Method for identifying high-temperature-resistant rice and application of high-temperature-resistant rice in breeding

Abstract

The invention relates to the technical field of high-temperature-resistant rice identification, and discloses a high-temperature-resistant rice identification method and application thereof in breeding. The identification method comprises the steps of rice seed pretreatment, three-leaf one-core stage seedling culture, seedling stage comprehensive scoring, booting stage gradient stress and parameter determination, pustulation stage gradient stress and parameter determination and rice identification grading. Gradient temperature stress is adopted to simulate field extreme high-temperature gradient, all index parameters at different gradient temperatures are combined, the high temperature resistance of the rice variety is jointly judged, the misjudgment probability is reduced, meanwhile, the conjoint analysis of the setting rate retention rate and the chalkiness increase value at the high temperature in the filling stage is conducted, the synergistic heat resistance indexes of the seedling stage and the adult-plant stage are integrated, and the high temperature resistance of the rice variety is obtained. And yield stability and quality under high temperature stress can be synchronously predicted.

Description

Technical Field

[0001] This invention relates to the field of heat-resistant rice identification technology, and more specifically, to a method for identifying heat-resistant rice and its application in breeding. Background Technology

[0002] Global warming has led to frequent extreme heat events, seriously threatening rice production. Statistics show that for every 1°C increase in the global average temperature, rice yields decrease by 6.6%–25%, accompanied by problems such as increased chalkiness and decreased palatability.

[0003] However, existing methods for identifying rice heat tolerance mostly rely on single temperature stress assessment during the greenhouse seedling stage, which differs significantly from the physiological response to actual high-temperature stress in the field (especially during the grain-filling stage), leading to a high misjudgment rate. Furthermore, they fail to integrate synergistic heat tolerance indicators from the seedling to mature plant stages, making it impossible to simultaneously assess the heat tolerance stability of yield and quality, thus hindering the breakthrough of the breeding bottleneck of high yield but low quality. Therefore, the present invention proposes a method for identifying heat-resistant rice, which has important practical significance. Summary of the Invention

[0004] In view of this, the present invention proposes a method for identifying heat-resistant rice and its application in breeding, aiming to solve at least one of the problems in the above-mentioned background art.

[0005] This invention proposes a method for identifying heat-resistant rice, comprising the following steps: After pretreatment, rice seeds of the variety to be identified were placed in an artificial climate chamber and cultured until the three-leaf-one-heart stage to obtain seedlings at the three-leaf-one-heart stage. Three-leaf-one-heart seedlings were subjected to high-temperature stress culture, while a control group was set up. The control group was cultured at room temperature. After the culture was completed, the seedling height growth rate, root-shoot ratio, proline content and relative conductivity of the stress group and the control group were measured. The seedlings were comprehensively scored based on the test results. After the scoring was completed, the plants were transplanted into pots, with 3 plants per pot, and then placed in a greenhouse for normal temperature cultivation. When the plants reached the booting stage, some plants were selected for gradient stress treatment during the booting stage. During this period, the first index parameter was measured on the plants subjected to gradient stress during the booting stage, and the first index parameter was obtained. The remaining plants were cultured normally. When the plants reached the grain-filling stage, a portion of the plants were selected for a gradient stress treatment during the grain-filling stage, and a normal temperature control group was set up. After the gradient stress treatment during the grain-filling stage was completed, the treated plants and the plants in the normal temperature control group were cultured at room temperature until maturity and the rice ears were harvested. During this period, the second index parameter of the gradient stress treatment plants and the normal temperature control group plants was measured to obtain the second index parameter. The weights of the temperature gradient parameters in the first and second index parameters are calculated to obtain the comprehensive values ​​of pollen vigor during the booting stage, Fv / Fm during the grain-filling stage, seed setting retention rate during the grain-filling stage, and chalkiness increase during the grain-filling stage for this rice variety. Based on the comprehensive values ​​of pollen vigor during the booting stage, Fv / Fm during the grain-filling stage, seed setting retention rate during the grain-filling stage, chalkiness increase during the grain-filling stage, and seedling stage comprehensive score, this rice variety is identified and graded.

[0006] Furthermore, the preprocessing is as follows: The rice seeds of the variety to be identified were disinfected by soaking in a 3% sodium hypochlorite solution for 20 minutes, rinsed with water three times, and then germinated at a constant temperature of 28°C for 48 hours before being sown in seedling trays containing seedling substrate.

[0007] 3. The method for identifying heat-resistant rice according to claim 2, characterized in that the high-temperature stress culture parameters are: The stress group was subjected to a temperature of 45℃ / 38℃ (day / night), a light exposure of 14 hours / day (light intensity 500 μmol·m⁻²·s⁻¹), a relative humidity of 65%, and a culture time of 7 days. The control group was maintained at a temperature of 28℃ / 22℃ (day / night), with a light exposure of 14 hours / day (light intensity of 500 μmol·m⁻²·s⁻¹), a relative humidity of 65%, and a culture time of 7 days.

[0008] Furthermore, the comprehensive seedling stage score is as follows: A comprehensive seedling score was calculated based on the seedling height growth rate, root-to-shoot ratio, proline content, and relative conductivity of the plants in the stress group and the control group. Among them, the seedling stage comprehensive score = (seedling height growth rate / 100 × 30) + (root-shoot ratio / 0.5 × 20) + (proline content / 200 × 30) + ((100 - relative conductivity) / 100 × 20).

[0009] Furthermore, the gradient stress treatment during the booting stage is as follows: When the sheathing rate of the sword leaves of the plants was ≥90%, 9 pots of plants were removed and transferred to an artificial climate chamber. Three temperature gradients were set up: T1 (34℃ / 27℃, day / night), T2 (36℃ / 29℃, day / night), and T3 (38℃ / 31℃, day / night). Three pots of plants were treated for each gradient. Each gradient stress lasted for 8 days, with 14 hours of light per day (light intensity 600 μmol·m-2·s-1), relative humidity 70%, and daily watering to maintain soil moisture content at 80% of field capacity.

[0010] Furthermore, the first index parameter is measured as follows: After the gradient stress treatment during the booting stage was completed, small florets from the middle of the panicles of rice plants at each temperature gradient were taken, stained with 1% iodine-potassium iodide solution, and observed under a microscope for pollen viability. The proportion of fertile pollen that stained dark blue was counted as pollen viability, and the pollen viability of plants at different temperature gradients was obtained.

[0011] Furthermore, the gradient stress treatment during the grouting period is as follows: Five days after more than 80% of the rice panicles had completed heading, nine pots of plants were removed and transferred to an artificial climate chamber. Three temperature gradients were set up: T1 (35℃ / 28℃, day / night), T2 (37℃ / 30℃, day / night), and T3 (39℃ / 32℃, day / night). Three pots of plants were treated for each gradient, and the remaining plants were cultured at room temperature as a control group. Each gradient stress lasted for 15 days. Strong light (light intensity ≥750μmol·m-2·s-1) was provided daily from 9:00 to 17:00, and light intensity was 600μmol·m-2·s-1 at other times. The relative humidity was 65%, and the soil moisture content was maintained at 80% of the field capacity by regular watering.

[0012] Furthermore, the second indicator parameter is measured as follows: At 10:00 AM each day during the last 5 days (days 11-15) of the gradient stress treatment during the grouting period, the Fv / Fm values ​​of the flag leaves of the plants at three temperature gradients were measured, and the average value was taken as the Fv / Fm value of the plants at the corresponding temperature gradients. After the gradient stress treatment during the grain filling period was completed, the plants were transferred to a greenhouse for normal temperature cultivation until maturity, and then the rice ears were harvested. The seed setting rate, thousand-grain weight, and chalkiness of the plants at the three temperature gradients and the normal temperature control group were measured respectively. The seed setting rate retention rate, thousand-grain weight retention rate, and chalkiness increase of the plants at the three temperature gradients relative to the normal temperature control group were calculated.

[0013] Furthermore, when the comprehensive score during the seedling stage is ≥80 points, the comprehensive value of pollen viability during the booting stage is ≥82%, the comprehensive value of Fv / Fm during the grain-filling stage is ≥0.82, the comprehensive value of seed setting retention rate during the grain-filling stage is ≥88%, and the comprehensive value of chalkiness increase during the grain-filling stage is ≤3.5%, this rice variety is judged to be a Grade 1 heat-resistant rice. When the comprehensive score during the seedling stage is 70-79 points, the comprehensive value of pollen viability during the booting stage is 65%-81%, the comprehensive value of Fv / Fm during the grain-filling stage is 0.76-0.81, the comprehensive value of seed setting retention rate during the grain-filling stage is 72%-87%, and the comprehensive value of chalkiness increase during the grain-filling stage is 3.6%-6.5%, this rice variety is judged to be a Grade 2 heat-resistant rice. When the comprehensive score during the seedling stage is <70 points, or the comprehensive value of pollen viability during the booting stage is <65%, or the comprehensive value of Fv / Fm during the grain-filling stage is <0.76, or the comprehensive value of seed setting retention rate during the grain-filling stage is <72%, or the comprehensive value of chalkiness increase during the grain-filling stage is >6.5%, this rice variety is judged to be a high-temperature sensitive rice.

[0014] This invention also proposes an application of a method for identifying heat-resistant rice in the breeding of heat-resistant rice.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention uses gradient temperature stress to simulate extreme high temperature gradients in the field, and combines various index parameters under different gradient temperatures to jointly determine the high temperature resistance of rice varieties, thereby reducing the probability of misjudgment.

[0016] 2. This invention combines the analysis of the retention rate of seed setting rate and the increase in chalkiness under high temperature during the grain filling period, and integrates the synergistic heat resistance index from the seedling stage to the mature plant stage, which can simultaneously predict the yield stability and quality under high temperature stress. Detailed Implementation

[0017] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0018] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0019] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0020] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0021] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0022] This invention provides a method for identifying heat-resistant rice, comprising the following preparation steps: After pretreatment, rice seeds of the variety to be identified were placed in an artificial climate chamber and cultured until the three-leaf-one-heart stage to obtain seedlings at the three-leaf-one-heart stage. Three-leaf-one-heart seedlings were subjected to high-temperature stress culture, while a control group was set up. The control group was cultured at room temperature. After the culture was completed, the seedling height growth rate, root-shoot ratio, proline content and relative conductivity of the stress group and the control group were measured. The seedlings were comprehensively scored based on the test results. After the scoring was completed, the plants were transplanted into pots, with 3 plants per pot, and then placed in a greenhouse for normal temperature cultivation. When the plants reached the booting stage, some plants were selected for gradient stress treatment during the booting stage. During this period, the first index parameter was measured on the plants subjected to gradient stress during the booting stage, and the first index parameter was obtained. The remaining plants were cultured normally. When the plants reached the grain-filling stage, a portion of the plants were selected for a gradient stress treatment during the grain-filling stage, and a normal temperature control group was set up. After the gradient stress treatment during the grain-filling stage was completed, the treated plants and the plants in the normal temperature control group were cultured at room temperature until maturity and the rice ears were harvested. During this period, the second index parameter of the gradient stress treatment plants and the normal temperature control group plants was measured to obtain the second index parameter. The weights of the temperature gradient parameters in the first and second index parameters are calculated to obtain the comprehensive values ​​of pollen vigor during the booting stage, Fv / Fm during the grain-filling stage, seed setting retention rate during the grain-filling stage, and chalkiness increase during the grain-filling stage for this rice variety. Based on the comprehensive values ​​of pollen vigor during the booting stage, Fv / Fm during the grain-filling stage, seed setting retention rate during the grain-filling stage, chalkiness increase during the grain-filling stage, and seedling stage comprehensive score, this rice variety is identified and graded.

[0023] Specifically, after pretreatment, the rice seeds of the varieties to be identified were cultured in an artificial climate chamber until the three-leaf-one-heart stage. The preferred culture conditions were a temperature of 28℃ / 22℃ (day / night) and 14 hours / day of light (light intensity of 500 μmol·m⁻¹). -2 ·s -1 ), relative humidity 70%.

[0024] Specifically, after the scoring was completed, the seedlings were transplanted into pots (30cm in diameter and 40cm in height) filled with paddy soil (2.5% organic matter content, pH 6.5), with 3 seedlings transplanted into each pot. They were then placed in a greenhouse for conventional cultivation under the following conditions: temperature 28℃ / 22℃ (day / night), natural light, relative humidity 75%, and regular watering and fertilization (using compound fertilizer at a ratio of N:P:K=1:0.5:1, 5g per pot per month). Specifically, after the gradient stress treatment during the grain-filling stage, the plants were transferred to a greenhouse for normal cultivation until maturity, with the same cultivation parameters as normal cultivation after the stress during the heading stage (temperature 28℃ / 22℃ (day / night), natural light + LED supplemental lighting (light intensity ≥400μmol·m). -2 ·s -1 (14 hours of sunlight / day, 75% relative humidity, 70%-80% soil moisture content, apply 3g of compound fertilizer per pot every 15 days) It is understandable that this invention uses gradient temperature stress to simulate extreme high temperature gradients in the field, and combines various index parameters under different gradient temperatures to jointly determine the high temperature resistance of rice varieties, thereby reducing the probability of misjudgment.

[0025] It is understandable that this invention combines the analysis of the retention rate of seed setting rate and the increase in chalkiness under high temperature during the grain filling period, and integrates the synergistic heat resistance index from the seedling stage to the mature plant stage, so as to simultaneously predict the yield stability and quality under high temperature stress.

[0026] In this invention, the preprocessing is as follows: The rice seeds of the variety to be identified were disinfected by soaking in a 3% sodium hypochlorite solution for 20 minutes, rinsed with water three times, and then germinated at a constant temperature of 28°C for 48 hours before being sown in seedling trays containing seedling substrate.

[0027] Specifically, rice seeds to be identified were selected, disinfected by soaking in a 3% sodium hypochlorite solution for 20 minutes, rinsed three times with distilled water, and placed in a 28℃ constant temperature incubator for 48 hours to germinate. After the seeds showed signs of sprouting, they were sown in seedling trays (50-well × 30-well) containing rice-specific seedling substrate (vermiculite: peat: perlite = 3:2:1), with one seed sown in each well. The trays were then placed in an artificial climate chamber for cultivation until the three-leaf stage.

[0028] Understandably, disinfection with 3% sodium hypochlorite removes pathogens from the seed surface to avoid seedling disease interference, constant temperature germination at 28℃ ensures uniform seed germination, a special seedling substrate (vermiculite: peat: perlite = 3:2:1) and standardized sowing methods provide a stable growth foundation, and artificial climate chamber cultivation can precisely control temperature, light and humidity conditions to reduce external fluctuations, ultimately cultivating seedlings with uniform growth at the three-leaf-one-heart stage, ensuring the reliability and repeatability of subsequent high-temperature pretreatment and various indicator measurements.

[0029] In this invention, the high-temperature stress culture parameters are: The stress group was subjected to a temperature of 45℃ / 38℃ (day / night) and a light exposure time of 14 hours / day (light intensity 500 μmol·m). -2 ·s -1 The relative humidity was 65%, and the incubation time was 7 days. The control group was maintained at a temperature of 28℃ / 22℃ (day / night) and a light exposure time of 14 hours / day (light intensity 500 μmol·m). -2 ·s -1 The relative humidity was 65%, and the incubation time was 7 days.

[0030] It is understandable that stress temperatures of 45℃ / 38℃ (day / night) can induce a heat-tolerant physiological response in seedlings, with a stress level of 14 hours / day and 500 μmol·m⁻². -2 ·s -1 The light intensity and 65% relative humidity ensured a stable and controllable stress environment, which lasted for 7 days and fully demonstrated the differences in heat resistance of the materials. At the same time, a normal temperature control group of 28℃ / 22℃ was set up. By comparing the seedling height growth rate, root-shoot ratio and other indicators between the stress group and the control group, materials with good growth and physiological status under high temperature can be accurately screened, providing a reliable early screening basis for subsequent identification of mature plants.

[0031] In this invention, the seedling stage comprehensive score is: A comprehensive seedling score was calculated based on the seedling height growth rate, root-to-shoot ratio, proline content, and relative conductivity of the plants in the stress group and the control group. Among them, the seedling stage comprehensive score = (seedling height growth rate / 100 × 30) + (root-shoot ratio / 0.5 × 20) + (proline content / 200 × 30) + ((100 - relative conductivity) / 100 × 20).

[0032] Specifically, the seedling height growth rate was calculated by measuring the distance from the base of the seedlings to the highest leaf tip in both the stress group and the control group using a ruler, and then calculating the average distance. The seedling height growth rate was calculated as follows: (average seedling height in the stress group / average seedling height in the control group) × 100%. Root-to-shoot ratio: Remove the seedlings from the substrate, rinse the roots with distilled water to remove impurities, absorb the moisture with filter paper, and weigh the average fresh weight of the underground part (roots) and the average fresh weight of the above-ground part (stems, sheaths and leaves) respectively. Calculate the root-to-shoot ratio = average fresh weight of roots / average fresh weight of above-ground part. Proline content: The acidic ninhydrin method was used. 0.5g leaf samples from the stress group were taken, and 3mL of 3% sulfosalicylic acid solution was added. The samples were homogenized, heated in a boiling water bath for 10 minutes, cooled, and centrifuged (3000rpm, 10 minutes). 1mL of the supernatant was collected, and 1mL of glacial acetic acid and 1mL of 2.5% ninhydrin reagent were added. The mixture was heated in a boiling water bath for 30 minutes, cooled, and extracted with toluene. The absorbance at 520nm was measured, and the proline content (unit: μg·g) was calculated using a standard curve. -1 FW), and calculate the average value; Relative conductivity: Take 0.2g leaf samples from the stress group plants, cut them into 0.5cm segments, add 20mL of distilled water, shake for 30 minutes, and then measure the initial conductivity (EC1) using a DDS-307 conductivity meter. Then, boil in water for 10 minutes, cool to room temperature, and measure the terminal conductivity (EC2). Calculate the relative conductivity = (EC1 / EC2) × 100%, and calculate the average value.

[0033] It is understandable that this invention incorporates seedling height growth rate (weight 30%), which reflects the aboveground growth status, root-shoot ratio (weight 20%), which reflects the coordination between the root system and the aboveground parts, proline content (weight 30%), which characterizes osmotic regulation capacity, and relative conductivity (weight 20%), which reflects cell membrane stability, into the calculation. By allocating reasonable weights, the role of key heat resistance indicators is highlighted, and the one-sidedness of evaluating a single indicator is avoided.

[0034] In this invention, the gradient stress treatment during the booting stage is as follows: When the sheathing rate of the sword leaves reached ≥90%, nine pots of plants were removed and transferred to an artificial climate chamber. Three temperature gradients were set up: T1 (34℃ / 27℃, day / night), T2 (36℃ / 29℃, day / night), and T3 (38℃ / 31℃, day / night). Three pots of plants were treated for each gradient, and the stress at each gradient lasted for 8 days, with a light intensity of 600 μmol·m⁻¹ per day (14 hours of light per day). -2 ·s -1 The relative humidity is 70%, and the soil moisture content is maintained at 80% of the field capacity by watering regularly every day.

[0035] Specifically, the period from when the flag leaf of the plant is fully unsheathed (unsheathing rate ≥90%) to 3 days before heading is the heading stage of the plant.

[0036] In this invention, the first index parameter is measured as follows: After the gradient stress treatment during the booting stage was completed, small florets from the middle of the panicles of rice plants at each temperature gradient were taken, stained with 1% iodine-potassium iodide solution, and observed under a microscope for pollen viability. The proportion of fertile pollen that stained dark blue was counted as pollen viability, and the pollen viability of plants at different temperature gradients was obtained.

[0037] Understandably, this study aims to systematically evaluate the heat tolerance of rice materials during the crucial reproductive growth stage—the booting stage—by simulating varying intensities of high temperature that may occur in the field, ranging from mild to extreme. Specifically, plants were treated for eight days under three increasing diurnal temperature gradients: T1 (34 / 27℃), T2 (36 / 29℃), and T3 (38 / 31℃), to precisely quantify the impact of high temperatures on pollen development. This design not only screens out valuable materials that maintain high pollen viability even under extreme high temperatures (T3), but also identifies the stability of their heat tolerance by analyzing their performance curves across different gradients. This provides crucial phenotypic data and scientific basis for breeding new rice varieties with reliable high-temperature adaptability.

[0038] In this invention, the gradient stress treatment during the grouting period is as follows: Five days after more than 80% of the rice panicles had completed heading, nine pots of plants were removed and transferred to an artificial climate chamber. Three temperature gradients were set up: T1 (35℃ / 28℃, day / night), T2 (37℃ / 30℃, day / night), and T3 (39℃ / 32℃, day / night). Three pots of plants were treated with each gradient, and the remaining plants were cultured at room temperature as a control group. Each gradient stress lasted for 15 days, with strong light (light intensity ≥750 μmol·m²) provided daily from 9:00 to 17:00. -2 ·s -1 The light intensity was 600 μmol·m for the rest of the time. -2 ·s -1 The relative humidity is 65%, and the soil moisture content is maintained at 80% of the field capacity by watering regularly every day.

[0039] In this invention, the gradient stress treatment during the grouting period is as follows: Five days after more than 80% of the rice panicles had completed heading, nine pots of plants were removed and transferred to an artificial climate chamber. Three temperature gradients were set up: T1 (35℃ / 28℃, day / night), T2 (37℃ / 30℃, day / night), and T3 (39℃ / 32℃, day / night). Three pots of plants were treated with each gradient, and the remaining plants were cultured at room temperature as a control group. Each gradient stress lasted for 15 days, with strong light (light intensity ≥750 μmol·m²) provided daily from 9:00 to 17:00. -2 ·s -1 The light intensity was 600 μmol·m for the rest of the time. -2 ·s -1 The relative humidity is 65%, and the soil moisture content is maintained at 80% of the field capacity by watering regularly every day.

[0040] Specifically, the plant enters the grain-filling stage on the 5th day after more than 80% of the rice panicles have completed heading (the top of the panicle emerges from the sheath of the sword leaf).

[0041] In this invention, the second index parameter is measured as follows: At 10:00 AM each day during the last 5 days (days 11-15) of the gradient stress treatment during the grouting period, the Fv / Fm values ​​of the flag leaves of the plants at three temperature gradients were measured, and the average value was taken as the Fv / Fm value of the plants at the corresponding temperature gradients. After the gradient stress treatment during the grain filling period was completed, the plants were transferred to a greenhouse for normal temperature cultivation until maturity, and then the rice ears were harvested. The seed setting rate, thousand-grain weight, and chalkiness of the plants at the three temperature gradients and the normal temperature control group were measured respectively. The seed setting rate retention rate, thousand-grain weight retention rate, and chalkiness increase of the plants at the three temperature gradients relative to the normal temperature control group were calculated.

[0042] Specifically, after maturity, the plants are harvested, and the seed setting rate (number of seeds / total number of florets × 100%) and the weight of 1,000 seeds are measured (1,000 plump seeds are randomly selected and weighed, and the average value is taken after repeating 3 times). Specifically, after the harvested grains are processed by rice hulling and polishing machines, the chalkiness (the percentage of chalky area to the area of ​​the rice grain) is measured using an SC-E type rice appearance quality tester.

[0043] Specifically, the retention rate or increase value of the index under each temperature gradient is calculated, where the seed setting rate retention rate = (seed setting rate of the stress group / seed setting rate of the control group) × 100%, the thousand-grain weight retention rate = (thousand-grain weight of the stress group / thousand-grain weight of the control group) × 100%, and the chalkiness increase value = chalkiness of the stress group - chalkiness of the control group.

[0044] Understandably, by simulating varying degrees of combined high temperature and intense light stress during the grain-filling stage, this study specifically evaluates the photosynthetic stability, grain filling capacity, and heat resistance of rice materials at the most critical stages of yield and quality formation. This design, by setting a temperature gradient from T1 (35 / 28℃) to T3 (39 / 32℃) supplemented with intense light, precisely quantifies the gradient effects of high temperature on the photosynthetic system (reflected by Fv / Fm values), yield composition (reflected by seed setting retention rate), and grain quality (reflected by chalkiness). This allows for the identification of valuable materials that can simultaneously maintain high yield and excellent appearance quality under extreme high temperatures, providing an identification process and scientific basis for breeding new rice varieties with comprehensive traits of "high yield, high quality, and resistance."

[0045] In this invention, when the seedling stage comprehensive score is ≥80 points, the pollen viability comprehensive value during the booting stage is ≥82%, the Fv / Fm comprehensive value during the grain filling stage is ≥0.82, the grain filling retention rate comprehensive value during the grain filling stage is ≥88%, and the chalkiness increase value during the grain filling stage is ≤3.5%, this rice variety is judged to be a Grade 1 heat-resistant rice. When the comprehensive score during the seedling stage is 70-79 points, the comprehensive value of pollen viability during the booting stage is 65%-81%, the comprehensive value of Fv / Fm during the grain-filling stage is 0.76-0.81, the comprehensive value of seed setting retention rate during the grain-filling stage is 72%-87%, and the comprehensive value of chalkiness increase during the grain-filling stage is 3.6%-6.5%, this rice variety is judged to be a Grade 2 heat-resistant rice. When the comprehensive score during the seedling stage is <70 points, or the comprehensive value of pollen viability during the booting stage is <65%, or the comprehensive value of Fv / Fm during the grain-filling stage is <0.76, or the comprehensive value of seed setting retention rate during the grain-filling stage is <72%, or the comprehensive value of chalkiness increase during the grain-filling stage is >6.5%, this rice variety is judged to be a high-temperature sensitive rice.

[0046] Specifically, the pollen viability during the booting stage was calculated using pollen viability measured at different temperature gradients under gradient stress treatments during the booting stage. The comprehensive pollen viability during the booting stage = pollen viability at temperature gradient T1 × 0.2 + pollen viability at temperature gradient T2 × 0.3 + pollen viability at temperature gradient T3 × 0.5. Specifically, the comprehensive values ​​of Fv / Fm, grain retention rate, and chalkiness increase during the grouting period were calculated using the Fv / Fm values, grain retention rate, and chalkiness increase value measured at different temperature gradients during the gradient stress treatment during the grouting period. Wherein, the comprehensive value of Fv / Fm during the grouting period = the average value of Fv / Fm of temperature gradient T1 × 0.2 + the average value of Fv / Fm of temperature gradient T2 × 0.3 + the average value of Fv / Fm of temperature gradient T3 × 0.5; The comprehensive value of the grouting period retention rate = retention rate of grouting under temperature gradient T1 × 0.2 + retention rate of grouting under temperature gradient T2 × 0.3 + retention rate of grouting under temperature gradient T3 × 0.5; The comprehensive increase in chalkiness during the grouting period = chalkiness increase during temperature gradient T1 × 0.2 + chalkiness increase during temperature gradient T2 × 0.3 + chalkiness increase during temperature gradient T3 × 0.5; Specifically, the heat resistance ranking is: Grade 1 high-temperature resistant rice > Grade 2 high-temperature resistant rice > High-temperature sensitive rice. Understandably, the following approach is adopted: First, the weighting is allocated according to T3 (extreme high temperature) > T2 (relatively high temperature) > T1 (moderate high temperature). The full gradient data of core indicators such as pollen viability during the booting stage and Fv / Fm value during the grain filling stage are calculated into a single comprehensive value. This retains the performance information under each temperature intensity and highlights the heat resistance limit by giving high weight to extreme high temperature. Then, based on the comprehensive score of the seedling stage and the comprehensive value of each indicator, a clear threshold is set. The high heat resistance, relatively heat resistance and sensitive levels are divided according to the standard of multiple indicators meeting the standard simultaneously. This achieves a systematic heat resistance judgment from the seedling stage to the mature stage, and the synergy between yield and quality, ensuring the scientific and practical nature of the identification results.

[0047] This invention also provides the application of the method for identifying heat-resistant rice in the breeding of heat-resistant rice.

[0048] The applications include providing identification methods and scientific basis for breeding new rice varieties with comprehensive superior traits such as "high yield, high quality and resistance".

[0049] Example 1 S1. Take 200 seeds of Liangyou 8022 rice, disinfect them by soaking in a 3% sodium hypochlorite solution for 20 minutes, rinse them three times with distilled water, and place them in a 28℃ constant temperature incubator for 48 hours to germinate. After the seeds show white sprouts, sow them in seedling trays (50-cell × 30-cell) filled with rice-specific seedling substrate (vermiculite: peat: perlite = 3:2:1), sowing one seed per cell. Place them in an artificial climate chamber for cultivation until the three-leaf stage. The cultivation conditions are: temperature 28℃ / 22℃ (day / night), light 14 hours / day (light intensity 500 μmol·m²). -2 ·s -1 ), relative humidity 70%; S2. Twenty seedlings at the three-leaf-one-heart stage were transferred to another artificial climate chamber as the stress group. The stress conditions were set as follows: 45℃ / 38℃ (day / night) and 14 hours / day of light (light intensity 500 μmol·m⁻¹). -2 ·s -1 The relative humidity was 65% for 7 days. The remaining three-leaf-one-heart seedlings were used as the control group. The culture conditions were 28℃ / 22℃ (day / night). Other environmental parameters were the same as those of the stress group. After the stress was over, the seedling height growth rate, root-shoot ratio, proline content and relative conductivity of the plants in the stress group and the control group were measured according to the above method. The comprehensive seedling score of the variety was calculated according to the formula. S3. Transplant the plants from the control group into pots (30cm in diameter and 40cm in height) filled with paddy soil (2.5% organic matter content, pH 6.5), with 3 plants per pot. Place them in a greenhouse for conventional cultivation under the following conditions: temperature 28℃ / 22℃ (day / night), natural light, relative humidity 75%. Water and fertilize regularly (apply compound fertilizer at a ratio of N:P:K=1:0.5:1, 5g per pot per month). S4. When the flag leaf of the plant has fully emerged from its sheath (emergence rate ≥90%), remove 9 pots of plants and transfer them to an artificial climate chamber. Set up 3 temperature gradients: T1 (34℃ / 27℃, day / night), T2 (36℃ / 29℃, day / night), and T3 (38℃ / 31℃, day / night). Treat 3 pots of plants for each gradient. Each gradient stress lasts for 8 days, with 14 hours of light per day (light intensity 600 μmol·m²). -2 ·s -1With a relative humidity of 70%, the soil was watered regularly every day to maintain the soil moisture content at 80% of the field capacity. After the stress ended, the pollen viability was observed under a microscope after staining with 1% iodine-potassium iodide solution. The percentage of fertile pollen that stained dark blue was counted as pollen viability, and the pollen viability of plants at different temperature gradients was obtained. S5. The remaining plants were cultured at room temperature in the greenhouse (28℃ / 22℃ day / night) until more than 80% of the panicles had completed heading (the panicle tip exposed the flag leaf sheath). On the 5th day after this, 9 pots of plants were randomly selected and transferred to the artificial climate chamber. The remaining plants were cultured at room temperature in the greenhouse as a control group. Three temperature gradients were set up in the artificial climate chamber: T1 (35℃ / 28℃, day / night), T2 (37℃ / 30℃, day / night), and T3 (39℃ / 32℃, day / night). Three pots of plants were treated for each gradient. Each gradient stress lasted for 15 days, with strong light (light intensity ≥750μmol·m²) provided daily from 9:00 to 17:00. -2 ·s -1 The light intensity was 600 μmol·m for the rest of the time. -2 ·s -1 With a relative humidity of 65%, the soil moisture content was maintained at 70%-80% of the field capacity by regular daily watering. During each temperature gradient, the Fv / Fm value of the flag leaf was measured at 10:00 am every day at 5 days after the stress period (days 11-15) using a PAM-2500 chlorophyll fluorometer (measured 30 minutes after dark adaptation). The average value was calculated as the representative Fv / Fm value for that gradient. S6. After completion, the plants were transferred to a greenhouse and cultured at room temperature with the remaining plants. The cultivation parameters were: temperature 28℃ / 22℃ (day / night), natural light + LED supplemental lighting (light intensity ≥400μmol·m). -2 ·s -1 The conditions were: 14 hours of light per day, 75% relative humidity, 70%-80% soil moisture content, and 3g of compound fertilizer per pot applied every 15 days. After the rice ears matured, the seed setting rate, thousand-grain weight, and chalkiness of the plants in the three temperature gradients and the normal temperature control group were measured. The seed setting rate retention rate, thousand-grain weight retention rate, and chalkiness increase of the three temperature gradient plants relative to the normal temperature control group were calculated according to the above formula. S7. The weights of the temperature gradient parameters in the first and second indicator parameters are calculated according to the above formula to obtain the comprehensive values ​​of pollen vigor during the booting stage, Fv / Fm during the grain-filling stage, seed setting retention rate during the grain-filling stage, and chalkiness increase during the grain-filling stage for this rice variety. Based on these comprehensive values, along with the seedling stage comprehensive score, the rice variety is identified and graded. The criteria are as follows: When the comprehensive score during the seedling stage is ≥80 points, the comprehensive value of pollen viability during the booting stage is ≥82%, the comprehensive value of Fv / Fm during the grain filling stage is ≥0.82, the comprehensive value of seed setting retention rate during the grain filling stage is ≥88%, and the comprehensive value of chalkiness increase during the grain filling stage is ≤3.5%, this rice variety is judged to be a Grade 1 heat-resistant rice. When the comprehensive score during the seedling stage is 70-79 points, the comprehensive value of pollen viability during the booting stage is 65%-81%, the comprehensive value of Fv / Fm during the grain-filling stage is 0.76-0.81, the comprehensive value of seed setting retention rate during the grain-filling stage is 72%-87%, and the comprehensive value of chalkiness increase during the grain-filling stage is 3.6%-6.5%, this rice variety is judged to be a Grade 2 heat-resistant rice. When the comprehensive score at the seedling stage is <70 points, or the comprehensive pollen viability value at the booting stage is <65%, or the comprehensive Fv / Fm value at the grain-filling stage is <0.76, or the comprehensive value of the grain-filling retention rate is <72%, or the comprehensive value of the chalkiness increase value at the grain-filling stage is >6.5%, this rice variety is judged to be a high-temperature sensitive rice. When the comprehensive score at the seedling stage is ≥80 points, the comprehensive pollen viability value at the booting stage is ≥82%, the comprehensive Fv / Fm value at the grain-filling stage is ≥0.82, the comprehensive value of the grain-filling retention rate is ≥88%, and the comprehensive value of the chalkiness increase value at the grain-filling stage is ≤3.5%, this rice variety is judged to be a Grade 1 high-temperature resistant rice. When the comprehensive score during the seedling stage is 70-79 points, the comprehensive value of pollen viability during the booting stage is 65%-81%, the comprehensive value of Fv / Fm during the grain-filling stage is 0.76-0.81, the comprehensive value of seed setting retention rate during the grain-filling stage is 72%-87%, and the comprehensive value of chalkiness increase during the grain-filling stage is 3.6%-6.5%, this rice variety is judged to be a Grade 2 heat-resistant rice. When the comprehensive score during the seedling stage is <70 points, or the comprehensive value of pollen viability during the booting stage is <65%, or the comprehensive value of Fv / Fm during the grain-filling stage is <0.76, or the comprehensive value of seed setting retention rate during the grain-filling stage is <72%, or the comprehensive value of chalkiness increase during the grain-filling stage is >6.5%, this rice variety is judged to be a high-temperature sensitive rice.

[0050] Example 2 The only difference from Example 1 is that the rice variety is Weiliangyou 2133.

[0051] Example 3 The only difference from Example 1 is that the rice variety is Nongxiang 32.

[0052] Test results: The seedling stage comprehensive scoring results of Examples 1-3 are shown in Table 1: Table 1. Comprehensive scoring results during the seedling stage

[0053] The results of the determination of the first index parameter (plant pollen viability) under different temperature gradients in the gradient stress treatments during the booting stage of Examples 1-3 are shown in Table 2: Table 2 Results of the first indicator parameter (plant pollen viability)

[0054] The results of measuring the second index parameters (Fv / Fm value, grain filling rate retention rate, thousand-grain weight retention rate, and chalkiness increase value) under different temperature gradients during the grouting period gradient stress treatments of Examples 1-3 are shown in Table 3: Table 3. Measurement results of the second indicator parameter

[0055] The final identification results of the rice varieties described in Examples 1-3 are shown in Table 4: Table 4. Identification Results

[0056] The rice varieties Liliangyou 8022 and Weiliangyou 2133 used in Examples 1 and 2 are high-temperature resistant rice varieties bred by the Hunan Hybrid Rice Research Center, while Nongxiang 32 is a high-temperature sensitive rice variety. It can be seen that the high-temperature resistant rice identification method of the present invention accurately identified the two high-temperature resistant varieties, Liliangyou 8022 and Weiliangyou 2133.

[0057] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A method for identifying high temperature tolerant rice, characterized by, The method comprises the following steps: The seeds of the rice variety to be identified are pretreated and then placed in a phytotron for culture to the three-leaf-one-heart stage to obtain seedlings at the three-leaf-one-heart stage; Part of the seedlings at the three-leaf-one-heart stage are taken out for high-temperature stress culture, and a control group is set up at the same time, the control group is cultured at normal temperature, and after the culture is completed, the seedling height growth rate, root-shoot ratio, proline content and relative conductivity of the stress group and the control group are detected, and a seedling stage comprehensive score is made according to the detection results; After the scoring is completed, the remaining plants are transplanted into pots, 3 plants per pot, and then placed in a greenhouse for normal temperature culture, when the culture reaches the booting stage, part of the plants are selected for gradient stress treatment at the booting stage, during which, the first index parameter of the plants at the booting stage is determined to obtain the first index parameter; The remaining plants are normally cultured, when the culture reaches the grain filling stage, part of the plants are selected for gradient stress treatment at the grain filling stage, and a normal temperature control group is set up, after the gradient stress treatment at the grain filling stage is completed, the treated plants and the plants of the normal temperature control group are cultured at normal temperature to maturity and the rice panicles are harvested, during which, the second index parameter of the plants at the grain filling stage and the plants of the normal temperature control group is determined to obtain the second index parameter; The index parameters of each temperature gradient in the first index parameter and the second index parameter are subjected to weight calculation to obtain the comprehensive value of pollen viability at the booting stage, the comprehensive value of Fv / Fm at the grain filling stage, the comprehensive value of seed setting rate retention rate at the grain filling stage and the comprehensive value of chalkiness increase at the grain filling stage of the rice variety, and the rice variety is identified and classified according to the comprehensive value of pollen viability at the booting stage, the comprehensive value of Fv / Fm at the grain filling stage, the comprehensive value of seed setting rate retention rate at the grain filling stage, the comprehensive value of chalkiness increase at the grain filling stage and the seedling stage comprehensive score.

2. The high temperature tolerant rice identification method of claim 1, wherein, The pretreatment comprises: The seeds of the rice variety to be identified are soaked in a 3% volume concentration sodium hypochlorite solution for disinfection for 20 minutes, then rinsed with water for 3 times, then incubated at 28°C for 48 hours, and then sown in a seedling tray filled with seedling substrate.

3. The high temperature tolerant rice identification method of claim 2, wherein, The high-temperature stress culture parameters comprise: The temperature of the stress group is 45℃ / 38℃ (day / night), the light time is 14 hours / day (light intensity 500 μmol·m -2 ·s -1 ), the relative humidity is 65%, and the culture time is 7 days; The temperature of the control group was 28℃ / 22℃ (day / night), the light time was 14 hours / day (light intensity 500 μmol·m -2 ·s -1 -2), the relative humidity was 65%, and the culture time was 7 days.

4. The high temperature tolerant rice identification method of claim 3, wherein, The seedling stage comprehensive score comprises: The seedling stage comprehensive score is made according to the seedling height growth rate, root-shoot ratio, proline content and relative conductivity of the plants of the stress group and the control group; The seedling stage comprehensive score = (seedling height growth rate / 100×30) + (root-shoot ratio / 0.5×20) + (proline content / 200×30) + ((100-relative conductivity) / 100×20).

5. The high temperature tolerant rice identification method of claim 4, wherein, The gradient stress treatment at the booting stage comprises: When the emergence rate of the plant's flag leaf is ≥ 90%, 9 pots of plants are removed and transferred to a phytotron, 3 temperature gradients are set, T1 (34℃ / 27℃, day / night), T2 (36℃ / 29℃, day / night), T3 (38℃ / 31℃, day / night), 3 pots of plants are treated in each gradient, each gradient stress lasts for 8 days, the light is 14 hours / day (light intensity 600 μmol·m -2 ·s -1 ), the relative humidity is 70%, and the soil water content is kept at 80% of the field maximum water holding capacity by timed watering every day.

6. The high-temperature tolerant rice identification method of claim 5, wherein, The first index parameter determination comprises: After the gradient stress treatment at the booting stage is completed, the middle flowers of the rice panicles of the plants at each temperature gradient are taken out, dyed with 1% iodine-potassium iodide solution, and then observed under a microscope to observe the pollen viability, and the percentage of fertile pollen dyed dark blue is taken as the pollen viability to obtain the pollen viability of the plants at different temperature gradients.

7. The high-temperature tolerant rice identification method of claim 6, wherein, The gradient stress treatment at the grain filling stage comprises: On the 5th day after more than 80% of the plants had completed heading, 9 pots of plants were removed and transferred to a phytotron. Three temperature gradients were set up, T1 (35℃ / 28℃, day / night), T2 (37℃ / 30℃, day / night), and T3 (39℃ / 32℃, day / night). Three pots of plants were treated for each gradient, and the remaining plants were cultured at normal temperature as the normal temperature control group. Each gradient stress lasted for 15 days, and strong light (light intensity ≥750 μmol·m -2 ·s -1 ) was supplemented from 9:00 to 17:00 every day. The light intensity was 600 μmol·m -2 ·s -1 for the rest of the time, the relative humidity was 65%, and the soil water content was maintained at 80% of the maximum field water holding capacity by watering at regular intervals every day.

8. The high temperature tolerant rice identification method of claim 7, wherein, The second index parameter determination comprises: The Fv / Fm value of the sword leaves of the plants at the three temperature gradients is measured at 10:00 every day in the last 5 days (11th-15th day) of the gradient stress treatment at the grain filling stage, and then the average value is taken as the Fv / Fm value of the plants at the corresponding temperature gradient; After the end of the irrigation period gradient stress treatment, the plants are transferred to the greenhouse for normal temperature culture until maturity, and then the rice ears are harvested. The seed setting rate, thousand-grain weight and chalkiness of the plants in three temperature gradients and the normal temperature control group are measured, and the seed setting rate retention rate, thousand-grain weight retention rate and chalkiness increase value of the plants in three temperature gradients relative to the normal temperature control group are calculated.

9. The high-temperature-resistant rice identification method according to claim 8, characterized in that, when the seedling stage comprehensive score is ≥80 points, the pollen viability comprehensive value at the booting stage is ≥82%, the Fv / Fm comprehensive value at the filling stage is ≥0.82, the seed setting rate retention rate comprehensive value at the filling stage is ≥88%, and the chalkiness increase value comprehensive value at the filling stage is ≤3.5%, the rice of the variety is judged to be a first-grade high-temperature-resistant rice; when the seedling stage comprehensive score is 70-79 points, the pollen viability comprehensive value at the booting stage is 65%-81%, the Fv / Fm comprehensive value at the filling stage is 0.76-0.81, the seed setting rate retention rate comprehensive value at the filling stage is 72%-87%, and the chalkiness increase value comprehensive value at the filling stage is 3.6%-6.5%, the rice of the variety is judged to be a second-grade high-temperature-resistant rice; when the seedling stage comprehensive score is <70 points, or the pollen viability comprehensive value at the booting stage is <65%, or the Fv / Fm comprehensive value at the filling stage is <0.76, or the seed setting rate retention rate comprehensive value at the filling stage is <72%, or the chalkiness increase value comprehensive value at the filling stage is >6.5%, the rice of the variety is judged to be a high-temperature-sensitive rice.

10. Application of the high-temperature-resistant rice identification method according to any one of claims 1-9 in high-temperature-resistant rice breeding.

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