Quantitative induction method for time point of tetraploid of crassostrea hongkongensis

Abstract

The application develops a quantitative induction method for tetraploid time point of Fujian oyster by taking the development stage of fertilized egg as a biological index. The application effectively eliminates the defects of poor gamete synchrony caused by traditional mixed gametes of multiple pairs of oysters by means of optimizing sperm and egg, obtaining sperm and egg by dry stripping method, and single pair fertilization. The application is different from the traditional method (after 8-10 min of fertilization, using reagent for continuous treatment for 15-20 min), and takes the development status of fertilized egg as a biological index. The time period from the beginning of fertilization of the control group to the appearance of the first first polar body (PB1) of the fertilized egg is defined as A min, and the time period from the appearance of the first PB1 of the fertilized egg of the control group to the development of PB1 of 50% of the fertilized eggs is defined as B min. When the fertilization time of the treatment group is (A-3) min, the inducer is added for continuous treatment for B min, which effectively avoids the problems of low and unstable induction rate caused by fixed induction time. The tetraploid rate of the Fujian oyster cultivated by the application is high and stable to the juvenile stage, which provides a new idea for polyploid breeding of Fujian oyster.

Description

Technical Field

[0001] This invention belongs to the field of shellfish breeding technology, specifically involving a time-point quantitative induction method for tetraploid oysters in Fujian. Background Technology

[0002] Compared to diploid oysters, triploid molluscs grow faster. Furthermore, due to their sterility, they do not consume glycogen, lipids, or other nutrients to produce gametes during their reproductive cycle, thus ensuring a year-round market supply with better taste and flavor. Since Stanley et al. (1981) first successfully induced triploid American oysters using drugs… C. virginica Subsequently, many scholars conducted extensive research on triploid mollusks, such as the Hong Kong giant oyster, the Pacific oyster, and the Fujian oyster. Although triploids can be obtained by treating diploid fertilized eggs with physical or chemical methods (such as cytochalasin B, CB, 6-dimethylaminopurine, 6-DMAP, temperature, hypotonicity, and caffeine), these methods often result in extremely unstable triploid rates and low survival rates. It wasn't until Guo and Allen (1994) developed a method for cultivating viable tetraploid Pacific oysters and successfully obtained 100% triploid offspring through hybridization of tetraploids and diploids that many researchers began to study the cultivation of tetraploids in different mollusks.

[0003] Although polyploid oyster breeding has been carried out for many years, very few polyploid oyster varieties have been industrialized. The main reason is the difficulty in cultivating tetraploid broodstock. Currently, tetraploids can be induced in three ways: (1) inhibiting the polar body release of diploid fertilized eggs; (2) inhibiting the polar body release of male diploid and female triploid fertilized eggs; and (3) inhibiting the polar body release of female diploid and male tetraploid fertilized eggs. Although the tetraploid induction rate by method (2) is higher than that by methods (1) and (3), the timing and duration of treatment are difficult to control, often resulting in a low tetraploid rate and low survival rate. Therefore, an efficient and highly stable induction method is needed. Summary of the Invention

[0004] To address the shortcomings of traditional tetraploid mollusc induction methods, such as low induction rate, unstable ploidy, and high mortality, this invention develops a time-quantitative treatment method using the developmental stage of the fertilized egg as a biological indicator. Tetraploids cultivated using this method exhibit stable ploidy, high tetraploidity, and good growth, providing a theoretical and practical basis for the cultivation of superior tetraploid Fujian oyster varieties and the industrialized production of triploid Fujian oysters.

[0005] The present invention provides a time-point quantitative induction method for tetraploid oysters from Fujian, characterized by comprising the following steps:

[0006] a. Selection of triploid maternal parents: After detecting ploidy using flow cytometry, weigh and select the top 10% of individuals with beautiful shells and strong viability based on wet weight; dissect and distinguish sex under an optical microscope, and retain individuals with ≥15 million eggs, uniform egg development, and deep yolk color as triploid maternal parents;

[0007] b. Obtaining triploid eggs: Oyster eggs from individual oysters were obtained using the "dry peeling method" to ensure synchronous development of gametes. After washing away impurities through a 500-mesh silk screen, the eggs were placed in seawater at a temperature of 25°C and a salinity of 28 psu for 40 minutes to mature. After the eggs were completely rounded, they were washed three more times using a 500-mesh silk screen. After maturation, a small number of eggs were placed in a 50 mL glass beaker as the control group, and the remaining eggs were defined as the treatment group.

[0008] c. Selecting diploid paternal parents: Select the top 10% of diploid Fujian oysters based on wet weight; dissect and distinguish sex under an optical microscope, and retain individuals with vigorous sperm motility and full gonads as diploid paternal parents;

[0009] d. Obtaining diploid sperm: When more than 90% of the total number of triploid eggs in step a has matured, sperm from a single oyster is obtained by the "dry peeling method" and activated in seawater for 3-5 minutes; after activation, a small amount of sperm is placed in a 50 mL glass beaker as the control group, and the remaining sperm is defined as the treatment group.

[0010] e. Experimental treatment: First, sperm and eggs from the control group were mixed at a sperm-egg ratio of 8-10:1 and timing was started. The time from the start of fertilization in the control group to the appearance of the first polar body of the fertilized egg was defined as A min. The time from the appearance of the first polar body of the fertilized egg to the appearance of the first polar body in 50% of the fertilized eggs was defined as B min. After 10 min of fertilization in the control group, the treatment group was fertilized at a sperm-egg ratio of 8-10:1 and timing was started. When the fertilization time was (A-3) min, the treatment group was induced with 0.75 mg / L cytochalasin B for a duration of B min.

[0011] f. Larval and Juvenile Shellfish Development: After induction, wash away cytochalasin B from the fertilized egg fluid, place in a cement tank for hatching, cultivate larvae using conventional methods, and transfer to the sea for further development after attachment and metamorphosis.

[0012] g. Ploidy detection: Ploidy of the larvae obtained in step c is detected by flow cytometry; ploidy analysis of adult shellfish is completed by double ploidy detection of hemolymph and gills.

[0013] The obtained larvae have a high hatching rate, high survival rate, and maintain a high tetraploidy rate while also maintaining stable ploidy. The obtained juvenile shellfish have a fast growth rate, high survival rate, high and stable tetraploidy rate during the sea recuperation stage.

[0014] The present invention differs from the traditional tetraploid oyster induction method, and its main innovations are as follows: (1) Different methods of obtaining sperm and eggs: The traditional method obtains sperm and eggs by hand squeezing and washing and filtering with seawater; while the present invention uses the "dry peeling method" to obtain sperm and eggs, and then places them in seawater at the same time to maximize their hydration time. (2) Different mating methods: The traditional method mixes the sperm and eggs of multiple oysters to induce tetraploids, and it is difficult to guarantee the synchronicity of their gamete development; while the present invention, based on the selection and selection of sperm and eggs, fertilizes one-to-one single oysters, resulting in good gamete development and extremely high synchronicity, effectively avoiding the problem of asynchronous gamete development caused by environmental factors or individual differences of oysters. (3) Different induction timing and continuous induction time: Traditional methods use an inducing agent to continuously treat for 15-20 minutes after fertilization for 8-10 minutes. The fixed induction timing and continuous induction time lead to large variations in the tetraploid rate of oysters induced in different environments, and the tetraploid rate is unstable. In contrast, this invention defines the time from the start of fertilization in the control group to the appearance of the first first polar body as A min, and the time from the appearance of the first first polar body in the fertilized eggs in the control group to the appearance of the first polar body in 50% of the fertilized eggs as B min. When the fertilization time in the treatment group is (A-3) min, 0.75 mg / L of cytochalasin B is added and continuously treated for B min. This invention uses the development status of the fertilized eggs as a biological indicator, which effectively avoids the problems of low and unstable induction rate caused by fixed induction time. (4) Different results of the offspring: the survival rate of tetraploid larvae obtained by traditional methods is less than 20%, and the tetraploidity rate is less than 50%; while the survival rate of tetraploid larvae obtained by the present invention is more than 28%, the tetraploidity rate of larvae is more than 70%, and the tetraploidity rate of adult shellfish is more than 90% at 360 days old.

[0015] This invention develops a quantitative induction method for tetraploid oysters from Fujian oysters by carefully selecting triploid maternal and diploid paternal parents, selecting superior sperm and eggs, employing a one-to-one single-pair oyster fertilization method, and using the developmental stage of the fertilized egg as a biological indicator. This method yields Fujian oyster tetraploids with high survival rate, high tetraploidity rate, and stable ploidy. By carefully selecting parental parents and superior sperm and eggs, obtaining sperm and eggs through a "dry peeling" method, and using single-pair fertilization, this invention maximizes the synchronicity of gamete development and effectively eliminates the defect of poor gamete synchronicity caused by mixing multiple pairs of oyster sperm and eggs in traditional methods. Unlike traditional induction methods (which involve continuous treatment with reagents for 15-20 minutes after 8-10 minutes of fertilization), this invention uses the developmental status of the fertilized egg as a biological indicator. The time period from the start of fertilization to the appearance of the first first polar body in the control group is defined as Amin, and the time period from the appearance of the first first polar body in 50% of the fertilized eggs in the control group to the appearance of the first polar body is defined as Bmin. When the fertilization time in the treatment group is (A-3) min, 0.75% of the fertilized egg is added to the treatment group. Continuous treatment with mg / L cytochalasin B for a duration of 1 minute effectively avoids problems such as low and unstable induction rates caused by fixed induction time; the tetraploid rate of the cultured larvae and adult oysters is high and stable; this invention provides a new approach for tetraploid breeding of Fujian oysters; this invention has the advantages of simple operation, high efficiency and ease of implementation. Attached Figure Description

[0016] Figure 1 This is a technical roadmap of the present invention, wherein... Figure 1 a represents the traditional tetraploid induction method. Figure 1 b is a technical roadmap of the present invention;

[0017] Figure 2 Peak diagram for ploidy detection of tetraploid Fujian oyster;

[0018] Figure 3 This shows the growth of tetraploid Fujian oysters at 180 days of age. Detailed Implementation

[0019] The present invention will be further illustrated below with specific embodiments, but these are not intended to limit the invention. Figure 1 Method a is the traditional method, which involves continuous treatment with reagents for 15-20 minutes after fertilization (8-10 minutes later). This method results in a low and unstable rate of tetraploid adult shellfish and a low survival rate. Figure 1b is the method of the present invention. The time period from the start of fertilization to the appearance of the first first polar body in the control group is defined as Amin. The time period from the appearance of the first first polar body in the fertilized eggs in the control group to the appearance of the first polar body in 50% of the fertilized eggs is defined as Bmin. When the fertilization time in the treatment group is (A-3) min, 0.75 mg / L of cytochalasin B is added and treated for B min. This effectively avoids problems such as asynchronous development caused by environmental factors or individual differences in oysters. The tetraploid rate of the larvae and adult oysters cultivated is high and stable. Example 1

[0020] a. Selection of triploid maternal parents: In mid-June 2022, triploid Fujian oysters from Putian City, Fujian Province were used as experimental parents. The top 10% of individuals (30 individuals) with beautiful shell shape and strong vitality were selected based on wet weight (wet weight ≥120.48g). They were dissected and sexed under an optical microscope. Individuals with ≥15 million eggs, uniform egg development, and deep yolk color were retained as triploid maternal parents (5 individuals).

[0021] b. Obtaining triploid eggs: Eggs from individual oysters were obtained using the "dry peeling method". After impurities were removed by washing through a 500-mesh silk screen, the eggs were placed in seawater at a temperature of 25℃ and a salinity of 28psu for 40 minutes to mature. After the eggs were completely rounded, they were washed three more times using a 500-mesh silk screen. After maturation, a small number of eggs were placed in a 50 mL glass beaker as the control group, and the remaining eggs were defined as the treatment group. The degree of maturation of the eggs and the presence of sperm contamination were examined under a microscope.

[0022] c. Selecting diploid paternal parents: Using diploid Fujian oysters from Putian City, Fujian Province as experimental parents, the top 10% of diploid Fujian oysters (wet weight ≥30.63g) were selected based on wet weight; the oysters were dissected and sexed under an optical microscope, and individuals with vigorous sperm motility and full gonads were retained as diploid paternal parents.

[0023] d. Obtaining diploid sperm: When more than 90% of the total number of triploid eggs in step a has matured, the sperm of a single oyster is obtained by the "dry peeling method" and activated in seawater for 3-5 minutes; after activation, a small amount of sperm is placed in a 50mL glass beaker and defined as the control group, and the remaining sperm is defined as the treatment group.

[0024] e. Experimental treatment: First, the sperm and eggs of the control group were mixed at a sperm-egg ratio of 8-10:1 and the timing was started. The time from the start of fertilization in the control group to the appearance of the first polar body of the fertilized egg was defined as A min (15 min). The time from the appearance of the first polar body of the fertilized egg in the control group to the appearance of the first polar body in 40%-50% of the fertilized eggs was defined as B min (23 min). After 10 min of fertilization in the control group, the treatment group was fertilized at a sperm-egg ratio of 8-10:1 and the timing was started. At the fertilization time of (A-3) min, that is, 12 min after fertilization, the treatment group was induced with 0.75 mg / L cytochalasin B for a duration of B min, that is, 23 min of treatment.

[0025] f. Larval and Juvenile Shellfish Development: After induction, wash away cytochalasin B from the fertilized egg fluid, place the fertilized eggs in a cement tank for incubation, cultivate the larvae using conventional methods, and transfer them to the sea for further development after attachment and metamorphosis.

[0026] g. Ploidy detection: Ploidy of the larvae obtained in step c was detected by flow cytometry and the tetraploidy rate of the larvae at 30 days of age was 71.28%; Ploidy of the juvenile shellfish cultivated in step d was detected by double ploidy of hemolymph and gills and the tetraploidy rate of the adult shellfish at 360 days of age was 91%.

[0027] The larvae obtained have a high hatching rate, high survival rate, and a high tetraploidy rate while maintaining stable ploidy. During the sea rearing stage, the juvenile shellfish grow rapidly, have a high survival rate, and maintain a high and stable tetraploidy rate. Example 2

[0028] a. Selection of triploid maternal parents: In mid-June 2022, triploid Fujian oysters from Zhangzhou City, Fujian Province were used as experimental parents. The top 10% of individuals (60 individuals) with beautiful shell shape and strong vitality were selected based on wet weight (wet weight ≥113.26g). They were dissected and sexed under an optical microscope. Individuals with ≥15 million eggs, uniform egg development, and deep yolk color were retained as triploid maternal parents (8 individuals).

[0029] b. Obtaining triploid eggs: Eggs from individual oysters were obtained using the "dry peeling method"; after being washed through a 500-mesh silk screen to remove impurities, the eggs were placed in seawater at a temperature of 25℃ and a salinity of 28 psu for 40 minutes to mature; after the eggs were completely rounded, they were washed three more times using a 500-mesh silk screen; after maturation, a small number of eggs were placed in a 50 mL glass beaker as the control group, and the remaining eggs were defined as the treatment group. The degree of maturation of the eggs and the presence of sperm contamination were examined under a microscope;

[0030] c. Selecting diploid paternal parents: Using diploid Fujian oysters from Zhangzhou City, Fujian Province as experimental parents, the top 10% of diploid Fujian oysters (wet weight ≥32.59g) were selected based on wet weight; the oysters were dissected and sexed under an optical microscope, and individuals with vigorous sperm motility and full gonads were retained as paternal parents.

[0031] d. Obtaining diploid sperm: When more than 90% of the total number of triploid eggs in step a has matured, sperm from a single oyster is obtained by the "dry peeling method" and activated in seawater for 3-5 minutes; after activation, a small amount of sperm is placed in a 50 mL glass beaker as the control group, and the remaining sperm is defined as the treatment group.

[0032] e. Experimental treatment: First, the sperm and eggs of the control group were mixed at a sperm-egg ratio of 8-10:1 and the timing was started. The time from the start of fertilization in the control group to the appearance of the first first polar body of the fertilized egg was defined as A min (8 min). The time from the appearance of the first first polar body of the fertilized egg in the control group to the appearance of the first polar body in 40%-50% of the fertilized eggs was defined as B min (15 min). After 10 min of fertilization in the control group, the treatment group was fertilized at a sperm-egg ratio of 8-10:1 and the timing was started. At the fertilization time (A-3) min, that is, 5 min after fertilization, the treatment group was induced with 0.75 mg / L cytochalasin B for a duration of B min, that is, a treatment duration of 15 min.

[0033] f. Larval and Juvenile Shellfish Development: After induction, wash away cytochalasin B from the fertilized egg fluid, place the fertilized eggs in a cement tank for incubation, cultivate the larvae using conventional methods, and transfer them to the sea for further development after attachment and metamorphosis.

[0034] g. Ploidy detection: Ploidy of the larvae obtained in step c was detected by flow cytometry and the tetraploidy rate of the larvae at 30 days of age was 73.64%; tetraploidy of the adults obtained in step d was detected by double ploidy of hemolymph and gills and the tetraploidy rate of the adults at 180 days of age was 93%.

[0035] The larvae obtained have a high hatching rate, high survival rate, and a high tetraploidy rate while maintaining stable ploidy. During the sea rearing stage, the juvenile shellfish grow rapidly, have a high survival rate, and maintain a high and stable tetraploidy rate.

Claims

1. A time-point quantitative induction method for tetraploid oysters in Fujian, characterized in that, Includes the following steps: a. Select triploid female parent and diploid male parent of Fujian oyster, and place the parents in the breeding pond for indoor synchronous maturation cultivation; b. After the parent oysters matured, well-developed parent oysters were selected by microscopic examination, and their eggs were dissected and removed. The eggs of individual oysters were placed in filtered seawater to mature. Appropriate eggs were used as the control group, and the remaining eggs were used as the treatment group. c. After maturation, the diploid oysters were dissected to obtain sperm from individual oysters, which were then activated before fertilization. The sperm were also divided into a control group and a treatment group. d. After sperm activation, the control group sperm and eggs were mixed in a one-to-one single-pair fertilization method and timed. The time from the start of fertilization of the control group fertilized egg to the appearance of the first first polar body was recorded as A min, and the time from the appearance of the first first polar body of the fertilized egg to the development of the first polar body of 50% of the fertilized eggs was recorded as B min. e. After fertilization of the control group for 10 minutes, the sperm and eggs of the treatment group were mixed and timed. When the fertilization time was (A-3) min, the treatment group was induced with 0.75 mg / L cytochalasin B for B min. After induction, the cytochalasin B in the fertilized egg fluid was washed away and the eggs were placed in a cement tank for incubation. f. Raise larvae using conventional methods, and after attachment metamorphosis, transfer them to the sea for further development.

2. The method for quantitative induction of tetraploid oysters at specific time points according to claim 1, characterized in that: In step (a), the parent parents are those with beautiful shells, good vitality and no damage. Weigh all the parent parents and select the top 10% of individuals by wet weight.

3. The method for quantitative induction of tetraploid oysters at specific time points according to claim 1, characterized in that: In step (b), individuals with uniform development, ≥15 million eggs, and dark yolk color are preferred as maternal parents.

4. The method for quantitative induction of tetraploid oysters at specific time points according to claim 1, characterized in that: In step (b), individuals with full gonads and vigorous sperm motility are selected as the fathers, and sperm and eggs are obtained using the "dry peeling method".

5. A method for quantitative induction of tetraploid oysters at specific time points according to claim 1, characterized in that: In steps (d) and (e), fertilization is performed using a one-to-one single-pair fertilization method. During this period, the temperature is controlled at 20-25℃, the salinity is controlled at 25 psu, and there are 5-8 sperm cells around each egg. The density of fertilized eggs is controlled at ≤15000 / mL. During hatching and larval rearing, the temperature is controlled at 25℃ and the salinity is controlled at 25-30 psu.

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