Artificial breeding method of epinephelus fuscoguttatus and pink pterus coerulescens hybrid

Abstract

The invention discloses an artificial breeding method of epinephelus fuscoguttatus and pink pterus coerulescens hybrid species, which comprises the following steps: screening healthy epinephelus fuscoguttatus as a female parent, screening healthy pink pterus coerulescens as a male parent, and carrying out enhanced breeding on the male parent and the female parent, the gonad maturation of the parent is promoted; the method comprises the following steps: collecting egg liquid of a female parent and semen of a male parent, inseminating to obtain a fertilized egg, incubating the fertilized egg to obtain a hybrid spot, performing authenticity identification on the hybrid spot by using one or more of sequences SEQ ID NO.1-SEQ ID NO.8, and determining that the hybrid spot inherits genetic information of the female parent and the male parent. The technical blank of crossbreeding of the pink pink bass as the parent grouper is filled, so that the hybrid obtained by hybridizing the epinephelus fuscoguttatus and the pink pink bass is reliable in authenticity, and inherits genetic information of male and female parents on the molecular level.

Description

Technical Field

[0001] This invention relates to the fields of aquaculture and molecular biology, specifically to a method for the artificial breeding of a hybrid of brown-spotted grouper and blue-spotted sea bass. Background Technology

[0002] Grouper (Epinephelus spp.) is a diverse species and an important fish resource for aquaculture in tropical and subtropical marine environments in my country. Interspecific hybridization holds promise for cultivating new varieties with faster growth rates, stronger disease resistance, and superior body shape.

[0003] The Blue-spotted Nine-spined Bass (Cephalopholis miniata), belonging to the order Perciformes, family Serranidae, and genus Cephalophorus, is also known as the Strawberry Spotted Bass, Red Bass, and Red Checkered Bass. It is distributed in tropical waters of the Pacific and Indian Oceans. It typically inhabits rocky crevices or coral reef slopes at depths of 2–150 m where corals are abundant. Its body length is generally 200–300 mm, with larger individuals reaching 400 mm. It has extremely vibrant colors, with an elongated, laterally compressed body, reddish in color but with numerous evenly distributed blue spots. It feeds on small fish and crustaceans.

[0004] The brown-spotted grouper (Epinephelus fuscoguttatus), belonging to the order Perciformes, family Serranidae, and genus Epinephelus, is also known as the tiger grouper or brown-spotted grouper. It is distributed in the Red Sea, the Indian Ocean off the east coast of Africa, the Pacific Polynesia, north to the Ryukyu Islands, and south to Australia and the Penghu Islands, among other waters. It inhabits waters 1-60 meters deep and can reach a length of 120 cm. A warm-water, nearshore, and coral reef fish, it lives in clear coral reefs and lagoons. Carnivorous, it feeds on fish, cephalopods, and crustaceans, and is used as an edible, farmed, and ornamental fish. The brown-spotted grouper is a voracious feeder and grows rapidly, making it widely used in aquaculture.

[0005] Currently, some artificial hybridization breeding methods for grouper have been publicly disclosed, and corresponding breeding procedures have been established. However, there are no publicly available reports on artificial breeding and hybridization of grouper using the blue-spotted nine-spined perch as a parent. Therefore, there is still a significant technological gap in the research on grouper hybridization breeding using the blue-spotted nine-spined perch as a parent. Existing technologies cannot effectively solve the relevant problems of utilizing this species as a parent, and further exploration and breakthroughs are needed. Summary of the Invention

[0006] The purpose of this invention is to provide an artificial breeding method for a hybrid of brown-spotted grouper and blue-spotted sea bass, to fill the technical gap in hybrid breeding of grouper with blue-spotted sea bass as parent, so that the hybrid obtained by crossing blue-spotted sea bass and brown-spotted grouper is authentic and reliable, and inherits the genetic information of the parents at the molecular level.

[0007] To solve this technical problem: This invention provides a method for artificially breeding a hybrid of brown-spotted grouper and blue-spotted sea bass, comprising the following steps: Step A: Select healthy brown-spotted grouper as female parent fish and healthy blue-spotted nine-spined perch as male parent fish; Step B: Enhanced cultivation of female and male parents, including nutritional enhancement and / or temperature induction, to promote gonadal maturation of the parents; Step C: Collect egg fluid from the female parent and sperm from the male parent, fertilize to obtain fertilized eggs, incubate the fertilized eggs to obtain hybrid spots, and use sequences SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO.8 to identify the authenticity of the hybrid spots and confirm that the hybrid spots inherit the genetic information of the female and male parents.

[0008] Furthermore, in step A, the standard for health is: no disease, no injury, no deformity, robust physique, and no hormone-induced labor or ovarian induction treatment; Female parents must weigh ≥4 kg, and male parents must weigh ≥0.5 kg.

[0009] Furthermore, step B includes: Step B-1: Nutritionally fortify the female parent to obtain fortified female parent; Step B-2: Nutritional fortification and temperature induction were performed on the male parent to obtain fortified male parent.

[0010] Furthermore, in step B-2, temperature induction includes low-temperature induction and rewarming activation.

[0011] Furthermore, the cooling rate during low-temperature induction is no more than 0.5~1℃ per day, the induction temperature is 23℃±0.5℃, and the induction time is 12~13 days, 13~15 days, or 15~16 days.

[0012] Furthermore, the heating rate during rewarming activation is no more than 0.5~1℃ per day, and the starting temperature for rewarming activation is 29℃±0.5℃; the rewarming activation time is 6~7 days, 7~9 days, or 9~10 days.

[0013] Furthermore, nutritional fortification involves feeding the animal to its full capacity with fortified feed. The fortified feed includes a basal feed and nutritional supplements. The nutritional supplements include fish oil at a weight ratio of 0.2-0.4% of the basal feed, arginine at a weight ratio of 0.2-0.5% of the basal feed, vitamin E at a weight ratio of 0.3-0.7% of the basal feed, lecithin at a weight ratio of 0.75-1.20% of the basal feed, and folic acid at a weight ratio of 0.05%-0.20% of the basal feed.

[0014] Furthermore, step C includes: Step C-1: Collect egg fluid from the female parent and sperm from the male parent, and perform dry fertilization on the egg fluid and sperm to obtain fertilized eggs; Step C-2: Incubate the fertilized eggs to obtain hybridization spots; Step C-3: The authenticity of the hybridization spots was determined using sequences SEQ ID NO.1 to SEQ ID NO.8. The hybridization spots inherited the genetic information of both the female and male parents.

[0015] Furthermore, in step C-1, the conditions for collecting egg fluid are: the female parent's upper abdomen is bulging and the genital pore is slightly red, indicating sexual maturity; the conditions for collecting semen are: the male parent's genital pore is slightly red, and milky white semen can be squeezed out when the abdomen is gently pressed, indicating sexual maturity.

[0016] Furthermore, in step C-2, the temperature conditions for incubating the fertilized eggs are 24~26℃, 26~29℃, or 29~32℃.

[0017] Furthermore, step C-3 specifically involves: extracting DNA from the female parent, male parent, and hybrid spot to obtain genomic DNA from the female parent, male parent, and hybrid spot, respectively, and then performing PCR identification using primer sequences; the primer sequences are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, or SEQ ID NO.8.

[0018] Furthermore, the fertilization rate of the fertilized eggs is ≥95%, and the hatching rate of the fertilized eggs is ≥89.5%.

[0019] This application provides an artificial breeding method for a hybrid of brown-spotted grouper and blue-spotted grouper. By using blue-spotted grouper as the male parent and brown-spotted grouper as the female parent, a hybrid grouper can be obtained. This application also uses specific primer pairs to further verify the genetic information of the parents in the hybrid, ensuring that the hybrid variety is a hybrid of brown-spotted grouper and blue-spotted grouper. The artificial breeding method at a temperature of 28℃ is used, and the spawning rate of the parent fish is high, reaching 95% or more, and the hatching rate of fertilized eggs reaches 90% or more. This fills the current technical gap in hybrid breeding of grouper using blue-spotted grouper as the parent. Moreover, the hybrid obtained by crossing blue-spotted grouper and brown-spotted grouper is authentic and reliable, inheriting the genetic information of the parents at the molecular level, laying the foundation for obtaining a superior grouper breed and improving the economic value of blue-spotted grouper and brown-spotted grouper. Attached Figure Description

[0020] The above description of the present invention and the following detailed embodiments will be better understood when read in conjunction with the accompanying drawings. It should be noted that the drawings are merely examples of the claimed technical solutions.

[0021] Figure 1 The image shown is of a female brown-spotted grouper laying eggs in the example (scale bar is 5 cm). Figure 2 The image shown is of a male Blue-spotted Nine-spined Bass ejaculating in the example (scale bar is 2 cm). Figure 3 This is a microscopic observation of the fertilized eggs of a female brown-spotted grouper and a male blue-spotted sea bass during the embryonic sac stage, as shown in the example (scale bar: 500 μm). Figure 4 This is a microscopic observation of the gastrula stage of fertilized eggs from a female brown-spotted grouper and a male blue-spotted sea bass in the example (scale bar: 500 μm). Figure 5 This is a microscopic observation of the somitotic stage of fertilized eggs from a female brown-spotted grouper and a male blue-spotted sea bass in the example (scale bar: 500 μm). Figure 6 The image shown is of a 35-day-old hybrid grouper obtained by crossing a female brown-spotted grouper with a male blue-spotted grouper in the example (scale bar: 500 mm). Figure 7The image shown is an agarose gel electrophoresis image used in the example to identify the authenticity of hybrid spots and female brown-spotted grouper and male blue-spotted sea bass (where M is the marker; lanes 1, 4, 7 and 10 are the PCR product results of female brown-spotted grouper, single band; lanes 2, 5, 8 and 11 are the PCR product results of male blue-spotted sea bass, single band; lanes 3, 6, 9 and 12 are the PCR product results of hybrid spots, double band, representing hybrid species; lanes 1-3 are for primer sequence detection SEQ ID NO.1-SEQ ID NO.2, lanes 4-6 are for primer sequence detection SEQ ID NO.3-SEQ ID NO.4, lanes 7-9 are for primer sequence detection SEQ ID NO.5-SEQ ID NO.6, and lanes 10-12 are for primer sequence detection SEQ ID NO.7-SEQ ID NO.8). Detailed Implementation

[0022] The detailed features and advantages of this application are described below in the specific embodiments. The content of this description is sufficient to enable any person skilled in the art to understand the technical content of this application and implement it accordingly. Based on the specification, claims and drawings disclosed in this specification, a person skilled in the art can easily understand the related objectives and advantages of this application.

[0023] In this specification and claims, several terms will be used, and unless otherwise indicated, these terms will be defined to have the following meanings: As used in this patent, "no disease, no injury, no deformity" refers to the broodstock having an intact body surface, no mechanical damage or infectious lesions, no ulcers on the skin, no broken fins, no obvious parasites, normal swimming posture, and active feeding response.

[0024] As used in this patent, "satisfying feeding" refers to feeding the parent fish in an amount that is sufficient to satisfy their hunger and prevent them from constantly searching for food due to hunger.

[0025] All other terms used herein for special definition are intended to have the general meaning understood by one of ordinary skill in the art, and in particular, meaning that one of ordinary skill in the art, upon reading the claims, specification and drawings of this patent, can directly and without doubt determine how the technical solution of this patent can be implemented.

[0026] Even if there are incomplete descriptions, omissions, or ambiguities in the grammar, words, punctuation, graphics, symbols, etc. of the claims, specification, and drawings of this patent, a person skilled in the art can still arrive at the only correct understanding by reading the claims, specification, and drawings as a whole without extensive reasoning or experimentation, and effectively exclude various incorrect interpretations that are not aimed at achieving the purpose of this patent.

[0027] The "range" disclosed herein is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60-120 and 80-110 are listed for a specific parameter, it is expected that ranges of 60-110 and 80-120 are also expected. Furthermore, if minimum range values ​​of 1 and 2 are listed, and if maximum range values ​​of 3, 4, and 5 are listed, then the following ranges are all expected: 1-3, 1-4, 1-5, 2-3, 2-4, and 2-5. In this application, unless otherwise stated, the numerical range "ab" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0-5" indicates that all real numbers between "0-5" have been listed in this article; "0-5" is simply a shortened representation of these numerical combinations. Furthermore, when a parameter is stated as an integer ≥2, it is equivalent to disclosing that the parameter is, for example, an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0028] Unless otherwise specified, all embodiments and preferred embodiments mentioned herein can be combined to form new technical solutions.

[0029] Unless otherwise specified, all the technical features and preferred features mentioned herein can be combined to form new technical solutions.

[0030] Unless otherwise specified, all steps mentioned herein may be performed sequentially or randomly, but are preferably performed sequentially. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, the mention that the method may also include step (c) indicates that step (c) may be added to the method in any order; for example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0031] Unless otherwise specified, the terms "comprising" and "including" as used herein can be open-ended or closed-ended. For example, "comprising" and "including" may mean that other components not listed may also be included, or that only the listed components may be included.

[0032] In the description of this article, it should be noted that, unless otherwise stated, "above" and "below" include the number itself, and "several" in "one or more" means two or more.

[0033] In this description, unless otherwise stated, the term "or" is inclusive. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, the condition "A or B" is satisfied by any of the following conditions: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0034] Unless otherwise specified, percentages (%) in this document refer to percentages by mass relative to the composition.

[0035] Unless otherwise stated herein, the sum of the contents of the components in the composition is 100%.

[0036] In this document, unless otherwise stated, “combination of” means a multi-component mixture of the elements, such as two, three, four, and up to the maximum possible multi-component mixture.

[0037] Unless otherwise specified, the term "a" as used in this specification means "at least one".

[0038] The terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0040] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0041] (1) Source of sample materials Brown-spotted grouper: purchased from Guangzhou Blue Ocean Marine Technology Co., Ltd. (Liwan District, Guangzhou).

[0042] Blue-spotted nine-spined perch: purchased from Guangzhou Blue Ocean Ocean Technology Co., Ltd. (Liwan District, Guangzhou).

[0043] Sequence synthesis: carried out by Sangon Biotech (Shanghai) Co., Ltd., hereinafter referred to as Sangon.

[0044] (2) Sources of reagents and consumables Table 1. Sources and Product Codes of Reagents and Consumables Required for the Experiment ;

[0045] (3) Source of instruments and equipment Table 2. Sources and Models of Instruments and Equipment Required for the Experiment ;

[0046] Example A method for artificially breeding a hybrid of Blue-spotted Nine-spined Perch and Brown-spotted Grouper includes the following steps: S1. Parent selection and feeding, the specific steps are as follows: (1) Select healthy, disease-free, mature brown spot grouper with a weight of more than 4 kg as candidate parent stock.

[0047] (2) Select healthy, disease-free, brightly colored, 500 g (0.5 kg) or more wild-type male blue-spotted nine-spined perch that have been successfully domesticated as candidate sires.

[0048] (3) Broodstock rearing: The above-mentioned candidate female and male populations were reared separately in a recirculating aquaculture system. The daily rearing pond temperature was set at 25-28℃, and the salinity was controlled at 28-32‰ to simulate the natural near-shore ecological environment. The pH was maintained between 7.8 and 8.3. The dissolved oxygen concentration (DO) was ≥6.0 mg / L, the ammonia nitrogen concentration was <0.2 mg / L, and the nitrite concentration was <0.01 mg / L. The indoor lighting was set to 12 h light / 12 h dark to simulate the tropical diurnal variation. The light intensity was 800-1000 lx, and the light was evenly distributed to avoid local strong light or shadow areas.

[0049] Meanwhile, a combined ultraviolet and biological filter water treatment device in the recirculating aquaculture system is used to maintain water quality cleanliness; grouper feed is given twice a day, supplemented with fresh small fish and squid; among them, the amount of fresh small fish is 10% to 15% of the total feed, and the amount of squid is 8% to 10% of the total feed.

[0050] S2, Parent Fish Enhancement, the specific steps are as follows: 25-30 days before artificial insemination, the parent fish fed in step S1 above are subjected to intensive culture.

[0051] (1) Preparation of fortified feed for broodstock: The basic feed consists of a coarsely ground mixture of triggerfish and deep-sea squid in a mass ratio of (5-8):1, serving as the main protein source and flavor enhancer. Molluscs, such as mussels, sandworms, and oysters, are added at a mass ratio of 10%. For example, per 100 parts of feed, the composition is: triggerfish 77.15 parts, squid 12.86 parts, oyster 3.33 parts, mussels 3.33 parts, and sandworms 3.33 parts.

[0052] Add 1.7% of the weight percentage of the basic feed to the basic feed. The nutrients include fish oil, arginine, vitamin E, lecithin, and folic acid. The amount of fish oil added is 0.2% of the weight percentage of the fortified feed, the amount of arginine added is 0.2% of the weight percentage of the fortified feed, the amount of vitamin E added is 0.3% of the weight percentage of the fortified feed, the amount of lecithin added is 0.9% of the weight percentage of the fortified feed, and the amount of folic acid added is 0.1% of the weight percentage of the fortified feed.

[0053] All feed should be refrigerated after preparation and thawed before each use, and the temperature should be controlled to not exceed room temperature to avoid loss of nutrients or spoilage.

[0054] In some other specific embodiments, the components and amounts of the added nutrients can be flexibly adjusted. The nutrients can also be one or more combinations of fish oil, arginine, vitamin E, lecithin, and folic acid, with the added amount being 1.5-3.0% of the basal feed. Specifically, the added amount of fish oil is 0.2%-0.4% of the weight percentage of the fortified feed, the added amount of arginine is 0.2%-0.5% of the weight percentage of the fortified feed, the added amount of vitamin E is 0.3%-0.7% of the weight percentage of the fortified feed, the added amount of lecithin is 0.75%-1.20% of the weight percentage of the fortified feed, and the added amount of folic acid is 0.05%-0.20% of the weight percentage of the fortified feed.

[0055] (2) Feeding the parent fish: The broodstock should be fed twice daily, following a "full-feeding" method. Feeding should be stopped for one day every 2-3 days to promote intestinal emptying, enhance feed absorption, and reduce the metabolic burden on the liver. Throughout the feeding period, the actual amount of feed given, uneaten feed, and the feeding behavior of the broodstock should be recorded daily. In the first stage, although the basal metabolic rate of the broodstock decreases, a stable feeding schedule should be maintained to ensure the accumulation of energy reserves. This helps the broodstock convert and store energy in muscles, hepatopancreas, and adipose tissue, building the necessary nutritional foundation for subsequent gonadal development.

[0056] (3) Water quality control: The daily water exchange rate in the aquaculture pond is maintained at around 50%, the salinity is controlled at 28‰, the pH is maintained between 8.0, and the dissolved oxygen concentration (DO) is maintained at 6.5 mg / L or above.

[0057] During the female parent strengthening period, the water temperature was maintained at 28~32℃, and the maturity was mainly judged by the state of the abdomen swelling, resulting in strengthened female parent fish (also known as female brown-spotted grouper). The male parent was induced to have gonads by low temperature stimulation-warming maturation method. During the low temperature stage, the water temperature was kept constant at 23.0℃ for 14 days, followed by the warming activation stage, with the temperature increased by 0.5℃ every day, gradually rising to 28.0℃, with temperature fluctuations not exceeding ±0.5℃. The maturity was judged by whether milky white semen flowed out when the abdomen was gently pressed, resulting in strengthened male parent fish (also known as male blue-spotted grouper).

[0058] S3. Labor induction and artificial insemination, the specific steps are as follows: (1) After the parent fish are strengthened in step S2 above, the gonads of female brown-spotted grouper can reach the level of natural ovulation. Figure 1 Male blue-spotted spiny perch also exhibited the ability to ejaculate naturally. Figure 2 ).

[0059] Both egg and semen collection were performed without anesthesia. Male semen was collected in sterile tubes and immediately placed on ice for preservation for no more than 12 hours.

[0060] (2) Dry insemination was used. Fertilized eggs from the same batch were randomly divided into 5 groups and incubated under different temperature conditions. The incubation temperatures were set at 24℃, 26℃, 28℃, 30℃, and 32℃. The insemination process was consistent for each group of fertilized eggs. After adding semen to the egg mixture, it was immediately mixed and then an appropriate amount of natural seawater was added for activation. The activation time was controlled within 1 minute. The fertilized eggs were then placed in the corresponding water temperature for 5 minutes to obtain fertilized eggs. The fertilized eggs were rinsed 2-3 times with sterile seawater and then transferred to the incubation tank for further culture. The fertilization rate was measured by the cleavage rate. Each group had 3 replicates, and the fertilization status of the fertilized eggs was observed under a microscope.

[0061] During hybridization, the fertilized eggs obtained from the hybridization of female brown-spotted grouper and male blue-spotted sea bass can develop successfully and go through typical embryonic development stages such as blastocyst stage, gastrula stage and somites stage.

[0062] After fertilization, the fertilized egg is like Figures 3-5 As shown: Among them, Figure 3 The fertilized egg is in the blastocyst stage. The fertilized egg has a regular shape, a transparent membrane, and a clearly visible blastocoel. The embryonic cells are evenly distributed at one end of the membrane. The overall development is good, and no deformities or fragmentation are observed. Figure 4As a fertilized egg in the gastrula stage, the cells gradually invaginate towards the center of the embryo, forming a distinct primitive opening. The cell arrangement gradually changes from scattered to regular, showing the process of early germ layer differentiation, and the embryonic development enters a critical stage. Figure 5 The fertilized egg is in the somite stage. The embryo gradually elongates and segments, with obvious somite differentiation bands visible. The embryonic axis is clear, and the head and tail are initially differentiated, indicating that the hybrid fertilized egg can normally enter the pre-organogenesis stage, laying the foundation for subsequent hatching.

[0063] The fertilization rate and hatching rate statistics are shown in Table 3. At 24℃ and 26℃, the fertilization rates of the fertilized eggs were 68% and 81%, respectively, which were relatively low. At 28℃, the fertilization rate of the fertilized eggs was the highest, reaching over 95%, and the hatching rate remained stable at around 90%. When the temperature rose to 30℃, the fertilization rate of the fertilized eggs decreased to approximately 84%. At 32℃, the fertilization rate of the fertilized eggs further decreased to approximately 70%. This indicates that the optimal temperature condition for hybridization fertilization of the fertilized eggs provided in this application is 28℃, under which the optimal fertilization rate and hatching rate can be obtained.

[0064] Table 3. Statistical results of fertilization rate and hatching rate of fertilized eggs under different temperature conditions. ;

[0065] S4. Seedling cultivation in the workshop, the specific steps are as follows: After the fertilized eggs obtained in step S3 above are hatched, they are fed with nutritionally fortified rotifers (rich in DHA, vitamins and other nutrients) starting 3 days after hatching, and the density is controlled at 10-20 rotifers / mL daily. Copepods and Artemia larvae are introduced between 7 and 20 days of age. Adult Artemia larvae are introduced between 20 and 30 days of age. After 30 days of age, micro-particle artificial feed with a protein content of more than 45% is gradually added until they are fully domesticated. At the same time, microecological preparations are added regularly to maintain intestinal health. Key parameters such as water temperature, salinity, ammonia nitrogen, pH, dissolved oxygen, and feeding rate are recorded daily to ensure good growth of fry and a deformity rate of less than 2%. The resulting offspring of female brown-spotted grouper and male blue-spotted grouper are called hybrid grouper.

[0066] like Figure 6 The image shown is a photograph of a hybrid spotted animal on its 35th day of age. The average body length is about 2 cm and the average weight is about 0.8 g. The body shape is streamlined, and the overall color is light brown to pale red. Slight pigmentation can be seen on the sides of the body, and the caudal peduncle area is lighter in color.

[0067] S5. Identification of hybridization spots, the specific steps are as follows: To determine whether the hybrid spot is a genuine hybrid offspring of a female brown-spotted grouper and a male blue-spotted grouper, the authenticity of the hybrid spot was verified.

[0068] 1. Reference genome alignment DNA extracted from the muscle tissue of the blue-spotted grouper was used to construct and sequence a third-generation genome library. The paternal genome of the blue-spotted grouper was used as a reference, and the maternal genome of the brown-spotted grouper was used as the comparison object. The minimap2 tool was used to perform whole-genome alignment and generate SAM format files.

[0069] 2. Mutation extraction and screening The SAM file is formatted, sorted, and indexed using samtools (sam tools), and then paftools.js is called to extract the missing insertion (InDel) information.

[0070] All InDel events longer than 20 bp were filtered out, and their coordinates and strand orientation (QSTRAND) in the parent genome were recorded.

[0071] 3. Flanking region sequence extraction For each InDel sequence in step 2 above, extract 150 bp flanking regions from the paternal genome upstream and downstream; extract the corresponding sequence from the maternal genome according to the alignment direction, and perform reverse complementation operation if it is located on the negative strand to ensure that the direction is consistent.

[0072] 4. Initial screening of conserved primer sequences The first and last 100 bp segments were extracted from the paternal flanking region sequence, and 25 bp candidate primer fragments were extracted using a sliding window. Only fragments that could be perfectly matched in the maternal genome were retained as "conserved fragments," and fragments with a GC content of 40%–60% were further screened to ensure primer stability.

[0073] 5. Primer design and optimization Primers were designed using Primer3 software for the highly conserved InDel region (more than 50 qualified fragments before and after each region). The product length was set to 100-300 bp, and the Tm value, GC content and secondary structure parameters were optimized to screen candidate primer pairs. The specific primer sequences are shown in Table 4.

[0074] Table 4 Primer sequence listing ;

[0075] 6. Specificity verification In step 5 above, all primer pairs were BLAST-aligned with the parent genome to ensure a unique matching site only in the target InDel region. Primer pairs with specificity and no non-specific amplification were used as the final genotyping primers.

[0076] (1) DNA was extracted from the above-mentioned brown-spotted grouper, blue-spotted sea bass, and 35-day-old hybrid grouper using a marine animal genome extraction kit (Tiangen Biochemical; all reagents and consumables below are from this kit). The DNA extraction steps included: 1) Sample pretreatment: Weigh 30 mg of muscle tissue, add 200 μL of GA buffer to a centrifuge tube, mix gently, and shake for 15 seconds to ensure complete infiltration of muscle tissue.

[0077] 2) Proteinase K digestion: Add 20 mL of Proteinase K solution (20 mg / mL) to a centrifuge tube, mix thoroughly, and incubate at 56°C until the tissue is completely dissolved.

[0078] 3) Pyrolysis and precipitation: Add 200 μL of GB lysis buffer, mix thoroughly, place in a 70℃ water bath for 10 minutes, then add 200 μL of anhydrous ethanol and mix well.

[0079] 4) Adsorption column purification: Transfer the mixture to a CB3 adsorption column, place it in a collection tube, centrifuge at 12000 rpm for 30 seconds, and discard the waste liquid. Then add 500 μL of GD buffer and 600 μL of PW wash buffer, centrifuge for 30 seconds each time, and wash the adsorption column to remove impurities.

[0080] 5) Drying the adsorption column: Place the cleaned adsorption column back into a centrifuge tube, centrifuge at 12,000 rpm for 2 minutes, and then let it stand at room temperature for 2-5 minutes to ensure that the ethanol in the adsorption column is completely evaporated.

[0081] 6) DNA elution: Place the adsorption column into a new centrifuge tube, add 50-200 μL of sterile water to the middle of the adsorption column, let it stand at room temperature for 2 minutes, then centrifuge at 12000 rpm for 2 minutes and collect the eluted DNA solution.

[0082] 7) Quality Inspection and Preservation: Use a spectrophotometer to detect the concentration and purity of DNA to ensure OD. 260 / OD 280 Between 1.8 and 2.0, OD 260 / OD 230 Greater than 2.0. Genomic DNA of brown-spotted grouper, blue-spotted sea bass, and hybrid grouper was obtained respectively.

[0083] (2) The total DNA of brown spot grouper, total DNA of blue-spotted nine-spined perch and total DNA of hybrid grouper obtained in step (1) above were amplified by PCR using the primer pairs in Table 4 above.

[0084] 1) The PCR amplification reaction system is as follows: Genomic DNA (100 ng / μL), 1 μL; Forward primer Fw (10 μM), 1 μL; Reverse primer Fw (10 μM), 1 μL; 2×Taq Plus Master Mix Ⅱ (Plus), 12.5 μL; Enzyme-free sterile water, 9.5 μL.

[0085] 2) The PCR amplification reaction procedure is as follows: Pre-denaturation at 95℃ for 3 min; Denaturation at 95℃ for 15 seconds; Anneal at 56℃ for 20 seconds; The denaturation and extension process was performed at 72°C for 40 seconds, with a total of 32 cycles. Extend the heat at 72℃ for 10 minutes. Store at 4℃.

[0086] After the PCR reaction was completed, PCR products of brown-spotted grouper, blue-spotted sea bass, and hybrid grouper were obtained. The PCR amplification products were detected by 1.5% agarose gel electrophoresis.

[0087] The results are as follows Figure 7 As shown, all hybrid individuals amplified allelic bands exhibiting characteristics of both the father and mother, clearly distinguishing and confirming their hybrid status. This demonstrates that the offspring individuals obtained by this invention inherit the genetic information of their parents at the molecular level, further verifying the reliability of the hybridization breeding results.

[0088] Therefore, it can be concluded that this application provides an artificial breeding method for a hybrid of brown-spotted grouper and blue-spotted sea bass. By using brown-spotted grouper as the female parent and blue-spotted sea bass as the male parent, a hybrid grouper can be obtained. This application also uses specific primer pairs to further verify the genetic information of the parents in the hybrid, ensuring that the hybrid variety is a hybrid of brown-spotted grouper and blue-spotted sea bass. Using an artificial breeding method at a temperature of 28℃, the spawning rate of the parent fish is high, reaching 95% or more, and the hatching rate of fertilized eggs reaches 90% or more. This fills the current technical gap in hybrid breeding of grouper using blue-spotted sea bass as the parent. Moreover, the hybrid obtained by crossing blue-spotted sea bass and brown-spotted grouper is authentic and reliable, inheriting the genetic information of the parents at the molecular level, laying the foundation for obtaining a superior grouper breed and improving the economic value of blue-spotted sea bass and brown-spotted grouper.

[0089] In the foregoing description of exemplary embodiments / specific implementations of this patent, various features of this patent are sometimes combined in a single embodiment / specific implementation or its figures and description, with the aim of simplifying the disclosure and aiding in the understanding of one or more of the various aspects of the invention. However, unless expressly stated otherwise or in obvious technical contradiction or exclusion, the descriptive method of this patent should not be construed as reflecting an intention that the claimed features of the invention are more than expressly stated in each claim. Rather, the inventive aspect reflected in the claims lies in not all the features of a single foregoing disclosed embodiment / specific implementation. Therefore, the claims following the detailed description are hereby expressly incorporated into this detailed description, each claim existing independently as a separate embodiment / specific implementation of this patent.

[0090] The terms and expressions used in this specification are for illustrative purposes and not for limitation. Their use is not intended to exclude any equivalents of the shown and described features or portions thereof, but rather to facilitate the understanding that various modifications may be possible within the scope of this patent claim. Therefore, it should be understood that while this patent has been specifically disclosed through preferred embodiments, exemplary embodiments, and optional features, variations or modifications of the concepts disclosed herein may be adopted by those skilled in the art, and such variations and modifications are therefore considered to be within the scope of this patent as defined by the appended claims. The specific embodiments given in this specification are examples of useful embodiments of this patent, and it will be apparent to those skilled in the art that this patent can be implemented using many variations of the devices, device components, and method steps disclosed herein.

[0091] The foregoing description of specific embodiments fully discloses the general features of this patent, enabling others to easily modify and / or adapt such embodiments for various applications by applying knowledge within the scope of the art, without excessive experimentation or deviation from the general concept of this patent. Therefore, based on the teachings and guidance provided herein, it is intended that such modifications and alterations be included within the meaning and scope of equivalents of the disclosed embodiments. It should be understood that the wording or terminology used herein is for descriptive purposes and not intended to be limiting; thus, the wording or terminology in this specification will be interpreted by those skilled in the art based on the foregoing teachings and guidance.

[0092] Furthermore, the scope of this patent should not be limited to any of the exemplary embodiments described above, but only to the appended claims and their equivalents.

Claims

1. A method for artificial breeding of a hybrid of Epinephelus fuscoguttatus and Plectropomus laevis, characterized by, The method comprises the following steps: Step A: screening healthy Epinephelus fuscoguttatus as female parent and screening healthy Plectryagus intermedius as male parent; Step B: intensively breeding the female parent and the male parent, wherein the intensive breeding comprises nutritional intensification and / or temperature induction to promote gonadal maturation of the parents; Step C: collecting egg liquid of the female parent and sperm of the male parent, fertilizing to obtain fertilized eggs, incubating the fertilized eggs to obtain hybrid grouper, and using a primer pair to identify the authenticity of the hybrid grouper to determine whether the hybrid grouper inherits genetic information of the female parent and the male parent; The primer pair is a sequence pair consisting of SEQ ID NO. 1 and SEQ ID NO. 2, a sequence pair consisting of SEQ ID NO. 3 and SEQ ID NO. 4, a sequence pair consisting of SEQ ID NO. 5 and SEQ ID NO. 6, or a sequence pair consisting of SEQ ID NO. 7 and SEQ ID NO.

8.

2. The artificial breeding method of the Pteragogon koreanus and Pseudogramma polyacanthum hybrid according to claim 1, wherein, In the step A, The health standard is: no disease, no injury, no deformity, strong constitution, and no hormone induced spawning or ovary induction treatment; The weight of the female parent is greater than or equal to 4 kg, and the weight of the male parent is greater than or equal to 0.5 kg.

3. The artificial breeding method of Pterocaesio marri and Pseudolabrus eoethinus according to claim 2, characterized in that, The step B comprises: Step B-1: intensively breeding the female parent to obtain an intensified female parent; Step B-2: intensively breeding the male parent to obtain an intensified male parent.

4. The artificial breeding method of Plectropomus leopardus and Pseudolabrus eoethro hybrid according to claim 3, characterized in that, In the step B-2, the temperature induction comprises low-temperature induction and rewarming activation, In the low-temperature induction, the cooling rate is not more than 0.5℃ per day, the temperature at the beginning of the low-temperature induction is 23℃±0.5℃, the time of the low-temperature induction is 12-13 days, 13-15 days or 15-16 days; In the rewarming activation, the heating rate is not more than 0.5℃ per day, the temperature at the beginning of the rewarming activation is 29℃±0.5℃, and the time of the rewarming activation is 6-7 days, 7-9 days or 9-10 days.

5. The artificial breeding method of the hybrid of Epinephelus fuscoguttatus and Plectryagus intermedius according to claim 3, wherein The nutritional intensification is satiation feeding of the intensified feed, and the intensified feed comprises a base feed and a nutritional agent, wherein the nutritional agent comprises fish oil with a weight ratio of 0.2-0.4% of the base feed, arginine with a weight ratio of 0.2-0.5% of the base feed, vitamin E with a weight ratio of 0.3-0.7% of the base feed, lecithin with a weight ratio of 0.75-1.20% of the base feed, and folic acid with a weight ratio of 0.05%-0.20% of the base feed.

6. The artificial breeding method of Pterocaesio marri and Pseudolabrus eoethinus according to claim 1, wherein, The step C comprises: Step C-1: collecting the egg liquid of the female parent and the sperm of the male parent respectively, and dry fertilizing the egg liquid with the sperm to obtain the fertilized eggs; Step C-2: incubating the fertilized eggs to obtain hybrid grouper; Step C-3: using the primer pair to identify the authenticity of the hybrid grouper to determine whether the hybrid grouper inherits genetic information of the female parent and the male parent.

7. The artificial breeding method of Pterocaesio marri and Pseudolabrus eozeni according to claim 6, characterized in that, Said step C-1 in, The collection condition of the egg liquid is that the female parent has a bulge on the apparent abdomen and the genital pore is slightly red, that is, reaching sexual maturity. The collection condition of the sperm is that the genital pore of the male parent is slightly red, the abdomen can be squeezed out of the milky white sperm, that is, reaching sexual maturity.

8. The artificial breeding method of the hybrid of Epinephelus fuscoguttatus and Plectropomus laevis according to claim 6, characterized in that, In step C-2, the temperature condition of the incubation of the fertilized eggs is 24-26℃, 26-29℃ or 29-32℃.

9. The artificial breeding method of the hybrid of Epinephelus fuscoguttatus and Plectropomus laevis according to claim 6, characterized in that, Step C-3 is specifically: DNA extraction is performed on the female parent, the male parent and the hybrid, to obtain female parent genomic DNA, male parent genomic DNA and hybrid genomic DNA, respectively, and then PCR identification is performed using the primer pair to determine that the hybrid inherits the genetic information of the female parent and the male parent.

10. The artificial breeding method of the Pterocaesio marri and the Pseudolabrus eoethinus hybrid according to any one of claims 1 to 9, characterized in that, The fertilization rate of the fertilized eggs is ≥95%, and the hatching rate of the fertilized eggs is ≥89.5%.

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