CN112535127A - Method for producing allotetraploid by hybridizing diploid of Crassostrea ampelopsis Grossdentata and triploid of Crassostrea hongkongensis - Google Patents
Method for producing allotetraploid by hybridizing diploid of Crassostrea ampelopsis Grossdentata and triploid of Crassostrea hongkongensis Download PDFInfo
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- CN112535127A CN112535127A CN202010673077.0A CN202010673077A CN112535127A CN 112535127 A CN112535127 A CN 112535127A CN 202010673077 A CN202010673077 A CN 202010673077A CN 112535127 A CN112535127 A CN 112535127A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/50—Culture of aquatic animals of shellfish
- A01K61/54—Culture of aquatic animals of shellfish of bivalves, e.g. oysters or mussels
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/10—Culture of aquatic animals of fish
- A01K61/17—Hatching, e.g. incubators
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Abstract
The invention discloses a method for producing allotetraploid by hybridizing a portuguese oyster diploid and a hong Kong giant oyster triploid. The invention takes the crassostrea hongkongensis triploid as a female parent, takes the full weight as a target, preferably selects the portuguese oyster diploid as a male parent, and produces the oyster allotetraploid by drug induced hybridization. The oyster allotetraploid obtained by the method has the characteristics of wide salt and high temperature resistance, is suitable for breeding in low-salt regions such as estuary and the like, is suitable for breeding in sea regions with different salinity in China by hybridizing with common portuguese oysters or hong Kong giant oyster diploids, and has wide popularization and application prospects.
Description
Technical Field
The invention relates to a method for producing heterotetraploid by shellfish hybridization, in particular to a method for producing oyster heterotetraploid by hybridizing a portuguese oyster and a hongkong giant oyster.
Background
Oyster, as an important marine organism, has delicious meat taste and high nutritional value and is called 'milk in the sea'. The oysters have wide geographical distribution, fast growth, high yield and high economic value, and are important mariculture objects in all countries in the world.
The growth advantage of the triploid oyster is obvious. Triploid oysters have 3 sets of chromosomes and are therefore extremely low in fertility, which means that on the one hand they can grow faster (their energy is used for growth and not for reproduction), and on the other hand they produce no or very little gametes during the summer, which ensures a stable quality. Triploid oysters are composed of tetraploid oysters and diploid oysters that are crossed, and thus it is important to obtain culturable tetraploid parents.
Traditional tetraploid oyster preparation employs homologous species oysters, wherein:
the crassostrea viticola serves as a main crassostrea gigas breeding variety in the south China coastal region, the growth speed is high, the breeding period is short, and the breeding risk is low. However, the oyster shells are thin and crisp, the meat yield is low, the selling price is low and the like because the growth speed of the portuguese oysters is too high;
the crassostrea hongkongensis is used as a main oyster breeding variety in coastal estuary areas (low salinity) in south China, and the oyster shell is firm and has high meat yield. However, the culture period of the crassostrea hongkongensis is too long, the crassostrea hongkongensis generally can be listed in 2-3 years, the survival rate is low, and the crassostrea hongkongensis can only be used for barbecue and the like due to poor taste caused by long-term life in estuary areas.
The allopolyploid is a polyploid formed by chromosome doubling of hybrid offspring generated by hybridization of different species, and has a whole set of chromosome sets of two or more different species and stronger natural selection advantage. Therefore, the heterotetraploid is bred by hybridization of the Ostrea vittata and the Ostrea hongkongensis, so that heterosis is utilized, and the problems of the two oysters can be effectively solved.
Disclosure of Invention
Technical problem to be solved
Traditional tetraploid oyster preparation uses homologous species oysters: the growth speed of the ostrea vittata is too high, so that the ostrea vittata shell is thin and crisp, and the meat yield is not high; the culture period of the crassostrea hongkongensis is too long, the crassostrea hongkongensis can be generally listed after 2-3 years, and the survival rate is low. Therefore, the invention performs hybridization on the two varieties to obtain the allotetraploid, utilizes the heterosis and solves the inherent problems of the oyster variety.
(II) technical scheme
The method for producing the allotetraploid by hybridizing the diploid of the crassostrea viticola and the triploid of the crassostrea hongkongensis is characterized by comprising the following steps of:
(a) selecting triploid of Crassostrea hongkongensis as a female parent for propagation, selecting diploid of common Portuguese oyster as a male parent for propagation, and respectively putting the breeding shells into a shell breeding pond for ripening;
(b) accelerating the maturation of the crassostrea hongkongensis until the gonads of the crassostrea hongkongensis mature, obtaining ova, and maturing the ova before the next step; dissecting the mature-promoted grapevine oyster diploid to obtain sperms, and activating the sperms before the next step;
(c) mixing the mature ovum and the activated sperm in water, wherein the ratio of the number of the sperm to the number of the ovum is 5-10: 1, controlling the density of fertilized eggs to be less than or equal to 1 ten thousand/ml;
(d) observing the development condition of the fertilized eggs, and when 50-75% of the first polar body appears, rapidly adding cytochalasin B (0.5-1.0 mg/L) or 6-dimethylaminopurine (15-30 mg/L) for treatment for 20-30 min;
(e) cleaning and filtering the drug immediately after the drug treatment of the fertilized eggs, setting the hatching density, and placing the fertilized eggs in a black barrel for hatching; after the larvae grow to D-type larvae, screening the larvae, setting the breeding density and putting the larvae into a black barrel for breeding; and after the larvae have eyespots, putting the attaching matrix.
Preferably, in the step (a), the breeding female parent is large in individual, good in vitality and free of damage, the breeding male parent measures the total weight of all individuals, and 10% of the individuals in the top of the total weight are intercepted.
Preferably, in the step (a), the biological bait in the shellfish culture pond is artificially cultured golden algae, Chaetoceros and Platymonas verrucosa or Tuchi algae water.
Preferably, in the step (b), the curing time is 0.5-1 h, and the activation time is 10-20 min.
Preferably, in step (c), the water temperature is maintained at 25 ℃ and the salinity is maintained at 20.
Preferably, in the step (e), the hatching density is kept between 1 and 3 per ml, and the breeding density is kept between 0.5 and 1 per ml.
(III) advantageous effects
The invention not only utilizes the heterosis between the portuguese oyster and the hong Kong giant oyster, but also induces the portuguese oyster diploid and the hong Kong giant oyster triploid to be hybridized and produce the oyster allotetraploid by medicaments, and the invention has the characteristics of wide salt and high temperature resistance. The oyster triploid produced by hybridization of the oyster allotetraploid and the common portuguese oyster or the hong Kong giant oyster diploid is suitable for cultivation in sea areas with different salinity in China, and has wide popularization and application prospect.
Drawings
FIG. 1 is a diagram of a common oyster diploid peak;
FIG. 2 is a graph of the peak values of larvae of D-type heterotetraploid of oysters produced by the method of the invention;
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to FIGS. 1-2, a first embodiment of the present invention is provided;
(a) selection and ripening of breeding shellfish: 100 triploids (with the shell height of 10-15 cm) of large-individual, high-activity and nondestructive triploids of Crassostrea hongkongensis are selected as breeding female parents, and 10% of individuals in the top of the total weight of 1000 diploids (with the shell height of 8-12 cm) of Portulaca grossedentata are cut out as breeding male parents. And then putting the breeding shells into a breeding shell culture pond respectively to promote the maturity by adopting a running water method, wherein the biological bait mainly comprises artificially cultured golden algae, Chaetoceros and Platymonas subcordiformis, and the soil pond algae water is used as a standby bait source.
(b) Obtaining triploid ovum: after the gonads of the crassostrea hongkongensis fingerlings have matured, 30 triploid individuals of the crassostrea hongkongensis are detected by a ploidy analyzer, 27 individuals with 3N are detected in total, and 8 female individuals with full glands and good egg development are screened out as female parents through microscope observation. Taking out soft parts of 8 female parents one by one, cleaning, extruding out ova, filtering large-particle impurities of the ova by using a 100-mesh silk net, filtering mucus by using a 500-mesh silk net, and placing the ova liquid in a 1L plastic cup. Placing the filtered egg liquid in seawater with salinity of 20 for curing for 0.5h, and performing next fertilization after the eggs become round;
obtaining diploid sperms: dissecting 30 diploid of Ostrea vittata, distinguishing male and female by microscope, selecting 8 individuals with plump gonads and strong sperm motility as male parents, extruding sperm 10min before artificial fertilization, filtering impurities by using 500-mesh silk net, activating in seawater with salinity of 20, and detecting sperm motility above 90% under microscope.
(c) Artificial insemination: according to a one-to-one principle, 8 parts of cured crassostrea hongkongensis triploid egg liquid is respectively added with activated grapevine oyster diploid sperms, the number ratio of sperms to eggs is 5:1, the density of fertilized eggs is kept to be less than or equal to 1 ten thousand/ml, the egg liquid is continuously stirred, the water temperature is kept to be about 25 ℃, and the salinity is 20.
(d) Drug induction: note that the development of fertilized eggs was observed, and when 50% of the first polar body appeared, 1.0mg/L cytochalasin B was added rapidly and treated for 30 min.
(e) Larva breeding: immediately cleaning fertilized eggs in 1% dimethyl sulfoxide for 2 times after the drug treatment, soaking for 15min each time, filtering with a 500-mesh silk screen to remove the drug, and then placing fertilized egg liquid in 8 black barrels of 500L respectively for hatching, wherein the hatching density is kept at 1/ml, the salinity is 20, and slight aeration is carried out. When fertilized eggs develop into D-type larvae, a ploidy analyzer is used for detecting the proportion of 4N, 5 groups with higher induced proportion of 4N are left, and a 400-mesh silk net is used for collecting the D-type larvae and putting the D-type larvae into 5 disinfected black barrels for cultivation. The larva breeding density is kept at 0.5/ml, and artificially cultured golden algae and chaetoceros are fed. After the larvae developed to eyespot, 5 groups of larvae were collected together and put into a black barrel, and oyster shell was put in as the attachment substrate.
The present invention also provides a second embodiment:
(a) selection and ripening of breeding shellfish: 100 triploids (with the shell height of 10-15 cm) of large-individual, high-activity and nondestructive triploids of Crassostrea hongkongensis are selected as breeding female parents, and 10% of individuals in the top of the total weight of 1000 diploids (with the shell height of 8-12 cm) of Portulaca grossedentata are cut out as breeding male parents. And then putting the breeding shells into a breeding shell culture pond respectively to promote the maturity by adopting a running water method, wherein the biological bait mainly comprises artificially cultured golden algae, Chaetoceros and Platymonas subcordiformis, and the soil pond algae water is used as a standby bait source.
(b) Obtaining triploid ovum: after the gonads of the crassostrea hongkongensis fingerlings have matured, 30 triploid individuals of the crassostrea hongkongensis are detected by a ploidy analyzer, 24 3N individuals are detected in total, and 10 female 3N individuals with plump individual glands and good ovum development are screened out as female parents through microscope observation. Taking out the soft parts of 10 female parents one by one, cleaning, extruding out ova, filtering large-particle impurities of the ova by using a 100-mesh silk net, filtering mucus by using a 500-mesh silk net, and placing the ova liquid in a 1L plastic cup. Placing the filtered egg liquid in seawater with salinity of 20 for curing for 1h, and performing next fertilization after the ovum becomes round;
obtaining diploid sperms: dissecting 30 diploid of the ostrea viticola, distinguishing male and female by using a microscope, selecting 10 individuals with plump gonads and strong sperm motility as male parents, extruding the sperms 20min before the start of artificial fertilization, filtering impurities by using a 500-mesh silk net, placing in seawater with salinity of 25 for activation, and detecting that the sperm motility is more than 90% under the microscope.
(c) Artificial insemination: according to a one-to-one principle, 10 parts of cured crassostrea hongkongensis triploid egg liquid is respectively added with activated grapevine oyster diploid sperms, the number ratio of sperms to eggs is 10:1, the density of fertilized eggs is kept to be less than or equal to 1 ten thousand/ml, the egg liquid is continuously stirred, the water temperature is kept to be about 25 ℃, and the salinity is 20.
(d) Drug induction: when 75% of the first polar body appears, 30mg/L of 6-dimethylaminopurine is rapidly added for 20 min.
(e) Larva breeding: immediately cleaning fertilized eggs in 1% dimethyl sulfoxide for 2 times after the drug treatment is finished, soaking for 15min each time, filtering with a 500-mesh silk screen to remove the drugs, then placing fertilized egg liquid in 16 black barrels of 500L respectively for hatching, and keeping the hatching density at 3/ml and the salinity at 20, and slightly inflating. When fertilized eggs develop into D-type larvae, a ploidy analyzer is used for detecting the proportion of 4N, 8 groups with higher induced proportion of 4N are left, and a 400-mesh silk net is used for collecting the D-type larvae and putting the D-type larvae into 12 disinfected black barrels for cultivation. The larva breeding density is kept at 1/ml, and artificially cultured golden algae and chaetoceros are fed. 8 group larvae die 1 group in the larva breeding process, when remaining 7 groups of larvae develop to eyepoint, 7 groups of larvae are collected together and put into a black barrel, and oyster shells are put in as an attaching matrix.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (6)
1. The method for producing the allotetraploid by hybridizing the diploid of the crassostrea viticola and the triploid of the crassostrea hongkongensis is characterized by comprising the following steps of:
(a) selecting triploid of Crassostrea hongkongensis as a female parent for propagation, selecting diploid of common Portuguese oyster as a male parent for propagation, and respectively putting the breeding shells into a shell breeding pond for ripening;
(b) accelerating the maturation of the crassostrea hongkongensis until the gonads of the crassostrea hongkongensis mature, obtaining ova, and maturing the ova before the next step; dissecting the mature-promoted grapevine oyster diploid to obtain sperms, and activating the sperms before the next step;
(c) mixing the mature ovum and the activated sperm in water, wherein the ratio of the number of the sperm to the number of the ovum is 5-10: 1, controlling the density of fertilized eggs to be less than or equal to 1 ten thousand/ml;
(d) observing the development condition of the fertilized eggs, and when 50-75% of the first polar body appears, rapidly adding cytochalasin B (0.5-1.0 mg/L) or 6-dimethylaminopurine (15-30 mg/L) for treatment for 20-30 min;
(e) cleaning and filtering the drug immediately after the drug treatment of the fertilized eggs, setting the hatching density, and placing the fertilized eggs in a black barrel for hatching; after the larvae grow to D-type larvae, screening the larvae, setting the breeding density and putting the larvae into a black barrel for breeding; and after the larvae have eyespots, putting the attaching matrix.
2. The method of claim 1, wherein: in the step (a), the breeding female parent is large in individual, good in vitality and free of damage, the breeding male parent measures the total weight of all individuals, and 10% of the individuals in the top of the total weight are intercepted.
3. The method of claim 1, wherein: in the step (a), the biological bait in the shellfish culture pond is artificial culture water of chrysophyceae, chaetoceros and platymonas or euglena.
4. The method of claim 1, wherein: in the step (b), the curing time is 0.5-1 h, and the activation time is 10-20 min.
5. The method of claim 1, wherein: in step (c), the water temperature is kept at 25 ℃ and the salinity is kept at 20.
6. The method of claim 1, wherein: in the step (e), the hatching density is kept at 1-3/ml, and the breeding density is kept at 0.5-1/ml.
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| CN202010673077.0A CN112535127A (en) | 2020-07-14 | 2020-07-14 | Method for producing allotetraploid by hybridizing diploid of Crassostrea ampelopsis Grossdentata and triploid of Crassostrea hongkongensis |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114208735A (en) * | 2021-12-22 | 2022-03-22 | 中国科学院南海海洋研究所 | Method for cultivating rapid-growth new strain of hong Kong oyster triploid by backcross breeding technology |
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| CN114208735B (en) * | 2021-12-22 | 2023-02-28 | 中国科学院南海海洋研究所 | Method for cultivating rapid-growth new strain of hong Kong oyster triploid by backcross breeding technology |
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Application publication date: 20210323 |