Breeding method of crassostrea gigas' Haida No.3 'fast-growing triploid

Abstract

The invention provides a breeding method of crassostrea gigas' Haida No.3 'fast-growing triploid, and belongs to the technical field of aquaculture genetic breeding. According to the method, a new variety of diploid crassostrea gigas' Haida No.3 'is taken as a basic population, crassostrea gigas' Haida No.3 'fast-growing diploid is generated through breeding, crassostrea gigas' Haida No.3 'triploid and tetraploid are generated through induction, and crassostrea gigas' Haida No.3 'fast-growing tetraploid is generated through breeding. Crassostrea gigas' Haida No.3 'fast-growing tetraploid and diploid are hybridized to generate Crassostrea gigas' Haida No.3 'fast-growing triploid. By evaluating the production performance, the ploidy stability and the genetic diversity of the crassostrea gigas' Haida No.3 'fast-growing triploid, the crassostrea gigas' Haida No.3 'triploid which is fast in growth, pure black in shell color, stable in ploidy and high in genetic diversity is obtained, and the crassostrea gigas' Haida No.3 'fast-growing triploid has important application value in the oyster breeding industry.

Description

Technical Field

[0001] The invention belongs to the technical field of shellfish breeding, and relates to a breeding method for a triploid long oyster, and specifically to a breeding method for a fast-growing triploid long oyster "Haida No. 3". Background Art

[0002] Oysters are an important marine shellfish, known as the "milk of the sea" for their delicious taste and rich nutrition. In 2023, my country's total oyster production was about 6.671 million tons, accounting for 40.5% of the total production of farmed shellfish and 27.9% of the total production of marine aquaculture (China Fisheries Statistical Yearbook, 2024). The main oyster species farmed in my country are long oysters ( Crassostrea gigas )、Fujian oysters( Crassostrea angulata ) and Hong Kong oysters ( Crassostrea hongkongensis ), the main species in the north of the Yangtze River is Crassostrea gigas. In recent years, the rapid development of the Crassostrea gigas industry has led to an urgent need for new fast-growing and highly resistant Crassostrea gigas varieties.

[0003] Selective breeding is a basic tool for selecting and improving specific phenotypic traits of populations and is a classic breeding method. Selective breeding is widely used in aquatic species such as fish, shrimp, crab, and shellfish, and has played an important role in increasing yield, improving quality, and enhancing stress resistance. In previous studies, 360 long oysters with black left shells were selected from the wild population of long oysters along the Shandong coast to construct a basic population. The black left and right shells and shell height were selected as selection indicators. After 4 generations of family selection and 2 generations of group selection, a fast-growing breeding variety, the long oyster "Haida No. 3" (variety registration number: GS-01-007-2018), with both left and right shells and mantles black, was obtained. However, the new variety of long oyster "Haida No. 3" is a diploid oyster. During the reproductive period, nutrients such as glycogen are consumed in large quantities for gonad development and gametogenesis, resulting in a decrease in taste and texture. After spawning, the soft part rapidly loses weight and cannot be supplied to the market.

[0004] Polyploid breeding refers to the use of artificial mutagenesis or natural variation to increase the number of chromosomes of an organism to obtain polyploid materials for the purpose of breeding excellent varieties that meet people's needs. In recent years, with the growing demand for high-quality oysters in my country, triploid oyster breeding and aquaculture have developed rapidly in China. Although the current scale of triploid oyster aquaculture has occupied a considerable proportion, there are still many problems in triploid oyster aquaculture. First, there is a lack of good varieties of triploid oysters, and their tetraploid fathers are often induced by unselected oysters, which leads to a large room for improvement in the production performance of triploid offspring. Secondly, the genetic diversity of tetraploid strains obtained by traditional induction methods is low, which may have an adverse effect on the growth and survival of tetraploid self-propagation and triploid hybrid populations. In addition, since triploid oysters are no different from ordinary diploid oysters in appearance, farmers cannot distinguish triploids from diploids with the naked eye during the aquaculture process. The triploid seed market is chaotic and mixed, and inferior seedlings and fake seedlings are often used as good ones and fake ones as real ones. Therefore, breeding a triploid long oyster "Haida No. 3" with excellent production performance, easy to distinguish appearance and high genetic diversity is of great significance for promoting the promotion of oyster varieties. Summary of the invention

[0005] The object of the present invention is to provide a method for breeding triploid Crassostrea gigas "Haida No. 3" which has rapid growth, pure black shell color, stable ploidy and high genetic diversity.

[0006] In order to achieve the above-mentioned invention object, the specific technical solution adopted by the present invention is:

[0007] a. Taking the long oyster "Haida No. 3" diploid as the basic population, with the left and right shells being black and the shell height as the selection index, and with a seed retention rate of 10%, after 5 consecutive generations of population selection, the long oyster "Haida No. 3" fast-growing diploid was obtained;

[0008] b. Taking the long oyster "Haida No. 3" diploid in step a as the basic population, an inducer is used to inhibit the discharge of the first polar body of the fertilized egg to obtain the long oyster "Haida No. 3" with high genetic diversity to induce triploid;

[0009] c. Using the triploid oyster "Haida No. 3" induced in step b as the female parent and the diploid oyster "Haida No. 3" as the male parent, using an inducer to inhibit the discharge of the first polar body of the fertilized egg to obtain the tetraploid oyster "Haida No. 3" with high genetic diversity;

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

[0011] e. The fast-growing diploid of the long oyster "Haida No. 3" in step a is used as the female parent, and the fast-growing tetraploid of the long oyster "Haida No. 3" in step d is used as the male parent, and hybridization is performed to obtain the fast-growing triploid of the long oyster "Haida No. 3";

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

[0013] Furthermore, in the step a, the long oyster "Haida No. 3" is the long oyster "Haida No. 3" (variety registration number: GS-01-007-2018) approved in 2018 after 4 generations of family breeding and 2 generations of group breeding.

[0014] Furthermore, in the step b, the long oyster "Haida No. 3" is induced to triploid: 300 diploid long oysters "Haida No. 3" are dissected, and 50 individuals with regular egg shape, rich nuclear material and uniform size are selected as maternal parents, and 50 individuals with active sperm are selected as paternal parents; 0.5 mg / L 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 the step c, the long oyster "Haida No. 3" induces tetraploidy: the long oyster "Haida No. 3" induced triploidy in step b is detected by flow cytometry, and individuals with regular, dense eggs and large egg diameters are selected after dissection. After filtering out impurities through a 160-mesh silk screen, the eggs are washed with a 500-mesh silk screen to remove components such as tissue fluid, and after maturation, they are fertilized with the long oyster "Haida No. 3" diploid with good sperm motility. 0.5 mg / L of cytochalasin B is added 8-10 minutes after fertilization to inhibit the release of the first polar body of the fertilized egg. When the first polar body of the control group is about 60% discharged, dimethyl sulfoxide solution (DMSO) is used to wash the eggs for 20 minutes.

[0016] Furthermore, in the step d, the long oyster "Haida No. 3" fast-growing tetraploid: use a flow cytometer to perform two non-lethal ploidy tests on the long oyster "Haida No. 3" induced tetraploid in step c, select the long oyster "Haida No. 3" tetraploid with consistent ploidy in the two tests, a single peak without impurities, and screen the top 10% of the tetraploids as broodstock, fertilize them in a conventional method at a ratio of egg: sperm = 1:10, and hatch and select the best.

[0017] Furthermore, in the step d, the two non-lethal ploidy tests: the tetraploid parents are tested twice using a flow cytometer (BeckmanCoulter CytoFLEX). The first time, 300 μL of hemolymph is extracted from the adductor muscle using a syringe for ploidy detection. The second time, a small part of the gills is clamped from the shell opening using forceps for ploidy detection. The relative DNA content of the diploid is used as a control for the analysis of the ploidy level of the seed shellfish, and the tetraploid parents with consistent test results in the two tests are used for population breeding.

[0018] Furthermore, in the step e, the long oyster "Haida No. 3" fast-growing triploid: the long oyster "Haida No. 3" fast-growing diploid with regular egg shape and dense egg quality is selected as the female parent, and the long oyster "Haida No. 3" fast-growing tetraploid measured by flow cytometry is selected, and the individuals with good sperm motility are dissected and selected as the male parent.

[0019] Furthermore, in the step f, the ploidy stability is evaluated by using a flow cytometer to evaluate the triploid rate of the fast-growing triploid of the long oyster "Haida No. 3" at the D-shaped larvae stage, the eyed larvae stage, 120 days of age, 360 days of age and 480 days of age, to determine the stability of its triploid rate.

[0020] Furthermore, in the step f, the molecular markers and genetic diversity: using mitochondrial COI sequences to detect polymorphic sites of the fast-growing triploid of the long oyster "Haida No. 3"; using microsatellite DNA molecules to detect the genetic diversity of the fast-growing triploid of the long oyster "Haida No. 3".

[0021] Beneficial effects of the present invention:

[0022] (1) The present invention uses the new variety of long oyster "Haida No. 3" (variety registration number: GS-01-007-2018) as the basic population, and induces and constructs the long oyster "Haida No. 3" fast-growing tetraploid through polyploid breeding technology, and produces the long oyster "Haida No. 3" fast-growing triploid through intraspecific ploidy hybridization. The growth advantage of the long oyster "Haida No. 3" diploid obtained through multiple generations of selective breeding is inherited to the long oyster "Haida No. 3" triploid, and the selection advantage and triploid advantage are superimposed, so that the long oyster "Haida No. 3" triploid has more excellent traits and commercial value.

[0023] (2) In the present invention, the fast-growing triploid long oyster "Haida No. 3" inherits the distinctive characteristics of the long oyster "Haida No. 3" diploid, which is fast-growing and has a pure black shell color. It can not only significantly increase the economic value of aquatic products, but also serve as a marker to effectively distinguish groups that have not been selected for shell color. By cultivating a new variety of triploid long oysters with a specific shell color, it can not only fill the gap in the summer long oyster market, but also meet the current industry's breeding needs for genetic improvement of long oysters.

[0024] (3) The present invention suppresses the first polar body of the diploid long oyster "Haida No. 3" to produce the induced triploid long oyster "Haida No. 3" with high genetic diversity, and then suppresses the first polar body of the induced triploid long oyster "Haida No. 3" to obtain the induced tetraploid long oyster "Haida No. 3" with high genetic diversity. Microsatellite DNA molecules are used to detect the genetic diversity of the fast-growing triploid long oyster "Haida No. 3", timely evaluate the inbreeding accumulation of the strain and make corresponding breeding strategies, so as to effectively avoid the inbreeding depression phenomenon. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 A breeding method roadmap for the fast-growing triploid Crassostrea gigas “Haida No. 3” of the present invention;

[0026] Figure 2 This is the peak graph of ploidy detection of fast-growing diploid, triploid and tetraploid oyster "Haida No. 3";

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

[0028] Figure 4 This is the capillary electrophoresis pattern of the microsatellite multiplex PCR combination of the fast-growing triploid Crassostrea gigas "Haida No. 3". DETAILED DESCRIPTION

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

[0030] Example:

[0031] A. Breeding of fast-growing diploid long oysters “Haida No. 3”: In 2018, 1,000 long oysters “Haida No. 3” were used as the basic population, with black shells and shell height as selection indicators, and a 10% seed retention rate to construct a fast-growing breeding line of long oysters “Haida No. 3”. Fertilized eggs were obtained through artificial insemination and placed in a 20 m 3 The larvae were cultured in the nursery pond until they were eyed larvae, and the attachment substrate was placed to allow the eyed larvae to attach and metamorphose. After one week, they were transferred to the sea area of ​​Rushan, Shandong for aquaculture. With the same breeding objectives, seedling raising techniques and aquaculture methods, the fast-growing diploid long oyster "Haida No. 3" was obtained through continuous breeding for 5 generations.

[0032] B. Induction of triploids in the long oyster "Haida No. 3": In 2018, 300 adult diploid long oysters "Haida No. 3" constructed in 2017 were dissected, and 50 individuals with regular egg shapes, rich nuclear substances, and uniform sizes were selected as female parents, and 50 individuals with active sperm were selected as male parents. After all egg cells were filtered through a 260-mesh silk screen to remove impurities, the eggs were washed with a 500-mesh silk screen to remove tissue fluid and other components, and the eggs were added to 24-25 ℃ sand-filtered seawater for 30 minutes. 0.5 mg / L cytochalasin B was added 6 minutes after fertilization and treated for 15-20 minutes to inhibit the release of the first polar body of the fertilized egg. The eggs were washed with dimethyl sulfoxide solution (DMSO) for 20 minutes and placed in a hatching pond for incubation and cultivation. The triploid induction rate was detected by flow cytometry and was above 85%. After attachment, the hanging rope method was used to culture the oysters in the Rongcheng waters of Shandong Province, and about 20,000 long oysters "Haida No. 3" were induced to become triploid shellfish.

[0033] C. Induction of tetraploidy in long oyster "Haida No. 3": In 2019, 2,000 one-year-old long oysters "Haida No. 3" were taken from the sea area to induce triploidy, and the long oysters were heated indoors for three months to promote maturation. The ploidy of the induced triploid long oysters "Haida No. 3" was detected using flow cytometry. After dissection, 44 female parents with regular and dense eggs and large egg diameters were selected. After filtering out impurities with a 160-mesh screen, the tissue fluid and other components were washed away with a 500-mesh screen. After maturation, they were fertilized with 50 diploid long oysters "Haida No. 3" with good sperm motility. 0.5 mg / L of cytochalasin B inhibited the release of the first polar body of the fertilized egg. When the first polar body of the control group was about 60% expelled, the eggs were washed with dimethyl sulfoxide solution (DMSO) for 20 minutes, placed in the hatching pond for incubation and cultured until attachment. After attachment, the hanging rope method was used to culture in the waters of Rongcheng, Shandong, and about 22,000 long oysters "Haida No. 3" were obtained to induce tetraploid shellfish.

[0034] D. Breeding of fast-growing tetraploids of long oysters "Haida No. 3": In 2020, 18,000 long oysters "Haida No. 3" tetraploids were selected from the long oysters "Haida No. 3" tetraploid population induced in 2019 through two non-lethal ploidy tests by flow cytometry as the basic population. The top 10% of tetraploids with pure black shell color were selected as broodstock. The population breeding method (50♀:50♂) was adopted, and the fertilization was carried out in a conventional way at a ratio of eggs: sperm = 1:10. The superior ones were hatched and selected. The larvae were cultivated in a conventional way until they attached. One week later, they were transferred to the Jiaonan waters of Shandong for cultivation, and the first generation of long oysters "Haida No. 3" tetraploids were obtained. With the same breeding objectives, seedling raising techniques and cultivation methods, three generations of long oysters "Haida No. 3" fast-growing tetraploids were obtained.

[0035] E. Cultivation of the fast-growing triploid of the long oyster "Haida No. 3": In 2023, the fast-growing diploid of the long oyster "Haida No. 3" constructed in 2022 was used as the female parent, and the fast-growing tetraploid of the long oyster "Haida No. 3" constructed in 2022 was used as the male parent. The intraspecific ploidy hybridization seed production method (♀:♂=50:50) was adopted, and fertilization was carried out at a ratio of egg: sperm=1:10. The larvae were hatched, selected and cultured until they attached. After attachment, they were cultured in the Jiaonan waters of Shandong using the hanging rope method. This is the fast-growing triploid of the long oyster "Haida No. 3".

[0036] F. Evaluation of the production performance, ploidy stability, molecular markers and genetic diversity of the fast-growing triploids of the long oyster "Haida No. 3": The growth traits of the fast-growing triploids of the long oyster "Haida No. 3" in step E were measured and statistically analyzed. At the same time, the growth data of the fast-growing triploids of the long oyster "Haida No. 3" were compared with those of the corresponding control group, and the production performance data were recorded. The triploid rate of the fast-growing triploids of the long oyster "Haida No. 3" at the D-shaped larvae stage, the eyed larvae stage, 120 days old, 360 days old and 480 days old was detected by flow cytometry to determine the stability of its triploid rate. The polymorphic sites of the fast-growing triploids of the long oyster "Haida No. 3" were detected using mitochondrial COI sequences: (1) DNA of the fast-growing triploids of the long oyster "Haida No. 3" was extracted. (2) DNA of the obtained long oyster 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. Detection of genetic diversity of the fast-growing triploid long oyster "Haida No. 3" using microsatellite DNA molecules: (1) Extract DNA of the fast-growing triploid long oyster "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) PCR amplification was performed on the obtained DNA. (4) The obtained PCR products were subjected to capillary electrophoresis using ABI 3730XL. (5) The capillary electrophoresis results were analyzed using the data analysis software popgene.

[0037] Practice has proved that the growth traits of the fast-growing triploid long oyster "Haida No. 3" have been significantly improved. Compared with the fast-growing diploid long oyster "Haida No. 3", its shell height, body weight, soft part 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 the unselected diploid oyster commercial seedlings, the average increases were 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 long oyster "Haida No. 3" was 100% at the D-shaped larvae stage, the eyed larvae stage, 120 days, 360 days and 480 days of age, and the ploidy was very stable. The mtCOI gene sequence of 47 long oyster "Haida No. 3" triploids was analyzed by molecular markers. The length was 637 bp, and the sequence contained 2 polymorphic sites without insertion and deletion. Two haplotypes, H2 (GenBank registration number: MH160013) and H3 (GenBank registration number: MH1600134), were detected. The two haplotypes differed by only one base. Among them, the H2 haplotype was a high-frequency haplotype, accounting for 87.23% of the total haplotypes. Twelve microsatellite markers were used to detect the genetic diversity of the fast-growing triploid population of the long oyster "Haida No. 3". The average number of alleles in the fast-growing triploid of the long oyster "Haida No. 3" was 4.528, which was significantly decreased compared with the wild population in the control group (average number of alleles was 8.778); the genetic differentiation index between the fast-growing triploid of the long oyster "Haida No. 3" and the wild population was 0.092, indicating a moderate degree of genetic differentiation.

[0038] The specific embodiments described above are only preferred implementations of the present invention and are not intended to limit the protection scope of the present invention. Any modification, supplement or replacement of the specific embodiments by a person skilled in the art to which the present invention belongs shall fall within the protection scope of the present invention.

Claims

1. A method for breeding fast-growing triploid Crassostrea gigas "Haida No. 3", characterized in that: The following steps are involved: a. Taking the long oyster "Haida No. 3" diploid as the basic population, and taking the black color of the left and right shells and the shell height as the selection indicators, after 5 consecutive generations of population selection, the long oyster "Haida No. 3" fast-growing diploid was obtained; b. Taking the long oyster "Haida No. 3" diploid in step a as the basic population, using an inducer to inhibit the discharge of the first polar body of the fertilized egg to obtain the long oyster "Haida No. 3" with high genetic diversity to induce triploid; c. Using the triploid oyster "Haida No. 3" induced in step b as the female parent and the diploid oyster "Haida No. 3" as the male parent, using an inducer to inhibit the discharge of the first polar body of the fertilized egg to obtain a tetraploid oyster "Haida No. 3" with high genetic diversity; d. Using the induced tetraploid of the long oyster "Haida No. 3" in step c as the basic population, after two non-lethal ploidy tests, black shell color and shell height were used as selection indicators to construct the third generation of long oyster "Haida No. 3" fast-growing tetraploid; e. The fast-growing diploid Crassostrea gigas "Haida No. 3" in step a is used as the female parent, and the fast-growing tetraploid Crassostrea gigas "Haida No. 3" in step d is used as the male parent, and hybridization is performed to obtain the fast-growing triploid Crassostrea gigas "Haida No. 3"; f. Evaluate the production performance, ploidy stability, molecular markers and genetic diversity of the fast-growing triploid Crassostrea gigas “Haida No. 3”.

2. The method for breeding fast-growing triploid Crassostrea gigas "Haida No. 3" according to claim 1, characterized in that: In step a, the long oyster "Haida No. 3" is the result of 4 generations of family breeding and 2 generations of group breeding. In 2018, the 6th generation of long oyster black shell breeding line was declared as a new variety, and the approved long oyster "Haida No. 3" (variety registration number: GS-01-007-2018).

3. The method for breeding fast-growing triploid Crassostrea gigas "Haida No. 3" according to claim 1, characterized in that: In step b, 300 diploid long oysters "Haida No. 3" were dissected, and 50 individuals with regular egg shape, rich nuclear material and uniform size were selected as maternal parents, and 50 individuals with active sperm were selected as paternal parents; 0.5 mg / L cytochalasin B was added 6 minutes after fertilization, and the treatment lasted 15-20 minutes to inhibit the release of the first polar body of the fertilized egg, and the eggs were washed with dimethyl sulfoxide solution (DMSO) for 20 minutes.

4. The method for breeding fast-growing triploid Crassostrea gigas "Haida No. 3" according to claim 1, characterized in that: In step c, flow cytometry is used to detect the ploidy of the induced triploids of the long oyster "Haida No. 3" in step b. After dissection, 50 female parents with regular, dense eggs and large egg diameters are selected. After maturation, they are fertilized with diploid long oysters "Haida No. 3" with good sperm motility. 0.5 mg / L cytochalasin B is added 8-10 minutes after fertilization to inhibit the release of the first polar body of the fertilized egg. When the first polar body of the control group is about 60% discharged, dimethyl sulfoxide solution (DMSO) is used to wash the eggs for 20 minutes.

5. The method for breeding fast-growing triploid Crassostrea gigas "Haida No. 3" according to claim 1, characterized in that: In step d, the induced tetraploid of the long oyster "Haida No. 3" in step c is subjected to two non-lethal ploidy tests using a flow cytometer, and the long oyster "Haida No. 3" tetraploid with consistent ploidy and a single peak without impurities in the two tests is selected, and the tetraploids in the top 10% of the shell height are selected as broodstock, and fertilized by conventional methods at a ratio of egg: sperm=1:10, and the best ones are hatched and selected.

6. The method for breeding fast-growing triploid Crassostrea gigas "Haida No. 3" according to claim 1, characterized in that: In step e, the fast-growing diploid long oyster "Haida No. 3" with regular egg shape and dense egg quality is selected by dissection as the female parent, the fast-growing tetraploid long oyster "Haida No. 3" measured by flow cytometry is selected, and the individuals with good sperm motility are dissected and selected as the male parent, and fertilization is carried out in a conventional manner at a ratio of egg: sperm = 1:10, and the best ones are selected by hatching.

7. The method for breeding fast-growing triploid Crassostrea gigas "Haida No. 3" according to claim 1, characterized in that: In step f, flow cytometry is used to evaluate the triploid rate of the fast-growing triploid of the long oyster "Haida No. 3" at the D-shaped larvae stage, the eyed larvae stage, and 120 days, 360 days, and 480 days of age to determine the stability of its triploid rate; mitochondrial COI sequences are used to detect polymorphic sites of the fast-growing triploid of the long oyster "Haida No. 3"; and microsatellite DNA molecules are used to detect the genetic diversity of the fast-growing triploid of the long oyster "Haida No. 3".

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