A specific molecular ID card for identifying Queshan black pig germplasm resources and its application
Through the SNP site as a specific molecular ID card, combined with the gene chip technology and the principal component analysis of PLINK software, the problem of difficulty in quickly and accurately identifying the germplasm resources of Queshan black pigs in the existing technology is solved, and efficient and accurate identification and protection are achieved.
Patent Information
- Application Number
- CN202210815775.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-07-12
- Publication Date
- 2025-06-06
- Estimated Expiration
- 2042-07-12
AI Technical Summary
The prior art is difficult to quickly and accurately identify and protect Queshan black pig germplasm resources, and there is a lack of effective methods to distinguish Queshan black pig from other local pig breeds.
The SNP site was used as the specific molecular ID card, and the genomic DNA of the pigs to be tested was SNP typing through gene chip technology, and the principal component analysis was combined with PLINK software to identify the specific molecular markers of the Queshan black pig breed.
The rapid and accurate identification of the germplasm resources of Queshan black pigs has been achieved, which reduces the identification time and cost, improves the identification efficiency, and provides a scientific basis for the intellectual property protection of the Queshan black pig breeds.
Smart Images
Figure CN114959067B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of pig breed identification, relates to Queshan black pig germplasm resources, and particularly refers to a specific molecular identity card for identifying Queshan black pig germplasm resources and an application thereof. Background Art
[0002] The bristles of Queshan black pigs are thick and short, and they are large in size, generally 136 cm long and about 70 cm tall. There are diamond-shaped wrinkles on the face, drooping ears, straight waist and back, slightly drooping abdomen, slightly sloping buttocks, and thick and long tails that exceed the hocks. Its biggest feature is that it is gentle and suitable for free-range breeding. The central production area of Queshan black pigs is in Zhugou, Wagang and Shiluohe townships in the two parts of Queshan. It is also distributed in the neighboring townships of Rendian, Yifeng, Humiao, Chengjiao, Zhugudong, Lixindian and Daluzhuang Laohe in Qinyang. According to a survey of nine townships, there are more than 18,900 Queshan black pigs.
[0003] In March 2009, the expert group of the Pig Special Committee of the National Committee for Livestock and Poultry Genetic Resources conducted an on-site inspection and identification of the Queshan Black Pig in Queshan County, Zhumadian City. After review, the Queshan Black Pig met the basic conditions for pig genetic resource identification in the "Measures for the Approval of New Livestock and Poultry Breeds and the Identification of Livestock and Poultry Genetic Resources" of the Ministry of Agriculture, and agreed to pass the preliminary review and report to the National Committee for Livestock and Poultry Genetic Resources for approval. The effective protection and rational development of local breeds 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 based on 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, using bioinformatics to identify the unique genetic characteristics of Henan local pig breeds is an important part of accurately protecting local pig germplasm genetic resources.
[0004] Single nucleotide polymorphism (SNP) markers are third-generation molecular markers, which refer to a polymorphism caused by a single base mutation in the genomic DNA sequence. This mutation includes single base transversion, conversion, insertion and deletion. SNP has the characteristics of wide distribution, high frequency and low mutation rate, and is widely used in genome analysis, bioinformatics automated detection, genetic research of diseases and livestock and poultry breeding markers.
[0005] Genome-wide association studies (GWAS) are an important and commonly used method for genome analysis of livestock and poultry genetic resources. 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 provide a method for batch identification of Queshan black pig germplasm resources, our research group conducted a long-term follow-up study. Summary of the invention
[0006] To achieve the above-mentioned purpose, the present invention proposes a specific molecular ID card for identifying Queshan black pig germplasm resources and its application.
[0007] The technical solution of the present invention is achieved in this way:
[0008] A specific molecular ID card for identifying Queshan black pig germplasm resources, wherein the SNP loci are a set of SNP loci with higher allele frequencies among Queshan black pig varieties.
[0009] Preferably, the SNP site is located in the pig reference genome EnsemblSscrofa 11.1 version.
[0010] Further, the set of SNP sites includes CNC10010832 site, CNC10012824 site, CNC10012972 site, CNC10013000 site, CNC10013872 site, CNC10040563 site, CNC10041271 site, CNC10041332 site, CNC10041501 site, CNC10041536 site, CNC10041599 site, CNC10041838 site, CNC10042298 site, CNC10060925 site, CNC10062082 site, CNC10062105 site, CNC10062442 site, CNC10062524 site, CNC10090798 site , CNC10091437 site, CNC10091643 site, CNC10092360 site, CNC10092481 site, CNC10092509 site, CNC10092580 site, CNC10130757 site, CNC10131664 site, CNC10133552 site, CNC10141731 site, CNC10141931 site, CNC10142249 site, CNC10142250 site, CNC10151846 site, CNC10152169 site, CNC10152617 site, CNC10170559 site, CNC10170888 site, CNC10170889 site and CNC10171158 site.
[0011] Preferably, the mutation type of the CNC10010832 site is T / C, the mutation type of the CNC10012824 site is T / G, the mutation type of the CNC10012972 site is G / A, the mutation type of the CNC10013000 site is A / G, the mutation type of the CNC10013872 site is C / T, the mutation type of the CNC10040563 site is T / G, the mutation type of the CNC10041271 site is C / T, the mutation type of the CNC10041332 site is T / C, the mutation type of the CNC10041501 site is T / C, and the mutation type of the CNC10041536 site is The mutation type of the site is C / T, the mutation type of the site CNC10041599 is G / A, the mutation type of the site CNC10041838 is C / T, the mutation type of the site CNC10042298 is C / T, the mutation type of the site CNC10060925 is C / T, the mutation type of the site CNC10062082 is A / G, the mutation type of the site CNC10062105 is T / C, the mutation type of the site CNC10062442 is A / G, the mutation type of the site CNC10062524 is A / G, the mutation type of the site CNC10090798 is T / C, and the mutation type of the site CNC10091 The mutation type of site 437 is C / T, the mutation type of site CNC10091643 is G / T, the mutation type of site CNC10092360 is T / C, the mutation type of site CNC10092481 is T / C, the mutation type of site CNC10092509 is A / G, the mutation type of site CNC10092580 is A / G, the mutation type of site CNC10130757 is G / A, the mutation type of site CNC10131664 is C / T, the mutation type of site CNC10133552 is A / G, the mutation type of site CNC10141731 is T / C, and the mutation type of site CNC10141732 is T / C. The mutation type at site 0141931 is C / T, the mutation type at site CNC10142249 is A / G, the mutation type at site CNC10142250 is G / T, the mutation type at site CNC10151846 is G / T, the mutation type at site CNC10152169 is G / C, the mutation type at site CNC10152617 is G / A, the mutation type at site CNC10170559 is G / A, the mutation type at site CNC10170888 is C / T, the mutation type at site CNC10170889 is T / C, and the mutation type at site CNC10171158 is C / T.
[0012] A gene chip used to identify the above-mentioned specific molecular ID card.
[0013] The application of the above gene chip in identifying the Queshan black pig variety.
[0014] The steps are:
[0015] (1) Collect tissue samples from the pigs to be tested and extract genomic DNA;
[0016] (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;
[0017] (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.
[0018] Furthermore, in step (1), the light absorption ratio of the genomic DNA at A260 / 280 is between 1.8 and 2.0, and the concentration is ≥50 ng / μl.
[0019] Furthermore, when the result of the principal component analysis of the SNP typing in step (3) is close to the genetic distance of the Queshan black pig population and clusters into a cluster, it is the Queshan black pig.
[0020] The present invention has the following beneficial effects:
[0021] 1. 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 Queshan black pig population is close and they are clustered together, Figure 3 , it can be determined that the pig to be tested is a Queshan black pig.
[0022] 2. The present invention discloses a method for rapidly and accurately identifying and evaluating the germplasm resources of Queshan black pigs and a Queshan black pig breed-specific molecular ID card. The Queshan black pig breed is studied through SNP molecular markers, and a comprehensive analysis is performed using bioinformatics technology. The research results are preliminarily verified to construct a breed-specific molecular marker for the Queshan black pig. The difference between the Queshan black pig and other local pig breeds can be simply and clearly distinguished, and can be used for the authenticity identification and genetic relationship analysis of the Queshan black pig breed, providing an effective scientific basis for the intellectual property protection of the Queshan black pig breed. When identifying and evaluating the Queshan black pig breed, it is only necessary to compare and analyze the Queshan black pig breed with the existing specific molecular ID card to determine whether it is a Queshan black pig breed. This greatly reduces the time and cost of identifying and evaluating the germplasm resources of the Queshan black pig, and improves the efficiency of identifying and evaluating the Queshan black pig breed. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] 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.
[0024] Figure 1 Manhattan plot and QQ plot of the GWAS analysis results of the Queshan black pig of the present invention.
[0025] Figure 2 This is the Manhattan plot of the selection signal analysis results of the Queshan black pig of the present invention.
[0026] Figure 3 This is the principal component analysis verification diagram of the Queshan black pig breed-specific molecular identity card of the present invention. DETAILED DESCRIPTION
[0027] 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
[0028] A method for screening specific molecular ID cards of Queshan black pig germplasm resources, comprising the following steps:
[0029] (1) Ear sample collection
[0030] 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.
[0031] (2) Total DNA extraction, quality testing and genotyping
[0032] Total DNA was extracted using an animal tissue genomic DNA extraction kit;
[0033] The results were detected by 1% agarose gel electrophoresis using a DYY-6C electrophoresis apparatus.
[0034] 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:
[0035] a. Use Tn5 transposase to establish a gene library for the sample pigs and perform a 50 K gene chip scan.
[0036] b. Use Beagle to fill in genotypes for the 50K chip and whole genome resequencing results in step (1).
[0037] c. Perform genome-wide association analysis and selection signal analysis on the genotype filling data of all individuals obtained through step b.
[0038] d. For the significant loci obtained in step c, calculate the allele frequencies among breeds, retain the SNP loci with higher allele frequencies for Queshan black pigs, and use the collection of SNP loci as the breed-specific molecular identity card for Queshan black pigs.
[0039] (3) Genotype data filling and quality control
[0040] A total of 1,117 heads and 51,315 SNPs were obtained by chip sequencing. The chip data were quality controlled using PLINK software. The genotype data were filtered using 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, and located on autosomes. The missing genotypes were filled in using the Hidden Markov Model (HMM) algorithm in BEAGLE software.
[0041] (4) Screening of SNP-specific loci by genome-wide association analysis
[0042] The whole genome association analysis was performed using GEMMA software. The experimental group consisted of 10 Queshan black pigs (case) and the control group consisted of the other 9 breeds (control). The Manhattan plot of Queshan black pigs is shown in 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.
[0043] (5) Select signal analysis to screen SNP-specific sites
[0044] 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.
[0045] (6) Allele frequency screening of SNP-specific sites
[0046] 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 Queshan black pig breed.
[0047] Table 1 Specific molecular marker set of Queshan black pig breed
[0048]
[0049] (7) The above 39 SNPs of 10 varieties were extracted using PLINK software and verified by principal component analysis.
[0050] (8) Application of the 39 SNP loci in identifying the Queshan black pig breed, wherein the SNP loci are located in the genome version EnsemblSscrofa 11.1.
[0051] Application Examples
[0052] A method for identifying a pig breed to be tested specifically comprises the following steps:
[0053] 1. Extract ear tissue samples from the pigs to be tested, extract genomic DNA from the tissue samples, and type the above 39 sites through the chip. The genomic DNA is sent to Beijing Compson Biotechnology Co., Ltd. for SNP typing using 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 colorimetric 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 colorimetric probe hybridizes to the DNA fragment in the second round of hybridization. Then, using the recognition effect of the ligase, only the colorimetric 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.
[0054] 2. Use PLINK software to merge the genotype data of the pig to be tested and the genotype data of the Queshan black pig, extract the above 39 loci in the data, and then perform principal component analysis. When the genetic distance between the individual pig to be tested and the Queshan black pig population is close, the pig to be tested can be determined to be a Queshan black pig.
[0055] 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 Queshan 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 gene chip contains a set of SNP sites that identify the following; the SNP sites are located in the pig reference genome EnsemblSscrofa 11.1 version; The set of SNP sites is:
3. Use of the gene chip described in claim 2 in identifying the Queshan 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 merged with the genotype data of Queshan black pigs 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 Queshan black pig population and clusters into a cluster, it is the Queshan black pig.