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CN-121970704-A - Artificial cultivation method for hybrid of black garrupa and garrupa

CN121970704ACN 121970704 ACN121970704 ACN 121970704ACN-121970704-A

Abstract

The invention discloses an artificial cultivation method of hybrid seeds of black lapis and Epinephelus coioides, which comprises the steps of taking healthy black lapis as female parent fish, carrying out operation and cultivation enhancement and artificial ripening to obtain enhanced female parent fish, taking healthy Epinephelus coioides as male parent fish, carrying out natural ripening to obtain sexually mature male parent fish, enabling ovulation time to be consistent with sperm discharge time, collecting egg liquid and seminal fluid, obtaining fertilized eggs after insemination, carrying out embryo hatching and opening and illumination regulation cultivation to obtain hybrid spots, carrying out authenticity identification on the hybrid spots by using a primer pair, and determining that the hybrid spots inherit genetic information of the female parent fish and the male parent fish, wherein the primer pair is a sequence pair consisting of SEQ ID NO.1 and SEQ ID NO.2 and a sequence pair consisting of SEQ ID NO.3 and SEQ ID NO. 4. Fills the technical blank of cross breeding by using black garrupa as parent garrupa, and the authenticity of the cross breeding is reliable.

Inventors

  • WANG BO
  • BAO ZHENMIN
  • ZHAI ZHONGYI
  • HU JINGJIE
  • JIN CHAOFAN
  • Tang Haizhan
  • ZHENG DA
  • LI BINGLIN
  • LI JIAHANG
  • ZHANG ZHIHAO

Assignees

  • 中国海洋大学三亚海洋研究院

Dates

Publication Date
20260505
Application Date
20260409

Claims (10)

  1. 1. An artificial cultivation method of a hybrid of black groupers and garrupa is characterized by comprising the following steps: the method comprises the steps of A, taking healthy black lapis as female parent fish, carrying out operation and cultivation strengthening and artificial ripening to obtain strengthened female parent fish, taking healthy Epinephelus lanceolatus as male parent fish, and carrying out natural ripening to obtain sexually mature male parent fish, wherein the ovulation time of the strengthened female parent fish is consistent with the sperm discharge time of the sexually mature male parent fish; The artificial ripening is that the temperature is reduced from 26 plus or minus 0.5 ℃ to 22.5 plus or minus 0.5 ℃ for 7-10 days at the cooling rate of 0.5 ℃ per day, and then the water temperature is regulated and controlled at the heating rate of 0.5 ℃ per day to 28.5 plus or minus 0.5 ℃; Step B, collecting the egg liquid of the reinforced female parent fish and the seminal fluid of the sexually mature male parent fish, inseminating to obtain fertilized eggs, and carrying out embryo hatching and opening and then light regulation and cultivation to obtain hybridization spots; Step C, carrying out authenticity identification on the hybridization spots by using a primer pair, and determining that the hybridization spots inherit genetic information of the female parent fish and the male parent fish; the primer pair is a sequence pair consisting of SEQ ID NO.1 and SEQ ID NO.2 and a sequence pair consisting of SEQ ID NO.3 and SEQ ID NO. 4.
  2. 2. The method for artificially breeding the hybrid of black garrupa and garrupa according to claim 1, wherein the step B comprises: step B-1, collecting egg liquid of the reinforced female parent fish and semen of the sexually mature male parent fish, and obtaining fertilized eggs after dry insemination and activation; And B-2, embryo hatching and staged illumination regulation and cultivation after opening are carried out on the fertilized eggs, so as to obtain hybridization spots.
  3. 3. The method for artificially breeding a hybrid of Epinephelus septemlobus and Epinephelus lanceolatus as claimed in claim 2, wherein in the step B-2, The embryo is hatched into the fertilized ovum from the cleavage stage to the embryo development stage, and the light-shielding or weak light condition is adopted, so that the illumination intensity is less than or equal to 100 lx.
  4. 4. The artificial cultivation method of the hybrid of black garrupa and garrupa according to claim 2, wherein the post-opening stage illumination regulation cultivation is specifically, The day-old of 0-3 is that the light-proof or weak light condition is adopted, so that the illumination intensity is less than or equal to 100 lx; the illumination intensity gradient is increased to 1600-1800 lx at the age of 4-8 days; 9-20 days old, namely reducing the illumination intensity from 1600-1800 lx gradient to 800-1200 lx; 21-37 days old, namely reducing the illumination intensity from 800-1200 lx gradient to 600-800 lx; the light intensity is increased from 600 to 800 lx to 800 to 1000 lx in a gradient way at the age of 38 to 45 days; The illumination period of 4-45 days is14 h illumination/10 h darkness, the gradient is reduced to reduce the illumination intensity by 100-200 lx per hour, and the gradient is increased to increase the illumination intensity by 100-200 lx per hour.
  5. 5. The artificial breeding method of the hybrid of black garrupa and garrupa according to claim 2, wherein, The fertilized egg has water temperature of 25-27 deg.c, salinity of 33-35%o, dissolved oxygen concentration of 6.5 mg/L or higher and pH value of 7.9-8.4 during the stage illumination regulation cultivation after embryo hatching and opening.
  6. 6. The method for artificially breeding the hybrid of black garrupa and garrupa according to claim 1, wherein the step C is specifically, Extracting DNA of the female parent fish, the male parent fish and the hybridization spots to obtain female parent fish genome DNA, male parent fish genome DNA and hybridization spot genome DNA respectively, and carrying out PCR identification by using the primer pair to determine that the hybridization spots inherit genetic information of the female parent fish and the male parent fish.
  7. 7. The artificial breeding method of hybrid of black garrupa and garrupa according to claim 6, wherein, The PCR identification system is a 25 mu L amplification system, wherein the 25 mu L amplification system comprises 1 mu L of genomic DNA with the concentration of 100 ng/mu L,1 mu L of forward primer Fw with the concentration of 10 mu M, 1 mu L of reverse primer Rw with the concentration of 10 mu M, 2X Taq Plus Master Mix II, 12.5 mu L and 9.5 mu L of sterile water without enzyme; The PCR identification procedure comprises, in order, pre-denaturation 95 ℃ 3min, denaturation 95 ℃ 15 s, annealing 56 ℃ 20 s, extension 72 ℃ 40 s, total 32 cycles from denaturation to extension, and full extension 72 ℃ 10 min.
  8. 8. The method for artificially breeding a hybrid of Epinephelus septemlobus and Epinephelus lanceolatus according to claim 1, wherein in the step A, The health standard is that no disease, no injury or deformity exists, the physique is strong, and hormone induced spawning or ovary induction treatment is not carried out; The weight of the female parent fish is more than or equal to 1.0 kg, and the weight of the male parent fish is more than or equal to 3.0 kg.
  9. 9. The artificial breeding method of hybrid of black garrupa and garrupa according to claim 8, wherein, The nutrition-enriched feed comprises a basic feed and a nutritional agent, wherein the addition amount of the nutritional agent is 3.93% -6.37% of the weight of the basic feed, and the nutritional agent comprises 0.8% -1.2% of deep sea fish oil, 0.4% -0.6% of L-arginine, 0.6% -0.9% of coated vitamin E, 0.2% -0.5% of taurine, 1.0% -1.5% of special lecithin for aquatic products, 0.15% -0.25% of microcapsule folic acid, 0.3% -0.5% of vitamin C, 0.2% -0.5% of arachidonic acid, 0.08% -0.12% of methionine zinc and 0.2% -0.3% of inositol.
  10. 10. The artificial breeding method of the hybrid of black garrupa and garrupa according to any one of claims 1 to 9, characterized in that, The fertility rate of the fertilized eggs is more than or equal to 95 percent, and the hatching rate of the fertilized eggs is more than or equal to 88 percent; The survival rate of the hybridization spots at the age of 35 days is more than or equal to 50 percent, and the survival rate of the hybridization spots at the age of 65 days is more than or equal to 40 percent.

Description

Artificial cultivation method for hybrid of black garrupa and garrupa Technical Field The invention relates to the technical field of aquaculture and genetic breeding, in particular to an artificial breeding method of hybrid seeds of black garrupa and garrupa. In particular to a method for improving hybridization fertilization rate, embryo development stability and early survival rate of fries by the synergistic effect of artificial maturation promotion regulation, dry artificial insemination and staged light regulation under the non-hormonal condition and identifying the authenticity of the hybrid by utilizing molecular biological means. Background The grouper (Epinephelus spp.) is rich in variety, and is one of fish resources with important economic value in the tropical and subtropical mariculture industry in China. The grouper has the advantages of tender fish, high nutritive value, stable market demand and continuous expansion of cultivation scale in coastal areas. The advantages of different groupers in growth speed, environmental adaptability, stress resistance and the like are integrated by an interspecific hybridization mode, and the method is considered as an important breeding way for cultivating a new high-performance grouper variety. The black rockfishes (Epinephelus corallicola) belong to the genus of the order of the weever, the family of the bass and the genus of the rockfishes, are mainly distributed in tropical coral reef sea areas of the Indian ocean and the western Pacific ocean, and often inhabit coral reef and rock reef environments with water depths of 5-80 m. The fish has moderate body shape, obvious mottled patterns on the body surface, strong environment adaptability and good meat quality, and has a certain resource foundation in partial sea areas. The black garrupa grows faster and adapts to artificial breeding conditions, but the artificial breeding and large-scale seedling raising technology is still to be further perfected. The Epinephelus lanceolatus (Epinephelus lanceolatus) belongs to the genus Epinephelus of the order Perciformes, the family bass, and the genus Epinephelus, and is widely distributed in the tropical and subtropical sea areas of the Indian ocean and the Pacific ocean, and is mostly inhabited in the coastal coral reef and rock reef areas. The fish body has large size, strong stress resistance, good tolerance to water environment change, high survival rate and stability in the cultivation process, and high cultivation utilization value. At present, a method for artificial cross breeding of partial groupers is disclosed, and a certain artificial propagation and seedling raising process is formed. However, no mature and stable public report has been found about the systematic study and technical scheme of carrying out artificial crossbreeding by taking black garrupa and garrupa as parents. In particular, the method has great technical difficulties in the aspects of parent gonad development rhythm difference, artificial maturation promotion control, hybridization fertilization efficiency, early development stability of hybridization embryo and the like. Therefore, in the aspect of carrying out artificial hybridization breeding by taking black garrupa and garrupa as parents, the prior art can not provide an effective and repeatable solution, and still has obvious technical blank, and needs to be further researched and broken through to promote the development of the hybridization breeding and the industrialization application of garrupa. Disclosure of Invention The invention aims to provide an artificial cultivation method of hybrid seeds of the brown spot groupers and the glabrous greenbrier, which fills the technical blank of hybridization breeding of the glabrous greenbrier as a parent grouper, so that the obtained hybrid seeds obtained by hybridization of the glabrous greenbrier and the glabrous greenbrier are reliable in authenticity, and inherit the genetic information of a parent and a female parent on a molecular level. To solve the technical problems: The patent provides an artificial cultivation method of black lapis and garrupa hybrid, which comprises the following steps: Step A, taking healthy black lapis as female parent fish, carrying out operation and cultivation strengthening and artificial ripening to obtain strengthened female parent fish, taking healthy Epinephelus lanceolatus as male parent fish, and carrying out natural ripening to obtain sexually mature male parent fish, so that the ovulation time of the strengthened female parent fish is consistent with the sperm discharging time of the sexually mature male parent fish; The artificial ripening is that the temperature is reduced from 26 plus or minus 0.5 ℃ to 22.5 plus or minus 0.5 ℃ for 7-10 days at the cooling rate of 0.5 ℃ per day, and then the water temperature is regulated and controlled at the heating rate of 0.5 ℃ per day to 28.5 plus or minus 0.5 ℃; step B, collecting egg liquid of the