A method for rapid identification of single seed of Medicago polymorpha and Melica princeps
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- LANZHOU UNIV
- Filing Date
- 2026-02-11
- Publication Date
- 2026-05-29
Smart Images
Figure CN122109364A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of agricultural testing technology, specifically to a rapid identification method for single seeds of alfalfa and sweet clover. Background Technology
[0002] Alfalfa (Medicago sativa L.) is a high-quality perennial leguminous forage crop, rich in protein and minerals, low in crude fiber, easily digestible, and its roots can form a symbiotic relationship with rhizobia, improving soil crop rotation efficiency. Therefore, it is widely used worldwide in agriculture, ecological restoration, and for food and medicinal purposes. However, during alfalfa seed harvesting, seeds from other species are often easily mixed in, with yellow sweet potato (Melilotus officinalis L.) being a major source of contamination. During its growth, yellow sweet potato often releases coumarins and other substances through allelopathic effects, inhibiting the growth of surrounding crops. Therefore, in agricultural production, the mixing of these two seeds will significantly impact the economic benefits of alfalfa cultivation. However, alfalfa and yellow sweet potato seeds are extremely similar in botanical morphology, often difficult to distinguish by sight and experience alone. Therefore, the rapid and accurate identification of individual alfalfa and yellow sweet potato seeds is crucial for improving forage planting efficiency in key areas and guiding agricultural production.
[0003] In light of this, several methods have been published for identifying these two types of seeds. Direct observation relies primarily on production experience to differentiate them based on odor, morphology, and color. However, due to the morphological similarity between the two, this method has a relatively large margin of error. Fluorescence identification uses an ultraviolet analyzer to observe the blue fluorescent spots remaining on culture filter paper after alfalfa seeds have absorbed moisture and germinated. However, this process causes leakage of seed metabolites, which can have unpredictable effects on subsequent germination, and the molecular mechanism of this method is currently unclear. Field identification involves direct sowing of seeds and continuous observation for 2-6 months after emergence. While this method can achieve accurate species identification, it is costly and extremely time-consuming. Molecular biology methods, represented by PCR technology, target the genetic information of the species for identification. This method is accurate and effective, but requires a clean experimental environment and can cause fundamental damage to the seeds.
[0004] Mass spectrometry fingerprinting refers to a spectral pattern obtained through mass spectrometry detection technology that represents the overall intrinsic molecular composition distribution characteristics of a specific sample. It can be used for sample identification, classification, or tracing. Direct injection mass spectrometry analysis, based on the coupling of a liquid chromatography (LC) injection unit and a high-resolution mass spectrometry (HR-MS) detection unit, can sensitively and effectively achieve rapid qualitative analysis of analytes over a wide mass range. Given the different metabolic characteristics among species, mass spectrometry fingerprinting technology is a powerful tool for rapidly analyzing metabolic characteristics in plant culture media and identifying species accordingly. Summary of the Invention
[0005] To address the aforementioned technical problems, the present invention aims to provide a rapid identification method for single-seed alfalfa and yellow sweet clover. Based on mass spectrometry analysis, the method rapidly acquires mass spectra of single-seed aqueous soaking solutions in positive / negative ion mass spectrometry modes. Principal component analysis and other data analysis algorithms are used to extract characteristic mass spectrometry peaks (m / z) of the target samples, accurately identifying the mass spectrometry fingerprint of the species' germination metabolic process. This method achieves rapid identification of single-seed alfalfa and yellow sweet clover without damaging the botanical function of the grass seeds and minimizing disturbance to the germination process.
[0006] The technical solution of this invention to solve the above-mentioned technical problems is as follows: A rapid identification method for single-seed alfalfa and yellow sweet clover is provided, comprising the following steps: S1. Soak the surface-sterilized single grass seeds in ultrapure water or nutrient solution to obtain a soaking solution. S2. Inject the soaking solution into the UPLC-QTOF-MS mass spectrometry system, test and record the mass spectrum data; S3. Align the peaks of the mass spectrum data, extract features from the high-dimensional mass spectrum data, and construct a mass spectrometry fingerprint with the mass-to-charge ratio m / z as the abscissa and the normalized ion intensity of the sample at that mass-to-charge ratio m / z as the ordinate.
[0007] Furthermore, in step S1, disinfection is carried out by immersion in a 75% ethanol aqueous solution for 0.5-2 minutes; Disinfect by soaking in a 1 wt% potassium permanganate solution for 5-15 minutes; Disinfect by soaking in 55℃ hot water for 10-20 minutes; Rinse with ultrapure water after disinfection.
[0008] Furthermore, in step S1, the seeds are soaked for 0.5-24 hours under relatively sealed and light-protected conditions.
[0009] Furthermore, in step S2, before the soaking solution is injected, it is filtered through a 0.22 µm pore size filter membrane or centrifuged at a speed of not less than 10,000 rpm.
[0010] Furthermore, in step S2, a blank control solution is used, which is the same as the grass seed culture medium and has undergone the same process as the grass seed culture.
[0011] Furthermore, in step S2, the test is performed in positive / negative ion mode using an electrospray ionization source as the mass spectrometry ionization source, with a mass scan range of 100-750 Da, an injection volume of 0.2-5 μL, and a mobile phase of 70-95 vt% methanol aqueous solution. The methanol-water solution contains 0.1 vt% formic acid.
[0012] Furthermore, in step S3, the high-dimensional mass spectrometry data includes mass-to-charge ratio m / z and ion intensity information.
[0013] Furthermore, in step S3, the mass spectrum data is peak-aligned and then feature extracted using MassHunter Profinder software; The ion peak intensity filtering threshold is 5000 counts, the charge state is 1-2, and the Mass offset is 15 ppm.
[0014] The adduct ion types are [M+H]+, [M+Na]+, [M+K]+ and [M+NH4]+ or the adduct ion type is [MH]-.
[0015] Furthermore, in step S3, principal component analysis is used to reduce the dimensionality and visualize the mass spectrum data based on the correlation matrix or covariance matrix; The number of principal components is 4.
[0016] The present invention has the following beneficial effects: 1. The rapid identification method of the present invention does not involve complex chemical methods such as molecular extraction, molecular amplification and molecular labeling. It is simple to operate and has relatively relaxed requirements for the site, environment and personnel of plant cultivation.
[0017] 2. The mass spectrometry detection method involved in this invention is sensitive and can quickly, with high throughput, and intuitively collect information on single grass seed samples.
[0018] 3. The mass spectrometry fingerprinting method involved in this invention is based on the molecular metabolic characteristics of the germination process of grass seeds. It only requires the extraction of a small amount of seed soaking solution for analysis, does not damage the botanical function of grass seeds, and causes minimal disturbance to the germination process of grass seeds. It is a non-destructive and mild identification method.
[0019] 4. The grass seeds involved in this invention have a high efficiency in releasing internal metabolites into the seed soaking culture medium, which can significantly shorten the identification time of the entire process. Attached Figure Description
[0020] Figure 1This is a mass spectrum of a representative single-seed soaking solution of alfalfa and sweet clover in positive ion mode in Example 1. Figure 2 The image shows the PCA score of the mass spectrometry peaks (m / z) obtained from the soaking solutions of 20 single alfalfa seeds and 20 single yellow sweet clover seeds in positive ion mode in Example 1. Figure 3 This is a normalized fingerprint heatmap of the characteristic mass spectrometry fingerprints (m / z) obtained from the soaking solutions of 20 single alfalfa seeds and 20 single yellow sweet clover seeds in positive ion mode in Example 1. Figure 4 This is a mass spectrum of a representative single-seed soaking solution of alfalfa and sweet clover in negative ion mode in Example 2; Figure 5 The PCA score diagram of the mass spectrometry peaks (m / z) obtained from the soaking solutions of 20 single alfalfa seeds and 20 single yellow clover seeds in negative ion mode in Example 2. Figure 6 This is a normalized fingerprint heatmap of the characteristic mass spectrometry fingerprints (m / z) obtained from the soaking solutions of 20 single alfalfa seeds and 20 single yellow clover seeds in negative ion mode in Example 2. Detailed Implementation
[0021] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.
[0022] Example 1: A rapid identification method for single-seed alfalfa and yellow sweet clover includes the following steps: S1. Place 20 seeds each of alfalfa and yellow sweet clover into 1.5 mL plastic centrifuge tubes, disinfect them by soaking in 75 vt% ethanol aqueous solution for 1 min and then wash them with ultrapure water. Then add 150 µL of seed soaking nutrient solution (containing vitamin B1, amino acids, plant growth regulators and other components) for soaking and culture in the dark for 12 h to obtain the soaking solution. S2. Filter 100 µL of the seed soaking solution through a 0.22 µm pore size filter membrane, and use a blank nutrient solution with the same culture medium and process as the grass seeds as the blank control solution; inject 0.5 µL of the seed soaking solution directly into an Agilent 1290 UHPLC-6560 Q-TOF / MS mass spectrometer system, test and record the mass spectrum data; the test is performed in positive ion mode using an electrospray ionization source (ESI ionization source) as the mass ionization source, with a mass scan range of 100-750 Da, and the mobile phase is 90 vt% methanol aqueous solution containing 0.1 vt% formic acid; the specific mass spectrum outline is as follows. Figure 1 As shown.
[0023] S3. Forty mass spectrometry data were processed using Agilent's MassHunter Profinder software. After peak alignment, feature extraction was performed. The ion peak intensity filtering threshold was 5000 counts. The adduct ion types were [M+H]+, [M+Na]+, [M+K]+, and [M+NH4]+, with charge states of 1-2 and a Mass offset of 15 ppm. Principal component analysis (PCA) was used to reduce the dimensionality of the mass spectrometry data based on the correlation matrix. The number of principal components was 4. The score plot is shown below. Figure 2 As shown, the differences in mass spectrometry peak characteristics between the two types of seeds are visually presented. A mass spectrometry fingerprint is constructed with the mass-to-charge ratio (m / z) as the abscissa and the normalized ion intensity at that mass-to-charge ratio (m / z) as the ordinate, visually presenting the differences in mass spectrometry fingerprints between individual seeds of alfalfa and sweet clover. Figure 3 As shown.
[0024] It can be seen that the mass-to-charge ratio m / z is 103.0996, 103.0999, 117.0790, 137.0478, 143.0946, 143.0947, 143.0948, 146.0366, 157.1104, 165.0768, 165.0773, 175.0035, 179.0919, 197.0648, 223.1206, 247.2003, 256.1405, 260.2102, 261.0768, 265.1524, 272.1132, 272. 1140, 291.1900, 292.1746, 300.0632, 300.2052, 302.0427, 304.1293, 308.1705, 310.1165, 322.0308, 322.0312, 322.1866, 338.0191, 338.1603, 338.1606, 350.0634, 350.0639, 350.0641, 350.1865, 352.1764, 352.1778, 353.2424 359.9870, 366.0689, 366.1811, 368.1955, 380.0718, 382.1751, 391.1393, 394.0849, 432.1063, 448.1006, 448.1012, 452.1601, 472.0404, 486.0606, 486.0615, 486.0764, 504.0576, 505.1977, 526.1492, 526.1494, 530.0828, 530. 0829, 534.1381, 540.1656, 540.1664, 542.1239, 544.3038, 556.1395, 557.1433, 558.2799, 572.0842, 576.3281, 592.3012, 688.2023, 688.2026, 700.4854, 702.2223, 704.1768, 706.1773, 716.4590, 716.4603, 718.1953, 721.2179. This can be used as a mass spectrometry fingerprint feature set in positive ion mode, enabling rapid identification of two types of grass seeds.
[0025] Example 2: A rapid identification method for single-seed alfalfa and yellow sweet clover includes the following steps: S1. Place 20 seeds each of alfalfa and sweet clover into 1.5 mL plastic centrifuge tubes, sterilize by soaking in 55 ℃ hot water for 10 min and rinse with ultrapure water, then add 300 µL of ultrapure water for soaking, and incubate in the dark for 3 h to obtain the soaking solution. S2. Centrifuge 200 µL of the seed soaking solution at 10,000 rpm. Use ultrapure water (same as the grass seed culture medium and following the same culture process) as the grass seed culture solution as a blank control. Inject 2 µL of the seed soaking solution directly into an Agilent 1290 UHPLC-6560 Q-TOF / MS mass spectrometer system, test and record the mass spectrum data. The test is performed in negative ion mode using an electrospray ionization source (ESI ionization source) with a mass scan range of 100-750 Da and a mobile phase of 80 vt% methanol aqueous solution. The specific mass spectrum outline is shown below. Figure 4 As shown.
[0026] S3. Forty mass spectrometry data were processed using Agilent's MassHunter Profinder software for peak alignment and feature extraction. The ion peak intensity filtering threshold was 5000 counts, the adduct ion type was [MH]-, the charge state was 1-2, and the Mass offset was 15 ppm. Principal component analysis (PCA) was used to reduce the dimensionality of the mass spectrometry data based on the covariance matrix. The number of principal components was 4. The score plot is shown below. Figure 5 As shown, the differences in mass spectrometry peak characteristics between the two types of seeds are visually presented. A mass spectrometry fingerprint is constructed with the mass-to-charge ratio (m / z) as the abscissa and the normalized ion intensity at that mass-to-charge ratio (m / z) as the ordinate, visually presenting the differences in mass spectrometry fingerprints between individual seeds of alfalfa and sweet clover. Figure 6 As shown.
[0027] It can be seen that the mass-to-charge ratio m / z 168.0286, 242.1146, 244.1305, 251.0782, 284.0764, 286.0474, 300.0636, 310.1217, 312.2286, 314.0776, 322.0803, 328.0577, 328.1138, 328.2239, 330.2387, 342.1153, 351.1669, 353.1823, 355.1977, 369.1775, 370.1256, 371.1927, 372.1055, 373.2083, 385.1725, 388.1725, 390.1865, 396.2161, 400.1713, 402.1874, 432.1075, 434.0843, 440.0895, 448.0988, 448.0994, 448.1071, 462.1144, 466.1433, 486.0740, 492.1270, 492.1808, 518.1819, 518.3068, 534.1361, 534.2291, 552.1094, 564.1462, 590.2354, 602.1386, 616.1526, 622.3165, 666.2183, 702.1974, 734.1473. It can be used as a mass spectrometry fingerprint feature recognition set in negative ion mode, and can be used to quickly identify two types of grass seeds.
[0028] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A rapid identification method for single-seed alfalfa and yellow sweet clover, characterized in that, Includes the following steps: S1. Soak the surface-sterilized single grass seeds in ultrapure water or nutrient solution to obtain a soaking solution. S2. Inject the soaking solution into the UPLC-QTOF-MS mass spectrometry system, test and record the mass spectrum data; S3. Align the peaks of the mass spectrum data, extract features from the high-dimensional mass spectrum data, and construct a mass spectrometry fingerprint with the mass-to-charge ratio m / z as the abscissa and the normalized ion intensity of the sample at that mass-to-charge ratio m / z as the ordinate.
2. The rapid identification method for single-seed alfalfa and yellow sweet clover as described in claim 1, characterized in that, In step S1, disinfection is carried out by immersion in a 75% ethanol aqueous solution for 0.5-2 minutes; Disinfect by soaking in a 1 wt% potassium permanganate solution for 5-15 minutes; Disinfect by soaking in 55℃ hot water for 10-20 minutes; Rinse with ultrapure water after disinfection.
3. The rapid identification method for single-seed alfalfa and yellow sweet clover as described in claim 1, characterized in that, In step S1, the seeds are soaked for 0.5-24 hours under relatively sealed and light-protected conditions.
4. The rapid identification method for single-seed alfalfa and yellow sweet clover as described in claim 1, characterized in that, In step S2, before the soaking solution is injected, it is filtered through a 0.22 µm pore size filter membrane or centrifuged at a speed of not less than 10,000 rpm.
5. The rapid identification method for single-seed alfalfa and yellow sweet clover as described in claim 1, characterized in that, In step S2, a blank control solution is used, which is the same as the grass seed culture medium and has undergone the same process as the grass seed culture.
6. The rapid identification method for single-seed alfalfa and yellow sweet clover as described in claim 1, characterized in that, In step S2, the test was performed in positive / negative ion mode using an electrospray ionization source as the mass spectrometry ionization source. The mass scan range was 100-750 Da, the injection volume was 0.2-5 μL, and the mobile phase was 70-95 vt% methanol aqueous solution. The methanol-water solution contains 0.1 vt% formic acid.
7. The rapid identification method for single-seed alfalfa and yellow sweet clover as described in claim 1, characterized in that, In step S3, the high-dimensional mass spectrometry data includes mass-to-charge ratio m / z and ion intensity information.
8. The rapid identification method for single-seed alfalfa and yellow sweet clover as described in claim 1, characterized in that, In step S3, the mass spectrum data is peak aligned and then feature extracted using MassHunter Profinder software; The ion peak intensity filtering threshold is 5000 counts, the charge state is 1-2, and the Mass offset is 15 ppm. The adduct ion types are [M+H]+, [M+Na]+, [M+K]+ and [M+NH4]+ or the adduct ion type is [MH]-.
9. The rapid identification method for single-seed alfalfa and yellow sweet clover as described in claim 1, characterized in that, In step S3, principal component analysis is used to reduce the dimensionality of the mass spectrum data and visualize it based on the correlation matrix or covariance matrix. The number of principal components is 4.