CN110849795B - Method for rapidly detecting Chinese cabbage ploidy by using flow cytometer - Google Patents

Abstract

The invention discloses a method for rapidly detecting Chinese cabbage ploidy by using a flow cytometer, belonging to the technical field of biology, wherein the method comprises the steps of preparing an extraction buffer solution; taking Chinese cabbage cotyledons, putting the Chinese cabbage cotyledons into a culture dish, adding an extraction buffer solution, dissociating, and cutting to obtain a mixture; sucking the mixture by a pipette gun, putting the mixture into a centrifuge tube, performing centrifugal treatment, and removing supernatant; adding staining solution, detecting the ploidy of Chinese cabbage with flow cytometry, and repeating for 3 times as control for each sample with diploid variety as control. According to the invention, the Chinese cabbage cotyledons are selected to carry out ploidy detection of the flow cytometer, so that the problem of ploidy drift in the process of identifying the Chinese cabbage ploidy by using the flow cytometer is successfully solved.

Description

Method for rapidly detecting Chinese cabbage ploidy by using flow cytometer

Technical Field

The invention relates to the technical field of biology, in particular to a method for rapidly detecting Chinese cabbage ploidy by using a flow cytometer.

Background

Chinese cabbage is one of the main cruciferous vegetables, and Chinese cabbage breeding comprises the following steps: two forms of crossbreeding and self-breeding. In view of the homogeneity and difficult innovation of the existing Chinese cabbage germplasm resources, and the urgent need of Chinese cabbage breeding due to the research and development of new means and new technology, people are beginning to research the tetraploid Chinese cabbage breeding at present.

In the process of tetraploid resource innovation, the identification of resource materials usually adopts a microscopic examination mode, and the method has the following defects: long time consumption, low efficiency, limited visual field, toxic hazard of medicines and the like.

Flow cytometers are currently widely used in animal and plant science. The flow cytometer is a simple, fast and high-throughput method for detecting the ploidy level, intraspecific hybridization, parthenogenesis, genome size, polyploid and chromosome of plants. The instrument for fast quantitative determination and analysis of high speed linear flowing cell or biological particle includes mainly sample liquid flow technology, cell counting and sorting technology, data acquisition and analysis technology in computer, etc. Is characterized in that: the measurement speed is fast, the measured population is large, and multi-parameter measurement can be carried out, namely multi-parameter measurement related to physical and biochemical characteristics can be carried out on the same cell. Flow cytometry plays an important role in the detection of crops.

The successful application of the flow cytometer in detecting other vegetables, such as cabbage, cauliflower, leaf lettuce, cucumber, tomato, potato, spinach, radish lettuce, pumpkin, fennel, strawberry and other crops, is compared. The peak value graphs (fig. 1-12) can show that the application result of the flow cytometer on other crops is relatively accurate, the peak value is clear, the impurities are few, and the ploidy of the flow cytometer can be obviously seen.

However, the flow cytometry has problems in Chinese cabbage ploidy detection: the flow cytometer has the problem of ploidy drift in the process of detecting the tetraploid Chinese cabbage. Such as: the tetraploid Chinese cabbage has a high diploid peak value in flow cytometry detection, and has the problems of ploidy drift, more miscellaneous peaks, relatively unobvious main peak values and the like in detection of different leaves (outer leaves, inner leaves and first true leaves).

Therefore, a reliable method for rapidly identifying the ploidy of the Chinese cabbage based on flow cytometry detection is urgently needed to be established.

Disclosure of Invention

The invention aims to provide a method for rapidly detecting the ploidy of Chinese cabbages by using a flow cytometer, so as to solve the problem of ploidy drift when the flow cytometer is used for detecting the ploidy of Chinese cabbages.

At present, the flow cytometer has successful application in detecting ploidy of other vegetables except Chinese cabbage, and the invention is used for the following vegetables: the ploidy detection of crops such as cabbage, cauliflower, leaf lettuce, cucumber, tomato, potato, spinach, radish lettuce, pumpkin, fennel, strawberry and the like is compared. From the following numerical value chart (table 1) and peak value chart (fig. 1 to fig. 12), it can be known that the operation result of the flow cytometer on other crops is relatively accurate, the peak value is clear, the impurities are few, and the ploidy of the flow cytometer can be obviously seen.

TABLE 1

The flow cytometer has the problems of ploidy drift in the process of detecting tetraploid Chinese cabbage: such as: the tetraploid Chinese cabbage has a high diploid peak value in flow cytometry detection, and has the problems of ploidy drift, more miscellaneous peaks, relatively unobvious main peak values and the like in detection of different leaves (outer leaves, inner leaves and first true leaves) (see table 2 and fig. 13-21).

TABLE 2

In order to solve the problem of ploidy drift when the flow cytometer detects the ploidy of the Chinese cabbage, the invention provides the following scheme:

the invention provides a method for rapidly detecting the ploidy of Chinese cabbage by using a flow cytometer, which uses cotyledons of the Chinese cabbage for detection.

Further, the method for rapidly detecting the ploidy of the Chinese cabbage by using the flow cytometer comprises the following steps:

(1) preparing an extraction buffer solution;

(2) putting the Chinese cabbage cotyledons into a culture dish, adding the extraction buffer solution prepared in the step (1), dissociating, and cutting to obtain a mixture;

(3) sucking the mixture obtained in the filtering step (2) by using a pipette gun, putting the mixture into a centrifuge tube, performing centrifugal treatment, and removing supernatant;

(4) adding staining solution, placing into CYTOFLEX flow cytometry to detect ploidy of Chinese cabbage, and repeating for 3 times with diploid variety "polyclonal antibody No. 4" as test control.

Furthermore, the cotyledon collecting period is between the expansion of the cotyledon of the Chinese cabbage and the expansion of the first true leaf.

Further, the extraction buffer solution is Tris-HCl solution, KCl solution, NaCl solution, EDTA-Na₂And a mixture of mercaptoethanol and Triton X-100.

Further, the extraction buffer solution is Tris-HCl solution, KCl solution, NaCl solution, EDTA-Na₂The mass ratio of each substance in the mixed solution of the solution, the mercaptoethanol solution and the Triton X-100 is 1:4:1:1:2: 1.

Further, the preparation method of the buffer extracting solution comprises the following steps:

(1) weighing 1.82g of Tris-HCl solution, adding water to 950ml, adjusting pH to 7.5, weighing 5.96g/L of KCl solution, weighing 1.17g/L of NaCl solution and EDTA-Na₂Weighing 7.45g/L, absorbing 1050ul/L by mercaptoethanol and absorbing 500ul/L by Triton X-100;

(2) and (2) pouring the weighed/sucked medicines in the step (1) into 950ML of solution in sequence, adding distilled water to a constant volume of 1000ML, stirring until the medicines are dissolved, and filtering by using a 300-mesh nylon filter screen for later use.

Further, every 1cm²Cotyledons, 1-3mL extraction buffer was added.

Further, the staining solution is a mixed solution composed of DAPI staining solution, ribonuclease A and 1XPBS buffer solution, the ratio of the staining solution DAPI to the ribonuclease A is 2000:1, and the volume of the 1XPBS buffer solution is 100mL for later use.

Further, the preparation process of the dyeing solution is as follows:

(1) taking 1mg/ml of DAPI staining solution; ribonuclease A0.05 mg/ml, 1XPBS buffer solution, constant volume;

(2) filtering twice with 300 mesh nylon filter screen until no precipitate is formed, and placing into a brown bottle for later use.

Further, the centrifugation time of the step (3) is 2-3 min.

The invention adopts a method of identifying different positions of the same species for identifying the phenomenon that the Chinese cabbage ploidy is subjected to ploidy drift by using a flow cytometer. In the selected position of the Chinese cabbage, the old mature leaves are selected for detection at first, the problems of more miscellaneous peaks, unobvious peak values, paradoxical repeated detection results and the like are generated in the result, then the new leaves are selected for detection, the phenomenon of unstable ploidy drift still occurs in the result, and finally the Chinese cabbage cotyledons are selected for detection, the phenomena of obvious peak values, less impurities and no ploidy drift are generated in the result, and the chromosome microscopy is combined to verify that the detection result is accurate and reliable. Therefore, the invention selects the Chinese cabbage cotyledons to carry out ploidy detection of the flow cytometer, and successfully solves the problem of ploidy drift of the Chinese cabbage identified by the flow cytometer.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without inventive exercise.

FIG. 1 shows the result of detecting spinach ploidy using a flow cytometer;

FIG. 2 shows the result of ploidy detection of cauliflower by flow cytometry;

FIG. 3 shows the result of strawberry ploidy detection using a flow cytometer;

FIG. 4 shows the results of ploidy detection of tomato by flow cytometry;

FIG. 5 shows the result of the ploidy detection of cabbage using a flow cytometer;

FIG. 6 shows the result of ploidy detection of cucumber by flow cytometry;

FIG. 7 shows the results of the ploidy test of fennel using a flow cytometer;

FIG. 8 shows the result of ploidy detection of radish by flow cytometry;

FIG. 9 shows the result of using flow cytometry to detect the ploidy of lettuce;

FIG. 10 shows the results of ploidy detection of potatoes by flow cytometry;

FIG. 11 shows the result of the ploidy detection of Cucurbita pepo by flow cytometry;

FIG. 12 shows the results of the detection of the ploidy of rye using a flow cytometer;

FIG. 13 shows the result of detecting ploidy of Chinese cabbage at the true leaf part of multi-dimensional 462 of Chinese cabbage by flow cytometry;

FIG. 14 shows the results of the detection of the ploidy of the leaf part of the multi-dimensional 462 outer leaf of Chinese cabbage by flow cytometry;

FIG. 15 shows the results of the detection of the ploidy of Chinese cabbage at the inner leaf part of the multi-dimensional 462 of Chinese cabbage by flow cytometry;

FIG. 16 shows the result of detecting the ploidy of Chinese cabbage at 505 true leaves by flow cytometry;

FIG. 17 shows the results of the detection of the ploidy of Chinese cabbage at the outer leaf part of multidimensional 505 using flow cytometry;

FIG. 18 shows the results of the detection of Chinese cabbage ploidy at the inner leaf part of Chinese cabbage multidimensional 505 by flow cytometry;

FIG. 19 shows the result of detecting the ploidy of cabbage at the true leaf part of the multi-breed No. 2 cabbage using a flow cytometer;

FIG. 20 shows the result of detecting the ploidy of the celery cabbage of the outer leaf part of the growing cabbage No. 2 by using a flow cytometer;

FIG. 21 shows the result of detecting the ploidy of cabbage at the inner leaf part of No. 2 of the multi-breed cabbage by flow cytometry;

FIG. 22 shows the result of detecting the ploidy of the control diploid cabbage against the leaf of the No. 4 cotyledon by flow cytometry;

FIG. 23 shows the result of detecting the ploidy of tetraploid cabbage with multi-dimensional 462 cotyledon part using flow cytometry;

FIG. 24 shows the result of measuring the ploidy of the tetraploid cabbage with 505 cotyledons by flow cytometry;

FIG. 25 is a graph showing the results of measuring the ploidy of the tetraploid cabbage with the 2 nd cotyledon thereof in the case of the 2 nd cotyledon thereof by using a flow cytometer;

in FIGS. 1-25, the COUNT value on the ordinate of the peak plots represents the relative value of the number of cells measured, the PB450-A value on the abscissa represents the channel value of fluorescence, and the position of the peak reflects the ploidy of the test sample.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

Example 1

(1) Preparing an extraction buffer solution: weighing 1.82g of Tris-HCl solution, adding water to 950ml, adjusting the pH value to 7.5, weighing 5.96g/L of KCl solution, weighing 1.17g/L of NaCl solution, weighing 7.45g/L of EDTA-2Na, sucking 1050ul/L of mercaptoethanol and sucking 500ul/L of Triton X-100; sequentially diluting the weighed/sucked medicines to 1000mL by using distilled water, stirring until the medicines are dissolved, and filtering by using a 300-mesh nylon filter screen for later use;

preparing a dyeing solution: taking a 1mg/mL DAPI staining solution 10 tube, taking 5ul ribonuclease A0.05 mg/mL and 1XPBS buffer solution for constant volume to 100 mL; filtering twice with 300 mesh nylon filter screen until no precipitate is formed, and placing into a brown bottle for later use;

(2) spreading cotyledon to the first true leaf, and taking 1cm²Putting cotyledons into a culture dish, adding 2mL of the extraction buffer solution prepared in the step (1), dissociating, and cutting a blade to obtain a mixture;

(3) sucking the mixture obtained in the step (2) by using a 1mL pipette, placing the mixture into a 2mL centrifuge tube, centrifuging by using a centrifuge at a centrifuging rate of 1000r/min for 3min, and removing the supernatant;

(4) adding 50 μ L staining solution, placing in CYTOFLEX flow cytometer to detect Chinese cabbage ploidy, using diploid variety "polyclonal antibody No. 4" as control, and repeating for 3 times for each test sample.

In the flow cytometry identification process, a diploid variety 'polyclonal antibody 4' is taken as a reference contrast, the ploidy of 'multidimensional 462', 'multidimensional 505' and 'fertile 2' is detected, the results of flow cytometry ploidy analysis are shown in a table 3 and figures 22-25, the control diploid in 500 cell nucleuses sucked by the flow cytometry accounts for more than 70% of the total father group percentage, the tetraploid can be between 60% and 70% of the total father group percentage, a chimera phenomenon appears in the tetraploid detection process, the detected neutron leaves relatively meet the requirement of normal ploidy characteristics, and the ploidy of the Chinese cabbage can be preliminarily determined according to the father group percentage.

TABLE 3

The peak plots in FIGS. 1-25 have the COUNT values on the ordinate representing the relative values of the number of cells measured, PB450-A values on the abscissa representing the channel values of fluorescence, and the position of the peak reflecting the ploidy of the test sample. As can be seen from FIGS. 22 to 25, the relative content of the chromosome of the control diploid "polyclonal antibody 4" (FIG. 22) is about 40, while the relative content of the chromosome of the variety to be tested "multidimensional 462", "multi 505" and "fertile 2" (FIG. 23, FIG. 24 and FIG. 25) is about 80, which is approximately 2 times the relative content of the diploid, indicating that the diploid is determined to be tetraploid.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Claims (9)

1. A method for rapidly detecting the ploidy of Chinese cabbage by using a flow cytometer is characterized in that cotyledons of the Chinese cabbage are used for detection;

the cotyledon collecting period is from the expansion of the cotyledon of the Chinese cabbage to the expansion of the first true leaf.

2. The method for rapidly detecting Chinese cabbage ploidy by using the flow cytometer as claimed in claim 1, comprising the following steps:

(1) preparing an extraction buffer solution;

(2) putting the Chinese cabbage cotyledons into a culture dish, adding the extraction buffer solution prepared in the step (1), dissociating, and cutting to obtain a mixture;

(3) sucking the mixture obtained in the filtering step (2) by using a pipette gun, putting the mixture into a centrifuge tube, performing centrifugal treatment, and removing supernatant;

(4) adding staining solution, placing into flow cytometer to detect Chinese cabbage ploidy, using diploid variety as control, and repeating for 3 times for each test sample.

3. The method for rapidly detecting Chinese cabbage ploidy by using flow cytometer as claimed in claim 2, wherein the extraction buffer solution is Tris-HCl solution, KCl solution, NaCl solution, EDTA-Na₂And a mixture of mercaptoethanol and Triton X-100.

4. The method for rapidly detecting Chinese cabbage ploidy by using flow cytometer as claimed in claim 3, wherein the extraction buffer solution is Tris-HCl solution, KCl solution, NaCl solution, EDTA-Na₂The mass ratio of each substance in the mixed solution of the solution, the mercaptoethanol solution and the Triton X-100 is 1:4:1:1:2: 1.

5. The method for rapidly detecting Chinese cabbage ploidy by using a flow cytometer as claimed in claim 4, wherein the preparation method of the buffer extract comprises:

(1) weighing 1.82g of Tris-HCl solution, adding water to 950ml, adjusting pH to 7.5, weighing 5.96g/L of KCl solution, weighing 1.17g/L of NaCl solution and EDTA-Na₂Weighing 7.45g/L, absorbing 1050ul/L by mercaptoethanol and absorbing 500ul/L by Triton X-100;

(2) pouring the weighed/sucked medicines in the step (1) into 950mL of solution with constant volume in sequence, adding distilled water to 1000mL, stirring until the distilled water is dissolved, and filtering by using a 300-mesh nylon filter screen for later use.

6. Use of a flow cytometer according to claim 2The method for rapidly detecting the ploidy of the Chinese cabbage is characterized in that each 1cm of the method²Cotyledons, 1-3mL extraction buffer was added.

7. The method for rapidly detecting ploidy of Chinese cabbage by using flow cytometer as claimed in claim 2, wherein the staining solution is a mixed solution of DAPI staining solution, ribonuclease A and 1 XPPBS buffer solution, the ratio of DAPI staining solution to ribonuclease A is 2000:1, and the volume of 1XPBS buffer solution is 100mL for future use.

8. The method for rapidly detecting the ploidy of the Chinese cabbage by using the flow cytometer as claimed in claim 7, wherein the dyeing solution is prepared by the following steps:

(1) taking the final concentration of the DAPI staining solution as 1 mg/ml; the final concentration of the ribonuclease A is 0.05mg/mL, and the volume of the 1XPBS buffer solution is 100 mL;

(2) filtering twice with 300 mesh nylon filter screen until no precipitate is formed, and placing into a brown bottle for later use.

9. The method for rapidly detecting the ploidy of the Chinese cabbage by using the flow cytometer as claimed in claim 2, wherein the centrifugation time in the step (3) is 2-3 min.

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