CN114128649A - Selective breeding method for distant hybridization offspring between megalobrama amblycephala and ancherythroculter nigrocauda - Google Patents
Selective breeding method for distant hybridization offspring between megalobrama amblycephala and ancherythroculter nigrocauda Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; 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
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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
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- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
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- Farming Of Fish And Shellfish (AREA)
Abstract
The invention discloses a selective breeding method of distant hybrid offspring between megalobrama amblycephala and ancherythroculter nigrocauda, which comprises the following steps: A. distant hybridization of megalobrama amblycephala and ancherythroculter nigrocauda: a. selecting hybrid parents; b. breeding hybrid parents: culturing parent strains of megalobrama amblycephala and ancherythroculter nigrocauda in separate ponds; c. and (3) hybridization induced spawning: selecting 5 months to 6 months in the middle trip of the Yangtze river; d. hybrid insemination: respectively fishing by netting when the parent fishes estrus, selecting megalobrama amblycephala female fishes and ancherythroculter nigrocauda male fishes, manually collecting sperm and eggs, and carrying out dry insemination, wherein the ratio of the female fishes to the male fishes is 1: 1-2; e. hatching the hybrid fertilized eggs: debonding fertilized eggs by yellow mud slurry or talcum powder slurry, or directly adhering the fertilized eggs to an artificial fish nest, placing the fertilized eggs into a flushing type hatching tank or a hatching jar and a hatching loop for hatching, moving out hatching equipment, and culturing fry and fingerlings and parent fishes in a pond; B. and (4) breeding hybrid offspring of the megalobrama amblycephala and the ancherythroculter nigrocauda. The method is easy to implement and simple and convenient to operate, and the excellent new culter bream variety with high yield, stress resistance and scale compactness is obtained.
Description
Technical Field
The invention belongs to the technical field of selective breeding of distant hybrid offspring of fishes in subfamily Cyprinidae and Erythroculter culter, and particularly relates to a selective breeding method of distant hybrid offspring between megalobrama amblycephala and Ancherythroculter nigrocauda.
Background
Crossbreeding refers to a method of crossing between individuals of different populations and different genotypes, and breeding a homozygous variety among the progeny of the hybrid. The hybridization can recombine the genes of parents to form various types, and provides abundant materials for selective breeding, the ideal breeding goal is to combine the excellent genes of parents controlling different characters into a whole, or to accumulate different micro-effect genes of parents controlling the same character to generate types exceeding the parents in each character, so the correct selection and reasonable combination of the parents are the key for hybridization breeding success or failure. Since the end of the 20 th century 50 years, China has carried out a large number of fish hybridization tests, such as Hibiscus carp (Spirosoma carpi female parent multiplied by Korysan carp red carp male parent), glume carp (Spirosoma carpi female multiplied by Carassius auratus trans-nuclear transfer fish), and the like, and the hybrids have the advantages of faster growth, higher nutrient content, stronger disease resistance and the like compared with the parents. How to use biotechnology to genetically improve the prior fishes to obtain new aquatic species with excellent characters is a long-standing problem in the field and needs to be solved.
The fishes of the subfamily culter are widely distributed groups in China, are mostly main objects for catching in rivers, lakes and reservoirs, have high economic value, are common in the genus of the Erythroculter and the genus of the bream, and comprise the Ancherythroculter nigrocauda, the Megalobrama amblycephala and the like.
The Ancherythroculter nigrocauda is also called as a high-shoulder Ancherythroculter nigrocauda and Ancherythroculter nigrocauda, belongs to Cyprinidae (Cyprinidae), cultilisidae (Cultrinae) and Ancherythroculter nigrocauda (Ancherythroculter), and is a special omnia carnivorous economic fish at the upstream of Yangtze river in China; due to the building of hydraulic engineering, the cold fishing and the like, natural resources are few, specifications are small, and learners are listed as 'three-level eager protection objects' in the last 80 th century. Researches show that the demand of protein of the Ancherythroculter nigrocauda feed is low (33.0-36.7%), the Ancherythroculter nigrocauda feed is absolutely unique in culter, and the Ancherythroculter nigrocauda feed has the characteristics of disease resistance, stress resistance and the like, and is suitable for living body transportation and live fish marketing; however, the variety is small in size, the sexual maturity in Yangtze river is only 100-200 g, the adult fish domesticated artificially is 400-600 g generally, and the variety is not suitable for the large-scale consumption habit of asparagus. In view of the excellent properties of the Ancherythroculter nigrocauda, the Ancherythroculter nigrocauda is considered to be an rare breeding material, and a living germplasm resource library is established; by utilizing the characteristics of low protein demand and disease resistance and stress resistance of the ancherythroculter nigrocauda feed, the male ancherythroculter nigrocauda bred by four generations is hybridized with the female culter alburnus for producing drifting eggs, and the new hybrid culter 'Pioneer 1' variety (GS-02-001 sulponicas 2012) with the breeding feed cost reduced by 50.0-52.4% compared with the female parent culter alburnus and the growth speed improved by 23.5-29.6% compared with the male parent ancherythroculter nigrocauda is cultivated, so that the industrial problems of high culter breeding cost, difficulty in catching and marketing of live fishes are solved.
Megalobrama amblycephala (Megalobrama ambycephala) also called Megalobrama amblycephala belongs to Cyprinidae (Cyprinidae), Erythroculter culteridae (Cultrinae) and Megalobrama (Megalobrama), and is one of important herbivorous economic fishes in China. The megalobrama amblycephala has high growth speed, the suitable protein content of the feed is 32 percent and is similar to the suitable protein content (33 percent) of the adult fish of the Ancherythroculter nigrocauda; however, the variety has poor resistance to aeromonas hydrophila, the survival rate of cultivation is greatly influenced by explosive hemorrhagic disease, and the variety has strong stress, so that the problems of unstable cultivation yield, increased labor intensity, increased production cost and the like are caused. In order to overcome the defects of the megalobrama amblycephala, the characteristics of aeromonas hydrophila resistance and stress resistance of the ancherythroculter nigricans are utilized, a good new strain is obtained by distant hybridization of the megalobrama amblycephala and the ancherythroculter nigricans, a method for distant hybridization of the megalobrama amblycephala and the ancherythroculter nigricans is disclosed by retrieval, and the application number is 201510950545.3, wherein the patent application mainly has the following defects: the core technologies of parent selection, parent cultivation, artificial induced spawning and the like of the megalobrama amblycephala are disclosed by a 'method for crossbreeding Erythroculter ilishaeformis and megalobrama amblycephala' (application number: 200610053442.8, application date: 09.18.2006), and the core technologies of parent selection, parent cultivation, artificial induced spawning and the like of the Ancherythroculter nigrocauda are disclosed by a 'method for distant crossbreeding between Ancherythroculter ilishaeformis and Ancherythroculter nigrocauda' (application number: ZL 201310119287.5, application date: 04.09.2013). Compared with the published patent ZL 201310119287.5, the technical problem actually solved by the application is that which fish species are selected for crossbreeding and how to hybridize, and a new excellent hybrid line can be obtained, but the basis for obtaining the excellent characters of the hybrid line is not specified through experiments or optimization and improvement, the technical measures cannot be implemented by the technical personnel in the field according to the disclosure, compared with the published patent 200610053442.8, the Erythroculter ilishaeformis Bleeker and the Ancherythroculter nigrocauder belong to subfamily fishes of Cyprinidae, the living habits and genetic relationship of the two are relatively similar, the technical personnel in the field cannot obtain effective technical inspiration from the patent 200610053442.8, and the Ancherythroculter nigrocauder is adopted to replace the Erythroculter ilishaeformis Bletilla to carry out distant hybridization to screen filial generations. The technical scheme does not have outstanding substantive features and remarkable progress by combining with a patent 200610053442.8 on the basis of a patent ZL 201310119287.5.
In summary, the three patent application numbers 200610053442.8, 201310119287.5 and 201510950545.3 have the common main problems that: only one filial generation is obtained by intergeneric distant hybridization, and a more excellent new variety is obtained by systematic breeding of filial generations.
Through retrieval, a selective breeding method of distant hybrid offspring between megalobrama amblycephala and ancherythroculter nigrocauda is not disclosed, reported or patented.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and the defects of varieties, and provides a selective breeding method of distant hybridization filial generation between megalobrama amblycephala and ancherythroculter nigrocauda, which is easy to implement, simple and convenient to operate and capable of obtaining a high-yield, stress-resistant and scale-compact excellent new variety of the megalobrama amblycephala.
In order to solve the technical problems, the invention adopts the following technical measures:
a selective breeding method for distant hybridization offspring between megalobrama amblycephala and Ancherythroculter nigrocauda comprises the following steps:
1. hybridizing the male parent of the megalobrama amblycephala and the male parent of the ancherythroculter nigrocauda;
1.1, selecting hybrid parents. Selectively mature and healthy megalobrama amblycephala and ancherythroculter nigrocauda serve as parents, female megalobrama amblycephala is 3-4 years old and has the weight of more than 1.0kg, and male ancherythroculter nigrocauda is 2-3 years old and has the weight of more than 0.4 kg.
1.2, breeding the hybrid parents. And (4) breeding the parent strains of the megalobrama amblycephala and the ancherythroculter nigrocauda in separate ponds. The stocking amount of parent fishes of megalobrama amblycephala is 150-200 pieces/mu, and special compound feed and fresh green grass are fed; the stocking amount of the parent fishes of the Ancherythroculter nigrocauda is 200-300/mu, and the special compound feed produced by regular manufacturers with the crude protein content of more than 33 percent is fed (the special compound feed is purchased in the market, and can be produced by a plurality of manufacturers, and the special compound feed is feasible as long as the protein content of the feed is met).
1.3, cross spawning induction. Selecting 5-6 months in midstream of Yangtze river, controlling water temperature at 20-28 deg.C, respectively injecting oxytocic drug Cyprinus Carpio Pituitary Gland (PG) or dopamine antagonist Diohonone (DOM), chorionic gonadotropin (HCG), and luteinizing hormone releasing hormone analog 2 (LRH-A) for fish2) Alone or in admixture; recommended dosage of female fish is PG 2-6 mg or DOM 2-8 mg, HCG 500-1200 IU, LRH-A per kilogram of parent fish22-10 μ g, and adjusting dosage (upper limit dosage for injection at 20 deg.C and lower limit dosage for injection at 28 deg.C) or making into reasonable formula (oxytocic drug including PG or DOM, HCG, LRH-A)2Dosages typically only give the range of use of each oxytocin, as dosages are not invariable); generally, the upper limit dose (PG 6mg or DOM 8mg, H) is injected at 20 deg.CCG 1200IU、LRH-A210 ug), lower limit dose (PG 2mg or DOM 2mg, HCG 500IU, LRH-A) at 28 deg.C22 μ g). The female fish adopts a one-needle injection method or a two-needle injection method, the needle pitch of the two-needle injection method is 6-10 h, and the first needle is 1/10 or 1-2 mu g LRH-A of the total dose2And the second needle injects the remaining amount. The injection dosage of the male fish is 1/3-1/2 of the female fish, and the male fish is injected immediately after the injection of the total dosage of the female fish is finished. The parents after the injection of the oxytocic are separately cultured in an oxytocic pool and sufficient dissolved oxygen is kept.
1.4, hybrid insemination. When the parent fish is in heat, the parent fish respectively catches the fish in a net, and the megalobrama amblycephala female fish and the ancherythroculter nigrocauda male fish are selected to artificially collect sperms and eggs for dry insemination or semi-dry insemination; the ratio of the male parent fish to the female parent fish is 1: 1-2.
1.5, hatching the hybrid fertilized eggs. Debonding fertilized eggs with yellow mud slurry or talc powder slurry, or directly adhering artificial fish nest (such as palm sheet, net sheet, etc.), placing in a flushing hatching tank or hatching jar, and hatching loop for hatching at a density of 50-80 ten thousand grains/m3Water body, hatching water flow takes the egg not deposited as degree; when the waist point appears, the hatching equipment is moved out, and fry and fingerling and parent fish culture are carried out in the pond.
2. Breeding filial generations of megalobrama amblycephala (male parent) and ancherythroculter nigrocauda (female parent);
and 2.1, parent selection of hybrid filial generation. And selecting healthy and sexually mature individuals with the weight of more than 1.0kg from hybrid offspring of megalobrama amblycephala (male parent) and ancherythroculter nigrocauda (female parent) as parents.
2.2, breeding the parents of the filial generation. The stocking amount of parent fishes of hybrid offspring of megalobrama amblycephala (male parent) and ancherythroculter nigrocauda (female parent) is 150-200/mu, and the special compound feed with the crude protein content of more than 33% is fed. Starting in the middle ten (4) months, the parent culture pond is changed for 1 time per week, and is flushed with water irregularly (generally 2-3 times per week) at the same time, so that the gonad development of the parent fish is promoted.
And 2.3, breeding filial generations by generations. The spawning induction of the filial generation by generation parent fish is the same as the spawning induction of the cross, the generation-by-generation artificial insemination of the filial generation is the same as the cross insemination, and the generation-by-generation breeding and hatching of the filial generation is the same as the cross fertilized egg hatching.
And 2.4, generation-by-generation breeding of hybrid filial generation. In hybrid offspring of megalobrama amblycephala (male parent) and ancherythroculter nigrocauda (female parent), taking the rhombus type and the growth speed as breeding targets, and continuously breeding for four generations by adopting a colony breeding technology; from fry rearing to parent fish, each generation is subject to two years and 3 times of selection. The breeding time is respectively 6 months old, 18 months old and 24 months old, the corresponding selection rates are respectively 20%, 10% and 5%, and the total selection rate is 0.1%.
2.5, detecting the character of the new variety of the culter bream obtained by continuously breeding the filial generation for four generations. Compared with the original parent megalobrama amblycephala and the ancherythroculter nigrocauda, the new variety character of the culter amblycephala obtained by hybrid offspring of the megalobrama amblycephala (male parent) and the ancherythroculter nigrocauda (female parent) through continuous four-generation breeding is detected:
(1) genetic characterization based on SSR markers: the genetic similarity and the genetic distance of three groups such as megalobrama amblycephala (male parent) and anchovy (female parent) as well as new culter amblycephala varieties bred by continuous four generations after hybridization are analyzed by using 11 pairs of microsatellite primers with higher polymorphism, and the results show that the genetic similarity of the new culter amblycephala and the female parent megalobrama amblycephala is 0.7627, is higher than the genetic similarity 0.5838 of the male parent anchovy amblycephala, and the genetic distance of the new culter amblycephala and the female parent megalobrama amblycephala is 0.2709 and is less than the genetic distance 0.5381 of the male parent anchovy amblycephala; shows that the new variety of the culter bream has closer genetic relationship with the female parent megalobrama bream and is more genetically biased to the female parent. The primer is as follows:
erythroculter nigrocauda microsatellite fluorescent primer and site characteristics
(2) Body type: measuring the quantitative and frame data of the three groups, analyzing the forms of the new culter breams, the megalobrama amblycephala and the ancherythroculter nigrocauda by using a clustering analysis and principal component analysis method, and showing that the genetic distances of the new culter breams, the megalobrama amblycephala and the ancherythroculter nigrocauda are respectively 0.18 and 0.26, and the genetic distances of the megalobrama amblycephala and the ancherythroculter nigrocauda are 0.39; the new species of the culter breams are in the shape of a rhombus as the megalobrama amblycephala.
(3) Asphyxia point: the asphyxiation points of the new culter breams, the megalobrama amblycephala and the juvenile ancherythroculter nigrocauda are researched by adopting a closed respiration experiment method, and the results show that the asphyxiation points of the new culter breams, the megalobrama amblycephala and the juvenile ancherythroculter nigrocauda with the body mass of 7.82g, the new culter breams, the megalobrama amblycephala with the body mass of 6.02g and the ancherythroculter nigrocauda with the body mass of 8.02g are respectively 0.49 +/-0.03 mg/L, 0.63 +/-0.02 mg/L and 0.41 +/-0.04 mg/L at the water temperature of 23-25.5 ℃ (24.7 +/-0.5 ℃); the suffocation point of the new culter bream variety is obviously lower (p is less than 0.05) than that of the female parent megalobrama amblycephala, and is reduced by 22.2 percent compared with that of the megalobrama amblycephala.
(4) Ability to resist infection by Aeromonas hydrophila: under the condition of water temperature of 24-26 ℃ (25 +/-1 ℃), an infection method of aeromonas hydrophila suspension by intraperitoneal injection is adopted to determine the aeromonas hydrophila infection resistance of new culter breams, megalobrama amblycephala and ancherythroculter nigrocauda; the results show that the aeromonas hydrophila infection resistance of the three varieties is remarkably different (p is less than 0.05), and the semi-lethal concentrations of the pathogenic bacteria to the new variety of the culter breams, the megalobrama amblycephala and the ancherythroculter nigrocauda are respectively 6.9 multiplied by 10 according to the calculation of the kowter method8CFU/mL、7.2×106CFU/mL and 3.6X 107CFU/mL, the sensitivity of the three fishes to Aeromonas hydrophila is from strong to if in turn: megalobrama amblycephala is more than Ancherythroculter nigrocauda and a new species of culter amblycephala.
(5) Growth rate and survival rate: under the same conditions, a new culter bream species is marked with megalobrama amblycephala and ancherythroculter nigrocauda and cultured in the same pond, and the bream material suitable for growth of the megalobrama amblycephala is fed, so that the result shows that the new culter bream species grows 21.9% faster than the megalobrama amblycephala on average and 84.8% faster than the ancherythroculter nigrocauda on average, and the culture survival rate is improved by 37.5% compared with the megalobrama amblycephala on average.
The method selects specific megalobrama amblycephala and ancherythroculter nigrocauda for distant hybridization, ensures that the hybridization fertility rate, the hatching rate and the emergence rate all reach more than 80 percent through the set hybridization method and parameter control, ensures that the offspring has excellent properties, ensures that the offspring with higher body type shows excellent properties such as high growth speed, strong disease resistance, more compact scales and the like, can effectively obtain breeding materials with excellent properties, lays a foundation for further breeding new varieties, has important significance and important application value on genetic breeding, and can not be obtained through the hybridization prediction of similar varieties but can be obtained through continuous tests and optimization improvement; the change of the breeding method, the used medicines and the parameters can generate great influence on the offspring, thereby changing the breeding time, the variety and the partial structure of the offspring.
The preferable scheme is as follows: the original parent is selected and hybridized with the male fishes of the megalobrama amblycephala and the ancherythroculter nigrocauda which are bred for four or more generations continuously to obtain hybrid offspring.
The preferred steps are as follows: the original parent megalobrama amblycephala and ancherythroculter nigrocauda are selected 3 times in two years per generation from fry breeding to parent fish by adopting a colony selection technology with the growth speed as a breeding target. The breeding time is respectively 6 months old, 18 months old and 24 months old, the corresponding selection rates are respectively 20%, 10% and 5%, and the total selection rate is 0.1%.
The obtained hybrid offspring is bred generation by generation, a new variety of excellent culter breams with high growth speed and stress resistance is cultivated, and the problem that the breams are susceptible to aeromonas hydrophila to cause fulminant hemorrhagic disease is solved.
Through the technical measures: the most important is the generation-by-generation breeding of hybrid offspring of megalobrama amblycephala (male parent) and ancherythroculter nigrocauda (female parent) in the step 2; the invention mainly solves the technical problems that hybrid offspring of megalobrama amblycephala (male parent) and Erythroculter nigrocauda (female parent) takes the rhombohedral type and the growth speed as breeding targets, obtains a new breeding variety of hybridized culter amblycephala with excellent properties, and solves the explosive hemorrhage and strong irritability caused by susceptibility of aeromonas hydrophila of the amblycephala; compared with the prior art, the technical scheme of the invention is a technical scheme for obtaining a good new variety by breeding hybrid offspring of megalobrama amblycephala (male parent) and ancherythroculter nigrocauda (male parent); compared with the prior art, the technical scheme of the invention adds the technical measures of hybrid offspring breeding of megalobrama amblycephala (male parent) and ancherythroculter nigrocauda (female parent).
Compared with the prior art, the invention has the following advantages: the same points are as follows: (1) the crossbreeding combination of megalobrama amblycephala (male parent) and Ancherythroculter nigrocauda (female parent) is selected. (2) Solves the cross breeding technology and obtains the rhombic excellent hybrid strain with fast growth.
Compared with the prior art, the invention has the following advantages: the difference is as follows: (1) in hybrid offspring of megalobrama amblycephala (male parent) and Ancherythroculter nigrocauda (female parent), the method continuously breeds four generations by adopting a population breeding technology by taking the rhomboid type and the growth speed as breeding targets; from fry rearing to parent fish, each generation is subject to two years and 3 times of selection. The breeding time is respectively 6 months old, 18 months old and 24 months old, the corresponding selection rates are respectively 20%, 10% and 5%, and the total selection rate is 0.1%. (2) The invention discloses a breeding technical scheme for obtaining a new product of megalobrama amblycephala by continuously breeding male and female hybridized filial generations of megalobrama amblycephala and Erythroculter nigrocauda for four generations and a detection scheme for excellent characters of the new product. (3) The obtained new species of the culter breams solves the industrial problem that the breams are easy to infect aeromonas hydrophila to cause explosive hemorrhagic disease.
Compared with the prior art, the invention has the beneficial effects that:
obtains a new culter bream variety (variety number: GS-01-005-2018) with stable and excellent properties of rhomboid, high growth speed, stress resistance and the like. According to the invention, the megalobrama amblycephala (male parent) and the ancherythroculter nigrocauda (female parent) are hybridized, and the obtained hybrid offspring is subjected to continuous four-generation breeding, so that the obtained excellent new variety of the megalobrama amblycephala has a rhombic body type, high growth speed, strong stress resistance, high survival rate, mild temperament and difficulty in scale falling. Compared with megalobrama amblycephala, the growth speed is averagely improved by 21.9 percent, the asphyxia point is averagely reduced by 22.2 percent, the resistance to aeromonas hydrophila is obviously enhanced, and the culture survival rate is averagely improved by 37.5 percent; compared with the Ancherythroculter nigrocauda, the growth speed is averagely improved by 84.8 percent. Solves the difficult problems of explosive hemorrhage and strong irritability caused by the susceptibility of the bream to aeromonas hydrophila, and promotes the green and healthy development of the aquaculture industry in China.
Detailed Description
Example 1:
hybridizing megalobrama amblycephala female fish and ancherythroculter nigrocauda male fish, and performing continuous four-generation colony breeding on obtained hybrid offspring to culture a new culter amblycephala variety with excellent characters;
a selective breeding method for distant hybridization offspring between megalobrama amblycephala and Ancherythroculter nigrocauda comprises the following steps:
1. selecting megalobrama amblycephala and ancherythroculter nigrocauda: selecting the individual with good body shape, health and maturity as parent in winter and spring.
2. Cultivating parent strains of megalobrama amblycephala and ancherythroculter nigrocauda: and (5) cultivating in separate pools. The stocking amount of parent fishes of megalobrama amblycephala is 150-200 pieces/mu, and special compound feed and fresh green grass are fed; the stocking amount of the parent fishes of the Ancherythroculter nigrocauda is 200-300/mu, and the special compound feed with the crude protein content of more than 33% is fed. And starting in the middle ten (4) months, changing water in the parent culture pond 1 time per week, and flushing water irregularly at the same time to promote the gonad development of the parent fishes of the megalobrama amblycephala and the Ancherythroculter nigrocauda.
3. Artificial spawning induction of megalobrama amblycephala female fish and ancherythroculter nigrocauda male fish: selecting 5-6 months in midstream of Yangtze river, controlling water temperature at 20-28 deg.C, respectively injecting oxytocic drug Cyprinus Carpio Pituitary Gland (PG) or dopamine antagonist Eremono (DOM), chorionic gonadotropin (HCG), and luteinizing hormone releasing hormone analog 2 (LRH-A) for fish into two parents2) Alone or in admixture; the injection is preferably carried out at the position without scales at the base of the pectoral fin, and the included angle between the needle insertion angle and the body of the parent fish is about 45 degrees. The recommended dosage of the female fish is PG 2-6 mg or DOM 2-8 mg, HCG 500-1200 IU and LRH-A used for each kilogram of parent fish22-10 mu g, adjusting the dosage or reasonably matching according to the gonad development condition and the water temperature, and adopting a one-needle injection method or a two-needle injection method for female fish; the needle pitch of the female fish two-needle injection method is 6-10 h, and the first needle is 1/10 or 1-2 mu g LRH-A of the total dose2And the second needle injects the remaining amount. The injection dosage of the male fish is 1/3-1/2 of the female fish, and the male fish is injected immediately after the injection of the total dosage of the female fish is finished. The parents after the injection of the oxytocic are separately cultured in an oxytocic pool, and the dissolved oxygen in water is kept above 5 mg/L.
4. Hybridizing megalobrama amblycephala (male parent) and ancherythroculter nigrocauda (female parent) for insemination: catching fish by netting when parent fish chase after or show impatience, selecting megalobrama amblycephala female fish to slightly press abdomen and have egg granules flowing out, selecting ancherythroculter nigrocauda male fish to slightly press abdomen and have semen flowing out, manually collecting the egg and sperm of the parent fish, and performing insemination by dry method or semi-dry method (an insemination mode, which is described in detail later); the ratio of the male parent fish to the female parent fish is 1: 1-2. Dry insemination: wiping off body surface water of megalobrama amblycephala female fish and ancherythroculter nigrocauda male fish, extruding megalobrama amblycephala ovum into a dry fertilization container, simultaneously extruding sperm of ancherythroculter nigrocauda male fish, and strictly prohibiting water from being brought in during the operation process; mixing sperm and egg with dry feather, adding 0.7% physiological saline, stirring, and immediately debonding or directly adhering to artificial fish nest. Semi-dry insemination: wiping off body surface water of megalobrama amblycephala female fish and ancherythroculter nigrocauda male fish, extruding ovum of megalobrama amblycephala female fish into a fertilization container filled with a small amount of 0.7% physiological saline, simultaneously extruding sperm of ancherythroculter nigrocauda male fish, uniformly stirring with dry feather, and immediately debonding or directly adhering to an artificial fish nest.
5. Artificially incubating hybrid fertilized eggs of megalobrama amblycephala (male parent) and ancherythroculter nigrocauda (female parent): dissolving yellow soil or talcum powder with water, filtering with 60 mesh net cloth, keeping certain viscosity, placing the hybridized fertilized eggs into filtered yellow mud or talcum powder slurry for debonding, or directly adhering artificial fish nests (such as palm sheets, net sheets and the like), and placing the fertilized eggs into a flushing type hatching tank or a hatching jar and a hatching loop for hatching, wherein the hatching density is 50-80 ten thousand grains/m3Water body, hatching water flow takes the egg not deposited as degree; when the waist spots (swim bladders) appear, the hatching equipment is moved out, and hybrid fry and parent fish cultivation is carried out in the pond. The waist point is commonly known as the swim bladder organ of the fish.
6. Breeding parent of hybrid offspring of megalobrama amblycephala (male parent) and ancherythroculter nigrocauda (female parent): the stocking amount of the parent fish of the hybrid offspring is 150-200 fish/mu, and the special compound feed with the crude protein content of more than 33% is fed. And starting in the middle ten (4) months, changing the water of the parent culture pond 1 time per week, and flushing water irregularly at the same time to promote the gonad development of the parent fish.
7. Breeding generations of hybrid offspring of megalobrama amblycephala (male parent) and ancherythroculter nigrocauda (female parent): the spawning induction of the filial generation by generations of parent fishes is the same as the artificial spawning induction of megalobrama amblycephala female fishes and ancherythroculter nigrocauda male fishes, the artificial insemination is the same as the hybrid insemination of megalobrama amblycephala female fishes and ancherythroculter nigrocauda male fishes, and the artificial incubation is the same as the artificial incubation of the megalobrama amblycephala female fishes and ancherythroculter nigrocauda male fertilized eggs.
8. And (3) breeding the hybrid offspring of the megalobrama amblycephala (male parent) and the ancherythroculter nigrocauda (female parent): the obtained hybrid offspring is selected 3 times from fry breeding to parent fish breeding by taking the rhomboid type and the growth speed as breeding targets; the first selection time is 6 months of age, the selection rate is 20%, the second selection time is 18 months of age, the selection rate is 10%, and the third selection time is 24 months of age, and the selection rate is 5%. And (5) continuously breeding the hybrid offspring for 4 generations to obtain a new excellent culter bream variety with diamond shape, fast growth, stress resistance and stress resistance.
9. The inventor determines the related character parameters of a new culter bream variety obtained by a selective breeding method of distant hybrid offspring between megalobrama amblycephala and ancherythroculter nigrocauda, and obtains remarkable technical progress. The specific data are shown in the following table I, table II, table III, table IV and table V.
Morphology difference cluster analysis results of three groups of new species of Meculter amblycephala, Megalobrama amblycephala and Ancherythroculter nigrocauda
| Group of people | Megalobrama amblycephala | Novel culter bream variety | Ancherythroculter nigrocauda |
| Megalobrama amblycephala | 0.00 | 0.18 | 0.39 |
| Novel culter bream variety | 0.18 | 0.00 | 0.26 |
| Erythroculter nigrocauda | 0.39 | 0.26 | 0.00 |
From the data in the table I, the genetic distance (0.18) between the new species of the culter breams and the megalobrama amblycephala is smaller than the genetic distance (0.26) between the culter nigrocauda and the culter nigrocauda based on morphological clustering analysis; the new variety of the culter bream is shown to be more similar to the megalobrama amblycephala in morphology.
Genetic similarity (on diagonal) and genetic distance (under diagonal) of Nei among three groups of novel Erythroculter amblycephala, megalobrama amblycephala and Ancherythroculter nigrocauda
| Group of people | Megalobrama amblycephala | Novel culter bream variety | Ancherythroculter nigrocauda |
| Megalobrama amblycephala | 0.7627 | 0.3184 | |
| Novel culter bream variety | 0.2709 | 0.5838 | |
| Ancherythroculter nigrocauda | 1.1446 | 0.5381 |
From the data of the two tables, the genetic similarity (0.7627) of the new variety of the culter bream and the megalobrama amblycephala based on SSR markers is higher than the genetic similarity (0.5838) of the ancherythroculter nigrocauda, and the genetic distance (0.2709) of the megalobrama amblycephala is smaller than the genetic distance (0.5381) of the ancherythroculter nigrocauda; the genetic relationship between the new variety of the culter bream and the female parent megalobrama amblycephala is closer, and the genetic relationship is more inclined to the female parent.
Asphyxia points of new variety of Metocrylobrama amblycephala, Metocrylobrama amblycephala and Ancherythroculter nigrocauda (water temperature 24.7 +/-0.5 ℃)
| Variety of (IV) C | Novel culter bream variety | Megalobrama amblycephala | Ancherythroculter nigrocauda |
| Body weight (g) | 7.82±0.69 | 6.02±0.57 | 8.02±0.78 |
| Suffocation point (mg/L) | 0.49±0.03b | 0.63±0.02a | 0.41±0.04c |
Note that the upper right-hand notation of the values of the same row of a, b and c with different letters represents significant difference (p < 0.05)
From the data of the three tables, when the water temperature is 24.7 +/-0.5 ℃, the asphyxia point of the megalobrama amblycephala is greater than the asphyxia point of a new culter megalobrama amblycephala variety is greater than the asphyxia point of an ancherythroculter nigrocauda variety, and the asphyxia point of the new culter megalobrama amblycephala variety is 0.49 +/-0.03 mg/L and has significant difference (p is less than 0.05) with 0.41 +/-0.04 mg/L of the asphyxia point of the ancherythroculter nigrocauda and 0.63 +/-0.02 mg/L of the asphyxia point of the megalobrama amblycephala; the suffocation point of the new variety of the culter bream is reduced by 22.2 percent compared with that of the megalobrama amblycephala.
Semilethal concentrations (water temperature 25 +/-1 ℃) of new species of ectorhynchophilus exigua and new species of culter breams, megalobrama amblycephala and ancherythroculter nigrocauda
| Variety of (IV) C | Novel culter bream variety | Megalobrama amblycephala | Ancherythroculter nigrocauda |
| LC50(CFU/mL) | 6.9×108 | 7.2×106 | 3.6×107 |
From the above data, it can be seen that the half-lethal concentrations of the new species of the culter breams, the megalobrama amblycephala and the ancherythroculter nigrocauda to aeromonas hydrophila at the water temperature of 25 +/-1 ℃ are respectively 6.9X 108CFU/mL、7.2×106CFU/mL and 3.6X 107CFU/mL; sensitization of three fish to aeromonas hydrophilaThe perception is from strong to if: megalobrama amblycephala is more than Ancherythroculter nigrocauda and a new species of culter amblycephala.
New variety of Megalobrama amblycephala, Megalobrama amblycephala and Ancherythroculter nigrocauda same-pond culture data
From the five data, the new species of the culter breams grow 21.9% faster than the megalobrama amblycephala on average and 84.8% faster than the ancherythroculter nigrocauda on average, and the culture survival rate is improved by 37.5% compared with the megalobrama amblycephala on average.
Claims (2)
1. A selective breeding method for distant hybridization offspring between megalobrama amblycephala and Ancherythroculter nigrocauda is characterized by comprising the following steps:
A. distant hybridization of megalobrama amblycephala and ancherythroculter nigrocauda:
a. and (3) hybrid parent selection: selecting megalobrama amblycephala and ancherythroculter nigrocauda as parents, wherein the female megalobrama amblycephala is 3-4 years old and 1.0kg in weight, and the male ancherythroculter nigrocauda is 2-3 years old and 0.4kg in weight;
b. breeding hybrid parents: the method comprises the following steps of breeding parent megalobrama amblycephala and ancherythroculter nigrocauda in separate ponds, feeding 150-200 fish/mu of parent megalobrama amblycephala with special compound feed and fresh green grass, feeding 200-300 fish/mu of parent ancherythroculter nigrocauda with special compound feed with the crude protein content of 33%;
c. and (3) hybridization induced spawning: selecting 5-6 months in midstream of Yangtze river, controlling water temperature at 20-28 ℃, respectively injecting oxytocic drug cyprinus carpio pituitary or dopamine antagonist diohonone, chorionic gonadotropin and fish luteinizing hormone releasing hormone analogue No. 2 into the two parents, and using the two parents singly or in a mixed way; recommended dosage of female fish is PG 2-6 mg or DOM 2-8 mg, HCG 500-1200 IU, LRH-A per kilogram of parent fish22-10 mug, adjusting the dosage or reasonably matching according to the gonad development condition and the water temperature, injecting the upper limit dosage at the water temperature of 20 ℃, injecting the lower limit dosage at the water temperature of 28 ℃, adopting a one-needle injection method or a two-needle injection method for the female fish, wherein the needle pitch of the two-needle injection method is 6-10 h, and the first needle is 1/10 or 1-2 mug LRH-A of the total dosage2Second, secondThe injection amount of the male fish is 1/3-1/2 of the female fish, the male fish is injected after the injection of the total dose of the female fish is finished, and parents injected with the oxytocic are separately cultured in an oxytocic pool and are kept with sufficient dissolved oxygen;
d. hybrid insemination: when the parent fishes are in heat, fishing by netting respectively, selecting megalobrama amblycephala female fishes and ancherythroculter nigrocauda male fishes for artificial sperm and egg collection, and carrying out dry insemination or semi-dry insemination, wherein the ratio of the female parent fishes to the male parent fishes is 1: 1-2;
e. hatching the hybrid fertilized eggs: debonding fertilized eggs by yellow mud slurry or talcum powder slurry, or directly adhering the fertilized eggs to an artificial fish nest, and then placing the fertilized eggs into a flushing type hatching tank or a hatching jar and a hatching loop for hatching, wherein the hatching density is 50-80 ten thousand grains/m3Water body, hatching water flow takes the egg not deposited as degree; when the waist point appears, moving out the hatching equipment, and culturing fry and fingerlings and parent fishes in a pond;
B. and (3) breeding hybrid offspring of megalobrama amblycephala and ancherythroculter nigrocauda:
(1) and parent selection of hybrid progeny: selecting healthy and sexually mature 3-4-year-old individuals with the weight of 1.0kg as parents from hybrid offspring of megalobrama amblycephala and ancherythroculter nigrocauda;
(2) and breeding parents of hybrid filial generations: the stocking amount of parent fishes of hybrid offspring of megalobrama amblycephala and ancherythroculter nigrocauda is 150-200/mu, special compound feed with the crude protein content of 33% is fed, the parent culture pond is changed with water 1 time every week starting in the middle ten (4) months, and meanwhile, water is flushed irregularly to promote the gonadal development of the parent fishes;
(3) and breeding filial generation by generation: the spawning induction of the filial generation by generation parent fish is the same as the spawning induction of the hybridization, the generation-by-generation artificial insemination of the filial generation is the same as the hybridization insemination, and the generation-by-generation breeding incubation of the filial generation is the same as the hybridization fertilized egg incubation;
(4) and generation-by-generation breeding of hybrid filial generation: in the hybrid offspring of the megalobrama amblycephala and the ancherythroculter nigrocauda, taking the rhombohedral type and the growth speed as breeding targets, and continuously breeding for four generations by adopting a colony breeding technology; from fry breeding to parent fish breeding, each generation is subject to two years and 3 times of selection, the breeding time is respectively 6 months old, 18 months old and 24 months old, the corresponding selection rates are respectively 20%, 10% and 5%, and the total selection rate is 0.1%;
(5) and detecting the character of the new variety of the culter bream obtained by continuously breeding the hybrid offspring for four generations: compared with the original parent megalobrama amblycephala and ancherythroculter nigrocauda, the new variety character of the culter amblycephala obtained by continuously breeding the hybrid offspring of the megalobrama amblycephala and the ancherythroculter nigrocauda for four generations is detected:
A. genetic characterization based on SSR markers: analyzing the genetic similarity and genetic distance of three groups of megalobrama amblycephala and ancherythroculter nigrocauda and new varieties of culter amblycephala bred by continuous four generations after hybridization by using polymorphic 11 pairs of microsatellite primers of ancherythroculter nigrocauda;
B. body type: measuring the quantitative and frame data of the three groups, analyzing the forms of the new culter breams, the megalobrama amblycephala and the ancherythroculter nigrocauda by using a clustering analysis and principal component analysis method, and showing that the genetic distances of the new culter breams, the megalobrama amblycephala and the ancherythroculter nigrocauda are respectively 0.18 and 0.26, and the genetic distances of the megalobrama amblycephala and the ancherythroculter nigrocauda are 0.39; the new species of the culter breams are in the shape of a rhombus as the megalobrama amblycephala;
C. asphyxia point: the asphyxiation points of new species of the culter breams, the megalobrama amblycephala and the ancherythroculter nigrocauda are explored by adopting a closed respiration experiment method;
D. ability to resist infection by Aeromonas hydrophila: under the condition of water temperature of 24-26 ℃, an infection method of injecting aeromonas hydrophila suspension into the abdominal cavity is adopted to determine the aeromonas hydrophila infection resistance of new varieties of culter breams, megalobrama amblycephala and ancherythroculter nigrocauda;
E. growth rate and survival rate: under the same condition, the new species of the culter bream is marked with the megalobrama amblycephala and the ancherythroculter nigrocauda and then cultured in the same pond.
2. The selective breeding method for the distant hybrid offspring between megalobrama amblycephala and ancherythroculter nigrocauda according to claim 1, characterized in that the polymorphic 11 pairs of microsatellite primers of ancherythroculter nigrocauda are utilized:
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