0), internal standard D2-JA (m / z 211.1 → 61.0).

[0196] (2214) Data processing: Calculate the jasmonic acid content in the tassel by the external standard method or the internal standard method, and the result is expressed as ng / g fresh weight.

[0197] Furthermore, the specific steps for the determination of ethylene content in the tassel in step (221) are as follows:

[0198] (s2211) Sample treatment

[0199] Sampling: Take 1 g of fresh tassel tissue, immediately put it into a 10 mL airtight syringe, and seal it;

[0200] Ethylene release: Place the syringe in a constant temperature incubator at 25 °C for dark incubation for 1 hour, and gently shake it once every 10 minutes during this period;

[0201] (s2212) ETH gas collection

[0202] Use a 1 mL airtight needle to draw 1 mL of the gas at the top of the syringe and inject it into the injection port of a gas chromatograph (GC), where:

[0203] The chromatographic conditions are as follows:

[0204] Chromatographic column: Porapak Q packed column (2m × 3mm); Carrier gas: High-purity nitrogen, flow rate 30 mL / min;

[0205] Detector: Flame ionization detector (FID), temperature 250 °C; Column temperature: 80 °C, injection port temperature 150 °C;

[0206] Standard curve: A standard curve was plotted using ethylene standard gas (10 - 100 μL / L);

[0207] (s2213) Data calculation

[0208] The ethylene content in the tassel was calculated according to the standard curve, and the result was expressed as μL / kg·h.

[0209] Furthermore, the specific steps for the determination of the polar auxin transport rate in the ear (fluorescence method) in step (222) are as follows:

[0210] (2221). Preparation of ear segments:

[0211] Ears with consistent growth were selected, and the upper and lower morphological segments with a length of 3 cm were cut with a blade and immediately immersed in the transport buffer, and pre-equilibrated in the dark at 25 °C for 30 minutes to restore tissue activity, where:

[0212] The pH value of the transport buffer was 5.5, and its composition was as follows: 1 mM KCl, 1 mM CaCl2, 1 mM MES, 1% sucrose, and the balance was water;

[0213] (2222) Application of the marker:

[0214] Fluorescently labeled IAA (Alexa Fluor 488-IAA) was dissolved in the transport buffer, and 10 μL of the fluorescent IAA solution was added dropwise to the cut surface at the base of the ear; Blank control: Only the transport buffer (without fluorescent IAA) was added;

[0215] Inhibitor control: A polar transport inhibitor was added to the buffer, and the treated ears were laid flat in a petri dish with moist filter paper and incubated at 25 °C for 2 - 4 hours in the dark, where: The polar transport inhibitor was NPA with a concentration of 10 μM;

[0216] (2223) Transport detection:

[0217] Terminate the transportation by quickly freezing the sample with liquid nitrogen, immediately perform cryosectioning, control the section thickness at 30 μm, fix the sections on glass slides, observe the distribution of the marker with a fluorescence microscope, and use the image analysis software ImageJ to measure the transportation distance of the fluorescence signal;

[0218] (2224) Data calculation: Calculate the polar auxin transport rate in the ear through the following formula:

[0219] Transport rate (mm / h) = Transport distance (mm) / Incubation time (h).

[0220] Furthermore, the specific steps for measuring the activity of SOD (superoxide dismutase) in the ear leaf at the ear position in step (231) are as follows:

[0221] (2311) Enzyme solution extraction: Weigh 0.5 g of fresh ear leaf of the test material, add liquid nitrogen and grind it into powder, add 5 mL of pre-cooled SOD extraction buffer to it, homogenize in an ice bath, centrifuge at 12,000×g at 4 °C for 20 minutes, take the supernatant and aliquot to obtain the enzyme solution, and store it at -20 °C for later use. Among them: The composition of the SOD extraction buffer is as follows:

[0222] 50 mM phosphate buffer (pH value is 7.8), and contains 1 wt% PVP (polyvinylpyrrolidone, to prevent phenolic interference);

[0223] 0.1 mM EDTA;

[0224] (2312) SOD activity measurement:

[0225] Establish a reaction system control group and a reaction system measurement group, where:

[0226] Reaction system control group: 0.1 mL SOD extraction buffer + 2.9 mL SOD reaction solution;

[0227] Reaction system measurement group: 0.1 mL SOD extraction buffer + 0.1 mL of the enzyme solution obtained in step (2311) + 2.8 mL SOD reaction solution;

[0228] Light reaction: Mix the reaction system control group and the reaction system measurement group respectively and react under 4000 lux light for 15 minutes;

[0229] Terminate the reaction: Let it stand in the dark for 5 minutes, and immediately measure the absorbance at 560 nm. Among them: The SOD reaction solution is made up to 3.0 mL / tube, and its components are as follows:

[0230] 1.5 mL 50 mM phosphate buffer (pH value is 7.8);

[0231] 0.3 mL of 130 mM methionine (final concentration 13 mM); 0.3 mL of 750 μM NBT (final concentration 75 μM); 0.3 mL of 100 μM EDTA (final concentration 10 μM); 0.3 mL of 100 μM riboflavin (final concentration 10 μM);

[0232] (2313) Calculate the SOD activity of the ear leaf, and its calculation formula is as follows:

[0233]

[0234] Furthermore, the specific steps for measuring the POD (peroxidase) activity of the ear leaf in step (232) are as follows:

[0235] (2321) Enzyme solution extraction: Weigh 0.5 g of fresh ear leaf of the test material, add liquid nitrogen and grind it into powder, add 5 mL of pre-cooled POD extraction buffer thereto, homogenize in an ice bath, centrifuge at 12,000×g for 20 minutes at 4 °C, take the supernatant and divide it into aliquots to obtain the enzyme solution, and store it at -20 °C for later use. Among them: The composition of the POD extraction buffer is as follows:

[0236] 50 mM phosphate buffer (pH value 6.0) and containing 1 wt% PVP (polyvinylpyrrolidone, to prevent phenolic interference);

[0237] 0.1 mM EDTA;

[0238] (2322) POD activity measurement:

[0239] Reaction system control group: 2 mL of phosphate buffer + 0.9 mL of o-methoxyphenol + 0.1 mL of hydrogen peroxide + 0.1 mL of ultrapure water;

[0240] Reaction system measurement group: 2 mL of phosphate buffer + 0.9 mL of o-methoxyphenol + 0.1 mL of hydrogen peroxide + 0.1 mL of the enzyme solution obtained in step (2321);

[0241] Reaction initiation and detection: 30 °C constant temperature water bath → immediately mix evenly → measure the absorbance at 470 nm (OD 470 ) for a total of 3 minutes;

[0242] (2323) Calculate the POD activity of the ear leaf, and its calculation formula is as follows:

[0243]

[0244] Six candidate lines with HTI≥75% were screened from 200 inbred lines, including PH6WC, HW2, HW9, HW18, HW23, and HW36.

[0245] Inbred lines with HTI ≥ 75% Indoor HTI Corresponding sister lines Indoor HTI of sister lines PH6WC 82％ PH6WM 68％ HW2 77％ HW5 60％ HW9 79％ HW10 64％ HW18 85％ HW17 66％ HW23 80％ HW26 68％ HW36 82％ HW34 69％

[0246] Hybrid combination and verification:

[0247] Subject the hybrid seeds obtained to high temperature treatment during the flowering stage: 4 days before and after pollen shedding. Use a self-made polyethylene plastic film greenhouse (light transmittance ≥ 95%) to increase the temperature for high temperature stress treatment of maize germplasm during the flowering stage. The high temperature treatment time is from 10:00 to 16:00 every morning for 8 consecutive days.

[0248] Daytime temperature 40 ± 1°C / nighttime temperature 28 ± 1°C. Starting from the 2nd - 3rd day after natural pollen shedding in the field, from 10:00 in the morning to 13:00 in the afternoon every day, use a movable infrared radiator and hang it 20 cm above the tassel. Real-time monitor the surface temperature of the tassel through a thermal imager, so that the surface temperature of the tassel is maintained at 41 ± 0.5°C for 3 hours. After the treatment, remove the high temperature greenhouse and let the hybrid seeds grow naturally to maturity.

[0249] Measure the flowering stage specific indicators of the hybrid seeds of the test materials described in "Step 2", and calculate the HTI of the hybrid seeds according to "Step 3". The results are as follows:

[0250]

[0251] If the HTI of the hybrid seeds obtained by crossing heat - tolerant inbred lines (PH6WC, HW2, HW9, HW18, HW23, HW36) ≥ (HTI of heat - tolerant maize inbred lines + HTI of Zheng58 maize inbred lines) × 0.5 × 110%,

[0252] That is: the HTI of the hybrid seeds obtained by crossing heat - tolerant inbred lines is 10% higher than the mean value of the parents, and the HTI of the hybrid seeds obtained by crossing the corresponding heat - sensitive sister lines (PH6WC sister line is PH6WM, HW2 sister line is HW5, HW9 sister line is HW10, HW18 sister line is HW17, HW23 sister line is HW26, HW36 sister line is HW34) is 10% lower than the mean value of the parents, then the field verification is qualified, and it is judged as a truly heat - tolerant maize inbred line;

[0253] If the HTI of the hybrid seeds obtained by crossing heat - tolerant inbred lines and their heat - sensitive sister lines are both 10% higher than the mean value of the parents, then the field verification is unqualified. Because there may be environmental and gene - environment interaction interference factors, and it is impossible to exclude "false heat - tolerant" materials, it is necessary to return to Step 1 to re - conduct indoor verification.

[0254] Conclusion: PH6WC, HW2, HW18, HW36 passed the double verification, and their heat - tolerance can be stably inherited to the hybrid seeds; while HW9 and HW23 did not pass the double verification, and their heat - tolerance needs to be re - evaluated.

[0255] Example 2

[0256] Double-verification screening method for heat tolerance of maize inbred lines based on multi-index joint evaluation during flowering stage, the steps are as follows:

[0257] Step 1: Material pretreatment and high-temperature stress:

[0258] (11) Select 300 maize inbred line varieties that have been self-purified for more than 6 generations as test materials, and conduct phenotypic consistency screening in an artificial climate chamber before sowing;

[0259] (12) Set the control group and treatment group for the test materials, and then conduct high-temperature treatment during the flowering stage of the test materials using the artificial climate chamber. Among them:

[0260] - Control group: Day temperature 28±2°C / night temperature 22±1°C, photoperiod 14h / 10h, lasting for 7 days;

[0261] - Treatment group: Day temperature 40±1°C / night temperature 28±1°C, lasting for 7 days; Starting from the second day after natural pollen scattering in the field, from 10:00 am to 1:00 pm every day, use a movable infrared radiator to hang 15 cm above the tassel, and monitor the surface temperature of the tassel in real time through an infrared thermal imager, so that the surface temperature of the tassel is maintained at 41±0.5°C for 3 hours;

[0262] Step 2: Determination of flowering-stage specific indicators of the test materials

[0263] (21) Determine the reproductive development indicators, which consist of the following:

[0264] Duration of tassel anthesis, in hours, used to characterize pollen viability;

[0265] Silking synchrony, referring to the number of days between tasseling and silking, in days, abbreviated as ASI;

[0266] Silk browning index, with the grading standard of 0 level no browning → 4 level full browning;

[0267] (22) Determination of hormones of the test materials:

[0268] (221) Determination of jasmonic acid (JA) and ethylene (ETH) contents in the tassel;

[0269] (222) Determination of the polar transport rate of auxin in the ear;

[0270] (23) Determination of antioxidant enzyme activities of the test materials:

[0271] (231) Determination of the activity of superoxide dismutase (SOD) in the ear leaf;

[0272] (232) Determination of the activity of peroxidase (POD) in the ear leaf;

[0273] Step 3: Use the entropy weight-TOPSIS model to assign weights to the indicators, construct the heat tolerance comprehensive index HTI, and then screen heat-tolerant inbred lines:

[0274] (31) Calculate the relative values of the indicators measured in Step 2

[0275] If the indicators measured in Step 2 are the anthesis duration of the tassel, the browning index of the silk, the jasmonic acid content in the tassel, the ethylene content in the tassel, the polar transport rate of auxin in the ear, the SOD activity of the ear leaf, and the POD activity of the ear leaf, their relative value RV i is calculated as follows:

[0276] where i is the sample number;

[0277] If the indicator measured in Step 2 is the silking synchrony, its relative value RV i is calculated as follows:

[0278] where: i is the sample number;

[0279] (32) Perform extreme value standardization on the relative value RV i obtained in Step (31):

[0280] Use the range method to standardize the relative value RV i obtained in Step (31), and the standardization calculation formula is as follows:

[0281] where:

[0282] Z ij is the standardized value of the i-th sample on the j-th indicator; RV ij is the relative value of the i-th sample on the j-th indicator; max(RV j ) is the maximum relative value among all samples on the j-th indicator; min(RV j ) is the minimum relative value among all samples on the j-th indicator; i is the sample number, i is a positive integer, i ≤ 300; j is the number of indicators, j is a positive integer, 1 ≤ j ≤ 8;

[0283] (33) Use the weight allocation method to calculate the weights of the data standardized in Step (32) through Formulas (4) to (6) respectively, and their calculation formulas are as follows:

[0284]

[0285] In the formula: p ij is the indicator probability matrix; n is the number of indicators, n = 300; Z ij is the standardized value of the i-th sample on the j-th indicator; ej is the information entropy of the j-th index; W j is the weight of the j-th index;

[0286] (34) Calculation of the heat resistance comprehensive index HTI, and its calculation formula is as follows:

[0287]

[0288] In the formula, HTI i is the heat resistance comprehensive index of the i-th sample; n is the number of indexes, n = 300;

[0289] (35) Grading: If the heat resistance comprehensive index HTI ≥ 75%, it is identified as a heat-resistant maize inbred line indoors and enters Step Four;

[0290] Otherwise, it is identified as a non-heat-resistant maize inbred line indoors and this operation ends;

[0291] Step Four: Field verification:

[0292] Field verification is carried out on the heat-resistant maize inbred lines identified indoors in Step Three. If the field verification meets the standards, it is judged as a truly heat-resistant maize inbred line; if the field verification does not meet the standards, it is necessary to return to Step One to re-conduct indoor verification.

[0293] Furthermore, the specific steps of Step Four are as follows:

[0294] (41) The heat-resistant maize inbred lines screened indoors are verified through sister line hybridization. The HTI of the hybrid seeds obtained by the heat-resistant maize inbred lines should be more than 10% higher than the average value of the heat-resistant maize inbred line parents;

[0295] (42) Set a treatment group and a control group for hybridization respectively, where:

[0296] Treatment group: heat-resistant maize inbred line × conventional maize inbred line;

[0297] Control group: sister line of heat-resistant maize inbred line × conventional maize inbred line, where:

[0298] The conventional maize inbred line is Zheng 58, and the indoor HTI is 65%;

[0299] (43) Evaluation of the heat resistance of hybrid seeds:

[0300] Carry out the same high-temperature stress treatment on the hybrid seeds of the treatment group and the control group as in Step One, and calculate their HTI,

[0301] If the hybrid HTI assembled by heat-tolerant inbred lines ≥ (HTI of heat-tolerant maize inbred lines + HTI of conventional maize inbred lines) × 0.5 × 110%, the field verification is up to standard, and it is determined to be a truly heat-tolerant maize inbred line; otherwise, if the field verification is not up to standard, it is necessary to return to step 1 to re-conduct indoor verification.

[0302] Furthermore, the specific steps of the phenotypic consistency screening in the artificial climate chamber before sowing in step (11) are as follows:

[0303] (111) Number the i test materials respectively, sow each test material evenly in flower pots, sow 3 seeds in each flower pot, with a sowing depth of 5 cm, then gently cover a thin layer of soil, water thoroughly, and then place them in the artificial climate chamber for cultivation, watering, fertilizing and pest control regularly, where: i is a positive integer, i ≤ 300;

[0304] The nutrient soil in the flower pot is evenly mixed by leaf mold soil, peat soil and perlite in a ratio of 3:2:1;

[0305] The temperature of the artificial climate chamber is controlled at 25 °C, the light intensity is 5000 lux, the light time is 12 hours / day, and the air humidity is maintained at 60%;

[0306] (112) According to the phenotypic characteristics of the test materials, determine the observation indexes, sort out the data of the indexes recorded in the observation, calculate the coefficient of variation of each index, and take the coefficient of variation ≤ 5% as the standard of good phenotypic consistency, where:

[0307] The indexes include plant height, ear height, number of leaves, leaf color, leaf shape, stem diameter, number of male spike branches, color of female spike filaments, and ear shape.

[0308] Furthermore, for qualitative traits such as leaf color, leaf shape, color of female spike filaments and ear shape, they need to be converted into quantifiable variation indexes, where: leaf color: light green = 1; green = 2; dark green = 3; purple = 4; leaf shape: lanceolate = 1, broad lanceolate = 2, oval = 3; color of female spike filaments: yellowish green = 1; light red = 2; dark red = 3; variegated = 4; ear shape: cylinder = 1; cone = 2.

[0309] Furthermore, in step (12), the daytime temperature of the treatment group reaches the peak high temperature from 10:00 to 16:00 every day;

[0310] In step (12), the wavelength of the movable infrared radiator is 800 nm, and the power density is 2.5 kW / m 2 .

[0311] In step (12), during the pulsed high-temperature treatment of the treatment group, the female ear is covered with a reflective aluminum foil sunshade to maintain the temperature around it ≤ 32 °C.

[0312] Further, the humidity of the control group and the treatment group in step (12) is uniformly controlled at 60%.

[0313] Further, the specific steps for measuring the jasmonic acid content in the tassel in step (221) are as follows:

[0314] (2211) Sampling: At the tasseling stage, collect fresh tassel tissue, quickly freeze it in liquid nitrogen, and store it at -80 °C for later use;

[0315] (2212) Extraction: Take 0.5 g of the frozen sample, place it in a pre-cooled mortar, add liquid nitrogen and grind it into a powder, add 3 mL of 80% methanol extract, extract at 4 °C for 4 h, centrifuge at 1000 g for 15 min, take the supernatant, and filter it through a 0.22 μm filter membrane to obtain the sample to be measured;

[0316] (2213) Place the sample to be measured obtained in step (2212) in a high performance liquid chromatograph for determination, where:

[0317] The chromatographic conditions are as follows: Chromatographic column: C18 reverse phase column (such as Waters Atlantis T3, 2.1×100 mm, 3 μm); Mobile phase: Phase A is an aqueous solution of formic acid with a mass concentration of 0.1%, and phase B is an acetonitrile solution of formic acid with a mass concentration of 0.1%;

[0318] Gradient elution: 0 - 5 min, 10% B → 90% B; 5 - 7 min, maintain 90% B; 7 - 7.1 min, 90% B → 10% B; 7.1 - 10 min, balance with 10% B; Flow rate: 0.3 mL / min, Column temperature: 40 °C;

[0319] Mass spectrometry conditions: Ion source: Electrospray ionization (ESI-); Monitoring mode: Multiple reaction monitoring (MRM); Quantitative ion pair: JA (m / z 209.1 → 59.0), internal standard D2-JA (m / z 211.1 → 61.0);

[0320] (2214) Data processing: Calculate the jasmonic acid content in the tassel by the external standard method or the internal standard method, and the result is expressed as ng / g fresh weight.

[0321] Further, the specific steps for measuring the ethylene content in the tassel in step (221) are as follows:

[0322] (s2211) Sample treatment

[0323] Sampling: Take 1 g of fresh tassel tissue, immediately put it into a 10 mL airtight syringe, and seal it;

[0324] Ethylene release: Place the syringe in an incubator at 25 °C and incubate it in the dark for 1 hour, gently shake it once every 10 minutes during this period;

[0325] (s2212)ETH gas collection

[0326] Use a 1 mL gastight syringe to extract 1 mL of gas from the top of the syringe and inject it into the injection port of a gas chromatograph (GC). Among them:

[0327] The chromatographic conditions are as follows: Chromatographic column: Porapak Q packed column (2 m × 3 mm); Carrier gas: High-purity nitrogen, flow rate 30 mL / min;

[0328] Detector: Flame ionization detector (FID), temperature 250 °C; Column temperature: 80 °C, injection port temperature 150 °C;

[0329] Standard curve: Use ethylene standard gas (10 - 100 μL / L) to draw the standard curve;

[0330] (s2213)Data calculation

[0331] Calculate the ethylene content in the tassel according to the standard curve, and the result is expressed in μL / kg·h (ethylene release rate per unit time and unit fresh weight).

[0332] Furthermore, the specific steps for measuring the polar transport rate of auxin in the ear (fluorescence method) in step (222) are as follows:

[0333] (2221). Preparation of ear segments:

[0334] Select ears with consistent growth, use a blade to cut them into segments with a length of 2 cm, retaining the morphological upper and lower ends, and immediately immerse the tissue segments in the transport buffer. Pre-equilibrate in the dark at 25 °C for 30 minutes to restore tissue activity. Among them:

[0335] The pH value of the transport buffer is 5.5, and its composition is as follows: 1 mM KCl, 1 mM CaCl2, 1 mM MES, 1% sucrose;

[0336] (2222) Application of the marker: Dissolve the fluorescently labeled IAA (Alexa Fluor 488-IAA) in the transport buffer, and add 10 μL of the fluorescent IAA solution dropwise to the cut surface at the base of the ear; Blank control: Only add the transport buffer (without fluorescent IAA);

[0337] Inhibitor control: Add a polar transport inhibitor to the buffer, lay the treated ear flat in a petri dish with moist filter paper, and incubate at 25 °C in the dark for 2 hours. Among them: The polar transport inhibitor is NPA with a concentration of 10 μM;

[0338] (2223)Transport detection: Rapidly freeze the sample with liquid nitrogen to terminate transportation, immediately perform frozen sectioning, control the section thickness at 20 - 50 μm, fix the sections on glass slides, observe the distribution of markers with a fluorescence microscope, and use the image analysis software ImageJ to measure the transport distance of the fluorescence signal;

[0339] (2224)Data calculation: Calculate the polar auxin transport rate in the ear through the following formula:

[0340] Transport rate (mm / h) = Transport distance (mm) / Incubation time (h).

[0341] Furthermore, the specific steps for measuring the SOD (superoxide dismutase) activity of the ear leaf in step (231) are as follows:

[0342] (2311)Enzyme solution extraction: Weigh 0.5 g of fresh ear leaf of the test material, add liquid nitrogen and grind it into powder, add 5 mL of pre-cooled SOD extraction buffer to it, homogenize in an ice bath, centrifuge at 12,000×g for 20 minutes at 4 °C, take the supernatant and divide it into aliquots to obtain the enzyme solution, and store it at -20 °C for later use. Among them: The composition of the SOD extraction buffer is as follows:

[0343] 50 mM phosphate buffer (pH value is 7.8), and contains 1 wt% PVP (polyvinylpyrrolidone, to prevent phenolic interference);

[0344] 0.1 mM EDTA;

[0345] (2312)SOD activity determination: Establish a reaction system control group and a reaction system determination group, where:

[0346] Reaction system control group: 0.1 mL of SOD extraction buffer + 2.9 mL of SOD reaction solution;

[0347] Reaction system determination group: 0.1 mL of SOD extraction buffer + 0.1 mL of the enzyme solution obtained in step (2311) + 2.8 mL of SOD reaction solution;

[0348] Light reaction: Mix the reaction system control group and the reaction system determination group respectively and react under 4000 lux light for 15 minutes;

[0349] Terminate the reaction: Let it stand in the dark for 5 minutes, and immediately measure the absorbance at 560 nm, where:

[0350] The SOD reaction solution is made up to 3.0 mL per tube, and its components are as follows:

[0351] 1.5 mL of 50 mM phosphate buffer (pH value is 7.8); 0.3 mL of 130 mM methionine (final concentration 13 mM);

[0352] 0.3 mL of 750 μM NBT (final concentration 75 μM); 0.3 mL of 100 μM EDTA (final concentration 10 μM);

[0353] 0.3 mL of 100 μM riboflavin (final concentration 10 μM);

[0354] (2313) Calculate the SOD activity of the ear leaf, and the calculation formula is as follows:

[0355]

[0356] Furthermore, the specific steps for measuring the POD (peroxidase) activity of the ear leaf in step (232) are as follows:

[0357] (2321) Enzyme solution extraction: Weigh 0.5 g of fresh ear leaf of the test material, add liquid nitrogen and grind it into powder, add 5 mL of pre-cooled POD extraction buffer thereto, homogenize in an ice bath, centrifuge at 12,000×g for 20 minutes at 4 °C, take the supernatant and divide it into aliquots to obtain the enzyme solution, and store it at -20 °C for standby. Among them, the composition of the POD extraction buffer is as follows:

[0358] 50 mM phosphate buffer (pH value 6.0) and containing 1 wt% PVP (polyvinylpyrrolidone, to prevent phenolic interference);

[0359] 0.1 mM EDTA;

[0360] (2322) POD activity measurement:

[0361] Reaction system control group: 2 mL of phosphate buffer + 0.9 mL of o-methoxyphenol + 0.1 mL of hydrogen peroxide + 0.1 mL of ultrapure water;

[0362] Reaction system measurement group: 2 mL of phosphate buffer + 0.9 mL of o-methoxyphenol + 0.1 mL of hydrogen peroxide + 0.1 mL of the enzyme solution obtained in step (2321);

[0363] Reaction start and detection: 30 °C constant temperature water bath → immediately mix evenly → measure the absorbance at 470 nm (OD 470 ) for a total of 3 minutes;

[0364] (2323) Calculate the POD activity of the ear leaf, and the calculation formula is as follows:

[0365]

[0366] Example 3

[0367] A double-verification screening method for the heat tolerance of maize inbred lines based on multi-index joint evaluation at the flowering stage, the steps are as follows:

[0368] Step 1: Material pretreatment and high-temperature stress:

[0369] (11) Select 500 maize inbred line varieties that have been self-purified for more than 6 generations as test materials, and conduct phenotypic consistency screening in an artificial climate chamber before sowing;

[0370] (12) Set the control group and treatment group for the test materials, and then use the artificial climate chamber to conduct high-temperature treatment during the flowering period of the test materials, where:

[0371] - Control group: Day temperature 28±2°C / night temperature 22±1°C, photoperiod 14h / 10h, lasting for 10 days;

[0372] - Treatment group: Day temperature 40±1°C / night temperature 28±1°C, lasting for 10 days; 3 days after the start of natural pollen shedding in the field, from 10:00 am to 13:00 pm every day, use a movable infrared radiator to hang 25 cm above the tassel, and monitor the surface temperature of the tassel in real time through a thermal imager, so that the surface temperature of the tassel is maintained at 41±0.5°C for 3 hours;

[0373] Step 2: Determination of flowering-stage specific indicators of the test materials

[0374] (21) Determine the reproductive development indicators, which consist of:

[0375] Duration of tassel pollen shedding, in hours, used to characterize pollen viability;

[0376] Silking synchrony, referring to the number of days between tasseling and silking, in days, abbreviated as ASI;

[0377] Silk browning index, with the grading standard of 0 level no browning → 4 level full browning;

[0378] (22) Determination of hormones in the test materials:

[0379] (221) Determination of the contents of jasmonic acid (JA) and ethylene (ETH) in the tassel;

[0380] (222) Determination of the polar transport rate of auxin in the ear;

[0381] (23) Determination of the antioxidant enzyme activities of the test materials:

[0382] (231) Determination of the activity of superoxide dismutase (SOD) in the ear leaf;

[0383] (232) Determination of the activity of peroxidase (POD) in the ear leaf;

[0384] Step 3: Use the entropy weight-TOPSIS model to assign weights to the indicators, construct the heat tolerance comprehensive index HTI, and then screen heat-tolerant inbred lines:

[0385] (31) Calculate the relative values of the indicators measured in Step 2

[0386] If the indicators measured in Step 2 are the duration of tassel anthesis, silk browning index, jasmonic acid content in the tassel, ethylene content in the tassel, polar transport rate of auxin in the ear, SOD activity in the ear leaf, and POD activity in the ear leaf, their relative value RV i The calculation formula is as follows:

[0387] Where i is the sample number;

[0388] If the indicator measured in Step 2 is silking synchrony, its relative value RV i The calculation formula is as follows:

[0389] Where: i is the sample number;

[0390] (32) Perform extreme value standardization on the relative value RV i obtained in Step (31):

[0391] Use the range method to standardize the relative value RV i obtained in Step (31), and the standardization calculation formula is as follows:

[0392] Where:

[0393] Z ij is the standardized value of the i-th sample on the j-th indicator; RV ij is the relative value of the i-th sample on the j-th indicator; max(RV j ) is the maximum relative value among all samples on the j-th indicator; min(RV j ) is the minimum relative value among all samples on the j-th indicator; i is the sample number, i is a positive integer, i ≤ 500; j is the number of indicators, j is a positive integer, 1 ≤ j ≤ 8;

[0394] (33) Use the weight distribution method to calculate the weights of the data standardized in Step (32) through Formulas (4) to (6) respectively, and the calculation formula is as follows:

[0395]

[0396] In the formula: p ij is the indicator probability matrix; n is the number of indicators, n = 500; Z ij is the standardized value of the i-th sample on the j-th indicator; ej is the information entropy of the j-th index; W j is the weight of the j-th index;

[0397] (34) Calculate the heat resistance comprehensive index HTI, and its calculation formula is as follows:

[0398]

[0399] In the formula, HTI i is the heat resistance comprehensive index of the i-th sample; n is the number of indexes, n = 500;

[0400] (35) Classification: If the heat resistance comprehensive index HTI ≥ 75%, it is identified as a heat-resistant maize inbred line indoors and enters step four;

[0401] Otherwise, it is identified as a heat-sensitive maize inbred line indoors and this operation ends;

[0402] Step four: Field verification:

[0403] Field verification is carried out on the heat-resistant maize inbred lines identified indoors in step three. If the field verification is qualified, it is judged as a truly heat-resistant maize inbred line; if the field verification is unqualified, it is necessary to return to step one to re-conduct indoor verification.

[0404] Furthermore, the specific steps of step four are as follows:

[0405] (41) The heat-resistant maize inbred lines screened indoors are verified through sister line hybridization. The HTI of the hybrid seeds obtained by the combination of heat-resistant maize inbred lines should be more than 10% higher than the average value of the heat-resistant maize inbred line parents;

[0406] (42) Set treatment groups and control groups for hybridization respectively, where: Treatment group: heat-resistant maize inbred line × conventional maize inbred line; Control group: sister line of heat-resistant maize inbred line × conventional maize inbred line, and the conventional maize inbred line is Zheng 58, with an indoor HTI of 65%;

[0407] (43) Evaluation of the heat resistance of hybrid seeds:

[0408] Perform the same high-temperature stress treatment on the hybrid seeds of the treatment group and the control group as in step one, and calculate their HTI;

[0409] If the HTI of the hybrid seeds obtained by the combination of heat-resistant inbred lines ≥ (HTI of heat-resistant maize inbred line + HTI of conventional maize inbred line) × 0.5 × 110%, the field verification is qualified and it is judged as a truly heat-resistant maize inbred line; otherwise, the field verification is unqualified and it is necessary to return to step one to re-conduct indoor verification.

[0410] Further, the specific steps of the pre-sowing phenotypic consistency screening in step (11) are as follows:

[0411] (111) Number the i test materials respectively, sow each test material evenly in flower pots, sow 3 seeds in each flower pot, with a sowing depth of 5 cm, then gently cover a thin layer of soil, water thoroughly, and then place them in an artificial climate chamber for cultivation, watering, fertilizing and pest control regularly, where: i is a positive integer, i ≤ 500;

[0412] The nutrient soil in the flower pot is evenly mixed by leaf mold soil, peat soil and perlite in a ratio of 3:2:1;

[0413] The temperature of the artificial climate chamber is controlled at 30 °C, the light intensity is 3000 lux, the light time is 14 hours / day, and the air humidity is maintained at 70%;

[0414] (112) Determine the observation indexes according to the phenotypic characteristics of the test materials, sort out the data of the observed and recorded indexes, calculate the coefficient of variation of each index, and take the coefficient of variation ≤ 5% as the standard of good phenotypic consistency, where:

[0415] The indexes include plant height, ear height, number of leaves, leaf color, leaf shape, stem diameter, number of male spike branches, color of female spike filaments, and ear shape.

[0416] Furthermore, for qualitative traits such as leaf color, leaf shape, color of female spike filaments and ear shape, they need to be converted into quantifiable variation indexes, where:

[0417] Leaf color: light green = 1; green = 2; dark green = 3; purple = 4; Leaf shape: lanceolate = 1, broad lanceolate = 2, oval = 3; Color of female spike filaments: yellowish green = 1; light red = 2; dark red = 3; variegated = 4; Ear shape: cylindrical = 1; conical = 2.

[0418] Further, in step (12), the daytime temperature of the treatment group reaches the peak high temperature from 10:00 to 16:00 every day;

[0419] In step (12), the wavelength of the movable infrared radiator is 1200 nm, and the power density is 2.5 kW / m 2 .

[0420] In step (12), during the pulse high temperature treatment, the female ear is covered with a reflective aluminum foil sunshade to keep the temperature around it ≤ 32 °C.

[0421] Further, in step (12), the humidity of the control group and the treatment group is uniformly controlled at 70%.

[0422] Furthermore, the specific steps for determining the jasmonic acid content in the tassel in step (221) are as follows:

[0423] (2211) Sampling: At the tasseling stage, collect fresh tassel tissues, quickly put them into liquid nitrogen for quick freezing, and store them at -80 °C for later use;

[0424] (2212) Extraction: Take 0.5 g of the frozen sample, place it in a pre-cooled mortar, add liquid nitrogen and grind it into powder, add 5 mL of 80% methanol extraction solution, extract at 4 °C for 4 h, centrifuge at 1000 g for 15 min, take the supernatant, and filter it through a 0.22 μm filter membrane to obtain the sample to be measured;

[0425] (2213) Place the sample to be measured obtained in step (2212) into a high performance liquid chromatograph for determination, where:

[0426] The chromatographic conditions are as follows: Chromatographic column: C18 reverse phase column (such as Waters Atlantis T3, 2.1×100 mm, 3 μm); Mobile phase: Phase A is an aqueous solution of formic acid with a mass concentration of 0.1%, and phase B is an acetonitrile solution of formic acid with a mass concentration of 0.1%; Gradient elution: 0 - 5 min, 10% B → 90% B; 5 - 7 min, 90% B is maintained; 7 - 7.1 min, 90% B → 10% B; 7.1 - 10 min, 10% B is balanced; Flow rate: 0.3 mL / min, Column temperature: 40 °C;

[0427] Mass spectrometry conditions: Ion source: Electrospray ionization (ESI-); Monitoring mode: Multiple reaction monitoring (MRM); Quantitative ion pairs: JA (m / z 209.1 → 59.0), internal standard D2-JA (m / z 211.1 → 61.0);

[0428] (2214) Data processing: Calculate the jasmonic acid content in the tassel by the external standard method or the internal standard method, and the result is expressed as ng / g fresh weight.

[0429] Furthermore, the specific steps for determining the ethylene content in the tassel in step (221) are as follows:

[0430] (s2211) Sample treatment: Sampling: Take 1 g of fresh tassel tissue, immediately put it into a 10 mL airtight syringe, and seal it;

[0431] Ethylene release: Place the syringe in a 25 °C incubator for dark culture for 1 hour, and gently shake it once every 10 minutes during this period;

[0432] (s2212) ETH gas collection

[0433] Use a 1 mL airtight needle to draw 1 mL of the gas at the top of the syringe and inject it into the injection port of the gas chromatograph (GC), where:

[0434] The chromatographic conditions are as follows: Chromatographic column: Porapak Q packed column (2m × 3mm); Carrier gas: High-purity nitrogen, flow rate 30 mL / min;

[0435] Detector: Flame ionization detector (FID), temperature 250 °C; Column temperature: 80 °C, injection port temperature 150 °C;

[0436] Standard curve: A standard curve was plotted using ethylene standard gas (10 - 100 μL / L);

[0437] (s2213) Data calculation: Calculate the ethylene content in the tassel according to the standard curve, and the result is expressed as μL / kg·h.

[0438] Furthermore, the specific steps for measuring the polar transport rate of auxin in the ear (fluorescence method) in step (222) are as follows:

[0439] (2221). Preparation of ear segments:

[0440] Select ears with consistent growth, cut them into segments with a length of 2 - 5 cm while retaining the morphological upper and lower ends using a blade, immediately immerse the tissue segments in the transport buffer, and pre-equilibrate them in the dark at 25 °C for 30 minutes to restore tissue activity, where:

[0441] The pH value of the transport buffer is 5.5, and its composition is as follows: 1 mM KCl, 1 mM CaCl2, 1 mM MES, 1% sucrose;

[0442] (2222) Application of the marker: Dissolve the fluorescently labeled IAA (Alexa Fluor 488-IAA) in the transport buffer, and add 10 μL of the fluorescent IAA solution dropwise to the cut surface at the base of the ear; Blank control: Only add the transport buffer (without fluorescent IAA);

[0443] Inhibitor control: Add a polar transport inhibitor to the buffer, lay the treated ear flat in a petri dish with moist filter paper, and incubate it at 25 °C in the dark for 4 hours, where: The polar transport inhibitor is NPA with a concentration of 10 μM;

[0444] (2223) Transport detection: Rapidly freeze the sample with liquid nitrogen to terminate the transport, immediately perform cryosectioning, control the section thickness at 20 - 50 μm, fix the sections on glass slides, observe the distribution of the marker using a fluorescence microscope, and measure the transport distance of the fluorescent signal using the image analysis software ImageJ;

[0445] (2224) Data calculation:

[0446] Calculate the polar transport rate of auxin in the ear through the following formula: Transport rate = Transport distance / Incubation time.

[0447] Further, the specific steps for measuring the SOD (superoxide dismutase) activity of the ear leaf in step (231) are as follows:

[0448] (2311) Enzyme solution extraction: Weigh 0.5 g of fresh ear leaf of the test material, add liquid nitrogen and grind it into powder, add 5 mL of pre-cooled SOD extraction buffer thereto, homogenize in an ice bath, centrifuge at 12,000×g for 20 minutes at 4 °C, take the supernatant, divide and store it to obtain the enzyme solution, and store it at -20 °C for standby. Among them, the composition of the SOD extraction buffer is as follows:

[0449] 50 mM phosphate buffer (pH value is 7.8), and contains 1 wt% PVP (polyvinylpyrrolidone, to prevent phenolic interference);

[0450] 0.1 mM EDTA;

[0451] (2312) SOD activity measurement: Establish a reaction system control group and a reaction system measurement group. Among them:

[0452] Reaction system control group: 0.1 mL of SOD extraction buffer + 2.9 mL of SOD reaction solution;

[0453] Reaction system measurement group: 0.1 mL of SOD extraction buffer + 0.1 mL of the enzyme solution obtained in step (2311) + 2.8 mL of SOD reaction solution;

[0454] Light reaction: Mix the reaction system control group and the reaction system measurement group respectively and react under 4000 lux light for 15 minutes;

[0455] Terminate the reaction: Let it stand in the dark for 5 minutes, and immediately measure the absorbance at 560 nm. Among them:

[0456] The SOD reaction solution is made up to 3.0 mL / tube, and its components are as follows:

[0457] 1.5 mL of 50 mM phosphate buffer (pH value is 7.8); 0.3 mL of 130 mM methionine (final concentration 13 mM);

[0458] 0.3 mL of 750 μM NBT (final concentration 75 μM); 0.3 mL of 100 μM EDTA (final concentration 10 μM);

[0459] 0.3 mL of 100 μM riboflavin (final concentration 10 μM);

[0460] (2313) Calculate the SOD activity of the ear leaf, and its calculation formula is as follows:

[0461]

[0462] Further, the specific steps for measuring the POD (peroxidase) activity of the ear leaf in step (232) are as follows:

[0463] (2321) Enzyme solution extraction: Weigh 0.5 g of fresh ear leaf of the test material, add liquid nitrogen and grind it into powder. Add 5 mL of pre-cooled POD extraction buffer thereto, homogenize in an ice bath, centrifuge at 12,000×g for 20 minutes at 4°C, take the supernatant, divide it into aliquots to obtain the enzyme solution, and store it at -20°C for later use. Among them, the composition of the POD extraction buffer is as follows:

[0464] 50 mM phosphate buffer (pH value 6.0) containing 1 wt% PVP (polyvinylpyrrolidone, to prevent phenolic interference);

[0465] 0.1 mM EDTA;

[0466] (2322) POD activity measurement:

[0467] Reaction system control group: 2 mL of phosphate buffer + 0.9 mL of o-methoxyphenol + 0.1 mL of hydrogen peroxide + 0.1 mL of ultrapure water;

[0468] Reaction system measurement group: 2 mL of phosphate buffer + 0.9 mL of o-methoxyphenol + 0.1 mL of hydrogen peroxide + 0.1 mL of the enzyme solution obtained in step (2321);

[0469] Reaction initiation and detection: 30°C constant temperature water bath → immediately mix evenly → measure the absorbance at 470 nm (OD 470 ) for a total of 3 minutes;

[0470] (2323) Calculate the POD activity of the ear leaf, and its calculation formula is as follows:

[0471]

[0472] The above has described the embodiments of the present invention in detail. However, the present invention is not limited to the above embodiments, and various changes can be made without departing from the gist of the present invention within the knowledge scope of those of ordinary skill in the art.

Claims

1. A dual-verification screening method for heat tolerance of maize inbred lines based on multi-index joint evaluation during the flowering period, characterized in that, The steps are as follows: Step 1: Material pretreatment and high-temperature stress: (11) Select maize inbred line varieties that have been self-purified for more than 6 generations as the test materials, and conduct phenotypic consistency screening in an artificial climate chamber before sowing; (12) Set a control group and a treatment group for the test materials, and then use the artificial climate chamber to conduct high-temperature treatment during the flowering period of the test materials, where: - Control group: Day temperature 28±2°C / night temperature 22±1°C, photoperiod 14h / 10h, lasting for 7-10 days; - Treatment group: Day temperature 40±1°C / night temperature 28±1°C, lasting for 7-10 days; Starting from the 2nd to 3rd day after natural pollination in the field, from 10:00 am to 1:00 pm every day, use a movable infrared radiator to hang 15-25 cm above the tassel, and use a thermal imager to monitor the surface temperature of the tassel in real time, so that the surface temperature of the tassel is maintained at 41±0.5°C for 3 hours; Step 2: Determination of the flowering period specific indicators of the test materials (21) Determine the reproductive development indicators, which consist of: Duration of tassel shedding, in hours; Silking synchrony, in days; Silk browning index, the grading standard is 0 level without browning → 4 level fully brown; (22) Determination of the hormones of the test materials: (221) Determination of the contents of jasmonic acid and ethylene in the tassel; (222) Determination of the polar transport rate of auxin in the ear; (23) Determination of the antioxidant enzyme activities of the test materials: (231) Determination of the SOD activity of the ear leaf; (232) Determination of the POD activity of the ear leaf; Step 3: Use the entropy weight-TOPSIS model to assign weights to the indicators, construct the heat tolerance comprehensive index HTI, and then screen heat-tolerant inbred lines: (31) Calculate the relative values of the indicators measured in Step 2 If the indexes measured in Step 2 are the duration of tassel anthesis, silk browning index, jasmonic acid content in the tassel, ethylene content in the tassel, polar auxin transport rate in the ear, SOD activity in the ear leaf at the ear position, POD activity in the ear leaf at the ear position, their relative value RV i is calculated as follows: Relative value where i is the sample number; If the index measured in Step 2 is the synchronization of silk spinning, its relative value RV i The calculation formula is as follows: Relative value Where: i is the sample number; (32) Normalize the relative value RV obtained in step (31). i Perform extreme value normalization: The relative value RV obtained in step (31) is normalized by the range method i for data standardization. The standardization calculation formula is as follows: Wherein: Z ij is the standardized value of the i-th sample on the j-th index; RV ij is the relative value of the i-th sample on the j-th index; max(RV j ) is the maximum relative value among the j-th indicators of all samples; min(RV j ) is the minimum relative value among the j-th indicators of all samples; i is the sample number, i is a positive integer, i≤500; j is the number of indicators, j is a positive integer, 1≤j≤8; (33) Use the weight distribution method to calculate the weights of the data standardized in Step (32) respectively through formulas (4) to (6), and its calculation formula is as follows: where: p ij is the index probability matrix; n is the number of indicators, 200≤n≤500; Z ij is the standardized value of the i-th sample on the j-th index; e j is the information entropy of the j-th index; W j is the weight of the j-th index; (34) Calculate the heat tolerance comprehensive index HTI, and its calculation formula is as follows: wherein, HTI i is the comprehensive heat resistance index of the i-th sample; n is the number of indicators, 200≤n≤500; (35) Grading: If the heat tolerance comprehensive index HTI≥75%, it is identified as a heat-tolerant maize inbred line indoors and enters Step 4; Otherwise, it is identified as a heat-intolerant maize inbred line indoors and this operation ends; Step 4: Field verification: Conduct field verification on the heat-tolerant maize inbred lines identified indoors in Step 3. If the field verification meets the standards, it is judged as a truly heat-tolerant maize inbred line; if the field verification does not meet the standards, it is necessary to return to Step 1 to conduct indoor verification again.

2. The dual-verification screening method for heat tolerance of maize inbred lines based on multi-index joint evaluation of flowering periods as claimed in claim 1, wherein The specific steps of Step 4 are as follows: (41) The heat-tolerant maize inbred lines screened indoors are verified through sister line hybridization. The HTI of the hybrids assembled by the heat-tolerant maize inbred lines needs to be more than 10% higher than the average value of the heat-tolerant maize inbred line parents; (42) Set a treatment group and a control group for hybridization respectively, where: Treatment group: Heat-tolerant maize inbred line × Conventional maize inbred line; Control group: Sibling lines of heat-tolerant maize inbred lines × Conventional maize inbred lines, where: The conventional maize inbred line is Zheng 58, and the indoor HTI is 65%; (43) Evaluation of heat tolerance of hybrids: Perform the same high-temperature stress treatment as in Step 1 on the hybrids of the treatment group and the control group, and calculate their HTI. If the HTI of the hybrid assembled by the heat-tolerant inbred line ≥ (HTI of heat-tolerant maize inbred line + HTI of conventional maize inbred line) × 0.5 × 110%, then the field verification is qualified, and it is judged as a truly heat-tolerant maize inbred line; otherwise, if the field verification is unqualified, it is necessary to return to Step 1 to re-perform the indoor verification.

3. The dual-verification screening method for heat tolerance of maize inbred lines based on multi-index joint evaluation during the flowering period as claimed in claim 1, wherein The specific steps of the phenotypic consistency screening in the artificial climate chamber before sowing in Step (11) are as follows: (111) Number the i test materials respectively, sow each test material evenly in flower pots, sow 3 seeds in each flower pot, with a sowing depth of 5 cm, then gently cover a thin layer of soil, water thoroughly, and then place them in the artificial climate chamber for cultivation, and water, fertilize, and prevent and control pests and diseases regularly, where: i is a positive integer, i ≤ 500; The nutrient soil in the flower pot is evenly mixed by leaf mold, peat soil, and perlite in a ratio of 3:2:1; The temperature of the artificial climate chamber is controlled at 25 - 30 °C, the light intensity is 3000 - 5000 lux, the light time is 12 - 14 hours / day, and the air humidity is maintained at 60% - 70%; (112) According to the phenotypic characteristics of the test materials, determine the observation indexes, sort out the data of the indexes recorded in the observation, calculate the coefficient of variation of each index, and take the coefficient of variation ≤ 5% as the standard of good phenotypic consistency, where: The indexes include plant height, ear height, number of leaves, leaf color, leaf shape, stem diameter, number of branches of male inflorescence, color of silk of female inflorescence, and ear shape.

4. The double-verification screening method for heat tolerance of maize inbred lines based on multi-index joint evaluation of flowering stages as claimed in claim 3, wherein For qualitative traits such as leaf color, leaf shape, color of silk of female inflorescence, and ear shape, they need to be converted into quantifiable variation indexes, where: Leaf color: light green = 1; green = 2; dark green = 3; purple = 4; Leaf shape: lanceolate = 1, broad lanceolate = 2, oval = 3; Color of silk of female inflorescence: yellowish green = 1; light red = 2; dark red = 3; variegated = 4; Ear shape: cylindrical = 1; conical = 2.

5. The double-verification screening method for heat tolerance of maize inbred lines based on multi-index joint evaluation during the flowering period as claimed in claim 1, wherein In Step (12), the daytime temperature of the treatment group reaches the peak high temperature from 10:00 to 16:00 every day; The wavelength of the movable infrared radiator described in step (12) is 800 - 1200 nm, and the power density is 2.5 kW / m 2 .

6. The dual-verification screening method for heat tolerance of maize inbred lines based on multi-index joint evaluation during the flowering period according to claim 1, characterized in that In Step (12) during the pulsed high-temperature treatment, the female inflorescence of the treatment group is covered with a reflective aluminum foil sunshade to maintain the temperature around it ≤ 32 °C; In Step (12), the humidity of the control group and the treatment group is uniformly controlled at 60 - 70%.

7. The double-verification screening method for heat tolerance of maize inbred lines based on multi-index joint evaluation during the flowering period as described in claim 1, wherein The specific steps of the determination of jasmonic acid content in the male inflorescence in Step (221) are as follows: (2211) Sampling: At the tasseling stage, collect fresh male inflorescence tissues, quickly freeze them in liquid nitrogen, and store them at -80 °C for later use; (2212) Extraction: Take 0.5 g of the frozen sample, place it in a pre-cooled mortar, add liquid nitrogen and grind it into powder, add 3 - 5 mL of 80% methanol extract, extract at 4 °C for 4 h, centrifuge at 1000 g for 15 min, take the supernatant, and filter it through a 0.22 μm filter membrane to obtain the sample to be tested; (2213) Place the test sample obtained in step (2212) into a high performance liquid chromatograph for determination, where: The chromatographic conditions are as follows: Chromatographic column: C18 reversed-phase column; Mobile phase: Phase A is an aqueous formic acid solution with a mass concentration of 0.1%, and Phase B is an acetonitrile solution of formic acid with a mass concentration of 0.1%; Gradient elution: 0 - 5 min, 10% B → 90% B; 5 - 7 min, 90% B is maintained; 7 - 7.1 min, 90% B → 10% B; 7.1 - 10 min, 10% B is balanced; Flow rate: 0.3 mL / min, column temperature: 40 °C; Mass spectrometry conditions: Ion source: Electrospray ionization; Monitoring mode: Multiple reaction monitoring; Quantitative ion pairs: JA——m / z 209.1 → 59.0; internal standard D2-JA——m / z 211.1 → 61.0; (2214) Data processing Calculate the content of jasmonic acid in the tassel by the external standard method or the internal standard method; Furthermore, the specific steps for the determination of the ethylene content in the tassel in step (221) are as follows: (s2211) Sample treatment Sampling: Take 1 g of fresh tassel tissue and immediately put it into a 10 mL airtight syringe and seal it; Ethylene release: Place the syringe in a 25 °C incubator for dark incubation for 1 hour, and gently shake it once every 10 minutes during this period; (s2212) ETH gas collection Use a 1 mL airtight needle to extract 1 mL of the gas at the top of the syringe and inject it into the injection port of the gas chromatograph, where: The chromatographic conditions are as follows: Chromatographic column: Porapak Q packed column; Carrier gas: High-purity nitrogen, flow rate 30 mL / min; Detector: Hydrogen flame ionization detector (FID), temperature 250 °C; Column temperature: 80 °C, injection port temperature 150 °C; Standard curve: Draw a standard curve with ethylene standard gas; (s2213) Data calculation Calculate the ethylene content in the tassel according to the standard curve.

8. The double-verification screening method for heat tolerance of maize inbred lines based on multi-index joint evaluation during the flowering period as claimed in claim 1, wherein The specific steps for the determination of the polar transport rate of auxin in the ear in step (222) are as follows: (2221). Preparation of ear segments: Select ears with consistent growth, use a blade to cut them into segments with a length of 2 - 5 cm, retaining the morphological upper and lower ends, and immediately immerse the tissue segments in the transport buffer and pre-equilibrate them in the dark at 25 °C for 30 minutes to restore tissue activity, where: The pH value of the transport buffer is 5.5, and its composition is as follows: 1 mM KCl, 1 mM CaCl2, 1 mM MES, 1% sucrose, and the balance is water; (2222) Application of the marker: Dissolve fluorescently labeled IAA in the transport buffer and add 10 μL of the fluorescent IAA solution dropwise to the cut surface at the base of the ear; Blank control: Only add the transport buffer; Inhibitor control: Add a polar transport inhibitor to the buffer, lay the treated ear flat in a petri dish with moist filter paper, and incubate it at 25 °C in the dark for 2 - 4 hours, where: The polar transport inhibitor is NPA with a concentration of 10 μM; (2223) Transport detection: Terminate the transportation by quickly freezing the sample with liquid nitrogen, immediately perform frozen sectioning, control the section thickness at 20 - 50 μm, fix the sections on glass slides, observe the distribution of the marker with a fluorescence microscope, and use the image analysis software ImageJ to measure the transportation distance of the fluorescence signal; (2224) Data calculation Calculate the polar auxin transport rate in the ear by the following formula: Transport rate = transport distance / incubation time.

9. The double-verification screening method for heat tolerance of maize inbred lines based on multi-index joint evaluation during the flowering period as claimed in claim 1, wherein The specific steps for measuring the SOD activity of the ear leaf in step (231) are as follows: (2311) Enzyme solution extraction: Weigh 0.5 g of fresh ear leaf of the test material, add liquid nitrogen and grind it into powder, add 5 mL of pre-cooled SOD extraction buffer thereto, homogenize in an ice bath, centrifuge at 12,000×g for 20 minutes at 4°C, take the supernatant and divide it into aliquots to obtain the enzyme solution, and store it at -20°C for later use, where: The composition of the SOD extraction buffer is as follows: 50 mM phosphate buffer (pH 7.8) and containing 1 wt% PVP; 0.1 mM EDTA; (2312) SOD activity measurement: Establish a reaction system control group and a reaction system measurement group, where: Reaction system control group: 0.1 mL SOD extraction buffer + 2.9 mL SOD reaction solution; Reaction system measurement group: 0.1 mL SOD extraction buffer + 0.1 mL of the enzyme solution obtained in step (2311) + 2.8 mL SOD reaction solution; Light reaction: Mix the reaction system control group and the reaction system measurement group respectively and react under 4000 lux light for 15 minutes; Terminate the reaction: Let it stand in the dark for 5 minutes, and immediately measure the absorbance at 560 nm, where: The SOD reaction solution is made up to 3.0 mL / tube, and its components are as follows: 1.5 mL 50 mM phosphate buffer, pH 7.8; 0.3 mL 130 mM methionine; 0.3 mL 750 μM NBT; 0.3 mL 100 μM EDTA; 0.3 mL 100 μM riboflavin; (2313) Calculate the SOD activity of the ear leaf, and its calculation formula is as follows:

10. The dual-verification screening method for heat tolerance of maize inbred lines based on multi-index joint evaluation during the flowering period according to claim 1, wherein, The specific steps for measuring the POD activity of the ear leaf in step (232) are as follows: (2321) Enzyme solution extraction: Weigh 0.5 g of fresh ear leaf of the test material, add liquid nitrogen and grind it into powder, add 5 mL of pre-cooled POD extraction buffer thereto, homogenize in an ice bath, centrifuge at 12,000×g for 20 minutes at 4°C, take the supernatant and divide it into aliquots to obtain the enzyme solution, and store it at -20°C for later use, where: The composition of the POD extraction buffer is as follows: 50 mM phosphate buffer (pH 6.0) and containing 1 wt% PVP; 0.1 mM EDTA; (2322) POD activity measurement: Reaction system control group: 2 mL phosphate buffer + 0.9 mL o-methoxyphenol + 0.1 mL hydrogen peroxide + 0.1 mL ultrapure water; Reaction system measurement group: 2 mL phosphate buffer + 0.9 mL o-methoxyphenol + 0.1 mL hydrogen peroxide + 0.1 mL of the enzyme solution obtained in step (2321); Reaction initiation and detection: 30°C constant temperature water bath → immediately mix well → measure the absorbance at 470 nm (OD 470 ) for a total of 3 minutes at intervals of 30 seconds; (2323) Calculate the POD activity of the ear leaf, and its calculation formula is as follows:

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