Method for rapid detection of alfalfa chromosome ploidy by flow cytometry

Abstract

The application discloses a method for rapidly detecting alfalfa chromosome ploidy by using flow cytometry, and belongs to the technical field of plant ploidy analysis, and the method comprises the following steps: taking fresh alfalfa leaves, placing the alfalfa leaves, LB01 dissociation solution and steel balls in a container, and using a ball mill to break the tissues at a high frequency to release cell nuclei; then filtering and centrifuging the mixed solution to obtain cell nucleus precipitates; adding pre-cooled dissociation solution and PI staining solution to the precipitates, mixing, and then staining under low-temperature and light-proof conditions, finally preparing stained cell nucleus suspension, and using a flow cytometer to detect, and determining the chromosome ploidy according to the peak value distribution of the fluorescence signal. The application realizes efficient and uniform release and purification of the cell nuclei by using mechanical grinding combined with LB01 dissociation solution, significantly improves the detection efficiency and the atlas quality, has the advantages of simple operation, small sample requirement, high throughput, accurate and reliable results, and is suitable for large-scale rapid identification of different ploidy of alfalfa plants.

Description

Technical Field

[0001] This invention relates to the field of plant ploidy analysis technology, specifically a method for rapidly detecting chromosome ploidy in alfalfa using flow cytometry. Background Technology

[0002] Alfalfa (Medicago spp.) is an important legume forage and ecological restoration plant with wide cultivation and application. Its germplasm resources exhibit rich ploidy variation, including diploid, tetraploid, and higher ploidy types. Chromosomal ploidy is a key fundamental trait for alfalfa germplasm resource identification, parent selection in hybridization breeding, and genetic and evolutionary research. Therefore, establishing an accurate, rapid, and efficient method for ploidy identification is of great significance for alfalfa germplasm resource evaluation, core germplasm bank construction, and early screening of breeding materials.

[0003] Traditional chromosome ploidy identification mainly relies on chromosome counting, which involves preparing slides during metaphase of mitosis and directly observing and counting the number of chromosomes under a microscope. However, this method depends on cells at a specific stage of division, has a long material preparation cycle, and its success rate is greatly affected by the growth state; it also has extremely low throughput, making it difficult to meet the needs of rapid identification of large-scale germplasm resources.

[0004] Therefore, there is an urgent need to develop a method for rapid detection of chromosome ploidy in alfalfa using flow cytometry. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a method for rapidly detecting chromosome ploidy in alfalfa using flow cytometry, comprising the following steps: S1. Sample disruption: Take fresh alfalfa leaves, place them in a container with dissociation solution and steel balls, and use a ball mill to shake and disrupt the leaf tissue to release the cell nuclei. S2, Filtration and Collection: Filter the shaken mixture and collect the filtrate containing cell nuclei; S3. Preparation of nuclear suspension: Centrifuge the filtrate, discard the supernatant, and obtain the cell nuclear precipitate; S4. Staining: Add dissociation solution and staining solution containing nucleic acid dye to the cell nucleus precipitate, mix well and stain under the dark at 3-5℃ to obtain a stained cell nucleus suspension. S5. Detection: The stained cell nucleus suspension is detected using a flow cytometer, and the chromosome ploidy of alfalfa is determined based on the peak distribution of the obtained fluorescence signal.

[0006] Preferably, in step S1, the steel balls have a diameter of 3mm and are used in quantities of 3-5.

[0007] Preferably, in step S1, the oscillation conditions are: oscillation frequency of 1600-2000 Hz and oscillation time of 1-3 minutes.

[0008] Preferably, in step S1, the dissociation liquid is LB01 dissociation liquid.

[0009] Preferably, in step S1, the amount of fresh alfalfa leaves and dissociation solution used is: 1 mL of dissociation solution is added for every 80-120 mg of fresh alfalfa leaves.

[0010] Preferably, in step S1, after shaking, the container is placed on ice and left to stand for 8-12 minutes.

[0011] Preferably, in step S2, a 300-mesh nylon mesh is used for filtration.

[0012] Preferably, in step S3, the centrifugation conditions are: centrifugation at 800-1200 rpm for 3-7 minutes at 4°C.

[0013] Preferably, in step S4, the nucleic acid dye is propidium iodide (PI); the dissociation solution is pre-cooled to 3-5°C; the solid-liquid mass ratio of the cell nucleus precipitate to the dissociation solution is 1:8-1:10; and the staining conditions are staining for 25-35 minutes in the dark at 3-5°C.

[0014] Preferably, in step S5, when using flow cytometry for detection, the detection is stopped after collecting 10,000 cell nuclei to obtain the optimal ploidy identification results.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Traditional methods involve cumbersome and time-consuming experimental procedures, long material preparation cycles, and extremely low throughput, making it difficult to meet the needs of rapid identification of large-scale germplasm resources. This invention utilizes flow cytometry, which enables batch processing. The overall process from sample disruption to detection is relatively simple, and detection can be completed in a short time with high throughput, enabling rapid identification of large-scale germplasm resources and improving detection efficiency.

[0016] 2. Traditional methods require high operator skills and rely on cells at a specific division stage. The success rate is greatly affected by the growth state. This invention uses a ball mill to break up leaf tissue and release cell nuclei. On average, breaking up a single leaf sample only takes 20-30 seconds. Subsequent steps such as filtration, centrifugation, and staining are relatively easy to master. The operation requires relatively low operator skills and does not rely on cells at a specific division stage, reducing the risk of detection failure due to the material's growth state.

[0017] 3. Using the method of the present invention, only a small amount (about 100mg) of fresh young leaves is needed to complete the test, which causes little damage to the plant material. It is especially suitable for early identification of rare or young plants. This method is applicable to the detection of different ploidy (such as diploid, tetraploid, hexaploid, etc.) of alfalfa (Medicago sativa) and other alfalfa plants.

[0018] 4. This invention uses mechanical oscillation, which has a more uniform and intense force than manual chopping. This ensures that the cell nuclei are released more completely from the tough plant tissue, reducing cell nucleus loss and result deviation caused by insufficient crushing, thereby improving the intensity of the flow cytometer detection signal.

[0019] 5. The present invention uses LB01 dissociation solution, which can more effectively purify cell nuclear suspension, reduce fluorescence interference caused by cell debris, chloroplasts and other impurities, make the main peak in the flow cytometry detection spectrum sharper and clearer, significantly reduce impurity peaks, facilitate the identification and interpretation of ploidy peaks, and reduce the risk of misjudgment of results. Attached Figure Description

[0020] Figure 1 Histograms showing the effects of different fragmentation methods and dissociation solutions on alfalfa chromosome ploidy detection results; Figure 2 The effect of different cell nucleus numbers on the signal-to-noise ratio of alfalfa chromosome ploidy histogram. Detailed Implementation

[0021] Example 1 A method for rapidly detecting chromosome ploidy in alfalfa using flow cytometry includes the following steps: S1. Sample disruption: Take fresh alfalfa leaves, place them in a container with dissociation solution and steel balls, and use a ball mill to shake and disrupt the leaf tissue to release the cell nuclei. S2, Filtration and Collection: Filter the shaken mixture and collect the filtrate containing cell nuclei; S3. Preparation of nuclear suspension: Centrifuge the filtrate, discard the supernatant, and obtain the cell nuclear precipitate; S4. Staining: Add dissociation solution and staining solution containing nucleic acid dye to the cell nucleus precipitate, mix well and stain under the dark at 3-5℃ to obtain a stained cell nucleus suspension. S5. Detection: The stained cell nucleus suspension is detected using a flow cytometer, and the chromosome ploidy of alfalfa is determined based on the peak distribution of the obtained fluorescence signal.

[0022] Moreover, in step S1, the steel ball has a diameter of 3mm and is used in quantities of 3.

[0023] Furthermore, in step S1, the oscillation conditions are: an oscillation frequency of 1600 Hz and an oscillation time of 1 minute.

[0024] Moreover, in step S1, the dissociation liquid is LB01 dissociation liquid.

[0025] Furthermore, in step S1, the amount of fresh alfalfa leaves and dissociation solution used is: 1 mL of dissociation solution is added for every 80 mg of fresh alfalfa leaves.

[0026] Furthermore, in step S1, after oscillation, the container is placed on ice and left to stand for 8 minutes.

[0027] Furthermore, in step S2, a 300-mesh nylon mesh is used for filtration.

[0028] Furthermore, in step S3, the centrifugation conditions are: centrifuging at 800 rpm for 3 minutes at 4°C.

[0029] Furthermore, in step S4, the nucleic acid dye is propidium iodide (PI); the dissociation solution is pre-cooled to 3°C; the solid-liquid mass ratio of the cell nucleus precipitate to the dissociation solution is 1:8; and the staining conditions are staining at 3°C ​​in the dark for 25-35 minutes.

[0030] Example 2 A method for rapidly detecting chromosome ploidy in alfalfa using flow cytometry includes the following steps: S1. Sample disruption: Take fresh alfalfa leaves, place them in a container with dissociation solution and steel balls, and use a ball mill to shake and disrupt the leaf tissue to release the cell nuclei. S2, Filtration and Collection: Filter the shaken mixture and collect the filtrate containing cell nuclei; S3. Preparation of nuclear suspension: Centrifuge the filtrate, discard the supernatant, and obtain the cell nuclear precipitate; S4. Staining: Add dissociation solution and staining solution containing nucleic acid dye to the cell nucleus precipitate, mix well and stain under the dark at 3-5℃ to obtain a stained cell nucleus suspension. S5. Detection: The stained cell nucleus suspension is detected using a flow cytometer, and the chromosome ploidy of alfalfa is determined based on the peak distribution of the obtained fluorescence signal.

[0031] Moreover, in step S1, the steel ball has a diameter of 3mm and is used in quantities of 5.

[0032] Furthermore, in step S1, the oscillation conditions are: an oscillation frequency of 2000 Hz and an oscillation time of 1-3 minutes.

[0033] Moreover, in step S1, the dissociation liquid is LB01 dissociation liquid.

[0034] Furthermore, in step S1, the amount of fresh alfalfa leaves and dissociation solution used is: 1 mL of dissociation solution is added for every 120 mg of fresh alfalfa leaves.

[0035] Furthermore, in step S1, after oscillation, the container is placed on ice and left to stand for 12 minutes.

[0036] Furthermore, in step S2, a 300-mesh nylon mesh is used for filtration.

[0037] Furthermore, in step S3, the centrifugation conditions are: centrifuging at 1200 rpm for 7 minutes at 4°C.

[0038] Furthermore, in step S4, the nucleic acid dye is propidium iodide (PI); the dissociation solution is pre-cooled to 5°C; the solid-liquid mass ratio of the cell nucleus precipitate to the dissociation solution is 1:10; and the staining conditions are staining for 35 minutes in the dark at 5°C.

[0039] Example 3 A method for rapidly detecting chromosome ploidy in alfalfa using flow cytometry includes the following steps: S1. Sample disruption: Take fresh alfalfa leaves, place them in a container with dissociation solution and steel balls, and use a ball mill to shake and disrupt the leaf tissue to release the cell nuclei. S2, Filtration and Collection: Filter the shaken mixture and collect the filtrate containing cell nuclei; S3. Preparation of nuclear suspension: Centrifuge the filtrate, discard the supernatant, and obtain the cell nuclear precipitate; S4. Staining: Add dissociation solution and staining solution containing nucleic acid dye to the cell nucleus precipitate, mix well and stain at 4°C in the dark to obtain a stained cell nucleus suspension. S5. Detection: The stained cell nucleus suspension is detected using a flow cytometer, and the chromosome ploidy of alfalfa is determined based on the peak distribution of the obtained fluorescence signal.

[0040] Moreover, in step S1, the steel ball has a diameter of 3mm and is used in quantities of 4.

[0041] Furthermore, in step S1, the oscillation conditions are: an oscillation frequency of 1800 Hz and an oscillation time of 2 minutes.

[0042] Moreover, in step S1, the dissociation liquid is LB01 dissociation liquid.

[0043] Furthermore, in step S1, the amount of fresh alfalfa leaves and dissociation solution used is: 1 mL of dissociation solution is added for every 100 mg of fresh alfalfa leaves.

[0044] Furthermore, in step S1, after oscillation, the container is placed on ice and left to stand for 10 minutes.

[0045] Furthermore, in step S2, a 300-mesh nylon mesh is used for filtration.

[0046] Furthermore, in step S3, the centrifugation conditions are: centrifuging at 1000 rpm for 5 minutes at 4°C.

[0047] Furthermore, in step S4, the nucleic acid dye is propidium iodide (PI); the dissociation solution is pre-cooled to 4°C; the solid-liquid mass ratio of the cell nucleus precipitate to the dissociation solution is 1:9; and the staining conditions are staining for 30 minutes in the dark at 4°C.

[0048] Experimental Section The dissociation solutions (LB01, WPB, Galbraith) used in the following experiments were all provided by Shanghai Yuanye Biotechnology Co., Ltd.

[0049] Experiment 1 Experimental group: 100 mg of fresh alfalfa leaves were placed in a 1.5 mL centrifuge tube, along with 3-5 3 mm steel balls and 1 mL of dissociation buffer (LB01, WPB, Galbraith). The mixture was then placed in a ball mill and shaken at 1800 Hz for 2 min, followed by 10 min on ice. The entire liquid was filtered through a 300-mesh nylon mesh into a 1.5 mL centrifuge tube, centrifuged at 1000 rpm for 5 min at 4 °C, and the supernatant was discarded. 200 μL of 4 °C dissociation buffer and PI staining solution were added, and the mixture was gently mixed and stained in the dark at 4 °C for 30 min.

[0050] Control group: Approximately 100 mg of fresh leaf material was rinsed thoroughly with running water and dried with filter paper. 1 mL of dissociation buffer (LB01, WPB, Galbraith) was added, and the mixture was quickly and vertically chopped using a single-edged razor blade. The mixture was then placed on ice for 10 min to allow cells to dissociate into the extract. All liquid was filtered through a 300-mesh nylon mesh into a 1.5 mL centrifuge tube. The tube was centrifuged at 1000 rpm for 5 min at 4°C, the supernatant was discarded, and 200 μL of 4°C dissociation buffer and PI staining solution were added. The mixture was gently mixed and stained in the dark at 4°C for 30 min. The stained cell nuclear suspension was then transferred to a flow cytometry sampler to detect ploidy.

[0051] The alfalfa leaves from the two treatment groups were analyzed using flow cytometry. Chromosomal ploidy of the alfalfa was determined based on the peak distribution of the obtained fluorescence signals. The results are as follows: Figure 1As shown in the figure, the peak values ​​of the chromosome ploidy histograms obtained by the experimental group using a grinder under different dissociation solutions were all higher than those obtained by the control group using blade chopping. At the same time, the width of the impurity peaks (cell fragments) in the experimental group was also finer than the histogram obtained by the control group using blade chopping, indicating that grinding with a grinder can obtain better ploidy identification results.

[0052] Figure 1 It can also be seen that when the same alfalfa sample was treated with the three dissociation solutions, the peak value of the chromosome ploidy histogram of alfalfa treated with LB01 dissociation solution was significantly higher than that of the other two dissociation solutions, and the width and height of the heterogeneous peak band on the left were also lower than those of the other two dissociation solutions.

[0053] Experiment 2 Take approximately 100 mg of fresh alfalfa leaves from the same batch and part as in Experiment 1. Using LB01 dissociation buffer, add 3-5 3 mm steel balls to a grinder, shake at 1800 Hz for 2 min to prepare a single-nucleus suspension. After PI staining, divide into three portions, filter through a 300-mesh sieve, and analyze using flow cytometry. Set parameters: stop the analysis after collecting 5000, 10000, 15000, and 20000 nuclei. Obtain chromosome ploidy histograms (…). Figure 2 As can be seen, with the increase in the number of cell nuclei collected, the height of the heterogeneous peak on the left side of the chromosome ploidy histogram first decreases and then increases, while the height of the target peak shows a trend of first increasing and then decreasing.

[0054] As the total number of cell nuclei collected increases, the signal intensity of the left-side clutter peaks in the chromosome ploidy histogram shows a trend of first decreasing and then increasing, while the height of the target main peak, representing DNA content, exhibits a pattern of first increasing and then decreasing. Specifically, considering both the significance of the main peak and the level of background noise, a ploidy identification histogram with the optimal signal-to-noise ratio and the sharpest and clearest main peak can be obtained when 10,000 cell nuclei are collected, providing the most reliable basis for determining ploidy.

Claims

1. A method for rapid detection of chromosome ploidy in alfalfa using flow cytometry, characterized in that, Includes the following steps: S1. Sample crushing: Take fresh alfalfa leaves, place them in a container with dissociation solution and steel balls, and use a ball mill to shake and crush the leaf tissue; the dissociation solution is LB01 dissociation solution. S2, Filtration and Collection: Filter the shaken mixture and collect the filtrate containing cell nuclei; S3. Preparation of nuclear suspension: Centrifuge the filtrate, discard the supernatant, and obtain the cell nuclear precipitate; S4. Staining: Add dissociation solution and staining solution containing nucleic acid dye to the cell nucleus precipitate, mix well and stain under the dark at 3-5℃ to obtain a stained cell nucleus suspension. S5. Detection: The stained cell nucleus suspension is detected using a flow cytometer. After collecting 10,000 cell nuclei, the detection is stopped. The chromosome ploidy of alfalfa is determined based on the peak distribution of the obtained fluorescence signal.

2. The method according to claim 1, characterized in that, In step S1, the steel balls have a diameter of 3mm and are used in quantities of 3-5.

3. The method according to claim 1, characterized in that, In step S1, the oscillation conditions are: oscillation frequency of 1600-2000 Hz and oscillation time of 1-3 minutes.

4. The method according to claim 1, characterized in that, In step S1, the amount of fresh alfalfa leaves and dissociation solution used is: 1 mL of dissociation solution is added for every 80-120 mg of fresh alfalfa leaves.

5. The method according to claim 1, characterized in that, In step S1, after shaking, the container is placed on ice and left to stand for 8-12 minutes.

6. The method according to claim 1, characterized in that, In step S2, a 300-mesh nylon mesh is used for filtration.

7. The method according to claim 1, characterized in that, In step S3, the centrifugation conditions are: centrifuging at 800-1200 rpm for 3-7 minutes at 4°C.

8. The method according to claim 1, characterized in that, In step S4, the nucleic acid dye is propidium iodide; the dissociation solution is pre-cooled to 3-5°C; the solid-liquid mass ratio of the cell nucleus precipitate to the dissociation solution is 1:8-1:10; and the staining conditions are staining at 3-5°C in the dark for 25-35 minutes.

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