An indoor identification method for the opening time of rice lemma and palea
A controlled environment method for rice flowering time determination using 32°C and 50% humidity with 10-minute intervals and 30% anthesis tracking improves data accuracy and efficiency for genetic analysis and hybrid breeding.
Patent Information
- Application Number
- CN202510499433.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-21
- Publication Date
- 2025-07-15
- Estimated Expiration
- 2045-04-21
AI Technical Summary
The prior art is difficult to efficiently and accurately identify the blooming time of the flower in rice fields, and it is greatly disturbed by the environment, resulting in unstable data and poor repetition, making it difficult to meet the needs of indica-japonica hybrid rice breeding.
In the indoor environment, the temperature and humidity are controlled at 32℃ and 50% RH. By recording the flowering of a single ear every 10 minutes, the flowering of 30% small flowers is used as the standard for the flowering time. Combined with repeated sampling to reduce individual differences, basic temperature control equipment and rooting canals are used for identification.
It has achieved efficient and accurate identification of the blooming time of rice flower, reduced environmental interference, improved identification efficiency and data stability, was suitable for large-scale germplasm resource screening, and provided materials for subsequent gene location research.
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Figure CN120009482B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of rice agricultural biotechnology, and specifically to a method for identifying the opening time of rice glume flowers based on environmental standardized control, which is applicable to the screening of rice glume opening time characteristics and genetic mapping research. Background Art
[0002] Rice is an important food crop, and the utilization of its heterosis is crucial for increasing yield. At present, among the rice varieties planted in China, the yield of hybrid rice (mainly intra-subspecies indica hybrids) is 10 - 20% higher than that of conventional rice. However, the yield increase of intra-subspecies indica hybrids is limited by the genetic diversity of existing germplasms, making it difficult to further improve the yield increase ability of hybrid rice. Inter-subspecies indica-japonica hybrids have stronger heterosis, and their yield potential is 30% higher than that of intra-subspecies indica hybrids. However, during indica-japonica hybrid seed production, the difference in glume opening time 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 glume opening time of rice also affects its tolerance to high temperature. Early glume opening can effectively avoid male sterility caused by high temperature. Although some loci controlling the daily glume opening time of rice have been mapped, the related regulatory genes have not been cloned yet. The main reason is that it is not easy to observe and count the glume opening of rice.
[0003] Rice is a self-pollinating crop, and the opening time of its glumes is very short. Generally, the time from the beginning of glume opening to complete closure of rice is about 200 minutes. There are significant differences in the glume opening time among different rice varieties. For example, the florets of Zhenshan 97A (sterile line) open after 09:30 in the morning, and the glume opening time is scattered. The whole plant will completely close its glumes at 14:00 in the afternoon. While the florets of Zhenshan 97B (maintaining line) open concentratedly from 09:30 to 12:00, and the flowering peak is obvious. Therefore, it is very difficult to quantify the time required for glume opening among different varieties when counting the glume opening time of rice. Currently, the methods for counting the glume opening time of rice are mainly field statistical methods, including: fixed-point visual inspection method, marked plant method, and hyperspectral method. The main steps of the fixed-point visual inspection method are: taking the plot population as the observation unit, fixedly selecting the observation sites. Generally, 5 plants are selected as an observation point, and visual observation and recording of the number of panicles with glumes opening are carried out at intervals of 5 - 15 minutes. Generally, it takes more than 2 hours of observation to estimate the full-bloom time of rice within the observation point. However, the disadvantages of this method are obvious, that is, multiple population observations cannot be carried out simultaneously, and the flowering time of the field population fluctuates greatly, making it difficult to stably record the flowering time of rice. The marked plant method, also known as the flower-pointing method, its main steps are: selecting the target single panicle to observe the glume opening situation of the florets on the single panicle. When the florets open, they are marked with a marker pen. When more than 50% of the florets on the single panicle have opened, it is the glume opening time of the single plant. Although this method has relatively high accuracy, its efficiency is very low, time-consuming and laborious, and it is very difficult to use this method for population flowering time positioning. The hyperspectral method is to assist the flowering time observation through precision instruments. It estimates the glume opening time by counting the number of glume openings of rice spikelets through new hyperspectral technology and machine learning technology. The biggest problem with this method is that the instrument cost is very expensive and it is very difficult to popularize.
[0004] Due to the short glume opening time and the large influence of the environment, the above several methods are not an ideal, economical and easy-to-implement method for identifying the glume opening time of rice. Therefore, it is very necessary to invent a simple, easy-to-implement, environmentally controllable and operationally standardized method for identifying the glume opening time of rice for genetic mapping. Summary of the Invention
[0005] The purpose of the present invention is to provide a simple, easy-to-implement, environmentally controllable and operationally standardized method for identifying the glume opening time of rice in view of the above technical problems. Using this method, a large number of screenings and identifications of the glume opening time of rice can be carried out, solving the environmental interference of field identification, and at the same time solving problems such as poor repeatability and low efficiency.
[0006] In order to achieve the above purpose, the technical solutions adopted by the present invention are as follows:
[0007] An indoor identification method for the glume opening time of rice, comprising the following steps:
[0008] Step 1, indoor environment pre-regulation:
[0009] Before sampling, pre-treat the indoor environment according to the following conditions:
[0010] Temperature and humidity parameters: Set the indoor temperature to 32 °C and control the relative humidity at 50%;
[0011] Environmental stability period: Maintain the stable operation of the temperature and humidity parameters for at least 2 h;
[0012] Process monitoring: Record the temperature and humidity data every 30 min during the environmental stability period to ensure the stability of the temperature and humidity parameters;
[0013] Step 2. Field sampling:
[0014] a. When the rice is at the full heading stage, sample before 8:30 in the morning. The full heading stage means that more than 50% of the individual plants in the plot start to head;
[0015] b. Select the rice panicles whose leaves are fully unfolded but not flowering after heading. Cut a single panicle 5 cm below the panicle neck node and immediately put it into a 50 ml rooting tube containing pure water. Below the panicle neck node refers to a single panicle including the flag leaf;
[0016] c. Mark the sample number at the base of the leaf and hang a sign for the corresponding individual plant to record the field position;
[0017] Step 3. Indoor observation:
[0018] Place the single panicle sample obtained in Step 2 in the pre-regulated indoor environment at 9 o'clock. Count the flowering situation of the single panicle every 10 min until the single panicle reaches the full flowering time. The full flowering time refers to the time required from when the single panicle is placed indoors and starts timing at 9 o'clock until 30% of the florets complete anthesis;
[0019] Step 4. Calculate the glume-opening time:
[0020] Take 30% of the florets completing anthesis as the glume-opening time. The glume-opening refers to the glume unfolding and the anthers being exposed;
[0021] The present invention reduces the influence of individual differences and accidental errors through repeated sampling. In Step 2, take 3 single panicle samples with similar physiological states from each individual rice plant, respectively count the glume-opening times of the samples obtained 3 times, and take the average value to obtain the glume-opening time of this rice plant.
[0022] Cut the flag leaf of each sample, quickly freeze it in liquid nitrogen, and store it in a -80 °C ultra-low temperature refrigerator for subsequent gene mapping analysis.
[0023] The present invention provides a simple, easy-to-implement, environmentally controllable and operationally standardized method for identifying the anthesis time of rice florets. The results show that the method for identifying the anthesis time of rice florets designed by the present invention can efficiently and accurately identify the flowering time of rice, which lays a reliable method for cloning genes regulating the anthesis time.
[0024] The whole process from rice flowering to floret closure is about 200 minutes. If the statistical interval is too long, key flowering nodes will be missed, resulting in missing or biased statistics of the number of flowers; if the interval is too short, the field operation cost and human error will increase. The present invention further improves that counting every 10 minutes just balances efficiency and accuracy: it not only ensures high-frequency capture of flowering dynamics within the flowering cycle, but also avoids waste of resources caused by overly dense statistics, which is a necessary time strategy for accurately recording the number of flowers.
[0025] Traditional field identification lacks a unified quantitative standard for anthesis time and relies on subjective visual inspection (such as "roughly flowering"), which is prone to data deviation due to differences in the experience of identification personnel. "Taking the anthesis time of rice florets as the time when 30% of the florets complete anthesis" provides a clear quantitative threshold: by converting the flowering process of a single panicle into a measurable "30% anthesis ratio", the determination of anthesis time is upgraded from a vague description to a precise index, solving the long-standing problem of inconsistent definition of anthesis time, which is the core standard to ensure the reliability of field identification results. In the embodiments of the present invention, technicians conducted a comparative experiment on the anthesis ratio of florets and found that when 30% of the florets are in anthesis, the opening state of the flowers is relatively easy to accurately judge, and the time difference between different flowers reaching this state is small, indicating that using this as an index can more accurately quantify the anthesis time. However, when 60% of the flowers are in anthesis, it may be affected by various factors, resulting in unclear judgment criteria and large differences in anthesis time; when 10% of the florets are in anthesis, the number of florets is too small, resulting in insignificant differences in anthesis time between varieties.
[0026] In the existing rice field identification technologies, there is no solution that combines the "10-minute statistical interval" with the "30% anthesis quantitative standard". The former optimizes the statistical rhythm for the short duration of rice flowering, and the latter establishes a quantitative rule for the ambiguity of judgment. The two cooperate to form a complete solution of "time frequency + judgment standard".
[0027] Compared with the prior art, the present invention mainly has the following advantages:
[0028] (1) Environmental controllability and anti-interference: Breaking through the limitations of the natural field environment, eliminating the interference of weather fluctuations (such as sudden temperature changes, rainfall, and changes in light intensity) on flowering time through pre-treatment at a constant temperature and humidity (32°C, 50% RH), ensuring data stability.
[0029] (2) High efficiency: It can process at least 15 rice panicles at a time (up to 5 panicles by field methods), with the efficiency increased by more than 3 times, suitable for large-scale screening of germplasm resources; the standardized process (counting every 10 minutes) reduces the frequency of manual observation and shortens the identification cycle of individual plants.
[0030] (3) Accuracy: Taking 30% of the florets on a single panicle blooming as the quantification index, replacing traditional visual estimation, reducing subjective errors; the blooming time is concentrated in the indoor environment (e.g., the indoor average value of NJ11 in the embodiment is 23 minutes vs. 40 minutes in the field), the data dispersion is reduced, and the comparability between varieties is enhanced.
[0031] (4) Cost advantage: It does not require expensive instruments (such as hyperspectral equipment), only basic temperature control equipment and rooting tubes, and is suitable for promotion in grass-roots laboratories.
[0032] (5) Data compatibility: Synchronously save leaf samples (quick-frozen in liquid nitrogen), providing materials for subsequent gene mapping research, and forming a closed-loop of "phenotype-genotype" correlation analysis.
[0033] (6) Time flexibility: Breaking through the limitation of natural flowering period, it can achieve identification in non-natural periods by adjusting the pretreatment duration (such as stabilizing the environment in advance), meeting special research needs. Description of the Drawings
[0034] Figure 1 It is the technical flow chart of this scheme.
[0035] Figure 2 It is the statistical chart of the flowering time of field parents.
[0036] Figure 3 It is the statistical chart of the flowering time of indoor parents.
[0037] Figure 4 It is the statistical chart of the flowering time of parents with the flowering time of 10%, 30%, and 60% of the florets blooming. Detailed Implementation Modes
[0038] The following describes the detailed implementation modes of the present invention to facilitate those skilled in the art of this technology to understand the present invention. However, it should be clear that the present invention is not limited to the scope of the detailed implementation modes. For those of ordinary skill in the art of this technology, as long as various changes are within the spirit and scope of the present invention defined and determined by the appended claims, these changes are obvious, and all inventions and creations using the concept of the present invention are within the scope of protection.
[0039] Example 1 Indoor Identification of the Flowering Time of Rice Florets
[0040] 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.
[0041] 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.
[0042] The specific steps of the identification method of the present invention are as follows Figure 1 As shown:
[0043] (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.
[0044] (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 after heading. 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, and 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.
[0045] (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;
[0046] (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.
[0047] (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.
[0048] 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.
[0049] Example 2 Indoor Identification of the Glume Opening Time of Rice Glumes
[0050] NJ11 and NIP were hybridized to obtain 50 F2 offspring, and the glume opening time of rice glumes was identified by the fixed-point visual observation method and the method of the present invention respectively.
[0051] The data of the glume opening time of rice glumes obtained by the fixed-point visual observation method are shown in Table 1: Timing started at 9:00 am.
[0052] Table 1 Statistical values of the glume opening time characteristics of 50 F2 rice plants based on the fixed-point observation method
[0053]
[0054] The time difference between the earliest flowering individual plant and the latest flowering individual plant was 120 min, and the individual plant differences were relatively large.
[0055] The specific steps of the identification method of the present invention are as Figure 1 shown:[[]]END]]
[0056] (1) The above 50 F2 generations were planted in the same plot, and samples were taken after heading. Before sampling, the indoor temperature was set to 32 °C, and the relative humidity was controlled at 50% and maintained for more than 2 hours. The temperature and humidity data were recorded every 30 min during the environmental stability period to ensure the stability of the temperature and humidity parameters.
[0057] (2) When the rice was in the full heading stage, sampling was carried out before 8:30 in the morning. The full heading stage was when more than 50% of the individual plants in the plot began to head; rice panicles with fully expanded leaves but not yet flowering after heading were selected, and single panicles were cut at 5 cm below the panicle neck node and immediately placed in a 50 ml rooting tube containing pure water. Three single panicles with similar physiological states were taken from each individual plant. The part below the panicle neck node refers to the single panicle including the flag leaf; the sample number was marked at the base of the leaf, and the field position was recorded by hanging a sign corresponding to the individual plant.
[0058] (3) The single panicle samples obtained in step (2) were placed in the indoor environment pre-regulated in step 1 at 9:00, and the flowering situation of the single panicles was counted every 10 min until the single panicle reached the full flowering time. The full flowering time refers to the time required from when the single panicle was placed indoors and timed at 9:00 until 30% of the florets completed anthesis.
[0059] (4) Taking 30% of the florets completing anthesis as the glume opening time of the rice glumes. The glume opening of the rice glumes means that the glumes unfold and the anthers are exposed. The sum of the glume opening times of the single panicle samples obtained 3 times was statistically analyzed, and the average value was taken to obtain the glume opening time of this rice plant.
[0060] (5) For each sample, the flag leaf blade was cut, quickly frozen in liquid nitrogen and stored in a -80 °C ultra-low temperature refrigerator for subsequent gene mapping analysis.
[0061] The data of the identification method of the present invention are shown in Table 2: The result trend of the identification method of the present invention is completely consistent with that of the fixed-point visual inspection method.
[0062] Table 2 Statistical values of the opening time characteristics of the glumes of 50 F2-generation rice plants based on the method of the present invention
[0063]
[0064] The difference between the earliest flowering time and the latest flowering time is 90 minutes, and the difference between individual plants is relatively small.
[0065] Example 3 Optimal ratio comparison experiment for quantifying the glume opening time
[0066] Comparative example 1:
[0067] Taking 10% of the florets completing anthesis as the glume opening time, and the remaining operation steps are the same as those in Example 1.
[0068] Comparative example 2:
[0069] Taking 60% of the florets completing anthesis as the glume opening time, and the remaining operation steps are the same as those in Example 1.
[0070] The data of the identification methods of Comparative example 1 and Comparative example 2 are as Figure 4 shown: When the proportion of the florets in anthesis is 10%, the change trends of the glume opening times of NJ11, ZH11, and NIP are inconsistent with the field data. While when the proportion of the florets in anthesis is 60%, the error bars of NJ11, ZH11, and NIP are relatively longer than those at 30%. This shows that at these two proportions of the florets in anthesis, the data dispersion degree of the flowering time is larger, the difference between measurement values is more obvious, and the flowering time is more difficult to judge. When 30% of the glumes open, the error bars are shorter, indicating that the data volatility of the flowering time at this proportion is small, the repeatability is good, the measurement values are more concentrated on the mean value, 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 has longer error bars (large data dispersion degree). This shows that the characteristics of the flowering time at 30% are more independent and stable, and can more accurately represent the law of the glume opening of the florets. Therefore, 30% is selected as the best proportion of the glume opening of the florets.
[0071] The above embodiments are only general descriptions made to clearly describe the present invention. On this basis, some modifications or improvements can be made to it. Any changes made without violating the principle of the present invention fall within the scope claimed by the present invention.
Claims
1. An indoor identification method for the opening time of rice lemma and palea, characterized in that: It includes the following steps: Step 1, Indoor environment pre-regulation: Before sampling, pre-treat the indoor environment according to the following conditions: Temperature and humidity parameters: Set the indoor temperature to 32 °C and control the relative humidity at 50%; Environmental stability period: Maintain the stable operation of the temperature and humidity parameters for at least 2 h; Process monitoring: Record the temperature and humidity data every 30 min during the environmental stability period to ensure the stability of the temperature and humidity parameters; Step 2, Field sampling: a. When the rice is at the full heading stage, sample before 8:30 in the morning. The full heading stage means that more than 50% of the individual plants in the plot start to head; b. Select rice panicles with fully expanded leaves but not yet flowering after heading. Cut a single panicle 5 cm below the panicle neck node and immediately put it into a 50 ml rooting tube containing pure water; c. Mark the sample number at the base of the leaf and hang a label corresponding to the individual plant to record the field position; Step 3, Indoor observation: Place the single panicle sample obtained in Step 2 in the pre-regulated indoor environment at 9 o'clock and count the flowering situation of the single panicle every 10 min until the single panicle reaches the full flowering time; the full flowering time is the time required for 30% of the florets to complete anthesis starting from 9 o'clock; Step 4, Calculate the glume-opening time: Take the time when 30% of the florets complete anthesis as the glume-opening time; In Step 2, take 3 single panicle samples with similar physiological states from each individual rice plant, respectively count the glume-opening times of the 3 obtained single panicle samples, and take the average value to obtain the glume-opening time of this rice plant.
2. The indoor identification method of the glume-opening time of rice florets according to claim 1, characterized in that: The part below the panicle neck node mentioned in Step b refers to the single panicle including the flag leaf.
3. The indoor identification method for the opening time of rice glume flowers according to claim 1, characterized in that: In Step 4, the glume-opening means that the glumes open and the anthers are exposed.