A method for cultivating an ovate fast-growing triploid mactra veneriformis "shibei No. 4"

Abstract

The application provides a cultivating method of an ovate fast-growing triploid Mactra veneriformis "Shibai No.4", and belongs to the technical field of aquaculture breeding. The application adopts a method combining group selection, polyploidy breeding and hybridization breeding to carry out selection and breeding of the ovate M. veneriformis fast-growing line. The fast-growing breeding line is induced by two methods of directly inducing tetraploidy from diploids and inducing tetraploidy from triploids, different tetraploid groups are directionally selected by taking shell length, weight and shell type as the selection targets, and the tetraploid M. veneriformis fast-growing breeding line with stable tetraploidization rate is bred through inter-group hybridization. The ovate fast-growing triploid M. veneriformis is bred through hybridization between the diploid and tetraploid fast-growing breeding lines. The fast-growing triploid M. veneriformis "Shibai No.4" has the advantages of regular shell type (ovate), fast growth speed, high meat yield, strong stress resistance and excellent quality, is very suitable for coastal aquaculture, and has wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture breeding technology, and in particular relates to a method for cultivating the oval-shaped, fast-growing triploid Crassostrea gigas 'Shibei 4'. Background Technology

[0002] The Crassostrea kojima, belonging to the class Bivalvia, order Pterioida, family Ostreidae, and genus Crassostrea, is a eurythermal and euryhaline marine economic shellfish, primarily inhabiting the subtidal estuarine areas of coastal estuaries in my country where salinity is low. Although the Crassostrea kojima possesses advantages such as rapid growth, eurythermal and euryhaline tolerance, strong disease resistance, and high survival rate, its low meat yield, red or yellow soft body, and irregular shell shape have resulted in low consumer market acceptance, hindering its widespread aquaculture.

[0003] Cultivating triploid oysters with three sets of chromosomes in their somatic cells using cell engineering technology can effectively improve their production performance. Because the three sets of chromosomes have high genetic heterozygosity and exhibit poor fertility due to disorder during meiosis, triploid oysters possess advantages such as rapid growth, high meat yield, strong resistance to adverse conditions, large size, low fertility, high glycogen content, and good meat quality, which can compensate for the inherent shortcomings of diploid *Crassostrea gigas* oysters. Therefore, cultivating new triploid *Crassostrea gigas* oyster strains with regular shell shape, rapid growth, high meat yield, short cultivation cycle, and high quality is of great significance for promoting the transformation and upgrading of my country's oyster farming industry and contributing to rural revitalization. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a method for cultivating the oval-shaped fast-growing triploid Crassostrea gigas 'Shibei 4'. Using the method of this invention, a new fast-growing triploid Crassostrea gigas oyster strain with regular shell shape, fast growth rate, high meat yield, strong resistance, and excellent quality can be produced on a large scale.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a method for cultivating the oval-shaped, fast-growing triploid Crassula ovata 'Sebei 4', comprising:

[0007] (1) Select 200-300g oysters with shell length of 8-10cm and oval shell as parents, and carry out directional breeding to obtain the fast-growing breeding line F of oysters;

[0008] (2) Using F as the parent, tetraploid Crassula ovata a was obtained by inhibiting the first polar body to induce direct induction; using F as the parent, triploid Crassula ovata was induced by inhibiting the second polar body, and then tetraploid Crassula ovata population b was obtained by inhibiting the first polar body to induce induction; through two consecutive generations of population selection, fast-growing tetraploid Crassula ovata breeding lines Ga and Gb were obtained respectively.

[0009] (3) Ga and Gb were crossed to obtain Gab, a ploidy-stable tetraploid fast-growing breeding line of Crassula ovata 'Oriole';

[0010] (4) Using Gab as the male parent and F as the female parent, a sex hybridization was carried out to cultivate oval-shaped fast-growing triploid oyster "Shibei 4".

[0011] Preferably, the oyster in step (1) is a 2-3 year old diploid oyster.

[0012] Preferably, the number of generations of directional breeding in step (1) is 4 or more.

[0013] Preferably, the induction method in step (2) is as follows: after treating the fertilized egg with cytochalasin B, it is soaked in dimethyl sulfoxide solution.

[0014] More preferably, the volume concentration of cytochalasin B is 0.6-0.8 mg / L; the inhibition time of the first polar body by cytochalasin B is 10 min or 15 min; and the inhibition time of the second polar body by cytochalasin B is 30 min or 45 min.

[0015] More preferably, the volume concentration of the dimethyl sulfoxide is 0.05% to 0.2%, and the soaking time is 15 to 25 minutes.

[0016] Preferably, the selection criteria in steps (1) and (2) are: weight 200-300g, shell length 8-10cm, and shell oval shape.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] This invention employs a combination of population selection, polyploid breeding, and hybridization breeding to cultivate a fast-growing line of oval-shaped Crassula ovata. The fast-growing line utilizes two methods: direct induction of tetraploids from diploids and induction of tetraploids from triploids to create different tetraploid populations. Shell length, weight, and shell shape are used as selection targets for these different tetraploid populations. Through inter-population hybridization, a fast-growing line of oval-shaped Crassula ovata with a stable tetraploid ploidization rate is cultivated. Furthermore, through hybridization between diploid and tetraploid fast-growing lines, the oval-shaped fast-growing Crassula ovata 'Shibei 4' triploid is cultivated. The fast-growing triploid Crassula ovata 'Shibei 4' cultivated using this method exhibits advantages such as regular shell shape (oval-shaped), rapid growth, high meat yield, strong resistance to adverse conditions, and excellent quality, making it highly suitable for coastal aquaculture and promising for widespread application. Attached Figure Description

[0019] Figure 1 Technical roadmap for the cultivation of the fast-growing triploid Crassostrea gigas oyster "Shibei 4";

[0020] Figure 2 Peak values ​​of diploid-induced tetraploids in fast-growing Crassula ovata were detected by flow cytometry.

[0021] Figure 3 Peak values ​​of the triploid-induced tetraploid oyster from fast-growing Crassula ovata were detected by flow cytometry.

[0022] Figure 4 Peak values ​​detected by flow cytometry for triploid fast-growing Crassula ovata. Detailed Implementation

[0023] This invention provides a method for cultivating the oval-shaped, fast-growing triploid Crassostrea gigas oyster, 'Sebei 4', comprising:

[0024] (1) Select 200-300g oysters with shell length of 8-10cm and oval shell as parents, and carry out directional breeding to obtain the fast-growing breeding line F of oysters;

[0025] (2) Using F as the parent, tetraploid Crassula ovata a was obtained by inhibiting the first polar body to induce direct induction; using F as the parent, triploid Crassula ovata was induced by inhibiting the second polar body, and then tetraploid Crassula ovata population b was obtained by inhibiting the first polar body to induce induction; through two consecutive generations of population selection, fast-growing tetraploid Crassula ovata breeding lines Ga and Gb were obtained respectively.

[0026] (3) Ga and Gb were crossed to obtain Gab, a ploidy-stable tetraploid fast-growing breeding line of Crassula ovata 'Oriole';

[0027] (4) Using Gab as the male parent and F as the female parent, a sex hybridization was carried out to cultivate oval-shaped fast-growing triploid oyster "Shibei 4".

[0028] The present invention does not specifically limit the source of the oyster, including cultured oysters and wild oysters. In the present invention, the oyster is preferably a 2-3 year old oyster, and more preferably a 2-year old oyster.

[0029] When screening parent oysters of Crassula ovata, the present invention uses body weight, shell length and shell shape as target traits. The preferred body weight is 200-300g, more preferably 230-270g, the preferred shell length is 8-10cm, more preferably 9cm, and the shell shape is oval.

[0030] The preferred method of targeted breeding described in this invention is to collect sperm and eggs from selected parent oysters, obtain D-shaped larvae through artificial insemination, cultivate the D-shaped larvae in an indoor culture tank until they develop into eye-spot larvae, introduce an attachment substrate, and transfer oyster juveniles that grow to a shell height of 2-3 mm to natural sea areas for culture until they become adult oysters. Then, they are screened again based on weight, shell length, and shell shape as target traits. This process is repeated continuously, which constitutes targeted breeding.

[0031] In this invention, the preferred number of generations for directional breeding is four, resulting in the fast-growing selective breeding line F of Crassula ovata. This invention uses body weight, shell length, and shell shape as breeding targets, selecting individuals with oval shell shape, the top 10% in body weight, and shell length as breeding parents. After four consecutive generations of population selection, the fast-growing selective breeding line F is obtained.

[0032] This invention induces tetraploidy through two methods:

[0033] (1) Tetraploid population a: Tetraploids are obtained by direct induction of the first polar body by using F as the parent. The preferred induction method is to treat the fertilized eggs with cytochalasin B and then soak them in dimethyl sulfoxide solution. The volume concentration of cytochalasin B is preferably 0.6-0.8 mg / L, more preferably 0.7 mg / L. The treatment time of the first polar body with cytochalasin B is preferably 10-15 min, more preferably 12 min. The volume concentration of the dimethyl sulfoxide solution is preferably 0.05%-0.2%. The soaking time is preferably 15-25 min, more preferably 20 min.

[0034] (2) Tetraploid population b: Using F1 as the parent, triploids are obtained by inhibiting the second polar body induction. The preferred induction method is to treat the fertilized eggs with cytochalasin B and then soak them in dimethyl sulfoxide solution. The volume concentration of cytochalasin B is preferably 0.6-0.8 mg / L, more preferably 0.7 mg / L. The treatment time for the second polar body is preferably 30-45 min, more preferably 40 min. The volume concentration of the dimethyl sulfoxide solution is preferably 0.05%-0.2%. The soaking time is preferably 15-25 min, more preferably 20 min. Then, using F1 as the male parent and triploids as the female parent, tetraploids are obtained by inhibiting the first polar body induction. The induction method is the same as the method of directly inducing tetraploids when F1 is the parent.

[0035] This invention does not specifically limit the source of cytochalasin B and dimethyl sulfoxide.

[0036] In this invention, the preferred method for treating fertilized eggs with cytochalasin B is as follows: Female oysters of the gonadally mature, fast-growing selective breeding line F, or triploid female oysters, and male oysters of the fast-growing selective breeding line F generation are dissected separately, and sperm and eggs are collected separately. Artificial insemination is performed, and when the fertilized eggs develop to the first or second polar body, they are treated with cytochalasin B. During artificial insemination, the egg-to-sperm ratio is preferably 1:5-10, more preferably 1:6-8, and most preferably 1:7. After treating the fertilized eggs with cytochalasin B, they are preferably soaked in a dimethyl sulfoxide solution before conventional hatching, larval rearing, and marine aquaculture. The volume concentration of the dimethyl sulfoxide solution is preferably 0.1%, and the soaking time is preferably 20 minutes.

[0037] After induction, the process preferably includes flow cytometry screening of the induced fertilized eggs to obtain triploid Crassostrea gigas with a ploidy of 3N and tetraploid Crassostrea gigas with a ploidy of 4N.

[0038] After obtaining tetraploid populations a and b, two generations of population selection were carried out using body weight, shell length, and shell shape as breeding targets to obtain tetraploid fast-growing oyster breeding lines Ga and Gb, respectively. Then, through hybridization between the tetraploid fast-growing oyster breeding lines Ga and Gb, a tetraploid fast-growing oyster breeding line Gab with stable ploidy (tetraploidity rate of over 90%) was obtained.

[0039] In this invention, the preferred method for population selection and breeding is to collect eggs and sperm from fast-growing tetraploid Crassula ovata Ga and Gb for hybridization. When the fertilized eggs develop into D-shaped larvae, the density is adjusted for cultivation. When the larvae develop to the eye point, an attachment substrate is introduced. When the juvenile oysters grow to a shell height of 2-3 mm, they are placed in an upwelling system for cultivation. When the juvenile oysters grow to a shell height of more than 1 cm, they are removed from the substrate, and individual oysters are caged and transferred to natural sea areas for cultivation. The preferred cultivation density of D-shaped larvae is 1-3 larvae / ml, more preferably 2 larvae / ml. The attachment substrate preferably includes oyster shells, scallop shells, plastic sheets, films, etc.

[0040] After obtaining the ploidy-stable tetraploid fast-growing oyster breeding line Gab, the tetraploid Gab was used as the male parent and the F1 generation as the female parent for hybridization to cultivate the oval-shaped fast-growing triploid oyster "Shibei 4". This invention does not impose any special limitations on the hybridization method; any in vitro hybridization method well-known in the art can be used.

[0041] This invention does not impose any special restrictions on the source of raw materials not mentioned; conventional commercially available products in the field can be used.

[0042] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0043] Unless otherwise specified, the following embodiments are all conventional methods.

[0044] Example 1

[0045] (1) Selection of Fast-Growing Crassula ovata F4 Breeding Line: 2000 2-3 year old populations of Crassula ovata cultured and wild were collected from coastal areas such as Guangxi and Shandong. 200 individuals with oval shells and the top 10% in weight and shell length were selected as breeding broodstock. These broodstock weighed 200-265g, had a shell length of 8-9cm, and an oval shell shape. D-shaped larvae were obtained through artificial insemination at a water temperature of 25℃ and a salinity of 20. These larvae were then cultured in indoor nursery ponds until they reached the eyepoint larval stage, and cement ropes were used as attachment substrates. When the juvenile oysters grew to 2-3mm, they were transferred to natural sea areas for further cultivation. Finally, using the same breeding objectives, nursery techniques, and cultivation methods, a fast-growing Crassula ovata F4 breeding line was developed through four consecutive generations of population selection.

[0046] (2) Induction of tetraploid Crassula ovata population a: 200 mature gonadal-developing Crassula ovata fast-growing breeding line F4 parents were dissected, and females (56) and males (88) were separated using a biological microscope; 30 pairs of male and female parents with the best gonadal development were selected, and sperm and eggs were extracted separately. Eggs were first filtered through a 150-mesh silk screen and then collected through a 450-mesh silk screen, while sperm were directly filtered through a 450-mesh silk screen; fertilization was performed using a one-to-one method, while controlling the number of sperm around each egg to be 5-10; 5 minutes after fertilization, 0.8 mg / L cytochalasin B was immediately added, and the fertilized eggs were treated for 10 minutes, followed by soaking in seawater containing 0.1% dimethyl sulfoxide for 20 minutes; at a water temperature of 25℃, Under salinity conditions of 20, fertilized eggs from each group were collected and placed in an indoor nursery for hatching and larval rearing. D-shaped larvae were detected using flow cytometry, and the group with a tetraploid induction rate of over 60% was retained. When the larvae developed to the eyespot larval stage, a film attachment substrate was introduced (using segmented attachment screening). The tetraploid induction rate was monitored daily. When the tetraploid induction rate was above 60%, the previously introduced film was removed and new film attachment substrates were introduced. The attachment process was stopped when the tetraploid induction rate fell below 60%. Film attachment substrates with an induction rate of 60% were transferred to an upwelling system for juvenile oyster rearing. When the juvenile oysters grew to a shell height of over 1 cm, they were removed from the substrate, and individual oysters were caged and transferred to natural sea areas for rearing, resulting in a population of approximately 2000 individuals.

[0047] Induction of tetraploid *Crassostrea gigas* population b: ① Triploid induction: 200 gonadally mature *Crassostrea gigas* fast-growing breeding line F4 parents were dissected, and females (43) and males (67) were separated using a biological microscope; 30 pairs of male and female parents with the best gonadal development were selected, and sperm and eggs were extracted separately. The eggs were first filtered through a 150-mesh silk screen and then collected through a 450-mesh silk screen, while the sperm were directly filtered through a 450-mesh silk screen; fertilization was performed using a one-to-one method, while controlling the number of sperm around each egg to be 5-10. When 50% of the fertilized eggs develop the first polar body, immediately add 0.8 mg / L cytochalasin B and treat the fertilized eggs for 30 min. Then soak them in seawater containing 0.1% dimethyl sulfoxide for 20 min. Under the conditions of water temperature 25℃ and salinity 20, collect the fertilized eggs from each group and put them into an indoor nursery for hatching and larval cultivation. Use flow cytometry to detect the induced group with a triploid induction rate of more than 90%. When they develop into eyespot larvae, oyster shell attachment substrates are introduced, and finally they are cultured in the sea area. ② Tetraploid Induction: Triploid *Crassostrea gigas* with mature gonads were dissected. Flow cytometry was used to identify 100 individuals with a ploidy of 3N. Microscopic observation was used to select 30 well-developed female 3N individuals as maternal parents. Sixty gonad-mature *Crassostrea gigas* F4 breeding lines with mature gonads were dissected. Sexing was performed using a microscopic microscope, and 30 individuals with plump gonads and strong sperm motility were selected as paternal parents. Sperm and eggs were extracted separately. Eggs were first filtered through a 150-mesh silk screen and then collected through a 450-mesh silk screen. Sperm were directly filtered through a 450-mesh silk screen. One-to-one fertilization was performed, with each egg surrounded by 5-10 sperm. Sperm; 10 minutes after fertilization, 0.7 mg / L cytochalasin B was added immediately and the fertilized eggs were treated for 20 minutes, followed by soaking in seawater containing 0.1% dimethyl sulfoxide for 20 minutes; under conditions of water temperature 25℃ and salinity 20, the fertilized eggs of each group were collected and placed in an indoor nursery for hatching and larval cultivation. The induced group with a tetraploid induction rate of more than 80% was detected by flow cytometry; when the larvae developed to the eyespot larvae, a film attachment substrate was placed, and after the attachment was completed, the larvae were transferred to an upwelling system for juvenile oyster cultivation; when the juvenile oysters grew to a shell height of more than 1 cm, they were de-subjected, and individual oysters were caged and transferred to natural sea areas for cultivation, resulting in a population of about 3000 individuals.

[0048] (3) Tetraploid Crassula ovata fast-growing breeding line Gab: During the Crassula ovata breeding season, tetraploid Crassula ovata population a or b was used as the parent. 200 individuals with oval shells and the top 10% in weight and shell length were selected as breeding parents. Flow cytometry analysis confirmed that all individuals were 4N. D-shaped larvae were obtained through artificial insemination at a water temperature of 25℃ and a salinity of 20. These larvae were cultured in an indoor nursery until they reached the eyepoint stage, and polyethylene plastic sheets were used as attachment substrates. When the juveniles grew to 2–3 mm, the polyethylene plastic sheets were dispersed into an upwelling system for further culture. When the juveniles grew to over 1 cm, individual oysters were obtained using a substrate removal method and then caged and transferred to natural sea areas for further culture. Two consecutive generations of tetraploid populations were selected using the same method to obtain the tetraploid Crassula ovata fast-growing breeding lines Ga and Gb. Finally, the tetraploid fast-growing oyster breeding lines Ga and Gb were used as parents for inter-population hybridization to obtain the tetraploid fast-growing oyster breeding line Gab with stable ploidy (tetraploidity rate of over 90%).

[0049] (4) Production of the new fast-growing triploid Crassula 'Shibei 4' strain: 1000 individuals of the fast-growing Crassula 'Shibei 4' strain F4 and 200 individuals of the tetraploid Crassula 'Shibei 4' strain Gab strain were transferred to an indoor parent stock culture tank for maturation (water temperature 23℃, salinity 20). The biological feed consisted of artificially cultivated golden algae, Chaetoceros, small diatoms, and flat algae. After the gonads of the parents matured, 90 male individuals of the tetraploid Crassula 'Shibei 4' strain Gab were obtained by flow cytometry (4N) and biological microscopy. 950 fast-growing F4 individuals of the Crassula ovata 'Oriole' breeding line were dissected and, through microscopic observation, 550 female individuals were selected for later use. Using male Gab individuals as the father and F4 female individuals as the mother, hybridization was performed. Specifically, sperm and eggs were extracted from the parents. Eggs were first filtered through a 150-mesh silk sieve and then collected through a 450-mesh silk sieve, while sperm were directly filtered through a 450-mesh silk sieve. During artificial insemination, the number of sperm cells around each egg was controlled to be 3-5. After fertilization, the fertilized eggs were evenly distributed into 11 30m... 2 Hatching takes place in a nursery pond at a density of 20-30 larvae / ml. When the larvae develop into D-shaped larvae, ploidy is checked (to prevent contamination), and then 2500g (approximately 12.5 billion D-shaped larvae) is collected using a 400-mesh silk screen. The D-shaped larvae are then evenly distributed into 100 sterilized 30ml... 2 Larvae are raised in a nursery pond at a density of 1-3 larvae / ml. When the larvae develop into eyed larvae, cement ropes are placed as attachment substrates, resulting in a larval metamorphosis rate of over 30%. When the juveniles grow to 2-3 mm, they are transferred to a natural sea area for temporary rearing. After 25 days of temporary rearing in the sea area, artificial separation is carried out, and the juveniles are then transferred to a natural sea area for further rearing.

[0050] Comparative Example 1

[0051] During the oyster breeding season, 200 mature oysters (both farmed and wild, aged 2-3 years) were collected from the coastal areas of Shandong. After one month of maturation in indoor cement tanks, the parent oysters were dissected to select 20 pairs of male and female oysters with the best gonad development, and their sperm and eggs were extracted. Eggs were first filtered through a 150-mesh silk sieve and then collected through a 450-mesh silk sieve, while sperm were directly filtered through a 450-mesh silk sieve. Fertilization was performed using a one-to-one method, with each egg surrounded by 5-10 sperm. When 50% of the fertilized eggs developed the first polar body, 0.8 mg / L cytochalasin B was immediately added and the eggs were treated for 30 minutes, followed by soaking in seawater containing 0.1% dimethyl sulfoxide for 20 minutes. Under conditions of 25°C and 20 salinity, the fertilized eggs from each group were collected and placed in an indoor nursery for hatching and larval rearing. Flow cytometry was used to detect the experimental group with a triploid induction rate of over 95%. When the larvae developed to the eyespot stage, oyster shell attachment substrates were introduced, and the larvae were finally cultured in the sea area, serving as the control group for ordinary triploid Crassula ovata.

[0052] Experimental Example 1

[0053] After one year of marine culture, the triploid Crassostrea gigas 'Shibei 4' obtained in Example 1 and the common triploid Crassostrea gigas in Comparative Example 1 were cultured together. The average total weight, shell length, meat yield and survival rate were statistically analyzed. The specific results are shown in Table 1.

[0054] Table 1 Comparison of growth indicators of common triploid oysters in Example 1 and Comparative Example 1

[0055]

[0056] As shown in Table 1, after one year of cultivation in the sea area, the average total weight of "Shibei No. 4" reached 71.54g, which was 20.78% higher than that of the common triploid oyster of Comparative Example 1; the average shell length of "Shibei No. 4" was 7.62cm, which was 19.62% higher than that of the common triploid oyster of Comparative Example 1; the average meat yield of "Shibei No. 4" was 19.68%, which was 45.35% higher than that of the common triploid oyster of Comparative Example 1; and the average survival rate of "Shibei No. 4" was 86.50%, which was 10.19% higher than that of the common triploid oyster of Comparative Example 1.

[0057] Example 2

[0058] (1) Breeding of the fast-growing Crassula ovata F4 line: 3000 2-3 year old Crassula ovata populations (cultured and wild) were collected from coastal areas such as Guangxi and Guangdong. 300 individuals with oval shells and in the top 10% of weight and shell length were selected as breeding broodstock. These broodstock weighed 220-300g, had shell lengths of 9-10cm, and oval shells. D-type larvae were obtained through artificial insemination at a water temperature of 25℃ and a salinity of 20. These larvae were then cultured in indoor nursery ponds until they reached the eyepoint stage, and cement ropes were used as attachment substrates. When the juvenile oysters grew to 2-3mm, they were transferred to natural sea areas for further cultivation. Finally, using the same breeding objectives, nursery techniques, and cultivation methods, the fast-growing Crassula ovata F4 line was developed through four consecutive generations of population selection.

[0059] (2) Induction of tetraploid Crassula ovata population a: 200 mature gonadal-developing Crassula ovata fast-growing breeding line F4 parents were dissected, and females (72) and males (56) were separated using a biological microscope; 30 pairs of male and female parents with the best gonadal development were selected, and sperm and eggs were extracted separately. Eggs were first filtered through a 150-mesh silk screen and then collected through a 450-mesh silk screen, while sperm were directly filtered through a 450-mesh silk screen; fertilization was performed using a one-to-one method, while controlling the number of sperm around each egg to be 5-10; 10 minutes after fertilization, 0.6 mg / L cytochalasin B was immediately added, and the fertilized eggs were treated for 20 minutes, followed by soaking in seawater containing 0.1% dimethyl sulfoxide for 20 minutes; under conditions of water temperature 25℃ and salinity 20, the eggs were... Fertilized eggs from each group were collected and placed in indoor rearing tanks for incubation and larval rearing. D-shaped larvae were detected using flow cytometry, and the group with a tetraploid induction rate of over 60% was retained. When the larvae developed to the eyespot larval stage, polyethylene plastic sheets were placed as attachment substrates (using segmented attachment screening). The tetraploid induction rate was monitored daily. When the tetraploid induction rate was over 60%, the previously placed polyethylene plastic sheets were removed and new polyethylene plastic sheets were placed as attachment substrates. This process was continued until the tetraploid induction rate fell below 60%, at which point attachment was terminated. The polyethylene plastic sheets with an induction rate of 60% were dispersed and transferred to the upwelling system for juvenile oyster rearing. When the juvenile oysters grew to a shell height of over 1 cm, they were removed from the substrate, and individual oysters were caged and transferred to natural sea areas for rearing, resulting in a population of approximately 5000 individuals.

[0060] Induction of tetraploid *Crassostrea gigas* population b: ① Triploid induction: Dissect 200 gonadally mature *Crassostrea gigas* fast-growing breeding line F4 parents, and separate females (60) and males (56) using a biological microscope; select 30 pairs of male and female parents with the best gonadal development, and extract sperm and eggs separately. The eggs were first filtered through a 150-mesh silk screen and then collected through a 450-mesh silk screen, while the sperm were directly filtered through a 450-mesh silk screen; fertilization was performed using a one-to-one method, while controlling the number of sperm around each egg to be 5-10. When 50% of the fertilized eggs develop the first polar body, immediately add 0.6 mg / L cytochalasin B and treat the fertilized eggs for 45 min. Then soak them in seawater containing 0.1% dimethyl sulfoxide for 20 min. Under the conditions of water temperature 25℃ and salinity 20, collect the fertilized eggs from each group and put them into an indoor nursery for hatching and larval cultivation. Use flow cytometry to detect the induced group with a triploid induction rate of more than 90%. When they develop into eyespot larvae, oyster shell attachment substrates are introduced, and finally they are cultured in the sea area. ② Tetraploid Induction: Triploid Crassula ovata individuals with mature gonads were dissected, and 150 individuals with a ploidy of 3N were identified using flow cytometry. 30 well-developed female 3N individuals were selected as maternal parents through microscopic observation. 80 F4 parents of the fast-growing Crassula ovata breeding line with mature gonads were dissected, and sexing was determined using a microscopic microscope. 30 individuals with plump gonads and strong sperm motility were selected as paternal parents. Sperm and eggs were extracted separately. Eggs were first filtered through a 150-mesh silk screen and then collected through a 450-mesh silk screen, while sperm were directly filtered through a 450-mesh silk screen. One-to-one fertilization was performed, with each egg surrounded by 5-10 sperm. Sperm; 8 minutes after fertilization, 0.7 mg / L cytochalasin B was added immediately and the fertilized eggs were treated for 15 minutes, followed by soaking in seawater containing 0.1% dimethyl sulfoxide for 20 minutes; under conditions of water temperature 25℃ and salinity 20, the fertilized eggs of each group were collected and placed in an indoor nursery for hatching and larval cultivation. The induced group with a tetraploid induction rate of more than 80% was detected by flow cytometry; when the larvae developed to the eyespot larvae, a film attachment substrate was placed, and after the attachment was completed, the larvae were transferred to an upwelling system for juvenile oyster cultivation; when the juvenile oysters grew to a shell height of more than 1 cm, they were de-subjected, and individual oysters were caged and transferred to natural sea areas for cultivation, resulting in a population of about 8,000.

[0061] (3) Tetraploid Crassula ovata fast-growing breeding line Gab: During the Crassula ovata breeding season, using tetraploid Crassula ovata population a or b as parents, 500 individuals with oval shells and the top 10% in weight and shell length were selected as breeding parents. Flow cytometry analysis showed that all individuals were 4N. D-shaped larvae were obtained through artificial insemination at a water temperature of 25℃ and a salinity of 20. These larvae were cultured in an indoor nursery until they reached the eyepoint stage, and polyethylene plastic sheets were placed as attachment substrates. When the juveniles grew to 2-3 mm, the polyethylene plastic sheets were dispersed into the upwelling system for culture. When the juveniles grew to more than 1 cm, individual oysters were obtained using a substrate removal method and then caged and transferred to natural sea areas for further culture. Two consecutive generations of tetraploid populations were selected using the same method to obtain the tetraploid Crassula ovata fast-growing breeding lines Ga and Gb. Finally, the tetraploid fast-growing oyster breeding lines Ga and Gb were used as parents for hybridization to obtain the tetraploid fast-growing oyster breeding line Gab with stable ploidy (tetraploidity rate of over 90%).

[0062] (4) Production of the new fast-growing triploid Crassula 'Shibei 4' strain: 1500 individuals of the fast-growing Crassula 'Shibei 4' strain F4 and 500 individuals of the tetraploid Crassula 'Shibei 4' strain Gab strain were transferred to an indoor parent stock culture tank for maturation (water temperature 24℃, salinity 19). The biological feed consisted of artificially cultivated golden algae, Chaetoceros, Chlorella, and Platycladus; after the gonads of the parents matured, 250 male individuals of the tetraploid Crassula 'Shibei 4' strain Gab were obtained by flow cytometry (4N) and biological microscopy. 1400 fast-growing F4 individuals of the Crassula ovata 'Omi' breeding line were dissected and, through microscopic observation, 800 female individuals were selected for later use. Using male Gab individuals as the father and F4 female individuals as the mother, hybridization was performed. Specifically, sperm and eggs were extracted from the parents. Eggs were first filtered through a 150-mesh silk sieve and then collected through a 450-mesh silk sieve, while sperm were directly filtered through a 450-mesh silk sieve. During artificial insemination, 3-5 sperm were controlled to surround each egg. After fertilization, the fertilized eggs were evenly distributed into 16 30m... 2 Hatching takes place in cement tanks at a density of 20-30 larvae / ml. When the larvae develop into D-shaped larvae, ploidy is checked (to prevent contamination), and then 3000g (approximately 15 billion D-shaped larvae) is collected using a 400-mesh silk screen. The D-shaped larvae are then evenly distributed into 120 sterilized 30ml tanks. 2 Larvae are raised in a nursery pond at a density of 1-3 larvae / ml. When the larvae develop into eyespot larvae, oyster shell attachment substrates are introduced, and the larval metamorphosis rate is over 30%. When the juveniles grow to 2-3 mm, they are transferred to a natural sea area for temporary rearing. After 20 days of temporary rearing in the sea area, artificial separation is carried out, and then they are transferred to a natural sea area for further rearing.

[0063] Comparative Example 2

[0064] During the oyster breeding season, 200 mature oysters (both farmed and wild, aged 2-3 years) were collected from the coastal areas of Guangxi. After one month of maturation in indoor cement tanks, the parent oysters were dissected to select 20 pairs of male and female oysters with the best gonad development, and their sperm and eggs were extracted. Eggs were first filtered through a 150-mesh silk sieve and then collected through a 450-mesh silk sieve, while sperm were directly filtered through a 450-mesh silk sieve. Fertilization was performed using a one-to-one method, with each egg surrounded by 5-10 sperm. When 50% of the fertilized eggs developed the first polar body, 0.8 mg / L cytochalasin B was immediately added and the eggs were treated for 30 minutes, followed by soaking in seawater containing 0.1% dimethyl sulfoxide for 20 minutes. Under conditions of 25°C and 20 salinity, the fertilized eggs from each group were collected and placed in an indoor nursery for hatching and larval rearing. Flow cytometry was used to detect the experimental group with a triploid induction rate of over 95%. When the larvae developed to the eyespot stage, oyster shell attachment substrates were introduced, and the larvae were finally cultured in the sea area, serving as the control group for ordinary triploid Crassula ovata.

[0065] Experimental Example 2

[0066] After one year of marine culture, the triploid Crassostrea gigas 'Shibei 4' obtained in Example 2 and the common triploid Crassostrea gigas in Comparative Example 2 were cultured together. The average total weight, shell length, meat yield and survival rate were statistically analyzed. The specific results are shown in Table 2.

[0067] Table 2 Comparison of growth indicators of common triploid *Crassostrea gigas* oysters in Example 2 and Comparative Example 2

[0068]

[0069]

[0070] As shown in Table 2, after one year of cultivation in the sea area, the average total weight of "Shibei No. 4" reached 78.26g, which was 30.04% higher than that of the control group 2, the common triploid oyster *Crassostrea gigas*. The average shell length of "Shibei No. 4" was 7.79cm, which was 26.87% higher than that of the control group 2. The average meat yield of "Shibei No. 4" was 17.25%, which was 39.34% higher than that of the control group 2. The average survival rate of "Shibei No. 4" was 80.65%, which was 12.09% higher than that of the control group 2.

[0071] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for cultivating the oval-shaped, fast-growing triploid Crassostrea gigas 'Sebei 4', characterized in that, include: (1) Select 200-300g oysters with shell length of 8-10cm and oval shell as parents, and carry out directional breeding to obtain the fast-growing breeding line F of oysters; (2) Using F as the parent, tetraploid Crassula ovata a was obtained by inhibiting the first polar body to induce direct induction; using F as the parent, triploid Crassula ovata was induced by inhibiting the second polar body, and then tetraploid Crassula ovata population b was obtained by inhibiting the first polar body to induce induction; through two consecutive generations of population selection, fast-growing tetraploid Crassula ovata breeding lines Ga and Gb were obtained respectively. (3) Ga and Gb were crossed to obtain Gab, a ploidy-stable tetraploid fast-growing breeding line of Crassula ovata 'Oriole'; (4) Using Gab as the male parent and F as the female parent, a sex hybridization was carried out to cultivate the oval-shaped fast-growing triploid oyster "Shibei 4". The oysters mentioned in step (1) are diploid oysters aged 2-3 years; The number of generations of targeted breeding mentioned in step (1) is 4 or more; The induction method in step (2) is as follows: after treating the fertilized egg with cytochalasin B, it is soaked in dimethyl sulfoxide solution; the volume concentration of cytochalasin B is 0.6-0.8 mg / L; the inhibition time of the first polar body by cytochalasin B is 10 min or 15 min; the inhibition time of the second polar body by cytochalasin B is 30 min or 45 min; the volume concentration of dimethyl sulfoxide is 0.05%~0.2%, and the soaking time is 15~25 min; The selection criteria in steps (1) and (2) are: weight 200-300g, shell length 8-10cm, and shell oval shape.

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