A method for establishing a fish germplasm resource bank

Abstract

The present invention relates to a method for establishing a fish germplasm resource bank, and belongs to the field of biological germplasm resource protection technology. The present invention discloses a method for establishing a fish germplasm resource bank, which establishes a complete fish germplasm resource bank by investigating fish germplasm resources and habitats, determining target fish, determining the type of fish germplasm resource bank sub-bank, determining the minimum sample storage volume of the fish germplasm resource bank sub-bank, sample collection, sample processing, sample storage, regular sample quality testing, establishing a fish germplasm resource database and fish germplasm resource data management, and preserving and sharing fish germplasm resource information. The present invention can realize the digitization and informatization of fish germplasm resources, and carry out hierarchical, multi-dimensional, and full-coverage management of information data of fish germplasm resources, facilitate the query, exchange, and sharing of resources, improve resource utilization efficiency, promote transparent information exchange and scientific research cooperation, and provide technical support for my country's seed source security and seed industry revitalization construction.

Description

Technical Field

[0001] The invention relates to a method for establishing a fish germplasm resource bank, and belongs to the technical field of biological germplasm resource protection. Background Art

[0002] Currently, with increasing human interference, climate change, and shifts in agricultural practices, the risk of local breeds disappearing is increasing, population numbers and regional distribution are shifting significantly, and wild resources are dwindling dramatically. Once they disappear, the superior genes and cultural heritage they contain will also vanish, impacting biodiversity and causing inestimable losses. As a crucial strategic resource, aquatic genetic resources have a significant impact on people's lives and socioeconomic development. Protecting aquatic genetic resources is crucial for preserving aquatic biodiversity and maintaining the sustainable development of the aquaculture industry, and is therefore of great significance and importance.

[0003] The development of fish germplasm banks is a key strategic measure for biodiversity conservation, particularly for rare and endangered species. In recent years, China has accelerated the development of germplasm banks for rare and endemic fish species. However, issues such as incomplete construction, lack of detailed data, unsystematic processes, low visibility, inefficient retrieval, and inadequate management exist in fish resource display and sample management. Summary of the Invention

[0004] In order to solve the problems existing in the above-mentioned prior art, the present invention provides a method for establishing a fish germplasm resource bank, which can collect and protect the fish germplasm resources in the river basin at multiple levels, and construct fish germplasm resource banks at different levels such as living banks, specimen banks, cell banks and nucleic acid banks. Moreover, the present invention can further strengthen the standardized construction of resource banks and their information display and management.

[0005] To achieve the above-mentioned purpose, the present invention provides a technical solution: a method for establishing a fish germplasm resource bank, comprising the following steps:

[0006] (1) Investigate fish germplasm resources and habitats: Conduct an investigation of fish germplasm resources and habitats in the basin by combining field investigations with the review of relevant data, and obtain information on fish species diversity, population structure, and temporal and spatial distribution; understand the living habits and hydrological characteristics of fish through historical data analysis and on-site surveys, and obtain an overview of the habitat distribution of fish spawning grounds, feeding grounds, and wintering grounds;

[0007] (2) Determine target fish species: select target fish species for the fish germplasm resource bank based on the species protection level, representativeness and relative importance index of fish species in the water area;

[0008] (3) Determine the sub-bank type of fish germplasm resource bank: Determine the sub-bank type of germplasm resource bank corresponding to each target fish species based on the endangered status of each target fish species;

[0009] (4) Determine the minimum sample storage volume of the fish germplasm resource bank sub-banks: Based on the difficulty of constructing each sub-bank, calculate the minimum sample storage volume of the seven sub-banks for each target species;

[0010] (5) Sample collection: Collect the fish samples selected in step (2) accordingly; collect fish samples from wild fishing, aquatic origin, breeding farms, seedling breeding farms and farms;

[0011] (6) Sample processing: The samples collected in step (5) are processed according to the different requirements of the living body bank, specimen bank, tissue bank, gene bank, sperm bank and cell bank to meet the sample preservation requirements of the living body bank, specimen bank, tissue bank, gene bank, sperm bank and cell bank and obtain corresponding information;

[0012] A. Sample processing of living body bank:

[0013] a. Transportation and transfer: Use appropriate containers and water quality conditions to safely transport collected fish individuals to live storage, avoiding overcrowding and prolonged transportation to reduce stress and mortality during transportation;

[0014] b. Temporary holding: Collected fish will be temporarily placed in suitable temporary holding facilities, ensuring that appropriate water conditions, diet and living space are provided to promote individual adaptation and health;

[0015] c. Health assessment and screening: Conduct a health assessment on each individual to check for diseases, injuries, or other health issues. Select healthy individuals for final storage, and avoid introducing sick or unsuitable individuals into the living bank.

[0016] d. Pre-banking adaptability assessment: Before individuals enter the live bank, an adaptability assessment is conducted to ensure they can adapt to the new environmental conditions and social structure. The adaptability assessment should at least include behavioral observations, food intake, and social interaction monitoring.

[0017] e. Entry and Identification: Individuals meeting the criteria are formally introduced into the living organism bank and identified. The living organism bank identification shall at least include the individual number, collection information, and health status;

[0018] f. Health Monitoring: Regularly inspect and evaluate the health and reproductive status of individuals in the live bank, and adjust feeding and management strategies based on the monitoring results to maximize the survival and reproductive success rates of individuals in the live bank;

[0019] B. Specimen library sample processing:

[0020] There are many types of fish specimens, mainly including immersed specimens, skinned specimens, skeletal specimens and transparent specimens. Immersed specimens are specimens in which the whole or part of the fish is directly placed in various chemicals for fixation and preservation; skinned specimens refer to specimens in which the fish skin is preserved by various methods and then filled with various materials to restore it to its original state; skeletal specimens refer to specimens in which the fish viscera and muscle tissue are removed by various methods, leaving only the bones, and the bones are degreased, bleached and reshaped in series by chemicals; transparent skeletal specimens refer to small fish that have been degreased, transparentized and stained with chemicals, so that their hard bones and cartilage tissues can be clearly observed; the processed specimens are placed in the specimen library and marked. The specimen library mark includes at least the specimen number, specimen type and specimen fish species;

[0021] C. Tissue Bank Sample Processing:

[0022] a. After putting on disposable rubber gloves, the collector first uses a flame to burn the front end of the scissors that have been cleaned with alcohol. After it cools, cut the fins or muscle tissue of the specimen;

[0023] b. Then, using alcohol-cleaned tweezers, place the sample into a pre-prepared 2ml centrifuge tube, screw on the tube cap, and place it in a cryostat. The tube should contain a label and at least 95% ethanol.

[0024] c. Repeatedly clean the tools before removing the next piece of molecular tissue material to avoid cross contamination;

[0025] D. Gene bank sample processing:

[0026] a. Sample sampling: For the aquatic organisms for which genomic DNA libraries are to be constructed, the number of samples to be sampled shall be determined in accordance with the provisions of GB / T18654.2;

[0027] b. Extraction of genomic DNA: Extract genomic DNA using traditional genomic DNA extraction methods or commercial genomic DNA extraction kits;

[0028] c. Genomic DNA processing: Depending on the selected vector, the genomic DNA is digested with appropriate restriction endonucleases to produce ends that are compatible with the vector;

[0029] d. Detection and recovery of DNA enzyme slices of appropriate size: DNA fragments obtained by cleavage were detected by agarose gel electrophoresis and observed with a gel imager. The enzyme-digested segments of appropriate size were recovered using low-melting-point agarose gel;

[0030] e. Vector selection: Select an appropriate vector based on the size of the target genomic region, the difficulty of screening the library, and the copy number of the vector;

[0031] f. Ligation of genomic DNA fragments and vectors: Use ligase to connect the above DNA fragments of appropriate size into the vector;

[0032] g. Transformation of the recombinant vector into host cells and identification of positive clones: The recombinant vector is transformed into competent host cells, and positive clones are screened using the blue-white color of the colonies as an indicator;

[0033] E. Sperm bank sample processing:

[0034] a. Use a towel or paper towel to dry the abdomen and genital area of mature male fish. Gently press the abdomen to expel urine. Use a dry pipette to collect the expelled semen and place it in a clean, dry, graduated glass bottle or test tube. Avoid direct sunlight during handling and transportation.

[0035] b. Microscopic examination: observe three times and take the average of the results. Semen with a sperm survival rate of 80% or above can be used for cryopreservation. The sperm survival rate is the percentage of motile sperm to total sperm.

[0036] c. Select appropriate cryopreservation diluent and antifreeze based on the fish species. Add the cryopreservation diluent after semen collection and microscopic examination. Mix the semen of different fish species with the pre-chilled diluent in appropriate proportions and place in a 4°C refrigerator for 20-30 minutes to equilibrate.

[0037] d. Aliquot the equilibrated semen with diluent in 1.0-1.5 mL portions into 2 mL cryovials and cryopreserve in liquid nitrogen using a three-step freezing procedure.

[0038] e. When thawing, first remove the cryovial from liquid nitrogen and place it in liquid nitrogen vapor for 2-3 minutes to equilibrate. Then, remove it from the liquid nitrogen tank and place it in a 37°C water bath for rapid thawing or thaw it at room temperature. After the frozen sperm is thawed, dry insemination is performed with fresh eggs. Pour an appropriate amount of frozen semen onto the eggs and gently stir to mix. Add fresh water or seawater equivalent to 2-3 times the volume of the eggs to activate the sperm. After standing for 10 minutes, add 5-10 times the volume of water to wash away dead eggs and excess sperm. Place the fertilized eggs in a thaw incubator for insemination.

[0039] F. Cell Bank Sample Processing:

[0040] a. Wash the collected fish tissue blocks with PBS at least three times in a clean bench;

[0041] b. Transfer the tissue to a penicillin vial containing L-15 medium. Cut the tissue into 1 mm pieces with scissors and wash three times with PBS.

[0042] c. Soak with a small amount of L-15 complete medium and inoculate evenly on a 25cm 2Culture flasks: record cell information on the side of the flask, including tissue type, time, and culture generation, and place it upside down in an incubator at a suitable temperature for culture;

[0043] d. After 10-12 hours, add 3 ml of complete culture medium and begin upright culture. Change the medium every 2-3 days during the primary culture period.

[0044] e. Subculture: The first subculture is performed when the cells have grown to 95% of the bottom of the cap and adhere to the wall. The first subculture is performed in the original bottle. Subculture is performed at a ratio of 1 to 2 for subsequent subcultures. The original culture medium is aspirated, and after washing with PBS, an appropriate amount of trypsin is added to digest the cells. When the cells shrink and become round, and are about to detach from the wall, the trypsin is aspirated and the culture medium is added and pipetted several times until the cells detach from the wall.

[0045] (7) Sample storage: The method for storing samples in a living body bank is to build a fish pond for ex situ conservation; the method for storing samples in a specimen bank is to place the specimen or organ in a 10% formalin solution, replace the solution several times until it is fixed, and then place it in a 5% formalin solution for another 2-3 weeks, and finally place it in a glass specimen bottle with a preservation solution, and seal it with vaseline or paraffin to prevent the fixative from evaporating or leaking; the method for storing samples in a tissue bank is to place the collected tissue sample in a 2ml centrifuge tube containing more than 95% ethanol and store it at -20℃ or -70℃; the method for storing samples in a gene bank is to dissolve the genomic DNA that meets the requirements in TE with a pH value of 8.0 Storage: The sperm bank sample storage method is to add the balanced semen to the diluent in 1.0mL~1.5mL portions into 2mL cryovials, and then freeze in liquid nitrogen according to the three-step cooling and freezing mode. The cell bank sample storage method is to aspirate the culture medium, rinse the cells with phosphate buffer, and then add an appropriate amount of trypsin for digestion. When the adherent cells are small and round and about to detach from the wall, aspirate the trypsin, add an appropriate amount of culture medium and pipette to detach the cells. The cell suspension after pipetting is mixed with an equal volume of cell cryopreservation culture medium, and the mixture is placed in a cryovial. The cells are first placed in a 4℃ refrigerator for 30 minutes, then in a -80℃ refrigerator for 2 hours to 4 hours, and finally the cell cryovials are stored in liquid nitrogen.

[0046] (8) Regular testing of sample quality: After samples are put into storage, three samples are randomly selected from each batch for testing. The quality of samples is regularly evaluated and identified, and samples that do not meet the requirements are eliminated and replenished;

[0047] Live stock library: Regularly test the health status, survival rate and growth rate of live fish. If the fish are sick, the samples in that batch will be removed and replenished;

[0048] Specimen library: regularly remove specimens from the specimen tank and observe changes in specimen morphology. If the morphology changes significantly or the specimens are severely decayed, the batch of samples will be discarded and replenished;

[0049] Tissue bank: Regularly check whether tissue samples in the refrigerator are sealed, stored at -20°C or -70°C, and whether they have been frozen and thawed multiple times. Repeated freezing and thawing will result in smaller genomic DNA fragments and reduced extraction quality. If the freezer is not sealed, the temperature does not meet the standard, or the number of freeze-thaw cycles exceeds 5, the batch of samples should be discarded and replenished.

[0050] Gene bank: DNA samples are regularly taken out of the refrigerator for concentration, purity and integrity testing;

[0051] The benzene ring structure of the bases on the DNA chain has strong absorption in the violet region, and its absorption peak is at 260nm. By detecting the absorbance values of DNA samples at wavelengths of 260nm and 280nm:

[0052] An A260 / A280 ratio of 1.8-2.0 indicates good purity; an A260 / A280 ratio < 1.8 indicates residual protein impurities in the nucleic acid extraction or a very low sample concentration; an A260 / A280 ratio > 2.0 indicates partial DNA degradation or RNA contamination.

[0053] The A260 / A230 ratio of pure nucleic acid should be greater than 2.0. The DNA concentration can be calculated based on the purity test. Generally, a DNA sample concentration of 100-300 ng / uL is conducive to long-term DNA storage.

[0054] The integrity of DNA was analyzed by gel electrophoresis. A bright band near the sample well without impurities or tailing indicated that the DNA molecule was intact and not degraded. A bright band in the sample well indicated that there was protein residue in the DNA solution. Smearing and tailing in the lane indicated that the DNA was degraded.

[0055] If the DNA sample concentration is lower than 100 ng / uL, it should be removed and replenished; if the A260 / A280 ratio in the DNA sample is greater than 2.0, it should be removed and replenished; if the DNA sample integrity is poor, such as diffusion or tailing in the lane, it should be removed and replenished;

[0056] Sperm bank: Sperm samples are taken out of the liquid nitrogen tank regularly for thawing and recovery, and the motility of the sperm after freezing and recovery is tested. If the recovery rate is less than 50%, the batch of samples will be discarded and replenished;

[0057] Cell bank: Cell samples are regularly taken out of the liquid nitrogen tank for thawing and recovery. The recovery method is to randomly select three samples from each generation of cells for recovery culture. After the cells have re-divided, the degree of cell deformity of the passaged cells is observed under a microscope. If all cells show deformity, all samples of the cell generation are discarded and replenished;

[0058] (9) Establishing a fish germplasm resource database: Based on the various data obtained in steps (1) to (7), a complete fish germplasm resource database is established. The fish germplasm resource database is constructed with the spatial distribution data of fish germplasm resources, the spatial distribution data of fish habitats, and the target fish germplasm resource bank data as the first-level categories. The specific construction method is as follows:

[0059] S1. Collect basic data on fish germplasm resources and habitats: Set the geographical area to be studied for the fish germplasm resources database to be constructed, and obtain basic data on fish germplasm resources and habitats within the geographical area through actual surveys;

[0060] S2. Establish spatial distribution data of fish germplasm resources: pre-process the fish germplasm resources data, form a basic database with spatial data as the main body, and separate and obtain the spatial distribution data of fish germplasm resources;

[0061] The spatial distribution data of fish germplasm resources are extracted according to the first-level fish germplasm resource spatial distribution data type and refined into second-level and third-level data types. The second-level data type includes all species in the study area, and the third-level data type is divided into abundance and biomass data according to the biological data type. The data structure of the spatial distribution data of fish germplasm resources set based on the basic database includes the name of the administrative region, the administrative region code, the type and code of fish germplasm resources, the second-level type and code of fish germplasm resources, the third-level type and code of fish germplasm resources spatial distribution data, and the tail number of catch per unit fishing effort.

[0062] S3. Establishing spatial distribution data of habitats: Pre-processing fish habitat data, forming a basic database with spatial data as the main body, and separating and obtaining spatial distribution data of fish habitats;

[0063] Fish habitat spatial distribution data are extracted according to the first-level fish habitat spatial distribution data type and refined into second-level and third-level data types. The second-level data type includes all species in the study area, and the third-level data type is divided into spawning grounds, feeding grounds, and wintering grounds according to the habitat data type. The data structure of fish habitat spatial distribution data set based on the basic database includes administrative region name, administrative region code, fish habitat spatial distribution data and code, fish habitat spatial distribution data second-level type and code, fish habitat spatial distribution data third-level type and code, and map area.

[0064] S4. Establish target fish germplasm resource bank data: process and analyze target fish germplasm resource bank data to obtain fish habitat spatial distribution data;

[0065] The spatial distribution data of fish habitats are extracted according to the data type of the first-level target fish germplasm resource bank and refined into second-level and third-level data types. The second-level data type includes living bank, specimen bank, tissue bank, gene bank, sperm bank and cell bank. The third-level data type is divided into sample marking information, sample processing information and sample genetic information.

[0066] (10) Fish germplasm resource data management: Based on the database in step (9), a fish germplasm resource data management system is established, including a container management system, a sample management system, a data management system and a security management system; the container management system is used to manage the equipment data for storing samples; the sample management system at least includes managing the increase, decrease and status of different types of samples, automating the sample information entry and maintenance process through fish wireless radio frequency identification chip technology, recording the use and maintenance history of samples, monitoring the status of the resource library in real time, recording the increase and decrease changes, and providing an interface for querying information of various resource libraries; the data management system implements the processing and storage logic of genetic and information data, designs the data distribution and access interface, and ensures the security and integrity of the data; realizes the structured and persistent storage of data, and designs and develops the logical functions of the online resource sharing platform; the security management system determines the security risks and protection requirements of the system through a comprehensive analysis of the architecture of the information system, the business carried and the system classification, and determines the security protection measures based on the corresponding protection requirements, balancing the relationship between security, cost and efficiency.

[0067] Further improvements to the above scheme are:

[0068] The type of germplasm resource bank sub-bank corresponding to each target species in step (3) is determined as follows:

[0069] Extinct species: building a specimen library;

[0070] Extinct species in the wild: building a specimen library;

[0071] Critically endangered species: building a specimen library;

[0072] Endangered species: building specimen and tissue banks;

[0073] Vulnerable species: build specimen banks, nucleic acid banks, and tissue banks;

[0074] Low-risk species: build specimen banks, nucleic acid banks, tissue banks, sperm banks, and cell banks;

[0075] Least Concern Species: Establish specimen bank, nucleic acid bank, tissue bank, sperm bank, cell bank, and living organism bank;

[0076] In step (4), except for extinct species, species extinct in the wild, and critically endangered species, the minimum sample storage volume of the germplasm resource bank sub-banks of other species is based on the minimum sample quantity of the specimen bank. The specific formula is:

[0077] The number of samples in a tissue bank = the number of samples in a specimen bank * 10; the number of samples in a gene bank = the number of samples in a specimen bank * 10; the number of samples in a sperm bank = the number of samples in a specimen bank; the number of samples in a cell bank = the number of samples in a specimen bank; for species of least concern, the minimum number of parents and backup parents in a live bank is the minimum production capacity requirement of the provincial aquatic breeding farm;

[0078] When processing tissue bank samples to obtain materials for transcriptome experiments in step (6), first use a scalpel or scissors combined with tweezers to quickly remove the gills, brain, heart, and liver of anesthetized or recently killed fish specimens and place them in pre-cooled cryovials. Then, place them in a small liquid nitrogen tank, a -80°C freezer, or dry ice for temporary storage. The entire process is performed under strict sterile conditions and on dry ice or ice. The obtained molecular experimental materials should be promptly placed in a -80°C refrigerator for long-term storage.

[0079] The process of generating the three-level class data in step (9) is as follows:

[0080] 1) Living Organ Bank:

[0081] Sample labeling information for the biobank: Assign a unique identifier to each sample to facilitate identification and management in the database; record the basic information, species name, common name, and subspecies information of the sample corresponding to each identifier; record the collection date, location, and collector of each sample; record the biological characteristics of each sample, including at least sex, age, and reproductive status; record the minimum sample storage volume data and actual sample storage volume data for each sample;

[0082] Live bank sample processing information: record the environment type and water quality when the sample was collected, the water environment conditions during transportation and transfer, the health status before storage, the adaptability assessment data before storage, the health monitoring data after storage, the water quality management after storage, the feed type and feeding frequency, disease inspection and nutrition control methods;

[0083] Genetic information of living bank samples: record genetic characteristics, breeding rules and reproduction strategy information;

[0084] 2) Specimen library:

[0085] Specimen library sample labeling information: Assign a unique identifier to each sample to facilitate identification and management in the database; record the basic information, species name, common name and subspecies information of the sample corresponding to each identifier; record the collection date, location and collector of each sample; record the minimum sample storage volume data and actual sample storage volume data of each sample;

[0086] Sample processing information of the specimen library: record the environment type and water quality when the sample was collected; record the specimen collection and preparation methods;

[0087] Genetic information of specimens in the specimen bank: record the taxonomic status, morphological characteristics and habitat preference of fish;

[0088] 3) Tissue Bank:

[0089] Tissue bank sample labeling information: Assign a unique identifier to each sample to facilitate identification and management in the database; record the basic information, species name, common name, and subspecies information of the sample corresponding to each identifier; record the collection date, location, and collector of each sample; record the fish sample part information; record the minimum sample storage volume data and actual sample storage volume data of each sample;

[0090] Tissue bank sample processing information: record the environment type and water quality when the sample was collected; record the tissue collection and freezing preservation methods;

[0091] Genetic information of tissue bank samples: records fish tissue structure, pathological signs and tissue-specific expression information;

[0092] 4) Gene Bank:

[0093] Gene bank sample identification information: Assign a unique identifier to each sample to facilitate identification and management in the database; record the basic information, species name, common name, and subspecies information of the sample corresponding to each identifier; record the collection date, location, and collector of each sample; record genotype information; record the minimum sample storage volume data and actual sample storage volume data of each sample;

[0094] Gene bank sample processing information: record the fish's length, weight, age, gonadal development, environment type and water quality at the time of sample collection; record the gene sample collection and freezing storage methods;

[0095] Genetic information of gene bank samples: record gene library information;

[0096] 5) Sperm Bank:

[0097] Sperm bank sample labeling information: Assign a unique identifier to each sample to facilitate identification and management in the database; record the basic information, species name, common name and subspecies information of the sample corresponding to each identifier; record the collection date, location and collector of each sample; record the sperm storage volume of each sample; record the minimum sample storage volume data; record the actual sample storage volume data;

[0098] Sperm bank sample processing information: record the fish's length, weight, age, gonadal development, environment type and water quality at the time of sample collection; record the sperm collection and freezing preservation methods;

[0099] Genetic information of sperm bank samples: record the normal sperm morphology rate, sperm motility, fertilization rate, hatching rate and sperm membrane integrity rate before and after cryopreservation;

[0100] 6) Cell Bank:

[0101] Cell bank sample labeling information: Assign a unique identifier to each sample to facilitate identification and management in the database; record the basic information, species name, common name, and subspecies information of the sample corresponding to each identifier; record the collection date, location, and collector of each sample; record cell type information; record the minimum sample storage volume data and actual sample storage volume data of each sample;

[0102] Cell bank sample processing information: record the fish's length, weight, age, gonadal development, environment type, and water quality at the time of sample collection; record the cell collection, cell culture, and cryopreservation methods;

[0103] Genetic information of cell bank samples: records fish cell morphological characteristics, cell growth characteristics and chromosome karyotype.

[0104] The container management system in step (10) at least includes daily data management of ultra-low temperature refrigerators, liquid nitrogen tanks and freezing tubes; monitoring the status, temperature, humidity and liquid level of storage devices; managing and allocating storage space; real-time status query and abnormal status alarm.

[0105] The specific solution for the security management system in step (10) is as follows:

[0106] 1) Authentication and Authorization: Use the Web Token mechanism to implement user authentication and authorization, ensuring that only legitimate users can access system functions;

[0107] 2) HTTPS: Use the HTTPS protocol in front-end and back-end communications to encrypt data transmission and prevent man-in-the-middle attacks;

[0108] 3) Input validation and SQL injection prevention: Strictly validate user input to prevent malicious input and SQL injection attacks;

[0109] 4) Cross-site scripting (XSS) and cross-site request forgery (CSRF) protection: Implementing Content Security Policy (CSP) to restrict how browsers load and execute external resources effectively prevents XSS attacks. Each user request includes a unique CSRF token, which the server verifies upon receiving the request to ensure the legitimacy of the request, effectively preventing CSRF attacks. These methods ensure user data and session security.

[0110] 5) Regular security reviews: Perform regular security reviews of the system to ensure that the system is consistent with the latest security standards and best practices.

[0111] In step (10), the fish germplasm resource data management system adopts a multi-layer architecture, where each layer is responsible for a specific function and communicates with other layers through clearly defined interfaces, thereby improving the modular maintainability and scalability of the system;

[0112] The system is divided into five levels based on the order of functional interaction;

[0113] 1) Access layer:

[0114] The user access device and the interaction between the information library and the system belong to the access layer. The components used in this layer include API interface and ID card authentication;

[0115] Users can access the system through multiple terminals, and administrators can manage and set various system functions through the management background;

[0116] The samples in the information library are generated into a QR code by the system according to the specified coding rules. The printing device prints it and sticks it on the corresponding sample container. When sampling, the QR code can be scanned by the scanning device to obtain the sample information;

[0117] 2) Gateway layer:

[0118] The information database transmits data through the local gateway and transmits the data to the server for processing;

[0119] The monitoring indicator data of the information database is transmitted to the server through the local gateway for comparison. If the monitoring data is abnormal, an early warning reminder will be issued in time;

[0120] After users visit the web page, they interact with the server through the RESTful API to achieve front-end and back-end separation;

[0121] 3) Web layer:

[0122] The web layer is the user interface layer in the system architecture. It is mainly responsible for processing requests from users and presenting information to users. It includes components such as the front-end framework, page routing, and template engines to provide a good user experience and a friendly user interface.

[0123] 4) Service layer:

[0124] The service layer is responsible for implementing core business logic, including at least database data retrieval and storage, access control logic, monitoring data recording, logging, message queue data processing, and data analysis;

[0125] 5) Data layer:

[0126] The data layer is responsible for storing and retrieving data and includes at least a database, cache, and communication methods:

[0127] The database is used to store the system's persistent data and uses a relational database such as PostgreSQL or MySQL.

[0128] Cache uses Redis to store frequently accessed data to improve system performance;

[0129] The communication method adopts a front-end and back-end separation method, and the front-end and back-end interact through the restful API to enhance the maintainability and scalability of the system; the information of the monitoring equipment is communicated with the server in a timely manner through the message queue to ensure the timeliness and stability of the transmitted data.

[0130] According to the above technical solution, the method for establishing a fish germplasm resource bank provided by the present invention collects fish germplasm resource information in a river basin at multiple levels, constructs fish germplasm resource banks at different levels, such as living body banks, specimen banks, cell banks, and nucleic acid banks, and utilizes the fish germplasm resource banks at different levels to construct a fish germplasm resource management system to store and share fish germplasm resource information. Compared with the existing technology, the present invention has the following advantages:

[0131] (1) Because the technical solution adopted by the present invention collects fish germplasm resource information at multiple levels within the basin, the present invention can ensure the comprehensiveness and diversity of resources and provide richer and more accurate original data for research and protection.

[0132] (2) Because the technical solution adopted by the present invention constructs a comprehensive fish germplasm resource library including a living library, a specimen library, a cell library and a nucleic acid library, which diversifies the resource preservation forms, the present invention can respond to different research needs and preservation conditions, thereby increasing the safety and availability of resources.

[0133] (3) Because the technical solution adopted by the present invention constructs a cell bank and a nucleic acid bank, the present invention can achieve long-term stable preservation of fish germplasm. At the same time, with the help of modern biotechnology, resources can be restored and regenerated, providing an effective means for the protection of rare and endangered fish species.

[0134] (4) Because the technical solution adopted by the present invention constructs a fish germplasm resource data management system, the present invention can realize the digital and information management of fish germplasm resources, facilitate the query, exchange and sharing of resources, improve the efficiency of resource utilization, and promote transparent information exchange and scientific research cooperation. BRIEF DESCRIPTION OF THE DRAWINGS

[0135] Figure 1 Flowchart of the method for establishing a fish germplasm resource bank;

[0136] Figure 2 Schematic diagram of the multi-layer architecture of the fish germplasm resources data management system; DETAILED DESCRIPTION

[0137] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited to the following embodiments. Figure 1 As shown, the following steps are included:

[0138] (1) Investigate fish germplasm resources and habitats: Conduct an investigation of fish germplasm resources and habitats in the basin by combining field investigations with the review of relevant data, and obtain information on fish species diversity, population structure, and temporal and spatial distribution; understand the living habits and hydrological characteristics of fish through historical data analysis and on-site surveys, and obtain an overview of the habitat distribution of fish spawning grounds, feeding grounds, and wintering grounds;

[0139] (2) Determine target fish species: select target fish species for the fish germplasm resource bank based on the species protection level, representativeness and relative importance index of fish species in the water area;

[0140] In this embodiment, the specific calculation method for selecting target fish is as follows:

[0141] X = fish species score = fish species protection level score + fish representativeness score + fish relative importance index score. Fish species with X ≥ 6 are listed as target fish.

[0142] Species protection level score: 5 points for national level 1, 4 points for national level 2, and 3 points for provincial or autonomous region level;

[0143] Representativeness score: 3 points for species endemic to China and 3 points for species endemic to the river basin;

[0144] Relative importance index score: IRI = (N + W) * F;

[0145] Where: N is the percentage of the number of tails of a species to the total number of tails; W is the percentage of the biomass of the species to the total biomass; F is the percentage between the frequency of the station where the corresponding species appears and the total number of stations surveyed; when IRI ≥ 10, it is scored as 1, 1 ≤ IRI < 10 is scored as 2, and IRI < 1 is scored as 3.

[0146] In the process of calculating X, the highest score is taken as the fish species protection level score, and the cumulative score is taken as the fish representativeness score.

[0147] (3) Determine the sub-bank type of fish germplasm resource bank: Determine the sub-bank type of germplasm resource bank corresponding to each target fish species based on the endangered status of each target fish species;

[0148] In this embodiment, the types of germplasm resource bank sub-banks corresponding to each target species are as follows:

[0149] Extinct species: building a specimen library;

[0150] Extinct species in the wild: building a specimen library;

[0151] Critically endangered species: building a specimen library;

[0152] Endangered species: building specimen and tissue banks;

[0153] Vulnerable species: build specimen banks, nucleic acid banks, and tissue banks;

[0154] Low-risk species: build specimen banks, nucleic acid banks, tissue banks, sperm banks, and cell banks;

[0155] Least Concern Species: Establish specimen bank, nucleic acid bank, tissue bank, sperm bank, cell bank, and living organism bank;

[0156] The above endangered status is determined according to national standards.

[0157] (4) Determine the minimum sample storage volume of the fish germplasm resource bank sub-banks: Based on the difficulty of constructing each sub-bank, calculate the minimum sample storage volume of the seven sub-banks for each target species;

[0158] In this embodiment, except for extinct species, species extinct in the wild, and critically endangered species, the minimum sample storage capacity of the germplasm resource bank sub-banks of other species is based on the minimum sample quantity of the specimen bank. The specific formula is:

[0159] The number of samples in the tissue bank = the number of samples in the specimen bank * 10; the number of samples in the gene bank = the number of samples in the specimen bank * 10; the number of samples in the sperm bank = the number of samples in the specimen bank; the number of samples in the cell bank = the number of samples in the specimen bank; for the living bank of non-endangered species, the minimum number of parents and backup parents is the minimum production capacity requirement of the provincial aquatic breeding farm.

[0160] (5) Sample collection: Collect the fish samples selected in step (2) accordingly; collect fish samples from wild fishing, aquatic origin, breeding farms, seedling breeding farms and farms;

[0161] (6) Sample processing: The samples collected in step (5) are processed according to the different requirements of the living body bank, specimen bank, tissue bank, gene bank, sperm bank and cell bank to meet the sample preservation requirements of the living body bank, specimen bank, tissue bank, gene bank, sperm bank and cell bank and obtain corresponding information;

[0162] A. Sample processing of living body bank:

[0163] a. Transportation and transfer: Use appropriate containers and water quality conditions to safely transport collected fish individuals to live storage, avoiding overcrowding and prolonged transportation to reduce stress and mortality during transportation;

[0164] b. Temporary holding: Collected fish will be temporarily placed in suitable temporary holding facilities, ensuring that appropriate water conditions, diet and living space are provided to promote individual adaptation and health;

[0165] c. Health assessment and screening: Conduct a health assessment on each individual to check for diseases, injuries, or other health issues. Select healthy individuals for final storage, and avoid introducing sick or unsuitable individuals into the living bank.

[0166] d. Pre-banking adaptability assessment: Before individuals enter the live bank, an adaptability assessment is conducted to ensure they can adapt to the new environmental conditions and social structure. The adaptability assessment should at least include behavioral observations, food intake, and social interaction monitoring.

[0167] e. Entry and Identification: Individuals meeting the criteria are formally introduced into the living organism bank and identified. The living organism bank identification shall at least include the individual number, collection information, and health status;

[0168] f. Health Monitoring: Regularly inspect and evaluate the health and reproductive status of individuals in the live bank, and adjust feeding and management strategies based on the monitoring results to maximize the survival and reproductive success rates of individuals in the live bank;

[0169] B. Specimen library sample processing:

[0170] There are many types of fish specimens, mainly including immersed specimens, skinned specimens, skeletal specimens and transparent specimens. Immersed specimens are specimens in which the whole or part of the fish is directly placed in various chemicals for fixation and preservation; skinned specimens refer to specimens in which the fish skin is preserved by various methods and then filled with various materials to restore it to its original state; skeletal specimens refer to specimens in which the fish viscera and muscle tissue are removed by various methods, leaving only the bones, and the bones are degreased, bleached and reshaped in series by chemicals; transparent skeletal specimens refer to small fish that have been degreased, transparentized and stained with chemicals, so that their hard bones and cartilage tissues can be clearly observed; the processed specimens are placed in the specimen library and marked. The specimen library mark includes at least the specimen number, specimen type and specimen fish species;

[0171] C. Tissue Bank Sample Processing:

[0172] a. After putting on disposable rubber gloves, the collector first uses a flame to burn the front end of the scissors that have been cleaned with alcohol. After it cools, cut the fins or muscle tissue of the specimen;

[0173] b. Then, using alcohol-cleaned tweezers, place the sample into a pre-prepared 2ml centrifuge tube, screw on the tube cap, and place it in a cryostat. The tube should contain a label and at least 95% ethanol.

[0174] c. Repeatedly clean the tools before removing the next piece of molecular tissue material to avoid cross contamination;

[0175] Among them, molecular materials of large fish can be taken in a small amount, enough to be completely immersed in the preservation solution; for small specimens, several tissue parts or the whole body are directly placed in a centrifuge tube filled with 95% or more ethanol solution;

[0176] When processing tissue bank samples to obtain materials for transcriptome experiments, first use a scalpel or scissors combined with tweezers to quickly remove the gills, brain, heart, and liver of anesthetized or recently killed fish specimens and place them into pre-cooled cryovials. Then, temporarily store them in a small liquid nitrogen tank, a -80°C freezer, or dry ice. The entire process must be performed under strict sterile conditions and on dry ice or ice cubes. The molecular experimental materials obtained should be promptly placed in a -80°C freezer for long-term storage.

[0177] D. Gene bank sample processing:

[0178] a. Sample sampling: For the aquatic organisms for which genomic DNA libraries are to be constructed, the number of samples to be sampled shall be determined in accordance with the provisions of GB / T18654.2;

[0179] b. Extraction of genomic DNA: Extract genomic DNA using traditional genomic DNA extraction methods or commercial genomic DNA extraction kits;

[0180] c. Genomic DNA processing: Depending on the selected vector, the genomic DNA is digested with appropriate restriction endonucleases to produce ends that are compatible with the vector;

[0181] d. Detection and recovery of DNA enzyme slices of appropriate size: DNA fragments obtained by cleavage were detected by agarose gel electrophoresis and observed with a gel imager. The enzyme-digested segments of appropriate size were recovered using low-melting-point agarose gel;

[0182] e. Vector selection: Select an appropriate vector based on the size of the target genomic region, the difficulty of screening the library, and the copy number of the vector;

[0183] f. Ligation of genomic DNA fragments and vectors: Use ligase to connect the above DNA fragments of appropriate size into the vector;

[0184] g. Transformation of the recombinant vector into host cells and identification of positive clones: The recombinant vector is transformed into competent host cells, and positive clones are screened using the blue-white color of the colonies as an indicator;

[0185] E. Sperm bank sample processing:

[0186] a. Use a towel or paper towel to dry the abdomen and genital area of mature male fish. Gently press the abdomen to expel urine. Use a dry pipette to collect the expelled semen and place it in a clean, dry, graduated glass bottle or test tube. Avoid direct sunlight during handling and transportation.

[0187] b. Microscopic examination: observe three times and take the average of the results. Semen with a sperm survival rate of 80% or above can be used for cryopreservation. The sperm survival rate is the percentage of motile sperm to total sperm.

[0188] c. Select appropriate cryopreservation diluent and antifreeze based on the fish species. Add the cryopreservation diluent after semen collection and microscopic examination. Mix the semen of different fish species with the pre-chilled diluent in appropriate proportions and place in a 4°C refrigerator for 20-30 minutes to equilibrate.

[0189] d. Aliquot the equilibrated semen with diluent in 1.0-1.5 mL portions into 2 mL cryovials and cryopreserve in liquid nitrogen using a three-step freezing procedure.

[0190] e. When thawing, first remove the cryovial from liquid nitrogen and place it in liquid nitrogen vapor for 2-3 minutes to equilibrate. Then, remove it from the liquid nitrogen tank and place it in a 37°C water bath for rapid thawing or thaw it at room temperature. After the frozen sperm is thawed, dry insemination is performed with fresh eggs. Pour an appropriate amount of frozen semen onto the eggs and gently stir to mix. Add fresh water or seawater equivalent to 2-3 times the volume of the eggs to activate the sperm. After standing for 10 minutes, add 5-10 times the volume of water to wash away dead eggs and excess sperm. Place the fertilized eggs in a thaw incubator for insemination.

[0191] F. Cell Bank Sample Processing:

[0192] a. Wash the collected fish tissue blocks with PBS at least three times in a clean bench;

[0193] b. Transfer the tissue to a penicillin vial containing L-15 medium. Cut the tissue into 1 mm pieces with scissors and wash three times with PBS.

[0194] c. Soak with a small amount of L-15 complete medium and inoculate evenly on a 25cm 2 Culture flasks: record cell information on the side of the flask, including tissue type, time, and culture generation, and place it upside down in an incubator at a suitable temperature for culture;

[0195] d. After 10-12 hours, add 3 ml of complete culture medium and begin upright culture. Change the medium every 2-3 days during the primary culture period.

[0196] e. Subculture: The first subculture is performed when the cells have grown to 95% of the bottom of the cap and adhere to the wall. The first subculture is performed in the original bottle. Subculture is performed at a ratio of 1 to 2 for subsequent subcultures. The original culture medium is aspirated, and after washing with PBS, an appropriate amount of trypsin is added to digest the cells. When the cells shrink and become round, and are about to detach from the wall, the trypsin is aspirated and the culture medium is added and pipetted several times until the cells detach from the wall.

[0197] (7) Sample storage: The method for storing samples in a living body bank is to build a fish pond for ex situ conservation; the method for storing samples in a specimen bank is to place the specimen or organ in a 10% formalin solution, replace the solution several times until it is fixed, and then place it in a 5% formalin solution for another 2-3 weeks, and finally place it in a glass specimen bottle with a preservation solution, and seal it with vaseline or paraffin to prevent the fixative from evaporating or leaking; the method for storing samples in a tissue bank is to place the collected tissue sample in a 2ml centrifuge tube containing more than 95% ethanol and store it at -20℃ or -70℃; the method for storing samples in a gene bank is to dissolve the genomic DNA that meets the requirements in TE with a pH value of 8.0 Storage: The sperm bank sample storage method is to add the balanced semen to the diluent in 1.0mL~1.5mL portions into 2mL cryovials, and then freeze in liquid nitrogen according to the three-step cooling and freezing mode. The cell bank sample storage method is to aspirate the culture medium, rinse the cells with phosphate buffer, and then add an appropriate amount of trypsin for digestion. When the adherent cells are small and round and about to detach from the wall, aspirate the trypsin, add an appropriate amount of culture medium and pipette to detach the cells. The cell suspension after pipetting is mixed with an equal volume of cell cryopreservation culture medium, and the mixture is placed in a cryovial. The cells are first placed in a 4℃ refrigerator for 30 minutes, then in a -80℃ refrigerator for 2 hours to 4 hours, and finally the cell cryovials are stored in liquid nitrogen.

[0198] (8) Regular testing of sample quality: After samples are put into storage, three samples are randomly selected from each batch for testing. The quality of samples is regularly evaluated and identified, and samples that do not meet the requirements are eliminated and replenished;

[0199] Live stock library: Regularly test the health status, survival rate and growth rate of live fish. If the fish are sick, the samples in that batch will be removed and replenished;

[0200] Specimen library: regularly remove specimens from the specimen tank and observe changes in specimen morphology. If the morphology changes significantly or the specimens are severely decayed, the batch of samples will be discarded and replenished;

[0201] Tissue bank: Regularly check whether tissue samples in the refrigerator are sealed, stored at -20°C or -70°C, and whether they have been frozen and thawed multiple times. Repeated freezing and thawing will result in smaller genomic DNA fragments and reduced extraction quality. If the freezer is not sealed, the temperature does not meet the standard, or the number of freeze-thaw cycles exceeds 5, the batch of samples should be discarded and replenished.

[0202] Gene bank: DNA samples are regularly taken out of the refrigerator for concentration, purity and integrity testing;

[0203] The benzene ring structure of the bases on the DNA chain has strong absorption in the violet region, and its absorption peak is at 260nm. By detecting the absorbance values of DNA samples at wavelengths of 260nm and 280nm:

[0204] An A260 / A280 ratio of 1.8-2.0 indicates good purity; an A260 / A280 ratio < 1.8 indicates residual protein impurities in the nucleic acid extraction or a very low sample concentration; an A260 / A280 ratio > 2.0 indicates partial DNA degradation or RNA contamination.

[0205] The A260 / A230 ratio of pure nucleic acid should be greater than 2.0. The DNA concentration can be calculated based on the purity test. Generally, a DNA sample concentration of 100-300 ng / uL is conducive to long-term DNA storage.

[0206] The integrity of DNA was analyzed by gel electrophoresis. A bright band near the sample well without impurities or tailing indicated that the DNA molecule was intact and not degraded. A bright band in the sample well indicated that there was protein residue in the DNA solution. Smearing and tailing in the lane indicated that the DNA was degraded.

[0207] If the DNA sample concentration is lower than 100 ng / uL, it should be removed and replenished; if the A260 / A280 ratio in the DNA sample is greater than 2.0, it should be removed and replenished; if the DNA sample integrity is poor, such as diffusion or tailing in the lane, it should be removed and replenished;

[0208] Sperm bank: Sperm samples are taken out of the liquid nitrogen tank regularly for thawing and recovery, and the motility of the sperm after freezing and recovery is tested. If the recovery rate is less than 50%, the batch of samples will be discarded and replenished;

[0209] Cell bank: Cell samples are regularly taken out of the liquid nitrogen tank for thawing and recovery. The recovery method is to randomly select three samples from each generation of cells for recovery culture. After the cells have re-divided, the degree of cell deformity of the passaged cells is observed under a microscope. If all cells show deformity, all samples of the cell generation are discarded and replenished;

[0210] (9) Establishing a fish germplasm resource database: Based on the various data obtained in steps (1) to (7), a complete fish germplasm resource database is established. In this embodiment, the fish germplasm resource database is constructed with the spatial distribution data of fish germplasm resources, the spatial distribution data of fish habitats, and the target fish germplasm resource library data as the first-level categories. The codes and definitions of the first-level categories are shown in Table 1.

[0211] Table 1 Codes and definitions of first-level categories

[0212]

[0213] The specific construction method is as follows:

[0214] S1. Collect basic data on fish germplasm resources and habitats: Set the geographical area to be studied for the fish germplasm resources database to be constructed, and obtain basic data on fish germplasm resources and habitats within the geographical area through actual surveys;

[0215] S2. Establish spatial distribution data of fish germplasm resources: pre-process the fish germplasm resources data, form a basic database with spatial data as the main body, and separate and obtain the spatial distribution data of fish germplasm resources;

[0216] The spatial distribution data of fish germplasm resources were extracted according to the first-level fish germplasm resource spatial distribution data type and refined into second-level and third-level data types. The second-level data type includes all species in the study area, and the third-level data type is divided into abundance and biomass data according to the biological data type, as shown in Table 2. The data structure of the spatial distribution data of fish germplasm resources set based on the basic database includes the administrative region name, administrative region code, fish germplasm resource type and code, fish germplasm resource second-level type and code, fish germplasm resource spatial distribution data third-level type and code, and the number of catch per unit fishing effort, as shown in Table 3.

[0217] Table 2 Codes and definitions of spatial distribution data of fish germplasm resources at all levels

[0218]

[0219] Table 3. Data structure of spatial distribution of fish germplasm resources

[0220]

[0221]

[0222] S3. Establish spatial distribution data of habitats: Pre-process the fish habitat data, form a basic database with spatial data as the main body, and separate and obtain spatial distribution data of fish habitats;

[0223] Fish habitat spatial distribution data were extracted based on the first-level fish habitat spatial distribution data type and refined into second-level and third-level data types. The second-level data type includes all species in the study area, and the third-level data type is divided into spawning grounds, feeding grounds, and wintering grounds according to the habitat data type, as shown in Table 4. The data structure of fish habitat spatial distribution data set based on the basic database includes administrative region name, administrative region code, fish habitat spatial distribution data and code, fish habitat spatial distribution data second-level type and code, fish habitat spatial distribution data third-level type and code, and map area.

[0224] Table 4. Codes and definitions of fish habitat spatial distribution data at all levels

[0225]

[0226]

[0227] Table 5. Data structure of spatial distribution of fish habitats

[0228]

[0229] S4. Establish target fish germplasm resource bank data: process and analyze target fish germplasm resource bank data to obtain fish habitat spatial distribution data;

[0230] The spatial distribution data of fish habitats are extracted according to the data type of the first-level target fish germplasm resource bank and refined into second-level and third-level data types. The second-level data type includes living bank, specimen bank, tissue bank, gene bank, sperm bank and cell bank. The third-level data type is divided into sample marking information, sample processing information and sample genetic information, as shown in Table 6.

[0231] Table 6 Codes and definitions of fish habitat spatial distribution data at all levels

[0232]

[0233] In this embodiment, the process of generating the three-level class data is as follows:

[0234] 1) Living Organ Bank:

[0235] Sample labeling information for the biobank: Assign a unique identifier to each sample to facilitate identification and management in the database; record the basic information, species name, common name, and subspecies information of the sample corresponding to each identifier; record the collection date, location, and collector of each sample; record the biological characteristics of each sample, including at least sex, age, and reproductive status; record the minimum sample storage volume data and actual sample storage volume data for each sample;

[0236] Live bank sample processing information: record the environment type and water quality when the sample was collected, the water environment conditions during transportation and transfer, the health status before storage, the adaptability assessment data before storage, the health monitoring data after storage, the water quality management after storage, the feed type and feeding frequency, disease inspection and nutrition control methods;

[0237] Genetic information of living bank samples: record genetic characteristics, breeding rules and reproduction strategy information;

[0238] 2) Specimen library:

[0239] Specimen library sample labeling information: Assign a unique identifier to each sample to facilitate identification and management in the database; record the basic information, species name, common name and subspecies information of the sample corresponding to each identifier; record the collection date, location and collector of each sample; record the minimum sample storage volume data and actual sample storage volume data of each sample;

[0240] Sample processing information of the specimen library: record the environment type and water quality when the sample was collected; record the specimen collection and preparation methods;

[0241] Genetic information of specimens in the specimen bank: record the taxonomic status, morphological characteristics and habitat preference of fish;

[0242] 3) Tissue Bank:

[0243] Tissue bank sample labeling information: Assign a unique identifier to each sample to facilitate identification and management in the database; record the basic information, species name, common name, and subspecies information of the sample corresponding to each identifier; record the collection date, location, and collector of each sample; record the fish sample part information; record the minimum sample storage volume data and actual sample storage volume data of each sample;

[0244] Tissue bank sample processing information: record the environment type and water quality when the sample was collected; record the tissue collection and freezing preservation methods;

[0245] Genetic information of tissue bank samples: records fish tissue structure, pathological signs and tissue-specific expression information;

[0246] 4) Gene Bank:

[0247] Gene bank sample identification information: Assign a unique identifier to each sample to facilitate identification and management in the database; record the basic information, species name, common name, and subspecies information of the sample corresponding to each identifier; record the collection date, location, and collector of each sample; record genotype information; record the minimum sample storage volume data and actual sample storage volume data of each sample;

[0248] Gene bank sample processing information: record the fish's length, weight, age, gonadal development, environment type and water quality at the time of sample collection; record the gene sample collection and freezing storage methods;

[0249] Genetic information of gene bank samples: record gene library information;

[0250] 5) Sperm Bank:

[0251] Sperm bank sample labeling information: Assign a unique identifier to each sample to facilitate identification and management in the database; record the basic information, species name, common name and subspecies information of the sample corresponding to each identifier; record the collection date, location and collector of each sample; record the sperm storage volume of each sample; record the minimum sample storage volume data; record the actual sample storage volume data;

[0252] Sperm bank sample processing information: record the fish's length, weight, age, gonadal development, environment type and water quality at the time of sample collection; record the sperm collection and freezing preservation methods;

[0253] Genetic information of sperm bank samples: record the normal sperm morphology rate, sperm motility, fertilization rate, hatching rate and sperm membrane integrity rate before and after cryopreservation;

[0254] 6) Cell Bank:

[0255] Cell bank sample labeling information: Assign a unique identifier to each sample to facilitate identification and management in the database; record the basic information, species name, common name, and subspecies information of the sample corresponding to each identifier; record the collection date, location, and collector of each sample; record cell type information; record the minimum sample storage volume data and actual sample storage volume data of each sample;

[0256] Cell bank sample processing information: record the fish's length, weight, age, gonadal development, environment type, and water quality at the time of sample collection; record the cell collection, cell culture, and cryopreservation methods;

[0257] Genetic information of cell bank samples: records fish cell morphological characteristics, cell growth characteristics and chromosome karyotype.

[0258] (10) Fish germplasm resource data management: Based on the database in step (9), a fish germplasm resource data management system is established, including a container management system, a sample management system, a data management system and a security management system; the container management system is used to manage the equipment data for storing samples; the sample management system at least includes managing the increase, decrease and status of different types of samples, automating the sample information entry and maintenance process through fish wireless radio frequency identification chip technology, recording the use and maintenance history of samples, monitoring the status of the resource library in real time, recording the increase and decrease changes, and providing an interface for querying information of various resource libraries; the data management system implements the processing and storage logic of genetic and information data, designs the data distribution and access interface, and ensures the security and integrity of the data; realizes the structured and persistent storage of data, and designs and develops the logical functions of the online resource sharing platform; the security management system determines the security risks and protection requirements of the system through a comprehensive analysis of the architecture of the information system, the business carried and the system classification, and determines the security protection measures based on the corresponding protection requirements, balancing the relationship between security, cost and efficiency.

[0259] In this embodiment, the container management system includes at least daily data management of ultra-low temperature refrigerators, liquid nitrogen tanks and freezing tubes; monitoring the status, temperature, humidity and liquid level of storage devices; managing and allocating storage space; real-time status query and abnormal status alarm.

[0260] The specific solutions for handling the security aspects of the safety management system are as follows:

[0261] 1) Authentication and Authorization: Use the Web Token mechanism to implement user authentication and authorization, ensuring that only legitimate users can access system functions;

[0262] 2) HTTPS: Use the HTTPS protocol in front-end and back-end communications to encrypt data transmission and prevent man-in-the-middle attacks;

[0263] 3) Input validation and SQL injection prevention: Strictly validate user input to prevent malicious input and SQL injection attacks;

[0264] 4) Cross-site scripting (XSS) and cross-site request forgery (CSRF) protection: Implementing Content Security Policy (CSP) to restrict how browsers load and execute external resources effectively prevents XSS attacks. Each user request includes a unique CSRF token, which the server verifies upon receiving the request to ensure the legitimacy of the request, effectively preventing CSRF attacks. These methods ensure user data and session security.

[0265] 5) Regular security reviews: Perform regular security reviews of the system to ensure that the system is consistent with the latest security standards and best practices.

[0266] like Figure 2 As shown in the figure, the fish germplasm resources data management system adopts a multi-layer architecture. Each layer is responsible for a specific function and communicates with other layers through clearly defined interfaces, which improves the modular maintainability and scalability of the system.

[0267] In this embodiment, the fish germplasm resource data management system is divided into five levels in the order of functional interaction;

[0268] 1) Access layer:

[0269] The user access device and the interaction between the information library and the system belong to the access layer. The components used in this layer include API interface and ID card authentication;

[0270] Users can access the system through multiple terminals, and administrators can manage and set various system functions through the management background;

[0271] The samples in the information library are generated into a QR code by the system according to the specified coding rules. The printing device prints it and sticks it on the corresponding sample container. When sampling, the QR code can be scanned by the scanning device to obtain the sample information;

[0272] 2) Gateway layer:

[0273] The information database transmits data through the local gateway and transmits the data to the server for processing;

[0274] The monitoring indicator data of the information database is transmitted to the server through the local gateway for comparison. If the monitoring data is abnormal, an early warning reminder will be issued in time;

[0275] After users visit the web page, they interact with the server through the RESTful API to achieve front-end and back-end separation;

[0276] 3) Web layer:

[0277] The web layer is the user interface layer in the system architecture. It is mainly responsible for processing requests from users and presenting information to users. It includes components such as the front-end framework, page routing, and template engines to provide a good user experience and a friendly user interface.

[0278] 4) Service layer:

[0279] The service layer is responsible for implementing core business logic, including at least database data retrieval and storage, access control logic, monitoring data recording, logging, message queue data processing, and data analysis;

[0280] 5) Data layer:

[0281] The data layer is responsible for storing and retrieving data and includes at least a database, cache, and communication methods:

[0282] The database is used to store the system's persistent data and uses a relational database such as PostgreSQL or MySQL.

[0283] Cache uses Redis to store frequently accessed data to improve system performance;

[0284] The communication method adopts a front-end and back-end separation method, and the front-end and back-end interact through the restful API to enhance the maintainability and scalability of the system; the information of the monitoring equipment is communicated with the server in a timely manner through the message queue to ensure the timeliness and stability of the transmitted data.

Claims

1. A method for establishing a fish germplasm resource bank, characterized in that: The following steps are involved: (1) Investigate fish germplasm resources and habitats: Conduct an investigation of fish germplasm resources and habitats in the basin by combining field investigations with the review of relevant data, and obtain information on fish species diversity, population structure, and temporal and spatial distribution; understand the living habits and hydrological characteristics of fish through historical data analysis and on-site surveys, and obtain an overview of the distribution of fish spawning grounds, feeding grounds, and wintering grounds; (2) Determine target fish species: select target fish species for the fish germplasm resource bank based on the species protection level, representativeness and relative importance index of fish species in the water area; (3) Determine the type of fish germplasm resource bank sub-bank: Determine the type of germplasm resource bank sub-bank corresponding to each target fish species based on the endangered status of each target fish species; (4) Determine the minimum sample storage volume of the fish germplasm resource bank sub-banks: Based on the difficulty of constructing each sub-bank, calculate the minimum sample storage volume of the seven sub-banks for each target species; (5) Sample collection: Collect the fish samples selected in step (2) accordingly; collect fish samples from wild fishing, aquatic origin, breeding farms, seedling breeding farms and farms; (6) Sample processing: The samples collected in step (5) are processed according to the different requirements of the living body bank, specimen bank, tissue bank, gene bank, sperm bank and cell bank to meet the sample preservation requirements of the living body bank, specimen bank, tissue bank, gene bank, sperm bank and cell bank and obtain the corresponding information; A. Sample processing of living body bank: a. Transport and transfer: Collected fish should be safely transported to the live bank using suitable containers and appropriate water quality conditions, avoiding overcrowding and prolonged transport to reduce stress and mortality during transport. b. Temporary holding: Collected fish will be temporarily placed in suitable temporary holding facilities, ensuring that appropriate water conditions, diet and living space are provided to promote individual adaptation and health; c. Health assessment and screening: Conduct a health assessment on each individual to check for illness, injury, or other health issues. Select healthy individuals for final storage, and avoid introducing sick or unsuitable individuals into the living bank. d. Pre-banking adaptability assessment: Before individuals enter the live bank, an adaptability assessment is conducted to ensure they can adapt to the new environmental conditions and social structure. The adaptability assessment should at least include behavioral observation, food intake, and social interaction monitoring. e. Entry and Identification: Individuals meeting the criteria are formally introduced into the living organism bank and identified. The living organism bank identification includes at least the individual number, collection information, and health status; f. Health Monitoring: Regularly inspect and evaluate the health and reproductive status of individuals in the live bank, and adjust feeding and management strategies based on the monitoring results to maximize the survival and reproductive success rates of individuals in the live bank; B. Specimen Bank Sample Processing: There are many types of fish specimens, mainly including immersion specimens, skinned specimens, skeletal specimens and transparent specimens. Immersion specimens are specimens in which the whole or part of the fish is directly placed in various chemicals for fixation and preservation; skinned specimens refer to specimens in which the fish skin is preserved by various methods and then filled with various materials to restore it to its original state; skeletal specimens refer to specimens in which the fish viscera and muscle tissue are removed by various methods, leaving only the bones, and the bones are degreased, bleached and reshaped in series with chemicals; transparent skeletal specimens refer to small fish that have been degreased, transparentized and stained with chemicals, so that their hard bones and cartilage tissues can be clearly observed; the processed specimens are placed in a specimen library and marked. The specimen library mark includes at least the specimen number, specimen type and specimen fish species; C. Tissue Bank Sample Processing: a. After putting on disposable rubber gloves, the collector first uses a flame to burn the front end of the scissors that have been cleaned with alcohol. After it cools, cut the fins or muscle tissue of the specimen; b. Then, using alcohol-cleaned tweezers, place the sample into a pre-prepared 2ml centrifuge tube, secure the tube cap, and place it in a cryovial. The tube should contain a label and at least 95% ethanol. c. Repeatedly clean the tools before removing the next piece of molecular tissue material to avoid cross contamination; D. Gene bank sample processing: a. Sample sampling: For the aquatic organisms for which genomic DNA libraries are to be constructed, determine the number of samples to be sampled according to the provisions of GB / T18654.2; b. Genomic DNA extraction: Extract genomic DNA using traditional genomic DNA extraction methods or commercial genomic DNA extraction kits; c. Genomic DNA processing: Depending on the selected vector, the genomic DNA is digested with an appropriate restriction endonuclease to produce ends that are compatible with the vector; d. Detection and recovery of DNA enzyme slices of appropriate size: DNA fragments obtained by attachment were subjected to agarose gel electrophoresis and observed using a gel imager. The appropriate size of the enzyme-digested fragments was recovered using a low-melting point agarose gel; e. Vector selection: Select an appropriate vector based on the size of the target genomic region, the difficulty of library screening, and the vector copy number; f. Ligation of genomic DNA fragments and vectors: Use ligase to connect the above DNA fragments of appropriate size into the vector; g. Transformation of the recombinant vector into host cells and identification of positive clones: The recombinant vector is transformed into competent host cells, and positive clones are screened using the blue-white color of the colonies as an indicator; E. Sperm Bank Sample Processing: a. Use a towel or paper towel to dry the abdomen and genital area of mature male fish. Gently press the abdomen to expel urine. Use a dry pipette to collect the expelled semen and place it in a clean, dry, graduated glass bottle or test tube. Avoid direct sunlight during handling and transportation. b. Microscopic examination: observe three times and take the average of the results. Semen with a sperm survival rate of 80% or above can be cryopreserved. The sperm survival rate is the percentage of motile sperm to total sperm. c. Select appropriate cryopreservation diluent and antifreeze based on the fish species. After semen collection and microscopic examination, add the cryopreservation diluent. Mix the semen of different fish species with the pre-chilled diluent in appropriate proportions and place in a 4°C refrigerator for 20-30 minutes to equilibrate. d. Aliquot the equilibrated semen with diluent in 1.0-1.5 mL portions into 2 mL cryovials. Cryopreserve in liquid nitrogen using a three-step freezing protocol. e. When thawing, first remove the cryovial from liquid nitrogen and place it in liquid nitrogen vapor to equilibrate for 2-3 minutes. Then remove it from the liquid nitrogen tank and place it in a 37°C water bath for rapid thawing or thaw it at room temperature. After the frozen sperm is thawed, dry insemination is performed with fresh eggs. Pour an appropriate amount of frozen semen onto the eggs and gently stir to mix. Add fresh water or seawater equivalent to 2-3 times the volume of eggs to activate the sperm. After standing for 10 minutes, add 5-10 times the volume of water to wash away dead eggs and excess sperm. Place the fertilized eggs in a thaw incubator for insemination. F. Cell Bank Sample Processing: a. Wash the collected fish tissue blocks with PBS at least three times in a clean bench; b. Transfer the tissue to a penicillin vial containing L-15 medium. Cut the tissue into 1 mm pieces with scissors and wash three times with PBS. c. Soak with a small amount of L-15 complete medium and inoculate evenly on a 25 cm 2 Culture flasks: record cell information on the side of the flask, including tissue type, time, and culture generation, and place it upside down in an incubator at a suitable temperature for culture; d. After 10-12 hours, add 3 ml of complete culture medium and begin upright culture. Change the medium every 2-3 days during the primary culture. e. Subculture: The first subculture is performed when the cells have grown to 95% of the bottom of the cap and adhere to the wall. The first subculture is performed in the original bottle. Subculture is performed at a ratio of 1 to 2 for subsequent subcultures. The original culture medium is aspirated, and after washing with PBS, an appropriate amount of trypsin is added to digest the cells. When the cells shrink and become round, indicating that they are about to detach from the wall, the trypsin is aspirated and the culture medium is added and pipetted several times until the cells detach from the wall. (7) Sample storage: The method for storing samples in a living body bank is to build a fish pond for ex situ conservation; the method for storing samples in a specimen bank is to place the specimen or organ in a 10% formalin solution, replace the solution several times until it is fixed, and then place it in a 5% formalin solution for another 2-3 weeks, and finally place it in a glass specimen bottle with a preservation solution, and seal it with vaseline or paraffin to prevent the fixative from evaporating or leaking; the method for storing samples in a tissue bank is to place the collected tissue sample in a 2ml centrifuge tube containing more than 95% ethanol and store it at -20℃ or -70℃; the method for storing samples in a gene bank is to dissolve the genomic DNA that has been tested to meet the requirements in TE with a pH value of 8.0 for storage; the method for storing samples in a sperm bank is to add the balanced semen to the diluent and divide it into 1.0mL to 1.5mL bottles in 2 mL cryopreservation tubes were placed in liquid nitrogen for cryopreservation according to a three-step cooling and freezing mode. The cell bank sample storage method was as follows: the culture medium was aspirated, the cells were rinsed with phosphate buffer, and an appropriate amount of trypsin was added for digestion. When the adherent cells were small and round and about to detach from the wall, the trypsin was aspirated and an appropriate amount of culture medium was added to perfuse the cells to detach from the wall. The perfusion cell suspension was mixed with an equal volume of cell cryopreservation culture medium, and the mixture was placed in a cryopreservation tube. The tubes were first placed in a 4°C refrigerator for 30 minutes, then in a -80°C refrigerator for 2 hours to 4 hours, and finally the cell cryopreservation tubes were stored in liquid nitrogen. (8) Regular testing of sample quality: After samples are put into storage, three samples are randomly selected from each batch for testing. The quality of samples is regularly evaluated and identified, and samples that do not meet the requirements are eliminated and replenished; Live stock library: Regularly test the health status, survival rate and growth rate of live fish. If the fish are sick, the samples in that batch will be removed and replenished; Specimen library: regularly remove specimens from the specimen tank and observe changes in specimen morphology. If there are significant changes in morphology or severe decay, discard the samples from that batch and replenish them. Tissue bank: Regularly check whether tissue samples in the refrigerator are sealed, stored at -20°C or -70°C, and whether they have been frozen and thawed multiple times. Repeated freezing and thawing will result in smaller genomic DNA fragments and reduced extraction quality. If the freezer is not sealed, the temperature does not meet the standard, or the number of freeze-thaw cycles exceeds 5, the batch of samples should be discarded and replenished. Gene bank: DNA samples are regularly taken out of the refrigerator for concentration, purity and integrity testing; The benzene ring structure of the bases on the DNA chain has strong absorption in the violet region, with its absorption peak at 260nm. By detecting the absorbance values of DNA samples at wavelengths of 260nm and 280nm: An A260 / A280 ratio of 1.8-2.0 indicates good purity; an A260 / A280 ratio < 1.8 indicates residual protein impurities in the nucleic acid extraction or a very low sample concentration; an A260 / A280 ratio > 2.0 indicates partial DNA degradation or RNA contamination. The A260 / A230 ratio of pure nucleic acid should be greater than 2.

0. The DNA concentration is calculated based on the purity test. Generally, a DNA sample concentration of 100-300 ng / uL is conducive to long-term DNA storage. The integrity of DNA was analyzed by gel electrophoresis. A bright band near the sample well without impurities or tailing indicated that the DNA molecule was intact and not degraded. A bright band in the sample well indicated that there was protein residue in the DNA solution. Smearing and tailing in the lane indicated that the DNA was degraded. If the DNA sample concentration is lower than 100 ng / uL, it should be removed and replenished; if the A260 / A280 ratio in the DNA sample is greater than 2.0, it should be removed and replenished; if the DNA sample integrity is poor, with diffusion or tailing in the lane, it should be removed and replenished; Sperm bank: Sperm samples are taken out of the liquid nitrogen tank regularly for thawing and recovery, and the motility of the sperm after freezing and recovery is tested. If the recovery rate is less than 50%, the batch of samples will be discarded and replenished; Cell bank: Cell samples are regularly taken out of the liquid nitrogen tank for thawing and recovery. The recovery method is to randomly select three samples from each generation of cells for recovery culture. After the cells have re-divided, the degree of cell deformity of the passaged cells is observed under a microscope. If all cells show deformity, all samples of the cell generation are discarded and replenished; (9) Establishing a fish germplasm resource database: Based on the various data obtained in steps (1) to (7), a complete fish germplasm resource database is established. The fish germplasm resource database is constructed with the spatial distribution data of fish germplasm resources, the spatial distribution data of fish habitats and the target fish germplasm resource bank data as the first-level categories. The specific construction method is as follows: S1. Collect basic data on fish germplasm resources and habitats: Set the geographical area to be studied for the fish germplasm resources database to be constructed, and obtain basic data on fish germplasm resources and habitats within the geographical area through actual surveys; S2. Establish spatial distribution data of fish germplasm resources: pre-process the fish germplasm resources data, form a basic database with spatial data as the main body, and separate and obtain the spatial distribution data of fish germplasm resources; The spatial distribution data of fish germplasm resources are extracted according to the first-level fish germplasm resource spatial distribution data type and refined into second-level and third-level data types. The second-level data type includes all species in the study area, and the third-level data type is divided into abundance and biomass data according to the biological data type. The data structure of the spatial distribution data of fish germplasm resources set based on the basic database includes the name of the administrative region, the administrative region code, the type and code of fish germplasm resources, the second-level type and code of fish germplasm resources, the third-level type and code of fish germplasm resources spatial distribution data, and the tail number of catch per unit fishing effort. S3. Establishing spatial distribution data of habitats: Pre-processing fish habitat data, forming a basic database with spatial data as the main body, and separating and obtaining spatial distribution data of fish habitats; Fish habitat spatial distribution data are extracted according to the first-level fish habitat spatial distribution data type and refined into second-level and third-level data types. The second-level data type includes all species in the study area, and the third-level data type is divided into spawning grounds, feeding grounds, and wintering grounds according to the habitat data type. The data structure of fish habitat spatial distribution data set based on the basic database includes administrative region name, administrative region code, fish habitat spatial distribution data and code, fish habitat spatial distribution data second-level type and code, fish habitat spatial distribution data third-level type and code, and map area. S4. Establish target fish germplasm resource bank data: process and analyze target fish germplasm resource bank data to obtain fish habitat spatial distribution data; The spatial distribution data of fish habitats are extracted according to the data type of the first-level target fish germplasm resource bank and refined into second-level and third-level data types. The second-level data type includes living bank, specimen bank, tissue bank, gene bank, sperm bank and cell bank. The third-level data type is divided into sample marking information, sample processing information and sample genetic information. (10) Fish germplasm resource data management: Based on the database in step (9), a fish germplasm resource data management system is established, including a container management system, a sample management system, a data management system, and a security management system; the container management system is used to manage the data of the equipment storing the samples; the sample management system at least includes the management of the increase, decrease, and status of different types of samples, and uses the fish wireless radio frequency identification chip technology to automate the sample information entry and maintenance process, record the use and maintenance history of the samples, monitor the status of the resource library in real time, record the increase and decrease changes, and provide an interface for querying the information of various resource libraries; the data management system implements the processing and storage logic of genetic and information data, designs the data distribution and access interface, and ensures the security and integrity of the data; Realize the structured and persistent storage of data, and design and develop the logical functions of the online resource sharing platform; the security management system determines the system's security risks and protection needs through a comprehensive analysis of the information system's architecture, the business it carries, and the system classification, and determines security protection measures based on the corresponding protection requirements, balancing the relationship between security, cost, and efficiency.

2. The method for establishing a fish germplasm resource bank according to claim 1, characterized in that The types of germplasm resource sub-banks corresponding to each target species in step (3) are as follows: Extinct species: building a specimen library; Extinct species in the wild: building a specimen library; Critically endangered species: building a specimen library; Endangered species: building specimen and tissue banks; Vulnerable species: build specimen banks, nucleic acid banks, and tissue banks; Low-risk species: build specimen banks, nucleic acid banks, tissue banks, sperm banks, and cell banks; Least-endangered species: construct specimen banks, nucleic acid banks, tissue banks, sperm banks, cell banks, and living organism banks.

3. The method for establishing a fish germplasm resource bank according to claim 1, wherein: In step (4), except for extinct species, species extinct in the wild, and critically endangered species, the minimum sample storage volume of the germplasm resource bank sub-banks of other species is based on the minimum sample quantity of the specimen bank. The specific formula is: The number of samples in the tissue bank = the number of samples in the specimen bank * 10; the number of samples in the gene bank = the number of samples in the specimen bank * 10; the number of samples in the sperm bank = the number of samples in the specimen bank; the number of samples in the cell bank = the number of samples in the specimen bank; for the living bank of non-endangered species, the minimum number of parents and backup parents is the minimum production capacity requirement of the provincial aquatic breeding farm.

4. The method for establishing a fish germplasm resource bank according to claim 1, wherein: When processing tissue bank samples to obtain materials for transcriptome experiments in step (6), first use a scalpel or scissors combined with tweezers to quickly remove the gills, brain, heart and liver of the anesthetized or recently killed fish specimens and place them in pre-cooled cryovials. Then, place them in a small liquid nitrogen tank, a -80°C freezer or dry ice for temporary storage. The entire process is carried out under strict sterile conditions and on dry ice or ice cubes. The obtained molecular experimental materials should be promptly placed in a -80°C refrigerator for long-term storage.

5. The method for establishing a fish germplasm resource bank according to claim 1, characterized in that The process of generating the three-level class data in step (9) is as follows: 1) Living Organ Bank: Sample labeling information for the biobank: Assign a unique identifier to each sample to facilitate identification and management in the database; record the basic information, species name, common name, and subspecies information of the sample corresponding to each identifier; record the collection date, location, and collector of each sample; record the biological characteristics of each sample, including at least sex, age, and reproductive status; record the minimum sample storage volume data and actual sample storage volume data for each sample; Live bank sample processing information: record the environment type and water quality when the sample was collected, the water environment conditions during transportation and transfer, the health status before storage, the adaptability assessment data before storage, the health monitoring data after storage, the water quality management after storage, the feed type and feeding frequency, disease inspection and nutrition control methods; Genetic information of living bank samples: record genetic characteristics, breeding rules and reproduction strategy information; 2) Specimen library: Specimen library sample identification information: assign a unique identifier to each sample to facilitate identification and management in the database; record the basic information, species name, common name and subspecies information of the sample corresponding to each identifier; Record the date, location, and collector of each sample; Record the minimum sample storage volume data and actual sample storage volume data for each sample; Sample processing information of the specimen library: record the environment type and water quality when the sample was collected; record the specimen collection and preparation methods; Genetic information of specimens in the specimen bank: record the taxonomic status, morphological characteristics and habitat preference of fish; 3) Tissue Bank: Tissue bank sample identification information: assign a unique identifier to each sample to facilitate identification and management in the database; record the basic information, species name, common name and subspecies information of the sample corresponding to each identifier; Record the date, location, and collector of each sample; Record the fish sample part information; Record the minimum sample storage volume data and actual sample storage volume data for each sample; Tissue bank sample processing information: record the environment type and water quality when the sample was collected; Document tissue collection and cryopreservation methods; Genetic information of tissue bank samples: records fish tissue structure, pathological signs and tissue-specific expression information; 4) Gene Bank: Gene bank sample identification information: assign a unique identifier to each sample to facilitate identification and management in the database; record the basic information, species name, common name, and subspecies information of the sample corresponding to each identifier; Record the date, location, and collector of each sample; Record genotype information; record the minimum sample storage volume data and actual sample storage volume data for each sample; Gene bank sample processing information: record the fish's length, weight, age, gonadal development, environment type and water quality at the time of sample collection; record the gene sample collection and freezing storage methods; Genetic information of gene bank samples: record gene library information; 5) Sperm Bank: Sperm bank sample identification information: assign a unique identifier to each sample to facilitate identification and management in the database; record the basic information, species name, common name and subspecies information of the sample corresponding to each identifier; Record the date, location, and collector of each sample; Record the sperm storage volume of each sample; record the minimum sample storage volume data; Record actual sample storage data; Sperm bank sample processing information: record the fish's length, weight, age, gonadal development, environment type and water quality at the time of sample collection; record the sperm collection and freezing preservation methods; Genetic information of sperm bank samples: record the normal sperm morphology rate, sperm motility, fertilization rate, hatching rate and sperm membrane integrity rate before and after cryopreservation; 6) Cell Bank: Cell bank sample labeling information: assign a unique identifier to each sample to facilitate identification and management in the database; record the basic information, species name, common name, and subspecies information of the sample corresponding to each identifier; Record the date, location, and collector of each sample; Record cell type information; record the minimum sample storage volume data and actual sample storage volume data for each sample; Cell bank sample processing information: record the fish's length, weight, age, gonadal development, environment type, and water quality at the time of sample collection; record the cell collection, cell culture, and cryopreservation methods; Genetic information of cell bank samples: records fish cell morphological characteristics, cell growth characteristics and chromosome karyotype.

6. The method for establishing a fish germplasm resource bank according to claim 1, characterized in that: The container management system in step (10) at least includes daily data management of ultra-low temperature refrigerators, liquid nitrogen tanks and freezing tubes; monitoring the status, temperature, humidity and liquid level of storage devices; managing and allocating storage space; real-time status query and abnormal status alarm.

7. The method for establishing a fish germplasm resource bank according to claim 1, characterized in that The specific solutions for handling the security aspects of the security management system in step (10) are as follows: 1) Authentication and Authorization: Use the Web Token mechanism to implement user authentication and authorization, ensuring that only legitimate users can access system functions; 2) HTTPS: Use the HTTPS protocol in front-end and back-end communications to encrypt data transmission and prevent man-in-the-middle attacks; 3) Input validation and SQL injection prevention: Strictly validate user input to prevent malicious input and SQL injection attacks; 4) Cross-site scripting (XSS) and cross-site request forgery (CSRF) protection: Implementing Content Security Policy (CSP) restricts how browsers load and execute external resources, effectively preventing XSS attacks. Each user request includes a unique CSRF token, which the server verifies upon receiving the request to ensure the legitimacy of the request, effectively preventing CSRF attacks. These methods ensure user data and session security. 5) Regular security reviews: Perform regular security reviews of the system to ensure that the system is consistent with the latest security standards and best practices.

8. The method for establishing a fish germplasm resource bank according to claim 1, characterized in that: In step (10), the fish germplasm resource data management system adopts a multi-layer architecture, where each layer is responsible for a specific function and communicates with other layers through clearly defined interfaces, thus improving the modular maintainability and scalability of the system. The system is divided into five levels based on the order of functional interaction; 1) Access layer: The access layer includes user access devices and the interaction between the information library and the system. Components used in this layer include API interfaces and ID card authentication. Users can access the system through multiple terminals, and administrators can manage and set various system functions through the management background; The samples in the information library are generated into a QR code by the system according to the specified coding rules. The printing device prints it and sticks it on the corresponding sample container. When sampling, the QR code can be scanned by the scanning device to obtain the sample information; 2) Gateway layer: The information database transmits data through the local gateway and transmits the data to the server for processing; The monitoring indicator data of the information database is transmitted to the server through the local gateway for comparison. If the monitoring data is abnormal, an early warning reminder will be issued in time; After users visit the web page, they interact with the server through the RESTful API to achieve front-end and back-end separation; 3) Web layer: The web layer is the user interface layer in the system architecture. It is mainly responsible for processing requests from users and presenting information to users. It includes front-end frameworks, page routing, and template engine components to provide a good user experience and a friendly user interface. 4) Service layer: The service layer is responsible for implementing core business logic, including at least database data retrieval and storage, access control logic, monitoring data recording, logging, message queue data processing, and data analysis; 5) Data layer: The data layer is responsible for storing and retrieving data and includes at least a database, cache, and communication methods: The database is used to store the system's persistent data and uses a relational database such as PostgreSQL or MySQL. Cache uses Redis to store frequently accessed data to improve system performance; The communication method adopts a front-end and back-end separation method, and the front-end and back-end interact through a restful API to enhance the maintainability and scalability of the system; the information of the monitoring equipment is communicated with the server in a timely manner through the message queue method to ensure the timeliness and stability of the transmitted data.

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