CN104255587A - Building method and application of Argopecten irradians irradians and Argopecten purpuratus hybrid scallop maintainer line - Google Patents

Abstract

The invention relates to a construction method and application of a sea purple hybrid scallop maintainer line. The maintainer line is a purple scallop line that can make the male sterile line progeny of the corresponding Haizi hybrid scallop still have the characteristics of male sterility and egg fertility, wherein the Haizi hybrid scallop is a hybrid offspring of bay scallop eggs and purple scallop sperm. The construction method is to first construct a large number of Haizi hybrid scallop offspring families, and find male sterile individuals from them for the selection of male sterile lines; The male sterile line-maintainer combination is a male sterile individual; finally, the male sterile line and the maintainer line are crossed to produce male sterile broodstock, which is used to produce sea purple hybrid scallop commercial seedlings. The invention establishes the maintainer line of scallops in animals for the first time, effectively overcoming the obstacle of self-fertilization in the hybridization of hermaphroditic scallops.

Description

Construction method and application of Haizi hybrid scallop maintainer line

technical field

The invention belongs to shellfish breeding technology-a method for cultivating commercial hybrid scallop seedlings, and in particular relates to the construction and application of a maintainer line of Haizi hybrid scallop (the hybrid offspring of bay scallop ova and purple scallop sperm).

Background technique

Bay scallops (Argopecten irradians irradians) are native to the Atlantic coast of the United States and were introduced to my country in 1982. The bay scallop grows fast, has a wide range of temperature adaptability, and has strong stress resistance. It is very suitable for farming in the northern seas of my country. At present, the output and farming area account for more than 50% of the scallop farming in my country. However, due to the long-term inbreeding after the introduction, the germplasm of bay scallops has been seriously degraded during the breeding process. The size of commercial shellfish is getting smaller and smaller, and the mortality rate in the breeding process is high. Therefore, the scallop farming industry urgently needs genetic improvement of bay scallops. , among which cross breeding is one of the most effective germplasm improvement methods.

For this reason, the inventor has introduced purple scallop (Argopecten purpuratus) from Peru for the first time since 2007. The bay scallop is a fast-growing medium-sized scallop native to the South Pacific. It and the bay scallop belong to the genus Argopecten. They have the same number of chromosomes and are both hermaphrodites. However, they complement each other in terms of individual size, growth, and temperature adaptability. Therefore, the inventors crossed the bay scallop with the purple scallop again, and cultivated two kinds of hybrid offspring generation: orthogonal (fertilization of purple scallop ovum and bay scallop sperm) and reverse cross (fertilization of bay scallop ovum and purple scallop sperm). The individual weight of the first generation of hybrids is about 100% higher than that of the purple scallops cultivated in the same period. It can grow to 7-8 cm in the first year, and can continue to be cultivated to a larger size in the second year. The individual weight can reach more than 200 grams, and its stress resistance is enhanced. , especially in Shandong, which can survive summer and winter smoothly, showing great industrial potential (Wang Chunde et al., 2009. Studies on interspecific hybridization of bay scallops and purple scallops. Marine Science, 33(10): 84-87; Chunde Wang et al., 2011. Introduction of the Peruvian scallop and its hybridization with the bay scallop in China, Aquaculture, 2011, 310:380-387).

Although the above-mentioned first generation of hybrids can produce greater heterosis, it is still difficult to popularize in a large area. This is because the purple scallop and the bay scallop are both hermaphroditic scallops, and they emit sperm and eggs at the same time during reproduction. Eggs are easily polluted by the same kind of sperm, and it is difficult to obtain a large number of unfertilized eggs for interspecific hybridization, resulting in a greatly reduced proportion of real hybrid offspring in the obtained commercial seedlings, thus greatly limiting the increase in yield.

The study found that the hermaphroditism of the above two species of Argopecten scallops is similar to that of many crop plants. In monoecious crops such as rice breeding, there is also the problem of same flower pollination, which requires artificial detasseling, which is similar to the self-fertilization problem encountered in the crossbreeding of bay scallops and purple scallops. The key to solving this problem in agriculture depends on the discovery of male sterile plants. Using male sterile plants to continuously backcross with their male parents, male sterile lines, maintainer lines and restorer lines can be selected to construct crop breeding. The three-line supporting breeding system in the system realizes the large-scale production of hybrids, thereby solving the problem of hybrid seed production. So far, a three-line matching breeding system has been established in many crops at home and abroad, and has been widely used in the production of hybrids, which has become the main way to greatly increase the yield of crops.

However, in animal breeding, there have been no reports of male sterile and female fertile individuals at home and abroad so far, so it is impossible to establish male sterile lines similar to crops and their maintainer and restorer lines for hybrid breeding. Obviously, if a similar three-line breeding system including male sterile lines, maintainer lines and restorer lines can be constructed in animal breeding, low-cost, large-scale interspecific hybridization of hermaphroditic animals can be realized, and hybridization efficiency can be improved , improve the stress resistance and yield of breeding animals, and thus achieve a major breakthrough in animal breeding technology. The male sterile line and its maintainer line are important components of the three-line breeding system and the key to large-scale production of male sterile line parents.

Contents of the invention

The purpose of the present invention is to realize in animal breeding, specifically to construct sea purple hybrid scallop maintainer line for the first time in hermaphroditic scallop, so as to overcome the deficiency of existing hybrid scallop breeding technology.

Another object of the present invention is to establish an application method for large-scale production of the sea purple hybrid scallop male sterile line by crossing the above-mentioned maintainer line with the male sterile line.

The above-mentioned male sterile line is a strain in which the male gonads are abnormally developed and cannot produce functional sperm, but its eggs develop normally, can receive normal sperm for fertilization, and can pass on its male sterile characteristics to offspring. Obviously, the male sterile line is the core line in the three-line system, and its main function is to provide a large number of unfertilized eggs in the commercial production of hybrid seedlings, and its breeding depends on the corresponding maintainer line. The male sterile line is the establishment of a backcross family by continuous backcrossing of accidentally discovered male sterile individuals with fertile eggs and their fathers, when all individuals in the backcross family are male sterile and fertile eggs Scallop strains.

The maintainer line is also called the male sterile line maintainer. When it is used as a male parent to cross with the male sterile line, it can make the offspring maintain the male sterile characteristics. The main function of the maintainer line is to provide the sperm necessary for the male sterile line to breed. The maintainer line is a purple scallop strain obtained by continuously backcrossing purple scallops with male sterile individuals, and when all the backcrossed offspring are male sterile and eggs are fertile. The maintainer line is a hermaphroditic strain capable of normal breeding, and the next generation of maintainer lines can be bred through selfing.

The present invention is based on the following ideas and practices: 1) The inventors have found that there are 2-10% male sterile individuals in the first generation of Haizi hybrid scallop (the hybrid offspring of bay scallop ovum and purple scallop sperm), and these individuals do not produce positive active sperm, but its eggs can be fertilized by the sperm of purple scallop, and the obtained fertilized eggs can develop normally, that is, the backcross scallop is obtained; 10% of the individuals are still male sterile and their eggs can be fertilized by the sperm of purple scallop, so as long as the eggs of the above male sterile individuals are backcrossed with the sperm of the self-bred offspring of the male parent purple scallop for multiple generations, they can be bred A male sterile line (female parent)-Purple scallop (male parent) combination in which all offspring are male sterile and eggs are fertile, and the purple scallop line in this combination has the ability to keep the offspring of the corresponding male sterile line male sterile The ability to obtain the male sterile line of Haizi hybrid scallop and the maintainer line of the male sterile line; 3) The hybridization of the maintainer line of Haizi hybrid scallop with its corresponding male sterile line can produce large-scale male sterile and Egg-fertile sea violet hybrid scallop male sterile line shellfish.

The construction method of the Haizi hybrid scallop maintainer line of the present invention is as follows:

1) Screening of male sterile individuals of Haizi hybrid scallops and breeding of their male parents: After selecting excellent bay scallops and purple scallops to promote ripening synchronously, the bay scallops and purple scallops were induced to produce sterility by conventional methods of drying in the shade and temperature stimulation of purple scallops. eggs, and collect the eggs of bay scallops and purple scallops respectively, induce purple scallops to ejaculate, and filter with 500 mesh sieves to obtain purple scallop sperm, and then add an appropriate amount of purple scallop sperm to the eggs of bay scallops, after the fertilized eggs develop normally That is, a first-generation family of sea purple scallops (sea scallop ova fertilized with purple scallop sperm) is obtained, which will be used to select male sterile individuals. At the same time, the purple scallop ova are fertilized with their own sperm to establish an inbred family of the purple scallop. Since the proportion of male sterile individuals in the first generation of hybrids is very low, it is necessary to construct a large number of families of the first generation of hybrids to find male sterile individuals for the selection of maintainer lines.

2) Breeding of the Haizi hybrid scallop maintainer line: Select the male sterile individuals in the above-mentioned Haizi hybrid scallop offspring family, and promote maturation synchronously with the excellent individuals in the male parent purple scallop inbred family. After the gonads mature, use conventional The method of drying in the shade and temperature stimulation of purple scallops induces ovulation of the above-mentioned first-generation hybrid scallops and collects eggs, and induces purple scallops to ejaculate sperm at the same time, and obtains purple scallop sperm by filtering with a 500-mesh sieve. Fertilization, collect fertilized and normally developed fertilized eggs to establish a backcross family, and at the same time, self-fertilize the donated purple scallop to establish a self-bred family; in the next year, the backcross family and its male purple scallop self-bred family will be matured, and the backcross family will be selected After the individuals who were still male sterile were stimulated to lay eggs by the conventional method of oviposition induction of purple scallop, they continued to backcross with the sperm of the self-bred offspring of the male parent purple scallop, and at the same time continued to self-seed the purple scallop that provided the sperm to establish the self-fertilization. Afterwards, the same method will be used for continuous backcross selection every year until all the offspring of the backcross are male sterile and fertile individuals, and the backcross family will become the male sterile line of Haizi hybrid scallop. The purple scallop inbred family whose offsprings of the male sterile line continue to maintain the male sterile property adopts the conventional method of raising seedlings of purple scallop to self-propagate to obtain the maintainer line of the male sterile line. The main function of the maintainer line is to provide the sperm necessary for the reproduction of the male sterile line, which is the male line necessary for the male sterile line to be passed on.

3) Application of the maintainer line of Haizi hybrid scallop: the male sterile line of Haizi hybrid scallop with fertile eggs can be bred on a large scale by crossing the maintainer line with the corresponding male sterile line of Haizi hybrid scallop scallop, and used in the production of sea purple hybrid scallop commercial seedlings.

The maintainer line construction method of the present invention is also applicable to Zihai hybrid scallop (the hybrid offspring of purple scallop egg and Gulf scallop sperm), Zimo hybrid scallop (the hybrid offspring of purple scallop egg and Mexico Gulf scallop sperm) and Mozi hybrid scallop ( The breeding practice of hybrid scallops of the genus Argopecten such as the hybrid progeny of Gulf of Mexico scallop eggs and purple scallop sperm, and is applied to the large-scale production method for establishing commercial seedlings of these hybrid scallops.

The method of the present invention is also applicable to the interspecific cross breeding of other hermaphroditic scallops, such as the interspecific cross breeding of scallops of the genus Pecten, and the basic method of the present invention is also applicable to the interspecific cross breeding of other hermaphroditic invertebrates.

The Haizi hybrid scallop maintainer line constructed by the present invention is the first maintainer line established in the animal kingdom, and should belong to a major breakthrough in animal breeding technology. The invention effectively overcomes the obstacle of self-fertilization in the hybridization process of hermaphroditic scallops, and makes it possible to produce complete hybrid offspring at low cost and on a large scale.

Detailed ways

The present invention constructs the maintainer line of the sea purple hybrid scallop, and establishes a method for large-scale production of sea purple hybrid scallop male sterile line seed shellfish with the maintainer line, and the specific steps are as follows:

Example 1

1) Screening of male sterile individuals of sea purple hybrid scallops and breeding of their male parents: select 50 sea scallops with mature gonads and 50 purple scallops in the first year, and place each scallop in the pre-added Spawn eggs and ejaculate in a separate container in seawater at 23°C, collect the ova of each bay scallop and the sperm and eggs of each purple scallop separately, add a small amount from a single purple scallop to the egg suspension of each bay scallop Stir to mix the sperm and eggs evenly, observe under a microscope so that there are 5-6 sperm around each egg, and place the fertilized eggs in a dark environment to hatch, that is, to obtain the first-generation family of Haizi hybrids. Establish at least 20 such hybrid families with the above method, and simultaneously fertilize the fertilized purple scallop sperm eggs to establish a corresponding number of purple scallop self-bred families, and carry out larvae cultivation, seedling preservation and cultivation according to the conventional scallop seedling cultivation method, Screening for male sterile individuals.

2) Breeding of the maintainer line of Haizi hybrid scallops: After the first-generation family of Haizi hybrids is matured in the spring of the second year, 50 male sterile individuals with excellent traits are selected, and egg-laying is stimulated by the conventional method of egg-laying induction of purple scallops. , respectively fertilized with the sperm of the male purple scallop, collected the normally fertilized and developed fertilized eggs to establish a backcross family, and collected the normally fertilized and developed fertilized eggs to establish a backcross family, and at the same time, the purple scallop that provided the sperm was self-propagated to establish a self-cross Family, establish at least 20 backcross families and corresponding purple scallop self-bred families, and carry out larva cultivation, seedling preservation, and cultivation according to conventional purple scallop seedling raising methods; After ripening, select 50 male sterile individuals in the backcross family with excellent traits, backcross with the self-bred progeny sperm of the male purple scallop, collect normally fertilized and developed fertilized eggs to continue to establish the backcross family, and continue to breed at the same time For the self-bred family of purple scallop, choose to establish more than 20 backcross families with excellent traits and their corresponding purple scallop self-bred families; in the future, select male sterile scallops with good traits and their male purple scallop self-bred families in the same way every year The family line is backcrossed until all the individuals of the backcrossed offspring show excellent traits, male sterility and fertile eggs, that is, the male sterile line of Haizi hybrid scallop is obtained; then the above-mentioned backcrossed offspring that can make the male sterile line All individuals showed excellent traits, male sterility and fertile eggs. The male purple scallop was self-bred in a large number according to the conventional purple scallop seedling breeding method, and the maintainer line of the male sterile line of the sea purple hybrid scallop was obtained.

3) The method of producing the male sterile line of Haizi hybrid scallop with the maintainer line: use the above-mentioned maintainer line to cross the corresponding male sterile line of Haizi hybrid scallop every year to obtain male sterile and fertile sea scallops. The male sterile line of purple hybrid scallop is used for large-scale production of sea purple hybrid scallop commercial seedlings.

Example 2

At the end of March 2008, the bay scallops with mature gonads were taken, and the eggs of bay scallops were obtained after temperature stimulation. After insemination with the sperm of purple scallops, the first-generation seedlings of hybrids of bay scallops and purple scallops were obtained and raised. In the spring of 2009, the first-generation scallops of the above-mentioned hybrids were matured, and the scallops that were large and only developed in the female area were selected from them. After 30 minutes of drying in the shade, the scallops were placed in a container filled with seawater at 23°C to stimulate spawning. The sperm of the scallop was mixed and fertilized, and the backcross population of the sea purple hybrid scallop and the purple scallop was bred. Most of the backcross populations were hermaphroditic after maturation in the spring of 2010, but <10% of the individuals were still male sterile.

Continue to backcross the above-mentioned male sterile individuals with the sperm of the self-bred progeny of the male parent purple scallop, and after one or more generations of selective breeding, a male sterile line (female parent) in which all offspring are male sterile and eggs are fertile can be obtained - Combination of maintainer lines (male parents) to obtain male sterile lines and maintainer lines respectively. After the gonads are stimulated to induce maturation, a large number of unfertilized eggs can be obtained by using the conventional purple scallop oviposition induction method to obtain a large number of unfertilized eggs, which can be fertilized with maintainer sperm, and large-scale production of sea purple hybrid scallop commercial seedlings can be obtained. Male sterile line of shellfish.

Example 3

In 2012, 20 male sterile individuals in the first-generation family of Haizi hybrids were stimulated to lay eggs. These eggs were backcrossed with the sperm of a single purple scallop, and 2 were successfully obtained. When the backcrossing families were harvested in mid-November 2012, compared with the bay scallops in the same period, the shell height, shell length, shell width and weight of one of the backcrossing families increased by 28.2%, 29.5%, 16.4% and 74.0%, respectively. After maturation in the spring of 2013, it was found that most of the offspring only developed in the female region and did not develop in the male region, which proved that the male sterile line and its maintainer line of Haizi hybrid scallop can be bred by backcrossing with purple scallop sperm and male sterile individuals.

Example 4

In 2013 and 2014, a total of 12 first-generation hybrid families of bay scallop eggs and purple scallop sperm were established using imported bay scallops and purple scallops, and self-bred families of purple scallops for sperm were established, according to the method in Example 1 , the male sterile line of Haizi hybrid scallop and its corresponding maintainer line can be selected and bred within 1-3 years, and the commercial production of the male sterile line of Haizi hybrid scallop can be realized.

Example 5

The present invention is used for the construction and application of Zihai hybrid scallop maintenance system

The method for constructing the maintainer line established by the present invention is also applied to the breeding of Zihai hybrid scallop (hybrid progeny of purple scallop egg and bay scallop sperm), and is applied to the large-scale production method for establishing Zihai hybrid scallop commercial seed.

Firstly, purple scallops and bay scallops are selected to promote maturation simultaneously and stimulate simultaneous oviposition and ejaculation. The eggs of purple scallops and the sperm and eggs of bay scallops are collected separately, and the eggs of purple scallops and the sperm of bay scallops are fertilized to establish the generation of purple sea hybrids. The family is used to select male sterile individuals. At the same time, the sperm and eggs of bay scallops are mixed and fertilized to establish a bay scallop inbred family; The sperm fertilizes it, and if the fertilized eggs develop normally, a backcross family is established to continue to select male sterile lines, and at the same time, the male parent bay scallop is self-fertilized to establish an inbred family; in this way, after one or more generations of selection, when all the backcross offspring are When male sterility is shown, male sterile lines and corresponding maintainer lines of Zihai hybrid scallops are obtained.

By hybridizing the egg of the male sterile line of the Zihai hybrid scallop with the sperm of the maintainer line, efficient, convenient and large-scale production of the male sterile line of the Zihai hybrid scallop can be realized.

Example 6

The invention is used for the construction and application of purple ink hybrid scallop maintainer system

The method for constructing the maintainer line established by the present invention is also applied to the breeding of Zimo hybrid scallops (hybrid progeny of purple scallop ova and Gulf of Mexico scallop sperm), and is applied to the large-scale production method for establishing commercial seedlings of Zimo hybrid scallops.

First, purple scallops and Gulf scallops are selected to promote maturation simultaneously and stimulate simultaneous oviposition and ovulation. The eggs of purple scallops and the sperm and eggs of Gulf scallops are collected separately, and the eggs of purple scallops are fertilized with the sperm of Gulf scallops to establish Zimo. The first-generation hybrid family is used to select male sterile individuals. At the same time, the sperm and eggs of Gulf scallops are mixed and fertilized to establish a self-bred family of Gulf scallops; male sterile individuals are selected from the first-generation hybrid Zimo family in the second year , use the sperm of the Gulf of Mexico scallop to fertilize it, and if the fertilized eggs develop normally, a backcross family is established to continue to select a male sterile family, and at the same time, the male parent of the Gulf of Mexico scallop is self-fertilized to establish an inbred family; in this way, after one or more generations Selection, when all the backcross offspring are male sterile, the male sterile line and the corresponding maintainer line of Zimo hybrid scallop are obtained.

By hybridizing the egg of the male sterile line of the Zimo hybrid scallop with the sperm of the maintainer line, efficient, convenient and large-scale production of the male sterile line of the Zimo hybrid scallop can be realized.

Example 7

The invention is used for the construction and application of the maintainer line of Mozi hybrid scallop

The method for constructing the maintainer line established in the present invention is also applied to the breeding practice of Mozi hybrid scallop (the hybrid offspring of Gulf of Mexico scallop egg and purple scallop sperm), and is applied to the large-scale production method for establishing commercial seedlings of Mozi hybrid scallop.

Firstly, purple scallops and Gulf scallops are selected to promote maturation simultaneously and stimulate simultaneous oviposition and sperm ovulation. The eggs of Gulf scallops and the sperm and eggs of purple scallops are collected separately, and the eggs of Gulf scallops and the sperm of purple scallops are fertilized to establish Mozi The first-generation family of hybrids is used to select male sterile individuals. At the same time, the sperm and eggs of purple scallops are mixed and fertilized to establish a self-bred family of purple scallops; The sperm of the purple scallop is fertilized with it. If the fertilized egg develops normally, a backcross family is established to continue to select a male sterile family. At the same time, the male parent purple scallop is self-fertilized to establish an inbred family; When all offspring showed male sterility, the male sterile line of Mozi hybrid scallop and its corresponding maintainer line were obtained.

By hybridizing the eggs of the male sterile line of the Mozi hybrid scallop with the sperm of the maintainer line, efficient, convenient and large-scale production of the male sterile hybrid scallop of the Mozi hybrid can be realized.

Claims (9)

1. the maintainer of the purple scallop hybrid in sea, it is characterized in that, this maintainer the male sterile line offspring of corresponding extra large purple scallop hybrid can be made still to have male sterile and ovum can educate the purple scallop strain of characteristic, and wherein extra large purple scallop hybrid is the filial generation of bay scallop ovum and purple scallop sperm.

2. the construction method of the purple scallop hybrid maintainer in the sea of claim 1, is characterized in that:

1) screening of extra large purple scallop hybrid male sterile individuality and breeding of male parent thereof: first build 20 with purple scallop hybrid first filial generation, Shanghai family for screening male sterile individuality, set up the purple scallop self-fertilization family (sff) of male parent for essence, for the seed selection of following maintainer simultaneously;

2) seed selection of extra large purple scallop hybrid maintainer: Second Year selects 50 male sterile individualities from the family of purple scallop hybrid first filial generation, above-mentioned sea, it is stimulated to ovulate after accelerating, with the sperm fertilization of defect individual in the purple scallop self-fertilization family (sff) of the male parent of this male sterile individuality, fertilized egg foundation of collecting normal development backcrosses family more than 20, will set up self-fertilization family (sff) for the purple scallop self-fertilization of the male parent of essence simultaneously; To select next year to backcross in family still for male sterile individual 50 acceleratings and after stimulating and laying eggs, continue to backcross with the sperm of the purple scallop self progeny of the male parent of this male sterile individuality, set up more than 20 familys that backcross, selfing will be continued for the purple scallop of the male parent of essence simultaneously and set up self-fertilization family (sff); Later annual continuous backcross seed selection after the same method, until backcross progeny is all male sterile and the individuality that can educate of ovum, namely obtains extra large purple scallop hybrid male sterile line; Male sterile line offspring can be made to continue to keep the conventional Process of Rearing Scallop Seeds method selfing of the purple scallop self-fertilization family (sff) of male sterile characteristic to expand numerous by above-mentioned again, namely obtain the maintainer of this extra large purple scallop hybrid male sterile line.

3. the purple scallop hybrid maintainer in the sea of the claims 1 and claim 2 is applied to the purple scallop hybrid male sterile line kind shellfish in large-scale production sea; namely hybridize with the purple scallop hybrid male sterile line in maintainer and corresponding sea, with propagation in scale male sterile and the ovum extra large purple scallop hybrid male sterile line kind shellfish of the next generation that can educate.

4. the construction method of the purple scallop hybrid maintainer in the sea of claim 2 foundation is also applied to the breeding of following each scallop hybrid:

(1) with the purple sea scallop hybrid that purple scallop ovum and bay scallop sperm fertilization are cultivated;

(2) with the purple ink scallop hybrid that purple scallop ovum and Gulf of Mexico scallop sperm fertilization are cultivated;

(3) with the violet black scallop hybrid that Gulf of Mexico scallop ovum and purple scallop sperm fertilization are cultivated.

5. the application method of the purple scallop hybrid maintainer in the sea of claim 3 foundation is also applied to the breeding of following each scallop hybrid:

(1) with the purple sea scallop hybrid that purple scallop ovum and bay scallop sperm fertilization are cultivated;

(2) with the purple ink scallop hybrid that purple scallop ovum and Gulf of Mexico scallop sperm fertilization are cultivated;

(3) with the violet black scallop hybrid that Gulf of Mexico scallop ovum and purple scallop sperm fertilization are cultivated.

6. the construction method of the purple scallop hybrid maintainer in the sea of claim 2 foundation is also applied to other hermaphroditism type scallops, and such as Argopecten genus and Pecten belong to the interspecific cross breeding of scallop.

7. the sea purple scallop hybrid maintainer application method that claim 3 is set up also is applied to other hermaphroditism type scallops, and such as Argopecten genus and Pecten belong to the interspecific cross breeding of scallop.

8. the basic construction method of the purple scallop hybrid maintainer in the sea of claim 2 foundation is also applied to the interspecific cross breeding of other hermaphroditism type invertebrates.

9. the application method of the purple scallop hybrid maintainer in the sea of claim 3 foundation is also applied to the interspecific cross breeding of other hermaphroditism type invertebrates.