CN117849038A - Method for identifying ploidy of young sporophytes of kelp single plant - Google Patents
Method for identifying ploidy of young sporophytes of kelp single plant Download PDFInfo
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- CN117849038A CN117849038A CN202410033336.1A CN202410033336A CN117849038A CN 117849038 A CN117849038 A CN 117849038A CN 202410033336 A CN202410033336 A CN 202410033336A CN 117849038 A CN117849038 A CN 117849038A
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- 241000512259 Ascophyllum nodosum Species 0.000 title claims abstract description 44
- 238000000034 method Methods 0.000 title claims abstract description 44
- 230000000366 juvenile effect Effects 0.000 claims abstract description 10
- 238000009826 distribution Methods 0.000 claims description 44
- 210000003855 cell nucleus Anatomy 0.000 claims description 23
- 239000000463 material Substances 0.000 claims description 23
- 239000007788 liquid Substances 0.000 claims description 11
- 239000012192 staining solution Substances 0.000 claims description 9
- 210000001519 tissue Anatomy 0.000 claims description 9
- PRDFBSVERLRRMY-UHFFFAOYSA-N 2'-(4-ethoxyphenyl)-5-(4-methylpiperazin-1-yl)-2,5'-bibenzimidazole Chemical compound C1=CC(OCC)=CC=C1C1=NC2=CC=C(C=3NC4=CC(=CC=C4N=3)N3CCN(C)CC3)C=C2N1 PRDFBSVERLRRMY-UHFFFAOYSA-N 0.000 claims description 8
- 239000000872 buffer Substances 0.000 claims description 7
- 208000026487 Triploidy Diseases 0.000 claims description 6
- 238000007664 blowing Methods 0.000 claims description 6
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 4
- 208000035199 Tetraploidy Diseases 0.000 claims description 4
- 239000007853 buffer solution Substances 0.000 claims description 4
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- 229930040373 Paraformaldehyde Natural products 0.000 claims description 2
- 239000000834 fixative Substances 0.000 claims description 2
- 229920002866 paraformaldehyde Polymers 0.000 claims description 2
- 239000011780 sodium chloride Substances 0.000 claims description 2
- 238000010186 staining Methods 0.000 claims description 2
- 238000011161 development Methods 0.000 abstract description 12
- 230000018109 developmental process Effects 0.000 abstract description 12
- 230000008186 parthenogenesis Effects 0.000 abstract description 12
- 238000011160 research Methods 0.000 abstract description 12
- 238000009395 breeding Methods 0.000 abstract description 10
- 208000020584 Polyploidy Diseases 0.000 abstract description 9
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- 230000001737 promoting effect Effects 0.000 abstract description 3
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- 241000756998 Alismatales Species 0.000 description 1
- 208000037088 Chromosome Breakage Diseases 0.000 description 1
- 241000195493 Cryptophyta Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000015177 Saccharina japonica Species 0.000 description 1
- 241001123264 Zosteraceae Species 0.000 description 1
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention provides a method for identifying the ploidy of a kelp single plant juvenile sporophyte, which is simpler and more convenient to operate. The invention is suitable for ploidy identification of different sporophytes in the process of cultivating polyploid kelp and in the process of parthenogenesis development research of gametophytes; has important significance for promoting the breeding application research of kelp polyploid and the research of kelp propagation development.
Description
Technical Field
The invention belongs to the field of molecular genetic breeding of algae, and particularly relates to a method for identifying ploidy of young sporophytes of kelp single plants.
Background
Kelp (Saccharina japonica) belongs to Phaeophyta, zosterales, zosteraceae, and is large-scale economic seaweed with longest artificial cultivation history, highest yield and widest cultivation area. Kelp has a typical alternate life history of abnormal generation and consists of tiny gametophyte generation and large sporophyte generation which are mutually alternated. The gametophyte is tiny filiform body, which is the object of artificial culture seedling. The sporophyte is large and is the object of artificial cultivation and commodity production. The gametophytes of kelp under natural conditions are considered to be haploids, and sporophytes obtained by sexual reproduction and hybridization of male and female gametophytes are diploids.
Kelp is used as large-scale economic seaweed with highest yield and the largest cultivation area in China, and the cultivation of main varieties applied to production at present adopts more traditional pedigree breeding and hybridization breeding, so that the defect of innovation of a breeding method gradually causes the kelp breeding to enter a bottleneck period. Ploidy breeding is a breeding method which is commonly applied to higher plants, has been tried in kelp breeding, but is not widely applied, and one of the reasons is that mature, efficient and credible kelp sporophyte ploidy identification technology is lacking at present. In addition, apomictic phenomena such as parthenogenesis and apomictic reproduction exist in the development process of kelp gametophyte besides sexual reproduction. There are a series of problems which are pending in the asexual reproduction process of kelp gametophytes, for example, whether kelp sporophytes formed by parthenogenesis and apomixis are subjected to a chromosome doubling process, how the proportion of sporophytes formed by chromosome doubling is, and whether a single plant sporophyte individual has a plurality of cells with different ploidy; the resolution of these problems requires the development of suitable ploidy identification techniques for individual sporophytes.
Current methods of ploidy identification include chromosome counting and flow cytometry. Chromosome counting can be used for observing single cells of a single organism, but the operation process is complex, the workload is large, time and labor are wasted, and a higher cytological operation technology is required; meanwhile, as the kelp chromosome is smaller, the interference of factors such as cell fragments, chromosome breakage and the like possibly exists in the counting process, so that a certain error exists in the process of identifying the ploidy of the kelp sporophyte in the chromosome counting method, and the reliability of the ploidy identification result is low. Flow cytometry is a rapid and accurate method currently used in plant and animal ploidy identification, but has the disadvantages of requiring expensive specialized equipment, requiring high levels of expertise by maintenance personnel and complex instrumentation; in addition, the flow cytometry method needs to separate out the nuclei of the identified species, while kelp is brown algae of polysaccharide polyphenol, the process of extracting the nuclei from sporophytes is difficult, and more fragments are generated, so that more impurity peaks are generated during flow cytometry analysis, and the flow cytometry analysis result is influenced; in addition, the flow cytometry method needs to obtain a certain amount of cell nuclei, and for ploidy identification of early stage small sporophytes in the kelp culture process, a plurality of small sporophytes need to be mixed, so that ploidy identification of single plant sporophytes cannot be realized by the flow cytometry method, and the flow cytometry method is not suitable for related propagation development researches such as solitary reproduction of kelp gametophytes.
Therefore, in order to perform ploidy identification on different sporophyte individuals in the process of polyploid kelp cultivation and in the process of gametophyte parthenogenesis development research, a kelp single plant juvenile sporophyte ploidy identification method which is simpler, more convenient, more economical and faster to operate is also needed.
Disclosure of Invention
The invention aims to provide a method for identifying the juvenile sporophyte ploidy of a kelp single plant, which is simpler and more convenient to operate, so that the juvenile sporophyte ploidy of the kelp single plant can be identified more effectively.
The method for identifying the ploidy of the young sporophytes of the kelp single plant provided by the invention comprises the following steps:
1) Filling young sporophyte leaves to be detected into a centrifuge tube, adding tissue fixing liquid into the centrifuge tube, and standing at room temperature;
the tissue fixing liquid is 4% paraformaldehyde
Standing at room temperature for 10-20min;
2) Removing tissue fixing liquid, adding PBS buffer liquid, slowly blowing back and forth, and removing the PBS buffer liquid after cleaning is finished;
the PBS buffer solution has the composition ratio of 0.027 percent KH 2 PO 4 ；0.11％Na 2 HPO 4 ；0.79％NaCl；0.02％KCl。
3) Adding a cell nucleus staining solution into the leaves treated in the step 2), and standing at room temperature in a dark place;
the cell nucleus staining solution is 100 mu M Hoechst33342 staining solution.
4) After the staining is finished, removing the cell nucleus staining solution, and cleaning with PBS buffer solution again;
5) Placing the cleaned sporophyte blades on a glass slide, covering the glass slide, and observing and photographing under a confocal microscope;
the confocal microscope was loaded with Hoechst33342 color filters and photographed under a 20-fold objective lens.
6) Confocal microscope shooting pictures, counting the size of the cell nucleus area by using Image J software, and determining the ploidy of a detection sample through the peak value of a normal distribution curve of the cell nucleus area;
wherein, taking the normal distribution curve peak value of the diploid sporophyte with known ploidy as a standard, and when the ratio of the normal distribution curve peak value of the identified sample to the normal distribution curve peak value of the diploid with known ploidy is 0.3-0.7, the identified material is haploid; the ratio is diploid at 0.8-1.2, triploid at 1.3-1.7, tetraploid at 1.8-2.2.
The invention uses Hoechst33342 to dye and identify the ploidy of the single plant juvenile sporophyte of the kelp, and the ploidy of the single plant juvenile sporophyte of the kelp is determined by detecting the cell nucleus area of different single plant juvenile sporophyte samples of the kelp and comparing the cell nucleus area of the single plant juvenile sporophyte of the kelp with the cell nucleus area of the sea weed sporophyte samples with known ploidy. The invention is suitable for ploidy identification of different sporophytes in the process of cultivating polyploid kelp and in the process of parthenogenesis development research of gametophytes; has important significance for promoting the breeding application research of kelp polyploid and the research of kelp propagation development.
Drawings
FIG. 1 is a graph comparing criteria and ploidy nuclei when selecting nuclei using Image J software;
FIG. 2 is a graph showing the distribution histogram and normal distribution of the nuclear area frequency of diploid sporophytes obtained by hybridization of haploid male and female gametophytes;
FIG. 3 is a histogram of the nuclear area frequency distribution of sporophytes numbered 1 and a normal distribution graph;
FIG. 4 is a histogram of the nuclear area frequency distribution of sporophytes numbered 2 and a normal distribution graph;
FIG. 5 is a histogram of the nuclear area frequency distribution of sporophytes numbered 3 and a normal distribution graph;
FIG. 6 is a histogram of the nuclear area frequency distribution and a normal distribution of sporophytes numbered 4;
FIG. 7 is a histogram of the nuclear area frequency distribution and a normal distribution of sporophytes numbered 5;
FIG. 8 is a graph of the normal distribution of nuclear areas of different ploidy sporozoites;
FIG. 9 is a histogram of the nuclear area frequency distribution and a normal distribution of sporophytes numbered 6-1;
FIG. 10 is a histogram of the nuclear area frequency distribution and a normal distribution of sporophytes numbered 6-2;
FIG. 11 is a histogram of the nuclear area frequency distribution and a normal distribution of sporophytes numbered 6-3;
FIG. 12 is a histogram of the nuclear area frequency distribution and a normal distribution of sporophytes numbered 6-4;
FIG. 13 is a graph showing the normal distribution of the nuclear area of the blade of different sporophytes during parthenogenesis.
Detailed Description
The instrument and reagent information used in the embodiment of the invention are as follows: zeiss LSM900 ultra-high resolution laser confocal microscope, PBS buffer (Solarbio), hoechst33342 (Thermo Fisher), tissue fixative (meilunbio).
The present invention will be described in detail with reference to the following examples and the accompanying drawings.
Example 1: counting the nuclear area of diploid sporophyte obtained by hybridization of haploid male and female gametophytes
And (3) carrying out statistics on the nuclear area of the diploid juvenile sporophyte obtained by hybridization of the haploid male and female gametophytes:
(1) Selecting materials: selecting a diploid sporophyte material obtained by hybridization of haploid male and female gametophytes, and placing 1 sporophyte blade with the length of 0.8-1.2cm into a 2ml centrifuge tube;
(2) Fixing materials: adding 1ml of tissue fixing solution into the centrifuge tube in the step (1), and standing for 15 minutes at room temperature;
(3) Washing off the fixing liquid: sucking the tissue fixing liquid in the centrifuge tube in the step (2) by using a pipetting gun, adding 1ml of PBS buffer, slowly blowing back and forth, maintaining the integrity of sporophyte blades as much as possible, sucking PBS out after blowing for more than ten times, and repeating the steps;
(4) Dyeing of materials: 1ml of 100 mu M Hoechst33342 staining solution is added into the centrifuge tube in the step (3), and the mixture is kept stand for 15 minutes at room temperature in a dark place;
(5) Washing off the staining solution: sucking away the nuclear staining solution in the centrifuge tube in the step (4) by using a pipetting gun, adding 1ml of PBS buffer solution, slowly blowing back and forth, maintaining the integrity of sporophyte blades as much as possible, sucking out PBS after blowing for more than ten times, and repeating the steps for two times;
(6) Observing and photographing under a microscope: clamping the sporophyte blade on a glass slide by using tweezers, covering a cover glass on the glass slide, and observing and photographing under a 20-time objective lens under a Hoechst33342 color filter by using a confocal microscope;
(7) Treatment of experimental data: confocal microscope photographs are taken, image J software is used for selecting cell nuclei which are approximately circular in shape after dyeing and clear in imaging, the size of the cell nuclei is counted, the ploidy of a detected sample is determined through the size of the cell nuclei, excel is utilized for processing the obtained data, and Origin is utilized for plotting the obtained data to more intuitively reflect the ploidy of the detected sample.
The nuclei with approximately circular morphology and sharp imaging (fig. 1) were selected for the photographed Image and the area was calculated under Image J software. The statistical results are shown in table 1.
Table 1: example 1 data sheet obtained after photographing statistics
The resulting data was plotted as a frequency distribution histogram and a normal distribution curve was plotted (fig. 2).
Successfully counting the size of the cell nucleus area and drawing a normal distribution curve graph, which shows that the experimental method for counting the size of the cell nucleus area in the experimental process is feasible. Ploidy identification can be performed on materials with unknown ploidy on the basis.
Example 2: identifying ploidy of different sporophyte individuals in the process of cultivating polyploid kelp
The cell nucleus area statistics of the sporophytes in the specific embodiment and the example 1 are the same, except that the materials are sporophytes with different ploidy obtained by hybridization of male and female gametophytes with different ploidy. Specific information for each material is shown in table 2.
Table 2: specific information table of materials used in example 2
For the photographed Image, nuclei with approximately circular morphology and clear Image are selected and the area is calculated under Image J software. The difference in nuclear size of the ploidy cells can also be seen from the effect plot of the treatment under software (fig. 1). The statistical results are shown in Table 3.
Table 3: example 2 data sheet obtained after photographing statistics
The resulting data were plotted as frequency distribution histograms and normal distribution curves (fig. 3-7).
By using the diploid sporophytes of known ploidy in example 1 as a control, the combination of normal distribution curves can be obtained:
the nuclear area of the sporophytes numbered 1 in example 2 (FIG. 3, table 3) was 1.95 times that of the diploid sporophytes, from which it can be deduced that the material numbered 1 should be tetraploid.
The nuclear area of the number 2 sporophyte in example 2 (fig. 4, table 3) is 1.01 times that of the diploid sporophyte, from which it can be deduced that the material of number 2 should be diploid.
The nuclear area of the number 3 sporophytes in example 2 (fig. 5, table 3) is 1.44 times that of diploid sporophytes, from which it can be deduced that the number 3 material should be triploid.
The nuclear area of the number 4 sporophyte in example 2 (fig. 6, table 3) is 1.47 times that of the diploid sporophyte, from which it can be deduced that the number 4 material should be triploid.
The nuclear area of the number 5 sporophytes in example 2 (fig. 7, table 3) is 1.37 times that of diploid sporophytes, from which it can be deduced that the number 5 material should be triploid.
By comparing the normal distribution curves of the cell nucleus areas of the sporophytes obtained by combining the hybridization of different ploidy, the difference of the materials with different ploidy can be intuitively seen, and the materials with the same ploidy are concentrated together.
Finally, determining that the material is haploid when the ratio of the peak value of the normal distribution curve of the sample to the peak value of the normal distribution curve of the diploid sporophyte with known ploidy is 0.3-0.7; the ratio is diploid at 0.8-1.2, triploid at 1.3-1.7, tetraploid at 1.8-2.2.
The deduced result is consistent with the theoretical ploidy of the sporophyte obtained by hybridization combination, so that the invention is suitable for ploidy identification of different sporophyte individuals in the process of cultivating polyploid kelp.
Example 3: identification of ploidy of individuals of different sporophytes in parthenogenesis development process of kelp gametophytes
The cell nucleus areas of the sporophytes in the specific embodiment and the example 1 are the same, except that the material is 4 young sporophytes obtained by parthenogenesis, and the nuclear area sizes of the young sporophytes are respectively counted.
For the photographed Image, nuclei with approximately circular morphology and clear Image are selected and the area is calculated under Image J software. The statistical results are shown in Table 4.
Table 4: example 3 data sheet obtained after photographing statistics
The resulting data were plotted as frequency distribution histograms and normal distribution curves (fig. 9-12).
By using the diploid sporophytes of known ploidy in example 1 as a control, the combination of normal distribution curves can be obtained:
the nuclear area of the sporophytes numbered 6-1 in example 3 (FIG. 9, table 4) was 0.46 times that of the diploid sporophytes, from which it can be deduced that the material numbered 6-1 should be haploid.
The nuclear area of the sporophytes numbered 6-2 in example 3 (FIG. 10, table 4) was 0.93 times that of the diploid sporophytes, from which it was deduced that the material numbered 6-2 should be diploid.
The nuclear area of the sporophytes numbered 6-3 in example 3 (FIG. 11, table 4) was 0.94 times that of the diploid sporophytes, from which it was deduced that the material numbered 6-3 should be diploid.
The nuclear area of the sporophytes numbered 6-4 in example 3 (FIG. 12, table 4) was 0.92 times that of the diploid sporophytes, from which it was deduced that the material numbered 6-4 should be diploid.
Comparing the normal distribution curves of the cell nucleus areas of different blades of the sporophytes obtained by parthenogenesis (figure 13), the difference of the blades with different ploidy can be intuitively seen, and the blades with the same ploidy are concentrated together. The result shows that the invention is suitable for ploidy identification of different sporophytes in the research process of parthenogenesis development of kelp gametophytes. The sporophytes generated by parthenogenesis of kelp female gametophytes have haploid individuals and diploid individuals.
From the results, the invention is suitable for ploidy identification of different sporophytes in the culture process of polyploid kelp and in the parthenogenesis development research process of gametophytes. Provides a simple, economical and rapid identification method for the ploidy of the young sporophytes of the kelp single plant, and has important significance for promoting the breeding application research of the kelp polyploid and the research of the kelp reproductive development.
Claims (7)
1. A method for identifying the ploidy of a single plant juvenile sporophyte of kelp, which is characterized by comprising the following steps:
1) Filling young sporophyte leaves to be detected into a centrifuge tube, adding tissue fixing liquid into the centrifuge tube, and standing at room temperature;
2) Removing tissue fixing liquid, adding PBS buffer liquid, slowly blowing back and forth, and removing the PBS buffer liquid after cleaning is finished;
3) Adding a cell nucleus staining solution into the leaves treated in the step 2), and standing at room temperature in a dark place;
4) After the staining is finished, removing the cell nucleus staining solution, and cleaning with PBS buffer solution again;
5) Placing the cleaned sporophyte blades on a glass slide, covering the glass slide, and observing and photographing under a confocal microscope;
6) Confocal microscope photographs were taken, and Image J software was used to count the size of the cell nucleus area, and the ploidy of the test sample was determined from the peak value of the normal distribution curve of the cell nucleus area size.
2. The method of claim 1, wherein the tissue fixative is 4% paraformaldehyde.
3. The method of claim 1, wherein the standing at room temperature is performed for a period of 10 to 20 minutes.
4. The method of claim 1, wherein the PBS buffer is formulated as follows: 0.027% KH 2 PO 4 ；0.11％Na 2 HPO 4 ；0.79％NaCl；0.02％KCl。
5. The method of claim 1, wherein the nuclear stain is 100 μm Hoechst33342 stain.
6. The method of claim 1, wherein the confocal microscope is loaded with Hoechst33342 color filters and photographed under a 20-fold objective.
7. The method of claim 1, wherein the determination of the ploidy of the test sample from the peak value of the normal distribution curve of the size of the cell nucleus is based on the peak value of the normal distribution curve of the diploid sporophyte of known ploidy, and the material is determined to be a haploid when the ratio of the peak value of the normal distribution curve of the test sample to the peak value of the normal distribution curve of the diploid of known ploidy is 0.3-0.7; the ratio is diploid at 0.8-1.2, triploid at 1.3-1.7, tetraploid at 1.8-2.2.
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