A specific SNP molecular marker combination of hypophthalmichthys nobilis, DNA fingerprint of hypophthalmichthys nobilis and application thereof

Abstract

The application discloses a kind of silver carp variety specific SNP molecular marker combination, silver carp DNA fingerprint and application, the SNP molecular marker combination includes 367 SNP sites, the site information of the SNP molecular marker combination is as shown in table 1.The application takes silver carp germplasm resource as research object, obtains genotype by whole genome resequencing method, and then filters out 367 specific SNP markers capable of distinguishing different silver carp varieties, and the DNA fingerprint based on these markers can be used to distinguish different silver carp varieties.Compared with traditional methods, the fingerprint has the characteristics of high accuracy and strong reliability when used for silver carp variety identification.This technology not only provides strong support for the specific evaluation of silver carp varieties, the protection and management of breed rights, and the optimization and structure adjustment of silver carp breeding industry, but also shows good application prospects in the classification of silver carp germplasm resources, the confirmation of breed rights and the tracing of individual sources.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, specifically to a silver carp-specific SNP molecular marker combination, and the silver carp DNA fingerprint constructed based thereon, and its applications. Background Technology

[0002] Silver carp (Hypophthalmichthys molitrix) is an important endemic economic fish species in my country and one of the famous "four major freshwater fish." It is highly adaptable and widely distributed in the Yangtze, Pearl, and Heilongjiang river basins. Currently, newly bred varieties include Changfeng silver carp, Tianjin silver carp, and the fast-growing new strain Changfeng Silver Carp No. 2 developed by the Yangtze River Fisheries Research Institute of the Chinese Academy of Fishery Sciences. Furthermore, through long-term domestication and breeding processes, different geographical groups, strains, and families have emerged, exhibiting certain characteristics in growth and adaptability, providing rich germplasm resources for the breeding of superior new silver carp varieties. However, in aquaculture practice, problems such as mixed varieties, low genetic purity, and difficulties in variety identification are becoming increasingly prominent.

[0003] Traditional methods for identifying silver carp varieties mainly rely on morphological characteristics and growth performance observation. This method is not only time-consuming and labor-intensive, but also easily affected by various factors such as environment and aquaculture conditions, leading to inaccurate identification results. With the development of next-generation sequencing technology, single nucleotide polymorphisms (SNPs) have been widely used as molecular markers in the identification of plant and animal varieties and population genetic evaluation, demonstrating significant advantages. DNA fingerprinting technology is a modern method based on DNA molecular markers that can be used for the classification and identification of germplasm resources. Constructing DNA fingerprints for fish varieties can overcome the limitations of identifying varieties solely based on morphological characteristics, which is crucial for the precise identification of fish germplasm resources and the discovery of superior genes. DNA fingerprinting technology has been widely used in aquaculture variety diversity research and variety identification, but its systematic application in silver carp research remains insufficient. Currently, most existing methods for identifying silver carp varieties rely on morphological observation or a few traditional molecular markers, with few methods based on SNP molecular markers to construct fingerprints for precise identification of silver carp varieties.

[0004] Because different silver carp varieties exhibit relatively small morphological differences and are easily affected by the aquaculture environment, traditional identification methods have limited accuracy. Furthermore, traditional molecular markers suffer from insufficient polymorphism and low coverage, making it difficult to meet the needs for rapid differentiation of silver carp varieties and detection of genetic purity. Therefore, constructing a DNA fingerprint of silver carp based on SNP molecular markers is of great significance for filling the gap in molecular identification technology for silver carp varieties and improving the management level of silver carp germplasm resources. Summary of the Invention

[0005] The main objective of this invention is to propose a combination of SNP molecular markers specific to silver carp varieties, and the DNA fingerprint of silver carp constructed based on this combination, as well as their applications. The aim is to provide a specific combination of SNP molecular markers that can distinguish different silver carp varieties, which can be used to construct a DNA fingerprint of silver carp, enabling rapid identification of silver carp varieties. This will provide support for germplasm resource identification, variety screening, and tracing, and provide an efficient and accurate technical tool for the development, utilization, and protection of silver carp germplasm resources.

[0006] To achieve the above objectives, this invention proposes a silver carp-specific SNP molecular marker combination, which contains 367 SNP sites. The site information of the SNP molecular marker combination is shown in Table 1 below.

[0007] This invention also proposes the application of the SNP molecular marker combination described above in the analysis of silver carp genetic diversity, identification of silver carp varieties, and construction of silver carp DNA fingerprinting.

[0008] The present invention also proposes a DNA fingerprint of silver carp, which is constructed based on the SNP molecular marker combination described above, and the DNA fingerprint can distinguish different silver carp species.

[0009] This invention also proposes a method for constructing a silver carp DNA fingerprint, comprising the following steps:

[0010] S1. Obtain genomic DNA from samples of different silver carp species;

[0011] S2. Using the SNP molecular marker combination described above, detect the genotype of each sample at the 367 SNP loci.

[0012] S3. Construct a silver carp DNA fingerprint map based on the genotype information of each sample at the 367 SNP sites.

[0013] Preferably, in step S1, the genomic DNA is extracted using the magnetic bead method.

[0014] Preferably, in step S2, the genotype of each sample at the 367 SNP loci is detected by whole-genome resequencing combined with Sentieon software analysis.

[0015] This invention also proposes a method for identifying silver carp varieties, comprising the following steps:

[0016] S1. Obtain the genomic DNA of the fish sample to be tested;

[0017] S2. Detect the genotype of the fish sample to be tested at 367 SNP loci of the SNP molecular marker combination as described above;

[0018] S3. Compare the genotype of the fish sample to be tested with the DNA fingerprint site, and determine whether the silver carp sample to be tested is a silver carp based on the comparison result;

[0019] S4. Calculate the genetic distance between samples and perform kinship analysis with known samples to determine the variety of the sample to be tested.

[0020] Preferably, in step S1, the genomic DNA is extracted using a high-throughput DNA extraction kit.

[0021] Preferably, in step S3, the judgment criterion is that when the genotype of the fish to be tested at 367 SNP loci matches the DNA fingerprint at a rate of ≥95%, the fish to be tested is considered to be silver carp.

[0022] Preferably, in step S4, the kinship analysis includes cluster analysis or PCA analysis between the silver carp sample to be tested and known silver carp species samples;

[0023] The known samples include Changfeng silver carp, the new Changfeng silver carp strain 2, Tianjin silver carp, and common silver carp.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] This invention focuses on silver carp germplasm resources. By performing whole-genome resequencing on 117 different silver carp varieties to obtain genotypes, 367 specific SNP markers capable of distinguishing different silver carp varieties were screened. A DNA fingerprint map constructed based on these markers can be effectively used to differentiate between different silver carp varieties. Compared with traditional methods, this fingerprint map has high accuracy and reliability in silver carp variety identification. Its principle is to detect the genotype of the sample at these loci and compare it with the corresponding specific nucleotides at the 367 loci in the DNA fingerprint database to determine whether the sample is a silver carp. By calculating the genetic distance between samples and performing phylogenetic analysis, the variety type of the sample can be identified. This technology not only provides strong support for the specificity evaluation of silver carp varieties, the protection and management of variety rights, and the optimization and structural adjustment of silver carp aquaculture, but also shows good application prospects in silver carp germplasm resource classification, variety rights confirmation, and individual source traceability. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 The similarity heatmap of 117 samples provided for this invention;

[0028] Figure 2 Clustering tree diagram of 117 samples provided by this invention;

[0029] Figure 3 The PCA distribution scatter plot provided by this invention;

[0030] Figure 4 DNA fingerprints of different silver carp species provided for this invention;

[0031] Figure 5 Clustering tree diagram of 11 unknown samples and Changfeng silver carp population provided by the present invention.

[0032] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially. Furthermore, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0035] Example 1: Screening of Silver Carp Variety-Specific SNP Marker Sets

[0036] Based on existing silver carp species information, 30 samples each of Changfeng silver carp (CF) and the new Changfeng silver carp strain 2 (CF2) were collected from Qianjiang, Hubei Province; 30 samples of Jinlian silver carp (Hmo-09) were collected from Tianjin Huanxin; and 27 samples of common silver carp (HB-Hmo-02) were collected from Jianli, Hubei Province. Fin rays or muscle tissue were collected from these 117 samples of different silver carp species, preserved in anhydrous ethanol, and subsequently subjected to genomic DNA extraction and whole-genome resequencing.

[0037] Whole-genome resequencing specifically includes the following steps: (1) DNA extraction from sample tissue using magnetic beads. (2) Fragmenting of DNA samples using dsDNAFragmentase, repairing the enzyme ends, and adding an A base to the 3' end. (3) Ligating sequencing adapters to fragmented DNA using ligase, and purifying the ligation product using magnetic beads. (4) Amplification of the ligation product using PCR, and fragment screening of the PCR product using magnetic beads. (5) Denaturation of the linear library to single strands followed by circularization, digestion of uncirculated linear DNA molecules to obtain a single-stranded circular library. (6) Single-stranded circular DNA molecules replicate through rolling circle replication to form a DNA nanosphere (DNB) containing more than 300 copies. (7) Loading the DNB into a sequencing chip using the MGIDL-T7 loading device, and sequencing using a combined probe anchoring polymerization technique with a sequencing strategy of PE150 and a sequencing depth of 10×.

[0038] The obtained resequencing data were analyzed. Sentieon was used to align and detect variants in the resequencing data. The analysis process was as follows: (1) Sentieon was used to align reads to the corresponding silver carp reference genome (GCA_041475455.1), orient the positions, and mark duplicate reads. (2) Sentieon was used to detect variant sites in each sample to obtain the variant information of each sample. (3) Sentieon was used to perform joint-calling and joint analysis of gVCF of all samples to obtain the variant results of each individual in the population. To ensure the accuracy of SNPs, the filtering criteria of "QD<2.0||FS>60.0||MQ<40.0||SOR>3.0||MQRankSum<-12.5||ReadPosRankSum<-8.0" were used to perform preliminary hard filtering on the SNP sites obtained after joint analysis, resulting in a vcf file containing genotype information of all individuals, containing 5,377,245 sites.

[0039] From the VCF file containing SNP variation information of all 117 samples, the MAF value, detection rate, heterozygosity, and sequencing depth of the loci were calculated and statistically analyzed. Genotype loci with MAF ≥ 0.05, SNP detection rate ≥ 90%, sequencing depth ≥ 10×, and dimorphism were selected. After extracting the flanking sequences of 100 bp before and after the loci, copy number analysis was performed using BLASTN software to remove multicopy loci. Then, based on the differences in allele distribution between any two varieties, differential loci between varieties were screened, ultimately resulting in 367 variety-specific differentiation loci. Each locus exhibits genotypic differences in at least two populations. Specific information about the loci is shown in Table 1.

[0040] Table 1367 SNP loci: chromosome number, location, and nucleotide information.

[0041]

[0042]

[0043]

[0044]

[0045] To verify the effectiveness of the 367 SNP loci set in silver carp variety identification, based on the genotypic information of these loci, the IBS genetic distance among 117 samples was calculated using Plink software, and a sample similarity heatmap was plotted. The results showed that these loci reflected low genetic similarity between different varieties and the highest genetic similarity among the same variety. Figure 1 Furthermore, based on genotypic information at 367 loci, Plink software was used to calculate the genetic distances among 117 samples and construct cluster trees. The results showed that the 117 samples clustered together according to their respective varieties. Figure 2 PCA analysis was performed using Plink software, and a scatter plot of the PCA distribution was plotted using R language. The results showed that the four varieties each clustered into one group. Figure 3 These results indicate that the 367 specific loci obtained through screening can effectively distinguish between different silver carp species.

[0046] Example 2: Construction of DNA fingerprinting of silver carp

[0047] The 367 specific loci selected in Example 1 were used to classify and identify 117 silver carp samples. Genotypic data at the 367 specific SNP loci were extracted from the 117 resequencing data, obtaining the fingerprint sequence for each individual in each variety. The base composition information at these loci was visualized using R language, with different colors representing different genotypic compositions. A heatmap was created showing the genotypic composition of each silver carp variety at these 367 loci, obtaining the DNA fingerprint profiles of different silver carp varieties, as shown below. Figure 4 As shown in the figure, each row represents a sample, and each column represents a SNP genotype. Based on the generated fingerprint sequence information, the composition of the fingerprint sequences for different varieties is different, indicating that these 117 materials can be effectively distinguished, achieving accurate differentiation between different silver carp varieties.

[0048] Example 3: Application of silver carp DNA fingerprinting in variety identification

[0049] Eleven fish samples from the same population were collected from a fish farm in Jingzhou, Hubei Province. After removing the caudal fins, DNA was extracted using a high-throughput DNA extraction kit, and the genotypes of these samples were subsequently obtained using resequencing. The genotypic composition information at the aforementioned 367 loci was extracted using Plink software and compared with the loci in the DNA fingerprint of this invention. The results showed that the genotypes of the 11 samples at the 367 loci were 95.2%-99.5% consistent with those in the DNA fingerprint of this invention, identifying them as silver carp. The genetic distances between these samples and the four silver carp varieties in this invention were calculated, and a clustering tree was constructed. The results showed that the 11 unknown samples clustered together with the Changfeng silver carp population (…). Figure 5 Therefore, it was determined that these 11 samples were Changfeng silver carp, which is consistent with the sampling results.

[0050] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the patent protection scope of the present invention.

Claims

1. The application of a SNP molecular marker combination in the analysis of genetic diversity of silver carp, identification of silver carp varieties, and construction of silver carp DNA fingerprinting, wherein the SNP molecular marker combination contains 367 SNP loci, which were determined by sequence alignment with reference to the silver carp genome GCA_041475455.

1. The locus information of the SNP molecular marker combination is shown in the table below: 。 2. The application of a silver carp DNA fingerprint in distinguishing different silver carp species, characterized in that, The DNA fingerprint is constructed based on the SNP molecular marker combination as described in claim 1.

3. A method for constructing a silver carp DNA fingerprint, characterized in that, Includes the following steps: S1. Obtain genomic DNA from samples of different silver carp species; S2. Using the SNP molecular marker combination as described in claim 1, detect the genotype of each sample at the 367 SNP loci; S3. Construct a silver carp DNA fingerprint map based on the genotype information of each sample at the 367 SNP sites.

4. The method according to claim 3, characterized in that, In step S1, the genomic DNA is extracted using the magnetic bead method.

5. The method according to claim 3, characterized in that, In step S2, the genotype of each sample at the 367 SNP loci is detected by whole-genome resequencing combined with Sentieon software analysis.

6. A method for identifying silver carp varieties, characterized in that, Includes the following steps: S1. Obtain the genomic DNA of the fish sample to be tested; S2. Detect the genotype of the fish sample to be tested at 367 SNP loci of the SNP molecular marker combination described in claim 1; S3. Compare the genotype of the sample to be tested with the loci of the DNA fingerprint pattern described in claim 3, and determine whether the sample to be tested is a silver carp based on the comparison results; S4. Calculate the genetic distance between samples and perform kinship analysis with known samples to determine the variety of the sample to be tested; In step S3, the criterion for determining whether the sample to be tested is a silver carp is that when the genotype of the fish to be tested at 367 SNP loci matches the DNA fingerprint at a rate of ≥95%, the fish to be tested is considered to be a silver carp.

7. The identification method according to claim 6, characterized in that, In step S1, the genomic DNA is extracted using a high-throughput DNA extraction kit.

8. The identification method according to claim 6, characterized in that, In step S4, the kinship analysis includes cluster analysis of the silver carp sample to be tested and samples of known silver carp species, or PCA analysis. The known samples include Changfeng silver carp, the new Changfeng silver carp strain 2, Tianjin silver carp, and common silver carp.

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