Specific molecular ID card for identifying the germplasm resources of Henan black pig and its application
Through specific molecular ID card and gene chip technology, the collection of SNP sites of Henann black pig breeds was screened, solving the problem of difficulty in quickly and efficiently identifying Henann black pigs in the existing technology, and achieving efficient and accurate breed identification and protection.
Patent Information
- Application Number
- CN202210816333.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-07-12
- Publication Date
- 2025-05-23
- Estimated Expiration
- 2042-07-12
AI Technical Summary
The existing technology is difficult to quickly and efficiently identify and identify the Henan agricultural black pig breeds, which affects the protection, development and utilization of local pig breeds.
A specific molecular ID card was used to identify it through the collection of SNP sites, and the principal component analysis was performed by combining gene chips and PLINK software to screen out the specific molecular ID card of the Henann black pig breed.
The rapid and efficient identification of Henan agricultural black pig breeds has been achieved, the identification time and cost have been reduced, the identification efficiency has been improved, and the scientific basis for the protection, development and utilization of varieties has been provided.
Smart Images
Figure CN114959068B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of pig breed identification, relates to the germplasm resources of Yunong black pigs, and particularly refers to a specific molecular identity card for identifying Yunong black pig breeds and its application. Background Art
[0002] The Yunnan Black Pig is distributed in Sanmenxia City, Henan Province. It is bred from generations with excellent local pigs: Nanyang Black Pig, Erhualian Pig, Laiwu Pig and Western commercial pig: Duroc Pig as the basic group. It has excellent characteristics such as strong fertility, good meat quality, high disease resistance and tolerance to roughage. In the process of selecting and breeding superior varieties, it is necessary to explore new technologies and means to provide guarantees for the breeding of excellent varieties. How to quickly and efficiently perform molecular identification and variety identification of livestock and poultry varieties has become the key to the development and utilization of varieties in production and sustainable development. The effective protection and rational development of local varieties will contribute to the sustainable development of the pig industry in Henan Province and the enrichment of livestock resource diversity. In particular, the study of the specific genetic structure and characterization of local pig breeds will help to formulate a protection plan for each breed according to the genetic situation of the breed and promote the specific protection of local pig breeds. Preserving the unique variations, genes and characteristics of each breed is extremely important for maintaining biodiversity and adapting to future environmental changes. Therefore, the use of bioinformatics to identify the unique genetic characteristics of Henan local pig breeds is an important part of accurately protecting the genetic resources of local pig germplasm.
[0003] With the development of sequencing technology, chip sequencing technology has become a powerful tool for high-throughput SNP typing. At the same time, when animals are subjected to long-term natural selection and artificial selection, corresponding genetic imprints will be left on their genomes. These genetic imprints are usually called selection signals. The study of selection signals is a research strategy based on the concept of genome to phenotype. Due to the lack of phenotypic records of local pig breeds in my country and the small population size, the analysis of livestock germplasm characteristics has become an increasingly important method. In order to achieve batch testing of Yunong Black Pigs, our research group has conducted long-term exploration. Summary of the invention
[0004] To achieve the above objectives, the present invention proposes a specific molecular ID card for identifying the germplasm resources of Henan Agricultural Black Pig and its application.
[0005] The technical solution of the present invention is achieved in this way:
[0006] A specific molecular ID card used to identify the germplasm resources of Yunong black pigs, wherein the SNP loci are a set of SNP loci with higher allele frequencies among Yunong black pig varieties.
[0007] Preferably, the SNP site is located in the pig reference genome EnsemblSscrofa 11.1 version.
[0008] Preferably, the set of SNP sites includes CNC10010909 site, CNC10011130 site, CNC10011533 site, CNC10012476 site, CNC10012481 site, CNC10012482 site, CNC10012483 site, CNC10013609 site, CNC10013901 site, CNC10013912 site, CNC10014023 site, CNC10014056 site, CNC10014645 site, CNC10020600 site, CNC10021264 site, CNC10021414 site, and CNC100215 34, CNC10021776, CNC10021861, CNC10022664, CNC10030888, CNC10031341, CNC10032139, CNC10032433, CNC10040109, CNC10041731, CNC10042349, CNC10050024, CNC10050222, CNC10050620, CNC10051703, CNC10060034, CNC10030065, CNC10060366, CNC100 61501 site, CNC10061519 site, CNC10061560 site, CNC10061562 site, CNC10061603 site, CNC10061804 site, CNC10070094 site, CNC10070095 site, CNC10070970 site, CNC10072223 site, CNC10072406 site, CNC10081349 site, CNC10081350 site, CNC10081506 site, CNC10081507 site, CNC10081568 site, CNC10082853 site, CNC10090019 site, CNC 10090805 site, CNC10091776 site, CNC10091860 site, CNC10091972 site, CNC10100994 site, CNC10111342 site, CNC10120314 site, CNC10120395 site, CNC10130054 site, CNC10130479 site, CNC10132801 site, CNC10133069 site, CNC10133411 site, CNC10133517 site, CNC10133915 site, CNC10134173 site, CNC10140073 site, CNC10140089 site,CNC10140088 site, CNC10140503 site, CNC10140928 site, CNC10142200 site, CNC10070140 site, CNC10142598 site, CNC10150082 site, CNC10150342 site, CNC10150857 site, CNC10150887 site, CNC10151225 site, CNC10151534 site, CNC10152380 site, CNC10152716 site, CNC10160964 site, CNC10161348 site, CNC10170850 site, CNC10180332 site.
[0009] Further, the mutation type of the CNC10010909 site is A / G, the mutation type of the CNC10011130 site is T / C, the mutation type of the CNC10011533 site is G / A, the mutation type of the CNC10012476 site is T / C, the mutation type of the CNC10012481 site is T / G, the mutation type of the CNC10012482 site is G / T, the mutation type of the CNC10012483 site is G / A, the mutation type of the CNC10013609 site is G / T, the mutation type of the CNC10013901 site is A / G, the mutation type of the CNC10013912 site is A / G, the mutation type of the CNC10014023 site is A / G, and the mutation type of the CNC10014023 site is A / G. The mutation type of NC10014056 is T / C, the mutation type of CNC10014645 is G / C, the mutation type of CNC10020600 is C / A, the mutation type of CNC10021264 is A / G, the mutation type of CNC10021414 is T / C, the mutation type of CNC10021534 is C / G, the mutation type of CNC10021776 is A / T, the mutation type of CNC10021861 is A / C, the mutation type of CNC10022664 is T / C, the mutation type of CNC10030888 is A / G, the mutation type of CNC10031341 is T / C, and the mutation type of CNC10032 The mutation type of CNC10050024 is C / T, the mutation type of CNC10050222 is T / C, the mutation type of CNC10050620 is A / G, the mutation type of CNC10051703 is C / A, the mutation type of CNC10060034 is G / A, the mutation type of CNC10030065 is T / C, the mutation type of CNC10060366 is The mutation type is A / G, the mutation type of CNC10061501 is T / C, the mutation type of CNC10061519 is C / T, the mutation type of CNC10061560 is T / C, the mutation type of CNC10061562 is T / C, the mutation type of CNC10061603 is T / C, the mutation type of CNC10061804 is A / G, the mutation type of CNC10070094 is C / T, the mutation type of CNC10070095 is A / G, the mutation type of CNC10070970 is A / G, the mutation type of CNC10072223 is C / T, the mutation type of CNC10072406 is G / A,The mutation type of CNC10081349 is G / A, the mutation type of CNC10081350 is T / C, the mutation type of CNC10081506 is G / A, the mutation type of CNC10081507 is G / A, the mutation type of CNC10081568 is G / A, the mutation type of CNC10082853 is T / A, the mutation type of CNC10090019 is T / C, the mutation type of CNC10090805 is T / C, the mutation type of CNC10091776 is G / A, the mutation type of CNC10091860 is G / A, and the mutation type of CNC10091972 is The mutation type of CNC10100994 is T / G, the mutation type of CNC10111342 is A / G, the mutation type of CNC10120314 is G / T, the mutation type of CNC10120395 is G / T, the mutation type of CNC10130054 is G / A, the mutation type of CNC10130479 is T / C, the mutation type of CNC10132801 is G / T, the mutation type of CNC10133069 is A / G, the mutation type of CNC10133411 is C / T, the mutation type of CNC10133517 is C / T, and the mutation type of CNC10133915 is C / T. The mutation type of the site is A / G, the mutation type of the site CNC10134173 is C / G, the mutation type of the site CNC10140073 is C / T, the mutation type of the site CNC10140089 is A / G, the mutation type of the site CNC10140088 is G / A, the mutation type of the site CNC10140503 is C / T, the mutation type of the site CNC10140928 is T / C, the mutation type of the site CNC10142200 is G / A, the mutation type of the site CNC10070140 is A / C, the mutation type of the site CNC10142598 is G / A, the mutation type of the site CNC10150082 is G / T, and the mutation type of the site CNC10140503 is C / T. The mutation type of the 150342 site is A / G, the mutation type of the CNC10150857 site is A / G, the mutation type of the CNC10150887 site is C / T, the mutation type of the CNC10151225 site is G / T, the mutation type of the CNC10151534 site is A / G, the mutation type of the CNC10152380 site is G / A, the mutation type of the CNC10152716 site is G / A, the mutation type of the CNC10160964 site is C / A, the mutation type of the CNC10161348 site is C / T, the mutation type of the CNC10170850 site is A / G, and the mutation type of the CNC10180332 site is T / C.
[0010] A gene chip used to identify the above-mentioned specific molecular ID card.
[0011] The application of the above gene chip in identifying the Henan black pig variety.
[0012] Preferably, the steps are:
[0013] (1) Collect tissue samples from the pigs to be tested and extract genomic DNA;
[0014] (2) Using a gene chip to perform SNP typing on the genomic DNA of step (1) to obtain genotype data of the pig to be tested;
[0015] (3) The genotype data of the pigs to be tested were combined with the genotypes of the SNP sites on the specific molecular ID card using PLINK software, and then principal component analysis was performed.
[0016] Furthermore, in step (1), the genomic DNA has an absorbance ratio at A260 / 280 between 1.8 and 2.0, and a concentration of ≥50 ng / μl.
[0017] Furthermore, when the result of the principal component analysis of the SNP typing in step (3) is close to the genetic distance of the Yunong Black Pig population and clusters into one cluster, it is the Yunong Black Pig.
[0018] The present invention has the following beneficial effects:
[0019] 1. This application uses PLINK software to merge the significant SNPs of group A and group B, and calculates the allele frequency of each SNP in 10 varieties, and selects the SNP set with a higher allele frequency in the experimental group than in the other 9 varieties as the specific molecular identity card of the Yunong black pig variety.
[0020] 2. This patent combines the whole genome association analysis and the analysis method of selection signal to determine the specific molecular identity card of the Henan Agricultural Black Pig variety, laying the foundation for the development, utilization and identification of the Henan Agricultural Black Pig variety. The Henan Agricultural Black Pig variety was studied through SNP molecular markers, and bioinformatics technology was used for comprehensive analysis. The research results were preliminarily verified to construct the breed-specific molecular markers of the Henan Agricultural Black Pig, which can simply and clearly distinguish the differences between the Henan Agricultural Black Pig and other local pig breeds. It can be used for the authenticity identification and genetic relationship analysis of the Henan Agricultural Black Pig variety, and provides an effective scientific basis for the intellectual property protection of the Henan Agricultural Black Pig variety. When identifying and evaluating the Henan Agricultural Black Pig variety, it is only necessary to compare and analyze the Henan Agricultural Black Pig variety with the existing specific molecular identity card to determine whether it is a Henan Agricultural Black Pig variety. This greatly reduces the time and cost of identification and evaluation of Henan Agricultural Black Pig germplasm resources, and improves the efficiency of identification and evaluation of Henan Agricultural Black Pig varieties. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.
[0022] Figure 1 Manhattan plot and QQ plot of the GWAS analysis results of the Yunong black pig of the present invention.
[0023] Figure 2 This is the Manhattan plot of the selection signal analysis results of the Yunong black pig of the present invention.
[0024] Figure 3 This is the principal component analysis verification diagram of the Yunong black pig breed-specific molecular identity card of the present invention. DETAILED DESCRIPTION
[0025] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Example
[0026] A method for screening the specific molecular identity card of Henan Agricultural Black Pig germplasm resources, comprising the following steps:
[0027] (1) Ear sample collection
[0028] The experimental population consisted of 1117 pigs of 10 pig breeds, including 7 Chinese pig breeds: Nanyang Black Pig (n=10, NY), Huainan Pig (n=10, HN), Yunong Black Pig (n=1036, YN), Queshan Black Pig (n=10, QS), Laiwu Pig (n=10, LWH), Erhualian Pig (n=10, EHL), Min Pig (n=6, MIN); and 3 Western commercial pig breeds: Duroc Pig (n=10, DU), Large White Pig (n=10, LW), Landrace Pig (n=5, LR). The ears of the pigs were cleaned with 75% alcohol, and a small amount of ear tissue was cut with ear sample forceps, placed in a 2ml centrifuge tube filled with 75% alcohol, and stored in a -20℃ refrigerator.
[0029] (2) Total DNA extraction, quality testing and genotyping
[0030] Total DNA was extracted using an animal tissue genomic DNA extraction kit;
[0031] The results were detected by 1% agarose gel electrophoresis using a DYY-6C electrophoresis apparatus.
[0032] The DNA concentration was detected by Nanodrop-2000 UV spectrophotometer, and the genomic DNA samples with a light absorption ratio (A260 / 280) between 1.8 and 2.0 and a concentration of ≥ 50 ng / μl were used for whole genome chip typing using Illumina Porcine SNP50BeadChip (Beijing Compson Biotechnology Co., Ltd., Zhongxin No. 1). The specific operation was as follows:
[0033] a. Use Tn5 transposase to establish a gene library for the sample pigs and perform a 50 K gene chip scan.
[0034] b. Use Beagle to fill in genotypes for the 50K chip and whole genome resequencing results in step (1).
[0035] c. Perform genome-wide association analysis and selection signal analysis on the genotype filling data of all individuals obtained through step b.
[0036] d. Calculate the allele frequencies among varieties for the significant loci obtained in step c, retain the SNP loci with higher allele frequencies for Yunong Black Pig, and use the collection of SNP loci as the variety-specific molecular ID card for Yunong Black Pig.
[0037] (3) Genotype data filling and quality control
[0038] A total of 1,117 heads and 51,315 SNPs were obtained by chip sequencing. PLINK software was used to perform quality control on chip data. The genotype data were filtered by the following parameters: individual genotype detection rate (--mind) > 90%, marker genotype detection rate (--geno) > 95%, minimum allele frequency (--maf) > 1%, minimum Hardy-Weinberg equilibrium (--hwe) of 10 -6 , located on the autosomes. The missing genotypes were filled in the BEAGLE software using the Hidden Markov Model (HMM) algorithm.
[0039] (4) Screening of SNP-specific loci by genome-wide association analysis
[0040] The GEMMA software was used for genome-wide association analysis. The experimental group consisted of 10 Yunong black pigs (case) and the control group consisted of the other 9 breeds (control). Figure 1 See the left and QQ diagrams Figure 1Right, there are two threshold lines in the Manhattan plot, where the solid line threshold is 0.05 / N (N is the number of chip sites used), and the sites above the solid line are at the genome-wide significant level; the dotted line threshold is 1 / N, and the sites above the dotted line are at the chromosome significant level. The closer the λ value in the QQ plot is to 1, the more reliable the results of the genome-wide association analysis are; the Banferonni correction method is used to identify SNPs that are significantly associated with the variety, and the set of significant SNPs is group A.
[0041] (5) Select signal analysis to screen SNP-specific sites
[0042] The genetic differentiation index (Fst) was calculated using VCFtools software, using the sliding window mean calculation method. The results are shown in Figure 2 , Figure 2 The threshold line in is the top 1% of the largest Fst values after sorting, and the sites above this threshold line are significant sites (marked in red). The specific parameters are as follows: the size of the sliding window (--fst-window-size) is 100,000 bp, and the step length of the sliding window (--fst-window-step) is 40,000 bp. The windows are sorted from large to small by Fst value, and the top 1% of windows are defined as significant windows. Then, PLINK software is used to extract SNPs in the significant windows. The set of significant SNPs is group B.
[0043] (6) Allele frequency screening of SNP-specific sites
[0044] PLINK software was used to merge the significant SNPs of group A and group B, and the allele frequency of each SNP in the 10 breeds was calculated. The SNP set with a higher allele frequency in the experimental group than in the other 9 breeds was selected as the specific molecular identity card of the Yunong black pig breed.
[0045] Table 1 Specific molecular marker set of Yunong black pig breed
[0046]
[0047] (7) The above 88 SNPs of 10 varieties were extracted using PLINK software and verified by principal component analysis.
[0048] (8) Application of the 88 SNP loci in identifying the Yunong black pig variety, wherein the SNP loci are located in the genome version EnsemblSscrofa 11.1.
[0049] Application Examples
[0050] A method for identifying a pig breed to be tested specifically comprises the following steps:
[0051] 1. Extract ear tissue samples from the pigs to be tested, extract genomic DNA from the tissue samples, and type the above 88 sites through the chip. The genomic DNA is sent to Beijing Compson Biotechnology Co., Ltd. for SNP typing on the "Axiom" chip for local pigs. The experimental principle of SNP typing on the chip is based on the ligation reaction, in which two probes play a role. The first is the capture probe on the chip, which plays the role of fixing the target DNA fragment to the surface of the chip. The second is the color probe, which is responsible for coloring the SNP chip (red and green fluorescence). The experiment is carried out in two rounds of hybridization. In the first round of hybridization, the target DNA is hybridized with the chip, and the capture probe will capture the matching target DNA fragment; the color probe hybridizes to the DNA fragment in the second round of hybridization. Then, using the recognition of the ligase, only the color probe complementary to the target DNA fragment will be connected to the capture probe. Through fluorescent labeling staining, SNP typing is performed under laser scanning to obtain the genotype data of the pigs to be tested.
[0052] 2. Use PLINK software to merge the genotype data of the pigs to be tested with the genotype data of the above 10 breeds, then perform principal component analysis and use R language to visualize the results. When the genetic distance between the individual pigs to be tested and the Yunong black pig population is close and they are clustered together, Figure 3 , it can be determined that the pig to be tested is a Yunong black pig.
[0053] Through the use of SNP molecular markers, the Henan Agricultural Black Pig breed was studied, and bioinformatics technology was used for comprehensive analysis. The research results were preliminarily verified to construct the breed-specific molecular markers of Henan Agricultural Black Pig, which can simply and clearly distinguish the differences between Henan Agricultural Black Pig and other local pig breeds. It can be used for the authenticity identification and genetic relationship analysis of Henan Agricultural Black Pig breeds, and provide an effective scientific basis for the intellectual property protection of Henan Agricultural Black Pig breeds. When identifying and evaluating the Henan Agricultural Black Pig breed, it is only necessary to compare and analyze the Henan Agricultural Black Pig breed with the existing specific molecular identity card to determine whether it is a Henan Agricultural Black Pig breed. This greatly reduces the time and cost of identification and evaluation of Henan Agricultural Black Pig germplasm resources, and improves the efficiency of identification and evaluation of Henan Agricultural Black Pig breeds.
[0054] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims
1. A method for identifying the variety of Henan Agricultural Black Pig, Features: The method is achieved by identifying a set of the following SNP sites; the SNP sites are located in the pig reference genome EnsemblSscrofa 11.1 version; The set of SNP sites is:
2. Gene chip for identifying a collection of SNP sites, It is characterized in that The SNP sites are located in the pig reference genome EnsemblSscrofa 11.1 version; the set of SNP sites is:
3. Application of the gene chip described in claim 2 in identifying the Yunong black pig variety.
4. The use according to claim 3, It is characterized in that The steps are: (1) Collecting tissue samples from the pigs to be tested and extracting genomic DNA; (2) performing SNP typing on the genomic DNA of step (1) using a gene chip to obtain genotype data of the pig to be tested; (3) The genotype data of the pigs to be tested were combined with the genotype data of the Henan Agricultural University black pig using PLINK software, and then principal component analysis was performed.
5. The use according to claim 4, Features: In the step (1), the genomic DNA has an A260 / 280 light absorption ratio between 1.8 and 2.0, and a concentration of ≥50 ng / μl.
6. The use according to claim 5, Features: When the result of the principal component analysis of the SNP typing in the step (3) is close to the genetic distance of the Yunong black pig population and clusters into one cluster, it is the Yunong black pig.