Preparation method of brachymystax lenok tetraploid fry

Abstract

The invention relates to the technical field of aquatic organism breeding, in particular to a preparation method of brachymystax lenok tetraploid offspring seeds. The method comprises the following steps: carrying out dry fertilization on a naturally mature brachymystax lenok parent to obtain a fertilized egg, inducing the brachymystax lenok fertilized egg after 6-8 h to obtain a tetraploid offspring seed, carrying out induction for 10-20 min, carrying out DNA content determination by using a flow cytometer to determine the chromosome ploidy of the fish body, and carrying out cross-linked immunosorbent assay on the chromosome ploidy of the fish body to obtain the chromosome ploidy of the brachymystax lenok. And identifying and screening tetraploid offspring seeds of brachymystax lenok for cultivation. According to the method disclosed by the invention, the induction rate and the survival rate of the tetraploid brachymystax lenok are improved by controlling the artificial fertilization time, inducing by adopting a physical or chemical method and controlling the induction duration and inhibiting the first mitosis time of fertilized eggs. According to experimental statistics, the induction rate of the obtained brachymystax lenok tetraploid is 20-26%, and the survival rate of offspring seeds is 80% or above.

Description

Technical Field

[0001] The invention relates to the technical field of aquatic biological breeding, and in particular to a method for preparing tetraploid salmonid seedlings. Background Art

[0002] Squid salmon Lenok ), belonging to the class Osteichthyes ( Osteichthyes ), Herringiformes ( Clupeomorpha ), Salmoniformes ( Salmoniformes ), Salmonidae( Salmonidae ), Salmonidae Brachymystax lenok ) is a precious land-locked cold-water fish, mostly living in clear streams with a water temperature of 9-22℃, and the dissolved oxygen content is generally required to be above 6 mg / L.

[0003] Fish polyploid breeding belongs to chromosome engineering technology. Since the chromosomes of triploid salmon cannot undergo normal synapsis and cannot form fertile female and male gametes, triploids are infertile. The energy required for gonadal development can be converted into growth, effectively avoiding the decline in growth rate and death during sexual maturity. Its growth rate is faster than that of diploids, shortening the breeding cycle, saving costs, and increasing breeding income. Triploid salmon cannot reproduce normally, and each generation of triploid seedlings needs to be artificially induced. However, the technology for directly preparing triploid seedlings is still immature, the survival rate of each batch of seedlings prepared is unstable, and the triploid rate cannot be guaranteed, which restricts the application and promotion of triploid fish. The reproductive cells of tetraploid salmon contain an even number of chromosome sets and are fertile. Mature tetraploid parents can be fertilized with normal diploid parents to stably and mass-produce seedlings with a triploid rate of 100%. Therefore, it is necessary to develop a mature and stable technology for preparing tetraploid salmon seedlings. By using tetraploid salmon and diploid salmon for breeding, it will be beneficial to achieve stable large-scale production of triploid salmon. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a method for preparing tetraploid seedlings of Salmo salar, which greatly improves the tetraploid rate and survival rate of Salmo salar by drug induction, establishes a mature and stable tetraploid seedling preparation technology of Salmo salar, and obtains a high-quality seedling production system of polyploid Salmo salar. Based on this, the following technical solution is proposed.

[0005] Firstly, the present invention provides the use of 6-dimethylaminopurine in inducing the preparation of tetraploid salmon seedlings in vitro.

[0006] Furthermore, the present invention provides a method for preparing tetraploid fry of Salmo salar, comprising: soaking fertilized eggs of Salmo salar in a 6-dimethylaminopurine solution.

[0007] In some embodiments, the preparation method includes: under the condition that the water temperature is 8.4°C to 8.6°C, after the eggs are fertilized for 6.0 to 7.2 h, soaking the fertilized eggs of Brachymystax lenok in a 6-dimethylaminopurine solution with a concentration of 90 to 210 mg / L (such as 90, 120, 150, 180, 210 mg / L) for 5 to 25 min, and then moving the fertilized eggs of Brachymystax lenok into water at 7.8°C to 8.2°C for hatching.

[0008] In some embodiments, the fertilization time of the eggs is 6.2 to 6.9 h (such as 6.0, 6.3, 6.6, 6.9, 7.2 h).

[0009] In some embodiments, the concentration of the 6-dimethylaminopurine solution is 120 to 150 mg / L.

[0010] In some embodiments, the soaking treatment time is 5 to 25 min (such as 5, 10, 15, 20, 25 min), preferably 10 to 15 min.

[0011] In some embodiments, the water body is hatching water that meets the fishery water quality standard.

[0012] Preferably, the preparation method includes: under the condition that the water temperature is 8.4°C to 8.6°C (most preferably 8.5°C), after the eggs are fertilized for 6.6 to 6.7 h (most preferably 6.6 h), soaking the fertilized eggs of Brachymystax lenok in a 6-dimethylaminopurine solution with a concentration of 115 to 125 mg / L (most preferably 120 mg / L) for 12 to 18 min (most preferably 15 min), and then moving the fertilized eggs of Brachymystax lenok into water at 7.8°C to 8.2°C (most preferably 8°C) for hatching.

[0013] Preferably, the hatching time is 20 to 25 days.

[0014] Preferably, the parent of the fertilized eggs is Brachymystax lenok.

[0015] In some embodiments, the eye emergence rate of the fertilized eggs of Brachymystax lenok is more than 41%, preferably more than 45%, more preferably more than 51%, and even more preferably more than 52%; and / or, the fry hatching rate is more than 75%, preferably more than 80%, more preferably more than 85%, and even more preferably more than 86%; and / or, the fry malformation rate is less than 1.3%, preferably less than 0.5%, more preferably less than 0.4%, and even more preferably less than 0.3%; and / or, the fry tetraploid rate is more than 13%, preferably more than 15%, more preferably more than 25%, and even more preferably more than 26%.

[0016] In addition, the present invention also provides an application of the preparation method in the tetraploid breeding of Brachymystax lenok.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention uses 6-dimethylaminopurine to induce fertilized eggs, greatly improving the tetraploid rate and survival rate of Brachymystax lenok. Compared with other methods, it has the advantages of simple equipment, short treatment time, stable effect, high hatching rate of fertilized eggs, high survival rate of fry, high tetraploid rate and low malformation rate. The hatching rate of fertilized eggs and the hatching rate of fry of the method of the present invention are close to the indexes of normal reproduction of Brachymystax lenok. The tetraploid rate of fry reaches the design requirements, which is beneficial to the large-scale production of tetraploid populations of Brachymystax lenok and lays a foundation for the subsequent stable and large-scale production of fry with a triploid rate of 100%, having high economic benefits and market value. Detailed implementation manners

[0018] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below. Apparently, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without making creative efforts shall fall within the protection scope of the present invention. In the embodiments provided in this specification, those not specifying specific techniques or conditions shall be carried out according to the techniques or conditions described in the literature in the art or according to the product specifications. Those reagents or instruments not indicating the manufacturer can be obtained as conventional products through regular channels. The Brachymystax lenok parents used in the following embodiments were provided by the Fisheries Science Research Institute of Beijing Academy of Agriculture and Forestry Sciences.

[0019] Example 1 This example provides a method for preparing tetraploid fry of Brachymystax lenok, and the steps are as follows: Select well-developed Brachymystax lenok parents for egg collection, and the fish eggs are fertilized by the dry method. First, squeeze the fish eggs of mature female fish into a clean porcelain basin by the extrusion method. Every 4 female fish are in a group, and then squeeze the semen of 2 male fish into the basin, immediately stir evenly, add hatching water, and the volume ratio of semen to hatching water is 1:3000. Gently stir, pour out the water after 1.5 min, repeat 4 times, wash the semen clean, and place it waiting for treatment.

[0020] Under the condition of water temperature of 8.5 °C, 6.6 h after the fertilization of the eggs, quickly transfer the fertilized eggs into a 6-dimethylaminopurine solution with a concentration of 120 mg / L that has been prepared and soak them. The duration of drug induction is 15 min; then quickly transfer the fertilized eggs into an 8 °C water body for normal hatching.

[0021] Example 2 This example provides a method for preparing fine-scale salmon tetraploid fry. The only difference in the steps from Example 1 is as follows: Under the condition of a water temperature of 8.5 °C, 6.2 h after egg fertilization, the fertilized eggs are quickly transferred into a pre-prepared 6-dimethylaminopurine solution with a concentration of 130 mg / L for soaking, and the duration of drug induction is 15 min; then the fertilized eggs are quickly transferred into an 8 °C water body for normal hatching.

[0022] Example 3 This example provides a method for preparing fine-scale salmon tetraploid fry. The only difference in the steps from Example 1 is as follows: Under the condition of a water temperature of 8.5 °C, 6.6 h after egg fertilization, the fertilized eggs are quickly transferred into a pre-prepared 6-dimethylaminopurine solution with a concentration of 150 mg / L for soaking, and the duration of drug induction is 10 min; then the fertilized eggs are quickly transferred into an 8 °C water body for normal hatching.

[0023] Example 4 This example provides a method for preparing fine-scale salmon tetraploid fry. The only difference in the steps from Example 1 is as follows: Under the condition of a water temperature of 8.5 °C, 6.9 h after egg fertilization, the fertilized eggs are quickly transferred into a pre-prepared 6-dimethylaminopurine solution with a concentration of 120 mg / L for soaking, and the duration of drug induction is 15 min; then the fertilized eggs are quickly transferred into an 8 °C water body for normal hatching.

[0024] Example 5 This example provides a method for preparing fine-scale salmon tetraploid fry. The only difference in the steps from Example 1 is as follows: Under the condition of a water temperature of 8.5 °C, 6.7 h after egg fertilization, the fertilized eggs are quickly transferred into a pre-prepared 6-dimethylaminopurine solution with a concentration of 120 mg / L for soaking, and the duration of drug induction is 15 min; then the fertilized eggs are quickly transferred into an 8 °C water body for normal hatching.

[0025] Test Example Using the preparation methods in the above examples, fry hatch normally after 20 - 25 days. The eye emergence rate of fertilized eggs, fry hatching rate, fry malformation rate, and tetraploid rate detected by flow cytometry after fry opening are counted during this period. The test results are shown in Table 1.

[0026] Table 1

[0027] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

Application of 1.6-dimethylaminopurine in inducing tetraploid seedlings of Salmo salar in vitro.

2. A method for preparing tetraploid salmon seedlings, characterized in that: include: The fertilized eggs of salmon larvae were immersed in 6-dimethylaminopurine solution.

3. The preparation method according to claim 2, characterized in that: include: Under water temperature of 8.4℃~8.6℃, 6.0~7.2 hours after the eggs were fertilized, the fertilized eggs of S. leucopsis were immersed in a 6-dimethylaminopurine solution with a concentration of 90~210 mg / L for 5~25 minutes, and then moved into water of 7.8℃~8.2℃ for incubation.

4. The preparation method according to claim 3, characterized in that: The time for egg fertilization is 6.2~6.9 hours.

5. The preparation method according to claim 3, characterized in that: The concentration of 6-dimethylaminopurine solution is 120~150 mg / L.

6. The preparation method according to claim 3, characterized in that: The soaking time is 5 to 25 minutes.

7. The preparation method according to claim 3, characterized in that: The water body is hatching water that meets the fishery water quality standards.

8. The preparation method according to claim 3, characterized in that: include: Under water temperature of 8.4℃~8.6℃, 6.6~6.7 hours after the eggs were fertilized, the fertilized eggs of S. leucopsis were immersed in a 6-dimethylaminopurine solution with a concentration of 115~125 mg / L for 12~18 minutes, and then moved into water of 7.8℃~8.2℃ for incubation.

9. The preparation method according to any one of claims 1 to 8, characterized in that The eye opening rate of fertilized eggs of salmon is 41% or more, preferably 45% or more, more preferably 51% or more, and even more preferably 52% or more; and / or, the hatching rate of seedlings is 75% or more, preferably 80% or more, more preferably 85% or more, and even more preferably 86% or more; and / or, the seedling deformity rate is below 1.3%, preferably below 0.5%, more preferably below 0.4%, and even more preferably below 0.3%; And / or, the tetraploid rate of seedlings is 13% or more, preferably 15% or more, more preferably 25% or more, and even more preferably 26% or more.

10. Use of the preparation method according to any one of claims 1 to 9 in tetraploid breeding of Salmo salar.

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