CN112931323A

CN112931323A - Induction method of high heterozygosity tetraploid of novel variety Haoda No. 3 of crassostrea gigas

Info

Publication number: CN112931323A
Application number: CN202110246934.3A
Authority: CN
Inventors: 李琪; 李永国
Original assignee: Ocean University of China
Current assignee: Ocean University of China
Priority date: 2021-03-05
Filing date: 2021-03-05
Publication date: 2021-06-11

Abstract

The invention provides an induction method of a high heterozygosity tetraploid of a new variety of crassostrea gigas, Haanda No. 3. The method comprises the following steps: the female triploid crassostrea gigas 'Haoda No. 3' obtained by inhibiting the first polar body of fertilized eggs of the diploid crassostrea gigas 'Haoda No. 3' is taken as a female parent; taking diploid 'Hailada No. 3' male as a male parent; after the female parent and the male parent are artificially matured and matured, the ovum is placed in seawater for maturation, and the maturation is completed when the ovum is round through microscopic examination; inseminating the matured ovum and sperm, mixing the sperm and the ovum, timing, treating the fertilized ovum with cytochalasin B to inhibit the first polar body from discharging, collecting the fertilized ovum, and soaking; transferring the fertilized eggs into a culture container for hatching and larva culture. The triploid female parent shellfish adopted by the invention is obtained by inhibiting the discharge of the first polar body of the fertilized egg of the 'Haima No. 3' of the diploid crassostrea gigas, and the tetraploid obtained by the invention also has higher growth advantage due to higher gene heterozygosity.

Description

Induction method of high heterozygosity tetraploid of novel variety Haoda No. 3 of crassostrea gigas

Technical Field

The invention belongs to the technical field of shellfish breeding, relates to a crassostrea gigas tetraploid induction method, and particularly relates to an induction method of a crassostrea gigas 'Haoda No. 3' new variety high heterozygosity tetraploid with the advantages of pure black shell color and rapid growth.

Background

The crassostrea gigas is also called pacific oyster, is a main oyster cultivation variety in the north of China, and becomes the economic shellfish which is the widest cultivation range in the world due to the characteristics of strong environmental adaptability, high growth speed, rich nutrition and the like. For a long time, because the egg laying amount of the crassostrea gigas is large, the parents used for producing the seedlings are limited and are all from breeding groups which are not bred, the germplasm degeneration phenomena of the breeding groups, such as reduction of genetic diversity, individual miniaturization and the like, occur, and the healthy and continuous development of the oyster breeding industry is restricted. In recent years, genetic improvement work of crassostrea gigas is carried out successively in China, America and other countries, new species of high-yield and high-quality oysters are cultivated, and important contribution is made to yield increase and income increase of the oyster cultivation industry.

The crassostrea gigas Hayada No. 3 (variety registration number: GS-01-007-2018) is prepared by adopting a mixed breeding technology combining family breeding and group breeding and carrying out continuous 6-generation breeding by taking the shell black color and the growth speed as target characters. Compared with the crassostrea gigas which is not bred, the crassostrea gigas has obvious growth advantages, and meanwhile, as the left and right shells and the mantle are black and transparent in black, the crassostrea gigas is commonly called as 'black golden crassostrea gigas'. The breeding of the crassostrea gigas 'Hailada No. 3' increases the quantity of improved varieties of oysters in China, promotes the continuous and healthy development of the oyster industry in China, meets the consumption requirements of consumers on high quality and high grade at the same time, and has important significance for opening up the high-end market of oysters and increasing the diversified development of the oyster industry. However, since the crassostrea gigas "haida No. 3" is diploid, it enters the summer every year and starts to enter the breeding period, and in order to meet the energy demand of breeding, glycogen content in the body is consumed in large amount, and the weight of soft body is lost by more than 60% after spawning, which is difficult to eat, thus leading to the failure of market supply in summer at least 3 months every year.

Researches show that the gonadal development retardation of the diploid oysters solves the problem that the diploid oysters are difficult to supply to markets due to poor taste and emaciation of soft bodies in summer, and provides guarantee for the oyster consumption markets all the year round. Meanwhile, the gonad of the triploid oyster is low in development degree in summer, so that more energy can be used for growth, and the growth speed of the triploid oyster is improved. Triploid oysters have already been industrialized in many countries and have a wide potential for development. The obtaining ways of the triploid oysters are mainly two, firstly, the triploid is induced directly through the sperm and egg of the diploid oyster, but the triploid rate is unstable and is difficult to reach 100 percent, and the method is difficult to be applied on a large scale; and secondly, triploid oysters and diploid oysters are hybridized to obtain triploid, 100% of triploid offspring can be obtained theoretically by the method, and the tetraploid population can be expanded by self-reproduction, so that the commercial production of the triploid is guaranteed. Therefore, the method realizes the triploid industrialization of the new breeding species of the oysters and is the most ideal way to obtain the tetraploid of the new breeding species. At present, there are two main methods for obtaining tetraploid oysters, and the most common method is to obtain triploid through fertilization of sperm and egg of diploid oyster and then fertilization of sperm of male diploid ostrea gigas and egg of female triploid, and induce tetraploid production through inhibition of first polar body discharge of fertilized egg. The research shows that the tetraploid obtained by the method has slow growth speed and obvious attenuation phenomenon of the tetraploid population chromosome, and when the tetraploid is used for tetraploid production, the later generation triploid gonad can develop to a certain degree. The other method is to directly induce and generate tetraploid oysters by utilizing sperms and ova of diploid oysters, the tetraploid obtained by the method is higher in sterility of triploid offspring generated by crossing the diploid oysters, theoretically, the tetraploid has higher growth advantages, but the method also has the problems of relatively lower tetraploid induction rate, low survival rate and the like. The triploid oyster is obtained by inhibiting the fertilized egg second polar body of the diploid oyster in the most common way, and the triploid used for inducing the tetraploid oyster is obtained by inhibiting the discharge of the fertilized egg second polar body of the diploid basically, so that the tetraploid has the problem of slow growth speed compared with the diploid and the triploid.

Therefore, there is a need to develop a method for inducing high-quality tetraploid suitable for crassostrea gigas "haida No. 3".

Disclosure of Invention

The invention aims to provide an induction method of high heterozygosity tetraploid of a new variety of Crassostrea gigas Hadama No. 3, which is used for making up the defects of the prior art.

In order to achieve the purpose, the invention adopts the following specific technical scheme:

a method for inducing high heterozygosity tetraploid of a new variety of crassostrea gigas, Haoda No. 3, comprises the following steps:

(1) selecting a female parent and a male parent: the female triploid crassostrea gigas 'Haoda No. 3' obtained by inhibiting the first polar body of fertilized eggs of the diploid crassostrea gigas 'Haoda No. 3' is taken as a female parent; taking diploid 'Hailada No. 3' male as a male parent;

(2) maturation of the ovum: after the female parent and the male parent are artificially matured and matured, the ovum is placed in seawater for maturation, and the maturation is completed when the ovum is round through microscopic examination;

(3) inseminating the matured ovum and sperm, timing after the sperm and the ovum are mixed, treating the fertilized ovum with cytochalasin B to inhibit the first polar body from discharging, collecting the fertilized ovum after the treatment is finished, and transferring the fertilized ovum into low-concentration ethanol solution for soaking;

(4) and finally transferring the fertilized eggs to a culture container for hatching and larva culture.

Further, in the step (1), the selection of the female parent is further limited to selecting a triploid crassostrea gigas 'Hailao No. 3' female with a large number of ova and an oval shape as the female parent after the dissection microscopic examination.

Further, in the step (1), the diploid crassostrea gigas 'Haoda No. 3' is a new subculture variety, and the left and right shells and the mantle are pure black; the diploid crassostrea gigas 'Hailao No. 3' is obtained by inhibiting the first polar body of fertilized eggs of the diploid crassostrea gigas 'Hailao No. 3' from being discharged, and the left and right shells and the mantle are pure black.

Further, in the step (2), the ovum is placed in filtered seawater at the temperature of 24-25 ℃ for curing for 40-60 minutes, and the curing is completed when the ovum is round through microscopic examination. The number of the eggs is at least 100 ten thousand, and the eggs are round or nearly round before insemination.

Further, in the step (3), inseminating the matured ovum and sperm in sand-filtered seawater at 24-25 ℃; timing after the sperm and the ovum are mixed, inseminating for 8-10 minutes, and treating the fertilized ovum for 15-20 minutes by 0.5mg of cytochalasin B to inhibit the first polar body from discharging; the beginning time of the cytochalasin B is 8-10 minutes after insemination, and the duration time of the cytochalasin B treatment is 15-20 minutes; collecting fertilized eggs, namely using 500-mesh bolting silk, separating the fertilized eggs from impurities and removing an inducer; the concentration of the ethanol solution is 0.2%, and the soaking time is 40-60 minutes.

Further, the water temperature is controlled to be 24-25 ℃ in the curing and treating processes in the whole steps (2) and (3).

Further, in the step (4), the larva breeding density is controlled not to be higher than 0.2/mL during the larva breeding process.

The method further comprises the following detection steps: detecting the ploidy of the induced tetraploid by using a flow cytometer, and separating out the surviving subculture breeding tetraploid crassostrea gigas 'Haoda No. 3' juvenile mollusk by adopting a non-lethal ploidy detection method.

The invention has the advantages and technical effects that:

the invention uses triploid crassostrea gigas 'Hailao No. 3' obtained by inhibiting the first polar body of fertilized eggs of 'Hailao No. 3' of diploid crassostrea gigas as a female parent and uses diploid crassostrea gigas 'Hailao No. 3' as a male parent to carry out artificial insemination, and obtains the tetraploid of a new variety of the crassostressostrea gigas 'Hailao No. 3' by a method of inhibiting the first polar body from discharging and inducing the high heterozygosity tetraploid of the new variety of the crassostressostrea gigas.

According to the invention, tetraploid induction is carried out by inhibiting the triploid obtained from the first polar body of the diploid oosperm, so that the gene heterozygosity of the tetraploid is improved, the problem of slow growth of the tetraploid is improved, and the problem of attenuation of the tetraploid chromosome of the oyster is solved. The triploid female parent shellfish is obtained by inhibiting the discharge of the first polar body of the fertilized egg of the 'Haoda No. 3' of the diploid crassostrea gigas, and the tetraploid obtained by the triploid female parent shellfish has higher growth advantage due to higher gene heterozygosity.

Detailed Description

The invention is further illustrated and described below by means of specific examples.

Example 1:

(1) selecting a diploid and a triploid crassostrea gigas of which the 2-year age is 2, the height of the shells is 8-12cm, and the left and right shells are pure black, and transferring the diploid and the triploid crassostrea gigas of the Haemao No. 3 from a culture sea area to a seedling raising workshop for gonad maturity promotion in advance by 3 months.

(2) Carrying out non-lethal ploidy detection on the Crassostrea gigas Hayao No. 3 by using a flow cytometer, then using the whole triploid, selecting an individual with large egg amount (at least 100 ten thousand grains) and better egg shape as a female parent after anatomical microscopy, dissecting and taking the egg, filtering the egg by using a 500-mesh silk screen, removing components such as tissue fragments, tissue fluid and the like, and then adding the egg into sand-filtered seawater at 24-25 ℃ for curing.

(3) The maturation of the eggs was examined microscopically for the presence of sperm contamination and the fully matured eggs from 12 triploid females were then inseminated with vigorous sperm from 6 diploid males in sand filtered seawater.

(4) Timing is started after sperm and egg are mixed, and after insemination is carried out for 8-10 minutes, cytochalasin B of 0.5mg/L is used for treating for 15 minutes, so that the first polar body of fertilized eggs is prevented from being discharged.

(5) After the treatment is finished, collecting fertilized eggs by using 500-mesh bolting silk, immediately fully washing the fertilized eggs by using filtered seawater, then transferring the fertilized eggs into 0.2% ethanol solution for soaking for 40 minutes, and finally filtering to remove residual cytochalasin B on the surfaces of the fertilized eggs. The water temperature is controlled to be 24-25 ℃ in the whole curing and treating process. The larvae are cultivated in a cement pond of a seedling raising workshop, and the cultivation density of the larvae is controlled to be not higher than 0.2 per mL.

The tetraploid rate and survival rate of the larval stage and the tetraploid rate of the juvenile stage were examined.

1. At 24h of fertilization, ploidy detection was performed using a flow cytometer. The detection result shows that the quadruple rate of the new variety of the crassostrea gigas, Hailao No. 3, is up to 42.6 percent; the survival rate of the larva is 22.2 percent at most.

2. When young shellfish grow to 180 days, the young shellfish is subjected to non-lethal ploidy detection by using a flow cytometer. The result shows that the rate of the bigelite of the crassostrea gigas, i.e. the Haima No. 3, is up to 25.0 percent.

Compared with the prior art: different from the problems of poor growth of tetraploid oysters compared with diploids and triploids in the existing report. At 180 days, the crassostrea gigas 'sea size No. 3' tetraploid obtained in this example has no significant growth difference in growth rate compared with the contemporaneous diploid and triploid crassostrea gigas 'sea size No. 3', which indicates that the tetraploid obtained in the present invention has significantly improved growth performance.

Example 2

(1) selecting diploid and triploid ostrea "Haoda No. 3" with 2 years old, 8-12cm high shell and pure black left and right shells as male and female parent shellfish respectively.

(2) Carrying out non-lethal ploidy detection on the Crassostrea gigas Hayao No. 3 by using a flow cytometer, then using the whole triploid female, selecting an individual with large egg amount (at least 100 ten thousand grains) and better egg shape as a female parent after anatomical microscopy, dissecting and taking the egg, filtering the egg by using a 500-mesh sieve silk, removing components such as tissue fragments, tissue fluid and the like, and then adding the egg into sand-filtered seawater at 24-25 ℃ for curing.

(3) The maturation of the eggs was examined microscopically for the presence of sperm contamination and the fully matured eggs from 14 triploid females were then inseminated with vigorous sperm from 6 diploid males in sand filtered seawater.

(4) Timing is started after sperm and egg are mixed, and after insemination is carried out for 8-10 minutes, cytochalasin B with the concentration of 0.5mg/L is used for treating for 20 minutes, so that the first polar body of fertilized eggs is prevented from being discharged.

1. At 24h of fertilization, ploidy detection was performed using a flow cytometer. The detection result shows that the quadruple rate of the new variety of the crassostrea gigas, Hailao No. 3, is 32.6 percent at most; the survival rate of the larva is 20.3 percent at most.

2. When young shellfish grow to 180 days, the young shellfish is subjected to non-lethal ploidy detection by using a flow cytometer. The result shows that the rate of the tetraploid of the crassostrea gigas 'Haoda No. 3' is 16.7 percent at most; the obtained ostrea crassipes 'sea size 3' tetraploid is detected to have no obvious growth difference in growth speed compared with the homoperiod diploid and triploid ostrea crassipes 'sea size 3'.

The practices in the above example 1 and example 2 prove that the tetraploid induction method provided by the invention can obtain the high-heterozygosity crassostrea gigas 'Haoda No. 3' tetraploid with stable induction rate, and compared with the growth disadvantage of the tetraploid oyster in the existing report, the growth performance of the tetraploid is obviously improved.

Claims

1. The method for inducing the high heterozygosity tetraploid of the new variety Haoda No. 3 of the crassostrea gigas is characterized by comprising the following steps of:

selecting a female parent and a male parent: the female triploid crassostrea gigas 'Haoda No. 3' obtained by inhibiting the first polar body of fertilized eggs of the diploid crassostrea gigas 'Haoda No. 3' is taken as a female parent; taking diploid 'Hailada No. 3' male as a male parent;

maturation of the ovum: after the female parent and the male parent are artificially matured and matured, the ovum is placed in seawater for maturation, and the maturation is completed when the ovum is round through microscopic examination;

inseminating the matured ovum and sperm, timing after the sperm and the ovum are mixed, treating the fertilized ovum with cytochalasin B to inhibit the first polar body from discharging, collecting the fertilized ovum after the treatment is finished, and transferring the fertilized ovum into low-concentration ethanol solution for soaking;

and finally transferring the fertilized eggs to a culture container for hatching and larva culture.

2. The method of inducing, according to claim 1, wherein in step (1), the selection of the female parent is further limited to selecting, as the female parent, a triploid crassostrea gigas "Hailao No. 3" female with a large number of ova and an oval shape after dissecting and microscopic examination.

3. The induction method according to claim 1, wherein in the step (1), the diploid crassostrea gigas "Hailao No. 3" is a new variety for subculture breeding, and the left and right shells and the mantle are pure black; the diploid crassostrea gigas 'Hailao No. 3' is obtained by inhibiting the first polar body of fertilized eggs of the diploid crassostrea gigas 'Hailao No. 3' from being discharged, and the left and right shells and the mantle are pure black.

4. The induction method according to claim 1, wherein in the step (2), the ova are aged in filtered seawater at 24-25 ℃ for 40-60 minutes, and the aging is completed when the ova are round under microscopic examination; the number of the eggs is at least 100 ten thousand, and the eggs are round or nearly round before insemination.

5. The induction method according to claim 1, wherein in the step (3), the matured ovum and sperm are inseminated in sand filtered seawater at 24-25 ℃; timing after the sperm and the ovum are mixed, inseminating for 8-10 minutes, and treating the fertilized ovum for 15-20 minutes by 0.5mg of cytochalasin B to inhibit the first polar body from discharging; the beginning time of the cytochalasin B is 8-10 minutes after insemination, and the duration time of the cytochalasin B treatment is 15-20 minutes; collecting fertilized eggs, namely using 500-mesh bolting silk, separating the fertilized eggs from impurities and removing an inducer; the concentration of the ethanol solution is 0.2%, and the soaking time is 40-60 minutes.

6. The induction method according to claim 1, wherein the water temperature is controlled to be 24-25 ℃ during the whole curing and treating process in the steps (2) and (3).

7. The induction method according to claim 1, wherein in the step (4), the larva breeding density is controlled not to be higher than 0.2/mL during the larva breeding process.

8. The induction method according to claim 1, further comprising the step of: detecting the ploidy of the induced tetraploid by using a flow cytometer, and separating out the surviving subculture breeding tetraploid crassostrea gigas 'Haoda No. 3' juvenile mollusk by adopting a non-lethal ploidy detection method.