SNP (Single Nucleotide Polymorphism) marker primer pair for distinguishing Heilongjiang and Beijing water system mandarin fish groups, kit, application and method

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) marker primer pair for distinguishing mandarin fish groups in Heilongjiang and Beijing water systems, a kit, application and a method. According to the invention, an SNP (Single Nucleotide Polymorphism) marker is found, the site is lgr4p.D874V and is located at the base 14537718 of a chromosome 1, and the base mutation type is T / A; different genotypes are shown in the two groups, and group verification shows that the site has remarkable group genetic differentiation characteristics and can be used as an effective molecular marker for subsequent group identification. On the basis, the invention also discloses a primer pair of the SNP marker, a kit containing the primer pair, and application of the SNP marker in molecular traceability, germplasm resource identification or population genetic characteristic analysis of a mandarin fish population. The invention further discloses a method for distinguishing siniperca chuatsi group sources in Heilongjiang and Beijing water systems and a molecular monitoring method for siniperca chuatsi local germplasm resources. The method can quickly and accurately identify the population source of the mandarin fish, judge the genetic background and guide the germplasm resource protection and improved variety breeding of the mandarin fish.

Description

Technical Field

[0001] This invention relates to the field of molecular genetics and germplasm resource identification technology for aquatic animals, and in particular to an SNP marker primer pair, kit, application and method for distinguishing mandarin fish populations in the Heilongjiang and Beijing water systems. Background Technology

[0002] Superior mandarin fish germplasm resources are the fundamental guarantee for the high-quality development of modern specialty freshwater fisheries, and are of key significance for improving industrial efficiency and promoting stable production and increased income. Therefore, systematically improving the flesh quality traits of native mandarin fish in my country has become one of the key directions of current mandarin fish genetic breeding research. The mandarin fish from the Heilongjiang River system, with its unique natural geographical environment, has developed excellent quality and is hailed as one of China's four famous freshwater fish.

[0003] Traditional methods for population differentiation rely heavily on morphological characteristics and geographical distribution records. However, these methods are susceptible to environmental influences, highly subjective, and struggle to identify individuals from hybrid or mixed origins. Molecular marker technology provides a stable and objective means for population identification. Among these, single nucleotide polymorphism (SNP) markers have become a powerful tool in population genetics and germplasm identification research due to their abundant quantity, wide distribution, high stability, and ease of high-throughput detection.

[0004] Currently, no specific SNP markers have been reported for effectively distinguishing different geographical populations of mandarin fish. Developing such markers would help establish a rapid and accurate molecular identification system, serving the healthy development of the mandarin fish industry and the revitalization of its seed industry. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide an SNP marker primer pair, kit, application and method for distinguishing mandarin fish populations in the Heilongjiang and Beijing water systems, so as to effectively distinguish mandarin fish populations in the Heilongjiang and Beijing water systems.

[0006] To solve the above technical problems, the following technical solution is adopted:

[0007] On one hand, this invention provides an SNP marker primer pair for distinguishing mandarin fish populations in the Heilongjiang and Beijing water systems. The SNP marker site is lgr4 p.D874V, located in the mandarin fish lgr4 gene. The sequence of the lgr4 gene is shown in SEQ ID NO:1 and is located at positions 14505615-14540530 on chromosome 1. The SNP marker site is located at base 14537718 on chromosome 1, with a T / A mutation type. This site belongs to a T or A nucleotide sequence polymorphism site, meaning that the corresponding homozygous genotypes are TT and AA. The primer pair is a primer pair that can specifically amplify nucleic acid fragments containing the SNP marker site.

[0008] The SNP marker is located in the coding region of the mandarin fish lgr4 gene (a G protein-coupled receptor 4 gene rich in leucine repeat sequences), and the sequence of the lgr4 gene is shown in SEQ ID NO:1, located at positions 14505615-14540530 on chromosome 1. This mutation causes the encoded amino acid 874 to change from aspartic acid (Asp, D) to valine (Val, V) (denoted as p.D874V).

[0009] Furthermore, the nucleotide sequence of the primer pair is as follows:

[0010] The forward primer (SEQ ID NO:2: lgr4-F1) is 5'-CTCTCCTCTCTGTCTGCCCT-3';

[0011] The reverse primer (SEQ ID NO:3: lgr4-R1) is 5'-CTGAACCTGTTCCGAGCTGT-3'.

[0012] On the other hand, the present invention also provides a kit for distinguishing mandarin fish populations in the Heilongjiang and Beijing water systems, comprising the above-mentioned SNP marker primer pairs for distinguishing mandarin fish populations in the Heilongjiang and Beijing water systems.

[0013] Furthermore, it also includes one or more of the following: DNA extraction reagents, PCR reaction solutions, and reagents required for sequencing.

[0014] Furthermore, the present invention also provides an application of SNP markers in mandarin fish, wherein the SNP marker site is: lgr4 p.D874V, located at base 14537718 on chromosome 1, and the base mutation type is T / A;

[0015] The applications include molecular tracing of mandarin fish populations, identification of germplasm resources, or analysis of population genetic characteristics;

[0016] The mandarin fish populations include those from the Heilongjiang River system and the Beijing River system.

[0017] On the other hand, the present invention also provides a method for distinguishing the origin of mandarin fish populations in the Heilongjiang and Beijing water systems, comprising the following steps: (1) providing mandarin fish samples to be tested; (2) detecting the genotype of the SNP marker site in the sample; the SNP marker site is: lgr4 p.D874V, located at base 14537718 on chromosome 1, with a base mutation type of T / A; (3) determining the origin of the mandarin fish population to be tested based on the detected genotype.

[0018] Furthermore, the dominant genotype of the SNP marker in the Beijing water system mandarin fish population is TT, with a genotype frequency of 100%, while in the Heilongjiang water system mandarin fish population, it is TT / TA / AA genotype.

[0019] Furthermore, the above-mentioned SNP-labeled primer pairs or kits are used in the detection step of step (2);

[0020] Furthermore, in the detection step of step (2), the genotype of the SNP marker site is determined by sequencing the amplification product;

[0021] Furthermore, in step (3), based on the detected genotype, the population origin of the mandarin fish to be tested is determined to include:

[0022] If the genotype is TT, it suggests that the individual may have originated from the Beijing water system mandarin fish population or its genetic background is similar to that of the Beijing water system mandarin fish population; if the genotype is TA or AA, it suggests that the individual may have originated from the Heilongjiang water system mandarin fish population or has genetic infiltration from the Heilongjiang water system mandarin fish population.

[0023] Furthermore, this invention also provides a molecular monitoring method for local germplasm resources of mandarin fish, comprising the following steps:

[0024] (1) Provide a sample of mandarin fish to be tested;

[0025] (2) Detect the genotype of the SNP marker site in the sample; the SNP marker site is: lgr4p.D874V, located at 14537718 bases on chromosome 1, and the base mutation type is T / A; (3) Compare the detected genotype with the standard genotype of the original species or target conservation population of mandarin fish in a specific aquatic system to monitor its genetic purity and whether there is any contamination from foreign genetic background;

[0026] The mandarin fish populations mentioned include those from the Heilongjiang River system and those from the Beijing River system.

[0027] Furthermore, the above-mentioned SNP-labeled primer pairs or kits are used in the detection step of step (2);

[0028] Furthermore, in step (2), the genotype of the SNP marker site is determined by sequencing the amplified product.

[0029] Compared with the prior art, the present invention has the following significant advantages:

[0030] 1. This invention is the first to discover and verify that there are significant differences in the genotype frequency of the p.D874V site of the lgr4 gene in mandarin fish populations from the Heilongjiang and Beijing water systems. The Beijing population is almost monomorphic, while the Heilongjiang population is rich in polymorphism, which gives the marker excellent population discrimination ability and the advantage of high specificity.

[0031] 2. The identification based on DNA sequence differences at SNP marker sites in this invention is objective and stable, and is not affected by superficial factors such as individual age, sex, and breeding environment, thus having the advantage of accurate and reliable results.

[0032] 3. The primer pairs provided by this invention have good specificity and high PCR amplification efficiency. Combined with conventional sequencing or commercial SNP genotyping platforms, batch sample detection can be completed in a short time, which has the advantages of being fast and convenient.

[0033] 4. The SNP markers of this invention can not only be used for simple population identification, but also be applied in depth to population genetic analysis, germplasm resource protection assessment and precision breeding practices, providing important molecular technology support for the sustainable development of the mandarin fish industry, and have the advantage of wide application. Attached Figure Description

[0034] The above is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0035] Figure 1 This is a nucleotide sequence diagram of the SNP-marked sites. Detailed Implementation

[0036] Exemplary embodiments of the invention will now be described in more detail with reference to the accompanying drawings. While exemplary embodiments of the invention are shown in the drawings, it should be understood that the invention may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that a more thorough understanding of the invention will be achieved and that the full scope of the invention will be conveyed to those skilled in the art.

[0037] This invention constructs two wild mandarin fish populations from Heilongjiang and Beijing, and uses whole-genome resequencing technology and whole-genome SNP analysis to perform genetic comparison and selection signal detection on wild mandarin fish populations from the Heilongjiang River system (30 individuals) and Miyun Reservoir in Beijing (30 individuals). It identifies genomic regions in the Beijing population that show significant selection relative to the Heilongjiang population. The results show that a missense mutation site, lgr4 p.D874V, located in the lgr4 gene, exhibits different genotypes in the two populations. For example, it shows a completely homozygous TT genotype in the Beijing population, while it shows a TT / TA / AA genotype in the Heilongjiang population. Population validation was then performed on 122 wild mandarin fish from the Heilongjiang River system and 81 wild mandarin fish from Miyun Reservoir in Beijing, and the validation results were consistent with the above findings. This indicates that the locus has significant population genetic differentiation characteristics and can be used as an effective molecular marker for subsequent population identification, enabling rapid and accurate identification of the population origin and genetic background of mandarin fish, guiding the protection of mandarin fish germplasm resources and the breeding of superior varieties, and providing scientific guidance for the strain improvement of farmed mandarin fish.

[0038] The discovery and verification of SNP markers will be explained in detail below through specific examples:

[0039] Example 1: Discovery of SNP markers

[0040] Thirty wild mandarin fish from the Heilongjiang River system and 30 wild mandarin fish from Miyun Reservoir in Beijing were collected. Muscle samples were taken from the mandarin fish, and genomic DNA was extracted. Based on genome-wide SNPs, the two wild mandarin fish populations were compared. Selection signal analysis revealed the selection window of the mandarin fish from Miyun Reservoir compared to those from the Heilongjiang River system, identifying a missense mutation site in the lgr4 gene, lgr4 p.D874V. The nucleotide sequence of the SNP-marked site is shown below. Figure 1 As shown in SEQ ID NO:1, the SNP marker is located in the coding region of the mandarin fish lgr4 gene (a G protein-coupled receptor 4 gene rich in leucine repeat sequences). The sequence of the lgr4 gene is shown in SEQ ID NO:1, located at positions 14505615-14540530 on chromosome 1. This mutation causes the encoded amino acid 874 to change from aspartic acid (Asp, D) to valine (Val, V) (denoted as p.D874V).

[0041] Based on the published lgr4 gene sequence, primers were designed and synthesized, and the lgr4-F1 and lgr4-R1 primer pairs were selected (as shown in Table 1). PCR amplification was performed (25 μL system: 12.5 μL 2×Mix, 0.5 μL each primer, 1 μL DNA, 10.5 μL water; program: 95℃ for 5 min; 35 cycles × [95℃ for 30 s, 56℃ for 30 s, 72℃ for 45 s]; 72℃ for 5 min). The PCR product was purified and sent to a sequencing company for bidirectional Sanger sequencing.

[0042] Sequencing results were compared with the reference sequence, revealing a T / A polymorphism at position 2621 of the coding region (corresponding to nucleotide 14,537,718 on chromosome 1). Statistical analysis showed that all 30 individuals from the Beijing population were homozygous for the T / T type (allele frequency T = 1.0). In the Heilongjiang population, 9 individuals were T / T, 16 were T / A, and 5 were A / A, with allele frequencies of T = 0.57 and A = 0.43. The heterozygosity (Ho) of this locus in the Heilongjiang population was 0.533, and the polymorphism information content (PIC) was 0.353, indicating moderate polymorphism and conforming to Hardy-Weinberg equilibrium. The genotype and allele frequencies of *S. mandarin fish* in different regions of the SNP locus are shown in Table 2. These results confirm that this SNP locus has significant population-distinguishing characteristics.

[0043] Table 1 Primer Information

[0044]

[0045] Table 2. Distribution of genotype and allele frequencies of SNP loci in different regions of Mandarin fish.

[0046]

[0047] Example 2: Verification of SNPs

[0048] To verify the feasibility of using the lgr4 p.D874V mutation site as a molecular marker, population genotyping analysis was conducted on 203 individuals. PCR amplification was performed using primers lgr4-F1 and lgr4-R1 (25 μL system: 12.5 μL 2×Mix, 0.5 μL each primer, 1 μL DNA, 10.5 μL water; program: 95℃ for 5 min; 35 cycles × [95℃ 30 s, 56℃ 30 s, 72℃ 45 s]; 72℃ for 5 min). The purified PCR products were sent to a sequencing company for bidirectional Sanger sequencing.

[0049] Sequencing results showed that all 81 individuals in the Beijing population were homozygous for the T / T type (allele frequency T=1.0). In the Heilongjiang population of 122 individuals, 46 were T / T, 44 were T / A, and 32 were A / A.

[0050] In the Beijing population, the dominant genotype at this locus was TT, with a genotype frequency of 100%. A χ² test showed a significant difference in genotype frequency between the two populations (P < 0.01), indicating that the lgr4 p.D874V locus exhibits significant population genetic differentiation characteristics and can serve as an effective molecular marker for subsequent population identification. The genotype and allele frequencies of different regions of the SNP locus in the validation experiment are shown in Table 3.

[0051] Table 3. Distribution of genotype and allele frequencies of SNP loci in different regions of mandarin fish in the validation experiment.

[0052]

[0053] Based on the above experimental results, this embodiment developed the following SNP-labeled primer pairs, kits, applications, and methods.

[0054] A primer pair for distinguishing mandarin fish populations in the Heilongjiang and Beijing water systems. The SNP marker site is lgr4 p.D874V, located at base 14537718 on chromosome 1, with a base mutation type of T / A. The primer pair is a primer pair that can specifically amplify nucleic acid fragments containing the SNP marker site.

[0055] Preferably, the nucleotide sequence of the primer pair is as follows:

[0056] The forward primer is 5'-CTCTCCTCTCTGTCTGCCCT-3'.

[0057] The reverse primer is 5'-CTGAACCTGTTCCGAGCTGT-3'.

[0058] The primer pairs described above can be used as part of a kit for distinguishing mandarin fish populations in the Heilongjiang and Beijing water systems. In addition to the primer pairs, the kit also includes one or more of the following: DNA extraction reagents, PCR reaction solution, and sequencing reagents. These components are standard choices in the field and will not be elaborated further here.

[0059] The aforementioned SNP marker for mandarin fish can be applied to molecular tracing, germplasm resource identification, or population genetic characteristic analysis of mandarin fish populations, including mandarin fish populations in the Heilongjiang River system and mandarin fish populations in the Beijing River system.

[0060] Molecular traceability can include the geographical origin and authenticity verification of mandarin fish products (such as fry and adult fish). Germplasm resource identification can include the identification of mandarin fish germplasm resources and the establishment of molecular identification; as well as its use in breeding to screen and preserve the pure genetic background of specific geographical populations (such as the Heilongjiang "Ao Hua") to prevent germplasm mixing during the breeding process; and molecular monitoring of local germplasm resources. Population genetic characteristic analysis can include the analysis of the genetic structure of mandarin fish populations and the assessment of genetic diversity, as well as the monitoring of gene flow between different populations.

[0061] This embodiment also provides a method for distinguishing the origin of mandarin fish populations in the Heilongjiang and Beijing water systems, including the following steps: (1) providing mandarin fish samples to be tested;

[0062] Genomic DNA was extracted from samples of mandarin fish to be tested (such as fins, blood, and muscle).

[0063] (2) Detect the genotype of the SNP marker sites in the sample;

[0064] First, using the extracted genomic DNA as a template, PCR amplification was performed using the aforementioned SNP marker primer pairs and kit.

[0065] The PCR amplification products are then sequenced (e.g., Sanger sequencing) to determine the genotype of the SNP marker site.

[0066] (3) Determine the population origin of the mandarin fish to be tested based on the genotype obtained from the test.

[0067] The dominant genotype of the above-mentioned SNP markers in the Beijing water system mandarin fish population is TT, with a genotype frequency of 100%. The Heilongjiang water system mandarin fish population exhibits polymorphism at this locus, including three genotypes: TT, TA, and AA, among which the A allele has a significant frequency.

[0068] Therefore, if the genotype is TT, it suggests that the individual may have originated from the mandarin fish population of the Beijing water system or its genetic background is similar to that of the mandarin fish population of the Beijing water system; if the genotype is TA or AA, it suggests that the individual may have originated from the mandarin fish population of the Heilongjiang water system or has genetic infiltration from the mandarin fish population of the Heilongjiang water system.

[0069] In addition, this embodiment also provides a molecular monitoring method for local germplasm resources of mandarin fish, including the following steps:

[0070] (1) Provide a sample of mandarin fish to be tested;

[0071] (2) Detect the genotype of the SNP marker site in the sample; wherein, the aforementioned SNP marker primer pair and kit are used in the detection step, and the genotype of the SNP marker site is determined by sequencing the amplification product in the detection step.

[0072] (3) Compare the detected genotypes with the standard genotypes of the original species or target conservation population of mandarin fish in a specific water system to monitor their genetic purity and whether there is any contamination from foreign genetic background;

[0073] The populations of mandarin fish include those in the Heilongjiang River system and those in the Beijing River system.

[0074] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, or alterations made by those skilled in the art using the disclosed technical content shall fall within the protection scope of the present invention.

Claims

1. A pair of SNP marker primers for distinguishing mandarin fish populations in the Heilongjiang and Beijing water systems, characterized in that, The SNP marker site is: lgr4 p.D874V, located at 14537718 bases on chromosome 1, with a base mutation type of T / A; The primer pair is a primer pair that can specifically amplify nucleic acid fragments containing the SNP marker.

2. The SNP marker primer pair for distinguishing mandarin fish populations in the Heilongjiang and Beijing water systems according to claim 1, characterized in that, The nucleotide sequences of the primer pair are as follows: The forward primer is 5'-CTCTCCTCTCTGTCTGCCCT-3'. The reverse primer is 5'-CTGAACCTGTTCCGAGCTGT-3'.

3. A reagent kit for distinguishing mandarin fish populations in the Heilongjiang and Beijing water systems, characterized in that, It includes the SNP marker primer pair described in claim 1 or 2 for distinguishing mandarin fish populations in the Heilongjiang and Beijing water systems.

4. The reagent kit for distinguishing mandarin fish populations in the Heilongjiang and Beijing water systems according to claim 3, characterized in that, It also includes one or more of the following: DNA extraction reagents, PCR reaction solutions, and sequencing reagents.

5. An application of SNP markers for mandarin fish, characterized in that, The SNP marker site is: lgr4 p.D874V, located at 14537718 bases on chromosome 1, with a base mutation type of T / A; The applications include molecular tracing of mandarin fish populations, identification of germplasm resources, or analysis of population genetic characteristics; The mandarin fish populations include those from the Heilongjiang River system and the Beijing River system.

6. A method for distinguishing the origins of mandarin fish populations in the Heilongjiang and Beijing water systems, characterized in that, The process includes the following steps: (1) providing a sample of mandarin fish to be tested; (2) detecting the genotype of the SNP marker site in the sample; the SNP marker site is: lgr4 p.D874V, located at base 14537718 on chromosome 1, with a base mutation type of T / A; (3) determining the population origin of the mandarin fish to be tested based on the detected genotype.

7. The method for distinguishing the origin of mandarin fish populations in the Heilongjiang and Beijing water systems according to claim 6, characterized in that, The SNP marker locus is dominant in the Beijing mandarin fish population with the TT genotype, which has a frequency of 100%, while in the Heilongjiang mandarin fish population, it is represented by the TT / TA / AA genotype.

8. The method for distinguishing the origin of mandarin fish populations in the Heilongjiang and Beijing water systems according to claim 6 or 7, characterized in that, The detection step (2) uses the SNP-labeled primer pair as described in claim 1 or 2 or the kit as described in claim 3 or 4; And / or, in the detection step of step (2), the genotype of the SNP marker site is determined by sequencing the amplification product; And / or, in step (3), the population origin of the mandarin fish to be tested is determined based on the detected genotype, including: If the genotype is TT, it suggests that the individual may have originated from the Beijing water system mandarin fish population or its genetic background is similar to that of the Beijing water system mandarin fish population; if the genotype is TA or AA, it suggests that the individual may have originated from the Heilongjiang water system mandarin fish population or has genetic infiltration from the Heilongjiang water system mandarin fish population.

9. A molecular monitoring method for local germplasm resources of mandarin fish, characterized in that, Includes the following steps: (1) Provide a sample of mandarin fish to be tested; (2) Detect the genotype of the SNP marker site in the sample; the SNP marker site is: lgr4 p.D874V, located at 14537718 bases on chromosome 1, and the base mutation type is T / A; (3) Compare the detected genotype with the standard genotype of the original species or target conservation population of mandarin fish in a specific aquatic system to monitor its genetic purity and whether there is any contamination from foreign genetic background; The mandarin fish populations mentioned include those from the Heilongjiang River system and those from the Beijing River system.

10. The molecular monitoring method for local germplasm resources of mandarin fish according to claim 9, characterized in that, The detection step in step (2) uses the SNP marker primer pair as described in claim 1 or 2 or the kit as described in claim 3 or 4; and / or, in the detection step in step (2), the genotype of the SNP marker site is determined by sequencing the amplification product.

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