CN117431325A - SNP locus combination for identifying Ronchang pig variety and application thereof - Google Patents
SNP locus combination for identifying Ronchang pig variety and application thereof Download PDFInfo
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- 238000012935 Averaging Methods 0.000 description 1
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Abstract
The invention discloses a SNP locus combination for identifying Rongchang pig breeds and application thereof, wherein the Rongchang pig breeds are researched through SNP loci, bioinformatics technology comprehensive analysis is adopted, the research result is initially verified, a Rong Chang pig breeds specificity SNP locus set is constructed, the SNP locus combination comprises 54 SNP loci on genome version EnsemblSscofa 11.1, the SNP locus combination can be used for authenticity identification and genetic relationship analysis of Rongchang pig breeds, and effective scientific basis is provided for intellectual property protection of Rongchang pig breeds. When the Rongchang pig variety is identified and evaluated, whether the Rongchang pig variety is the Rongchang pig variety can be determined only by comparing and analyzing the Rongchang pig variety with the existing specific SNP locus, so that the efficiency of the Rongchang pig variety identification and evaluation is greatly reduced, the efficiency of the Rongchang pig variety identification and evaluation is improved, and the method has remarkable application value.
Description
Technical Field
The invention relates to the field of molecular biology identification, in particular to SNP locus combination for identifying Ronchang pig breeds and application thereof.
Background
Rong Changzhu, which is named as native to Chongqing Rong Chang, is one of the eight excellent breeds of pigs in the world, and the variety formation of which has been 400 years old. Rong Chang pigs were first loaded into the world domestic animal breed and breed dictionary published in the united kingdom in 1957 and became an internationally recognized precious pig breed resource. Later, the strain is incorporated into a national breeding scientific research cooperation plan in 1972, the strain is listed in a famous local fine breed pig in China in 1984, the strain is listed as a national first-grade protection variety in 1985, the national standard of Rong Chang pigs is issued in 1987, the strain is listed in a first national livestock and poultry variety protection directory in 2000, a national grade resource protection farm is established in 2001, and a national grade resource protection region is established in 2008. Rong Chang pigs are developed into one of the local pig species with the largest popularization area and the most influence in the pig industry in China.
The Rong Chang pig has the advantages of large size, symmetrical structure, coarse feeding resistance, strong adaptability, good meat quality, high lean meat percentage, good matching force, excellent bristle quality, stable genetic performance and the like. Effective protection and reasonable development of local varieties are facilitated, and sustainable development of pig industry in China and enrichment of livestock resource diversity are facilitated. Particularly, the research on the specific genetic structure and characterization of the local pig breeds is helpful for making a protection plan for each breeds according to the breeds genetic condition and promoting the specific protection of the local pig breeds. Preserving the unique variations, genes and characteristics of each variety is extremely important to maintain biodiversity and to accommodate future environmental changes. Thus, the identification of the unique genetic characteristics of Rong Chang pigs by bioinformatics is an important part of accurately protecting local pig germplasm genetic resources.
Single nucleotide polymorphism (single nucleotide ploymophism, SNP) refers to a variation of the base of a single nucleotide at a specific position in the genome, including single base transversions, transitions, insertions and deletions. SNPs are a common form of genetic variation and are also the most common genetic markers in the genome. In livestock breeding, SNPs are widely used for genetic markers and genomic analysis. Through SNP markers, the genotypes of animal individuals can be accurately identified and distinguished, so that researchers are helped to know important genetic characteristics such as disease resistance, production performance, quality characteristics and the like of animals.
The continuous development of high-throughput genotyping technologies such as gene chips greatly promotes the study of genetic characteristics at the genome level. During the long term natural and artificial selection of animals, they leave corresponding genetic traces, also known as selection signals, on their genome. The study of selection signals is a strategy based on genome-to-phenotype concepts. Due to the lack of phenotype records of local pigs in China and small population scale, analysis of germplasm characteristics of livestock is increasingly becoming an important method. In order to provide a method for mass identification of Ronchang pig breeds, the subject group has been studied for a long time.
Disclosure of Invention
The invention aims to provide a SNP locus combination for identifying the Rongchang pig variety, which can be used for identifying the Rongchang pig variety, can be used for identifying the Rongchang pig variety and analyzing the genetic relationship, provides effective scientific basis for the intellectual property protection of the Rongchang pig variety, improves the identification and evaluation efficiency of the Rongchang pig variety, and has remarkable application value.
In order to achieve the above object, the present invention provides an application of a SNP locus combination related to Ronchang pig variety identification in Ronchang pig variety identification, wherein the SNP locus combination comprises 54 SNP loci as shown in the following table 1:
TABLE 1 54 SNP site information
Wherein the SNP site combination is located on genomic version EnsemblSscofa 11.1.
The invention also provides a gene chip for Ronchang pig variety identification, which is prepared by developing SNP locus combinations related to Ronchang pig variety identification, wherein the SNP locus combinations comprise 54 SNP loci, specific information is shown in the table 1, and the SNP locus combinations are positioned on genome version EnsemblSscofa 11.1.
The invention also provides a kit for identifying the Ronchang pig variety, which comprises the gene chip.
The gene chip or the kit provided by the invention can be applied to the variety identification of Yu Rongchang pigs and the genetic relationship analysis of Rong Chang pigs, and has remarkable application value.
The SNP locus combination for identifying the Rongchang pig variety solves the problem of identification of the Rongchang pig variety and has the following advantages:
the method combines the genotype data of the pigs to be detected with the genotype data of the 5 varieties by using PLINK software, then performs principal component analysis and visualizes the results by using R language, and can clearly and accurately judge that the varieties of the pigs to be detected are Ronchang pigs when the genetic distances between the individual pigs to be detected and Rong Chang pig groups are relatively close and are clustered.
The invention discloses a method for rapidly and accurately identifying and evaluating Rongchang pig breeds and SNP locus combinations of Rongchang pig breeds, wherein the Rongchang pig breeds are researched through SNP molecular markers, bioinformatics technology comprehensive analysis is adopted, the research results are initially verified, the breed specific molecular markers of Rong Chang pigs are constructed, the differences between Rong Chang pigs and other local pig breeds can be simply and clearly distinguished, the method can be used for authenticity identification and genetic relationship analysis of Rongchang pig breeds, and effective scientific basis is provided for intellectual property protection of Rongchang pig breeds. When the Ronchang pig variety is identified and evaluated, whether the Ronchang pig variety is the Ronchang pig variety can be determined only by comparing and analyzing the Ronchang pig variety with the existing specific molecular marker. The time and the cost for identifying and evaluating the Ronchang pig variety are greatly reduced, and the efficiency of identifying and evaluating the Ronchang pig variety is improved.
Drawings
FIG. 1 is a Manhattan and QQ plot of the results of a GWAS analysis of a Rong Chang pig of the present invention.
FIG. 2 is a Manhattan plot showing the results of analysis of selection signals from Rong Chang pigs according to the present invention.
FIG. 3 shows the results of principal component analysis verification of the combination of SNP loci according to the invention selected from Rong Changzhu.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1 specific SNP site screening of Ronchang pigs
A specific SNP locus screening method for identifying Ronchang pig breeds comprises the following steps:
(1) Ear sample collection
The experimental group contained in the experiment is ear tissues of 250 pigs, and the total number of the pig breeds is 5, including 3 Chinese pig breeds: rong Changzhu (n=131, rcz), ba Ma Zhu (n=85, bmxxz), dao xi pig (n=21, qxz); western commercial pigs 2: long white pigs (n=7, cbz), yoke pigs (n=6, ykz). All ear tissue samples were labeled and placed in 2ml centrifuge tubes containing 75% alcohol, respectively, and stored in a-20 ℃ freezer.
(2) Total DNA extraction, quality detection and genotyping
Extracting total DNA by using an animal tissue genome DNA extraction kit; detecting the DNA quality by adopting a DYY-6C type electrophoresis apparatus and using 1% agarose gel electrophoresis; detecting the concentration of DNA by using a Nanodrop-2000 ultraviolet spectrophotometer, and reserving a genome DNA sample with the light absorption ratio (A260/280) between 1.8 and 2.0 and the concentration of more than or equal to 50 nanograms per microliter, wherein the genome DNA sample is used for carrying out whole genome chip typing by using a Illumina Porcine SNP50Beadchip (Beijing Kang Pusen Biotechnology Co., central core number one), and the specific operation is as follows:
a. a gene library was created for the sample pigs using Tn5 transposase and a 50K gene chip scan was performed.
b. Genotyping was performed on the 50K chip and whole genome resequencing results in step (1) using Beagle.
c. Performing whole genome association analysis and selection signal analysis on the genotype filling data obtained in step b for all individuals.
d. C, calculating allele frequency among varieties of the obvious loci obtained in the step c, and reserving SNP loci with higher allele frequency of Rong Changzhu, wherein the SNP loci are collected and used as a Ronchang pig variety specific SNP locus collection.
(3) Genotype data filling and quality control
32,457 SNPs in a total of 250 samples were obtained by chip sequencing. Chip data were quality controlled using PLINK software. Genotype data was filtered by the following parameters: the detection rate of the individual genotypes (- -mini) >90%, the detection rate of the marker genotypes (- -geno) is >95%, the minimum allele frequency (- -maf) >1%, and the minimum Hardy-Guerbet balance (- -hwe) is 1.0E-6, and the marker is located on an autosome. Filling of the missing genotypes was performed in the BEAGLE software using a hidden Markov model (Hidden Markov Model, HMM) algorithm.
(4) Screening SNP specific sites by whole genome association analysis
The whole genome association analysis was performed using GEMMA software, and the test group was 131 Ronchang pigs (case) and the control group was the remaining 4 varieties (control) in (1). Wherein, the manhattan diagram of the GWAS analysis Rong Chang pig is shown as A in fig. 1, the QQ diagram of the Rong Chang pig is shown as B in fig. 1, two threshold lines are shown in the manhattan diagram, wherein the threshold of the solid line is 0.05/N (N is the number of used chip sites), the sites above the solid line are all genome significant levels, the threshold of the dotted line is 1/N, the sites above the dotted line are chromosome significant levels, and the lambda value in the QQ diagram is closer to 1, indicating that the result of the all genome association analysis is more reliable; SNPs significantly related to variety were identified using the Banferoni correction method, with the pool of significant SNPs being group A.
(5) Selection signal analysis for screening SNP specific sites
The calculation of the genetic differentiation index (genetic differentiation index, fst) is performed by using VCFtools software, and a calculation method of sliding window averaging is used to obtain a manhattan diagram of the analysis result of the selection signal of the rongchun pig, as shown in fig. 2, it is known that the threshold line in fig. 2 is the first 1% of the Fst value which is larger after being sequenced, and the sites above the threshold line are significant sites (red marks). The specific parameters are as follows: the size of the sliding window (- -fst-window-size) is 100,000bp, and the step size of the sliding window (- -fst-window-step) is 40,000bp. Ordering the windows from big to small according to Fst values, defining the first 1% of windows as significant windows, and extracting SNPs in the significant windows by PLINK software, wherein the collection of the significant SNPs is group B.
(6) Allele frequency screening SNP specific loci
Combining the remarkable SNPs of the A group and the B group by using PLINK software, calculating the allele frequency of each SNP in 5 varieties, screening a SNP set with higher distribution of the allele frequency in a test group than that of other 4 varieties as specific SNP locus combinations of Ronchang pig varieties to obtain 54 SNP loci, wherein specific SNP locus information is shown in the table 1, wherein Chr1, chr2, … … and Chr16 represent chromosomes where the loci are located, subsequent numerical values represent the physical positions of the loci on the genome of the chromosomes, and the SNP loci are located on a genome version EnsemblSscofa 11.1.
The 54 SNPs of 5 varieties are extracted by PLINK software, and principal component analysis (PCA analysis) verification is carried out, and the result is shown in figure 3, so that all Rong Chang pig individuals are obviously different from other pig individuals, and the SNP loci provided by the invention can be used for identifying the varieties of pigs and the genetic relationship of the varieties.
Application example 1 SNP locus combination in Ronchang pig variety identification
The application example identifies the variety of the pig to be tested, and comprises the following steps:
1. extracting an ear tissue sample of a pig to be detected, extracting genome DNA of the tissue sample, sending the genome DNA to Beijing Kang Pusen biotechnology limited company, and carrying out SNP typing on the 54 SNP loci by using an Axiom chip aiming at local pigs so as to obtain genotype data of the pig to be detected.
2. And combining the genotype data of the pig to be detected and the genotype data of the Rong Chang pig by using PLINK software, extracting the above 54 SNP locus information in the data, and then carrying out principal component analysis, wherein when the genetic distance between the individual pig to be detected and the 131 Ronchang pig group in the figure 3 is short, the pig to be detected can be judged to be Ronchang pig.
In conclusion, the SNP locus combination obtained by screening can be used for variety identification of Rongchang pigs, so that a gene chip for identifying Rongchang pig varieties can be developed and prepared according to the obtained SNP locus combination, a kit for identifying Rongchang pig varieties can be prepared, the variety or germplasm resources of pigs can be detected in batches, and the kit has remarkable application value.
While the present invention has been described in detail through the foregoing description of the preferred embodiment, it should be understood that the foregoing description is not to be considered as limiting the invention. Many modifications and substitutions of the present invention will become apparent to those of ordinary skill in the art upon reading the foregoing. Accordingly, the scope of the invention should be limited only by the attached claims.
Claims (5)
1. The application of SNP locus combination related to Ronchang pig variety identification in Ronchang pig variety identification is characterized in that the SNP locus combination comprises 54 SNP loci, which are respectively as follows:
wherein the SNP locus combination is positioned on a genome version EnsemblSscofa 11.1.
2. The gene chip for Ronchang pig variety identification, which is prepared by SNP locus combination development related to Ronchang pig variety identification, is characterized in that the SNP locus combination is as follows:
wherein the SNP locus combination is positioned on a genome version EnsemblSscofa 11.1.
3. A kit for identifying a rongchun pig breed, comprising the gene chip of claim 2.
4. Use of the gene chip according to claim 2 or the kit according to claim 3 for the identification of Ronchang swine breeds.
5. Use of the gene chip of claim 2 or the kit of claim 3 in Rong Chang pig genetic relationship analysis.
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