SNP (Single Nucleotide Polymorphism) molecular marker for identifying different geographical populations of Nile tilapia and application of SNP molecular marker
By designing SNP molecular markers and specific primers, the problem of identifying geographical populations of Nile tilapia was solved, enabling efficient and accurate germplasm resource management and source tracing, and ensuring the safety and breeding quality of germplasm resources.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
- Filing Date
- 2026-01-22
- Publication Date
- 2026-04-21
AI Technical Summary
Current technologies lack effective molecular tools to identify different geographical populations of Nile tilapia, making it difficult to solve the problem of mixed germplasm and affecting the protection of germplasm resources and the breeding of superior varieties.
Using SNP molecular markers, including SNP chr4-17660871, SNP chr7-30426048 and SNP chr16-3192004, specific primers were designed for PCR amplification, and the geographic populations of Nile tilapia were identified by combining genotype determination.
This method enables efficient and accurate identification of geographical populations of Nile tilapia, solves the problem of mixed germplasm sources, and improves the accuracy of germplasm resource identification and management reliability.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of molecular marker technology, specifically relating to an SNP molecular marker for identifying different geographical populations of Nile tilapia and its application. Background Technology
[0002] Nile tilapia ( Oreochromis niloticus Nile tilapia, native to Africa, is a rapidly growing, highly adaptable, and easily reproduced important freshwater economic fish. Taxonomically, it belongs to the order Perciformes, family Cichlidae, and genus *Carassius*, and is now widely farmed globally. As a major Nile tilapia farming country, my country's industry continues to expand. To achieve sustainable utilization of germplasm resources and healthy industry development, it is urgent to strengthen the discovery and protection of core germplasm and establish a molecular technology system capable of rapidly and accurately identifying different geographical populations.
[0003] Single nucleotide polymorphisms (SNPs), as third-generation genetic markers, are characterized by their abundance, high polymorphism, and strong genetic stability, and have been applied to rapid, high-throughput genotyping in various organisms. With the maturity of high-throughput sequencing technology, genome-wide SNP screening has become a routine method for germplasm resource identification and population genetic structure analysis, efficiently identifying molecular markers closely related to target traits or population differentiation. Compared to earlier genetic markers, SNPs have significant advantages in resolution, detection throughput, and result reproducibility, and are therefore widely used in fields such as plant and animal genetics breeding and germplasm resource conservation, providing key technical support for the development of modern seed industry. However, there are currently no molecular tools for identifying different geographical populations of Nile tilapia. Summary of the Invention
[0004] The purpose of this invention is to provide an SNP molecular marker for identifying different geographical populations of Nile tilapia, which can efficiently and accurately identify different geographical populations of Nile tilapia, effectively solve the problem of mixed germplasm, and promote the breeding of superior varieties and the protection of germplasm resources.
[0005] This invention provides an SNP molecular marker, including SNP chr4-17660871, SNP chr7-30426048 and SNP chr16-3192004; The nucleotide sequence of SNP chr4-17660871 is shown in SEQ ID NO:1, and a C / T polymorphism site exists at position 49 in SEQ ID NO:1. The nucleotide sequence of SNP chr7-30426048 is shown in SEQ ID NO:2, and an A / C polymorphism site exists at position 220 in SEQ ID NO:2. The nucleotide sequence of SNP chr16-3192004 is shown in SEQ ID NO:3, and an A / G polymorphism site exists at position 105 in SEQ ID NO:3.
[0006] This invention provides primers for amplifying the aforementioned SNP molecular markers, including primer pairs for amplifying SNP chr4-17660871, SNP chr7-30426048, and SNP chr16-3192004; The nucleotide sequences of the primer pairs used for amplifying SNP chr4-17660871 are shown in SEQ ID NO:4 for the forward primer and SEQ ID NO:5 for the reverse primer. The nucleotide sequences of the primer pairs used for amplifying SNP chr7-30426048 are shown in SEQ ID NO:6 for the forward primer and SEQ ID NO:7 for the reverse primer. The nucleotide sequences of the primer pairs used for amplifying SNP chr16-3192004 are shown in SEQ ID NO:8 for the forward primer and SEQ ID NO:9 for the reverse primer.
[0007] This invention provides a kit for identifying Nile tilapia from different geographical locations, including the primers mentioned above.
[0008] Preferably, it also includes PCR amplification premix.
[0009] This invention provides the application of the primers or the kit in identifying geographical populations of Nile tilapia.
[0010] Preferably, the geographical populations of Nile tilapia include the Alexandria lineage, the Ismailia lineage, and the Senegalese lineage.
[0011] This invention provides a method for identifying geographical populations of Nile tilapia, comprising the following steps: The DNA of the Nile tilapia sample to be tested was amplified by PCR using the primers described above to obtain the PCR amplification product; The geographical population to which the Nile tilapia belongs is determined based on the PCR amplification products: When the genotype of SNP Chr4-17660871 is C / C, and the genotypes of SNP Chr7-30426048 and SNP Chr16-3192004 are A / A, the Nile tilapia to be tested is of the Alexandrine strain. When the genotypes of SNP Chr4-17660871 and SNP Chr7-30426048 are C / C, and the genotype of SNP Chr16-3192004 is G / G, the Nile tilapia to be tested is the Ismailia strain. When the genotype of SNP Chr4-17660871 is T / T, the genotype of SNP Chr7-30426048 is C / C, and the genotype of SNPChr16-3192004 is A / A, the Nile tilapia to be tested is of the Senegalese strain.
[0012] Preferably, the PCR amplification reaction system has a total volume of 20 μl, containing 10 μl Premix Ex Taq, 8 μl ddH2O, 0.5 μl 10 μM forward primer, 0.5 μl 10 μM reverse primer, and 1 μl DNA template.
[0013] Preferably, the PCR amplification reaction program is as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 52℃ annealing for 30 s, 72℃ extension for 50 s, 35 cycles; 72℃ final extension for 5 min.
[0014] Preferably, the Nile tilapia sample to be tested includes at least one of the following: fin tissue, blood, and fish meat tissue.
[0015] This invention provides an SNP molecular marker, including SNP chr4-17660871, SNP chr7-30426048, and SNP chr16-3192004; the nucleotide sequence of SNP chr4-17660871 is shown in SEQ ID NO:1, and a C / T polymorphism site is present at position 49 in SEQ ID NO:1; the nucleotide sequence of SNP chr7-30426048 is shown in SEQ ID NO:2, and an A / C polymorphism site is present at position 220 in SEQ ID NO:1; the nucleotide sequence of SNP chr16-3192004 is shown in SEQ ID NO:3, and an A / G polymorphism site is present at position 105 in SEQ ID NO:1. Based on the above three SNP molecular markers, it is possible to effectively identify Nile tilapia individuals of the Alexandria (AiT), Ismailia (IsT), and Senegalese (SeT) strains with high identification accuracy. Experiments showed that detecting the genotypes of the above three SNP molecular markers and determining the geographical population of Nile tilapia based on the genotypes of the SNP loci: when the genotype of SNP Chr4-17660871 is C / C, the genotype of SNP Chr7-30426048 is A / A, and the genotype of SNP Chr16-3192004 is A / A, the tested individual belongs to the Alexandrian lineage; when the genotypes of SNP Chr4-17660871 are C / C, the genotypes of SNP Chr7-30426048 are C / C, and the genotype of SNP Chr16-3192004 is G / G, the tested individual belongs to the Ismailia lineage; when the genotypes of SNPChr4-17660871 are T / T, and the genotypes of SNP Chr7-30426048 are C / C and SNP... When the genotype of Chr16-3192004 is A / A, the tested individual is of the Senegalese line. The SNP molecular markers provided by this invention can effectively overcome the subjectivity and error of manual phenotypic judgment, and achieve stable, specific and accurate identification of germplasm resources. Detailed Implementation
[0016] This invention provides an SNP molecular marker, including SNP chr4-17660871, SNP chr7-30426048 and SNP chr16-3192004.
[0017] In this invention, SNP chr4-17660871 is a polymorphic site C / T at position 17660871 on chromosome 4 of the reference genome; SNP chr7-30426048 is a polymorphic site A / C at position 30426048 on chromosome 7 of the reference genome; and SNP chr16-3192004 is a polymorphic site A / G at position 3192004 on chromosome 16 of the reference genome. The reference genome is the Nile tilapia genome GCF_001858045.2 published in the NCBI database.
[0018] In this invention, the identification of geographical groups can be completed based on any one location, and the accuracy of the identification can be effectively improved based on three locations, as shown in Table 1.
[0019] Table 1. Genotypes of geographic populations corresponding to the three SNP molecular markers
[0020] This invention provides primers for SNP molecular marker amplification, including primer pairs for SNP chr4-17660871, SNP chr7-30426048, and SNP chr16-3192004; the nucleotide sequences of the SNP chr4-17660871 amplification primer pair are shown in SEQ ID NO:4 (forward primer) and SEQ ID NO:5 (reverse primer); the nucleotide sequences of the SNP chr7-30426048 amplification primer pair are shown in SEQ ID NO:6 (forward primer) and SEQ ID NO:7 (reverse primer); and the nucleotide sequences of the SNP chr16-3192004 amplification primer pair are shown in SEQ ID NO:8 (forward primer) and SEQ ID NO:9 (reverse primer).
[0021] In this invention, the primers are designed based on SNP molecular markers and can specifically amplify SNP molecular markers. This invention does not impose special restrictions on the source of the primers; primer synthesis methods well-known in the art can be used, such as artificial synthesis. In this embodiment of the invention, the primers were synthesized by Yixin Biotechnology (Shanghai) Co., Ltd.
[0022] This invention provides a kit for identifying Nile tilapia from different geographical locations, including the primers mentioned above.
[0023] In this invention, a PCR amplification premix is also preferably included. The PCR amplification premix preferably includes Premix ExTaq, purchased from Takara Bio Engineering (Dalian) Co., Ltd., catalog number RR003A.
[0024] This invention provides the application of the primers or the kit in identifying geographical populations of Nile tilapia.
[0025] In this invention, the preferred geographical populations of Nile tilapia include the Alexandrian, Ismailian, and Senegalese strains. The kit is capable of distinguishing individual Nile tilapia from these three strains.
[0026] This invention provides a method for identifying geographical populations of Nile tilapia, comprising the following steps: The DNA of the Nile tilapia sample to be tested was amplified by PCR using the primers described above to obtain the PCR amplification product; The geographical population to which the Nile tilapia belongs is determined based on the PCR amplification products: When the genotype of SNP Chr4-17660871 is C / C, and the genotypes of SNP Chr7-30426048 and SNP Chr16-3192004 are A / A, the Nile tilapia to be tested is of the Alexandrine strain. When the genotypes of SNP Chr4-17660871 and SNP Chr7-30426048 are C / C, and the genotype of SNP Chr16-3192004 is G / G, the Nile tilapia to be tested is the Ismailia strain. When the genotype of SNP Chr4-17660871 is T / T, the genotype of SNP Chr7-30426048 is C / C, and the genotype of SNPChr16-3192004 is A / A, the Nile tilapia to be tested is of the Senegalese strain.
[0027] This invention does not impose any particular limitation on the method for extracting DNA from the Nile tilapia sample to be tested; any DNA extraction method well-known in the art can be used, such as using the Tiangen DNA extraction kit to extract tilapia genomic DNA. This invention also does not impose any particular limitation on the type of Nile tilapia sample to be tested; any sample type well-known in the art can be used, such as fin tissue, blood, and fish meat tissue.
[0028] In this invention, the PCR amplification reaction system preferably has a total volume of 20 μl, comprising 10 μl PremixEx Taq, 8 μl ddH2O, 0.5 μl 10 μM forward primer, 0.5 μl 10 μM reverse primer, and 1 μl DNA template. The preferred PCR amplification reaction program is: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 52℃ annealing for 30 s, 72℃ extension for 50 s, for 35 cycles; and a final extension at 72℃ for 5 min. This invention does not impose any special limitations on the PCR amplification instrument; any PCR instrument well-known in the art can be used.
[0029] In this invention, the preferred method for analyzing the genotype of the PCR amplification product is to determine the base type of the SNP site based on the sequencing results of the PCR amplification product. If only one base type A appears, the genotype is AA; if both base types A and C appear, the genotype is AC, and so on.
[0030] In one embodiment of the present invention, to evaluate the ability of the SNP molecular marker to distinguish different geographical populations of Nile tilapia in mixed culture, F1 generations of Nile tilapia from the Alexandria, Ismailia, and Senegalese strains were mixed-cultured. The primers or their kits were used to identify their geographical strain origins. The results showed that the origin of individuals in a randomly sampled group of 30 F1 offspring could be accurately identified with an accuracy rate of 100%. Therefore, the results of this invention verify that the SNP can accurately and efficiently identify the geographical origin of Nile tilapia, providing reliable technical support for the safe management and traceability of germplasm resources.
[0031] The following detailed description, in conjunction with embodiments, illustrates an SNP molecular marker for identifying different geographical populations of Nile tilapia provided by the present invention and its application, but these should not be construed as limiting the scope of protection of the present invention.
[0032] Example 1 Whole-genome resequencing analysis and screening of SNPs for identification The Nile tilapia strains involved in this embodiment—the Alexandria (AiT), Ismailia (IsT), and Senegalese (SeT)—are all deposited at the Freshwater Fisheries Research Center-Tilapia Genetic and Breeding Center, Chinese Academy of Fishery Sciences. Fifteen individuals from each population were collected, and their caudal fin tissues were preserved in anhydrous ethanol. These 45 samples were then sent to Nanjing Paiseno Biotechnology Co., Ltd. for whole-genome resequencing. After obtaining the raw data, GATK software was used for genotyping to obtain SNPs. Finally, Plink software was used for quality control filtering of the genotyping data to generate a high-quality dataset for subsequent analysis.
[0033] The screening of SNP loci followed this procedure: First, loci on the chromosome were preserved, and then those with MAF > 0.2, het < 0.05, and geno < 0.05 were screened sequentially. Next, Beagle (v5.1) was used for genotyping, and SNPs located in intergenic regions were removed. Subsequently, stricter screening criteria (MAF > 0.3, het < 0.05) were applied, and linkage disequilibrium filtering (LDR) was performed. 2 <0.2). After the above steps, 85 core SNP sites were finally obtained (Table 2).
[0034] Table 2. Core SNP locus information
[0035] Among the core SNP sites, SNP sites with high population differentiation were further screened and refined, and three SNP sites that may be used to identify different geographical populations of Nile tilapia were initially identified. Their physical locations and sequence information are shown in Table 2. In Table 2, SNP site chr4-17660871 is located at position 17660871 on chromosome 4 of Nile tilapia, which is position 49 of the corresponding sequence in Table 2. The base at position 49 is either C (cytosine) or T (thymine). SNP site chr7-30426048 is located at position 30426048 on chromosome 7 of Nile tilapia, which is position 220 of the corresponding sequence in Table 2. The base at position 220 is either C (cytosine) or A (adenine). SNP site chr16-3192004 is located at position 3192004 on chromosome 16 of Nile tilapia, which is position 105 of the corresponding sequence in Table 3. The base at position 105 is either G (guanine) or A (adenine).
[0036] Table 3. SNP locus information for identifying different geographical populations of tilapia
[0037] Example 2 Reliability verification of identification using SNP loci in different geographic populations Based on the three SNP sites located in Example 1 of this invention, corresponding amplification primers were developed, and the primer information is shown in Table 4.
[0038] Table 4 Primer sequences for identifying SNP sites
[0039] Caudal fin tissues were collected from 30 individuals from each of the three geographic populations' conservation ponds to verify the reliability of the selected loci. Genomic DNA was extracted from tilapia using the Tiangen DNA Extraction Kit, and PCR amplification was performed using primers listed in Table 5. The total volume of the PCR reaction system was 20 μl, containing 10 μl Premix Ex Taq, 8 μl ddH2O, 0.5 μl forward primer (10 μM), 0.5 μl reverse primer (10 μM), and 1 μl DNA template (50 ng). The amplification program was set as follows: 95℃ pre-denaturation for 5 min; followed by 35 cycles of amplification, including 95℃ denaturation for 30 s, 52℃ annealing for 30 s, 72℃ extension for 50 s; and a final extension at 72℃ for 5 min.
[0040] Table 5. Genotyping of SNP loci in individuals from different geographic populations.
[0041] Genotyping results of 90 individuals from the three populations showed that the genotypes of SNPs chr4-17660871, chr7-30426048, and chr16-3192004 were stable and consistent with the sequencing results. These three loci effectively classified Nile tilapia into the Alexandrian, Ismailian, and Senegalese strains (Table 5).
[0042] Example 3 Application of SNP loci in the identification of offspring in different geographical populations Two hundred F1 generation Nile tilapia from each of the Alexandria, Ismailia, and Senegalese strains were co-cultured in a mixed tank. Prior to co-culturing, each fish was intraperitoneally tagged with a PIT tag. Subsequently, 30 individuals were randomly selected from the mixed-culture tank to identify their geographical strain origin. Caudal fin tissue samples were collected from each fish, and DNA extraction, PCR amplification, and Sanger sequencing analysis were performed using the appropriate reagents and primers as described in Example 2.
[0043] Table 6. Genotyping of SNP loci in offspring of different geographical populations.
[0044] Genotyping results of the mixed-culture population showed that among 30 randomly sampled F1 offspring, 6 individuals belonged to the Alexander strain, 14 to the Ismailia strain, and 10 to the Senegalese strain (Table 6). Three SNP molecular markers demonstrated clear discriminative ability in the mixed samples, successfully distinguishing different geographical strains. These results validate that the aforementioned SNP marker set can accurately and efficiently identify the geographical provenance of Nile tilapia, providing reliable technical support for the safe management and traceability of germplasm resources.
[0045] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. An SNP molecular marker, characterized in that, Including SNP chr4-17660871, SNP chr7-30426048 and SNP chr16-3192004; The nucleotide sequence of SNP chr4-17660871 is shown in SEQ ID NO:1, and a C / T polymorphism site exists at position 49 in SEQ ID NO:
1. The nucleotide sequence of SNP chr7-30426048 is shown in SEQ ID NO:2, and an A / C polymorphism site exists at position 220 in SEQ ID NO:
2. The nucleotide sequence of SNP chr16-3192004 is shown in SEQ ID NO:3, and an A / G polymorphism site exists at position 105 in SEQ ID NO:
3.
2. A primer for SNP molecular marker amplification as described in claim 1, characterized in that, This includes primer pairs for amplifying SNP chr4-17660871, SNP chr7-30426048, and SNP chr16-3192004; The nucleotide sequences of the primer pairs used for amplifying SNP chr4-17660871 are shown in SEQ ID NO:4 for the forward primer and SEQ ID NO:5 for the reverse primer. The nucleotide sequences of the primer pairs used for amplifying SNP chr7-30426048 are shown in SEQ ID NO:6 for the forward primer and SEQ ID NO:7 for the reverse primer. The nucleotide sequences of the primer pairs used for amplifying SNP chr16-3192004 are shown in SEQ ID NO:8 for the forward primer and SEQ ID NO:9 for the reverse primer.
3. A kit for identifying Nile tilapia from different geographical locations, characterized in that, Includes the primers described in claim 2.
4. The kit for identifying Nile tilapia from different geographical locations according to claim 3, characterized in that, It also includes PCR amplification premix.
5. The use of the primers of claim 2 or the kits of claim 3 or 4 in identifying geographical populations of Nile tilapia.
6. The application according to claim 5, characterized in that, The geographical populations of Nile tilapia include the Alexandrian lineage, the Ismailian lineage, and the Senegalese lineage.
7. A method for identifying geographical populations of Nile tilapia, characterized in that, Includes the following steps: The DNA of the Nile tilapia sample to be tested was amplified by PCR using the primers described in claim 2 to obtain the PCR amplification product; The geographical population to which the Nile tilapia belongs is determined based on the PCR amplification products: When the genotype of SNP Chr4-17660871 is C / C, and the genotypes of SNP Chr7-30426048 and SNP Chr16-3192004 are A / A, the Nile tilapia to be tested is of the Alexandrine strain. When the genotypes of SNP Chr4-17660871 and SNP Chr7-30426048 are C / C, and the genotype of SNP Chr16-3192004 is G / G, the Nile tilapia to be tested is the Ismailia strain. When the genotype of SNP Chr4-17660871 is T / T, the genotype of SNP Chr7-30426048 is C / C, and the genotype of SNPChr16-3192004 is A / A, the Nile tilapia to be tested is of the Senegalese strain.
8. The method according to claim 7, characterized in that, The PCR amplification reaction system has a total volume of 20 μl, containing 10 μl Premix Ex Taq, 8 μl ddH2O, 0.5 μl 10 μM forward primer, 0.5 μl 10 μM reverse primer, and 1 μl DNA template.
9. The method according to claim 7, characterized in that, The PCR amplification reaction procedure was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 52℃ annealing for 30 s, 72℃ extension for 50 s, 35 cycles; 72℃ final extension for 5 min.
10. The method according to any one of claims 7 to 9, characterized in that, The Nile tilapia sample to be tested includes at least one of the following: fin tissue, blood, and fish meat tissue.