Specific SNP site combination for identifying dunchou pig breed and application thereof

Abstract

The application belongs to the field of molecular biology identification, and particularly relates to a specific SNP site combination for identifying Duntou pig breeds and application. The specific SNP site combination for identifying Duntou pig breeds comprises 40 SNP sites, and the physical position is based on the reference genome of pigs Sscrofa11.1 The sequence comparison is determined. The specific SNP site combination is screened based on the method of whole genome association analysis combined with selection signal analysis, so as to ensure the accuracy of site selection. The specific SNP site combination provided by the application can rapidly realize the accurate identification of Duntou pig breeds at the gene level, and has significant application value in the accurate protection of Hainan local pig genetic resources and breed utilization.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology identification, specifically relating to a specific SNP site combination for identifying the Duntou pig breed and its application. Background Technology

[0002] Hainan Island's unique geographical and ecological conditions have fostered numerous local pig genetic resources, including Wenchang pig, Tunchang pig, Lingao pig, Ding'an pig, Duntou pig, and Wuzhishan pig. Among them, the Duntou pig is a local pig breed unique to Hainan Province, renowned for its excellent characteristics such as tolerance to roughage, resistance to heat and humidity, and delicious meat. It is an important component of Hainan's local pig genetic resources.

[0003] In recent years, with the increasing awareness of local pig breed resource protection and the growing demand for industrial development, the collection and preservation of Duntou pig germplasm resources has been gradually carried out. However, in the process of resource protection, population restoration, and subsequent breeding and industrial promotion, a key technical bottleneck is faced: the lack of efficient and accurate genetic identification methods for Duntou pigs to distinguish them from other similar-looking local pig breeds and imported commercial pig breeds in and outside Hainan Island, thus ensuring the purity and genetic integrity of germplasm resources.

[0004] The key to building a precise genetic identification system lies in screening a set of core single nucleotide polymorphism (SNP) markers with high breed differentiation capabilities. This set must achieve precise identification at three levels: first, it must be able to clearly distinguish Duntou pigs from introduced commercial pig breeds such as Duroc, Landrace, and Large White; second, it must be able to effectively distinguish them from other major local pig breeds in China (such as Min pigs and Jinhua pigs); and third, and most importantly, it must achieve specific identification among Hainan native pig breeds (including Lingao pigs and Wuzhishan pigs) with highly similar genetic backgrounds, in order to solve the practical problem of difficulty in distinguishing Hainan local pig breeds due to their close genetic distance and similar phenotypes.

[0005] Genome-wide association analysis (GWAS) and selection signal analysis are core techniques for identifying such discriminative loci. GWAS can systematically analyze genetic loci closely related to typical traits of Duntou pigs (such as tolerance to roughage and strong stress resistance); selection signal analysis helps reveal the genomic imprints formed by the breed during long-term natural selection and artificial domestication. Combining these two methods organically, from the perspectives of "phenotypic correlation" and "selective imprinting," can accurately identify SNP markers with high discriminative power, laying the foundation for constructing a "molecular identity map" of Duntou pigs and establishing a standardized breed identification and genetic tracing system. This provides crucial support for the protection of its germplasm resources, the breeding of superior traits, and subsequent industrial development. Summary of the Invention

[0006] In order to overcome the shortcomings and disadvantages of the existing technology, the primary objective of this invention is to provide a specific SNP locus combination for identifying the Duntou pig breed, which can achieve rapid and accurate identification of the Duntou pig at the genetic level.

[0007] Another object of the present invention is to provide the application of the above-mentioned specific SNP locus combinations for identifying the Duntou pig breed.

[0008] Another object of the present invention is to provide a method for rapid identification of pigs with a thick head.

[0009] The objective of this invention is achieved through the following technical solution:

[0010] A specific SNP locus combination for identifying the Duntou pig breed comprises 40 SNP loci, named SNP loci 1-40. The physical locations of the 40 SNP loci were determined based on sequence alignment of the pig reference genome Sscrofa11.1. The locations and variation information of the 40 SNP loci are shown below:

[0011] SNP site 1 is located at 71291093 bp on chromosome 1, and its mutation type is C / G.

[0012] SNP site 2 is located at 219372828bp on chromosome 1, and its mutation type is T / C.

[0013] SNP site 3 is located at 221005301 bp on chromosome 1, and its mutation type is G / A.

[0014] SNP site 4 is located at 221005824bp on chromosome 1, and its mutation type is A / G.

[0015] SNP site 5 is located at 221008294bp on chromosome 1, and its mutation type is A / G.

[0016] SNP locus 6 is located at 221008321 bp on chromosome 1, and its mutation type is G / A.

[0017] SNP site 7 is located at 221008537bp on chromosome 1, and its mutation type is T / C.

[0018] SNP site 8 is located at 221008568 bp on chromosome 1, and its mutation type is C / T.

[0019] SNP locus 9 is located at 221008656 bp on chromosome 1, and its mutation type is A / T.

[0020] SNP locus 10 is located at 221011244bp on chromosome 1, and its mutation type is T / C.

[0021] SNP locus 11 is located at 221013180 bp on chromosome 1, and its mutation type is T / G.

[0022] SNP locus 12 is located at 221039447bp on chromosome 1, and its mutation type is C / T.

[0023] SNP locus 13 is located at 221048804 bp on chromosome 1, and its mutation type is A / C.

[0024] SNP locus 14 is located at 31714266 bp on chromosome 4, and its mutation type is G / A.

[0025] SNP locus 15 is located at 52659120 bp on chromosome 7, and its mutation type is G / A.

[0026] SNP locus 16 is located at 52663754 bp on chromosome 7, and its mutation type is C / G.

[0027] SNP locus 17 is located at 58983433 bp on chromosome 7, and its mutation type is T / C.

[0028] SNP locus 18 is located at 59543790 bp on chromosome 7, and its mutation type is G / A.

[0029] SNP locus 19 is located at 59601220 bp on chromosome 7, and its mutation type is G / A.

[0030] SNP site 20 is located at 59610710 bp on chromosome 7, and its mutation type is C / T.

[0031] SNP site 21 is located at 59612202 bp on chromosome 7, and its mutation type is C / T.

[0032] SNP locus 22 is located at 59612309 bp on chromosome 7, and its mutation type is G / A.

[0033] SNP site 23 is located at 59612325 bp on chromosome 7, and its mutation type is C / T.

[0034] SNP locus 24 is located at 59612409 bp on chromosome 7, and its mutation type is C / T.

[0035] SNP site 25 is located at 59612520 bp on chromosome 7, and its mutation type is C / T.

[0036] SNP locus 26 is located at 59613024 bp on chromosome 7, and its mutation type is T / G.

[0037] SNP site 27 is located at 65815519 bp on chromosome 9, and its mutation type is C / T.

[0038] SNP site 28 is located at 77504853 bp on chromosome 9, and its mutation type is C / T.

[0039] SNP 29 is located at 97702199 bp on chromosome 9, and its mutation type is A / G.

[0040] SNP site 30 is located at 97718522 bp on chromosome 9, and its mutation type is G / A.

[0041] SNP site 31 is located at 97719064 bp on chromosome 9, and its mutation type is G / A.

[0042] SNP site 32 is located at 60464546 bp on chromosome 10, and its mutation type is T / G.

[0043] SNP site 33 is located at 49,874,673 bp on chromosome 11, and its mutation type is T / G.

[0044] SNP site 34 is located at 118786842 bp on chromosome 13, and its mutation type is T / A.

[0045] SNP site 35 is located at 118804557bp on chromosome 13, and its mutation type is G / T.

[0046] SNP site 36 is located at 118882958 bp on chromosome 13, and its mutation type is T / G.

[0047] SNP site 37 is located at 52911684 bp on chromosome 16, and its mutation type is C / G.

[0048] SNP site 38 is located at 52912528 bp on chromosome 16, and its mutation type is G / A.

[0049] SNP site 39 is located at 52912973 bp on chromosome 16, and its mutation type is A / G.

[0050] SNP locus 40 is located at 52913937 bp on chromosome 16, and its mutation type is G / A.

[0051] The application of the specific SNP locus combination in identifying Duntou pigs or in the analysis of Duntou pig genetic relationships.

[0052] Application of a product for detecting the above-mentioned specific SNP locus combinations in the identification of Duntou pigs or in the analysis of Duntou pig genetic relationships.

[0053] The products mentioned are reagents, kits, or gene chips, etc.

[0054] The reagents include at least one of primers, probes, etc.

[0055] A molecular probe array for identifying the specific SNP site combination of the pig, the molecular probe array targeting the aforementioned specific SNP site combination for detection of the aforementioned specific SNP site combination.

[0056] A gene chip for identifying Duntou pigs, the chip being loaded with the aforementioned combination of molecular probes.

[0057] A method for rapid identification of Duntou pigs preferably includes the following steps:

[0058] (1) Collect tissue samples from the pigs to be tested, extract DNA from the tissue samples, and obtain DNA samples from the pigs to be tested;

[0059] (2) Perform gene sequencing on the DNA sample of the pig to be tested obtained in step (1) to obtain the genotype data of the pig to be tested;

[0060] (3) Combine the genotype data of the pigs to be tested obtained in step (2) with the genotype data of the known dwarf pig population and the control population, extract the genotype data of the above-mentioned specific SNP locus combination (40 SNP loci), perform principal component analysis on the combined and extracted genotype data, and determine whether the pigs to be tested are dwarf pigs.

[0061] The DNA sample mentioned in step (2) needs to be quality inspected first, and gene sequencing can be performed after the quality inspection is qualified.

[0062] The absorbance ratio of the DNA sample nucleic acid purity detection A260 / 280 in step (2) is between 1.8 and 2.0, and the concentration is ≥50 ng / μL.

[0063] The known group of Duntou pigs mentioned in step (3) is a group composed of multiple (≥15) purebred Duntou pigs. It can be the group of Duntou pigs in the example, or a group of Duntou pigs selected by oneself, or a group of Duntou pigs in a public database.

[0064] The control group mentioned in step (3) is a group of other pigs that are not chubby pigs (≥5 pigs in a single breed group, ≥30 pigs in the whole group). It can be the control group in the example, or other non-chubby pig groups selected by oneself, or non-chubby pig groups in a public database.

[0065] The control group is preferably at least one of the following: Wuzhishan pig, white Wuzhishan pig, Lingao pig, Bama fragrant pig, Yunnan small-eared pig, Anqing pig, Erhualian pig, Meishan pig, Jinhua pig, Min pig, Landrace pig, Large White pig, and Duroc pig.

[0066] The merging and extraction in step (3) are preferably performed using Plink software.

[0067] The principal component analysis described in step (3) is preferably visualized using the ggplot2 package in R language.

[0068] The method for judgment described in step (3) is as follows:

[0069] When the principal component analysis results show that the pig to be tested is genetically close to the known Duntou pig population, i.e., they cluster together, then the pig to be tested is a Duntou pig.

[0070] The present invention has the following advantages and effects compared with the prior art:

[0071] (1) Based on resequencing data, this invention screens a specific SNP locus combination for identifying the Duntou pig breed by combining genome-wide association analysis with selection signal analysis. This screening method further significantly improves the specificity and accuracy of screening specific SNP locus combinations for identifying Duntou pigs, and provides reliable molecular marker support for the identification of Duntou pig breeds.

[0072] (2) The SNP locus combination provided by the present invention contains 40 SNP loci, which has the advantages of simple operation, low detection cost, fast analysis speed, strong specificity and high accuracy. It can be used for the scientific and accurate identification and genetic relationship analysis of purebred pigs of Duntou pig, which is conducive to promoting the protection and development of Hainan's characteristic local pig breeds.

[0073] (3) The SNP locus combination provided by this invention has good universality and identification accuracy. It is suitable for accurately identifying multiple ecological groups such as Duntou pig and Hainan local pig breeds, South China, Jianghai, Central China, North China and Western introduced breeds (e.g., Wuzhishan pig, white Wuzhishan pig, Lingao pig, Bama fragrant pig, Diannan small-eared pig, Anqing pig, Erhualian pig, Meishan pig, Jinhua pig, Min pig, Landrace pig, Large White pig and Duroc pig, etc.). It has a wide coverage, high identification accuracy and a wider range of applications and scenarios.

[0074] (4) This invention provides a method for rapid identification of purebred pig breeds of Duntou pigs. The method is simple and convenient to operate, and the results are clear and easy to read. It can intuitively, quickly and efficiently reveal the overall relationship structure and relationship between samples / groups.

[0075] (5) This invention utilizes genomics and bioinformatics technologies to achieve accurate identification of Duntou pigs, providing an effective scientific basis for the protection of the Duntou pig breed from the genetic level. Attached Figure Description

[0076] Figure 1 This is the principal component analysis diagram of the genotype data in Embodiment 1 of the present invention.

[0077] Figure 2 This is an phylogenetic tree analysis diagram of the genotype data in Embodiment 1 of the present invention.

[0078] Figure 3 This is a Manhattan plot of the GWAS analysis results of the Duntou pig in Embodiment 1 of the present invention.

[0079] Figure 4 This is a Manhattan plot of the selection signal analysis results of the pig in Embodiment 1 of the present invention.

[0080] Figure 5 This is a genotype distribution map of the specific SNP locus combination (C2 group) provided by the present invention in different populations.

[0081] Figure 6 This is a graph showing the validation results of principal component analysis of the specific SNP loci combination of Duntou pig (C1 group, threshold 1E-20) and other pig breeds.

[0082] Figure 7 This is a graph showing the validation results of principal component analysis of the specific SNP loci combination of Duntou pig (C2 group, threshold 1E-12) and other pig breeds. Detailed Implementation

[0083] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0084] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise stated, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field.

[0085] Example 1: Screening of SNP loci combinations specific to Duntou pigs

[0086] 1. Genotype Data Acquisition

[0087] (1) The experimental group consisted of 725 pigs from 14 different breeds (strains). Among them, 11 were local Chinese pig breeds, including: Hainan local breeds: Duntou pig (n=20), Wuzhishan pig (a nationally recognized breed with a coat color of black and white, https: / / zypc.nahs.org.cn / pzml / classify.html) (n=73), white Wuzhishan pig (n=20), and Lingao pig (n=24); South China type pig breeds: Bama fragrant pig (n=6) and Yunnan small-eared pig (n=33); Jianghai type pig breeds: Anqing pig (n=20), Erhualian pig (n=24), and Meishan pig (n=44); Central China type pig breeds: Jinhua pig (n=12); and North China type pig breeds: Min pig (n=18). The introduced breeds were: Landrace pig (n=58), Large White pig (n=198), and Duroc pig (n=175).

[0088] Table 1. Statistical table of experimental population varieties, types and quantities

[0089]

[0090] (2) The genotype data of the Duntou pig, Lingao pig and white Wuzhishan pig populations were obtained directly through resequencing, and the specific method is as follows:

[0091] Ear tissue samples were collected from the Duntou pig herd, and total DNA was extracted from the samples using a tissue DNA extraction kit. The integrity and purity of the DNA in each sample were checked by agarose gel electrophoresis and ultraviolet spectrophotometry. The absorbance ratio (A260 / 280) of the DNA samples was required to be between 1.8 and 2.0, and the concentration was ≥50 ng / μL. The DNA samples that passed the quality check were then subjected to genome resequencing using the DNBSEQ sequencing platform (Shenzhen BGI Genomics Co., Ltd.).

[0092] (3) Genotypic data for other pig breeds were obtained from the PGIDB database.

[0093] 2. Genotype Data Processing

[0094] (1) Genotype data merging and quality control

[0095] Genotype data from each population were merged using Plink software according to standard methods. The merged dataset was then subjected to quality control, and SNPs with a detection rate of less than 95% and individuals with a genotyping rate of less than 90% were removed. Loci with a minimum allele frequency of less than 1% and sex chromosome loci were also removed. A total of 725 individuals and 6,733,908 high-quality SNP loci were retained for subsequent analysis.

[0096] (2) Principal Component Analysis (PCA)

[0097] PCA analysis was performed on the above SNP dataset using Plink software. The output results were prefixed with "pca" and generated files ending in ".eigenval" and ".eigenvec". ".eigenval" represents the weight of each PCA, and the other file records the feature vectors used for plotting the PCA graph. The relevant code is shown below:

[0098] plink --bfile data --pca 3 --out pca

[0099] plink: Used to invoke Plink;

[0100] --bfile data: Used to read binary format genotype files;

[0101] --pca 3: Used to perform principal component analysis and obtain the eigenvector values ​​of the first 3 columns;

[0102] --out pca: Outputs the principal component analysis results, with the file prefixed with pca.

[0103] (3) Evolutionary tree analysis

[0104] Phylogenetic analysis was performed on the above SNP dataset using VCF2Dis and FastMe software. Specifically, VCF2Dis was used to calculate genetic distances, and FastMe was used to construct the phylogenetic tree based on the distance matrix. The output results were prefixed with "data" and generated files ending in ".mat" and ".nwk". The ".mat" file represents the genetic distance between each individual, and the ".nwk" file records the feature vectors used for drawing the phylogenetic tree. The relevant code is shown below:

[0105] VCF2Dis -InPut data.vcf -OutPut data_dis.mat

[0106] fastme -i data_dis.mat -m N -o data.nwk

[0107] VCF2Dis\fastme: Calls the corresponding software;

[0108] -InPut data.vcf: Reads the input file data (vcf format);

[0109] -OutPut data_dis.mat: Outputs genetic distance results, with the file prefixed with "data";

[0110] -i data_dis.mat: Reads the output file from the previous step;

[0111] -m N: The adjacency-based method is selected to construct the tree;

[0112] -o data.nwk: Outputs a phylogenetic tree file.

[0113] (4) Results visualization

[0114] ①PCA Analysis Visualization: The output results of step (2) are formatted using Excel. Specifically, the first column is set as breed, the second column as eigenvector value 1 (PC1), and the third column as eigenvector value 2 (PC2). The formatted data (the data is named pca) is read into R language, and the ggplot2 package is used to plot the PCA analysis chart based on the eigenvector values ​​of the first two columns (PC1 and PC2). The X-axis is set as PC1, the Y-axis as PC2, and different breeds are represented by different colors. The relevant code is shown below:

[0115] ggplot(pca,aes(x=PC1,y=PC2,color=Breed))+

[0116] geom_point(size=3)+

[0117] theme_bw() +

[0118] theme(panel.border=element_blank(), panel.grid.major=element_blank(), panel.grid.minor=element_blank(), axis.line=element_line(colour="black"))

[0119] ggplot(): Creates a base layer;

[0120] geom_point(): Draws a scatter plot layer;

[0121] theme_bw(): Draws the theme.

[0122] The PCA analysis results are shown below. Figure 1 As can be seen from the figure, there is some overlap between Duntou pigs and local Hainan pig breeds (such as white Wuzhishan pigs, Wuzhishan pigs, and Lingao pigs), showing a relatively close genetic distance. This indicates that Duntou pigs and local Hainan pigs have similar genetic characteristics, making identification more difficult.

[0123] ② Visualization of phylogenetic tree analysis: The output of step (3) is visualized using iTOL. Specifically, the result file with the suffix .nwk is uploaded, and different colors are used to annotate the individual species information.

[0124] See the results of the phylogenetic tree analysis. Figure 2 As can be seen from the figure, the branching distance between Duntou pig and Hainan local pig breed is relatively close, indicating a high degree of similarity in genetic characteristics between the groups.

[0125] 3. Genome-wide association analysis to screen for SNP loci specific to Duntou pigs.

[0126] Based on step 2 (1), the GEMMA software was used to conduct a genome-wide association analysis based on a mixed linear model. The specific method was as follows: the Duntou pig was used as the experimental group, and other breeds were uniformly set as the control group. The genotype data in step 2 was converted into a GEMMA-compatible Plink binary format file. First, the genetic relationship matrix between samples was calculated using the GEMMA software. Then, the phenotype file was specified (the experimental group was assigned a value of 1, and the control group was assigned a value of 0) for association analysis. The threshold for significant sites was set to 1E-20 or 1E-12.

[0127] like Figure 3 As shown, based on the significant site threshold setting, a total of two SNP site combinations were obtained. Among them, the significant SNP site combination above the threshold line 1E-20 was named the Duntou pig breed-specific SNP site group A1, and the significant SNP site combination above the threshold line 1E-12 was named the Duntou pig breed-specific SNP site group A2. Figure 3 ).

[0128] 4. Select signal analysis to screen for SNP sites specific to Duntou pigs.

[0129] Based on step 2, the quality-controlled genotype data were converted to VCF format using Plink software, and then the fixed index (F) was calculated using VCFtools software. ST The specific method is as follows: The Duntou pig was used as the experimental group, and other breeds were used as the control group. The sliding window size was set to 50,000 bp, and the sliding step size was 20,000 bp. The F of the window was set... ST Sort the values ​​from largest to smallest.

[0130] like Figure 4 As shown, filter F ST The SNP sites included in the top 1% window are designated as Group B of pig-specific SNP sites.

[0131] 5. Genome-wide association analysis and selection signal analysis: Screening for common-specific SNP sites.

[0132] The shared SNP loci (20 SNP loci) between groups A1 and B were retained as group C1, and the shared SNP loci (40 SNP loci) between groups A2 and B were retained as group C2. The combination of SNP loci in groups C1 and C2 represents the specific SNP loci of the Duntou pig.

[0133] The specific SNP loci screened in group C2 are shown in Table 2. The distribution of each specific SNP locus in the genome and the genotype distribution of each specific SNP locus in different populations are shown in Table 2. Figure 5 The rows in the figure represent different SNP loci, and the columns represent the experimental individuals. SNP loci are annotated in the vcf genotype file: homozygous wild type 0 / 0, gray; heterozygous 1 / 0, yellow; homozygous mutant 1 / 1, red. (Example) Figure 5 The results showed that the genotypes of the specific SNP loci in Duntou pigs differed significantly from those in other populations.

[0134] Table 2. Information on 40 specific SNP molecular marker combinations from Duntou pigs.

[0135] Serial Number chromosome Physical location (bp) Mutation type (ref / alt) 1 1 71291093 C / G 2 1 219372828 T / C 3 1 221005301 G / A 4 1 221005824 A / G 5 1 221008294 A / G 6 1 221008321 G / A 7 1 221008537 T / C 8 1 221008568 C / T 9 1 221008656 A / T 10 1 221011244 T / C 11 1 221013180 T / G 12 1 221039447 C / T 13 1 221048804 A / C 14 4 31714266 G / A 15 7 52659120 - 16 7 52663754 - 17 7 58983433 T / C 18 7 59543790 G / A 19 7 59601220 G / A 20 7 59610710 C / T 21 7 59612202 C / T 22 7 59612309 G / A 23 7 59612325 C / T 24 7 59612409 C / T 25 7 59612520 C / T 26 7 59613024 T / G 27 9 65815519 C / T 28 9 77504853 C / T 29 9 97702199 A / G 30 9 97718522 G / A 31 9 97719064 G / A 32 10 60464546 T / G 33 11 49874673 T / G 34 13 118786842 T / A 35 13 118804557 G / T 36 13 118882958 - 37 16 52911684 C / G 38 16 52912528 G / A 39 16 52912973 A / G 40 16 52913937 G / A

[0136] Example 2: Visual analysis of PCA in the identification of Duntou pigs using specific SNP loci combinations.

[0137] 1. Processing of genotype data

[0138] Referring to Example 1, using Plink software, the SNP locus genotype data of the 14 pig breeds / strains in Example 1 (data of 725 individuals merged in step 2) were extracted to the corresponding C1 and C2 groups (Table 2) (named D1 and D2 respectively).

[0139] 2. Application in variety identification

[0140] (1) PCA analysis

[0141] PCA analysis was performed on the genotype data D1 and D2 using Plink software. The output results were prefixed with "pca", resulting in two files ending with ".eigenval" and ".eigenvec". ".eigenval" represents the weight of each PCA, while the other file (".eigenvec") records the eigenvectors used for plotting the PCA graph. The relevant code is shown below:

[0142] plink --bfile D --pca 3 --out pca

[0143] plink: Used to launch Plink;

[0144] --bfile D: Used to read the binary format genotype file D;

[0145] --pca 3: Used to perform principal component analysis and obtain the eigenvector values ​​of the first 3 columns;

[0146] --out pca: Outputs the principal component analysis results, with the file prefixed with pca.

[0147] (2) Visualization

[0148] The output of step (1) is formatted using Excel. Specifically, the first column is set to breed, the second column to eigenvector value 1 (PC1), and the third column to eigenvector value 2 (PC2). The formatted data (named pca) is then read into R. The ggplot2 package is used to plot the PCA analysis based on the eigenvector values ​​of the first two columns (PC1 and PC2). The X-axis is set to PC1, the Y-axis to PC2, and different breeds are represented by different colors. The relevant code is shown below:

[0149] ggplot(pca,aes(x=PC1,y=PC2,color=Breed))+

[0150] geom_point(size=3)+

[0151] theme_bw() +

[0152] theme(panel.border=element_blank(), panel.grid.major=element_blank(), panel.grid.minor=element_blank(), axis.line=element_line(colour="black"))

[0153] ggplot(): Creates a base layer;

[0154] geom_point(): Draws a scatter plot layer;

[0155] theme_bw(): Draws the theme.

[0156] The PCA analysis results of the genotype data D1 corresponding to the SNP locus combinations in group C1 are shown below. Figure 6 As can be seen from the figure, the clustering effect is poor, and it is impossible to clearly separate Duntou pigs from other pig breeds, indicating that the SNP locus combination of group C1 is not suitable for identifying Duntou pig breeds.

[0157] The PCA analysis results of the genotype data corresponding to the SNP loci combinations in group C2 for D2 are shown below. Figure 7 As can be seen from the figure, the SNP locus combination of group C2 has better application potential for identifying Duntou pigs. The Duntou pigs in Table 1 are clustered together, showing a relatively close genetic distance; while they are clearly separated from other pig breeds, indicating that they have a relatively far genetic distance from other pig breeds.

[0158] The above results demonstrate that PCA analysis using 40 specific SNP loci in group C2 can accurately identify chubby pigs. Therefore, the combination of 40 specific SNP loci provided in this invention can be used to identify chubby pigs.

[0159] Example 3: Application of specific SNP locus combinations of Duntou pigs in the identification of Duntou pigs

[0160] 1. Genotype Data Acquisition

[0161] Tissue samples were collected from the pigs to be tested, and DNA was extracted from the tissue samples to obtain DNA samples from the pigs to be tested.

[0162] 2. Obtaining SNP data from the pigs to be tested

[0163] The DNA samples of the pigs to be tested undergo quality inspection. Once the quality inspection is passed, the samples are sent to a sequencing company for genotyping to obtain the genotype data of the pigs to be tested.

[0164] 3. Processing of genotype data

[0165] Referring to Example 1, the genotype data of the pigs to be tested obtained in step 2 were merged with the genotype data of the known stunted pig population (Table 1) and the control population (non-stunted pig population in Table 1) using Plink software, and the genotype data of the corresponding 40 specific SNP loci were extracted in conjunction with Table 2.

[0166] 4. Application in variety identification

[0167] (1) PCA analysis

[0168] The specific method is the same as in Example 2.

[0169] (2) Visualization

[0170] The specific method is the same as in Example 2.

[0171] (3) Result determination

[0172] When the principal component analysis results show that the genetic distance between the pig to be tested and the known Duntou pig population (Duntou pig population in Table 1) is close, i.e. they cluster together, the pig to be tested is a Duntou pig; otherwise, it is another breed of pig.

[0173] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of a product containing a specific SNP locus combination for identifying the Duntou pig breed in the identification of Duntou pigs, characterized in that... The specific SNP locus combination consists of 40 SNP loci, the physical locations of which are based on a pig reference genome. Sscrofa11.1 The locations and variation information of the 40 SNP sites, determined through sequence comparison, are shown below: SNP site 1 is located at 71291093 bp on chromosome 1, and its mutation type is C / G. SNP site 2 is located at 219372828bp on chromosome 1, and its mutation type is T / C. SNP site 3 is located at 221005301 bp on chromosome 1, and its mutation type is G / A. SNP site 4 is located at 221005824bp on chromosome 1, and its mutation type is A / G. SNP site 5 is located at 221008294bp on chromosome 1, and its mutation type is A / G. SNP locus 6 is located at 221008321 bp on chromosome 1, and its mutation type is G / A. SNP site 7 is located at 221008537bp on chromosome 1, and its mutation type is T / C. SNP site 8 is located at 221008568 bp on chromosome 1, and its mutation type is C / T. SNP locus 9 is located at 221008656 bp on chromosome 1, and its mutation type is A / T. SNP locus 10 is located at 221011244bp on chromosome 1, and its mutation type is T / C. SNP locus 11 is located at 221013180 bp on chromosome 1, and its mutation type is T / G. SNP locus 12 is located at 221039447bp on chromosome 1, and its mutation type is C / T. SNP locus 13 is located at 221048804 bp on chromosome 1, and its mutation type is A / C. SNP locus 14 is located at 31714266 bp on chromosome 4, and its mutation type is G / A. SNP locus 15 is located at 52659120 bp on chromosome 7, and its mutation type is G / A. SNP locus 16 is located at 52663754 bp on chromosome 7, and its mutation type is C / G. SNP locus 17 is located at 58983433 bp on chromosome 7, and its mutation type is T / C. SNP locus 18 is located at 59543790 bp on chromosome 7, and its mutation type is G / A. SNP locus 19 is located at 59601220 bp on chromosome 7, and its mutation type is G / A. SNP site 20 is located at 59610710 bp on chromosome 7, and its mutation type is C / T. SNP site 21 is located at 59612202 bp on chromosome 7, and its mutation type is C / T. SNP locus 22 is located at 59612309 bp on chromosome 7, and its mutation type is G / A. SNP site 23 is located at 59612325 bp on chromosome 7, and its mutation type is C / T. SNP locus 24 is located at 59612409 bp on chromosome 7, and its mutation type is C / T. SNP site 25 is located at 59612520 bp on chromosome 7, and its mutation type is C / T. SNP locus 26 is located at 59613024 bp on chromosome 7, and its mutation type is T / G. SNP site 27 is located at 65815519 bp on chromosome 9, and its mutation type is C / T. SNP site 28 is located at 77504853 bp on chromosome 9, and its mutation type is C / T. SNP 29 is located at 97702199 bp on chromosome 9, and its mutation type is A / G. SNP site 30 is located at 97718522 bp on chromosome 9, and its mutation type is G / A. SNP site 31 is located at 97719064 bp on chromosome 9, and its mutation type is G / A. SNP site 32 is located at 60464546 bp on chromosome 10, and its mutation type is T / G. SNP site 33 is located at 49,874,673 bp on chromosome 11, and its mutation type is T / G. SNP site 34 is located at 118786842 bp on chromosome 13, and its mutation type is T / A. SNP site 35 is located at 118804557bp on chromosome 13, and its mutation type is G / T. SNP site 36 is located at 118882958 bp on chromosome 13, and its mutation type is T / G. SNP site 37 is located at 52911684 bp on chromosome 16, and its mutation type is C / G. SNP site 38 is located at 52912528 bp on chromosome 16, and its mutation type is G / A. SNP site 39 is located at 52912973 bp on chromosome 16, and its mutation type is A / G. SNP locus 40 is located at 52913937 bp on chromosome 16, and its mutation type is G / A.

2. The application according to claim 1, characterized in that: The products mentioned are reagent kits or gene chips.

3. The application according to claim 1, characterized in that: The product described is a reagent.

4. The application according to claim 3, characterized in that: The reagents include at least one of primers and probes.

5. A method for rapid identification of Duntou pigs, characterized in that... It includes the following steps: (1) Collect tissue samples from the pigs to be tested, extract DNA from the tissue samples, and obtain DNA samples from the pigs to be tested; (2) Perform gene sequencing on the DNA sample of the pig to be tested obtained in step (1) to obtain the genotype data of the pig to be tested; (3) The genotype data of the pig to be tested obtained in step (2) is merged with the genotype data of the known dwarf pig population and the control population, and the genotype data of the specific SNP site combination described in claim 1 is extracted. The merged and extracted genotype data is subjected to principal component analysis, and it is determined whether the pig to be tested is a dwarf pig. The known group of Duntou pigs mentioned in step (3) is a group composed of multiple purebred Duntou pigs; The control group mentioned in step (3) is other pig groups with multiple non-slender pigs; The principal component analysis described in step (3) is visualized using the ggplot2 package in R language; The method for judgment described in step (3) is as follows: When the principal component analysis results show that the pig to be tested is genetically close to the known Duntou pig population, i.e., they cluster together, then the pig to be tested is a Duntou pig.

6. The method for rapid identification of Duntou pigs according to claim 5, characterized in that: The control group is at least one of the following: Wuzhishan pig, white Wuzhishan pig, Lingao pig, Bama fragrant pig, Yunnan small-eared pig, Anqing pig, Erhualian pig, Meishan pig, Jinhua pig, Min pig, Landrace pig, Large White pig, and Duroc pig.

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