CN113278711B - Gene chip, molecular probe combination, kit and application for sheep germplasm resource identification and pedigree reconstruction - Google Patents
Gene chip, molecular probe combination, kit and application for sheep germplasm resource identification and pedigree reconstruction Download PDFInfo
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Abstract
The invention discloses a gene chip, a molecular probe combination, a kit and an application for sheep germplasm resource identification and pedigree reconstruction, relates to the technical field of biology, and provides 4213 SNP site combinations and a gene chip, a molecular probe combination and a kit prepared based on the SNP site combinations, wherein the physical positions of the sites are shown in table 1, so that the gene chip, the molecular probe combination and the kit can be used for sheep germplasm resource identification and sheep pedigree reconstruction, and can be used for variety identification, variety traceability and sheep breeding control of sheep individuals and products thereof.
Description
Technical Field
The invention relates to the technical field of biology, in particular to the technical field of biological detection, and more particularly relates to a gene chip, a kit and application for sheep germplasm resource identification and pedigree reconstruction.
Background
For thousands of years, humans have applied selective pressure during animal breeding to produce livestock that exhibit certain beneficial characteristics. These features are selected to meet aesthetic, technical, religious, social and economic needs. The result is the production of a large number of different animal species. The term "breed" is a specialized term used to define a homogeneous, sub-population of livestock within which the livestock have definable and identifiable external characteristics that enable them to be distinguished from other similarly defined populations in the same species by visual assessment. Thus, this term defines a group of animals to which humans apply selective pressure to obtain the same external characteristics that are heritable and distinguishable from other members of the species. As breeds are established, their integrity is maintained by breed populations, stock pedigrees and pedigree records. Traditional breed selection methods are based on direct determination of the phenotype of the animal and/or its relatives. Therefore, the completion of breeding strategies requires the maintenance of extensive phenotypic records. For example, dairy livestock improvement programs in the united states and western europe rely in part on the collection of millions of individual records of cows (milk yield and composition, species traits, health traits, etc.) on a monthly basis. However, some important features do not immediately manifest at the level of surviving animals. For example, some meat quality parameters are determined by subtle physiological or biochemical characteristics that are not readily observable and therefore cannot be used as a basis for effective manual selection. Traditional breeding selection methods are limited by the fact that some phenotypes are expressed in only one sex or at a specific developmental stage. In addition, some phenotypes are difficult to determine and expensive to test.
With the continuous progress of biological theory and technology, the identification method of livestock and poultry varieties has been developed into a method for judging the sources, the affiliations, the genetic relationship between the sources and the affiliations according to the marker characteristics of animal groups such as morphology, cytology, biochemistry and molecules. The molecular genetic marker is a genetic marker based on nucleotide sequence difference in genetic materials among individuals, is a direct reflection of genetic variation on a DNA level, has extremely large quantity of markers, has high polymorphism and stable heredity throughout the whole genome, is not limited by environment and whether gene expression or not, and can take tissues, organs and even cells in different development stages when detecting the markers. Therefore, compared with morphological, cytological and biochemical markers, the molecular marker is a more common marking mode for identifying the livestock and poultry varieties at present. Molecular marker detection technology has been developed over three generations, namely, the first generation based on restriction fragment length polymorphism markers, randomly amplified polymorphic DNA, amplified fragment length polymorphism, the second generation based on microsatellite markers, and the third generation based on single nucleotide polymorphism. The DNA structure directly determines the genetic material, is the root cause of the species identity, and genetic differences of different species or classes may be reflected to varying degrees in their sequence. DNA molecular genetic markers provide an effective solution to overcome this problem.
Disclosure of Invention
In order to meet the detection requirements of the current sheep variety research, variety identification, pedigree reconstruction and breeding in China, the invention provides the locus combination only consisting of 4213 loci, and the molecular probe combination, the gene chip and the kit which are prepared based on the locus combination.
In order to achieve the purpose of the invention, the first aspect of the invention provides a molecular probe combination for identifying sheep germplasm resources, wherein the molecular probe combination detects SNP locus combinations shown in Table 1 in a sample to be detected:
TABLE 14213 site information
The physical location information for the site combinations in table 1 is determined based on sheep v4.0 genomic sequence alignment.
In order to achieve the purpose of the invention, the second aspect of the invention provides a gene chip for identifying sheep germplasm resources, wherein the gene chip is loaded with the molecular probe combination of the first aspect.
In order to achieve the purpose of the invention, the third aspect of the invention provides a kit for identifying sheep germplasm resources, which comprises the molecular probe combination of the first aspect or the gene chip of the second aspect.
In order to achieve the purpose of the invention, the fourth aspect of the invention provides a molecular probe combination for sheep genealogy reconstruction, wherein the molecular probe combination detects SNP locus combinations shown in table 1 in a sample to be detected, and the physical position information of the locus combinations in table 1 is determined based on sheep v4.0 genome sequence alignment.
In order to achieve the object of the present invention, the fifth aspect of the present invention provides a gene chip for sheep pedigree reconstruction, wherein the gene chip is loaded with the molecular probe combination described in the fourth aspect.
In order to achieve the object of the present invention, the sixth aspect of the present invention provides a kit for sheep pedigree reconstitution, which has the molecular probe combination of the fourth aspect or the gene chip of the fifth aspect
In order to achieve the purpose of the invention, the seventh aspect of the invention provides a method for identifying sheep germplasm resources, which uses the molecular probe combination of the first aspect or the gene chip of the second aspect or the kit of the third aspect to detect sheep samples.
In order to achieve the object of the present invention, the eighth aspect of the present invention provides a method for identifying a sheep product, which uses the molecular probe combination of the first aspect or the gene chip of the second aspect or the kit of the third aspect to detect the sheep product.
In order to achieve the object of the present invention, a ninth aspect of the present invention provides a method for determining the breed origin of a sheep product, wherein the molecular probe combination of the first aspect or the gene chip of the second aspect or the kit of the third aspect is used for detecting the sheep product.
The sheep products include, but are not limited to, commercially available sheep meat and processed products thereof, sheep fat and processed products thereof, sheep wool and processed products thereof, and the like.
The molecular probe combination of the first aspect, the gene chip of the second aspect, or the kit of the third aspect has the use of any one of the following: (1) the application in sheep germplasm resource identification; (2) the application in sheep pedigree reconstruction; (3) the application in screening sheep varieties; (4) the application in sheep variety identification; (5) the application in tracing sheep varieties; (6) the application in sheep breeding; (7) the application in sheep germplasm resource protection; (8) the application in sheep germplasm resource improvement; (9) the application in identification of sheep products; (10) the application in determining the variety source of sheep products.
Has the advantages that:
based on the genetic resource research on more than 97 percent of sheep varieties in China, the invention discovers that the sheep varieties can be identified by utilizing 4213 SNP site combinations, the method is used for sheep genealogy reconstruction, and the sheep individuals or products thereof are subjected to variety or source identification, so that the method is beneficial to shortening the breeding process, ensures the quality of the sheep products, and simultaneously provides technical support for germplasm resource protection and germplasm resource improvement.
Compared with other chips, the probe combination, the gene chip and the kit which are made based on the sites provided by the invention have more uniform distance distribution of each SNP site, and improve the efficiency of the sites in research such as correlation analysis, selected signal positioning and the like; and the method also has the characteristics of small flux, low cost, easier analysis, wide universality and wide market prospect.
The site information provided by the invention, the prepared probe combination, the gene chip and the kit can also be used for quality control of sheep product raw materials.
Drawings
FIG. 1 shows the number of individuals identified for 47 sheep breeds studied in accordance with the present invention, wherein 47 colors represent 47 breeds, which are abbreviated as representing different sheep breeds, respectively, as shown in Table 2: TABLE 247 sheep breed information
Detailed Description
The invention is further illustrated by reference to the following detailed description of specific embodiments, which are intended to be illustrative only and not to be construed as limiting the invention. Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art, and can be performed with reference to the third edition of the original book "bioinformatics and functional genomics" or related books, and bioinformatics software and products used therein are commercially available. Various procedures and methods not described in detail are conventional methods well known in the art, and the sources of materials used, trade names, and components thereof, if necessary, are indicated at the time of first appearance, and the same reagents used thereafter, if not specifically indicated, are the same as those indicated at the time of first appearance.
In addition, it should be noted that the site combination and application provided by the present invention are completed by the inventors of the present application through hard creative work and optimization work.
The features and advantages described in the site combination section above are also applicable to the molecular probe combination, gene chip, kit and application thereof formed based on the site combination, and are not described herein again.
Example 14213 acquisition of combination of SNP sites
1 sample Collection
The invention collects 41 native sheep varieties in China, including 6 breeding varieties (including Charolay, Dorper, Australian merino, poll Dorsey, Wu-Dong, Yunan, Guizhou and Jiangsu) from Xinjiang, Tibet, Ningxia, Henan, inner Mongolia, Shanxi, Qinghai, Shandong, Yunnan, Guizhou and Jiangsu, and the like, and totally 47 varieties, 2012 sheep individuals, and variety information is shown in Table 2.
2. Extraction of DNA
The method comprises the steps of collecting auricular tissues of sheep samples to extract DNA of each sheep variety, collecting the auricular tissues of sheep in Xinjiang, Tibet, Ningxia, Henan, inner Mongolia, Shanxi, Qinghai, Shandong, Yunnan, Guizhou, Jiangsu and the like, preserving the collected auricular tissues in a tube by using 95% -100% ethanol, preserving the auricular tissues at-80 ℃, extracting genome DNA from the auricular tissues according to a phenol-chloroform extraction program to generate a complete genome data set, wherein 2012 DNA samples are obtained in total, comparing the genome DNA with a sheep 4.0 reference genome (obtained from NCBI) released in 2015 by using SAMtols and GATK, and obtaining a common result by using the two modes to form an SNP set to obtain 48201 SNP sites in total.
3. SNP BeadChip genotyping
2012 samples were genotyped with Illumina Ovine SNP50 developed by the international sheep genomics society (ISGC) to generate ped and map files.
4. Quality control
Quality control of the data was performed using plink1.9 software, according to: 1) deleting SNP sites with high SNP deletion rate, wherein geno is more than 0.05; 2) deleting SNP with low frequency of minor allele, wherein MAF is more than 0.01; 3) deletion of SNPs that do not meet Hardy-Weinberg equilibrium, hwe > 0.00001; 4) the deletion of the point of Linkage Disequilibrium (LD), indep-pair 100250.05, when r2>0.05, this function calculates a pair of LD estimates r2 moving at the speed of 25 SNPs, excluding one of a pair of SNPs, in the 100-SNPs window. Variability estimates based on the bearchipsnps may have definite biases in SNP discovery. The elimination of the snp in the high level LD has been shown to offset the effects of the determination bias, so that the determination bias can be greatly reduced by pruning the LD in the quality control program. 5) Chromosomes 0 and 27 are deleted. Through the above operations, 20748 SNPs and 2012 sheep individual samples were obtained.
5. Selection signal analysis
SNPs with significantly higher Fst values (the first 0.1% of the total number of SNPs) are considered as predictive signals under strong manual selection. The method comprises the steps of firstly converting an original data file into a vcf format by using vcftools software, then calculating a group fst value by using a shell script, and deleting 0.1% of sites before top to obtain 20727 SNPs and 2012 individuals.
6. Screening of the sites for chip formation data
And uniformly distributing and screening according to the chromosome base distance to finally obtain 4213 SNP loci.
4213 sites of ped and map files were extracted using the plink1.9 software- -extract command.
7. Genetic relationships and population architecture
Distance-matrix generation using the distance-matrix command of plink1.9 software followed by construction of neighbor-join trees for all individuals with split 5 and visualization through Figtree1.4.4 resulted in individual trees for 2012 sheep individuals.
Deleting the individuals which cannot be gathered together, continuing mapping, repeating for a plurality of times, and obtaining 1524 sheep individuals through 7 times of deletion, wherein 4213 sites are obtained, finally, the chip comprises 4213 SNP sites, 1524 sheep individuals, the obtained sites are shown in the table 1, and the number of the sheep individuals is shown in the table 1.
Based on the site information provided by the present invention, those skilled in the art can prepare molecular probes, gene chips, and kits according to the conventional methods in the art, but products prepared based on the conventional methods in the art and the site information provided by the present invention are within the scope of the present invention.
Example 2 sheep breed identification method
Collecting individual samples of sheep to be detected, collecting peripheral blood or tissues to extract DNA, obtaining ped and map files after DAN sequencing analysis and calculation by a biological company, combining the files with the site information data provided by the invention, using split tree5 and figtree software to make individual trees, and determining the varieties of the individual trees according to the gathering of the samples to be detected on the branches of the varieties.
Industrial applications
The locus information provided by the invention can be applied to sheep germplasm resource identification, sheep pedigree reconstruction, sheep variety screening, sheep variety identification, sheep variety traceability, sheep breeding, sheep germplasm resource protection and sheep germplasm resource improvement, and can also be used for sheep product identification and sheep product variety source determination.
The above description is only a preferred example for helping understanding the present invention, and is not intended to limit the present invention, and those skilled in the art can make various changes and modifications to the present invention without departing from the spirit of the present invention, and those skilled in the art should make various changes and modifications to the present invention without departing from the spirit of the present invention.
Claims (10)
1. Identifying a molecular probe combination of sheep germplasm resources, wherein the molecular probe combination detects 4213 SNP site combinations shown in table 1 in a sample to be detected, and the physical position information of the site combinations in table 1 is determined based on sheep v4.0 genome sequence alignment.
2. A gene chip for identifying sheep germplasm resources, wherein the gene chip is loaded with the molecular probe combination of claim 1.
3. A kit for identifying sheep germplasm resources, which comprises the molecular probe combination of claim 1 or the gene chip of claim 2.
4. The molecular probe combination for sheep genealogy reconstruction is characterized in that the molecular probe combination detects SNP locus combinations shown in table 1 in a sample to be detected, and the physical position information of the locus combinations in table 1 is determined based on sheep v4.0 genome sequence alignment.
5. A gene chip for sheep pedigree reconstruction, said gene chip loaded with the molecular probe combination of claim 4.
6. A kit for sheep pedigree reconstitution, which comprises the molecular probe combination of claim 4 or the gene chip of claim 5.
7. A method for identifying sheep germplasm resources, which uses the molecular probe combination of claim 1 or the gene chip of claim 2 or the kit of claim 3 to detect sheep samples.
8. A method for identifying sheep products, which comprises detecting sheep products by using the molecular probe combination of claim 1 or the gene chip of claim 2 or the kit of claim 3.
9. A method for determining the breed origin of sheep products, which comprises the step of detecting the sheep products by using the molecular probe combination as claimed in claim 1, the gene chip as claimed in claim 2 or the kit as claimed in claim 3.
10. The use of the molecular probe combination of claim 1, the gene chip of claim 2 or the kit of claim 3, which is any one of the following:
(1) the application in sheep germplasm resource identification;
(2) the application in sheep pedigree reconstruction;
(3) the application in screening sheep varieties;
(4) the application in sheep variety identification;
(5) the application in tracing sheep varieties;
(6) the application in sheep breeding;
(7) the application in sheep germplasm resource protection;
(8) the application in sheep germplasm resource improvement;
(9) the application in identification of sheep products;
(10) the application in determining the variety source of sheep products.
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| PCT/CN2022/106905 WO2023001210A1 (en) | 2021-07-21 | 2022-07-20 | Gene chip, and kit for sheep germplasm resource identification and pedigree reconstruction, and use |
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