Miniature pig SNP (Single Nucleotide Polymorphism) molecular marker combination and application thereof

Abstract

The invention relates to the technical field of biological gene detection, and particularly discloses a miniature pig SNP (Single Nucleotide Polymorphism) molecular marker combination and application thereof. The miniature pig SNP molecular marker combination disclosed by the invention comprises at least one of 221 SNP molecular markers, the physical positions of the 221 SNP molecular markers are determined by performing sequence alignment on whole genome sequencing with the coverage rate of 10 * on the basis of miniature pigs, and the site information of the 221 SNP molecular markers is shown in a table 1. The miniature pig SNP molecular marker combination provided by the invention is used for detecting the SNP genotyping of miniature pigs, and has the properties of novelty, stability, economy and high efficiency.

Description

Technical Field

[0001] The present application relates to the technical field of biological gene detection, and specifically to a miniature pig SNP molecular marker combination and its application. Background Art

[0002] The genetic quality monitoring of experimental animals is to conduct consistency tests on the genetic background of inbred animals and population genetics analysis of closed group animals through scientific methods to evaluate whether they meet the characteristics of the strain or group, and ensure the inherent genetic composition and biological characteristics of the breed and strain of animals. With the development of life sciences, the genetic quality detection technology and methods of experimental animals have also been continuously improved. From the initial morphology, chromosome technology, protein molecules and immunology technology to molecular biology gene detection, it has experienced a development process from extensive to fine, from qualitative to quantitative, and from cumbersome to simple. The above developments have not only significantly improved the accuracy and reliability of genetic quality evaluation of experimental animals, reduced workload and shortened the detection cycle, but also ensured the reliability of genetic quality of experimental animals through the improvement of genetic detection technology, and played an important role in the classification of experimental animals, the maintenance of varieties and strains, the preservation, development and utilization of resources, and the selection and breeding of animals.

[0003] Genetic markers refer to genes used as markers in genetic analysis, mainly including morphological markers, cytological markers, biochemical markers and molecular markers. The continuous development of biological science and technology has also put forward higher requirements and challenges for genetic markers. Ideal genetic markers should have the characteristics of high density, strong genetic stability, simple genetic behavior, no influence from internal and external environment, and easy to realize analysis automation. Under this premise, DNA molecular markers become the most ideal genetic markers. DNA molecular markers are based on DNA sequence polymorphism. The earliest restriction fragment length polymorphism (RFLP) was identified as the first generation of DNA molecular markers. The second generation of DNA molecular markers are microsatellites, which are mainly composed of tandem repeats of 2, 3 or 4 nucleotides. As a third generation genetic marker, SNP is easier to be confirmed in the genome, and can be stably inherited between parents and offspring. Therefore, it is widely used in clinical and scientific research.

[0004] Single nucleotide polymorphism (SNP) refers to the polymorphic changes in DNA sequences caused by the variation of a single nucleotide. The widespread existence of genomic variation was first discovered in 1960. In 1978, Kan and Dozy announced that SNP could be used to explain the differences in the origins of different populations, susceptibility to various genetic diseases, and physiological characteristics such as appearance at the genomic level. After restriction fragment length polymorphism and simple repeat sequence polymorphism, the emergence of the third-generation genetic marker SNP plays an important role in biomedical research.

[0005] Miniature pigs belong to the class Mammalia, order Artiodactyla, family Sus, genus Sus, and species Sus. They have a chromosome number of 2n=38. There are many varieties of miniature pigs, and their growth and reproductive performance vary due to differences in genetic background, geographical environment, and other factors. Miniature pigs have strong resistance to adversity and are highly adaptable to adverse ecological and feed conditions. They have simple feed requirements and are kept in a relatively extensive manner. Their average lifespan in the natural environment is more than 10 years, and is around 15 years[5]. Miniature pigs and micro-pigs are differentiated by weight. Generally, pigs weighing 45 to 65 kg at 12 months of age are miniature pigs, and pigs weighing 25 to 35 kg at 12 months of age are called micro-pigs. Compared with rodents and non-human primates, miniature pigs are not as small in size, grow faster, and are easier to operate in experiments, but they also have some irreplaceable advantages as model animals. Compared with rodents, miniature pigs are more closely related to humans. In terms of organ size and physiological structure, miniature pigs can supplement many animal model experiments that rodents are not suitable for. Food and Drug Administrations around the world stipulate that before clinical trials of new drugs, safety and toxicity assessments must be conducted on at least one large non-rodent animal (usually monkeys, dogs, and pigs). Compared with non-human primates, although non-human primates are the closest group to humans in evolutionary history, they are highly homologous to humans in terms of cognitive ability, reproduction, and fertility. However, non-human primate resources are relatively scarce, resulting in high experimental costs. In addition, only a few laboratories have the technology to genetically modify them, and many ethical issues need to be considered. In comparison, miniature pigs have more abundant genetic resources and numbers, making them easier to use widely.

[0006] The existing GB14923-2022 "Genetic Quality Control of Laboratory Animals" standard was revised in 2022. The standard recommends microsatellite marker genes suitable for miniature pigs, but does not clearly specify SNP genetic quality detection methods and standard detection systems applicable to miniature pigs.

[0007] Therefore, it is urgent to build an internationally advanced SNP genetic quality detection method and standard technical system suitable for miniature pigs. This can not only fill the gaps in relevant domestic and international standards, but also has important significance for improving the level of genetic detection technology for miniature pig populations and ensuring the quality of miniature pig populations. Summary of the invention

[0008] In order to solve the above technical problems, the present application provides a miniature pig SNP molecular marker combination and its application.

[0009] In the first aspect, the present application provides a miniature pig SNP molecular marker combination, including at least one of 221 SNP molecular markers, the physical positions of the 221 SNP molecular markers are determined based on sequence alignment of whole genome sequencing of miniature pigs with a coverage of 10×, and the site information of the 221 SNP molecular markers is shown in Table 1.

[0010] In view of the shortcomings of existing miniature pig genetic quality detection methods, such as complex operation, time-consuming and labor-intensive, difficult result analysis, and unsuitability for high-throughput detection, the purpose of this application is to establish a SNP site genetic detection method suitable for miniature pig germplasm resources. The system is novel, stable, economical and efficient for SNP site detection in miniature pigs.

[0011] In a second aspect, the present application provides a method for screening the loci of the miniature pig SNP molecular marker combination, comprising the following steps: (1) Whole-genome sequencing and sequencing quality control of miniature pigs: Whole-genome sequencing of miniature pigs was performed with a coverage of 10×. Raw reads were filtered using fastp software. Paired-end mapping to MunDraft was performed using BWA software. SNP calling and filtering were performed using GATK4.1 software. Data quality control was performed using BCFtools software, and the following criteria were set: SNP sites with a depth of less than 2.0 were removed, SNP sites with a fisherman strand of less than 60.0 were removed, SNP sites with an RMS mapping quality of less than 30.0 were removed, and SNP sites with a depth of less than 8.0 were discarded. (2) Locus screening was performed according to the following five criteria: SNP loci were screened from different genomic fragments; the genotype frequency of the SNP loci was between 25% and 75%; there was no complete linkage between the SNP loci; no other SNPs or Indels were found within the 200 bp region upstream or downstream of the selected SNP loci; the SNP loci existed in the missense mutation, nonsense mutation and non-interrupted mutation regions of the gene region.

[0012] In a third aspect, the present application provides a primer set for detecting the miniature pig SNP molecular marker combination, and the position information of the primer set is shown in Table 4.

[0013] In a fourth aspect, the present application provides a miniature pig SNP detection kit, which is used to detect the miniature pig SNP molecular marker combination.

[0014] Preferably, the kit comprises a primer set and / or a probe for detecting the miniature pig SNP molecular marker combination.

[0015] In a fifth aspect, the present application provides a miniature pig SNP gene detection system, including the miniature pig SNP molecular marker combination or the primer set of the miniature pig SNP molecular marker combination or the miniature pig SNP detection kit.

[0016] Preferably, the miniature pig SNP gene detection system comprises the following steps in sequence: collecting miniature pig samples; extracting and authenticating miniature pig DNA; resequencing the whole genome of miniature pigs; comparing sequencing data, performing bioinformatics analysis, and screening SNP candidate sites located in gene regions; establishing SNP screening criteria to form a set of high-quality SNP site combinations; multiplex PCR primer design and quality verification of all SNP sites; forming a miniature pig multi-target region capture kit to perform genotyping on all SNP sites; multi-target region capture kit verification; and population genetic structure analysis.

[0017] In a sixth aspect, the present application provides the application of at least one of the miniature pig SNP molecular marker combination, the primer set, the miniature pig SNP detection kit, and the miniature pig SNP gene detection system in a miniature pig SNP genetic quality detection method and standard technical system. In summary, the technical solution of this application has the following effects: This study developed a multi-target region capture technology based on second-generation sequencing for SNP genotyping; this application discloses a miniature pig gene detection system using SNP markers, and developed a multi-target region capture technology based on second-generation sequencing for SNP genotyping. Based on the results of second-generation sequencing, 221 SNPs sites were preliminarily screened out. Through genotyping, a multiplex PCR amplification system including 207 SNP sites was established and optimized; a repeatable screening standard was established to preliminarily screen out SNPs that are widely distributed in the genome and have high polymorphism levels. The detection system of this application has novel, stable, cost-effective and efficient properties for SNP genotyping of miniature pigs. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the specific experimental process of the small and medium-sized pig SNP gene detection system in this application.

[0019] Figure 2This is the screening process for SNP molecular markers in this application.

[0020] Figure 3 This is the genetic tree diagram of the four closed groups of miniature pigs in this application.

[0021] Figure 4 Two-dimensional (A) and three-dimensional (B) principal component analysis plots of the four miniature pig populations for the 207 SNPs in this application. DETAILED DESCRIPTION

[0022] The present application is further described in detail below in conjunction with embodiments. These embodiments should not be construed as limiting the scope of protection claimed in the present application. Example

[0023] This embodiment provides a miniature pig SNP gene detection system, the experimental process is as follows Figure 1 As shown, the specific steps are described as follows.

[0024] 1. Collection of Animal Samples Four commonly used closed groups of miniature pigs in China were selected, namely, Hainan Wuzhishan closed group of miniature pigs (HN, 31), Tongzhou Bama closed group of miniature pigs (TZ, 29), Harbin Veterinary Research Institute Large White Pig Closed Group (HSY, 27), Yunnan Banna closed group of miniature pigs (YNF, 30). This experiment has been approved by the Animal Experimentation and Experimental Animal Welfare Committee of Capital Medical University (AEEI-2021-309).

[0025] 2. Preparation of Genomic DNA The DNA was extracted from the ear tissue of miniature pigs using the FastPure Cell / Tissue DNA Isolation Mini Kit DC102 from Vazyme. The absorbance A260 / A280 value was between 1.8 and 2.0 as detected by Nanodrop 2000c micro-spectrophotometer, and the DNA quality inspection was qualified, and it was stored at -20°C for future use.

[0026] 3. Whole genome sequencing and sequencing quality control This application performed whole genome sequencing with a coverage of 10× on 20 Hainan Wuzhishan miniature pigs and 20 Tongzhou Bama miniature pigs, aiming to find potential polymorphic SNP sites through sequence alignment. The whole genome sequencing of 20 Hainan Wuzhishan miniature pigs and 20 Tongzhou Bama miniature pigs was performed with a coverage of 10×.

[0027] Fastp software was used to filter raw reads. Paired-end mapping to MunDraft was performed using BWA software. SNP calling and filtering were performed using GATK4.1 software. BCFtools software was used for data quality control, and the following criteria were set: (1) SNP sites with a depth of less than 2.0 were removed; (2) SNP sites with a fisherman strand less than 60.0 were removed; (3) SNP sites with an RMS mapping quality less than 30.0 were removed; (4) SNP sites with a depth less than 8.0 were discarded.

[0028] 4.SNP locus screening The SNP locus screening criteria for this study include the following five points: (1) Screening SNP loci from different genomic fragments; (2) the genotype frequency of the SNP locus is between 25% and 75%; (3) There is no complete linkage between SNP loci; (4) no other SNPs or Indels were found within the 200 bp region upstream or downstream of the selected SNP locus; (5) These SNP loci exist in the missense mutation, nonsense mutation and non-interrupting mutation regions of the gene region.

[0029] This study followed this strict screening process to screen out high-quality SNP loci combinations suitable for genetic quality identification of miniature pigs.

[0030] By analyzing the second-generation sequencing data of 40 miniature pigs, 221 high-quality SNP sites were finally identified for the development of a miniature pig SNP detection kit ( Figure 2 , Table 1).

[0031] Table 1 Information on 221 SNP loci in miniature pigs 5. Kit Primer Design and Genotyping (1) After obtaining the candidate high-quality SNPs loci, the applicant will submit the region of interest (ROI), i.e., the physical location information of the SNPs, to Beijing Ajitaikang (Jiaxing) Biotechnology Co., Ltd.

[0032] (2) Using MFEprimer v3.1 software, highly specific multiplex PCR primers were designed at both ends of the ROI region based on thermodynamic stability; the length of the amplified sequence was in the range of 160 bp-260 bp.

[0033] (3) The quality of PCR primer pairs was verified, primer dimerization and nonspecific amplification were evaluated, the designed and synthesized primers were tested, and primers with poor detection effects were replaced.

[0034] (4) On the NovaSeqTM 6000 system, PE150 paired-end sequencing with multi-target region capture sequencing technology was used to perform genotyping of SNP loci.

[0035] (5) The instructions for the miniature pig SNP detection kit are as follows: The first round of multiplex PCR reaction is: Take out the Enhancer buffer NB (1N), Primer pool, and IGT-EM808 polymerase mixture in the kit from the -20°C refrigerator in advance, place them on an ice box to melt, vortex briefly to mix, centrifuge them instantaneously, and place them on an ice box for later use.

[0036] Take out Enhancer buffer M from the 4°C refrigerator and thaw at room temperature.

[0037] Prepare the reaction system according to Table 2 on an ice box: Table 2 First round multiplex PCR reaction system Reactants Volume (μL) <![CDATA[ddH2O]]> 9-x Enhancer buffer NB(1N) 3.5 Enhancer buffer M 2.5 Primer pool 5 The sample starting amount is 40ng / reaction tube x IGT-EM808polymerase mixture 10 total 30 For the multiplex PCR reaction, the PCR instrument program was as follows: heating, 105°C; 95°C, 3 min 30 s; 98°C, 20 s; 55°C, 1 min; 60°C, 1 min; 65°C, 2 min; 72°C, 5 min; for 18 cycles.

[0038] Magnetic bead purification product: Prepare 80% ethanol with anhydrous ethanol and Nuclease-Free Water in advance and keep it at room temperature for later use. Please try to use freshly prepared 80% ethanol for magnetic bead purification.

[0039] Take out the purified magnetic beads from the 4°C refrigerator in advance, mix them and place them at room temperature for equilibration for 30 minutes; vortex the purified magnetic beads that have been equilibrated to room temperature and set aside.

[0040] Add 0.9 times the volume of magnetic beads (27 μL) to the 30 μL reaction system, pipette or vortex to mix, and let stand at room temperature for 5 minutes.

[0041] Centrifuge briefly and place the PCR tube on a magnetic rack for 3 minutes to wait for the solution to clarify.

[0042] Remove the supernatant completely, remove the PCR tube from the magnetic stand, add 50 μL YF buffer B to the tube, pipette to mix, and let stand at room temperature for 5 min.

[0043] Centrifuge briefly and place the PCR tube on the DynaMag-96 Side magnetic stand for 3 minutes.

[0044] Keep the PCR tube on the magnetic rack, carefully remove the supernatant, add 180 μL 80% ethanol solution into the PCR tube, and let it stand for 30 seconds.

[0045] Keep the PCR tube on the magnetic rack, discard the supernatant, add 180 μL 80% ethanol solution to the PCR tube again, let it stand for 30 seconds, and discard the supernatant.

[0046] Put the cap on the tube and centrifuge briefly to remove the residual ethanol to the bottom of the tube. Place the PCR tube on the magnetic rack and carefully use a 10μL pipette to remove the residual ethanol at the bottom, being careful not to suck up the magnetic beads.

[0047] Keep the PCR tube on the magnetic rack and let it stand at room temperature for 3 to 5 minutes to dry the magnetic beads and allow the residual ethanol to evaporate completely.

[0048] Add 24 μL Nuclease-Free Water, remove the PCR tube from the magnetic stand, pipette or vortex to mix, and let stand at room temperature for 2 minutes.

[0049] Centrifuge briefly and place the PCR tube on a magnetic rack for 2 minutes to wait for the solution to clarify.

[0050] Use a pipette to take up 13.5 μL of supernatant and transfer it to a new PCR tube. The supernatant in the tube is the purified multiple PCR product, which is marked and used for PCR reaction.

[0051] The second round of adapter sequence PCR reaction is: Reaction system: Prepare the reaction system according to Table 3 on an ice box: Table 3 Second round of linker sequence PCR reaction system The reaction conditions were as follows: heating at 105°C, 95°C for 3 min 30 s, 98°C for 20 s, 58°C for 1 min, 72°C for 30 s, and 72°C for 5 min for 9 cycles.

[0052] Second round of magnetic bead purification Prepare 80% ethanol with anhydrous ethanol and Nuclease-Free Water in advance and keep it at room temperature for later use. Please try to use freshly prepared 80% ethanol for magnetic bead purification.

[0053] Take out the purified magnetic beads from the 4°C refrigerator in advance, mix them and place them at room temperature for equilibration for 30 minutes; vortex the purified magnetic beads that have been equilibrated to room temperature and set aside.

[0054] Add 0.9 times the volume of magnetic beads (27 μL) to the 30 μL reaction system, pipette or vortex to mix, and let stand at room temperature for 5 minutes.

[0055] Centrifuge briefly and place the PCR tube on a magnetic rack for 3 minutes to wait for the solution to clarify.

[0056] Remove the supernatant completely, remove the PCR tube from the magnetic stand, add 50 μL YF buffer B to the tube, pipette to mix, and let stand at room temperature for 5 min.

[0057] Centrifuge briefly and place the PCR tube on the DynaMag-96 Side magnetic stand for 3 minutes.

[0058] Keep the PCR tube on the magnetic rack, carefully remove the supernatant, add 180 μL 80% ethanol solution into the PCR tube, and let it stand for 30 seconds.

[0059] Keep the PCR tube on the magnetic rack, discard the supernatant, add 180 μL 80% ethanol solution to the PCR tube again, let it stand for 30 seconds, and discard the supernatant.

[0060] Put the cap on the tube and centrifuge briefly to remove the residual ethanol to the bottom of the tube. Place the PCR tube on the magnetic rack and carefully use a 10μL pipette to remove the residual ethanol at the bottom, being careful not to suck up the magnetic beads.

[0061] Keep the PCR tube on the magnetic rack and let it stand at room temperature for 3 to 5 minutes to dry the magnetic beads and allow the residual ethanol to evaporate completely.

[0062] Add 24 μL Nuclease-Free Water, remove the PCR tube from the magnetic stand, pipette or vortex to mix, and let stand at room temperature for 2 minutes.

[0063] Centrifuge briefly and place the PCR tube on a magnetic rack for 2 minutes to wait for the solution to clarify.

[0064] Use a pipette to take 20 μL of supernatant and transfer it to a new PCR tube. The supernatant in the tube is the prepared multiplex PCR library.

[0065] Take 1 μL of library for concentration measurement and record the library concentration (the concentration range of normal library is 20 ng / μL to 40 ng / μL, and the library concentration is mainly related to the quality of the template.

[0066] (6) The quality of PCR primers for miniature pigs was verified, primer dimerization and nonspecific amplification were evaluated, the designed and synthesized primers were tested, and primers with poor detection effects were replaced. Finally, the matching degree of 221 primers on the genome reached 100% (Table 4).

[0067] Table 4 Minipig PCR primer positions and predicted coverage 6. Validation of Multi-target Region Capture Sequencing Kit Three companies (Aijitaikang, Tianyi Huiyuan, and Zhongke Guobang) simultaneously verified the miniature pig SNP locus genetic detection kit, as shown in Table 5 below, and verified the sensitivity, specificity, and repeatability of the miniature pig kit: Table 5 Minipig kit validation The experimental results of the three units showed that when the miniature pig DNA concentration was as low as 10 μg / mL, the kit could still perform multiple PCR amplification on the miniature pig SNP combination and accurately type the SNP loci, which showed that the miniature pig kit had good sensitivity; the typing results of the SNP loci of the four miniature pig DNA samples with a concentration of 40 μg / mL were completely consistent, which showed that the miniature pig kit had strong repeatability (Table 6); the experiment used 40 μg / mL miniature pig DNA to verify the specificity of the miniature pig kit and found that the miniature pig kit could not amplify and type the SNP loci of the Chinese hamster DNA sample, which showed that the miniature pig kit had good specificity. In summary, the miniature pig kit performed well in terms of sensitivity, specificity and repeatability.

[0068] Table 6 Minipig test kit validation results 7. Popgen.32 population genetic structure analysis In order to further optimize an efficient combination of SNP loci, the present application performed genotyping tests on 221 SNP loci from 5 miniature pig populations. The results showed that the MAPQ (mapping quality) values ​​of three SNP loci (MG160, MG166 and MG218) were less than 59, indicating that the reads at these three loci were not unique when compared to the reference genome, and the typing results were unreliable. The present application also found that one SNP locus, MP131, was monomorphic in all miniature pig samples, with a genotype of T / T. The present application believes that such a locus with limited polymorphism is not suitable for subsequent population analysis.

[0069] In addition, this study also found several groups of linkage sites MP36, MP39, MP53, MP54, MP189, MP221, MP222, as well as sites with poor sequencing quality MP21 and indel sites MP76, which were subsequently optimized. In summary, this application finally screened out 207 high-quality SNP sites and recommended them for genetic evaluation of miniature pig populations.

[0070] The present application uses Popgen.32 software to analyze the genetic structure of four closed groups of miniature pigs using 207 SNP loci with high amplification quality and good polymorphism in the kit (Table 7). The research results of the present application show that the HN and TZ groups have large values ​​of various group genetic parameters, including the average effective number of alleles (ne*), Shannon index (I*), observed heterozygosity (ObsHet*), expected heterozygosity (Exp Het*), average heterozygosity (Ave Het*) and polymorphic information content (PIC). Then comes the HSY group, and finally the YNF group.

[0071] Table 7 Genetic analysis of four closed populations of miniature pigs This application constructed a genetic tree diagram of four closed groups of miniature pigs based on Nei genetic distance and UPGMA method, and found that the genetic distance between HN group and TZ group was the closest, and they were the farthest from YNF group ( Figure 3 ).

[0072] 8. PCA Principal Component Analysis In this study, principal component analysis was performed on the dimensionality-reduced data to calculate the genetic distance between complex samples. The results of principal component analysis based on the allele frequency distribution of 207 polymorphic SNP loci in four closed populations of miniature pigs are shown in Figure 2. Figure 4 In the PCA results based on genotype, HN and TZ groups are difficult to separate, while YNF and HSY groups can be separated well from other groups ( Figure 4 A). The PCA three-dimensional graph can distinguish the four closed groups of small pigs, but the distance between the HN and TZ groups is still relatively close ( Figure 4 B) These are consistent with the results of the genetic tree diagram.

[0073] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A miniature pig SNP molecular marker combination, characterized in that: Including at least one of 221 SNP molecular markers, the physical positions of the 221 SNP molecular markers are determined by sequence alignment based on whole genome sequencing of miniature pigs with a coverage rate of 10×, and the site information of the 221 SNP molecular markers is shown in Table 1: Table 1 Information on 221 SNP loci in miniature pigs 2. The method for screening loci of miniature pig SNP molecular marker combination according to claim 1, characterized in that: The following steps are involved: (1) Whole-genome sequencing and sequencing quality control of miniature pigs: Whole-genome sequencing of miniature pigs was performed with a coverage of 10×. Raw reads were filtered using fastp software. Paired-end mapping to MunDraft was performed using BWA software. SNP calling and filtering were performed using GATK4.1 software. Data quality control was performed using BCFtools software, and the following criteria were set: SNP sites with a depth of less than 2.0 were removed, SNP sites with a fisherman strand of less than 60.0 were removed, SNP sites with an RMS mapping quality of less than 30.0 were removed, and SNP sites with a depth of less than 8.0 were discarded. (2) Locus screening was performed according to the following five criteria: SNP loci were screened from different genomic fragments; the genotype frequency of the SNP loci was between 25% and 75%; there was no complete linkage between the SNP loci; no other SNPs or Indels were found within the 200 bp region upstream or downstream of the selected SNP loci; the SNP loci existed in the missense mutation, nonsense mutation and non-interrupted mutation regions of the gene region.

3. A primer set for detecting the miniature pig SNP molecular marker combination according to claim 1, characterized in that: The position information of the primer set is shown in Table 4: Table 4 Minipig PCR primer positions and predicted coverage 4. A miniature pig SNP detection kit, characterized in that: The kit is used to detect the miniature pig SNP molecular marker combination described in claim 1.

5. The miniature pig SNP detection kit according to claim 4, characterized in that: The kit comprises a primer set and / or a probe for detecting the miniature pig SNP molecular marker combination according to claim 1.

6. A miniature pig SNP gene detection system, characterized in that: It comprises the miniature pig SNP molecular marker combination according to claim 1 or the primer set of the miniature pig SNP molecular marker combination according to claim 3 or the miniature pig SNP detection kit according to any one of claims 4-5.

7. The miniature pig SNP gene detection system according to claim 6, characterized in that: The method includes the following steps: collecting miniature pig samples; extracting and identifying the quality of miniature pig DNA; and resequencing the whole genome of miniature pigs; Compare sequencing data, perform bioinformatics analysis, and screen SNP candidate sites located in gene regions; Establish SNP screening standards to form a set of high-quality SNP loci combinations; design and quality verification of multiplex PCR primers for all SNP loci; develop a multi-target region capture kit for miniature pigs to perform genotyping on all SNP loci; Validation of multi-target region capture kit; analysis of population genetic structure.

8. Application of at least one of the miniature pig SNP molecular marker combination of claim 1, the primer set of claim 3, the miniature pig SNP detection kit of claim 4, and the miniature pig SNP gene detection system of any one of claims 6-7 in the SNP genetic quality detection method and standard technical system of miniature pigs.

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