Identification of the relationship between red nine-spined perch broodstock and the method of indoor seedling rearing

Abstract

The present invention belongs to the technical field of aquatic organisms, and specifically relates to the identification of kinship between broodstock of red nine-spined perch and a method for fully indoor seedling cultivation. The fully indoor seedling cultivation method of red nine-spined perch described in the present invention includes the selection and cultivation of broodstock of red nine-spined perch, artificial induction of spawning of red nine-spined perch, incubation and seedling cultivation of fertilized eggs of red nine-spined perch in an indoor seedling pond, and the method described in the present invention is to cultivate the seedlings of red nine-spined perch in an indoor factory workshop throughout the whole process. The method is advanced, the breeding effect is stable, and it is convenient for daily management. The present invention proposes a method for identifying the kinship between broodstock of red nine-spined perch for the first time, which can effectively avoid the problem of inbreeding in the process of artificial breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of aquatic organisms, and in particular relates to a method for identifying the kinship of red nine-spined perch broodstock and a fully indoor seedling cultivation method. Background Art

[0002] Red nine-spined perch ( Cephalopholissonnerati ), also known as Song's nine-thorn sardine, netted bream, and red melon seed spot, belongs to the genus Nine-thorn Sardine of the family Serranidae, order Perciformes, suborder Perciformes, class Actinopterygii, and is a tropical marine fish. The red nine-thorn sardine has delicate meat and delicious taste, and is widely praised by consumers. It has a high market value and is sold at a price of about 500 yuan per catty. It has high economic and aquaculture value, but the artificial breeding and breeding technology of red nine-thorn sardine is still immature at this stage. In 2023, it was reported that a small number of red melon seed spot seedlings were cultivated in outdoor open-air ponds, but outdoor pond seedlings are easily affected by weather, heavy rain, pathogenic microorganisms and ambient temperature, and are prone to infection and outbreaks of infectious diseases, resulting in seedling failure, affecting the enthusiasm and economic income of fishermen. Therefore, the establishment of fully indoor seedling and breeding technology for red nine-thorn sardine is an important issue that needs to be overcome in the field of marine fish farming.

[0003] In the process of artificial breeding of red nine-spined perch, the selection of broodstock is crucial. Individuals with distant kinship need to be selected for breeding to avoid problems such as inbreeding depression and germplasm degradation caused by inbreeding. At present, the artificial breeding of red nine-spined perch is in its infancy, and it is necessary to collect wild red nine-spined perch germplasm resources as a breeding population. However, the kinship between wild red nine-spined perch individuals is unclear, and there is a lack of corresponding identification methods. So far, there have been no research reports on the identification of kinship between red nine-spined perch. Therefore, the establishment of kinship identification technology for red nine-spined perch broodstock is also a key link in the red nine-spined perch seedling technology. Summary of the invention

[0004] In order to solve the above technical problems, the present invention proposes a method for identifying the kinship of broodstock of red nine-spined seabass and a fully indoor seedling cultivation method. The method of the present invention carries out seedling cultivation and breeding of red nine-spined seabass in an indoor factory workshop throughout the entire process. The method is advanced, the breeding effect is stable, and the daily management is convenient. A method for identifying the kinship of broodstock of red nine-spined seabass is proposed for the first time, which can effectively avoid the problem of inbreeding in the process of artificial breeding.

[0005] The technical solution of the present invention is as follows:

[0006] The method for identifying the kinship of red nine-spined seabass broodstock of the present invention comprises the following steps:

[0007] A1. Obtain the single nucleotide polymorphism (SNP) sites of candidate broodstock of red ninethorn perch. The number of SNP sites ≥ the total number of candidate broodstock. The SNP sites meet the following conditions: evenly distributed in the genome, no genotype missing, conforming to Hardy-Weinberg equilibrium (p=0.0001), the minimum allele frequency>0.1, biallelic sites, and located on autosomes;

[0008] A2, using the SNP sites obtained in A1 to calculate the kinship coefficients between candidate parent fish of red nine-spined seabass, and construct a molecular kinship matrix;

[0009] The molecular kinship matrix needs to calculate the minimum allele frequency. To avoid the calculation bias caused by insufficient number of individuals, the genotype matrix can be added to the pedigree-based kinship matrix. The regression is:

[0010] Formula 1;

[0011] Among them, the matrix for The genotype matrix contains Individual The genotype information of the SNP loci, genotype AA / Aa / aa is represented by 0 / 1 / 2 respectively; the matrix For the matrix The transpose of and are the intercept and slope of formula 1 respectively; the matrix is a kinship matrix based on pedigree information. If the kinship between individuals is unknown or there is no pedigree record, this matrix can be used The identity matrix representation of is the residual term. Since the matrix The expectation is the matrix , and the matrix It follows a normal distribution with a mean of 0. Therefore, the molecular kinship matrix It can be calculated using formula 2:

[0012] Formula 2;

[0013] According to formula 2, we can know that the calculation matrix We only need to solve for the coefficients and Therefore, multiply both sides of equation 1 by the matrix , combined with the matrix properties, we can get:

[0014] Formula 3;

[0015] Among them, in formula 3 represents the total number of individuals used for analysis; the matrix Represents an individual and individuals Pedigree-based kinship matrices;

[0016] Solve the coefficients according to formula 3 and , and then use formula 2 to calculate the molecular affinity matrix . According to the calculated matrix , select individuals with small inter-individual kinship coefficient to construct breeding groups, and the inter-individual kinship coefficient is ≤1 / 16, and avoid placing individuals with inter-individual kinship coefficient>1 / 16 (0.0625) in the same breeding group.

[0017] The method for raising red nine-spined perch indoors comprises the following steps: selecting and raising red nine-spined perch broodstock, artificially inducing spawning of red nine-spined perch, and incubating and raising fertilized red nine-spined perch eggs in an indoor breeding pool. The specific method is as follows:

[0018] (1) Selection and cultivation of red nine-spined perch broodstock, including the following steps:

[0019] B1. Select 2-5 years old, 1.5-4 kg in weight, healthy, smooth, free of injuries, deformities, and vigorous red nine-spined perch females and males as candidate parents;

[0020] B2. Using a PIT electronic tag with a unique number to mark each broodstock, and using the red nine-spined seabass broodstock relationship identification method to select broodstock with distant genetic relationship;

[0021] B3. Place the selected broodstock in sea cages for intensive cultivation and feed them fresh squid, bonito flakes and shelled oysters twice a day at a feeding amount of 3% to 5% of the broodstock body weight to accelerate gonadal maturation.

[0022] (2) Artificial induction of spawning of red nine-spined perch, comprising the following steps:

[0023] C1. Artificial induction of spawning was performed on male and female red snapper with mature gonads. The oxytocin for female fish was 200-300 IU / kg human chorionic gonadotropin (HCG) and 20-25 μg / kg luteinizing hormone-releasing hormone analog (LHRH-A2). The oxytocin dose for male fish was halved.

[0024] C2. After 30 hours of artificial induction of labor, egg collection and fertilization are carried out. There are two ways of egg collection and fertilization: the first way is artificial squeezing of eggs and sperm, and the dry method is used for artificial insemination. Successfully fertilized eggs will float on the water surface, while dead eggs, bad eggs, and eggs of poor quality will sink to the bottom of the water. Use a scoop net or an egg collection net to collect the floating fertilized eggs, place them in an incubation bucket for incubation, and then directly spread them into the indoor nursery pond. The proportion of floating fertilized eggs is 80-85% of the total eggs. The second way is to collect the fertilized embryos produced by natural spawning in the broodstock cages, wash them with fresh seawater, and then spread them into the nursery pond.

[0025] (3) Hatching and raising fertilized eggs of red nine-spined perch in an indoor nursery pond includes the following steps:

[0026] D1. Disinfect with bleach for 16-40 minutes before placing eggs 2 The nursery pond is filled with 1m deep filtered and disinfected fresh seawater (filtered with a 100-mesh filter bag), and aerated using an air pipe and air stones. The density of air stones is 1 / m 2 ; 12 hours before laying eggs, the water quality is regulated by spraying organic acid throughout the pond; water quality indicators are: water temperature 25~29℃, seawater specific gravity 1.022~1.024 (salinity 29~32), pH 7.5~7.8; artificial light source is installed above the nursery pond, and the light intensity is controlled at 6000~8000lx; according to 5~10g / m 3 The density of the water body will collect the fertilized eggs and place them in the nursery pond;

[0027] The nursery pond is a 4m×4m×1.2m square cement pond with rounded corners, and the egg laying time is 2 to 16 hours after fertilization.

[0028] D2, fertilized eggs can hatch and become fry after 24 hours of incubation. At this time, the gas volume should be reduced in time to make the water in the pond slightly boiling, reduce the energy consumption of fry, and avoid damage to fry due to excessive gas volume. Add oyster fertilized eggs and SS-type rotifers to the nursery pond as starter bait and fresh Chlorella to maintain the rotifer density 2 days after the fry hatch. Eye spots appear 3 days after the fry hatch, the yolk sac is consumed, the mouth cleft is formed, and the nutrition mode changes from endogenous nutrition to exogenous nutrition, and begins to feed on plankton in the pond.

[0029] D3. 3-7 days after the fry hatch, feed fertilized oyster eggs and SS-type rotifers daily at a density of 3-5 pieces / mL; for fry on day 8-12, feed L-type rotifers at a density of 2-3 pieces / mL; for fry on day 13-18, the feed gradually changes from L-type rotifers to copepods at a density of 1-2 pieces / mL; for fry on day 18-24, feed copepods and Artemia nauplii at a density of 1-2 pieces / mL; for juveniles on day 25-34, feed Artemia nauplii and adult copepods at a density of 1-2 pieces / mL; after day 35, feed adult Artemia at a density of 1-2 pieces / mL; for juveniles on day 40, start to add grouper compound feed, and the feeding amount is calculated based on 8-10% of fish body weight. The water quality needs to be kept stable during the fry cultivation period. The water quality management requirements are: dissolved oxygen > 5 mg / L, ammonia nitrogen < 0.5 mg / L, nitrite < 0.05 mg / L. Photosynthetic bacteria can be sprayed regularly to adjust the water quality.

[0030] D4. When the fry are 10 days old, the light intensity in the nursery pond begins to be gradually reduced, and when the fry are 15 to 18 days old, the pond enters a state of complete lightlessness.

[0031] D5. When the fry grow to 45 days old (body length is about 3~5cm), they can be acclimated and routinely cultured in the cement pool of the indoor workshop. Before the fry completely consume artificial compound feed, they need to be gradually acclimated by mixing biological bait (copepods, adult Artemia, etc.) with artificial compound feed. After the fry completely consume artificial compound feed, they can be routinely cultured to commercial fish specifications. During this period, attention should be paid to daily management such as disease prevention and control. The daily feeding amount is 8~10% of the fish body weight.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1. The method described in the present invention proposes a method for identifying the kinship of red nine-spined seabass broodstock. Starting from the source, SNP loci are used to identify the kinship between red nine-spined seabass broodstock, and individuals with distant kinship are used as breeding broodstock, thereby avoiding the problem of germplasm decline caused by inbreeding and ensuring the excellent germplasm and sustainable development of breeding.

[0034] 2. The present invention establishes a standard method for fully indoor factory cultivation of early red nine-spined seabass seedlings, clarifies the nutrients required for red nine-spined seabass fry and juveniles at different developmental stages, and provides palatable bait and appropriate bait density, which can effectively reduce the probability of large size differences among the same batch of fry due to insufficient feeding and cannibalism, as well as the probability of enteritis caused by excessive feeding, and further ensures the quality and survival rate of the fry.

[0035] 3. Compared with the traditional outdoor pond seedling raising method, the present invention reduces the adverse effects of external uncertain factors such as temperature changes, heavy rains, pathogenic microorganisms, etc. on seedling raising work, and can accurately regulate aquaculture water, biological bait, daily management, etc., which is conducive to the smooth hatching and seedling cultivation of red nine-spined seabass, and thus realizes large-scale fully indoor artificial seedling raising and breeding to meet market demand.

[0036] 4. The fully indoor seedling raising method of the present invention has the advantages of simple operation, easy standardization, prevention of neural necrosis virus infection in external ponds, cost savings, etc., and can improve the survival rate of seedling raising. The present invention can be implemented in modern factory workshops, which is conducive to the mass production and promotion of red nine-spined seabass seedlings. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The embryonic development process of red nine-spined seabass, including: a. 4-cell stage; b. 8-cell stage; c. 16-cell stage; d. 32-cell stage; e. 64-cell stage; f. multi-cell stage; g. mulberry stage; h. high blastocyst stage; i. low blastocyst stage; j. early gastrula stage; k. mid-gastrula stage; l. late gastrula stage; m. embryonic body formation stage; n. blastopore closure stage; o. optic sac formation stage; p. myometrium appearance stage; q. otic sac formation stage; r. brain vesicle formation stage; s. heart formation stage; t. tail bud stage; u. crystal formation stage; v. heart beating stage; w. hatching stage; x. hatching stage; y. newly hatched fry; a~w scale is 100μm, x and y scale is 500μm;

[0038] Figure 2 This is the development process of red nine-spined perch larvae, juveniles and young fish, including: a. 1-day-old larvae; b. 2-day-old larvae; c. 3-day-old larvae; d. 4-day-old larvae; e. 7-day-old larvae; f. 11-day-old larvae; g. 14-day-old larvae; h. 18-day-old larvae; i. 24-day-old larvae; j. 29-day-old young fish; k. 37-day-old young fish; l. 45-day-old young fish; m. 60-day-old young fish;

[0039] Figure 3 This is a chart showing the changes in the total length of red nine-spined perch larvae, juveniles and young fish. DETAILED DESCRIPTION

[0040] To further illustrate the present invention, the present invention will be described more comprehensively and systematically in conjunction with the application examples below. The technical terms involved in the present invention are all common terms that can be understood by those skilled in the art. In addition, unless otherwise specified, the materials and reagents used in the present invention can be purchased or prepared by themselves in a conventional manner.

[0041] Example 1

[0042] Identification of the relationship between red nine-spined perch broodstock

[0043] The DNBSEQ-T7 sequencing platform was used to obtain the sequencing reads of candidate broodstock of red nine-spined seabass. After the obtained reads were quality controlled by fastp software, the sequencing reads were aligned to the red nine-spined seabass reference genome using BWA and Samtools software. Finally, the SNP variation was detected using GATK software. The above software used default parameters. The obtained SNP variation was quality controlled using plink2 software. The quality control parameters were: --autosome-num 24, --geno 0, --maf0.1, --max-alleles 2, --hwe 0.0001. Finally, --bp-space was used to adjust the number of SNPs used for analysis to 3000. The quality-controlled genotypes were converted to 0, 1, and 2 encoding modes, and stored as a binary file "geno.bin" readable by R with a matrix data type, and the variable was named M. Run the following command in R to calculate the kinship matrix:

[0044] load("geno.bin")

[0045] MM<- M %*% t(M)

[0046] A<- diag(1, nrow(M))

[0047] LHS<- matrix(c(nrow(M)^2, sum(A), sum(A), sum(A^2)), nrow = 2, ncol =2, byrow = T)

[0048] RHS<- matrix(c(sum(MM), sum(MM * A)), nrow = 2, ncol = 1)

[0049] a<- solve(LHS, RHS)[1]

[0050] b<- solve(LHS, RHS)[2]

[0051] G<- round((MM - matrix(a, nrow = nrow(MM), ncol = ncol(MM))) / b, 3)

[0052] After running the above command, the kinship between each individual can be checked in G. The present invention uses 9 red nine-spined perch and 1 red nine-spined perch offspring to calculate the results. After running the above command, the kinship between the above 10 red nine-spined perch is shown in Table 1.

[0053] Table 1 The relationship among 10 red nine-spined sea bass

[0054]

[0055] According to the breeding records, individuals I1~I9 are wild red nine-spined seabass broodstock, I10 is the offspring of individuals I3 and I8, and the molecular relatedness coefficients between I10 and I3 and I8 are 0.252 and 0.231, respectively, indicating that the method of the present invention can determine the relatedness between individuals.

[0056] In order to avoid the problem of germplasm decline caused by inbreeding, 1 / 16 (0.0625) is used as a threshold. Individuals above this threshold are brothers and sisters and should not be bred together. In this embodiment, individuals I1 to I9 can all be used as parents for breeding, and inbreeding will not occur.

[0057] Example 2

[0058] Parent selection and breeding of red nine-spined perch

[0059] In October 2021, 2-5-year-old wild red nine-spined perch germplasm resources were introduced from the Nansha Islands, and broodstock were cultivated in a land-based cement pond with a size of 8m×8m×1.5m. The water depth is 1.2m, the water temperature is 25~29℃, and the stocking density is 1~3 / m 3 Under the conditions of natural light rhythm and flowing water aeration, the broodstock are cultured to promote maturation. After one year of culture, red nine-spined perch weighing 1.5-4kg, with strong physique, smooth body, no injuries, no deformities, strong vitality, and distant relatives are selected as parents and moved to Lingshui fish rafts in Hainan in March 2023. High-protein fresh squid, bonito and shelled oysters are fed twice a day for nutritional enhancement, with a feeding amount of 5% of the broodstock body weight to promote the maturation of gonads.

[0060] Example 3

[0061] Artificial induced spawning of red snapper

[0062] Select male and female red nine-spined perch with mature gonads. The abdomen of mature female fish is swollen and soft, with obvious ovarian outlines and rosy and everted cloaca; the abdomen of mature male fish is smaller, and a small amount of milky white semen flows out when lightly pressed. Artificial induction of labor is performed on male and female red nine-spined perch with mature gonads. The oxytocin for female fish is 300IU / kg human chorionic gonadotropin HCG and 25μg / kg luteinizing hormone-releasing hormone analog LHRH-A2; the oxytocin dose for male fish is half of that for female fish. 30h after induction of labor, artificial egg and sperm are squeezed, and dry method is used for artificial insemination. Clean sperm is collected during the collection process to prevent contamination by urine and feces. Dry the fish body before collecting sperm and eggs, and wipe the utensils used with paper to prevent contact with seawater to activate sperm and eggs prematurely. After the sperm and eggs are evenly stirred, they are activated with fresh disinfected seawater. The successfully fertilized eggs will float on the water surface, while dead eggs, bad eggs, and eggs of poor quality will sink to the bottom of the water. The proportion of floating fertilized eggs is 82% of the total eggs. Use a scoop net or egg collection net to collect the fertilized eggs and place them in a hatching bucket. After washing with fresh seawater, they are placed in the pool.

[0063] Example 4

[0064] Hatching and raising fertilized eggs of red nine-spined bass in indoor nursery ponds

[0065] (1) Preparation of nursery ponds and layout of fertilized eggs: Pay attention to adjusting the amount of air in the early stage of fertilized egg hatching. The amount of air should not be too small during the hatching process, which will lead to insufficient dissolved oxygen in the water. The nursery ponds are 4 cement pools in Workshop No. 2 of Linlan Aquatic Products Co., Ltd., Wanning City, Hainan Province. The specifications are 4m×4m×1.2m. Natural seawater is used as water for nursery after filtration, sedimentation and disinfection. The water depth is maintained at 1m. Air pipes and air stones are used for inflation. The density of air stones is 1m 2 / pc. 12h before laying eggs, the water quality is regulated by spraying 1ppm of organic acid throughout the pond. Water quality indicators are: water temperature 26℃, seawater specific gravity 1.022, pH value 7.5. Artificial light source is installed above the nursery pond, and the light intensity is controlled at 6000~8000lx. According to 10g / m 3 The collected fertilized eggs are carefully placed into the pool at a density of 1.

[0066] (2) Hatching of fertilized eggs and rearing of fry: After the fertilized eggs were placed in the pond, the embryonic development process was observed. The embryonic development sequence is shown in Table 2, and the embryonic development process is shown in Figure 1. It was found that the red nine-spined perch hatched 24 hours after fertilization. The newly hatched fry had an oval translucent yolk sac at the front of the body and a small oil ball at the back of the yolk sac. The newly hatched fry floated on the water surface due to the effect of the oil ball and occasionally moved vertically upward. Pay attention to adjusting the inflation volume in the early stage of hatching. The inflation volume should not be too small during the hatching process, which will lead to insufficient dissolved oxygen in the water. The fertilized eggs can hatch and become fry after 24 hours of hatching. At this time, the gas volume should be reduced in time to make the water in the pond slightly boiling, reduce the physical energy consumption of the fry, and avoid damaging the fry due to excessive gas volume. When the fry hatched 2 days ago, oyster fertilized eggs and SS-type rotifers were added to the nursery pond as the starter bait and fresh Chlorella was used to maintain the rotifer density. Pay attention to maintaining the water transparency of 35~40cm. 3 days after the fry hatched, the yolk sac became significantly smaller and gradually disappeared, eye spots appeared, mouth cracks formed, and the nutrition mode changed from endogenous nutrition to exogenous nutrition, and began to feed on plankton in the pond. Reduce the light intensity when the fry are 10 days old, and stop the light when the fry are 15-18 days old.

[0067] Table 2 Time sequence of embryonic development of red nine-spined perch

[0068] Time after fertilization Embryonic development period Water temperature (℃) Main morphological features Survival rate (%) 56min 4-cell stage 25.5 The second cleavage is perpendicular to the first cleavage 99.4 1h8min 8-cell stage 25.6 The third cleavage is parallel to the first cleavage 98.8 1h27min 16-cell stage 25.3 The fourth cleavage 98.8 1h50min 32-cell stage 25.3 The fifth cleavage 98.4 2h16min 64-cell stage 25.3 The sixth cleavage has a disordered cleavage plane 98.4 2h40min Multicellular stage 25.3 The cells become smaller and arranged irregularly 98.0 3h8min Mulberry stage 25.1 The number of cells increases, and the cell clusters resemble mulberry balls 97.7 3h50min High blastocyst stage 24.7 The blastocyst bulges on the yolk, forming a high hat shape 97.4 5h10min Low blastocyst stage 24.2 The blastoderm becomes lower and moves toward the vegetal pole 96.8 7h10min Early gastrula 24.2 The embryonic ring and crescent-shaped embryonic shield can be seen from the side 95.5 9h32min Mid-gastrula 24.9 The germ layer covers 1 / 2 of the yolk, and the embryo shield becomes longer 93.0 10h20min end gastrula 25.4 The germ layer covers 3 / 4 of the yolk, and the embryo shield grows 91.7 11h15min Embryo formation stage 25.4 A notochord appears in the central line, forming the embryonic body. 90.7 12h10min Blastopore closure stage 25.4 The germ layer is enclosed, and the blastopore is closed 90.0 13h2min Optic capsule formation period 25.7 Optic vesicle appears in the embryo head 89.6 14h20min Myometrium emergence 25.7 Myotome 87.7 15h16min Ottocyst formation stage 26.3 A pair of auditory vesicles appear behind the optic vesicle of the embryo 87.5 17h22min Brain vesicle formation stage 26.3 Brain vesicles form in the middle of the optic vesicle 87.0 18h11min Heart formation period 26.3 Heart formation, clear outline 86.8 19h18min tail bud stage 26.3 Separation of tail bud and yolk sac 86.3 21h26min Crystal formation period 25.6 The crystal outline gradually becomes clear, and the embryo begins to vibrate. 84.5 22h20min Heart beat period 25.6 The heart starts beating 84.2 23h1min Incubation period 25.6 The embryo vibrates frequently and with large amplitude 83.4 24h05min incubation period 25.6 The fry breaks through the egg membrane from the head 83.2 24h30min Newly hatched fish 25.6 -- 83.0

[0069] (3) Feeding method: For 3-7 day old fry, fertilized oyster eggs with a size of less than 100 μm and SS-type rotifers are fed daily at a feeding density of 3-5 cells / mL; fresh Chlorella is sprayed throughout the pond twice a day to maintain the rotifer density and improve water quality, with a Chlorella concentration of 300,000-400,000 cells / mL. For 8-12 day old fry, L-type rotifers that have been fortified with Schizochytrium salina are fed at a density of 2-3 cells / mL. For 13-18 day old fry, the feed is gradually changed from L-type rotifers to copepods at a feeding density of 1-2 cells / mL. When the fry are 18-24 days old, they are fed with adult copepods and Artemia nauplii, with a density maintained at 1-2 cells / mL. For 25-34 day old juveniles, adult Artemia and adult copepods are fed at a density of 1-2 cells / mL. After 35 days of age, fry are fed with 1-2 Artemia adults / mL. At 40 days of age, grouper compound feed is added to fry, and the feeding amount is calculated based on 8-10% of the fish body weight.

[0070] (4) Preparation of aquaculture ponds: fresh seawater is taken, filtered, precipitated and disinfected as aquaculture water. Two days before stocking, organic acid is added to adjust the water quality, and a chelating agent is used to chelate heavy metal ions in the water. In the subsequent water management process, pathogen and water quality testing should be carried out regularly to avoid threats to aquaculture work caused by aquaculture water carrying pathogens or unqualified water quality, and to ensure the safety of aquaculture water. Water quality testing indicators are: pH 7.5-7.9, dissolved oxygen>5mg / L, ammonia nitrogen<0.5mg / L, nitrite<0.05mg / L; in case of rainy weather or drastic temperature changes, aquatic VC should be used appropriately to prevent or relieve fry stress and avoid adverse effects. The daily water change volume depends on the growth of the fry. During the frying period, the aquaculture workshop, frying tools and frying workers must be strictly disinfected to prevent cross contamination and disease.

[0071] Through the above embodiments, the hatching rate of fertilized eggs reached 80%, the average total length of 30-day-old red nine-spined seabass fry was 3.0±0.15 cm, the average weight was 0.93±0.18 g, and the average total length of 60-day-old fry was 6.3±0.35 cm, and the average weight was 9.83±1.55 g; this shows that the indoor breeding method for red nine-spined seabass described in the present invention has achieved good results.

Claims

1. A method for identifying the relationship of red nine-spined sea bass broodstock, characterized in that: The following steps are involved: A1. Obtaining single nucleotide polymorphism sites of candidate broodstock of red nine-spined perch; A2, using the single nucleotide polymorphism sites obtained in A1 to calculate the kinship coefficients between candidate parent fish of red nine-spined seabass, and construct a molecular kinship matrix; 1) Convert the genotype matrix into a pedigree-based kinship matrix Regression in the middle, we get formula 1: Formula 1; Among them, the matrix for The genotype matrix contains Individual The genotype information of the single nucleotide polymorphism site, the genotype AA / Aa / aa is represented by 0 / 1 / 2 respectively; the matrix For the matrix The transpose of and are the intercept and slope of formula 1 respectively; the matrix is a kinship matrix based on pedigree information. If the kinship between individuals is unknown or there is no pedigree record, the matrix is The identity matrix representation of is the residual term; 2) Matrix The expectation is the matrix , and the matrix It obeys a normal distribution with a mean of 0, and the molecular kinship matrix Calculate using formula 2 to get formula 2: Formula 2; 3) Multiply both sides of equation 1 by the matrix , combined with the matrix properties, we get formula 3: Formula 3; Among them, in formula 3 represents the total number of individuals used for analysis; the matrix Represents an individual and individuals Pedigree-based kinship matrices; 4) Solve the coefficients according to formula 3 and , and then use formula 2 to calculate the molecular affinity matrix ; According to the calculated matrix , select individuals with small inter-individual kinship coefficient to construct a breeding population; In step A2, the breeding population is constructed to meet the following conditions: the coefficient of relatedness between individuals is ≤ 1 / 16.

2. The method for identifying the relationship of red nine-spined seabass broodstock according to claim 1, characterized in that: In step A1, the single nucleotide polymorphism site meets the following conditions: the number of sites ≥ the total number of candidate broodstock, evenly distributed in the genome, no genotype missing, conforming to Hardy-Weinberg equilibrium, the minimum allele frequency > 0.1, being a biallelic site, and located on an autosome.

3. A method for raising red nine-spined sea bass in indoors, comprising selecting and raising red nine-spined sea bass broodstock, artificially inducing spawning of red nine-spined sea bass, and incubating and raising fertilized red nine-spined sea bass eggs in an indoor breeding pool, characterized in that: In the selection and cultivation of red nine-spined seabass broodstock, broodstock of 2 to 5 years old, weighing 1.5 to 4 kg, and with an inter-individual kinship coefficient of ≤1 / 16 are selected, and the kinship is obtained by the red nine-spined seabass broodstock kinship identification method described in claim 1; the broodstock are subjected to intensive cultivation, and the feed includes squid, bonito flakes and shelled oysters, and the feeding amount is 3% to 5% of the broodstock body weight.

4. The method for raising red nine-spined sea bass indoors according to claim 3, characterized in that: In the artificial induction of red ninethorn seabass, the oxytocin for female fish is 200~300IU / kg human chorionic gonadotropin and 20~25μg / kg luteinizing hormone-releasing hormone analogue; the oxytocin for male fish is the same as that for female fish, and the dosage is 1 / 2 of that for female fish.

5. The method for raising red nine-spined sea bass indoors according to claim 3, characterized in that: During the hatching and raising of fertilized eggs of red nine-spined seabass in the indoor nursery pond, the preparation work for the nursery pond includes: disinfection with bleaching powder before laying eggs, injecting fresh seawater that has been filtered and disinfected, and inflating with an air tube and air stone; the water quality is adjusted by spraying organic acid throughout the pond, and the water quality indicators are: water temperature 25~29℃, seawater specific gravity 1.022~1.024, pH 7.5~7.8; an artificial light source is installed above the nursery pond, and the light intensity is 6000~8000lx.

6. The method for raising red nine-spined sea bass indoors according to claim 5, characterized in that: The egg laying time is 2 to 16 hours after fertilization, and the egg laying density is 5 to 10 grams of fertilized eggs per cubic meter of water.

7. The method for raising red nine-spined sea bass indoors according to claim 6, characterized in that: When the fertilized eggs hatch and become fry, the gas volume is reduced; 2 days before the fry hatch, oyster fertilized eggs and SS-type rotifers are added to the nursery pond as starter bait, and fresh Chlorella is used to maintain the rotifer density.

8. The method for raising red nine-spined sea bass indoors according to claim 7, characterized in that: The feeding method for fry is as follows: for 3-7 day old fry, feed oyster fertilized eggs and SS-type rotifers at a density of 3-5 pieces / mL; for 8-12 day old fry, feed L-type rotifers at a density of 2-3 pieces / mL; for 13-18 day old fry, feed copepods at a density of 1-2 pieces / mL; for 18-24 day old fry, feed copepods and Artemia nauplii at a density of 1-2 pieces / mL; for 25-34 day old juveniles, feed Artemia nauplii and copepod adults at a density of 1-2 pieces / mL; after 35 days old, juveniles are fed Artemia adults at a density of 1-2 pieces / mL; for 40 day old juveniles, grouper compound feed is added, and the feeding amount is 8-10% of the fish body weight.

9. The method for raising red nine-spined sea bass indoors according to claim 8, characterized in that: The light control method during fry cultivation is as follows: the light intensity is 6000~8000lx when the fry are 1 to 10 days old; the light intensity begins to gradually decrease after the fry are 10 days old, and there is no light when the fry are 15 to 18 days old; the water quality management requirements during fry cultivation are: dissolved oxygen>5mg / L, ammonia nitrogen<0.5mg / L, and nitrite<0.05mg / L.

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