A method for cultivating fast-growing, low-salt-adaptable ternary hybrid triploid oysters

Abstract

The application firstly adopts selection breeding technology combined with ploidy breeding technology to construct and cultivate long oyster tetraploid population and Fujian oyster tetraploid population which are genetically stable, fast in growth speed and high in survival rate; constructs hybrid oyster tetraploid population (GGAA) through hybridization of long oyster tetraploid and Fujian oyster tetraploid; hybridizes near Jiang oyster diploid breeding population as a female parent and GGAA as a male parent to obtain "near Jiang oyster-long oyster-Fujian oyster" three-way hybrid triploid oyster (RGA). The cultivated three-way hybrid triploid oyster can keep fast growth, high survival rate and high stress resistance under the condition of low salt sea area (<20), and at the same time, shows a relatively high proportion of infertile individuals in the breeding season. The application is suitable for large-scale preparation of three-way hybrid triploid oyster seedlings, provides matching breeding technology of three-way hybrid triploid oyster suitable for estuary, gulf and other medium and low salinity breeding sea areas of oyster industry, and has good industrial application value.

Description

Technical Field

[0001] This invention belongs to the field of shellfish breeding technology, specifically relating to a method for cultivating triploid oysters that are fast-growing and highly adaptable to low salinity. Background Technology

[0002] In recent years, the integration and application of technologies such as polyploid breeding, hybridization breeding, and selective breeding have become important methods for the genetic improvement of shellfish. Among them, polyploid oysters, especially triploid oysters, occupy an important position in industrial breeding and production applications due to their characteristics such as rapid growth, extended market period, and relatively stable commercial traits. At the same time, interspecific hybridization can introduce tolerance traits and improve overall production performance through heterosis and genetic recombination, and is therefore widely used to improve key traits such as growth, survival, and stress resistance.

[0003] The oysters farmed along my country's coast are mainly of the genus *Crassostrea*. Among them, the giant oyster (*Crassostrea gigas*) is mainly distributed along the coast north of the Yangtze River, characterized by rapid growth, strong environmental adaptability, and high yield; the Fujian oyster (*Crassostrea angulata*) is mainly produced in coastal areas such as Fujian, and typically exhibits strong heat tolerance, maintaining relatively stable growth and survival even during hot seasons; the ark oyster (*Crassostrea ariakensis*) is a common giant oyster in my country's estuaries and nearshore brackish water areas, distributed in estuaries and bays significantly affected by freshwater runoff. It is relatively large, with thick flesh, and has strong tolerance to salinity fluctuations, maintaining relatively stable feeding and growth under low to medium salinity conditions.

[0004] Existing technologies still have limitations when facing aquaculture environments with low to medium salinity (< 20) and significant salinity fluctuations, such as brackish water in estuaries. Triploid or hybrid triploid oysters constructed primarily from Pacific oysters or Fujian oysters often exhibit certain production advantages in sea areas with normal salinity. However, in low-salinity or salinity-fluctuating environments, their growth advantages are difficult to maintain consistently. Furthermore, during the high-temperature summer period, they are prone to problems such as decreased survival rates and unstable stress resistance, making it difficult to meet the actual demand for "fast-growing + high survival rate" seedlings in low-salinity aquaculture areas.

[0005] Therefore, there is an urgent need to establish a scalable triploid oyster cultivation method that can achieve rapid growth and high survival rate during the high-temperature summer period in low-salinity (< 20) aquaculture environments such as brackish water in estuaries, thus providing triploid oysters and their supporting cultivation technologies that are more suitable for promotion and application in low-salinity marine aquaculture. Summary of the Invention

[0006] To overcome the defects and shortcomings of the traditional triploid oysters in my country described in the background art, this invention develops a method for cultivating triploid oysters that are fast-growing and highly adaptable to low salinity.

[0007] A method for cultivating triploid oysters that are fast-growing and highly adaptable to low salinity, characterized by comprising the following steps:

[0008] a. Using the diploid of the new cultivar "Haida No. 1" (GS-01-005-2013) as the base population, triploid of "Haida No. 1" oyster was induced to obtain the base population; using the triploid of "Haida No. 1" oyster as the female parent and the diploid of "Haida No. 1" oyster as the male parent, tetraploid of "Haida No. 1" oyster was induced to obtain the base population; the tetraploid population was continuously selected and bred for 2 to 3 generations to obtain the tetraploid breeding population of "Haida No. 1" oyster.

[0009] b. Using a wild diploid population of *Crassostrea gigas* as the base population, and with rapid growth as the breeding objective, 2-3 generations of selective breeding were conducted to obtain a selected diploid population of *Crassostrea gigas*. The selected diploid population of *Crassostrea gigas* was then used as the parent to induce the production of triploid *Crassostrea gigas*. A tetraploid base population of *Crassostrea gigas* was then induced using the triploid population as the female parent and the selected diploid population as the male parent. The tetraploid population was then subjected to 2-3 generations of selective breeding to obtain a selected tetraploid population of *Crassostrea gigas*.

[0010] c. Using the tetraploid oyster breeding population of the Pacific oyster as the female parent and the tetraploid oyster breeding population of the Fujian oyster as the male parent, hybridization was carried out to obtain a tetraploid oyster population of Pacific oyster and Fujian oyster, denoted as GGAA.

[0011] d. Using the wild diploid population of Crassula ovata as the base population, and with rapid growth as the breeding objective, the population was continuously bred for 2-3 generations to obtain a diploid breeding population of Crassula ovata. The diploid breeding population of Crassula ovata was used as the female parent and the tetraploid hybrid was used as the male parent to obtain a ternary hybrid triploid of Crassula ovata, Crassula longifolia and Crassula fukiensis, denoted as RGA.

[0012] Furthermore, the triploid and tetraploid induction in steps (a) and (b) includes: dissecting the parent shellfish and selecting no fewer than 50 males and 50 females for artificial insemination; after fertilization, observing under a microscope, and when 20% of the fertilized eggs show the first polar body, adding cytochalasin B at a concentration of 0.5 mg / L for 10-15 min; after treatment, soaking in seawater containing 1% dimethyl sulfoxide (DMSO) for 30 min and rinsing thoroughly with filtered seawater; using flow cytometry to detect the ploidy of the larvae, screening batches with an induction rate ≥80%, and using conventional methods for larval rearing and juvenile rearing.

[0013] Furthermore, in steps (a) and (b), when selecting individuals from the tetraploid population, the top 10% of individuals in terms of shell height and wet weight are selected as candidate parents; before fertilization, ploidy is determined, and individuals with a ploidy of 4N are selected as parents, with no fewer than 50 males and 50 females.

[0014] Furthermore, in steps (b) and (d), when selecting the diploid population, individuals in the top 10% of shell height and wet weight are selected as candidate parents; genetic identification is performed using COⅠ and ITS2 primers before fertilization to ensure that the parents used in step (b) are purebred Fujian oysters and the parents used in step (d) are purebred Ostridium pistemon; there are no fewer than 50 males and 50 females.

[0015] Furthermore, in step (c), the ploidy of the candidate parents is determined before fertilization, and individuals with a ploidy of 4N are selected as parents, with no fewer than 50 males and 50 females.

[0016] Furthermore, in step (d), individuals with well-developed gonads, uniform egg development, and deep yolk color are selected from the diploid oyster breeding population as candidate maternal parents, and individuals with vigorous sperm motility are selected from the tetraploid hybrid (GGAA) as candidate paternal parents; before fertilization, ploidy is determined using flow cytometry, and individuals with maternal ploidy of 2N and paternal ploidy of 4N are selected for hybridization; during artificial insemination, the seawater salinity is controlled at 20-25‰ to ensure that there are 10-15 sperm cells around each egg; larval rearing and juvenile rearing are carried out using conventional methods to obtain a fast-growing, low-salinity adapted ternary hybrid triploid oyster.

[0017] Beneficial effects of the present invention

[0018] The technical solution provided by this invention has the following advantages compared with known technologies.

[0019] (1) This invention constructs and selects tetraploid parent systems for both Pacific oyster and Fujian oyster, and obtains GGAA paternal populations through hybridization. This is beneficial to improving the genetic diversity and comprehensive production performance potential of the paternal parents, providing a stable genetic basis for the offspring of the three-way hybrids. (2) This invention uses growth as the breeding target and constructs a female parent system for Pacific oyster, ensuring the stability of the female parent in the rapid growth trait. This allows the three-way hybrid triploids to maintain high growth performance and improve stress resistance in low-salinity environments. (3) The three-way hybrid triploids obtained by this invention are suitable for aquaculture in low-salinity fluctuating sea areas such as estuaries and bays. While maintaining the triploid gonadal development inhibition and high sterility rate, they grow rapidly in low-salinity sea areas (about 20‰) and have a high survival rate in summer. This makes them suitable for the mass production of triploid seedlings and has good prospects for industrial application. Attached Figure Description

[0020] Figure 1This is a technical roadmap for the cultivation method of triploid oysters with rapid growth and strong adaptability to low salt conditions, as described in this invention.

[0021] Figure 2 Flow cytometry results of a selected population of Crassula ovata 'Omi'.

[0022] Figure 3 This is a flow cytometry image of a hybrid tetraploid population.

[0023] Figure 4 Flow cytometry results of triploid oysters that are fast-growing, adaptable to low salt conditions, and are a three-way hybrid. Detailed Implementation

[0024] The following examples further illustrate the cultivation method of a fast-growing, low-salt-adaptable triploid oyster according to the present invention, which is not intended to limit the present invention.

[0025] Example:

[0026] (1) Establishment of the tetraploid breeding population (GGGG) of the Pacific oyster “Haida No. 1”

[0027] a. Triploid Induction in the "Haida No. 1" Crassula 'Grass Oyster': Mature individuals from the diploid breeding population of "Haida No. 1" Crassula 'Grass Oyster' were dissected, and sexes were identified under a microscope. Sperm and eggs were collected separately. The sperm and eggs were filtered through a silk screen to remove impurities and tissue fluid, and then added to seawater for maturation for 30–60 minutes. During fertilization, approximately 5–10 sperm cells were controlled to surround each egg. When approximately 20% of the fertilized eggs showed the first polar body (approximately 15 minutes), 0.5 mg / L cytochalasin B (CB) was added for treatment for 15 minutes. After treatment, the eggs were soaked in seawater containing 1% dimethyl sulfoxide (DMSO) for 30 minutes, and then thoroughly rinsed with filtered seawater. Flow cytometry was used to detect ploidy. Batches with a triploid induction rate ≥80% were selected and further cultured until the eyespot larvae attached, then released into the sea for further growth, thus obtaining the "Haida No. 1" Crassula 'Grass Oyster' triploid population.

[0028] b. Tetraploid induction of Crassula 'Haida No. 1': Dissected triploid individuals of Crassula 'Haida No. 1' and selected mature female triploids as maternal parents; dissected diploid individuals of Crassula 'Haida No. 1' and selected male individuals with full gonads and strong sperm motility as paternal parents; the treatment, maturation and fertilization control of sperm and eggs were the same as (1)a. When about 20% of the fertilized eggs showed the first polar body (about 15 min), 0.5 mg / L cytochalasin B (CB) was added for 15 min; after treatment, the eggs were soaked in seawater containing 1% dimethyl sulfoxide (DMSO) for 30 min, and then rinsed thoroughly with filtered seawater. Flow cytometry was used to detect ploidy, and batches with a triploid induction rate ≥80% were selected for further cultivation until the eyespot larvae attached and released into the sea to grow, thus obtaining a tetraploid population of Crassula 'Haida No. 1'.

[0029] c. Breeding of tetraploid population of Crassula 'Haida No. 1': Using the tetraploid population of Crassula 'Haida No. 1' as the base population, select tetraploid individuals from the top 10% of shell height and wet weight as parents, with no fewer than 50 males and 50 females, and fertilize them at a sperm-to-egg ratio of 50:1. Continue the above process for three generations to obtain a tetraploid breeding population of Crassula 'Haida No. 1' with stable ploidy and growth advantage, denoted as GGGG.

[0030] (2) Establishment of the Fujian Oyster Tetraploid Breeding Population (AAAA)

[0031] a. Establishment of a diploid breeding population of Fujian oysters: Collect approximately 3,000 wild 1-year-old Fujian oysters. After removing individuals with deformed shells, damage, or poor vitality, retain approximately 1,500 individuals with uniform shell shapes. Select individuals, approximately 150, from the top 10% in terms of shell height and wet weight as candidate parents, with wet weight as the primary selection criterion and shell height as a secondary consideration. Genetic identification of the candidate parents is performed using COⅠ and ITS2 primers. Individuals that are not Fujian oysters or suspected hybrids are removed, and at least 50 male and 50 female parents are retained. Continuous breeding of the population for 3 generations will yield a diploid breeding population of Fujian oysters.

[0032] b. Induction of triploid Fujian oysters: Dissect diploid Fujian oysters, and follow the procedure described in (1)a to collect sperm and eggs, filter, mature, artificially fertilize and induce; select batches with triploid induction rate ≥80% and continue to cultivate until attachment and sea aging to obtain triploid Fujian oyster population.

[0033] c. Induction of tetraploid oysters in Fujian: Using triploid female oysters as the mother and male diploid oysters as the father, fertilization was carried out. Following the procedure described in (1)b, sperm and eggs were collected, filtered, matured, artificially inseminated, and induced. Batches with a tetraploid induction rate of ≥80% were selected and continued to be cultivated until they attached and were released into the sea to grow, thus obtaining a tetraploid population of Fujian oysters.

[0034] d. Breeding of tetraploid population of Fujian oyster: Based on the tetraploid population of Fujian oyster, the tetraploid population was continuously bred for 3 generations according to the tetraploid population breeding process described in (1)c, and a tetraploid population of Fujian oyster with stable ploidy and growth advantage was obtained, which was denoted as AAAA.

[0035] (3) Establishment of hybrid tetraploid oyster (GGAA)

[0036] Dissect tetraploid oysters (GGGG) and select mature male and female individuals as maternal parents; dissect tetraploid oysters (AAAA) and select male individuals with strong sperm motility as paternal parents; each group should have no fewer than 50 male and female individuals, and fertilize them at a sperm-to-egg ratio of 50:1; follow the conventional hatching and larval rearing process to obtain a hybrid tetraploid population, denoted as GGAA.

[0037] (4) Preparation of triploid oysters (RGA)

[0038] a. Establishment of the diploid breeding population (RR) of Crassula ovata: Collect approximately 3,000 wild 1-year-old Crassula ovata individuals. After removing individuals with deformed shells, damaged shells, and poor vitality, retain approximately 1,500 individuals with uniform shells. Select approximately 150 individuals from the top 10% in shell height and wet weight as candidate parents. Use COⅠ and ITS2 primers for genetic identification, remove non-Crassostrea ovata individuals or suspected hybrid individuals, and retain no less than 50 male and female parents. Continuously select and breed the population for 3 generations to obtain the diploid breeding population of Crassula ovata, denoted as RR.

[0039] b. Preparation of triploid oysters (RGA): Individuals with regular shell shape and good vitality were selected from the diploid breeding population (RR) and the tetraploid hybrid population (GGAA) of Crassula ovata. Parents were screened by combining flow cytometry and microscopic observation. 2N female Crassula ovata were selected as the maternal parent and 4N males from the GGAA population were selected as the paternal parent for hybridization. During artificial insemination, the salinity of the seawater was controlled at 20-25‰ to ensure that there were 10-15 sperm cells around each egg. The larvae were cultured and the juveniles were raised using conventional methods to obtain triploid oysters, which were denoted as RGA.

[0040] (6) Comparative test:

[0041] The triploid oysters (RGA) from (5) above were set as the experimental group. Purebred triploid *Crassostrea gigas* (GGG) and purebred triploid *Crassostrea kwangsiensis* (AAA) were constructed with the same number of parents (GGG group: diploid *Crassostrea gigas* ♀ × tetraploid *Crassostrea gigas* ♂; AAA group: diploid *Crassostrea kwangsiensis* ♀ × tetraploid *Crassostrea kwangsiensis* ♂) as control group 1 and control group 2. They were bred using conventional cultivation methods. After attaching and reaching a certain size, they were transferred to the Lianyungang aquaculture area in Jiangsu Province. The salinity of the aquaculture area was continuously recorded during the cultivation period, fluctuating steadily around 20‰. The hanging cage method was used for cultivation, and the production data of the three groups of triploid oysters were statistically analyzed (Table 1). In September of the following year, the wet weight and survival rate of the three groups of oysters were recorded. During the breeding season (May–August), at least 100 individuals from each group (total ≥400) were randomly selected each month for gonadal histological section observation. The proportion of individuals without mature gametes or with inhibited gonadal development was used as the infertile ratio.

[0042] As shown in Table 1, under low-salinity aquaculture conditions (salinity approximately 20‰) in Lianyungang, Jiangsu Province, the overall production performance of the RGA group of triploid oysters was superior to that of the two control groups. The average wet weight of the RGA group was 77.28 g, which was 14.17 g and 8.82 g higher than that of the AAA group (63.11 g) and the GGG group (68.46 g), respectively. The survival rate of the RGA group was 68.61%, which was 14.74 and 5.35 percentage points higher than that of the AAA group (53.87%) and the GGG group (63.26%), respectively. At the same time, the proportion of sterile individuals in the RGA group was 83.02%, which was 27.00 and 8.02 percentage points higher than that of the AAA group and the GGG group, respectively. This indicates that the RGA group of triploid oysters not only maintained better growth performance and higher survival stability under low-salinity conditions, but also had more significantly inhibited gonadal development, and the proportion of sterile phenotypes in the population was higher. In summary, the RGA group triploid hybrids bred in this invention have the combined advantages of "rapid growth and high survival" under low-salinity marine aquaculture conditions, and also have a high proportion of sterile individuals, which is conducive to stabilizing commercial traits during the breeding season. They have good prospects for large-scale promotion and industrial application.

[0043] Table 1. Comparison of production performance between triploid oysters (RGA) and the control group. Group Wet weight (g) Survival rate (%) Percentage of infertile individuals (%) RGA Group 77.28 68.61 83.02 Group AAA 63.11 53.87 56.02 GGG Group 68.46 63.26 75.00

[0044] The above description is only used to illustrate the technical solutions and features of the present invention, and is not intended to limit the scope of protection of the present invention. Any simple modifications or equivalent substitutions made by those skilled in the art to the technical solutions of the present invention should fall within the scope of protection of the technical solutions of the present invention.

Claims

1. A method for cultivating a fast-growing, low-salt-adaptable, three-way hybrid triploid oyster, characterized in that... It includes the following steps: a. Using the diploid of the new cultivar "Haida No. 1" (GS-01-005-2013) as the base population, triploid of "Haida No. 1" oyster was induced to obtain the base population; using the triploid of "Haida No. 1" oyster as the female parent and the diploid of "Haida No. 1" oyster as the male parent, tetraploid of "Haida No. 1" oyster was induced to obtain the base population; the tetraploid population was continuously selected and bred for 2 to 3 generations to obtain the tetraploid breeding population of "Haida No. 1" oyster. b. Using a wild diploid population of Crassula fruticosa as the base population, and with rapid growth as the breeding objective, the population is continuously bred for 2-3 generations to obtain a selected diploid population of Crassula fruticosa; using the selected diploid population of Crassula fruticosa as the parent, triploid Crassula fruticosa is induced to obtain a base population of Crassula fruticosa; using the triploid population of Crassula fruticosa as the female parent and the selected diploid population of Crassula fruticosa as the male parent, a basic tetraploid population of Crassula fruticosa is induced to obtain a base population of Crassula fruticosa; the tetraploid population is continuously bred for 2-3 generations to obtain a selected tetraploid population of Crassula fruticosa. c. Using the tetraploid oyster breeding population of the Pacific oyster as the female parent and the tetraploid oyster breeding population of the Fujian oyster as the male parent, hybridization was carried out to obtain a tetraploid oyster population of Pacific oyster and Fujian oyster, denoted as GGAA. d. Using the wild diploid population of Crassula ovata as the base population, and with rapid growth as the breeding target, the population was continuously bred for 2-3 generations to obtain the diploid breeding population of Crassula ovata. e. Using the diploid oyster breeding population as the female parent and the tetraploid oyster GGAA as the male parent, hybridization was carried out to obtain a triploid hybrid of oyster, crane oyster and Fujian oyster, denoted as RGA.

2. The breeding method according to claim 1, characterized in that, In steps (a) and (b), triploid and tetraploid induction was performed by microscopic observation after fertilization. When 20% of the fertilized eggs showed the first polar body, 0.5 mg / L cytochalasin B was added for 10-15 min. After treatment, the eggs were soaked in seawater containing 1% dimethyl sulfoxide for 30 min and then thoroughly rinsed with filtered seawater. Flow cytometry was used to detect the ploidy of the larvae, and batches with an induction rate ≥80% were selected for larval rearing and juvenile rearing.

3. The breeding method according to claim 1, characterized in that, Both diploid and tetraploid populations were selected for breeding. Individuals with good vitality, intact shell shape, and no damage were chosen, and the top 10% of individuals in terms of shell height and wet weight were selected as candidate parents. For diploid candidate parents, genetic identification was performed using COⅠ and ITS2 before fertilization to select purebred parents. For tetraploid candidate parents, ploidy was determined before fertilization, and individuals with ploidy of 4N were selected as parents. There were no fewer than 50 male and female parents, and high-intensity breeding was carried out for 2 to 3 generations.

4. The breeding method according to claim 1, characterized in that, In step (e), during the three-way hybridization, individuals with well-developed glands, uniform egg development, and dark yolk color were selected from the diploid oyster breeding population as candidate maternal parents, and individuals with vigorous sperm motility were selected from the tetraploid oyster population GGAA as candidate paternal parents. Before fertilization, ploidy was determined using flow cytometry, and individuals with maternal 2N and paternal 4N were selected for hybridization. During artificial insemination, the seawater salinity was controlled at 20-25‰, and the sperm-egg ratio was controlled so that there were 10-15 sperm around each egg.

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