CN117121853A - Preparation method of scallop solitary haploid embryo - Google Patents
Preparation method of scallop solitary haploid embryo Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/50—Culture of aquatic animals of shellfish
- A01K61/54—Culture of aquatic animals of shellfish of bivalves, e.g. oysters or mussels
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
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Abstract
The invention provides a preparation method of a scallop solitary haploid embryo, which comprises the steps of using an ovum of a bay scallop and an ovum of a full female individual in a child generation cultivated by sperm fertilization of a purple scallop, and performing backcross fertilization with the sperm of the bay scallop to obtain the solitary haploid embryo; the screening of the total female individuals is to screen the total female individuals with the proportion of the nuclear genome of the bay scallop source to the nuclear genome of the purple scallop source in the genome lower than 1.5. The method for obtaining the scallop solitary haploid embryo provided by the invention is to use the ovum of the bay scallop and the ovum of the full female in the sperm fertilization cultivation sub-generation of the purple scallop to carry out backcross fertilization with the sperm of the bay scallop, has the advantages of natural fertilization, low cost, simple operation, cleanness, no damage, large obtaining amount and the like, is suitable for large-scale application, and provides a method support for the germplasm creation and the species protection of the scallop industry in China.
Description
Technical Field
The invention belongs to the technical field of shellfish genetic breeding in marine agriculture, and particularly relates to a preparation method of a scallop solitary haploid embryo.
Background
Androgenetic development (Androgenesis) refers to a special reproductive mode in which development is only genetically controlled by the male parent, either due to the disappearance of the nucleus or complete inactivation of the genetic material of the ovum. The reproductive mode relative to Gynogenesis (Gynogenesis) appears to be more specific and rare. The nuclear genetic material of male nuclear development is completely derived from male parents, usually only contains a set of male gamete chromosomes, is difficult to develop into adults due to the lack of maternal nuclear genome, but can often obtain a solitary haploid embryo. The solitary haploid embryo is not only an ideal material for constructing a haploid embryo stem cell line, but also an intermediate carrier for obtaining protection of homozygous sports parents and endangered species, and has important value in life science research.
The existing technology for male nuclear development generally obtains a haploid for male nuclear development by inactivating genetic materials of ova through ultraviolet rays, X rays and gamma rays, then taking part in fertilization, injecting mature sperm heads into enucleated oocytes, or removing female prokaryotes in fertilized ova, and then utilizing chromosome doubling technology (methods such as temperature shock or hydrostatic pressure treatment and the like) to inhibit first cleavage of the zygotes, so as to recover the diploidy.
Although the traditional androgenetic method lays a certain theoretical foundation for constructing the solitary haploid embryo, ultraviolet rays, X rays and gamma rays may cause irreversible damage to embryo development, and the processes of injecting mature sperm heads into enucleated oocytes and removing female prokaryotes in fertilized eggs need to use a plurality of chemical reagents/culture solutions and be provided with a micromanipulation platform, so that the method is high in price and complex in operation, and limits the large-scale acquisition and application of the solitary haploid embryo.
Scallop culture is an important component of the mariculture industry in China, the annual output of the scallops in China is 182.8-200.8 ten thousand tons in 2017-2021, and the scallops are stable in the first world. The shellfish solitary haploid embryo is not only a convenient material for forward and reverse genetic screening, but also an important object for gene editing, and has wide application prospect in aspects of shellfish functional genome research, important economic character excavation and the like. There is an urgent need in the shellfish industry for a method that is inexpensive, efficient, clean, and capable of obtaining a solitary haploid embryo on a large scale.
Disclosure of Invention
The invention aims to provide a preparation method of scallop solitary haploid embryos, which can solve the problems that the number of scallop solitary haploid embryos obtained at the current stage is small, the operation is complicated in the preparation process, and the fertilized eggs are damaged due to physical and chemical factor induction.
The preparation method of the scallop solitary haploid embryo provided by the invention comprises the steps of screening a full female individual in a child generation cultivated by fertilization of ova of a bay scallop and sperms of a purple scallop, and performing backcross fertilization on the ova of the full female individual obtained by screening and the sperms of the bay scallop to obtain the solitary haploid embryo; the screening of the total female individuals is to screen the total female individuals with the proportion of the nuclear genome of the bay scallop source to the nuclear genome of the purple scallop source in the genome lower than 1.5;
further, the screening of the whole female individuals is to screen the whole female individuals with the proportion of the nuclear genome of the bay scallop source to the nuclear genome of the purple scallop source in the genome of 1.04-1.18;
the ovum of the whole female is prepared by adopting a method of heating stimulation after drying in the shade to make the whole female spawn in seawater;
the shade drying is specifically described as an example, and the time is 30min;
the temperature rise is to raise the temperature by 3-6 ℃ from the shade drying temperature;
the breeding conditions of the obtained solitary haploid embryo method are as follows: the seawater temperature is 24+ -1deg.C, salinity is 25-35ppt, and culturing is carried out for 24-30h after fertilization.
Compared with the traditional preparation method of shellfish solitary haploid embryo, the invention has the following different points:
1) The method is different: in the prior art, the method mainly adopts ultraviolet rays, X rays and gamma rays to completely inactivate ovum genetic materials and then participate in fertilization, infuses the mature sperm heads into enucleated oocytes, or removes female and prokaryotes in fertilized eggs to obtain scallop solitary haploid embryos; the invention screens bay scallop (female) x purple scallop (female) first-generation total female individuals (sea purple scallop (female), diploid) (the proportion of the nuclear genome of bay scallop source to the nuclear genome of purple scallop source is lower than 1.5) for spawning, and naturally fertilizes with the sperm of bay scallop (diploid).
2) Operational feasibility is different: the traditional method is generally divided into two types, one is to select proper ultraviolet, X-ray and gamma-ray irradiation dose to completely inactivate ovum genetic material and participate in fertilization; the other is to pour the mature sperm head into the enucleated oocyte or remove the female prokaryote in the fertilized egg, the enucleation accuracy is required to be detected, and the operation is very complicated. The method of the invention is natural fertilization when the sea purple scallop (male) and the Xuewan scallop (female) are hybridized, and the operation is simple and efficient.
3) The obtained amount of the solitary haploid embryo is different: conventional methods are carried out on conventional plastic dishes (ovum density 2.0X10) 4 About 45% of the obtained solitary haploid embryos are cultivated in the culture medium of about 8 ten thousand eggs per mL, and finally about 3.6 ten thousand solitary haploid embryos are obtained; while the sea purple scallop (female) is multiplied by seaThe haploid rate of the female parent filial generation larvae (30 hours after fertilization) of the bay scallops is about 50%, the number of the male haploid embryos obtained by hybridization of one family is about 40-60 ten thousand grains, and the male haploid embryos are greatly reduced>10-fold) increase in the number of solitary haploid embryos obtained.
4) Based on traditional backcross breeding, the method for obtaining the shellfish parents is optimized: namely, female parents are screened according to the proportion of the nuclear genome of the source of the bay scallop and the source of the purple scallop in the whole female individuals in the filial generation (the ratio of the bay scallop to the purple scallop is <1.5 times).
The invention creates a new method for preparing scallop solitary haploid embryo through the technical links of spawning of sea purple scallop (female parent) and natural fertilization with sperm of bay scallop (diploid). The invention opens up a brand new method for obtaining the scallop solitary haploid embryo by utilizing the characteristic of haploid in the hybridized filial generation of sea purple scallop (female) X bay scallop (female), and being different from the traditional method for obtaining the scallop solitary haploid embryo by utilizing ultraviolet rays, X rays and gamma rays to completely inactivate ovum genetic materials, taking part in fertilization, injecting mature sperm heads into enucleated oocytes, removing female prokaryotes in fertilized eggs and the like. The method for obtaining the scallop solitary haploid embryo provided by the invention is to use the ovum of the bay scallop and the ovum of the full female in the sperm fertilization cultivation sub-generation of the purple scallop to carry out backcross fertilization with the sperm of the bay scallop, has the advantages of natural fertilization, low cost, simple operation, cleanness, no damage, large obtaining amount and the like, is suitable for large-scale application, and provides a method support for the germplasm creation and the species protection of the scallop industry in China.
Drawings
Fig. 1: ratio (A) and duty ratio (B) of two sets of nuclear genome in sea purple scallop (male) (diploid); the black rectangular frame (individuals 1-14) is used for screening total female parents for preparing the solitary haploid embryo;
fig. 2: a flow cytometry detection result diagram of bay scallop offspring and sea purple scallop (male) hybrid offspring larvae (30 h after fertilization); wherein A is a bay scallop (H1×H2) offspring larva (30H after fertilization); b is the filial generation larva of sea purple scallop (male parent) x bay scallop (female parent) hybrid (K4X H1); c is the filial generation larva of sea purple scallop (male parent) x bay scallop (female parent) hybrid (K5 XH 1); d is the filial generation larva of sea purple scallop (male parent) x bay scallop (female parent) hybrid (K9X H1); 2n represents that the bay scallop offspring larva is diploid, n ',2n ',3n ' respectively represent that the sea purple scallop (male parent) ×bay scallop (female parent) hybrid offspring larva is haploid, diploid and triploid;
fig. 3: results of the detection of bay scallop and sea purple scallop (male) adult gill silk flow cytometry are shown in the figure, wherein: A. bay scallop (H1) adult gill wires; B. sea purple scallop (male) (K4) adult gill wires; C. sea purple scallop (male) (K5) adult gill wires; D. sea purple scallop (male) (K9) adult gill wires; 2n represents that the adult Argopecten irradians is diploid, and 2n' represents that the adult Argopecten irradians is diploid
Fig. 4: microscopic observation of fertilized eggs (male haploids) of the filial generation of the sea purple scallop (male) x bay scallop (female) is carried out (1000×), wherein the optical microscope (A, B, C) and the fluorescence microscope are used for observing the fertilized eggs (male haploids) of the filial generation of the sea purple scallop (female) x bay scallop (male); A. d is derived from K4XH 1; B. e is derived from K5×H2; C. f is derived from K9×H2; the dye liquor used for fluorescence microscopic observation is Hoechst33258, cytoplasm is dyed blue, and chromosome is dyed bright blue; SN stands for seminal nuclei, PB stands for polar bodies.
Detailed Description
The bay scallop and the purple scallop are hermaphrodite, the gonad is limited to the abdomen, the male area (testis) is positioned at the outer periphery of the abdomen, and the mature scallop and the purple scallop are milky white; the female (ovary) is located inside the male, and exhibits pink/orange color when mature; normally, the surface of the gonad part is provided with a layer of black film, and in the process of gradually maturing the gonad, the black film gradually fades, so that the male region and the female region can be obviously distinguished. When gonads begin to develop, a method of drying in the shade and heating stimulation is adopted to stimulate parent Bei Chanluan/seminal emission: drying parent shellfish in the shade for 30-40 min before parturition, and then placing in seawater with the temperature raised by 3-6 ℃. The parent scallops mainly discharge white smoke sperms within the first 20-30 min; then ovulation is carried out successively, spawning can be carried out for 4-5 times within 15-20 min, and mature ova in the female area are exhausted. The ovum/fertilized ovum can be filtered out by 500 mesh (aperture 30 μm) bolting silk to obtain parent sperm.
The method of the invention is to use the ovum of the bay scallop and the ovum of the full female in the child generation cultivated by the sperm fertilization of the purple scallop, and then to carry out backcross fertilization with the sperm of the bay scallop to obtain the solitary haploid embryo.
The identification method of the total female individuals (sea purple scallop () (diploid)) in the child generation cultivated by fertilization of the ova of bay scallops and the sperm of purple scallops is as follows:
female individuals with pink/orange gonads in the child generation of the bay scallop (male) x purple scallop (female) are selected, a small amount of tissue samples are taken in a living state, and the ploidy of the female individuals is determined by a flow cytometer (CytoFLEX), and the specific operation is as follows: 2-3 gills were picked up with sterilized forceps, thoroughly dissociated by repeated blowing with a sterilized syringe containing 1mL of 1 XPBS, filtered through a 500 mesh (30 μm pore size) sieve, and the single cell suspension after filtration was stained with DAPI (1.5. Mu.g/mL, 50. Mu.L) in the absence of light for 30min. The cells treated in the above manner can be subjected to ploidy measurement by a flow cytometer (CytoFLEX) (control group is a common diploid bay scallop).
In order to increase the obtaining amount of the solitary haploid embryo, the sea purple scallop (female parent) is screened, and the specific method is as follows:
extracting DNA of offspring female parents identified by flow cytometry ploidy through a non-lethal method, constructing a resequencing library and performing high-throughput sequencing through an Illunima Hiseq sequencing platform; after sequencing data are obtained, statistics is carried out on the ratios of the ready-to-use nuclear genomes of bay scallops and purple scallops in the genome of each of the filial generation total females to be screened, and two sets of filial generation total females with the ready ratios of less than 1.5 times of the nuclear genomes of bay scallops (ready ratio < 60%) and purple scallops (ready ratio > 40%) are screened out to serve as female parents of subsequent backcross.
In the artificial accumulated temperature ripening season (1-3 months) of the screened sea purple scallop (female parent), selecting individuals with the gonad types of total females, regular gonad morphology and full development and pink/orange red color as female parents; the screened whole female individuals lay eggs in the sea water by a method of drying in the shade (30 min) and heating (heating to 3-6 ℃) stimulation.
Selecting a diploid bay scallop which is synchronously accumulated with a full female parent and is mature-promoted, wherein the gonad type of the diploid bay scallop is hermaphrodite, the gonad is regular in morphology and full in development, the color of a female region is pink/orange, and the color of a male region is milky; the bay scallop individual can lay eggs and discharge sperms normally in the sea water by using the same method of drying in the shade and heating stimulation as the method for obtaining the full female parent ovum. Filtering ovum of bay scallop with 500 mesh (aperture 30 μm) sieve silk to obtain sperm;
mixing the filtered sperms with the eggs of the whole female, controlling the density of 3-5 sperms around each egg to be 50-80/mL; after fertilization, the fertilized eggs are hatched in normal seawater to obtain a solitary haploid embryo.
Ploidy detection is carried out on the filial generation larvae (30 h after fertilization) of the bay scallop filial generation and the sea purple scallop (male parent) of the Xuewan scallop; ploidy detection is carried out on adult gill wires of bay scallops and sea purple scallops; microscopic observation (1000×) was made on the development process of fertilized eggs of the hybridized filial generations of sea purple scallop (male) x bay scallop (female).
In the method of the present invention, conventional methods may be employed for stimulating spawning/refining of bay scallops, and methods of fertilization and cultivation, and are not limited to the specific description of the examples.
The method of the present invention does not use ultraviolet rays, X rays and gamma rays to irradiate the ovum genetic material to completely inactivate and participate in fertilization, inject the mature sperm head into the enucleated oocyte, or remove the female prokaryote in the fertilized ovum to treat the oocyte/fertilized ovum.
The present invention will be described below with reference to examples and drawings.
Example 1: preparation of scallop solitary haploid embryo
1) Obtaining identified and screened ova of all female individuals (diploid) of the first generation of Argopecten irradias (female) of Argopecten irradias (male)
In the early 4 th ten days of 2022, in the first generation group of bay scallop (male) x purple scallop (female) of limited company of tobacco desk and sea in Shandong province, selecting individuals with full gonad types, regular gonad morphology and pink/orange red color as female parents (52 sea purple scallops), identifying that the 52 sea purple hybrid offspring individuals are diploid by using a flow cytometry, randomly selecting 2-3 gills of 26 individuals by using a non-lethal method, namely using sterilized tweezers, extracting genome DNA of the 2-3 gills, constructing 26 heavy sequencing libraries, performing high-throughput sequencing by using an Illima Hiseq sequencing platform, and counting the proportion of the bay scallops and the nuclear genome reads of the purple scallops in the genome of each of the hybrid offspring to be screened, wherein the proportion of the nuclear genome of the bay scallops and the purple scallops in the whole female individuals to be screened is 1.04 times that of the genome of the 14 sea scallops and the nuclear genome of the purple scallops in the genome is 1.04 times that of the whole female scallops and 1.5 times that of the genome of the whole sea scallops in the genome to be screened is 1.5 times that of the genome of the whole female scallops and the nuclear genome of the whole scallops to be screened, and the nuclear genome of the whole scallops to be 1.5 times that the whole scallops to be screened.
The 14 selected filial generation total female individuals (the ratio of the two sets of nuclear genome reads of the bay scallop and the purple scallop is 1.04-1.18) are used as the subsequent backcross female parent (figure 1). Heating (heating to 3-6deg.C) by drying in the shade (30 min) to stimulate, and allowing the female to stand in seawater (spawning barrel, 2.5X10) -3 m 3 ) Normal spawning in the middle. The ovum is full in egg quality and round in shape, and the ovum is not fertilized by microscopic examination.
2) Obtaining sperm of Bay scallop (diploid)
14 bay scallops (diploids) which are synchronously accumulated with female parents and promote maturation are selected, the gonad types of the bay scallops are hermaphrodite, the gonad forms are regular and full in development, the female zone is pink/orange, and the male zone is milky; the bay scallop individual is subjected to sea water (semen discharging barrel, 2.5X10) at 24 ℃ by the same method of drying in the shade and heating stimulation as in step 1) -3 m 3 ) Normal spawning and semen discharge. Filtering ovum/fertilized ovum of the above bay scallop with 500 mesh bolting silk (aperture 30 μm) to obtain sperm; and (3) performing microscopic examination again, ensuring that more than or equal to 90 percent of sperms move and no pollution is caused to ova/fertilized ova of the bay scallops.
3) Fertilization
Adding sperms obtained by filtering from the semen discharging barrel into a spawning barrel, and controlling the density of 3-5 sperms around each ovum to be 53+/-3/mL; after fertilization, the fertilized eggs are hatched in normal seawater, the temperature of the seawater is 24.2 ℃ and the salinity is 27.2ppt, and the scallop solitary haploid embryos can be obtained after 24-30h of culture after fertilization.
Detecting parent ploidy: ploidy test was performed on adult gills of Argopecten irradias (male) (K4, K5, K9) and Argopecten irradias (H1) in steps 1) and 2) at month 4 of 2022, resulting in diploid (FIG. 3).
Detecting offspring ploidy: in 2022, for month 4, ploidy test was performed on the larvae of the filial generation (30H after fertilization) (N > 1000) of the seaviolet scallop (female) ×gulf scallop (male) (k4×h1, k5×h1, k9×h1) in step 3), and it was found that the haploids of the two families of k4×h1 and k5×h1 occupy the main peak compared with the larvae of the filial generation (30H after fertilization) of the seaviolet scallop (h1×h1) (diploid) (fig. 2).
Table 1: K4×H1, K5×H1, K9×H1 maternal genome composition and solitary haploid embryo acquisition scale
After statistics, it is found that the proportion of two sets of nuclear genome ready of the sea bay scallop and the purple scallop in the female parent of sea bay scallop and the purple scallop of K4 XH 1 and K9 XH 1 is 1.10,1.06 and 1.18 respectively, the number of the solitary haploid embryos in the filial generation larvae of the corresponding families is 43.25 ten thousand, 60.43 ten thousand and 12.51 ten thousand respectively, which is far higher than the obtained amount of 3-5 ten thousand in the traditional method, and the more the proportion of the nuclear genome ready of the sea bay scallop and the purple scallop of the female parent is close to 1, the higher the number of the solitary haploid embryos in the backcross family is, and the Pearson correlation coefficient is as high as 0.987. The result shows that the method can obtain a large number of solitary haploid embryos.
Detecting a haploid source: microscopic observation was carried out on fertilized eggs of the hybridized filial generations of sea purple scallop (male parent) x bay scallop (female parent) obtained in the step 3) at 6 months of 2022, so that a solitary haploid (sperm in-egg and primary oocyte chromosome completely discharged) was found (fig. 4), and the authenticity of the solitary haploid embryo was proved from the cytology level.
In summary, the invention opens up a brand-new method for obtaining the solitary haploid embryo, namely, the method uses the ovum of the bay scallop and the ovum of the full female individual in the child generation cultivated by the sperm fertilization of the purple scallop, and then carries out backcross fertilization with the sperm of the bay scallop to obtain the solitary haploid embryo, and has the advantages of low price, simple operation, cleanness, no damage, large obtaining amount, suitability for large-scale application and the like, and provides a method support for the germplasm creation and species protection of the scallop industry in China.
Claims (6)
1. A preparation method of a scallop solitary haploid embryo is characterized by screening full female individuals in a child generation cultivated by fertilization of ova of bay scallops and sperms of purple scallops, and then backcrossing and fertilizing the ova of the full female individuals obtained by screening with the sperms of the bay scallops to obtain the solitary haploid embryo; the screening of the total female individuals is to screen the total female individuals with the proportion of the nuclear genome of the bay scallop source to the nuclear genome of the purple scallop source in the genome lower than 1.5.
2. The method of claim 1, wherein the selected total female is a total female having a ratio of a nuclear genome derived from bay scallop to a nuclear genome derived from purple scallop in the selected genome of 1.04 to 1.18.
3. The method of claim 1, wherein the egg of the whole female is laid in sea water by heating stimulation after drying in the shade.
4. A method according to claim 3, wherein the drying in the shade takes about 30 minutes.
5. A method according to claim 3, wherein the temperature is raised from the drying temperature to a temperature of 3-6 ℃.
6. The method according to claim 1, wherein the cultivation conditions are as follows: the seawater temperature is 24+ -1deg.C, salinity is 25-35ppt, and culturing is carried out for 24-30h after fertilization.
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| CN104160998A (en) * | 2014-08-27 | 2014-11-26 | 青岛海弘达生物科技有限公司 | Construction method and application of male sterility line of hybrid scallops of purple scallops and Argopecten irradians |
| CN106498086A (en) * | 2016-12-30 | 2017-03-15 | 青岛农业大学 | Purple scallop and bay scallop and its authentication method in the maternal source of backcross progeny |
| CN115413613A (en) * | 2022-09-19 | 2022-12-02 | 中国海洋大学 | Preparation method of scallop androgenesis haploid embryo |
| CN115669615A (en) * | 2022-06-08 | 2023-02-03 | 中国海洋大学 | Method for preparing allotriploid scallop |
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| CN104160998A (en) * | 2014-08-27 | 2014-11-26 | 青岛海弘达生物科技有限公司 | Construction method and application of male sterility line of hybrid scallops of purple scallops and Argopecten irradians |
| CN106498086A (en) * | 2016-12-30 | 2017-03-15 | 青岛农业大学 | Purple scallop and bay scallop and its authentication method in the maternal source of backcross progeny |
| CN115669615A (en) * | 2022-06-08 | 2023-02-03 | 中国海洋大学 | Method for preparing allotriploid scallop |
| CN115413613A (en) * | 2022-09-19 | 2022-12-02 | 中国海洋大学 | Preparation method of scallop androgenesis haploid embryo |
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