Method for inducing triploid and identifying male and female of dwarf clam

Abstract

The present invention provides a method for inducing triploid and identifying male and female of dwarf clams. The method includes selecting appropriate dwarf clam parents, and then artificially inducing spawning by means of drying in the shade and raising the temperature. After obtaining sperm and eggs and removing impurities, artificial insemination is carried out at a ratio of 1:3. When the water temperature is controlled at 22 °C, 1 mg / L of cytochalasin B is added 25 minutes after fertilization (and treated for 10 minutes. After the treatment is completed, the reagent is immediately washed off, and incubation is continued until the trochophore larvae or D-shaped larvae stage, and then triploid seedlings can be obtained. The present invention not only has a high induction rate and strong stability, but also the triploid induction rate can reach 100% or be close to 100%. By detecting the difference in the expression level of the FOXL2 gene in the gonad tissue of dwarf clams by qPCR, triploid male and female individuals that are difficult to identify with the naked eye can be determined.

Description

Technical Field

[0001] The present invention belongs to the technical field of shellfish genetic breeding, and specifically relates to a method for inducing triploid and identifying male and female of dwarf clams (Mulinia lateralis). Background Art

[0002] The research on polyploidy is a field of extensive exploration and practice, providing a good material opportunity for the analysis of functional genes and variety improvement of shellfish. Triploid shellfish have various advantages such as fast growth rate, high survival rate, and large size. Therefore, the establishment and improvement of triploid induction technology are of great significance for the industrial development of aquatic shellfish.

[0003] The artificial induction methods of polyploidy can be roughly divided into three types: physical, chemical, and biological methods. Physical methods mainly interfere with the release of polar bodies of fertilized eggs through methods such as temperature shock, hydrostatic pressure, and osmotic pressure, and then obtain triploids; biological methods are through hybridization of tetraploids and diploids, and theoretically 100% triploid offspring can be obtained; chemical methods are the most widely used polyploid induction methods at present, mainly using reagents such as cytochalasin B (CB) and 6-dimethylaminopurine (6-DMAP) to inhibit polar body release.

[0004] At present, triploid induction has been carried out in many shellfish such as oysters, scallops, abalones, and clams through attempts, and great progress has been made. However, it has only been successfully industrialized in a few species such as oysters. The reasons are, on the one hand, that the induction technology has long-term problems such as low efficiency and poor stability; on the other hand, because the reproductive cycle of shellfish is usually long, research cannot be carried out quickly and in large quantities. These factors seriously restrict the progress of shellfish polyploid breeding research and the development and utilization of polyploid varieties.

[0005] The dwarf clam (Mulinia lateralis) is a small buried shellfish, which has advantages such as rapid growth and short generation cycle, and can be used as an ideal material for shellfish polyploid research. Previously, our team has systematically carried out research on introduction and domestication, artificial breeding, construction of germplasm resource bank, and development of genetic platform with the dwarf clam as the object, and built a shellfish model research platform, providing materials and technical support for the polyploid research of dwarf clams. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for inducing triploid and identifying male and female 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 method for inducing triploid of dwarf clams, including the following steps:

[0008] 1) Promote the sexual maturity of dwarf clam parent shells at 22°C, and use the methods of drying in the shade and increasing temperature to make the parents naturally release gametes;

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

[0010] 3) Mix the sperm processed in step 3 above with the eggs, and control the sperm-egg ratio to be 1:3;

[0011] 4) When 25 minutes after fertilization, when 80% of the fertilized eggs extrude the first polar body, add 1 mg / L of cytochalasin B and treat for 10 minutes;

[0012] 5) Rinse successively with 0.01% DMSO seawater solution and filtered seawater to remove colchicine CB from the fertilized eggs, and incubate in filtered seawater at 22 °C to obtain triploid dwarf clams.

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

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

[0015] Detect the relative expression content of FOXL2 in gonadal tissue by qPCR method. It is found that the expression level of triploid females is 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 method for inducing triploid in dwarf clams, which is significantly different from the existing research on inducing triploid in shellfish, mainly manifested in the following aspects:

[0022] 1. High stability of triploid rate: In the existing research on inducing triploid in other shellfish, the induction rate usually ranges from 40% to 80%. However, the triploid induction rate of the present invention is almost stable at about 100%, significantly reducing the emergence rate of diploids, tetraploids and aneuploids;

[0023] 2. Identification method for triploid male and female: In the juvenile stage, it is difficult to identify the male and female genders because the gonads of triploids have not yet developed. The present invention utilizes the significant difference in the expression level of the FOXL2 gene between males and females to provide a new method for identifying the gender of triploid dwarf clams. Brief Description of the Drawings

[0024] Figure 1 : Induction effect diagrams of different reagents;

[0025] Figure 2 : Expression level diagram of the FOXL2 gene. Detailed Embodiments

[0026] The present invention provides a method for inducing triploid in dwarf clams, which has better effects compared to the commonly used reagent 6-DMAP in aquaculture shellfish breeding and general polyploid breeding methods. The dwarf clams used are all from the Laboratory of Marine Biology Genetics and Breeding, Ocean University of China.

[0027] Comparative Example 1

[0028] 1. Select 4-month-old dwarf clam parents with a shell length of 5.8 ± 0.5 mm and sexual maturity, including 12 females and 8 males. First, perform air-drying treatment for 1 - 2 hours, and then place the parents in filtered seawater at 27°C to promote the natural release of sperm and eggs. During the process of gamete release, select the parents that produce gametes, wash them and place them separately in new beakers for spawning induction. After the spawning induction is completed, 10 cups of eggs and 5 cups of sperm are obtained respectively.

[0029] 2. Examine the eggs under a microscope, select 6 cups of eggs with regular and plump morphology for merging, filter out impurities with a 300-mesh sieve, and enrich them with a 500-mesh sieve to finally obtain about 500,000 eggs; at the same time, filter out impurities from the produced sperm with a 300-mesh sieve and merge them for standby.

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

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

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

[0033]

[0034] 5. The fertilized eggs were rinsed successively with 0.01% DMSO seawater solution and filtered seawater. After that, the fertilized eggs were hatched in filtered seawater to obtain triploid dwarf clam larvae. After 12 hours, the ploidy was measured by a flow cytometer, and the results of the triploid rate are shown in Table 2 below and Figure 1 as shown.

[0035] Table 2: Data table of triploid rate

[0036] experimental group Triploid rate of CB group / % Triploid rate of 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 dwarf clam parents with a shell length of 5.5 ± 0.6 mm and sexual maturity, including 10 females and 5 males. First, perform air-drying treatment for 1 - 2 hours, and then place the parents in filtered seawater at 27°C to promote the natural release of sperm and eggs. During the process of gamete release, select the parents that produce gametes, wash them and place them separately in a new beaker for induced spawning. After the induced spawning is completed, 9 cups of eggs and 5 cups of sperm are obtained respectively.

[0039] 2. Examine the eggs under a microscope, select 6 cups of eggs with regular and plump morphology for merging. After filtering out impurities with a 300-mesh sieve, enrich them with a 500-mesh sieve to finally obtain about 600,000 eggs; at the same time, filter out impurities from the produced sperm with a 300-mesh sieve and then merge them for standby.

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

[0041] 4. At 25 minutes after fertilization, when 80% of the fertilized eggs had released the first polar body (PB1), 50 μL of cytochalasin B stock solution (CB was dissolved in DMSO and prepared as a 1 mg / mL stock solution) was added to the treatment group to achieve a final concentration of 1 mg / L, and the solution was gently stirred with a pipette to mix evenly.

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

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

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

[0045] The above results indicate that, by taking advantage of the significant difference in the expression level of the FOXL2 gene between males and females, a new method for identifying the sex of triploid dwarf clams is provided.

Claims

1. A method for detecting triploid male and female individuals of dwarf clams, characterized in that: The method described is to identify male and female individuals by detecting the expression level of FOXL2 gene in triploid individuals of dwarf clams; The method is to extract total RNA from gonadal tissue when the individual to be detected 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 amount of the target gene; and the relative expression level of the FOXL2 gene is detected by qPCR to detect the triploid male and female individuals of the dwarf clam; The primer pair used to detect the relative expression level of the FOXL2 gene has an upstream primer sequence of SEQ ID NO: 1 and a downstream primer sequence of SEQ ID NO: 2; the primer pair used to detect the EF1A gene has an upstream primer sequence of SEQ ID NO: 3 and a downstream primer sequence of SEQ ID NO:

4.

2. The method for detecting triploid male and female individuals of dwarf clams as claimed in claim 1, characterized in that: The method for inducing triploid dwarf clams comprises the following steps: 1) Promote sexual maturity of dwarf clam broodstock, and then use shade drying and temperature raising methods to allow the broodstock to naturally release gametes; 2) Use 300-mesh and 500-mesh sieves to filter out impurities and enrich eggs to obtain eggs; similarly, filter out impurities from sperm through a 500-mesh sieve to obtain sperm; 3) Mix the sperm and eggs processed in step 2 above; 4) 25 minutes after fertilization, add cytochalasin B for 10 minutes; 5) The fertilized eggs were rinsed with a 0.01% DMSO seawater solution and then filtered seawater to remove cytochalasin B, and then incubated in filtered seawater at 22°C to obtain triploid dwarf clams.

3. The method for detecting triploid male and female individuals of dwarf clams as claimed in claim 2, characterized in that: In step 1), sexual maturity is promoted at 22°C.

4. The method for detecting triploid male and female individuals of dwarf clams as claimed in claim 2, characterized in that: In step 3), the sperm-egg ratio is controlled to be 1:

3.

5. The method for detecting triploid male and female individuals of dwarf clams as claimed in claim 2, characterized in that: In step 4), 1 mg / L cytochalasin B was added.

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