Method for identifying difficulty of japonica rice sterile line seed production based on flowering related gene expression

Abstract

This invention discloses a method for identifying the ease of seed production of japonica rice male-sterile lines based on the expression levels of flowering-related genes, belonging to the field of plant molecular breeding technology. The method includes: collecting spikelet samples from japonica rice male-sterile lines at their peak flowering period; extracting RNA and reverse transcribing it; using qPCR to detect the expression levels of flowering-promoting genes (OsMYC2, OsMYB8, OsOPR7) and flowering-inhibiting genes (OsJAZ7, OsJAZ9, OsHAN1); and calculating the ratio of the sum of the relative expression levels of these genes, i.e., the "promotion-inhibition ratio". This ratio is significantly correlated with the flowering habits and ease of seed production of the male-sterile line; the higher the ratio, the easier the seed production. This method allows for rapid and objective identification in the laboratory at the early stages of breeding, overcoming the shortcomings of traditional field phenotypic observation, which has a long cycle and is easily affected by the environment. It provides a powerful molecular tool for the efficient selection of superior japonica rice male-sterile lines.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular breeding technology, specifically relating to a method for identifying a male-sterile line of japonica rice, and more specifically, relating to a method for identifying the ease of seed production of a male-sterile line of japonica rice by quantitatively detecting the expression level of specific flowering-related genes and calculating the "promotion-inhibition ratio". Background Technology

[0002] Utilizing heterosis between indica and japonica subspecies is an important way to further increase rice yield, and the "japonica-sterile-indica restorer line" model is currently the main mode for realizing this advantage. In this model, the flowering habits of japonica rice male-sterile lines (such as the time of initial flowering and the concentration of flowering) are key limiting factors that directly affect their flowering time encounter with indica rice restorer lines, pollination efficiency, and final seed production yield. Therefore, rapidly and accurately identifying the quality of flowering habits of japonica rice male-sterile lines is of great significance for efficiently breeding male-sterile lines that are easy to produce seeds and reducing the production cost of hybrid rice seeds.

[0003] Currently, the evaluation of flowering habits of Japonica rice male-sterile lines mainly relies on field phenotypic observations, such as recording traits like initial flowering time, peak flowering time, pre-morning flowering rate, and glenching angle. While these methods are intuitive, they have significant shortcomings: the evaluation cycle is long, requiring waiting for the plants to reach the full flowering stage; they are easily affected by the climate conditions of the experimental year, resulting in large fluctuations and poor repeatability; and they are post-hoc evaluations, unable to pre-screen selected lines in the early stages of breeding, leading to a large amount of resources being invested in subsequent ineffective seed production trials of materials with poor flowering habits.

[0004] The development of molecular breeding technology has made early selection of crop traits possible. Previous studies have reported several genes related to the regulation of daily flowering time in rice, such as OsMYC2, OsMYB8, OsOPR7, OsJAZ7, OsJAZ9, OsHAN1, and OsJAR1. These genes participate in the jasmonic acid signaling pathway, positively or negatively regulating sclerotium cell swelling to control floret opening and closing. However, current techniques are limited to individual elucidations of the functions of these genes; no molecular identification method has yet been developed to predict the flowering habits and seed production ease of japonica rice male-sterile lines by quantifying their expression balance, treating them as a synergistic regulatory network. Summary of the Invention

[0005] To address the aforementioned shortcomings of existing technologies, the present invention aims to provide a method for identifying the ease of seed production of japonica rice male-sterile lines based on the expression levels of flowering-related genes. This method can be rapidly completed in the laboratory during the early heading and flowering stage, yielding objective and accurate results unaffected by environmental interference, thus greatly improving breeding and screening efficiency.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a method for identifying the ease of seed production of japonica rice male-sterile lines based on the expression levels of flowering-related genes, comprising the following steps:

[0008] S1. Collect spikelet samples from the male-sterile lines of japonica rice at the peak flowering time during the heading and flowering stages;

[0009] S2. Extract total RNA from the spikelet sample and reverse transcribe it into cDNA;

[0010] S3. Using the cDNA as a template, and with specific primers, the mRNA expression levels of a set of flowering-promoting genes and a set of flowering-inhibiting genes are detected by real-time quantitative PCR. The flowering-promoting genes include OsMYC2, OsMYB8, and OsOPR7, and the flowering-inhibiting genes include OsJAZ7, OsJAZ9, and OsHAN1.

[0011] S4. Using the expression level of the internal reference gene OsActin1 as a benchmark, calculate the relative expression level of each gene at the peak flowering time relative to its pre-flowering time.

[0012] S5. Calculate the promotion-inhibition ratio, which is the ratio of the sum of the relative expression levels of all flowering-promoting genes to the sum of the relative expression levels of all flowering-inhibiting genes in step S3.

[0013] S6. Evaluate the seed production difficulty of the tested japonica rice male-sterile line based on the promotion-inhibition ratio, wherein the larger the promotion-inhibition ratio, the lower the seed production difficulty.

[0014] Furthermore, in step S1, the sampling time for the peak flowering period is the moment when the flowering rate of the japonica rice male-sterile line is the highest each day.

[0015] Furthermore, the method also includes collecting control spikelet samples before flowering and calculating the relative gene expression levels of samples during the peak flowering period based on the gene expression levels of the samples before flowering.

[0016] Furthermore, step S3 also includes detecting the relative expression level of the gene OsJAR1, and its expression trend is used as an auxiliary identification indicator.

[0017] Furthermore, the reference threshold for determining the promotion-inhibition ratio is: when the promotion-inhibition ratio is greater than 0.6, the difficulty of indicator seed production is low; when the promotion-inhibition ratio is less than 0.4, the difficulty of indicator seed production is high.

[0018] Furthermore, step S1 is performed on consecutive sunny mornings.

[0019] Furthermore, the pre-flowering samples were collected 1 hour before the expected start of flowering.

[0020] Furthermore, the specific primers comprise a set of primers with nucleotide sequences as shown in SEQ ID NO.1 to SEQ ID NO.16.

[0021] Secondly, the present invention provides the application of the above method in the early screening of japonica rice male-sterile line breeding, wherein the application is to select breeding materials with a promotion-inhibition ratio greater than 0.6 as materials with excellent flowering habits for advancement.

[0022] Furthermore, the application also includes: selecting breeding materials with a promotion-inhibition ratio greater than 0.6 and a relative expression level of the OsJAR1 gene greater than 1 as materials with excellent flowering habits for advancement.

[0023] The beneficial effects of this invention are:

[0024] (1) The present invention can complete sampling and identification during the heading and flowering stage of rice, which is much earlier than the traditional method that relies on observation of the entire flowering cycle. This enables early screening of breeding materials and saves a lot of field trial time and cost.

[0025] (2) This invention quantitatively detects the expression levels of the core promoting and inhibiting module genes in the jasmonic acid signaling pathway and proposes the quantitative index of "promotion-inhibition ratio". It accurately reflects the balance of the signal network regulating flowering at the molecular level. The identification results are objective and stable and are not affected by subjective observation errors or weather conditions.

[0026] (3) The reference threshold for the ratio of promotion to inhibition established by this invention (e.g., >0.6 is good and <0.4 is bad) provides breeders with a clear and operable molecular discrimination standard, which facilitates rapid decision-making in actual breeding.

[0027] (4) This invention not only provides an identification method, but also clarifies the intrinsic relationship between the level of the promotion-inhibition ratio and the flowering phenotype (such as the time of first flowering and the concentration of flowering) through a molecular model, so that the identification results have a reliable theoretical basis. Attached Figure Description

[0028] Figure 1 Molecular models of flowering habits of Japonica rice male-sterile lines; (a) is the molecular model of Zhejing 12A in spikelets during the peak flowering period in Hangzhou, and (b) is the molecular model of Zhenuo 1A in spikelets during the peak flowering period in Hangzhou. ↑ indicates upregulation, and ↓ indicates downregulation. Detailed Implementation

[0029] To enable those skilled in the art to better understand the technical solutions of this invention, the present application will be further described in detail below with reference to embodiments.

[0030] Example 1: Detection of expression levels of flowering-related genes and calculation of the promotion-inhibition ratio in japonica rice male-sterile lines

[0031] This embodiment details how to obtain gene expression levels in a Japonica rice male-sterile line and calculate the "promotion-inhibition ratio".

[0032] 1. Experimental materials

[0033] The three-line sterile rice lines Zhejing 12A (superior) and Zhenuo 1A (inferior), with known flowering habits, were used as experimental materials. They were sown and cultivated in Hangzhou, Zhejiang Province, with field management in the same manner as conventional high-yield cultivation.

[0034] 2. Sample Collection

[0035] (1) Sampling period: During the heading and flowering stage of the main stem spike of the sterile line, and on consecutive sunny mornings, to reduce the impact of environmental fluctuations on gene expression. Precise sampling was carried out on the date when the growth and development of the main stem spike were consistent and about 1 / 3 of the spikelets had opened.

[0036] (2) Sampling time points and methods

[0037] Pre-flowering samples: About one hour before the start of flowering each day (estimated based on the material's past flowering patterns), select the spikelets from the upper part of the spike that are expected to open that day, quickly cut them off with pre-cooled scissors, and immediately place them into cryovials containing liquid nitrogen. Each material should have at least two biological replicates.

[0038] Peak flowering samples: The time of day with the highest flowering rate of the material was determined by fixed-point observation (usually between 10:00 AM and 12:00 PM). At this time, the open spikelets were cut and quickly placed in liquid nitrogen. At least two biological replicates were set up for each material.

[0039] Post-flowering samples: At the end of the flowering period on the same day, the closed spikelets after opening were cut off and flash-frozen in liquid nitrogen. This sample can be used to assist in the analysis of the complete dynamics of the signaling pathway.

[0040] (3) After all samples are collected, they are quickly transferred to an ultra-low temperature freezer at -80℃ for long-term storage and are used for subsequent RNA extraction.

[0041] 3. RNA extraction and quality testing

[0042] (1) The frozen sample was ground into a fine powder in liquid nitrogen and the total RNA was extracted using the TIANGEN RNAprep Pure Plant Total RNA Extraction Kit (centrifuge column type). The specific operation was strictly in accordance with the kit instructions.

[0043] (2) Use a micro spectrophotometer to determine the concentration and purity of the extracted RNA. The A260 / A280 ratio should be between 1.8 and 2.1, and the A260 / A230 ratio should be greater than 2.0.

[0044] (3) Take about 1 μg of total RNA for 1.2% agarose gel electrophoresis to check RNA integrity. A qualified RNA sample should show clear 28S and 18S rRNA bands without obvious degradation.

[0045] 4. cDNA first-strand synthesis

[0046] Using Toyobo Corporation's ReverTra Ace TM The qPCR RT Master Mix reverse transcription kit is used for reverse transcription to synthesize cDNA.

[0047] (1) Prepare the RNA heat denaturation reaction system (12 μL) in a nuclease-free PCR tube: less than 1 μg of total RNA, Primer Oligo(dT) 20 Add 1 μL of RNase-Free ddH2O to a final volume of 12 μL.

[0048] (2) React at 65℃ for 5 minutes, then immediately place on ice.

[0049] (3) Add reverse transcription reaction solution (8 μL) directly to the denatured RNA solution: 5× RT Buffer 4 μL, dNTP Mixture 2 μL, RNase Inhibitor 1 μL, ReveTra Ace 1 μL.

[0050] (4) Perform reverse transcription reaction: 42℃ for 50 min, 99℃ for 5 min, 4℃ for 5 min. After the reaction, centrifuge briefly and store at -20℃ for later use.

[0051] 5. Real-time quantitative PCR (qPCR)

[0052] (1) Primers: The primer sequences used are shown in Table 1. The specificity of all primers was verified by NCBI Primer-BLAST and the amplification product was confirmed to be a single specific band by melting curve analysis.

[0053] Table 1 Quantitative PCR Primers

[0054]

[0055] (2) Reaction system: Using Vazyme ChamQ Universal SYBR qPCR Master Mix, a 20 μL reaction system was prepared on ice: 10 μL of 2× ChamQ Universal SYBR qPCR Master Mix, 0.4 μL of forward primer (10 μM), 0.4 μL of reverse primer (10 μM), 2 μL of diluted cDNA template, and 7.2 μL of RNase-Free ddH2O.

[0056] (3) Reaction procedure: Run on a real-time PCR instrument. The program is set as follows: 95℃ pre-denaturation for 30 seconds; followed by 40 cycles of 95℃ denaturation for 10 seconds and 60℃ annealing / extension for 30 seconds (fluorescence signal is collected in this step); finally, melting curve analysis is performed to confirm amplification specificity.

[0057] (4) Each sample and each gene should have at least 3 technical replicates, and a template-free control (NTC) should also be set up.

[0058] 6. Data Calculation and Analysis

[0059] (1) Adopt Methods for calculating the relative expression level of the target gene:

[0060] 1) Calculate the average Ct value of the internal reference gene (OsActin1) and the target gene for each replicate.

[0061] 2) Calculation : (Target gene) = Ct (Target gene) - Ct (OsActin1).

[0062] 3) The expression level of samples from the same material before flowering was used as a calibration control. Calculation : = (Peak flowering sample) - (Pre-flowering control sample).

[0063] 4) Calculate relative expression level: Relative expression level = .

[0064] (2) Calculate the "promotion-inhibition ratio":

[0065] 1) The sum of expression levels of the promoting modules (Sum_P) = relative expression level (OsMYC2) + relative expression level (OsMYB8) + relative expression level (OsOPR7).

[0066] 3) The sum of expression levels of the suppression modules (Sum_S) = relative expression level (OsJAZ7) + relative expression level (OsJAZ9) + relative expression level (OsHAN1).

[0067] 3) Promotion / Suppression Ratio (P / S Ratio) = Sum_P / Sum_S.

[0068] (3) The relative expression level of the OsJAR1 gene was calculated in the same way as above and used as a separate analytical indicator.

[0069] Example 2: Identification and comparison of male-sterile lines of japonica rice with different flowering habits and establishment of thresholds

[0070] Using the method described in Example 1, two japonica rice male-sterile lines, Zhejing 12A and Zhenuo 1A, were tested and analyzed, and a reference for judging the ease of seed production was established.

[0071] 1. Gene expression and the ratio of promotion to suppression

[0072] Quantitative PCR results (mean of two biological replicates) showed that during the peak flowering period, the relative expression level of the OsJAR1 gene in Zhejing 12A was upregulated by 1.48-fold, while the relative expression level of the OsJAR1 gene in Zhenuo 1A was downregulated to 0.61-fold of the control. The calculated P / S ratio for Zhejing 12A was 0.71, and for Zhenuo 1A it was 0.39.

[0073] 2. Establishment of a reference range for the pro-inhibition ratio threshold

[0074] Based on the analysis of multiple known field seed production performances of japonica rice male-sterile lines, including the materials mentioned above, this invention proposes the following reference thresholds for the seed-promoting / inhibiting ratio to assist in judging the ease of seed production:

[0075] (1) The ratio of promoting to inhibiting is >0.6: This indicates that the sterile line has excellent flowering habits, the promoting signal is dominant, the jasmonic acid pathway is smooth, and the seed production is easy.

[0076] (2) 0.4 ≤ Promotion-inhibition ratio ≤ 0.6: indicates moderate flowering habit, and needs to be comprehensively evaluated in combination with the expression of OsJAR1 gene (preferably upregulated expression) and other agronomic traits (such as stigma exposure rate).

[0077] (3) The ratio of promotion to inhibition < 0.4 indicates that the sterile line has poor flowering habits, the inhibition signal is too strong, the jasmonic acid signaling pathway is blocked, and the seed production is difficult.

[0078] This threshold range provides a clear quantitative benchmark for the practical application of this method.

[0079] 3. Molecular mechanism and phenotypic association analysis

[0080] (1) For Zhejing 12A (promotion-inhibition ratio 0.71, >0.6)

[0081] Molecular characteristics indicate a relative balance between promoting and inhibiting signaling modules, with the promoting signal slightly dominant. Simultaneously, upregulation of OsJAR1 expression facilitates the effective establishment of positive feedback in the jasmonic acid signaling pathway and the activation of downstream cell wall remodeling genes. This process relies on the sufficient synthesis of JA-Ile to ensure smooth signal transmission from upstream to downstream, thereby driving the sapstone to absorb water and swell, ultimately resulting in early flowering and concentrated blooming. Figure 1 a).

[0082] The superior phenotypes include: early flowering (10:04), high pre-morning flowering rate (80.44%), and concentrated flowering (cumulative flowering rate during peak bloom is 88.41%) (Table 2). These traits significantly improve the flowering time encounter rate with the restorer line, making seed production easier.

[0083] (2) For Zhe Nuo 1A (promotion-inhibition ratio 0.39, <0.4)

[0084] Molecular characterization revealed abnormally strong expression of inhibitory signaling modules (especially OsJAZ7 / 9), far exceeding that of promoting signals, resulting in a low promoter-inhibition ratio. OsJAZ7 / 9 was over-upregulated by more than 40-fold, preventing complete degradation of OsJAZ protein and potentially leading to sustained inhibition of OsMYC2 activity. Simultaneously, downregulation of OsJAR1 expression resulted in insufficient JA-Ile synthesis, further weakening the effective establishment of positive feedback in the signaling pathway. These two factors combined to obstruct the upstream-to-downstream transmission of jasmonic acid signaling, consequently hindering the activation of cell wall remodeling genes, ultimately manifesting as delayed flowering of Zhe Nuo 1A. Figure 1 b).

[0085] Unfavorable phenotypes include late initial flowering (10:55) and extremely low pre-morning flowering rate (3.45%) (Table 2). These traits result in a high risk of not coinciding with the flowering time of the restorer line, making seed production difficult.

[0086] Table 2 Comparison of Japonica rice male-sterile lines with superior and inferior flowering habits

[0087]

[0088] Example 3: Application of this method in early screening of new strains

[0089] In the breeding of new male-sterile lines of japonica rice, Zhejing 7A was used as the male-sterile cytoplasm donor and crossed with the high-quality disease-resistant intermediate material "F2007". Then, "F2007" was used as the male parent for gradual backcrossing. Each backcross involved self-pollination bags on both the male-sterile line and the maintainer line. Seeds of the male-sterile line with zero self-pollination set rate and the corresponding bagged maintainer line seeds were harvested to ensure the restorer-maintainer relationship and the purity of the maintainer line in the next generation. The method of this invention was applied in the BC2F1 generation for early pre-screening in the laboratory to identify potential plants with excellent flowering habits. The specific steps are as follows:

[0090] (1) On a sunny day during the heading and flowering period, samples of flowering spikes were collected from each candidate BC2F1 line at the peak of flowering, according to the method in Example 1.

[0091] (2) RNA extraction, reverse transcription and qPCR detection were performed according to the procedure in Example 1 to calculate the ratio of promotion to inhibition and the relative expression level of OsJAR1 in this strain.

[0092] (3) Filter according to the threshold established in Example 2.

[0093] Priority advancement: Lines with a growth-inhibition ratio > 0.6 and a relative OsJAR1 expression level > 1 (i.e., upregulated expression) can directly enter small-scale seed production trials or high-level yield comparison trials.

[0094] Careful evaluation is needed for lines with a growth-inhibition ratio between 0.4 and 0.6. Further examination of key outcrossing traits such as stigma exposure rate and glume opening angle is required before deciding whether to advance them.

[0095] Prioritize the elimination of lines with a growth-inhibition ratio < 0.4. The investment of significant field seed production resources in these lines can be terminated earlier.

[0096] This method was used to screen and eliminate individual plants with poor flowering habits, obtain BC2F1 individual plants with excellent flowering habits, and continue to conduct early generation combining ability testing. The population was then backcrossed until it was uniform, with zero seed setting rate after bagging self-pollination, and agronomic traits and fertility were basically stable and consistent, thus breeding a new Japonica rice male-sterile line, Zhejing 14A, with excellent flowering habits. This molecular identification method can efficiently eliminate materials with defective flowering habits in the early stages of breeding, significantly improving breeding efficiency and reducing the cost of blindly conducting seed production trials.

Claims

1. A method for identifying the ease of seed production of japonica rice male-sterile lines based on the expression levels of flowering-related genes, characterized in that, Includes the following steps: S1. Collect spikelet samples from the male-sterile lines of japonica rice at the peak flowering time during the heading and flowering stages; S2. Extract total RNA from the spikelet sample and reverse transcribe it into cDNA; S3. Using the cDNA as a template, and with specific primers, the mRNA expression levels of a set of flowering-promoting genes and a set of flowering-inhibiting genes are detected by real-time quantitative PCR. The flowering-promoting genes include OsMYC2, OsMYB8, and OsOPR7, and the flowering-inhibiting genes include OsJAZ7, OsJAZ9, and OsHAN1. S4. Using the expression level of the internal reference gene OsActin1 as a benchmark, calculate the relative expression level of each gene at the peak flowering time relative to its pre-flowering time. S5. Calculate the promotion-inhibition ratio, which is the ratio of the sum of the relative expression levels of all flowering-promoting genes to the sum of the relative expression levels of all flowering-inhibiting genes in step S3. S6. Evaluate the seed production difficulty of the tested japonica rice male-sterile line based on the promotion-inhibition ratio, wherein the larger the promotion-inhibition ratio, the lower the seed production difficulty.

2. The method according to claim 1, characterized in that, In step S1, the sampling time for the peak flowering period is the time when the flowering rate of the japonica rice male-sterile line is the highest each day.

3. The method according to claim 1, characterized in that, The method also includes collecting control spikelet samples before flowering and calculating the relative gene expression levels of samples during the peak flowering period based on the gene expression levels of the samples before flowering.

4. The method according to claim 1, characterized in that, Step S3 also includes detecting the relative expression level of the gene OsJAR1, and its expression trend is used as an auxiliary identification indicator.

5. The method according to claim 1, characterized in that, The reference threshold for determining the promotion-inhibition ratio is as follows: when the promotion-inhibition ratio is greater than 0.6, the difficulty of indicator seed production is low; when the promotion-inhibition ratio is less than 0.4, the difficulty of indicator seed production is high.

6. The method according to claim 1, characterized in that, Step S1 is performed on consecutive sunny mornings.

7. The method according to claim 3, characterized in that, The pre-flowering samples were collected 1 hour before the expected start of flowering.

8. The method according to claim 1, characterized in that, The specific primers include primer sets with nucleotide sequences as shown in SEQ ID NO.1 to SEQ ID NO.

16.

9. The application of the method according to any one of claims 1-8 in early screening of japonica rice male-sterile lines, characterized in that, The application involves selecting breeding materials with a promotion-inhibition ratio greater than 0.6 as materials with excellent flowering habits for further advancement.

10. The application according to claim 9, characterized in that, The application also includes: selecting breeding materials with a promotion-inhibition ratio greater than 0.6 and a relative expression level of the OsJAR1 gene greater than 1 as materials with excellent flowering habits for advancement.

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