Induction method and application of triploid phoxinus lagowskii

Abstract

The invention discloses an induction method of triploid phoxinus lagowskii and application thereof, and belongs to the technical field of polyploid breeding of phoxinus lagowskii. The invention aims to produce triploid phoxinus lagowskii in a high proportion. The invention provides a method for inducing triploid phoxinus lagowskii, which comprises the following steps of: inseminating sperms and ova on the phoxinus lagowskii to obtain fertilized ova, performing hydrostatic pressure treatment on the fertilized ova, performing temperature shock and drug combined induction treatment, and incubating in culture water. The production method suitable for the triploid phoxinus lagowskii is established.

Description

Technical Field

[0001] The invention belongs to the technical field of polyploid breeding of dacey, and in particular relates to an induction method of triploid dacey and application thereof. Background Art

[0002] The Lasberg's gizzard bream is a fish of the Cyprinidae family and the genus Macrorhinus, also known as the willow root fish. As a native fish in Northeast China, the Lasberg's gizzard bream is increasingly popular among consumers due to its tender meat, delicious taste, few bones and high nutritional value, and has great market development potential.

[0003] Most fishes are diploid in nature. Diploid fish mature eggs are in the second meiotic metaphase. There are 2 sets of chromosomes in the eggs. After fertilization, after sperm (1 set of chromosomes) enters the eggs, 1 set of chromosomes in the eggs is stimulated to be discharged from the eggs as the second polar body, completing the second meiotic division. At this time, 1 set of chromosomes in the fertilized egg and 1 set of chromosomes brought in by sperm combine to develop into a normal diploid. If the sperm enters the eggs and the second polar body is not yet discharged, the fertilized eggs are treated with high temperature, low temperature, high pressure or chemical drugs to prevent the second meiotic division from being carried out, and the second polar body just cannot be discharged from the eggs, then the individuals with 3 sets of chromosomes are induced, which is called artificial induced triploid. No matter which method is used for induction, the starting time and duration of the treatment must be strictly controlled. At present, there are many reports at home and abroad on the production of triploid fish using methods such as temperature shock, hydrostatic pressure, and chemical treatment.

[0004] Due to the imbalance of chromosome composition, gonads of triploid fish cannot mature, which greatly reduces the adverse effects of growth stagnation and decreased fish meat quality during gonad development, and can increase the growth rate and meat quality of fish. Therefore, conducting research on triploid fish and cultivating infertile triploid populations will help increase the aquaculture output and commodity value of fish, and is also crucial for protecting my country's fishery germplasm resources. So far, there has been no report on the production method of triploid Lasbergi. Due to species differences, when the technical parameters for triploid induction of marine fish and most freshwater fish that have been reported are used for the induction of triploid Lasbergi, the induction rate of triploid Lasbergi is not high, which is difficult to meet the needs of industrial production. Therefore, it is necessary to establish a production method suitable for triploid Lasbergi. Summary of the invention

[0005] The present invention aims at producing triploid mullet at a high rate.

[0006] The invention provides a method for inducing triploid mullet, comprising the steps of obtaining fertilized eggs after fertilization of sperm and eggs of mullet on a net, subjecting the fertilized eggs to hydrostatic pressure treatment, and then subjecting them to temperature shock and drug combined induction treatment, and completing hatching in aquaculture water.

[0007] It is further specified that the hydrostatic pressure treatment occurs 6-10 minutes after fertilization.

[0008] It is further defined that the hydrostatic pressure is 5000-8000PSI and the action time is 2-5 minutes.

[0009] Further limiting the scope, drug treatment refers to colchicine treatment.

[0010] It is further defined that the temperature shock and drug combined induction treatment means that the concentration of colchicine is 200-500 μg / mL, the temperature of the aqueous solution is 5-25° C., and the treatment time is 15-45 minutes.

[0011] It is further defined that the culture water temperature is 12-15°C and the incubation time is 72 hours.

[0012] It is further defined that 8 minutes after fertilization, the fertilized eggs are placed in a hydrostatic pressure chamber for hydrostatic pressure treatment, the pressure is 7000PSI, and the treatment time is 3 minutes; after hydrostatic pressure treatment, the fertilized eggs are immersed in a 200μg / ml colchicine aqueous solution, the water temperature is 20°C, and the treatment time is 25 minutes; finally, the fertilized eggs are transferred to the breeding water, the temperature is kept constant at 12-15°C, and the hatching is completed in 72 hours.

[0013] It is further defined that the method for obtaining fertilized eggs is to selectively mature the herring, collect mature eggs and sperm, place the eggs in a dry container at 12-15°C, add semen, the fertilization volume ratio of semen to eggs is 100μL:15ml, mix the sperm and eggs, then add breeding water at a temperature of 12-15°C, and shake to complete the sperm and egg fertilization process.

[0014] It is further defined that the volume of the breeding water is 50 times the volume of the eggs.

[0015] The present invention provides an application of the above-mentioned induction method in breeding triploid mullet.

[0016] Beneficial effect: The present invention determines a scheme of combined treatment of hydrostatic pressure, temperature shock and chemical substance treatment in a production method for inducing chromosome triploidization of fertilized eggs of mullet: 8 minutes after fertilization, the fertilized eggs are placed in a hydrostatic pressure chamber for hydrostatic pressure treatment, the pressure is 7000PSI, and the treatment time is 3 minutes; after the hydrostatic pressure treatment, the fertilized eggs are immersed in a 200μg / ml colchicine aqueous solution, the water temperature is 20°C, and the treatment time is 25 minutes; finally, the fertilized eggs are transferred to the breeding water, the temperature is kept constant at 12-15°C, and the hatching is completed. This method has the highest triploidization ratio, which is 91%. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1This is the result diagram of the relative content of diploid mullet DNA; the horizontal axis is the relative content, and the vertical axis is the half peak value CV;

[0018] Figure 2 This is the result diagram of the relative content of triploid mullet DNA; the horizontal axis is the relative content, and the vertical axis is the half peak value CV;

[0019] Figure 3 This is the result graph of chicken DNA relative content; the horizontal axis is the relative content, and the vertical axis is the half-peak value CV;

[0020] Figure 4 This is the result of the metaphase division of the chromosomes of diploid mullet;

[0021] Figure 5 This is the result of the metaphase division of the chromosomes of the triploid Larvae.

[0022] Figure 6 This is the result of the chromosome karyotype composition of diploid mullet;

[0023] Figure 7 This is the result of the chromosome karyotype composition of the triploid Larvae. DETAILED DESCRIPTION

[0024] Determination of the starting time of triplicate:

[0025] Example 1: Induction method of triploid dace

[0026] 1) Artificial insemination of mullet:

[0027] Selectively mature the Lasbergii, collect mature eggs and sperm, place the eggs in a dry container at 12-15℃, slowly add semen, the fertilization volume ratio of semen to eggs is 100μL:15ml, slowly operate to mix the sperm and eggs, then add 50 times the volume of the eggs and the temperature of 12-15℃ breeding water, slowly shake to complete the sperm and egg fertilization process.

[0028] 2) Induction of triploid mullet: 6 minutes after fertilization, the hydrostatic pressure is 6000PSI, the action time is 4 minutes, the fertilized eggs are transferred to the breeding water, the temperature is kept constant at 12-15℃, and the hatching is completed in 72 hours.

[0029] Embodiment 2:

[0030] 1) Artificial insemination of mullet:

[0031] Selectively mature the Lasbergii, collect mature eggs and sperm, place the eggs in a dry container at 12-15℃, slowly add semen, the fertilization volume ratio of semen to eggs is 100μL:15ml, slowly operate to mix the sperm and eggs, then add 50 times the volume of the eggs and the temperature of 12-15℃ breeding water, slowly shake to complete the sperm and egg fertilization process.

[0032] 2) Induction of triploid mullet: 7 minutes after fertilization, the hydrostatic pressure is 6000PSI, the action time is 4 minutes, the fertilized eggs are transferred to the breeding water, the temperature is kept constant at 12-15℃, and the hatching is completed in 72 hours.

[0033] Embodiment 3:

[0034] 1) Artificial insemination of mullet:

[0035] Selectively mature the Lasbergii, collect mature eggs and sperm, place the eggs in a dry container at 12-15℃, slowly add semen, the fertilization volume ratio of semen to eggs is 100μL:15ml, slowly operate to mix the sperm and eggs, then add 50 times the volume of the eggs and the temperature of 12-15℃ breeding water, slowly shake to complete the sperm and egg fertilization process.

[0036] 2) Induction of triploid mullet: 8 minutes after fertilization, the hydrostatic pressure is 6000PSI, the action time is 4 minutes, the fertilized eggs are transferred to the breeding water, the temperature is kept constant at 12-15℃, and the hatching is completed in 72 hours.

[0037] Embodiment 4:

[0038] 1) Artificial insemination of mullet:

[0039] Selectively mature the Lasbergii, collect mature eggs and sperm, place the eggs in a dry container at 12-15℃, slowly add semen, the fertilization volume ratio of semen to eggs is 100μL:15ml, slowly operate to mix the sperm and eggs, then add 50 times the volume of the eggs and the temperature of 12-15℃ breeding water, slowly shake to complete the sperm and egg fertilization process.

[0040] 2) Induction of triploid mullet: 9 minutes after fertilization, the hydrostatic pressure is 6000PSI, the action time is 4 minutes, the fertilized eggs are transferred to the breeding water, the temperature is kept constant at 12-15℃, and the hatching is completed in 72 hours.

[0041] Embodiment 5:

[0042] 1) Artificial insemination of mullet:

[0043] Selectively mature the Lasbergii, collect mature eggs and sperm, place the eggs in a dry container at 12-15℃, slowly add semen, the fertilization volume ratio of semen to eggs is 100μL:15ml, slowly operate to mix the sperm and eggs, then add 50 times the volume of the eggs and the temperature of 12-15℃ breeding water, slowly shake to complete the sperm and egg fertilization process.

[0044] 2) Induction of triploid mullet: 10 minutes after fertilization, the hydrostatic pressure is 6000PSI for 4 minutes, the fertilized eggs are transferred to the breeding water, the temperature is kept constant at 12-15℃, and the hatching is completed in 72 hours.

[0045] The triploid ratios obtained in the above embodiments were statistically analyzed. The triploid ratio in Embodiment 1 was 36%, the triploid ratio in Embodiment 2 was 42%, the triploid ratio in Embodiment 3 was 49%, the triploid ratio in Embodiment 4 was 42%, and the triploid ratio in Embodiment 5 was 44%. Among them, the triploid ratio in Embodiment 3 was the highest. Therefore, the starting time for inducing triploidization was determined to be 8 minutes after fertilization.

[0046] Determination of hydrostatic pressure treatment time:

[0047] Embodiment 6:

[0048] 1) Artificial insemination of mullet:

[0049] Selectively mature the Lasbergii, collect mature eggs and sperm, place the eggs in a dry container at 12-15℃, slowly add semen, the fertilization volume ratio of semen to eggs is 100μL:15ml, slowly operate to mix the sperm and eggs, then add 50 times the volume of the eggs and the temperature of 12-15℃ breeding water, slowly shake to complete the sperm and egg fertilization process.

[0050] 2) Induction of triploid mullet: 8 minutes after fertilization, the hydrostatic pressure is 6000PSI, the action time is 2 minutes, the fertilized eggs are transferred to the breeding water, the temperature is kept constant at 12-15℃, and the hatching is completed in 72 hours.

[0051] Embodiment 7:

[0052] 1) Artificial insemination of mullet:

[0053] Selectively mature the Lasbergii, collect mature eggs and sperm, place the eggs in a dry container at 12-15℃, slowly add semen, the fertilization volume ratio of semen to eggs is 100μL:15ml, slowly operate to mix the sperm and eggs, then add 50 times the volume of the eggs and the temperature of 12-15℃ breeding water, slowly shake to complete the sperm and egg fertilization process.

[0054] 2) Induction of triploid mullet: 8 minutes after fertilization, the hydrostatic pressure is 6000PSI, the action time is 3 minutes, the fertilized eggs are transferred to the breeding water, the temperature is kept constant at 12-15℃, and the hatching is completed in 72 hours.

[0055] Embodiment 8:

[0056] 1) Artificial insemination of mullet:

[0057] Selectively mature the Lasbergii, collect mature eggs and sperm, place the eggs in a dry container at 12-15℃, slowly add semen, the fertilization volume ratio of semen to eggs is 100μL:15ml, slowly operate to mix the sperm and eggs, then add 50 times the volume of the eggs and the temperature of 12-15℃ breeding water, slowly shake to complete the sperm and egg fertilization process.

[0058] 2) Induction of triploid mullet: 8 minutes after fertilization, the hydrostatic pressure is 6000PSI, the action time is 5 minutes, the fertilized eggs are transferred to the breeding water, the temperature is kept constant at 12-15℃, and the hatching is completed in 72 hours.

[0059] The triploid ratios obtained in the above examples were statistically analyzed, and it was found that the triploid ratio of Example 6 was 51%, the triploid ratio of Example 7 was 55%, the triploid ratio of Example 3 was 49%, and the triploid ratio of Example 8 was 52%. Therefore, the time of the hydrostatic pressure treatment was determined to be 3 minutes.

[0060] Determination of pressure during hydrostatic treatment:

[0061] Embodiment 9:

[0062] 1) Artificial insemination of mullet:

[0063] Selectively mature the Lasbergii, collect mature eggs and sperm, place the eggs in a dry container at 12-15℃, slowly add semen, the fertilization volume ratio of semen to eggs is 100μL:15ml, slowly operate to mix the sperm and eggs, then add 50 times the volume of the eggs and the temperature of 12-15℃ breeding water, slowly shake to complete the sperm and egg fertilization process.

[0064] 2) Induction of triploid mullet: 8 minutes after fertilization, the hydrostatic pressure is 5000PSI, the action time is 3 minutes, the fertilized eggs are transferred to the breeding water, the temperature is kept constant at 12-15℃, and the hatching is completed in 72 hours.

[0065] Embodiment 10:

[0066] 1) Artificial insemination of mullet:

[0067] Selectively mature the Lasbergii, collect mature eggs and sperm, place the eggs in a dry container at 12-15℃, slowly add semen, the fertilization volume ratio of semen to eggs is 100μL:15ml, slowly operate to mix the sperm and eggs, then add 50 times the volume of the eggs and the temperature of 12-15℃ breeding water, slowly shake to complete the sperm and egg fertilization process.

[0068] 2) Induction of triploid mullet: 8 minutes after fertilization, the hydrostatic pressure is 7000PSI, the action time is 3 minutes, the fertilized eggs are transferred to the breeding water, the temperature is kept constant at 12-15℃, and the hatching is completed in 72 hours.

[0069] Embodiment 11:

[0070] 1) Artificial insemination of mullet:

[0071] Selectively mature the Lasbergii, collect mature eggs and sperm, place the eggs in a dry container at 12-15℃, slowly add semen, the fertilization volume ratio of semen to eggs is 100μL:15ml, slowly operate to mix the sperm and eggs, then add 50 times the volume of the eggs and the temperature of 12-15℃ breeding water, slowly shake to complete the sperm and egg fertilization process.

[0072] 2) Induction of triploid mullet: 8 minutes after fertilization, the hydrostatic pressure is 8000PSI, the action time is 3 minutes, the fertilized eggs are transferred to the breeding water, the temperature is kept constant at 12-15℃, and the hatching is completed in 72 hours.

[0073] The triploid ratios obtained in the above embodiments were statistically analyzed. The highest triploid ratio among the embodiments was Example 10, which was 65%. Therefore, the induction conditions were determined as follows: 8 minutes after sperm and egg fertilization, the fertilized eggs were treated at a hydrostatic pressure of 7000PSI for 3 minutes, the fertilized eggs were transferred to the breeding water, the temperature was kept constant at 12-15°C, and the hatching was completed in 72 hours.

[0074] Determine whether to conduct temperature shock and chemical combined induction treatment time:

[0075] Embodiment 12:

[0076] 1) Artificial insemination of mullet:

[0077] Selectively mature the Lasbergii, collect mature eggs and sperm, place the eggs in a dry container at 12-15℃, slowly add semen, the fertilization volume ratio of semen to eggs is 100μL:15ml, slowly operate to mix the sperm and eggs, then add 50 times the volume of the eggs and the temperature of 12-15℃ breeding water, slowly shake to complete the sperm and egg fertilization process.

[0078] 2) Induction of triploid mullet: 8 minutes after fertilization, hydrostatic pressure was 7000PSI for 3 minutes. After hydrostatic pressure treatment, the fertilized eggs were immersed in a 15°C, 300μg / mL colchicine aqueous solution for 15 minutes (the fertilized eggs were subjected to temperature shock and chemical induction treatment). The fertilized eggs were transferred to the breeding water, the temperature was kept constant at 12-15°C, and the hatching was completed in 72 hours.

[0079] Embodiment 13:

[0080] 1) Artificial insemination of mullet:

[0081] Selectively mature the Lasbergii, collect mature eggs and sperm, place the eggs in a dry container at 12-15℃, slowly add semen, the fertilization volume ratio of semen to eggs is 100μL:15ml, slowly operate to mix the sperm and eggs, then add 50 times the volume of the eggs and the temperature of 12-15℃ breeding water, slowly shake to complete the sperm and egg fertilization process.

[0082] 2) Induction of triploid mullet: 8 minutes after fertilization, hydrostatic pressure was 7000PSI for 3 minutes. After hydrostatic pressure treatment, the fertilized eggs were immersed in a 15°C, 300μg / mL colchicine aqueous solution for 25 minutes (the fertilized eggs were subjected to temperature shock and chemical induction treatment). The fertilized eggs were transferred to the breeding water, the temperature was kept constant at 12-15°C, and the hatching was completed in 72 hours.

[0083] Embodiment 14:

[0084] 1) Artificial insemination of mullet:

[0085] Selectively mature the Lasbergii, collect mature eggs and sperm, place the eggs in a dry container at 12-15℃, slowly add semen, the fertilization volume ratio of semen to eggs is 100μL:15ml, slowly operate to mix the sperm and eggs, then add 50 times the volume of the eggs and the temperature of 12-15℃ breeding water, slowly shake to complete the sperm and egg fertilization process.

[0086] 2) Induction of triploid mullet: 8 minutes after fertilization, hydrostatic pressure was 7000PSI for 3 minutes. After hydrostatic pressure treatment, the fertilized eggs were immersed in a 15°C, 300μg / mL colchicine aqueous solution for 35 minutes (the fertilized eggs were subjected to temperature shock and chemical induction treatment). The fertilized eggs were transferred to the breeding water, the temperature was kept constant at 12-15°C, and the hatching was completed in 72 hours.

[0087] Embodiment 15:

[0088] 1) Artificial insemination of mullet:

[0089] Selectively mature the Lasbergii, collect mature eggs and sperm, place the eggs in a dry container at 12-15℃, slowly add semen, the fertilization volume ratio of semen to eggs is 100μL:15ml, slowly operate to mix the sperm and eggs, then add 50 times the volume of the eggs and the temperature of 12-15℃ breeding water, slowly shake to complete the sperm and egg fertilization process.

[0090] 2) Induction of triploid mullet: 8 minutes after fertilization, hydrostatic pressure was 7000PSI for 3 minutes. After hydrostatic pressure treatment, the fertilized eggs were immersed in a 15°C, 300μg / mL colchicine aqueous solution for 45 minutes (the fertilized eggs were subjected to temperature shock and chemical induction treatment). The fertilized eggs were transferred to the breeding water, the temperature was kept constant at 12-15°C, and the hatching was completed in 72 hours.

[0091] The triploid ratios obtained in the above embodiments were statistically analyzed, and the one with the highest triploid ratio among all the embodiments was Embodiment 13.

[0092] Temperature determination for temperature shock and chemical combination induction treatment:

[0093] Embodiment 16:

[0094] 1) Artificial insemination of mullet:

[0095] Selectively mature the Lasbergii, collect mature eggs and sperm, place the eggs in a dry container at 12-15℃, slowly add semen, the fertilization volume ratio of semen to eggs is 100μL:15ml, slowly operate to mix the sperm and eggs, then add 50 times the volume of the eggs and the temperature of 12-15℃ breeding water, slowly shake to complete the sperm and egg fertilization process.

[0096] 2) Induction of triploid mullet: 8 minutes after fertilization, hydrostatic pressure of 7000PSI for 3 minutes. After hydrostatic pressure treatment, the fertilized eggs were immersed in a 300μg / mL colchicine aqueous solution at 5°C for 25 minutes (the fertilized eggs were subjected to temperature shock and combined induction treatment with chemicals). The fertilized eggs were transferred to the breeding water, the temperature was kept constant at 12-15°C, and the hatching was completed in 72 hours.

[0097] Embodiment 17:

[0098] 1) Artificial insemination of mullet:

[0099] Selectively mature the Lasbergii, collect mature eggs and sperm, place the eggs in a dry container at 12-15℃, slowly add semen, the fertilization volume ratio of semen to eggs is 100μL:15ml, slowly operate to mix the sperm and eggs, then add 50 times the volume of the eggs and the temperature of 12-15℃ breeding water, slowly shake to complete the sperm and egg fertilization process.

[0100] 2) Induction of triploid mullet: 8 minutes after fertilization, hydrostatic pressure of 7000PSI for 3 minutes. After hydrostatic pressure treatment, the fertilized eggs were immersed in a 10°C, 300μg / mL colchicine aqueous solution for 25 minutes (the fertilized eggs were subjected to temperature shock and chemical induction treatment). The fertilized eggs were transferred to the breeding water, the temperature was kept constant at 12-15°C, and the hatching was completed in 72 hours.

[0101] Embodiment 18:

[0102] 1) Artificial insemination of mullet:

[0103] Selectively mature the Lasbergii, collect mature eggs and sperm, place the eggs in a dry container at 12-15℃, slowly add semen, the fertilization volume ratio of semen to eggs is 100μL:15ml, slowly operate to mix the sperm and eggs, then add 50 times the volume of the eggs and the temperature of 12-15℃ breeding water, slowly shake to complete the sperm and egg fertilization process.

[0104] 2) Induction of triploid mullet: 8 minutes after fertilization, hydrostatic pressure was 7000PSI for 3 minutes. After hydrostatic pressure treatment, the fertilized eggs were immersed in a 300μg / mL colchicine aqueous solution at 20°C for 25 minutes (the fertilized eggs were subjected to temperature shock and chemical induction treatment), and then transferred to breeding water to complete hatching.

[0105] Embodiment 19:

[0106] 1) Artificial insemination of mullet:

[0107] Selectively mature the Lasbergii, collect mature eggs and sperm, place the eggs in a dry container at 12-15℃, slowly add semen, the fertilization volume ratio of semen to eggs is 100μL:15ml, slowly operate to mix the sperm and eggs, then add 50 times the volume of the eggs and the temperature of 12-15℃ breeding water, slowly shake to complete the sperm and egg fertilization process.

[0108] 2) Induction of triploid mullet: 8 minutes after fertilization, hydrostatic pressure of 7000PSI for 3 minutes. After hydrostatic pressure treatment, the fertilized eggs were immersed in a 300μg / mL colchicine aqueous solution at 25°C for 25 minutes (the fertilized eggs were subjected to temperature shock and combined induction treatment with chemicals). The fertilized eggs were transferred to the breeding water, the temperature was kept constant at 12-15°C, and the hatching was completed in 72 hours.

[0109] The triploid ratios obtained in the above embodiments were statistically analyzed. Among the embodiments, the one with the highest triploid ratio was Embodiment 18, which was 84%.

[0110] Determination of drug concentration for combined induction treatment with temperature shock and chemical substances:

[0111] Embodiment 20:

[0112] 1) Artificial insemination of mullet:

[0113] Selectively mature the Lasbergii, collect mature eggs and sperm, place the eggs in a dry container at 12-15℃, slowly add semen, the fertilization volume ratio of semen to eggs is 100μL:15ml, slowly operate to mix the sperm and eggs, then add 50 times the volume of the eggs and the temperature of 12-15℃ breeding water, slowly shake to complete the sperm and egg fertilization process.

[0114] 2) Induction of triploid mullet: 8 minutes after fertilization, hydrostatic pressure of 7000PSI for 3 minutes. After hydrostatic pressure treatment, the fertilized eggs were immersed in a 100μg / mL colchicine aqueous solution at 20°C for 25 minutes (the fertilized eggs were subjected to temperature shock and combined induction treatment with chemicals). The fertilized eggs were transferred to the breeding water, the temperature was kept constant at 12-15°C, and the hatching was completed in 72 hours.

[0115] Embodiment 21:

[0116] 1) Artificial insemination of mullet:

[0117] Selectively mature the Lasbergii, collect mature eggs and sperm, place the eggs in a dry container at 12-15℃, slowly add semen, the fertilization volume ratio of semen to eggs is 100μL:15ml, slowly operate to mix the sperm and eggs, then add 50 times the volume of the eggs and the temperature of 12-15℃ breeding water, slowly shake to complete the sperm and egg fertilization process.

[0118] 2) Induction of triploid mullet: 8 minutes after fertilization, hydrostatic pressure of 7000PSI for 3 minutes. After hydrostatic pressure treatment, the fertilized eggs were immersed in a 200μg / mL colchicine aqueous solution at 20°C for 25 minutes (the fertilized eggs were subjected to temperature shock and combined induction treatment with chemicals). The fertilized eggs were transferred to the breeding water, the temperature was kept constant at 12-15°C, and the hatching was completed in 72 hours.

[0119] Embodiment 22:

[0120] 1) Artificial insemination of mullet:

[0121] Selectively mature the Lasbergii, collect mature eggs and sperm, place the eggs in a dry container at 12-15℃, slowly add semen, the fertilization volume ratio of semen to eggs is 100μL:15ml, slowly operate to mix the sperm and eggs, then add 50 times the volume of the eggs and the temperature of 12-15℃ breeding water, slowly shake to complete the sperm and egg fertilization process.

[0122] 2) Induction of triploid mullet: 8 minutes after fertilization, hydrostatic pressure of 7000PSI for 3 minutes. After hydrostatic pressure treatment, the fertilized eggs were immersed in a 400μg / mL colchicine aqueous solution at 20°C for 25 minutes (the fertilized eggs were subjected to temperature shock and combined induction treatment with chemicals). The fertilized eggs were transferred to the breeding water, the temperature was kept constant at 12-15°C, and the hatching was completed in 72 hours.

[0123] Embodiment 23:

[0124] 1) Artificial insemination of mullet:

[0125] Selectively mature the Lasbergii, collect mature eggs and sperm, place the eggs in a dry container at 12-15℃, slowly add semen, the fertilization volume ratio of semen to eggs is 100μL:15ml, slowly operate to mix the sperm and eggs, then add 50 times the volume of the eggs and the temperature of 12-15℃ breeding water, slowly shake to complete the sperm and egg fertilization process.

[0126] 2) Induction of triploid mullet: 8 minutes after fertilization, hydrostatic pressure of 7000PSI for 3 minutes. After hydrostatic pressure treatment, the fertilized eggs were immersed in a 500μg / mL colchicine aqueous solution at 20°C for 25 minutes (the fertilized eggs were subjected to temperature shock and combined induction treatment with chemicals). The fertilized eggs were transferred to the breeding water, the temperature was kept constant at 12-15°C, and the hatching was completed in 72 hours.

[0127] The triploid ratios obtained in the above embodiments are statistically analyzed, as shown in Table 1. Among the embodiments, the one with the highest triploid ratio is Embodiment 21, which is 91%.

[0128] Table 1 Triploid ratios obtained in Example

[0129] Induction method Triploid ratio Induction method Triploid ratio Induction method Triploid ratio Example 1 36％ Example 9 56％ Embodiment 17 78％ Example 2 42％ Example 10 65％ Embodiment 18 84％ Example 3 49％ Embodiment 11 60％ Embodiment 19 82％ Example 4 42％ Example 12 65％ Embodiment 20 88％ Example 5 44％ Embodiment 13 72％ Embodiment 21 91％ Example 6 51％ Embodiment 14 68％ Embodiment 22 86％ Example 7 55％ Embodiment 15 69％ Embodiment 23 85％ Example 8 52％ Example 16 73％

[0130] Comprehensively analyzing the experiments of various conditional treatment groups, the scheme of combined treatment of hydrostatic pressure, temperature shock and chemical treatment in the production method of inducing chromosome triploidization of fertilized eggs of mullet was determined: 8 minutes after fertilization, the fertilized eggs were placed in a hydrostatic pressure chamber for hydrostatic pressure treatment, the pressure was 7000PSI, and the treatment time was 3 minutes; after hydrostatic pressure treatment, the fertilized eggs were subjected to temperature shock and chemical combined induction treatment, and the fertilized eggs were immersed in 200μg / ml colchicine aqueous solution, the water temperature was 20℃, and the treatment time was 25 minutes; finally, the fertilized eggs were transferred to the breeding water, the temperature was kept constant at 12-15℃, and the hatching was completed. This method has the highest triploidization rate, which is 91%.

[0131] The triploid mullet obtained in Examples 1-23 can be distinguished between triploid and diploid by the following identification means: In the induction method, if the water temperature is low, for example, below 20 degrees, or the concentration of colchicine is higher than 500 μg / ml, or the pressure is higher than 8000 PSI, any of these conditions will cause the fish to die easily.

[0132] Identification method of triploid mullet:

[0133] 1. Red blood cell volume identification method: Use a blood collection needle to collect blood from diploid Lasbergia serrata and triploid Lasbergia serrata produced in Example 21, prepare blood smears, HE stain, observe under an optical microscope, measure the length of the long and short diameters of 10 red blood cell nuclei in each smear, and calculate the volume of red blood cells and nuclei. The present invention proves that the red blood cell volume of diploid Lasbergia serrata is in the range of 58.21±3.45μm 3 The range of red blood cell nuclear volume is 39.21±5.32μm 3 The volume of triploid erythrocytes ranged from 94.17±9.43μm 3 The range of red blood cell nuclear volume is 62.66±7.34μm 3 between.

[0134] It is generally believed that cell size is related to chromosome and DNA content. In a single cell, because the number of triploid chromosomes is more than that of diploid, in order to maintain the constant ratio of nucleus to cytoplasm, the volume of triploid cells is generally larger than that of diploid cells. Different fish species have large differences in the volume of triploid and diploid red blood cells and the volume of red blood cell nuclei. Theoretically, the red blood cell nucleus volume of diploid fish and triploid fish is 1:1.5. In the present invention, a large number of samples were used for measurement and calculation to obtain a red blood cell volume ratio of diploid mullet and triploid mullet of 1:1.62, and a red blood cell nucleus volume ratio of 1:1.60.

[0135] The results of various parameters of erythrocytes and nuclei of diploid and triploid erythrocytes are shown in Table 2. As can be seen from Table 2, the values ​​of triploids are significantly higher than those of diploids. The long diameter, short diameter, erythrocyte area, and erythrocyte volume of triploid cells are 1.49, 1.45, 1.57, and 1.62 times those of diploid cells, respectively. The long diameter, short diameter, nuclear area, and erythrocyte nuclear volume of triploid erythrocytes are 1.58, 1.54, 1.67, and 1.650 times those of diploid cells, respectively. There are significant differences in various parameters between diploid and triploid (P﹤0.05). The range of diploid erythrocyte volume is 58.21±3.45μm 3 The range of red blood cell nuclear volume is 39.21±5.32μm 3 The volume range of triploid red blood cells is 94.17±9.43μm 3The range of red blood cell nuclear volume is 62.66±7.34μm 3 between.

[0136] Table 2 Erythrocyte and nuclear parameters of diploid and triploid Lasbergi

[0137]

[0138]

[0139] 2. DNA content identification method: Blood was collected from the caudal vein of diploid Lasbergi and triploid Lasbergi produced by this method, and the blood and 0.75% saline were fully mixed in a ratio of 1:5. Lymphocytes were separated with lymphocyte separation solution, PI was stained at 4°C in the dark for 30 minutes, and ploidy was identified by filtering with a PARTEC ploidy determination instrument with 400 mesh. The present invention proves that the DNA content of triploid Lasbergi is about 1.55 times that of diploid Lasbergi, and the absolute DNA content of diploid Lasbergi somatic cells is 3.53 pg.N -1 The absolute DNA content of triploid larvae somatic cells is 5.46 pg.N -1 .

[0140] 100 samples were tested, and chicken blood with known DNA content was used as a control. Flow cytometry was used to analyze the relative content of DNA in diploid scorpionfish and triploid scorpionfish produced by this method to determine ploidy. The results of the flow cytometry experiment are shown in Figure 1 , Figure 2 , Figure 3 The relative DNA content of diploid Lasbergi was 146.22±21.32, and that of triploid Lasbergi was 226.29±37.21. The DNA content of triploid Lasbergi was about 1.55 times that of diploid Lasbergi, which was consistent with the results of erythrocyte size determination, Table 3. It is known that the DNA content of chicken erythrocytes is 2.3 pg / N -1 , the absolute DNA content of diploid larvae somatic cells is 3.53 pg.N -1 The absolute DNA content of triploid larvae somatic cells is 5.46 pg.N -1 .

[0141] Table 3 DNA content of diploid, triploid mullet and chicken

[0142]

[0143] 3. Chromosome counting and analysis method: The chromosome metaphase phase is prepared by in vivo culture of kidney cells, Giemsa staining, and observation under a microscope. The present invention proves that the number of diploid chromosomes is 2n=50, and the karyotype formula is 2n=24sm+14st+12t. The number of triploid chromosomes is 3n=75, and the karyotype formula is 3n=36sm+21st+18t.

[0144] Metaphases of chromosomes were prepared by in vivo culture of kidney cells, stained with Giemsa, and observed under a microscope. The number of chromosomes in 100 metaphases of diploid and triploid Lasbergi was counted. The counting results showed that the number of diploid chromosomes was 2n=50, and the karyotype formula was 2n=24sm+14st+12t. The number of triploid chromosomes was 3n=75, and the karyotype formula was 3n=36sm+21st+18t. Metaphases of chromosomes of diploid and triploid Lasbergi were as follows: Figure 4 , Figure 5 As shown in Figure 2, the karyotype composition of diploid and triploid scorpions is as follows: Figure 6 , Figure 7 As shown in Figure 2, the chromosome numbers of diploid and triploid mullets are consistent with the ploidy relationship.

[0145] Polyploidization increases the number of chromosomes, the DNA content also increases proportionally, and the cell nucleus volume also increases. There are significant differences in the red blood cells and red blood cell nuclear area and volume, DNA content, chromosome number, etc. between diploid and triploid Lasbergi, and the data differences are consistent with the ploidy relationship.

[0146] In this experiment, the identification of triploid mullet was mainly based on the determination of red blood cells and red blood cell nuclear volume, assisted by the identification methods of chromosome counting and DNA content. Red blood cell determination can be conveniently and quickly identified, and the experimental process is relatively simple, which is suitable for large-scale determination at the breeding site. Chromosome karyotype analysis can very intuitively observe the ploidy of cells, but the operation is relatively complicated and time-consuming. The flow cytometer method for identifying DNA content requires professional experimental instruments and is relatively limited. In summary, the red blood cell determination method can be used as the preferred method for large-scale identification of ploidy, and the accuracy of the identification can be improved by chromosome counting and DNA content identification methods, so that large quantities of fish can be quickly and effectively identified for ploidy.

Claims

1. A method for inducing triploid dace, characterized in that: After fertilization of sperm and eggs on the net of the fish, the fertilized eggs are obtained, and the fertilized eggs are subjected to hydrostatic pressure treatment, and then subjected to temperature shock and drug combined induction treatment, and hatched in the breeding water.

2. The method according to claim 1, characterized in that Hydrostatic pressure treatment takes place 6-10 minutes after fertilization.

3. The method according to claim 1, characterized in that The hydrostatic pressure is 5000-8000PSI and the action time is 2-5 minutes.

4. The method according to claim 1, characterized in that: Drug treatment refers to colchicine treatment.

5. The method according to claim 1, characterized in that The temperature shock and drug combined induction treatment means that the concentration of colchicine is 200-500 μg / mL, the temperature of the aqueous solution is 5-25°C, and the treatment time is 15-45 minutes.

6. The method according to claim 1, characterized in that The breeding water temperature is 12-15℃ and the incubation time is 72 hours.

7. The method according to claim 1, characterized in that 8 minutes after fertilization, the fertilized eggs are placed in a hydrostatic pressure chamber for hydrostatic pressure treatment at a pressure of 7000PSI for 3 minutes; after hydrostatic pressure treatment, the fertilized eggs are immersed in a 200μg / ml colchicine aqueous solution at a water temperature of 20°C for 25 minutes; finally, the fertilized eggs are transferred to breeding water at a constant temperature of 12-15°C, and hatching is completed in 72 hours.

8. The method according to claim 1, characterized in that: The method for obtaining fertilized eggs is to selectively mature the mullet, collect mature eggs and sperm, place the eggs in a dry container at 12-15°C, add semen, the fertilization volume ratio of semen to eggs is 100μL:15ml, mix the sperm and eggs, then add breeding water at a temperature of 12-15°C, and shake to complete the sperm and egg fertilization process.

9. The method according to claim 8, characterized in that The volume of the breeding water is 50 times the volume of the eggs.

10. Use of the induction method according to any one of claims 1 to 9 in breeding triploid mullet.

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