Ctenopharyngodon idellus parent salinity tolerance related molecular marker suitable for fry salinity domestication and application of molecular marker in salt-tolerant breeding of ctenopharyngodon idellus

Abstract

The invention belongs to the technical field of aquatic animal breeding, and particularly relates to a grass carp parent salinity tolerance related molecular marker suitable for fry salinity domestication and application of the grass carp parent salinity tolerance related molecular marker in grass carp salt tolerance breeding. The nucleotide sequence of the SNP molecular marker related to the salinity tolerance character of the grass carp is as shown in SEQ ID NO: 1; a / C polymorphism exists at the 111st site in the DNA fragment shown in the nucleotide sequence, and when the genotype of the SNP molecular marker is CC or AC, the to-be-detected sample is the salinity-tolerant grass carp; when the genotype of the SNP molecular marker is AA, the to-be-detected sample is the salinity-sensitive grass carp. The SNP molecular marker closely related to the salt tolerance character is screened through a salinity tolerance test. Therefore, the molecular marker provided by the invention can be used for carrying out breeding work and detection work of the salt-tolerant grass carp.

Description

Technical Field

[0001] The invention belongs to the technical field of aquatic animal breeding, and in particular relates to a grass carp parent salinity tolerance-related molecular marker suitable for fry salinity acclimation and an application thereof in grass carp salt-tolerant breeding. Background Art

[0002] Grass carp (Ctenopharyngodon idellus) is the most productive freshwater commercial fish species, occupying a vital position in aquaculture due to its rapid growth, strong adaptability, and delicious meat. However, with the ban on cage aquaculture in many waters and the tightening of ecological protection and tailwater treatment standards, the area of ​​grass carp aquaculture in traditional major production areas has been shrinking. This has necessitated the expansion of grass carp aquaculture and the search for new breeding areas. Currently, saline-alkali regions such as the Northwest and the eastern coastal areas are gradually becoming new aquaculture targets. These areas generally have salinity levels of 5‰-15‰, to which traditional grass carp species may not be able to adapt. As a typical freshwater fish, grass carp exhibits significantly reduced survival and growth rates in high-salinity aquaculture environments, resulting in a significant decline in aquaculture profitability. Therefore, selecting grass carp parents with salinity tolerance to produce seed adapted to saline-alkali environments has become an important direction in grass carp breeding.

[0003] Traditional methods for selecting grass carp parents rely primarily on phenotypic observation and manual selection, which have significant drawbacks such as low efficiency, long selection cycles, and susceptibility to environmental interference. Therefore, how to quickly and accurately select grass carp parents adapted to high-salinity environments is a key technical challenge in improving the production of salinity-acclimated grass carp seedlings.

[0004] In recent years, with the rapid development of molecular biology techniques, molecular marker-based selection methods have gradually become an important tool in animal and plant breeding. The complete genome sequence of grass carp has now been published, providing an important technical foundation for the development of molecular markers. By utilizing molecular marker-assisted breeding techniques, it is possible to quickly and accurately identify grass carp parents suitable for salinity acclimation, thereby enabling the large-scale production of high-quality seedlings and significantly improving grass carp's salinity tolerance and aquaculture efficiency. Summary of the Invention

[0005] The present invention discloses a method for selecting grass carp parents based on molecular marker technology. In order to overcome the shortcomings of existing breeding technologies, the invention aims to efficiently screen out grass carp parents that are adaptable to high-salinity environments for use in the production of salinity-adapted seedlings.

[0006] The present invention provides a SNP molecular marker related to the salinity tolerance trait of grass carp, the nucleotide sequence of which is shown in SEQ ID NO: 1;

[0007] Wherein, there is an A / C polymorphism at position 111 in the DNA fragment shown in the nucleotide sequence;

[0008] When the genotype of the SNP molecular marker is CC or AC, the sample to be tested is salinity-tolerant grass carp; when the genotype of the SNP molecular marker is AA, the sample to be tested is salinity-sensitive grass carp.

[0009] The invention provides a grass carp salinity tolerance detection kit, which comprises amplification primers of SNP molecular markers.

[0010] Furthermore, the amplification primers are the upstream primer shown in SEQ ID NO: 2 and the nucleotide sequence is the downstream primer shown in SEQ ID NO: 3.

[0011] Furthermore, the specific sequence of the amplification primer is: upstream primer SEQ ID NO: 2, AGTAGTTTGCGATTTCTGAGTAAGA

[0012] Downstream primer SEQ ID NO: 3, GCACTGTCGTGACGTTTGCCCAA.

[0013] The present invention provides an application of a grass carp salinity tolerance detection kit in detecting the salinity tolerance of grass carp, comprising the following steps:

[0014] The grass carp genomic DNA of the test sample is used as a template and the amplification primers are used to perform PCR amplification to obtain an amplified product.

[0015] Furthermore, the reaction system for PCR amplification is: 20 μL, including 10 μL Premix Ex Taq, 8 μL dH2O, 1 μL 10 μMs upstream and downstream primers, and 1 μL 50 ng / μL DNA template.

[0016] Furthermore, the reaction procedure of the PCR amplification is: 95°C for 4 min; 95°C for 30 s, 56°C for 30 s, 72°C for 45 s, 35 cycles; 72°C for 8 min.

[0017] Furthermore, the upstream and downstream primers are consistent with the amplification primers.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The method of the present invention is applicable to all species of grass carp.

[0020] The present invention provides a molecular marker associated with the salinity tolerance trait of grass carp. The SNP molecular marker is an A / C polymorphism at position 111 in the DNA fragment with the nucleotide sequence shown in SEQ ID NO: 1. The present invention screens and obtains SNP molecular markers closely associated with the salt tolerance trait through salinity tolerance testing. Therefore, the molecular markers provided by the present invention can be used in the breeding and detection of salt-tolerant grass carp.

[0021] The present invention provides primers for amplifying molecular markers associated with the salinity tolerance trait of grass carp. A pair of primers is designed for two SNP molecular markers. The primer pair has strong specificity and can accurately amplify sequences containing the SNP molecular marker sites of the present invention. This primer pair can be used to assist in selective breeding, efficiently identify salinity-tolerant grass carp parents, and accelerate the progress of salt-tolerant grass carp breeding.

[0022] The present invention discloses a method for selecting grass carp parents based on molecular marker technology. In order to overcome the shortcomings of existing breeding technologies, the method aims to efficiently screen out grass carp parents that are adapted to high-salinity environments for the production of salinity-adapted seedlings. The method first understands the tolerance of common grass carp to salinity through salinity stress, obtains grass carp populations that are sensitive and tolerant to salinity, further develops the association between the molecular characteristics of grass carp and salinity adaptability, and screens and obtains the key salt-resistance regulatory gene stat3 and SNP sites related to salt tolerance and sensitivity traits. On this basis, specific molecular markers are designed to achieve rapid and accurate parent selection. Compared with traditional methods, the present invention significantly improves selection efficiency, shortens the breeding cycle, and has higher accuracy, providing reliable technical support for the large-scale production of grass carp salinity-adapted seedlings. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a flow chart for screening salinity tolerance / sensitive gene loci in Example 2.

[0024] Figure 2 This is the melting curve of the β-actin gene in Example 5;

[0025] Figure 3 This is the melting curve of the Stat3 gene in Example 5;

[0026] Figure 4 is the β-actin gene amplification efficiency in Example 5;

[0027] Figure 5 is the Stat3 gene amplification efficiency in Example 5. DETAILED DESCRIPTION

[0028] Example 1

[0029] Sample collection and processing

[0030] 1,400 grass carp were randomly collected from a natural population of grass carp at the seedling stage (10m-15cm) and their phenotypic characteristics were recorded. The collected grass carp were divided into 7 groups and placed in water bodies with different salinity gradients (such as 0‰, 2.5‰, 5.0‰, 7.5‰, 10‰, 12.5‰, and 15‰). 200 grass carp were placed in each salinity gradient. Initially, each gradient was 0‰, and the salinity was increased by 2.5‰ per day to reach the set concentration. Then the experiment began to conduct an adaptability test: the salinity was gradually increased by 2.5‰ per day. After stabilization, the culture was continued for 30 days. During this period, appropriate feed was fed, the dissolved oxygen in the water was maintained above 5mg / L, and the temperature was kept at 25℃-26℃.

[0031] As shown in Table 1 , the survival rate and other indicators of grass carp cultured under different salinity gradient conditions within 30 days were recorded, and individuals with outstanding performance were selected.

[0032] Table 1 Survival rate of grass carp cultured under different salinity gradient conditions within 30 days

[0033] salinity 0‰ 2.5‰ 5.0‰ 7.5‰ 10‰ 12.5‰ 15‰ Survival rate 100％ 99％ 98.5％ 98.5％ 81.5％ 65％ 14％

[0034] Example 2

[0035] Screening of salinity tolerance / sensitivity gene loci

[0036] The tail fins of 30 grass carp individuals that survived at a salinity of 15‰ and 30 individuals that died at a salinity of 10‰ were collected, and the total DNA of the tail fins was extracted. The grouped sample DNA was resequenced to filter out low-quality genotype data, such as SNPs with high deletion rate (deletion rate>10%), low allele frequency (minor allele frequency<5%), and SNPs that did not conform to the Hartmann-Wenzhou equilibrium (p<0.000001). Based on survival / death as binary phenotypic data, logistic regression was selected to perform GWAS association analysis on SNPs. The GWAS results were corrected for multiple tests, and SNPs with significant correlation with phenotypic data were screened out, and their positions on the genome were analyzed. Through bioinformatics analysis, the key gene Stat3 related to salinity resistance was further screened out, and the corresponding molecular marker SNPs were designed based on the coding region and promoter region. The specific process is as follows Figure 1 shown.

[0037] Example 3

[0038] Molecular marker design and validation

[0039] In the Stat3 gene, key SNPs related to salinity resistance and sensitivity were found in the promoter region (-929) before the transcription start site (+1). Primers were designed based on the above information, wherein the upstream primer is such as SEQ ID NO: 2: AGTAGTTTGCGATTTCTGAGTAAGA; the downstream primer is such as SEQ ID NO: 3: GCACTGTCGTGACGTTTGCCCAA, which are used for the detection of molecular markers and molecular marker detection of individuals in natural grass carp populations;

[0040] The grass carp genomic DNA of the sample to be tested in Example 2 was used as a template and PCR amplification was performed using the above primers.

[0041] The PCR amplification reaction system was as follows: 20 μL, including 10 μL Premix Ex Taq, 8 μL dH2O, 1 μL 10 μMs upstream and downstream primers (upstream primer: SEQ ID NO: 2; downstream primer 1R SEQ ID NO: 3), and 1 μL 50 ng / μL DNA template. The PCR amplification reaction program was as follows: 95°C for 4 min; 35 cycles of 95°C for 30 s, 56°C for 30 s, and 72°C for 45 s; and 72°C for 8 min.

[0042] PCR products are obtained and the SNP genotypes in the PCR products are tested. The salinity tolerance of grass carp is determined based on the genotype of the SNP site: when the genotype of the SNP molecular marker is CC or AC, the sample to be tested is salinity-tolerant grass carp; when the genotype of the SNP molecular marker is AA, the sample to be tested is salinity-sensitive grass carp. Individuals with the genotype CC are selected as excellent parents;

[0043] The 111th base of the fragment SEQ ID NO: 1 (represented by M in the following sequence) is a CC, AC, or AA genotype (in the parental breeding population of grass carp caught in the Yangtze River, the AA genotype is 67.0%, the AC genotype is 27.1%, and the CC genotype is 5.9%). Compared to the AA genotype, the CC genotype mutation results in an additional binding site for the homeobox transcription factor (HOXC8) in the stat3 gene promoter region, which has the function of promoting gene transcription. This improves the binding efficiency of HOXC8, greatly enhances the stat3 promoter activity, and increases the expression of stat3, thereby stimulating the fish's adaptation to salinity and improving the growth rate of CC haplotype individuals in the breeding population. The CT genotype is intermediate between the two.

[0044] SEQ ID NO: 1

[0045] AGTAGTTTGCGATTTCTGAGTAAGAAAGGACTGTGGTTAAAATTACTTGAAGAGACTTTTGTTTGTTCTACAATATAAATTGCAGACTCACCTGAAACATGAATTTTAAG M CCATTGATAAAATATAAAAACAAACAAACAAAAATGTATATTATAAAAAATGCATATATACATATATACATATATATACTTAAATACATATATACACACATACTTAAATAAAAAATTGCTAATTCTAAAAACCCATTAGAAATTCCTAAGGGATATTATACTGTGTATATATACAGTCAAACCAAAATTTATTTGGACACCTTGAACATTTCATTCATTAATACAGGTATAATATGTCACAGTTTACTTTATTTTGCAGTCCTCACTTACATAAATGAACTATAGTGTCCTGCACCAACGAGTAAAAATATATCAAAAAAATCTAAAATGTCTGAATCATTTTTGGTTTGACTGTATGTCTATATATATATATATATATATATATATATATATATATAAAGCTAATACTATTCCAGGTTTTACAAATAGTCCAGTCAAGAACCTTTATTGATTTGTTAGCTTTATATTTTGCCATTTTTGAAAATGATGTTTTAAACAGGACTATCCATTATTTCGTAAGTCTCAGTTTTCTGTAAAATAATGTTAAAATGCACTTTGAGTTCATATTAATTTAGTTTCTCTCTGAGATTTGTGCTCACACTTAAACATACACAGTGTTATAAAAAAAGGGGGGATGATTGATTTGGGCAAACGTCACGACAGTGC。

[0046] Example 4

[0047] Parent selection and verification

[0048] Multiple four-year-old grass carp families were randomly selected. RFID tags were injected into each parent and kept in the same pond to ensure individual fish traceability. A 0.5g caudal fin of the grass carp was minced and placed in a 1.5mL centrifuge tube. DNA was extracted using a commercial kit with 500μL of DNA extraction buffer and dissolved in 50μL of pure water. The DNA template was amplified by PCR using 20μL of 10μL Premix Ex Taq, 8μL of dH2O, 1μL of 10μM upstream and downstream primers (upstream primer: SEQ ID NO: 2; downstream primer: SEQ ID NO: 3), and 1μL of 50ng / μL DNA template. The PCR reaction conditions were: 95°C for 5min, 94°C for 30s, 56°C for 30s, 72°C for 45s for 35 cycles, and 72°C for 8min. In this way, the fragment where the SNP marker of the present invention is located can be amplified efficiently and accurately and the target amplification product can be obtained, which is convenient for the subsequent steps; the obtained PCR amplification fragment is sequenced by Sanger sequencing to obtain the gene sequence of the amplified fragment, and the sequencing results are batch compared using SnapGene software, and the individuals with the CC genotype at the 111bp site of the amplified sequence are retained as salt-tolerant grass carp parents for seedling breeding.

[0049] Parents with salt-tolerant genotypes (CC genotype) were screened by molecular markers for mating (the male-female ratio was controlled at 2:1). The parents were bred by injecting hormones when the water temperature was stable at 23°C. Through parent breeding, 30 seedlings from the selected group were randomly collected and placed in a salinity of 7.5‰ (the safe concentration for grass carp farming was established as 7.5‰ in the first step, and the survival rate was above 98% at this concentration) for farming for 3 months. At the same time, 30 unselected grass carp seedlings were collected for farming control (also at 7.5‰). During this period, they were fed 32% protein pellet feed, the water temperature was maintained at 25-28°C, and the dissolved oxygen was maintained at above 5.0 mg / L. After 90 days of farming, the growth rates of the selected and control groups were verified. The average weight of the selected seedlings (CC) was 97.6g, and the average weight of the control group seedlings (AA) was 66.3g. The weight of the selected group was significantly higher than that of the control group (P<0.05).

[0050] Example 5

[0051] Analysis of Stat3 gene expression levels

[0052] After 90 days of culture, nine grass carp of each of the three genotypes were randomly collected based on genotyping results. The fish were anesthetized by rapid immersion in 100 mg / L MS222. The fish were dissected and 0.2 g of gill tissue was collected. Total RNA from the grass carp gill tissue was extracted using Trizol reagent (Invitrogen). Purity and concentration were determined by 2% agarose gel electrophoresis and a NanoDrop-Lite UV spectrophotometer (Thermo Scientific). 1 μg of RNA was used to synthesize cDNA using a PrimeScript™ Reverse Transcription Kit (TaKaRa) and stored at −20°C.

[0053] Using actin β-actin as a reference gene, Stat3 gene primers were designed and selected. Specific primers were designed using the biological software Snapgene. The primer sequence information is shown in Table 2. The primer sequences were commissioned to Sangon Biotech (Shanghai) Co., Ltd. for synthesis.

[0054] Table 2 Primer sequences for real-time fluorescence quantitative analysis of Stat3 and β-actin

[0055]

[0056] Fluorescence quantitative PCR was performed on an ABI 7900HT PCR instrument using cDNA from grass carp gill tissue as a template.

[0057] The reaction system is 20 μL: Green Realtime PCR Master Mix (Toyobo) 10 μL, upstream and downstream primers of the target gene 1.6 μL each, cDNA template 1 μL, RNase Free Water 5.8 μL, and repeat each sample 3 times.

[0058] The reaction program was as follows: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 15 s, annealing at 59°C for 20 s, and extension at 72°C for 30 s, for 40 cycles.

[0059] After the experiment, melting curve analysis was performed. Figure 2-5 The specificity of each reaction was verified as shown.

[0060] The relative expression data of fluorescence quantitative PCR were analyzed using 2 -ΔΔct Method for analysis.

[0061] In the correlation analysis between genotype and relative mRNA expression, the dependent variable was the relative mRNA expression level, and the independent variable was the genotypes of the A-929C locus. LSD analysis was performed on the three genotypes.

[0062] The results showed that the relative expression of Stat3 gene in individuals with CC genotype at A-929C locus was 1.96 times that of individuals with AA genotype (P<0.05), and the relative expression in individuals with AC genotype was 1.85 times that of individuals with AA genotype (P<0.05).

[0063] The present invention provides a molecular marker-based method for selecting salinity-acclimated grass carp parents, which can significantly improve the salinity resistance and breeding efficiency of grass carp fry. This method is suitable for grass carp breeding and selection, and has important application value in improving the breeding efficiency of grass carp in high-salinity environments.

[0064] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A SNP molecular marker related to the salinity tolerance trait of grass carp, characterized in that: The nucleotide sequence is shown in SEQ ID NO: 1; Wherein, there is an A / C polymorphism at position 111 in the DNA fragment shown in the nucleotide sequence; When the genotype of the SNP molecular marker is CC or AC, the sample to be tested is salinity-tolerant grass carp; when the genotype of the SNP molecular marker is AA, the sample to be tested is salinity-sensitive grass carp.

2. A grass carp salinity tolerance detection kit, characterized in that: The invention also provides a SNP amplification primer comprising the SNP molecular marker as claimed in claim 1.

3. The kit according to claim 2, wherein The amplification primers are the upstream primer shown in SEQ ID NO: 2 and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:

3.

4. The kit according to claim 3, wherein The specific sequence of the amplification primer is: upstream primer SEQ ID NO: 2, AGTAGTTTGCGATTTCTGAGTAAGA Downstream primer SEQ ID NO: 3, GCACTGTCGTGACGTTTGCCCAA.

5. Use of a grass carp salinity tolerance detection kit in detecting grass carp salinity tolerance, characterized in that: The following steps are involved: The grass carp genomic DNA of the test sample is used as a template and the amplification primers are used to perform PCR amplification to obtain an amplified product.

6. The use according to claim 5, characterized in that The PCR amplification reaction system is: 20 μL, including 10 μL Premix Ex Taq, 8 μL dH2O, 1 μL 10 μMs upstream and downstream primers, and 1 μL 50 ng / μL DNA template.

7. The use according to claim 5, characterized in that The reaction program of the PCR amplification was: 95°C for 4 min; 95°C for 30 s, 56°C for 30 s, 72°C for 45 s, 35 cycles; 72°C for 8 min.

8. The use according to claim 6, characterized in that The upstream and downstream primers are consistent with the amplification primers described in claim 3.

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