Method for physically inducing tetraploid of haliotis discus hannai inked

Abstract

The application discloses a method for inducing tetraploid of Haliotis discus hannai Ino, and relates to a method for cultivating tetraploid of shellfishes, which comprises the following steps: 1) selecting and accelerating maturation of seed abalones: selecting healthy and active male and female seed abalones without external damage, and placing the seed abalones in a seed abalone cultivation pool to accelerate maturation simultaneously; 2) artificially inducing spawning: selecting diploid abalones with well-developed gonads to induce spawning, and stopping until the male and female seed abalones spawn and ejaculate; 3) selecting and mixedly fertilizing sperm and eggs: filtering impurities in sperm and eggs respectively, and synchronously mixedly fertilizing the sperm and the eggs by adding the sperm into the egg liquid; 4) cold shock induction: when 60% to 80% of the fertilized eggs are ready to start the first cleavage and elongation, the fertilized eggs are immediately collected into pre-cooled seawater with a temperature of 2-8 DEG C to be treated for 10-15 min, so as to inhibit the first cleavage; and 5) incubation: immediately placing the fertilized eggs into an incubation water tank with a temperature of 18-22 DEG C to be incubated after the cold shock is finished. The method for inducing tetraploid of Haliotis discus hannai Ino realizes efficient and stable obtaining of tetraploid of Haliotis discus hannai Ino by improving the method for inducing spawning of seed abalones and optimizing the inducing conditions.

Description

[0001] SPECIFICATION TECHNICAL FIELD

[0002] The present application belongs to the technical field of genetic breeding of aquaculture species, and particularly relates to a breeding method of tetraploid abalone haptocnus conchophilus. BACKGROUND

[0003] The induction of tetraploid abalone usually adopts physical and chemical means, and its basic mechanism is similar to that of triploid induction. In theory, tetraploid abalone has the ability to reproduce and can mate with diploids to produce high-yield triploid offspring. Compared with direct artificial induction of triploids, this approach has significant advantages in terms of embryo development and survival rate. At the same time, this method is simple to operate and the induction results are stable, which helps to overcome the bottleneck problem of large-scale production of artificially induced triploid abalone. Although tetraploid early life stages, including embryos, larvae, and even juvenile and juvenile bivalves, have been successfully bred in many bivalve studies, few cases have been able to be bred to the adult stage. Currently, tetraploid adults of pacific oyster and abalone have been successfully bred, and hybridization with diploids has been achieved to breed triploids.

[0004] The generation of tetraploids usually involves three strategies: direct application of chemical or physical intervention to inhibit the first cleavage of fertilized eggs; use of nuclear transfer and cell fusion techniques; and indirect acquisition of tetraploids by inhibiting polar body release in the offspring of triploid females and diploid males. However, although research on tetraploid abalone began in 1995, the success rate of tetraploids is still limited through direct methods such as the use of cytochalasin B or colchicine, and the survival rate of adults is not high and mass reproduction is difficult.

[0005] For example, Guo obtained 45% tetraploid embryos in pacific oyster using heat shock treatment; Cai Guoxiong induced 82.8% and 58.6% tetraploid embryos in purple mussels at the first and second cleavage, respectively, using 6-DMAP; Yang Huiping et al. detected 5 tetraploid larvae in Chlamys farreri by inhibiting the first cleavage with CB, but unfortunately these larvae did not survive after a year. In theory, inhibiting the first or second division of fertilized eggs can cause chromosome doubling without cytoplasmic division, thereby inducing tetraploids. This strategy is common in fish research, but in bivalves, although attempts are frequent, there are few records of successfully breeding surviving tetraploid adults. The suspected reason may involve the homozygous effect of lethal genes and potential damage to cleavage regulatory substances during induction.

[0006] Up to now, the research on the induction of H. discus hannai Ino tetraploid still focuses on direct induction technology, but the survival rate of tetraploid adults is not high. The cultivation of abalone tetraploid still faces challenges, such as the scarcity of triploid mature oocytes, immature induction conditions, low efficiency, inconsistent results and low survival rate of larvae, which seriously hinders the maturation and stable development of H. discus hannai Ino tetraploid breeding technology. SUMMARY

[0007] In order to solve the above technical problems, the present application provides a method for cultivating H. discus hannai Ino tetraploid, which uses a cold shock induction method: when 60% to 80% of the fertilized eggs are ready to start the first cleavage elongation, the fertilized eggs are immediately collected and treated in pre-cooled seawater with a temperature of 2 to 8℃ for 10 to 15 minutes to inhibit the first cleavage. The method can successfully cultivate to adulthood, greatly increasing the proportion of H. discus hannai Ino tetraploid adults, and is beneficial to the triploidization of H. discus hannai Ino industry.

[0008] The present application comprises the following steps:

[0009] 1) Selection and maturation of seed abalones: healthy and vigorous female and male seed abalones without external damage are selected and placed in seed abalone cultivation pools for synchronous maturation;

[0010] 2) Artificial induction of spawning: diploid seed abalones with well-developed gonads are selected for artificial induction of spawning until the female and male seed abalones spawn and release sperm;

[0011] 3) Selection and mixed fertilization of eggs and sperm: impurities in the eggs of step 2) are filtered out, and sperm is added to the egg solution for mixed fertilization to obtain fertilized eggs;

[0012] 4) Cold shock induction: when 60% to 80% of the fertilized eggs are ready to start the first cleavage elongation, the fertilized eggs are immediately collected and treated in pre-cooled seawater with a temperature of 2 to 8℃ for 10 to 15 minutes to inhibit the first cleavage of the fertilized eggs;

[0013] 5) Incubation: after the cold shock is completed, the fertilized eggs are placed in an incubation water tank at 18 to 22℃ for incubation. According to the conventional incubation and later cultivation method, H. discus hannai Ino tetraploid seedlings can be obtained, the hatching rate of trochophore larvae can be counted 14 hours after fertilization, and the polyploidization rate of trochophore larvae (10 to 14 hours) can be determined by flow cytometry.

[0014] In step 1), the specific method for selection and maturation of seed abalones can be: selecting H. discus hannai Ino with good individual vitality and no external damage as seed abalones, and placing male and female abalones in seed abalone cultivation pools for synchronous maturation; the seed abalones can be 3 years old with a shell length of 65mm to 80mm; the water temperature of the seed abalone cultivation pool is 20 to 22℃.

[0015] In step 2), the specific method of artificial induction of spawning can be: using the method of sea water stimulation by means of sun drying combined with ultraviolet irradiation to induce the spawning of the diploid individuals with well-developed gonads and determined ploidy until the female and male abalone spawn eggs and sperm, respectively; the gonads of the female individuals with well-developed gonads are close to dark green, and the gonads are full; the method of determining the ploidy is to cut part of the abalone upper tentacle for flow cytometry detection.

[0016] In step 3), the specific method of selection and mixed fertilization of the sperm and eggs can be: using a 300-mesh screen gauze to filter out the impurities in the sperm and eggs of step 2), respectively, adding the sperm with good motility determined by microscopy into the high-quality egg liquid for mixed fertilization, and dividing into a treatment group and a control group; the sperm with good motility refers to sperm with good motility and dispersibility; the high-quality eggs refer to eggs with intact yolk membranes, regular shape and no accidental fertilization; the quality ratio of the sperm to the eggs can be (9-11):1; the temperature of the fertilization can be 18-22℃, and the salinity can be 30-35.

[0017] In step 4), the specific method of cold shock induction can be: taking the development of the fertilized eggs in the control group of step 3) as a reference, when 60%-80% of the fertilized eggs in the control group are ready to start the first cleavage, the fertilized eggs in the treatment group are immediately placed in seawater with a temperature of 2-8℃ for 10-15 min.

[0018] In step 5), the specific method of conventional incubation can be: after the cold shock induction treatment is completed, the conventional incubation and later cultivation methods are used for incubation, and the flow cytometry can be used to determine the tetraploid rate at the trochophore larva stage.

[0019] The significant effect of the present application is that:

[0020] Compared with the existing preparation method of abalone tetraploid, the present application realizes the efficient and stable obtaining of the H. discus hannai tetraploid by improving the induction method of the abalone and optimizing the induction conditions (cold shock treatment temperature and time), and the tetraploid rate of the adult H. discus hannai is about 33.33% under the condition of cold shock treatment in seawater at 4℃ for 15 min. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 For the flow cytometry detection of the tetraploid ploidy of the juvenile H. discus hannai, 2N is diploid, and 4N is tetraploid;

[0022] Figure 2 For the flow cytometry detection of the tetraploid ploidy of the adult H. discus hannai, 2N is diploid, and 4N is tetraploid. DETAILED DESCRIPTION

[0023] The following examples will further illustrate the present application.

[0024] Example 1

[0025] 1) Take no external damage, healthy and vigorous Haliotis discus hannai Ino as seed abalone, the seed abalone is 3 years old, the shell length is between 71.4±5.7mm, the shell width is 45.8±4.9mm, and the wet weight is 44.18±10.32g, respectively, and is placed in a 20-22℃ seed abalone culture tank for synchronous maturation.

[0026] 2) After the gonad of the seed abalone matures, the gonad is stimulated by means of air-drying combined with ultraviolet irradiation of seawater until the sperm and eggs are discharged.

[0027] 3) The impurities in the sperm and eggs are filtered out using a 300-mesh silk screen. After the quality of the eggs and the motility of the sperm are determined by microscopy, the sperm and eggs are mixed for fertilization, and the ratio of the number of sperm to the number of eggs is (9-11):1. The fertilized eggs are equally divided into two parts and labeled as the control group and the experimental group.

[0028] 4) The development of the fertilized eggs in the control group is observed in real time, and the observation is performed by using an ordinary optical microscope. When 60%-80% of the fertilized eggs are preparing to start the first cleavage and elongation, the fertilized eggs in the experimental group are quickly placed in 2-8℃ seawater that has been precooled and treated for 10-15min.

[0029] 5) After the timer ends, the fertilized eggs in the experimental group are placed in 21℃ seawater filtered by a 300-mesh silk screen to terminate the induction.

[0030] 6) The tetraploid seedlings of Haliotis discus hannai Ino can be obtained by following the conventional hatching and later cultivation methods. The hatching rate of trochophore larvae can be counted 14h after fertilization, and the polyploidization rate of trochophore larvae (10-14h) can be determined by flow cytometry.

[0031] 7) The seawater temperature during the fertilization and larval hatching process is 18-22℃.

[0032] 8) Table 1 shows the results of the cold shock induction of tetraploid Haliotis discus hannai Ino.

[0033] Table 1 Results of the cold shock induction of tetraploid Haliotis discus hannai Ino

[0034]

[0035] Example 2

[0036] 1) Take no external damage, healthy and vigorous 3-year-old female and male Haliotis discus hannai Ino as seed abalone, and place them in a 20-22℃ seed abalone culture tank for synchronous maturation.

[0037] 2) After the gonad of the seed abalone matures, 55 female abalones with well-developed gonads and dark green gonad color are selected, and 7 male abalones with mature gonads are selected. The gonads are stimulated by means of air-drying combined with ultraviolet irradiation of seawater until the sperm and eggs are discharged.

[0038] 3) Using 300 mesh sieve silk to filter out the impurities in the sperm and egg respectively. After the quality of the egg and the activity of the sperm were determined by microscopy, the sperm and the egg were mixed for fertilization. The ratio of the number of sperm to the number of egg was (9-11): 1.

[0039] 4) The development of the fertilized eggs in the control group was observed in real time. When 60% to 80% of the fertilized eggs were ready to start the first cleavage and elongation, the experimental group was divided into two groups. One group was: the fertilized eggs were added into seawater with a temperature of 4°C and 8°C respectively for 15 min. The other group was: the fertilized eggs were added into seawater with a temperature of 4°C and 8°C respectively for 10 min.

[0040] 5) The tetraploid seedlings of H. discus can be obtained by following the conventional hatching and post-cultivation methods. The fertilization rate and the trochophore hatching rate were statistically analyzed by microscopy observation. The polyploidization rate of the trochophore (10-14 h) was determined by flow cytometry.

[0041] 6) The seawater temperature during fertilization and larval hatching was 19.8-20.8°C.

[0042] 7) To preliminarily explore the induction conditions, the fertilization rate, the trochophore hatching rate, and the tetraploid rate of the cold-shocked H. discus were statistically analyzed as follows:

[0043] The fertilization rate of the normal H. discus control group and the tetraploid group was basically above 80%. Under the conditions of cold shock treatment temperature of 4°C and 8°C, the hatching rate and the tetraploid rate of the 10 min continuous treatment group were 68.19±3.12% and 67.57±2.56% at 4°C, and 75.76±0.87% and 51.5±1.56% at 8°C. The hatching rate and the tetraploid rate of the 15 min continuous treatment group were 63.28±3.20% and 73.07±1.22% at 4°C, and 71.38±4.21% and 55.56±2.47% at 8°C.

[0044] It was preliminarily explored that the cold shock temperature and the treatment time affected the hatching rate and the tetraploid rate. The tetraploid rate of the 15 min treatment group was significantly higher than that of the 10 min treatment group under the condition of ensuring the basic hatching rate.

[0045] Example 3

[0046] 1) Take 3-year-old healthy and active female and male H. discus as seed abalones, and place them in 20-22°C seed abalone cultivation pools for synchronous maturation.

[0047] 2) After the gonad of the seed abalone is mature, 71 female abalones with well developed gonad and dark green gonad color are selected, and 16 male abalones with mature gonad are used for inducing spawning by means of dry mating combined with ultraviolet irradiation of seawater until the sperm and egg are discharged.

[0048] 3) The impurities in the sperm and egg are filtered out by using a 300-mesh silk screen. After the quality of the egg and the activity of the sperm are determined by microscopy, the sperm and egg are mixed for fertilization. The ratio of the number of sperm to the number of egg is (8-14): 1. The fertilized eggs are divided into two groups, which are marked as a control group and an experimental group.

[0049] 4) The development of the fertilized eggs in the control group is observed in real time. When 60%-80% of the fertilized eggs are ready to start the first cleavage and elongation, the fertilized eggs are added into seawater with a gradient temperature of 2°C, 4°C, 6°C and 8°C respectively for 15 minutes.

[0050] 5) The tetraploid seedlings of H. discus can be obtained by using the conventional hatching and later cultivation methods. The fertilization rate and the hatching rate of trochophore larvae are observed by microscopy, and the polyploidization rate of the trochophore larvae (10-14h) is determined by flow cytometry.

[0051] 6) The temperature of seawater during the fertilization and the hatching of the larvae is 19.3-20.6°C.

[0052] 7) The statistics of the fertilization rate and the hatching rate of the cold-shocked H. discus obtained in step 5) are as follows:

[0053] The fertilization rate of the normal H. discus in the control group and the tetraploid group is basically above 80%. The hatching rate of the 2°C group is only 48.36±3.37%, which indicates that 2°C is the limit temperature for induction. The hatching rates of the other three groups are 63.28±1.26%, 65.45±3.16% and 71.38±1.83% respectively. The survival rate of the fertilized eggs is affected by the cold-shock treatment temperature.

[0054] Example 4

[0055] 1) The 3-year-old female and male H. discus diploids without external damage are taken as seed abalones and are placed in 20-22°C seed abalone cultivation pools for synchronous maturation.

[0056] 2) After the gonad of the seed abalone is mature, 71 female abalones with well developed gonad and dark green gonad color are selected, and 16 male abalones with mature gonad are used for inducing spawning by means of dry mating combined with ultraviolet irradiation of seawater until the sperm and egg are discharged.

[0057] 3) Using 300 mesh screen silk to filter out the impurities in the sperm and egg. After the quality of the egg and the activity of the sperm were determined by microscopy, the sperm and the egg were mixed for fertilization. The ratio of the number of sperm to the number of egg was (8-14): 1. The fertilized eggs were divided into two groups, which were labeled as the control group and the experimental group.

[0058] 4) Real-time observation of the development of the control group of fertilized eggs. When 60% to 80% of the fertilized eggs were ready to start the first cleavage and elongation, the fertilized eggs were added to seawater with a temperature gradient of 2°C, 4°C, 6°C, and 8°C for 15 minutes.

[0059] 5) The tetraploid seedlings of H. discus can be obtained by following the conventional hatching and post-cultivation methods. The polyploidization rate of the trochophore larvae (10-14h) was determined by flow cytometry.

[0060] 6) The temperature of the seawater during fertilization and larval hatching was 19.3-20.6°C.

[0061] 7) The tetraploid rate of step 5) was: 2°C group 77.62±5.27%, 4°C group 73.07±2.07%, 6°C group 67.89±3.39%, and 8°C group 55.56±2.67%. The cold shock induction temperature has an effect on the tetraploid rate of the trochophore larvae. To ensure the tetraploid rate, the temperature of the cold shock treatment is preferably 4-6°C.

[0062] Example 5

[0063] The method for detecting the ploidy of the H. discus larvae as described in Examples 1 and 4 has the following detailed steps:

[0064] 1) Take the trochophore larvae fertilized for 12-14h in a centrifuge tube, add 1mL of 1×PBS (Phosphate buffered saline) buffer, and mix several times with a pipette gun. Filter with a 300 mesh screen silk.

[0065] 2) Fixation: Take 4ml of centrifuge tube and add 3ml of anhydrous ethanol. Mix the cell suspension with 1ml of pipette gun, then suck out and filter (300 mesh) into a 4ml centrifuge tube for storage for more than 24h.

[0066] 3) After centrifugation (300×g), discard the supernatant, add 500μL of 1×PBS, and finally add 33.33μL of PI staining for 10 minutes. After filtering with a 300 mesh screen silk, the DNA content of the cells is detected by flow cytometry.

[0067] The normal breeding group is the diploid control, and the samples in the experimental group are randomly detected. The tetraploid rate and the diploid rate are calculated. For example, Figure 1 The DNA content of the abalone larvae in the cold shock treatment group is about twice that of the diploid abalone larvae.

[0068] Example 6

[0069] The detailed steps of the method for detecting the ploidy of adult H. discus hannai are as follows:

[0070] 1) Clean the slide and drop a drop of PBS buffer in the center of the slide.

[0071] 2) Take H. discus hannai, cut part of the upper foot tentacles, and chop the tentacles on the PBS in the center of the slide with a knife for about 30 times.

[0072] 3) Transfer the chopped tissue to a 1.5 ml centrifuge tube, add PBS buffer to 1 ml, and gently blow and crush the tissue with a pipette.

[0073] 4) Filter the crushed tissue into a 1.5 ml centrifuge tube, centrifuge at 300 g for 7 min, remove the supernatant, add 500 μl of PBS buffer, and then add 33.33 μl of PI staining solution and mix well, avoid light, and stain for 10 min before machine detection of ploidy.

[0074] According to the method for inducing tetraploid H. discus hannai described in Example 1, the adult H. discus hannai is subjected to cold shock treatment at 4℃ for 15 min, the rate of tetraploid adult H. discus hannai is about 33.33%, and 38 adult tetraploid abalones are obtained. Figure 2 For the results of detecting the ploidy of diploid and tetraploid by flow cytometry, the DNA content of the adult abalones subjected to cold shock treatment is about 2 times that of the diploid adult abalones.

[0075] The above only describes the preferred embodiments of the present application, and any equivalent changes and modifications made within the scope of the patent application of the present application shall be included in the scope of the present application.

Claims

1. A method for inducing tetraploid of Haliotis discus hannai Ino, comprising the steps of, The method of cold shock induction is as follows: when 60% to 80% of the fertilized eggs begin to extend for the first time, the fertilized eggs are collected and treated in pre-cooled seawater with a temperature of 2 to 8 ℃ for 10 to 15 min to inhibit the first cleavage; the method comprises the following steps: ​ 1) selection and maturation of the abalones; 2) artificial induction of spawning; 3) selection and mixed fertilization of the sperm and eggs; 4) cold shock induction; 5) incubation; In step 2), the specific method of artificial induction of spawning is as follows: the gonads of the diploid individuals with mature gonad development and determined ploidy are stimulated by the method of dryness combined with ultraviolet irradiation of seawater until the female and male abalones spawn and ejaculate; the gonads of the female individuals with mature gonad development are close to dark green, and the gonads are full; The method of dryness combined with ultraviolet irradiation is as follows: after dryness for 120 min to 150 min, the female and male individuals are strictly separated and placed in different containers with clear marks, and the treatment seawater with an ultraviolet irradiation intensity of 800 to 1200 mWh / L is injected, and the seawater is replaced every 1 h until the gametes are discharged, and attention is paid to prevent accidental fertilization during the period.

2. a method for inducing a tetraploid of Haliotis discus hannai according to claim 1, characterized in that, In step 1), the specific method of selection and maturation of the abalones is as follows: the wrinkled abalones with a shell length of 65 mm to 80 mm, good individual vitality and no external damage are selected as the abalones, and the abalones are placed in the abalone cultivation pool for maturation according to the female and male; the abalones are 3-year-old wrinkled abalones; and the gender characteristics in the abalone cultivation pool must be single.

3. a method for inducing a tetraploid of Haliotis discus hannai according to claim 1, characterized in that, In step 3), the specific method of selection and treatment of the gametes is as follows: the impurities in the gametes in step 2) are filtered by using a 300-mesh screen, and the quality of the eggs and the vitality of the sperm are determined by microscopy, and the eggs with high quality and the sperm with good vitality are selected; The eggs with high quality refer to the eggs in fresh egg liquid with complete egg membranes, regular shapes and no accidental fertilization; and the sperm with good vitality refer to fresh sperm with good activity and dispersibility.

4. A method for inducing a tetraploid of Haliotis discus hannai according to claim 1, characterized in that, In step 3), the specific method of selection and mixed fertilization of the sperm and eggs is as follows: the sperm and eggs with good vitality and high quality determined in step 3) are mixed and divided into a treatment group and a control group; the quality ratio of the sperm to the eggs is (9 to 11) to 1; and the temperature of fertilization is 18 to 22 ℃, and the salinity is 30 to 35.

5. A method for inducing a tetraploid of Haliotis discus hannai according to claim 1, characterized in that: In step 4), the temperature stability of the cold shock treatment liquid is continuously monitored by periodic sampling and microscopic observation to prevent missing the induction time.

6. A method for inducing a tetraploid of Haliotis discus hannai according to claim 1, characterized in that, In step 5), the specific method of incubation is as follows: after the cold shock is completed, the fertilized eggs are immediately collected in an incubation water tank with a temperature of 18 to 22 ℃; the routine incubation and later cultivation methods are used, and the tetraploid rate of the trochophore larvae is determined by using a flow cytometer.

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