Triploid induction and sex identification method for dwarf

Abstract

The invention provides a triploid induction and sex identification method for dwarf, which comprises the following steps: selecting a proper dwarf parent, and then performing artificial spawning induction by adopting a shade drying and heating means. After sperms and ova are obtained and impurities are removed, artificial insemination is carried out according to the proportion of 1: 3. When the water temperature is controlled to be 22 DEG C, 1 mg / L of cytochalasin B is added 25 minutes after fertilization, and treatment is conducted for 10 minutes. And after the treatment is completed, immediately cleaning and removing the reagent, and continuously incubating to trochophore or D-type larva stage to obtain triploid seedlings. The induction rate is high, the stability is high, and the triploid induction rate can reach 100% or close to 100%. The expression level difference of the FOXL2 gene in the Dwarf gonad tissue is detected through qPCR, and triploid male and female individuals which are difficult to identify by naked eyes can be determined.

Description

Technical Field

[0001] The invention belongs to the technical field of shellfish genetic breeding, and in particular relates to a method for triploid induction and sex identification of dwarf clams. Background Art

[0002] Polyploid research is a field of extensive exploration and practice, providing a good material opportunity for functional gene analysis and variety improvement of shellfish. Triploid shellfish have many advantages such as fast growth rate, high survival rate and large individuals, so the establishment and improvement of triploid induction technology is of great significance to the development of aquatic shellfish industry.

[0003] There are three ways to artificially induce polyploidy: physical, chemical and biological. The physical method mainly interferes with the release of fertilized egg polar bodies through temperature shock, hydrostatic pressure, osmotic pressure, etc., thereby obtaining triploids; the biological method is to hybridize tetraploids with diploids, and theoretically 100% of triploid offspring can be obtained; the chemical method is currently the most widely used polyploidy induction method, mainly using reagents such as cytochalasin B (CB) and 6-dimethylaminopurine (6-DMAP) to inhibit the release of polar bodies.

[0004] At present, triploid induction has been attempted in many shellfish, such as oysters, scallops, abalone, clams, etc., and great progress has been made. However, it has only been successfully industrialized in a few species, such as oysters. The reason is that, on the one hand, the induction technology has long suffered from problems such as low efficiency and poor stability; on the other hand, the reproduction cycle of shellfish is usually long, and research cannot be carried out quickly and in large quantities. These factors have seriously restricted the progress of shellfish polyploid breeding research and the development and utilization of polyploid varieties.

[0005] The dwarf clam (Mulinia lateralis) is a small burrowing shellfish with the advantages of rapid growth and short generation cycle, making it an ideal material for shellfish polyploidy research. In the early stage, our team has systematically carried out research on the introduction and domestication, artificial breeding, germplasm resource bank construction, and genetic platform development of dwarf clams, and established a shellfish model research platform to provide material and technical support for the study of dwarf clam polyploidy. Summary of the invention

[0006] The invention aims to provide a method for triploid induction and sex identification of dwarf clams, which can achieve stable and efficient induction of triploid dwarf clams and can efficiently detect triploid dwarf clams.

[0007] The present invention first provides a triploid induction method for dwarf clams, comprising the following steps:

[0008] 1) The dwarf clam broodstock were placed in an environment of 22°C to promote their sexual maturity, and the parents were dried in the shade and heated to allow them to naturally release gametes;

[0009] 2) Using 300-mesh and 500-mesh sieves to filter out impurities and enrich eggs to obtain eggs; similarly, passing sperm through a 500-mesh sieve to filter out impurities to obtain sperm;

[0010] 3) Mixing the sperm and eggs treated in step 3 above to control the sperm-egg ratio to 1:3;

[0011] 4) 25 minutes after fertilization, when 80% of the fertilized eggs have released the first polar body, 1 mg / L cytochalasin B is added for 10 minutes;

[0012] 5) The fertilized eggs were washed with a 0.01% DMSO seawater solution and filtered seawater to remove the colchicine CB in the fertilized eggs, and then incubated in filtered seawater at 22° C. to obtain triploid dwarf clams.

[0013] In another aspect, the present invention provides a method for detecting male and female individuals of dwarf clams, wherein the method is to identify male and female individuals by detecting the expression level of FOXL2 gene in dwarf clams;

[0014] As a specific record of an embodiment, when the individual to be tested develops to the juvenile or adult stage, the gonadal tissue is taken to extract total RNA, and cDNA is obtained by reverse transcription; EF1A is used as an internal reference gene to correct the expression level of the target gene; and the relative expression level of the FOXL2 gene is detected by qPCR.

[0015] The relative expression of FOXL2 in gonadal tissue was detected by qPCR. The results showed that the expression level of triploid females was significantly higher than that of triploid males.

[0016] The primer pair sequence information for detecting the FOXL2 gene is as follows:

[0017] FOXL2_F: 5′-AGTCCAGCGGACATACTGACAAGA-3′ (SEQ ID NO: 1);

[0018] FOXL2_R: 5'-CATGGCAATGAGGGCAACGTAAGA-3' (SEQ ID NO: 2).

[0019] EF1A_F: 5′-TCATCATTGCCGTCAACAAGAT-3′ (SEQ ID NO: 3);

[0020] EF1A_R: 5'-GTTGGATACTTCAGTGGAGATT-3' (SEQ ID NO: 4).

[0021] The present invention discloses for the first time a triploid induction method suitable for dwarf clams. This method is significantly different from the existing research on triploid induction of shellfish, mainly in the following aspects:

[0022] 1. High stability of triploid rate: In other studies on triploid induction of shellfish, the induction rate is usually between 40% and 80%. However, the triploid induction rate of the present invention is almost stable at around 100%, significantly reducing the occurrence rate of diploid, tetraploid and aneuploid;

[0023] 2. Method for identifying triploid males and females: In the juvenile stage, since the triploid gonads have not yet developed, it is difficult to identify the males and females. The present invention provides a new method for identifying the sex of triploid dwarf clams by utilizing the fact that the expression level of the FOXL2 gene is significantly different between males and females. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 :The induction effect diagram of different reagents;

[0025] Figure 2 : Expression level graph of FOXL2 gene. DETAILED DESCRIPTION

[0026] The invention provides a triploid induction method suitable for dwarf clams, which has better effect than using 6-DMAP, a reagent commonly used in aquatic shellfish breeding, and general polyploid breeding methods. The dwarf clams used are all from the Marine Biological Genetics and Breeding Laboratory of Ocean University of China.

[0027] Comparative Example 1

[0028] 1. Select 4-month-old, 5.8±0.5mm shell length, sexually mature dwarf clam parents, including 12 females and 8 males. First, dry them in the shade for 1-2 hours, then place them in filtered seawater at 27°C to induce natural discharge of sperm and eggs. During the process of discharging gametes, the gamete-producing parents are selected, washed, and placed in a new beaker for induction of production. After the induction of production, 10 cups of eggs and 5 cups of sperm are obtained.

[0029] 2. Check the eggs under a microscope, select 6 cups of eggs with regular shape and fullness, combine them, use a 300-mesh sieve to filter out impurities, and then use a 500-mesh sieve to enrich them, eventually obtaining about 500,000 eggs; at the same time, use a 300-mesh sieve to filter out impurities from the produced sperm and combine them for later use.

[0030] 3. Add sperm to eggs and mix thoroughly. Observe under a microscope to ensure that there are 3-5 sperm attached to each egg. Then add filtered seawater at 22°C and adjust the density of fertilized eggs to about 10 4 / mL, and divided them into a treatment group and a control group, where the volume of the treatment group was 40mL and the volume of the control group was 10mL.

[0031] 4. At different treatment periods and treatment times, different concentrations of cytochalasin B (CB) and 6-DMAP were used to treat fertilized eggs, as shown in Table 1.

[0032] Table 1: Data of fertilized eggs treated with different concentrations of CB and 6-DMAP

[0033]

[0034] 5. Use 0.01% DMSO seawater solution and filtered seawater to rinse the fertilized eggs in turn. After that, the fertilized eggs are hatched in filtered seawater to obtain triploid dwarf clam larvae. After 12 hours, the ploidy is determined by flow cytometry. The triploid rate results are shown in Table 2 and Figure 1 shown.

[0035] Table 2: Triploidy rate data table

[0036] Experimental Group Triploidy rate in CB group / % Triploidy rate in 6-DMAP group / % 1 55.05±14.21 13.60±1.98 2 83.20±6.22 26.80±6.65 3 82.05±21.14 13.15±1.06 4 92.75±0.78 32.80±4.38 5 95.10±6.93 37.20±1.56 6 99.95±0.07 40.50±0.71 7 25.50±13.01 8.00±3.25 8 57.20±4.10 10.76±1.76 9 50.70±3.25 16.80±10.61 Control group 0.00±0.00 0.00±0.00

[0037] Example 1

[0038] 1. Select 3-month-old, 5.5±0.6mm shell length, sexually mature dwarf clam parents, including 10 females and 5 males. First, dry them in the shade for 1-2 hours, then place them in filtered seawater at 27°C to induce natural discharge of sperm and eggs. During the process of discharging gametes, select the gamete-producing parents, wash them, and place them in a new beaker for induction of production. After the induction of production, 9 cups of eggs and 5 cups of sperm were obtained.

[0039] 2. Check the eggs under a microscope, select 6 cups of eggs with regular shape and fullness, combine them, use a 300-mesh sieve to filter out impurities, and then use a 500-mesh sieve to enrich them, and finally obtain about 600,000 eggs; at the same time, use a 300-mesh sieve to filter out impurities from the produced sperm and combine them for later use.

[0040] 3. Add sperm to eggs and mix thoroughly. Observe under a microscope to ensure that there are 3-5 sperm attached to each egg. Then add filtered seawater at 22°C and adjust the density of fertilized eggs to about 10 4 / mL, and divided them into a treatment group and a control group, where the volume of the treatment group was 50mL and the volume of the control group was 10mL.

[0041] 4. 25 minutes after fertilization, when 80% of the fertilized eggs have released the first polar body (PB1), add 50 μL of cytochalasin B stock solution (CB is dissolved in DMSO and prepared as 1 mg / mL stock solution) to the treatment group to reach a final concentration of 1 mg / L, and use a pipette to gently blow and mix well.

[0042] 5. After 10 minutes of treatment (i.e. 35 minutes after fertilization), the fertilized eggs were rinsed with 0.01% DMSO seawater solution and filtered seawater in turn. After that, the fertilized eggs were hatched in filtered seawater to obtain triploid dwarf clam larvae. After 12 hours, the ploidy was determined by flow cytometry, and the triploid rate was found to be 100.0±0.0%.

[0043] During fertilization and incubation, the water temperature was maintained at a constant 22°C.

[0044] After the triploid dwarf clams developed to the juvenile stage (after 40 days), their gonadal tissues were taken and total RNA was extracted. Reverse transcriptase was used to reverse the RNA to obtain cDNA. The relative expression level of FOXL2 in the gonadal tissues was detected by qPCR. The results showed that the expression level of triploid females was significantly higher than that of triploid males ( Figure 2 ).

[0045] The above results indicate that the significant difference in FOXL2 gene expression between males and females can be used to provide a new method for identifying the sex of triploid dwarf clams.

Claims

1. A triploid induction method for dwarf clams, characterized in that: The method comprises the following steps: 1) Promoting the sexual maturity of dwarf clam broodstock, using shade drying and temperature raising methods to allow the broodstock to naturally release gametes; 2) Using 300-mesh and 500-mesh sieves to filter out impurities and enrich eggs to obtain eggs; similarly, passing sperm through a 500-mesh sieve to filter out impurities to obtain sperm; 3) mixing the sperm and eggs processed in step 3 above; 4) 25 minutes after fertilization, add cytochalasin B for 10 minutes; 5) The fertilized eggs were washed with a 0.01% DMSO seawater solution and filtered seawater to remove the colchicine CB in the fertilized eggs, and then incubated in filtered seawater at 22° C. to obtain triploid dwarf clams.

2. The method according to claim 1, characterized in that In the above 1), sexual maturity is promoted in an environment of 22°C.

3. The method according to claim 1, characterized in that In the above 3), the sperm-egg ratio is controlled to be 1:

3.

4. The method according to claim 1, characterized in that 1 mg / L cytochalasin B was added to the above 4).

5. A method for detecting triploid male and female individuals of dwarf clams, characterized in that: The method is to identify male and female individuals by detecting the expression level of FOXL2 gene in triploid individuals of dwarf clams.

6. The method according to claim 5, characterized in that The method is to extract total RNA from gonadal tissue when the individual to be tested develops to the juvenile or adult stage, and obtain cDNA by reverse transcription; EF1A is used as an internal reference gene to correct the expression level of the target gene; and the relative expression level of the FOXL2 gene is detected by qPCR.

7. The method according to claim 6, characterized in that The primer pair for detecting the FOXL2 gene has an upstream primer sequence of SEQ ID NO: 1 and a downstream primer sequence of SEQ ID NO:

2.

8. The method according to claim 6, characterized in that The primer pair for detecting the EF1A gene has an upstream primer sequence of SEQ ID NO: 3 and a downstream primer sequence of SEQ ID NO: 4.

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