A specific SNP locus combination and its application for identifying Tunchang pig breeds

Abstract

The present invention belongs to the field of molecular biological identification, and specifically relates to a specific SNP site combination and application for identifying Tunchang pig breeds. The specific SNP site combination for identifying Tunchang pig breeds includes 62 SNP sites, and the physical location is based on the reference genome of the pig. Sscrofa11 .1 The specific SNP locus combination was screened based on the whole genome association analysis combined with the selection signal analysis method to ensure the accuracy of the locus selection. The selected specific SNP locus combination can quickly realize the accurate identification of the Tunchang pig breed at the gene level, and has significant application value in the accurate protection of the genetic resources and breed utilization of the Tunchang pig in Hainan.

Description

Technical Field

[0001] The invention belongs to the field of molecular biological identification, and specifically relates to a specific SNP site combination and application for identifying Tunchang pig breeds. Background Art

[0002] Tunchang pig is a major group of Hainan local pigs, originating from Tunchang County, Hainan Province. It has typical advantages of Chinese local pig breeds, such as early maturity, strong fertility, tolerance to roughage, strong disease resistance, and good meat quality. It was included in the National List of Livestock and Poultry Genetic Resources Protection in 2014. Tunchang pig is one of the characteristic local pig breeds in Hainan Province. The Tunchang black pig, which is bred by hybridization based on the Tunchang pig mother, has formed an important characteristic agricultural product industry chain and was selected into the second batch of national famous, special and new agricultural products in 2024.

[0003] As one of Hainan's excellent local pig breeds, it has received more extensive attention in recent years. The new Tunchang black pig breed bred with Tunchang pigs as the maternal group has also been widely recognized in the market. Further research on the characteristic genetic resources of Tunchang pigs based on modern genomic technology and the promotion of the group protection of purebred Tunchang pigs are of great significance to the characteristic development of my country's pig breeding industry and the maintenance of the diversity of germplasm resource banks. Among them, the use of bioinformatics technology to carry out the mining of Tunchang pig characteristic variety sites and realize the precise identification of the unique genetic characteristics of Tunchang pigs at the gene level is an important part of the precise protection of Tunchang pig genetic resources.

[0004] Single nucleotide polymorphism (SNP) is one of the most common forms of genetic variation. As a type of genetic marker, it is widely used in genome research. It has important application value in many aspects such as genetic relationship analysis of biological populations, genetic structure analysis, and pathogenic gene mining.

[0005] In the long-term breeding of Chinese local pig breeds, a large number of selection marks are usually left in the genome. These selection marks can be used as unique gene markers of the breed in the genome and applied to the purity identification of the breed. Genome wide association study (GWAS) and selection signal analysis are effective methods for screening differential loci. The combination of the two can further ensure the reliability of screening differential loci between populations. Summary of the invention

[0006] In order to overcome the deficiencies and shortcomings of the prior art, the primary purpose of the present invention is to provide a specific SNP site combination for identifying the Tunchang pig breed, which can achieve rapid and accurate identification of purebred Tunchang pigs at the genetic level.

[0007] Another object of the present invention is to provide an application of the above-mentioned specific SNP site combination for identifying the Tunchang pig breed.

[0008] Another object of the present invention is to provide a method for quickly identifying the Tunchang pig breed.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] A specific SNP site combination for identifying the Tunchang pig breed includes 62 SNP sites, named SNP sites 1-62, the physical positions of the 62 SNP sites are determined based on sequence comparison of the reference genome Sscrofa11.1 of the pig, and the positions and variation information of the 62 SNP sites are as follows:

[0011] SNP1 is located at 76530269bp on chromosome 1, and its mutation type is G / A;

[0012] SNP2 is located at 76530641bp on chromosome 1, and its mutation type is A / G;

[0013] SNP3 is located at 103689663bp on chromosome 1, and its mutation type is A / G;

[0014] SNP4 is located at 103695747bp on chromosome 1, and its mutation type is T / C;

[0015] SNP5 is located at 132219294bp on chromosome 1, and its mutation type is T / C;

[0016] SNP6 is located at 132221789bp on chromosome 1, and its mutation type is C / T;

[0017] SNP7 is located at 152270968bp on chromosome 1, and its mutation type is T / C;

[0018] SNP8 is located at 152294579bp on chromosome 1, and its mutation type is G / T;

[0019] SNP9 is located at 172260410bp on chromosome 1, and its mutation type is G / A;

[0020] SNP10 is located at 172260954bp on chromosome 1, and its mutation type is G / A;

[0021] SNP11 is located at 184586884bp on chromosome 1, and its mutation type is G / A;

[0022] SNP12 is located at 184671184bp on chromosome 1, and its mutation type is A / C;

[0023] SNP13 is located at 230688597bp on chromosome 1, and its mutation type is T / C;

[0024] SNP14 is located at 72175633bp on chromosome 2, and its mutation type is A / G;

[0025] SNP15 is located at 109815414bp on chromosome 2, and its mutation type is A / G;

[0026] SNP16 is located at 143211293bp on chromosome 2, and its mutation type is A / G;

[0027] SNP17 is located at 143220436bp on chromosome 2, and its mutation type is T / C;

[0028] SNP18 is located at 1880826bp on chromosome 4, and its mutation type is G / T;

[0029] SNP19 is located at 42245420bp on chromosome 4, and its mutation type is T / C;

[0030] SNP20 is located at 53354488bp on chromosome 4, and its mutation type is A / T;

[0031] SNP21 is located at 53390725bp on chromosome 4, and its mutation type is T / G;

[0032] SNP22 is located at 73746508bp on chromosome 4, and its mutation type is T / A;

[0033] SNP23 is located at 77277851bp on chromosome 4, and its mutation type is A / T;

[0034] SNP24 is located at 284024bp on chromosome 6, and its mutation type is C / G;

[0035] SNP25 is located at 331712bp on chromosome 6, and its mutation type is A / T;

[0036] SNP26 is located at 336611bp on chromosome 6, and its mutation type is T / C;

[0037] SNP27 is located at 342900bp on chromosome 6, and its mutation type is C / G;

[0038] SNP28 is located at 348013bp on chromosome 6, and its mutation type is T / A;

[0039] SNP29 is located at 354872bp on chromosome 6, and its mutation type is A / T;

[0040] SNP30 is located at 366515bp on chromosome 6, and its mutation type is C / T;

[0041] SNP31 is located at 367880bp on chromosome 6, and its mutation type is A / G;

[0042] SNP32 is located at 382542bp on chromosome 6, and its mutation type is T / C;

[0043] SNP33 is located at 386275bp on chromosome 6, and its mutation type is T / C;

[0044] SNP34 is located at 389423bp on chromosome 6, and its mutation type is A / G;

[0045] SNP35 is located at 401555bp on chromosome 6, and its mutation type is T / C;

[0046] SNP36 is located at 402714bp on chromosome 6, and its mutation type is A / G;

[0047] SNP37 is located at 34514268bp on chromosome 6, and its mutation type is A / G;

[0048] SNP38 is located at 37934666bp on chromosome 6, and its mutation type is G / A;

[0049] SNP39 is located at 38004132bp on chromosome 6, and its mutation type is T / C;

[0050] SNP40 is located at 89493269bp on chromosome 6, and its mutation type is T / A;

[0051] SNP41 is located at 118923976bp on chromosome 7, and its mutation type is T / G;

[0052] SNP42 is located at 133435bp on chromosome 8, and its mutation type is G / A;

[0053] SNP43 is located at 47612827bp on chromosome 8, and its mutation type is T / A;

[0054] SNP44 is located at 115190887bp on chromosome 8, and its mutation type is G / A;

[0055] SNP45 is located at 56049266bp on chromosome 9, and its mutation type is G / A;

[0056] SNP46 is located at 78396948bp on chromosome 11, and its mutation type is C / A;

[0057] SNP47 is located at 78396993bp on chromosome 11, and its mutation type is A / G;

[0058] SNP48 is located at 30660582bp on chromosome 13, and its mutation type is G / C;

[0059] SNP49 is located at 43982773bp on chromosome 13, and its mutation type is C / T;

[0060] SNP50 is located at 47468808bp on chromosome 13, and its mutation type is T / C;

[0061] SNP51 is located at 51220907bp on chromosome 13, and its mutation type is T / C;

[0062] SNP52 is located at 51222109bp on chromosome 13, and its mutation type is C / T;

[0063] SNP53 is located at 57409689bp on chromosome 15, and its mutation type is C / T;

[0064] SNP54 is located at 57431817bp on chromosome 15, and its mutation type is G / T;

[0065] SNP55 is located at 63107876bp on chromosome 15, and its mutation type is A / G;

[0066] SNP56 is located at 72985805bp on chromosome 15, and its mutation type is A / T;

[0067] SNP57 is located at 72991321bp on chromosome 15, and its mutation type is T / G;

[0068] SNP58 is located at 42990525bp on chromosome 17, and its mutation type is A / G;

[0069] SNP59 is located at 43005390bp on chromosome 17, and its mutation type is A / C;

[0070] SNP60 is located at 43008076bp on chromosome 17, and its mutation type is T / C;

[0071] SNP61 is located at 32181137bp on chromosome 18, and its mutation type is G / A;

[0072] SNP62 is located at 32193213bp on chromosome 18, and its mutation type is T / C;

[0073] Application of the specific SNP locus combination in identifying Tunchang pig breeds;

[0074] Application of a product for detecting the above-mentioned specific SNP locus combination in identifying Tunchang pig breeds;

[0075] The product is a reagent, a kit or a gene chip, etc.;

[0076] The reagents include at least one of primers, probes, etc.;

[0077] A molecular probe combination for identifying Tunchang pig breeds, the molecular probe combination targets the above-mentioned specific SNP site combination and is used to detect the above-mentioned specific SNP site combination;

[0078] A gene chip for identifying Tunchang pig breeds, the chip being loaded with the above molecular probe combination;

[0079] A method for quickly identifying Tunchang pig breeds preferably comprises the following steps:

[0080] (1) Collecting tissue samples from the pigs to be tested, extracting DNA from the tissue samples, and obtaining DNA samples from the pigs to be tested;

[0081] (2) performing gene sequencing on the DNA sample of the pig to be tested obtained in step (1) to obtain genotype data of the pig to be tested;

[0082] (3) The genotype data of the pig to be tested obtained in step (2) are combined with the genotype data of the known Tunchang pig population and the control population, and the genotype data of the above-mentioned specific SNP site combination (62 SNP sites) are extracted. The combined and extracted genotype data are subjected to principal component analysis, and the principal component analysis graph is visualized by using the R language ggplot2 package to determine whether the pig to be tested is a Tunchang pig;

[0083] The DNA sample described in step (2) must first undergo quality inspection, and then undergo gene sequencing after passing the quality inspection;

[0084] The DNA sample nucleic acid purity test in step (2) has an A260 / 280 light absorption ratio between 1.8 and 2.0, and a concentration of ≥50 ng / μl;

[0085] The known Tunchang pig group described in step (3) is a group consisting of multiple (≥15) purebred Tunchang pigs, which may be the group consisting of the Tunchang pigs in the embodiment, or a group consisting of Tunchang pigs selected by oneself, or a group of Tunchang pigs in a public database;

[0086] The control group described in step (3) is a group of multiple non-Tunchang pigs (≥5 pigs for a single breed group and ≥30 pigs for the entire group), which can be the control group group in the embodiment, or other non-Tunchang pig groups selected by the user, or non-Tunchang pig groups in a public database.

[0087] The control group is preferably at least one of Lingao pig, Ding'an pig, Wuzhishan pig, Bama Xiang pig, Diannan small-eared pig, Erhualian pig, Meishan pig, Anqing pig, Min pig, Tibetan Xiang pig, Landrace pig, Large White pig, Duroc pig and Asian wild boar;

[0088] The extraction and merging described in step (3) are preferably performed using Plink software;

[0089] The judgment method described in step (3) is:

[0090] When the principal component analysis results show that the genetic distance between the pig to be tested and the known Tunchang pig population is close, that is, they are clustered in one cluster, the pig to be tested is a Tunchang pig; otherwise, it is a pig of other breeds.

[0091] Compared with the prior art, the present invention has the following advantages and effects:

[0092] (1) Based on resequencing data, the present invention obtains a specific SNP locus combination for identifying the Tunchang pig breed by combining whole genome association analysis with selection signal analysis. This method further improves the accuracy of identifying the specific SNP locus combination. The selected specific SNP locus combination is more reliable in the identification of the Tunchang pig breed.

[0093] (2) The SNP locus combination provided by the present invention has strong specificity and can be used for the scientific and accurate identification and genetic relationship analysis of Tunchang purebred pigs, which is conducive to promoting the protection and development and utilization of Hainan's characteristic local pig varieties.

[0094] (3) The present invention provides a method for rapidly identifying the purebred Tunchang pig breed. The method uses genomics and bioinformatics technology to achieve accurate identification of purebred Tunchang pigs, providing an effective scientific basis for the protection of the Tunchang pig breed at the genetic level. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] Figure 1 It is a Manhattan plot of the GWAS analysis results of the Tunchang pig of the present invention.

[0096] Figure 2 It is a Manhattan plot of the selection signal analysis results of the Tunchang pig of the present invention.

[0097] Figure 3 It is a distribution diagram of each specific SNP site disclosed in the present invention in each chromosome.

[0098] Figure 4 It is a genotype comparison diagram of different groups at each specific SNP site disclosed in the present invention.

[0099] Figure 5 This is a diagram showing the verification results of the principal component analysis of the Tunchang pig-specific SNP site combination and other breeds disclosed in the present invention. DETAILED DESCRIPTION

[0100] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0101] Example 1 Screening of Tunchang pig-specific molecular marker SNP sites

[0102] 1. Genotype data acquisition

[0103] (1) The experimental population included 809 pigs of 15 breeds, including 11 local Chinese breeds, including 4 Hainan local breeds, namely Tunchang pig (n=29, TC), Lingao pig (n=27, LG), Dingan pig (n=24, DA), and Wuzhishan pig (n=26, WZS); 2 South China breeds, namely Bama Xiang pig (n=6, BMX) and Diannan small-eared pig (n=33, DNXE); 2 Jianghai type breeds, namely Erhualian pig (n=24, EHL) and Meishan pig (n=43, MS); 1 East China breed, namely Anqing pig (n=18, AQ); 1 North China breed, namely Min pig (n=18, MIN); and 1 plateau breed, namely Tibetan Xiang pig (n=118, ZX). There are three Western pig breeds, including Landrace (n=52, LR), Large White (n=177, LW), and Duroc (n=172, DU), and one wild boar breed, the Asian wild boar (n=42, AWB).

[0104] Table 1 Statistical table of species, types and numbers of experimental populations

[0105] Serial number variety type Quantity (head) 1 Tunchang Pig Hainan local pig 29 2 Lingao Pig Hainan local pig 27 3 Dingan Pig Hainan local pig 24 4 Wuzhishan Pig Hainan local pig 26 5 Bama Xiang pig South China Pig 6 6 Yunnan Small-eared Pig South China Pig 33 7 Two-faced pig Jianghai Pig 24 8 Meishan Pig Jianghai Pig 43 9 Anqing Pig East China Pig 18 10 Minzhu North China Pig 18 11 Tibetan Pig Plateau Pig 118 12 Landrace Pig Western breed pigs 52 13 Large White Pig Western breed pigs 177 14 Duroc pig Western breed pigs 172 15 Asian wild boar Wild Boar 42

[0106] (2) The genotype data of Tunchang pig, Lingao pig, Ding'an pig and Wuzhishan pig populations were directly obtained by resequencing. The specific method is as follows:

[0107] Ear tissue samples were collected from Tunchang pigs, Lingao pigs, Ding'an pigs, and Wuzhishan pigs, and the total DNA of the samples was extracted using the Omega tissue DNA extraction kit. The integrity and purity of the DNA of each sample were inspected by agarose gel electrophoresis and UV spectrophotometer. The DNA sample light absorption ratio (A260 / 280) was required to be between 1.8 and 2.0, and the concentration was ≥50 ng / μL. The DNA samples that passed the quality inspection were genotyped using the DNBSEQ sequencing platform (Shenzhen BGI Genomics Service Co., Ltd.) to obtain whole-genome genotype (SNP) data. The sequencing depth was 10×, and a total of 40,201,194 SNP sites were obtained for 106 samples.

[0108] (3) The genotype data of other pigs were obtained from the PGIDB database.

[0109] 2. Genotype data processing

[0110] (1) The genotype data of each group were merged according to the conventional method using Plink software (command: --bmerge), and the merged genetic data were quality controlled. SNPs with a site detection rate lower than 95% (command: --geno0.05) and individuals with an individual detection rate lower than 90% (command: --mind 0.1) were removed. A total of 12,633,058 SNP sites shared by 809 individuals were obtained.

[0111] (2) A secondary quality control was then performed. Based on the original quality control conditions, loci with a minimum allele frequency of less than 1% and sex chromosome loci were removed (command: --maf 0.1 --chr 1-18). Finally, 12,631,911 high-quality SNP loci from 809 individuals were used for subsequent analysis.

[0112] 3. Screening of Tunchang pig-specific loci by genome-wide association analysis

[0113] On the basis of step 2, GEMMA software was used to conduct genome-wide association analysis based on a mixed linear model. The specific method was as follows: Tunchang pigs were used as the experimental group, and other breeds were uniformly set as the control group. Association analysis was carried out, and the threshold of significant sites was set to 1e-20.

[0114] like Figure 1 As shown, the significant SNP sites above the threshold line were selected as Tunchang pig breed-specific SNP site group A.

[0115] 4. Select signal analysis to screen Tunchang pig specific loci

[0116] Based on step 2, Plink software was used to convert the quality-controlled genotype data into vcf format (command: --recode vcf-iid), and VCFtools software was used to calculate the genetic differentiation index (Fst). The specific method was as follows: Tunchang pigs were used as the experimental group, and other breeds were uniformly set as the control group. The sliding window size was set to 50,000 bp (command: --fst-window-size 50,000), and the sliding step was set to 10,000 bp (command: --fst-window-step 10,000), and the Fst values ​​of the analysis results were sorted from large to small.

[0117] like Figure 2 As shown, the loci in the top 1% window with the largest Fst value are group B of Tunchang pig breed-specific SNP loci.

[0118] 5. Screening of common specific loci for whole genome association analysis and genetic differentiation index

[0119] The common SNP sites of group A and group B were retained as group C. The SNP sites of group C were sorted according to their position in the genome, and the SNP sites with a distance of less than 1Mb were regarded as a significant QTL region. The top 1-8 sites with the most significant P value (obtained from the whole genome association analysis in step 3) in each QTL region were retained as the specific SNP sites of Tunchang pigs.

[0120] The distribution of each specific SNP site screened in the genome is as follows Figure 3 The specific site information is shown in Table 2. The genotypes of different populations at each specific SNP site are shown in Figure 4 As shown in the figure, the rows represent different specific SNP sites, the columns represent all individuals used for analysis, and different breeds are represented by different colors above each column. The higher the frequency of the major allele of each SNP site, the closer it is to red. Figure 4 The results showed that the genotypes of the specific SNP loci of Tunchang pigs were significantly different from those of other populations.

[0121] Table 2 Combination information of 62 specific SNP molecular markers of Tunchang pig

[0122] Serial number chromosome Physical Location Mutation type 1 1 76530269 G / A 2 1 76530641 A / G 3 1 103689663 A / G 4 1 103695747 T / C 5 1 132219294 T / C 6 1 132221789 C / T 7 1 152270968 T / C 8 1 152294579 G / T 9 1 172260410 G / A 10 1 172260954 G / A 11 1 184586884 G / A 12 1 184671184 A / C 13 1 230688597 T / C 14 2 72175633 A / G 15 2 109815414 A / G 16 2 143211293 A / G 17 2 143220436 T / C 18 4 1880826 G / T 19 4 42245420 T / C 20 4 53354488 A / T 21 4 53390725 T / G 22 4 73746508 T / A 23 4 77277851 A / T 24 6 284024 C / G 25 6 331712 A / T 26 6 336611 T / C 27 6 342900 C / G 28 6 348013 T / A 29 6 354872 A / T 30 6 366515 C / T 31 6 367880 A / G 32 6 382542 T / C 33 6 386275 T / C 34 6 389423 A / G 35 6 401555 T / C 36 6 402714 A / G 37 6 34514268 A / G 38 6 37934666 G / A 39 6 38004132 T / C 40 6 89493269 T / A 41 7 118923976 T / G 42 8 133435 G / A 43 8 47612827 T / A 44 8 115190887 G / A 45 9 56049266 G / A 46 11 78396948 C / A 47 11 78396993 A / G 48 13 30660582 G / C 49 13 43982773 C / T 50 13 47468808 T / C 51 13 51220907 T / C 52 13 51222109 C / T 53 15 57409689 C / T 54 15 57431817 G / T 55 15 63107876 A / G 56 15 72985805 A / T 57 15 72991321 T / G 58 17 42990525 A / G 59 17 43005390 A / C 60 17 43008076 T / C 61 18 32181137 G / A 62 18 32193213 T / C

[0123] Example 2 PCA visualization analysis of specific SNP loci combination identification of Tunchang pigs

[0124] 1. Processing of genotype data

[0125] Referring to Example 1, the genotype data of 15 pig breeds (data of 809 individuals merged in step 2) in Example 1 were extracted using Plink software according to Table 2 to obtain genotype data corresponding to 62 specific SNP sites (named: D).

[0126] 2. Variety identification application

[0127] (1) Principal component analysis (PCA)

[0128] Use Plink software to perform PCA analysis on the above genotype data D. The output result prefix is ​​named tcpca. The relevant code is as follows:

[0129] plink --bfile D --pca 3 --out tcpca

[0130] plink: used to start Plink;

[0131] --bfile D: used to read the genotype file D in binary format;

[0132] --pca 3: used to perform principal component analysis and obtain the first three columns of eigenvector values;

[0133] --out tcpca: Output principal component analysis results, the file prefix is ​​named tcpca.

[0134] (2) Visualization

[0135] The output result (tcpca.eigenvec) of step (1) is sorted in the data format through Excel. Specifically, the first column is set as the breed (Breed), the second column is the eigenvector value 1 (PC1), and the third column is the eigenvector value 2 (PC2).

[0136] Among them, in the tcpca.eigenvec file, the second column is the sample id, the third column is the eigenvector value 1, and the fourth column is the eigenvector value 2. When sorting data in Excel, the variety can be determined according to the sample id, and finally sorted into drawing data with the first column as the variety (Breed), the second column as the eigenvector value 1 (PC1), and the third column as the eigenvector value 2 (PC2).

[0137] Read the sorted data (named pca in this example) into R language, and use the ggplot2 package to draw a PCA analysis chart based on the eigenvector values ​​of the principal component analysis of the first two columns (PC1 and PC2). Set the X-axis to PC1 and the Y-axis to PC2, and different varieties (Breed) are represented by different colors. The relevant code is as follows:

[0138] ggplot(pca,aes(x=PC1,y=PC2,color=Breed))+

[0139] geom_point(size=3)+

[0140] theme_bw() +

[0141] theme(panel.border=element_blank(), panel.grid.major=element_blank(), panel.grid.minor=element_blank(), axis.line=element_line(colour="black"))

[0142] ggplot(pca, aes(x=PC1, y=PC2, color=Breed)):

[0143] ggplot(): The function of the ggplot2 library is used to create graphic objects;

[0144] pca: object / data frame defining the data, containing the result data of principal component analysis;

[0145] aes(x=PC1, y=PC2, color=Breed): Aesthetic mapping function aes(), specifies how to map data to graphic attributes; x=PC1: maps the PC1 column in the data to the x-axis; y=PC2: maps the PC2 column in the data to the y-axis; color=Breed: uses the Breed column in the data to determine the color of the scatter points.

[0146] geom_point(size=3):

[0147] geom_point(): This is a function that adds a scatter point layer to the graph; size=3: sets the size of the scatter points to 3.

[0148] theme_bw():

[0149] Apply a theme, theme_bw() represents the "black and white" theme, which is a simple theme with a white background and grid lines;

[0150] theme(panel.border=element_blank(), panel.grid.major=element_blank(), panel.grid.minor=element_blank(), axis.line=element_line(colour="black")):

[0151] theme(): used to set or adjust non-data elements of the graphic; panel.border=element_blank(): removes the border of the graphic panel; panel.grid.major=element_blank(): removes major grid lines; panel.grid.minor=element_blank(): removes minor grid lines; axis.line=element_line(colour="black"): sets the axis line to black.

[0152] The results are as follows Figure 5 As shown in Table 1, the 29 Tunchang pigs in Table 1 clustered together, showing a close genetic distance, and a long genetic distance from the other 14 pig breeds, indicating that the combination of 62 specific SNP loci combined with PCA analysis can accurately identify Tunchang pigs. Therefore, the 62 specific SNP loci combination provided by the present invention can be used to identify the breed of Tunchang pigs.

[0153] Example 3 Application of Tunchang pig-specific SNP locus combination in identification of purebred Tunchang pigs

[0154] 1. Genotype data acquisition

[0155] 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.

[0156] 2. Obtaining SNP data of the pigs to be tested

[0157] The DNA samples of the pigs to be tested are quality inspected, and after passing the quality inspection, they are sent to a gene sequencing company for gene sequencing to obtain the genotype data of the pigs to be tested.

[0158] 3. Processing of genotype data

[0159] 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 Tunchang population (Table 1) and the control population (non-Tunchang population in Table 1) through Plink software, and combined with Table 2, the genotype data corresponding to 62 specific SNP sites were extracted.

[0160] 4. Variety identification application

[0161] (1) Principal component analysis (PCA)

[0162] The specific method is the same as in Example 2;

[0163] (2) Visualization

[0164] The specific method is the same as Example 2.

[0165] (3) Result determination

[0166] When the results of principal component analysis show that the genetic distance between the pig to be tested and the known Tunchang pig population (Tunchang pig population in Table 1) is close, that is, they are clustered in one cluster, the pig to be tested is Tunchang pig; otherwise, it is other breeds of pig.

[0167] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Application of a product for detecting a specific SNP locus combination in identifying Tunchang pig breeds, characterized in that The specific SNP locus combination consists of 62 SNP loci, and the physical positions of the 62 SNP loci are based on the reference genome of the pig Sscrofa11.1 The positions and variation information of the 62 SNP sites were determined by sequence comparison as follows: SNP1 is located at 76530269bp on chromosome 1, and its mutation type is G / A; SNP2 is located at 76530641bp on chromosome 1, and its mutation type is A / G; SNP3 is located at 103689663bp on chromosome 1, and its mutation type is A / G; SNP4 is located at 103695747bp on chromosome 1, and its mutation type is T / C; SNP5 is located at bp 132219294 on chromosome 1, and its mutation type is T / C; SNP6 is located at bp 132221789 on chromosome 1, and its mutation type is C / T; SNP7 is located at bp 152270968 on chromosome 1, and its mutation type is T / C; SNP8 is located at 152294579bp on chromosome 1, and its mutation type is G / T; SNP9 is located at 172260410bp on chromosome 1, and its mutation type is G / A; SNP10 is located at bp 172260954 on chromosome 1, and its mutation type is G / A; SNP11 is located at bp 184586884 on chromosome 1, and its mutation type is G / A; SNP12 is located at bp 184671184 on chromosome 1, and its mutation type is A / C; SNP13 is located at bp 230688597 on chromosome 1, and its mutation type is T / C; SNP14 is located at 72175633bp on chromosome 2, and its mutation type is A / G; SNP15 is located at bp 109815414 on chromosome 2, and its mutation type is A / G; SNP16 is located at 143211293bp on chromosome 2, and its mutation type is A / G; SNP17 is located at bp 143220436 on chromosome 2, and its mutation type is T / C; SNP18 is located at 1880826bp on chromosome 4, and its mutation type is G / T; SNP19 is located at 42245420bp on chromosome 4, and its mutation type is T / C; SNP20 is located at 53354488bp on chromosome 4, and its mutation type is A / T; SNP21 is located at 53390725bp on chromosome 4, and its mutation type is T / G; SNP22 is located at 73746508bp on chromosome 4, and its mutation type is T / A; SNP23 is located at 77277851bp on chromosome 4, and its mutation type is A / T; SNP24 is located at bp 284024 on chromosome 6, and its mutation type is C / G; SNP25 is located at bp 331712 on chromosome 6, and its mutation type is A / T; SNP26 is located at 336611bp on chromosome 6, and its mutation type is T / C; SNP27 is located at 342900bp on chromosome 6, and its mutation type is C / G; SNP28 is located at 348013bp on chromosome 6, and its mutation type is T / A; SNP29 is located at bp 354872 on chromosome 6, and its mutation type is A / T; SNP30 is located at bp 366515 on chromosome 6, and its mutation type is C / T; SNP31 is located at bp 367880 on chromosome 6, and its mutation type is A / G; SNP32 is located at bp 382542 on chromosome 6, and its mutation type is T / C; SNP33 is located at bp 386275 on chromosome 6, and its mutation type is T / C; SNP34 is located at bp 389423 on chromosome 6, and its mutation type is A / G; SNP35 is located at 401555bp on chromosome 6, and its mutation type is T / C; SNP36 is located at 402714bp on chromosome 6, and its mutation type is A / G; SNP37 is located at bp 34514268 on chromosome 6, and its mutation type is A / G; SNP38 is located at bp 37934666 on chromosome 6, and its mutation type is G / A; SNP39 is located at 38004132bp on chromosome 6, and its mutation type is T / C; SNP40 is located at bp 89493269 on chromosome 6, and its mutation type is T / A; SNP41 is located at 118923976bp on chromosome 7, and its mutation type is T / G; SNP42 is located at 133435bp on chromosome 8, and its mutation type is G / A; SNP43 is located at 47612827bp on chromosome 8, and its mutation type is T / A; SNP44 is located at 115190887bp on chromosome 8, and its mutation type is G / A; SNP45 is located at 56049266bp on chromosome 9, and its mutation type is G / A; SNP46 is located at 78396948bp on chromosome 11, and its mutation type is C / A; SNP47 is located at 78396993bp on chromosome 11, and its mutation type is A / G; SNP48 is located at bp 30660582 on chromosome 13, and its mutation type is G / C; SNP49 is located at 43982773bp on chromosome 13, and its mutation type is C / T; SNP50 is located at 47468808bp on chromosome 13, and its mutation type is T / C; SNP51 is located at 51220907bp on chromosome 13, and its mutation type is T / C; SNP52 is located at bp 51222109 on chromosome 13, and its mutation type is C / T; SNP53 is located at bp 57409689 on chromosome 15, and its mutation type is C / T; SNP54 is located at 57431817bp on chromosome 15, and its mutation type is G / T; SNP55 is located at bp 63107876 on chromosome 15, and its mutation type is A / G; SNP56 is located at 72985805bp on chromosome 15, and its mutation type is A / T; SNP57 is located at 72991321bp on chromosome 15, and its mutation type is T / G; SNP58 is located at 42990525bp on chromosome 17, and its mutation type is A / G; SNP59 is located at 43005390bp on chromosome 17, and its mutation type is A / C; SNP60 is located at 43008076bp on chromosome 17, and its mutation type is T / C; SNP61 is located at bp 32181137 on chromosome 18, and its mutation type is G / A; SNP62 is located at 32193213bp on chromosome 18, and its mutation type is T / C.

2. The use according to claim 1, characterized in that: The product is a kit or a gene chip.

3. The use according to claim 1, characterized in that: The product described is a reagent; The reagents include at least one of primers and probes.

4. A method for rapid identification of Tunchang pig breeds, characterized in that The following steps are included: (1) Collecting tissue samples from the pigs to be tested, extracting DNA from the tissue samples, and obtaining DNA samples from the pigs to be tested; (2) performing gene sequencing on the DNA sample of the pig to be tested obtained in step (1) to obtain genotype data of the pig to be tested; (3) merging the genotype data of the pig to be tested obtained in step (2) with the genotype data of the known Tunchang pig population and the control population, and extracting the genotype data of the specific SNP site combination described in claim 1, performing principal component analysis on the combined and extracted genotype data, and visualizing the principal component analysis graph using the R language ggplot2 package to determine whether the pig to be tested is a Tunchang pig; The judgment method described in step (3) is: When the principal component analysis results show that the genetic distance between the test pig and the known Tunchang pig population is close, that is, they are clustered together, the test pig is a Tunchang pig; otherwise, it is a pig of other breeds; the known Tunchang pig population is a group composed of multiple purebred Tunchang pigs; the control group is a group of other pigs consisting of multiple non-Tunchang pigs.

5. The method for rapid identification of Tunchang pig breeds according to claim 4, characterized in that: The control group is at least one of Lingao pig, Ding'an pig, Wuzhishan pig, Bama Xiang pig, Diannan small-eared pig, Erhualian pig, Meishan pig, Anqing pig, Min pig, Tibetan Xiang pig, Landrace pig, Large White pig, Duroc pig and Asian wild boar.

6. The method for rapid identification of Tunchang pig breeds according to claim 4, characterized in that: The extraction and merging described in step (3) were performed using Plink software.

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