CN114959067A - Specific molecular identity card for identifying germplasm resources of Ningshan black pigs and application of specific molecular identity card - Google Patents

Specific molecular identity card for identifying germplasm resources of Ningshan black pigs and application of specific molecular identity card Download PDF

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CN114959067A
CN114959067A CN202210815775.9A CN202210815775A CN114959067A CN 114959067 A CN114959067 A CN 114959067A CN 202210815775 A CN202210815775 A CN 202210815775A CN 114959067 A CN114959067 A CN 114959067A
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李新建
李秀领
李聪
段栋栋
韩雪蕾
王克君
乔瑞敏
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Henan Agricultural University
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Abstract

The invention belongs to the field of pig breed identification, relates to a germplasm resource of a Ningshan black pig, and particularly relates to a specific molecular identity card for identifying the germplasm resource of the Ningshan black pig and application thereof. The research on the variety of the black pig in the Jiang shan is carried out through the SNP molecular marker, the bioinformatics technology is adopted for comprehensive analysis, the research result is preliminarily verified, and the variety specific molecular marker of the black pig in the Jiang shan is constructed, so that the difference between the variety of the black pig in the Jiang shan and the variety of other local pigs can be simply and clearly distinguished, the method can be used for the authenticity identification and genetic relationship analysis of the variety of the black pig in the Jiang shan, and an effective scientific basis is provided for the intellectual property protection of the variety of the black pig in the Jiang shan. When the variety of the black pig in the Ningshan mountain is identified and evaluated, whether the variety of the black pig in the Ningshan mountain is the variety of the black pig in the Ningshan mountain can be determined only by comparing and analyzing the variety of the black pig in the Ningshan mountain and the existing specific molecular identity card. The method greatly reduces the time and cost for identifying and evaluating the germplasm resources of the black pig in the Shashan mountain, and improves the efficiency for identifying and evaluating the breed of the black pig in the Shashan mountain.

Description

Specific molecular identity card for identifying germplasm resources of Ningshan black pigs and application of specific molecular identity card
Technical Field
The invention belongs to the field of pig breed identification, relates to a germplasm resource of a Ningshan black pig, and particularly relates to a specific molecular identity card for identifying the germplasm resource of the Ningshan black pig and application thereof.
Background
The bristles of the black pig in the mountain are thick and short, and have a large body, wherein the body length is 136 cm, and the body height is about 70 cm. The face has diamond-shaped wrinkles, two ears droop, the waist and the back are flat, the abdomen of the sow slightly droops, the hip is slightly declined, and the tail is thicker and longer than the festivals. It features gentle sexual condition and suitable for stocking. The central producing area of the black pig is in three villages of Zhugou, Wagang and Shizhao in the two parts of the Jian. The adjacent countries such as the ren shop, the ant bee, the temple, the suburb, the chocolate cave, the Lixin shop and the Xinyang road, the old river and the like are also distributed. According to the investigation of nine villages, the existing Jianshan black pig has more than ten thousand, eight thousand and nine hundred heads.
In 2009 and 3 months, the national livestock and poultry genetic resource committee pig professional committee expert group performed on-site investigation and identification on the black pig in the county of the Zhengshan of the Zhima shop. Through examination, the confirmed black pig meets the basic conditions about pig genetic resource identification in the complete set line examination and livestock genetic resource identification method of new livestock and poultry of the Ministry of agriculture, and agrees to pass the preliminary examination and go to the approval of the committee of livestock and poultry genetic resources. The effective protection and reasonable development of local varieties are beneficial to the sustainable development of the pig industry in Henan province and the abundance of livestock resource diversity. Particularly, the research on the specific genetic structure and the characteristics of the local pig breed is helpful for making a protection plan for each breed according to the genetic condition of the breed, and promoting the specific protection of the local pig breed. Preserving the unique variations, genes and characteristics of each variety is extremely important for maintaining biodiversity and adapting to future environmental changes. Therefore, the identification of the unique genetic characteristics of the local pig breed in Henan by using the bioinformatics technology is an important part for accurately protecting the genetic resources of the local pig breed.
Single Nucleotide Polymorphism (SNP) markers are third-generation molecular markers, and refer to a polymorphism generated by mutation of a single base on a genomic DNA sequence, wherein the mutation comprises single base transversion, conversion, insertion and deletion. The SNP has the characteristics of wide distribution, high frequency, low mutation rate and the like, and is widely applied to genome analysis, biological information automatic detection, genetic research of diseases, livestock breeding markers and other researches.
Genome-wide association assays (GWAS) are an important method commonly used for livestock genetic resource Genome analysis. With the development of sequencing technology, chip sequencing technology becomes a powerful tool for high-throughput SNP typing. At the same time, when animals are subjected to long-term natural and artificial selection, corresponding genetic imprints are left on their genomes. These genetic imprints are commonly referred to as selection signals. The study of selection signals is a research strategy based on the concept of genome-to-phenotype. Due to the lack of local pig species phenotype records and small population scale in China, the analysis of the germplasm characteristics of livestock increasingly becomes an important method. In order to provide a method for batch identification of germplasm resources of black pigs in the mountains, the subject group has been subjected to long-term follow-up research.
Disclosure of Invention
In order to realize the purpose, the invention provides a specific molecular identity card for identifying the germplasm resources of the Ningshan black pig and application thereof.
The technical scheme of the invention is realized as follows:
a specific molecular identity card for identifying germplasm resources of a Ningshan black pig is disclosed, wherein the SNP loci are a set of SNP loci with higher allele frequency among Ningshan black pig breeds.
Preferably, the SNP site is located in the porcine reference genome EnsemblScrofa version 11.1.
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, CNC 10010090798 site, CNC10091437 site, CNC10091643 site, CNC10092360 site, CNC10092481 site, CNC10092509 site, CNC10092580 site, CNC10130757 site, CNC 1013131664 site, CNC10133552 locus, CNC 41731 site, CNC10141931 locus, CNC 10142931 locus, CNC 42249 site, CNC 101846 site, CNC 101525152515252617 site, CNC 10110152559 site, CNC 101707088158 site, CNC 88158 site and CNC 10041138158 site.
Preferably, the mutant of CNC10010832 site is T/G, CNC10012972 site mutant is G/A, CNC10013000 site mutant is A/G, CNC10013872 site mutant is C/T, CNC10040563 site mutant is T/G, CNC10041271 site mutant is C/T, CNC10041332 site mutant is T/C, CNC10041501 site mutant is T/C, CNC10041536 site mutant is C/T, CNC10041599 site mutant is G/A, CNC10041838 site mutant is C/T, CNC10042298 site mutant is C/T, CNC 60925 site mutant is C/T, CNC10062082 site mutant is A/G, CNC10062105 site mutant is T/C, CNC10062442 site mutant is A/3638 site mutant is A/G100G, CNC site mutant is A/G1004238 site, The mutant of CNC10090798 site is T/C, CNC10091437 site, the mutant is C/T, CNC10091643 site, the mutant is T/T, CNC10092360 site, the mutant is T/C, CNC10092481 site, the mutant is T/C, CNC10092509 site, the mutant is A/G, CNC10092580 site, the mutant is G/G, CNC10130757 site, the mutant is G/A, CNC 101664 site, the mutant is C/T, CNC10133552 site, the mutant is A/G, CNC10141731 site, the mutant is T/10141931 locus, the mutant is C/T, CNC10142249 site, the mutant is A/G, CNC10142250 site, the mutant is G/T, CNC 1015152169 site, the mutant is G/5852617 site, the mutant is G/58709 site, the mutant is G/101552 888 site, the mutant is C/T/10170709 site, The mutant at the CNC10170889 site is T/C, CNC10171158 site, and the mutant is C/T.
The gene chip is used for identifying the specific molecular identity card.
The gene chip is applied to identifying the variety of the Ningshan black pig.
The method comprises the following steps:
(1) collecting a tissue sample of a pig to be detected, and extracting genome DNA;
(2) carrying out SNP typing on the genome DNA in the step (1) by using a gene chip to obtain the genotype data of the pig to be detected;
(3) and combining the genotype data of the pig to be detected with the genotype of the SNP locus on the specific molecular identity card by utilizing PLINK software, and then carrying out principal component analysis.
Further, in the step (1), the light absorption ratio of the genome DNA at A260/280 is between 1.8 and 2.0, and the concentration is more than or equal to 50 ng/microliter.
Further, the result of the main component analysis of SNP typing in the step (3) is close to the genetic distance of the black pig group in the Jianshan, and when the result is gathered into a cluster, the black pig in the Jianshan is obtained.
The invention has the following beneficial effects:
1. and combining the genotype data of the to-be-detected pig and the genotype data of the above 10 varieties by using PLINK software, then performing principal component analysis and visualizing the result by using R language, and when the genetic distance between the individual to-be-detected pig and the pan-chung pig group is short and the individual to-be-detected pig is gathered into a cluster, as shown in figure 3, judging that the to-be-detected pig is the pan-chung pig.
2. The invention discloses a method for rapidly and accurately identifying and evaluating the germplasm resources of a Jian shan black pig and a Jian shan black pig breed specific molecular identity card. When the variety of the black pig in the Ningshan mountain is identified and evaluated, whether the variety of the black pig in the Ningshan mountain is the variety of the black pig in the Ningshan mountain can be determined only by comparing and analyzing the variety of the black pig in the Ningshan mountain and the existing specific molecular identity card. The method greatly reduces the time and cost for identifying and evaluating the germplasm resources of the black pig in the Shashan mountain, and improves the efficiency for identifying and evaluating the breed of the black pig in the Shashan mountain.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 shows Manhattan chart and QQ chart of GWAS analysis results of the Kingshan black pig of the present invention.
FIG. 2 is a Manhattan plot of the results of the selective signal analysis of the Gongshan black pig of the present invention.
FIG. 3 is a principal component analysis and verification chart of the molecular identity card specific to the variety of the black pig in the Chun mountain of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.
Examples
A method for screening a specific molecular identity card of a germplasm resource of a Ningshan black pig comprises the following steps:
(1) ear sample collection
The test population is 1117 pigs, and the total number of the pig breeds is 10, including 7 Chinese pig breeds: black southern yang pigs (n =10, NY), huai nan pigs (n =10, HN), yunong black pigs (n =1036, YN), chun black pigs (n =10, QS), lekuwa pigs (n =10, LWH), dihedral face pigs (n =10, EHL), MIN pigs (n =6, MIN); 3 commercial western pig breeds: duroc pigs (n =10, DU), large white pigs (n =10, LW), long white pigs (n =5, LR). Cleaning pig ear with 75% alcohol, cutting a small amount of ear tissue with ear-like forceps, placing in 2ml centrifuge tube filled with 75% alcohol, and storing in-20 deg.C refrigerator.
(2) Total DNA extraction, quality detection and genotyping
Extracting total DNA by using an animal tissue genome DNA extraction kit;
detecting with DYY-6C electrophoresis apparatus by 1% agarose gel electrophoresis;
the method adopts a Nanodrop-2000 ultraviolet spectrophotometer to detect the concentration of DNA, reserves 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 ng/microliter, is used for carrying out whole genome chip typing on Illumina Portine SNP50 Beadchip (Beijing Congpson biotechnology, Inc., center core I), and has the specific operation that:
a. a gene library is established for the sample pig by using Tn5 transposase, and 50K gene chip scanning is carried out.
b. And (3) carrying out genotype filling on the 50K chip and the whole genome re-sequencing result in the step (1) by using Beagle.
c. And c, performing genome-wide association analysis and selection signal analysis on the genotype filling data obtained by the step b on all individuals.
d. And c, calculating the allele frequency among the breeds of the remarkable sites obtained in the step c, reserving SNP sites with higher allele frequency of the black pig in the Jianshan, and collecting the SNP sites as the specific molecular identity card of the black pig breed in the Jianshan.
(3) Genotype data filling and quality control
A total of 1,117 SNPs of 51,315 were obtained by chip sequencing. Quality control is carried out on chip data by using PLINK software. The genotype data was filtered by the following parameters: the detection rate of individual genotypes (-mind) is more than 90%, the detection rate of marker genotypes (-gene) is more than 95%, the minimum allele frequency (-maf) is more than 1%, and the minimum Hawth Winberg balance (-hwe) is 10-6, and is located in an autosome. The filling of the missing genotypes is performed in the BEAGLE software using the Hidden Markov Model (HMM) algorithm.
(4) Whole genome correlation analysis and screening of SNP specific sites
The GEMMA software was used for whole genome association analysis, and the test group was 10 Touchai black pigs (case) and the control group was the remaining 9 breeds (control). Wherein the Manhattan graph of the Shashan black pig is shown in the left side of figure 1 and the QQ graph is shown in the right side of figure 1, two threshold lines are arranged in the Manhattan graph, wherein the threshold value of a solid line is 0.05/N (N is the number of chip sites used), the site above the solid line is the significant level of a whole genome, the threshold value of a dotted line is 1/N, the site above the dotted line is the significant level of a chromosome, and the more the lambda value in the QQ graph is close to 1, the more credible the result of the whole genome association analysis is shown; identifying SNPs significantly related to the breed using a Bancost correction method, the set of significant SNPs being group A.
(5) Selection signal analysis and screening of SNP specific sites
The VCFtools software is used to calculate the genetic differentiation index (Fst), and a calculation method of averaging with a sliding window is used, and the result is shown in fig. 2, where the threshold line in fig. 2 is the first 1% of the Fst value after sorting, 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 100,000 bp, and the step size of the sliding window (- -fst-window-step) is 40,000 bp. Sorting the windows according to the Fst value from large to small, defining the first 1 percent of windows as significant windows, and extracting the SNPs in the significant windows by utilizing PLINK software, wherein the set of the significant SNPs is a B group.
(6) Allele frequency screening of SNP specific sites
And combining the significant SNPs of the group A and the group B by utilizing PLINK software, calculating the allele frequency of each SNP in 10 varieties, and screening SNP sets with higher distribution of the allele frequency in a test group than other 9 varieties to serve as specific molecular identity cards of the variety of the black pig in the Chun mountain.
TABLE 1 specific molecular marker set for the variety of the black pig in the mountains
Figure DEST_PATH_IMAGE001
(7) The 39 SNPs of 10 varieties were extracted by PLINK software, and principal component analysis and verification were performed.
(8) The 39 SNP loci are applied to identifying the variety of the Ningshan black pig, and the SNP loci are positioned in the genome version EnsemblSscrofa 11.1.
Application example
The identification method of the pig breed to be detected specifically comprises the following steps:
1. extracting an ear tissue sample of a pig to be detected, extracting genome DNA of the tissue sample, and typing the 39 sites through a chip. The genomic DNA was sent to Beijing Congpson Biotechnology Ltd for SNP typing on the "center-in-the-core No. (Axiom) chip of the local pig. The principle of the SNP typing assay performed on the chip is based on a ligation reaction in which two probes function. The first is capture probes on a chip, which serve to immobilize target DNA fragments to the chip surface. The second is a chromogenic probe, responsible for staining the SNP chip (red and green fluorescence). The assay was run in two rounds of hybridization. The first round of hybridization is that the target DNA is hybridized with the chip, and the capture probe can grab the matched target DNA segment; the chromogenic probe hybridizes to the DNA fragment in a second round of hybridization. Then, only the chromogenic probe complementary to the target DNA fragment is ligated to the capture probe by the recognition of the ligase. And carrying out SNP typing under laser scanning by dyeing of a fluorescent marker to obtain the 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 black pig in the Jian mountain by using PLINK software, extracting the above 39 sites in the data, and then performing principal component analysis.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims (9)

1. A specific molecular identity card for identifying germplasm resources of a Kuishan black pig is characterized in that: the SNP loci are a SNP locus set with high allele frequency among varieties of the Ningshan black pigs.
2. The specific molecular identity card for identifying germplasm resources of black hogs in the mountains of claim 1, which is characterized in that: the SNP site is located in the porcine reference genome EnsemblScrofa version 11.1.
3. The specific molecular identity card for identifying germplasm resources of black hogs in the mountains of claim 2, which is characterized in that: 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, CNC 62082 site, CNC10062105 site, CNC10062442 site, CNC10062524 site, CNC 10010090798 site, CNC10091437 site, CNC 1009191919191site, CNC10092360 site, CNC10092481 site, CNC 100509 92509 site, CNC 92580 site, CNC10130757 site, CNC10131664 site, CNC10133552 locus, CNC10141731 site, CNC 41931 locus, CNC 41931, CNC10142249 site, CNC10142250 site, CNC 10152846 site, CNC 5252525152169 site, CNC 10110110110110110110110110110152617, 10110110110110110110110171552 site, CNC 7088158 site, CNC 10041158 site.
4. The specific molecular identity card for identifying germplasm resources of Chinemys reevesii as claimed in claim 3, wherein: the mutant of the CNC10010832 locus is T/C, CNC10012824 locus, the mutant of the T/G, CNC10012972 locus is T/A, CNC10013000 locus, the mutant of the A/G, CNC10013872 locus is C/T, CNC10040563 locus, the mutant of the T/G, CNC10041271 locus is C/T, CNC10041332, the mutant of the T/C, CNC10041501 locus, the mutant of the T/C, CNC10041536 locus is C/T, CNC10041599, the mutant of the G/100A, CNC 41838 locus is C/T, CNC10042298, the mutant of the C/T, CNC10062082 locus is A/G, CNC 62105, the mutant of the T/581006226 62442 locus is A/5862524, the mutant of the A/10062524 locus is A/G, CNC 100798 locus, the mutant of the T/100798 locus is T/1007972, and the mutant of the A/1004188 locus is C/1004172 locus, The mutant at the CNC10091437 site is C/T, CNC10091643 site, the mutant at the G/T, CNC10092360 site is T/C, CNC10092481 site, the mutant at the T/C, CNC10092509 site is A/G, CNC10092580 site, the mutant at the A/G, CNC10130757 site is G/A, CNC10131664 site, the mutant at the C/T, CNC10133552 site, the mutant at the A/G, CNC10141731 site is T/C, CNC10141931 site, the mutant at the C/10142249 site is A/G, CNC10142250 site, the mutant at the G/3985 10151846 locus, the mutant at the G/3910152169 site is G/C, CNC10152617 site, the mutant at the G/58101709 site, the mutant at the G/5810170888 site is C/T, CNC 101889 site, the mutant at the T/C T, CNC10092481 site is T/10092481 site, the mutant at the T/G, CNC site is T/101309 site, The mutant of the CNC10171158 locus is C/T.
5. A gene chip for identifying the specific molecular identity card of any one of claims 1 to 4.
6. The use of the gene chip of claim 5 in identifying a variety of a black pig.
7. Use according to claim 6, characterized by the steps of:
(1) collecting a tissue sample of a pig to be detected, and extracting genome DNA;
(2) carrying out SNP typing on the genome DNA in the step (1) by using a gene chip to obtain the genotype data of the pig to be detected;
(3) and combining the genotype data of the pig to be detected with the genotype of the SNP locus on the specific molecular identity card by utilizing PLINK software, and then carrying out principal component analysis.
8. Use according to claim 7, characterized in that: in the step (1), the light absorption ratio of the genome DNA at A260/280 is between 1.8 and 2.0, and the concentration is more than or equal to 50 ng/microliter.
9. Use according to claim 7, characterized in that: and (4) the result of the SNP typing principal component analysis in the step (3) is close to the genetic distance of the Jian black pig population, and the Jian black pigs are obtained when the result is gathered into a cluster.
CN202210815775.9A 2022-07-12 2022-07-12 Specific molecular identity card for identifying germplasm resources of Ningshan black pigs and application of specific molecular identity card Pending CN114959067A (en)

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