Fish hypoxic adaptation genotypes, detection methods and uses

Abstract

The present invention discloses a nucleotide sequence determining the hypoxic adaptation phenotype of fish, and the base sequence is as shown in SEQ ID NO: 1. The present invention also discloses a method for detecting the hypoxic adaptation genotype of fish, including: extracting genomic DNA of the fish to be tested; using the genomic DNA as a template and performing PCR amplification with the primer pair as shown in SEQ ID NO: 3 and 4; if a product with a size conforming to the first detection value is detected in the PCR amplification product, it is determined that the individual to be tested has a hypoxic adaptation genotype, and if only a product with a size conforming to the second detection value is detected in the PCR amplification product, it is determined that the individual to be tested has a hypoxia-sensitive genotype. The present invention also discloses the use of the nucleotide sequence determining the hypoxic adaptation phenotype of fish or the detection primer described above in the detection, identification and cultivation of hypoxia-tolerant fish varieties. The present invention can be used for the early screening of breeding candidate parents and the design of parental mating groups, with simple operation and low detection cost.

Description

Technical Field

[0001] The present invention belongs to the technical field of fish genetic breeding, and relates to fish hypoxia adaptation genotypes, detection methods and uses thereof. Background Art

[0002] Aquaculture is the fastest-growing food production sector globally and plays an important role in ensuring human food security and nutritional security. Compared with crops and livestock, aquaculture species are generally considered to be in the initial stage of domestication, with rich genetic diversity within the species and good breeding potential. However, the lack of understanding of genes regulating important economic traits and their related molecular markers has become a bottleneck restricting the genetic improvement of aquaculture species and further molecular design breeding.

[0003] In the aquatic environment, the oxygen saturation concentration is only 1 / 30 of that in the same volume of air, with a slow diffusion rate and uneven spatial distribution. After an individual is exposed to a hypoxic environment, a series of compensatory hypoxic regulatory responses are mobilized to restore homeostasis and maintain the survival ability of the body, which is called "hypoxic acclimation". Through multiple generations of natural or artificial selection, genotypes that are beneficial to resisting hypoxic stress are fixed in the genome, forming an inheritable hypoxic tolerance phenotype, which is called "hypoxia adaptation". The hypoxic adaptation phenotype is heritable and has corresponding sequence differences or epigenetic modification characteristics, that is, the hypoxia adaptation genotype, which can be used as the genetic basis for breeding hypoxic-tolerant varieties.

[0004] To adapt to the insufficient oxygen supply under intensive aquaculture conditions, hypoxia has become one of the high-intensity environmental pressures in aquaculture. In the breeding program of cultured fish, exploring excellent alleles and haplotypes suitable for molecular design breeding, detecting the hypoxia adaptation genotypes of candidate parent populations, and avoiding direct hypoxic stress trait testing can effectively prevent lethal damage to candidate parents, screen parent individuals with hypoxic adaptation phenotypes in advance, and facilitate the calculation of breeding values and the formulation of parent mating plans in combination with other important economic traits, so as to breed cultured varieties with excellent hypoxic adaptation traits.

[0005] However, the screening of hypoxia adaptation genotypes and molecular markers at the genomic sequence level is still in its infancy, and the sequence and structural variation information that can explain high phenotypic variation is very limited. For example, the research group of Professor Xia Junhong at Sun Yat-sen University located two hypoxia-related loci in tilapia through QTL-seq and ddRAD-seq, which are located in the genes GPR132 (G protein coupled receptor 132) and ABCG4 (ATP binding cassette subfamily G member 4) respectively. The research group of Professor Liu Zhanjiang at Auburn University in the United States carried out genome-wide association analysis in different families using a high-throughput single nucleotide polymorphism (SNP) chip, located multiple hypoxia-related loci, and focused on 6 pathways including VEGF, mTOR, PI3K-AKT, DNA damage checkpoint, P53-mediated apoptosis, and MAPK. Carp (Cyprinus carpio) is an indigenous fish species native to China, with a breeding history of more than 8,000 years in China. Now it has developed into a worldwide aquaculture species, with its farming areas covering Eurasia and an annual output of 4.1895 million tons. At present, the understanding of the hypoxia adaptation genotype and molecular markers of carp is almost blank.

[0006] The development of hypoxia adaptation genotypes in fish and their detection technologies will be of great significance for achieving the goal of genetic improvement of aquaculture that is "resource-saving and widely adaptable to the environment", coping with the realistic challenges of the continuous compression of land-based aquaculture space, and promoting the green transformation of the aquatic food system.

[0007] In summary, it is urgent to obtain a batch of reliable hypoxia adaptation genotypes in fish and develop simple and efficient detection methods, and apply them to the cultivation of hypoxia-tolerant aquaculture varieties. Summary of the Invention

[0008] One object of the present invention is to solve at least the above problems and / or deficiencies, and provide at least the advantages described hereinafter.

[0009] Another object of the present invention is to provide a nucleotide sequence that determines the hypoxia adaptation phenotype of fish and a nucleotide sequence that determines the hypoxia-sensitive phenotype of farmed fish.

[0010] Another object of the present invention is to provide a primer pair for detecting the hypoxia adaptation genotype of fish.

[0011] Another object of the present invention is to provide a detection method for the hypoxia adaptation genotype of fish.

[0012] Another object of the present invention is to provide a detection kit for the hypoxia adaptation genotype of fish.

[0013] Another object of the present invention is to provide the use of the nucleotide sequence or the detection primer in the detection, identification and cultivation of hypoxia-tolerant fish varieties.

[0014] For this purpose, the technical solution provided by the present invention is as follows:

[0015] The nucleotide sequence determining the hypoxia adaptation phenotype of fish comprises the base sequence shown in SEQ ID NO: 1. Both the heterozygote and the homozygote of the base sequence shown in SEQ ID NO: 1 show hypoxia adaptation.

[0016] The nucleotide sequence determining the hypoxia-sensitive phenotype of cultured fish has the base sequence shown in SEQ ID NO: 2.

[0017] The primer pair for detecting the hypoxia adaptation genotype of fish is: the base sequence shown in SEQ ID NO: 3 and the base sequence shown in SEQ ID NO: 4.

[0018] A method for detecting the hypoxia adaptation genotype of fish comprises the following steps:

[0019] 1) Extract the genomic DNA of the fish to be tested;

[0020] 2) Using the genomic DNA in step 1) as a template, perform PCR amplification with a pair of primers shown in SEQ ID NO: 3 and SEQ ID NO: 4;

[0021] 3) Determine the length of the PCR amplification product obtained in step 2). If a product conforming to the first detection value size is detected in the PCR amplification product, it is determined that the individual to be tested of the template genomic DNA is of the hypoxia adaptation genotype. Both the heterozygote and the homozygote of the base sequence shown in SEQ ID NO: 1 show hypoxia adaptation. If only a product conforming to the second detection value size is detected in the PCR amplification product, it is determined that the individual to be tested of the template genomic DNA is of the hypoxia-sensitive genotype, wherein the first detection value is greater than the second detection value.

[0022] Preferably, in the method for detecting the hypoxia adaptation genotype of fish, the fish is a common carp.

[0023] Preferably, in the method for detecting the hypoxia adaptation genotype of fish, in step 1), the fish to be tested is in the juvenile stage.

[0024] Preferably, in the method for detecting the hypoxia adaptation genotype of fish, in step 1), fin rays or blood samples of the fish to be tested are taken to extract genomic DNA.

[0025] Preferably, in the method for detecting the fish hypoxic adaptation genotype, in step 3), the first detection value is 358 bp and the second detection value is 179 bp.

[0026] A kit for detecting the fish hypoxic adaptation genotype, which comprises the primer pair.

[0027] Use of the nucleotide sequence or the detection primer in the detection, identification and cultivation of fish varieties with hypoxia tolerance.

[0028] The present invention has at least the following beneficial effects:

[0029] The hypoxic adaptation genotype provided by the present invention is derived from a significant association locus of a large-scale genome-wide association study (GWAS), located in the calsequestrin-2-like gene on chromosome 2 of carp. This gene encodes a calsequestrin-like protein, which is expressed in the myocardium and skeletal muscle, has the function of regulating calcium ion concentration, and plays an important role in hypoxic signal transduction.

[0030] The nucleotide sequence difference provided by the present invention is located in the upstream promoter region of the calsequestrin-2-like gene, including an insertion / deletion mutation of up to 179 bp in the promoter region, which directly determines the transcription level of the calsequestrin-2-like gene, has a high phenotypic correlation with the hypoxic adaptation trait of carp, and has a certain generality in other cultured fish.

[0031] The method for detecting the hypoxic adaptation genotype provided by the present invention can use fin rays or blood samples without damaging the health of the fish body, effectively avoiding the health damage and lethal consequences caused by measuring the hypoxic adaptation phenotype of fish individuals through hypoxic stress experiments.

[0032] The method for detecting the hypoxic adaptation genotype provided by the present invention can effectively distinguish the hypoxic adaptation phenotype of candidate individuals at the juvenile stage, without the need to grow to commercial size or sexual maturity, and can be used for early screening of breeding candidate parents and parental mating group design.

[0033] The method for detecting the hypoxic adaptation genotype provided by the present invention is based on the sequence difference of long fragment insertion / deletion, designs PCR primers in its conserved flanking sequence, and the result can be judged by simple PCR amplification and agarose gel electrophoresis. The operation is simple and the detection cost is low.

[0034] Other advantages, objectives and features of the present invention will be partially reflected by the following description, and partially will also be understood by those skilled in the art through the research and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a DNA sequence alignment diagram of the hypoxia-adapted genotype and hypoxia-sensitive genotype of carp in the embodiments of the present invention. Among them, HR-cis1_Resistant_C.Carpio represents the hypoxia-adapted genotype, and HS-cis2_Sensitive_C.Carpio represents the hypoxia-sensitive genotype.

[0036] Figure 2 This is an agarose gel electrophoresis diagram of the method for detecting the hypoxia-adapted genotype in the embodiments of the present invention. Among them, Sample 1 is the DNA molecular weight standard, Samples 2 and 3 are the PCR amplification products of two hypoxia-sensitive individuals respectively, and the product length is 179 bp. Samples 4 and 5 are the PCR amplification products of two hypoxia-adapted individuals respectively, and the product length is 358 bp. Detailed implementation manners

[0037] The following further elaborates the present invention in conjunction with the attached drawings, so that those skilled in the art can implement it with reference to the text of the specification.

[0038] It should be understood that terms such as "having", "comprising", and "including" used herein do not preclude the presence or addition of one or more other elements or their combinations.

[0039] The hypoxia-adapted genotype provided by the present invention is derived from the significant associated locus of a large-scale genome-wide association study (GWAS), which is located in the calsequestrin-2-like gene on chromosome 2 of carp. This gene encodes a calsequestrin-like protein, is expressed in the myocardium and skeletal muscle, has the function of regulating calcium ion concentration, and plays an important role in hypoxia signal transduction.

[0040] The present invention provides a nucleotide sequence that determines the hypoxia-adapted phenotype of fish, including the base sequence shown in SEQ ID NO: 1. Both the heterozygote and homozygote of the base sequence shown in SEQ ID NO: 1 show hypoxia adaptation.

[0041] The present invention also discloses a nucleotide sequence that determines the hypoxia-sensitive phenotype of farmed fish, and its base sequence is shown in SEQ ID NO: 2.

[0042] The nucleotide sequence difference provided by the present invention is located in the upstream promoter region of the calsequestrin-2-like gene, including an insertion / deletion mutation with a length of up to 179 bp in the promoter region, which directly determines the transcription level of the calsequestrin-2-like gene, has a high phenotypic correlation with the hypoxia-adapted trait of carp, and has a certain generality in other farmed fish.

[0043] The present invention also provides a primer pair for detecting the hypoxia adaptation genotype of fish, and the primer pair is: the base sequence shown in SEQ ID NO: 3 and the base sequence shown in SEQ ID NO: 4.

[0044] In the present invention, SEQ ID NO: 1 represents the nucleotide sequence of the hypoxia adaptation genotype of Cyprinus carpio, and SEQ ID NO: 2 represents the nucleotide sequence of the hypoxia-sensitive genotype of Cyprinus carpio. The sequence similarity comparison results are as Figure 1 shown. Among them, the detection object of the hypoxia adaptation genotype detection method provided by the present invention is located at the 323-503 bases of SEQ ID NO: 1, corresponding to the 323-324 bases of SEQ ID NO: 2. The detection upstream primer is located at the 199-220 bases of SEQ ID NO: 1 and 2, and the detection downstream primer is located at the 536-556 bases of SEQ ID NO: 1 and the 357-377 bases of SEQ ID NO: 2.

[0045] The present invention also provides a method for detecting the hypoxia adaptation genotype of fish, comprising the following steps:

[0046] 1) Extract the genomic DNA of the fish to be tested;

[0047] 2) Using the genomic DNA in step 1) as a template, perform PCR amplification with a pair of primers such as SEQ ID NO: 3 and SEQ ID NO: 4;

[0048] 3) Determine the product length of the PCR amplification product obtained in step 2). If a product with a size conforming to the first detection value is detected in the PCR amplification product, it is determined that the individual to be tested of the template genomic DNA is of the hypoxia adaptation genotype. Both the heterozygote and homozygote of the base sequence shown in SEQ ID NO: 1 show hypoxia adaptation. If only a product with a size conforming to the second detection value is detected in the PCR amplification product, it is determined that the individual to be tested of the template genomic DNA is of the hypoxia-sensitive genotype, where the first detection value is greater than the second detection value.

[0049] In the above solution, preferably, the fish is Cyprinus carpio.

[0050] In the above solution, preferably, in step 1), the fish to be tested is in the juvenile stage.

[0051] In the above solution, preferably, in step 1), fin rays or blood samples of the fish to be tested are taken to extract genomic DNA.

[0052] In the above solution, preferably, in step 3), the first detection value is 358 bp, and the second detection value is 179 bp.

[0053] Specifically, it includes the following steps:

[0054] Collect tissue samples of the carp individuals to be tested, and extract genomic DNA respectively; the tissue samples are fin rays or blood. Dissolve the genomic DNA in Tris-EDTA buffer to prepare a solution with a concentration of about 100 ng / μL for storage.

[0055] Using the genomic DNA as a template, using the nucleotide sequence shown in SEQ ID NO: 3 as the upstream primer and the nucleotide sequence shown in SEQ ID NO: 4 as the downstream primer, perform a polymerase chain reaction (PCR) amplification reaction to obtain a PCR product.

[0056] The reaction system of the PCR amplification reaction is 20 μL: that is, add 0.1 μL of Taq DNA polymerase with a concentration of 2,500 units / mL, 2.0 μL of 10× buffer PCR buffer, 2.0 μL of dNTPs with a concentration of 10 μM, 1.2 μL of 25 mM MgCl2, 2.0 μL of the genomic DNA, 0.5 μL of the upstream primer with a concentration of 10 μM, 0.5 μL of the downstream primer with a concentration of 10 μM, and 11.7 μL of sterile water into the PCR reaction tube.

[0057] The reaction conditions of the PCR amplification reaction are pre-denaturation at 94°C for 5 min, then pre-heating and denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 30 s. A total of 30 cycles are carried out from pre-heating, annealing to extension, and then extension at 72°C for 10 min. After the reaction is completed, it is stored at 4°C.

[0058] Determine the length of the PCR product to identify the individual's hypoxia adaptation genotype. If the PCR product band is 358 bp, it indicates that the individual to be tested is a hypoxia adaptation genotype. If the PCR product band is 179 bp, it indicates that the individual to be tested is a hypoxia-sensitive genotype.

[0059] The determination of the product length is carried out by agarose gel electrophoresis detection method.

[0060] The present invention also provides a detection kit for fish hypoxia adaptation genotype, which contains the primer pair described above.

[0061] The present invention also provides the use of the nucleotide sequence or the detection primer in the detection, identification and cultivation of hypoxia-tolerant fish varieties. The base sequence of the nucleotide sequence is as shown in SEQ ID NO: 1 or 2.

[0062] Example

[0063] Determination of Hypoxia Adaptation Genotype of Candidate Parents for Carp Breeding

[0064] 1. Sample Collection

[0065] Select 30-day-old carp fry, cut about 0.5 cm of fin tissue from each fish for genomic DNA extraction. After fin cutting, the fry are returned to the culture pond. The fin tissue samples are dried at 55 °C and stored at room temperature.

[0066] 2. Genomic DNA Extraction from Carp Tissue Samples

[0067] Use a marine animal genomic DNA extraction kit (Tiangen DP324-03) to extract genomic DNA according to the instructions of the manual. The DNA is dissolved in 100 μL of Tris-EDTA buffer, and the concentration is measured by a UV spectrophotometer.

[0068] 3. PCR Amplification

[0069] The amplification reaction system for the sample to be tested is as follows:

[0070]

[0071]

[0072] Put the PCR tube containing the above amplification reaction system into a PCR instrument for amplification. The reaction conditions are: pre-denaturation at 94 °C for 5 min, then denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 30 s. The denaturation, annealing and extension are repeated for 30 cycles, and then extension at 72 °C for 10 min. After the reaction is completed, the temperature of the system is lowered to 4 °C for storage for later use.

[0073] 4. Detection by Agarose Gel Electrophoresis

[0074] Prepare an agarose electrophoresis gel with a concentration of 2.0%, use ethidium bromide as the dye, and perform electrophoresis at 80 V for 30 min. The electrophoresis result is photographed using a gel imager, and the result is as Figure 2 shown. In the figure, sample 1 is the molecular weight standard, and the band sizes from top to bottom are 2000 bp, 1000 bp, 750 bp, 500 bp, 200 bp and 100 bp respectively; the amplified product bands of samples 2 and 3 are less than 200 bp, and they are determined to be hypoxia-sensitive genotypes; the amplified product bands of samples 4 and 5 are greater than 200 bp, and they are determined to be hypoxia-adapted genotypes.

[0075] 5. Determination of Hypoxia Adaptation Phenotype

[0076] The sampled juvenile fish were normally reared until they reached 3 months old, and then a hypoxia stress experiment was conducted. The tested individuals were transferred into an indoor temporary rearing tank, and the dissolved oxygen concentration was controlled at about 6.0 mg / L by aerating with an air pump. They were reared at 25 °C for 7 days to adapt to the environment. After fasting for 24 h, the aeration was stopped, and the dissolved oxygen concentration was allowed to decrease naturally. The time of hypoxia-induced coma and the dissolved oxygen value of each individual were recorded. Individuals 2 and 3 lost their balance and floated on the water surface at 3 h of hypoxia stress when the dissolved oxygen value was 0.96 mg / mL. Individuals 4 and 5 still maintained a normal swimming state at 24 h of hypoxia stress when the dissolved oxygen value was 0.64 mg / mL. The determination results of the hypoxia adaptation phenotype were consistent with the aforementioned detection results of the hypoxia adaptation genotype.

[0077] The present invention discloses a nucleotide sequence that determines the hypoxia adaptation phenotype of cultured fish. The base sequence of the nucleotide sequence is shown as SEQ ID NO:1 or SEQ ID NO:2. This hypoxia adaptation genotype is derived from a significant association locus in a genome-wide association study, located in the upstream region of the calsequestrin-2-like gene on chromosome 2 of common carp, and contains an insertion / deletion mutation with a length of 179 bp in the promoter region. It directly determines the transcription level of a gene encoding a calsequestrin-like protein, is highly related to the hypoxia signal transduction pathway in common carp, and has a certain generality in other cultured fish. The present invention also discloses a method for detecting this hypoxia adaptation genotype. Using genomic DNA as a template, a pair of amplification primers located at the conserved sequences on both sides of the long fragment insertion / deletion mutation are used for genotype typing through PCR amplification and product length discrimination. The present invention also discloses the use of this hypoxia adaptation genotype and its detection method in the cultivation of hypoxia-tolerant varieties of cultured fish.

[0078] Although the embodiments of the present invention have been disclosed as above, it is not limited to the applications listed in the specification and embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the field, additional modifications can be easily made. Therefore, without departing from the general concept defined by the claims and the equivalent scope, the present invention is not limited to the specific details and the embodiments shown and described here.

Claims

1. A polynucleotide that determines the hypoxia adaptation phenotype of fish, characterized in that, It is the base sequence shown in SEQ ID NO:

1.

2. A polynucleotide that determines the hypoxia-sensitive phenotype of cultured fish, characterized in that, It is the base sequence shown in SEQ ID NO:

2.

3. Detection primer pairs for fish hypoxia adaptation genotypes, characterized in that, The primer pair is: the base sequence shown in SEQ ID NO: 3 and the base sequence shown in SEQ ID NO:

4.

4. A method for detecting fish hypoxic adaptation genotypes, characterized in that, It includes the following steps: 1) Extract the genomic DNA of the carp to be tested. 2) Using the genomic DNA in step 1) as a template, perform PCR amplification with a pair of primers such as SEQ ID NO: 3 and SEQ ID NO:

4. 3) Determine the length of the PCR amplification product obtained in step 2). If a product with a size conforming to the first detection value is detected in the PCR amplification product, it is determined that the individual to be tested of the template genomic DNA is of the hypoxia-adapted genotype. If only a product with a size conforming to the second detection value is detected in the PCR amplification product, it is determined that the individual to be tested of the template genomic DNA is of the hypoxia-sensitive genotype. Among them, the first detection value is 358 bp, and the second detection value is 179 bp.

5. The detection method of fish hypoxic adaptation genotype according to claim 4, characterized in that, In step 1), the carp to be tested is in the juvenile stage.

6. The detection method of the fish hypoxic adaptation genotype according to claim 4, characterized in that In step 1), take the fin rays or blood samples of the carp to be tested to extract genomic DNA.

7. A detection kit for the hypoxia-adapted genotype of carp, which contains the primer pair described in claim 3.

8. The use of the nucleotide described in claim 1 or the detection primer described in claim 3 in the detection, identification and cultivation of hypoxia-tolerant carp varieties.

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