Cultivation method of high-yield low-salt-tolerant razor clam

Abstract

The invention discloses a method for rapidly cultivating high-yield and low-salt-tolerant razor clams, particularly relates to a method for rapidly cultivating high-yield and low-salt-tolerant razor clams through interspecific hybridization of sinonovacula constricta and razor clams, and belongs to the field of shellfish genetic breeding. The method comprises the steps of parent selection, matching scheme formulation, parent shellfish optimization, spawning induction, artificial insemination, fry breeding and the like. Based on the salinity tolerance difference of sinonovacula constricta and sinonovacula constricta, an interspecific hybridization technology, a synchronous ovulation induction technology, an efficient artificial insemination technology and the like are comprehensively utilized, interspecific reproductive disorders are broken through, and high-yield and low-salt-tolerant sinonovacula constricta is cultivated through genome recombination of filial generations. Compared with common sinonovacula constricta species for production, the growth speed of the hybrid sinonovacula constricta is increased by 31.97%, and the half lethal salinity in 96 h is reduced from 3.03 to 1.11. The bred hybrid razor clams are excellent in production character and stable in heredity, razor clam germplasm resources are greatly enriched, the razor clam breeding area is widened, and the method has potential application value for saline-alkali soil ecological environment restoration.

Description

Technical Field

[0001] The invention relates to the technical field of marine aquaculture shellfish breeding, and in particular to a method for cultivating high-yield, low-salt-tolerant razor clams. Background Art

[0002] Sinonovacula constricta is one of the four traditional marine aquaculture shellfish in my country. It is mainly distributed in the middle and low intertidal zones of coastal inner bays and estuaries. It has a short breeding cycle, delicious taste and high market value. According to statistics, the annual output of razor clams in recent years is about 850,000 tons, and the output of Sinonovacula constricta accounts for more than 95% of razor clams. In the past 20 years, with the breakthrough of Sinonovacula constricta factory seedling technology and the maturity of fully artificial breeding technology, the aquaculture industry has shown a rapid development trend. The breeding area has rapidly expanded from the coastal areas of Zhejiang and Fujian to the north and south coasts of Jiangsu, Shandong, Liaoning, Guangdong, Guangxi, etc., and the development prospects are very broad. However, in low-salinity estuary areas, Sinonovacula constricta are prone to slow growth, low survival rate, and long breeding cycle due to their intolerance to low salt. In addition, the salinity of seawater has decreased due to summer typhoons and heavy rains in the southeast coast, which has led to large-scale deaths of Sinonovacula constricta from time to time. Therefore, under the new aquaculture industry layout, the razor clam aquaculture industry urgently needs new germplasms that grow fast and tolerate low salt.

[0003] Sinonovaculatus (Sinonovacularivularis) is naturally distributed in the Minjiang River estuary in Fujian. It is similar to Sinonovaculatus constricta, but has significant differences in sperm morphology and genetic genes. "A New Species of Sinonovaculatus" written by Huang Rui et al. mentioned that Sinonovaculatus was officially established as a new species of Sinonovaculatus in 2002. Compared with Sinonovaculatus constricta, Sinonovaculatus has the advantages of strong tolerance to low salt (specific gravity 1.001-1.015, salinity 2-19) and fast growth rate, but Sinonovaculatus will die quickly in a high-salt environment, which limits the breeding area of ​​Sinonovaculatus ("Aquatic Science and Technology Information" 2010 No. 37, Volume 5, Huang Rui et al., "Preliminary Experiment on the Effect of Environmental Factors on the Survival Rate and Growth of Sinonovaculatus Juveniles"). At present, the artificial seedling technology of the Yangtze razor clam has been broken through, and large-scale breeding has been promoted in Fujian, Tianjin, and Binzhou, Shandong estuary waters. It has also been introduced to saline-alkali ponds in Xinxiang, Henan, Lanzhou, Gansu, Yinchuan, Ningxia, etc. for breeding, all of which have achieved good results. In view of the complementary characteristics of the two species in terms of growth and salt tolerance, the present invention conducts interspecific hybridization of the two species in order to obtain a new variety with fast growth and strong low-salt tolerance.

[0004] Compared with traditional selective breeding, interspecific hybridization has the advantages of rich sources of genetic variation and fast seed production. Through hybridization, the hybrid advantages obtained can be directly used to develop new varieties or provide intermediate materials for breeding. Interspecific hybridization has played a huge role in increasing the growth rate of hybrid offspring, improving meat quality, and enhancing stress resistance, and is an important means of genetic improvement of aquatic animals. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for cultivating high-yield and low-salt-tolerant razor clams to fill the gap in the existing technical field. The method of the present invention for rapidly cultivating high-yield and low-salt-tolerant razor clams by interspecific hybridization of Sinonovacula constricta and Sinonovacula ovata can solve the problems that the current Sinonovacula constricta are prone to slow growth, low survival rate and long breeding cycle due to their intolerance to low salt.

[0006] In order to solve the above technical problems, the present invention provides a method for cultivating high-yield and low-salt-tolerant razor clams, comprising the following steps:

[0007] S1: Parent selection: Taking growth traits as breeding indicators, select Sinonovacula constricta and Sinonovacula ovata breeding lines / varieties as the source of hybrid parent populations;

[0008] S2: Formulation of mating plan: Using the male Sinonovacula constricta and the female Sinonovacula ovata in S1 as hybrid parents, the male and female parents are intensively cultivated in ponds of the same region with suitable salinity, and the temperature and other breeding environments are controlled to be consistent, so as to regulate the synchronous development and maturation of the gonads of the male and female broodstock;

[0009] S3: Selection of broodstock: Select individuals of Sinonovacula constricta and Sinonovacula ovata with excellent growth traits as the preferred broodstock; to ensure the purity of the germplasm, use PCR-RFLP technology to conduct molecular genetic identification.

[0010] S4: Inducing labor: using a combination of physical and chemical methods such as shade drying, running water stimulation, and injection of 5-hydroxytryptamine into the axe foot to induce labor on the preferred parent shellfish in step S3;

[0011] S5: Artificial insemination: extracting sperm and eggs after the induction of labor in step S4, performing artificial insemination, and transferring the embryos to an incubation bucket for incubation after more than 20% of the fertilized eggs have two cells, and the incubation process is oxygenated by bottom aeration;

[0012] S6: Seedling cultivation: According to the conventional seedling cultivation method, the hybrid offspring seedlings of female Omi razor clam × female Sinonovacula constricta can be obtained, and the seedlings of high-yield and low-salt resistant razor clam can be obtained.

[0013] In the step S5, after the artificial insemination is completed, an oxygenation step is also included. After 20% of the fertilized eggs have two cells, the embryos are transferred to the hatching bucket for hatching, and oxygenation is performed by bottom aeration during the hatching process. During the hatching process, stirring and aeration are continuously performed to prevent hypoxia caused by excessive density due to the sinking of the fertilized eggs. Through the above embodiment, after adding sperm to the fertilization container, stirring and aeration are continuously performed to improve the hybrid fertilization rate and reduce interspecific reproductive barriers, so as to prevent hypoxia caused by excessive density due to the sinking of the fertilized eggs, thereby improving the breeding efficiency.

[0014] Compared with the prior art, the high-yield low-salt-tolerant razor clam cultivation method of the present invention has the following advantages: in view of the complementary traits of Sinonovacula constricta and Sinonovacula nervosa in growth, salt tolerance and other aspects, the present invention performs interspecific hybridization on the two species in order to obtain a new variety with fast growth and strong low-salt tolerance. The present invention develops a method for rapidly cultivating low-salt-tolerant razor clams by constructing a Sinonovacula nervosa (♀)×Sinonovacula constricta (♂) hybrid system, and the hybrid F1 generation shows obvious low-salt tolerance and growth advantages. Compared with the ordinary production seed Sinonovacula constricta, the growth rate of the hybrid razor clam is greatly improved, and the 96h semi-lethal salinity is also greatly reduced. In addition, the hybrid razor clam obtained by the cultivation method of the present invention has excellent phenotype and genetic stability, enriches the germplasm resources of razor clams, broadens the razor clam breeding area, and also has potential application value for the restoration of saline-alkali land ecological environment.

[0015] In one possible embodiment, in step S1, the hybrid parent population is derived from a genetically improved preferred population with fast growth characteristics and stable inheritance, including breeding with growth traits such as shell length or weight as the breeding target, with a selection pressure of no more than 10%, and obtaining a preferred population of Sinonovacula constricta and Sinonovacula ovata after 3 to 4 consecutive generations of genetic improvement.

[0016] Through the above possible implementation methods, the present invention optimizes the selection of hybrid parent groups, lays a foundation for subsequent cultivation and breeding, and improves the stability of hybridization.

[0017] In a possible embodiment, in step S2, the enhanced cultivation step includes: intensively cultivating the female and male parents in ponds of the same area with suitable salinity, respectively, and controlling the temperature and other breeding environments to be consistent, so as to regulate the synchronous development and maturation of the gonads of the female and male broodstock.

[0018] In a possible embodiment, in step S2, the conditions for enhanced cultivation are: the salinity for cultivating Sinonovacula constricta is 15-30, and the salinity for cultivating Sinonovacula ovata is 3-10, and the breeding environment is consistent including temperature, bait, and breeding density, so that the nutritional accumulation, effective accumulated temperature, etc. of the parents are consistent, thereby regulating the synchronous development and maturation of the gonads.

[0019] Through the above possible implementation methods, the present invention further optimizes the cultivation conditions of Sinonovacula constricta and Sinonovacula ovata, so that the nutritional accumulation, effective accumulated temperature, etc. of the parents are consistent during the cultivation process, thereby further regulating the synchronous development and maturation of the gonads and improving the stability of the cultivation.

[0020] In a possible implementation, in step S3, the preferred broodstock include Sinonovacula constricta and Sinonovacula ovalis individuals that are large, have strong water tube expansion and contraction, and have full gonads.

[0021] In a possible embodiment, in step S3, after the preferred parent shellfish is selected, the molecular genetic identification is also carried out using PCR-RFLP technology, including: extracting DNA from the water pipe tissue of the parent shellfish, and then amplifying the DNA by PCR with COI universal primers, and then digesting it with restriction endonuclease PCIL. If the electrophoresis result of the PCR product after digestion shows that there is a band near 230bp and 420bp, the sample to be tested is Sinonovacula ovata; otherwise, if there is only one band after digestion and the size is consistent with the PCR product band before digestion, the sample to be tested is Sinonovacula constricta.

[0022] In a possible implementation, the sequences of the COI upstream primer COI-F and the downstream primer COI-R are respectively:

[0023] COI-F: 5'-GGTCAACAAATCATAAAGATATTGG-3';

[0024] COI-R: 5'-TAAACTTCAGGGTGACCAAAAAATCA-3'.

[0025] In a possible embodiment, in step S4, the step of inducing labor includes: inducing labor by combining physical and chemical methods, wherein the pre-inducing labor time is from 15:00 to 17:00 in the afternoon, firstly, the parent shellfish are dried in the shade for 3 hours, and then stimulated with running water for 1 to 2 hours; after dark, 2 to 5 mL of 0.02 mM 5-hydroxytryptamine is injected into the parent's axolotl tissue to induce labor individually, and individuals with an egg-laying capacity of not less than 200,000 and good sperm motility are selected as candidate parent shellfish.

[0026] In a possible implementation, in step S5, the step of inducing labor includes: the step of performing artificial insemination includes: using a microscope to accurately identify high-quality, uncontaminated sperm and eggs, and selecting more than 50 female razor clams and male razor clams for artificial insemination; adding razor clams to the razor clam eggs for fertilization, and controlling the sperm:egg ratio at (10-20):1. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The electrophoresis diagrams of COIPCR products and PCR-RFLP are shown in Figure 1. The left diagram is the COIPCR product diagram, and the right diagram is the PCR-RFLP diagram. Figure 1 In the figure, lanes 1 to 4 are samples of Sinonovacula constricta; lanes 5 to 8 are samples of Sinonovacula ovata; and M is the DNA molecular weight standard DL2000. DETAILED DESCRIPTION

[0028] First, those skilled in the art should understand that these implementations are only used to explain the technical principles of the embodiments of the present application, and are not intended to limit the protection scope of the embodiments of the present application. Those skilled in the art can make adjustments to them as needed to adapt to specific application scenarios.

[0029] The present invention provides a method for cultivating high-yield and low-salt-tolerant razor clams, comprising the following steps:

[0030] S1: Parent selection: Taking growth traits as breeding indicators, select Sinonovacula constricta and Sinonovacula ovata breeding lines / varieties as the source of hybrid parent populations;

[0031] S2: Formulation of mating plan: Using the male Sinonovacula constricta and the female Sinonovacula ovata in S1 as hybrid parents, the male and female parents are intensively cultivated in ponds of the same region with suitable salinity, and the temperature and other breeding environments are controlled to be consistent, so as to regulate the synchronous development and maturation of the gonads of the male and female broodstock;

[0032] S3: Selection of broodstock: Select individuals of Sinonovacula constricta and Sinonovacula ovata with excellent growth traits as the preferred broodstock; to ensure the purity of the germplasm, use PCR-RFLP technology to conduct molecular genetic identification.

[0033] S4: Inducing labor: using a combination of physical and chemical methods such as shade drying, running water stimulation, and injection of 5-hydroxytryptamine into the axe foot to induce labor on the preferred parent shellfish in step S3;

[0034] S5: Artificial insemination: extracting sperm and eggs after the induction of labor in step S4, performing artificial insemination, and transferring the embryos to an incubation bucket for incubation after more than 20% of the fertilized eggs have two cells, and the incubation process is oxygenated by bottom aeration;

[0035] S6: Seedling cultivation: According to the conventional seedling cultivation method, the hybrid offspring seedlings of female Omi razor clam × female Sinonovacula constricta can be obtained, and the seedlings of high-yield and low-salt resistant razor clam can be obtained.

[0036] As a preferred scheme, in step S1, the source of the hybrid parent population is a preferred population that has been genetically improved and has fast growth characteristics and stable inheritance, including taking growth traits such as shell length or body weight as breeding targets, and breeding with a selection pressure of no more than 10%, and obtaining a preferred population of Sinonovacula constricta and Sinonovacula ovata after 3 to 4 consecutive generations of genetic improvement.

[0037] As a preferred scheme, in step S2, the conditions for enhanced cultivation are: the salinity for cultivating Sinonovacula constricta is 15-30, and the salinity for cultivating Sinonovacula ovata is 3-10, and the breeding environment is consistent including temperature, bait, and breeding density, so that the nutritional accumulation, effective accumulated temperature, etc. of the parents are consistent, thereby regulating the synchronous development and maturation of the gonads.

[0038] As a preferred solution, in step S3, the preferred broodstock include Sinonovacula constricta and Sinonovacula ovale individuals that are large, have strong water tube expansion and contraction, and have full gonads.

[0039] As a preferred scheme, in step S3, after the preferred parent shellfish is selected, it also includes using PCR-RFLP technology to perform molecular genetic identification, including: extracting DNA from the water pipe tissue of the parent shellfish, then amplifying the DNA by PCR with COI universal primers, and then digesting it with restriction endonuclease PCIL. If the electrophoresis results of the PCR product after digestion show that there is a band near 230bp and 420bp, respectively, the sample to be tested is Sinonovacula ovata; otherwise, if there is only one band after digestion and the size is consistent with the PCR product band before digestion, the sample to be tested is Sinonovacula constricta.

[0040] As a preferred solution, the sequences of the COI upstream primer COI-F and the downstream primer COI-R are respectively:

[0041] COI-F: 5'-GGTCAACAAATCATAAAGATATTGG-3';

[0042] COI-R: 5'-TAAACTTCAGGGTGACCAAAAAATCA-3'.

[0043] As a preferred solution, in step S4, the step of inducing labor includes: inducing labor by combining physical and chemical methods, the purpose of which is to increase the ovulation rate and ovulation volume. The pre-inducing labor time is from 15:00 to 17:00 in the afternoon. First, the parent shellfish is dried in the shade for 3 hours, and then stimulated with running water for 1 to 2 hours; after dark, 2 to 5 mL of 0.02 mM 5-hydroxytryptamine is injected into the parent's axolotl tissue to induce labor individually, and individuals with an egg-laying capacity of not less than 200,000 and good sperm motility are selected as candidate parent shellfish.

[0044] As a preferred solution, in step S5, the step of artificial insemination includes: using a microscope to accurately identify high-quality, uncontaminated sperm and eggs, and selecting more than 50 female razor clams and male razor clams for artificial insemination; adding razor clams to the razor clam eggs for fertilization, and controlling the sperm:egg ratio at (10-20):1.

[0045] The following provides an embodiment in combination with the above data range to further explain the above technical solution of the present invention:

[0046] Embodiment 1:

[0047] This embodiment provides a method for cultivating high-yield low-salt resistant clams, comprising the following steps:

[0048] S1. Parent selection: The selected lines / varieties of Sinonovacula constricta and Sinonovacula nervosa obtained by optimizing the growth traits for 4 generations are used as the source of the hybrid parent population; the selection of the Sinonovacula constricta and Sinonovacula nervosa parents is a preferred population with rapid growth characteristics and stable inheritance after genetic improvement, specifically, the growth traits such as shell length or body weight are selected as the breeding target, and the selection pressure does not exceed 10%, and the preferred population of Sinonovacula constricta and Sinonovacula nervosa is obtained after 4 consecutive generations of genetic improvement;

[0049] S2. Formulation of mating plan: The male Sinonovacula constricta (♂) and the female Sinonovacula nerii (♀) in S1 are used as hybrid parents, and the male and female parents are intensively cultivated in ponds of the same region with suitable salinity, and the temperature and other breeding environments are controlled to be consistent, so as to regulate the synchronous development and maturation of the gonads of the female and male broodstock. The suitable breeding salinity for Sinonovacula constricta is 25, and that for Sinonovacula nerii is 5. The breeding environment includes temperature, bait, breeding density, etc., so as to make the nutrient accumulation, effective accumulated temperature, etc. of the parents consistent, and regulate the synchronous development and maturation of the gonads.

[0050] S3, parent shellfish selection: select large individuals, strong water tube expansion, and full gonads of Sinonovacula constricta and Sinonovacula nervosa individuals as preferred parent shellfish; to ensure the purity of the germplasm, use molecular genetic identification technology to carry out molecular genetic identification, and the molecular genetic identification method is to use PCR-RFLP technology to identify Sinonovacula constricta and Sinonovacula nervosa parent shellfish, specifically including: extracting parent shellfish water tube tissue DNA, and then amplifying the DNA by PCR with COI universal primers, and then digesting it with restriction endonuclease PCIL. If the electrophoresis results after digestion of the PCR product show that there is a band of about 230bp and 420bp respectively, the sample to be tested is Sinonovacula nervosa; on the contrary, if there is only one band after digestion and it is consistent with the size of the PCR product (before digestion) band (about 650bp), the sample to be tested is Sinonovacula constricta. The sequences of the COI upstream primer COI-F and the downstream primer COI-R are respectively:

[0051] COI-F: 5'-GGTCAACAAATCATAAAGATATTGG-3';

[0052] COI-R: 5'-TAAACTTCAGGGTGACCAAAAAATCA-3'.

[0053] S4, inducing labor: using a combination of physical and chemical methods such as shade drying, running water stimulation, and injection of 5-hydroxytryptamine into the axe foot, respectively, the selected parent individuals of Sinonovacula constricta and Sinonovacula nervosa in S3 were induced to labor; the purpose of the physical and chemical combined induction method is to increase the ovulation rate and the amount of ovulation. The pre-induction time is 16:00 in the afternoon. First, the parent shellfish are shade dried for 3 hours, and then stimulated with running water for 1.5 hours; after dark, 3.5mL of 5-hydroxytryptamine (0.02mM) is injected into the parent axe foot tissue for single induction of labor, and individuals with an egg production of not less than 200,000 and good sperm motility are selected as candidate parent shellfish.

[0054] S5. Artificial insemination: Use a microscope to accurately identify high-quality, uncontaminated sperm and eggs, and select more than 50 female Omi clam egg fluids and male Sinonovacula constricta semen for artificial insemination. Add Sinonovacula constricta semen to Omi clam egg fluid for fertilization, and control the sperm:egg ratio at 15:1; after artificial insemination, when 20% of the fertilized eggs have two cells, transfer the embryos to the hatching bucket for incubation, and use bottom aeration to increase oxygen during the incubation process; after adding sperm to the fertilization container, in order to increase the hybrid fertilization rate and reduce interspecies reproductive barriers, the fertilization time is extended to 20% of the fertilized eggs to develop into two cells, and the incubation period is continuously stirred and aerated to prevent hypoxia caused by sinking of fertilized eggs due to high density.

[0055] S6. Seedling cultivation: The hybrid progeny seedlings of Sinonovacula ovata (♀) and Sinonovacula constricta (♂) can be obtained by conventional seedling cultivation methods.

[0056] Embodiment 2:

[0057] This embodiment provides a method for cultivating high-yield low-salt resistant clams, comprising the following steps:

[0058] S1. Parent selection: The selected lines / varieties of Sinonovacula constricta and Sinonovacula nervosa obtained by optimizing the growth traits for three generations are used as the source of the hybrid parent population; the selection of the Sinonovacula constricta and Sinonovacula nervosa parents is a preferred population with rapid growth characteristics and stable inheritance after genetic improvement, specifically, the growth traits such as shell length or body weight are selected as the breeding target, and the selection pressure does not exceed 10%, and the preferred population of Sinonovacula constricta and Sinonovacula nervosa is obtained after three consecutive generations of genetic improvement;

[0059] S2. Formulation of mating plan: The male Sinonovacula constricta (♂) and the female Sinonovacula nerii (♀) in S1 are used as hybrid parents, and the male and female parents are intensively cultivated in ponds of the same region with suitable salinity, and the temperature and other breeding environments are controlled to be consistent, so as to regulate the synchronous development and maturation of the gonads of the female and male broodstock. The suitable breeding salinity for Sinonovacula constricta is 15, and that for Sinonovacula nerii is 3. The breeding environment includes temperature, bait, breeding density, etc., so as to make the nutrient accumulation, effective accumulated temperature, etc. of the parents consistent, and regulate the synchronous development and maturation of the gonads.

[0060] S3, parent shellfish selection: select large individuals, strong water tube expansion, and full gonads of Sinonovacula constricta and Sinonovacula nervosa individuals as preferred parent shellfish; to ensure the purity of the germplasm, use molecular genetic identification technology to carry out molecular genetic identification, and the molecular genetic identification method is to use PCR-RFLP technology to identify Sinonovacula constricta and Sinonovacula nervosa parent shellfish, specifically including: extracting parent shellfish water tube tissue DNA, and then amplifying the DNA by PCR with COI universal primers, and then digesting it with restriction endonuclease PCIL. If the electrophoresis results after digestion of the PCR product show that there is a band of about 230bp and 420bp respectively, the sample to be tested is Sinonovacula nervosa; on the contrary, if there is only one band after digestion and it is consistent with the size of the PCR product (before digestion) band (about 650bp), the sample to be tested is Sinonovacula constricta. The sequences of the COI upstream primer COI-F and the downstream primer COI-R are respectively:

[0061] COI-F: 5'-GGTCAACAAATCATAAAGATATTGG-3';

[0062] COI-R: 5'-TAAACTTCAGGGTGACCAAAAAATCA-3'.

[0063] S4, inducing labor: using a combination of physical and chemical methods such as shade drying, running water stimulation, and injection of 5-hydroxytryptamine into the axe foot, respectively, the selected parent individuals of Sinonovacula constricta and Sinonovacula nervosa in S3 were induced to labor; the purpose of the physical and chemical induction method was to increase the ovulation rate and the amount of ovulation. The pre-induction time was 15:00 in the afternoon. First, the parent shellfish were shade dried for 3 hours, and then stimulated with running water for 1 hour; after dark, 2mL of 5-hydroxytryptamine (0.02mM) was injected into the parent axe foot tissue for single induction of labor, and individuals with an egg production of not less than 200,000 and good sperm motility were selected as candidate parent shellfish.

[0064] S5. Artificial insemination: Use a microscope to accurately identify high-quality, uncontaminated sperm and eggs, and select more than 50 female Omi clam egg fluids and male Sinonovacula constricta semen for artificial insemination. Add Sinonovacula constricta semen to Omi clam egg fluid for fertilization, and control the sperm:egg ratio at 10:1; after artificial insemination, when 20% of the fertilized eggs have two cells, transfer the embryos to the hatching bucket for hatching, and use bottom aeration to increase oxygen during the hatching process; after adding sperm to the fertilization container, in order to increase the hybrid fertilization rate and reduce interspecies reproductive barriers, the fertilization time is extended to 20% of the fertilized eggs to develop into two cells, and the incubation period is continuously stirred and aerated to prevent hypoxia caused by sinking of the fertilized eggs due to high density.

[0065] S6. Seedling cultivation: The hybrid progeny seedlings of Sinonovacula ovata (♀) × Sinonovacula constricta (♂) can be obtained by conventional seedling cultivation methods, and the low-salt tolerance evaluation and productivity comparison test of the hybrid clams can be carried out.

[0066] Embodiment 3:

[0067] This embodiment provides a method for cultivating high-yield low-salt resistant clams, comprising the following steps:

[0068] S1. Parent selection: The selected lines / varieties of Sinonovacula constricta and Sinonovacula nervosa obtained by optimizing the growth traits for 4 generations are used as the source of the hybrid parent population; the selection of the Sinonovacula constricta and Sinonovacula nervosa parents is a preferred population with rapid growth characteristics and stable inheritance after genetic improvement, specifically, the growth traits such as shell length or body weight are selected as the breeding target, and the selection pressure does not exceed 10%, and the preferred population of Sinonovacula constricta and Sinonovacula nervosa is obtained after 4 consecutive generations of genetic improvement;

[0069] S2. Formulation of mating plan: The male Sinonovacula constricta (♂) and the female Sinonovacula nerii (♀) in S1 are used as hybrid parents, and the male and female parents are intensively cultivated in ponds of the same region with suitable salinity, and the temperature and other breeding environments are controlled to be consistent, so as to regulate the synchronous development and maturation of the gonads of the female and male broodstock. The suitable breeding salinity for Sinonovacula constricta is 30, and that for Sinonovacula nerii is 10. The breeding environment includes temperature, bait, breeding density, etc., so as to make the nutrient accumulation, effective accumulated temperature, etc. of the parents consistent, and regulate the synchronous development and maturation of the gonads.

[0070] S3, parent shellfish selection: select large individuals, strong water tube expansion, and full gonads of Sinonovacula constricta and Sinonovacula nervosa individuals as preferred parent shellfish; to ensure the purity of the germplasm, use molecular genetic identification technology to carry out molecular genetic identification, and the molecular genetic identification method is to use PCR-RFLP technology to identify Sinonovacula constricta and Sinonovacula nervosa parent shellfish, specifically including: extracting parent shellfish water tube tissue DNA, and then amplifying the DNA by PCR with COI universal primers, and then digesting it with restriction endonuclease PCIL. If the electrophoresis results after digestion of the PCR product show that there is a band of about 230bp and 420bp respectively, the sample to be tested is Sinonovacula nervosa; on the contrary, if there is only one band after digestion and it is consistent with the size of the PCR product (before digestion) band (about 650bp), the sample to be tested is Sinonovacula constricta. The sequences of the COI upstream primer COI-F and the downstream primer COI-R are respectively:

[0071] COI-F: 5'-GGTCAACAAATCATAAAGATATTGG-3';

[0072] COI-R: 5'-TAAACTTCAGGGTGACCAAAAAATCA-3'.

[0073] S4, inducing labor: using a combination of physical and chemical methods such as shade drying, running water stimulation, and injection of 5-hydroxytryptamine into the axe foot, respectively, the selected parent individuals of Sinonovacula constricta and Sinonovacula nervosa in S3 were induced to labor; the purpose of the physical and chemical combined induction method was to increase the ovulation rate and the amount of ovulation. The pre-induction time was 17:00 in the afternoon. First, the parent shellfish were shade dried for 3 hours, and then stimulated with running water for 2 hours; after dark, 5mL of 5-hydroxytryptamine (0.02mM) was injected into the parent axe foot tissue for single induction of labor, and individuals with an egg production of not less than 200,000 and good sperm motility were selected as candidate parent shellfish.

[0074] S5. Artificial insemination: Use a microscope to accurately identify high-quality, uncontaminated sperm and eggs, and select more than 50 female Omi clam egg fluids and male Sinonovacula constricta semen for artificial insemination. Add Sinonovacula constricta semen to Omi clam egg fluid for fertilization, and control the sperm:egg ratio at 20:1; after artificial insemination, when 20% of the fertilized eggs have two cells, transfer the embryos to the hatching bucket for hatching, and use bottom aeration to increase oxygen during the hatching process; after adding sperm to the fertilization container, in order to increase the hybrid fertilization rate and reduce interspecies reproductive barriers, the fertilization time is extended to 20% of the fertilized eggs to develop into two cells, and the incubation period is continuously stirred and aerated to prevent hypoxia caused by the sinking of fertilized eggs due to high density.

[0075] S6. Seedling cultivation: The hybrid progeny seedlings of Sinonovacula ovata (♀) × Sinonovacula constricta (♂) can be obtained by conventional seedling cultivation methods, and the low-salt tolerance evaluation and productivity comparison test of the hybrid clams can be carried out.

[0076] The following provides a method for cultivating high-yield, low-salt-tolerant razor clams in combination with specific selected species, cultivation dates, and experimental conditions to further illustrate the above embodiments of the present invention:

[0077] Embodiment 4:

[0078] This embodiment provides a method for cultivating high-yield low-salt resistant clams, comprising the following steps:

[0079] S1. Parent selection: In 2012, the wild population of Sinonovacula constricta in Changle, Fujian was used as the base population, and body weight and low-salt tolerance were used as breeding traits. Four generations of population breeding were carried out, and in 2019, a new high-yield and low-salt-tolerant variety of Sinonovacula constricta "Yongle No. 1" was bred; in 2019, the wild population of Jinjiang razor clams in Zhangzhou, Fujian was used as the base population, and body weight was used as the breeding target. By 2022, the G3 generation of the Jinjiang razor clam fast-growing strain was obtained.

[0080] S2. Formulation of mating plan: Select male individuals of the "Yongle No. 1" variety of Sinonovacula constricta (♀) and female individuals of the G3 generation of Sinonovacula nervosa (♂) as hybrid parents. The hybridization method of group hybridization can improve the genetic diversity of the first generation, and at the same time construct self-propagating populations of Sinonovacula constricta and Sinonovacula nervosa.

[0081] S3. Selection of broodstock: On October 1, 2022, the new variety of Sinonovacula constricta "Yongle No. 1" and the G3 generation broodstock of the fast-growing strain of Sinonovacula nervosa were collected at the multi-model base of seawater pond aquaculture of Ningbo Institute of Oceanography and Fisheries. 15 kg of parent groups with strong retractable water tubes, large individuals and full gonads were selected, and molecular genetic identification of Sinonovacula constricta and Sinonovacula nervosa parents was carried out using PCR-RFLP technology.

[0082] S4. Induction of spawning: After drying in the shade for 3 hours in the afternoon, the broodstock of Sinonovacula constricta and Sinonovacula ovalis were stimulated with running water for 2 hours at 16:00 for pre-induction of spawning. At 18:00, a chemical method of injecting 5-hydroxytryptamine into the axe foot was used to induce spawning of the selected broodstock individually. After microscopic examination of sperm and eggs for contamination, a total of 145 sperms of Sinonovacula constricta and 159 eggs of Sinonovacula ovalis were collected.

[0083] S5, artificial insemination: collect sperm of Sinonovacula constricta and eggs of Sinonovacula ovata in S4 into 30L plastic buckets for artificial insemination, and the sperm:egg ratio should be controlled at 10:1-20:1; after artificial insemination, when 20% of the fertilized eggs have two cells, transfer all the fertilized eggs to a 500L hatching bucket and use bottom aeration for oxygenation. During the period of induction of spawning, fertilization and hatching, the temperature is controlled at 22-24℃ and the salinity is controlled at 9-12.

[0084] S6. Seed cultivation: The 19 million D-shaped larvae of Sinonovacula ovata (♀) × Sinonovacula constricta (♂) obtained in S5 were cultured in seawater with a salinity of 9 to 12, and intermediate cultivation was performed in seawater with a salinity of 12 to 20.

[0085] Calculation of half-lethal salinity at low salt end:

[0086] The half-lethal salinity of hybrid razor clams, Sinonovacula constricta and Omi River razor clams was determined by designing five salinity gradients (0.40, 0.77, 1.52, 2.16, 3.00) using the equal logarithmic interval method. The experiment lasted for 96 hours, the water temperature was 22-23℃, and the mortality was recorded every 12 hours after the start of the experiment (Table 1). The Probit regression calculation results based on the summary data showed that the half-lethal low salt of hybrid razor clams (Omi River razor clams♀× Sinonovacula constricta♂) at 96 hours was 1.11, that of Sinonovacula constricta was 3.03, and that of Omi River razor clams was 0.47.

[0087] Table 1 Average survival rates of hybrid clams (Rn1a natans♀×Rn1a constricta♂), Rn1a natans and Rn1a constricta at different salinities for 96 h

[0088] salinity Hybrid razor clam Sinonovacula constricta Omi razor clam 0.40 0.00±0.00 0.00±0.00 43.33±2.35 0.77 42.86±1.83 0.00±0.00 60.00±3.64 1.52 62.50±2.64 21.80±2.94 90.62±2.65 2.16 90.21±3.04 30.00±2.51 100.00±0.00 3.00 100.00±0.00 43.75±3.66 100.00±0.00

[0089] Test on growth characteristics of hybrid razor clams in low-salt environment:

[0090] From April 2023 to February 2024, the low-salt tolerance growth performance of hybrid razor clams, Sinonovacula constricta and Omijima razor clams was tested under pond culture conditions of salinity 5.00±1.25, 12.00±2.53 and 20.00±3.00, respectively. The results showed that the Omijima razor clam (♀) × Sinonovacula constricta (♂) hybrid group showed obvious growth advantages in different low-salt environments. Compared with Sinonovacula constricta, the shell length and body weight of the hybrid group increased by an average of 11.55% and 31.97%; compared with Omijima razor clams, the shell length and body weight of the hybrid group increased by an average of 17.39% and 37.14%.

[0091] Table 2 Growth indices of hybrid clams (Rn1a natans♀×Rn1a constricta♂), Rn1a natans and Rn1a constricta at different salinities

[0092]

[0093] Through further verification of the above embodiments, it is also explained that the present invention provides a method for cultivating high-yield and low-salt-tolerant razor clams based on the difference in salinity tolerance between Sinonovacula constricta and Sinonovacula nervosa, comprehensively utilizing interspecific hybridization technology, synchronous ovulation induction technology, efficient artificial insemination technology, etc., breaking through interspecific reproductive barriers, and cultivating high-yield and low-salt-tolerant razor clams through hybrid offspring genome recombination. Compared with the ordinary production Sinonovacula constricta species, the growth rate of hybrid razor clams increased by 31.97%, and the 96h half-lethal salinity was reduced from 3.03 to 1.11, solving the problems existing in the background technology. In addition, the hybrid razor clams cultivated by the present invention have excellent production traits and genetic stability, which greatly enriches the germplasm resources of razor clams, broadens the razor clam breeding area, and also has potential application value for the restoration of saline-alkali land ecological environment.

[0094] In the description of the embodiments of the present application, it should be noted that in the description of the present application, terms such as "inside" and "outside" indicating directions or positional relationships are based on the directions or positional relationships shown in the drawings. This is only for the convenience of description, and does not indicate or imply that the device or component must have a specific orientation, be constructed and operated in a specific orientation. Therefore, it cannot be understood as a limitation on the present application.

[0095] In the description of the present application, the description with reference to the terms "one embodiment", "some embodiments", "in the present embodiment", "specific example", or "some examples" etc. means that the specific features, mechanisms, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, mechanisms, materials or characteristics described may be combined in a suitable manner in any one or more embodiments or examples. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0096] The above is only a specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed in the present application should be included in the protection scope of the present application. Therefore, the protection scope of the present application shall be based on the protection scope of the claims.

Claims

1. A method for cultivating high-yield, low-salt-tolerant razor clams, characterized in that: The following steps are involved: S1: Parent selection: Taking growth traits as breeding indicators, select Sinonovacula constricta and Sinonovacula ovata breeding lines / varieties as the source of hybrid parent populations; S2: Formulation of mating plan: Using the male Sinonovacula constricta and the female Sinonovacula ovata in S1 as hybrid parents, the male and female parents are intensively cultivated in ponds of the same region with suitable salinity, and the temperature and other breeding environments are controlled to be consistent, so as to regulate the synchronous development and maturation of the gonads of the male and female broodstock; S3: Selection of broodstock: Select individuals of Sinonovacula constricta and Sinonovacula ovata with excellent growth traits as the preferred broodstock; to ensure the purity of the germplasm, use PCR-RFLP technology to conduct molecular genetic identification. S4: Inducing labor: using a combination of physical and chemical methods such as shade drying, running water stimulation, and injection of 5-hydroxytryptamine into the axe foot to induce labor on the preferred parent shellfish in step S3; S5: Artificial insemination: extracting sperm and eggs after the induction of labor in step S4, performing artificial insemination, and transferring the embryos to an incubation bucket for incubation after more than 20% of the fertilized eggs have two cells, and the incubation process is oxygenated by bottom aeration; S6: Seedling cultivation: According to the conventional seedling cultivation method, the hybrid offspring seedlings of female Omi razor clam × female Sinonovacula constricta can be obtained, and the seedlings of high-yield and low-salt resistant razor clam can be obtained.

2. The method for cultivating high-yield and low-salt resistant razor clams according to claim 1, characterized in that: In step S1, the hybrid parent population is derived from a preferred population that has been genetically improved and has fast growth characteristics and stable inheritance, including taking growth traits such as shell length or body weight as breeding targets, and breeding with a selection pressure of no more than 10%, and obtaining a preferred population of Sinonovacula constricta and Sinonovacula ovata after 3 to 4 consecutive generations of genetic improvement.

3. The method for cultivating high-yield and low-salt resistant razor clams according to claim 2, characterized in that: In step S2, the conditions for enhanced cultivation are: the salinity for cultivating Sinonovacula constricta is 15-30, and the salinity for cultivating Sinonovacula ovata is 3-10. The breeding environment is consistent including temperature, bait, and breeding density, so that the nutritional accumulation, effective accumulated temperature, etc. of the parents are consistent, thereby regulating the synchronous development and maturation of the gonads.

4. The method for cultivating high-yield and low-salt resistant razor clams according to claim 1, characterized in that: In step S3, the preferred broodstock include Sinonovacula constricta and Sinonovacula ovale individuals that are large, have strong water tube expansion and contraction, and have full gonads.

5. The method for cultivating high-yield and low-salt resistant razor clams according to claim 1, characterized in that: In the step S3, after the preferred parent shellfish is selected, the molecular genetic identification is also carried out using PCR-RFLP technology, including: extracting DNA from the water pipe tissue of the parent shellfish, and then amplifying the DNA by PCR with COI universal primers, and then digesting it with restriction endonuclease PCIL. If the electrophoresis result of the PCR product after digestion shows that there is a band near 230bp and 420bp, the sample to be tested is Sinonovacula ovata; otherwise, if there is only one band after digestion and the size is consistent with the PCR product band before digestion, the sample to be tested is Sinonovacula constricta.

6. The method for cultivating high-yield and low-salt resistant razor clams according to claim 5, characterized in that: The sequences of the COI upstream primer COI-F and the downstream primer COI-R are respectively: COI-F: 5'-GGTCAACAAATCATAAAGATATTGG-3'; COI-R: 5'-TAAACTTCAGGGTGACCAAAAAATCA-3'.

7. The method for cultivating high-yield and low-salt resistant razor clams according to claim 1, characterized in that: In step S4, the step of inducing labor includes: inducing labor by combining physical and chemical methods, the purpose of which is to increase the ovulation rate and the amount of ovulation. The pre-inducing labor time is from 15:00 to 17:00 in the afternoon. First, the parent shellfish is dried in the shade for 3 hours, and then stimulated with running water for 1 to 2 hours; after dark, 2 to 5 mL of 0.02 mM 5-hydroxytryptamine is injected into the parent's axe foot tissue to induce labor individually, and individuals with an egg-laying capacity of not less than 200,000 and good sperm motility are selected as candidate parent shellfish.

8. The method for cultivating high-yield and low-salt resistant razor clams according to claim 1, characterized in that: In step S5, the step of artificial insemination includes: using a microscope to accurately identify high-quality, uncontaminated sperm and eggs, selecting more than 50 female razor clams and male razor clams for artificial insemination; adding razor clams to the razor clam eggs for fertilization, and controlling the sperm:egg ratio at (10-20):1.

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