Breeding method of long oyster "haida 3" fast-growing triploid

Abstract

The application provides a breeding method of long oyster Crassostrea gigas "Haida No.3" fast-growing triploid, and belongs to the technical field of aquaculture genetics and breeding. The application takes the new variety of diploid long oyster "Haida No.3" as a basic population, produces long oyster "Haida No.3" fast-growing diploid through breeding, induces long oyster "Haida No.3" triploid and tetraploid, and produces long oyster "Haida No.3" fast-growing tetraploid. The long oyster "Haida No.3" fast-growing triploid is produced by hybridization of the long oyster "Haida No.3" fast-growing tetraploid and the diploid. The production performance, ploidy stability and genetic diversity of the long oyster "Haida No.3" fast-growing triploid are evaluated, and the long oyster "Haida No.3" triploid which is fast-growing, has pure black shell color, stable ploidy and high genetic diversity is obtained, and has important application value in oyster breeding industry.

Description

Technical Field

[0001] This invention belongs to the field of shellfish breeding technology and relates to a method for breeding triploids of the Pacific oyster, specifically a method for breeding a fast-growing triploid of the Pacific oyster "Haida No. 3". Background Technology

[0002] Oysters are an important marine shellfish, known as "milk of the sea" for their delicious taste and rich nutrition. In 2023, my country's total oyster production was approximately 6.671 million tons, accounting for 40.5% of the total farmed shellfish production and 27.9% of the total marine aquaculture production (China Fisheries Statistical Yearbook, 2024). In recent years, the rapid development of the Pacific oyster industry has created an increasingly urgent need for new fast-growing and highly resistant Pacific oyster varieties.

[0003] Selective breeding is a fundamental tool for selecting and improving specific phenotypic traits in a population, and it is a classic breeding method. Selective breeding is widely used in aquatic species such as fish, shrimp, crabs, and shellfish, playing a significant role in increasing yield, improving quality, and enhancing stress resistance. In previous studies, 360 oysters with black left shells were selected from a wild population of *Crassostrea gigas* along the Shandong coast to construct a basic population. Using black left and right shells and shell height as selection indicators, after four generations of family selection and two generations of population selection, a superior breed of *Crassostrea gigas*, "Haida No. 3" (variety registration number: GS-01-007-2018), with black left and right shells and mantle, and rapid growth, was obtained. However, the new *Crassostrea gigas* variety "Haida No. 3" is a diploid oyster. During the reproductive period, a large amount of nutrients such as glycogen are consumed for gonadal development and gamete formation, leading to a decline in taste and texture. After spawning, the soft body rapidly thins, making it unsuitable for the market.

[0004] Polyploid breeding refers to obtaining polyploid materials by increasing the number of chromosomes in an organism through artificial mutagenesis or natural variation, in order to select superior varieties that meet human needs. In recent years, with the increasing demand for high-quality oysters in my country, triploid oyster breeding and aquaculture have developed rapidly domestically. Although triploid oyster farming currently accounts for a considerable proportion, the industry still faces many problems. First, there is a lack of high-quality triploid oyster breeds; their tetraploid sires are often induced from unselected oysters, resulting in significant room for improvement in the production performance of triploid offspring. Second, tetraploid strains obtained through traditional induction methods have low genetic diversity, which may adversely affect the growth and survival of tetraploid self-propagation and triploid hybrid populations. Furthermore, because triploid oysters are indistinguishable from ordinary diploid oysters in appearance, farmers cannot visually differentiate them during cultivation. The triploid seedling market is chaotic, with inferior and counterfeit seedlings frequently being sold as superior or genuine. Therefore, breeding a triploid of the Pacific oyster "Haida No. 3" with excellent production performance, easily distinguishable appearance, and high genetic diversity is of great significance for promoting the dissemination of superior oyster varieties. Summary of the Invention

[0005] The purpose of this invention is to provide a method for breeding triploid oysters of the Pacific oyster “Haida No. 3” that exhibit rapid growth, pure black shell color, stable ploidy, and high genetic diversity.

[0006] To achieve the above-mentioned objectives, the specific technical solution adopted by the present invention is as follows:

[0007] a. Using the diploid oyster "Haida No. 3" as the base population, and with the black color of the left and right shells and shell height as selection indicators, and with a seed retention rate of 10%, the fast-growing diploid oyster "Haida No. 3" was obtained after five consecutive generations of population selection.

[0008] b. Using the diploid population of the Pacific oyster “Haida No. 3” from step a as the base population, an inducing agent was used to inhibit the expulsion of the first polar body of the fertilized egg to obtain a high genetic diversity Pacific oyster “Haida No. 3” induced triploid.

[0009] c. Using the triploid induced oyster "Haida No. 3" from step b as the maternal parent and the diploid oyster "Haida No. 3" as the paternal parent, an inducing agent was used to inhibit the expulsion of the first polar body of the fertilized egg to obtain a high genetic diversity induced tetraploid oyster "Haida No. 3".

[0010] d. Using the induced tetraploid of the Pacific oyster “Haida No. 3” from step c as the base population, after two non-lethal ploidy tests, with black shell color and shell height as selection indicators, and with a selection intensity of 1.6-1.9, the population selection method (50♀:50♂) was used to construct the third generation of fast-growing tetraploid Pacific oyster “Haida No. 3”.

[0011] e. Using the fast-growing diploid of Crassula 'Haida No. 3' from step a as the female parent and the fast-growing tetraploid of Crassula 'Haida No. 3' from step d as the male parent, hybridize to obtain the fast-growing triploid of Crassula 'Haida No. 3'.

[0012] f. Evaluate the production performance, ploidy stability, molecular markers, and genetic diversity of the fast-growing triploid of the Pacific oyster “Haida No. 3”.

[0013] Furthermore, in step a, the "Haida No. 3" long oyster is the long oyster "Haida No. 3" (variety registration number: GS-01-007-2018) that was approved in 2018 after four generations of family selection and two generations of population selection.

[0014] Furthermore, in step b, the triploid of the Pacific oyster "Haida No. 3" is induced as follows: 300 diploid Pacific oysters "Haida No. 3" are dissected, and 50 individuals with regular egg cell shape, abundant nuclear material, and uniform size are selected as the maternal parent, and 50 individuals with active sperm are selected as the paternal parent; 0.5 mg / L of cytochalasin B is added 6 minutes after fertilization, and the treatment is carried out for 15-20 minutes to inhibit the release of the first polar body of the fertilized egg, and the eggs are washed with dimethyl sulfoxide solution (DMSO) for 20 minutes.

[0015] Furthermore, in step c, the induced tetraploids of the Pacific oyster "Haida No. 3" are: the ploidy of the induced triploids of the Pacific oyster "Haida No. 3" in step b is detected by flow cytometry. After dissection, individuals with regular, dense eggs and large egg diameter are selected. After filtering out impurities through a 160-mesh silk screen, the eggs are washed with a 500-mesh silk screen to remove tissue fluid and other components. After maturation, the eggs are fertilized with diploid Pacific oysters "Haida No. 3" with good sperm motility. 8-10 minutes after fertilization, 0.5 mg / L of cytochalasin B is added to inhibit the release of the first polar body of the fertilized egg. When about 60% of the first polar body is expelled in the control group, the eggs are washed with dimethyl sulfoxide (DMSO) solution for 20 minutes.

[0016] Furthermore, in step d, the fast-growing tetraploids of the Pacific oyster "Haida No. 3" are: the induced tetraploids of the Pacific oyster "Haida No. 3" in step c are subjected to two non-lethal ploidy tests using flow cytometry. The tetraploids of the Pacific oyster "Haida No. 3" with consistent ploidy in the two tests and a single peak without impurities are selected. The top 10% of the tetraploids in terms of growth are selected as parent oysters and fertilized with eggs at a ratio of 1:10 using conventional methods. Hatching and selection are then carried out.

[0017] Furthermore, in step d, the two non-lethal ploidy tests are performed using flow cytometry (Beckman-Coulter CytoFLEX) on the tetraploid parents. The first test involves drawing 300 μL of hemolymph from the adductor muscle using a syringe for ploidy testing. The second test involves using tweezers to collect a small portion of the gills from the shell opening for ploidy testing. The relative DNA content of the diploid serves as a control for ploidy level analysis of the breeding stock. Tetraploid parents with consistent results from both tests are used for population selection.

[0018] Furthermore, in step e, the fast-growing triploid of the Pacific oyster "Haida No. 3" is selected by dissecting and selecting fast-growing diploids of Pacific oyster "Haida No. 3" with regular oval shape and dense oval tissue as the maternal parent, and fast-growing tetraploids of Pacific oyster "Haida No. 3" with good sperm motility are selected by dissecting and selecting individuals as the paternal parent.

[0019] Furthermore, in step f, the assessment of its ploidy stability involves using flow cytometry to evaluate the triploid rate of the fast-growing triploids of the Crassula 'Haida No. 3' at the D-shaped larval stage, the eyespot larval stage, and at 120, 360, and 480 days of age, thereby determining the stability of its triploid rate.

[0020] Furthermore, in step f, the molecular markers and genetic diversity are as follows: the polymorphic sites of the fast-growing triploid of the Pacific oyster “Haida 3” are detected using mitochondrial COI sequences; and the genetic diversity of the fast-growing triploid of the Pacific oyster “Haida 3” is detected using microsatellite DNA molecules.

[0021] The beneficial effects of this invention are:

[0022] (1) This invention uses the new variety of Pacific oyster “Haida No. 3” (variety registration number: GS-01-007-2018) as the base population. Through polyploid breeding technology, a fast-growing tetraploid of Pacific oyster “Haida No. 3” is induced and constructed. Through intraspecific hybridization, a fast-growing triploid of Pacific oyster “Haida No. 3” is produced. The growth advantages obtained by the diploid of Pacific oyster “Haida No. 3” through multiple generations of selective breeding are inherited by the triploid of Pacific oyster “Haida No. 3”. The combination of selective advantage and triploid advantage makes the triploid of Pacific oyster “Haida No. 3” more superior in traits and commercial value.

[0023] (2) In this invention, the fast-growing triploid of the Pacific oyster “Haida No. 3” inherits the distinctive characteristics of the diploid Pacific oyster “Haida No. 3”, namely rapid growth and pure black shell color. This not only significantly enhances the economic value of aquatic products but also serves as a marker to effectively distinguish populations that have not undergone shell color selection. By cultivating new Pacific oyster varieties with specific shell color triploids, the market gap for Pacific oysters in summer can be filled, and the current industry's breeding needs for genetic improvement of Pacific oysters can be met.

[0024] (3) This invention produces highly genetically diverse induced triploids of the Pacific oyster “Haida 3” by inhibiting the first polar body of the diploid of the Pacific oyster “Haida 3”, and then inhibits the first polar body of the induced triploid of the Pacific oyster “Haida 3” to obtain highly genetically diverse induced tetraploids of the Pacific oyster “Haida 3”. The genetic diversity of the fast-growing triploids of the Pacific oyster “Haida 3” is detected by microsatellite DNA molecules, and the inbreeding accumulation of this strain is assessed in a timely manner and corresponding breeding strategies are made to effectively avoid inbreeding depression. Attached Figure Description

[0025] Figure 1 This is a flowchart illustrating the breeding method for the fast-growing triploid of the Pacific oyster "Haida No. 3" according to the present invention.

[0026] Figure 2 Peak values ​​for ploidy detection in fast-growing diploid, triploid, and tetraploid forms of the Pacific oyster “Haida No. 3”;

[0027] Figure 3 The sequences of haplotypes H2 and H3 of the mtDNA COI gene of the fast-growing triploid oyster “Haida No. 3” were obtained.

[0028] Figure 4 Capillary electrophoresis pattern of multiplex PCR combinations of the fast-growing triploid Pacific oyster “Haida No. 3”. Detailed Implementation

[0029] The following detailed description of a breeding method for a fast-growing triploid of the Pacific oyster "Haida No. 3" provided by the present invention is illustrated through specific embodiments.

[0030] Example:

[0031] A. Breeding of fast-growing diploid oysters 'Haida No. 3': In 2018, using 1000 'Haida No. 3' oysters as the base population, and selecting based on the blackness of the left and right shells and shell height, a fast-growing breeding line of 'Haida No. 3' oysters was constructed with a 10% seed retention rate. Fertilized eggs were obtained through artificial insemination and placed in a 20 m... 3 The larvae were cultivated in the nursery ponds until they reached the eyepoint larvae stage. An attachment substrate was then introduced to induce the eyepoint larvae to attach and metamorphose. One week later, they were transferred to the sea area off Rushan, Shandong Province for further cultivation. Using the same breeding objectives, nursery techniques, and cultivation methods, five generations of fast-growing diploid oysters, known as "Haida No. 3," were obtained through continuous breeding.

[0032] B. Induction of triploid oysters in the "Haida No. 3" variety: In 2018, 300 diploid adult oysters of the "Haida No. 3" variety, established in 2017, were dissected. Fifty individuals with regularly shaped, richly nucleated, and uniformly sized oocytes were selected as maternal parents, and 50 individuals with active sperm were selected as paternal parents. All oocytes were filtered through a 260-mesh silk screen to remove impurities, then washed with a 500-mesh silk screen to remove tissue fluid and other components. The oocytes were then added to sand-filtered seawater at 24-25°C for 30 minutes to mature. Six minutes after fertilization, 0.5 mg / L of cytochalasin B was added, and the treatment lasted for 15-20 minutes to inhibit the release of the first polar body of the fertilized egg. The eggs were washed with dimethyl sulfoxide (DMSO) solution for 20 minutes, then placed in an incubation tank for hatching and cultivation. The triploid induction rate was found to be above 85% by flow cytometry. After attachment, the eggs were cultured in the waters off Rongcheng, Shandong Province using the rope hanging method, resulting in approximately 20,000 triploid oysters of the "Haida No. 3" variety.

[0033] C. Induction of tetraploids in the Pacific Oyster “Haida No. 3”: In 2019, 2000 one-year-old Pacific Oyster “Haida No. 3” induced triploids were collected from the sea area and matured indoors for three months. Flow cytometry was used to detect the ploidy of the adult “Haida No. 3” induced triploids. After dissection, 44 female oysters with regular, dense eggs and large egg diameter were selected. Impurities were filtered through a 160-mesh silk screen, and tissue fluid and other components were washed away through a 500-mesh silk screen. After maturation, the oysters were fertilized with 50 diploid “Haida No. 3” Pacific Oysters with good sperm motility. 0.5% of the sperm was added 8-10 minutes after fertilization. cytochalasin B at mg / L inhibited the release of the first polar body from fertilized eggs. When the first polar body of the control group was about 60% expelled, the eggs were washed with dimethyl sulfoxide (DMSO) solution for 20 minutes, placed in the hatching pond for incubation and cultured until attachment. After attachment, the eggs were cultured in the waters off Rongcheng, Shandong using the rope method, and about 22,000 tetraploid oysters of the "Haida No. 3" variety were obtained.

[0034] D. Breeding of fast-growing tetraploids of the Pacific oyster "Haida No. 3": In 2020, using the tetraploid population of Pacific oyster "Haida No. 3" induced in 2019, 18,000 adult tetraploid oysters of "Haida No. 3" were selected as the base population through two non-lethal ploidy tests by flow cytometry. The top 10% of tetraploids with pure black shells were selected as parent oysters. Using a population selection method (50♀:50♂), fertilization was carried out at a conventional ratio of egg:sperm = 1:10. After hatching and selection, the larvae were raised to attachment using conventional methods. One week later, they were transferred to the Jiaonan sea area of ​​Shandong for cultivation, obtaining the first generation of Pacific oyster "Haida No. 3" tetraploids. Using the same breeding objectives, seedling technology and cultivation methods, three generations of fast-growing tetraploids of Pacific oyster "Haida No. 3" were continuously bred.

[0035] E. Cultivation of the fast-growing triploid of Crassula longifolia 'Haida No. 3': In 2023, using the fast-growing diploid of Crassula longifolia 'Haida No. 3' constructed in 2022 as the female parent and the fast-growing tetraploid of Crassula longifolia 'Haida No. 3' constructed in 2022 as the male parent, the intraspecific hybridization method (♀ : ♂=50 : 50) was adopted, and fertilization was carried out at an egg:sperm ratio of 1:10. The hatched larvae were selected and cultivated until attachment. After attachment, they were raised in the Jiaonan sea area of ​​Shandong using the rope hanging method. This is the fast-growing triploid of Crassula longifolia 'Haida No. 3'.

[0036] F. Evaluation of the production performance, ploidy stability, molecular markers and genetic diversity of the fast-growing triploid of Crassostrea gigas 'Haida No. 3': The growth traits of the fast-growing triploid of Crassostrea gigas 'Haida No. 3' in step E were measured and statistically analyzed. At the same time, the growth data of the fast-growing triploid of Crassostrea gigas 'Haida No. 3' were compared with the corresponding control group, and the production performance data were recorded. The triploidity of the fast-growing triploid of Crassostrea gigas 'Haida No. 3' at the D-shaped larval stage, the eyespot larval stage, and at 120 days, 360 days and 480 days of age was determined by flow cytometry. The polymorphic sites of the fast-growing triploid of Crassostrea gigas 'Haida No. 3' were detected by mitochondrial COI sequence: (1) DNA was extracted from the fast-growing triploid of Crassostrea gigas 'Haida No. 3'. (2) The DNA of the obtained Crassostrea gigas parents and offspring individuals was amplified by PCR. (3) The obtained PCR products were detected by 1% agarose gel electrophoresis, and individuals with bright bands were selected for first-generation sequencing and analysis. Genetic diversity of fast-growing triploids of Crassula 'Haida No. 3' was detected using microsatellite DNA molecules: (1) DNA was extracted from fast-growing triploids of Crassula 'Haida No. 3'. (2) The 12 pairs of microsatellite primers selected were otgfa0_0129_E11, Crgi39, CgEH67; Cgsili39, Cgsili50, Cgsili4; CGE009, Cgsili46, CGE005; Cgsili50, ucdCg-109, CgEH173. (3) The obtained DNA was amplified by PCR. (4) The obtained PCR products were subjected to capillary electrophoresis on an ABI 3730XL. (5) The capillary electrophoresis results were analyzed using the data analysis software popgene.

[0037] Practice has proven that the growth traits of the fast-growing triploid 'Haida No. 3' Pacific oyster have been significantly improved. Compared with the fast-growing diploid 'Haida No. 3', its shell height, body weight, soft body weight, meat yield, and survival rate have increased by an average of 15.03–20.70%, 33.20–48.64%, 60.21–68.24%, 16.71–18.16%, and 27.59–36.29%, respectively. Compared with unselected diploid oyster seedlings, the average increases are 29.21–38.63%, 66.32–74.39%, 78.27–88.94%, 13.71–16.24%, and 30.25–37.19%, respectively. The triploid rate of the fast-growing triploids of the Pacific oyster "Haida No. 3" was 100% at the D-shaped larval stage, the eyespot larval stage, and at 120, 360, and 480 days of age, indicating very stable ploidy. Molecular marker analysis revealed 637 bp mtCOI gene sequences from 47 triploids of the Pacific oyster "Haida No. 3". These sequences contained two polymorphic sites with no insertions or deletions. Two haplotypes, H2 (GenBank accession number: MH160013) and H3 (GenBank accession number: MH1600134), were detected. These two haplotypes differed by only one base, with haplotype H2 being the most frequent, accounting for 87.23% of all haplotypes. The genetic diversity of the fast-growing triploid population of Crassula 'Haida 3' was examined using 12 microsatellite markers. The average number of alleles in the fast-growing triploid population of Crassula 'Haida 3' was 4.528, which was significantly lower than that in the control wild population (average number of alleles 8.778). The genetic differentiation index between the fast-growing triploid population of Crassula 'Haida 3' and the wild population was 0.092, indicating a moderate degree of genetic differentiation.

[0038] The specific embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, additions, or substitutions made by those skilled in the art to which this invention pertains to, thereby falling within the scope of protection of the present invention, should be considered valid.

Claims

1. A method for breeding a fast-growing triploid of the Pacific oyster "Haida No. 3", characterized in that, Includes the following steps: a. Using 1,000 diploid oysters of the "Haida No. 3" variety as the base population, and taking the black color of the left and right shells and shell height as selection indicators, five generations of population selection were carried out continuously with a seed retention rate of 10% to obtain the fast-growing diploid oyster "Haida No. 3". b. Using the fast-growing diploid of the Pacific oyster "Haida No. 3" obtained in step a as the base population, an inducing agent was used to inhibit the expulsion of the first polar body of the fertilized egg to obtain a high genetic diversity Pacific oyster "Haida No. 3" induced triploid; c. Using the induced triploid of Crassula 'Haida No. 3' obtained in step b as the maternal parent and the diploid of Crassula 'Haida No. 3' as the paternal parent, an inducing agent was used to inhibit the expulsion of the first polar body of the fertilized egg to obtain a induced tetraploid of Crassula 'Haida No. 3' with high genetic diversity. d. The induced tetraploids of the Pacific oyster "Haida No. 3" obtained in step c were cultured to adulthood. Each candidate tetraploid adult oyster was subjected to two non-lethal ploidy tests using flow cytometry. The first test involved extracting 300 μL of hemolymph from the adductor muscle, and the second test involved taking a small amount of gill tissue from the shell opening. Only individuals with tetraploid results in both tests and a single peak without any impurities were retained to form a basic population of 18,000 Pacific oyster "Haida No. 3" tetraploid adults. From the basic population, tetraploids with shell height in the top 10% and pure black shell color were selected as parents. A population selection configuration of 50♀:50♂ was used, and fertilization was carried out at an egg:sperm ratio of 1:

10. After three generations of continuous selection, fast-growing tetraploid Pacific oyster "Haida No. 3" was obtained. e. Using the fast-growing diploid of Crassula 'Haida No. 3' obtained in step a as the female parent and the fast-growing tetraploid of Crassula 'Haida No. 3' obtained in step d as the male parent, a 50♀:50♂ intraspecific hybridization breeding configuration was adopted, and fertilization was carried out at a ratio of egg:sperm = 1:

10. After hatching, the best were selected and the larvae were raised until attachment, thus obtaining the fast-growing triploid of Crassula 'Haida No. 3'. f. The production performance, ploidy stability, mitochondrial COI molecular markers, and microsatellite DNA genetic diversity of the fast-growing triploid oyster "Haida No. 3" obtained in step e were evaluated. The triploidity rate at the D-shaped larval stage, eyespot larval stage, 120 days old, 360 days old, and 480 days old was detected by flow cytometry. When the triploidity rate was 100% at all five stages, it was identified as a ploidy-stable fast-growing triploid oyster "Haida No. 3".

2. The breeding method according to claim 1, characterized in that, Step b includes: dissecting 300 fast-growing diploid oysters of the "Haida No. 3" variety; selecting 50 individuals with regular egg cell shape, abundant nuclear material, and uniform size as maternal parents; and selecting 50 individuals with active sperm as paternal parents; filtering impurities through a 260-mesh silk screen, then washing the eggs through a 500-mesh silk screen to remove tissue fluid; maturing the eggs in sand-filtered seawater at 24-25 ℃ for 30 min; adding 0.5 mg / L cytochalasin B 6 min after fertilization; treating for 15-20 min; and washing the eggs with dimethyl sulfoxide solution for 20 min. The resulting induced triploids had a triploid induction rate of over 85%.

3. The breeding method according to claim 1, characterized in that, Step c includes: from 2000 one-year-old Pacific oysters "Haida No. 3" induced triploids, after maturation and ploidy testing, 44 individuals with regular, dense eggs and large egg diameters were selected as maternal parents. After filtering impurities through a 160-mesh silk screen and washing away tissue fluid through a 500-mesh silk screen, the maternal parents were fertilized with 50 Pacific oyster "Haida No. 3" diploid paternal parents with good sperm motility. 8-10 minutes after fertilization, 0.5 mg / L cytochalasin B was added. When about 60% of the first polar body was expelled in the control group, the eggs were washed with dimethyl sulfoxide solution for 20 minutes. The eggs were then hatched and cultured until attachment, thus obtaining Pacific oyster "Haida No. 3" induced tetraploid adult oysters.

4. The breeding method according to claim 1, characterized in that, In step f, the shell height, body weight, soft tissue weight, meat yield, and survival rate of the fast-growing triploid of the Pacific oyster "Haida No. 3" were increased by an average of 15.03-20.70%, 33.20-48.64%, 60.21-68.24%, 16.71-18.16%, and 27.59-36.29% respectively compared with the fast-growing diploid of the Pacific oyster "Haida No. 3". Its polymorphic sites and haplotypes were detected using mitochondrial COI sequences, and its genetic diversity and genetic differentiation were detected using 12 pairs of microsatellite DNA primers.

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