A method for inducing distant hybrid triploid grouper

Abstract

The present invention relates to an induction method for distant hybridization triploid grouper, and belongs to the technical field of genetic breeding of grouper. The present invention adopts hydrostatic pressure combined with chemical drug induction method to cultivate triploid grouper. The method is to obtain fertilized eggs of female brown-spotted grouper and male blue-bodied big-spotted grouper, record the starting time of fertilization, put the fertilized eggs into a hydrostatic pressure instrument for hydrostatic shock treatment, and inject natural seawater containing 6-dimethylaminopurine into the hydrostatic pressure instrument in advance. After the hydrostatic pressure treatment, the fertilized eggs are moved into a 6-DMAP solution to continue drug induction, and finally the fertilized eggs are transferred to aquaculture water for hatching. The present invention obtains the induction parameters of distant hybridization grouper triploid for the first time. The method is simple to operate, short in processing time, low in damage to fertilized eggs, and the induction rate is more than 60%.

Description

Technical Field

[0001] The invention relates to the technical field of genetic breeding of groupers, and in particular to a method for inducing distant hybridization triploid groupers, wherein the distant hybridization is distant hybridization of brown-spotted groupers and blue-bodied big-spotted groupers. Background Art

[0002] Grouper is widely distributed in tropical and subtropical waters of the Pacific, Atlantic, and Indian Oceans. It is an important economic fish with delicious meat, rich nutrition and low fat content. In recent years, the breeding volume of grouper has steadily increased, and the breeding area has also expanded to the north, such as Shandong, Hebei, Tianjin and other places. In recent years, the breeding volume of grouper ranks among the top three in marine fish breeding. With the rapid expansion of grouper breeding, there are also problems of germplasm degradation in the breeding process, such as slow growth, high deformity rate, and serious diseases. In order to promote the healthy and sustainable development of grouper breeding industry, the cultivation of new varieties is one of the ways to solve the problem of germplasm degradation.

[0003] Hybrid breeding and polyploid breeding are commonly used technical means in fish breeding. Fish hybrid breeding combines the excellent genes of the parents through mating between parents with different genetic traits, and screens out phenotypic traits that meet the needs of aquaculture production. The hybrid offspring often have hybrid advantages, fast growth rate, strong stress resistance and other advantages, and are widely used in fish breeding. The induction mechanism of triploidy is to inhibit the discharge of the second polar body of the oocyte after the sperm and egg combine. Triploid fish generally have the advantages of fast growth rate, good meat quality, strong stress resistance and the like due to their infertility or low fertility. The methods for artificially inducing triploidy mainly include physical methods such as temperature shock and hydrostatic pressure shock, and chemical methods induced by drugs. Compared with the temperature shock method, the hydrostatic pressure method has the characteristics of less damage to the fertilized egg and uniform pressure transmission. In addition, chemical drugs also have the characteristics of good induction effect and less damage. The present invention combines distant hybridization with polyploidy induction, and combines hydrostatic pressure shock with drug induction for breeding, which is technically innovative.

[0004] Brown-spotted grouper (Epinephelus fuscoguttatus), commonly known as tiger grouper, grows slowly and has a long breeding cycle, but it can produce a large number of high-quality eggs throughout the year. Blue-bodied big-spotted grouper (Epinephelus fuscoguttatus), commonly known as golden-bodied big-spotted grouper, is one of the larger species of grouper, with the characteristics of fast growth and strong resistance to adversity. In the early stage, the brown-spotted grouper was used as the female parent and the blue-bodied big-spotted grouper was used as the male parent for hybridization. The offspring "Golden Tiger Hybrid Grouper" has the advantages of fast growth, delicious meat and strong resistance to adversity, and has been widely cultivated in the north and south. However, the gonadal development of the hybrid offspring may affect the wild fish resources. Therefore, in order to cultivate a new germplasm of grouper with faster growth and high infertility, the breeding of distant hybrid triploid grouper was carried out. Summary of the invention

[0005] The invention discloses a method for inducing distant hybrid triploid grouper, that is, a method for increasing chromosome sets by combining hydrostatic pressure induction and drug induction to obtain hybrid grouper triploid.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The invention discloses an induction method of distant hybrid triploid grouper, comprising the steps of obtaining fertilized eggs of female brown-spotted grouper and male blue-bodied big-spotted grouper, recording the starting time of fertilization, placing the fertilized eggs in a hydrostatic pressure instrument for hydrostatic shock treatment, wherein natural seawater containing 6-dimethylaminopurine (6-DMAP) is injected into the hydrostatic pressure instrument in advance, transferring the fertilized eggs into a 6-DMAP solution after the hydrostatic pressure treatment to continue drug induction, and finally transferring the fertilized eggs to a culture water body for hatching.

[0008] Preferably, the method for obtaining fertilized eggs is to collect eggs of mature brown-spotted grouper, thaw and examine under a microscope the frozen sperm of blue-bodied big-spotted grouper, use sperm with a sperm motility of more than 80% to fertilize the eggs of brown-spotted grouper, mix the sperm and eggs, and record the starting time of fertilization; wash the fertilized eggs with excess seawater 3 minutes after fertilization, and then put the fertilized eggs into fresh seawater, let them stand, and wait for processing.

[0009] Preferably, the hydrostatic pressure treatment starts 4 to 8 minutes after fertilization.

[0010] Preferably, the hydrostatic pressure is 35-55 MPa and the treatment duration is 3-7 min.

[0011] Preferably, the concentration of 6-DMAP in the hydrostatic pressure instrument is 200 mg / L.

[0012] Preferably, during the pressurization stage of the hydrostatic pressure instrument, the pressure is increased from 0 MPa to 35-55 MPa within 15-20 seconds; and during the pressure relief stage, the pressure is reduced to 0 MPa within 15-20 seconds.

[0013] Preferably, after the hydrostatic pressure treatment is completed, the fertilized eggs are transferred to a seawater solution containing 6-DMAP at a concentration of 200 mg / L and aerated for 8 to 12 minutes.

[0014] Preferably, the insemination water temperature, the water temperature in the hydrostatic pressure instrument, the 6-DMAP solution, and the incubation water temperature are all 27-28°C.

[0015] The principle of the present invention is to perform interspecific distant hybridization of two groupers with different geographical distributions and use polyploidy induction to obtain triploid hybrid offspring with excellent traits.

[0016] The beneficial effects of the present invention compared with the prior art are as follows:

[0017] 1. There is geographical isolation and reproductive isolation between brown-spotted grouper and blue-bodied grouper. The present invention utilizes the blue-bodied grouper sperm bank to break through the geographical isolation between the two fish; the frozen sperm is applied to the production of grouper fry, which greatly facilitates the production and accelerates the process of breeding new grouper varieties.

[0018] 2. Provides an induction method for efficiently producing hybrid triploids of brown-spotted grouper♀×blue-spotted grouper♂, involving a method of combined induction of hydrostatic pressure shock and chemical drug treatment. It is an efficient, novel, simple to operate, and highly practical induction method. This method has opened up a new technical approach for polyploid breeding of hybrid progeny of grouper, which has not been reported at home and abroad;

[0019] 3. The speed of embryonic development is greatly affected by temperature, which in turn affects the best time to inhibit the release of the second polar body, ultimately resulting in unstable induction effects and poor repeatability. Therefore, the fertilization water temperature, hydrostatic pressure treatment water temperature, drug treatment water temperature, and incubation water temperature are strictly regulated to 27-28°C;

[0020] 4. Different grouper species have different embryonic development times, and the optimal triploid induction conditions vary greatly. Reports on distant hybrid triploid grouper have not yet involved the relevant parameters of hydrostatic pressure combined with chemical drug induction. There are differences between the principles of hydrostatic pressure induction, chemical drug induction and cold shock induction. In addition, the induction conditions of different fish have their species specificity and cannot be simply used as a reference. Therefore, the present invention obtains the triploid induction parameters of distant hybrid grouper for the first time. This method is simple to operate, short in processing time, low in damage to fertilized eggs, and an induction rate of more than 60%, which is suitable for industrial promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a triploid red blood cell of brown-spotted grouper♀×blue-bodied grouper♂;

[0022] Figure 2 It is the diploid brown-spotted grouper♀×blue-bodied big-spotted grouper♂ red blood cell;

[0023] Figure 3 This is a DNA content analysis chart of triploid brown-spotted grouper♀×blue-bodied big-spotted grouper♂;

[0024] Figure 4 This is the DNA content analysis diagram of diploid brown-spotted grouper♀×blue-bodied big-spotted grouper♂;

[0025] Figure 5 It is the metaphase of chromosomes of triploid brown-spotted grouper♀×blue-bodied big-spotted grouper♂;

[0026] Figure 6This is the metaphase of chromosome division of diploid brown-spotted grouper♀×blue-spotted grouper♂. DETAILED DESCRIPTION

[0027] The present invention will be further described below in conjunction with specific embodiments. The advantages and features of the present invention will become clearer as the description proceeds, but the protection scope of the present invention is not limited to the following embodiments.

[0028] Example 1

[0029] (1) Artificial insemination of grouper: Select female brown-spotted grouper with a bulging abdomen and slightly reddish genital opening after induction of spawning. First, wipe the fish body surface with a dry towel, then squeeze the eggs and place the mature eggs in a plastic beaker; at the same time, thaw the frozen sperm of blue-bodied big-spotted grouper. The thawing method is to take out the cryotube from the liquid nitrogen tank and place it in a 37℃ water bath to thaw. When the solid semen in the cryotube has just melted, take it out and the thawing is completed; then examine it under a microscope. When the sperm motility is greater than 80%, artificial insemination is performed with sperm and eggs at a volume ratio of 1:700. Pour the sperm into seawater with a volume of one-tenth of the egg volume, stir it gently, and then pour it into the egg and stir to mix. This moment is the start time of fertilization. 3 minutes after fertilization, wash the fertilized eggs with excess seawater, then put the fertilized eggs into fresh seawater, let it stand and wait for processing. In this embodiment, the fertilization temperature, as well as the subsequent pressure treatment water temperature, drug treatment water temperature, and incubation water temperature are all 27-28°C.

[0030] (2) Screening of induction parameters for distant hybridization triploid grouper:

[0031] ① Screening of the duration of 6-DMAP treatment for triploid induction: 6 minutes after fertilization, the fertilized eggs were placed in fresh seawater containing 200 mg / L 6-dimethylaminopurine for 10, 15 and 20 minutes, respectively, and then rinsed with fresh seawater and poured into the pool for hatching. The normal development rate, hatching rate, and triploid induction rate were statistically analyzed. It was found that the normal development rate and hatching rate of the induction group with a continuous treatment time of 15 minutes were 41.5±10.5% and 38.5±8.6%, respectively, which were slightly lower than those of the 10-minute treatment group, but there was no significant difference; each induction group was found to have a split phase, and the triploid induction rate of the 15-minute treatment group was relatively high, at 21.5±5.4%.

[0032] ②Screening of the starting time of hydrostatic treatment combined with chemical drug treatment for triploid induction: At different times (4, 6, and 8 minutes) after fertilization, the fertilized eggs were placed in a hydrostatic pressure instrument containing 6-DMAP (200 mg / L) seawater for hydrostatic shock and drug induction. The hydrostatic pressure was 45 MPa and the treatment duration was 5 minutes. After decompression, the fertilized eggs were taken out and immersed in 200 mg / L 6-DMAP seawater solution for 10 minutes (the total drug treatment time was 15 minutes). During the pressurization stage, the pressure was increased from 0 MPa to 45 MPa within 15 to 20 seconds; during the decompression stage, the pressure was reduced to 0 MPa within 15 to 20 seconds; finally, they were transferred to normal fresh seawater for hatching. The normal development rate, hatching rate, and polyploidy of newly hatched fry were statistically analyzed, and it was found that the normal development rate and hatching rate of the induced group 6 minutes after fertilization were the highest, which were 23.5±5.4% and 14.5±2.6%, respectively. Triploid fission phases were found in each induced group, among which the 8-min treatment group had a higher proportion of triploid fission phases, but its fry survival rate was extremely low, so the 6-min treatment group (triploid induction rate was 34.5±6.9%) was selected as the induction condition with better results.

[0033] ③ Screening of triploid induction treatment pressure: 6 minutes after fertilization, the fertilized eggs were placed in a hydrostatic pressure instrument containing 6-DMAP (200 mg / L) seawater for hydrostatic shock and drug induction. The hydrostatic pressure was 35, 45, and 55 MPa, respectively, and the treatment time was 5 minutes. After decompression, the fertilized eggs were taken out and immersed in a 200 mg / L 6-DMAP seawater solution for 10 minutes (the total drug treatment time was 15 minutes), and finally transferred to normal fresh seawater for hatching. The normal development rate, hatching rate, and polyploidy of newly hatched fry were statistically analyzed, and it was found that the normal development rate and hatching rate of the induced group with a treatment pressure of 45 MPa were higher, at 22.5±4.1% and 13.4±3.2%, respectively. Triploid division phases were found in each induced group. Although the triploid rate of the 55MPa induced group was relatively high, its hatching rate was very low and the fry had more deformities. Therefore, the 45MPa induced group (triploid rate was 43.7±5.2%) was selected as the optimal treatment pressure.

[0034] ④ Screening of the duration of triploid hydrostatic induction: 6 minutes after fertilization, the fertilized eggs were placed in a hydrostatic pressure instrument containing 6-DMAP (200 mg / L) seawater for combined hydrostatic shock and drug induction treatment. The hydrostatic pressure was 45 MPa and the treatment time was 3, 5, and 7 minutes. After decompression, the fertilized eggs were taken out and immersed in a 200 mg / L 6-DMAP seawater solution for 12, 10, and 8 minutes (the total drug treatment time was 15 minutes). Finally, they were transferred to normal fresh seawater for hatching. The normal development rate, hatching rate, and triploid rate were statistically analyzed, and it was found that the normal development rate, hatching rate, and triploid rate were the highest when the hydrostatic pressure was continuously induced for 5 minutes, which were 32.9±3.1%, 18.5±2.4%, and 61.5±5.7%, respectively. Therefore, the duration of triploid hydrostatic induction was selected to be 5 minutes.

[0035] (3) Ploidy identification of distantly hybridized triploid grouper

[0036] ① Determination of red blood cell parameters: Compare the blood cells of grouper fry with a total length of 5 to 6 cm. Take a drop of blood and drop it on a clean slide, use another slide to push the slide, wait for the blood film to dry, fix it with methanol for 2 minutes, stain it with Giemsa dye for 30 minutes, and finally rinse it gently with running water, dry it at room temperature, observe it under a microscope, take pictures, and measure 20 red blood cells for each smear.

[0037] The results showed that the long axis of the erythrocytes of the hybrid triploid grouper was 16.78±1.35μm, which was 1.34 times that of the erythrocytes of the hybrid diploid grouper (12.49±0.91μm). The surface area of ​​the erythrocytes of the hybrid triploid grouper was 144.80±14.78μm. 2 , is the surface area of ​​hybrid diploid grouper (94.24±9.23μm 2 ) is 1.54 times. The red blood cell image results are shown in Figure 1 and Figure 2 .

[0038] ②DNA content determination: The ploidy of grouper fry with a body length of 5 to 6 cm was identified. The method used was to take blood from the tail vein, use 15 mg / L potassium ethylenediaminetetraacetic acid for anticoagulation, the ratio of anticoagulant: blood was 1:3-5, then take 10 μL of blood cells and add them to 0.5 mL of 0.01 M PBS, then add the cell suspension dropwise to 1.5 ml of pre-cooled anhydrous ethanol for fixation, the fixation time was at least 4 hours; the cell suspension was centrifuged at 3000 r / min for 3 minutes, the supernatant was discarded, and it was washed twice with PBS, centrifuged at 6000 r / min for 3 minutes, then 10 μL of 1 mg / ml RNaseA solution was added, treated at room temperature for 30 minutes, then filtered with a 300 mesh silk screen, and then 5 μL of 1 mg / ml PI solution was added, treated at 4°C in the dark for 30 minutes, and then tested on the machine. The blood samples of the untreated group of golden tiger hybrid spots were used as controls, and the DNA content of the blood samples of the induced group was analyzed, and the data were processed using ModFitLT5.0 software.

[0039] The results showed that the relative DNA content of hybrid triploid grouper was 33.8±1.3, and that of hybrid diploid grouper was 22.4±2.1. The relative DNA content of hybrid triploid grouper was 1.5 times that of hybrid diploid grouper. Figure 3 and Figure 4 .

[0040] ② Chromosome analysis: Chromosomes were prepared using the head kidney-colchicine method, after injection of phytohemagglutinin (PHA) and colchicine, preparation of kidney cell suspension, hypotonicity, fixation, drop slices and staining steps, and observation under a microscope. 100 metaphases were counted.

[0041] The results showed that the number of chromosomes in the diploid golden tiger hybrid spot was 2n=48, and the number of chromosomes in the triploid golden tiger hybrid spot was 3n=72. Figure 5 and Figure 6 shown.

[0042] Example 2

[0043] (1) Artificial insemination of grouper: Place mature eggs in a plastic beaker and thaw frozen sperm of blue-bodied grouper at the same time; when the sperm motility is greater than 85%, artificial insemination is performed with sperm and eggs at a volume ratio of 1:700. Pour the sperm into seawater with a volume of one-tenth of the egg volume and gently stir evenly; then pour it into the egg and stir to mix evenly. This moment is the start time of fertilization. 3 minutes after fertilization, wash the fertilized eggs with excess seawater, then put the fertilized eggs into fresh seawater and let it stand for processing.

[0044] (2) Induction of triploid grouper: Collect the floating fertilized eggs, and 6 minutes after fertilization, place the fertilized eggs in a hydrostatic pressure instrument with fresh seawater for hydrostatic shock, the hydrostatic pressure is 45MPa, the treatment duration is 5 minutes, and the fertilized eggs are taken out after decompression, and then transferred to normal fresh seawater for hatching. In this embodiment, the fertilization temperature, the subsequent pressure treatment water temperature, and the hatching water temperature are all 27-28°C.

[0045] (3) In this example, the normal development rate and hatching rate of the triploid induced group were 28.3±3.5% and 23.6±2.9%, respectively, and the triploid rate was 32.8±3.7%.

[0046] Example 3

[0047] (1) Artificial insemination of grouper: Place mature eggs in a plastic beaker and thaw frozen sperm of blue-bodied grouper at the same time; when the sperm motility is greater than 85%, artificial insemination is performed with sperm and eggs at a volume ratio of 1:700. Pour the sperm into seawater with a volume of one-tenth of the egg volume and gently stir evenly; then pour it into the egg and stir to mix evenly. This moment is the start time of fertilization. 3 minutes after fertilization, wash the fertilized eggs with excess seawater, then put the fertilized eggs into fresh seawater and let it stand for processing.

[0048] (2) Induction of triploid grouper: Collect the floating fertilized eggs, and 6 minutes after fertilization, place the fertilized eggs in a hydrostatic pressure instrument with fresh seawater for hydrostatic shock, the hydrostatic pressure is 45MPa, and the treatment duration is 5 minutes. After the pressure is released, take out the fertilized eggs, immerse them in a 200mg / L 6-DMAP seawater solution for 15 minutes, and finally transfer them to normal fresh seawater for hatching. In this embodiment, the fertilization temperature, the subsequent pressure treatment water temperature, the chemical drug treatment water temperature, and the hatching water temperature are all 27-28°C.

[0049] (3) In this example, the normal development rate and hatching rate of the triploid induced group were 23.3±4.3% and 19.6±3.1%, respectively, and the triploid rate was 27.8±2.9%.

[0050] Example 4

[0051] (1) Artificial insemination of grouper: Mature eggs were placed in a plastic beaker to obtain a total of 2L of high-quality eggs; while squeezing the eggs, the frozen sperm of blue-bodied grouper was thawed by taking out the cryovials from the liquid nitrogen tank and placing them in a 37°C water bath to thaw; when the sperm motility was greater than 85%, the sperm and eggs were artificially inseminated at a volume ratio of 1:700, and the sperm was poured into seawater with a volume of one-tenth the volume of the eggs, gently stirred evenly, and then poured into the eggs and stirred to mix evenly. This moment was the start time of fertilization. 3 minutes after fertilization, the fertilized eggs were washed with excess seawater, and then the fertilized eggs were placed in fresh seawater and allowed to stand for processing.

[0052] (2) Induction of triploid grouper: Collect the floating fertilized eggs, and 6 minutes after fertilization, place the fertilized eggs in a hydrostatic pressure instrument containing 6-DMAP (200 mg / L) seawater for hydrostatic shock and drug induction combined treatment, the hydrostatic pressure is 45 MPa, the treatment duration is 5 minutes, and the fertilized eggs are taken out after decompression, and immersed in a 200 mg / L 6-DMAP seawater solution for 10 minutes (the total drug treatment time is 15 minutes). During the pressurization stage, the pressure is increased from 0 MPa to 45 MPa within 15 to 20 seconds; during the decompression stage, the pressure is reduced to 0 MPa within 15 to 20 seconds; finally, it is transferred to normal fresh seawater to complete the incubation. In this embodiment, the fertilization temperature, the subsequent pressure treatment water temperature, the chemical drug treatment water temperature, and the incubation water temperature are all 27 to 28°C.

[0053] (3) In this embodiment, the normal development rate and hatching rate of the triploid batch induced group were 35.2% and 32.8% respectively, and the triploid rate of the 2-month-old golden tiger hybrid spot was 63.6%.

[0054] Although the present invention has been disclosed as above in the form of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, these improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for inducing distant hybridization triploid grouper, characterized in that: Fertilized eggs of female brown-spotted grouper and male blue-bodied big-spotted grouper were obtained, and the starting time of fertilization was recorded. The fertilized eggs were placed in a hydrostatic pressure instrument for hydrostatic shock treatment. During the pressurization stage of the hydrostatic pressure instrument, the pressure was increased from 0 MPa to 35-55 MPa within 15-20 s; during the depressurization stage, the pressure was reduced to 0 MPa within 15-20 s. The hydrostatic pressure instrument had natural seawater containing 6-dimethylaminopurine. After hydrostatic pressure treatment, the fertilized eggs were moved into 6-dimethylaminopurine solution for continued drug induction, and finally the fertilized eggs were transferred to the aquaculture water body for hatching.

2. The method for inducing distant hybrid triploid grouper according to claim 1, characterized in that: The hydrostatic pressure treatment started 4 to 8 minutes after fertilization.

3. The method for inducing distant hybrid triploid grouper according to claim 1, characterized in that: The hydrostatic pressure is 35~55 Mpa, and the treatment duration is 3~7 min.

4. The method for inducing distant hybrid triploid grouper according to claim 1, characterized in that: The concentration of 6-dimethylaminopurine in the hydrostatic pressure instrument was 200 mg / L.

5. The method for inducing distant hybridization triploid grouper according to claim 1, characterized in that: After the hydrostatic pressure treatment, the fertilized eggs were transferred to a seawater solution containing 200 mg / L 6-dimethylaminopurine and aerated for 8 to 12 min.

6. The method for inducing distant hybridization triploid grouper according to claim 1, characterized in that: The water temperature for insemination, the water temperature in the hydrostatic pressure instrument, the 6-dimethylaminopurine solution, and the incubation water were all 27-28°C.

7. The method for inducing distant hybridization triploid grouper according to claim 1, characterized in that: Method for obtaining fertilized eggs: Collect eggs from mature brown-spotted grouper, thaw and examine the frozen sperm of blue-bodied grouper, use sperm with a motility of more than 80% to fertilize the eggs of brown-spotted grouper, mix the sperm and eggs, and record the starting time of fertilization; wash the fertilized eggs with excess seawater 3 minutes after fertilization, then put them in fresh seawater, let them stand, and wait for processing.

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