A method for breeding a paralichthys olivaceus clone improved strain
Through a series of breeding steps, a fast-growing and highly fertile improved clonal line of turbot was bred, solving the problems of slow growth and weak fertility of double haploid and clonal lines of turbot, and realizing the large-scale application of double haploid fish breeding.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- BEIDAIHE CENT EXPERIMENTAL STATION OF CHINESE ACAD OF FISHERY SCI
- Filing Date
- 2024-10-10
- Publication Date
- 2026-06-26
AI Technical Summary
Existing double haploid and clonal lines of turbot exhibit slow growth, weak reproductive capacity, and poor stress resistance, which restricts the large-scale application of double haploid breeding in fish.
Through a series of breeding steps, including the preparation of highly homozygous turbot gynogenetic diploids, the preparation of turbot double haploid DH, the preparation of double clonal hybrid lines, the preparation of recombinant double haploid rDH, the cultivation of turbot recombinant double haploid rDH, and the preparation of clonal improved lines, a turbot clonal improved line with fast growth and high reproductive capacity has been bred.
The development of a fast-growing, highly fertile clonal improved strain of turbot has been achieved, solving the practical application problem of double haploid breeding in fish, significantly improving the growth and reproductive performance of turbot, and supporting the large-scale application of double haploid breeding in fish.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of fish genetic breeding technology, specifically relating to a method for breeding a clonal improved line of turbot. Background Technology
[0002] Existing double haploid (DH) and clonal lines of turbot exhibit problems such as slow growth, low reproductive capacity, and poor stress resistance. These issues hinder the large-scale application of double haploid fish breeding. Therefore, breeding faster-growing, more reproductive clonal lines is crucial for solving the practical application problems of double haploid fish breeding. Summary of the Invention
[0003] The technical problem to be solved by the present invention is to provide a method for breeding improved clonal lines of turbot, which addresses the shortcomings of the prior art. This method breeds improved clonal lines with fast growth and high reproductive capacity, and can solve the problem of practical application of double haploid breeding in fish.
[0004] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a method for breeding improved clonal lines of turbot, the method being as follows:
[0005] S1. Preparation of high-homozygous turbot gynogenetic diploids
[0006] From the core germplasm population, select turbot with superior traits as parents, and induce meiotic gynogenesis in turbot using the cold shock method to obtain turbot gynogenic diploids. After homozygosity identification by microsatellite molecular markers, select 1,000 turbot gynogenic diploids with homozygosity greater than 0.8 as reserve broodstock.
[0007] S2, Preparation of double haploid DH in turbot
[0008] Ten female individuals with a mature weight >3.0 kg, well-developed gonads, and no disease or injury were selected from the diploid mitotic development of turbot in S1. Mitotic gynogenesis was induced, and the first cleavage was suppressed by hydrostatic pressure to achieve chromosome doubling, thus obtaining diploid mitotic gynogenesis of turbot, namely double haploid DH. After identification by microsatellite markers with a high recombination rate, 1,000 double haploid DH individuals with a homozygosity of 1.0 were selected as reserve broodstock.
[0009] S3, preparation of double-clonal hybrid lines
[0010] After the selected double haploid DH individuals from S2 have matured, 10 female and 10 male double haploid DH individuals with well-developed gonads and no diseases or injuries on their bodies are selected for hybridization to obtain a double clone hybrid line. During the breeding process, the growth performance of the double clone hybrid line is evaluated, and 500 individuals with a weight of more than 500g at 12 months of age are selected as reserve broodstock.
[0011] S4, Preparation of recombinant double haploid rDH
[0012] After the selected biclonal hybrid line in S3 reaches sexual maturity, 10 individuals with a sexual maturity weight >3.0 kg, well-developed gonads, and no disease or injury on their body surface are selected as parents during the breeding season. Mitosis and gynogenesis are induced again, and the first cleavage is suppressed by hydrostatic pressure to achieve chromosome doubling, resulting in a homozygous recombinant double haploid population of turbot, which is the turbot rDH population.
[0013] Cultivation of S5 and turbot recombinant double haploid rDH
[0014] The turbot rDH population obtained from S4 was cultured according to the standardized turbot seedling cultivation procedure. After 6 months of age, individuals were marked with PIT. After marking, phenotypic tests were conducted monthly during the cultivation process, and growth performance was evaluated. At 12 months of age, 500 individuals with a weight >500g and a purity of 1.0 were selected to recombinant double haploid turbot rDH.
[0015] S6. Preparation and cultivation of clonal improvement lines
[0016] When the recombinant double haploid rDH of turbot selected from S5 is cultured to sexual maturity, 10 female individuals with a weight >3.0kg, well-developed gonads, and no disease or injury on their body surface are selected for maturation during the breeding season and used as parents to induce meiotic gynogenesis to obtain turbot clonal lines.
[0017] The turbot clones were cultured according to the standardized breeding specifications for turbot seedlings. During the culture process, the growth performance of different clones was evaluated, and families with an average weight of more than 500g at 12 months of age were selected as candidate clones. At sexual maturity, families with a weight of more than 3.0kg, good gonad development, and a fertility rate of more than 80% were selected as clone improvement lines.
[0018] Preferably, the species of turbot mentioned in S1 is the native economic fish of the Bohai Sea, the turbot (Paralichthysolivaceus).
[0019] Preferably, the cold shock method described in S1 is as follows: after fertilizing the turbot eggs in vitro with inactivated red sea bream sperm, the eggs are subjected to cold shock treatment in seawater at a temperature of 0±0.5℃ for 45 minutes, and then transferred to seawater at a temperature of 15℃ for incubation.
[0020] Preferably, the hydrostatic pressure method described in S2 and S4 is as follows: after turbot eggs are fertilized in vitro with inactivated red sea bream sperm, they are incubated in seawater at 17°C for 60 minutes, and then treated with hydrostatic pressure of 65 MPa for 6 minutes.
[0021] Compared with the prior art, the present invention has the following advantages:
[0022] The fast-growing, highly reproductive turbot clonal improvement line bred in this invention can solve the problem of practical application of double haploid breeding in fish.
[0023] The present invention will be further described in detail below with reference to the embodiments. Detailed Implementation
[0024] Example 1
[0025] The method for hybrid clonal selection and breeding of improved turbot clonal lines in this embodiment is as follows:
[0026] S1. Preparation of high-homozygous turbot gynogenetic diploids
[0027] From the core germplasm population of the Beidaihe Experimental Station of the Chinese Academy of Fishery Sciences, superior broodstock of *Paralichthys olivaceus*, a local economic fish species in the Bohai Sea, were selected. Meiotic gynogenesis was induced in the *Paralichthys olivaceus* using the cold shock method to obtain diploid *Paralichthys olivaceus*. After homozygosity identification by microsatellite molecular markers (Table 1), 1000 individuals with homozygosity greater than 0.8 were selected as diploid *Paralichthys olivaceus*. The cold shock method was as follows: after in vitro fertilization of *Paralichthys olivaceus* eggs with inactivated *Paralichthys spp.* sperm, the eggs were subjected to cold shock treatment in seawater at a temperature of 0±0.5℃ for 45 minutes, and then transferred to seawater at a temperature of 15℃ for incubation.
[0028] In this embodiment, sperm and eggs were obtained as follows: The experimental fish used were female turbot and male red sea bream artificially bred at the Beidaihe Experimental Station of the Chinese Academy of Fishery Sciences. Sperm was obtained by gently squeezing the abdomen of the male red sea bream and stored in a syringe at 4°C in the dark. Eggs were obtained by gently squeezing the abdomen of the female turbot and stored in a beaker at room temperature in the dark. The collected sperm and eggs were processed or fertilized as needed.
[0029] Sperm UV irradiation inactivation: Sea bream semen was diluted with Ringer's solution at a ratio of 1:50. Irradiation times were set at 11 gradients: 0, 1, 2, 5, 10, 20, 30, 45, 60, 75, and 90 seconds. The actual radiation dose at each set dose was measured using an ultraviolet radiometer (VLX-3W, Cole Parmer Instrument Company, Illinois, USA), with doses of 0, 1.4, 3.4, 7.2, 13.0, 25.0, 37.0, 55.0, 73.0, 92.0, and 110.0 mJ / cm³. 2 After irradiation, the semen was examined under a microscope to confirm sufficient sperm activity. Then, 0.1 mL of the irradiated semen from each group was fertilized with 1 mL of eggs from the same turbot. The fertilization water temperature was (16±0.5)℃. The fertilized eggs were then incubated in 1 L beakers at (17±0.5)℃. Three replicates were set for each group, and the hatching rate was statistically analyzed. The optimal sperm inactivation parameters were obtained through the above experiments. The optimal irradiation conditions were: irradiation time 60 s, irradiation distance 28–30 cm, and irradiation dose 73 mJ / cm². 2 After irradiation, the semen was examined under a microscope to confirm sperm motility and then stored at room temperature away from light for later use.
[0030] S2, Preparation of double haploid DH in turbot
[0031] Ten individuals with a mature body weight >3.0 kg, well-developed gonads, and no diseases or injuries were selected from the gynogenic diploids of turbot in S1. Mitotic gynogenesis was induced in these individuals, and the first mitosis was inhibited by hydrostatic pressure to obtain turbot mitotic gynogenic diploids, i.e., double haploids DH. Homozygosity was determined by high recombination rate microsatellite markers (recombination rate 1.0) (Table 2). 1000 turbot double haploid DH individuals with a homozygosity of 1.0 were selected as backup parents.
[0032] S3, preparation of double-clonal hybrid lines
[0033] After the selected double haploid DHs from S2 mature, 10 female and 10 male double haploid DHs with well-developed gonads and no diseases or injuries are selected for hybridization to obtain a double clone hybrid line. During the breeding process, the growth performance of the double clone hybrid line is evaluated, and 500 individuals with a weight of more than 500g at 12 months of age are selected as reserve broodstock.
[0034] S4, Preparation of recombinant double haploid rDH
[0035] After the selected biclonal hybrid line in S3 reaches sexual maturity, 10 female individuals with a weight >3.0 kg, well-developed gonads, and no disease or injury on their body surface are selected for sexual maturity during the breeding season and used as parents. Mitosis and gynogenesis are induced again, and the first mitosis is suppressed by hydrostatic pressure to obtain a homozygous recombinant double haploid population of turbot, which is the turbot rDH population.
[0036] The hydrostatic pressure method described in S2 and S4 is as follows: after the turbot eggs are fertilized in vitro with inactivated red sea bream sperm, they are incubated in seawater at 17°C for 60 minutes, and then treated with hydrostatic pressure of 65MPa for 6 minutes.
[0037] Cultivation of S5 and turbot recombinant double haploid rDH
[0038] The rDH population of turbot obtained from S4 was cultured according to the standardized breeding procedure for turbot seedlings. After 6 months of age, individuals were marked with PIT. After marking, phenotypic tests were conducted monthly during the breeding process, and growth performance was evaluated. At 12 months of age, 500 individuals with a weight >500g and a purity of 1.0 were selected as recombinant double haploid rDH turbot as reserve parents.
[0039] S6. Preparation and cultivation of clonal improvement lines
[0040] When the recombinant double haploid rDH of turbot selected from S5 is cultured to sexual maturity, 10 female individuals with a weight >3.0kg, well-developed gonads, and no disease or injury on their body surface are selected for maturation during the breeding season and used as parents to induce meiotic gynogenesis to obtain turbot clonal lines.
[0041] The turbot clones were cultured according to the standardized breeding specifications for turbot seedlings. During the culture process, the growth performance of different clones was evaluated, and families with an average weight of more than 500g at 12 months of age were selected as candidate clones. At sexual maturity, families with a weight of more than 3.0kg, good gonad development, and a fertility rate of more than 80% were selected as clone improvement lines.
[0042] Table 1. Microsatellite molecular markers used for DH homozygosity identification in turbot.
[0043]
[0044]
[0045] Table 2 Microsatellite marker sequences with high recombination rates
[0046]
[0047] The experimental method for identifying homozygosity using microsatellite molecular markers in this embodiment.
[0048] DNA extraction:
[0049] Genomic DNA was extracted using a marine animal DNA extraction kit (TIANGEN, DP324). After extraction, the quality and concentration of the DNA were determined by agarose gel electrophoresis and UV spectrophotometry, respectively, and stored at -20℃ for later use.
[0050] PCR amplification:
[0051] The DNA of turbot was amplified by PCR using the SSR primer pairs synthesized in Tables 1 and 2. When using fluorescent primers for PCR amplification, the upstream primer with a fluorescent group at the 5' end and the downstream primer directly amplified the DNA template to obtain PCR products with fluorescent groups.
[0052] The microsatellite loci poli1076TUF, poli1077TUF, Poli1831TUF, Poli148TUF, poli9TUF, poli107TUF, poli159TUF, poli1825TUF, poli1936TUF, Po15, poli907TUF, and poli212TUF have a fluorescent group (FAM) added to the 5' end of the upstream primers.
[0053] The 5' end of the upstream primers for microsatellite sites poli966TUF, poli1819TUF, Po13, Po25A, Poli1915TUF, poli1010TUF, Poli1490TUF, Poli1838TUF, Poli1925TUF, poli1498TUF, Poli-RC27-TUF, Poli1872TUF, and poli139tuf was supplemented with the fluorescent group HEX.
[0054] The PCR system and reaction procedure are as follows:
[0055] 1) Prepare the PCR system:
[0056] substance Dosage Template DNA 1μl 2XPCRMIX 7.5μl Upstream primer F (10 μM) 0.3μl Downstream primer R (10 μM) 0.3μl Ultrapure water 5.9μl Total volume 15μl
[0057] 2) PCR cycling conditions
[0058]
[0059]
[0060] 3) Take 1 μL of product and add 9 μL of HiDi sample. Denature at 95°C for 3 min, immediately in an ice-water bath, and then perform electrophoresis detection on an ABI 3730XL sequencer.
[0061] 4) Data Analysis
[0062] The data was analyzed using GeneMarker software, and the resulting genotype fragment values were statistically analyzed and stored in Excel. Genotypes with one allele fragment were classified as homozygous, and those with two allele fragments were classified as heterozygous. PopGene software was then used to perform statistical analysis on the genotype data to calculate the homozygosity of each individual. For example, in step S1, if the proportion of homozygous loci across all loci in an individual is 0.80, its homozygosity is 0.80; in step S2, if the genotypes at all three high recombination rate loci are homozygous, its homozygosity is 1.00.
[0063] In summary, through the above six breeding processes, superior clonal improved lines of turbot with excellent growth traits were finally cultivated. These clonal improved lines meet or exceed the growth and reproductive traits of ordinary turbot families, significantly outperforming general clonal lines. By inducing some individuals in these clonal improved lines into pseudo-males through sex control technology, hybridization between different clonal improved lines can be carried out to obtain biclonal hybrid lines with significant heterosis, solving key technologies in fish double haploid breeding technology, and ultimately realizing the large-scale production of turbot biclonal hybrid lines.
[0064] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.
Claims
1. A method for breeding improved clonal lines of turbot, characterized in that, The method is as follows: S1. Preparation of high-homozygous turbot gynogenetic diploids From the core germplasm population, select turbot with superior traits as parents, and induce meiotic gynogenesis in turbot using the cold shock method to obtain turbot gynogenic diploids. After homozygosity identification by microsatellite molecular markers, select 1,000 turbot gynogenic diploids with homozygosity greater than 0.8 as reserve broodstock. The turbot mentioned is a local economic fish species from the Bohai Sea, namely turbot ( Paralichthys olivaceus ); The cold shock method is as follows: after fertilizing the turbot eggs in vitro with inactivated red sea bream sperm, the eggs are subjected to cold shock treatment in seawater at a temperature of 0±0.5℃ for 45 minutes, and then transferred to seawater at a temperature of 15℃ for incubation. S2, Preparation of double haploid DH in turbot Ten individuals with mature body weight >3.0 kg, well-developed gonads, and no disease or injury were selected from the gynogenic diploids of turbot in S1. Mitotic gynogenesis was induced, and the first cleavage was suppressed by hydrostatic pressure to achieve chromosome doubling, thus obtaining diploids of turbot mitotic gynogenesis, namely double haploids (DH). After identification by microsatellite markers with a high recombination rate, 1,000 double haploid DH individuals with a homozygosity of 1.0 were selected as reserve broodstock. S3, preparation of double-clonal hybrid lines After the selected double haploid DHs from S2 mature, 10 female and 10 male double haploid DHs with well-developed gonads and no diseases or injuries are selected as parents and hybridized to obtain a double clone hybrid line. During the breeding process, the growth performance of the double clone hybrid line is evaluated, and 500 individuals with a weight of more than 500g at 12 months of age are selected as reserve broodstock. S4, Preparation of recombinant double haploid rDH After the selected biclonal hybrid line in S3 reaches sexual maturity, 10 female individuals with a weight >3.0 kg, well-developed gonads, and no disease or injury on their body surface are selected for sexual maturity during the breeding season and used as parents. Mitosis and gynogenesis are induced again, and the first cleavage is suppressed by hydrostatic pressure to achieve chromosome doubling, resulting in a homozygous recombinant double haploid population of turbot, which is the turbot rDH population. The hydrostatic pressure method described in S2 and S4 is as follows: after the turbot eggs are fertilized in vitro with inactivated red sea bream sperm, they are incubated in seawater at 17°C for 60 minutes, and then treated with hydrostatic pressure of 65MPa for 6 minutes. Cultivation of S5 and turbot recombinant double haploid rDH The rDH population of turbot obtained from S4 was cultured according to the standardized breeding procedure for turbot seedlings. After 6 months of age, individuals were marked with PIT. After marking, phenotypic tests were conducted monthly during the breeding process, and growth performance was evaluated. At 12 months of age, 500 individuals with a weight >500g and a purity of 1.0 were selected as recombinant double haploid rDH turbot as reserve broodstock. S6. Preparation and cultivation of clonal improvement lines When the recombinant double haploid rDH of turbot selected from S5 is cultured to sexual maturity, 10 female individuals with a weight >3.0kg, well-developed gonads, and no disease or injury on their body surface are selected for maturation during the breeding season and used as parents to induce meiotic gynogenesis to obtain turbot clonal lines. The turbot clones were cultured according to the standardized breeding specifications for turbot seedlings. During the culture process, the growth performance of different clones was evaluated, and families with an average weight of more than 500g at 12 months of age were selected as candidate clones. At sexual maturity, families with a weight of more than 3.0kg, good gonad development, and a fertility rate of more than 80% were selected as clone improvement lines.
2. The method for breeding a clonal improvement line of turbot according to claim 1, characterized in that, The microsatellite loci for the homozygosity identification of microsatellite molecular markers described in S1 are: poli1076TUF, poli1077TUF, Poli1831TUF, Poli148TUF, poli9TUF, poli107TUF, poli159TUF, poli1825TUF, poli1936TUF, Po15, poli907TUF, poli212TUF, poli966TUF, poli1819TUF, Po13, Po25A, Poli1915TUF, poli1010TUF, Poli1490TUF, Poli1838TUF, Poli1925TUF, poli1498TUF, Poli-RC27-TUF, and Poli1872TUF; The primer nucleotide sequences for amplifying the microsatellite locus poli1076TUF are shown in SEQ ID NO.1-2; The primer nucleotide sequences for amplifying the microsatellite locus poli1077TUF are shown in SEQ ID NO.3-4; The primer nucleotide sequences for amplifying the microsatellite locus Poli1831TUF are shown in SEQ ID NO.5-6; The primer nucleotide sequences for amplifying the microsatellite locus Poli148TUF are shown in SEQ ID NO.7-8; The primer nucleotide sequences for amplifying the microsatellite locus poli9TUF are shown in SEQ ID NO. 9-10; The primer nucleotide sequences for amplifying the microsatellite locus poli107TUF are shown in SEQ ID NO.11-12; The primer nucleotide sequences for amplifying the microsatellite locus poli159TUF are shown in SEQ ID NO. 13-14; The primer nucleotide sequences for amplifying the microsatellite locus poli1825TUF are shown in SEQ ID NO.15-16; The primer nucleotide sequences for amplifying the microsatellite locus poli1936TUF are shown in SEQ ID NO.17-18; The primer nucleotide sequences for amplifying the microsatellite site Po15 are shown in SEQ ID NO.19-20; The primer nucleotide sequences for amplifying the microsatellite locus poli907TUF are shown in SEQ ID NO.21-22; The primer nucleotide sequences for amplifying the microsatellite locus poli212TUF are shown in SEQ ID NO. 23-24; The primer nucleotide sequences for amplifying the microsatellite locus poli966TUF are shown in SEQ ID NO.25-26; The primer nucleotide sequences for amplifying the microsatellite locus poli1819TUF are shown in SEQ ID NO. 27-28; The primer nucleotide sequences for amplifying the microsatellite locus and Po13 are shown in SEQ ID NO.29–30; The primer nucleotide sequences for amplifying the microsatellite locus Po25A are shown in SEQ ID NO. 31-32; The primer nucleotide sequences for amplifying the microsatellite locus Poli1915TUF are shown in SEQ ID NO. 33-34; The primer nucleotide sequences for amplifying the microsatellite locus poli1010TUF are shown in SEQ ID NO. 35-36; The primer nucleotide sequences for amplifying the microsatellite locus Poli1490TUF are shown in SEQ ID NO.37-38; The primer nucleotide sequences for amplifying the microsatellite locus Poli1838TUF are shown in SEQ ID NO.39–40; The primer nucleotide sequences for amplifying the microsatellite locus Poli1925TUF are shown in SEQ ID NO.41-42; The primer nucleotide sequences for amplifying the microsatellite locus poli1498TUF are shown in SEQ ID NO.43-44; The primer nucleotide sequences for amplifying the microsatellite locus Poli-RC27-TUF are shown in SEQ ID NO.45-46; The primer nucleotide sequences for amplifying the microsatellite locus Poli1872TUF are shown in SEQ ID NO.47-48.
3. The method for breeding a clonal improvement line of turbot according to claim 1, characterized in that, The microsatellite loci in the high recombination rate microsatellite marker identification described in S2 are: poli9TUF, poli107TUF, Poli139TUF ; Amplifying microsatellite loci poli9TUF The primer nucleotide sequences are shown in SEQ ID NO. 9-10; Amplifying microsatellite loci poli107TUF The primer nucleotide sequences are shown in SEQ ID NO. 11-12; amplification of microsatellite sites. Poli139TUF The primer nucleotide sequences are shown in SEQ ID NO.49-50.