SNP site combination for identifying shenxian pigs, kit and application thereof

Abstract

The application provides a SNP site combination for identifying Shenxian pigs, application and a screening method of the SNP site combination, and relates to the technical field of molecular biology. The SNP site combination is composed of 10 SNP sites. The Shenxian pigs can be identified from the populations of Large White pigs, Duroc pigs, Lantang pigs, Min pigs and Beijing black pigs by using the SNP characteristic site combination.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to the identification of SNP locus combinations in Shenxian pigs, their application, and a screening method for said SNP locus combinations. Background Technology

[0002] Shenxian pig is an ancient domestic pig species distributed in the south-central part of Hebei Province, my country. Purebred Shenxian pigs possess breed advantages such as tolerance to roughage, strong disease resistance, tolerance to inbreeding, excellent meat quality, and high intramuscular fat content. However, the current population of purebred Shenxian pigs is small, making effective breeding and selection difficult. While utilizing their germplasm resources, there is an urgent need for conservation and propagation.

[0003] SNPs are a class of DNA sequence variations widely distributed in the genome. They are point mutations caused by the switching or transversion of a single base, are stable and reliable, and usually appear as digeles. As genetic markers, the allele frequencies of SNPs vary among different populations, thus they are widely used in species identification and other fields. Currently, breed identification based on whole-genome resequencing or high-density SNP microarray data is relatively expensive. Therefore, it is necessary to develop a new method suitable for identifying Shenxian pigs.

[0004] In view of this, the present invention screens a combination of SNP feature sites for the identification of Shenxian pigs, as well as its screening method and application. Summary of the Invention

[0005] The purpose of this invention is to provide SNP locus combinations for identifying Shenxian pigs, their application, and a screening method for these SNP locus combinations. This invention, for the first time, involves in-depth research, screening from a large population of SNP loci (139,547) to obtain SNP characteristic locus combinations suitable for identifying Shenxian pig germplasm resources. Applying these SNP characteristic locus combinations can distinguish Shenxian pigs from Large White, Duroc, Lantang, Min, and Beijing Black pig populations.

[0006] In one aspect, the present invention provides a combination of SNP loci for identifying Shenxian pigs, the combination of SNP loci consisting of the following 10 SNP loci:

[0007] SNP locus 1: C or T at position chr1:93005281

[0008] SNP site 2: C or G at position chr1:252504281

[0009] SNP site 3: C or T at position chr1:2876173

[0010] SNP site 4: A or G at position chr2:11972846

[0011] SNP locus 5: C or T at position chr2:24851224.

[0012] SNP locus 6: A or G at position chr3:45739362

[0013] SNP site 7: T or G at position chr4:13264289

[0014] SNP locus 8: chr9: C or T at position 111271153;

[0015] SNP locus 9: chr10:3286345, C or T, and

[0016] SNP locus 10: chr10: C or G at position 15307369;

[0017] The location of the SNP site was determined based on the pig Sscrofa11.1 reference genome sequence.

[0018] In one aspect, the present invention provides a reagent for detecting said SNP site combinations, said reagent being used to detect single nucleotide polymorphisms of said SNP sites; preferably, said reagent comprises PCR primers for amplifying products containing said SNP sites.

[0019] In this invention, primers can be designed for a gene sequence containing the aforementioned SNP sites, and the fragment can be amplified using quantitative real-time PCR. The SNP sites can then be determined based on the amplification results.

[0020] In one aspect, a gene chip for detecting said SNP site combinations, the gene chip comprising nucleotide probes for detecting said SNP sites; preferably, the nucleotide probes are immobilized on a solid support.

[0021] In the prior art, various detection methods for detecting polymorphisms of SNP molecular markers (SNP sites) are applicable to this invention. These include, but are not limited to, one or more of the following: SNP detection methods based on gel electrophoresis, DNA sequencing, DNA microarrays, denaturing high-performance liquid chromatography, or mass spectrometry.

[0022] In one aspect, the present invention provides the application of the said SNP site combination, the said reagent, or the said gene chip in pig breed identification and / or pig breeding.

[0023] In one implementation, the pig breed identification includes identifying Shenxian pigs from non-Shenxian pig breeds.

[0024] In one implementation, the non-Shenxian pig breeds include one or more of the following: Large White, Duroc, Beijing Black, Lantang, and Min pigs.

[0025] In this invention, conventional products can be designed based on the combinations of the aforementioned SNP loci for pig germplasm identification and typing (distinguishing or assisting in the identification of whether the tested pig is a Shenxian pig or a non-Shenxian pig). The SNP loci information provided by this invention can also be used for applications in pig germplasm resource improvement and breeding.

[0026] For example, a method for pig genetic improvement based on SNP locus combinations includes: determining the SNP locus information of breeding pigs and making corresponding selections based on the polymorphism of the SNP loci; culling breeding pig individuals with non-target genotypes at these loci.

[0027] In practice, pigs with the target genotype can also be selected for mating and breeding based on the genotype of the SNP site combination detected in the genome of the pig to be tested.

[0028] In one aspect, the present invention provides a method for identifying Shenxian pigs from Large White pigs, Duroc pigs, Beijing Black pigs, Lantang pigs, and Min pigs based on the aforementioned SNP locus combinations, comprising:

[0029] (a) Extracting genomic DNA from the pigs to be tested as a template;

[0030] (b) Amplify and sequence the target fragment containing the SNP site combination;

[0031] (c) Determine the breed of the pig based on the genotype of the corresponding SNP locus.

[0032] In one implementation, when the genotype results of the SNP sites show that the bases corresponding to SNP sites 1 to SNP sites 10 are CT / TT, GC / CC, CT / TT, GG / AG, CC / TC, AA / GA, GG / TG, TT / CT, TC / CC, and GG / CG, then it is a Shenxian pig.

[0033] In one aspect, the present invention provides a method for screening the aforementioned SNP site combinations, comprising the following steps:

[0034] (a) The whole genome SNP data obtained from the resequencing of Shenxian pigs were quality controlled and merged with the whole genome SNP data of Large White pigs, Duroc pigs, Beijing Black pigs, Lantang pigs and Min pigs in known public databases;

[0035] (b) Divide 70% of the individuals of different varieties in step (a) into the training set and the remaining 30% into the test set;

[0036] (c) Select SNPs using the maximum classification ability method;

[0037] (d) Combine the logistic regression classifier and the Bayesian classifier in machine learning methods to perform feature selection on the SNP information selected in step (c);

[0038] (e) Validate the feature sites selected in step (d) in the test set using principal component analysis.

[0039] In one implementation, step (a) involves selecting 39 Shenxian pigs to obtain whole-genome SNP data, and selecting and merging whole-genome SNPs from 16 Large White pigs, 22 Duroc pigs, 22 Beijing Black pigs, 5 Lantang pigs, and 5 Min pigs.

[0040] 70% (87 individuals in total) of the 6 breeds in step (a) were randomly assigned to the training set, and the remaining 30% (22 individuals in total) were assigned to the test set.

[0041] Preferably, the quality control standards are: SNP elimination detection rate < 99%, minimum allele frequency < 0.05, and Hardy-Weinberg equilibrium test p-value < 10. -50 And SNP sites that do not have chromosomal location information.

[0042] Beneficial effects:

[0043] This invention provides the first SNP locus combination for identifying Shenxian pigs. By applying this SNP characteristic locus combination, Shenxian pigs can be distinguished from Large White pigs, Duroc pigs, Lantang pigs, Min pigs, and Beijing Black pigs.

[0044] The SNP locus combination of this invention can distinguish Shenxian pigs from five other pig breeds with high accuracy;

[0045] The SNP locus combinations of this invention can be used as molecular markers for pig germplasm resource identification, assisted breeding, and other technologies.

[0046] The method of this invention can further collect suspected Shenxian pig individuals, prevent hybrid individuals from contaminating the bloodline, effectively improve the economic benefits of breeding pigs, and is of great significance for expanding the scale of the Shenxian pig population and its subsequent rational development and utilization. Attached Figure Description

[0047] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0048] Figure 1 The following is a graph showing the results of principal component analysis of Shenxian pigs, Large White pigs, Duroc pigs, Beijing Black pigs, Lantang pigs, and Min pigs based on 139,547 SNP loci, provided for embodiments of the present invention: where pc1: principal component 1; pc2: principal component 2;

[0049] Figure 2 The following is a graph showing the results of principal component analysis of Shenxian pigs, Large White pigs, Duroc pigs, Beijing Black pigs, Lantang pigs, and Min pigs based on 10 selected SNP feature sites of Shenxian pigs, provided for embodiments of the present invention: where pc1: principal component 1; pc2: principal component 2;

[0050] Figure 3 This is a clustering diagram of genomic kinship among 109 individuals calculated based on 10 SNP feature sites selected by the present invention, provided for an embodiment of the present invention. Detailed Implementation

[0051] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0052] Example 1. Screening of SNP sites

[0053] Experimental pig herd: The materials used in this invention include 39 Shenxian pigs, 16 Large White pigs, 22 Duroc pigs, 22 Beijing Black pigs, 5 Lantang pigs, and 5 Min pigs.

[0054] Next-generation resequencing was performed on 39 Shenxian pig individuals using a DNBseq PE150 manufactured by BGI. Low-quality sequences and adapter sequences were removed using TrimGalore 0.6.5 with the following parameters: --quality 20, --phred33, --stringency 3, --length 20, -e 0.1.

[0055] The quality-controlled data were aligned to the reference genome of pig version 11.1 (Sus scrofa 11.1) using BWA 0.7.17, and population SNPs were detected in Shenxian pigs using GATK 4.2.0.0.

[0056] The whole genome SNP data of Shenxian pigs were combined with the whole genome SNP data of 70 other pig breeds downloaded from public databases for quality control. The quality control criteria were: SNP removal detection rate <99%, minimum allele frequency <0.05, and Hardy-Weinberg equilibrium test p-value <10. -50In addition to SNP loci without chromosomal location information, a total of 139,547 SNP loci were obtained. Principal component analysis was performed on 109 individuals from 6 varieties using these loci, and the results are as follows: Figure 1 As shown, SX represents Shenxian pig, YY represents Large White pig, DD represents Duroc pig, BJ represents Beijing Black pig, LT represents Lantang pig, and MIN represents Min pig. Figure 1 It is known that Shenxian pigs can be distinguished from Large White pigs, Beijing Black pigs, and Duroc pigs, but cannot be distinguished from Lantang pigs and Min pigs.

[0057] 70% (87 individuals) of 6 breeds were randomly assigned to the training set, and the remaining 30% (22 individuals) were assigned to the test set. The maximum classification ability method was used to select informative SNPs in the training set, resulting in 1792 SNPs with strong classification ability. Then, a logistic regression classifier was used for regularization to select 9 SNP loci. Based on the previously selected 9 loci, a Naive Bayes classifier was used to further select loci using a wrapper method, finally obtaining a total of 10 SNP loci, at which point the accuracy reached its maximum.

[0058] The accuracy of the 10 SNP loci was calculated using Naive Bayes, Logistic Regression, and k-Nearest Neighbor classifiers in the test set, and the results were 0.9545, 0.9545, and 1.0000, respectively.

[0059] The 10 selected SNP loci are: C or T at position chr1:93005281, C or G at position chr1:252504281, C or T at position chr1:2876173, A or G at position chr2:11972846, C or T at position chr2:24851224, A or G at position chr3:45739362, T or G at position chr4:13264289, C or T at position chr9:111271153, C or T at position chr10:3286345, and C or G at position chr10:15307369.

[0060] Table 1 shows the genotypes of the 10 SNP loci in Shenxian pigs and five other breeds. SX represents Shenxian pigs, YY represents Large White pigs, DD represents Duroc pigs, BJ represents Beijing Black pigs, LT represents Lantang pigs, and MIN represents Min pigs. Taking the SNP locus chr3:45739362 as an example, the proportion of AA genotypes is very high in Shenxian pigs (32 / 39), while in other breeds, the genotype at this locus is AG or GG. The predominant genotype of each SNP locus in Shenxian pigs differs from that of other breeds. Therefore, the combination of these 10 characteristic loci can distinguish Shenxian pigs from Large White pigs, Duroc pigs, Beijing Black pigs, Lantang pigs, and Min pigs.

[0061] Table 1. Distribution of 10 SNP characteristic sites in 6 varieties

[0062]

[0063]

[0064]

[0065] Example 2. Application of 10 SNP loci in differentiating Shenxian pigs from other pigs

[0066] To verify whether these 10 SNP characteristic loci could distinguish Shenxian pigs from Large White, Duroc, Beijing Black, Lantang, and Min pigs, principal component analysis was performed on all 109 individuals using these 10 SNP loci. The results are as follows: Figure 2 As shown in the figure, these 10 SNP loci can distinguish Shenxian pigs from five other breeds, indicating that the 10 SNP characteristic loci screened in this invention can be used for the identification of Shenxian pigs. Furthermore, based on these 10 loci, the genomic relationships between 109 individuals were calculated using GCTA software, and a clustering diagram was drawn, as shown below. Figure 3 The clustering results show that 39 Shenxian pigs clustered into a large group, which can be well distinguished from other pig breeds.

[0067] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of SNP locus combinations, reagents, or gene chips in pig breed identification and / or pig breeding; The pig breed identification refers to the identification of Shenxian pigs and non-Shenxian pig breeds; the non-Shenxian pig breeds are one or more of the following: Large White, Duroc, Beijing Black, Lantang, and Min pigs. The SNP locus combination consists of the following 10 SNP loci: SNP locus 1: C or T at position chr1:93005281 SNP site 2: C or G at position chr1:252504281 SNP site 3: C or T at position chr1:2876173 SNP site 4: A or G at position chr2:11972846 SNP locus 5: C or T at position chr2:24851224. SNP locus 6: A or G at position chr3:45739362 SNP site 7: T or G at position chr4:13264289 SNP locus 8: chr9: C or T at position 111271153; SNP locus 9: C or T at position chr10:3286345 and C or G at SNP site 10: chr10:15307369; The location of the SNP site was determined based on the pig Sscrofa11.1 reference genome sequence; The reagent is used to detect single nucleotide polymorphisms at the SNP site; the reagent includes PCR primers for amplifying products containing the SNP site. The gene chip contains nucleotide probes for detecting the SNP sites; The nucleotide probe is immobilized on a solid support.

2. A method for identifying Shenxian pigs from Large White pigs, Duroc pigs, Beijing Black pigs, Lantang pigs, and Min pigs based on the SNP locus combinations described in claim 1, characterized in that, include: (a) Extracting genomic DNA from the pig to be tested as a template; (b) Amplify and sequence the target fragment containing the SNP site combination; (c) Determine the breed of the pig based on the genotype of the corresponding SNP locus; When the genotype results of the SNP sites show that the bases corresponding to SNP sites 1 to SNP sites 10 are CT / TT, GC / CC, CT / TT, GG / AG, CC / TC, AA / GA, GG / TG, TT / CT, TC / CC, and GG / CG, then it is a Shenxian pig.

3. The method for screening SNP site combinations according to claim 1, characterized in that, Includes the following steps: (a) The whole genome SNP data obtained from the resequencing of Shenxian pigs were quality controlled and merged with the whole genome SNP data of Large White pigs, Duroc pigs, Beijing Black pigs, Lantang pigs and Min pigs in known public databases; (b) Divide 70% of the individuals of different varieties in step (a) into the training set and the remaining 30% into the test set; (c) Select SNPs using the maximum classification ability method; (d) Combine the logistic regression classifier and the Bayesian classifier in machine learning methods to perform feature selection on the SNP information selected in step (c); (e) Validate the feature sites selected in step (d) on the test set using principal component analysis.

4. The screening method according to claim 3, characterized in that, In step (a), whole genome SNP data of 39 Shenxian pigs were obtained, and whole genome SNP data of 16 Large White pigs, 22 Duroc pigs, 22 Beijing Black pigs, 5 Lantang pigs and 5 Min pigs were selected for quality control and merging. The quality control standards are: SNP detection rate <99%, minimum allele frequency <0.05, Hardy-Weinberg equilibrium test P value <10-50, and SNP sites without chromosomal location information.

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