Preparation method of high-yield high-temperature-resistant homologous triploid argopecten irradians

Abstract

The invention provides a preparation method of high-yield high-temperature-resistant homologous triploid argopecten irradians. The preparation method comprises the following steps: screening parent argopecten irradians with high-temperature-resistant characteristics through a molecular marker-assisted breeding technology; after induced spawning of the parent shellfish, fertilized eggs are treated with cytochalasin B, the fertilized eggs are induced to generate the homologous triploid, the cleavage rate, the hatching rate, the metamorphosis rate and the triploid larva rate are comprehensively considered in the process, and efficient induction of the homologous triploid of the hermaphrodite shellfish is achieved. The high-temperature-resistant molecular marker is developed through SNP sites which are identified through whole genome association analysis and are most remarkably related to the high-temperature-resistant performance of the argopecten irradians, and individuals with high-temperature-resistant genotypes are screened out for spawning and fertilization. Compared with common diploid larvae, the triploid larvae obtained through the method have the higher growth and development speed, and the artificial breeding cost can be greatly reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of shellfish genetic breeding in marine agriculture, and specifically relates to a preparation method of high-yield, high-temperature-resistant homologous triploid bay scallops. Background Art

[0002] Bay scallops are the scallop species with the highest annual production in my country, with an annual production of up to 800,000 tons, and are one of the important economic shellfish. However, in recent years, the bay scallop farming industry has faced many industrial problems, such as the miniaturization of individuals and large-scale deaths caused by high temperatures in summer, which pose a severe challenge to the sustainable and healthy development of the industry. Therefore, cultivating high-yield and high-temperature resistant bay scallop varieties has become an important means to solve the above industrial problems.

[0003] In aquatic shellfish, polyploid individuals usually show significant growth advantages over diploid individuals. Therefore, polyploid induction technology has become an important method for genetic improvement of aquatic shellfish. It regulates chromosome ploidy through physical and chemical methods to improve target traits. However, in the current practice of polyploid breeding of shellfish, trait improvement is mainly focused on growth performance, and less attention is paid to stress resistance traits such as high temperature resistance. Shellfish such as polyploid oysters also suffer from large-scale deaths in summer, which has become an important bottleneck restricting the development of the industry. Therefore, how to improve the growth traits of shellfish while enhancing their stress resistance through polyploid breeding technology has become an important issue that breeders need to solve urgently.

[0004] Molecular marker breeding (MAS) can significantly improve breeding efficiency and shorten breeding cycles by screening out individuals with corresponding genotypes of excellent traits through specific molecular markers. MAS is an efficient breeding method for complex traits that cannot be directly measured, such as stress resistance. Through technologies such as genome-wide association analysis (GWAS), key genetic loci related to target traits are screened and identified, and corresponding molecular markers are developed, such as microsatellites (SSR), insertions or deletions (Indel), single nucleotide polymorphisms (SNP), etc. The rapid development of MAS provides an efficient solution for aquaculture, especially trait improvement.

[0005] As a hermaphroditic economic shellfish, bay scallops currently do not have an efficient polyploid induction technology system. Therefore, establishing and improving the polyploid induction technology for bay scallops is of great significance to its industrial development. In the early stage, the research team used GWAS technology to identify SNP sites significantly associated with the high temperature resistance traits of bay scallops, providing reliable molecular markers for MAS breeding of high temperature resistant and excellent bay scallops. The present invention further combines MAS technology with polyploid induction technology to successfully prepare high-yield, high temperature resistant homologous triploid bay scallops. The specific method is: using MAS to screen bay scallop parents with high temperature resistant genotypes for spawning and fertilization, and using cytochalasin B to inhibit the emission of the second polar body of fertilized eggs, thereby efficiently obtaining homologous triploids. This method has the advantages of simplicity, efficiency, low price, and green environmental protection, and provides certain technical support and innovation paths for the sustainable and healthy development of my country's bay scallop industry. Summary of the invention

[0006] The present invention provides a method for preparing high-yield and high-temperature resistant homologous triploid bay scallops, and prepares high-yield and high-temperature resistant homologous triploid bay scallops by combining molecular marker-assisted breeding (MAS) technology with polyploid induction technology. In the induction process, the cleavage rate, hatching rate, metamorphosis rate and triploid larvae rate are comprehensively considered, and the existing chemical induction method of triploid shellfish is optimized, so as to provide an innovative solution for the efficient seed production of fast-growing and stress-resistant bay scallops.

[0007] The present invention first provides a SNP site related to the high temperature resistance trait of bay scallops, wherein the SNP site is located at the 234th position of the nucleotide fragment of SEQ ID NO: 1, and the base thereof is T / C;

[0008] (SEQ ID NO:1)。

[0009] The present invention also provides a method for screening bay scallop individuals with high temperature resistance traits, wherein the method comprises detecting the genotype of the above-mentioned SNP site in the bay scallop individuals to be screened, and screening individuals with TT / TC genotypes;

[0010] Furthermore, the method is to use a live sampling method to extract DNA from the gill filaments of the bay scallop to be tested, amplify it with PCR primers, and then sequence the amplified product to determine the genotype;

[0011] As a specific record of the embodiment, the PCR primer pair, a specific sequence information is as follows:

[0012] Upstream primer (SEQ ID NO: 2): 5′-TGAAACTTGTCATCACGCAAT-3′;

[0013] Downstream primer: 5′-ACACTGTTAGCCTACTTATCCGA-3′.

[0014] The present invention also provides an induction and cultivation method for homologous triploid bay scallops, which uses the bay scallop individuals with high temperature resistance selected by the above method as parents to induce and cultivate homologous triploid bay scallops;

[0015] The induction cultivation method comprises the following steps:

[0016] 1) The broodstock that have been matured in 20°C seawater are taken out, dried in the shade at room temperature, and then transferred to warmed seawater for spawning induction;

[0017] 2): After the parent shellfish ovulates, record the ovulation time as the fertilization time, and observe the samples under a microscope. When 30% of the fertilized eggs show the first polar body, add cytochalasin B mother solution for treatment;

[0018] 3) After the treatment is completed, the fertilized eggs are collected by filtration, and are rinsed with 24° C. seawater to remove residual cytochalasin B (CB), and the collected fertilized eggs are cultured for embryonic development.

[0019] Furthermore, the broodstock is a bay scallop having a high temperature resistance trait obtained by screening using the above-mentioned method for screening bay scallops with a high temperature resistance trait.

[0020] Preferably, the treatment concentration of cytochalasin B is 0.5 mg / L, and the treatment time is 15 minutes.

[0021] The present invention is different from the traditional chemical induction method of homologous triploid shellfish and has the following innovative features:

[0022] 1) Innovation of the triploid induction method for hermaphroditic scallops: The present invention establishes a highly efficient homologous triploid induction technology for hermaphroditic bay scallops, which is significantly innovative in the induction technical parameters and provides a new solution for the triploid research of hermaphroditic shellfish;

[0023] 2) Innovation of polyploid induction efficiency evaluation indicators: The efficiency evaluation of traditional chemical induction methods is often based on only a single indicator of triploid larvae rate. The present invention has made innovations in the evaluation system, comprehensively considering multiple key characteristics of embryonic development, including cleavage rate, hatching rate, metamorphosis rate and triploid larvae rate, and established a more comprehensive and reliable new induction efficiency evaluation method.

[0024] 3) Innovation in shellfish combined breeding technology: Traditional breeding technology usually applies molecular marker-assisted breeding and polyploid induction technology separately, while the present invention combines molecular marker-assisted breeding with triploid induction technology through innovative combination, successfully breeding triploid bay scallops with high yield and high temperature resistance. This technology combination innovation provides an innovative path for the sustainable and healthy development of my country's bay scallop industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 : Orthogonal design experiment diagram of CB concentration and treatment time for inducing homologous triploid bay scallops, wherein (A) the cleavage rate of triploid larval embryos under different conditions; (B) the triploid rate of D-shaped larvae under different conditions; (C) the hatching rate of triploid embryos under different conditions; (D) the metamorphosis rate of triploid larvae under different conditions; (E) the flow cytometer ploidy detection results of diploid group larvae; (F) the flow cytometer ploidy detection results of D-shaped larvae using the reported conditions (0.1 mg / L CB treatment for 20 minutes); (G) the flow cytometer ploidy detection results of D-shaped larvae using the induction conditions optimized by the present invention (0.5 mg / L CB treatment for 15 minutes).

[0026] Figure 2 : Chromosome analysis diagram of trochophore larvae of bay scallop in control group (diploid) and induced group (triploid), including (A) chromosome observation of diploid trochophore larvae; (B) chromosome number statistics of trochophore larvae in control group; (C) chromosome observation of homologous triploid trochophore larvae; (D) chromosome number statistics of trochophore larvae in experimental group.

[0027] Figure 3 : Growth and development characteristics of embryos and larvae of bay scallops in the control group (diploid) and the induced group (triploid), where (A) the time taken for diploid and triploid bay scallop embryos to develop to different stages before hatching; (B) the growth changes of shell length of diploid and triploid bay scallop shell top larvae after hatching, and the arrows indicate the time of attachment substrate placement.

[0028] Figure 4 : Appearance, ploidy and growth characteristics of adult bay scallops in the control group (diploid) and induced group (triploid), including (A) appearance of adult diploid bay scallops; (B) appearance of adult triploid bay scallops; (C) ploidy detection results of gill filaments of diploid bay scallops by flow cytometry; (D) ploidy detection results of gill filaments of triploid bay scallops by flow cytometry; (E) growth trait statistics of 8-month-old diploid and triploid bay scallops; (F) dynamic changes in adductor muscle weight growth of diploid and triploid bay scallops.

[0029] Figure 5 : Differences in the ABT index of the temperature tolerance of bay scallops among the control group (diploid), the traditional group (triploids prepared by randomly selecting broodstock for spawning and induction) and the experimental group (triploids prepared by spawning and induction of high-temperature resistant broodstock). DETAILED DESCRIPTION

[0030] The present invention firstly adopts molecular marker-assisted breeding technology, and screens out bay scallop parents with high temperature resistance characteristics based on the results of genome-wide association analysis (GWAS), providing an excellent germplasm basis for subsequent polyploid induction. And through GWAS, the SNP site most significantly associated with the high temperature resistance index Arrhenius break temperature (ABT) of bay scallops was identified, and based on this, SNP molecular markers associated with temperature resistance traits were developed (Table 1). The screened SNP site is located at position 234 of the following sequence (Arg0230340.1, SEQ ID NO: 1), and its base is T / C;

[0031] Table 1: SNP marker information most significantly associated with the temperature tolerance trait ABT of bay scallop

[0032]

[0033] The live sampling method was used to clamp 1 to 2 gill filaments of the selected broodstock with tweezers, and the scallop gill filament DNA was extracted. The upstream and downstream primers of the SNP marker were designed (Table 2) for PCR amplification, and individuals with high temperature resistant genotypes were screened as broodstock through Sanger sequencing.

[0034] Table 2: Sequence list of primers used for detecting SNP sites and Sanger sequencing

[0035]

[0036] The selected heat-resistant broodstock are subjected to a temperature-accumulated ripening treatment. The water temperature is raised by 1°C per day on the basis of the natural water temperature until the water temperature reaches 20°C and is kept constant for spawning, so that the accumulated temperature of the broodstock reaches 180-200°C per day.

[0037] Orthogonal experiments were designed to study the induction effects of cytochalasin B (CB) at different concentrations (0.1 mg / L, 0.3 mg / L, 0.5 mg / L and 0.7 mg / L) and treatment times (10, 15, 20 and 25 minutes) on the fertilized eggs of bay scallops, explore the induction efficiency of autologous triploids under different conditions, and determine the best treatment plan:

[0038] 1) Inducing spawning of broodstock by drying in the shade and raising the temperature: The broodstock that have been matured in 20°C seawater are taken out and dried in the shade at room temperature (23-24°C) for 40 minutes, and then transferred to a 2000 mL beaker filled with 24°C seawater for spawning induction. Each broodstock is placed in a separate beaker.

[0039] 2) Record the fertilization time and control the sperm-egg ratio: After the parent shellfish ovulates, record the ovulation time as the fertilization time, and observe the samples under a microscope to control the sperm-egg ratio to 3:1 to 5:1. Monitor the fertilization progress of the fertilized eggs in real time. When 30% of the fertilized eggs have the first polar body, add the pre-configured 1mg / mL cytochalasin B mother solution to make the fertilized egg system reach the target treatment concentration.

[0040] 3) Collection of fertilized eggs and embryo hatching after CB treatment: After the chemical treatment, the fertilized eggs were collected by filtration using a sieve and rinsed three times with 24°C seawater to remove residual CB. Subsequently, the collected fertilized eggs were transferred to a 100L culture barrel and aerated culture was started. The culture water temperature was always maintained at 24±1°C to ensure stable embryonic development.

[0041] The different induction conditions were evaluated by comprehensively considering the cleavage rate, hatching rate, metamorphosis rate and triploidy rate to obtain the optimal induction conditions for homologous triploidy of bay scallops. The specific method is: observe and count the cleavage rate, hatching rate and metamorphosis rate of embryos / larvae during the breeding process, and collect samples for flow cytometry ploidy detection after the embryos develop into D-shaped larvae. The calculation formulas for each indicator are as follows:

[0042]

[0043] The cleavage rate, hatching rate, metamorphosis rate and triploid rate were used to comprehensively evaluate the different cytochalasin B treatment conditions. The formula is as follows:

[0044]

[0045] The comprehensive evaluation indexes of different conditions are shown in Table 3. Treatment with 0.5 mg / L CB for 15 minutes is the optimal condition for inducing homologous triploidy in bay scallops, and the comprehensive evaluation index (31.26%) is significantly higher than that of the traditional method (0.1 mg / L CB for 20 minutes, comprehensive evaluation index 14.97%).

[0046] Table 3: Comprehensive evaluation index table for different conditions

[0047]

[0048] The steps of measuring the specific growth performance in the specific embodiment of the present invention are as follows:

[0049] 1) Larval culture stage: Before the larvae develop to the blastocyst stage, the development of the larvae in the control group and the induced group was sampled and examined under a microscope at intervals of 10 minutes, and the time of the larvae developing to each stage was recorded; between the blastocyst stage and the D-shaped larvae stage, the larval development time was recorded by sampling and examination under a microscope every 1 hour; after hatching, the shell length of the shell-top larvae was counted every day. The development speed of the larvae in the induced group and the control group and the growth rate of the shell length of the shell-top larvae were compared to evaluate the growth performance of the two groups of larvae.

[0050] 2) Marine culture stage: From June to November, individuals in the induced group and control group (diploid) were randomly selected every month and their gill filaments were obtained. The ploidy was determined by flow cytometry. The growth traits of the triploid and diploid individuals with determined ploidy were measured, including shell height, shell length, shell width, live weight, adductor muscle weight, and the growth performance of the two groups was compared and evaluated.

[0051] The heat resistance performance determination steps in the specific embodiment of the present invention are as follows:

[0052] During the offshore aquaculture stage from June to November, the temperature tolerance of triploid and diploid individuals with confirmed ploidy was measured at the same time as the growth performance measurement: the scallops were placed in aerated seawater, and the water temperature was raised from 20°C to 38°C at a rate of 1°C / minute, with each 2°C increase set as a temperature detection point. The heart rate data of the scallops were collected using an infrared photoelectric sensor, and the waveform was analyzed by a computer to calculate the temperature tolerance index ABT: that is, the horizontal coordinate of the intersection of the linear fitting formula of the scatter plot formed by the temperature and heart rate rising stage and the linear fitting formula of the heart rate decreasing stage.

[0053] The present invention is described below in conjunction with embodiments and drawings.

[0054] Example 1

[0055] In early March 2024, the inventors started the preparation of high-yield, high-temperature resistant homologous triploid bay scallops at Shandong Yantai Haiyi Seedlings Co., Ltd. The specific process includes the following steps: MAS technology screening of high-temperature resistant bay scallop parents, accumulating temperature to promote ripening of bay scallop parents, optimizing cytochalasin B treatment conditions, induction effect detection, growth performance determination and temperature resistance determination.

[0056] 1. MAS technology to screen high temperature resistant bay scallop parents

[0057] 120 individuals of similar size (shell height of about 6 cm) were randomly selected from the common bay scallop population. One to two gill filaments were taken from each individual using tweezers for DNA extraction, and PCR amplification was performed using upstream and downstream primers designed based on the high temperature resistance SNP marker (Table 2). The high temperature resistant individuals with genotype of TT / TC at 234 bases were screened by Sanger sequencing results, and a total of 38 individuals with high temperature resistant genotypes were screened as broodstock in the experimental group. The remaining 82 individuals were divided into traditional group (n=41) and control group (n=41) broodstock.

[0058] 2. Accumulated temperature to promote the ripening of bay scallop broodstock

[0059] Each group of broodstock was placed in the same environment for ripening treatment until the gonads were well developed (the distal male area of ​​the gonad was milky white and the proximal female area was orange-red), and was temporarily raised in seawater with a temperature of 20°C and a salinity of 30‰. Subsequently, each group of broodstock was shade-dried for 40 minutes at room temperature of 24°C. After the shade drying was completed, 20 broodstock were randomly selected from each group and placed in a beaker with a volume of 2000mL. 24°C seawater was added to the cup to promote spermatogenesis and ovulation of bay scallops. The seawater temperature was measured regularly throughout the process, and the seawater was replaced regularly to keep the temperature within the range of 24±1°C.

[0060] 3. Optimizing cytochalasin B treatment conditions

[0061] After ovulation (mixed ovulation of sperm and eggs) of the experimental and traditional groups, the seawater was stirred in time to ensure that the eggs were fully fertilized. The ovulation time was recorded as the fertilization time, and samples were taken immediately for observation. The sperm-egg ratio was checked under a microscope to ensure that the sperm:egg ratio was 5:1. Subsequently, samples were taken every 1 minute to observe the development of the fertilized eggs under a microscope.

[0062] Experimental group: When 30% of the fertilized eggs showed the first polar body under the microscope, CB mother solution was added to the seawater enriched with fertilized eggs to make the final concentration of CB reach 0.5 mg / L. After 15 minutes of treatment, the fertilized eggs were collected with a 500-mesh sieve and rinsed with 24°C seawater to remove residual cytochalasin B.

[0063] Traditional group: According to the previously reported method, CB mother solution was added to the fertilized eggs seawater to make the concentration reach 0.1 mg / L. After 20 minutes of treatment, the fertilized eggs were collected with a 500-mesh sieve and rinsed with 24°C seawater.

[0064] Control group: No cytochalasin B was added, and the fertilized eggs were directly transferred to the breeding tank for subsequent incubation

[0065] Transfer the fertilized eggs of each group to a 100L breeding barrel (mark each group of breeding barrels). The breeding conditions are a water temperature of 24°C and a salinity of 30‰. During the first 10 hours of incubation, gently stir the breeding barrel every 0.5 hours to prevent the embryos from settling. After 1 hour of development: the cleavage rate of each group is counted; during the hatching stage: the hatching rate of each group of embryos is recorded; during the metamorphosis stage: 3 days after the attachment substrate is placed, the metamorphosis rate of each group is counted. The cleavage rate and hatching rate of the experimental group and the traditional group are similar, with no significant difference ( Figure 1 A&B); the triploid rate (75.75±6.51%) and abnormal rate (78.14±7.02%) of the experimental group were significantly (P<0.05) higher than those of the traditional group (triploid rate 40.35±6.11%, abnormal rate 59.61±3.84%) ( Figure 1 C&D).

[0066] 4. Induction effect detection

[0067] 1) Detection of larval ploidy by flow cytometry. After 18–24 hours of hatching, D-shaped larvae were selected. After selection, 300-mesh silk screen was used to enrich D-shaped larvae (N>500), and flow cytometry was used to detect embryo ploidy ( Figure 1 ). The specific operation is as follows: the larvae collected by 300 mesh silk filtration are placed in 1mL 4×PBS buffer; collagenase (working concentration 1×) is added to lyse at room temperature for 10 minutes; the cell suspension after larval lysis is collected by filtration using a cell sieve; the number of cells is counted under a microscope using a hemocytometer to ensure that the cell concentration reaches more than 1000 cells / μL; 20μL 1× propidium iodide (PI) is added to the cell suspension and gently mixed; the sample is stained in the dark for 30 minutes and then placed on a flow cytometer to detect the cell DNA content; according to the flow cytometry test results, the embryo ploidy distribution is analyzed and the proportion of triploid larvae is calculated. The rate of triploid larvae in the experimental group after the improved method (0.5mg / L CB treatment for 15 minutes) (75.75±6.51%) was significantly (P<0.05) higher than that in the traditional group (40.35±6.11%).

[0068] 2) Observation and analysis of chromosomes in trochophore larvae cells. When the larvae develop to the trochophore larvae stage, the trochophore larvae are enriched with a 500-mesh silk sieve (N>500), and the chromosomes of the cells are observed by the following steps: the enriched trochophore larvae are transferred to a 2 mL centrifuge tube; a 0.01% colchicine solution prepared with sterile seawater is added and treated for 1.5 hours; the sample is centrifuged at 240 g for 5 minutes and the supernatant is discarded; a hypotonic solution with a concentration of 0.075 MKCl is added to the larval sample and the treatment time is 20 minutes; the sample is centrifuged at 240 g for 5 minutes and the supernatant is discarded again; a -20°C precooled Calomel fixative (ethanol: acetic acid = 3:1) is added and the fixation treatment time is 15 minutes; the fixative is replaced and the fixation step is repeated 3 times; during the entire fixation process, ensure that the centrifuge tube remains horizontal to avoid cell compression or deformation. Transfer the fixed larval samples to a 1.5 mL centrifuge tube; centrifuge at 3500 g for 10 minutes and discard the supernatant; add 50% acetic acid solution to dissociate the cells for 2 minutes; drop the cell suspension on a pre-warmed slide at 56°C and dry naturally; drop 30 μL of 4',6-diamino-2-phenylindole (DAPI) on the dry slide and stain at room temperature in the dark for 10 minutes; wash off the excess dye with PBS or deionized water; cover with glycerol-gelatin mounting medium; observe the stained samples under a fluorescence microscope ( Figure 2 ), record and analyze chromosome morphology and structural information. Microscopic observation revealed that most of the diploid trochophore larvae cells had a typical 32 chromosome division phase, accounting for 76.06% of the total number of observed cells ( Figure 2 B); In contrast, most of the triploid trochophore larvae cells showed mitotic phases with 48 chromosomes, accounting for 52.54% of the total number of observed cells ( Figure 2 D).

[0069] 5. Growth performance determination

[0070] 1) Determination of growth performance during embryonic development

[0071] Before the larvae develop to the blastocyst stage, the development of the larvae in the control group and the experimental group is sampled and examined at intervals of 10 minutes, and the time when the larvae develop to each stage is recorded; between the blastocyst stage and the D-shaped larvae stage, the larval development time is recorded every 1 hour. The specific operation is as follows: use a disposable pipette to draw 1mL of embryo system into the embryo dish, and examine it under an optical microscope. If 80% of the embryos in the field of view are at the same developmental stage, it is considered that the embryos in this group have developed to that period. After hatching, the shell-top larvae are drawn into the embryo dish (N>30) every day, and the shell length of the shell-top larvae is counted under an optical microscope.

[0072] The results showed that from the fertilized egg to the middle stage of the shell top larvae (shell length 150mm), the larvae in the experimental group developed slower than those in the control group. In the late stage of the shell top larvae, the larvae in the experimental group showed a significant growth advantage over the control group, and the eye spots appeared 2 days earlier, and the attachment base was placed 2 days earlier ( Figure 3 ).

[0073] 2) Determination of growth performance in adult stage

[0074] The experimental group (N>50), traditional group (N>50) and control group (N>50) individuals cultured in the same batch were collected, and the gill filaments were taken for flow cytometry ploidy determination. The specific operation steps are as follows: the gill filaments were placed in 1mL 4×PBS buffer, collagenase (working concentration 1×) was added, and lysed at room temperature for 10 minutes. Then, the cell suspension was filtered using a cell sieve, and the cells were counted under a microscope using a hemocytometer to ensure that the number of cells was greater than 1000 / μL. Then, 20μL 1×PI was added to the cell solution, and flow cytometry was performed after staining in the dark for 30 minutes. After determining the ploidy, triploid (N=12) and diploid (N=12) individuals were selected from the experimental group and the control group, respectively, and their growth traits, including shell height, shell length, shell width, live weight and adductor muscle weight, were determined.

[0075] The results showed that triploids had significant (P<0.05) growth advantages over diploids in terms of shell height, shell length, shell width, live weight, and adductor muscle weight. Figure 4 E), and the adductor muscle weight of triploids increases faster than that of diploids ( Figure 4 F). In the first seedling division (May 16, 2024), the number of individuals in the experimental group of the same batch was about 4,000, the number of individuals in the traditional group was about 3,000, and the number of individuals in the control group was about 5,000; after cultivation until October 2024, the number of individuals in the experimental group was about 3,500 (survival rate 87.5%), the number of individuals in the traditional group was about 2,000 (survival rate 66.7%), and the number of individuals in the control group was about 4,200 (survival rate 84.0%). The survival rate of the experimental group was higher than that of the traditional group and the control group of the same batch.

[0076] 6. Determination of temperature resistance

[0077] Bay scallops from the experimental group (N=12), traditional group (N=12) and control group (N=12) were randomly selected for cardiac performance measurement to evaluate the temperature tolerance of the triploid individuals in the experimental group. The specific operations were as follows: an infrared photoelectric sensor was attached to the scallop shell near the heart, the collected signal was processed through a signal amplifier and a filter, and the cardiac waveform was analyzed using a computer. During the experiment, the seawater was continuously ventilated, the initial water temperature was set at 20°C, and the temperature was gradually increased to 38°C at a rate of 1°C / minute. During the experiment, each increase of 2°C was set as a temperature detection point. At each temperature point, cardiac waveform data was collected after the water temperature was stable for 5 minutes, and the data was recorded continuously for 5 minutes. The calculation method of Arrhenius breakthrough temperature (ABT) is as follows: During the cardiac performance measurement process, the scattered data formed by the increase and decrease of heart rate with temperature were linearly fitted to obtain two linear fitting formulas. The horizontal coordinate of the intersection of the two formulas is ABT. Through the above experimental process, the pattern of changes in heart rate of individuals in each group with temperature was obtained, and the corresponding ABT value was calculated. The results are as follows Figure 5 As shown, the ABT of triploid bay scallops in the experimental group (33.76±1.76℃) was higher than that in the traditional group (31.23±1.24℃) and the control group (31.94±0.94℃), showing good temperature tolerance.

[0078] The present invention uses the MAS technology to screen high-temperature resistant bay scallop parents, combines the optimized cytochalasin B to treat the fertilized eggs of bay scallops, and successfully prepares high-yield, high-temperature resistant triploid bay scallops. The experimental process covers parent scallop screening, accumulated temperature to promote ripening, cytochalasin B optimization treatment, induction effect detection, and growth and temperature resistance performance determination. The present invention comprehensively considers multiple key characteristics of embryonic development and establishes a more comprehensive and reliable new induction efficiency evaluation method. The triploid individuals obtained by the present invention are superior to diploids in growth performance and temperature resistance, providing innovative methods and technical support for the cultivation of fast-growing and stress-resistant bay scallop varieties.

Claims

1. A SNP locus associated with the high temperature resistance trait of bay scallop, characterized in that: The SNP site is located at the 234th position of the nucleotide fragment of SEQ ID NO: 1, and its base is T / C.

2. Use of the SNP site described in claim 1 as a molecular marker in screening bay scallop individuals with high temperature resistance traits.

3. A method for screening bay scallop individuals for high temperature resistance traits, characterized in that: The method is to detect the genotype of the SNP site of claim 1 in the bay scallop individuals to be screened, and screen individuals with the TT / TC genotype.

4. The method according to claim 3, characterized in that The method is to use a live sampling method to extract DNA from the gill filaments of the bay scallop to be tested, amplify the DNA using a PCR primer pair, and then sequence the amplified product to determine the genotype.

5. The method according to claim 4, characterized in that The upstream primer of the primer pair has a sequence of SEQ ID NO: 2, and the downstream primer has a sequence of SEQ ID NO:

3.

6. A method for inducing and cultivating homologous triploid bay scallops, characterized in that: The method is to use the bay scallop individuals with high temperature resistance selected by the method of claim 3 as parents to induce the cultivation of homologous triploid bay scallops.

7. The induction cultivation method according to claim 6, characterized in that: The method comprises the following steps: 1) The broodstock that have been matured in seawater are taken out, dried in the shade at room temperature, and then transferred to warmed seawater for spawning induction; 2): After the parent shellfish ovulates, record the ovulation time as the fertilization time, and observe the samples under a microscope. When 30% of the fertilized eggs show the first polar body, add cytochalasin B mother solution for treatment; 3) After the treatment is completed, the fertilized eggs are collected by filtration, and are rinsed with seawater to remove residual cytochalasin B, and the collected fertilized eggs are cultured for embryonic development.

8. The method according to claim 7, characterized in that In the method, step 1) uses heated seawater at 24°C.

9. The method according to claim 7, characterized in that In the method, the treatment concentration of cytochalasin B is 0.5 mg / L and the treatment lasts for 15 minutes.

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