A method for breeding a high-sterility triploid oyster of three species

Abstract

This invention first involves intensive population selection of diploid populations of *Crassostrea gigas*, *Crassostrea kwangsiensis*, and *Crassostrea hongkongensis* to obtain diploid parent populations with rapid growth, strong resistance, excellent traits, and genetic stability. Then, based on these superior diploid parents of *Crassostrea gigas* and *Crassostrea kwangsiensis*, ploidy breeding combined with selective breeding techniques is used to construct tetraploid populations of *Crassostrea gigas* and *Crassostrea kwangsiensis* with high ploidy rates, genetic stability, and high survival rates. Furthermore, a hybrid tetraploid population is constructed by crossing tetraploid *Crassostrea gigas* with tetraploid *Crassostrea kwangsiensis*, and then crossbed using tetraploid *Crassostrea hongkongensis* as the male parent and diploid *Crassostrea hongkongensis* as the female parent to obtain ternary hybrid triploid oysters. The cultivated ternary hybrid triploid oysters maintain rapid growth, high survival rates, and high resistance while significantly improving sterility. The highly sterile ternary hybrid triploid oysters obtained by this invention can significantly reduce problems such as decreased plumpness and high mortality rates during the breeding season due to gonadal development and ovulation. This invention is applicable to the large-scale preparation of superior three-way hybrid triploid seedlings, providing the oyster industry with a supporting breeding technology for three-way hybrid triploids with more stable sterility and excellent comprehensive production performance, and has good prospects for industrial application.

Description

Technical Field

[0001] This invention belongs to the field of shellfish breeding technology, specifically relating to a method for cultivating highly sterile triploid oysters. Background Technology

[0002] The oysters farmed along my country's coast are mainly of the genus *Crassostrea*, with the Pacific oyster (*Crassostrea gigas*), Fujian oyster (*Crassostrea fuscinata*), and Hong Kong oyster (*Crassostrea hongkongensis*) being important species. The Pacific oyster (*Crassostrea gigas*), also known as the giant oyster, is mainly distributed along the coast north of the Yangtze River in my country. It is characterized by its rapid growth, large size, strong environmental adaptability, and delicious meat. The Fujian oyster (*Crassostrea angulata*), mainly produced in coastal areas such as Fujian, is characterized by its small size, rapid growth, stronger heat resistance, and almost year-round fertility. The Hong Kong oyster (*Crassostrea hongkongensis*) is an important farmed species along the coast of South my country, mainly distributed in estuaries and bays in Guangdong, Guangxi, and parts of Fujian. It is commonly found in brackish water areas at the confluence of fresh and estuaries, has a wide salinity tolerance range (suitable salinity for larvae is approximately 15-30, and for adults approximately 5-25), and is characterized by high nutritional value and strong low-salinity tolerance.

[0003] In recent years, the comprehensive application of polyploid breeding, hybridization breeding, and selective breeding techniques has become an important method for the genetic improvement of shellfish. The application of polyploid technology has significantly increased oyster yield per unit area and promoted industrial development. Among them, triploid oysters have attracted much attention from the industry due to their advantages such as rapid growth, rich nutrition, and relatively stable commercial traits, and have become an important direction in oyster breeding. Typically, triploid oysters, due to inhibited gonadal development or reduced fertility, can achieve year-round market availability to some extent, thus compensating for the insufficient summer supply of diploid oysters. On the other hand, interspecific hybridization is an important way to obtain heterosis and introduce tolerance traits. However, due to the influence of genetic differences and reproductive isolation between species, hybrid offspring are prone to phenomena such as asymmetrical fertilization, increased mortality rates in the embryonic or larval stages, and abnormal gonadal development or reduced fertility in the adult stage. Therefore, a stable parental system and breeding process are needed to ensure this.

[0004] In oyster production practice, traditional triploid oysters are often constructed using a "diploid maternal × tetraploid paternal" method. While this type of triploid exhibits certain growth advantages under normal conditions, it still has shortcomings in industrial applications: some individuals may still experience gonadal development and even spawning during the breeding season, leading to a shift in nutrient input towards reproduction, resulting in decreased plumpness and fluctuations in marketable traits; under summer high-temperature stress conditions, its stress resistance is insufficient, easily leading to increased mortality. Therefore, there is an urgent need to further improve the traditional triploid oyster, maintaining or enhancing growth and stress resistance potential while further reducing fertility and improving sterility stability, in order to reduce breeding season risks and improve overall aquaculture efficiency.

[0005] Based on the above needs, a three-way hybrid triploid pathway of "a third species diploid maternal parent × a hybrid tetraploid paternal parent" was constructed, providing a new breeding strategy for obtaining comprehensive advantages. By first integrating the rapid growth or stress resistance genetic basis of two parents at the tetraploid level, and then introducing a third oyster species, the mechanism of inhibited gonadal development in triploids combined with reduced fertility in distant hybridization is utilized. While retaining rapid growth and stress resistance, fertility is further reduced, resulting in a new three-way hybrid triploid oyster strain with significantly improved sterility, significantly enhanced stress resistance, and more stable commercial traits. Furthermore, a supporting breeding method more suitable for large-scale promotion is developed. 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 cultivation method for highly sterile ternary hybrid triploid oysters.

[0007] A method for cultivating highly sterile triploid oysters, 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 the wild diploid population of Fujian oysters as the base population, select the top 10% of individuals in terms of shell height and wet weight as parents, and conduct continuous breeding for 2-3 generations to obtain a Fujian oyster diploid breeding population.

[0010] c. Using the diploid Fujian oyster breeding population obtained in step (b) as the parent, induce triploid Fujian oysters; using triploid Fujian oysters as the female parent and the diploid Fujian oyster breeding population obtained in step (b) as the male parent, induce tetraploid Fujian oyster basic population; conduct continuous 2-3 generations of population selection to obtain tetraploid Fujian oyster breeding population.

[0011] d. Using the tetraploid oyster breeding population obtained in step (a) as the female parent and the tetraploid oyster breeding population obtained in step (c) as the male parent, hybridize to obtain tetraploid oysters hybridized with oysters of ...

[0012] e. Using the Hong Kong oyster diploid population as the base population, select the top 10% of individuals in terms of shell height and wet weight as parents, and conduct continuous 2-3 generations of population selection to obtain the Hong Kong oyster diploid breeding population.

[0013] f. Using the diploid breeding population of Hong Kong oysters obtained in step (e) as the female parent and the tetraploid oyster hybridized with Fujian oysters obtained in step (d) as the male parent, hybridization is carried out to obtain triploid oysters of Hong Kong oysters, long oysters and Fujian oysters.

[0014] Furthermore, in steps (a) and (c), triploid induction is performed by continuously treating the larvae with 0.75 mg / L cytochalasin B for 15 min after fertilization; after induction, the larvae are soaked in 1% DMSO for 30 min and thoroughly rinsed with filtered seawater; the ploidy of the larvae is detected, and batches with a triploidity rate ≥95% are selected for cultivation and rearing.

[0015] Furthermore, in steps (a) and (c), tetraploid induction is performed by continuously treating the larvae with 1 mg / L cytochalasin B for 8–10 min after fertilization; after induction, the larvae are soaked in 1% DMSO for 30 min and then thoroughly rinsed with filtered seawater; the ploidy of the larvae is detected, and batches with a tetraploidity rate ≥80% are selected for subsequent cultivation and breeding.

[0016] Furthermore, in steps (a) and (c), the tetraploid population selection prioritizes individuals with intact shells and no damage, selects the top 10% of individuals in terms of shell height and wet weight as parents, selects no less than 100 males and 100 females for breeding in each generation, and conducts continuous high-intensity population selection for 2 to 3 generations.

[0017] Furthermore, in steps (b) and (e), the diploid population selection involves genetic identification using COⅠ and ITS2 before fertilization to ensure that the parents are purebred Fujian oysters or Hong Kong oysters. Each generation selects no fewer than 100 males and females for breeding, and the high-intensity population selection continues for 2-3 generations.

[0018] Furthermore, in step (d), female oysters with well-developed gonads, uniform egg development, and deep yolk color are selected from the tetraploid breeding population of Pacific oysters, while male oysters with vigorous sperm motility are selected from the tetraploid breeding population of Fujian oysters. The number of parents is no less than 50 males and 50 females.

[0019] Furthermore, in step (f), the female parent is selected from the diploid population of Hong Kong oysters with well-developed gonads, uniform egg development, and deep yolk color, and the male parent is selected from the tetraploid population with vigorous sperm motility. The ploidy of the three-way hybrid parents is determined before fertilization, and individuals with a female parent of 2N and a male parent of 4N are selected for hybridization. The larvae are raised and the adult oysters are grown using conventional methods, and a highly sterile three-way hybrid triploid oyster can be obtained.

[0020] Beneficial effects of the present invention

[0021] The technical solution provided by this invention has the following advantages compared with known technologies.

[0022] (1) This invention involves multi-generational high-intensity population selection of diploid Crassostrea gigas 'Haida No. 1', Crassostrea gigas 'Fujian', and Crassostrea gigas 'Hong Kong' populations, as well as tetraploid Crassostrea gigas 'Haida No. 1' and Crassostrea gigas 'Fujian' populations. This allows the parental populations to accumulate and fix additive genetic effects in terms of growth traits, thereby providing an excellent genetic basis for subsequent hybridization and improving the stability and uniformity of offspring growth performance. (2) This invention involves hybridizing a tetraploid Crassostrea gigas population with a tetraploid Crassostrea gigas population to obtain hybrid tetraploids, which significantly improves the genetic diversity and comprehensive production performance potential of the parental populations and provides an excellent male parent for subsequent ternary hybridization. (3) This invention involves hybridizing a diploid Crassostrea gigas population as the female parent and a hybrid tetraploid (GGAA) as the male parent to obtain ternary hybrid triploid offspring. The three-way hybridization introduces a distant third parent's genetic background, preserving the growth advantages of the Pacific oyster and the excellent environmental adaptability of the Fujian oyster, while further reducing fertility and increasing the stability of sterility. This effectively reduces industry problems such as decreased individual gamete formation and spawning-related decline in plumpness during the breeding season (especially the hot summer season). The method described in this invention is suitable for the mass production of three-way hybrid triploid seedlings, providing the oyster market with a more stable selection of sterile seedlings, helping to improve sterility stability and aquaculture efficiency, and has broad prospects for industrial application. Attached Figure Description

[0023] Figure 1 This is a technical roadmap for a method of cultivating highly sterile triploid oysters using a three-way hybridization method according to the present invention.

[0024] Figure 2 Flow cytometry analysis of a selected population of Hong Kong oysters.

[0025] Figure 3 This is a flow cytometry image of a hybrid tetraploid population.

[0026] Figure 4 This is a flow cytometry image of highly sterile triploid oysters. Detailed Implementation

[0027] The following examples further illustrate the method for cultivating highly sterile triploid oysters according to the present invention, but are not intended to limit the scope of the invention.

[0028] Example:

[0029] (1) Establishment of the tetraploid breeding population (GGGG) of the Pacific oyster “Haida No. 1”

[0030] a. Triploid Induction in the 'Haida No. 1' Crab Oyster: Mature individuals from the diploid breeding population of the 'Haida No. 1' Crab Oyster were dissected, and sexes were identified under a microscope. Sperm and eggs were collected separately. Sperm were filtered through 160-mesh and 500-mesh silk screens to remove impurities; eggs were filtered through 120-mesh and 300-mesh silk screens. Before fertilization, eggs were continuously rinsed through a 500-mesh silk screen to remove tissue fluid and then matured in seawater for 30-60 minutes. During fertilization, approximately 5-10 sperm were positioned around each egg. When approximately 20%-30% of the fertilized eggs showed the first polar body (approximately 15 minutes), 0.75 mg / L cytochalasin B (CB) was immediately added and treated for 15 minutes. After treatment, the eggs were soaked in seawater containing 1% dimethyl sulfoxide (DMSO) for 30 minutes, followed by rinsing 2-3 times with filtered seawater. Flow cytometry was used to detect the ploidy of the D-shaped larvae. Batches with a triploid induction rate of ≥95% were retained for further cultivation to obtain the triploid population of Crassula 'Haida No. 1'.

[0031] b. Tetraploid Induction of Crassula 'Haida No. 1': Dissect triploid individuals of Crassula 'Haida No. 1' and select mature female triploids as maternal parents; dissect diploid individuals of Crassula 'Haida No. 1' and select individuals with full gonads and strong sperm motility as paternal parents. The sperm and egg treatment and maturation methods are the same as above. Fertilization is performed, controlling approximately 5-10 sperm cells around each egg. When the first fertilized egg is observed to form the first polar body (8-10 min), immediately add 1 mg / L CB and continue treatment for 8-10 min. After treatment, soak in seawater containing 1% DMSO for 30 min and rinse 2-3 times with filtered seawater. Flow cytometry is used to detect the ploidy of the D-shaped larval stage. Batches with a tetraploid induction rate ≥80% are retained for further cultivation to obtain tetraploid Crassula 'Haida No. 1'.

[0032] c. Breeding of the tetraploid population of Crassula 'Haida No. 1': Using the tetraploid population of Crassula 'Haida No. 1' as the base population, individuals ranking in the top 10% by shell height and wet weight were selected as parents; at least 100 male and 100 female individuals were selected, and fertilization was carried out at a sperm-to-egg ratio of 50:1. Three generations of continuous breeding were conducted to obtain a tetraploid breeding population of Crassula 'Haida No. 1' with stable ploidy and growth advantages, denoted as GGGG.

[0033] (2) Establishment of the Fujian Oyster Tetraploid Breeding Population (AAAA)

[0034] a. Selection and breeding of diploid Fujian oyster population: Using wild Fujian oyster diploids as the base population, individuals in the top 10% of shell height and wet weight were selected as candidate parents; identification was performed using COⅠ and ITS2 primers, and non-Fujian oysters or suspected hybrid individuals were eliminated, retaining purebred individuals for breeding; at least 100 male and 100 female individuals were used for fertilization at a sperm-to-egg ratio of 50:1. Three generations of continuous population selection were conducted to obtain a selected Fujian oyster diploid breeding population.

[0035] b. Triploid Induction in Fujian Oysters: Dissect mature individuals from the diploid breeding population of Fujian oysters, identify males and females under a microscope, and collect sperm and eggs separately. The sperm and egg treatment and maturation methods are the same as above. During fertilization, control the number of sperm cells around each egg to approximately 5-10. When approximately 20%-30% of the fertilized eggs show the first polar body (approximately 15 minutes), immediately add 0.75 mg / L cytochalasin B (CB) and continue treatment for 15 minutes. After treatment, soak in seawater containing 1% dimethyl sulfoxide (DMSO) for 30 minutes, then rinse continuously with filtered seawater 2-3 times. Flow cytometry is used to detect the ploidy of the D-shaped larvae. Batches with a triploid induction rate ≥95% are retained for further cultivation to obtain a triploid population of Fujian oysters.

[0036] c. Induction of tetraploid oysters in Fujian: Dissect triploid individuals of Fujian oysters and select female triploids with mature gonads; simultaneously dissect diploid individuals of Fujian oysters and select individuals with strong sperm motility as paternal parents; sperm and egg treatment, maturation methods, and fertilization methods are the same as above; when the first fertilized egg is observed to form the first polar body (8-10 min), immediately add 1 mg / L CB and treat for 8-10 min. After treatment, soak in seawater containing 1% DMSO for 30 min and rinse 2-3 times with filtered seawater. Flow cytometry is used to detect the ploidy of the D-shaped larvae. Batches with a tetraploid induction rate ≥80% are retained for further cultivation to obtain tetraploid Fujian oysters.

[0037] d. Breeding of tetraploid Fujian oyster populations: Using tetraploid Fujian oysters as the base population, individuals in the top 10% of shell height and wet weight are selected as parents; there are no fewer than 100 male and female individuals, and fertilization is carried out at a sperm-to-egg ratio of 50:1. Continuous breeding of the population is carried out for 3 generations to obtain a tetraploid Fujian oyster breeding population with stable ploidy and growth advantage, denoted as AAAA.

[0038] (3) Establishment of hybrid tetraploid oyster (GGAA)

[0039] Dissect tetraploid oysters (GGGG), distinguish males and females under a microscope, and select mature male and female individuals as maternal parents; dissect tetraploid oysters (AAAA), distinguish males and females under a microscope, and select male individuals with strong sperm motility as paternal parents; each group consists of no fewer than 50 individuals, and fertilization is performed at a sperm-to-egg ratio of 50:1; follow the conventional hatching and larval rearing procedures to obtain a hybrid tetraploid population (GGAA).

[0040] (4) Establishment of diploid Hong Kong oyster breeding population

[0041] Using wild-caught Hong Kong oyster diploids as the base population, individuals in the top 10% of shell height and wet weight were selected as parents. Identification was performed using COⅠ and ITS2 primers to remove non-Hong Kong oysters or suspected hybrids. At least 100 male and 100 female individuals were fertilized at a sperm-to-egg ratio of 50:1. This population was continuously bred for three generations to obtain a Hong Kong oyster diploid breeding population (HH).

[0042] (5) Preparation of triploid oysters (HGA)

[0043] Dissecting diploid (HH) oysters, sexing was determined under a microscope, and mature male and female individuals were selected as maternal parents; dissecting tetraploid (GGAA) oysters, sexing was determined under a microscope, and male individuals with strong sperm motility were selected as paternal parents; after determining ploidy, 300 diploid Hong Kong oysters were retained. Figure 2 ), 50 hybrid tetraploids ( Figure 3 Fertilization was performed at a sperm-to-egg ratio of 50:1; a triploid population of three hybrids was obtained following standard hatching and larval rearing procedures. Figure 4 ), denoted as HGA.

[0044] (6) Comparative test:

[0045] The high-sterility triploid oysters (HGA) in (5) above were set as the experimental group. Purebred triploid Pacific oysters (GGG) and purebred triploid Fujian oysters (AAA) were constructed with the same number of parents (GGG group: diploid Pacific oyster ♀ × tetraploid Pacific oyster ♂; AAA group: diploid Fujian oyster ♀ × tetraploid Fujian oyster ♂) as control group 1 and control group 2. They were bred according to conventional culture methods and transferred to the main aquaculture areas of Rongcheng City and Rushan City, Shandong Province, and cultured using the hanging cage method. The production data of the three groups of triploid oysters are recorded in Table 1. During the breeding season (May-August), no less than 30 individuals (n≥30) were randomly selected from each group in the two locations, and the gonads were dissected and histological sections were observed. The proportion of individuals without mature gametes or with inhibited gonadal development was used as the sterile individual ratio.

[0046] Table 1 shows that the sterile ratio of the HGA group was 92.50% in Rongcheng and 90.00% in Rushan. Compared with the GGG group (75.00% in Rongcheng and 71.67% in Rushan), the HGA group increased by 17.50 and 18.33 percentage points, respectively; compared with the AAA group (55.83% in Rongcheng and 52.50% in Rushan), the HGA group increased by 36.67 and 37.50 percentage points, respectively. The survival rate of the HGA group was 73.20% in Rongcheng and 70.40% in Rushan, which increased by 5.10 and 4.40 percentage points compared with the GGG group (68.10% in Rongcheng and 66.00% in Rushan), respectively; and increased by 1.90 and 1.50 percentage points compared with the AAA group (71.30% in Rongcheng and 68.90% in Rushan), respectively. The wet weight of the HGA group was 73.60 g in Rongcheng and 72.80 g in Rushan, which was 3.50 g and 4.30 g higher than the AAA group (70.10 g in Rongcheng and 68.50 g in Rushan), respectively. Compared with the GGG group (75.20 g in Rongcheng and 73.50 g in Rushan), the wet weight was slightly lower, by 1.60 g and 0.70 g, respectively, but still at a relatively high level overall. The HGA group triploid hybrids bred in this invention showed a significantly higher proportion of sterile individuals, a better survival rate than the control, and maintained a high wet weight in both locations, demonstrating good comprehensive production and application value.

[0047] Table 1. Comparison of production performance between highly sterile three-way hybrid triploid oysters (HGA) and the control group. Place Group Wet weight (g) Survival rate (%) Percentage of infertile individuals (%) Rongcheng HGA group 73.60 73.20 92.50 Group AAA 70.10 71.30 55.83 GGG Group 75.20 68.10 75.00 Rushan HGA group 72.80 70.40 90.00 Group AAA 68.50 68.90 52.50 GGG Group 73.50 66.00 71.67

[0048] 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 highly sterile triploid oysters, characterized in that, Includes the following steps: a. Using the diploid 'Haida No. 1' (GS-01-005-2013) of the Pacific oyster as the base population, triploid 'Haida No. 1' of the Pacific oyster was induced to be obtained; using the triploid 'Haida No. 1' of the Pacific oyster as the female parent and the diploid 'Haida No. 1' of the Pacific oyster as the male parent, tetraploid 'Haida No. 1' of the Pacific oyster was induced to be a base population; the tetraploid 'Haida No. 1' of the Pacific oyster was subjected to continuous 2-3 generations of population selection and breeding to obtain the tetraploid breeding population of 'Haida No. 1' of the Pacific oyster; b. Using the wild diploid population of Fujian oysters as the base population, select the top 10% of individuals in terms of shell height and wet weight as parents, and conduct continuous 2-3 generations of population selection to obtain the Fujian oyster diploid breeding population; c. Using the diploid Fujian oyster breeding population obtained in step (b) as the parent, induce the production of triploid Fujian oysters; using the triploid Fujian oysters as the female parent and the diploid Fujian oyster breeding population obtained in step (b) as the male parent, induce the production of a tetraploid Fujian oyster basic population; conduct continuous 2-3 generations of population selection on the tetraploid Fujian oyster basic population to obtain a tetraploid Fujian oyster breeding population; d. Using the tetraploid oyster breeding population obtained in step (a) as the female parent and the tetraploid oyster breeding population obtained in step (c) as the male parent, hybridize to obtain a tetraploid oyster hybrid of oyster and oyster (GGAA). e. Using the diploid population of Hong Kong oysters as the base population, select the top 10% of individuals in terms of shell height and wet weight as parents, and conduct continuous 2-3 generations of population selection to obtain the Hong Kong oyster diploid breeding population; f. Using the diploid oyster breeding population obtained in step (e) as the female parent and the hybrid tetraploid oyster GGAA obtained in step (d) as the male parent, hybridization was carried out to obtain the three-way hybrid triploid oyster (HGA).

2. The breeding method according to claim 1, characterized in that, The triploid induction involved treating the larvae with 0.75 mg / L cytochalasin B for 15 minutes after fertilization to inhibit polar body expulsion. After treatment, the larvae were soaked in seawater containing 1% dimethyl sulfoxide for 30 minutes and thoroughly rinsed with filtered seawater. Flow cytometry was used to detect the ploidy of the larvae, and the triploid rate was selected to be ≥95%. The tetraploid induction involved determining the ploidy of the maternal parent before fertilization and selecting individuals with a ploidy of 3N as the maternal parent. After fertilization, the larvae were treated with 1 mg / L cytochalasin B for 8–10 minutes after fertilization. After treatment, the larvae were soaked in seawater containing 1% dimethyl sulfoxide for 30 minutes and thoroughly rinsed with filtered seawater. Flow cytometry was used to detect the ploidy of the larvae, and batches with a tetraploid rate of ≥80% were selected for cultivation and breeding.

3. The breeding method according to claim 1, characterized in that, Both diploid and tetraploid populations were selected for breeding. Individuals with good vigor, 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. Diploid candidate parents were genetically identified using COⅠ and ITS2 before fertilization to select purebred parents. Tetraploid candidate parents were ploidy determined before fertilization, and individuals with a ploidy of 4N were selected as parents. Each generation of breeding used no fewer than 100 male and female parents for reproduction, and high-intensity breeding was carried out for 2 to 3 consecutive generations.

4. The breeding method according to claim 1, characterized in that, In step (d), the ploidy of the hybrid parents is determined before fertilization, and parents with a ploidy of 4N are selected for hybridization, with no less than 50 males and 50 females.

5. The breeding method according to claim 1, characterized in that, In step (f), the ploidy of the three-way hybrid parents is determined before fertilization, and individuals with a maternal ploidy of 2N and a paternal ploidy of 4N are selected for hybridization.

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