Indoor identification method for rice spikelet blooming time

Abstract

The invention relates to the technical field of agricultural biology, in particular to an indoor identification method for rice spikelet blooming time, which comprises the following steps: placing a sample in a constant-temperature and constant-humidity environment of 32 DEG C, and observing the full-blooming time of each spike in real time, so that the flowering time of 30 percent of florets of each spike is finished, namely the spikelet blooming time. By controlling sampling environment conditions, standardizing operation procedures and repeatedly verifying, the rice spikelet blooming time can be quickly and accurately measured. According to the method, the influence of environmental fluctuation on glume opening time is effectively eliminated by standardizing environmental conditions and a sampling process, the accuracy and comparability of an identification result are remarkably improved, and the method is suitable for large-scale rice flowering characteristic screening.

Description

Technical Field

[0001] The invention relates to the field of rice agricultural biotechnology, in particular to a method for identifying rice spikelet opening time based on environmental standardization control, which is suitable for rice spikelet opening time characteristic screening and genetic positioning research. Background Art

[0002] As an important food crop, the utilization of hybrid vigor is crucial to increasing rice yield. At present, among the rice grown in China, hybrid rice (mainly intra-subspecies hybrids of indica rice) has a yield 10-20% higher than conventional rice, but the yield increase of intra-subspecies hybrids of indica rice is limited by the genetic diversity of existing germplasm, making it difficult to further improve the yield-increasing capacity of hybrid rice. Hybrids between indica and japonica subspecies have stronger hybrid vigor and a yield potential 30% higher than that of intra-subspecies hybrids of indica rice. However, in the production of indica-japonica hybrid seeds, the difference in the time of glume opening between parental varieties seriously reduces the efficiency of cross-pollination and the yield of hybrids, becoming one of the main limiting factors for the development of indica-japonica hybrid rice breeding. In addition, the time of rice glume opening also affects its tolerance to high temperature. Early glume opening can effectively avoid male sterility caused by high temperature. Although some studies have located some sites that control the daily glume opening time of rice, the relevant regulatory genes have not yet been cloned. The main reason is that the glume opening of rice is not easy to observe and count.

[0003] Rice is a self-pollinating crop, and its glume opening time is very short. Generally, the time from the beginning of glume opening to the complete glume closing of rice is about 200 minutes. The glume opening time of different rice varieties varies greatly. For example, the florets of Zhenshan 97A (sterile line) open after 09:30 in the morning, and the opening time is scattered. The whole plant will not be completely closed until 14:00 in the afternoon, while the florets of Zhenshan 97B (maintaining line) open in a concentrated manner from 09:30 to 12:00, and the flowering peak is obvious. Therefore, when counting the opening time of rice glume, it is difficult to quantify the time required for opening glume between different varieties. At present, the methods for counting the opening time of rice glume are mainly field statistical methods, including: fixed-point visual method, plant marking method and hyperspectral method. The main steps of the fixed-point visual method are: taking the plot group as the observation unit, selecting a fixed observation site, generally selecting 5 plants as one observation point, visually observing at intervals of 5-15 minutes and recording the number of opened spikes. Generally, it takes more than 2 hours of observation to estimate the flowering time of rice in the observation point. However, the disadvantages of this method are obvious, that is, it is not possible to observe multiple groups at the same time, and the flowering time of the field group fluctuates greatly, making it difficult to stably record the flowering time of rice. The plant marking method is also called the flower spotting method. Its main steps are: select the target single spike to observe the opening of the florets on the single spike, mark it with a marker after the florets open, and when more than 50% of the florets in the single spike open, it is the opening time of the single plant. Although this method is relatively accurate, it is very inefficient, time-consuming and labor-intensive, and it is difficult to use this method when locating the flowering time of the group. The hyperspectral method is to use precision instruments to assist in flowering time observation. It uses new hyperspectral technology and machine learning technology to count the number of rice spikelets that open to estimate the opening time. The biggest problem with this method is that the instrument cost is very expensive, and it is difficult to popularize.

[0004] Since the time of glume opening is short and greatly affected by the environment, the above methods are not an ideal, economical and feasible method for identifying the time of glume opening of rice. Therefore, it is very necessary to invent a simple, environmentally controllable and standardized method for identifying the time of glume opening of rice for genetic positioning. Summary of the invention

[0005] The purpose of the present invention is to provide a simple, environmentally controllable, and standardized rice spikelet opening time identification method for the above technical problems. The method can be used to screen and identify a large number of rice spikelets opening time, solve the environmental interference of field identification, and solve the problems of poor repeatability and low efficiency.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows: An indoor identification method for the opening time of rice spikelets comprises the following steps: Step 1: Pre-regulation of indoor environment: Before sampling, pre-treat the indoor environment according to the following conditions: Temperature and humidity parameters: Set the indoor temperature to 32°C and the relative humidity to 50%; Environmental stabilization period: maintain the temperature and humidity parameters stable for at least 2 hours; Process monitoring: record temperature and humidity data every 30 minutes during the environmental stabilization period to ensure that temperature and humidity parameters are stable; Step 2: Field sampling: a. When the rice heading peak period is selected, sampling is performed before 8:30 in the morning, and the heading peak period is when more than 50% of the individual plants in the plot begin heading; b. Select rice ears with fully expanded leaves but not flowering after heading, cut a single ear 5 cm below the ear neck node, and immediately place it in a 50 ml rooting tube containing pure water, wherein the ear neck node refers to a single ear containing a flag leaf; c. Mark the sample number at the base of the leaf and record the field location of the corresponding plant on a label; Step 3: Indoor observation: The single ear sample obtained in step 2 is placed in the indoor environment pre-regulated in step 1 at 9 o'clock, and the flowering situation of the single ear is counted every 10 minutes until the single ear reaches the full flowering time, and the full flowering time refers to the time required for 30% of the florets to complete flowering when the single ear is placed in the room and the timing starts at 9 o'clock; Step 4: Calculate the opening time: The time of glume opening is when 30% of the florets have finished flowering, and the glume opening means that the glume shell opens and the anthers are exposed; The present invention reduces the influence of individual differences and accidental errors by repeated sampling. In step 2, three single ear samples with similar physiological states are taken from each individual rice plant, and the ear opening time of the samples obtained three times is counted respectively, and the average value is taken to obtain the ear opening time of the rice plant.

[0007] The flag leaf was cut from each sample, quickly frozen in liquid nitrogen and stored in a -80°C ultra-low temperature freezer for subsequent gene positioning analysis.

[0008] The present invention provides a method for identifying the opening time of rice spikelets that is simple, easy to operate, and has a controllable environment and standardized operation. Results show that the method for identifying the opening time of rice spikelets designed by the present invention can efficiently and accurately identify the flowering time of rice, which lays a reliable foundation for cloning genes that regulate the opening time of rice spikelets.

[0009] The whole process from rice flowering to glume closure is about 200 minutes. If the statistical interval is too long, the key flowering node will be missed, resulting in missing or deviation in the number of flowerings. If the interval is too short, the field operation cost and human error will increase. The present invention further improves the statistics every 10 minutes to balance efficiency and accuracy: it ensures that the flowering dynamics are captured at a high frequency during the flowering cycle, and avoids the waste of resources caused by over-intensive statistics. It is a necessary time strategy for accurately recording the number of flowerings.

[0010] Traditional field identification lacks a unified quantitative standard for the time of glume opening, and relies on subjective visual inspection (such as "approximately flowering"), which is prone to data deviation due to differences in experience of identification personnel. "Taking 30% of the small flowers to complete flowering as the time of glume opening" provides a clear quantitative threshold: by converting the flowering process of a single ear into a measurable "30% flowering ratio", the judgment of the time of glume opening is upgraded from a fuzzy description to a precise indicator, solving the long-standing problem of inconsistent definition of the time of glume opening, and is the core standard to ensure the reliability of field identification results. In an embodiment of the present invention, technicians conducted a comparative test of the ratio of glume opening of small flowers and found that: when 30% of the small flowers opened, the state of flower opening was relatively easy to accurately judge, and the time difference between different flowers to reach this state was small, indicating that this indicator can be used to quantify the time of glume opening more accurately. However, 60% of the flowers may be affected by multiple factors, resulting in unclear judgment criteria and large differences in the time of glume opening; 10% of the small flowers opened, the number of glume flowers was too small, resulting in insignificant glume opening time between varieties.

[0011] In the existing rice field identification technology, there is no solution that combines the "10-minute statistical interval" with the "30% flowering quantification standard". The former optimizes the statistical rhythm for the short flowering period of rice, and the latter establishes quantitative rules for the ambiguity of judgment. The two work together to form a complete solution of "time frequency + judgment standard".

[0012] Compared with the prior art, the present invention has the following advantages: (1) Environmental controllability and anti-interference: Break through the limitations of the natural environment in the field and eliminate the interference of weather fluctuations (such as sudden temperature changes, rainfall, and changes in light intensity) on flowering time through constant temperature and humidity (32°C, 50% RH) pretreatment to ensure data stability.

[0013] (2) High efficiency: At least 15 rice ears can be processed at a time (up to 5 ears in the field method), which increases the efficiency by more than 3 times and is suitable for large-scale germplasm resource screening; the standardized process (statistics every 10 minutes) reduces the frequency of manual observation and shortens the single plant identification cycle.

[0014] (3) Accuracy: The quantitative index of 30% florets per ear is used to replace the traditional visual estimation, thereby reducing subjective errors. The flowering time is concentrated under indoor conditions (for example, the indoor mean of NJ11 in the example is 23 minutes vs. 40 minutes in the field), the data dispersion is reduced, and the comparability between varieties is enhanced.

[0015] (4) Cost advantage: No expensive instruments (such as hyperspectral equipment) are required, only basic temperature control equipment and rooting tubes are needed, which is suitable for promotion in grassroots laboratories.

[0016] (5) Data compatibility: Leaf samples are simultaneously preserved (quickly frozen in liquid nitrogen) to provide materials for subsequent gene localization research, forming a closed loop of "phenotype-genotype" association analysis.

[0017] (6) Time flexibility: Breaking through the limitation of natural flowering period, identification during non-natural periods can be achieved by adjusting the pretreatment time (such as stabilizing the environment in advance) to meet special research needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the technical flow chart of this solution.

[0019] Figure 2 This is a statistical chart of flowering time of field parents.

[0020] Figure 3 This is a statistical chart of flowering time for indoor parents.

[0021] Figure 4 This is a statistical chart of the flowering time of the parents with 10% of the florets opening at the glumes, 30% of the florets opening at the glumes, and 60% of the florets opening at the glumes. DETAILED DESCRIPTION

[0022] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0023] Example 1 Indoor identification of rice spikelet opening time

[0024] The rice varieties Nipponbare (NIP), Zhonghua 11 (ZH11) and Nanjing 11 (NJ11) were identified for the time of the opening of rice spikelets using the fixed-point visual inspection method and the method of the present invention, respectively.

[0025] Obtained by fixed-point visual inspection Figure 2 Data on the opening time of rice spikelets: starting at 9 a.m., the average flowering time of NJ11 is 40 minutes, that of ZH11 is 107 minutes, and that of NIP is 170 minutes.

[0026] The specific steps of the identification method of the present invention are as follows Figure 1 As shown: (1) The above three rice varieties were planted in the same plot, with 30 plants of each variety planted, and samples were taken after heading. Before sampling, the indoor temperature was set to 32°C and the relative humidity was controlled at 50% for more than 2 hours. During the environmental stabilization period, the temperature and humidity data were recorded every 30 minutes to ensure that the temperature and humidity parameters were stable.

[0027] (2) When the rice heading peak period was selected, sampling was carried out before 8:30 in the morning. The heading peak period was when more than 50% of the plants in the plot began to head. The rice ears with fully expanded leaves but not yet bloomed were selected. Single ears were cut 5 cm below the ear neck node and immediately placed in a 50 ml rooting tube containing pure water to remove the edge effect. Three plants were randomly selected. Three ears with similar physiological conditions were taken from each plant. The ear neck node refers to the ear containing the flag leaf. The sample number was marked on the base of the leaf and the field position was recorded by hanging a sign corresponding to the plant.

[0028] (3) placing the single ear sample obtained in step (2) in the indoor environment pre-conditioned in step 1 at 9 o'clock, and counting the flowering status of the single ear every 10 minutes until the single ear reaches the full flowering time, wherein the full flowering time refers to the time required for 30% of the florets to complete flowering after the single ear is placed in the room and the timing starts at 9 o'clock; (4) The time when 30% of the florets have completed flowering is defined as the time when the lemma opens and the anthers are exposed. The time when the lemma opens of the single ear samples obtained three times is counted and the average value is taken to obtain the time when the lemma opens of the rice plant.

[0029] (5) For each sample, the flag leaf was cut, quickly frozen in liquid nitrogen, and stored in a -80°C ultra-low temperature freezer for subsequent gene localization analysis.

[0030] The data of the identification method of the present invention are as follows Figure 3 As shown: the average flowering time of NJ11 is 23 minutes, ZH11 is 83 minutes, and NIP is 136 minutes. The results of the identification method of the present invention are completely consistent with the results of the fixed-point visual inspection method.

[0031] Example 2 Indoor identification of rice spikelet opening time

[0032] NJ11 and NIP were hybridized to obtain 50 F2 progeny, and the opening time of rice spikelets was identified by the fixed-point visual inspection method and the method of the present invention, respectively.

[0033] The data of rice spikelet opening time obtained by fixed-point visual observation are shown in Table 1: The timing starts at 9 am.

[0034] Table 1 Statistics of characteristic values ​​of glume opening time of 50 F2 rice plants based on fixed-point observation method

[0035] The time difference between the earliest flowering plant and the latest flowering plant was 120 min, and the difference between individual plants was relatively large.

[0036] The specific steps of the identification method of the present invention are as follows Figure 1 As shown: (1) Plant the 50 F2 generations in the same plot and take samples after heading. Before sampling, set the indoor temperature to 32°C and the relative humidity to 50% for more than 2 hours. Record the temperature and humidity data every 30 minutes during the environmental stabilization period to ensure that the temperature and humidity parameters are stable.

[0037] (2) When the rice is at its peak heading stage, sampling should be carried out before 8:30 in the morning. The peak heading stage refers to when more than 50% of the plants in the plot begin to head. Select rice ears with fully expanded leaves but not yet blooming after heading. Cut a single ear 5 cm below the ear neck node and immediately place it in a 50 ml rooting tube containing pure water. Take three ears with similar physiological conditions from each plant. The ear neck node refers to the ear with the flag leaf. Mark the sample number at the base of the leaf and record the field position of the corresponding plant with a sign. (3) placing the single ear sample obtained in step (2) in the indoor environment pre-conditioned in step 1 at 9 o'clock, and counting the flowering status of the single ear every 10 minutes until the single ear reaches the full flowering time, wherein the full flowering time refers to the time required for 30% of the florets to complete flowering after the single ear is placed in the room and the timing starts at 9 o'clock; (4) The time when 30% of the florets have completed flowering is defined as the time when the lemma opens and the anthers are exposed. The sum of the lemma opening times of the single ear samples obtained three times is counted and the average value is taken to obtain the lemma opening time of the rice plant.

[0038] (5) For each sample, the flag leaf was cut, quickly frozen in liquid nitrogen, and stored in a -80°C ultra-low temperature freezer for subsequent gene localization analysis.

[0039] The data of the identification method of the present invention are shown in Table 2: The results of the identification method of the present invention are completely consistent with the results of the fixed-point visual inspection method.

[0040] Table 2 Statistics of characteristic values ​​of glume opening time of 50 F2 rice plants based on the method of the present invention

[0041] The earliest and latest flowering times differed by 90 min, and the difference between individual plants was relatively small.

[0042] Example 3 Comparative experiment on the optimal ratio of quantified opening time

[0043] Comparative Example 1: The time when 10% of the florets have completed flowering is defined as the time when the spikelets open. The remaining steps are the same as those in Example 1.

[0044] Comparative Example 2: The time when 60% of the florets have completed flowering is considered as the time when the spikelets open, and the remaining operation steps are the same as those in Example 1.

[0045] The data of comparative example 1 and comparative example 2 identification method are as follows Figure 4 As shown in the figure, when the proportion of small flowers is 10%, the change trend of the opening time of NJ11, ZH11 and NIP is inconsistent with the field data, while when the proportion of small flowers is 60%, the error bars of NJ11, ZH11 and NIP are longer than 30%, indicating that the data dispersion of flowering time is larger under these two proportions of small flowers, the difference between the measured values ​​is more obvious, and the flowering time is more difficult to judge. When 30% of small flowers open, the error bar is shorter, indicating that the volatility of flowering time data is small, the repeatability is good, the measured values ​​are more concentrated on the mean, and the change trend is consistent with the field data, and the data reliability is higher. Comparing 10% and 60%, the former data is inaccurate (inconsistent with the field trend), and the latter error bar is longer (the degree of data dispersion is large), indicating that the characteristics of flowering time at 30% are more independent and stable, and can more accurately represent the law of small flowers opening, so 30% is selected as the proportion of small flowers opening.

[0046] The above embodiments are only general descriptions for the purpose of clearly describing the present invention, on which basis some modifications or improvements may be made, and any changes made without violating the principles of the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. An indoor identification method for the opening time of rice spikelets, characterized in that: The following steps are involved: Step 1: Pre-regulation of indoor environment: Before sampling, pre-treat the indoor environment according to the following conditions: Temperature and humidity parameters: Set the indoor temperature to 32°C and the relative humidity to 50%; Environmental stabilization period: maintain the temperature and humidity parameters stable for at least 2 hours; Process monitoring: record temperature and humidity data every 30 minutes during the environmental stabilization period to ensure that temperature and humidity parameters are stable; Step 2: Field sampling: a. Select the peak heading period of rice and take samples before 8:30 in the morning; b. Select rice ears with fully expanded leaves but not yet blooming after heading, cut a single ear 5 cm below the ear neck node, and immediately place it in a 50 ml rooting tube containing pure water; c. Mark the sample number at the base of the leaf and record the field location of the corresponding plant on a label; Step 3: Indoor observation: The single ear sample obtained in step 2 is placed in the indoor environment pre-conditioned in step 1 at 9 o'clock, and the flowering status of the single ear is counted every 10 minutes until the single ear reaches the full flowering time; Step 4: Calculate the opening time: The time when 30% of the florets have finished flowering is considered as the time when the spikelets open. In step 2, three single ear samples with similar physiological states are taken from each individual rice plant, and the glume opening time of the three obtained single ear samples are counted respectively, and the average value is taken to obtain the glume opening time of the rice plant.

2. The indoor identification method for rice spikelet opening time according to claim 1, characterized in that: The peak heading period in step a is when more than 50% of the plants in the plot begin to head.

3. The indoor identification method for rice spikelet opening time according to claim 1, characterized in that: The area below the ear neck node in step b refers to a single ear including the flag leaf.

4. The indoor identification method for rice spikelet opening time according to claim 1, characterized in that: The time for a single ear to reach full flowering in step 3 refers to the time required for a single ear to be placed in the room, starting at 9 o'clock, until 30% of the florets have completed flowering.

5. The indoor identification method for rice spikelet opening time according to claim 1, characterized in that: In step 4, the glume opening means that the glume shell opens and the anthers are exposed.

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