TWI407903B - Breeding method of compound diploid fish - Google Patents

Description

Breeding method of compound diploid fish

The invention relates to a compound diploid aquatic animal such as fish, shellfish and crustacean, and a breeding method thereof, in particular to a compound diploid aquatic animal which does not produce genetic separation and can stably breed a hybrid next generation.

Aquatic animals such as fish are fertilized first for interspecies or intergeneric crosses before they begin to produce embryos. However, hybrids will almost always die. Even if they survive, they will be incompletely combined due to the heterogeneity of chromosomes, unable to form a partner, and eventually cause infertility. Even if the hybrids between close relatives can be combined and have fertility, the next generation will also produce hereditary separation. For each generation, the excellent traits (hybrid strength) will be reduced compared with the parents.

Fig. 11 and Fig. 12 are diagrams showing a combination pattern of genomes at the time of hybrid breeding and the first generation of hybrids. The patent scope, specification and drawings of the present invention denote a general term for various (A, B) genomic groups (chromosomes) by A and B, and X and Y represent sex chromosomes. Further, when various genomic groups such as A species and B species and their sex chromosomes are clearly indicated, A chromosomes are represented by A _X or A _Y , and B chromosomes are represented by B _X or B _Y . Since the eggs before fertilization of the fish are the eggs before the second polar body is discharged, even if the eggs are reduced, the eggs produced by the individuals of the chromosome AA are still AA, and the second polar body is discharged for the first time after the fertilization. Egg (A). In addition to preventing the discharge of the second polar body, even if the egg is not fertilized, it is regarded as A.

In Fig. 11, the chromosome of the first generation of the hybrid is A _X B _X or A _X B _Y , and regardless of the constitution of the parent genome A and the father chromosome B, it will eventually die or become pregnant. As shown in Figure 12, genetic separation occurs even if the surviving hybrid has fertility. It is assumed that the chromosomes A and B constituting the F1 hybrid (AB) are a1, a2, a3, and b1, b2, and b3, respectively. When normal meiosis is possible, the first set of chromosomes produces a couple of a1 or b1, the second set of chromosomes produces a couple of a2 or b2, and the third set of chromosomes produces a partner of c1 or c2, for a total of 8 genomic groups Spouse. Therefore, F2 will produce 64 combinations, and the first to third sets of chromosomes will be observed. The chromosomes between the a1a1 or b3b3 equivalents will produce two combinations. The extreme case is that, like a1a1a2a2a3a3, only the A chromosome is returned. The characteristic of this hybrid is that each chromosome (also arguably each genetic) is separated into A or B species. To avoid the death of interspecific or intergeneric hybrids, it is best to perform a doubling of the chromosome combination. The doubling treatment, especially the doubling based on the prevention of somatic cell division, is widely used in plants, but it is rare in animals.

In the case of the aquatic animal, in particular, the breeding technique of the homogenous triploid of the fish, there is a patent document of the Japanese version of the Japanese Patent Publication No. Hei 10-150883 and Japanese Patent Application Laid-Open No. Hei 10-327706. The latter is "Patent Document 2") for reference. However, in the case of heterogeneous triploids and heterotetraploids (hereinafter referred to as "complex diploids"), it has been reported that heterogeneous triploids can be obtained from salmon and trout (for example, the Japanese Fisheries Society supervises "aquaculture and breeding" Chromosome manipulation, published by the stellar society Houshengge, 87-92. hereinafter referred to as "Non-Patent Document 1"), but except for special examples, it is only a general technique for theoretically proposed compound diploids (refer to the non-patent literature). 1) Only. The dioecious aquatic animals in nature differ from the self-fertilized plants, and there are currently no examples of the presence of complex diploids.

Next, the fish breeding method of the heterologous triploid and the compound diploid is schematically illustrated in FIG. Both Fig. 13 and the breeding method are based on Fig. 8-2 and the description thereof described in Non-Patent Document 1.

To breed a heterologous triploid, you must first fertilize the eggs of type A (the eggs during the second mature division, the chromosomes are A _X A _X ) and the sperm of type B (the chromosomes are B _X or B _Y ). Step 1), then warming or pressurizing, can suppress the discharge of the second polar body (step 2 in the figure). Eggs in which the second polar body is inhibited, the chromosomes of which are tripled (AAB) (steps 3 to 5 in the figure), eventually become heterogeneous triploid fish (A _X A _X B _X or A _X A _X B _Y ).

To cultivate a heterotetraploid (hereinafter referred to as "complex diploid"), the eggs of the A species and the sperm of the B species must be fertilized first, and then the second polar body is not inhibited, but the step 5 is prevented or inhibited. One egg cut (the inventors believe that the first egg cut is not prevented, but the second egg cut is prevented, as will be described later in detail). The chromosome of the egg is tetraploid (AABB) and eventually becomes a compound diploid fish.

Crossing Oryzias Luzonensis and Oryzias Curvinotus is a specific example of cultivating a compound diploid fish (This Journal of the Fisheries Society 1993 59:373. Hereinafter referred to as "non-profit literature 2"). This technique is not directly inherited Non-Patent Document 1 disclosed technique, but the γ rays, ultraviolet rays, or X-ray irradiation of the non-hybrid subtraction egg (A _X B _X) (discharge electrode precursor as in A _X A _X On B _X B _X ), the genetically inactivated sperm mediator prevents the second mature division of the cytoplasmic egg to produce a composite diploid.

The chromosome of this composite diploid is A _X A _X B _X B _X , and the sister chromosome exerts the same function as the same chromosome, restoring fertility. Unless a female diploid breeding egg is used to breed a female, the offspring will hereditarily appear male.

The chromosome that can solve the problem of heterogeneous triploid or inter-hybrid death is A _X A _X B _X or A _X A _X B _Y , which usually has no fecundity due to incomplete chromosome combination during meiosis. Therefore, if you want to cultivate a specific interspecific hybrid, you must repeat the above method. It can be seen that the technique of triploidization cannot cultivate the offspring by natural mating, and the utilization of breeding is limited.

In theory, composite diploid aquatic animals are feasible, but the specific successful examples are only the above cases. And since the successful example is through female breeding, the cultivated individuals are all females (A _X A _X B _X B _X ). The composite diploid cultivated in Figure 13 is a female having the A _X A _X B _X B _X chromosome or a male having the A _X A _X B _Y B _Y chromosome, and the composite diploid currently cultivated by the theoretical method is still not There is a conclusion. Therefore, it can only be assumed that the male compound diploid does not exist in reality, or is theoretically A _X A _X B _Y B _Y type.

It can be seen that the breeding and utilization technology of the composite diploid aquatic animals is more closed than the heterologous triploid.

The object of the present invention is to provide a composite diploid aquatic animal and its breeding method, the hybrid does not die and has fertility. Even the hybrid offspring can maintain the hybrid strength, and through normal mating, can produce stable offspring, completely eliminating the inbreeding weakness caused by inbreeding.

In order to achieve the above object, the present invention has the following constitution.

That is, the present invention is a composite diploid aquatic animal having a heterogeneous genome group AB, and a composite diploid aquatic animal having a fertile XXYY type sex chromosome.

The animals to which the present invention is applied are limited to fish, and all aquatic animals such as shellfish and crustaceans are suitable, and in particular, aquatic animals capable of producing seedlings for discharge or culture. For example, flounder, squid, squid, squid, squid, squid, squid, shrimp, crab, abalone, oyster, mother-of-pearl, medlar, clam, sea urchin, sea cucumber and other interspecies or intergeneric hybrids. The combination of species or genus is not limited to the range that can be hybridized, and even if it is lethal, it is also possible to restore the survivability by doubling.

So far, there is no composite diploid with the A _X A _X B _Y B _Y chromosome in reality or in the literature. The inventors have cultivated new composite diploids to provide new varieties that benefit greatly from the industry. Whether or not there is fertility is determined by whether or not the fertilization can be performed and whether the partner who has undergone the subsequent cultivation process is smoothly formed. There are no specific restrictions on the cultivation or cultivation process.

The composite diploid of the present invention is to fertilize the male non-subtractive sperm of the hybrid first-generation aquatic animal having the heterogeneous genome AB and the female non-subtractive egg of the hybrid first-generation aquatic animal having the same genomic group AB, thereby cultivating A compound diploid aquatic animal of fertile XXYY sex chromosome. Figure 1 is a schematic diagram of the cultivation process of the composite diploid.

Subtractive sperm refers to a sperm whose chromosome composition is consistent with that of the parental individual. For example, to breed male sperm with A _X B _Y chromosome, it is usually ploided from the spermatogonia of the A _X B _Y chromosome and split twice, and constitutes (A+B)/2 _X or A+B)/2 _Y chromosome. Non-subtractive sperm are not bound by doubling and division, meaning that non-subtractive sperm are sperm that retain the parental chromosome A _X B _Y during fertilization.

The non-subtractive egg is an egg whose chromosome composition is identical to that of the parental individual at the time of fertilization (when the second polar body is discharged). For example, having a female chromosome A _X B _X doubly performed when the secondary polar body and configured fertilization (A + B) / 2 _X A _X B _X chromosome from the chromosome of oogonia. Non-subtractive eggs are not bound by double and polar discharge, meaning that non-subtractive eggs are eggs that retain the parental chromosome A _X B _X when fertilized. Let the first generation of the non-subtractive egg A _X B _{X of the} hybrid and the first generation of the non-subtractive sperm A _X B _{Y of the} hybrid fertilize, then the chromosome of the fertilized egg is A _X A _X B _X B _Y . The last individuals developed were male composite diploids with the A _X A _X B _X B _Y chromosome.

The closed system is home to many hybrid first-generation aquatic animals. After reaching the mature age, the male's abdomen is pressed during the spawning period to confirm the presence or absence of sperm, and the sperm DNA is measured and confirmed as non-subtractive sperm. The male and mature females were moved to the same sink to induce spawning. The amount of DNA in the nuclei of juvenile fish is measured, and if the juvenile is tetraploid, the probability of being a compound diploid is quite high. Male parents who produce non-reduced sperm can be reused as parent fish after the next year. The ploidy is confirmed by laying eggs in a closed system and determining the ploidy according to the amount or size of the DNA of the nucleus of the juvenile fish. If the juvenile is a tetraploid, both the fertilized egg and the sperm are non-subtractive partners. By isolating the spawning parent fish, the parent fish producing the non-subtractive partner can be screened.

The present invention is a compound diploid aquatic animal of fertility type XXXY sex chromosome, and the breeding method thereof is to prevent the hybrid egg of the aquatic species A having the genomic group AA and the aquatic animal B having the genomic group BB for the second time. The eggs are cut, and then the meiotic and sub-fertile sperm produced by the first-generation complex diploid aquatic animals with XXXX or XXYY sex chromosomes are fertilized.

Once the composite diploid is cultivated, the first generation of composite diploids can be fertilized to reduce the number of eggs or subtotal sperm, and the second generation can be cultivated. Figure 2 is a schematic diagram of the cultivation process.

The second egg cutting prevention performed when cultivating the first generation complex doubled is to correct the doubling treatment of the first egg cutting prevention so far. The details are explained in the second embodiment. The composite diploids cultivated by the above process are complex diploids having a sex chromosome of XXXY type.

The present invention is a composite diploid aquatic animal of fertility A _X A _X B _X B _Y chromosome, which is to breed a homotetraploid of aquatic species B with genomic group BB, and then let chromosomes Group AA aquatic animals A eggs and sperm are fertilized and prevent the discharge of the second polar body. Figure 3 is a schematic diagram of the cultivation process.

A homogenous tetraploid of the aquatic animal B species having the genomic group BB can be cultivated by a known method. However, the egg cutting prevention means of the fertilized egg when cultivating the same tetraploid is blocked as described in Example 2 for the second egg cutting.

As shown in Fig. 3, the composite diploid cultivated by this process has A _X A _X B _X B _X or A _X A _X B _X B _Y chromosome. Another composite diploid having the A _X A _X B _X B _Y chromosome corresponds to the composite diploid of the present invention.

The present invention is a compound diploid aquatic animal of fertility type XXXY sex chromosome, which is a non-subtractive egg A _X A _X B _{X of} a hybrid triploid aquatic animal having fertility and chromosome AAB. Attenuated sperm B _{Y of} fertile animals with genomic group BB is fertilized.

Generally, a heterologous triploid has no fecundity, but the composite diploid of the present invention uses a non-subtractive egg of a heterogeneous triploid having fertility. Figure 4 is a schematic diagram of the cultivation process.

The meaning and screening of non-subtractive eggs are the same as in the application item 2 of the present invention.

Techniques for preventing the discharge of the second polar body when cultivating a heterogeneous triploid are used to utilize pressure or temperature.

The present invention is a compound diploid aquatic animal of fertility type XXXY sex chromosome, which is a method of letting the egg A _X A _X and the chromosome group BB of the second polar body of the aquatic animal having the genome AA discharge. The spermatozoa B _X or B _{Y of the} aquatic animal is fertilized, then the second polar body is prevented from being discharged, and the B _Y or B _{X is} fertilized by a micro injection operation.

This composite diploid is characterized by direct fertilization through microinjection operations.

The present invention is also a compound diploid aquatic animal of fertility type XXXY sex chromosome, and the breeding method is the egg A _X A _X and sperm produced by homologous tetraploids (AAAA and BBBB) of type A and B species. B _X B _Y fertilization.

This composite diploid is characterized in that once a homotetraploid is produced, eggs and sperm can be used to breed the offspring.

The breeding method of the present invention is characterized in that male non-subtractive sperm and female non-subtractive eggs are screened from the first generation of aquatic animals in the closed system, and non-subtractive and non-reduced sperm are fertilized for breeding. A compound diploid aquatic animal of the XXXY type sex chromosome.

Characteristics breeding methods of the present invention is to allow the eggs chromosome minuend A _X B _X chromosome with a composite diploid A _X A _X B _X B _X aquatic animal is generated A _X A _X B _X B _Y complex double The body water-sucking sperm A _X B _X or A _X B _{Y is} fertilized to produce a male with A _X A _X B _X B _Y chromosome and a female with A _X A _X B _X B _X chromosome in a ratio of 1:1. Compound diploid.

The method of the present invention adopts a composite diploid having an A _X A _X B _X B _Y chromosome, and after F3, a composite diploid having an A _X A _X B _X B _X chromosome is cultured in a ratio of 1:1 and has A Complex diploid of _X A _X B _X B _Y chromosome. Figure 5 is a process of cultivating the method of the present invention.

The method of the present invention shows that if a composite diploid having a XXXY sex chromosome can be cultured, the second generation will definitely exhibit a composite diploid in a ratio of 1:1 male to female.

The two species of AABB of the composite diploid of the present invention have two sets of chromosomes, respectively.

The chromosomes of each species can be combined in pairs to restore fertility.

Since it has a sex chromosome of the XXXY type, it can be mated with the egg A _X B _X of the hybrid F1 which produces non-subtractive eggs, and the next generation of the composite diploid can be stably cultivated by natural mating.

Moreover, when meiosis is performed, each group of the same species of chromosomes is indeed assigned, so the nature of the first generation (F1) can be maintained forever.

Because there is no hereditary separation, the heterogeneous genome will be distributed every two groups. Even if the inbreeding is repeated, it will not show the weakness of inbreeding.

Example 1 is a composite double map of goldfish and koi hybrids.

The inventor crossed the mother Jin Li and the male goldfish and successfully developed a compound diploid fish of the fertile XXXY sex chromosome.

〔background〕

The koi has a variety of colors that goldfish such as gold and silver do not have. Therefore, we hope to transplant the color of the koi to the goldfish. Because of the intergeneric relationship between goldfish and carp, hybrids cannot form normal partners. However, female hybrids can produce non-subtractive eggs with no decrement in the chromosome. On the other hand, male hybrids are unable to produce non-reduced sperm, but investigations have found that many hybrids F1 produce reduced sperm.

None of the seven female individuals surveyed produced non-subtractive eggs, but none of the 15 male individuals surveyed had fertility, and it was known that males who occasionally lay eggs could produce non-subtractive sperm. Therefore, among the hybrids of goldfish and carp, females produce non-reduced eggs, while males are quite rare.

〔technique〕

Hytone or Wakin (and fish) is crossed with black carp or brocade. After 6 months, the gonads were removed and their histological spouses were examined. After about 3 years, the genital nest was taken out from 7 individuals to check the formation of GSI and the partner. For normal occurrences, measure the relative DNA amount of the father's sperm and the child's blood cells. After 1 year, the sex of the child was examined, and the genetic relationship between the children was reviewed through fish scale transplantation and DNA fingerprinting.

〔result〕

Compared with the control group, the 6-month-old hybrid had abnormal gonads. Oocytes that enter the diplotene stage in the ovary are completely unrecognizable or sparsely present. All male germ cells end in the state of spermatogonia and do not enter meiosis.

However, the gonads of juveniles around 3 years old were examined, resulting in a male to female ratio of 4:3. The male and female gonads are well developed, with a female GSI of 7.2 to 10.0 and a male GSI of 0.9 to 2.9 (Table 1).

Ovarian eggs are quite developed and any individual has the ability to lay eggs. The males did not expel the sperm, and no sperm was found even under the microscope. Therefore, it is judged that males have no fecundity.

Six groups of fish and one male fish were placed in a concrete water tank filled with water, and after adding water plants, it was confirmed that there were 5 groups of eggs. However, except for the 1 group, no embryos were formed, and the eggs were judged to be unfertilized. On the other hand, a certain group of eggs are normally produced, and the fertility situation after hatching is also very smooth. When the relative DNA amount of the blood cell is set to 2, the relative DNA amount of the male sperm is measured to be about 2 (refer to Fig. 6), and it is judged to be a non-subtractive sperm. When the amount of DNA of F2 is 4, it is a peak, and it is judged that it is a tetraploid (refer FIG. 7).

These F2 are male and look similar to each other. As can be seen from the results of fish scale transplantation and DNA fingerprinting, these fish are genetically identical clones. The gonads are still immature after one year and cannot be confirmed to be mature. However, in the 3-year-old fish population, sperm-producing fish can be found to determine their fertility.

The significance of this result is as follows.

Hybrids of goldfish and squid could not form normal partners, but it was observed that a small number of oocytes that had entered the double-line phase appeared in the ovaries of young individuals. On the other hand, the male meiosis testis could not be found. In the 3-year-old fish population, almost all of the female fish are mature, but only one of the male fish can be discharged. Measuring the amount of DNA reveals that the sperm is a non-subtractive sperm. The next generation produced by mating between F1 is a tetraploid, and all individuals are male replicators. This result suggests that the chromosomes of the germ cells before meiosis will be multiplied and complex doubled.

Non-subtractive partners will occasionally multiply the chromosomes before the germ cells enter meiosis, and the copied sister chromosomes will be combined as the same chromosome. The male and female partners each have a chromosome of goldfish and carp, and the genetics are identical to those of the parent. Since the sex chromosomes X and Y between the heterogeneous species cannot be combined, in the male F1, the goldfish X and the squid Y will be combined with a subtraction, so the sex chromosomes of the sperm are all XY. The second generation (F2) sex chromosome is XXXY, X of 2 goldfish, X and Y of 1 carp. The male sperm is XX or XY, and a new variety is fixed after the third generation (F3). The male and female are produced in a ratio of 1:1, and each has two sets of goldfish and carp chromosomes (application 7 of the present invention). Since the genetics of goldfish and squid are opposite genetics, even if the siblings are repeated, there will be no weakness of inbreeding.

Example 2

In the present example, the red carp and the carp were crossed to produce the composite diploid of the present application 3 of the present invention.

The XXXY type compound diploid described in Patent Application No. 2 prevents the hybrid egg of the hybrid from undergoing the second egg cutting, and when the first generation compound doubled body is cultivated, the reduced egg and the reduced sperm are fertilized. The inventors have found that although the tetraploid is produced, in fact, the doubling treatment does not prevent the first egg cutting, but the second egg cutting. This can be proved by the following experiment.

The fertilized eggs of red carp or the hybrid eggs of red carp and carp were cultured at a water temperature of 10 degrees, and after 5 hours and 15 minutes, they were treated under water pressure of 650 atmospheres for 6 minutes, and then cultured at 10 degrees.

At regular intervals before and after treatment, fixed eggs were used as tissue specimens to track the activity of the nucleus. The results are shown in Fig. 8. The fertilized eggs of red carp are used as a control group, and the hybrid eggs of red carp and carp are treated as a treatment group.

The region I of Fig. 8 is the nuclear activity of the first cell cycle (before the completion of the first egg-cutting). The region II of Fig. 8 is the nuclear activity of the second cell cycle (before the completion of the second egg cutting of the control group). The region III of Figure 8 is the nuclear activity of the third cell cycle. Except for Figure i, the upper diagrams of each zone (a~d in zone I, j~n in zone II, and t and u in zone III) are the control group, the following figure (e~h of zone I, zone of zone II) The ~s, III regions of v and w) are water pressure treatment groups. The i in zone I is the water pressure treatment group.

Nuclear activity in the first cell cycle (region I): control group: a: nuclear image of the middle and middle of the control group after 5 hours and 15 minutes of fertilization. The nuclear membrane has largely disappeared and the chromosomes are visible. The chromosome is stellate or a spindle surrounding the nuclear membrane.

b: Image of the mid-group of the control group after 5 hours and 45 minutes of fertilization. The chromosomes are arranged on the equatorial plate and are known to be spindles.

c: After 6 hours and 15 minutes of fertilization, the images of the late group of the control group. The chromosome begins to separate in the direction of the poles.

d: After 6 hours and 30 minutes of fertilization, the images of the late group of the control group. The isolated chromosome forms the nucleus. The arrow indicates the image of the first egg-cutting groove.

Water pressure treatment: e: image after the water pressure treatment in the middle and the middle of the control group (the above a) after 5 hours and 15 minutes of fertilization. The stellate and centrosome disappear.

f: Image after 15 minutes of water pressure treatment. Star and body regeneration.

g: Image after 30 minutes of water pressure treatment. 30 minutes later than the graph b of the control group, indicating a normal mid-term image.

h: After the mid-term, enter the normal late stage.

i: 30 minutes later than the map d of the control group, entering the final stage of normal. The two mother cores formed by the two poles and the first egg-cutting groove in the center (see arrows).

From the above, it can be seen that the spindle disappeared in batches by water pressure treatment (Fig. e), but it was immediately regenerated, and a normal first egg cut occurred.

Nuclear activity in the second cell cycle (region II): Control group: j~n: indicates the nucleus of the early, early, middle, late, and late phases of the second cell cycle. In Figure n, the second egg-cutting groove is located below.

o: indicates the nucleus of the pre-second cell cycle of the water-treated embryo. Did not become a control group (Figure i).

p: indicates the pre-intermediate image of the water-treated embryo. Originally there should be only one of the two stelloids.

q: indicates a mid-term image of the water-treated embryo. Since only one star is present, a monopolar spindle is formed.

r: indicates the late image of the water-treated embryo. Since it is a monopolar spindle, the chromosome and the control group (Fig. m) are different and cannot be separated by two poles.

s: indicates the final image of the water-treated embryo. Chromosomes cannot be separated by two poles, forming a nucleus. The second egg cut was not formed and the two cells were maintained. The nucleus becomes larger compared to the final image.

In the second cell cycle, the hydrostatic treatment group does not form a normal bipolar spindle, but forms a monopolar spindle. If the chromosome is not separated, the second egg cutting is prevented at the same time, and the chromosome is multiplied.

Nuclear activity in the third cell cycle (region III): Control group: t and u: represent the mid-term image and post-image of the third cell cycle, respectively.

Water pressure treatment group: v and w: indicates the mid-term image and post-image of the water-treated embryo. After the third cell cycle, the egg is cut normally, the nuclear plate is enlarged, and the chromosome is multiplied.

In the third cell cycle, the embryos in the sputum are also normally egg-cut, but due to ploidy, the nuclear plate in the medium term becomes larger.

According to the results of Fig. 8, it is found that there is no difference in the initial production process of the fertilized egg and the hybrid egg of the red dragonfly. In the control group, 5 hours and 15 minutes before the start of the treatment, most of the eggs were in the middle of the first egg cutting, 5 hours and 45 minutes into the medium term, 6 hours and 15 minutes into the late stage, and 6 hours and 30 minutes into the final stage. . In the control group, the spindle and stellate completely disappeared after treatment. However, 30 minutes after the end of the treatment, the spindle was regenerated and the appearance entered a normal medium term. Thereafter, about 30 minutes later than the control group, the late stage, the end stage, and normal egg cutting were performed.

Significant differences between control and treatment groups were found in the second cell cycle. In the control group, 9 hours after fertilization entered the middle of the second egg cutting, each cell formed a bipolar spindle, and a chromosome was placed on the equatorial plate. In the treatment group, after 10 hours, each cell has a monopolar spindle, and each spindle faces the center side of the embryo, and the chromosomes are arranged at an arc of about 120 degrees at the front end of the spinning system. At the end of the period, only one nucleus is formed and there is no cytoplasmic division. The size of the nucleus at the end of the period was significantly larger than that of the control group.

The use of temperature or water pressure to prevent egg cutting is a widely used method of chromosome doubling. Since these physical treatments allow the microtubules to be depolymerized and the spindle disappears, the two poles of the chromosome are prevented from moving, and the copied chromosomes cannot be separated and multiplied. Therefore, it is also called the first egg cutting prevention method.

However, this idea is obviously wrong.

The spindle is regenerated shortly after the water pressure treatment, and the first egg cutting will be delayed with some delay. In the second cell cycle, any nucleus of the treated population forms a monopolar spindle forming a nucleus. Since this nucleus is significantly larger than the control group, it has been accompanied by chromosome replication. In the third cell cycle, bipolar division occurs in any cell nucleus, and cell division returns to normal. Therefore, the water pressure treatment in the middle of the first egg cutting prevents the second division from being correct. The temperature treatment and the water pressure treatment are basically the same. These results suggest that water pressure treatment during egg cutting may affect the centrosome from the spindle. In step 4 of Fig. 13, water pressure or temperature treatment is applied, and the first egg cutting will proceed normally, but the bipolar spindle will not be formed in step 6, but a monopolar spindle will be formed, and the chromosome will not be separated. There will be no egg cutting, so the two cells will each become a tetraploid nucleus. That is, the second egg cut is prevented and a tetraploid is formed.

Hydrogen treatment was used to cultivate red carp and carp composite diploids. From 4 hours 45 minutes to 5 hours 45 minutes after fertilization, the hybrid eggs of red carp and carp were treated with water pressure every 15 minutes to obtain the nucleus at the beginning of treatment. The relationship between period and doubling. Next, the chromosomes of 2n-4n male and female mosaics appearing on the treatment group were analyzed to confirm the genomic composition of 2n cells.

Figure 9 shows the relationship between processing start and multiplication.

4 hours and 45 minutes is equivalent to the first stage of the first egg cutting, 5 hours and 15 minutes is equivalent to the first medium term, and 5 to 30 minutes to 45 minutes. The maximum doubling was found at 5 hours and 15 minutes, and nearly 40% of the eggs were multiplied. The results of the karyotyping analysis show that these are positive tetraploids. The ratio of cultivating the tetraploid at the beginning of the intermediate treatment was lower than at the beginning of the treatment.

Figure 10 shows the nucleotypes of the composite diploid and the untreated hybrid diploid of red carp and carp appearing on the treatment group.

Repeatedly study the relationship between the start time and the doubling, and conclude that the best time to start processing is better than the medium term. Further comparison of the mid-term and the early period is better in the previous period. If the water pressure treatment only produces a multiplication by destroying the spindle, the intermediate treatment result should be good. The above results suggest that the physical treatment of the doubling has a greater influence on the centrosome than on the spindle.

The centrosome consists of two central organs. The two central organs are separated from each other before cell division, and then each bud is replicated to replicate a central organ. Before the middle of the split, the mother center will continue to form. The center is also a kind of neutrino tube composed of γ micro tubes. The mature center is affected by water pressure treatment. When the replication process is affected, the relationship between the start time and the doubling of the water pressure treatment can be clearly stated. At the initial stage of replication, even if the center of the mother is damaged due to water pressure, it can be replicated again after treatment, so the effect of water pressure treatment is not significant. However, if a certain degree of copying is performed, it is impossible to form again, and a center can complete the first split.

On the other hand, after the middle of the split, the copying of the center has been completed and is not easily affected by the water pressure, so the ratio of the doubling will fall again. The center of the copy is destroyed, and the center of replication failure is changed to the second cell cycle to replicate the mother center to form a complete centrosome. Therefore, the inventors believe that a monopolar spindle is formed.

The present invention is a composite diploid of a fertile XXXY type sex chromosome, and is also a composite diploid cultivated by the above-described embodiment. For example, a non-subtractive egg A _X A _X B _{X of} a fertile hybrid 3 ploid aquatic animal and a reduced sperm B _{Y of} an aquatic animal having a chromosome BB are fertilized to cultivate a composite diploid. Or fertilize the eggs A _X A _X of aquatic animals with chromosome AA and the sperm B _X and B _Y of aquatic animals with chromosome BB, and fertilize B _Y or B _X respectively by microinjection to prevent the second pole The body is discharged to cultivate a composite diploid. Either the eggs (A _X A _X ) and sperm (B _X B _Y ) produced by the homotetraploid (AAAA and BBBB) are fertilized to cultivate the composite diploid.

The above examples are all related to fish, but are equally applicable to all aquatic animals with the same elements in the aquaculture industry, such as most shellfish and crustaceans. In general, since the number of eggs produced by an individual of a marine animal is quite large, it is a source of screening objects, and the same fertilized egg or juvenile fish can be effectively produced by the same method. Therefore, when cultivating compound diploids, the techniques or ideas applied to fish are directly applicable to other aquatic animals.

The following two papers can prove that chromosome manipulation is not only effective for fish, but shellfish or crustaceans are equally effective.

(1) Cuo, X. and Allen, Jr. S.R. 1994 Viable tetraploids in the Pacific oyster (Crssostrea gigas Thunberg) produced by inhibiting polar body 1 in eggs from triploids. Molecular marine Biology Biotechnology, 3, 42-50.

(2) Li, F., Xiange, J., Zou, L., Wu, C. and Zhang, X. 2003 Optimization of triploid induction by heat shock in Chinese shrimp Fenneropenaeus chinensis. Aquaculture, 219, 221-231.

In summary, the present invention is a composite diploid composed of chromosomes that has not been considered in nature and in theory. The inventors have developed a composite diploid by pressure. The stable supply of composite diploids through natural mating, especially for the aquaculture industry, has an inestimable application value.

1 is a diagram showing a process of compound diploid cultivation according to an embodiment of the present invention.

2 is a diagram showing a process of compound diploid cultivation according to another embodiment of the present invention.

Figure 3 is a diagram showing the process of compound diploid cultivation according to another embodiment of the present invention.

4 is a diagram showing a process of compound diploid cultivation according to still another embodiment of the present invention.

Figure 5 is an explanatory view of the breeding method of the present invention.

Fig. 6 is a graph showing the relative DNA amount of the sperm produced by F1 of Example 1, and showing the value when the relative DNA amount of the parental individual is 2.

Fig. 7 is a graph showing the relative DNA amount of the red blood cell nuclei of F2 of Example 1, and showing the value when the relative DNA amount of the parental individual is 2.

Fig. 8 is a graph showing the comparison of the nuclear activities of the control group and the treatment group of the water treatment in Example 2.

Fig. 9 is a graph showing the relationship between the start time and the doubling of the water pressure treatment in the second embodiment.

Figure 10 is a nucleus diagram of the compound diploid of red carp and carp in Example 2. The above figure shows the hybrid of red carp and carp. The following figure shows the compound diploid, red carp and carp cultured by the second egg cutting prevention of hybrid eggs. The chromosomes become two groups.

Figure 11 is an explanatory diagram of a hybrid breeding process.

Figure 12 is a diagram showing the combination of chromosomes when the first generation of hybrids is mated. Figure 13 is a theoretical diagram of the cultivation of heterotetraploid and compound diploid fish.

Claims (6)

A breeding method for a compound diploid fish of the XXXY type sex chromosome having fertility, which is to determine whether the sperm is a non-subtractive sperm by measuring the DNA amount of the sperm of the male first-generation fish male having the heterogeneous genome AB Using a microinjection procedure, the non-reduced sperm of the male individual confirmed to be a non-subtractive sperm is fertilized with the non-decimated egg of the first female mature female having the heterogeneous genome AB, according to the The amount of nuclear DNA in the juvenile fish confirmed that the juvenile was tetraploid. A method for breeding a compound diploid fish is to use a water pressure treatment or a temperature treatment to prevent the hybrid egg of the fish A species having the genome AA and the fish B having the chromosome group BB from undergoing the second egg cutting, and then using the micro The injection operation fertilizes the eggs and sperm produced by the first generation of the compound diploid fish having the XXXX or XXYY sex chromosomes, thereby cultivating the compound diploid fish of the fertile XXXY sex chromosome. A method for breeding a compound diploid fish is to fertilize the egg A _X A _X of the fish having the genome AA and the sperm B _X of the fish having the genome group BB, and then use the water pressure treatment or the temperature treatment to block the second pole. Excretion of the body, using the microinjection operation, the non-subtractive egg A _X A _X B _X of the hybrid triploid fish of the genus AAB having the fertility and the sperm B _Y of the fish having the genome BB Fertilization, thereby breeding a compound diploid fish of the fertile XXXY sex chromosome. A method for breeding a compound diploid fish is to fertilize the egg A _X A _X before discharge of the second polar body of the fish having the genome AA and the sperm B _X or B _Y of the fish having the chromosome group BB, and then prevent the first The 2-pole body is discharged, and the sperm B _Y or B _{X is} fertilized by a micro-injection operation, thereby cultivating a compound diploid fish of the fertile XXXY sex chromosome. A breeding method for compound diploid fish is to fertilize eggs A _X A _X and sperm B _X B _Y produced by homologous tetraploids (AAAA and BBBB) of type A and B by microinjection operation, thereby cultivating A compound diploid fish of the fertile XXXY sex chromosome. A composite of diploid fish breeding methods, using microinjection makes subtraction with the resulting composite fish diploid chromosome A _X A _X B _X B _X A _X B _X eggs and patent range 1 to 5 The spermatozoa A _X B _X or A _X B _Y of the compound diploid fish having the chromosome A _X A _X B _X B _Y cultivated by any of the breeding methods is fertilized and cultured in a ratio of 1:1 A composite diploid of a male of chromosome A _X A _X B _X B _{Y and} a female having chromosome A _X A _X B _X B _X .