CN109187673B - Plant genetic relationship identification method based on pollen - Google Patents
Plant genetic relationship identification method based on pollen Download PDFInfo
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- CN109187673B CN109187673B CN201811242374.9A CN201811242374A CN109187673B CN 109187673 B CN109187673 B CN 109187673B CN 201811242374 A CN201811242374 A CN 201811242374A CN 109187673 B CN109187673 B CN 109187673B
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Abstract
The invention discloses a plant genetic relationship identification method based on pollen. The method comprises the following steps: respectively adding the plant pollen of the n samples into m different solvents for extraction to obtain nm pollen extract liquor; wherein n is more than or equal to 2, and m is more than or equal to 2; respectively mixing the nm pollen extract liquid with the graphene dispersion liquid for embedding to obtain nm embedding systems; respectively coating the nm embedding systems on the surface of a screen printing electrode to obtain nm modified electrodes; respectively placing the nm modified electrodes in a phosphate buffer solution for pulse voltammetry scanning, and constructing a multi-loop electrochemical signal obtained by each of the nm modified electrodes to obtain an electrochemical map; comparing the similarity of electrochemical spectra of different samples in the same solvent, wherein the higher the contact ratio of the electrochemical spectra is, the closer the plant genetic relationship is. The method has the advantages of simple and easy identification process, high identification speed, low cost and the like.
Description
Technical Field
The invention belongs to the technical field of plant identification, and particularly relates to a plant genetic relationship identification method based on pollen.
Background
Plant taxonomy is a fundamental discipline that mainly studies the genesis, genetic relationship, and evolutionary development laws of different groups throughout the plant kingdom. That is, the complicated plant kingdoms are classified into different categories and arranged according to a system so as to be convenient for people to know and utilize the plants.
According to incomplete statistics, there are more than 50 ten thousand plants (about 250000 seed plants) living on the earth. However, the genetic relationship of plants has been mainly studied from the aspects of plant morphology and DNA molecular markers. Starting from morphology, the error of naked eye judgment is too large, the efficiency and the accuracy are not high when the genetic relationship is identified, and the method is unfamiliar to the plant captain of which the morphology is incomplete and even only has a pollen sample; the DNA molecular marking method can sequence and compare the DNA of two plants, but obviously, the method has complicated process and high cost.
Seeds are the reproductive system of seed plants, and retain the genetic material of the plants as much as possible, but many plants bred by natural or artificial hybridization are triploid plants, for example, lycoris, which normally flower but have aborted seeds. Therefore, the method starts with the plant pollen and is more scientific and accurate for identifying the genetic relationship among plants.
For example, CN 107488712a discloses a multicolor in situ hybridization method for rapidly analyzing and detecting chromosome configuration of wheat plants, which applies mc-GISH to the analysis of chromosome pairing behavior during meiosis of pollen mother cells, and can effectively identify the chromosome pairing condition of different genomes of wheat plants during meiosis by one-time hybridization, and can be used for analyzing the genetic relationship between different genomes of wheat plants. However, the method has complicated identification process and high cost, and is not favorable for large-scale identification.
In summary, the present invention provides a method for identifying plant genetic relationship based on pollen, which uses electrochemical means to realize rapid identification of plant genetic relationship by simple and easy method.
Disclosure of Invention
The invention aims to provide a method for identifying plant genetic relationship based on pollen. The method has the advantages of simple and easy identification process, high identification speed, low cost and the like.
In order to achieve the purpose, the invention adopts the following technical scheme:
a plant genetic relationship identification method based on pollen comprises the following steps:
(1) respectively adding the plant pollen of the n samples into m different solvents for extraction to obtain nm pollen extract liquor; wherein n is more than or equal to 2, and m is more than or equal to 2. The pollen grains have no special requirements, and the higher the integrity degree is, the better the integrity degree is;
(2) and (3) mixing the nm pollen extract liquid with the graphene dispersion liquid respectively for embedding to obtain nm embedding systems. In the invention, the specific surface area of the graphene dispersion liquid is far larger than that of a plant tissue, so that the plant tissue can be embedded to form a uniform dispersion liquid;
(3) and respectively coating the nm embedding systems on the surface of the screen printing electrode to obtain nm modified electrodes. The coating mode has no special requirement, and a dry modified electrode is obtained after the solvent is naturally evaporated;
(4) respectively placing the nm modified electrodes in a phosphate buffer solution for pulse voltammetry scanning, and constructing a multi-loop electrochemical signal obtained by each of the nm modified electrodes to obtain an electrochemical map;
(5) comparing the similarity of electrochemical spectra of different samples in the same solvent, wherein the higher the contact ratio of the electrochemical spectra is, the closer the plant genetic relationship is.
Preferably, the ratio of the number of the plant pollen grains of the sample in the step (1) to the volume of the solvent is 1 (30-200) muL.
Preferably, the solvent in step (1) includes one of water, absolute ethanol, methanol, ethylene glycol, N-Dimethylformamide (DMF) or N-hexane.
Preferably, the concentration of the graphene dispersion liquid in the step (2) is 0.1-1 mg/mL.
Preferably, the volume ratio of the pollen extraction liquid to the graphene dispersion liquid in the step (2) is (50-120): 5-12.
Preferably, in the step (3), 1-8 μ L of embedding system is coated on the surface of each screen electrode.
Preferably, the phosphate buffer solution in the step (4) is prepared by dissolving potassium dihydrogen phosphate and sodium dihydrogen phosphate in water, wherein the concentration of the potassium dihydrogen phosphate and the concentration of the sodium dihydrogen phosphate are 0.03-0.3 mol/L independently.
Preferably, the sweep interval of the pulse voltammetry sweep in the step (4) is 1.2V to-0.2V; the scanning speed is 80-150 mV/s; the sampling interval is 1-5 mV.
The invention provides a plant genetic relationship analysis method based on electrochemical analysis and using different plant pollen as an implementation object. Extracting plant pollen with different solvents to obtain different extracts, modifying the extracts on the surface of an electrode, performing pulse voltammetry scanning on the modified electrode, converting the obtained electrochemical signals into electrochemical maps, and judging the distance of the relationship between the two by comparing the similarity of the electrochemical maps of different samples in the same solvent. The identification method provided by the invention is simple and feasible, and can be used for quickly identifying the genetic relationship of plants with similar shapes.
The invention has the beneficial effects that:
1. the whole analysis process provided by the invention does not need expensive reagents and instruments, and the cost for identifying the genetic relationship of the congeneric plants is greatly reduced.
2. By using plant pollen as a detection sample, the difference of plant tissue components caused by the growth environment is greatly avoided.
3. The plant genetic analysis method provided by the invention can be potentially manufactured into a miniature kit product, and has the potential of field detection.
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FIG. 1 shows standard electrochemical spectra of Lycoris chinensis, Lycoris radiata, Lycoris neglecta, Lycoris cantoniensis and Lycoris rosea in ethylene glycol.
FIG. 2 shows standard electrochemical spectra of Lycoris chinensis, Lycoris radiata, Lycoris neglecta, Lycoris cantoniensis and Lycoris rosea in water.
FIG. 3 is a standard electrochemical spectrum of Lycoris longituba, Lycoris radiata, and Lycoris anhua in ethylene glycol.
FIG. 4 is a standard electrochemical spectrum of Lycoris longituba, Lycoris radiata, and Lycoris anhua in water.
Detailed Description
The following specific examples are further illustrative of the methods and techniques provided by the present invention and should not be construed as limiting the invention thereto.
Example 1: the genetic relationship of 10 known lycoris plants was identified.
(1) Preparing a buffer solution:
dissolving potassium dihydrogen phosphate and disodium hydrogen phosphate in water to prepare a phosphate buffer solution; the concentration of potassium dihydrogen phosphate and sodium dihydrogen phosphate is 0.1 mol/L;
(2) preparing a plant pollen extract:
in ten pollen samples of Lycoris chinensis, Lycoris radiata, Lycoris aurea, Lycoris cantoniensis, Lycoris rosea, Lycoris longiradiata, Lycoris radiata and Lycoris anhua, two pollen grains are respectively added into 200 μ L of ethylene glycol and 200 μ L of water, and the mixture is sufficiently shaken to obtain 20 pollen extracts of the 10 Lycoris pollen in water and methanol.
(3) Embedding graphene:
taking 100 mu L of the pollen extract liquid of the lycoris, respectively and independently adding the pollen extract liquid into 10 mu L of graphene dispersion liquid, and quickly shaking up to fully embed the pollen extract liquid into the graphene dispersion liquid to obtain an embedding system of 20 kinds of lycoris pollen.
(4) Electrode modification:
and (3) respectively and independently dripping 2 mu L of the embedding systems of the 20 lycoris pollen on the surface of the screen printing electrode, and naturally drying to obtain 20 modified electrodes dripped with different embedding systems.
(5) Electrochemical analysis:
and independently placing 20 modified electrodes into a phosphate buffer solution to form a loop, and performing pulse voltammetry scanning. Wherein the scanning interval of the pulse voltammetry scanning is 1.2V to-0.2V; the scanning speed is 100 mV/s; the sampling interval was 1 mV.
Each Lycoris leaf obtains 2 different groups of electrochemical signals (one group is extracted by ethylene glycol and the other group is extracted by water), and electrochemical spectrums of samples are respectively constructed to obtain the electrochemical spectrums, and the results are shown in attached figures 1-4.
As can be seen from the comparison of FIGS. 1 and 2 and FIGS. 3 and 4, the Lycoris radiata pollen treated with different extracts has different electrochemical spectra. In addition, as can be seen from comparison of the electric spectra of different types of lycoris radiata in the group images, the electrochemical spectra obtained by different types of lycoris radiata pollen after the same type of extract liquid is treated are similar and different, and the closer the relationship is, the greater the contact ratio of the electrochemical spectra is. For example, it can be seen from FIG. 1 that the Lycoris rosea and Lycoris straw have two current peaks between 0.0-0.2V and one current peak between 0.9V, but the peak heights are different, but the peak shapes are similar. Meanwhile, in FIG. 2, it can be seen that there is a current peak at about 0.25V for Lycoris roseris and Lycoris radiata. The results of the two figures show that the electrochemically active substances of Lycoris rosea and Lycoris straw are similar and thus have close relationship with each other. For another example, in FIG. 3, there are distinct current peaks between 0-0.4V and between 0.8-1.0V for Lycoris longiradiata and Lycoris radiata. Meanwhile, in FIG. 4, it can be seen that the Lycoris longiradiata and Lycoris radiata have similar current peaks between 0.1V and 0.8V. The results of the two figures show that the electrochemical active substances of Lycoris longiradiata and Lycoris radiata are similar and are close to each other.
As is apparent from the above examples, the present invention provides a method for analyzing plant genetic relationship based on electrochemical analysis using different plant pollen as an object. Extracting plant pollen with different solvents to obtain different extracts, modifying the extracts on the surface of an electrode, performing pulse voltammetry scanning on the modified electrode, converting the obtained electrochemical signals into electrochemical maps, and judging the distance of the relationship between the two samples by comparing the similarity of the electrochemical maps of the two samples in the same solvent. The identification method provided by the invention is simple and feasible, and can be used for quickly identifying the genetic relationship of plants with similar shapes.
The above description of the embodiments is only intended to facilitate the understanding of the method of the invention and its core ideas. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.
Claims (7)
1. A plant genetic relationship identification method based on pollen is characterized by comprising the following steps
The method comprises the following steps:
(1) respectively adding the plant pollen of the n samples into m different solvents for extraction to obtain nm pollen extract liquor; wherein n is more than or equal to 2, and m is more than or equal to 2; the ratio of the number of the plant pollen grains of the sample to the volume of the solvent is 1 (30-200) mu L;
(2) respectively mixing the nm pollen extract liquid with the graphene dispersion liquid for embedding to obtain nm embedding systems;
(3) respectively coating the nm embedding systems on the surface of a screen printing electrode to obtain nm modified electrodes;
(4) respectively placing the nm modified electrodes in a phosphate buffer solution for pulse voltammetry scanning, and constructing a multi-loop electrochemical signal obtained by each of the nm modified electrodes to obtain an electrochemical map;
(5) comparing the similarity of electrochemical spectra of different samples in the same solvent, wherein the higher the contact ratio of the electrochemical spectra is, the closer the plant genetic relationship is.
2. The method according to claim 1, wherein the solvent in step (1) is selected from water, absolute ethanol, methanol, ethylene glycol, N-Dimethylformamide (DMF), and N-hexane.
3. The method for identifying plant genetic relationship based on pollen according to claim 1, wherein the concentration of the graphene dispersion liquid in the step (2) is 0.1-1 mg/mL.
4. The method for identifying plant genetic relationship based on pollen according to claim 1, wherein the volume ratio of the pollen extraction liquid to the graphene dispersion liquid in the step (2) is (50-120) to (5-12).
5. The method for identifying plant genetic relationship based on pollen according to claim 1, wherein in the step (3), 1-8 μ L of embedding system is coated on the surface of each screen printing electrode.
6. The method as claimed in claim 1, wherein the phosphate buffer solution of step (4) is prepared by dissolving potassium dihydrogen phosphate and sodium dihydrogen phosphate in water, wherein the concentration of potassium dihydrogen phosphate and sodium dihydrogen phosphate is 0.03-0.3 mol/L.
7. The method for identifying plant genetic relationship based on pollen according to claim 1, wherein the sweep interval of pulse voltammetry scan in the step (4) is 1.2V to-0.2V; the scanning speed is 80-150 mV/s; the sampling interval is 1-5 mV.
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| CN111128310B (en) * | 2019-12-24 | 2022-05-24 | 国家林业和草原局桉树研究开发中心 | Method for identifying eucalyptus tree species and genetic relationship between species |
| CN111413392B (en) * | 2020-03-25 | 2022-05-13 | 杭州电子科技大学 | Method for collecting electrochemical spectrum of lycoris seeds |
| CN111398398A (en) * | 2020-04-01 | 2020-07-10 | 杭州电子科技大学 | Method for constructing camellia nitidissima phylogenetic tree |
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| CN105936936A (en) * | 2016-06-22 | 2016-09-14 | 陕西师范大学 | Method for detecting Psa in kiwi fruit pollens |
| CN107488712A (en) * | 2017-08-07 | 2017-12-19 | 山东农业大学 | A kind of quick analysis and the polychrome in-situ hybridization method of detection Tribe Triticeae chromosome configuration |
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Effective date of registration: 20220217 Address after: 230000 Anhui Hefei high tech Zone Innovation Industrial Park two phase J2 District C block 18 floor. Patentee after: HEFEI LONGTUTEM INFORMATION TECHNOLOGY Co.,Ltd. Address before: 310018 no.1158, No.2 street, Baiyang street, Hangzhou Economic and Technological Development Zone, Zhejiang Province Patentee before: HANGZHOU DIANZI University |
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