A specific molecular ID card for identifying the Nanyang black pig breed and its application

Abstract

The present invention belongs to the field of pig breed identification, relates to Nanyang black pig germplasm resources, and particularly refers to a specific molecular ID card for identifying Nanyang black pig breeds and its application. The SNP site is a set of SNP sites with higher allele frequencies among Nanyang black pig breeds. Further, the SNP site is located in the pig reference genome EnsemblSscrofa 11.1 version. The breed-specific molecular markers screened at the genome level by molecular biology technology in this application can reveal the landmark genetic differences between breeds. As an important part of whole genome association analysis, genotype filling aims to predict SNPs that have not been typed in the research sample, increase the number of SNPs that can be used to detect associations, thereby improving the detection ability of GWAS, and the combined strategy of whole genome association analysis and selection signal analysis, further improve the efficiency and accuracy of identifying molecular markers, so as to achieve the effect of mutual verification.

Description

Technical Field

[0001] The invention belongs to the field of pig breed identification, relates to Nanyang black pig germplasm resources, and particularly refers to a specific molecular identity card for identifying the Nanyang black pig breed and an application thereof. Background Art

[0002] 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 protection plans 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, 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] Nanyang Black Pig is mainly distributed in Neixiang, Xichuan, Zhenping and Dengzhou in the western part of Nanyang. It was once widely raised. The central production areas are Shigang and Wating in Neixiang County, Houpo and Xianghua in Xichuan County, and Zhangcun in Dengzhou City. Nanyang Black Pig has black hair all over its body, a big pig head, wrinkles on its face, and a gentle personality. It eats chaff, bran, leftovers, rotten vegetables, etc. It is easy to raise and is known for its firm meat and delicious taste. The breeding range covers the entire Nanyang, Shangnan, Hubei and other places. It is an important pig breed in Henan Province. In 2015, Nanyang Black Pig was selected as a "Chinese Geographical Indication". Nanyang Black Pig has the same identity as Nanyang Yellow Cattle, Xinyang Maojian, Fuling Zhacai, etc. With the gradual improvement of bioinformatics technology, whole genome association analysis technology has been widely used to analyze livestock and poultry genome information. In the process of selecting local pig breeds, a large number of selection marks are left in their genomes, forming unique breed characteristics, which give them certain advantages in meat quality, disease resistance and reproductive performance. Therefore, protecting local pig breeds can greatly enrich the genetic resources of Henan Province, protect the diversity of local pigs, and retain the excellent genes of local pig breeds. In order to further accurately identify the Nanyang Black Pig, the combination of whole genome association analysis and selection signals as a research strategy for identifying local pig breed-specific molecular markers can clarify the genetic background of local pig breeds, locate the selected areas, promote the development of wild Ade in livestock and poultry, and provide technical support for the identification and protection of Henan local pigs. Summary of the invention

[0004] To achieve the above object, the present invention proposes a specific molecular ID card for identifying the Nanyang black pig breed and its application.

[0005] The technical solution of the present invention is achieved in this way:

[0006] A specific molecular ID card for identifying Nanyang black pig varieties, wherein the SNP loci are a set of SNP loci with higher allele frequencies among Nanyang black pig varieties.

[0007] Furthermore, the SNP site is located in the pig reference genome EnsemblSscrofa 11.1 version.

[0008] Further, the set of SNP sites includes CNC10010199, CNC10010544, CNC10010543, CNC10010542, CNC10010547, CNC10013108, CNC10013746, CNC10013844, CNC10013968, CNC10013970, CNC10014393, CNC10014425, CNC10020711, CNC10030907, CNC10030909, CNC10031532, CNC10031554, CNC10031731, CNC 10031777, CNC10031778, CNC10032256, CNC10032268, CNC10032663, CNC10040006, CNC10042293, CNC10042504, CNC10042521, CNC10051167, CNC10051960, CNC10052002, CNC10052067, CNC10060233, CNC10060234, CNC10060443, CNC10060560, CNC10063140, CNC10070127, CNC1007014 3. CNC10070170, CNC10071425, CNC10071434, CNC10071469, CNC10071481, CNC10071485, CNC10071484, CNC10071550, CNC10080294, CNC1008 0699, CNC10080999, CNC10081833, CNC10081992, CNC10082297, CNC10082394, CNC10090107, CNC10090690, CNC10100622, CNC10100950, CNC10 101273, CNC10120872, CNC10130429, CNC10131081, CNC10131956, CNC10131957, CNC10131963, CNC10131996, CNC10132031, CNC10132077, CNC10132124, CNC10132139, CNC10140369, CNC10141484, CNC10150308, CNC10150397, CNC10151078, CNC10152528, CNC10170142 and CNC10170555.

[0009] Further, the mutation type of the CNC10010199 site is C / A, the mutation type of the CNC10010544 site is A / G, the mutation type of the CNC10010543 site is C / T, the mutation type of the CNC10010542 site is C / T, the mutation type of the CNC10010547 site is C / T, the mutation type of the CNC10013108 site is A / C, the mutation type of the CNC10013746 site is G / C, the mutation type of the CNC10013844 site is C / A, the mutation type of the CNC10013968 site is G / A, the mutation type of the CNC10013970 site is A / G, the mutation type of the CNC10014393 site is G / A, C The mutation type of NC10014425 is G / A, the mutation type of CNC10020711 is C / T, the mutation type of CNC10030907 is C / T, the mutation type of CNC10030909 is T / C, the mutation type of CNC10031532 is C / T, the mutation type of CNC10031554 is A / G, the mutation type of CNC10031731 is G / C, the mutation type of CNC10031777 is G / A, the mutation type of CNC10031778 is T / A, the mutation type of CNC10032256 is A / G, the mutation type of CNC10032268 is T / C, and the mutation type of CNC10032 The mutation type of CNC10040006 is T / C, the mutation type of CNC10042293 is G / A, the mutation type of CNC10042504 is C / T, the mutation type of CNC10042521 is C / T, the mutation type of CNC10051167 is G / A, the mutation type of CNC10051960 is C / T, the mutation type of CNC10052002 is A / G, the mutation type of CNC10052067 is T / C, the mutation type of CNC10060233 is G / A, the mutation type of CNC10060234 is T / C, and the mutation type of CNC10060443 is The mutation type of the CNC10060560 site is C / T, the mutation type of the CNC10063140 site is C / T, the mutation type of the CNC10070127 site is G / A, the mutation type of the CNC10070143 site is G / A, the mutation type of the CNC10070170 site is T / C, the mutation type of the CNC10071425 site is A / C, the mutation type of the CNC10071434 site is C / T, the mutation type of the CNC10071469 site is A / G, the mutation type of the CNC10071481 site is T / C, the mutation type of the CNC10071485 site is C / A, the mutation type of the CNC10071484 site is C / T,The mutation type of CNC10071550 is T / C, the mutation type of CNC10080294 is A / G, the mutation type of CNC10080699 is G / T, the mutation type of CNC10080999 is G / A, the mutation type of CNC10081833 is C / T, the mutation type of CNC10081992 is C / T, the mutation type of CNC10082297 is T / C, the mutation type of CNC10082394 is T / G, and the mutation type of CNC10081833 is C / T. The mutation type of 090107 is G / A, the mutation type of CNC10090690 is A / T, the mutation type of CNC10100622 is G / T, the mutation type of CNC10100950 is A / G, the mutation type of CNC10101273 is A / C, the mutation type of CNC10120872 is T / G, the mutation type of CNC10130429 is T / C, the mutation type of CNC10131081 is C / T, and the mutation type of CNC10131956 is The mutation type of the site is A / G, the mutation type of the site CNC10131957 is G / A, the mutation type of the site CNC10131963 is G / T, the mutation type of the site CNC10131996 is T / C, the mutation type of the site CNC10132031 is T / G, the mutation type of the site CNC10132077 is C / A, the mutation type of the site CNC10132124 is T / C, the mutation type of the site CNC10132139 is T / G, and the mutation type of the site CNC10140369 is The mutation type of the CNC10141484 site is T / C, the mutation type of the CNC10150308 site is A / G, the mutation type of the CNC10150397 site is A / G, the mutation type of the CNC10151078 site is C / T, the mutation type of the CNC10152528 site is G / A, the mutation type of the CNC10152540 site is C / A, the mutation type of the CNC10170142 site is A / C, and the mutation type of the CNC10170555 site is G / T.

[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 Nanyang black pig variety.

[0012] For the above application, 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] In the step (1), the genomic DNA has an A260 / 280 absorbance ratio between 1.8 and 2.0, and a concentration ≥ 50 ng / μl.

[0017] When the result of the principal component analysis of the SNP typing in the step (3) is close to the genetic distance of the Nanyang black pig population and clusters into one cluster, it is the Nanyang black pig.

[0018] The present invention has the following beneficial effects:

[0019] 1. Breed-specific molecular markers screened at the genome level through molecular biology techniques can reveal the iconic genetic differences between breeds. As an important part of whole-genome association analysis, genotype filling aims to predict SNPs that have not been typed in the research samples, increase the number of SNPs that can be used to detect associations, thereby improving the detection capability of GWAS, and the combined strategy of whole-genome association analysis and selection signal analysis, further improving the efficiency and accuracy of identifying molecular markers to achieve the effect of mutual verification. The present invention uses biotechnology to explore the characteristics of precious genetic resources of local pigs, which is conducive to promoting the protection and innovative use of local pig genetic resources in Henan Province and promoting the high-quality development of the seed industry.

[0020] 2. Use PLINK software to merge the genotype data of the pig to be tested and the genotype data of Nanyang black pig, extract the above 78 loci in the data, and then perform principal component analysis. When the principal component 1 and principal component 2 of the individual pig to be tested and the Nanyang black pig group are visualized through R language, the degree of clustering is high, indicating that the genetic distance between the individual pig to be tested and the Nanyang black pig is close. At this time, it can be determined that the pig to be tested is Nanyang black pig. 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 Nanyang black pig provided by the present invention.

[0023] Figure 2 This is a Manhattan plot of the selection signal analysis results of the Nanyang black pig provided by the present invention.

[0024] Figure 3 This is a principal component analysis verification diagram of the Nanyang black pig breed-specific molecular identity card provided by 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 ID card of the Nanyang black pig breed, 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. For the significant loci obtained in step c, calculate the allele frequencies among breeds, retain the SNP loci with higher allele frequencies of Nanyang black pigs, and use the collection of SNP loci as the breed-specific molecular ID card of Nanyang black pigs.

[0037] (3) Genotype data filling and quality control

[0038] 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.

[0039] (4) Screening of SNP-specific loci by genome-wide association analysis

[0040] The whole genome association analysis was performed using GEMMA software. The experimental group consisted of 10 Nanyang black pigs (case) and the control group consisted of the other 9 breeds (control). Figure 1 See the left and QQ diagrams Figure 1 Right, 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 2The 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 Nanyang Black Pig breed.

[0045] Table 1. Specific molecular marker set of Nanyang black pig breed

[0046]

[0047] (7) The above 78 SNPs of 10 varieties were extracted using PLINK software and verified by principal component analysis.

[0048] (8) The application of the 78 SNP sites in identifying the Nanyang black pig variety is characterized in that the SNP sites 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 78 loci 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.

[0052] 2. Use PLINK software to merge the genotype data of the pig to be tested and the genotype data of Nanyang black pig, extract the above 78 loci in the data, and then perform principal component analysis. When the genetic distance between the individual pig to be tested and the Nanyang black pig group is close, the pig to be tested can be determined to be Nanyang black pig.

[0053] 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 Nanyang black pig breed, 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. Use of the gene chip according to claim 2 in identifying the Nanyang 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 the Nanyang 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 Nanyang black pig population and clusters into one cluster, it is the Nanyang black pig.

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