CN111700007B - Method for distant hybridization between broccoli and bighead carp subfamilies and application of tetraploid broccoli - Google Patents

Method for distant hybridization between broccoli and bighead carp subfamilies and application of tetraploid broccoli Download PDF

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CN111700007B
CN111700007B CN202010582326.5A CN202010582326A CN111700007B CN 111700007 B CN111700007 B CN 111700007B CN 202010582326 A CN202010582326 A CN 202010582326A CN 111700007 B CN111700007 B CN 111700007B
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carp
fish
bighead
water
male
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CN111700007A (en
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刘少军
王余德
姚嘉骏
谭慧芳
罗凯坤
覃钦博
张纯
陶敏
汤陈宸
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Hunan Normal University
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/454—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. pimozide, domperidone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A61K38/09—Luteinising hormone-releasing hormone [LHRH], i.e. Gonadotropin-releasing hormone [GnRH]; Related peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/04—Drugs for genital or sexual disorders; Contraceptives for inducing labour or abortion; Uterotonics
    • 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 distant hybridization between koi and bighead subfamily, which comprises the following steps: carrying out artificial induced spawning by taking fancy carps as female parents and bighead carps as male parents, and injecting mixed oxytocin containing LRH-A, HCG and diosone into the female parent carps by adopting a one-needle injection method to carry out induced spawning; injecting mixed oxytocin containing LRH-A and HCG into male parent fish by adopting a two-needle injection method for induced spawning, performing artificial dry insemination on ovum and sperm obtained after artificial induced spawning, hatching fertilized ovum with running water, and obtaining filial generation of koi and bighead carp after successful hatching. The obtained new filial generation provides new resources and basis for the genetic breeding of the freshwater fish, the whole hybridization process is simple to operate, the breeding period is short, and the hatching rate and the survival rate of the filial generation are high. The invention also discloses application of the filial generation tetraploid variegated carp, can obtain a large amount of triploid fishes with high growth speed and strong stress resistance, and has great industrial application prospect.

Description

Method for distant hybridization between broccoli and bighead carp subfamilies and application of tetraploid broccoli
Technical Field
The invention belongs to the field of freshwater fish hybridization, and particularly relates to a distant hybridization method between koi and bighead subfamily and application of tetraploid koi.
Background
Distant hybridization refers to hybridization between and above species of related lines, and has many applications in fish genetic breeding. The distant hybridization can integrate the exogenous gene of the parent, so that the advantages of the parent are integrated in the hybrid, the heterosis is generated, and the hybrid has more excellent performances in the aspects of growth cycle, disease resistance and the like. For example, distant hybridization between the subfamilies of koi carp and megalobrama amblycephala and distant hybridization between the red crucian carp and the Xiangjiang wild carp generate more excellent hybrid offspring. However, a long-standing problem in the field of distant hybridization is that the hybrid trait is highly segregating and not easily stable. Therefore, how to select a suitable parent fish can improve the genes of the filial generation, and has important research significance.
The fancy carp (cyprinus carpio haematopterus) is a diploid fish (2n is 100) of cypriniformes, cyprinidae and subfamily carpinidae, is bright in color and beautiful in posture, is more advantageous than carps in the aspect of appreciation, but is fragile and strict in requirement on water temperature, so that the cultivation cost is high. The breeding water temperature of the koi is required to be above 21 ℃, and if the water temperature fluctuates and drops during spawning, spawning can be interrupted. Bighead carp (Hypophthalmichthys nobilis, 2n is 48) is a diploid fish in Cyprinaceae and Hypophthalmichthys subfamily, is mostly distributed in the middle and upper layers of a freshwater area and is warm water fish, the water temperature suitable for growth is 25-30 ℃, the requirement on the water temperature is not strict, the Bighead carp can adapt to a fertile water body environment, the resistance is strong, the Bighead carp can be suitable for different water bodies, and the Bighead carp has high nutritional value. If distant hybridization is carried out on the fancy carp and the bighead carp, hybridized offspring fish which can not only keep the ornamental value of the fancy carp but also overcome the defect that the requirement of the fancy carp on water temperature is high is expected to be cultivated. However, the difference between the chromosome numbers of koi carp and bighead carp is large, and the fold relation does not exist, so that the filial generation is difficult to obtain smoothly by the conventional method.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects and shortcomings in the background technology and provide a method for distant hybridization between the subfamilies of koi and bighead carp and application of tetraploid koi and bighead carp.
In order to solve the technical problems, the technical scheme provided by the invention is as follows:
a method for distant hybridization between the subfamilies of koi and bighead carp comprises the following steps: carrying out artificial induced spawning by taking koi as a female parent and bighead carp as a male parent, and injecting mixed oxytocin containing luteinizing hormone releasing hormone analogue (LRH-A), chorionic gonadotropin (HCG) and diosdone into the female parent and carrying out induced spawning by adopting a one-needle injection method, wherein the injection dosage is 12-15 mu g/kg of luteinizing hormone releasing hormone analogue (LRH-A), 800-1000 IU/kg of chorionic gonadotropin (HCG) and 1-2 mu g/kg of diosone; injecting mixed oxytocin containing luteinizing hormone releasing hormone analogue (LRH-A) and chorionic gonadotropin (HCG) into the male parent fish by adopting a two-needle injection method for induced spawning, wherein the total injection dosage of the mixed oxytocin containing the luteinizing hormone releasing hormone analogue (LRH-A) is 6-8 mug/kg, and the total injection dosage of the chorionic gonadotropin (HCG) is 400-600 IU/kg, the male parent fish is injected for the first time while the female parent fish is induced spawning, the first injection dosage is 20% of the total injection dosage volume, the male parent fish is injected for the second time after 4-5 h, and the second injection dosage is 80% of the total injection dosage volume; and (3) carrying out artificial dry insemination on the ovum and the sperm obtained after artificial spawning induction, then hatching the fertilized ovum in running water, and obtaining the filial generation of the koi and the bighead carp after successful hatching.
The luteinizing hormone-releasing hormone analogue (LRH-A) can promote the oestrus and oviposition of parent fish, and chorionic gonadotropin (HCG) can not only promote ovulation of parent fish, but also accelerate gonad maturation. In addition, semen can be obtained from male parent bighead carp by one-time injection method, but the sperm is rare, mainly the sperm quality and concentration are low, and distant hybridization offspring is difficult to obtain effectively. Through long-term systematic groping, the method adopts secondary injection for inducing spawning when the bighead carp is induced to spawn, which is beneficial to obtaining a large amount of high-quality semen, and simultaneously adjusts the spawning inducing dose of the fancy carp, thereby being beneficial to reaching the maximum spawning inducing rate and improving the hatching rate and the survival rate of filial generations.
Preferably, the method comprises the steps of selecting female koi and male bighead carp which are healthy, strong and normal in development as female parent fish and male parent fish to move out of a fish pond for independent feeding 3-4 months before induced spawning of the female parent fish and the male parent fish, scientifically feeding the female koi and the male bighead carp by using concentrated feed 1-2 months before induced spawning, and periodically stimulating the fish by running water; raising female koiThe water quality indexes are as follows: the pH value is 7.0-7.4, the hardness is less than 15 ℃, and the concentration of iron ions is 0.3 multiplied by 10-6Less than mol/L, sulfate ion concentration < 15X 10-6mol/L, chloride ion concentration < 19X 10-6mol/L, free chlorine and other toxic and harmful substances, dissolved oxygen of more than 5 × 10-6mg/L, ammonia concentration < 0.1X 10-6mg/L, nitrite concentration < 0.1X 10-6mol/L, nitrate concentration at 5.5X 10-6mg/L～6.0×10-6mg/L, hydrogen sulfide free; the water quality index for raising male bighead carp is as follows: the pH value is 6.5-8.5, the dissolved oxygen is more than or equal to 4mg/L, the ammonia nitrogen content is less than 0.025mg/L, and the biochemical oxygen demand is less than 5 mg/L. Scientifically breeding the fancy carp: the water quality standard for feeding the fancy carps is colorless, tasteless and odorless, and the transparency of water is high; the moss on the wall of the pond grows normally; the method comprises the following steps that a fish pond for breeding fancy carps needs to have good sunshine and ventilation conditions, the area of the fish pond is not smaller than 30 square meters, and the depth of the fish pond is about 2 meters; the fancy carp is fed by adopting animal feed at regular time, the feeding time is 9:00-10:00 in the morning, 12:00-13:00 in the noon and 6:00-8:00 in the afternoon, and the regular feeding is favorable for training the conditioned reflex of the fancy carp. Scientific breeding of bighead carps: before stocking bighead carps, base fertilizer is applied to the pond, and after stocking, additional fertilizer is applied according to seasons and specific measures of the pond. The normal development refers to obvious sexual characteristics, and the absence of physical attack refers to the absence of infection and the absence of physical trauma.
Preferably, the spawning induction is carried out on the parent fish when the water temperature is stabilized above 20 ℃ in 5 and 6 months of the year.
Preferably, after injecting the mixed oxytocic into the female parent fish and the male parent fish, respectively putting the parent fish into different spawning ponds at 25 ℃ according to the quantity ratio of 1:2 between the koi carp and the bighead carp, culturing, keeping the water temperature constant, periodically stimulating the parent fish by running water, and fishing when the parent fish is in heat and tailgating; female fancy carps and male fancy carps are put into the spawning pond of the female parent fancy carps according to the quantity ratio of 1:2 of the male parent fancy carps to induce the female fancy carps to spawn; the size of the spawning pond is 30-40 m2. The time for injecting the mixed oxytocin is 18: 00-20: 00 in the evening, and the mixed oxytocin is injected into the abdominal cavity at the base part of the pectoral fin of the parent fish without scales; the fry is produced 8: 00-9: 00 in the morning on the next day. We have shown the relationship between fancy carp and bighead carpThe fish is spawned by separating ponds, which is favorable for reducing the mechanical damage to the fancy carps and the bighead carps caused by netting.
Preferably, the specific operation of artificial dry insemination comprises the following steps: grasping the tail handle of the parent fish with the left hand, holding the back of the parent fish with the right hand, cleaning the body surface of the parent fish, extruding the fish belly, respectively extruding the ovum and the sperm into a dry ceramic basin, slightly stirring the dried feather for 2-3 min, and then adding clear water into the ceramic basin to activate the sperm for insemination.
Preferably, the water temperature is controlled to be 24-26 ℃ when the fertilized eggs are incubated in running water. The invention adopts the novel fish viscous egg hatching device to hatch in running water, and the running water hatching has the advantages that: 1) continuously supplementing running water to ensure that the water quality and oxygen demand is more than 5 mg/L; 2) after the fry is taken out of the membrane, the egg membrane can be conveniently and timely removed, and the water quality is prevented from being polluted; 3) the hatched seedlings have stronger vitality and high survival rate.
Preferably, the fry obtained by the running water hatching is continuously cultured in a hatching tank for 3 days, the fry is transferred to a pond with pre-fertilization for fine breeding after waist spots appear, soybean milk is splashed for 1-3 days after the fry is placed in the pond, and the soybean milk is splashed for 1-2 times every day.
Preferably, the depth of the fine breeding water is controlled to be 0.7-0.8 m, the breeding water needs to be exposed for 2 days if tap water is used, the breeding water does not need to be exposed if well water is used, and 200-300 mL of green algae and 200-300 mL of blue algae are added after the tap water is exposed or directly added into the well water.
Preferably, the gray diploid bighead carp, the anthocyanin diploid bighead carp, the triploid variegated carp (variegated bighead carp with whiskers) and the tetraploid variegated carp (variegated bighead carp without obvious whiskers) hybrid fish are obtained after detection and screening of the filial generations of the fancy carp and the bighead carp, and the detection and screening method comprises fish body appearance measurement, flow cell DNA content detection, kidney tissue chromosome ploidy detection and blood cell morphology identification.
Based on a general inventive concept, the invention also provides an application of the tetraploid variegated carp obtained by the method, and the triploid fish is obtained by hybridizing the tetraploid variegated carp with the diploid fish and cultivating.
In the application, preferably, female diploid crucian with enlarged and soft abdomen is selected as a female parent, artificial oxytocin is performed by adopting a needle injection method, and the injected oxytocin comprises 500 mu g/kg of chorionic gonadotropin, 4 mu g/kg of luteinizing hormone releasing hormone analogue and 1 mu g/kg of diutanone; selecting strong, well-developed and slender male tetraploid brocade bighead carps as male parents, carrying out artificial induced spawning by adopting a one-needle injection method, wherein the injected oxytocin comprises 200 mu g/kg of chorionic gonadotropin and 3 mu g/kg of luteinizing hormone releasing hormone analogues, matching the male and female fishes according to the quantity ratio of 1:2 after injection, putting the male and female fishes into a spawning pond for induced spawning, putting a net sheet and a palm sheet into the pond, when egg grains appear on the net sheet or the palm sheet, netting and catching parent fishes after half an hour, collecting the sperms and the eggs for artificial dry insemination, then uniformly spreading fertilized eggs on the net sheet, putting the net sheet into inflow water for incubation, controlling the water temperature to be 24-25 ℃, and obtaining the triploid fishes after incubation.
Compared with the prior art, the invention has the beneficial effects that:
1. according to the method, the excellent properties of the fancy carp and the bighead carp are integrated through distant hybridization, so that a hybrid offspring has the advantages of two parents, the ornamental value of the fancy carp is kept, and the advantages of low requirement on water temperature and low culture cost of the bighead carp are inherited; in addition to obtaining gray diploid bighead carp and anthocyanin diploid bighead carp, hybrid offspring such as triploid variegated carp and tetraploid variegated carp are obtained, so that not only is a new ornamental fish germplasm resource obtained, but also an important role is played in improving the original ornamental fish in the market, and the obtained polyploid fish (triploid variegated carp and tetraploid variegated carp) also provides more excellent fish breeds for the market and also provides new resources and basis for the genetic breeding of fresh water fish; and the whole hybridization process is simple to operate, the breeding period is short, and the hatching rate (90.25%) and the survival rate (86.42%) of filial generations are high.
2. The mixed oxytocin adopted by the invention, namely the mixed oxytocin of the lutein release hormone analogue and the chorionic gonadotropin, takes the physical conditions of the koi into consideration and the requirements on the environment, and the mixed oxytocin with proper dosage can obviously improve the hatching rate and the survival rate of filial generations under the precondition of ensuring the physical health of parents.
3. The tetraploid variegated carp obtained by the invention is an excellent tetraploid fish, has beautiful body shape and excellent properties, provides a good model for rapid change of each level of genome in the initial period of polyploidization of vertebrates, has clear genetic background, plays an important role in analyzing the change of polyploidization of new vertebrates, and provides evidence support for explaining the rare reason of polyploidization phenomenon in the vertebrates; the triploid fish with high growth speed and strong stress resistance can be obtained in large quantity by utilizing the hybridization of the tetraploid variegated carp and the diploid fish, and has great industrial application prospect.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a gray diploid bighead carp in distant hybridization offspring of female parent koi and male parent bighead carp in example 1;
FIG. 2 shows the anthocyanin diploid bighead carp in distant hybridization offspring of female parent koi carp and male parent bighead carp in example 1;
FIG. 3 shows triploid fancy bighead carp in distant hybridization offspring of female parent fancy carp and male parent bighead carp in example 1;
FIG. 4 shows tetraploid fancy bighead carp in distant hybridization offspring of female parent fancy carp and male parent bighead carp in example 1;
FIG. 5 is a flow cytogram of gray diploid bighead carp in offspring of distant hybridization between female parent koi carp and male parent bighead carp in example 1;
FIG. 6 is a flow cytogram of an anthocyanin diploid bighead carp in distant hybridization offspring of female parent koi carp and male parent bighead carp in example 1;
FIG. 7 is a flow cytogram of triploid fancy bighead carp in offspring of distant hybridization of female parent fancy carp and male parent bighead carp in example 1;
FIG. 8 is a flow cytogram of tetraploid fancy bighead carp in offspring of distant hybridization of female parent fancy carp and male parent bighead carp in example 1;
FIG. 9 is a blood cell diagram of gray diploid bighead carp in distant hybridization offspring of female parent koi carp and male parent bighead carp in example 1;
FIG. 10 is a blood cell diagram of an anthocyanin diploid bighead carp in distant hybridization offspring of female parent koi carp and male parent bighead carp in example 1;
FIG. 11 is a blood cell diagram of triploid kochia carp in the offspring of distant hybridization between female parent kochia carp and male parent kochia carp in example 1;
FIG. 12 is a blood cell diagram of tetraploid kohlrabi among distant hybridization offspring of female parent kohlrabi and male parent kohlrabi in example 1;
FIG. 13 is a chromosome map of gray diploid bighead carp in offspring of distant hybridization between female parent koi carp and male parent bighead carp in example 1;
FIG. 14 is a chromosome map of an anthocyanin diploid bighead carp in distant hybridization offspring of female parent koi carp and male parent bighead carp in example 1;
FIG. 15 is a chromosome map of triploid fancy bighead carp in offspring of distant hybridization of female parent fancy carp and male parent bighead carp in example 1;
fig. 16 is a chromosome map of tetraploid fancy bighead carp in offspring of distant hybridization of female parent fancy carp and male parent bighead carp in example 1.
Detailed Description
In order to facilitate understanding of the invention, the invention will be described more fully and in detail with reference to the accompanying drawings and preferred embodiments, but the scope of the invention is not limited to the specific embodiments below.
Unless otherwise defined, all terms of art used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.
Example 1:
the invention relates to a method for distant hybridization between koi and bighead subfamily, which comprises the following steps:
(1) the parent selection of distant hybridization is that female fancy carps and male bighead carps which are physically careless, robust and normally developed are selected as female parent fish and male parent fish respectively 3-4 months before the breeding period of the fish, and the selected parent fish is moved out of a fish pond to be independently and specially cultured, so that accidents are avoided; scientifically feeding the parent fishes by using concentrated feed 1-2 months before the breeding period, and regularly stimulating the fishes with running water during the period to promote the maturity of the gonads of the parent fishes; the normal development refers to obvious sexual characteristics, and the physical absence refers to the absence of infection and trauma;
the water quality index for breeding female fancy carps is as follows: the pH value is 7.0-7.4, the hardness is less than 15 ℃, and the concentration of iron ions is 0.3 multiplied by 10-6Less than mol/L, sulfate ion concentration < 15X 10-6mol/L, chloride ion concentration < 19X 10-6mol/L, free chlorine and other toxic and harmful substances, dissolved oxygen of more than 5 × 10-6mg/L, ammonia concentration < 0.1X 10-6mg/L, nitrite concentration < 0.1X 10-6mol/L, nitrate concentration at 5.5X 10-6mg/L～6.0×10-6mol/L, no hydrogen sulfide;
the water quality index for raising male bighead carp is as follows: the pH value is 6.5-8.5, the dissolved oxygen is more than or equal to 4mg/L, the ammonia nitrogen content is less than 0.025mg/L, and the biochemical oxygen demand is less than 5 mg/L;
scientifically breeding the fancy carp: the water quality standard for feeding the fancy carps is colorless, tasteless and odorless, and the transparency of water is high; the moss on the wall of the pond grows normally; the method comprises the following steps that a fish pond for breeding fancy carps needs to have good sunshine and ventilation conditions, the area of the fish pond is not smaller than 30 square meters, and the depth of the fish pond is about 2 meters; the fancy carp is fed regularly by adopting animal feed, the feeding time is 9:00-10:00 in the morning, 12:00-13:00 in the noon and 6:00-8:00 in the afternoon, and the regular feeding is favorable for training the conditioned reflex of the fancy carp;
scientific breeding of bighead carps: before stocking bighead carps, applying base fertilizer to the pond, and after stocking, applying additional fertilizer according to seasons and specific measures of the pond;
(2) in 5 and 6 months of the year, when the water temperature is basically stabilized above 22 DEG CThe spawning induction can be carried out on the parent fish; injecting mixed oxytocin of luteinizing hormone releasing hormone analogue (LRH-A), chorionic gonadotropin (HCG) and diosone into the female parent fish obtained in the step (1) by adopting a needle injection method for oxytocin, wherein the injection dosage is 15 mu g/kg of luteinizing hormone releasing hormone analogue (LRH-A), 900IU/kg of chorionic gonadotropin (HCG) and 2 mu g/kg of diosone; injecting mixed oxytocin containing luteinizing hormone releasing hormone analogue (LRH-A) and chorionic gonadotropin (HCG) into the male parent fish by adopting a two-needle injection method for induced spawning, wherein the total injection dosage of the oxytocin is 6 mug/kg of the luteinizing hormone releasing hormone analogue (LRH-A) and 400IU/kg of the chorionic gonadotropin (HCG), the male parent fish is injected for the first time while the female parent fish is induced to spawn, the first injection dosage is 20% of the total injection dosage volume, the male parent fish is injected for the second time after 4-5 h, and the second injection dosage is 80% of the total injection dosage volume; the mixed oxytocin is injected at 18: 00-20: 00 evening, and the mixed oxytocin is injected in an abdominal cavity at the base part of the pectoral fin of the parent fish without scales; after the preparation, the carp and bighead carp are respectively thrown into the carps with the area of 30-40 m according to the quantity ratio of 1:22Temporarily culturing in different spawning ponds, then observing the conditions of the ponds more, flushing the ponds in time according to water conditions, periodically stimulating the parent fishes with running water, fishing when the parent fishes feel heat and chase tail, until the female parent fishes, namely the brocade carps, spawn and the male parent fishes, namely the bighead carps, produce sperms; the time for injecting the mixed oxytocic is 18: 00-20: 00 in the evening generally;
(3) putting female parent fancy carp and male parent bighead carp in water pools at different temperatures of 25 ℃, keeping the water temperature constant, observing the activity condition of the fish, and fishing when male fish fancy carp knocks back to female fancy carp; grasping a tail handle by a left hand, holding the tail handle by a right hand, cleaning the body surface of a fish after the belly is obliquely upwards at about 45 degrees, extruding eggs and sperms into a dry ceramic basin, slightly stirring the dried feathers for 2-3 min, then adding clear water into the ceramic basin to activate the sperms for insemination, then quickly transferring fertilized eggs into an incubation pipeline of a special fish viscous egg incubation device for running water incubation, preparing the incubation device at the temperature of 20 ℃ in advance, and obtaining hybrid offspring fries of koi carps and bighead carps after incubation, wherein the fries are produced 8: 00-9: 00 in the morning of the next day;
(4) fertilizing a pond, preparing a good environment for fry growth, controlling the water depth to be 0.7-0.8 m, and directly adding 200-300 mL of green algae and 200-300 mL of blue algae in well water for fertilizing; culturing the fry obtained in the step (3) in a hatching tank for 3 days, and transferring the fry into a fertilizing pond for fine feeding after the fry appears a waist point; observing the development condition of the fish fry, splashing soybean milk after 2 days until the fish fry has the capability of independently ingesting food, wherein the splashing frequency is 1-2 times/day, and the obtained offspring hybrid fish is gray diploid bighead carp, anthocyanidin diploid bighead carp, triploid brocade bighead carp (brocade bighead carp with mouth beard) and tetraploid brocade bighead carp (brocade bighead carp without obvious mouth beard), and the appearances of the offspring hybrid fish are respectively shown in figures 1, 2, 3 and 4;
after the obtained filial generations are bred for 3-4 months, the data related to the biological shape of each filial generation are randomly detected and compared with the male parent and the female parent respectively, and the results are shown in table 1:
table 1: comparison of countable characters (unit: sheet or strip) of fancy carp, bighead carp and filial generation
Figure GDA0003173972590000061
Figure GDA0003173972590000071
As can be seen from Table 1, the four filial generations are basically different from female parent koi and male parent bighead carp in appearance (side line scale, dorsal fin line, ventral fin line and hip fin line), which indicates that the filial generations have morphological variation characteristics.
Detecting and screening filial generations of the koi and the bighead carp:
the method comprises the following steps of measuring the DNA content of gray diploid bighead carp, anthocyanin diploid bighead carp, triploid variegated bighead carp (chinch with whiskers) and tetraploid variegated bighead carp (chinch without obvious whiskers) by using a flow cytometer, wherein the operation method for detecting the DNA content of the flow cytometry comprises the following steps: wetting a disposable syringe by using a heparin sodium solution, extracting 0.1mL of blood sample from a fish tail vein by using the disposable syringe, storing the obtained blood sample in a microcentrifuge tube (Eppendorf tube) with 0.8% of normal saline, adding 500 mu L of cell nucleus extract DAPI-A (nuclear extraction solution, provided by Partec Gmbh, Germany) into the microcentrifuge tube, and treating for 10-12 min; the sample was then filtered using a 20 μm nylon filter (supplied by Partec Gmbh, germany); the samples were stained with a DNA staining solution (DAPI-B, supplied by Partec Gmbh, Germany) for about 10min in the absence of light and then subjected to on-machine detection. The results are shown in fig. 5, 6, 7 and 8, and it is known that gray bighead carp and variegated carp are diploid fish, chinch with whiskers is triploid fish, and chinch without obvious whiskers is tetraploid fish.
Performing blood cell morphology identification on gray diploid bighead carp, anthocyanidin diploid bighead carp, triploid brocade bighead carp (chinch with oral beard) and tetraploid brocade bighead carp (chinch without obvious oral beard), wherein the blood smear step is as follows: 1) a small amount of blood was drawn with a syringe and dropped one drop onto the slide; 2) quickly and uniformly smearing the whole glass slide in a direction that a clean glass slide is inclined by 45 degrees; 3) adding phosphate buffer (phosphoric acid A, B liquid mixed at a ratio of 1: 1), standing for 10 min; 4) washed from the back with a thin stream of water, dried and then microscopically examined. As shown in fig. 9, 10, 11 and 12, it can be found that the red cell nuclei of gray diploid bighead carp and anthocyanidin diploid bighead carp are similar in size, but the red cell nuclei of triploid variegated bighead carp (chinch bighead carp with whisker) and tetraploid variegated bighead carp (chinch bighead carp without obvious whisker) are significantly larger than those of the former two. In addition, the triploid variegated carp also has abnormal binuclear red blood cells with the proportion of about 12.5 percent, the tetraploid variegated carp also has abnormal binuclear red blood cells with the proportion of 10.2 percent, and the number of blood cell nuclei of the gray diploid variegated carp crucian carp and the anthocyanin diploid variegated carp crucian carp is normal.
The method comprises the following steps of (1) detecting chromosome ploidy numbers of gray diploid bighead carp, anthocyanidin diploid bighead carp, triploid jin bighead carp (chinch with whisker) and tetraploid chinch bighead carp (chinch without obvious whisker) by using a kidney tissue chromosome ploidy detection method, wherein the kidney tissue chromosome ploidy detection operation steps are as follows: after the experimental fish is cultivated at the water temperature of 20-22 ℃ for 2-3 days, injecting PHA for 1-3 times into the experimental fish, wherein the dosage of each time is 2-8 mug/g of body weight, the interval time is 12-24 hours, and colchicine is injected 2-6 hours before anatomical material taking, and the dosage is 2-4 mug/g of body weight; taking out kidney tissue, shearing the kidney tissue under physiological saline water, performing hypotonic treatment with 0.075mol/LKCL at 25 deg.C for about 45 min; fixing kidney tissues 1-3 times by using Carnot's fixing solution (glacial acetic acid: methanol: 1: 3); dropping the slide on the frozen slide; and dyeing by using a Giemsa (Giemsa) dye solution for 55 minutes, washing the back surface of the glass slide by using a fine water flow to remove the dye solution, and performing microscopic examination and photographing after air drying. As shown in fig. 13, 14, 15 and 16, it can be seen that the number of chromosomes of the gray diploid bighead carp and the anthocyanin diploid bighead carp is 100, the number of chromosomes of the triploid bighead carp (chinch with whisker) is 124, and the number of chromosomes of the tetraploid chinch bighead carp (chinch without significant whisker) is 148. The technical results of flow cytometry, blood smear and chromosome slide show that the gray diploid bighead carp and the anthocyanin diploid bighead carp are diploid, the triploid variegated bighead carp (chinch bighead carp with mouth beard) is a triploid hybrid fish, and the tetraploid variegated bighead carp (chinch bighead carp without obvious mouth beard) is a tetraploid hybrid fish.
In general, the excellent properties of the koi and the bighead carp are integrated through distant hybridization, so that the filial generation has the advantages of two parents, the ornamental value of the koi can be kept, and the advantages of low requirement on water temperature, low requirement on water quality and low culture cost of the bighead carp can be inherited; besides obtaining gray diploid bighead carp and anthocyanin diploid bighead carp, hybrid offspring such as triploid variegated bighead carp and tetraploid variegated bighead carp are obtained, on one hand, not only is a new ornamental fish germplasm resource obtained, and plays an important role in improving the original ornamental fish in the market, but also polyploid fishes (triploid variegated bighead carp and tetraploid variegated bighead carp) are obtained, so that more excellent fish breeds are provided for the market, and new resources and foundations are provided for genetic breeding of freshwater fish; and the whole hybridization process is simple to operate, the breeding period is short, and the hatching rate (90.25%) and the survival rate (86.42%) of filial generations are high. The invention obtains four types of hybrid fish: the fish bred with gray diploid bighead carp, anthocyanidin diploid bighead carp, triploid variegated carp and tetraploid variegated carp has beautiful appearance and high ornamental value, has variation phenomenon obviously different from parents, and has good economic value and market prospect.
On one hand, the generation of the tetraploid variegated carp hybrid fish enriches the germplasm resources of the tetraploid fish and provides a new tetraploid source for preparing the triploid on a large scale.
Example 2:
the application of the tetraploid Jinbiao hybrid fish obtained in the embodiment 1 in cultivating the triploid fish comprises the following steps: selecting female diploid crucian with enlarged and soft abdomen as a female parent, and performing artificial induced spawning by adopting a needle injection method, wherein the injected oxytocin comprises 500 mu g/kg of chorionic gonadotropin, 4 mu g/kg of luteinizing hormone releasing hormone analogue and 1 mu g/kg of diosone; meanwhile, selecting strong, well-developed and slender male tetraploid broccoli bighead carps as male parents, carrying out artificial induced spawning by adopting a one-needle injection method, wherein the injected oxytocin comprises 200 mu g/kg of chorionic gonadotropin and 3 mu g/kg of luteinizing hormone releasing hormone analogues, matching the injected androgenic hormones and the androgenic hormones according to the number ratio of 1:2, putting the male and female fishes into a spawning pond for induced spawning, putting a net sheet and a palm sheet into the pond, when egg grains appear on the net sheet or the palm sheet, fishing out nets and catching parent fishes for half an hour, collecting sperms and eggs for artificial dry insemination, then uniformly spreading fertilized eggs on the net sheet, putting the fertilized eggs into inflow water for hatching, controlling the water temperature to be 24-25 ℃, and obtaining the triploid fishes after hatching.

Claims (2)

1. A method for distant hybridization between the subfamilies of koi and bighead carp is characterized by comprising the following steps: carrying out artificial induced spawning by taking fancy carps as female parents and bighead carps as male parents, and injecting mixed oxytocin containing luteinizing hormone releasing hormone analogues, chorionic gonadotropins and diospyrone into the female parent carps by adopting a one-needle injection method to carry out induced spawning, wherein the injection dosage is 12-15 mug/kg of the luteinizing hormone releasing hormone analogues, 900-1000 IU/kg of the chorionic gonadotropins and 1-2 mug/kg of the diospyrones; injecting mixed oxytocin containing a luteinizing hormone releasing hormone analogue and chorionic gonadotropin to the male parent fish by adopting a two-needle injection method for induced spawning, wherein the total injection dose of the oxytocin is 6-8 microgram/kg of the luteinizing hormone releasing hormone analogue and 400-600 IU/kg of the chorionic gonadotropin, performing first injection on the male parent fish while performing induced spawning on the female parent fish, the first injection dose is 20% of the total injection dose volume, performing second injection on the male parent fish after 4-5 h, and the second injection dose is 80% of the total injection dose volume; carrying out artificial dry insemination on the ovum and the sperm obtained after artificial spawning induction, then hatching the fertilized ovum in running water, and obtaining the filial generation of the koi and the bighead carp after successful hatching;
selecting female fancy carps and male bighead carps which are physically innoxious, robust and normally developed as female parent fish and male parent fish respectively to move out of a fishpond for independent feeding 3-4 months before the spawning induction of the female parent fish and the male parent fish, scientifically feeding the female parent fish and the male parent fish by using concentrated feed 1-2 months before the spawning induction, and periodically stimulating the fish by running water; the water quality index for breeding female fancy carps is as follows: the pH value is 7.0-7.4, the hardness is less than 15 ℃, and the concentration of iron ions is 0.3 multiplied by 10-6Less than mol/L, sulfate ion concentration < 15X 10-6 mol/L, chloride ion concentration < 19X 10-6 mol/L, free chlorine, dissolved oxygen >5mg/L, ammonia concentration < 0.1X 10-6 mol/L, nitrite concentration < 0.1X 10-6 mol/L, nitrate concentration at 5.5X 10-6 mg/L~6.0×10-6 mol/L, no hydrogen sulfide; the water quality index for raising male bighead carp is as follows: the pH value is 6.5-8.5, the dissolved oxygen is more than or equal to 4mg/L, the ammonia nitrogen content is less than 0.025mg/L, and the biochemical oxygen demand is less than 5 mg/L;
hastening spawning the parent fish when the water temperature is stabilized above 20 ℃ in 5 and 6 months in the year; the specific operation of the artificial dry insemination comprises the following steps: grasping the tail handle of the parent fish with the left hand, holding the back of the parent fish with the right hand, cleaning the body surface of the parent fish, extruding the fish belly, respectively extruding the ovum and the sperm into a dry ceramic basin, slightly stirring the dried feather for 2-3 min, and then adding clear water into the ceramic basin to activate the sperm for insemination;
controlling the water temperature to be 24-26 ℃ when the fertilized eggs are incubated in running water;
continuously culturing the fries obtained by running water incubation in an incubation tank for 3 days, transferring the fries to a pond with pre-fertilization for fine feeding after waist spots appear, splashing soybean milk 1-3 days after the fries are put in the pond, and splashing 1-2 times every day; the water depth of the fine breeding is controlled to be 0.7-0.8 m, the breeding water needs to be exposed for 2 days if running water is used, the breeding water does not need to be exposed if well water is used, and 200-300 mL of green algae and 200-300 mL of blue algae are added after the running water is exposed or directly added into the well water;
and the detection and screening method comprises the steps of fish body appearance measurement, flow cell DNA content detection, kidney tissue chromosome ploidy detection and blood cell morphology identification.
2. The application of the tetraploid variegated carp obtained by the method for distant hybridization between the variegated carp and the variegated carp subfamily in claim 1 is characterized in that the tetraploid variegated carp is hybridized with a diploid fish to breed the triploid fish.
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