Induction method and application of jellyfish polyploidy

Abstract

The invention discloses a jellyfish polyploid induction method and application thereof, and belongs to the technical field of marine ornamental animal breeding. According to the induction method, the polyploidy jellyfish is obtained by taking the scyphistoma stage of the jellyfish as an induction object and carrying out induction treatment through the combination of colchicine and dimethyl sulfoxide, and a plurality of polyploidy new germplasms are obtained through one-time treatment under the condition that the use amount of an inducer is small. The obtained polyploidy scyphistoma can rapidly proliferate a new individual with polyploidy characteristics through asexual propagation, and a jellyfish body with polyploidy characters such as high growth speed, long life cycle, beautiful form and enlarged individual can be obtained through transverse fissure; meanwhile, the method also has the characteristics of simple operation and low cost. Therefore, the induction method disclosed by the invention has important application value and wide popularization and application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of marine ornamental animal breeding, and in particular to a method for inducing jellyfish polyploidy and application thereof. Background Art

[0002] Jellyfish, a collective term for a diverse group of animals from the phyla Cnidaria and Ctenophora, are important plankton in aquatic environments and are also important marine economic and ornamental animals in my country. Their life cycle consists of a sexual generation (medusa) and an asexual generation (polyp). Due to the short life cycle of jellyfish and the difficulty of sexual reproduction, artificial aquaculture has long relied on wild resources, making the development and promotion of improved varieties crucial.

[0003] Polyploidy refers to organisms with three or more sets of chromosomes. It is a key mechanism for speciation, genomic evolution, and the maintenance of biodiversity, and is a classic strategy for de novo domestication. Breeding polyploids to improve target traits is widely used in plant breeding. However, compared to plant breeding, polyploidy in animals is difficult because most animals produce offspring exclusively through sexual reproduction.

[0004] Among the few reported polyploids of aquatic economic animals, their offspring have advantages over conventional diploids, such as larger size and faster growth. Therefore, polyploid breeding has become an important approach to obtaining new aquatic species in aquatic breeding and production. However, research on polyploids in aquatic species has primarily focused on improving the germplasm resources of aquatic economic organisms, enhancing energy balance, and increasing the quality and yield of aquatic products. Reports on polyploid breeding of ornamental aquatic animals are rare.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The object of the present invention is to provide a method for inducing polyploidy in jellyfish and its application. Through the induction method of the present invention, a plurality of new polyploid germplasms can be obtained through a single treatment at a relatively low amount of inducer. The polyploid polyps can not only rapidly proliferate through asexual reproduction while completely retaining the polyploid characteristics, but also produce jellyfish with polyploid traits such as fast growth rate, long life cycle, beautiful morphology, and enlarged individuals. Therefore, the induction method of the present invention lays an experimental foundation for the application of polyploidy induction in jellyfish.

[0007] The present invention is achieved in that: In a first aspect, the present invention provides a method for inducing polyploidy in jellyfish, comprising: induction culture and screening and identification; wherein the induction culture is to place jellyfish in the polyp stage in seawater containing colchicine and dimethyl sulfoxide for culturing; the screening and identification includes morphological screening, chromosome identification, and flow cytometry ploidy analysis; The mass volume concentration ratio of colchicine in seawater is 0.01%-0.05%, and the volume concentration of dimethyl sulfoxide is 0.1%-0.5%.

[0008] In a second aspect, the present invention provides jellyfish polyploids obtained by the above-mentioned induction method.

[0009] In a third aspect, the present invention provides the application of the above-mentioned induction method in jellyfish polyploid breeding.

[0010] The present invention has the following beneficial effects: (1) The present invention establishes for the first time a method for inducing polyploid jellyfish by treating the polyp stage of jellyfish with colchicine and dimethyl sulfoxide in combination. The method uses a small amount of mutagen, has low mutagen toxicity, a high induction rate, and a short induction time, and can quickly and efficiently obtain tetraploid polyps.

[0011] (2) The tetraploid polyps induced by the method of the present invention are in the asexual reproduction stage, so they can reproduce rapidly and maintain stable inheritance of excellent traits. At the same time, their jellyfish bodies have the characteristics of long life cycle, beautiful morphology, large individual size and easy breeding.

[0012] (3) The induction method of the present invention can be applied to jellyfish tetraploid breeding. This method can avoid the common difficulties of haploid sterility and chromosome ploidy differentiation in sexually reproduced offspring in animals, and avoid the difficulty of re-breeding each generation, thereby improving the utilization rate of high-quality germplasm resources and reducing induction costs. Therefore, the induction method of the present invention has significant application value and broad prospects for promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0014] Figure 1 The polyp morphological identification results obtained in Example 1; Figure 2 The polyp chromosome counting result obtained in Example 1; Figure 3 The figure shows the relative DNA content detection results of polyp somatic cells and stinging cells after treatment with different inducer solution concentrations in Experimental Example 1. From left to right, the colchicine volume concentrations are 0, 0.01%, and 0.02%; Figure 4 This is a stained image of the two main cells in the jellyfish. DETAILED DESCRIPTION

[0015] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0016] First, the present invention provides a method for inducing jellyfish polyploidy, which mainly consists of two processes: induction culture and screening and identification. The induction culture uses jellyfish in the polyp stage as the material and cultures it in seawater containing an inducer. The screening and identification is to perform morphological screening, chromosome counting and flow cytometry ploidy analysis on the induced polyp to obtain jellyfish polyploidy.

[0017] Scyphozoan jellyfish have a typical alternation of generations life cycle, including a polyp stage (asexual, sessile) and a medusa stage (sexual, planktonic). The complete cycle of alternation of generations is as follows: Fertilized eggs develop into planula larvae ( Planula ) - Planula larvae attach - metamorphosis into polyps ( Polyp , asexual generation) - polyps reproduce by transverse fission - releasing disc-shaped larvae ( Ephyra ) - disc-shaped larvae grow - mature into medusae ( Medusa , sexual generation) - the jellyfish lays eggs / sperm - fertilization becomes zygotes, completing the cycle.

[0018] The present invention chooses polyps as the induction object because jellyfish at this stage can not only proliferate rapidly through asexual reproduction and completely retain polyploid characteristics, but also avoid the common difficulties of haploid infertility in animals and chromosome multiplicity differentiation of sexually reproduced offspring, overcome the difficulty of re-breeding each generation, thereby improving the utilization rate of high-quality germplasm resources and reducing induction costs.

[0019] Specifically, the above-mentioned induction method comprises the following steps: S1. Prepare a 20x inducer solution by mixing colchicine with dimethyl sulfoxide and purified water.

[0020] In the induction method of the present invention, the inducer consists of colchicine and dimethyl sulfoxide.

[0021] Among them, colchicine is the most widely used chemical mutagen, which can inhibit mitosis, destroy the spindle, cause chromosomes to stagnate in metaphase, and double chromosomes. However, it is a highly toxic substance, and the occurrence of the above situation in most animal cells is fatal. However, the inventors found in the research process that polyps have formed super endurance in long-term evolution, and because they have asexual reproduction characteristics similar to plants, they have created conditions for the induction of polyploidy. Therefore, the present invention induces polyps by colchicine, so that when polyps undergo asexual reproduction of new individuals, the tubulin in the intracellular microtubules is bonded, preventing the polymerization of microtubules, thereby inhibiting the formation of spindles, preventing mitosis as the cell scaffold changes, and then causing chromosome doubling in the cell. It is precisely because of the asexual reproduction characteristics of polyps and the super endurance to colchicine that the goal of inducing diploid jellyfish into tetraploid jellyfish is achieved.

[0022] In order to increase the solubility of colchicine, the present invention adds dimethyl sulfoxide to achieve a concentration of colchicine within the range of 20 times the inducer solution prepared using pure water, thereby avoiding the adverse effects of excessive fresh water on polyp rearing. At the same time, the combination of colchicine and dimethyl sulfoxide can effectively enhance the mutagenic effect of the mutagen.

[0023] S2. Add the inducer solution prepared in S1 to seawater, and culture Atlantic golden jellyfish in the polyp stage in the seawater as the first-generation polyps; subculture the first-generation polyps until the second-generation polyps are obtained.

[0024] The concentration of the inducer solution and the treatment time will significantly affect the polyploid induction effect. Higher colchicine and dimethyl sulfoxide concentrations and treatment times can improve the induction efficiency of polyploidy, but too high a concentration can lead to problems such as developmental stagnation and organ malformations, and increase the mortality rate of the test materials. Therefore, in actual research, appropriate inducer concentration and treatment time are important influencing factors for the successful doubling. During the research process in the induction system of the present invention, it was found that the dimethyl sulfoxide concentration remained unchanged at 0.1%-0.5%. When the concentration of colchicine in seawater was ≤0.01%, newly bred polyps did not produce polyploidy, which may be related to the high tolerance and permeability of the polyps. When the concentration of colchicine in seawater was greater than 0.05%, the polyps of the Atlantic golden jellyfish quickly became smaller, turned white, shrank and disappeared, and the mortality rate was high due to multiple factors such as the freshwater in which the colchicine was dissolved and the concentration of the inducer. When the concentration of colchicine in seawater was 0.01%<≤0.05%, newly produced polyps through asexual reproduction were more likely to obtain polyploid traits, and no obvious chimeras or other multiple chromosomes were produced.

[0025] Based on this, the present invention selects the concentration of colchicine in seawater to be 0.01%-0.05%, preferably 0.02%. Under this condition, the appropriate volume concentration of dimethyl sulfoxide is 0.1%-0.5%.

[0026] In S2, the second generation of polyps is obtained, marked by the first generation of polyps' foot capsules, budding, or the emergence of small buds from the runners. These buds gradually develop into the second generation of polyps. The first generation of polyps is diploid, and the second generation of polyps obtained by further culturing them in an inducing agent is tetraploid.

[0027] In the present invention, the second generation polyp is derived from the asexual differentiation of cells in a certain part of the ectoderm of the first generation polyp, thus avoiding the generation of chimeras and mixed ploids, stabilizing the genetic traits of the second generation polyp and reducing the incidence of malformations in the split medusa.

[0028] Throughout the induction process, the temperature, salinity, and inducer concentration of the induction system remain constant. Ideally, the temperature is maintained between 20°C and 24°C, the salinity of the seawater is between 20‰ and 32‰, and the induction process lasts for 20-30 days. Furthermore, the induction culture environment is sealed to minimize evaporation.

[0029] In order to meet the normal growth and reproduction requirements of polyps during the induction process, it is necessary to feed Artemia nauplii and change the water every 5 days.

[0030] During the research process, the present invention induced different jellyfish respectively and found that the induction effect of the above-mentioned induction method on different jellyfish was different. The jellyfish species that can achieve polyploidy induction include the Atlantic golden jellyfish and some jellyfish species that can be cultured in low salinity, such as the leaf saltpeter jellyfish, the moon jellyfish, the sea jellyfish and all other Scyphozoan jellyfish. Therefore, the induction method provided by the present invention can be extended to the polyploidy induction research of related jellyfish.

[0031] S3. Screen out the second generation of polyps with significantly enlarged individuals and those with the excellent trait of jellyfish that can split transversely, and then perform chromosome counting and flow cytometry ploidy analysis on them to screen out tetraploid polyps.

[0032] Identifying the doubling effect of induced polyps is also a crucial step in the overall process. Morphological identification is the simplest and fastest method for identifying polyploids, and can be used for preliminary screening of variant organisms, significantly reducing workload. However, conclusions obtained through morphological identification require more accurate chromosome counting or DNA content determination. Because the chromosomes of the jellyfish in this invention are relatively small, they are not clearly visible under an optical microscope, and their number varies greatly between cells, potentially leading to inaccurate statistical results. Therefore, the present invention combines chromosome counting with flow cytometric ploidy analysis. Flow cytometry is used to examine newly bred polyps, accurately identifying polyploid individuals within variants and facilitating later jellyfish breeding and screening for superior traits.

[0033] Through the above induction method, multiple new polyploid germplasms can be obtained through one treatment with a smaller amount of inducer. The polyploid can not only rapidly proliferate through asexual reproduction while fully retaining the polyploid characteristics, but also obtain jellyfish with polyploid traits such as fast growth rate, long life cycle, beautiful morphology, and enlarged individuals, and has a high application prospect.

[0034] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0035] Example 1 This embodiment is a method for inducing polyploidy in jellyfish, and the specific steps are as follows: (1) Prepare a 20-fold inducer solution by mixing colchicine with dimethyl sulfoxide and purified water.

[0036] (2) The inducer solution prepared in (1) was added to seawater to prepare an induction system with a colchicine concentration of 0.02% and a dimethyl sulfoxide concentration of 0.4% in seawater. The Atlantic golden jellyfish in the polyp stage were placed in the induction system and cultured for 30 days. During this period, Artemia nauplii were fed and the water was changed every 5 days.

[0037] The culture conditions are: the temperature is maintained at 20℃-24℃, the salinity of seawater is 25‰, and the environment for induced culture is in a sealed state.

[0038] From the second generation of polyps, the polyps with obviously enlarged individuals and the polyps with excellent traits of medusa that can split transversely were screened out, and then they were counted by improved hypotonic method and analyzed by flow cytometry for ploidy, and tetraploid polyps were screened out.

[0039] The improved hypotonic method refers to the research on the chromosome karyotype of silver pomfret by Zhou Jianguang et al. The specific operation method is as follows: (1) Prepare a 0.02% colchicine solution by adding 4 ml of 32‰ salinity seawater to 1 ml of 0.1% colchicine solution. Add the test material and culture normally for 24 hours.

[0040] (2) To prevent interference from stinging cells, cut off the tentacles of the polyp in advance. Dissociate the tissue with 50% glacial acetic acid, gently pipette and let it stand at 37°C for 1 hour. Centrifuge the centrifuge tube containing the experimental materials at 12,000 rpm for 2 minutes, then gently remove the supernatant by aspiration, leaving about 0.5 ml of cell pellet.

[0041] (3) Fix the cells with freshly prepared Carnoy's fixative for 1.5 h (0°C to -4°C). To ensure the concentration of the fixative, change the fixative twice. Add 0.075 mol / L KCl solution as a hypotonic solution and disperse the cells with a pipette. After hot-slide mounting, air-drying, Giemsa staining, and microscopic examination, select well-dispersed metaphases for microscopic photography.

[0042] Flow cytometric ploidy analysis: Jindi Future Biotechnology (Beijing) Co., Ltd. uses the Sysmex Partec CyFlow Space flow cytometer for polyp ploidy analysis. The specific operation method is as follows: 0.2 g of newly propagated robust polyps were selected and placed in a culture dish. 500 μl of the nuclear lysis buffer in the CyStain UV Precise P kit was added around the sample and minced with a sharp blade to fully extract the intact cell nucleus. The liquid in the culture dish was filtered through a 50 μm celltrics filter into a sample tube. 2000 μl of the DAPI fluorescent stain in the CyStain UV Precise P kit was added to the sample tube. The tube was stained in the dark for 2 minutes. Ploidy was detected using a Sysmex Partec CyFlowSpace flow cytometer.

[0043] Figure 1 For morphological identification, Figure 2 is the result of chromosome counting, Figure 3 This is the flow cytometry test diagram of colchicine solution at different concentrations (0, 0.01%, 0.02% from left to right). Figure 4 There are two main types of cells in jellyfish.

[0044] Figure 1 These are polyps of the Atlantic golden jellyfish, reared under the same conditions. These fertile tetraploid polyps exhibit the characteristics of a new tetraploid germplasm: large size, strong mitotic activity, and ease of rearing. These polyps also exhibit polyploid traits such as rapid growth, a long life cycle, beautiful morphology, and increased size.

[0045] Figure 2 This is the result of chromosome staining of a large polyp. A large number of well-dispersed metaphases were obtained from the experimental material, which is about twice the number of chromosomes in the polyp before treatment.

[0046] Flow cytometry Figure 3 The left figure shows that the fluorescence intensity of the stained bases in the free cell nuclei has two peaks. The first main peak is located near the fluorescence intensity of 19, which is the DNA content. 77 fluorescently labeled cells were detected. The second main peak is located near the fluorescence intensity of 55, and the number of detectable cells is 238. This is consistent with the result that the polyp mainly contains two different sizes of cells, stinging cells and somatic cells (see Figure 4 ). Figure 3 The intermediate image results are the same as Figure 3 The left figure is similar to that of the previous figure, with two peaks near the fluorescence intensity of 19 and 55, both of which are diploid. Figure 3 In the right figure, the two peaks are located near the fluorescence intensity of 38 and 105 respectively, and both the stinging cells and the body cells doubled under the action of the drug.

[0047] Example 2 The difference from Example 1 is that the induced object in this experiment is the polyp of the Androsa jellyfish.

[0048] Example 3 The difference from Example 1 is that the induced object in this experiment is the polyp of the moon jellyfish.

[0049] Comparative Example 1 The difference from Example 1 is that no colchicine solution was added in this experiment.

[0050] Comparative Example 2 The difference from Example 1 is that the concentration of the colchicine solution in this experiment is 0.01%.

[0051] Comparative Example 3 The difference from Example 1 is that the concentration of the colchicine solution in this experiment is 0.05%.

[0052] Experimental Example 1 The polyps obtained in Example 1 and Comparative Examples 1-3 were screened and identified, and the results of flow cytometry ploidy analysis were as follows: Figure 3 shown.

[0053] The results show that polyps of the Atlantic golden jellyfish cultured in a 0.01% colchicine solution for 30 days reproduced more individuals through asexual reproduction, with a survival rate of 126.7%. Each polyp produced an average of 4.50 discs through multiple transverse fission, similar to the survival rate of 132% and the average production of 4.67 discs per polyp in natural seawater. Long-term culture with 0.01% colchicine did not alter polyp ploidy. Flow cytometry analysis revealed DNA content similar to that of naturally cultured polyps, with two main peaks located near fluorescence intensities of 19 and 55, respectively.

[0054] Under 0.02% colchicine, the survival rate of polyps was 40%, but some polyps produced new polyps through asexual reproduction. Flow cytometric analysis showed that the two main peaks of DNA content in the stinging cells and somatic cells of the newly produced polyps were located near fluorescence intensities of 38 and 105, respectively, indicating that almost all of them were tetraploid. Under 0.02% colchicine, each polyp produced 2.27 discs, mainly due to the large number of polyps undergoing multiple transverse fissions in the initial stage of the experiment, which may be related to the rapid change in the breeding environment. In this example, some tetraploid polyps underwent transverse fission, producing vigorous and fast-growing discs.

[0055] During the research, the induction effect of colchicine beyond the range of 0.01%-0.05% was also compared. When the concentration of colchicine in seawater was ≤0.01%, no polyploidy was produced in the newly bred polyps. When the concentration of colchicine in seawater was ≥0.05%, affected by multiple factors such as the freshwater in which colchicine was dissolved and the concentration of the inducer, the polyps of the Atlantic golden jellyfish quickly became smaller, turned white, shrank and disappeared, and the mortality rate was high.

[0056] The above results show that the polyploid hydra obtained by the present invention can reproduce normally asexually and produce disc-shaped bodies by transverse fission, the offspring can grow normally, and the asexually reproduced individuals of the hydra can maintain their polyploid traits to obtain polyploid hydra, the hydra have no mechanical damage, the survival rate of the first generation is more than 60%, the survival rate of the second generation is 100%, tetraploids appear in the second generation, and the polyploid induction rate is 100%.

[0057] Experimental Example 2 The polyps obtained in Example 1 and Example 2 were compared, and the results showed that the polyps of the Androsa jellyfish reproduced asexually by budding, and were cultured in a seawater induction system with a colchicine concentration of 0.02% and a dimethyl sulfoxide concentration of 0.4% for 30 days, during which time they were fed with Artemia nauplii and the water was changed once every 5 days, and tetraploids were produced.

[0058] Experimental Example 3 The polyps obtained in Example 1 and Example 3 were compared, and the results showed that the polyps of the moon jellyfish reproduced asexually by various means, including stoloniferous reproduction, foot sac reproduction, and budding reproduction. They were cultured at 20-25°C in a seawater induction system with a colchicine concentration of 0.02% and a dimethyl sulfoxide concentration of 0.4% for 30 days, during which they were fed with Artemia nauplii and the water was changed once every 5 days, and tetraploids were able to be produced.

[0059] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for inducing polyploidy in jellyfish, characterized in that: include: Induction culture and screening identification; The induction culture is to place the jellyfish in the polyp stage in seawater containing colchicine and dimethyl sulfoxide for culture; The screening and identification includes morphological screening, chromosome counting and flow cytometry ploidy analysis; The mass volume concentration ratio of colchicine in seawater is 0.01%-0.05%, and the volume concentration of dimethyl sulfoxide is 0.1%-0.5%.

2. The induction method according to claim 1, characterized in that The induction culture comprises: placing the polyp stage of the jellyfish in seawater containing colchicine and dimethyl sulfoxide to raise them as the first generation of polyps; subculturing the first generation of polyps and asexually reproducing the new generation of polyps in the mutagen to produce the second generation of polyps; and then transversely splitting the second generation of polyps to produce medusae.

3. The induction method according to claim 2, characterized in that The induction culture conditions are: temperature of 20°C-24°C, time of 20-40 days, salinity of seawater of 20‰-32‰, and the induction culture environment is in a sealed state.

4. The induction method according to claim 3, characterized in that During the induction culture, Artemia nauplii were fed and the water was changed once every 5 days.

5. The induction method according to claim 4, characterized in that The colchicine solution used in the induction culture is a 20-fold solution prepared by dissolving dimethyl sulfoxide and purified water, and the mass volume concentration ratio of the colchicine in seawater is 0.02%.

6. The induction method according to claim 1, characterized in that The screening and identification comprises the following steps: selecting the obviously enlarged polyps and the polyps with excellent traits of medusa that can split transversely from the obtained second-generation polyps, and then performing chromosome counting and flow cytometer ploidy analysis to select tetraploid polyps.

7. The induction method according to claim 1, characterized in that The jellyfish is a jellyfish of the class Scyphozoa, including the Atlantic golden jellyfish, moon jellyfish and medusa.

8. The jellyfish polyploid obtained by the induction method according to any one of claims 1 to 7.

9. Use of the induction method according to any one of claims 1 to 7 in polyploid breeding of jellyfish.

10. The use according to claim 9, characterized in that The polyploid breeding is a tetraploid breeding.

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